Experience

Publications

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• The death effector domain PEA-15 negatively regulates T cell receptor signaling

  FASEB J.

  PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• The death effector domain PEA-15 negatively regulates T cell receptor signaling

  FASEB J.

  PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

• PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

  Oncogene

  PMID: 22105357

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• The death effector domain PEA-15 negatively regulates T cell receptor signaling

  FASEB J.

  PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

• PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

  Oncogene

  PMID: 22105357

• RSK2 Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

  The Journal of Biological Chemistry

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• The death effector domain PEA-15 negatively regulates T cell receptor signaling

  FASEB J.

  PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

• PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

  Oncogene

  PMID: 22105357

• RSK2 Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

  The Journal of Biological Chemistry

• PEA15 impairs cell migration and correlates with clinical features predicting good prognosis in neuroblastoma.

  Int J Cancer

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• The death effector domain PEA-15 negatively regulates T cell receptor signaling

  FASEB J.

  PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

• PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

  Oncogene

  PMID: 22105357

• RSK2 Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

  The Journal of Biological Chemistry

• PEA15 impairs cell migration and correlates with clinical features predicting good prognosis in neuroblastoma.

  Int J Cancer

• Englerin A selectively induces necrosis in human renal cancer cells

  PLoS ONE

  PMID: 23144724

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• The death effector domain PEA-15 negatively regulates T cell receptor signaling

  FASEB J.

  PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

• PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

  Oncogene

  PMID: 22105357

• RSK2 Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

  The Journal of Biological Chemistry

• PEA15 impairs cell migration and correlates with clinical features predicting good prognosis in neuroblastoma.

  Int J Cancer

• Englerin A selectively induces necrosis in human renal cancer cells

  PLoS ONE

  PMID: 23144724

• Neopetrocyclamines A and B, Polycyclic Diamine Alkaloids from the Sponge Neopetrosia cf exigua

  J. Nat. Prod., Epub: January 13, 2015

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• The death effector domain PEA-15 negatively regulates T cell receptor signaling

  FASEB J.

  PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

• PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

  Oncogene

  PMID: 22105357

• RSK2 Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

  The Journal of Biological Chemistry

• PEA15 impairs cell migration and correlates with clinical features predicting good prognosis in neuroblastoma.

  Int J Cancer

• Englerin A selectively induces necrosis in human renal cancer cells

  PLoS ONE

  PMID: 23144724

• Neopetrocyclamines A and B, Polycyclic Diamine Alkaloids from the Sponge Neopetrosia cf exigua

  J. Nat. Prod., Epub: January 13, 2015

• R-Ras regulates migration through an interaction with filamin A in melanoma cells.

  PLoS One

• Bit-1 mediates integrin-dependent cell survival through activation of the NFKB pathway

  The Journal of Biological Chemistry

• Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter

  Small GTPases

  PMID: 22694972

• Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

  Molecular Biology of the Cell

  Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

• RSK Isoforms in Cancer Cell Invasion and Metastasis

  Cancer Research

  PMID: 24097826

• RSK2 activity mediates glioblastoma invasiveness and is a potential target for new therapeutics

  Oncotarget

  In glioblastoma (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Indeed, patients respond poorly to the current therapies of tumor resection followed by chemotherapy with radiotherapy and have only a 16-month median survival. It is therefore imperative to develop novel therapies. RSK2 is a kinase that regulates proliferation and adhesion and can promote metastasis. We demonstrate that active RSK2 regulates GBM cell adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. RSK2 control of adhesion and migration is mediated in part by its effects on integrin-Filamin A complexes. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. In agreement with the in vitro data, using public datasets, we find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy.

• Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

  Organometallics. 2014 Oct, 33(22), 6669-6681.

• RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

  Proc Natl Acad Sci U S A.

  doi: 10.1073/pnas.1708584115 Cell motility is a dynamic process that requires the directed application of force and continuous coordinated changes in cell adhesion and cytoskeletal architecture often in response to extracellular stimuli. Here we have defined a mechanism by which RSK2 can promote cell migration and invasion in response to promotility stimuli. We show that in response to these signals RSK2 directly binds the RhoGEF LARG and phosphorylates it, thereby promoting LARG activation of RhoA GTPases. Moreover, we find that RSK2 is important for epidermal growth factor activation of Rho GTPases. These results advance our understanding of cell motility, RSK kinase function, and LARG/RhoA activation by revealing that these pathways are integrated and the precise mechanism by which that is accomplished.

• RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration.

  JBC

• Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo.

  Chemical Science

  Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO-) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds.

• PTRH2 gene mutation causes progressive congenital skeletal muscle pathology

  Human Molecular Genetics

  eptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

• The death effector domain PEA-15 negatively regulates T cell receptor signaling

  FASEB J.

  PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

• PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

  Oncogene

  PMID: 22105357

• RSK2 Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

  The Journal of Biological Chemistry

• PEA15 impairs cell migration and correlates with clinical features predicting good prognosis in neuroblastoma.

  Int J Cancer

• Englerin A selectively induces necrosis in human renal cancer cells

  PLoS ONE

  PMID: 23144724

• Neopetrocyclamines A and B, Polycyclic Diamine Alkaloids from the Sponge Neopetrosia cf exigua

  J. Nat. Prod., Epub: January 13, 2015

• R-Ras regulates migration through an interaction with filamin A in melanoma cells.

  PLoS One

• ERK MAP kinase is targeted to RSK2 by the phosphoprotein PEA-15

  Proceedings of the National Academy of Sciences of the United States of America

  The ERK pathway responds to extracellular stimuli and oncogenes by modulating cellular processes, including transcription, adhesion, survival, and proliferation. ERK has diverse substrates that carry out these functions. The processes that are modulated are determined in part by the substrates that ERK phosphorylates. We demonstrate that PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) targets ERK to RSK2 and thereby enhances RSK2 activation. PEA-15 independently bound ERK and RSK2 and increased ERK association with RSK2 in a concentration-dependent manner. PEA-15 increased RSK2 activity and CREB-mediated transcription, and this process was regulated by phosphorylation of PEA-15. Finally, phorbol ester stimulation of PEA-15-null lymphocytes resulted in impaired RSK2 activation that was rescued by exogenous PEA-15 expression. Therefore, PEA-15 functions as a scaffold to enhance ERK activation of RSK2, and this activity is regulated by phosphorylation. Thus, PEA-15 can integrate signal transduction to provide a specific physiological outcome from activation of the multipotent ERK MAP kinase pathway.