Protein Kinase C Substrates in Human Breast Cancer

Summary

Principal Investigator: Susan A Rotenberg
Abstract: DESCRIPTION (provided by applicant): Protein kinase C (PKC) is an upstream component of the EGF-stimulated (erbB2) and IL-8-dependent signaling pathways whose activity has been linked to motile behavior of cultured human breast cells and to the acquisition of metastatic breast cancer. Therefore, in addition to PKC itself, identification of PKC substrate proteins would offer potential targets for developing anti-metastasis chemotherapies. This laboratory recently discovered that [unreadable]-tubulin and Cdc42 effector protein-4 (CEP4) are substrates of PKC in human breast cells. Phosphorylation of [unreadable]-tubulin at Ser-165 modulates microtubule dynamics such that the growth phase is prolonged, which correlates with cell movement. The significance of [unreadable]-tubulin phosphorylation to microtubule dynamics and cell movement will be investigated by testing phosphorylation site mutants of [unreadable]6-tubulin both in non-transformed MCF-10A cells and in highly metastatic human cancer cells (Specific Aim 1). The mechanistic significance of CEP4 phosphorylation will be explored with phosphorylation site-specific mutants in phenotypes of human breast cells (Specific Aim 2). Since CEP4 binds Cdc42 with high affinity, special emphasis will be on determining how CEP4 phosphorylation affects this binding interaction and consequently how Cdc42 signaling is affected. In addition, a role for IQGAP will be explored in the PKC signaling pathway. IQGAP, a known PKC substrate that requires activated Cdc42 for signaling, is also a required component for MT capture at the cell cortex. Re-localization of IQGAP to lamellapodia occurs in response to PKC activation and be used as a read-out for pro-motility signals arising from CEP4 phosphorylation and activated Cdc42. Thus, IQGAP may provide a nexus at which elongating microtubules and the CEP4-Cdc42 complex converge to promote motility. The significance of the three PKC substrates to tumor growth and metastasis will be explored with phosphorylation site-specific mutants expressed by MDA-MB-231 cells in three dimensional culture assays in vitro (Specific Aim 3). The 3-D assays will guide the selection of which substrate to analyze in an orthotopic nude mouse model. For the in vivo studies, the impact of a phosphorylation-resistant PKC substrate will be evaluated in transfectants of luciferase-MDA-MB-231 cells implanted in the fat pads of nude mice. Tumor formation, growth, and metastasis will be tracked by whole animal bioluminescence (Specific Aim 3). Overall, the results of these studies will establish a coherent mechanistic model that incorporates three PKC substrates ([unreadable]-tubulin, CEP4, and IQGAP), and provides a foundation for further investigations of the motility pathway. The anticipated findings are likely to inform strategies for design of novel chemotherapeutics for breast cancer, and to provide new bio-markers for predicting metastatic potential.
Funding Period: 2007-02-01 - 2016-03-31
more information: NIH RePORT

Top Publications

  1. pmc Conformationally constrained analogues of diacylglycerol. 29. Cells sort diacylglycerol-lactone chemical zip codes to produce diverse and selective biological activities
    Dehui Duan
    Laboratory of Medicinal Chemistry, National Cancer Institute at Frederick, National Institutes of Health, 376 Boyles Street, Frederick, Maryland 21702, USA
    J Med Chem 51:5198-220. 2008
  2. pmc Phosphorylation of alpha6-tubulin by protein kinase Calpha activates motility of human breast cells
    Thushara P Abeyweera
    Department of Chemistry and Biochemistry of Queens College of the City University of New York, Flushing, NY 11367 1597, USA
    J Biol Chem 284:17648-56. 2009
  3. pmc PhosphoMARCKS drives motility of mouse melanoma cells
    Xiangyu Chen
    Department of Chemistry and Biochemistry, Queens College, The City University of New York, NY 11367, USA
    Cell Signal 22:1097-103. 2010
  4. pmc Analysis of substrates of protein kinase C isoforms in human breast cells by the traceable kinase method
    Xiangyu Chen
    Department of Chemistry and Biochemistry, Queens College, The City University of New York, 65 30 Kissena Boulevard, Flushing, NY 11367, USA
    Biochemistry 51:7087-97. 2012
  5. pmc Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells
    Shatarupa De
    Department of Chemistry and Biochemistry, Queens College, The City University of New York, New York, New York The Graduate Center, The City University of New York, New York
    Cytoskeleton (Hoboken) 71:257-72. 2014

Detail Information

Publications5

  1. pmc Conformationally constrained analogues of diacylglycerol. 29. Cells sort diacylglycerol-lactone chemical zip codes to produce diverse and selective biological activities
    Dehui Duan
    Laboratory of Medicinal Chemistry, National Cancer Institute at Frederick, National Institutes of Health, 376 Boyles Street, Frederick, Maryland 21702, USA
    J Med Chem 51:5198-220. 2008
    ..Multiple cellular bioassays show that DAG-lactones, which bind in vitro to PKCalpha to varying degrees, expand their biological repertoire into a larger domain, eliciting distinct cellular responses...
  2. pmc Phosphorylation of alpha6-tubulin by protein kinase Calpha activates motility of human breast cells
    Thushara P Abeyweera
    Department of Chemistry and Biochemistry of Queens College of the City University of New York, Flushing, NY 11367 1597, USA
    J Biol Chem 284:17648-56. 2009
    ..These findings support a model in which PKC phosphorylates alpha-tubulin at Ser165, leading to microtubule elongation and motility...
  3. pmc PhosphoMARCKS drives motility of mouse melanoma cells
    Xiangyu Chen
    Department of Chemistry and Biochemistry, Queens College, The City University of New York, NY 11367, USA
    Cell Signal 22:1097-103. 2010
    ..These results demonstrate that phosphoMARCKS contributes to the metastatic potential of melanoma cells, and reveal a previously undocumented signaling role for this cytoplasmic phospho-protein...
  4. pmc Analysis of substrates of protein kinase C isoforms in human breast cells by the traceable kinase method
    Xiangyu Chen
    Department of Chemistry and Biochemistry, Queens College, The City University of New York, 65 30 Kissena Boulevard, Flushing, NY 11367, USA
    Biochemistry 51:7087-97. 2012
    ..These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms...
  5. pmc Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells
    Shatarupa De
    Department of Chemistry and Biochemistry, Queens College, The City University of New York, New York, New York The Graduate Center, The City University of New York, New York
    Cytoskeleton (Hoboken) 71:257-72. 2014
    ..These findings call attention to PKC-mediated phosphorylation of α-tubulin as a novel mechanism for controlling the dynamics of MTs that result in cell movement...

Research Grants30

  1. Molecular Response and Imaging-based Combination Strategies for Optimal PDT
    Tayyaba Hasan; Fiscal Year: 2013
    ..NMSC on the other hand has many options but the high incidence puts a heavy burden on society in terms of cost and suffering. ..
  2. Targeting FGFR to prevent the outgrowth of metastatic breast cancer
    MICHAEL KEITH WENDT; Fiscal Year: 2013
    ..More importantly, our studies will establish a diagnostic platform and therapeutic protocol for the inhibition of FGFR as an effective treatment option for TNBC patients undergoing metastatic tumor recurrence. ..
  3. The Role of HIF-1alpha in Breast Cancer Stem Cell Activity
    TIFFANY NICOLE SEAGROVES; Fiscal Year: 2013
    ....
  4. Targeted Intervention of Breast Oncogenic Pathways
    Said M Sebti; Fiscal Year: 2013
    ..Clinical trials will be performed to evaluate the efficacy of the drug combinations. ..
  5. Chemotherapeutic Agents with Enhanced Selectivity for the Tumor Microenvironment
    Mary Lynn Trawick; Fiscal Year: 2013
    ..Therefore, bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) will be used in this project to evaluate new VDAs. ..
  6. Role of 11q23 Chromosome Abnormalities in the Causation of Acute Leukemia
    Carlo M Croce; Fiscal Year: 2013
    ..abstract_text> ..
  7. INTEGRATIVE PATHOPHYSIOLOGY OF SOLID TUMORS
    Rakesh K Jain; Fiscal Year: 2013
    ..abstract_text> ..
  8. Interrogating Epigenetic Changes in Cancer Genomes
    Tim H M Huang; Fiscal Year: 2013
    ..abstract_text> ..
  9. Mechanistic Pharmacology of Anti-Mitotics and Apoptosis Regulation
    Timothy J Mitchison; Fiscal Year: 2013
    ..In aim 4 we will pursue several approaches towards translating mechanistic understanding from aims 1-3 into improved patient care. ..
  10. Model-based predictions of responses RTK Pathway therapies
    Joe W Gray; Fiscal Year: 2013
    ..abstract_text> ..