Final Report Summary - BCIMPYK2 (Regulation of breast cancer invasion and metastasis by the non-receptor tyrosine kinase Pyk2)
Project overview: Dissemination of cancer cells from the primary tumor and their metastatic spread to distant sites of the body is the leading cause of mortality in breast cancer patients. Metastatic cancer cells invade surrounding tissues and blood vessels by forming F-actin rich protrusions called invadopodia that degrade the extracellular matrix and enable invasion through it. A major challenge in breast cancer research is to elucidate the mechanisms that underlie invadopodia formation and function. The non-receptor tyrosine kinase Pyk2 is highly expressed in invasive cancers, but the signaling mechanism by which Pyk2 regulates invasion and metastasis were unknown. Based on the documented involvement of Pyk2 in cancer and invasion, its localization and important role in podosomes which are surprisingly similar to invadopodia, and the similarity of Pyk2 to FAK that is involved in invadopodia regulation, we hypothesized that Pyk2 has a critical role in invadopodia formation and function as well as in regulation of invasion and metastasis in vivo.
Achievements: Our recent research proposal, which is now in its last year of funding by ISF, aimed to elucidate the molecular, cellular, and whole organism mechanisms by which Pyk2 regulates tissue invasiveness and metastatic potential of breast cancer cells.
Our first goal in the recent research proposal was to elucidate the molecular mechanisms and signaling pathways by which Pyk2 regulates invadopodia formation and/or function. Although a correlation between Pyk2 and cancer invasiveness is now established, it is not clear yet how signaling via this protein leads to the subsequent cytoskeletal rearrangement that are needed for invasion of breast cancer cells to neighboring tissues and for their subsequent metastasis. To explore new signaling pathways that are regulated by Pyk2 in invadopodia, we performed high-throughput protein-protein and kinase-substrate proteomic screens. Bioinformatic analysis of the results from these screens revealed cortactin as novel substrate and interactor of Pyk2. The direct interaction between Pyk2 and cortactin was verified by both in vitro binding and kinase assays and by co-immunoprecipitation and FRET in cells. Additionally, high-resolution 2D and 3D microscopy revealed co-localization of Pyk2 with cortactin to matrix degrading invadopodia. Knockdown of Pyk2 in breast cancer cells showed reduced extracellular matrix degradation and barbed end formation in invadopodia, suggesting that Pyk2 regulates invadopodia maturation and activation by controlling MMP-mediated matrix degradation and actin polymerization in invadopodia. Together, these data indicate that Pyk2 regulates cortactin recruitment and phosphorylation to promote invadopodia maturation in breast cancer cells. Results of this study are described in our manuscript submitted to JCB (Genna et al.).
Our second goal was to compare the overlapping and unique roles of Pyk2 and FAK in invadopodia regulation. Previous studies have shown that FAK can negatively regulate invadopodia downstream of integrins by sequestering active Src kinase to focal adhesions. Accordingly, FAK deletion results transition of active Src from focal adhesions to invadopodia, where it recruits and phosphorylates downstream effectors that are necessary for invadopodia formation and function. However, the molecular mechanism and the identity of the component that localizes and activates Src at invadopodia as a response to extracellular cues were unknown. Using invadopodia formation, immunofluorescence localization, and FRET assays we have shown that Pyk2 regulates invadopodia formation in breast cancer cells by recruiting and activating Src kinase to these structures. Collectively, our data indicate that Pyk2 and FAK regulate the balance between focal adhesions and invadopodia formation in breast cancer cells by competing over Src kinase. Knockdown of Pyk2 significantly reduced MMP-mediated matrix degradation, actin polymerization in invadopodia, and consequent Matrigel invasion, while knockdown of FAK showed increased matrix degradation and actin polymerization in invadopodia, but decreased Matrigel invasion. We conclude that both Pyk2 and FAK regulate tumor cell invasion, albeit via different mechanisms: Pyk2 regulates invadopodia formation by recruitment of Src kinase, and MMP-mediated matrix degradation and actin-polymerization dependent invasion via recruitment and activation of cortactin, while FAK regulates focal adhesion-mediated motility and consequent breast cancer cell invasion via recruitment and activation of Src kinase and cortactin to focal adhesions. Results of this study are described in our manuscript submitted to JCB (Genna et al.).
Our third goal in the recent research proposal was to determine how deletion of Pyk2 affects the tumorigenic, invasive, and metastatic potential of breast cancer cells in vivo. To examine the role of Pyk2 in invadopodia-mediated metastasis in vivo, we generated a xenograft model of MDA-MB-231 metastatic breast cancer cells that were knocked down for either Pyk2 or FAK. Our findings indicated that knockdown of either Pyk2 or FAK reduced local invasiveness as measured by the in vivo invasion assay. Moreover, both Pyk2 knockdown or FAK knockdown showed significant reduction of lung metastasis. Using the mammary imaging window combined with Dendra2 photoswitching in intravital imaging, we have shown that knockdown of FAK significantly reduces motility of the cells, while Pyk2 knockdown does not interfere with cell motility. Immunofluorescence staining of primary MDA-MB-231 breast tumors showed localization of Pyk2 to cortactin-containing, matrix degrading invadopodia in vivo. Additionally, intravital live imaging revealed that formation of invadopodial protrusions in Pyk2 knockdown cells is significantly lower, while protrusion formation in FAK knockdown cells is significantly higher than control. In agreement with our in vitro and intravital imaging experiments, in situ degradation assay on primary tumor slices revealed increased extracellular matrix degradation in FAK knockdown tumors, and less degradation in Pyk2 knockdown tumors. Together, these observations indicate that both Pyk2 and FAK regulate breast cancer metastasis albeit via different mechanisms. Pyk2 regulates metastatic dissemination by controlling invadopodia-mediated invasion, while FAK mediates tissue invasiveness by controlling focal adhesion-mediated motility. Overall, our data suggest that Pyk2 and FAK may be controlling the in vivo phenotypic switch between fast-locomoting and slow-locomoting, matrix degrading tumor cells. Results of this study are described in our manuscript submitted to Cancer Research (Alter et al.).
Our long-term goal was to use our findings in order to develop a strategy for inhibiting breast cancer metastasis. Our studies identified a novel signaling pathway by which Pyk2 regulates invadopodial function and consequent breast cancer metastasis. We now aim to use the information gained from these studies in order to develop novel inhibitors for breast cancer metastasis. Using an in silico high-throughput screen, we have identified five highly specific kinase inhibitors for Pyk2 (Meirson et al., in review at Scientific Reports), that we intend to test in vitro by invadopodia assays and in vivo using mouse models. In addition, we have now solved the crystal structure of the free, unbound SH3 domain of cortactin (Twafra et al. Submitted to Acta Crystallographica), and the NMR structures of cortactin SH3 with proline-rich peptides of Pyk2. We intend to use this information in order to design peptide-mimetic and small molecule protein-protein interaction inhibitors of the Pyk2-cortactin interaction, which will be later tested for their ability to inhibit invadopodia-mediated breast cancer metastasis.
Additional achievements: In collaboration with Dr. Anthony Koleske from Yale University, we have identified the Rho-family guanine nucleotide exchange factor (GEF) Vav2 as a new interactor of cortactin and a regulator of invadopodia maturation. Our data indicate that cortactin recruits Vav2 to activate Rac3 and promote invadopodial maturation in invasive breast cancer cells. Results of this study are described in our manuscript submitted to MBOC (Rosenberg, Gil-Henn, et al). In addition, we are now revising a detailed protocol for the in vitro quantitative pull-down assay, which enables quantitative measurement of binding between two purified proteins (Lapetina & Gil-Henn, accepted for publication).
Our studies resulted two manuscripts that are currently under review and four additional manuscripts that have now been submitted to Molecular Biology of the Cell, Journal of Cell Biology, Cancer Research, and Acta Crystallographica. In addition, we are now preparing a manuscript for submission, which focuses on comparative bioinformatic analysis of Pyk2 and FAK signaling pathways.
Achievements: Our recent research proposal, which is now in its last year of funding by ISF, aimed to elucidate the molecular, cellular, and whole organism mechanisms by which Pyk2 regulates tissue invasiveness and metastatic potential of breast cancer cells.
Our first goal in the recent research proposal was to elucidate the molecular mechanisms and signaling pathways by which Pyk2 regulates invadopodia formation and/or function. Although a correlation between Pyk2 and cancer invasiveness is now established, it is not clear yet how signaling via this protein leads to the subsequent cytoskeletal rearrangement that are needed for invasion of breast cancer cells to neighboring tissues and for their subsequent metastasis. To explore new signaling pathways that are regulated by Pyk2 in invadopodia, we performed high-throughput protein-protein and kinase-substrate proteomic screens. Bioinformatic analysis of the results from these screens revealed cortactin as novel substrate and interactor of Pyk2. The direct interaction between Pyk2 and cortactin was verified by both in vitro binding and kinase assays and by co-immunoprecipitation and FRET in cells. Additionally, high-resolution 2D and 3D microscopy revealed co-localization of Pyk2 with cortactin to matrix degrading invadopodia. Knockdown of Pyk2 in breast cancer cells showed reduced extracellular matrix degradation and barbed end formation in invadopodia, suggesting that Pyk2 regulates invadopodia maturation and activation by controlling MMP-mediated matrix degradation and actin polymerization in invadopodia. Together, these data indicate that Pyk2 regulates cortactin recruitment and phosphorylation to promote invadopodia maturation in breast cancer cells. Results of this study are described in our manuscript submitted to JCB (Genna et al.).
Our second goal was to compare the overlapping and unique roles of Pyk2 and FAK in invadopodia regulation. Previous studies have shown that FAK can negatively regulate invadopodia downstream of integrins by sequestering active Src kinase to focal adhesions. Accordingly, FAK deletion results transition of active Src from focal adhesions to invadopodia, where it recruits and phosphorylates downstream effectors that are necessary for invadopodia formation and function. However, the molecular mechanism and the identity of the component that localizes and activates Src at invadopodia as a response to extracellular cues were unknown. Using invadopodia formation, immunofluorescence localization, and FRET assays we have shown that Pyk2 regulates invadopodia formation in breast cancer cells by recruiting and activating Src kinase to these structures. Collectively, our data indicate that Pyk2 and FAK regulate the balance between focal adhesions and invadopodia formation in breast cancer cells by competing over Src kinase. Knockdown of Pyk2 significantly reduced MMP-mediated matrix degradation, actin polymerization in invadopodia, and consequent Matrigel invasion, while knockdown of FAK showed increased matrix degradation and actin polymerization in invadopodia, but decreased Matrigel invasion. We conclude that both Pyk2 and FAK regulate tumor cell invasion, albeit via different mechanisms: Pyk2 regulates invadopodia formation by recruitment of Src kinase, and MMP-mediated matrix degradation and actin-polymerization dependent invasion via recruitment and activation of cortactin, while FAK regulates focal adhesion-mediated motility and consequent breast cancer cell invasion via recruitment and activation of Src kinase and cortactin to focal adhesions. Results of this study are described in our manuscript submitted to JCB (Genna et al.).
Our third goal in the recent research proposal was to determine how deletion of Pyk2 affects the tumorigenic, invasive, and metastatic potential of breast cancer cells in vivo. To examine the role of Pyk2 in invadopodia-mediated metastasis in vivo, we generated a xenograft model of MDA-MB-231 metastatic breast cancer cells that were knocked down for either Pyk2 or FAK. Our findings indicated that knockdown of either Pyk2 or FAK reduced local invasiveness as measured by the in vivo invasion assay. Moreover, both Pyk2 knockdown or FAK knockdown showed significant reduction of lung metastasis. Using the mammary imaging window combined with Dendra2 photoswitching in intravital imaging, we have shown that knockdown of FAK significantly reduces motility of the cells, while Pyk2 knockdown does not interfere with cell motility. Immunofluorescence staining of primary MDA-MB-231 breast tumors showed localization of Pyk2 to cortactin-containing, matrix degrading invadopodia in vivo. Additionally, intravital live imaging revealed that formation of invadopodial protrusions in Pyk2 knockdown cells is significantly lower, while protrusion formation in FAK knockdown cells is significantly higher than control. In agreement with our in vitro and intravital imaging experiments, in situ degradation assay on primary tumor slices revealed increased extracellular matrix degradation in FAK knockdown tumors, and less degradation in Pyk2 knockdown tumors. Together, these observations indicate that both Pyk2 and FAK regulate breast cancer metastasis albeit via different mechanisms. Pyk2 regulates metastatic dissemination by controlling invadopodia-mediated invasion, while FAK mediates tissue invasiveness by controlling focal adhesion-mediated motility. Overall, our data suggest that Pyk2 and FAK may be controlling the in vivo phenotypic switch between fast-locomoting and slow-locomoting, matrix degrading tumor cells. Results of this study are described in our manuscript submitted to Cancer Research (Alter et al.).
Our long-term goal was to use our findings in order to develop a strategy for inhibiting breast cancer metastasis. Our studies identified a novel signaling pathway by which Pyk2 regulates invadopodial function and consequent breast cancer metastasis. We now aim to use the information gained from these studies in order to develop novel inhibitors for breast cancer metastasis. Using an in silico high-throughput screen, we have identified five highly specific kinase inhibitors for Pyk2 (Meirson et al., in review at Scientific Reports), that we intend to test in vitro by invadopodia assays and in vivo using mouse models. In addition, we have now solved the crystal structure of the free, unbound SH3 domain of cortactin (Twafra et al. Submitted to Acta Crystallographica), and the NMR structures of cortactin SH3 with proline-rich peptides of Pyk2. We intend to use this information in order to design peptide-mimetic and small molecule protein-protein interaction inhibitors of the Pyk2-cortactin interaction, which will be later tested for their ability to inhibit invadopodia-mediated breast cancer metastasis.
Additional achievements: In collaboration with Dr. Anthony Koleske from Yale University, we have identified the Rho-family guanine nucleotide exchange factor (GEF) Vav2 as a new interactor of cortactin and a regulator of invadopodia maturation. Our data indicate that cortactin recruits Vav2 to activate Rac3 and promote invadopodial maturation in invasive breast cancer cells. Results of this study are described in our manuscript submitted to MBOC (Rosenberg, Gil-Henn, et al). In addition, we are now revising a detailed protocol for the in vitro quantitative pull-down assay, which enables quantitative measurement of binding between two purified proteins (Lapetina & Gil-Henn, accepted for publication).
Our studies resulted two manuscripts that are currently under review and four additional manuscripts that have now been submitted to Molecular Biology of the Cell, Journal of Cell Biology, Cancer Research, and Acta Crystallographica. In addition, we are now preparing a manuscript for submission, which focuses on comparative bioinformatic analysis of Pyk2 and FAK signaling pathways.