FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2006163902> ?p ?o ?g. }

• W2006163902 endingPage "1550" @default.
• W2006163902 startingPage "1540" @default.
• W2006163902 abstract "Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer. Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer. Malignant tumor cells invade surrounding normal tissue and disseminate to distant organs through a multistep and multifactorial process. In general, the acquisition of early autonomous motile property involves cell polarization toward blood and lymphatic vessels in response to chemotactic signals, cell cytoskeleton remodeling, and formation of cell plasma membrane protrusions. The latter are the site of dynamic turnover of multiple focal adhesion molecules, which allow formation of stable cell-matrix attachments near the leading edge of the protrusions, forward movement of the cell body, and disassembly of focal-matrix adhesions and retraction at the trailing edge. These processes are the driving force for early cancer cell migration and invasion. Central to the regulation of the cell migration cycle is the focal adhesion kinase (FAK) and its homologous FAK-related proline-rich tyrosine kinase 2 (Pyk2).1Du QS Ren XR Xie Y Wang Q Mei L Xiong WC Inhibition of PYK2-induced actin cytoskeleton reorganization: PYK2 autophosphorylation and focal adhesion targeting by FAK.J Cell Sci. 2001; 114: 2977-2987PubMed Google Scholar FAK and Pyk2 share 45% amino acid sequence homology with a highly conserved central catalytic kinase domain. The noncatalytic regions of both proteins contain proline-rich residues with binding motifs for Src homology (SH) 3 domain-containing proteins, such as Crk-associated substrate, GTPase regulator associated with FAK, and Pleckstrin homology, and SH3 domain containing Arf-GAP proteins, along with a focal adhesion-targeting domain that is critical for FAK recruitment to focal adhesions and for its association with paxillin and talin.2Chen HC Appeddu PA Parsons JT Hildebrand JD Schaller MD Guan JL Interaction of focal adhesion kinase with cytoskeletal protein talin.J Biol Chem. 1995; 270: 16995-16999Crossref PubMed Scopus (328) Google Scholar In addition, both Pyk2 and FAK contain several tyrosine autophosphorylation sites, including an autophosphorylation site (Y397 for FAK and Y402 for Pyk2), which acts as a binding site for the SH2 site of Src tyrosine kinases. Pyk2 and FAK are activated after integrin clustering in response to various extracellular matrix components, such as fibronectin, and by a number of growth factor receptors, including ErbB tyrosine kinases.3Vadlamudi RK Adam L Nguyen D Santos M Kumar R Differential regulation of components of the focal adhesion complex by heregulin: role of phosphatase SHP-2.J Cell Physiol. 2002; 190: 189-199Crossref PubMed Scopus (46) Google Scholar After activation, both Pyk2 and FAK undergo multiple phosphorylations and engage in protein-protein interactions with several cytoskeletal proteins, such as paxillin; tyrosine kinases, such as Src and C-terminal src-kinase; serine/threonine kinases, such as integrin-linked kinase and p21-activated kinase; and modulators of small GTPases of the Rho family.4Giancotti FG Ruoslahti E Integrin signaling.Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3821) Google Scholar These multiple interactions have been established as critical regulators of early cell invasion signaling, promoting cancer cell migration, and the initiation of metastasis formation.5Sieg DJ Hauck CR Ilic D Klingbeil CK Schaefer E Damsky CH Schlaepfer DD FAK integrates growth-factor and integrin signals to promote cell migration.Nat Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1064) Google Scholar However, increasing evidence points toward distinct and possibly antagonistic functions for Pyk2 and FAK. For instance, FAK is reported to be predominantly localized to focal adhesions,6Andreev J Simon JP Sabatini DD Kam J Plowman G Randazzo PA Schlessinger J Identification of a new Pyk2 target protein with Arf-GAP activity.Mol Cell Biol. 1999; 19: 2338-2350Crossref PubMed Scopus (146) Google Scholar whereas Pyk2 is largely cytosolic,6Andreev J Simon JP Sabatini DD Kam J Plowman G Randazzo PA Schlessinger J Identification of a new Pyk2 target protein with Arf-GAP activity.Mol Cell Biol. 1999; 19: 2338-2350Crossref PubMed Scopus (146) Google Scholar, 7Sieg DJ Ilic D Jones KC Damsky CH Hunter T Schlaepfer DD Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (289) Google Scholar, 8Zheng C Xing Z Bian ZC Guo C Akbay A Warner L Guan JL Differential regulation of Pyk2 and focal adhesion kinase (FAK): the C-terminal domain of FAK confers response to cell adhesion.J Biol Chem. 1998; 273: 2384-2389Crossref PubMed Scopus (127) Google Scholar, 9Sabri A Govindarajan G Griffin TM Byron KL Samarel AM Lucchesi PA Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle.Circ Res. 1998; 83: 841-851Crossref PubMed Scopus (137) Google Scholar, 10Sasaki H Nagura K Ishino M Tobioka H Kotani K Sasaki T Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily.J Biol Chem. 1995; 270: 21206-21219Crossref PubMed Scopus (365) Google Scholar and unlike FAK, its phosphorylation is independent of cell adhesion.10Sasaki H Nagura K Ishino M Tobioka H Kotani K Sasaki T Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily.J Biol Chem. 1995; 270: 21206-21219Crossref PubMed Scopus (365) Google Scholar FAK and Pyk2 contain conserved sites within their COOH-terminal domain for paxillin binding.11Liu S Thomas SM Woodside DG Rose DM Kiosses WB Pfaff M Ginsberg MH Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses.Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar However, FAK but not Pyk2 binds to talin.8Zheng C Xing Z Bian ZC Guo C Akbay A Warner L Guan JL Differential regulation of Pyk2 and focal adhesion kinase (FAK): the C-terminal domain of FAK confers response to cell adhesion.J Biol Chem. 1998; 273: 2384-2389Crossref PubMed Scopus (127) Google Scholar Pyk2 but not FAK associates with Hic-5, Nir (Pyk2 N-terminal domain-interacting receptors), and pancreatic associated protein (Pap) proteins.6Andreev J Simon JP Sabatini DD Kam J Plowman G Randazzo PA Schlessinger J Identification of a new Pyk2 target protein with Arf-GAP activity.Mol Cell Biol. 1999; 19: 2338-2350Crossref PubMed Scopus (146) Google Scholar, 12Lev S Hernandez J Martinez R Chen A Plowman G Schlessinger J Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein.Mol Cell Biol. 1999; 19: 2278-2288Crossref PubMed Google Scholar, 13Matsuya M Sasaki H Aoto H Mitaka T Nagura K Ohba T Ishino M Takahashi S Suzuki R Sasaki T Cell adhesion kinase beta forms a complex with a new member: Hic-5, of proteins localized at focal adhesions.J Biol Chem. 1998; 273: 1003-1014Crossref PubMed Scopus (116) Google Scholar Similarly, Pyk2 but not FAK binds to PSGAP, a Pleckstrin homology and SH3 domain containing Rho GTPase activating protein (RhoGAP) protein capable of stimulating Cdc42 and RhoA, and inhibits its effect on Cdc42.14Ren XR Du QS Huang YZ Ao SZ Mei L Xiong WC Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel Pleckstrin homology and Src homology 3 domain containing rhoGAP protein.J Cell Biol. 2001; 152: 971-984Crossref PubMed Scopus (101) Google Scholar Moreover, expression of Pyk2 induces apoptosis in several cell lines15Xiong W Parsons JT Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase.J Cell Biol. 1997; 139: 529-539Crossref PubMed Scopus (151) Google Scholar, 16Frisch SM Vuori K Ruoslahti E Chan-Hui PY Control of adhesion-dependent cell survival by focal adhesion kinase.J Cell Biol. 1996; 134: 793-799Crossref PubMed Scopus (998) Google Scholar and negatively regulates cell cycle progression,17Zhao J Pestell R Guan JL Transcriptional activation of cyclin D1 promoter by FAK contributes to cell cycle progression.Mol Biol Cell. 2001; 12: 4066-4077Crossref PubMed Scopus (163) Google Scholar, 18Zhao J Zheng C Guan J Pyk2 and FAK differentially regulate progression of the cell cycle.J Cell Sci. 2000; 113: 3063-3072PubMed Google Scholar whereas FAK can protect against apoptosis and promotes cell cycle progression.17Zhao J Pestell R Guan JL Transcriptional activation of cyclin D1 promoter by FAK contributes to cell cycle progression.Mol Biol Cell. 2001; 12: 4066-4077Crossref PubMed Scopus (163) Google Scholar, 19Lunn JA Jacamo R Rozengurt E Preferential phosphorylation of focal adhesion kinase tyrosine 861 is critical for mediating an anti-apoptotic response to hyperosmotic stress.J Biol Chem. 2007; 282: 10370-10379Crossref PubMed Scopus (29) Google Scholar These distinct mechanisms could explain the discrepant functional assignments and biological implications reported for FAK versus Pyk2.20Lipinski CA Tran NL Menashi E Rohl C Kloss J Bay RC Berens ME Loftus JC The tyrosine kinase pyk2 promotes migration and invasion of glioma cells.Neoplasia. 2005; 7: 435-445Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 21Bogenrieder T Herlyn M Axis of evil: molecular mechanisms of cancer metastasis.Oncogene. 2003; 22: 6524-6536Crossref PubMed Scopus (500) Google Scholar, 22de Amicis F Lanzino M Kisslinger A Cali G Chieffi P Ando S Mancini FP Tramontano D Loss of proline-rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells.J Cell Physiol. 2006; 209: 74-80Crossref PubMed Scopus (21) Google Scholar, 23Stanzione R Picascia A Chieffi P Imbimbo C Palmieri A Mirone V Staibano S Franco R De Rosa G Schlessinger J Tramontano D Variations of proline-rich kinase Pyk2 expression correlate with prostate cancer progression.Lab Invest. 2001; 81: 51-59Crossref PubMed Scopus (41) Google Scholar, 24Yano H Mazaki Y Kurokawa K Hanks SK Matsuda M Sabe H Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion.J Cell Biol. 2004; 166: 283-295Crossref PubMed Scopus (207) Google Scholar, 25Yano H Uchida H Iwasaki T Mukai M Akedo H Nakamura K Hashimoto S Sabe H Paxillin alpha and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation.Proc Natl Acad Sci USA. 2000; 97: 9076-9081Crossref PubMed Scopus (77) Google Scholar In this study, we investigated the functional role of Pyk2 versus FAK in ErbB-2-positive breast cancer cell proliferation, migration, and invasion and its correlation to human breast cancer progression. We report that both Pyk2 and FAK are co-overexpressed in early-stage breast cancer and in ErbB-2-positive human breast cancers, supporting a function in breast cancer progression. In contrast to FAK, we demonstrate that Pyk2 has a distinct function in the regulation of cell proliferation of ErbB2-positive cells but little impact on cell invasion; this mechanism involves at least in part modulation of the mitogen-activated protein kinase (MAPK) pathway. We demonstrate that dual down-regulation of Pyk2 and FAK results in a potent inhibition of breast cancer progression in a preclinical invasive breast cancer model. The following antibodies were used: Monoclonal anti-ErbB-2 (Ab-3, clone 3B5), polyclonal anti-ErbB-2 (Ab-1), and polyclonal anti-ErbB-3 (clone C-17) were from Oncogene Science (Cambridge, MA); anti-FAK (clone 4.47) was from Biosource International (Camarillo, CA); anti-phosphotyrosine antibody (4G10) and anti-extracellular signal-regulated kinase 2 (Erk2; clone B3B9) were obtained from Upstate (Lake Placid, NY); anti-Pyk2 was obtained from BD Bioscience Transduction Laboratories (Lexington, KY); anti-phospho-MAPK was obtained from New England Biolabs Inc. (Ipswich, MA); rhodamine conjugated to phalloidin was from Sigma-Aldrich (St. Louis, MO), and anti-glyceraldehyde-3-phosphate dehydrogenase was from Cedarlane (Burlington, ON, Canada). Heregulin (HRG) was from Neomarkers (Fremont, CA), and the mitogen-activated protein kinase kinase 1 inhibitor UO126 was from Alexis Biochemicals (Lausen, Switzerland). Mouse embryonic fibroblast (FAK+/+ or FAK−/−) cells were kindly provided by Dr. Dusko Ilic (University of California, San Francisco, CA). The breast adenocarcinoma cell lines MDA 231-AP2, MDA 231-ErbB-2, and MDA 231-M were described previously.26Benlimame N He Q Jie S Xiao D Xu YJ Loignon M Schlaepfer DD Alaoui-Jamali MA FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.J Cell Biol. 2005; 171: 505-516Crossref PubMed Scopus (116) Google Scholar FAK+/+ or FAK−/− cells were cultured in DMEM (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 1% (v/v) nonessential amino acids, 100 μmol/L 2-mercaptoethanol, and penicillin/streptomycin. Human breast adenocarcinoma cells were maintained in RPMI 1640 (Mediatech, Washington, DC) supplemented with 10% fetal bovine serum and penicillin/streptomycin. ErbB-2 and ErbB-3 receptors were expressed in polyclonal cell populations using bicistronic retrovectors that express each of the receptors with the enhanced green fluorescent protein (EGFP) as described previously.27Yen L Benlimame N Nie ZR Xiao D Wang T Al Moustafa AE Esumi H Milanini J Hynes NE Pages G Alaoui-Jamali MA Differential regulation of tumor angiogenesis by distinct ErbB homo- and heterodimers.Mol Biol Cell. 2002; 13: 4029-4044Crossref PubMed Scopus (121) Google Scholar A specific 21-nucleotide sequence of mouse Pyk2 gene (GenBank accession no. NM_172498) (5′-GACCUGUAAGAAAGACUGU-3′), human Pyk2 gene (5′-GTTGCTATAGAAGCAGACC-3′),20Lipinski CA Tran NL Menashi E Rohl C Kloss J Bay RC Berens ME Loftus JC The tyrosine kinase pyk2 promotes migration and invasion of glioma cells.Neoplasia. 2005; 7: 435-445Abstract Full Text PDF PubMed Scopus (114) Google Scholar and human FAK gene (5′-GCATGTGGCCTGCTATGGA-3′)26Benlimame N He Q Jie S Xiao D Xu YJ Loignon M Schlaepfer DD Alaoui-Jamali MA FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.J Cell Biol. 2005; 171: 505-516Crossref PubMed Scopus (116) Google Scholar were cloned as inverted repeats into pSuper-retro puro vector according to the manufacturer's instructions (Oligoengine, Seattle, WA). Control retroviral vector pRetro-Super puro alone or expressing target siRNA was transfected into Phoenix cells using Genejuice (Novagen, Darmstadt, Germany). Forty-eight hours after transfection, the supernatant of phoenix cells was filtered through 0.45-μm filter and used to infect target cell lines twice, 24 hours apart, in the presence of 8 μg/ml polybrene. Forty-eight hours after infection, polyclonal populations were selected for resistance to 1 μg/ml puromycin for 2 weeks to generate stable siRNA-expressing cells and matched controls. Exponentially growing cells were seeded in 96-well plates at a density of 1 × 103 cells per 100 μl completed well and left undisturbed for 96 hours. Cell proliferation was evaluated 96 hours later using the 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide metabolic assay as described previously.27Yen L Benlimame N Nie ZR Xiao D Wang T Al Moustafa AE Esumi H Milanini J Hynes NE Pages G Alaoui-Jamali MA Differential regulation of tumor angiogenesis by distinct ErbB homo- and heterodimers.Mol Biol Cell. 2002; 13: 4029-4044Crossref PubMed Scopus (121) Google Scholar Cell motility was examined using the phagokinetic track assay. Briefly, sterile coverslips coated with 1% bovine serum albumin and then a uniform carpet of gold colloidal solution containing 150 μmol/L HAuCl4-4H2O and 10 mmol/L Na2CO3 was applied. Serum-starved cells were plated at low density (0.5 × 103 per coverslip), allowed to attach for 1 hour, and then placed in 35-mm tissue culture dishes. Cells were then allowed to migrate for 8 hours in presence of serum-free medium and in the absence and presence of 10 ng of heregulin. Cells were then fixed by 0.1% formaldehyde, and the areas cleared of gold particles were examined under microscope and quantified by NIH image processing; 50 cells were examined per condition, and the results are presented as the average area free of gold colloidal particles in square millimeters ±SD. After digitization, color images were processed using specialized functions from Photoshop imaging software (Adobe Systems, Mountain View, CA), and results are reported as an area per square millimeter. Cell invasion experiments were performed with 8-μm porous chambers coated with Matrigel (Becton Dickinson, Franklin Lakes, NJ) according to the manufacturer's recommendations. Serum-starved cells were placed into the upper compartment (30,000 cells), and the chambers were placed into 24-well culture dishes containing 400 μl of DMEM 0.2% BSA with or without 20 ng/ml HRG (lower compartment). Cells were allowed to invade through the Matrigel membrane for 48 hours. The invasive cells underneath the membrane were fixed and stained. Filters were viewed under bright-field 40X objective and the counting was performed for three fields in each sample. Each experiment was done at least three times, and results are expressed as means ± SEM. Cells were grown to approximately 70 to 80% confluence after 24 hours of serum-starvation and treatment with HRG when indicated. Total cell lysates were prepared, blotted, detected using appropriate antibody, and visualized using enhanced chemiluminescence detection. Antibodies used are the following: ErbB-2 (antibody-3, clone 3B5; Oncogene Science), ErbB-3 (clone C-17; Santa Cruz Biotechnology, Santa Cruz, CA), Pyk2 (BD Bioscience Transduction Laboratories), glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Danvers, MA), FAK (clone 4.47; Biosource International), Erk1/2 (p44/42 mitogen-activated protein kinase), and phospho-Erk1/2-Y202/204 (Cell Signaling Technology). For immunoprecipitation, 200 μg of protein was immunoprecipitated with anti-ErbB-2 (antibody-3) and anti-ErbB-3 (clone C-17). Immunoprecipitated samples were then blotted on nitrocellulose and detected with antibody against phosphotyrosine (4G10; Upstate Biotechnology). Blots were then stripped and immunoblotted with antibodies specific for each immunoprecipitated receptor, as described above. To determine Erk activation, protein from cells was stimulated with HRG for 10 minutes and then immunoblotted with antibody specific for phosphorylated Erk1/2. Blots were then stripped and reprobed to recognize total Erk1/2. A preliminary Western assay was performed at 15 seconds, 30 seconds, 5 minutes, 10 minutes, 30 minutes, 1 hour, and 2 hours after HRG stimulation of serum-starved cells. Ten minutes was chosen as the optimal time point for ERK activation (not shown). Cells overexpressing ErbB receptors were processed for immunofluorescence based on similar conditions described above.27Yen L Benlimame N Nie ZR Xiao D Wang T Al Moustafa AE Esumi H Milanini J Hynes NE Pages G Alaoui-Jamali MA Differential regulation of tumor angiogenesis by distinct ErbB homo- and heterodimers.Mol Biol Cell. 2002; 13: 4029-4044Crossref PubMed Scopus (121) Google Scholar ErbB-2 (antibody-3, clone 3B5; Oncogene Science) antibody was used. After labeling, the cells were viewed in a Zeiss Axiophot fluorescent microscope (Carl Zeiss, Thornwood, NY) equipped with a 63× Plan Apochromat objective and selective filters. Images were acquired from a cooled CCD camera and displayed on a high-resolution monitor. Images were analyzed by Northern Eclipse Image analysis system (Carl Zeiss). In vivo studies were approved by the McGill Animal Care Committee (protocol no. 4101) and were conducted in accordance with institutional and Canadian federal guidelines. Female Scid mice were obtained from Charles River Laboratories (St. Zotique, PQ, Canada). For primary tumors, ErbB-FAK+/+ and ErbB-FAK−/− cells (1 × 106 cells) were implanted subcutaneously in the flank of female Scid mice. Parental cells expressing empty retroviral particles were used as controls. MDA 231-M cells expressing siRNA against FAK, Pyk2, or both were injected into the mammary fat pad of mice. Control cells expressed empty pSuper-retro puro vector. These cells maintained the same growth rate as the parental cells. Tumor volumes were measured every 2nd or 3rd day using a caliper, and tumor volumes were calculated as volume = π/6 (length × width2). For tumor invasion, the lungs were fixed in 10% Bouin's fixative, and lung surface metastases were counted using a stereomicroscope. In all cases, eight mice were used per condition. Tissue microarrays (TMAs) from breast cancer patients cohort were assembled using a manual tissue arrayer (Beecher Instruments, Silver Spring, MD) as previously described.28Rubin MA Dunn R Strawderman M Pienta KJ Tissue microarray sampling strategy for prostate cancer biomarker analysis.Am J Surg Pathol. 2002; 26: 312-319Crossref PubMed Scopus (269) Google Scholar All TMA cores were assigned a diagnosis (ie, benign, carcinoma in situ, invasive, and lymph node metastatic breast cancer) by the study pathologist. Tissue cores from circled areas were targeted for transfer to a recipient array paraffin block. Three to five replicate tissue cores on average were sampled from each patient for the tissue microarray. TMA cores (0.6 mm in diameter) were each spaced at 0.8 mm from core center to core center. Three TMA blocks with a total of 202 cores belonging to 75 patients were constructed. All blocks contained benign breast tissue, ductal in situ carcinoma (DCIS), invasive carcinoma, and lymph node metastatic tumors. After construction, 4-μm sections were cut and stained with H&E to verify initial diagnosis. The study was approved by the Jewish General Hospital institutional review board. Immunohistochemistry was performed using the NexES immunostainer (Ventana Medical Systems, Tuscon, AZ). Immunostaining was performed on 4-μm silane-coated slides (Sigma, St. Louis, MO), dried overnight at 37°C, and then dewaxed, rehydrated, and boiled (microwave) in EDTA (pH 7.0) or citrate buffer (pH 6.0) for antigen retrieval. Slides were incubated for 32 minutes at 37°C using the primary antibodies. Primary antibodies [Pyk2 (1:25; 610548; BD Bioscience Transduction Laboratories); and FAK (1:25; clone 4.47; catalog no. 05-537; Biosource International)] were incubated for 40 minutes at room temperature using the NexES automated immunostainer (Ventana Medical Systems). Antibodies were first titrated using test arrays of several samples representing various tissue types and various antigen retrieval methods. The titration showing the best staining with no background in epithelial breast tissue was used. The automated Ventana system then uses an indirect biotin-avidin system with a universal biotinylated immunoglobulin secondary antibody. Diaminobenzidine was used as a chromogen. Slides were subsequently counterstained with hematoxylin before mounting. Staining procedures were performed according to the manufacturer's recommendations. Negative controls were obtained by omitting the specific primary antibodies. Protein expression was assessed using a four-tiered system (0, negative; 1, weak; 2, moderate; and 3, high expression). To analyze tumor growth, unpaired Student's t-test was used to compare significance between groups. Data were then analyzed using analysis of variance comparing all groups, with group as an independent variable and volume as a repeated measure as a function of time, and Dunnett's and Bonferroni's tests. All statistical tests were two-sided and were considered to be significant at the 0.05 level. For human tissue data, analyses were performed using a computer-based statistical package of Statistical Product and Service Solution version 5.1 for Windows (SPSS, Chicago, IL). Results are reported as means ± SE. Differences among the four groups (benign, DCIS, invasive, and lymph node metastases) were compared with one-way analysis of variance and the post hoc Tukey honestly significantly different test for multiple comparisons. A value of P < 0.05 was considered significant. Only significant correlation coefficients are reported. To examine the significance of Pyk2 expression compared with FAK for human breast disease, we performed immunohistochemical study on a high-density tissue microarray from a total of more than 200 breast cancer cases of various stages of progression. The pathology of each tissue microarray element was confirmed by an experienced pathologist and scored for staining (scale of 1 to 4) per cell type considered (epithelial, myoepithelial, and stromal). As shown in Figure 1, Pyk2 protein expression was significantly increased in the various stages of breast cancer progression compared with benign (P < 0.05). The average expression was highest in in situ cancer and invasive cancer compared with benign tissue (P < 0.05). Lymph node metastases showed a wider expression range compared with invasive breast cancer (Figure 1; see Supplemental Table S1 at http://ajp.amjpathol.org). Protein expression for FAK showed similar trends, with increased expression in in situ, invasive, and a wider expression in lymph nodes metastasis versus benign breast tissue (Figure 1; see Supplemental Table S2 at http://ajp.amjpathol.org). Moreover, co-expression of Pyk2 and FAK was observed in more than 70% of cases (Table 1). In ErbB-2-positive breast cancer tissues (>grade 1), Pyk2/FAK co-overexpression was seen in 14 to 25% of cases (Table 1).Table 1Percent Co-expression of FAK, Pyk2, and ErbB-2 in TMA Specimens with Grade >1Tissue typeFAK/ErbB-2 (%)Pyk2/ErbB-2 (%)FAK/Pyk2 (%)Benign0/29 (0)0/23 (0)0/23 (0)In situ6/33 (18.2)6/42 (14.3)24/33 (72.7)Invasive23/119 (19.3)27/120 (22.5)76/119 (63.9)Lymph node metastasis4/17 (23.5)5/20 (25)13/17 (76.5) Open table in a new tab To investigate the role of Pyk2 versus FAK in ErbB-induced cancer progression, we initially used paired FAK-proficient and -deficient mouse embryonic fibroblasts transformed by overexpression of the ErbB-2/3 tyrosine kinase receptors. We have reported that co-expression of ErbB-2 and ErbB-3 in these cells promotes potent oncogenic transformation, tumor growth, and progression to metastases.26Benlimame N He Q Jie S Xiao D Xu YJ Loignon M Schlaepfer DD Alaoui-Jamali MA FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.J Cell Biol. 2005; 171: 505-516Crossref PubMed Scopus (116) Google Scholar ErbB-2 is an orphan receptor that is preferentially transactivated through receptor heterodimeriz" @default.
• W2006163902 created "2016-06-24" @default.
• W2006163902 creator A5012063931 @default.
• W2006163902 creator A5032122708 @default.
• W2006163902 creator A5040747953 @default.
• W2006163902 creator A5050326141 @default.
• W2006163902 creator A5057745939 @default.
• W2006163902 creator A5063482458 @default.
• W2006163902 creator A5067268235 @default.
• W2006163902 date "2008-11-01" @default.
• W2006163902 modified "2023-09-25" @default.
• W2006163902 title "Focal Adhesion Kinase-Related Proline-Rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Co-Overexpressed in Early-Stage and Invasive ErbB-2-Positive Breast Cancer and Cooperate for Breast Cancer Cell Tumorigenesis and Invasiveness" @default.
• W2006163902 cites W1525629072 @default.
• W2006163902 cites W1577573926 @default.
• W2006163902 cites W1582187753 @default.
• W2006163902 cites W1682186954 @default.
• W2006163902 cites W1893360284 @default.
• W2006163902 cites W1975004220 @default.
• W2006163902 cites W1979933401 @default.
• W2006163902 cites W1981493299 @default.
• W2006163902 cites W1982287353 @default.
• W2006163902 cites W1985961293 @default.
• W2006163902 cites W1988336550 @default.
• W2006163902 cites W1989776883 @default.
• W2006163902 cites W1994480447 @default.
• W2006163902 cites W2001587874 @default.
• W2006163902 cites W2005519312 @default.
• W2006163902 cites W2011562199 @default.
• W2006163902 cites W2012713087 @default.
• W2006163902 cites W2013353890 @default.
• W2006163902 cites W2017397076 @default.
• W2006163902 cites W2028936118 @default.
• W2006163902 cites W2028970216 @default.
• W2006163902 cites W2029531377 @default.
• W2006163902 cites W2029720743 @default.
• W2006163902 cites W2030192350 @default.
• W2006163902 cites W2034771296 @default.
• W2006163902 cites W2043503095 @default.
• W2006163902 cites W2057097544 @default.
• W2006163902 cites W2060097272 @default.
• W2006163902 cites W2065088072 @default.
• W2006163902 cites W2073513388 @default.
• W2006163902 cites W2074878830 @default.
• W2006163902 cites W2078660746 @default.
• W2006163902 cites W2079414703 @default.
• W2006163902 cites W2084867085 @default.
• W2006163902 cites W2086992732 @default.
• W2006163902 cites W2088129765 @default.
• W2006163902 cites W2091506455 @default.
• W2006163902 cites W2093223195 @default.
• W2006163902 cites W2097601945 @default.
• W2006163902 cites W2103363752 @default.
• W2006163902 cites W2104353597 @default.
• W2006163902 cites W2110841670 @default.
• W2006163902 cites W2112194601 @default.
• W2006163902 cites W2115100948 @default.
• W2006163902 cites W2116608087 @default.
• W2006163902 cites W2118105104 @default.
• W2006163902 cites W2119921106 @default.
• W2006163902 cites W2120324368 @default.
• W2006163902 cites W2121220704 @default.
• W2006163902 cites W2122667134 @default.
• W2006163902 cites W2125967276 @default.
• W2006163902 cites W2128554762 @default.
• W2006163902 cites W2132739803 @default.
• W2006163902 cites W2143167773 @default.
• W2006163902 cites W2144109765 @default.
• W2006163902 cites W2148340118 @default.
• W2006163902 cites W2155883999 @default.
• W2006163902 cites W2158769524 @default.
• W2006163902 cites W2159695799 @default.
• W2006163902 cites W2161873278 @default.
• W2006163902 cites W2165845069 @default.
• W2006163902 cites W2165905376 @default.
• W2006163902 cites W2165988906 @default.
• W2006163902 cites W2283549843 @default.
• W2006163902 cites W2388898124 @default.
• W2006163902 cites W4213141530 @default.
• W2006163902 doi "https://doi.org/10.2353/ajpath.2008.080292" @default.
• W2006163902 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2570143" @default.
• W2006163902 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18832579" @default.
• W2006163902 hasPublicationYear "2008" @default.
• W2006163902 type Work @default.
• W2006163902 sameAs 2006163902 @default.
• W2006163902 citedByCount "59" @default.
• W2006163902 countsByYear W20061639022012 @default.
• W2006163902 countsByYear W20061639022013 @default.
• W2006163902 countsByYear W20061639022014 @default.
• W2006163902 countsByYear W20061639022015 @default.
• W2006163902 countsByYear W20061639022016 @default.
• W2006163902 countsByYear W20061639022018 @default.
• W2006163902 countsByYear W20061639022019 @default.
• W2006163902 countsByYear W20061639022020 @default.
• W2006163902 countsByYear W20061639022021 @default.
• W2006163902 countsByYear W20061639022022 @default.
• W2006163902 countsByYear W20061639022023 @default.
• W2006163902 crossrefType "journal-article" @default.
• W2006163902 hasAuthorship W2006163902A5012063931 @default.

• next