Analyses of merlin/NF2 connection to FAK inhibitor responsiveness in serous ovarian cancer.
ABSTRACT: Focal adhesion kinase (FAK) is overexpressed in serous ovarian cancer. Loss of merlin, a product of the neurofibromatosis 2 tumor suppressor gene, is being evaluated as a biomarker for FAK inhibitor sensitivity in mesothelioma. Connections between merlin and FAK in ovarian cancer remain undefined.Nine human and two murine ovarian cancer cell lines were analyzed for growth in the presence of a small molecule FAK inhibitor (PF-271, also termed VS-6062) from 0.1 to 1 ?M for 72 h. Merlin was evaluated by immunoblotting and immunostaining of a human ovarian tumor tissue array. Growth of cells was analyzed in an orthotopic tumor model and evaluated in vitro after stable shRNA-mediated merlin knockdown.Greater than 50% inhibition of OVCAR8, HEY, and ID8-IP ovarian carcinoma cell growth occurred with 0.1 ?M PF-271 in anchorage-independent (p<0.001) but not in adherent culture conditions. PF-271-mediated reduction in FAK Y397 phosphorylation occurred independently of growth inhibition. Suspended growth of OVCAR3, OVCAR10, IGROV1, IGROV1-IP, SKOV3, SKOV3-IP, A2780, and 5009-MOVCAR was not affected by 0.1 ?M PF-271. Merlin expression did not correlate with serous ovarian tumor grade or stage. PF-271 (30 mg/kg, BID) did not inhibit 5009-MOVCAR tumor growth and merlin knockdown in SKOV3-IP and OVCAR10 cells did not alter suspended cell growth upon PF-271 addition.Differential responsiveness to FAK inhibitor treatment was observed. Intrinsic low merlin protein level correlated with PF-271-mediated anchorage-independent growth inhibition, but reduction in merlin expression did not induce sensitivity to FAK inhibition. Merlin levels may be useful for patient stratification in FAK inhibitor trials.
Project description:Ovarian cancer ascites fluid contains matrix proteins that can impact tumor growth via integrin receptor binding. In human ovarian tumor tissue arrays, we find that activation of the cytoplasmic focal adhesion (FAK) tyrosine kinase parallels increased tumor stage, ?5 integrin, and osteopontin matrix staining. Elevated osteopontin, ?5 integrin, and FAK mRNA levels are associated with decreased serous ovarian cancer patient survival. FAK remains active within ovarian cancer cells grown as spheroids, and anchorage-independent growth analyses of seven ovarian carcinoma cell lines identified sensitive (HEY, OVCAR8) and resistant (SKOV3-IP, OVCAR10) cells to 0.1 ?mol/L FAK inhibitor (VS-4718, formerly PND-1186) treatment. VS-4718 promoted HEY and OVCAR8 G0-G1 cell-cycle arrest followed by cell death, whereas growth of SKOV3-IP and OVCAR10 cells was resistant to 1.0 ?mol/L VS-4718. In HEY cells, genetic or pharmacological FAK inhibition prevented tumor growth in mice with corresponding reductions in ?5 integrin and osteopontin expression. ?5 knockdown reduced HEY cell growth in soft agar, tumor growth in mice, and both FAK Y397 phosphorylation and osteopontin expression in spheroids. FAK inhibitor-resistant (SKOV3-IP, OVCAR10) cells exhibited anchorage-independent Akt S473 phosphorylation, and expression of membrane-targeted and active Akt in sensitive cells (HEY, OVCAR8) increased growth but did not create a FAK inhibitor-resistant phenotype. These results link osteopontin, ?5 integrin, and FAK in promoting ovarian tumor progression. ?5 integrin expression may serve as a biomarker for serous ovarian carcinoma cells that possess active FAK signaling.
Project description:Recurrence and spread of ovarian cancer is the 5th leading cause of death for women in the United States. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase located on chromosome 8q24.3 (gene is Ptk2), a site commonly amplified in serous ovarian cancer. Elevated FAK mRNA levels in serous ovarian carcinoma are associated with decreased (logrank P = 0.0007, hazard ratio 1.43) patient overall survival, but how FAK functions in tumor progression remains undefined. We have isolated aggressive ovarian carcinoma cells termed ID8-IP after intraperitoneal (IP) growth of murine ID8 cells in C57Bl6 mice. Upon orthotopic implantation within the peri-ovarian bursa space, ID8-IP cells exhibit greater tumor growth, local and distant metastasis, and elevated numbers of ascites-associated cells compared to parental ID8 cells. ID8-IP cells exhibit enhanced growth under non-adherent conditions with elevated FAK and c-Src tyrosine kinase activation compared to parental ID8 cells. In vitro, the small molecule FAK inhibitor (Pfizer, PF562,271, PF-271) at 0.1 uM selectively prevented anchorage-independent ID8-IP cell growth with the inhibition of FAK tyrosine (Y)397 but not c-Src Y416 phosphorylation. Oral PF-271 administration (30 mg/kg, twice daily) blocked FAK but not c-Src tyrosine phosphorylation in ID8-IP tumors. This was associated with decreased tumor size, prevention of peritoneal metastasis, reduced tumor-associated endothelial cell number, and increased tumor cell-associated apoptosis. FAK knockdown and re-expression assays showed that FAK activity selectively promoted anchorage-independent ID8-IP cell survival. These results support the continued evaluation of FAK inhibitors as a promising clinical treatment for ovarian cancer.
Project description:Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1β (IL-1β) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1β stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1β signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE-/- mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.
Project description:Ovarian clear cell carcinoma (OCCC) displays a higher resistance to first line chemotherapy, requiring the development of new therapeutics. We previously identified a frequent chromosomal gain at 8q24 that harbors the focal-adhesion kinase (FAK) gene; the potential of this gene as a therapeutic target remains to be evaluated in OCCCs. We first examined the dependence of OCCCs on FAK and the PI3K/AKT signaling pathway. FAK was overexpressed in 20% of 67 OCCC samples, and this overexpression was correlated with its copy number gain. FAK copy number gains and mutations in PIK3CA accounted for about 40% of OCCC samples, suggesting that the FAK/PI3K/AKT axis is an attractive candidate for targeted therapeutics. We, therefore, treated ovarian cancer cell lines, including OCCC subtypes, with the FAK inhibitors PF-562,271 (PF271), and PF-573,228 (PF228). Ovarian cancer cells were more sensitive to PF271 than PF228. We then searched for single agents that exhibited a synergistic effect on cell death in combination with PF271. We found that co-treatment of PF271 with ABT-737, a BCL-2/BCL-XL antagonist, was profoundly effective at inducing apoptosis. RMGI and OVISE cells were more sensitive to ABT-737 than OVMANA and SKOV3 cells, which have PIK3CA mutations. Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway. Therefore, PF271/ABT-737 treatment led to the inhibition of the anti-apoptotic proteins MCL1 and BCL-XL/BCL-2. We suggest that pharmacological inhibition of BCL-XL and FAK/PYK2 can be a potential therapeutic strategy for the treatment of OCCC.
Project description:Antagonizing the oncogenic effects of human epidermal growth factor receptor 2 (HER2) with current anti-HER2 agents has not yet yielded major progress in the treatment of advanced HER2-positive epithelial ovarian cancer (EOC). Using preclinical models to explore alternative molecular mechanisms affecting HER2 overexpression and oncogenicity may lead to new strategies for EOC patient treatment. We previously reported that phosphatidylcholine-specific phospholipase C (PC-PLC) exerts a pivotal role in regulating HER2 overexpression in breast cancer cells. The present study, conducted on two human HER2-overexpressing EOC cell lines - SKOV3 and its in vivo-passaged SKOV3.ip cell variant characterized by enhanced in vivo tumorigenicity - and on SKOV3.ip xenografts implanted in SCID mice, showed: a) about 2-fold higher PC-PLC and HER2 protein expression levels in SKOV3.ip compared to SKOV3 cells; b) physical association of PC-PLC with HER2 in non-raft domains; c) HER2 internalization and ca. 50% reduction of HER2 mRNA and protein expression levels in SKOV3.ip cells exposed to the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609); d) differential effects of D609 and trastuzumab on HER2 protein expression and cell proliferation; e) decreased in vivo tumor growth in SKOV3.ip xenografts during in vivo treatment with D609; f) potential use of in vivo magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters as biomarkers of EOC response to PC-PLC inhibition. Overall, these findings support the view that PC-PLC inhibition may represent an effective means to target the tumorigenic effects of HER2 overexpression in EOC and that in vivo MR approaches can efficiently monitor its effects.
Project description:Gene expression analysis was also performed on 13 primary and established human ovarian cancer cell lines (A2008, OAW42, OVCAR2, OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVCAR10, OV7M, OV95, PE01, PE04, SKOV3) using Affymetrix Human Gene 1.0 ST Arrays The study focused on ovarian cancer chemokine expressions
Project description:BACKGROUND AND PURPOSE: As prognosis for patients with metastatic ovarian cancer is generally poor, advances in treatment are needed. Here, we studied the mechanism of action of a recombinant viral capsid protein (rVP1) and explored its effect against ovarian tumour growth and metastasis in vivo. EXPERIMENTAL APPROACH: The human ovarian cancer cell line SKOV3 and BALB/cAnN-Foxn1 female nude mice were used. Effects of rVP1 on the viability, invasive ability, matrix metalloproteinase (MMP)-2 activity and cancer cell proliferation and metastasis were determined by cell proliferation assay, Matrigel invasion assay, gelatin zymographic analysis, as well as bioluminescence imaging and immunohistological analysis in xenograft mouse models respectively. Levels of total and phosphorylated focal adhesion kinase (FAK), PKB/Akt, phosphatase and tensin homologue (PTEN) and glycogen synthase kinase-3? (GSK-3?) were detected by Western blotting. KEY RESULTS: rVP1 promoted apoptosis and decreased invasion of human ovarian cancer cells. This effect of rVP1 was accompanied by activation of PTEN and GSK-3? as well as down-regulation of FAK, Akt and MMP-2. Anti-integrin antibodies or overexpression of constitutively active Akt reversed the cellular effects of rVP1. Orthotopic and intraperitoneal xenograft mouse models demonstrated that rVP1 attenuated survival and metastasis of human ovarian cancer SKOV3 cell line in vivo through selective regulation of Akt and GSK-3? activity as shown by bioluminescence imaging of mice and immunohistochemical analysis. CONCLUSION AND IMPLICATIONS: These results indicate that negative regulation of Akt signalling and MMP-2 by rVP1 may have the potential to suppress ovarian tumour growth and metastasis in vivo.
Project description:Ovarian cancer is the most fatal gynecologic cancer with poor prognosis. Etiological factors underlying ovarian cancer genesis and progression are poorly understood. Previously, we have shown that JNK-associated Leucine zipper Protein (JLP), promotes oncogenic signaling. Investigating the role of JLP in ovarian cancer, our present study indicates that JLP is overexpressed in ovarian cancer tissue and ovarian cancer cells. Transient overexpression of JLP promotes proliferation and invasive migration of ovarian cancer cells. In addition, ectopic expression of JLP confers long-term survival and clonogenic potential to normal fallopian tube-derived epithelial cells. Coimmunoprecipitation and colocalization analyses demonstrate the in vivo interaction of JLP and JNK, which is stimulated by lysophosphatidic acid (LPA), an oncogenic lipid growth factor in ovarian cancer. We also show that LPA stimulates the translocation of JLP-JNK complex to the perinuclear region of SKOV3-ip cells. JLP-knockdown using shRNA abrogates LPA-stimulated activation of JNK as well as LPA-stimulated proliferation and invasive migration of SKOV3-ip cells. Studies using ovarian cancer xenograft mouse model indicate that the mice bearing JLP-silenced xenografts exhibits reduced tumor volume. Analysis of the xenograft tumor tissues indicate a reduction in the levels of JLP, JNK, phosphorylated-JNK, c-Jun and phosphorylated-c-Jun in JLP-silenced xenografts, thereby correlating the attenuated JLP-JNK signaling node with suppressed tumor growth. Thus, our results identify a critical role for JLP-signaling axis in ovarian cancer and provide evidence that targeting this signaling node could provide a new avenue for therapy.
Project description:Generally, most of ovarian cancer cannot be detected until large scale and remote metastasis occurs, which is the major cause of high mortality in ovarian cancer. Therefore, it is urgent to discover metastasis-related biomarkers for the detection of ovarian cancer in its occult metastasis stage. Altered glycosylation is a universal feature of malignancy and certain types of glycan structures are well-known markers for tumor progressions. Thus, this study aimed to reveal specific changes of N-glycans in the secretome of the metastatic ovarian cancer. We employed a quantitative glycomics approach based on metabolic stable isotope labeling to compare the differential N-glycosylation of secretome between an ovarian cancer cell line SKOV3 and its high metastatic derivative SKOV3-ip. Intriguingly, among total 17 N-glycans identified, the N-glycans with bisecting GlcNAc were all significantly decreased in SKOV3-ip in comparison to SKOV3. This alteration in bisecting GlcNAc glycoforms as well as its corresponding association with ovarian cancer metastatic behavior was further validated at the glycotransferase level with multiple techniques including real-time PCR, western blotting, transwell assay, lectin blotting and immunohistochemistry analysis. This study illustrated metastasis-related N-glycan alterations in ovarian cancer secretome in vitro for the first time, which is a valuable source for biomarker discovery as well. Moreover, N-glycans with bisecting GlcNAc shed light on the detection of ovarian cancer in early peritoneal metastasis stage which may accordingly improve the prognosis of ovarian cancer patients.
Project description:Ovarian cancer is the most lethal gynecological malignancy, characterized by a high rate of chemoresistance. Current treatment strategies for ovarian cancer focus on novel drug combinations of cytotoxic agents and molecular targeted agents or novel drug delivery strategies that often involve intraperitoneal (IP) injection. Poly(ethylene glycol)-block-poly(?-caprolactone) (PEG-b-PCL) micelles were loaded with paclitaxel (cytotoxic agent), cyclopamine (hedgehog inhibitor), and gossypol (Bcl-2 inhibitor). After physicochemical studies focusing on combination drug solubilization, 3-drug PEG-b-PCL micelles were evaluated in vitro in 2-D and 3-D cell culture and in vivo in xenograft models of ovarian cancer, tracking bioluminescence signals from ES-2 and SKOV3 human ovarian cancer cell lines after IP injection. 3-Drug PEG-b-PCL micelles were not significantly more potent in 2-D cell culture in comparison to paclitaxel; however, they disaggregated ES-2 tumor spheroids, whereas single drugs or 2-drug combinations only slowed growth of ES-2 tumor spheroids or had no noticeable effects. In ES-2 and SKOV3 xenograft models, 3-drug PEG-b-PCL micelles had significantly less tumor burden than paclitaxel based on bioluminescence imaging, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET imaging, and overall survival. (18)F-FLT-PET images clearly showed that 3-drug PEG-b-PCL micelles dramatically reduce tumor volumes over paclitaxel and vehicle controls. In summary, PEG-b-PCL micelles enable the IP combination drug delivery of paclitaxel, cyclopamine and gossypol, resulting in tumor growth inhibition and prolonged survival over paclitaxel alone. These results validate a novel treatment strategy for ovarian cancer based on drug combinations of cytotoxic agents and molecular targeted agents, delivered concurrently by a nanoscale drug delivery system, e.g. PEG-b-PCL micelles.