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Purpose: Aberrant activation of the intracellular tyrosine kinase Src has been implicated as a mechanism of acquired chemotherapy resistance in metastatic colorectal cancer (mCRC). Here, the oral tyrosine kinase Src inhibitor, dasatinib, was investigated in combination with FOLFOX and cetuximab.Experimental Design: We performed a phase IB/II study of 77 patients with previously treated mCRC. Primary objectives were to determine the maximum tolerated dose, dose-limiting toxicities (DLT), pharmacodynamics, and efficacy. Using a 3 + 3 design, patients received FOLFOX6 with cetuximab and escalating doses of dasatinib (100, 150, 200 mg daily), followed by a 12-patient expansion cohort at 150 mg. Phase II studies evaluated FOLFOX plus dasatinib 100 mg in KRAS c12/13mut patients or in combination with cetuximab if KRAS c12/13WT. FAK and paxillin were utilized as surrogate blood biomarkers of Src inhibition, and paired biopsies of liver metastases were obtained in patients in the expansion cohort.Results: In phase IB, the DLTs were grade 3/4 fatigue (20%) and neutropenia (23%). In phase II, grade 3/4 fatigue (23%) and pleural effusions (11%) were present. Response rates were 20% (6 of 30) in the phase IB escalation and expansion cohort and 13% (3 of 24) and 0% (0 of 23) in the KRAS c12/13WT and mutant cohorts of phase II, respectively. Median progression-free survival was 4.6, 2.3, and 2.3 months, respectively. There was no evidence of Src inhibition based on surrogate blood biomarkers or paired tumor biopsies.Conclusions: The combination of dasatinib plus FOLFOX with or without cetuximab showed only modest clinical activity in refractory colorectal cancer. This appears to be primarily due to a failure to fully inhibit Src at the achievable doses of dasatinib. The combination of dasatinib plus FOLFOX with or without cetuximab did not show meaningful clinical activity in refractory colorectal cancer due to failure to fully inhibit Src. Clin Cancer Res; 23(15); 4146–54. ©2017 AACR.

Supplemental Figure 1 (fig. S1). Representative whole body NaF-18 PET scan images after 44 days of (A) vehicle-treated and (B) cabozantinib-treated animals. Supplemental Figure 2 (fig. S2). Effect of cabozantinib on PDX tumor growth, animal weight, and survival. Supplemental Figure 3 (fig. S3). Representative images of viable islets of tumor cells in (A) Patients; (B) MDA PCa-118b grown intrafemurally; and (C) MDA PCa- 118b grown subcutaneously. Supplemental Figure 4 (fig. S4). Time-dependent effect of cabozantinib on expression and phosphorylation of primary and MET downstream targets. Supplemental Figure 5 (fig. S5). Relative expression of c-met mRNA in NT and knockdown cells as determined by qRT-PCR. Supplemental Figure 6 (fig. S6). MET expression and activity in NT and knockdown tumors. Supplemental Figure 7 (fig. S7): Quantitation of CD31 staining on bone tumors. Supplemental Figure 8 (fig. S8). Effects of cabozantinib on bone remodeling markers in the phase-2 clinical trial and growth and differentiation of primary osteoblasts. Supplemental Figure S9 (fig S9). Effect of cabozantinib on bone turnover in the Phase 2 trial and PDX grown intrafemurally. Supplemental Figure 10 (fig. S10). Quantitation of phospho-histone H3 staining.

Effects of integrin ligands on C4-2B4 soft agar colony formation.

Resistance to currently available targeted therapies significantly hampers the survival of patients with prostate cancer with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a “resistance niche” adjacent to prostate cancer-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed “osteocrines”) found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to the newly formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic prostate cancer. Cancer Res; 75(22); 4949–59. ©2015 AACR.

Validation of peripheral blood mononuclear cells as a biomarker of Src inhibition. Freshly collected peripheral blood mononuclear cells from a healthy volunteer were treated ex-vivo with dasatinib 300 nM or hydrogen peroxide 0.05 mM. The peripheral blood mononuclear cell lysates were generated and immunoblotted for phosphoFAK [Y861] and phophopaxillin [Y118].

Purpose: We performed parallel investigations in cabozantinib-treated patients in a phase II trial and simultaneously in patient-derived xenograft (PDX) models to better understand the roles of MET and VEGFR2 as targets for prostate cancer therapy.Experimental Design: In the clinical trial, radiographic imaging and serum markers were examined, as well as molecular markers in tumors from bone biopsies. In mice harboring PDX intrafemurally or subcutaneously, cabozantinib effects on tumor growth, MET, PDX in which MET was silenced, VEGFR2, bone turnover, angiogenesis, and resistance were examined.Results: In responsive patients and PDX, islets of viable pMET-positive tumor cells persisted, which rapidly regrew after drug withdrawal. Knockdown of MET in PDX did not affect tumor growth in mice nor did it affect cabozantinib-induced growth inhibition but did lead to induction of FGFR1. Inhibition of VEGFR2 and MET in endothelial cells reduced the vasculature, leading to necrosis. However, each islet of viable cells surrounded a VEGFR2-negative vessel. Reduction of bone turnover was observed in both cohorts.Conclusions: Our studies demonstrate that MET in tumor cells is not a persistent therapeutic target for metastatic castrate-resistant prostate cancer (CRPC), but inhibition of VEGFR2 and MET in endothelial cells and direct effects on osteoblasts are responsible for cabozantinib-induced tumor inhibition. However, vascular heterogeneity represents one source of primary therapy resistance, whereas induction of FGFR1 in tumor cells suggests a potential mechanism of acquired resistance. Thus, integrated cross-species investigations demonstrate the power of combining preclinical models with clinical trials to understand mechanisms of activity and resistance of investigational agents. Clin Cancer Res; 22(1); 107–21. ©2015 AACR.

Operating characteristics. A summary of the probability of stopping the trial early for the indicated true response rates.

Supplemental material

Lack of correlation between Src activity in PBMCs and hepatic tumors. For the patients in the expansion cohort, the change in the expression of pSrc and total Src was not correlated with a change in the phosphorlyation of the Src substrate paxillin. Patients with no change or a decrease in the expression of pSrc or Src from baseline to C2D8 were included in the "no increase" category.

Supplemental Figure Legends

Mass spectrometry of Bone-CM from PCa-118b tumor-associated bone.

supplementary materials and methods, supplementary tables and supplementary figure legends Supplemental Table 1: Patient Characteristics. Supplemental Table 2: Targeting sequences for c-met. Supplemental Table 3: Antibodies and Sources. Supplemental Table 4: Oligonucleotides for PCR Amplification of Mouse Alkaline Phosphatase and Osteonectin.

Prespecified dose modifications for adverse events related to study drug in Phase IB and Phase II.

Symptom-specific dose adjustment tables.

Supplementary Figures 1-3 from Synergistic Activity of the Src Family Kinase Inhibitor Dasatinib and Oxaliplatin in Colon Carcinoma Cells Is Mediated by Oxidative Stress

Supplementary Figure Legends 1-3 from Synergistic Activity of the Src Family Kinase Inhibitor Dasatinib and Oxaliplatin in Colon Carcinoma Cells Is Mediated by Oxidative Stress

Chemotherapeutic regimens for the treatment of colorectal cancer generally include oxaliplatin, although inherent and acquired resistance is common. One potential mediator of oxaliplatin sensitivity is the nonreceptor protein tyrosine kinase, Src, the activity of which correlates with disease stage and patient survival. Therefore, we investigated the effects of Src inhibition using the tyrosine kinase inhibitor dasatinib on oxaliplatin sensitivity. We show that oxaliplatin acutely activates Src and that combination treatment with dasatinib is synergistic in a cell-line dependent manner, with the level of Src activation correlating with extent of synergy in a panel of six cell lines. Intracellular reactive oxygen species (ROS) are generated after oxaliplatin treatment, and ROS potently activates Src. Pretreatment with antioxidants inhibits oxaliplatin-induced Src activation. In oxaliplatin-resistant cell lines, Src activity is constitutively increased. In a mouse model of colorectal liver metastases, treatment with oxaliplatin also results in chronic Src activation. The combination of dasatinib and oxaliplatin results in significantly smaller tumors compared with single-agent treatment, corresponding with reduced proliferation and angiogenesis. Therefore, we conclude that oxaliplatin activates Src through a ROS-dependent mechanism. Src inhibition increases oxaliplatin activity both in vitro and in vivo. These results suggest that Src inhibitors combined with oxaliplatin may have efficacy in metastatic colon cancer and may provide the first indication of a molecular phenotype that might be susceptible to such combinations. [Cancer Res 2009;69(9):3842–9]

Purpose: Radium-223 prolongs survival in a fraction of men with bone metastatic prostate cancer (PCa). However, there are no markers for monitoring response and resistance to Radium-223 treatment. Exosomes are mediators of intercellular communication and may reflect response of the bone microenvironment to Radium-223 treatment. We performed molecular profiling of exosomes and compared the molecular profile in patients with favorable and unfavorable overall survival. Experimental Design: We performed exosomal transcriptome analysis in plasma derived from our preclinical models (MDA-PCa 118b tumors, TRAMP-C2/BMP4 PCa) and from the plasma of 25 patients (paired baseline and end of treatment) treated with Radium-223. All samples were run in duplicate, and array data analyzed with fold changes +2 to −2 and P < 0.05. Results: We utilized the preclinical models to establish that genes derived from the tumor and the tumor-associated bone microenvironment (bTME) are differentially enriched in plasma exosomes upon Radium-223 treatment. The mouse transcriptome analysis revealed changes in bone-related and DNA damage repair–related pathways. Similar findings were observed in plasma-derived exosomes from patients treated with Radium-223 detected changes. In addition, exosomal transcripts detected immune-suppressors (e.g., PD-L1) that were associated with shorter survival to Radium-223. Treatment of the Myc-CaP mouse model with a combination of Radium-223 and immune checkpoint therapy (ICT) resulted in greater efficacy than monotherapy. Conclusions: These clinical and coclinical analyses showed that RNA profiling of plasma exosomes may be used for monitoring the bTME in response to treatment and that ICT may be used to increase the efficacy of Radium-223.

The overall goal of this study was to elucidate the role of FGFR1 induction in acquired resistance to MET and VEGFR2 inhibition by cabozantinib in prostate cancer (PCa) and leverage this understanding to improve therapy outcomes. The response to cabozantinib was examined in mice bearing patient-derived xenografts in which FGFR1 was overexpressed. Using a variety of cell models that reflect different PCa disease states, the mechanism underpinning FGFR1 signaling activation by cabozantinib was investigated. We performed parallel investigations in specimens from cabozantinib-treated patients to confirm our in vitro and in vivo data. FGFR1 overexpression was sufficient to confer resistance to cabozantinib. Our results demonstrate transcriptional activation of FGF/FGFR1 expression in cabozantinib-resistant models. Further analysis of molecular pathways identified a YAP/TBX5-driven mechanism of FGFR1 and FGF overexpression induced by MET inhibition. Importantly, knockdown of YAP and TBX5 led to decreased FGFR1 protein expression and decreased mRNA levels of FGFR1, FGF1, and FGF2. This association was confirmed in a cohort of hormone-naïve patients with PCa receiving androgen deprivation therapy and cabozantinib, further validating our findings. These findings reveal that the molecular basis of resistance to MET inhibition in PCa is FGFR1 activation through a YAP/TBX5-dependent mechanism. YAP and its downstream target TBX5 represent a crucial mediator in acquired resistance to MET inhibitors. Thus, our studies provide insight into the mechanism of acquired resistance and will guide future development of clinical trials with MET inhibitors.

Resistance to currently available targeted therapies significantly hampers the survival of patients with prostate cancer with bone metastasis. Here we demonstrate an important resistance mechanism initiated from tumor-induced bone. Studies using an osteogenic patient-derived xenograft, MDA-PCa-118b, revealed that tumor cells resistant to cabozantinib, a Met and VEGFR-2 inhibitor, reside in a “resistance niche” adjacent to prostate cancer-induced bone. We performed secretome analysis of the conditioned medium from tumor-induced bone to identify proteins (termed “osteocrines”) found within this resistance niche. In accordance with previous reports demonstrating that activation of integrin signaling pathways confers therapeutic resistance, 27 of the 90 osteocrines identified were integrin ligands. We found that following cabozantinib treatment, only tumor cells positioned adjacent to the newly formed woven bone remained viable and expressed high levels of pFAK-Y397 and pTalin-S425, mediators of integrin signaling. Accordingly, treatment of C4-2B4 cells with integrin ligands resulted in increased pFAK-Y397 expression and cell survival, whereas targeting integrins with FAK inhibitors PF-562271 or defactinib inhibited FAK phosphorylation and reduced the survival of PC3-mm2 cells. Moreover, treatment of MDA-PCa-118b tumors with PF-562271 led to decreased tumor growth, irrespective of initial tumor size. Finally, we show that upon treatment cessation, the combination of PF-562271 and cabozantinib delayed tumor recurrence in contrast to cabozantinib treatment alone. Our studies suggest that identifying paracrine de novo resistance mechanisms may significantly contribute to the generation of a broader set of potent therapeutic tools that act combinatorially to inhibit metastatic prostate cancer. Cancer Res; 75(22); 4949–59. ©2015 AACR.

Purpose: Aberrant activation of the intracellular tyrosine kinase Src has been implicated as a mechanism of acquired chemotherapy resistance in metastatic colorectal cancer (mCRC). Here, the oral tyrosine kinase Src inhibitor, dasatinib, was investigated in combination with FOLFOX and cetuximab. Experimental Design: We performed a phase IB/II study of 77 patients with previously treated mCRC. Primary objectives were to determine the maximum tolerated dose, dose-limiting toxicities (DLT), pharmacodynamics, and efficacy. Using a 3 + 3 design, patients received FOLFOX6 with cetuximab and escalating doses of dasatinib (100, 150, 200 mg daily), followed by a 12-patient expansion cohort at 150 mg. Phase II studies evaluated FOLFOX plus dasatinib 100 mg in KRAS c12/13mut patients or in combination with cetuximab if KRAS c12/13WT. FAK and paxillin were utilized as surrogate blood biomarkers of Src inhibition, and paired biopsies of liver metastases were obtained in patients in the expansion cohort. Results: In phase IB, the DLTs were grade 3/4 fatigue (20%) and neutropenia (23%). In phase II, grade 3/4 fatigue (23%) and pleural effusions (11%) were present. Response rates were 20% (6 of 30) in the phase IB escalation and expansion cohort and 13% (3 of 24) and 0% (0 of 23) in the KRAS c12/13WT and mutant cohorts of phase II, respectively. Median progression-free survival was 4.6, 2.3, and 2.3 months, respectively. There was no evidence of Src inhibition based on surrogate blood biomarkers or paired tumor biopsies. Conclusions: The combination of dasatinib plus FOLFOX with or without cetuximab showed only modest clinical activity in refractory colorectal cancer. This appears to be primarily due to a failure to fully inhibit Src at the achievable doses of dasatinib. The combination of dasatinib plus FOLFOX with or without cetuximab did not show meaningful clinical activity in refractory colorectal cancer due to failure to fully inhibit Src. Clin Cancer Res; 23(15); 4146–54. ©2017 AACR.

The receptor tyrosine kinase, MET, has been implicated in tumorigenesis and metastasis of many solid tumors, by multiple mechanisms, including cross talk with epidermal growth factor receptor. In this study, we examined the role of insulin-like growth factor receptor-1 (IGF-1R) signaling in MET activation, focusing on prostate cancer cells. Stimulation of the prostate cancer cell line PC3 with IGF-1 induces a delayed phosphorylation of MET at multiple sites (indicative of full activation), reaching a maximum 18 hr after IGF-1 addition. MET activation does not require the sole MET ligand hepatocyte growth factor (HGF), but does require transcription to occur. Furthermore, direct injection of IGF-1 is sufficient to induce MET activation in vivo, in a PC3 xenograft model. Pharmacologic or genetic inhibition of the tyrosine kinase, Src, abolishes MET phosphorylation, and expression of activated Src is sufficient to induce Met phosphorylation in the absence of IGF-1 stimulation. Activated MET is essential for IGF-1-mediated increased migration of PC3 cells, demonstrating an important biologic effect of IGF-1-mediated MET activation. Finally, we demonstrate that IGF-1-induced delayed MET activation occurs in multiple cell lines which express both the receptors, suggesting that IGF-1R-mediated MET activation may contribute to tumorigenic properties of multiple cancer types when both growth factor receptors are expressed. The results further suggest that MET may be activated by multiple receptor tyrosine kinase receptors, and dual targeting of these receptors may be important therapeutically.

Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation.Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC(50) of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts, and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release.We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases.Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.

Chemotherapeutic regimens for the treatment of colorectal cancer generally include oxaliplatin, although inherent and acquired resistance is common. One potential mediator of oxaliplatin sensitivity is the nonreceptor protein tyrosine kinase, Src, the activity of which correlates with disease stage and patient survival. Therefore, we investigated the effects of Src inhibition using the tyrosine kinase inhibitor dasatinib on oxaliplatin sensitivity. We show that oxaliplatin acutely activates Src and that combination treatment with dasatinib is synergistic in a cell-line dependent manner, with the level of Src activation correlating with extent of synergy in a panel of six cell lines. Intracellular reactive oxygen species (ROS) are generated after oxaliplatin treatment, and ROS potently activates Src. Pretreatment with antioxidants inhibits oxaliplatin-induced Src activation. In oxaliplatin-resistant cell lines, Src activity is constitutively increased. In a mouse model of colorectal liver metastases, treatment with oxaliplatin also results in chronic Src activation. The combination of dasatinib and oxaliplatin results in significantly smaller tumors compared with single-agent treatment, corresponding with reduced proliferation and angiogenesis. Therefore, we conclude that oxaliplatin activates Src through a ROS-dependent mechanism. Src inhibition increases oxaliplatin activity both in vitro and in vivo. These results suggest that Src inhibitors combined with oxaliplatin may have efficacy in metastatic colon cancer and may provide the first indication of a molecular phenotype that might be susceptible to such combinations. [Cancer Res 2009;69(9):3842–9]

Pancreatic cancer is an exceptionally lethal disease with an annual mortality nearly equivalent to its annual incidence. This dismal rate of survival is due to several factors including late presentation with locally advanced, unresectable tumors, early metastatic disease, and rapidly arising chemoresistance. To study the mechanisms of chemoresistance in pancreatic cancer we developed two gemcitabine-resistant pancreatic cancer cell lines.Resistant cells were obtained by culturing L3.6pl and AsPC-1 cells in serially increasing concentrations of gemcitabine. Stable cultures were obtained that were 40- to 50-fold increased in resistance relative to parental cells. Immunofluorescent staining was performed to examine changes in beta-catenin and E-cadherin localization. Protein expression was determined by immunoblotting. Migration and invasion were determined by modified Boyden chamber assays. Fluorescence-activated cell sorting (FACS) analyses were performed to examine stem cell markers.Gemcitabine-resistant cells underwent distinct morphological changes, including spindle-shaped morphology, appearance of pseudopodia, and reduced adhesion characteristic of transformed fibroblasts. Gemcitabine-resistant cells were more invasive and migratory. Gemcitabine-resistant cells were increased in vimentin and decreased in E-cadherin expression. Immunofluorescence and immunoblotting revealed increased nuclear localization of total beta-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells were activated in the receptor protein tyrosine kinase, c-Met and increased in expression of the stem cell markers CD (cluster of differentiation)24, CD44, and epithelial-specific antigen (ESA).Gemcitabine-resistant pancreatic tumor cells are associated with EMT, a more-aggressive and invasive phenotype in numerous solid tumors. The increased phosphorylation of c-Met may also be related to chemoresistance and EMT and presents as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

While several new therapies are FDA-approved for bone-metastatic prostate cancer (PCa), patient survival has only improved marginally. Here, we report that chitosan nanoparticle-mediated delivery of miR-34a, a tumor suppressive microRNA that downregulates multiple gene products involved in PCa progression and metastasis, inhibited prostate tumor growth and preserved bone integrity in a xenograft model representative of established PCa bone metastasis. Expression of miR-34a induced apoptosis in PCa cells, and, in accord with downregulation of targets associated with PCa growth, including MET and Axl and c-Myc, also induced a form of non-canonical autophagy that is independent of Beclin-1, ATG4, ATG5 and ATG7. MiR-34a-induced autophagy is anti-proliferative in prostate cancer cells, as blocking apoptosis still resulted in growth inhibition of tumor cells. Thus, combined effects of autophagy and apoptosis are responsible for miR-34a-mediated prostate tumor growth inhibition, and have translational impact, as this non-canonical form of autophagy is tumor inhibitory. Together, these results provide a new understanding of the biological effects of miR-34a and highlight the clinical potential for miR-34a delivery as a treatment for bone metastatic prostate cancer.

Background Src has a critical role in tumor cell migration and invasion. Increased Src activity has been shown to correlate with disease progression and poor prognosis, suggesting Src could serve as a therapeutic target for kinase inhibition. Saracatinib (AZD0530) is a novel selective oral Src kinase inhibitor. Methods Metastatic colorectal cancer patients who had received one prior treatment and had measurable disease were enrolled in this phase 2 study. Saracatinib was administered at 175 mg by mouth daily for 28 day cycles until dose-limiting toxicity or progression as determined by staging every 2 cycles. The primary endpoint was improvement in 4 month progression-free survival. Design of Thall, Simon, and Estey was used to monitor proportion of patients that were progression free at 4 months. The trial was opened with plan to enroll maximum of 35 patients, with futility assessment every 10 patients. Results A total of 10 patients were enrolled between January and November 2007. Further enrollment was stopped due to futility. Median progression-free survival was 7.9 weeks, with all 10 patients showing disease progression following radiographic imaging. Median overall survival was 13.5 months. All patients were deceased by time of analysis. Observed adverse events were notable for a higher than expected number of patients with grade 3 hypophosphatemia (n = 5). Conclusion Saracatinib is a novel oral Src kinase inhibitor that was well tolerated but failed to meet its primary endpoint of improvement in 4 month progression-free survival as a single agent in previously treated metastatic colorectal cancer patients.

Contains fulltext : 154473.pdf (Publisher’s version ) (Open Access) To study the role of FAK signaling complexes in promoting metastatic properties of prostate cancer (PCa) cells, we selected stable, highly migratory variants, termed PC3 Mig-3 and DU145 Mig-3, from two well-characterized PCa cell lines, PC3 and DU145. These variants were not only increased migration and invasion in vitro, but were also more metastatic to lymph nodes following intraprostatic injection into nude mice. Both PC3 Mig-3 and DU145 Mig-3 were specifically increased in phosphorylation of FAK Y861. We therefore examined potential alterations in Src family kinases responsible for FAK phosphorylation and determined only Yes expression was increased. Overexpression of Yes in PC3 parental cells and src-/-fyn-/-yes-/- fibroblasts selectively increased FAK Y861 phosphorylation, and increased migration. Knockdown of Yes in PC3 Mig-3 cells decreased migration and decreased lymph node metastasis following orthotopic implantation of into nude mice. In human specimens, Yes expression was increased in lymph node metastases relative to paired primary tumors from the same patient, and increased pFAK Y861 expression in lymph node metastases correlated with poor prognosis. These results demonstrate a unique role for Yes in phosphorylation of FAK and in promoting PCa metastasis. Therefore, phosphorylated FAK Y861 and increased Yes expression may be predictive markers for PCa metastasis.

Background EphA2 is an oncoprotein and tyrosine kinase receptor that is overexpressed in ovarian and many other cancers. We investigated the effects of reduced EphA2 levels on tumor growth and the tumor microenvironment in an orthotopic ovarian cancer model. Methods The effect of the EphA2-agonistic monoclonal antibody EA5, alone or in combination with paclitaxel, on the growth of ovarian cancer cells (SKOV3ip1, HeyA8, and HeyA8MDR [taxane-platinum resistant]) was determined in vitro and in vivo by immunoblotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and immunohistochemical analysis. Expression of EphA2 and markers of angiogenesis (CD31, vascular endothelial growth factor [VEGF], and basic fibroblast growth factor), proliferation (proliferating cell nuclear antigen), and endothelial cell apoptosis (CD31-terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick-end labeling colocalization) and phosphorylation of Src were analyzed by immunoblotting, immunohistochemistry, immunofluorescence, and in situ hybridization in tumors from treated mice. Statistical tests were two-sided. Results EA5 antibody treatment led to a more than 90% reduction in EphA2 expression in HeyA8 tumors in vivo. In mice bearing orthotopic SKOV3ip1 or HeyA8 tumors, 4 weeks of EA5 treatment resulted in tumors that weighed 31% and 45% less, respectively, than those in control (IgG-treated) mice (95% confidence interval [CI] = -0.09% to 71% and 20% to 70%, P = .27 and .01, respectively). Combination therapy with EA5 and paclitaxel reduced tumor weight by 77% and 80% (95% CI = 63% to 91% and 68% to 91%), respectively, compared with paclitaxel alone and by 92% and 88% (95% CI = 87% to 97% and 80% to 94%), respectively, compared with IgG alone. Combination therapy also reduced the weight of HeyA8MDR tumors by 47% (95% CI = 24% to 72%) compared with paclitaxel. Mice bearing SKOV3ip1 or HeyA8 tumors that were treated with combination therapy survived longer than those treated with paclitaxel alone (median survival = 144 versus 69 days and 46 versus 37 days, respectively). EA5-treated tumors had reduced microvascular density, proliferation, and VEGF protein and mRNA levels, with increased endothelial cell apoptosis. EphA2 was associated with Src, which was rapidly dephosphorylated after EA5 treatment. Conclusions EA5 in combination with paclitaxel decreased tumor growth in an orthotopic ovarian cancer mouse model through antiangiogenic mechanisms associated with reduced levels of VEGF and phosphorylated Src. Humanized antibody constructs against EphA2 are worthy of future study.

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process.

The nonreceptor protein tyrosine kinase Src is overexpressed in 70% of pancreatic adenocarcinomas. Here, we describe the effect of molecular and pharmacological down-regulation of Src on incidence, growth, and metastasis of pancreatic tumor cells in an orthotopic model. Src expression in L3.6pl human pancreatic tumor cells was reduced by stable expression of a plasmid encoding small interfering RNA (siRNA) to c-src. In stable siRNA clones, Src expression was reduced >80%, with no change in expression of the related kinases c-Yes and c-Lyn, and proliferation rates were similar in all clones. Phosphorylation of Akt and p44/42 Erk mitogen-activated protein kinase and production of VEGF and IL-8 in culture supernatants were also reduced (P < 0.005). On orthotopic implantation of varying cell numbers into nude mice, tumor incidence was unchanged; however, in the siRNA clones, large tumors failed to develop, and incidence of metastasis was significantly reduced, suggesting that c-Src activity is critical to tumor progression. To examine this possibility further, animals bearing established wild-type tumors were treated with the Src/Abl-selective inhibitor BMS-354825 (dasatinib). Tumor size was decreased, and incidence of metastases was significantly reduced in treated mice compared with controls. These results demonstrate that Src activation contributes to pancreatic tumor progression in this model, offering Src as a candidate for targeted therapy.

Experimental evidence suggests that CXCR4, a Gi protein–coupled receptor for the ligand CXCL12/stromal cell–derived factor-1α (SDF-1α), plays a role in breast cancer metastasis. Transactivation of HER2-neu by G protein–coupled receptor activation has been reported as a ligand-independent mechanism of activating tyrosine kinase receptors. We found that SDF-1α transactivated HER2-neu in the breast cancer cell lines MDA-MB-361 and SKBR3, which express both CXCR4 and HER2-neu. AMD3100, a CXCR4 inhibitor, PKI 166, an epidermal growth factor receptor/HER2-neu tyrosine kinase inhibitor, and PP2, a Src kinase inhibitor, each blocked SDF-1α–induced HER2-neu phosphorylation. Blocking Src kinase, with PP2 or using a kinase-inactive Src construct, and inhibiting epidermal growth factor receptor/HER2-neu signaling with PKI 166 each inhibited SDF-1α–stimulated cell migration. We report a novel mechanism of HER2-neu transactivation through SDF-1α stimulation of CXCR4 that involves Src kinase activation.

In 2004, there were an estimated 147,000 new cases of colorectal carcinoma (CRC) and 57,000 deaths from this disease, ranking it third among causes of cancer-related death in the United States.1 Significant advances in systemic therapy for metastatic CRC, including targeted therapies, have improved survival, but even with combination therapy the median survival is only about 15 to 21 months.2,3 To continue to improve our therapies for metastatic CRC, we need a better understanding of the factors that lead to tumor progression and metastasis. In particular, the mechanisms regulating CRC cell invasion through the basement membrane of the colon and migration of the cells to form metastases need to be further investigated. Insulinlike growth factor-I (IGF-I) and its tyrosine kinase receptor (IGF-IR) have been implicated in the development and progression of a variety of human cancers,4–12 including CRC.13–15 IGF-I has been shown to be an important mediator of tumor cell migration and invasion,16–20 but the downstream pathways by which IGF-I induces these processes have not been fully elucidated. Hepatocyte growth factor/scatter factor (HGF) and its tyrosine kinase receptor c-Met have also been implicated in the pathogenesis of a wide variety of human malignancies,21–25 including CRC.26 Similar to IGF-I, HGF/c-Met signaling is known to induce tumor-cell migration and invasion.11,25,27–30 Recently, cooperation between receptors and their signaling pathways has been shown to be important in regulating cellular responses to various ligands. We theorized that IGF-IR and c-Met cooperate in mediating migration and invasion of human CRC cells, given the following findings. First, IGF-I and HGF lead to activation of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system in various malignancies.4,11,17,27,31,32 This is central to our hypothesis in that uPA has been shown to cleave pro-HGF to active HGF.33 Second, IGF-I signaling results in induction of hypoxia inducible factor-1α in pancreatic carcinoma cells,10 and hypoxia, likely acting via hypoxia inducible factor-1α, has been shown to increase c-Met levels in human lung, hepatocellular, and other carcinomas.34 Third, growth factor receptor-binding protein 2-associated binder-1 functions as the main substrate and docking protein regulating downstream signaling by c-Met and has been shown to function as a signaling intermediate for IGF-I as well.35 Fourth, IGF-I and HGF have been shown to function as comitogens in a rat hepatoma cell line.36 In the current study, we investigated the hypotheses that IGF-IR and c-Met cooperate to mediate migration and invasion of human CRC cells and that uPA/uPAR activation is required for IGF-I- and HGF-mediated migration and invasion. We used a c-Met ribozyme to inhibit c-Met function in KM12L4 human CRC cells and performed transwell migration and invasion assays. The c-Met ribozyme experiments demonstrated that c-Met function is critical for IGF-I-mediated cell migration and invasion and for constitutive invasion. In experiments inhibiting uPA or uPAR, we demonstrated that migration and invasion mediated by IGF-I and HGF are dependent on uPA/uPAR activation. This suggests that uPA and uPAR are downstream of IGF-I and IGF-IR and of HGF and c-Met. To our knowledge, this study is the first to identify tyrosine kinase receptor cooperation between IGF-IR and c-Met in human CRC and the role of uPA/uPAR in mediating IGF-I and HGF/c-Met effects.

Purpose: The urokinase-type plasminogen activator receptor (u-PAR) contributes to colon cancer invasion and metastases. We have shown previously that u-PAR expression in colon cancer is driven by the Src tyrosine kinase. In the current study, we determined the ability of PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a Src kinase inhibitor, to reduce u-PAR expression and colon cancer invasion. Experimental Design: Western blotting, Northern blotting, and u-PAR promoter-reporter assays were performed to determine whether PP2 represses u-PAR expression. In vitro invasion assays were used to determine whether this kinase inhibitor, with or without a novel u-PAR antagonist, diminished cultured colon cancer invasiveness. Results: A constitutively active c-Src increased in vitro invasiveness of SW480 cells, whereas HT-29 cells expressing antisense c-Src showed diminished invasiveness, validating c-Src as a target for low molecular weight compound(s). The Src inhibitor PP2 reduced u-PAR transcription in HT-29 cells over the concentration range that blocked Src kinase activity. PP2 also reduced u-PAR protein amounts in three other colon cancer cell lines with modest to high constitutive Src activity. Treatment of HT-29 cells and 2C8 cells (a SW480 clone expressing a constitutively active Src) with PP2 diminished their in vitro invasiveness. Furthermore, combination of the Src inhibitor with a novel u-PAR peptide antagonist (NI-5.12) proved superior to the individual agents in suppressing invasiveness. Conclusions: A c-Src kinase inhibitor represses u-PAR expression and, alone or in combination with a u-PAR antagonist, diminishes colon cancer invasiveness. Thus, concurrent targeting of c-Src expression and pharmacological blockade of the u-PAR may represent a novel means of controlling colon cancer spread.

Objective. The objective of this study was to determine if the Src tyrosine kinase is overexpressed and activated in late-stage human ovarian cancers. Methods. Western analysis and immune complex kinase assays were performed on a panel of human ovarian cancer cell lines and normal ovarian epithelial cell cultures, and immunohistochemical analysis for Src and activated Src were performed on a panel of late-stage human ovarian tumors. Results and conclusions. Src is overexpressed and activated in a majority of late-stage ovarian tumors as well as in a panel of cultured malignant human ovarian epithelium grown in vitro, but not in normal ovarian epithelium (NOE) or immortalized NOE. Src overexpression was found to be frequently, but not always, associated with HER-2/ neu overexpression, but no statistical association between Src and Her-2/ neu overexpression could be demonstrated.

Src is a non-receptor protein tyrosine kinase, the expression and activity of which is increased in >80% of human colon cancers with respect to normal colonic epithelium. Previous studies from this and other laboratories have demonstrated that Src activity contributes to tumorigenicity of established colon adenocarcinoma cell lines. Src participates in the regulation of many signal transduction pathways, among which are those leading to cellular survival. In this study, we addressed the potential role of Src activation to a specific aspect of tumor cell survival, resistance to detachment-induced apoptosis (anoikis). Using five colon tumor cell lines with different biologic properties and genetic alterations, we demonstrate that expression and activity of Src corresponds with resistance to anoikis. Enforced expression of activated Src in subclones of SW480 cells (of low intrinsic Src expression and activity) increases resistance to anoikis; whereas decreased Src expression in HT29 cells (of high Src expression and activity) by transfection with anti-sense Src expression vectors increases susceptibility to anoikis. In contrast, increasing or decreasing Src expression had no effect on susceptibility to staurosporine-induced apoptosis in attached cells. PD173955, a Src family-specific tyrosine kinase inhibitor, increases the susceptibility of HT29 cells to anoikis in a dose- and time-dependent manner. Increasing Src expression and activity led to increased phosphorylation of Akt, a mediator of cellular survival pathways, whereas decreasing Src activity led to decreased Akt phosphorylation. In colon tumor cells with high Src activity, the PI3 kinase inhibitor LY 294002 sensitized cells to anoikis. These results suggest that Src activation may contribute to colon tumor progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis.

Overexpression and/or activation of c-Met, the protein tyrosine kinase receptor for the hepatocyte growth factor/scatter factor (HGF/SF), and the protein tyrosine kinase, Src, have been implicated in the progression and metastasis of human colorectal carcinoma (CRC). Previously, through ribozyme-mediated c-Met downregulation, we demonstrated a causal role for c-Met in CRC tumorigenesis and the production of liver metastases from the highly metastatic CRC cell line, KM20. Analysis of signaling intermediates downstream of c-Met demonstrated a specific reduction of Src activity with minimal effect on Erk1/2 and Akt following c-Met downregulation. As Src has been shown to upregulate Vascular Endothelial Growth Factor (VEGF), we sought to determine if the reduced tumor size and incidence could be due to reduced vessel formation in the c-Met downregulated clones. Here we show that tumors produced from c-Met downregulated cells have significantly reduced vessel density in tumors, correlating with a reduction in VEGF production. Additionally, the Src selective inhibitor, PP2, significantly reduced basal VEGF production in KM20 parental cells, and this effect could be rescued by HGF treatment. PP2 reduced basal proliferation, migration, and anchorage-independent growth. Furthermore, PP2 treatment demonstrated a dose-dependent decrease in HGF induced migration. In contrast, PP2 treatment only had a minor effect on HGF induced anchorage-independent growth. Collectively, these data demonstrate a significant role for c-Met-mediated Src activation in processes involved in CRC tumorigenesis and liver metastasis.

The purpose of this study was to determine the effects of adenoviral transgene expression of MMAC/PTEN on the in vitro and in vivo growth and survival of PC3 human prostate cancer cells.Adenoviruses expressing MMAC/PTEN or green fluorescent protein as a control were introduced into PC3 cells, and effects on signal transduction pathways and growth of tumors in an orthotopic nude mouse model were determined.MMAC/PTEN expression in PC3 cells decreased the level of phospho Akt but not that of phospho Mapk or FAK. Expression of MMAC/PTEN inhibited the in vitro growth of PC3 cells primarily by blocking cell cycle progression. Ex vivo introduction of MMAC/PTEN expression did not inhibit the tumorigenicity of orthotopically implanted PC3 cells, but it did significantly reduce tumor size and completely inhibited the formation of metastases. In vivo treatment of pre-established orthotopic PC3 tumors with adenoviral MMAC/PTEN did not significantly reduce local tumor size, but it did diminish metastasis formation.MMAC/PTEN functionally regulates prostate cancer cell metastatic potential in an in vivo model system and may be an important biological marker and therapeutic target for human prostate cancer.

Metastatic Prostate Cancer (PCa) is the second leading cause of cancer related-deaths in men in the United States. Understanding the molecular events critical to PCa metastasis should lead to novel therapies for treatment of metastatic disease. To assess molecular alterations required for increased PCa cell migration, a critical step in metastasis, multiple rounds of selection of PC3 and DU145 PCa cells were performed using a modified Boyden chamber assay. Highly migratory cells (termed PC3-Mig-3 and DU145-Mig-3) were obtained, a phenotype that has remained stable. PC3-Mig-3 and DU145-Mig-3 cells were also increased in invasiveness, decreased in adhesion and proliferation; thus acquiring additional properties of metastatic cells. Following orthotopic injection into nude mice, migratory variants were significantly increased in number of lymph node metastases (P Citation Format: Tanushree Chatterji, Jian H. Song, Nila U. Parikh, Chien-Jui Cheng, Sue-Hwa Lin, Gary E. Gallick. Differential phosphorylation of focal adhesion kinase and activation of Yes kinase are associated with increased metastatic potential of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4056. doi:10.1158/1538-7445.AM2014-4056

The c-Yes proto-oncogene (pp62c-Yes) encodes a non-receptor-type protein tyrosine kinase (NRPTK) of the Src family. c-Yes activities and protein levels are elevated in human melanoma and melanocyte cell lines. Because the neurotrophins (NT) are important in the progression of melanoma to the brain-metastatic phenotype, we determined whether NT stimulate c-Yes activity in human MeWo melanoma cells and two variant sublines with opposite metastatic capabilities, 3 S 5 and 70W. The highly brain-metastatic 70W subline had an intrinsically higher c-Yes activity than parental MeWo or poorly metastatic 3 S 5 cells. c-Yes kinase was further induced by the prototypic human NT, nerve growth factor (NGF) in a dose and time-dependent manner. In contrast, c-Src activity (pp60-Src) was similar in all these cells and unaffected by NGF exposure. Additionally, human NGF and neurotrophin-3 stimulated c-Yes in brain-metastatic 70W cells. The magnitude of c-Yes activation correlated with the degree of invasion of 70 W cells following incubation of these neurotrophins. To further examine NT stimulation of c-Yes in melanoma cells, three additional cell lines were examined. Metastatic TXM-13 and TXM-18 increased c-Yes activity in response to NGF. In contrast, no increase was observed in low-metastatic TXM-40 cells. Together, these data suggest that altered c-Yes expression may play a role in the malignant progression of the human melanocyte towards the brain-metastatic phenotype and that NT enhance the activity of c-Yes in signaling penetration into the matrix of NT-rich stromal microenvironments such as the brain.

Vascular endothelial growth factor (VEGF) is implicated in the angiogenesis of human colon cancer. Recent evidence suggests that factors that regulate VEGF expression may partially depend on c-src-mediated signal transduction pathways. The tyrosine kinase activity of Src is activated in most colon tumors and cell lines. We established stable subclones of the human colon adenocarcinoma cell line HT29 in which Src expression and activity are decreased specifically as a result of a transfected antisense expression vector. This study determined whether VEGF expression is decreased in these cell lines and whether the smaller size and reduced growth rate of antisense vector-transfected cell lines in vivo might result, in part, from reduced vascularization of tumors. Northern blot analysis of these cell lines revealed that VEGF mRNA expression was decreased in proportion to the decrease in Src kinase activity. Under hypoxic conditions, cells with decreased Src activity had a2-fold increase in VEGF expression, whereas parental cells had a50-fold increase. VEGF protein in the supernatants of cells was also reduced in antisense transfectants compared with that from parental cells. In nude mice, subcutaneous tumors from antisense transfectants showed a significant reduction in vascularity. These results suggest that Src activity regulates the expression of VEGF in colon tumor cells.

Background. The c-src protooncogene encodes a protein tyrosine kinase, pp60c-src, that is a mediator in many signal transduction pathways. One pathway in which pp60c-src protein tyrosine kinase activity is implicated involves regulation of vascular endothelial growth factor (VEGF), an angiogenic factor important to neovascularization of growing tumors. Recently we demonstrated that decreased activity of pp60c-src in colon tumor cells contributes to decreased expression of VEGF. This study examined the relationship between pp60c-src activation, cell density, and VEGF production in a colon tumor cell line. Methods. Parental HT-29 colon adenocarcinoma cells and stable subclones created by transfection with c-src antisense and sense (control) expression vectors were plated under sparse (2 × 104 cells/cm2) and confluent (20 × 104 cells/cm2) conditions and grown for 36 hours. Protein and RNA were extracted from cells to determine pp60c-src levels, c-Src tyrosine kinase activity, and VEGF mRNA expression. Results. The pp60c-src kinase activity of HT-29 cells and control sense-transfected clones grown under confluent conditions was increased threefold to fivefold compared with cells grown under sparse conditions. In contrast, the ability of confluent culture conditions to increase pp60c-src activity was blunted in antisense transfectants. By regression analysis, VEGF expression was found to vary directly with pp60c-src levels (r2 = 0.886). Conclusions. Cell density contributes to the regulation of c-src kinase activity and VEGF expression in HT-29 cells. When the steady-state level of pp60c-src is reduced in antisense transfectants, not only is the steady-state level of VEGF reduced, but the ability of confluence to stimulate pp60c-src activity and VEGF production is too. These data suggest that c-src may be an intermediary of both constitutive and inducible pathways for VEGF production in colon tumor cells.

Background: Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways. Materials and Methods: Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively). Results: In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers. Conclusions: Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth.

3561 Background: The nonreceptor tyrosine kinase Src regulates pathways critical to tumor proliferation, chemoresistance, and epithelial-to-mesenchymal transition. In vitro, Src is activated after acute oxaliplatin exposure and in acquired oxaliplatin resistance, but not after 5-FU alone. Activation of Src and its substrate FAK in metastatic colorectal cancer treated with oxaliplatin has not been studied in human specimens. Methods: Samples from 170 hepatic resections from two cohorts of patients with metastatic colorectal cancer were examined by IHC for expression of activated Src (pSrc) and FAK (total and pFAK). In the first cohort (n=50), tissue was collected at consecutive hepatic resections before and after oxaliplatin. Patients in the second cohort (n=120) were compared based on whether or not oxaliplatin was administered after resection. IHC was graded semi-quantitatively, 0 to 4 based on intensity (first cohort), and by automated image analysis (second cohort). Results: In the first cohort, pFAK expression increased after oxaliplatin exposure (mean IHC score 2.04 vs. 1.18, p

61 Background: The Src and IGF-1R axes are aberrantly activated in both PCa and the microenvironment of bone metastases. Dasatinib and BMS-754807 are clinically promising small molecule inhibitors with high potency against Src family kinases (SFK) and IR/IGF-1R, respectively. Based on a phase I/II clinical trial in which 9/19 pts treated with docetaxel + dasatinib were increased in serum IGF-1 levels after one cycle, the aim of this study was to establish potential antitumor cooperativity of inhibiting both IGF-1R and Src in experimental PCa models in vitro and in mice. Methods: Inhibition of Src and IGF-1R pathways was accomplished by pharmacologic agents (dasatinib against Src and BMS-754807 against IR/IGF-1R) as well as by shRNA, in PC3 and LNCaP cells. In vivo studies were done after orthotopic and intratibial injection of PC3 cells in nude mice. Results: SFK inhibition decreased proliferation and migration of PCa cells whereas IGF-1R blockade induced apoptosis. All anti-tumor effects were enhanced by dual blockade. IGF-1 induced phosphorylation of Akt1 and 2. Only Akt 1 phosphorylation was decreased by dasatinib; whereas Akt 1 and 2 phosphorylation were completely abrogated by the combination. Dasatinib and BMS-754807 inhibited orthotopic in vivo tumor growth of PC3 cells more potently than either inhibitor alone. Similarly, intratibial tumor growth and bone destruction was significantly reduced with the drug combination, accompanied by a decrease in serum bone turnover markers alkaline phosphatase and N-telopeptide. Conclusions: Dual inhibition of Src and IGF-1R has greater anti-tumor effect in PCa cells compared to inhibiting either alone. In the presence of IGF-1, dasatinib and BMS-754807 are necessary to inhibit IGF-1-induced phosphorylation of Akt1 and 2 in tumor cells in culture. In intratibial models, decreased bone turnover markers in serum support the concept of targeting both the epithelial and bone microenvironment. The combination of dasatinib and BMS-754807 may be a rational therapeutic approach in PCa by blocking complementary processes of tumor growth and progression.

Background: Prostate cancer (PCa) is the second leading cause of cancer deaths. Deaths invariably result from metastasis, most commonly to the bone. Tumor-microenvironment interactions of metastatic PCa in the bone are critical for growth at the metastatic site. These interactions suggest that therapies will need to target both tumor cells and the microenvironment to be successful. One target in both tumor and microenvironment is Src family kinases (SFKs), for which a phase three trial in combination with docetaxel is completed. Better targeting of SFKs will depend on understanding the tumor-stromal interactions that lead to its activation in both compartments. Aberrant Src activation de-represses cytokines including IL-8, contributing to the “vicious cycle” of metastatic growth in the bone. Stromal cells are a rich source of IL-8, but there is little information on how they affect tumor cells and viceversa. Here, we describe a feed-forward loop and paracrine mechanism of IL-8-mediated Src activation in PCa, both of which lead to increased tumor cell migration and invasion. Methods/Results: We demonstrate IL-8 and Src are overexpressed in more metastatic PC3-MM2 cells versus less metastatic PC3 cells. Previous work in the laboratory has demonstrated activated Src increases IL-8 expression. Through lentiviral knockdown of IL-8 in PC3-MM2 (PC3-MM2shIL-8) cells, we demonstrate that decreased IL-8 expression decreases Src activity in tumor cells. We also demonstrate that decreased IL-8 expression decreases migration and invasion while exogenous IL-8 rescues this phenotype. We next asked the effect of immortalized bone marrow-derived human stromal cells (HS5) on tumor cells. HS5 cells secrete high levels of IL-8. To determine if stromal-produced IL-8 affects Src activity in PCa, conditioned media from HS5 cells was added to PC3, PC3- MM2 and PC3-MM2shIL-8 cells. Src activity was increased in all cell lines. Conditioned media also increased migration and invasion of these cell lines. To determine if these increases were due, in part to IL-8, HS5 cells were infected with a lentivirus driving the expression of shIL-8 (HS5shIL-8) and conditioned media was collected. Addition of conditioned media from HS5shIL-8 cells to PC3 or PC3-MM2shIL-8 cell lines resulted in reduced Src activation, migration and invasion relative to HS-5 cells infected with a control vector. Conclusions/Future Directions: These results demonstrate stromal production of IL-8 increases Src activity and that stromal cells play a major role in metastatic properties of PCa cells. To confirm these in vitro studies, in vivo studies are being performed using co-injections of stromal cells and PCa cells. We will determine if: (1) stromal cells co-inoculated with tumor cells increase tumor growth and/or metastatic potential of PC3; (2) if knockdown of Src and/or IL-8 in stromal cells inhibits the ability to increase metastatic potential induced by stromal cells. Recent data from the Phase I/II dasatinib + docetaxel trial suggest that decreases in IL-8 levels after 21 days of treatment are associated with patient response (Araujo et al., unpublished). These results suggest that IL-8 from both tumor and stromal cells may be important inmetastatic potential and growth of prostate tumor cells in the bone. Understanding the roles of IL-8 and Src in PCa and the stromal microenvironment are important in better understanding PCa metastasis. Citation Format: Lynnelle Thorpe, John C. Araujo, Nila U. Parikh, Menashe Bar Eli, Gary E. Gallick. Contribution of stromal-derived IL-8 on prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A65.

e15004 Background: The Src and IGF-1R axes are aberrantly activated in both PCa and the microenvironment of bone metastases. Dasatinib and BMS-754807 are clinically promising small molecule inhibit...