Antiproliferative Effects Of Sorafenib Research Articles

Contrary influence of clinically applied sorafenib concentrations among hepatocellular carcinoma patients.

The treatment responses of sorafenib in hepatocellular carcinoma are modest which may be due to different characteristics of cancer cells or insufficient therapeutic concentrations. This study was to clarify this issue. The anti-proliferative effects and differential expressions of 8 genes related to sorafenib anti-cancer mechanisms (tyrosine kinase receptor genes: KDR, PDGFRB; RAF cascade: RAF1, BRAF, MAP2K1, MAP2K2, MAPK1, MAPK3) were investigated in primary cultured hepatocellular carcinoma cells collected from 8 patients using clinically applied sorafenib concentrations (5, 10μg/mL). The anti-proliferative effects of sorafenib at either 5 or 10μg/mL, which were related to down-regulations of KDR, PDGFRB and/or genes in the RAF cascade, were achieved only in one patient (HCC38/KMUH). However, either 5 or 10μg/mL sorafenib promoted proliferation in 4 patients (HCC29/KMUH, HCC62/KMUH, HCC87/KMUH, HCC98/KMUH). Among them, the RAF cascade, PDGFRB and/or KDR were up-regulated in 3 patients but no gene was differentially expressed in the remaining one patient (HCC87/KMUH). Increase the sorafenib concentration to 10μg/mL paradoxically up-regulated and/or obliterated the previously down-regulated genes in the RAF cascade and/or KDR in 4 patients (HCC29/KMUH, HCC76/KMUH, HCC87/KMUH, HCC98/KMUH). Significant down-regulations of the RAF cascade and PDGFRB by sorafenib but without anti-proliferative effects were detected in one patient (HCC54/KMUH). In conclusion, influence of sorafenib on proliferation is not simply through the RAF cascade. The responses of KDR, PDGFRB and the RAF cascade to sorafenib among patients are diverse or even contrary. Increase the sorafenib concentration has potential to up-regulate genes favored angiogenesis and proliferation.

Antitumor activity of sorafenib on colorectal cancer

Abstract In spite of progress in treatment on colorectal cancer, it is not still convincing. Because of that, there is a pursuit to find a better treatment option. Our goal of this study is to research antiproliferative effect and mechanism of action of sorafenib on colorectal cancer cell line. Antiproliferative effects of sorafenib have been evaluated by using colorectal DLD-1 cell line. Materials and Methods: For determining the effects of sorafenib on colorectal cell, Real time analysis was performed. Chorioallantoic membrane model (CAM) was used for assessing angiogenesis. Different concentrations of sorafenib (500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.9, 1.95, 0.97 and 0.48 nM/well) applied on DLD-1 colorectal cancer cells, caused strong antiproliferative effect in a concentration manner. IC50 value of sorafenib was 1.26 × 10-9 M. Sorafenib (100, 10 and 1 nM) also showed strong angiogenic effect in CAM model with scores 1.05, 0.75 and 0.55. The present study indicates that sorafenib has strong antitumor and antiangiogenic affect. These important features make it valuable treatment agent for colorectal cancer which is strongly connected to angiogenic factors. In order to verify and enlighten antitumor mechanism of sorafenib, there is still need for further investigations.

Evaluation of antiangiogenic and antiproliferative effects of sorafenib by sequential histology and intravoxel incoherent motion diffusion-weighted imaging in an orthotopic hepatocellular carcinoma xenograft model.

To investigate the effectiveness of intravoxel incoherent motion (IVIM) in the assessment of the therapeutic efficacy of sorafenib in an orthotopic hepatocellular carcinoma (HCC) xenograft model. Thirty-five HCC nude mouse models were established. IVIM was performed on a 1.5T MR scanner at baseline (n = 5) and serially at 7, 14, and 21 days after sorafenib treatment. The apparent diffusion coefficient (ADCtotal ), true diffusion coefficient (D), pseudodiffusion coefficient (D*), and perfusion fraction (f) at these timepoints were measured and compared between the treated (n = 15) and control group (n = 15). Differences in measurements among different timepoints were evaluated. Correlations between IVIM parameters and histologic features including necrotic fraction (NF) and microvessel density (MVD) were analyzed. Compared to the control group, ADCtotal and D were significantly higher at each timepoint (P = 0.009), while f significantly decreased at 7 days (P = 0.009) and increased at 21 days (P = 0.028) in the treated group. Serial measurements in the treated group showed that both ADCtotal and D increased significantly at 7, 14, and 21 days compared to baseline (P < 0.05), while f significantly declined at 7 days (P = 0.016) and increased at 21 days (P = 0.009). Significant correlations were found between ADCtotal and NF (r = 0.811, P < 0.001), D and NF (r = 0.838, P < 0.001), and between f and NF (r = 0.528, P = 0.017) in the treated group. IVIM may provide useful biomarkers for evaluating the therapeutic effects of sorafenib on HCC. 1 J. Magn. Reson. Imaging 2017;45:270-280.

Abstract 5336: Improved antitumor effect of combining WNT/beta-catenin inhibition with sorafenib in hepatocellular carcinoma

Abstract [Background] Sorafenib is currently the only approved systemic agent for advanced hepatocellular carcinoma (HCC); however, its efficacy is limited. Aberrant activation of WNT/β-catenin signaling pathway has been shown to mediate resistance to various types of anti-cancer therapy. We hypothesized that inhibition of WNT/β-catenin signaling pathway would improve the anti-tumor effect of sorafenib in HCC. [Materials and Methods] Human HCC cell lines, including Huh7, HepG2, PLC5, and Hep3B, were included. Inhibition of WNT/β-catenin pathway was achieved by ICG-001, a small molecule disrupting the interaction of cAMP-responsive element binding (CREB)-binding protein (CBP) and β-catenin and inhibiting the β-catenin-mediated transcription activities, or by RNA interference (RNAi)- downregulation of β-catenin. The efficacy of sorafenib combined with WNT/β-catenin pathway inhibition in HCC cells was determined by MTT for cell viability, and by flow cytometry and Western blotting for apoptosis. The in vivo efficacy was evaluated in a subcutaneous xenograft mode of Huh7 cells. [Results] In multiple HCC cell lines, ICG-001 enhanced the anti-proliferative effect of sorafenib dose-dependently; the combination of ICG-001 and sorafenib showed a synergistic antitumor effect. Downregulation of β-catenin by RNAi increased sorafenib sensitivity, whereas overexpression of β-catenin decreased sorafenib sensitivity in Huh7 cells. Furthermore, the sorafenib-sensitization effect by short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was offset by β-catenin overexpression. Mechanistically, sorafenib in combination of ICG-001 or shRNA- mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a more prominent downregulation of Mcl-1 in HCC cells. Finally, ICG-001 in combination of sorafenib showed a more significantly growth-retarding effect in Huh7 xenograft than either drug alone. [Conclusion] Our data indicate that inhibition of WNT/β- catenin signaling pathway improves the antitumor effect of sorafenib against HCC in vitro and in vivo.(This study was supported by grants NRPB-100CAP1020-2 and NSC-101-2314-B-002-141, 100CAP1020-2). Citation Format: Hsiao-Hui Lin, Wen-Chi Feng, Li-Chun Lu, Yu-Yun Shao, Ann-Lii Cheng, Chih-Hung Hsu. Improved antitumor effect of combining WNT/beta-catenin inhibition with sorafenib in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2015-5336

Abstract 2052: WNT/beta-catenin signaling inhibitors improve the anti-proliferative effect of sorafenib against hepatocellular carcinoma (HCC) cells.

Abstract [Background] Sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is currently the only indicated therapy for patients with advanced hepatocellular carcinoma (HCC). The aberrant activation of WNT/β-catenin signaling pathway, which is associated with the carcinogenetic process of HCC, has been shown to mediate the resistance to various types of anti-cancer therapy. We hypothesized that inhibition of WNT/β-catenin signaling pathway might improve the anti-tumor effect of sorafenib in HCC. [Materials and Methods] Huh7, HepG2, PLC5, and Hep3B HCC cells were treated with sorafenib with or without inhibitors of WNT/β-catenin pathway, and evaluated for viability in vitro. WNT/β-catenin signaling inhibitors included ICG-001 which down-regulates β-catenin/TCF signaling by specifically binding to cyclic AMP response element-binding protein, and XAV939 which targets tankyrases and facilitates degradation of β-catenin. [Results] Sorafenib induced an anti-proliferative effect in HCC cells, with IC50s of 3.5±0.3 μM, 3.7±0.4 μM, 7.7±0.8 μM and 14±0.3 μM for HepG2, Huh7, PLC5, and Hep3B cells, respectively. Upon treated with 10 μM sorafenib for 24 hours, the total cellular amounts of β-catenin were significantly decreased in HepG2 and Huh7 cells, moderately decreased in PLC5 cells, and unchanged in Hep3B cells. In all HCC cells, XAV939 or ICG-001 enhanced the anti-proliferative effect of sorafenib dose-dependently. The median effect analysis showed a synergistic anti-proliferative effect for the combination of ICG-001 plus sorafenib in all HCC cells. [Conclusion] Inhibitors of WNT/β- catenin signaling pathway in combination with sorafenib resulted in an improved anti-tumor effect against HCC in vitro. (The study was supported by the grant NSC 100-2325-B-002-043 from National Science Council of Taiwan) Citation Format: Hsiao-Hui Lin, Wen-Chi Feng, Li-Chun Lu, Yu-Yun Shao, Ann-Lii Cheng, Chih-Hung Hsu. WNT/beta-catenin signaling inhibitors improve the anti-proliferative effect of sorafenib against hepatocellular carcinoma (HCC) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2052. doi:10.1158/1538-7445.AM2013-2052

Multimodal imaging of tumor response to sorafenib combined with radiation therapy: comparison between diffusion‐weighted MRI, choline spectroscopy and 18F‐FLT PET imaging

The purpose of this study was to determine the value of different imaging modalities, that is, magnetic resonance imaging/spectroscopy (MRI/MRS) and positron emission tomography (PET), to assess early tumor response to sorafenib with or without radiotherapy. Diffusion-weighted (DW)-MRI, choline (1)H MRS at 11.7 T, and (18)F-FLT PET imaging were used to image fibrosarcoma (FSaII) tumor-bearing mice over time. The imaging markers were compared with apoptosis cell death and cell proliferation measurements assessed by histology. Anti-proliferative effects of sorafenib were evidenced by (1)H MRS and (18)F-FLT PET after 2 days of treatment with sorafenib, with no additional effect of the combination with radiation therapy, results that are in agreement with Ki67 staining. Apparent diffusion coefficient calculated using DW-MRI was not modified after 2 days of treatment with sorafenib, but showed significant increase 24 h after 2 days of sorafenib treatment combined with consecutive irradiation. The three imaging markers were able to show early tumor response as soon as 24 h after treatment initiation, with choline MRS and (18)F-FLT being sensitive to sorafenib in monotherapy as well as in combined therapy with irradiation, whereas DW-MRI was only sensitive to the combination of sorafenib with radiotherapy.

Effects of Sorafenib on C-Terminally Truncated Androgen Receptor Variants in Human Prostate Cancer Cells

Recent evidence suggests that the development of castration resistant prostate cancer (CRPCa) is commonly associated with an aberrant, ligand-independent activation of the androgen receptor (AR). A putative mechanism allowing prostate cancer (PCa) cells to grow under low levels of androgens, is the expression of constitutively active, C-terminally truncated AR lacking the AR-ligand binding domain (LBD). Due to the absence of a LBD, these receptors, termed ARΔLBD, are unable to respond to any form of anti-hormonal therapies. In this study we demonstrate that the multikinase inhibitor sorafenib inhibits AR as well as ARΔLBD-signalling in CRPCa cells. This inhibition was paralleled by proteasomal degradation of the AR- and ARΔLBD-molecules. In line with these observations, maximal antiproliferative effects of sorafenib were achieved in AR and ARΔLBD-positive PCa cells. The present findings warrant further investigations on sorafenib as an option for the treatment of advanced AR-positive PCa.

Abstract A54: Potential mechanisms of resistance to targeted agents in human clear cell renal cell carcinoma

Abstract Targeted therapy with multiple receptor tyrosine kinase inhibitors (RTKI) has led to a substantial improvement in the standard of care for patients with advanced or metastatic clear cell renal cell carcinoma (CCRCC). We have previously reported that using a phosphoproteomics approach, Ras and Rab interactor 1 (RIN1) was identified as a potential target modulating the antiproliferative effects of sorafenib and sunitinib in a panel of human CCRCC (Proc. AACR 51: Abst. #1626, 2010). While both 10 μM sorafenib and 7.5 μM sunitinib were effective in down-regulating RIN1 protein levels in CCRCC following treatment for 48h, during subsequent recovery for 48h in drug free medium, recovery of RIN1 protein levels was observed in sorafenib but not sunitinib treated cells. mRNA expression profiling revealed that: (a) VEGF-A and HIF-1α levels were unaffected during 48h treatment with sorafenib or sunitinib or subsequent recovery; and (b) while HIF-2α levels were increased &amp;gt;2-fold following 48h exposure to sorafenib or sunitinib, during subsequent recovery in drug-free medium, the increased levels of HIF-2α persisted in cells treated with sorafenib but not sunitinib. Compared to CCRCC cells that were growth inhibited in vitro at concentrations of 5–10 μM sorafenib or sunitinib, in 27 patient CCRCC samples evaluated, the median mRNA levels of RIN1 was 7.2-fold lower in 22 samples (Range 1.6–22.3) and 2.2-fold higher in 5 samples (Range 1.2–27.1). Stable over-expression and down-regulation of RIN1 in CCRCC led to resistance or sensitivity respectively, to the anti-proliferative effects of sorafenib and sunitinib but not the mTOR inhibitor temsirolimus. In contrast, CCRCC cells adapted to proliferate in 4 μM everolimus exhibited a &amp;gt;3-fold increase in IC50, and cross-resistance to temsirolimus but not sorafenib or sunitinib. Evaluation in vitro of other targeted agents, demonstrated dose-dependent anti-proliferative effects with the PI3K/mTOR dual inhibitor BEZ235 (&amp;gt;80% growth inhibition at 10nM) but not the pan-PI3K inhibitor BKM120 (&amp;lt;30% growth inhibition at 10–500 nM) with CCRCC cells. The observed anti-proliferative effects of BEZ235 or BKM120 were comparable in CCRCC cells harboring the wild type or mutant von Hippel-Lindau gene. While BEZ235 (1–5 nM) or everolimus (100 nM) alone led to &amp;lt;40% growth inhibition, the combination significantly enhanced anti-proliferative effects (&amp;gt;85% growth inhibition) in CCRCC cells expressing resistance to everolimus. These results suggest the PI3K/mTOR dual inhibitor BEZ235 may possibly improve the treatment efficacy of mTOR inhibitors for CCRCC. The findings suggest RTKI and mTOR inhibitors have distinct mechanisms of resistance, and combinations may be useful to improve disease control of metastatic CCRCC.

Sorafenib augments cytotoxic effect of S-1 in vitro and in vivo through TS suppression

Sorafenib, a multikinase and tyrosine-kinase inhibitor, has anti-tumor activity in patients with advanced renal cell carcinoma (RCC). Recently, we reported that S-1 was active and well tolerated for the treatment of cytokine-refractory metastatic RCC. Therefore, we hypothesized that S-1 might be a good candidate for combination therapy with molecular targeting agents. In this study, we examined the mechanisms underlying for the synergism between S-1 and Sorafenib for RCC treatment in vitro and in tumor-bearing murine models. Human RCC cell lines were used for the in vitro cell proliferation assay. ACHN and 786-O tumors were subcutaneously transplanted into NCr-nu/nu-mice. Mice were treated with S-1 and/or Sorafenib, and tumor growth and side effects were monitored. Synergistic anti-proliferative effects of Sorafenib and S-1 were clearly demonstrated in ACHN and 786-O cell lines in vitro due to the suppression of TS and E2F-1 expression. In the NCr-nu/nu model, the synergistic anti-tumor effects of S-1 and Sorafenib were again clearly seen, indicating direct synergistic effects of each drug on tumor growth. Our results demonstrate the synergistic activity of S-1 and Sorafenib and provided the rationale for combination therapy with S-1 and Sorafenib for the treatment of patients with advanced RCC.

Abstract 3828: RDEA119 synergy the anti-tumor effect of sorafenib in renal cell carcinoma cells

Abstract The MEK signaling pathway is over-expressed in more than 30 percentages of cancers, leading to cell growth and proliferation. Renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor where the Raf/MEK/ERK pathway is activated; suggesting that inhibition of this pathway may have therapeutic targets. Sorafenib as a multi-kinase inhibitor against Raf, VEGFR-2, PDGFR, c-kit, and FLT3 approved by the FDA for treatment of advanced renal cell and hepatocellular carcinomas. Since it was reported recently that combination treatment with sorafenib and MEKI exerted synergistic effect on cell proliferation in HCC, we studied the anti-proliferative effect of RDEA119 as a single agent or in combination with sorafenib for the treatment of RCC and HCC. We examined cell death effect on cell viability in 8 kinds of RCC cells and 6 kinds HCC cells. Treatment with RDEA119 inhibited proliferation of TK-10, UO31, A497 (RCC) and HepG2 (HCC) cells. In the case of combination treatment, pre-treatment with RDEA119 potentiated anti-proliferative effect of sorafenib in A498 cells (ccRCC and VHL-/-). This combination showed synergism in combination index and isobolograms analyzed by CalcuSyn software. Furthermore, the combination treatment activated caspases-3, -9 and -8, which resulted in intrinsic and extrinsic apoptosis. To figure out cell death mechanism activated by this combination, we analyzed the phosphorylation of phospho-ERK (p-ERK) and p-AKT in A498 cells. Single treatment with RDEA119 decreased p-ERK effectively, but increased p-AKT. On the other hand, sorafenib alone decreased p-AKT, but increased p-ERK. However, combination treatment with RDEA119 and sorafenib inhibited phosphorylation of MEK and AKT completely. The combination treatment decreased the level of p-GSK3β and anti-apoptotic protein, MCL-1 which are related to survival. Our study indicates that MEK inhibitor (MEKI) alone suppressed cell proliferation and the combination of MEKI with sorafenib increased apoptosis synergistically by decreasing activation of AKT pathway as well as ERK pathway in A498 cells. These results suggest that it may be a new therapeutic modality for treatment of ccRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3828. doi:10.1158/1538-7445.AM2011-3828

The multikinase inhibitor Sorafenib displays significant antiproliferative effects and induces apoptosis via caspase 3, 7 and PARP in B- and T-lymphoblastic cells

BackgroundTargeted therapy approaches have been successfully introduced into the treatment of several cancers. The multikinase inhibitor Sorafenib has antitumor activity in solid tumors and its effects on acute lymphoblastic leukemia (ALL) cells are still unclear.MethodsALL cell lines (SEM, RS4;11 and Jurkat) were treated with Sorafenib alone or in combination with cytarabine, doxorubicin or RAD001. Cell count, apoptosis and necrosis rates, cell cycle distribution, protein phosphorylation and metabolic activity were determined.ResultsSorafenib inhibited the proliferation of ALL cells by cell cycle arrest accompanied by down-regulation of CyclinD3 and CDK4. Furthermore, Sorafenib initiated apoptosis by cleavage of caspases 3, 7 and PARP. Apoptosis and necrosis rates increased significantly with most pronounced effects after 96 h. Antiproliferative effects of Sorafenib were associated with a decreased phosphorylation of Akt (Ser473 and Thr308), FoxO3A (Thr32) and 4EBP-1 (Ser65 and Thr70) as early as 0.5 h after treatment. Synergistic effects were seen when Sorafenib was combined with other cytotoxic drugs or a mTOR inhibitor emphasizing the Sorafenib effect.ConclusionSorafenib displays significant antileukemic activity in vitro by inducing cell cycle arrest and apoptosis. Furthermore, it influences PI3K/Akt/mTOR signaling in ALL cells.

133 mTOR is a druggable molecule in Malignant Pleural Mesothelioma targeted therapy: antiproliferative effect of sorafenib and everolimus in preclinical models

Sorafenib induces apoptosis and enhances Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-induced cell killing of tumoural cells. We have investigated the effects of the multikinase inhibitor Sorafenib alone or in combination with TRAIL and agonistic Fas antibodies on endometrial carcinoma cells. We have also focused on the search of the differential molecular mechanisms by which Sorafenib induces cell death and the ones involved in sensitisation to TRAIL. In the present study, we show that Sorafenib induces apoptosis of both endometrial cancer cell lines and human primary cultures and sensitises these cells to TRAIL and agonistic Fas antibodies (aFas)-induced apoptosis. However, Raf/MEK/ERK inhibition by Sorafenib was not responsible for Sorafenib cell death or TRAIL sensitisation of endometrial cancer cells. Sorafenib treatment correlated with a downregulation of both FLICE-Inhibitory Protein (FLIP) and myeloid cell leukaemia-1 (Mcl-1), caused by a proteasomal degradation of both proteins. We evaluated the contribution of FLIP and Mcl-1 downregulation in apoptosis triggered by Sorafenib alone or Sorafenib plus TRAIL. Interestingly, cell death caused by Sorafenib was mediated by downregulation of Mcl-1, but not by FLIP. In contrast, we found that Sorafenib sensitisation of endometrial carcinoma cells to TRAIL- and Fas-induced apoptosis was dependent on FLIP but not on Mcl-1 downregulation. Altogether, we discern the dual mechanisms by which Sorafenib causes cell death from those involved in death receptor sensitisation.

Abstract 1626: Tyrosine phosphorylation of RIN1 modulates the anti-proliferative effects of sorafenib and sunitinib in renal cell carcinoma (RCC)

Abstract Receptor-tyrosine kinase inhibitors (TKI), sorafenib (SRF) and sunitinib (SNT) are clinically used in treatment of RCC. Both drugs have anti-proliferative effects in several tumor types with inhibition of angiogenesis being a major mechanism of action in RCC. The intracellular molecular effects of SRF and SNT have not been completely described. Thus the identification of targets mediating the anti-proliferative effects of SRF and SNT could be useful to improve efficacy and to reduce toxicity. In this work we used immunoaffinity isolation for enrichment of phosphotyrosine containing proteins in control or SRF-treated CAKI-1 cells. To identify tyrosine-phosphorylated proteins that were differentially affected by SRF treatment, we performed liquid chromatography - tandem mass spectrometry (MS) followed by quantitative analysis using peptide counts and native peptide reference MS methods. In CAKI-1 cells, MS results revealed that signal transducer and activator of transcription 1 (STAT1) and Ras and Rab interactor 1 (RIN1) were the predominant proteins which exhibited tyrosine hypophosphorylation following treatment for 48 h with 10 μM SRF, when compared to the untreated control. Western blot analysis with phosphospecific antibody indicated a potential role for tyrosine 36 phosphorylation in RIN1 (pY36-RIN1) in CAKI-1, RC-45 and 786-O clear cell RCC. Up to a 8-fold SRF dose-dependent (5-15 µM) decrease in pY36-RIN1, but not in total RIN1 expression was also observed in a panel of RCC cell lines. Hypophosphorylation of Y36-RIN1 was also detected in CAKI-1 cells treated with 5 µM SNT but not with imatinib (≤ 10 µM), suggesting that pY36-RIN1 is a selective target for agents SRF and SNT that are also clinically effective in treatment of RCC. A ∼2-fold higher expression of RIN1 (total and phosphorylated) was observed in CAKI-1 cells following continuous exposure (∼ 6 weeks) to 7.5 µM SRF. However, unlike parent CAKI-1 cells, in cells adapted to 7.5 μM SRF, exposure to 10 μM SRF did not completely abrogate phosphorylation of Y36-RIN1. Stable expression of RIN1 targeted shRNA in CAKI-1 cells led to a 2-4 fold down-regulation of RIN1 protein and significantly (p &amp;lt; 0.002) enhanced sensitivity to the anti-proliferative effects of 7.5 μM SRF or SNT compared to scrambled shRNA. In contrast, overexpression of RIN1 in RC-45 or 786-O RCC cells resulted in significantly (p &amp;lt; 0.05) increased resistance to 10 μM SRF or 7.5 μM SNT compared to vector control. These results demonstrate that pY36-RIN1 is targeted by SRF and SNT and strongly suggest that anti-proliferative effects of these TKI drugs in clear cell RCC is mediated by inhibitory effects on RIN1 tyrosine phosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1626.

Target identification by phosphoproteomics: RIN1 modulation of sorafenib-induced cytotoxicity in renal cell carcinoma

e14543 Background: Sorafenib (SFB) is a multi-tyrosine kinase inhibitor clinically useful in treatment of metastatic renal cancer. While the inhibition of angiogenesis is considered a major mechanism of action, identification of targets regulating growth inhibitory effects of SFB is necessary to further improve its efficacy and reduce toxicity. Methods: In this study we used targeted phosphoproteomics to identify tyrosine phosphorylated proteins that are differentially affected in control and SFB-treated human CAKI-1 renal cell carcinoma cells. The strategy involved immunoaffinity isolation of phosphotyrosine containing proteins and liquid chromatography - tandem mass spectrometry (MS) for identification of candidate proteins. Results: Among identified proteins, signal transducer and activator of transcription 1 (STAT1) and Ras and Rab interactor 1 (RIN1) were found to be hypophosphorylated in SFB-treated compared to untreated CAKI-1 cells based on quantitative MS analysis, by peptide counts and native peptide reference method. A ∼4-fold decrease in expression and phosphorylation of STAT1 was observed in cells treated with 10 μM SFB for 48h. Up to 8-fold SFB dose-dependent (5–15 μM) decrease in phosphorylation of RIN1 at tyrosine 36, but not in total RIN1 expression, was observed. Similar effects on decreased phosphorylation of STAT1 and RIN1 were also observed in 786-O renal cell carcinoma treated with SFB. Hypophosphorylation of RIN1 at tyrosine 36 was observed in CAKI-1 cells treated with 5 μM sunitinib but not with imatinib (≤ 10 μM). Treatment of CAKI-1 cells with RIN1 targeted, but not control si-RNA led to down-regulation of RIN1 expression and attenuation of antiproliferative effects of SFB. Notably, ∼2-fold higher expression of RIN1 protein (total and phosphorylated) was observed in CAKI-1 cells selected for resistance following continuous exposure to 7.5 μM SFB. However, unlike parent CAKI-1 cells, prolonged exposure of these SFB-resistant CAKI-1 cells to 7.5 μM SFB did not completely abrogate phosphorylation of RIN1 at tyrosine 36. Conclusions: These results demonstrate that RIN1, a Ras effector protein with multiple biochemical functions, is a target for the anti-tumor effects of SFB in kidney cancer cells. [Table: see text]

Anti-proliferative effects of sorafenib in clear cell renal cell carcinoma (CCRCC) cell lines: Relationship to von Hippel Lindau protein (pVHL) expression and hypoxia

4601 Background: Sorafenib, a multikinase inhibitor targeting raf kinase and several receptor tyrosine kinases including VEGFR-2 and PDGFR, has demonstrated clinical activity in the treatment of metastatic CCRCC. To further characterize the molecular basis of response to this agent, we tested the functional role of pVHL and hypoxia in the anti-tumor activity of sorafenib in a panel of CCRCC cell lines. Methods: CCRCC cell lines CAKI-1 (wild-type pVHL), CAKI-2 (mutant VHL) and the isogenic pair, 786–0-neo (vector control) and 786–0-VHL (pVHL expressing) cells were treated with sorafenib (2.5 - 20 μM) for 24 - 96 hours at 37°C in a atmosphere of either normoxia (21% oxygen) or hypoxia (1% oxygen), 5% carbon dioxide and remainder nitrogen. Gene expression analysis of control and sorafenib treated CCRCC cell lines was carried out using a custom cancer cDNA array containing probes to 9240 cDNA clones corresponding to 4900 Unigenes or unclustered ESTs relevant to cancer or kidney development. Results: Anti-proliferative effects of sorafenib were both concentration and time of treatment dependent; the IC50 of sorafenib (inhibitory concentration for 50% cell kill) ranged from 7.5-10 μM and required continuous treatment for at least 48-72 hours for maximal cell kill. In contrast to CAKI-2 and 786–0-neo cells harboring mutant VHL, both CAKI-1 and 786–0-VHL cells expressing wild type VHL were ∼2-fold more resistant to the anti-proliferative effects of sorfaenib when treated under hypoxic conditions. No difference in resistance was observed under normoxic conditions. In contrast to 786–0-neo cells, there were ∼40 genes overexpressed (&gt;5-fold) under hypoxia in control 786–0-VHL cells, that were predominantly related to anti-apoptosis (e.g. bcl2-associated athanogene) or angiogenesis (e.g. PDGF beta). Bcl2-associated athanogene and PDGF beta were down regulated &gt;2-fold in hypoxia with sorafenib treatment. Conclusions: CCRCC with wild type VHL under hypoxic conditions can promote overexpression of anti-apoptotic and angiogenesis genes that attenuate the anti-proliferative effects of sorafenib. pVHL and hypoxia related genes may be important biomarkers for assessing the clinical efficacy of sorafenib in CCRCC. [Table: see text]