Antitumor Effects Of Sunitinib Research Articles

Abstract 4132: Anti-tumor and anti-metastatic effect of sunitinib in a patient derived metastatic clear cell renal cell carcinoma xenograft model

Abstract Background: Sunitinib, a multi-tyrosine kinase inhibitor, is considered first-line therapy for patient with advanced renal cell carcinoma. The mechanism by which sunitinib harnesses angiogenesis is by targeting receptors of pro-angiogenic growth factors such as VEGF, PDGF and kit. Recently, it has been speculated that sunitinib may have a direct anti-tumor effect. Our previous studies have shown that tumors resistant to sunitinib still present decreased tumor vasculature compared to untreated tumors, suggesting that sunitinib anti-tumor effect may be in part independent from its anti-angiogenic effect. Hence, we wanted to further investigate the anti-tumor effect of sunitinib in human renal cell carcinoma (RCC) cell lines and a patient derived clear cell RCC xenograft model. In addition, we examined the effect of sunitinib on early and late stage metastasis in a spontaneous metastatic human ccRCC model. Methods: Human ccRCC PDX RP-R-02 was implanted into SCID mice subcutaneously. Spontaneous metastatic ccRCC model, RP-R-02LM was developed from RP-R-02. RP-R-02LM tumor bearing mice were treated with sunitinib (40mg/kg; 5days/week). Tumor tissues and lungs were collected for immunohistochemistry analysis. In parallel, human RCC cell lines were treated in vitro with varying concentrations of sunitinib and cell viability was assessed. Results: Human RCC cells lines treated in vitro with sunitinib at pharmacological achievable concentrations showed a decrease in cell proliferation, suggesting a direct anti-tumor effect of sunitinib in RCC. RP-R-02LM but not parental RP-R-02 implanted either subcutaneous or orthotopically in the kidney, spontaneously metastasize to the lungs and closely mimics what is seen in the clinic. PCR results indicated that both xenografts still maintain human origin. In our in vivo system, RP-R-02LM treated with sunitinib had no anti-tumor effect on tumor at the primary site; however, we observed an inhibition of dissemination to the metastatic lung site as indicated by significantly low numbers of metastasis compared to the controls. Immunohistochemical analysis showed decrease in tumor vasculature with sunitinib treatment compared to the control, with increased tumor cell proliferations. Conclusion: Our studies suggest that sunitinib has a direct anti-tumor in vitro and eventually tumor cells acquire drug resistance. Our in vivo studies show that tumors resistant to sunitinib have decreased vessel density as compared to the untreated tumors, suggesting that sunitinib is still a potent anti-angiogenic agent. In addition, we show the anti-metastatic effect of sunitinib in a spontaneous metastatic human clear cell renal cell carcinoma. Overall our data suggest that sunitinib maintains an anti-metastatic effect in sunitinib resistant tumor bearing animals that is independent from its anti-angiogenic effect. Citation Format: Remi M. Adelaiye-Ogala, Li Shen, Sreenivasulu Chintala, Eric Ciamporcero, Ashley Orillion, May Elbanna, Shengyu Ku, Kiersten Marie Miles, Bryan Gillard, Michael Buck, Roberto Pili. Anti-tumor and anti-metastatic effect of sunitinib in a patient derived metastatic clear cell renal cell carcinoma xenograft model. [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 4132. doi:10.1158/1538-7445.AM2015-4132

Abstract 296: Synergistic actions of oncolytic vaccinia virus and sunitinib on pancreatic neuroendocrine tumors in RIP-Tag2 mice

Abstract Pexa-Vec (JX-594) is an engineered oncolytic vaccinia virus that lacks thymidine kinase and expresses human granulocyte-macrophage colony stimulating factor. In addition to having oncolytic effects on tumor cells and promoting an immune response, Pexa-Vec has been found to cause rapid reduction of tumor blood flow in preclinical models and in clinical trials. The angiogenesis inhibitor sunitinib reduces tumor vascularity and can augment the activity of vesicular stomatitis virus by suppressing two interferon-induced anti-viral host proteins, RNA-dependent protein kinase and ribonuclease L. We explored the interaction of mpJX-594, a mouse-adapted version of Pexa-Vec, and sunitinib. The mouse-adapted version, mpJX-594, was engineered from Western Reserve strain vaccinia virus by deletion of thymidine kinase and addition of human GM-CSF, yellow fluorescent protein, and luciferase genes. The effects of mpJX-594 or sunitinib given individually on pancreatic neuroendocrine tumors in RIP-Tag2 mice were compared to those of the two agents given together. Treatment with mpJX-594 alone reduced tumor vascularity and induced widespread apoptosis in the tumors. After treatment with sunitinib alone, tumor vascularity was decreased, but apoptosis was much less than after mpJX-594 alone. When mpJX-594 was given together with sunitinib, the amount of viral infection in tumors, assessed by vaccinia immunoreactivity, was much more than after mpJX-594 alone. Vaccinia staining was detected in most of the tumor blood vessels and in large patches of tumor cells. The reduction of tumor vascularity and amount of apoptosis were greater after both agents than after either of them given alone. To determine whether mpJX-594 increased the efficacy of sunitinib in tumors or vice versa, we examined the effects of sequential administration, where mpJX-594 or sunitinib was given first and then followed 5 days later by the other. In both cases, the reduction in tumor vascularity after sequential treatment was greater than after either agent given alone. When sunitinib was given first, tumor vascularity was less throughout tumors, and the surviving vessels appeared more normal. By comparison, when mpJX-594 was given before sunitinib, the tumor vasculature was greatly reduced in some regions and less so in others, and the remaining blood vessels were abnormal. Apoptotic cells were much more numerous and widespread when mpJX-594 preceded sunitinib than after either agent given alone. In contrast, when sunitinib was given first, tumor apoptosis was greater than after sunitinib alone but not after mpJX-594 alone. Together these findings indicate that mpJX-594 amplifies the anti-vascular and anti-tumor effects of sunitinib and vice versa in RIP-Tag2 pancreatic neuroendocrine tumors, regardless of the sequence of administration, but the magnitude of the changes differs according to the sequence of administration. Citation Format: Corry E. McDonald, Barbara Sennino, Brian J. Schriver, John C. Bell, David H. Kirn, Caroline J. Breitbach, Donald M. McDonald. Synergistic actions of oncolytic vaccinia virus and sunitinib on pancreatic neuroendocrine tumors in RIP-Tag2 mice. [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 296. doi:10.1158/1538-7445.AM2015-296

Abstract 3508: Inhibition of EZH2 overcomes resistance to sunitinib in clear cell renal cell carcinoma models

Abstract Background: Alterations in epigenetic mechanisms including histone modification and hyper-methylation at gene promoter regions have been implicated as mechanisms of drug resistance in cancer. Alternation of epigenetic regulators such histone methyltransferase, EZH2, has been reported in numerous cancer types including advanced renal cell carcinoma (RCC). Previous studies suggest that sunitinib may have a direct anti-tumor effect and that acquired sunitinib resistance may be induced in tumor cells rather than just in endothelial cells. In our study, we investigated the role of EZH2 in sunitinib resistance in clear cell renal cell carcinoma. Methods: Human RCC cell lines 786-0 were treated and exposed to increasing concentrations of sunitinib to develop a resistant cell line, 786-0R. Parental and resistant cell lines were treat with either sunitinib, GSK126 (EZH2 inhibitor) or both. In parallel, EZH2 was knocked down in 786-0 cells and exposed to increasing concentrations of sunitinib. Cell viability was quantitated by absorbance of crystal violet stained cells using a spectrometer at 570nm. In a second set of experiments, control and treated cells were collected for western analysis. Mice bearing human ccRCC patient derived xenograft (PDXs); RP-R-01, RP-R-02 and RP-R-02LM (a metastatic ccRCC model established from RP-R-02) were implanted into SCID mice either subcutaneously or orthotopic in the kidney (sub-renal). When tumors reached an average volume of 50mm3, mice were randomly grouped into 2 arms; control and sunitinib treatment (40mg/kg, 5days/week). Tumors volumes and body weight were assessed once per week. Tumor tissues and lungs were collected for immunohistochemistry analysis. All assessments and quantification were done blindly. Results: Our in vitro and in vivo data showed an increased expression of EZH2 with resistance to sunitinib. Furthermore, inhibition of EZH2 in our in vitro studies correlated with a significant decrease in the anti-tumor effect of sunitinib in both parental and resistant cell lines. Conclusion: Overall our data suggest the potential role of epigenetic alterations, specifically EZH2 overexpression and its association with resistance to sunitinib. We are currently assessing the effect of EZH2 inhibition with sunitinib resistance in our in vivo system using metastatic ccRCC PDX models. Citation Format: Remi M. Adelaiye-Ogala, Sreenivasulu Chintala, Li Shen, Ashley Orillion, Eric Ciamporcero, May Elbanna, Kiersten Marie Miles, Bryan Gillard, Michael Buck, Roberto Pili. Inhibition of EZH2 overcomes resistance to sunitinib in clear cell renal cell carcinoma models. [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 3508. doi:10.1158/1538-7445.AM2015-3508

Sunitinib significantly suppresses the proliferation, migration, apoptosis resistance, tumor angiogenesis and growth of triple-negative breast cancers but increases breast cancer stem cells.

The majority of triple-negative breast cancers (TNBCs) are basal-like breast cancers. However there is no reported study on anti-tumor effects of sunitinib in xenografts of basal-like TNBC (MDA-MB-468) cells. In the present study, MDA-MB-231, MDA-MB-468, MCF-7 cells were cultured using RPMI 1640 media with 10% FBS. Vascular endothelia growth factor (VEGF) protein levels were detected using ELISA (R & D Systams). MDA-MB-468 cells were exposed to sunitinib for 18 hours for measuring proliferation (3H-thymidine incorporation), migration (BD Invasion Chamber), and apoptosis (ApopTag and ApoScreen Anuexin V Kit). The effect of sunitinib on Notch-1 expression was determined by Western blot in cultured MDA-MB-468 cells. 106 MDA-MB-468 cells were inoculated into the left fourth mammary gland fat pad in athymic nude-foxn1 mice. When the tumor volume reached 100 mm3, sunitinib was given by gavage at 80 mg/kg/2 days for 4 weeks. Tumor angiogenesis was determined by CD31 immunohistochemistry. Breast cancer stem cells (CSCs) isolated from the tumors were determined by flow cytometry analysis using CD44+/CD24- or low. ELISA indicated that VEGF was much more highly expressed in MDA-MB-468 cells than MDA-MB-231 and MCF-7 cells. Sunitinib significantly inhibited the proliferation, invasion, and apoptosis resistance in cultured basal like breast cancer cells. Sunitinib significantly increased the expression of Notch-1 protein in cultured MDA-MB-468 or MDA-MB-231 cells. The xenograft models showed that oral sunitinib significantly reduced the tumor volume of TNBCs in association with the inhibition of tumor angiogeneisis, but increased breast CSCs. These findings support the hypothesis that the possibility should be considered of sunitinib increasing breast CSCs though it inhibits TNBC tumor angiogenesis and growth/progression, and that effects of sunitinib on Notch expression and hypoxia may increase breast cancer stem cells. This work provides the groundwork for an innovative therapeutic strategy in TNBC therapy by using sunitinib plus γ-secretase inhibitor to simultaneously target angiogenesis and CSC.

Abstract 860: The multi-targeted kinase inhibitor Sunitinib induces apoptosis in colon cancer cells via PUMA

Abstract Key words: Sunitinib; PUMA; FoxO3a; Bcl-2 family; colon cancer Purpose: Sunitinib is a FDA approved multi-targeted receptor tyrosine kinase (RTK) inhibitor for treating solid tumors. Inhibition of angiogenesis, immune modulation and induction of apoptosis has been suggested to mediate the anti-tumor effects of sunitinib. However, its cell killing mechanisms are not well-understood. In this study, we investigated the mechanisms and significance of sunitinib-mediated apoptosis in its antitumor activities using colon cancer cells and xenografts. Experimental Design: The expression of various Bcl-2 family proteins, apoptosis and long-term growth in response to sunitinib were examined in colon cancer cells. The mechanism of PUMA induction and effects of PUMA deficiency, BH3 mimetics and 5-FU on sunitinib-induced apoptosis were investigated. The effects of PUMA deficiency in responses to sunitinib were determined in xenografts. Results: We found that consistent PUMA induction mediated by transcription factor FoxO3a plays an important role in the apoptosis induced by sunitinib. PUMA deficiency led to impaired apoptosis and caspase activation in colon cancer cells with either wildtype or mutant p53. Small molecule BH3 mimetics or elevated PUMA levels sensitized colon cancer cells to sunitinib-induced apoptosis. PUMA deficiency significantly blocked apoptosis and therapeutic responses to sunitinib in xenografts. Conclusions: Our results establish a critical role of PUMA in mediating sunitinib sensitivity in colon cancer cells and lack of apoptosis induction as a potential resistance mechanism. Our study suggests modulations of Bcl-2 family proteins as potential biomarkers, and combination of sunitinib with BH3 mimetics or chemotherapeutic agents might improve the treatment efficacy in colon cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 860. doi:1538-7445.AM2012-860

Sunitinib enhances antitumor effects against chemotherapy-resistant bladder cancer through suppression of ERK1/2 phosphorylation

Bladder cancer patients who are refractory to chemotherapy have a poor prognosis. Furthermore, additional chemotherapies provide little benefit to patients who have relapsed after an initial response. Recently, it was reported that several molecular pathways are implicated in bladder carcinogenesis, including the epidermal growth factor receptor (EGFR) pathway, the vascular endothelial growth factor (VEGF) pathway and the Ras-MAPK pathway. We hypothesized that sunitinib would be effective in bladder cancer as it is an oral inhibitor of multiple receptor tyrosine kinases, including VEGF receptors, platelet derived growth factor (PDGF) receptors and stem cell factor receptor (c-KIT), and is a standard first-line treatment of advanced clear cell renal carcinoma. In the present study, the antiproliferative effects of sunitinib were clearly demonstrated in KK47, KK47/DDP20 and KK47/ADR cell lines in vitro due to the suppression of ERK1/2 phosphorylation. In a mouse model, the antitumor effects of sunitinib were again clearly seen. Also, treatment with sunitinib decreased the abundance of regulatory T cells (Tregs). However, cytotoxic T lymphocyte (CTL) activity was not induced sufficiently as compared with an IFN-α-treated group. Our results suggested that sunitinib was effective in chemotherapy-resistant bladder cancer patients. On the other hand, these findings provided the rationale for combination therapy with sunitinib and immune-based cancer therapy for advanced malignancies to prevent the occurrence of rebound phenomena.

Antitumor effect of sunitinib against skeletal metastatic renal cell carcinoma through inhibition of osteoclast function

We investigated the inhibitory effect of sunitinib, a newly approved multitargeted tyrosine kinase inhibitor, against the progression of renal cell cancer (RCC) bone metastases in vivo. In vitro cell proliferation was determined using the MTS assay. To investigate the inhibitory effects of sunitinib in vivo, we established luciferase-labeled ACHN(Luc) cells derived from papillary RCC. Mice in which ACHN(Luc) cells had been transplanted into the left ventricle to establish bone metastases were treated orally with 40 mg/kg/day sunitinib or vehicle control for 3 weeks. Growth of the cancer cells was monitored using an in vivo imaging system. In addition, 16 patients with metastatic RCC were treated with sunitinib, and serum and urine levels of amino-terminal telopeptide (NTx) were measured as markers of bone resorption. Sunitinib did not inhibit the growth of RCC cells in vitro at clinically or experimentally achievable serum levels (100 nM-1 μM). To investigate the inhibitory effect of sunitinib in vivo, we established luciferase-labeled human RCC cells (ACHN(Luc) ). Sunitinib prevented the growth of ACHN(Luc) RCC cells in the bone metastatic mouse model. The number of osteoclasts in sunitinib-treated mice was significantly less than that in control mice. Serum and urine levels of NTx in patients with metastatic RCC declined significantly during the first 4 weeks of sunitinib treatment (p = 0.027). Sunitinib is a potent anticancer agent for RCC bone metastases, at least for papillary RCC.

Abstract 388: Epithelial to mesenchymal transition is associated with acquired resistance to sunitinib in patients with renal cell carcinoma: evidence from a xenograft study

Abstract Purpose: Tyrosine kinase inhibitors (TKIs) targeting angiogenesis via inhibition of the vascular endothelial growth factor (VEGF) pathway have changed the medical management of metastatic renal cell carcinoma. While the treatment with TKIs has demonstrated clinical benefit these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. Experimental Design: To address this question we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response on sunitinib and eventually progressed on therapy. Tumor pieces were grafted subcutaneously in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor derived cell line was established and assessed in vitro for a potential direct antitumor effects of sunitinib. Results: To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histological examination of the original skin metastasis revealed evidence of epithelial-to-mesenchymal-transition while the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. Conclusions: The histological examination in this xenograft study suggests that reversible epithelial-to mesenchymal-transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma. 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 388.

Sequential Sorafenib and Sunitinib for Renal Cell Carcinoma

Sorafenib and sunitinib are 2 tyrosine kinase inhibitors that were recently approved for renal cell carcinoma. In many patients sequential administration of the 2 drugs occurs because of the lack of sustained efficacy of the first agent. We determined the efficacy and safety of sequential administration. To determine whether cross-resistance occurs between these 2 drugs we analyzed the outcome in 90 consecutive patients with renal cell carcinoma from 4 sites in France who had received the 2 drugs sequentially. All patients received sorafenib followed by sunitinib or vice versa. From 2003 to 2006, 68 patients received sorafenib, while 22 received sunitinib first. In the sorafenib-sunitinib group median progression-free survival was 26 weeks with sorafenib and 28 with sunitinib. In the sunitinib-sorafenib group median progression-free survival was 22 weeks with sunitinib and 17 with sorafenib. Median overall survival was 135 weeks in the sorafenib-sunitinib group and 82 weeks in the sunitinib-sorafenib group (HR 0.49, 95% CI 0.16 to 0.96, p = 0.04). The average duration of sequential administration was 61 and 49 weeks, respectively, in the sorafenib-sunitinib and sunitinib-sorafenib groups. Each sequence was well tolerated and no increase in grade 3-4 toxicity was observed. Overall this retrospective study supports the conclusion of the lack of absolute cross-resistance between tyrosine kinase inhibitors. In this renal cell carcinoma population sorafenib followed by sunitinib was associated with longer survival than sunitinib followed by sorafenib. However, this observation needs further confirmation.

Antitumor Effects of Sunitinib or Sorafenib in Patients With Metastatic Renal Cell Carcinoma Who Received Prior Antiangiogenic Therapy

Antiangiogenic therapy with sunitinib and sorafenib has become the standard of care for patients with advanced renal cell carcinoma. However, the clinical benefit of these agents after prior antiangiogenic therapy has not been defined. Currently, several agents with a putative antiangiogenic mechanism exist and they are often being used in sequence with little to no data regarding activity in a second line or later setting. Patients with advanced renal cell carcinoma currently being treated with either sunitinib or sorafenib after receiving 1 or more prior antiangiogenic agent(s) were investigated in a retrospective analysis. Time to progression and the overall response rate by Response Evaluation Criteria in Solid Tumors were evaluated. Thirty patients receiving current sunitinib (16 patients) or sorafenib (14 patients) were identified. Patients received 1 or more prior antiangiogenic therapies: thalidomide, lenalidomide, bevacizumab, volociximab, AG13736, sorafenib or sunitinib. Of 16 patients treated with sunitinib 13 had some degree of tumor shrinkage, including 9 with a partial response by Response Evaluation Criteria in Solid Tumors. Of 14 patients treated with sorafenib 10 had some degree of tumor shrinkage, including 1 with a partial response. The median time to progression for the entire cohort was 10.4 months. Significant antitumor activity is observed when sorafenib or sunitinib are used in patients who have failed prior therapy with an antiangiogenic agent. Prior response to an antiangiogenic agent does not appear to predict subsequent clinical benefit to either sunitinib or sorafenib.

The antitumor effects of sunitinib (formerly SU11248) against a variety of human hematologic malignancies: enhancement of growth inhibition via inhibition of mammalian target of rapamycin signaling

We studied antitumor effects of receptor tyrosine kinase inhibitor sunitinib (formerly SU11248) against a variety of hematologic malignancies including the following leukemias: eosinophilic (EOL-1), acute myeloid (THP-1, U937, Kasumi-1), biphenotypic (MV4-11), acute lymphoblastic (NALL-1, Jurkat, BALL-2, PALL-1, PALL-2), blast crisis of chronic myeloid (KU812, Kcl-22, K562), and adult T-cell (MT-1, MT-2, MT-4), as well as non-Hodgkin's lymphoma (KS-1, Dauji, Akata) and multiple myeloma (U266). Thymidine uptake studies showed that sunitinib was active against EOL-1, MV4-11, and Kasumi-1 cells, which possessed activating mutations of the PDGFRalpha, FLT-3, and c-KIT genes, respectively, with IC(50)s of <30 nmol/L. In addition, sunitinib inhibited the proliferation of freshly isolated leukemia cells from patients possessing mutations in FLT3 gene. Annexin V staining showed that sunitinib induced apoptosis of these cells. Sunitinib inhibited phosphorylation of FLT3 and PDGFRalpha in conjunction with blockade of mammalian target of rapamycin signaling in MV4-11 and EOL-1 cells, respectively. Interestingly, rapamycin analogue RAD001 enhanced the ability of sunitinib to inhibit the proliferation of leukemia cells and down-regulate levels of mammalian target of rapamycin effectors p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 in these cells. Taken together, sunitinib may be useful for treatment of individuals with leukemias possessing activation mutation of tyrosine kinase, and the combination of sunitinib and RAD001 represents a promising novel treatment strategy.