Effects Of SU11274 Research Articles

Tyrosine kinase inhibitor SU11274 increased tumorigenicity and enriched for melanoma-initiating cells by bioenergetic modulation.

BackgroundSmall molecule inhibitor of tyrosine kinase activity, compound SU11274, was reported to have antitumorigenic and antimetastatic effect in melanoma. In this study, we evaluated, whether similar effect could be achieved also in other melanoma cells including highly tumorigenic and hypermetastatic variant.MethodsThe effect of SU11274 was evaluated in adherent and non-adherent melanosphere cultures of human melanoma cells M14, M4Beu, A375 and EGFP-A375/Rel3. Tumorigenicity of SU11274-treated cells was tested by limiting dilution assay in xenograft model in vivo.ResultsHere we show that SU11274 enriched for melanoma-initiating cells in vivo. SU11274 substantially decreased number of cells in adherent and spheroid cultures, but increased their tumorigenic potential as determined by higher frequency of tumor-initiating cells in vivo. SU11274 treatment was not associated with any significant alteration in the expression of stem cell markers, but the inhibitor stimulated higher level of pluripotent markers. SU11274-treated melanoma cells exhibited higher ATP content and lactate release indicative of increased glycolysis. Our data suggest that the SU11274 altered bioenergetic state of the cells. Indeed, pharmacological intervention with a glycolytic inhibitor dichloroacetate significantly reduced SU11274-promoted increase in melanoma-initiating cells and decreased their tumorigenicity.ConclusionsOur data suggest critical role of glycolysis regulation in melanoma-initiating cells. Moreover, these data unravel substantial plasticity of melanoma cells and their adoptive mechanisms, which result in ambivalent response to therapeutic targeting.

Meis1 Is Required for c-Met Inhibition to Suppress Cell Proliferation of Skin Squamous Cell Carcinoma Cells

Previous studies have shown that Meis1 plays an important role in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Meis1 belongs to the TALE family, the members of which are used as biomarkers for AML. Meis1 has been shown to play a functional role in epithelial tissues, such as skin. However, its functions in skin carcinogenesis remain poorly understood. On the other hand, the c-Met inhibitor SU11274 has been identified through drug screening with HOXA9/Meis1-induced AML cell lines. SU11274 altered cell proliferation and the cell cycle status in human AML cell lines. Thus, we hypothesized that the effects of SU11274 are dependent on Meis1 and that its knockdown may diminish the effects of SU11274 not only in AML cell lines, but also in skin cancer cell lines. In order to test our hypothesis, we established Meis1 knockdown cell lines using two skin squamous cell carcinoma cell lines (B9 and D3) and treated these cell lines with SU11274. The results obtained showed that SU11274 suppressed cell proliferation by modulating cell cycle progression in the presence of Meis1, but not in its absence. Furthermore, an expression analysis showed that SU11274 activated the transcription of Meis1, which led to the transcription of Hif1α and Cdkn2a (p16Ink4a and p19Arf). These results suggest that Meis1 is required for the c-Met inhibitor SU11274 to suppress the proliferation of the skin squamous cell carcinoma cell lines.

Abstract C268: MET-inhibition using SU11274 and HER-inhibitions with lapatinib impair proliferation, motility and invasion in hepatocellular carcinoma (HCC) and exhibits an increased efficacy un sorafenib-tolerant model.

Abstract Introduction: Sorafenib significantly improved overall survival in advanced HCC. However patients may display primary resistance and a majority of them will develop acquired resistance over time when treated with sorafenib. Resistance to sorafenib has been correlated with upregulation of angiogenic pathways, including the c-MET pathway (Roskoski, 2007). In the SHARP study, patients exhibiting high HGF levels at baseline had a poor prognosis and appeared to benefit less from sorafenib in terms of survival than those with low levels of HGF (Pena, 2008). Therefore, MET appears to be a potential target to counteract sorafenib resistance. In this study we evaluate MET and EGFR expression and their sensitivity towards MET and EGFR inhibitors in terms of signaling, viability, and invasion in two HCC cell lines, one sensitive to sorafenib and with acquired sorafenib-tolerance. Methods: SU11274 is a specific inhibitor of overexpressed and activated MET. Antiproliferative effects of SU11274 (MET inhibitor) and lapatinib an (EGFR/HER2 inhibitor) (lapatinib) were evaluated in the human HCC cell lines, SK-HEP1 and its sorafenib-tolerant counterpart, SK-Sora, using MTT assays. mRNA expression and protein levels were assessed by qRT-PCR and Western blot, respectively. Cell motility was investigated by wound-healing and matrigel invasion assays. Results: SK-Sora was characterized by a 2-fold higher MET expression but a 3-fold lower EGFR expression respect to SK-HEP1. This differential expression of MET/EGFR between the two cell lines was associated with their sensitivity to SU11274 and lapatinib in proliferation assays. Whereas 5µM SU11274 or 20µM lapatinib induced 50 % growth inhibition of SK-HEP1 cells, SK-Sora cells displayed 86% and 27% of growth inhibition, respectively in the same conditions. These results indicated that baseline MET and EGFR expression levels may be good biomarkers to predict increased sensitivity or resistance to MET or EGFR inhibitors. Interestingly, SU11274 also more potently decreased HGF-dependent invasion in SK-Sora (80%) cells than in SK-HEP1 (50%) cells. To understand the differences observed regarding proliferation and invasion between SK-HEP1 and SK-Sora cells, we assessed MET-dependent signaling in both cell lines. HGF-induced MET activation was associated with differential pattern of p-METTyr1234/35, p-GAB1, p-ERK1/2, and p-AKTser473 between both cell lines, with higher phosphorylation of MET and GAB1 in SK-Sora than in SK-HEP1. Interestingly, we showed that SK-Sora cells were more sensitive to the inhibition of HGF-induced signaling by SU11274 than SK-HEP1 cells. Differential effects of lapatinib on cell signaling and invasion will be detailed. Conclusion: MET inhibition showed more potent effects in the sorafenib-tolerant cell line, which had increased MET expression levels, as compared to the parental cell line SK-HEP1. These results suggest that MET could be an attractive target in HCC patients particularly in the context of sorafenib resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C268. Citation Format: Annemilai Tijeras-Raballand, Eugenia Riveiro, Maria Serova, Cindy Neuzillet, Matthieu Martinet, Valérie Paradis, Mohamed Bouattour, Eric Raymond, Sandrine Faivre, Armand de Gramont. MET-inhibition using SU11274 and HER-inhibitions with lapatinib impair proliferation, motility and invasion in hepatocellular carcinoma (HCC) and exhibits an increased efficacy un sorafenib-tolerant model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C268.

Abstract 2178: MET inhibition using SU11274 impairs cellular activities in hepatocellular and Cholangiocarcinomas with high levels of MET and low levels of EGFR expressions.

Abstract Introduction: c-MET pathway is often deregulated in human cancer progression including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CK). Moreover, high level of HGF, and c-MET have been correlated with poor prognosis, aggressiveness and metastasis in HCC and CK (Kaposi-Novak et al., 2006; Miyamoto et al., 2011). We aimed to characterize the expression pattern of MET in HCC/CK specimens, to evaluate the effects of MET inhibition on HCC/CK cell properties, and to identify potential biomarkers of sensitivity and resistance to SU11274. Methods: SU11274 is a specific inhibitor of overexpressed and activated MET. MET expression was assessed on human paraffin-embedded liver section by IHC. Antiproliferative effects of SU11274 and an EGFR/HER2 inhibitor (lapatinib) were evaluated in human HCC (Sk-Hep1 and its sunitinib-tolerant counterpart SK-Suni) and CK (Mz-ch-A1, Mz-ch-A2, and SK-CH) cell lines using MTT assay. mRNA expression and protein levels were assessed by qRT-PCR and Western blot, respectively. Cell motility was investigated by wound-healing and matrigel invasion assays. Results: MET expression level was higher in HCC and CK tumor samples than adjacent normal liver tissue. Characterization of cell lines showed that MET-protein and -mRNA expression were detectable in all cell lines, Mz-ch-A1, Mz-ch-A2 and SK-HEP1 expressing higher MET levels (high-MET) compared to the other cell lines (low-MET). HGF-induced MET activation was associated with differential pattern of p-METTyr1234/35, p-GAB1, p-ERK1/2, and p-AKTser473 between high-MET and low-MET cell lines. We showed that SU11274 inhibits HGF-induced downstream signaling in all HCC and CK cell lines at 0.5-2μM. However, antiproliferative effects were only observed in the high-MET cell lines; inhibition rates were 83%, 53% and 48% in Mz-chA-1, Mz-chA-2 and SK-Hep1, respectively. Interestingly, SU11274 also potently decreased HGF-dependent cell motility (75-90% inhibition) in high-MET cells but not in low-MET cells. Since cells with low-MET showed high EGFR expression, we tested the EGFR/HER2 inhibitor lapatinib on cell proliferation. In contrast to SU11274, lapatinib inhibited proliferation in low-MET/high-EGFR cells (64% and 60% inhibition rate in SK-CH and SK-Suni, respectively) but not in high-MET/low-EGFR cell lines. Complementary results with a sorafenib-tolerant cell line, SK-sora and two intra-hepatic cholangiocarcinoma cell lines will be added. Conclusion: In HCC and CK cells, inhibition of MET-signaling by SU11274 results in decreased cell proliferation, motility and invasion restricted to high-MET/low-EGFR cell lines. Thus, high-MET/low EGFR HCC and CK might be the candidate tumor types to evaluate novel MET inhibitors, whereas low-MET/high-EGFR HCC and CK may be preferentially tested for EGFR pathway inhibition. Citation Format: Annemilai Tijeras-Raballand, Camille Serrate, Maria E. Riveiro, Maria Serova, Cindy Neuzillet, Matthieu Martinet, Valérie Paradis, Ivan Bièche, Eric Raymond, Armand de Gramont, Sandrine Faivre. MET inhibition using SU11274 impairs cellular activities in hepatocellular and Cholangiocarcinomas with high levels of MET and low levels of EGFR expressions. [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 2178. doi:10.1158/1538-7445.AM2013-2178

Study of critical role of c-Met and its inhibitor SU11274 in colorectal carcinoma

c-Met plays an important role in colorectal tumorigenesis and disease progression and thus is believed to be an attractive inhibitory target for receptor molecular therapeutic. SU11274 was identified as a small molecule, ATP competitive inhibitor of the catalytic activity of the c-Met kinase. Our study had investigated the relationship between the high expression of c-Met and colorectal carcinoma and the effect of c-Met inhibitor SU11274 in colorectal carcinoma in vitro and vivo. Immunohistochemistry was used to detect the expression of c-Met in 60 patients with colorectal cancer and 20 patients with benign adenoma and surrounding normal colon tissues. The effect of SU11274 on human colorectal carcinoma LoVo cells was detected by Western blot and MTT. And the influence of SU11274 on cell cycle was determined by flow cytometry. In addition, LoVo cell-transplanted tumor growth and expression of c-Met in nude mice was examined for inhibition of SU11274 in vivo. We found c-Met had high expression and was closely related to lymph node metastasis and TNM stage in colorectal carcinoma tissues. SU11274 significantly suppressed the phosphorylation of c-Met as well as the survival and proliferation of LoVo cell lines. G1-phase arrest was also induced by SU11274. SU11274 apparently restrained the growth of the xenograft tumor in nude mice. Our data suggest developing therapies that specifically inhibit the activation of c-Met may represent a novel therapeutic modality for patients with colorectal carcinoma expressing high levels of c-Met.

Abstract A188: MET inhibition by SU11274 impairs proliferation, motility, and invasion in hepatocellular and cholangiocarcinomas.

Abstract Background: Hepatocellular carcinomas (HCC) and cholangiocarcinomas (CK) are the most common primary tumors of the liver and are recognized among the most difficult tumors to treat. Signaling mediated by hepatocyte growth factor (HGF)/MET promotes multiple biological activities, including cell proliferation, motility, invasion, angiogenesis, and morphogenesis. Advanced HCC and CK are often displaying aberrant MET signaling and MET overexpression was observed in many tumors resistant to tyrosine kinase inhibitors (TKI). The aim of present study was to evaluate the effects of MET inhibition by SU11274 on HCC and CK cell proliferation and invasion. Materials and Methods: SU11274 is a pyrrole indolinone that specifically inhibits overexpressed and oncogenic MET activation at nanomolar concentrations. MET expression was assessed on human paraffin-embedded liver section by immunohistochemistry. Antiproliferative effects of SU11274, were evaluated in human HCC (SK-Hep1 and its sunitinib-tolerant counterpart SK-Suni) and CK (Mz-chA-1, Mz-chA-2, SK-ch) cell lines using a MTT assay. mRNA expression and protein levels were respectively assessed by qRT-PCR and Western blot. Cell mobility was investigated by wound-healing and matrigel invasion assays. Results: Higher MET protein expression was observed in human HCC and CK samples respect to normal tissues. MET-protein and -mRNA expression were detectable in all tested cell lines, Mz-ChA-1 and Mz-ChA-2 expressing higher MET levels. HGF-induced MET activation was associated with increase of p-METTyr1234/35, p-GAB1, p-ERK1/2, and p-AKTser473 in SK-CH, Mz-chA-2, SK-Hep1 and SK-Suni cells. We show that SU11274 inhibited HGF-induced downstream signaling by reducing p-METTyr1234/35, p-GAB1, p-AKT473 and p-ERK1/2 in HCC and CK cells at concentrations ranging from 0.5 to 2μM. However, antiproliferative effects were only observed for concentration above 2 μM (2,5 and 5 μM), In SK-Suni cells that are tolerant to high dose of sunitinib, proliferation was not inhibited even at the higher SU11274 dose (5μM). Interestingly, SU11274 potently decreased the spontaneous and HGF-dependent cell motility and invasion of Mz-chA-2 cells. Conclusion: In HCC and CK cells, inhibition of MET-signaling by SU11274 resulted in inhibition of cell proliferation, motility and invasion. HCC and CK may be interesting tumor types to evaluate novel MET inhibitors either as single agents and/or in combination with other targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A188.

Abstract 5372: Target proteins involved in resistance to c-Met inhibitors and their therapeutic role in human melanoma

Abstract c-Met is activated by binding with its ligand, hepatocyte growth factor (HGF), and is an attractive target for molecular therapeutic inhibition. c-Met is over-expressed and mutated in human melanoma and plays a role in tumorigenecity and metastasis. c-Met overexpression could be one of the factors responsible for the severe invasiveness of metastatic melanoma resulting in poor patient prognosis. We found overexpression of c-Met in 6 out of 7 melanoma cell lines studied by immunoblotting. MU, WK, RU, MC, EP and LH over-expressed c-Met, however, in AN we found minimal c-Met expression. Small molecule tyrosine kinase inhibitors (TKI's), such as SU11274, which is an ATP-competitive small molecule inhibitor, can inhibit phosphorylation, activation and tumorigenecity of c-Met. We found that SU11274 inhibited c-Met phosphorylation and cell growth in all cell lines by 85-98% at 96 hrs with an IC50 between 1 and 2.5 μM and caused apoptosis (12-58%) in all cell lines except AN. To test the therapeutic effects of SU11274 in vivo, five million RU melanoma cells were injected subcutaneously into the hind flanks of RAG (−/-) mice. Tumors were allowed to develop for a week after which daily intratumoral injections of 50μg of SU11274 or equal volumes of vehicle control were given. We found that SU11274 decreased the size of MU Melanoma tumor xenografts in RAG (−/-) mice by 6.2 fold compared to mice treated with a vehicle control. While c-Met inhibitors are currently in clinical trials, resistance to TKI's has been observed in cancer patients. To determine the mechanism of c-Met inhibitor resistance, four melanoma lines (MU, RU, WK, and EP) were exposed to increasing concentrations of SU11274 to develop drug resistance. The resistant melanoma cell lines proliferated in the presence of 7-9 fold higher concentration of SU11274. After generating cell lines resistant to SU11274, the expression levels of AKT and ERK were determined by immunoblotting to compare expression in parental vs. resistant cells. It was discovered that MU and EP drug resistant melanoma cells exhibited upregulation of p-AKT (up to 2.8 fold) and ERK (up to1.5 Fold) in the presence of SU11274. This upregulation may be through a c-Met independent pathway, possibly through the PI3K or HER-2/neu pathway, or there may be multiple pathways mediating resistance to c-Met TKI's. Conversely, RU and WK resistant lines showed down regulation of p-AKT (up to 7.3), however these cell lines showed upregulation of p-S6-kinase (up to1.3 fold) indicating that cell survival in these cell lines maybe mediated by p-S6- kinase. Additionally, WK and EP lines showed upregulation of p-4E-BP1 (up to 5.8 fold). Since both p-4E-BP1 and p-S6-Kinase are phosphorylated by mTOR, in human melanoma these results indicate that the mTOR pathway may play a key role in mediating resistance and further studies are being conducted to break SU11274 resistance in melanoma by rapamycin which targets the mTOR pathway. 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 5372. doi:10.1158/1538-7445.AM2011-5372

Abstract 5027: SU11274 induced apoptosis through accumulated p53-mediated pathway in NSCLC cells and xenograft model

Abstract Recent advances in molecular understanding of pathogenesis of non-small cell lung cancer (NSCLC) at molecular level improved treatment outcome. However, the overall 5 year survival rate of individuals with lung cancer remains at < 15%. Therefore, novel therapeutic strategies are still needed to improve the survival rate of lung cancer patients. MET, the tyrosine kinase receptor for hepatocyte growth factor (HGF), is involved in mediating tumorigenesis, cell motility, invasion and metastasis and is frequently deregulated in various human cancers including NSCLC. The MET receptor is an attractive potential target for novel therapeutic inhibition in human cancer. Recently, the MET tyrosine kinase inhibitors (TKIs), such as PHA-665752 and SU11274, have been most extensively evaluated in preclinical model, and have been known to be effective in growth inhibition of various malignancies. However, the mechanisms of MET TKIs on the growth-inhibitory effect on cancers are not clear. There have been a few reports that MET-induced oncogensis was accelerated by the loss of wild-type p53. In this study, we investigated the role of p53 in growth-inhibitory effects of MET TKI (SU11274) in the NSCLC cells. We used A549 cells (p53 wild-type) and Calu-1 cells (p53 null-type) and these cells were known to have wild-type MET. SU11274 showed more profound growth inhibition and apoptosis in A549 cells than in Calu-1 cells. The expression of p53 protein by modulation of p53 transitional level was observed by SU11274 treatment in A549 cells. Accumulated p53 protein after SU11274 treatment induced up-regulation of Bax, PUMA proteins and down-regulation of Bcl-2 protein and subsequently the activation of caspase 3 in p53 wild type A549 cells only. Inhibition of p53 expression using siRNA technology in A549 cells caused significant decrease of apoptosis by SU11274 and inactivation of p53-mediated apoptosis. The introduction of p53 into Calu-1 effectively leaded to SU11274-induced apoptosis through p53-mediated pathway. As same as in A549 cells to confirm the anti-tumor effect of SU11274 in vivo, A549 xenograft model was used and SU11274 effectively shrunk the tumor volume and also induced apoptosis via the increases of p53 protein. Taken together, SU11274 significantly induced apoptosis in wild-type p53 NSCLC cells through the increased expression p53-mediated apoptotic pathway but not in the p53-null NSCLC cells. These results suggest that p53 play an important role in SU11274-induced apoptosis and p53 status might be related with the sensitivity of SU11274 in NSCLC. * This study was supported by a grant of the Korea Health 21 R&D project, the Ministry of Health & Welfare, Republic of Korea. (A010250) 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 5027.

C-Met Is a Potentially New Therapeutic Target for Treatment of Human Melanoma

c-Met is a receptor tyrosine kinase involved in cell growth, invasion, metastases, and angiogenesis. In this study, we investigated the role of c-Met in melanoma biology using a novel small-molecule tyrosine kinase inhibitor SU11274 and small interfering (si) RNA against the receptor. The effects of SU11274 and c-Met siRNA were studied on proliferation, apoptosis, differentiation, reactive oxygen species, and intracellular signaling. c-Met mutations were examined, and the expression of c-Met and activated c-Met was studied in nevi, primary, and metastatic melanoma. c-Met was expressed in 6:7 melanoma cell lines by immunoblotting. SU11274 inhibited cell growth in all melanoma cell lines by 85% to 98% with an IC(50) between 1 and 2.5 mumol/L and caused apoptosis (12-58%) in five out of six cell lines. siRNA against c-Met inhibited proliferation of melanoma cells by 60%. This is the first study that shows that SU11274 and siRNA induced microphthalmia-associated transcription factor (MITF) and several other melanoma differentiation proteins and a morphologically differentiated phenotype. SU11274 also inhibited reactive oxygen species formation and phosphorylation of c-Met receptor, AKT and S-6 kinase by the hepatocyte growth factor. A new missense c-Met mutation N948S was identified in cell lines and R988C in tumor tissue in the juxtamembrane domain of c-Met. It was found that c-Met was expressed in 88% of melanomas and 15% of nevi, and that c-Met (pY1003) was activated in 21% of human melanomas. These results support the role of c-Met in proliferation, apoptosis, differentiation, and tumor progression of melanoma. SU11274 could be used in the therapeutic inhibition of melanoma.