A tightly controlled Src-YAP signaling axis determines therapeutic response to dasatinib in renal cell carcinoma.
ABSTRACT: Over the past decade, therapies targeting the VEGF/VEGFR and mTOR pathways have served as the standard of care for the clinical management of renal cell carcinoma (RCC) patients. Albeit promising, these targeted drugs have attained only modest clinical benefits with limited prolonged progression-free survival. Therefore, alternative reasonable and applicable therapeutic approaches should be introduced to improve the clinical outcome of RCC patients. Methods: FDA approved kinase inhibitors were screened to evaluate their abilities to suppress the proliferation of RCC cells. Then, the downstream effector, therapeutic target and signaling pathway of the selected drug were identified by gene expression array, RNAi, kinase profile and rescue verification. Finally, the in vivo effectiveness of the drug was assessed in cell line-based xenograft models and patient-derived xenograft models. Results: In this study, we discovered that dasatinib is a potent agent that can impair RCC cell viability in vitro and decrease tumor growth in vivo. Mechanistically, we improved the understanding of the precise mechanistic role of YAP as a pivotal effector of dasatinib-induced anti-proliferation through Src-JNK-LIMD1-LATS signaling cascade in RCC cells. Meanwhile, our results indicated that the alteration of p-YAP is closely correlated to the growth inhibition caused by dasatinib in sensitive RCC models. Conclusion: Our findings provide evidence that dasatinib may serve as a powerful drug candidate to treat subgroups of RCC patients with hyper-activated Src-YAP signaling axis, and the alteration of p-YAP could serve as a functional response biomarker of dasatinib in RCC.
Project description:The intracytoplasmic tyrosine kinase Src serves both as a conduit and a regulator for multiple processes required for the proliferation and survival cancer cells. In some cancers, Src engages with receptor tyrosine kinases to mediate downstream signaling and in other cancers, it regulates gene expression. Src therefore represents a viable oncologic target. However, clinical responses to Src inhibitors, such as dasatinib have been disappointing to date. We identified Stat3 signaling as a potential bypass mechanism that enables renal cell carcinoma (RCC) cells to escape dasatinib treatment. Combined Src-Stat3 inhibition using dasatinib and CYT387 (a JAK/STAT inhibitor) synergistically reduced cell proliferation and increased apoptosis in RCC cells. Moreover, dasatinib and CYT387 combine to suppress YAP1, a transcriptional co-activator that promotes cell proliferation, survival and organ size. Importantly, this combination was well tolerated, and caused marked tumor inhibition in RCC xenografts. These results suggest that combination therapy with inhibitors of Stat3 signaling may be a useful therapeutic approach to increase the efficacy of Src inhibitors.
Project description:Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.
Project description:Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15-20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.
Project description:Mechanical forces affect all the tissues of our bodies. Experiments conducted mainly on cultured cells have established that altering these forces influences cell behaviors, including migration, differentiation, apoptosis, and proliferation [1, 2]. The transcriptional coactivator YAP has been identified as a nuclear relay of mechanical signals, but the molecular mechanisms that lead to YAP activation were not identified . YAP is the main transcriptional effector of the Hippo signaling pathway, a major growth regulatory pathway within metazoa , but at least in some instances, the influence of mechanical strain on YAP was reported to be independent of Hippo signaling [5, 6]. Here, we identify a molecular pathway that can promote the proliferation of cultured mammary epithelial cells in response to cyclic or static stretch. These mechanical stimuli are associated with increased activity of the transcriptional coactivator YAP, which is due at least in part to inhibition of Hippo pathway activity. Much of this influence on Hippo signaling can be accounted for by the activation of c-Jun N-terminal kinase (JNK) activity by mechanical strain and subsequent inhibition of Hippo signaling by JNK. LATS1 is a key negative regulator of YAP within the Hippo pathway, and we further show that cyclic stretch is associated with a JNK-dependent increase in binding of a LATS inhibitor, LIMD1, to the LATS1 kinase and that reduction of LIMD1 expression suppresses the activation of YAP by cyclic stretch. Together, these observations establish a pathway for mechanical regulation of cell proliferation via JNK-mediated inhibition of Hippo signaling.
Project description:Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKC?), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTP?). The pY313 site PKC? was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKC? pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKC? pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.
Project description:Src kinase inhibition has been shown to augment the efficacy of chemotherapy. Dasatinib, a dual Src/Abl kinase inhibitor approved for the treatment of CML, is under investigation as monotherapy for tumors with abnormal Src signaling, such as melanoma. The goal of this study was to determine if Src kinase inhibition using dasatinib could enhance the efficacy of regionally administered melphalan in advanced extremity melanoma.The mutational status of c-kit and patterns of gene expression predictive of dysregulated Src kinase signaling were evaluated in a panel of 26 human melanoma cell lines. The effectiveness of dasatinib was measured by quantifying protein expression and activation of Src kinase, focal adhesion kinase, and Crk-associated substrate (p130(CAS)), in conjunction with in vitro cell viability assays using seven melanoma cell lines. Utilizing a rat model of regional chemotherapy, we evaluated the effectiveness of systemic dasatinib in conjunction with regional melphalan against the human melanoma cell line, DM443, grown as a xenograft.Only the WM3211 cell line harbored a c-kit mutation. Significant correlation was observed between Src-predicted dysregulation by gene expression and sensitivity to dasatinib in vitro. Tumor doubling time for DM443 xenografts treated with systemic dasatinib in combination with regional melphalan (44.8 days) was significantly longer (p = 0.007) than either dasatinib (21.3 days) or melphalan alone (24.7 days).Systemic dasatinib prior to melphalan-based regional chemotherapy markedly improves the efficacy of this alkylating agent in this melanoma xenograft model. Validation of this concept should be considered in the context of a regional therapy clinical trial.
Project description:BACKGROUND:Papillary thyroid carcinoma is the most common thyroid malignancy. Most papillary thyroid carcinomas contain BRAF mutations or RET/PTC rearrangements, thus providing targets for biologic therapy. Our previous studies had suggested papillary thyroid carcinomas (PTCs) with a BRAF mutation and the RET/PTC1 rearrangement have different sensitivities to MEK1/2 inhibitors, suggesting different signaling transduction pathways were involved. METHODS:Src signaling transduction pathway in PTC cells was examined using Src inhibitors (PP2, SU6656, or dasatinib) and si-Src RNA in vitro by Western blot analysis and proliferation analysis. An orthotopic xenograft mouse model was used for the in vivo studies using dasatinib. RESULTS:In PTC cells, Src inhibitors suppressed p-Src and p-FAK and inhibited cell growth. In addition, significant suppression and extension of the p-ERK1/2 dephosphorylation were detected in RET/PTC1-rearranged cells in combination with an MEK inhibitor (CI-1040). The Src family kinase/ABL inhibitor, dasatinib, significantly decreased tumor volume in mice inoculated with PTC cells carrying the RET/PTC1 rearrangement. In BRAF-mutated PTC cells, Src inhibitors effectively suppressed p-Src expression and dasatinib significantly decreased tumor volume with twice daily treatment. CONCLUSION:Src inhibitors effectively inhibited the Src signaling transduction pathway in PTC cells in vitro and dasatinib suppressed tumor growth in vivo. These results suggested that Src signaling transduction pathway plays an important role in regulating growth in PTC cells. Combination of Src and MEK1/2 inhibitors extended the dephosphorylation of extracellular signal-regulated kinase (ERK)1/2 in PTCs carrying the RET/PTC1 rearrangement suggesting that combination therapy with complementary inhibitors of other signaling transduction pathways may be needed to effectively suppress growth and induce apoptosis in these cells.
Project description:Muscle-invasive bladder cancer (MIBC) consists of a heterogeneous group of tumors with a high rate of metastasis and mortality. To facilitate the in-depth investigation and validation of tailored strategies for MIBC treatment, we have developed an integrated approach using advanced high-throughput drug screening and a clinically relevant patient-derived preclinical platform. We isolated patient-derived tumor cells (PDCs) from a rare MIBC case (BD-138T) that harbors concomitant epidermal growth factor receptor (EGFR) amplification and phosphatase and tensin homolog (PTEN) deletion. High-throughput in vitro drug screening demonstrated that dasatinib, a SRC inhibitor, and PKI-587, a dual PI3K/mTOR inhibitor, exhibited targeted anti-proliferative and pro-apoptotic effects against BD-138T PDCs. Using established patient-derived xenograft models that successfully retain the genomic and molecular characteristics of the parental tumor, we confirmed that these anti-tumor responses occurred through the inhibition of SRC and PI3K/AKT/mTOR signaling pathways. Taken together, these experimental results demonstrate that dasatinib and PKI-587 might serve as promising anticancer drug candidates for treating MIBC with combined EGFR gene amplification and PTEN deletion.
Project description:The main reasons for failure of cancer chemotherapy are intrinsic and acquired drug resistance. The Hippo pathway effector Yes-associated protein (YAP) is associated with resistance to both cytotoxic and molecular targeted drugs. Several lines of evidence indicate that YAP activates transcriptional programmes to promote cell cycle progression and DNA damage responses. Therefore, we hypothesised that YAP is involved in the sensitivity of cancer cells to small-molecule agents targeting cell cycle-related proteins. Here, we report that the inactivation of YAP sensitises the OVCAR-8 ovarian cancer cell line to AZD1775, a small-molecule WEE1 kinase inhibitor. The accumulation of DNA damage and mitotic failures induced by AZD1775-based therapy were further enhanced by YAP depletion. YAP depletion reduced the expression of the Fanconi anaemia (FA) pathway components required for DNA repair and their transcriptional regulator E2F1. These results suggest that YAP activates the DNA damage response pathway, exemplified by the FA pathway and E2F1. Furthermore, we aimed to apply this finding to combination chemotherapy against ovarian cancers. The regimen containing dasatinib, which inhibits the nuclear localisation of YAP, improved the response to AZD1775-based therapy in the OVCAR-8 ovarian cancer cell line. We propose that dasatinib acts as a chemosensitiser for a subset of molecular targeted drugs, including AZD1775, by targeting YAP.