MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies

MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.</p

Network Modeling Identifies Molecular Functions Targeted by miR-204 to Suppress Head and Neck Tumor Metastasis

Due to the large number of putative microRNA gene targets predicted by sequence-alignment databases and the relative low accuracy of such predictions which are conducted independently of biological context by design, systematic experimental identification and validation of every functional microRNA target is currently challenging. Consequently, biological studies have yet to identify, on a genome scale, key regulatory networks perturbed by altered microRNA functions in the context of cancer. In this report, we demonstrate for the first time how phenotypic knowledge of inheritable cancer traits and of risk factor loci can be utilized jointly with gene expression analysis to efficiently prioritize deregulated microRNAs for biological characterization. Using this approach we characterize miR-204 as a tumor suppressor microRNA and uncover previously unknown connections between microRNA regulation, network topology, and expression dynamics. Specifically, we validate 18 gene targets of miR-204 that show elevated mRNA expression and are enriched in biological processes associated with tumor progression in squamous cell carcinoma of the head and neck (HNSCC). We further demonstrate the enrichment of bottleneckness, a key molecular network topology, among miR-204 gene targets. Restoration of miR-204 function in HNSCC cell lines inhibits the expression of its functionally related gene targets, leads to the reduced adhesion, migration and invasion in vitro and attenuates experimental lung metastasis in vivo. As importantly, our investigation also provides experimental evidence linking the function of microRNAs that are located in the cancer-associated genomic regions (CAGRs) to the observed predisposition to human cancers. Specifically, we show miR-204 may serve as a tumor suppressor gene at the 9q21.1–22.3 CAGR locus, a well established risk factor locus in head and neck cancers for which tumor suppressor genes have not been identified. This new strategy that integrates expression profiling, genetics and novel computational biology approaches provides for improved efficiency in characterization and modeling of microRNA functions in cancer as compared to the state of art and is applicable to the investigation of microRNA functions in other biological processes and diseases.</p

CBL Is Frequently Altered in Lung Cancers: Its Relationship to Mutations in MET and EGFR Tyrosine Kinases

Background: Non-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC. Methods and Findings: Using archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility. Conclusions: Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis

The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target

Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability in vitro, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.</p

Network Modeling Identifies Molecular Functions Targeted by miR-204 to Suppress Head and Neck Tumor Metastasis

Due to the large number of putative microRNA gene targets predicted by sequence-alignment databases and the relative low accuracy of such predictions which are conducted independently of biological context by design, systematic experimental identification and validation of every functional microRNA target is currently challenging. Consequently, biological studies have yet to identify, on a genome scale, key regulatory networks perturbed by altered microRNA functions in the context of cancer. In this report, we demonstrate for the first time how phenotypic knowledge of inheritable cancer traits and of risk factor loci can be utilized jointly with gene expression analysis to efficiently prioritize deregulated microRNAs for biological characterization. Using this approach we characterize miR-204 as a tumor suppressor microRNA and uncover previously unknown connections between microRNA regulation, network topology, and expression dynamics. Specifically, we validate 18 gene targets of miR-204 that show elevated mRNA expression and are enriched in biological processes associated with tumor progression in squamous cell carcinoma of the head and neck (HNSCC). We further demonstrate the enrichment of bottleneckness, a key molecular network topology, among miR-204 gene targets. Restoration of miR-204 function in HNSCC cell lines inhibits the expression of its functionally related gene targets, leads to the reduced adhesion, migration and invasion in vitro and attenuates experimental lung metastasis in vivo. As importantly, our investigation also provides experimental evidence linking the function of microRNAs that are located in the cancer-associated genomic regions (CAGRs) to the observed predisposition to human cancers. Specifically, we show miR-204 may serve as a tumor suppressor gene at the 9q21.1–22.3 CAGR locus, a well established risk factor locus in head and neck cancers for which tumor suppressor genes have not been identified. This new strategy that integrates expression profiling, genetics and novel computational biology approaches provides for improved efficiency in characterization and modeling of microRNA functions in cancer as compared to the state of art and is applicable to the investigation of microRNA functions in other biological processes and diseases