Ben Staal

Evidence that MIG-6 is a tumor-suppressor gene.

Mitogen-inducible gene 6 (MIG-6) is located in human chromosome 1p36, a locus frequently associated with human lung cancer. MIG-6 is a negative regulator of epidermal growth factor (EGF) signaling, and we show that Mig-6 – like EGF – is induced by hepatocyte growth factor/scatter factor (HGF/SF) in human lung cancer cell lines. Frequently, the receptors for both factors, EGFR and Met, are expressed in same lung cancer cell line, and MIG-6 is induced by both factors in a mitogen-activated protein kinase-dependent fashion. However, not all tumor lines express MIG-6 in response to either EGF or HGF/SF. In these cases, we find missense and nonsense mutations in the MIG-6 coding region, as well as evidence for MIG-6 transcriptional silencing. Moreover, germline disruption of Mig-6 in mice leads to the development of animals with epithelial hyperplasia, adenoma, and adenocarcinoma in organs like the lung, gallbladder, and bile duct. These data suggests that MIG-6 is a tumor-suppressor gene and is therefore a candidate gene for the frequent 1p36 genetic alterations found in lung cancer.

MET Kinase Inhibitor SGX523 Synergizes with Epidermal Growth Factor Receptor Inhibitor Erlotinib in a Hepatocyte Growth Factor–Dependent Fashion to Suppress Carcinoma Growth

The hepatocyte growth factor (HGF)-MET pathway supports several hallmark cancer traits, and it is frequently activated in a broad spectrum of human cancers (http://www.vai.org/met/). With the development of many cancer drugs targeting this pathway, there is a need for relevant in vivo model systems for preclinical evaluation of drug efficacy. Here, we show that production of the human HGF ligand in transgenic severe combined immunodeficient mice (hHGF(tg)-SCID mice) enhances the growth of many MET-expressing human carcinoma xenografts, including those derived from lung, breast, kidney, colon, stomach, and pancreas. In this model, the MET-specific small-molecule kinase inhibitor SGX523 partially inhibits the HGF-dependent growth of lung, breast, and pancreatic tumors. However, much greater growth suppression is achieved by combinatorial inhibition with the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. Together, these results validate the hHGF(tg)-SCID mouse model for in vivo determination of MET sensitivity to drug inhibition. Our findings also indicate that simultaneously targeting the MET and EGFR pathways can provide synergistic inhibitory effects for the treatment of cancers in which both pathways are activated.

Cartilage-specific deletion of Mig-6 results in osteoarthritis-like disorder with excessive articular chondrocyte proliferation.

Significance Mitogen-inducible gene 6 (Mig-6) was found to be an important factor in maintaining joint homeostasis, and its loss in mice results in the development of an osteoarthritis (OA)-like disorder in multiple synovial joints. However, it was unclear in what cells Mig-6 was expressed and what cells were causal for developing the OA-like phenotype. Here we report that Mig-6 is uniquely expressed in the cells surrounding entire surface of the synovial joint, including chondrocytes in the superficial zone of the articular cartilage and in the meniscus, and synovial lining cells. We found that although chondrocytes play a critical role in developing the OA-like disorder in the knees, other cell types are likely required for full development of the Mig-6–deficient joint phenotype. A deficiency of mitogen-inducible gene-6 (Mig-6) in mice leads to the development of an early-onset, osteoarthritis (OA)-like disorder in multiple synovial joints, underlying its importance in maintaining joint homeostasis. Here we determined what joint tissues Mig-6 is expressed in and what role chondrocytes play in the Mig-6–deficient OA-like disorder. A Mig-6/lacZ reporter mouse strain expressing β-galactosidase under the control of the Mig-6 gene promoter was generated to determine Mig-6 expression in joint tissues. By β-galactosidase staining, we demonstrated that Mig-6 was uniquely expressed in the cells across the entire surface of the synovial joint cavity, including chondrocytes in the superficial zone of articular cartilage and in the meniscus, as well as synovial lining cells. By crossing Mig-6–floxed mice to Col2a1-Cre transgenic mice, to generate cartilage-specific deletion of Mig-6, we demonstrated that deficiency of Mig-6 in the chondrocytes results in a joint phenotype that only partially recapitulates the OA-like disorder of the Mig-6–deficient mice: Ubiquitous deletion of Mig-6 led to the OA-like disorder in multiple joints, whereas cartilage-specific deletion affected the knees but rarely other joints. Furthermore, chondrocytes with Mig-6 deficiency showed excessive proliferative activities along with enhanced EGF receptor signaling in the articular cartilage and in the abnormally formed osteophytes. Our findings provide insight into the crucial requirement for Mig-6 in maintaining joint homeostasis and in regulating chondrocyte activities in the synovial joints. Our data also suggest that other cell types are required for fully developing the Mig-6–deficient OA-like disorder.

Strengthening Context-Dependent Anticancer Effects on Non–Small Cell Lung Carcinoma by Inhibition of Both MET and EGFR

The MET and EGFR receptor tyrosine kinases (RTK) are often coexpressed and may cross-talk in driving the development and progression of non–small cell lung carcinoma (NSCLC). In addition, MET amplification is an alternative resistance mechanism for escaping EGFR-targeted therapy. To assess the benefits of combined targeting of MET and EGFR for treating NSCLCs, we investigated the activities of these two RTK pathways in NSCLC cell lines and evaluated their responses to SGX523 and erlotinib, the small-molecule kinase inhibitors of MET and EGFR, respectively. We showed that MET interacts with and cross-activates EGFR in MET-amplified or -overexpressed cells. The inhibition of both MET and EGFR results in maximal suppression of downstream signaling and of cell proliferation when their ligands are present. Furthermore, we showed that SGX523 plus erlotinib strengthens anticancer activity in vivo in a cellular context–dependent manner. The combination led to the regression of H1993 tumors by enhancing the suppression of proliferation and inducing apoptosis, whereas H1373 tumor growth was significantly reduced by the combination via suppression of proliferation without inducing apoptosis. SGX523 alone was sufficient to achieve near-complete regression of EBC-1 tumors; its combination with erlotinib strongly inhibited the viability of a population of insensitive cells emerging from an SGX523-treated EBC-1 tumor recurrence. Our data suggest that inhibition of both MET and EGFR can enhance anticancer effects against NSCLCs in a context-dependent manner and thus provide a strong rationale for combining MET and EGFR inhibitors in treating NSCLCs. Mol Cancer Ther; 12(8); 1429–41. ©2013 AACR.

Cancer-type regulation of MIG-6 expression by inhibitors of methylation and histone deacetylation.

Epigenetic silencing is one of the mechanisms leading to inactivation of a tumor suppressor gene, either by DNA methylation or histone modification in a promoter regulatory region. Mitogen inducible gene 6 (MIG-6), mainly known as a negative feedback inhibitor of the epidermal growth factor receptor (EGFR) family, is a tumor suppressor gene that is associated with many human cancers. To determine if MIG-6 is inactivated by epigenetic alteration, we identified a group of human lung cancer and melanoma cell lines in which its expression is either low or undetectable and studied the effects of methylation and of histone deacetylation on its expression. The DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) induced MIG-6 expression in melanoma cell lines but little in lung cancer lines. By contrast, the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induced MIG-6 expression in lung cancer lines but had little effect in melanoma lines. However, the MIG-6 promoter itself did not appear to be directly affected by either methylation or histone deacetylation, indicating an indirect regulatory mechanism. Luciferase reporter assays revealed that a short segment of exon 1 in the MIG-6 gene is responsible for TSA response in the lung cancer cells; thus, the MIG-6 gene can be epigenetically silenced through an indirect mechanism without having a physical alteration in its promoter. Furthermore, our data also suggest that MIG-6 gene expression is differentially regulated in lung cancer and melanoma.

Differential responses of MET activations to MET kinase inhibitor and neutralizing antibody

BackgroundAberrant MET tyrosine kinase signaling is known to cause cancer initiation and progression. While MET inhibitors are in clinical trials against several cancer types, the clinical efficacies are controversial and the molecular mechanisms toward sensitivity remain elusive.MethodsWith the goal to investigate the molecular basis of MET amplification (METamp) and hepatocyte growth factor (HGF) autocrine-driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies, we compared cancer cells harboring METamp (MKN45 and MHCCH97H) or HGF-autocrine (JHH5 and U87) for their sensitivity and downstream biological responses to a MET-TKI (INC280) and an anti-MET monoclonal antibody (MetMab) in vitro, and for tumor inhibition in vivo.ResultsWe find that cancer cells driven by METamp are more sensitive to INC280 than are those driven by HGF-autocrine activation. In METamp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although MetMab failed to inhibit METamp cell proliferation and tumor growth, both INC280 and MetMab reduced HGF-autocrine tumor growth. In addition, we also show that HGF stimulation promoted human HUVEC cell tube formation via the Src pathway, which was inhibited by either INC280 or MetMab. These observations suggest that in HGF-autocrine tumors, the endothelial cells are the secondary targets MET inhibitors.ConclusionsOur results demonstrate that METamp and HGF-autocrine activation favor different molecular mechanisms. While combining MET TKIs and ATM inhibitors may enhance the efficacy for treating tumors harboring METamp, a combined inhibition of MET and angiogenesis pathways may improve the therapeutic efficacy against HGF-autocrine tumors.

Abstract LB-015: Differential therapeutic responses of MET oncogenic activations to Met kinase inhibitor and neutralizing antibody

Purpose: MET inhibitors are in clinical trials against several cancer types, but the mechanisms toward vulnerability remain elusive. Here we characterized the molecular basis of MET amplification (METamp) and HGF-autocrine driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies. Experimental Design: METamp (MKN45 and MHCC97H) and HGF-autocrine activation (JHH5 and U87) cells were treated by the MET kinase inhibitor (INC280) and the anti-MET monoclonal antibody (MetMab) to determine the sensitivity and biological responses in vitro. Tumor inhibition was evaluated in vivo using SCID and SCIDhgf mouse models, respectively. HGF-mediated angiogenesis was measured by using the human endothelial cells (HUVEC) tube formation assay. Results: MKN45 and MHCC97H cells are more sensitive than JHH5 and U87 cells to INC280 treatment but are unresponsive to MetMab. In METamp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although INC280 and MetMab showed a moderate inhibitory effect on JHH5 and U87 cells in vitro, both treatments potently suppressed tumor growth in mouse models. We found that HGF stimulation promotes human HUVEC cell tube formation via the Src pathway. INC280 or MetMab inhibited tube formation; thus in HGF-autocrine tumors, the endothelial cells are the secondary targets of tumor-derived HGF and MET inhibition. Conclusion: METamp and HGF-autocrine activation favor different molecular mechanisms, such as a DNA damage response or angiogenesis. Because individual types of MET oncogenic activation may respond to MET inhibitors differently, combination strategies should be developed based upon the molecular subtypes of the tumors.

Abstract 2167: Strengthening anti-cancer effects on non-small cell lung carcinoma by dual blockage of MET and EGFR in a context-dependent manner.

Non-small cell lung carcinoma (NSCLC) accounts for more than 80% of lung cancer, the leading cause of death among all cancer casualties. The MET receptor tyrosine kinase (RTK) is often co-expressed with EGFR in the NSCLC cells, and is an important alternative resistant mechanism for escaping EGFR-targeted therapy. To assess the benefit of dual blockage of MET and EGFR for NSCLC therapy, we investigated the activities of these two RTK pathways in several NSCLC cell lines carrying distinct cellular contexts, and determined their responses to SGX523 and erlotinib, the small molecule kinase inhibitors of MET and EGFR, respectively. Here, we showed that MET can cross-activate EGFR in MET-amplified or overexpressing cells via hetero-receptor dimerization, and the MET-dependent phosphorylation of EGFR can be abolished by SGX523 but not by erlotinib. More importantly, combined inhibition of MET and EGFR in vitro results in a maximal suppression of downstream ERK and AKT activation and of cell proliferation when their ligands (HGF and EGF) are present. Furthermore, we demonstrated that SGX523 and erlotinib combination strengthens anti-cancer activity in vivo in a cellular context-dependent manner. The combination led to regression of MET-amplified H1993 tumors by enhancing suppression of proliferation and inducing apoptosis, whereas significantly reduced MET-non-amplified H1373 tumor growths by suppression of proliferation without inducing apoptosis. Although SGX523 alone was sufficient to achieve near complete regression of MET-addicted EBC-1 tumors, its combination with erlotinib strongly inhibited viability of a population of SGX523-insensitive cells emerging from SGX523-treated EBC-1 tumor relapse. Our data suggest that dual blockage of MET and EGFR can enhance anti-cancer effects on NSCLC in a context-dependent manner, and provide a strong rationale and mechanisms for combining MET and EGFR inhibitors for NSCLC therapy. Citation Format: Yu-Wen Zhang, Ben Staal, Curt Essenburg, Steven Lewis, Dafna Kaufman, George F. Vande Woude. Strengthening anti-cancer effects on non-small cell lung carcinoma by dual blockage of MET and EGFR in a context-dependent manner. [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 2167. doi:10.1158/1538-7445.AM2013-2167

Abstract 4092: Cancer-type regulation of MIG-6 expression by 5-Aza-2′-deoxycytidine and Trichostatin A

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Mitogen inducible gene 6 (MIG-6) is a tumor suppressor gene located in chromosome 1p36 which is frequently lost in many human cancers. It is mainly viewed as a negative feedback regulator of epidermal growth factor receptor (EGFR) family members by directly binding to the receptors and thereby negatively regulating the signaling. Mice with Mig-6 deficiency are prone to develop epithelial hyperplasia or tumors in various tissues including lung, gallbladder, bile duct, uterus and skin. Previously, we reported a few mutations in MIG-6 gene in human lung cancer, but such event is rare. To determine if MIG-6 is regulated by epigenetic silencing, we identified a group of cancer cell lines with low or no MIG-6 expression and investigated the status of DNA methylation and histone deacetylation in MIG-6 promoter. We found that DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) induced MIG-6 expression in the melanoma but not in the lung cancer cell lines, while histone deacetylase inhibitor Trichostatin A (TSA) induced MIG-6 expression in lung cancer lines but not in melanoma cell lines. However, the MIG-6 promoter itself does not seem to be directly affected by methylation or histone deacetylation, indicating they are indirectly regulated and most likely mediated by transcription factor(s). Luciferase reporter assays revealed that a short segment of exon-1 in MIG-6 gene is responsible for the induction of its expression by TSA in the lung cancer cells. Our data suggest that MIG-6 gene expression is differentially regulated by the inhibitors of methylation and histone deacetylation in lung cancer and melanoma cells respectively, without directly modifying the promoter. 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 4092. doi:1538-7445.AM2012-4092

Abstract LB-305: Why two are better than one: Suppression of carcinoma cell growth in vitro and in human HGF/SF transgenic SCID by inhibitors of MET (SGX523) and EGFR (erlotinib)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The signaling cascade originating from hepatocyte growth factor/scatter factor (HGF/SF)-MET ligand-receptor pair is frequently observed in a broad spectrum of human cancers. Inappropriate activation of this pathway has been shown to occur via: paracrine/autocrine activation; by amplification or mutation in the MET gene; or through ligand independent mechanisms (www.vai.org/met). Various MET drugs targeting this pathway are under development and in need of relevant in vivo tumor models to allow assessment of drug efficacy alone or in combination against human MET expressing tumors. Conventional mouse models post a significant limitation on fulfilling such demand as mouse HGF/SF has low affinity/activity on the human MET. Such limitations are abrogated in human HGF/SF transgenic SCIDs (designated hHGFtg-SCID), which, unlike its non-transgenic littermates, produce human HGF/SF to allow human MET signaling on cells of human cancer xenografts. Here, we show that the hHGFtg-SCID mouse significantly enhances tumor xenograft growth of many MET-expressing human carcinoma cell lines including lung, breast, kidney, colon, gastric and pancreatic cancers. Using the hHGFtg-SCID mouse model systems, we demonstrated that SGX523, a MET small molecule kinase inhibitor which suppressed MET phosphorylation and downstream signaling activation in vitro, partially inhibits the growth of xenografts derived from NCI-H596 lung cancer cells, HCC1954 breast cancer cells and HPAF II pancreatic cancer cells. Furthermore, we found that the partial tumor growth inhibition by SGX523 was significantly enhanced by co-administration of erlotinib, the EGFR small molecule kinase inhibitor. In vitro, erlotinib also enhanced SGX523 inhibitory activity in cell proliferation and G1/S cell cycle progression assays. Our data suggest that the hHGFtg-SCID mouse is a valuable system for evaluating MET drugs, and simultaneously targeting MET and EGFR pathways may provide a better treatment for cancers harboring inappropriate activation of both pathways. 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 LB-305.