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2009 Scientific Report

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  • Michigan
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Van Andel Research

Van Andel Research Institute | Scientific Report Research Interests The Laboratory of Molecular Oncology is focused on understanding the numerous and diverse roles that MET and HGF/SF play in malignant progression and metastasis. Our work involves a wide variety of cancers, animal models, and drug therapies. The combination of studies, coupled with our examination of MET signaling, will lead to a greater understanding of tumor progression and new knowledge for developing and delivering novel targeted therapies. Tumor phenotypic switching Malignant progression leading to metastasis is the primary cause of death due to cancer. Metastasis begins with proliferating tumor cells that become invasive and detach from the primary tumor mass, invading the extracellular matrix, entering the bloodstream or lymphatic vessels, and establishing metastases or secondary tumors as proliferating colonies at distant sites. Since phenotypic switching from proliferative to invasive and the return to proliferative is crucial for malignant progression, we use in vitro and in vivo methods to select proliferative and invasive subclones from tumor cell populations. We explored the signal pathway underlying phenotypic switching by integrative genomic studies including gene expression analysis, spectral karyotyping (SKY), and fluorescent in situ hybridization (FISH). We observed that subtle and specific changes in chromosome content ratio are virtually the same as the changes in the chromosome transcriptome ratio, showing that major changes in gene expression are mediated by gains or losses in chromosome content. Importantly, a significant number of the genes whose expression change is greater than twofold are functionally consistent with changes in the proliferative or invasive phenotypes. Our results imply that chromosome instability can provide the diversity of gene expression that allows a tumor to switch between proliferative and invasive phenotypes during tumor progression. Met in murine mammary tumors and human basal breast cancers Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. The oncogene MET is associated with decreased survival in breast cancer, yet the role it plays in the various breast cancer subtypes is unclear. We are investigating the role that this oncogene plays in breast cancer progression and metastasis by using a novel mouse model of mutationally activated Met (Met mut ). We discovered that mutationally activated Met induces a high incidence of diverse mammary tumors in mice, and these Met mut mice tumors have several characteristics similar to those of aggressive human breast cancers, such as the absence of progesterone receptor and ERBB2 expression. These results led us to examine how MET is associated with the various human breast cancer subtypes. With gene expression and tissue microarray analysis, we observed that high MET expression in human breast cancers significantly correlated with estrogen receptor–negative/ERBB2-negative tumors and with basal breast cancers. Few treatment options exist for breast cancers of the basal or trastuzumab-resistant ERBB2 subtypes. We conclude from these studies that MET is a key oncogene in the development of the most aggressive breast cancer subtypes and may be a significant therapeutic target. Currently, we are investigating the similarities and differences in signaling pathways involved in MET-driven versus ERBB2-driven breast cancers. Tumor xenograft models for preclinical testing of MET drugs Aberrant activation of the HGF-Met signaling pathway is one of the causal events in cancer development and progression and is frequently observed in almost all types of human cancers. MET is becoming an ideal target for cancer intervention, and the movements toward developing MET drugs are very active. In the past several years, many drugs targeting the HGF-MET pathway have been developed, including neutralizing antibodies against HGF or MET and various small-molecule kinase inhibitors of MET. This has resulted in the need for suitable animal models for preclinically testing the drug efficacies in vivo. 56

VARI | 2009 We had previously generated a transgenic mouse that produces human HGF in the severe combined immune deficiency (SCID) background. This animal model provides species-compatible ligand for human MET and is ideal for investigating paracrine MET signaling in human cancer cells (mouse HGF has very low activity on human MET). We found that, compared with control SCID mice, the human HGFtg-SCID (huHGFtg-SCID) mice significantly enhance tumor xenograft growth of many MET-positive human cancer cells derived from lung, breast, stomach, colon, kidney, and pancreas. Currently, we are using those xenograft models established in the huHGFtg-SCID mice for testing MET drugs alone or in combination with other cancer drugs. Meanwhile, we are also developing metastatic models in the huHGFtg-SCID mice. In vivo modeling of glioblastoma multiforme Glioblastoma multiforme (GBM) is one of the most devastating cancers. The hallmark of GBM is the invasiveness of the tumor cells infiltrating into normal brain parenchyma, making it virtually impossible to remove the tumor completely by surgery and inevitably leading to recurrent disease. Progress in understanding GBM pathobiology and in developing novel antitumor therapies could be greatly accelerated with animal model systems that display characteristics of human GBM and that enable tumor monitoring through noninvasive imaging in real time. Subjecting human cancer cells to an experimental metastasis assay (ELM) often yields highly metastatic cells with higher proliferative and invasive potential. However, the ELM assay has not been tested previously with GBM, most likely because extracranial metastases of human GBM are clinically rare. In this study, we used ELM to enrich metastatic cell populations and found that three of four commonly used GBM lines (U251, U87, and DBTRG-05MG) were highly metastatic after repeated (M2) ELM selection. These GBM-M2 lines grew more aggressive orthotopically, and all showed significant multifold increases in IL6, IL8, MCP-1, and GM-CSF, which are cytokines and factors associated with poor GBM prognosis. DBM2 cells, derived from the DBTRG-05MG cell line, are highly invasive when grown as an orthotopic tumor (with areas of central necrosis, vascular hyperplasia, and intracranial dissemination), and also erode the skull, permitting the use of high-resolution micro-ultrasound in real time to non-invasively observe tumor growth and vascularization. We conclude that commonly used GBM cells have intrinsic metastatic potential which can be selected for in ELM assays. When implanted in the brain, the metastatic potential of GBM cells can be realized as a highly invasive phenotype. The DBM2 mouse model has characteristics that mimic the aggressively invasive behavior of clinical GBM, providing a valuable tool for investigating the factors that modulate glioblastoma growth, assessing invasion and vascularity, and evaluating novel therapeutic agents in real time. Currently we are in the process of using this model to test MET drugs and possible combinations for the purpose of blocking GBM invasion and studying the micro-environment of the host-tumor response to the treatment. The role of Mig-6 in cancer and joint disease The signaling mediated by receptor tyrosine kinases such as Met and EGFR plays a very important role in many developmental and physiological processes, and it is fine-tuned by many factors for proper action. Mitogen-inducible gene-6 (Mig-6), a scaffolding molecule, is one of the factors that can regulate Met and EGFR signaling through a negative feedback loop. Mig-6 is an immediate early response gene that can be rapidly up-regulated by growth factors like HGF and EGF, as well as by many stress stimuli such as mechanical stress. The Mig-6 gene locus is at human chromosome 1p36 that is frequently associated with various cancers. Studies in both humans and mice indicate that Mig-6 is a tumor suppressor gene. Decreased expression of Mig-6 is observed in several human cancers including breast, skin, pancreatic, and ovarian cancers, while targeted elimination of Mig-6 in mice leads to the development of neoplasms in the lung, gallbladder, bile duct, and skin. We also identified several Mig-6 gene mutations in lung cancer, even though mutation in Mig-6 seems to be a rare event. Besides its role in cancer, Mig-6 also plays an important role in maintaining normal joint function: its deficiency in mice results in the development of early-onset degenerative joint disease. Currently, we are investigating what roles Mig-6 may play in cancer development and in maintenance of joint function, and the mechanism of how losing Mig-6 activity leads to the pathological conditions of cancer and joint disease. 57

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