Angiogenic and Metastatic Determinants of Malignant Melanoma

Cutaneous melanoma or malignant melanoma of the skin is a highly metastatic disease, with an increasing rate of incidence, poor prognosis and high resistance to therapeutic intervention. Although early diagnosis and surgical resection of the primary lesion could significantly improve survival, the high propensity of melanomas to disseminate through intradermal, haematogenous and lymphatic routes to regional and visceral sites leads to poor prognosis and high mortality rates. While the clinical staging and progression of melanoma is well defined, the molecular etiology of the disease is less well characterized. The principal goal of this thesis was to obtain novel mechanistic insights into melanoma biology and identify molecular determinants of this disease. We identified the capacity of melanoma-derived small molecules to promote long-term survival of endothelial cells under severe hypoxic conditions. This observation excludes known proangiogenic molecules which have been the focus of anti-angiogenic therapies and sets the stage for the identification of novel regulators with therapeutic potential. Moreover, TIMP3 was identified as a dominant negative regulator of melanoma development. The inhibitory role of TIMP3 in melanoma angiogenesis was validated and further extended to clinical samples from melanoma patients. We showed that promoter methylation mediated TIMP3 silencing impacts clinical outcome in melanoma and also evaluated the implications of the tumor suppressor role of TIMP3 in melanoma metastasis, using matched samples from melanoma patients. A decrease in TIMP3 expression with observed with disease progression and further TIMP3 inhibited melanoma cell migration and invasion. To characterize core mediators of the metastatic cascade of melanomas, we determined the migratory profile of melanoma cell lines and performed correlation analysis to identify potential genetic modulators. WNT5A was identified as a dominant regulator of the metastatic cascade in melanoma and further we show that WNT5A inhibition decreases the migratory and metastatic potential of melanoma cells. Additionally, this thesis describes novel tools to quantitatively characterize several biological processes. The ring barrier-based migration assay enables the quantitative assessment of several parameters of cell migration. The eNOS-Tag-GFP mouse model provides a platform for the in vivo and ex vivo study of early angiogenic events in physiological and pathological conditions. Collectively, the results presented in this thesis identify crucial pathophysiological determinants of melanoma. These insights and tools may further guide the discovery of novel regulators of melanoma biology and result in the implementation of new treatment rationales for therapeutic benefit

Melanomas prevent endothelial cell death under restrictive culture conditions by signaling through AKT and p38 MAPK/ ERK-1/2 cascades

Although melanoma progression and staging is clinically well characterized, a large variation is observed in pathogenesis, progression, and therapeutic responses. Clearly, intrinsic characteristics of melanoma cells contribute to this variety. An important factor, in both progression of the disease and response to therapy, is the tumor-associated vasculature. We postulate that melanoma cells communicate with endothelial cells (ECs) in order to establish a functional and supportive blood supply. We investigated the angiogenic potential of human melanoma cell lines by monitoring the survival of ECs upon exposure to melanoma conditioned medium (CM), under restrictive conditions. We observed long-term (up to 72 h) EC survival under hypoxic conditions upon treatment with all melanoma CMs. No such survival effect was observed with the CM of melanocytes. The CM of pancreatic and breast tumor cell lines did not show a long-term survival effect, suggesting that the survival factor is specific to melanoma cells. Furthermore, all size fractions (up to < 1 kDa) of the melanoma CM induced long-term survival of ECs. The survival effect observed by the < 1 kDa fraction excludes known pro-angiogenic factors. Heat inactivation and enzymatic digestion of the CM did not inactivate the survival factor. Global gene expression and pathway analysis suggest that this effect is mediated in part via the AKT and p38 MAPK/ ERK-1/2 signaling axis. Taken together, these data indicate the production of (a) survival factor/s (< 1 kDa) by melanoma cell lines, which enables long-term survival of ECs and promotes melanoma-induc

Lipid phosphatase SHIP2 functions as oncogene in colorectal cancer by regulating PKB activation

Colorectal cancer (CRC) is the second most common cause of cancer-related death, encouraging the search for novel therapeutic targets affecting tumor cell proliferation and migration. These cellular processes are under tight control of two opposing groups of enzymes; kinases and phosphatases. Aberrant activity of kinases is observed in many forms of cancer and as phosphatases counteract such "oncogenic" kinases, it is generally assumed that phosphatases function as tumor suppressors. However, emerging evidence suggests that the lipid phosphatase SH2-domain-containing 5 inositol phosphatase (SHIP2), encoded by the INPPL1 gene, may act as an oncogene. Just like the well-known tumor suppressor gene Phosphatase and Tensin Homolog (PTEN) it hydrolyses phosphatidylinositol (3,4,5) triphosphate (PI(3,4,5)P3). However, unlike PTEN, the reaction product is PI(3,4)P2, which is required for full activation of the downstream protein kinase B (PKB/Akt), suggesting that SHIP2, in contrast to PTEN, could have a tumor initiating role through PKB activation. In this work, we investigated the role of SHIP2 in colorectal cancer. We found that SHIP2 and INPPL1 expression is increased in colorectal cancer tissue in comparison to adjacent normal tissue, and this is correlated with decreased patient survival. Moreover, SHIP2 is more active in colorectal cancer tissue, suggesting that SHIP2 can induce oncogenesis in colonic epithelial cells. Furthermore, in vitro experiments performed on colorectal cancer cell lines shows an oncogenic role for SHIP2, by enhancing chemoresistance, cell migration, and cell invasion. Together, these data indicate that SHIP2 expression contributes to the malignant potential of colorectal cancer, providing a possible target in the fight against this devastating disease