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Abstract

Melanoma belongs to the most aggressive forms of skin cancer. While the primary tumor can generally be controlled, most patients develop metastasis upon tumor resection. Multiple reports show that the primary tumor secretes certain factors that limit metastatic outgrowth by restricting angiogenesis. Angiopoietin-like 4 (ANGPTL4) represents one of these primary tumor-derived factors as its expression enhances with malignancy. However, the published literature on ANGPTL4 function during angiogenesis and metastasis is highly controversial. While some studies describe ANGPTL4 as a stimulator of metastasis, other studies show that ANGPTL4 can also restrict tumor growth and secondary colonization. Given the conflicting literature, our lab has previously overexpressed the two cleavage fragments of ANGPTL4, namely nANGPTL4 and cANGPTL4, in tumor cells. The results indicated that overexpression of cANGPTL4 in tumor cells enhances metastasis, whereas nANGPTL4 overexpression reduces metastatic dissemination. However, the mechanism of nANGPTL4 action as well as its therapeutic and clinical potential, remain elusive. Therefore, the present thesis was aimed at mechanistically understanding how nANGPTL4 limits metastatic outgrowth and corroborate the clinical relevance of cleavage fragments of ANGPTL4 in tumor patients. Further, the study aimed to design and evaluate the efficacy of nANGPTL4 treatment in clinically relevant mouse models. Assessing the levels of ANGPTL4 cleavage fragments in primary tumors and serum samples of melanoma and lung cancer patients depicted that nANGPTL4 and cANGPTL4 were differentially distributed in primary tumor and serum samples. While cANGPTL4 was shown to be localized in the primary tumor, nANGPTL4 was highly expressed in the systemic circulation. Additionally, longitudinal studies of melanoma patient sera revealed that the expression of nANGPTL4 negatively correlated with disease progression whereas increasing nANGPTL4 levels in the serum correlated with higher overall survival in melanoma patients. In line, pre-clinical analyses showed that systemic administration of nANGPTL4 reduced metastasis formation and increased the overall survival of mice in both experimental and spontaneous metastasis models. In vitro studies negated a direct effect of nANGPTL4 on cancer cell behavior. Mechanistically, global gene expression profiling of endothelial cells from the metastatic site unveiled that nANGPTL4 specifically downregulated Wnt signaling and thereby reduced vascularity at the metastatic site. Correspondingly, in vitro spheroid sprouting assays and in vivo cornea pocket assays revealed that nANGPTL4 inhibited sprouting angiogenesis. This effect depends on the interaction of nANGPTL4 and its downstream receptor SDC4. As nANGPTL4 is a potent inhibitor of LPL, high levels of nANGPTL4 were previously shown to upregulate plasma triglyceride levels, thereby increasing the risk of developing cardiovascular disease. Recent studies have shown that a missense mutation in nANGPTL4 (E40K) strongly correlates with lower TG levels. As increased triglyceride levels may represent a significant side effect of nANGPTL4 treatment, the second part of this study was aimed at analyzing whether the E40K mutated nANGPTL4 affected tumor development and metastasis. Primary tumor experiments revealed that overexpression of E40K mutated nANGPTL4 did not affect primary tumor growth and the vasculature. In addition, overexpression of E40K mutated nANGPTL4 in the primary tumor and systemic administration of E40K mutated nANGPTL4 had no effect on metastasis and survival in pre-clinical mouse models. In conclusion, this study identified nANGPTL4 as an endogenous primary tumor-derived inhibitor of metastasis and substantiated the therapeutic efficacy of nANGPTL4 treatment. Further, the data unveil that oligomerization of nANPGTL4 is required to sustain its anti-metastatic activity. Moreover, the results show that serum nANGPTL4 levels can be a biomarker to determine disease progression in cancer patients.