Extracellular interactions as key regulators of oncogenic signaling in ovarian cancer and melanoma aggressiveness

Growth of metastatic tumors and therapy resistance – often arising in metastatic tumors – are the main causes of cancer mortalities. Early identification of cases with a potential to disseminate is important, as it greatly improves the prognosis. The microenvironment has a multifaceted effect on the signaling and survival of cancer cells, with cancer cells extensively modifying their microenvironment and thereby promoting a constantly evolving oncogenic landscape. Eph receptors and their membrane bound ephrin ligands possess the unique capability for bidirectional signaling. Eph-ephrin signaling is crucial in development of the nervous system, while modulating wound healing and tissue boundary maintenance among other functions in physiology. Eph-ephrin signaling has been implicated in the aggressiveness of multiple cancer types including ovarian cancer and melanoma, but the mechanics of Eph-ephrin signaling outside the physiological setting require further studies. Membrane type matrix metalloproteases (MT-MMPs) are the transmembrane members of the MMP family, which are the main proteolytic remodelers of the extracellular matrix (ECM). MMPs are widely associated with cancers, originally due to their prominent ECM degradation capabilities, but their capability for proteolytic regulation of various proteins has become a focus for their function. These targets include Eph-ephrin family receptors and ligands and even other members of the MMP family, forming a complex regulatory network between MMPs and pericellular proteins. These findings implicate MT-MMPs as key regulators of tumor microenvironment (TME)-related signaling transduction. My research in this thesis focused on ovarian cancer and melanoma, two cancer types that share multiple features in their disease progression: a propensity for lymphatic invasion, early dissemination, common emergence of therapy resistance, and the association of Eph-ephrin and MT-MMP expression in aggressiveness. In the first study of the thesis, I present ephrin-A5 as an atypical ligand for EphA2, leading to the increased maintenance of ligand independent oncogenic signaling of EphA2. Ephrin-A5 expression was found to associate with high-grade serous ovarian cancer (HGSOC), the most aggressive and common subtype of ovarian cancer, and ephrin-A5 expression increased at both mRNA and protein level during disease progression in the longitudinal HGSOC sample sets. In the second study the cytoskeleton remodeling proteins ezrin and formin-like 2 were investigated for their utility to assist in melanoma staging, but were discovered not to correlate with positive sentinel lymph node biopsy (SNB) status. However, we observed a correlation between wild type BRAF and a negative SNB result, indicating limited metastatic potential in the absence of constitutive activation of BRAF and subsequent MAPK signaling. In the third study of this thesis, I made a fascinating discovery on MMP16 in the SNB negative recurrent melanomas: MMP16 expression at the invasive front of the primary melanoma tumor correlated significantly with distant organ metastases and involvement of brain metastases analyzed by immunohistochemistry, and the staining was validated using in situ hybridization. Upon analyzing the metastatic samples of this cohort, MMP16-expressing melanoma cells were found aggressively invading especially in the prototypically fibrin rich, hemorrhaging microenvironment of brain metastases. Global gene expression analysis of melanoma cells with transient ectopic expression or siRNA mediated depletion of MMP16 revealed associations between MMP16 and numerous important oncogenic pathways. Several transcription factors were also among the differentially expressed genes. These findings shed additional light on the importance of MMP16 in melanoma aggressiveness, implicating it as an important factor in the regulation of oncogenic signaling. Using a zebrafish embryo model, we found that MMP16 increases the cluster size of melanoma cells in vivo, further connecting MMP16 with the highly aggressive collective invasion type. Additionally, MMP16 expression increased invasion towards the thoracic cavity which hosts a hub of the developing lymphatic system of the zebrafish. In summary, the work of this thesis elucidates mechanisms behind oncogenic factors that have known associations with cancer aggressiveness, reveals additional facets of the signaling related to them, and highlights the effects of extracellular signaling events on intracellular signaling. The context dependence of Eph-ephrin signaling is highlighted in the first study, as EphA2-mediated intracellular signaling is significantly dependent on the ephrin ligand bound to the receptor. Thus, further studies should not focus only on the Eph receptor facet of Eph-ephrin signaling, but rather on the wider context in which the signaling occurs. The second study displays that known factors associated with melanoma aggressiveness are important already at the beginning of the metastatic cascade, as wild type BRAF limited the capacity for dissemination. However, a panel of known intracellular melanoma oncogenes that are extensively associated with disease aggressiveness did not correlate with a positive SNB result, highlighting the complexity of the metastatic process. The third study further highlights the importance and extent of MMP function in cancer aggressiveness through the discovered connection between MMP16 and the metastatic potential of melanoma, reinforced by the cell-based assays uncovering the extensive effects MMP16 expression has on melanoma cell phenotype and transcriptome.