Exploring mechanisms of metastasis suppression in metastatic melaoma

Abstract

Melanoma is responsible for 76% of deaths from skin cancer, making it the deadliest form of commonly diagnosed skin cancer. The deadly nature of melanoma is due to its tendency towards rapid, early metastasis. Metastasis, the process of cells exiting the primary tumor and forming secondary tumors in other parts of the body, accounts for the majority of morbidity and mortality associated with cancer. Therapeutically targeting and treating melanoma metastases is a challenging clinical goal, as metastatic cells are heterogeneous and can be morphologically and genetically distinct from the primary tumor. This dissertation examines two approaches for prevention or treatment of disseminated melanoma metastases: 1) Re-introduction of metastasis suppressor protein fragments to prevent metastatic colonization, and 2) Treating disseminated metastases with a targeted small molecule treatment. By examining two discrete approaches of treating metastatic melanoma, this work sheds light on the clinical viability of using metastasis suppressors or metastasis-targeting drugs in patients with metastatic melanoma. To examine strategies for metastasis suppression through metastasis suppressor proteins, we examined cleavage products of the metastasis suppressor KISS1, a metastasis suppressor protein. Expression of KISS1 inhibits metastatic colonization at secondary sites, rendering disseminated cells dormant. KISS1 must be secreted outside of the cell to suppress metastasis, where furin cleaves KISS1 into kisspeptins at three dibasic sites. This cleavage liberates an internal kisspeptin, Kisspeptin-54 (KP54, amino acid K67 to F121), which is amidated and can bind a Gq/11-coupled protein receptor KISS1R. The mechanism of action for KISS1 metastasis suppression has long been assumed to be KP54 interacting with KISS1R. However, expression of KISS1R is not necessary for KISS1 metastasis suppression, and the extracellular processing of KISS1 hints at an alternative hypothesis: a different kisspeptin may be responsible for suppressing metastasis. To test this hypothesis, all possible kisspeptins (KISS1 Manufactured Peptides, or KMP) were generated based on known dibasic cleavage sites (M1–Q145; M1–R56; M1–R67; M1–R124; R56–R66; R67-F121; R56–F121; R56–Q145; R67–Q145; R124–Q145) and were used in an experimental metastasis assay to characterize their abilities to suppress metastasis. We found that while KP54 suppressed metastasis, additional KMP lacking the KISS1R binding site (LRF-NH2) were able to completely suppress metastasis (p<0.05). In particular, one kisspeptin (KMP2, M1 – R56) suppressed metastatic traits in vitro as well as completely suppressing metastasis in vivo. To identify the signaling pathways used by KMP2 to suppress metastasis, a genome wide CRISPR/Cas9 screen was performed in KMP2-expressing B16-F10 melanoma cells. As a whole, these data suggest that metastasis suppression by KISS1 is not necessarily contingent on KISS1R activation, and also supports investigation into additional receptors. To investigate the efficacy of targeting metastases with small molecules, we also investigated the impact of ML246 (AKA metarrestin). Metarrestin was discovered by a high throughput assay for molecules which disassemble the perinucleolar compartment (PNC). Perinucleolar compartments are structures composed of RNA and RNA binding proteins near the nucleolus. These structures are enriched in metastatic cells and are druggable targets which target metastases and not normal epithelium. We examined the impact of metarrestin treatment on orthotopic tumor growth, microscopic metastasis formation, and macroscopic metastasis formation. We found that metarrestin treatment had no significant impact on metastatic outgrowth, but suppressed intradermal tumor growth. Based on these data, we can infer that PNC-positive metastases may be too small a population to effectively target in this model. This treatment paradigm may be more effective in conjunction with a more potent approach to metastasis suppression. Overall, the work in this dissertation identified a potent metastasis suppressing fragment of KISS1, KMP2, and described the efficacy of metarrestin treatment of disseminated metastases. The metastasis suppression induced by KMP2 expression was far more potent than the effects of metarrestin treatment on suppressing metastatic colonization and outgrowth, suggesting that treatment deliveries and targets are critical considerations in the development of anti-metastatic therapeutics.