Role of Rhophilin2, a RhoB GTPase effector, in melanoma metastasis: zebrafish as a model

Supervisor: Dr. Isabelle Pirson
Collaborator: Dr. Ievgenia Pastushenko

RhoGTPases regulate cytoskeleton dynamics and vesicular trafficking and play key roles in cancer development and metastasis through activation of specific effectors. Rhophilin-2 (RHPN2), one of these effectors, is overexpressed in various human cancers and promotes their survival but less is known about its molecular mechanisms of action. Zebrafish has emerged as a major model for studying developmental processes and cancer development. The objective of the present project is, using zebrafish as a model, to evaluate the role of RHPN2 in the progression of melanoma, one of the most metastatic and aggressive human cancer. Interestingly, we recently identified a tetraspanin, involved in exosome generation, and a protein playing an important role in apoptosis as new Rhpn2 proteic partners. These partners play a role in melanoma as well and RhoB-KO mice are strikingly more susceptible to develop skin cancer. In addition, our previous data and a two-hybrid screen showed a link of Rhpn2 with vesicular trafficking. The project propose to investigate the physiological role of RHPN2 both in vivo and through biochemical experiments.

In the present project, we propose:

  • In zebrafish, to induce melanoma in already available Rhpn2KO fishes compared to wt to evaluate the role of Rhpn2 in tumor growth and metastasis. Primary tumors of the different genotypes will be studied morphologically. The tumors will be transplanted in wt fish to study the metastatic tendency. We will also use transgenic models expressing fluorochrome reporters under the control of Rhpn2 promoters to detail tissue and subcellular expression profiles of Rhpn2 in the tumor. A leading experiment of tumor transplantation in irradiated transparent fish was successful and suggested that the wt tumor grew faster than rhpn2-/- one.
  • In human melanoma cancer cells lines, to investigate the role of Rhpn2 in cellular migration/invasion processes and in exosome synthesis. Stable RHPN2 knock-down (shRNA) melanoma cell clones (already established) and overexpressing clones (lentiviral) of Rhpn2 will be studied. The metastatic properties of these cells could also be further assessed through in vivo xenografts experiments in zebrafish. To address the relevance of our findings for human disease, we will assess the expression of RHPN2 in paraffin-embedded human skin, normal and dysplastic nevi, and different stages of melanoma by immunostaining. We will determine whether there are differences in the expression of RHPN2 during tumor progression.
  • By biochemical experiments, to unravel the proteic partners of Rhpn2 to decipher the molecular function of this protein. We will further characterize the interaction with the JNKK using kinase dead mutant or other functional mutants of the JNKK already available in the lab. We will confirm the interaction in melanoma cells and measure the impact of hormones (EGF, TNF) on the interaction. Caspase 3 activation can be measured by Western blot to validate an implication of RHPN2 on apoptosis in transfected cells. The expression, localization or regulation of the convincing RHPN2 protein partner(s) identified could be further evaluated in KO fish compare to wild-type fish through WIF (whole-mount immunoflurescence) or WISH.

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