Studying a possible functional circuitry involving microRNAs, MITF, MYO5A/DLC2, RAB27A and connections with the invasion and metastasis cascade

Abstract

Gain of invasive capacity and resistance to cell death are the major requirements for metastasis and although several lines of evidence suggest a role for myosin-Va in this process, the relevance in vivo and mechanisms remain elusive. Recent work from our laboratory showed that myosin-Va is overexpressed during tumor progression and RNAi-mediated knockdown of myosin-Va leads to reduced anchorage-independent growth, cell migration and invasion in vitro (Alves et al., J. Inv. Dermatol., 2013). Also, overexpression of a myosin-Va tail fragment (MVaf) carrying the DLC2-binding site, in melanoma cells, induces apoptosis, which is at least partially dependent on the proapoptotic factors Bmf and Bim, as well as Bax and Bak, and attenuates tumor growth in mouse (Izidoro-Toledo and Borges et al., Cell Death & Dis., 2013). Recent unpublished data from our laboratory shows that myosin-Va interacts with FAK and is involved in focal adhesion dynamics. Moreover, MYO5A gene expression is activated by MITF, a transcription factor involved in melanoma progression, and MYO5A mRNA is targeted by miR-145, a metastasis suppressor microRNA. Other groups showed that the depletion of DLC2 causes a selective death of cells carrying KRAS oncogenic mutations. Mutations in the MYO5A gene have been revealed in many types of cancer and myosin-Va enhances cell migration and drug resistance in melanoma. Therefore, the main goal of this project is to gain novel insights into the role of myosin-Va in the invasion and metastasis cascade and its involvement in drug resistance in cancer. To assess the requirements of myosin-Va or DLC2, we will interfere in their functions by using specific shRNA-mediated knockdown, as well as by delivery of DNA vectors to overexpress proapoptotic fragments or mutated forms of myosin-Va and DLC2, or by means of penetrating peptides. We will use bioluminescent non-invasive imaging on the IVIS spectrum system and fluorescence imaging by multiphoton intravital microscopy to analyze the patterns and dynamics of tumor growth, invasion and metastasis in engrafted or transgenic mice. Virtual molecular docking to tridimensional atomic structures will be performed for the selection of natural or synthetic compounds aiming at dissociating the ternary complex myosin-Va/DLC2/Bmf or other interactions involving myosin-Va, such as those with melanofilin/Rab27a, PTEN and FAK. We will also explore the mechanisms underlying the role of myosin-Va in cell survival, cell migration and invasion, using live cell microscopy and other in vitro molecular and biochemical approaches to assess the role of myosin-Va in focal adhesion and invadopodia dynamics, endosomal and lysosomal trafficking, autophagy, as an effector of RAB27A in the release of exosomes, as well as in functions involving ABC transporters and drug resistance, and to understand the relationship with MITF and miR-145. Concluding, this project will contribute for the understanding of how myosin-Va influences malignant properties and may lead to the development of novel molecules and strategies with therapeutic potential. (AU)