Analysis of S100P - myosin IIA interaction in cells undergoing Epithelial Mesenchymal Transition

Abstract

Intoduction: Metastasis is associated with around the 90% of all deaths related with cancer. In metastasis, cancer cells can dissociate from the primary tumour mass and invade surrounding tissues, in a process called epithelial-mesenchymal transition (EMT). The EMT changes the epithelial characteristics of the tumour cells modifying the cytoskeleton configuration to alter the cell morphology and acquire migratory capacity, give more invasiveness capacity and increase the resistance to apoptosis. Non-muscle myosin II (NM II) is considered one of the major cytoskeletal proteins and an important regulator of cell adhesion, cell migration and cell division. Is reported that NM II can interact with several S100 proteins such S100A4 affecting cell motility and cytoskeletal dynamics. Furthermore, S100 family proteins such as S100A4 or S100A6 are overexpressed in diverse types of tumours and play an important role in cancer progression and tumorigenesis. Hypothesis: Since, S100P overexpression is described in multiple cancers, such as colon and pancreas carcinomas, is possible to hypothesize that an abnormal expression of S100P can induce to EMT, in a similar way of S100A4 does, including its binding activity with NM IIA. Materials and methods: In order to prove the hypothesis, human epidermoid carcinoma cells (A431 cell line) with inducible expression of the transcription factor ZEB2 by Doxycycline, a repressor of E-cadherin and inducer of EMT, were selected for the experiments. The expression of S100P was assessed by Immunofluorescence and Western Blotting. Additionally, S100P-NM IIA interactions were evaluated by Immunoprecipitation, Western Blotting, Mass Spectrometry Analysis and Blot Overlay. Results: The results of the characterization of S100P expression showed lower expression levels of the protein both in nucleus and cytoplasm in EMT-induced cells. S100P was successfully cloned and expressed in bacterial cells. Recombinant S100P was isolated and used in blot-overlay assays. Furthermore, the performed experiments revealed lack of direct interactions between S100P and NM IIA, suggesting that S100P plays a role in NM IIA regulation cascade, but the direct binding has not been confirmed.