Loss of Two-Pore Channel 2 (TPC2) Expression Increases the Metastatic Traits of Melanoma Cells by a Mechanism Involving the Hippo Signalling Pathway and Store-Operated Calcium Entry.

John Parrington,Giovanni Blandino,Andrea Sacconi,Ali Ahmed Hanbashi,Antonio Filippini,Aniruddha Voruganti,Antonella D’Amore,Marilena Taggi,Fioretta Palombi,Rita Canipari,Silvia Di Agostino

doi:10.3390/cancers12092391

Abstract

Melanoma is one of the most aggressive and treatment-resistant human cancers. The two-pore channel 2 (TPC2) is located on late endosomes, lysosomes and melanosomes. Here, we characterized how TPC2 knockout (KO) affected human melanoma cells derived from a metastatic site. TPC2 KO increased these cells’ ability to invade the extracelullar matrix and was associated with the increased expression of mesenchymal markers ZEB-1, Vimentin and N-Cadherin, and the enhanced secretion of MMP9. TPC2 KO also activated genes regulated by YAP/TAZ, which are key regulators of tumourigenesis and metastasis. Expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-βII, part of the HIPPO pathway that negatively regulates YAP/TAZ activity, were reduced by TPC2 KO and RNA interference knockdown. We propose a cellular mechanism mediated by ORAI1/Ca2+/PKC-βII to explain these findings. Highlighting their potential clinical significance, patients with metastatic tumours showed a reduction in TPC2 expression. Our research indicates a novel role of TPC2 in melanoma. While TPC2 loss may not activate YAP/TAZ target genes in primary melanoma, in metastatic melanoma it could activate such genes and increase cancer aggressiveness. These findings aid the understanding of tumourigenesis mechanisms and could provide new diagnostic and treatment strategies for skin cancer and other metastatic cancers.

Highlights

Melanoma originates from the tumoral transformation of melanocytes in the skin [1]
Target genes and N-Cadherin in ultra-low cell attachment conditions and found no such activation in these circumstances (Figure S2). These findings suggest that the loss of two-pore channel 2 (TPC2) may lead to the activation of yes association protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) target genes, and increased metastasis, because of changes in the capacity of both store-operated Ca2+ entry (SOCE) and the cytoskeleton to mediate the inhibition of the HIPPO pathway
Our data indicate that a loss of, or reduction of, TPC2 expression in a metastatic model of human melanoma increased the aggressiveness of melanoma cells

Summary

Introduction

Melanoma originates from the tumoral transformation of melanocytes in the skin [1]. The depth of the invasion and the metastasis of the lymph nodes are very important prognostic features for the classification of melanoma; typically, this type of cancer is associated with a very heterogeneous tumour characterized by high mutational burden (e.g., BRAF, NRAS, PTEN, TP53, CDKN2A are the most frequently muted genes) and because of this, it is considered a aggressive type of cancer [2]. NAADP-evoked TPC2 Na+ currents [12]; different experimental conditions may influence the mode of TPC activation In line with such a possibility, a recent study demonstrated the different effect of two novel TPC2 agonists; one of which induced Ca2+ release and the other Na+ release. Following the activation of the HIPPO pathway, MST1/2 is phosphorylated and activates LATS1/2, which can phosphorylate the yes association protein (YAP) and its paralogue, the transcriptional coactivator with PDZ-binding motif (TAZ), resulting in the inhibition of the transcriptional activity of YAP/TAZ [20] This phosphorylation promotes YAP/TAZ cytoplasmic localisation; when not inhibited in this way, the translocation of YAP/TAZ to the nucleus promotes the activation of their target genes which play key roles in cell.

Results

TPC2 expression in the melanoma patient human melanoma melanoma

TPC2 and HIPPO Pathway Connection in CHL1 and MeWo Metastatic Cell Lines

PD-L1 Induced by INTERFERON-γ Is Significantly Higher in CHL1 TPC2 KO Cells

Possible mechanism involved

Discussion

Cell Lines

Western Blot Analysis

CRISPR Design

Cell Transfection

Adhesion Assay

Invasion Assay