A Targeted Quantitative Proteomic Approach Assesses the Reprogramming of Small GTPases during Melanoma Metastasis

Abstract

Small GTPases of the Ras superfamily are master regulators of intracellular trafficking and constitute essential signaling components in all eukaryotes. Aberrant small GTPase signaling is associated with a wide spectrum of human diseases, including cancer. Here, we developed a high-throughput, multiple reaction monitoring-based workflow, coupled with stable isotope labeling by amino acids in cell culture, for targeted quantification of approximately 100 small GTPases in cultured human cells. Using this method, we investigated the differential expression of small GTPases in three pairs of primary and metastatic melanoma cell lines. Bioinformatic analyses of The Cancer Genome Atlas data and other publicly available data as well as cell-based assays revealed previously unrecognized roles of RAB38 in promoting melanoma metastasis. Diminished promoter methylation and the subsequent augmented binding of transcription factor MITF contributed to elevated expression of gene in metastatic versus primary melanoma cells. Moreover, RAB38 promoted invasion of cultured melanoma cells by modulating the expression and activities of matrix metalloproteinases-2 and -9. Together, these data establish a novel targeted proteomic method for interrogating the small GTPase proteome in human cells and identify epigenetic reactivation of RAB38 as a contributing factor to metastatic transformation in melanoma. A novel quantitative proteomic method leads to the discovery of RAB38 as a new driver of metastasis in melanoma. .

Subversion of Ras Small GTPases in Cutaneous Melanoma Aggressiveness

“…However, their role in tumor progression emerged more recently. A broad study using a directed proteomic quantification approach evaluated the differential expression of Ras small GTPases in primary and metastatic melanoma cell lines, in order to establish a general profile of the most relevant ones (Huang et al, 2018). Based on this, RAB27A and RAB38 were found upregulated in metastatic melanoma, relative to the matched primary melanoma cell lines derived from human samples (Huang et al, 2018).…”

“…On the other hand, RAB38 regulates melanoma cell invasion by increasing the expression and activity of two MMPs, MMP-2 and -9 (Huang et al, 2018). This effect is also mediated by the MITF, the master regulator of melanocyte development, which leads to an increase in RAB38 expression in metastatic melanoma (Huang et al, 2018). Considering the role of these proteins in melanoma invasion and the observation that RAB27A and RAB38 expression were associated with shorter OS of melanoma patients, these GTPases were proposed as drivers of melanoma metastasis (Huang et al, 2018;Guo et al, 2019) (Table 1).…”

“…This effect is also mediated by the MITF, the master regulator of melanocyte development, which leads to an increase in RAB38 expression in metastatic melanoma (Huang et al, 2018). Considering the role of these proteins in melanoma invasion and the observation that RAB27A and RAB38 expression were associated with shorter OS of melanoma patients, these GTPases were proposed as drivers of melanoma metastasis (Huang et al, 2018;Guo et al, 2019) (Table 1). Similarly, RAB22A expression is also a prognostic factor of poor outcome in melanoma patients, showing an increased expression in primary melanomas compared with benign nevi (Su et al, 2016;Zhou et al, 2017).…”

“…Several studies have reported the dysregulation of Rab proteins in melanoma, although few have yet elucidated the molecular networks involved, which could be important to identify new potential therapeutic targets (Huang et al, 2018). It is widely accepted that exosomes are mediators of cell-cell communication and the ones derived from tumor cells can carry factors that induce malignant transformation (Maia et al, 2018).…”

“…et al, 2017;Liu et al, 2019;Wen et al, 2017;Huang et al, 2018). Therefore, the normal functions of small GTPases can be subverted by melanoma cells to spread and invade, leading to metastasis (Wong and Ribas, 2016;Huang et al, 2018). As such, melanoma, which is one of the most invasive types of cancer, is a suitable and useful model to explore the mechanisms underlying the roles of Ras superfamily members in cancer aggressiveness.…”