Cancer Letters

Cancer Letters

Volume 407, 28 October 2017, Pages 66-75
Cancer Letters

Original Article
P-REX1 amplification promotes progression of cutaneous melanoma via the PAK1/P38/MMP-2 pathway

https://doi.org/10.1016/j.canlet.2017.08.001Get rights and content

Highlights

  • PREX-1 gene amplification promotes cutaneous melanoma progression.

  • PAK1/P38/MMP-2 pathway activated by PREX-1 amplification.

  • Apoptosis inhibition caused by loss of P-REX1 was related to expression of survivin.

  • P-REX1 regulates cell migration/invasion via P-REX1/RAC1/PAK1/p38/MMP-2 pathway.

  • High PREX-1 expression was related to poorer disease outcome in stage III patients.

Abstract

P-REX1 (PIP3-dependent Rac exchange factor-1) is a guanine nucleotide exchange factor that activates Rac by catalyzing exchange of GDP for GTP bound to Rac. Aberrant up-regulation of P-REX1 expression has a role in metastasis however, copy number (CN) and function of P-REX1 in cutaneous melanoma are unclear. To explore the role of P-REX1 in melanoma, SNP 6.0 and Exon 1.0 ST microarrays were assessed. There was a higher CN (2.82-fold change) of P-REX1 in melanoma cells than in melanocytes, and P-REX1 expression was significantly correlated with P-REX1 CN. When P-REX1 was knocked down in cells by P-REX1 shRNA, proliferation, colony formation, 3D matrigel growth, and migration/invasiveness were inhibited. Loss of P-REX1 inhibited cell proliferation by inhibiting cyclin D1, blocking cell cycle, and increased cell apoptosis by reducing expression of the protein survivin. Knockdown of P-REX1 expression inhibited cell migration/invasiveness by disrupting P-REX1/RAC1/PAK1/p38/MMP-2 pathway. Assessment of patient tumors and disease outcome demonstrated lower distant metastasis-free survival among AJCC stage I/II/III patients with high P-REX1 expression compared to patients with low P-REX1 expression. These results suggest P-REX1 plays an important role in tumor progression and a potential theranostic target.

Introduction

Cutaneous melanoma is the most devastating form of skin cancer [1]. The incidence has increased rapidly in recent years in western countries [2], [3], [4], [5]. Five-year survival rate for patients with distant metastatic disease remains below 20% [6]. Gene mutations in cutaneous melanoma are infrequent and less than 35% in general other than BRAFV600E [7], [8], [9]. There is a need to identify new genomic aberrations, other than driver mutations, that contribute to melanoma progression. Copy numbers of specific genes have been shown to be related to abnormal signal transduction in epithelial-derived cancers and are significant factors in tumor progression [7]. A limited number of genomic amplification changes related to functional signal transduction pathways in cutaneous melanoma have been identified. High-throughput microarrays for genome-wide-DNA aberrations can be used to screen gene copy number (CN) amplifications, allowing for the discovery of new markers by moving from hypothesis-driven, targeted-research to unbiased screening of the whole genetic spectrum [10]. Chromosomal aberrations are critical events in tumorigenesis, and genomic regions harboring DNA gains/losses have been identified in many cancers, such as breast cancer and colorectal cancer [11]. DNA amplification that involves tumor-related genes can support tumor progression [12]. Genomic instability often plays an important role in the development, growth, invasiveness, and eventual metastasis of tumor cells.

Changes in the extent of whole-genome CN may predict aggressive features in primary melanomas [13]. Higher amplification of N-myc is correlated with very poor outcome in melanoma [14]. Amplification of the c-kit gene was found in certain subgroups of malignant melanoma at a low frequency [15]. GAB2 (GRB2-Associated Binding Protein 2) amplification was used to refine molecular classification of melanoma [16]. Changes of gene CN have been associated with response to treatment of drugs [17]. YAP1 is an evolutionarily conserved transcription cofactor of the Hippo pathway, and the Hippo signal pathway is affected by CN amplification of YAP1 [18]. The MAPK signaling pathway is activated by CN amplification of MEK1 and ERK1 genes in melanoma [19].

P-REX1 is a Rac-specific guanine nucleotide exchange factor (GEF) that specifically activates Rac by catalyzing exchange of GDP for GTP bound to Rac. Exchange is driven by PI3K-stimulated phosphatidylinositol [3], [4], [5]-trisphos¬phate production and the β-gamma subunits of the heterotrimeric-G proteins (Gβγ), both of which bind to P-REX1 [20].

P-REX1 is a key component of a positive feedback loop that drives oncogenic signaling. Aberrant up-regulation of P-REX1 expression plays important roles in epithelial cancer progression and metastasis [21]. P-REX1 is also overexpressed in estrogen receptor-positive and/or ErbB2-positive breast cancers, and increased expression of P-REX1 correlates with poor patient outcome in breast cancer [22]. P-REX1 mRNA levels were predictive of sensitivity to PI3K inhibition [23].

P-REX1 expression is elevated in human melanoma cell lines and tumor tissues [24] and plays an important role in melanoblast migration and metastasis in mice and humans [25]. However, the molecular mechanism for function of P-REX1 and DNA aberrations of P-REX1 in cutaneous melanoma are relatively unknown. It is unclear in melanoma which pathway(s) are affected by amplification of P-REX1 CN. In our recent study demonstrated that RAC1 expression and mutation was associated with melanoma progression [7], implicating the P-REX1 activated downstream pathway.

In the present study we have investigated, for the first time, the increased CN of P-REX1 in metastatic melanoma cells compared with melanocytes, the promotion of melanoma cell proliferation by regulating expression of survivin, and the migration and invasiveness of metastatic melanoma cells via the P-REX1/RAC1/PAK1/p38/MMP-2 pathway in vitro.

Section snippets

Cell culture

M219 and Wp-0614 melanoma cell lines were established from AJCC stage IV melanoma patients who received elective surgery at John Wayne Cancer Institute (JWCI) at Providence Saint John's Health Center (SJHC). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 100U/ml penicillin, and 100 μg/ml streptomycin, and were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Established cell lines were used at early passages.

Tumor specimens

Approval for the use of human tissues

P-REX1 CN and mRNA expression in melanoma

Previously we have identified DNA gains/losses in cutaneous melanoma which suggest these events are important in tumor progression [7], [36]. To assess DNA CN changes in cutaneous melanoma tissues during metastasis, we performed the genome-wide Human SNP Array 6.0 assay screening in 42 patients melanomas (AJCC stage III and IV). We observed that P-REX1 CN was amplified significantly in melanoma patients (Fig. 1A). To further explore the relation between DNA CN and mRNA expression, Human Exon

Discussion

Aberrant expression of cell functional genes in growth, invasion, and migration often are associated with cancer progression. CN variations involving genes and expression have not been well studied during melanoma metastasis progression. In this study, we analyzed P-REX1 CN, expression of P-REX1, function role of P-REX1 and clinical significance of P-REX1 in cutaneous melanoma. We have shown, for the first time, that CN of P-REX1 was increased in cutaneous melanomas and that CN of P-REX1 was

Acknowledgments

This work was supported by the National Institute of Health; National Cancer Institute [grant number 1R01CA167967-01A1 (D.H.)], the Adelson Medical Research Foundation (D.H.), China National Natural Science Foundation [81573454 (JW)], Beijing Natural Science Foundation (7172142) and CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-3-007). The authors would like to thank Mrs. Nousha Javanmardi for her invaluable editorial assistance.

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      However, the PREX1 depletion-induced antioxidant effect and inhibition of lipid accumulation were not merely mediated by the NF-κB signaling. Accumulating evidence has suggested that PREX1 is involved in the p38 mitogen-activated protein pinase (MAPK) signaling and phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, which can regulate oxidative stress or/and hepatic lipid metabolism (Dillon et al., 2015; N. Li et al., 2019; Liao et al., 2018; Matsuda et al., 2013; Wang et al., 2017). The involvement of the p38 MAPK and PI3K/AKT signaling pathway in PREX1 depletion-induced antioxidant effect and inhibition of lipid accumulation needs to be explored in our subsequent works.

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