Overexpression of RACK1 Predicts Poor Prognosis in Melanoma

Melanoma is a highly malignant skin cancer with limited treatment options, the mechanism of the occurrence and development of melanoma is still unclear till now. Receptor for activated C kinase 1 (RACK1) is a scaffolding protein that mediates multiple signaling pathways; it interconnects distinct signaling pathways to control essential cellular processes. RACK1 was reported as an oncogene in human tumorigenesis, but little is known about its role in melanoma. This study aimed to investigate the expression of RACK1 in patients with melanoma and to reveal its possible functions in melanoma cells. The expression profiles of RACK1 detected in tumor tissues from melanoma patients showed that RACK1 was higher in tumor tissues, and its expression level was well associated with the clinical progression of melanoma (TNM stage, P=0.009). Furthermore, RNA interfering (RNAi) knockdown of RACK1 could efficiently suppress the proliferation, migration and invasion of A375 and A875 cells and promote their apoptosis. Taken together, these results suggest that RACK1 may be a poor prognostic factor in human melanoma, and it may be a new therapeutic target for melanoma treatment.


Introduction
Melanoma is a malignant skin cancer with aggressive forms, originating from pigmentcontaining cells known as melanocytes in the basal layer of the epidermis, being responsible for high mortality of skin cancers [1,2]. The main cause of melanoma is due to the DNA damage resulting from exposure to the sun or other sources of ultraviolet light in people with low skin pigmentation, genetic factors also plays a role in melanoma occurrence. In recent years, many studies have been reported on the occurrence and development of melanoma, but its molecular pathological mechanism is still unclear [3][4][5]. In current, due to the high malignancy of melanoma, there are few treatment options. Although target therapy strategies have been widely used in the treatment of many human cancers, its application in melanoma still has many limitations, many patients with advanced melanoma cannot achieve tumor regression [6,7]. Therefore, it is urgent to clarify the molecular mechanism of melanoma development and to develop novel gene targets for its clinical diagnosis and therapy [8].
Receptor for activated C kinase 1 (RACK1) was first cloned from the cDNA library of a chicken genomic DNA clusters and human B-lymphoblastoid cell [9], it is a 36 kDa cytosolic protein which adopts a highly conserved 7-bladed β-propeller structure [10], significant homology to the G protein β-subunit [11], and was originally identified as an intracellular protein receptor for protein kinase C. As a member of the tryptophan-aspartate (WD) repeat protein family, RACK1 serves as a scaffold protein for many kinases and receptors, and thus plays a pivotal role in shuttling proteins in intercellular space, anchoring Ivyspring International Publisher proteins at particular locations, participating in transcriptional/translational events, and stabilizing protein activity. Therein, RACK1 acts as a mediator in multiple signaling pathways, which interconnects distinct signaling pathways to control essential cellular processes, i.e. cell growth, proliferation, migration, adhesion, differentiation, signal transduction, and immune responses [12,13]. It has been reported that RACK1 plays as an oncogene in various tumor types , such as prostate cancer [14], ovarian cancer [15], esophageal squamous cell carcinoma [16], glioma [17], nasopharyngeal carcinoma [18], oral squamous-cell carcinoma (OSCC) [19], breast cancer [20], esophageal carcinoma [21], acute promyelocytic leukemia [22], myeloma [23], colorectal carcinoma [24,25], non-small cell lung cancer [26,27], hepatocellular carcinoma (HCC) [28,29], and etc.
However, the role of RACK1 in melanoma has not yet been reported. The present work demonstrates that RACK1 is highly expressed in melanoma tissues and its expression level is well correlated with the clinical progression of melanoma. Moreover, depletion of endogenous RACK1 lead to decreased proliferation, migration and increased apoptosis of melanoma cells. Thus, our results indicate that RACK1 contributes to the tumorigenesis and progression of melanoma.

Clinical patient samples
The tissue microarray was obtained from Xi'an Alenabio.com (China), including 67 cases of malignant melanoma tissues, 23 cases of normal skin tissues and 17 cases of benign tissues. 21 cases of fresh human acral melanoma tumor tissues and peripheral non-tumor skin tissues were collected from Affiliated Hospital of Nantong University (Nantong, Jiangsu, China). The study was approved by the Ethics Committee of Affiliated Hospital of Nantong University.

Western blot analysis
Western blot was performed according to the standard procedures. Briefly, tissues and cell lysates were extracted using RIPA buffer (Promega, USA), the protein concentrations were determined by a BCA™ Protein Assay Kit (Pierce, USA). Proteins were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto PVDF membranes (Merk-Millipore, USA). The membranes were first incubated with indicated primary antibodies overnight (4 °C), and then followed by HRP-conjugated secondary antibodies for 2 h at room temperature. Blots were detected with ECL Western Blotting Substrate (Promega, USA) and quantized by Image J software (NIH, USA), β-actin was used as an internal control. The following antibodies were included: a rabbit anti-human RACK1 antibody (Abcam, USA, 1:500 dilution), a mouse anti-human β-actin antibody (Abcam, USA, 1:1,000 dilution), a horseradish peroxidase (HRP)-conjugated IgG (Abcam, USA, 1:2,000 dilution).

Immunocytochemistry staining
Briefly, 2×10 5 cells were plated into 24-well plates with a round slide in each well and cultured at 37 °C with 5% CO 2 overnight. After treated for 48 h as described above, the cells were fixed with 4% paraformaldehyde for 30 min at 4 °C, rinsed with PBS, and incubated with 0.5% Triton X-100 for 10 min at room temperature, incubated with the block solution for 30 min at 4 °C, incubated with a rabbit anti-human RACK1 antibody (1:50 dilution) (Abcam, USA) at 4 °C overnight, rinsed with PBS, incubated with IgG-TRITC (1:1,000 dilution) (Abcam, USA) for 2 h at room temperature, rinsed with PBS again and followed by Hoechst 33258 staining (Sigma-Aldrich, USA) for 10 min. Finally, the cells were mounted and observed under an immunofluorescence microscopy protect from light.

Cell proliferation assay
Cell proliferation was detected with MTT assay. In brief, cells were treated differently as indicated above. After been treated for 24 h, 48 h and 72 h, the cells were incubated with 10 µl MTT (Promega, USA) at 37 °C for 4 h protected from light, followed by 150 µl DMSO incubation at 37 °C for 10 min. Finally, the fluorescence intensity of each well was measured using a microplate reader (Bio-Rad, USA) at spectrometric absorbance of 490 nm.

Wound-healing assay
The migration of cells was assessed by wound-healing assay. In brief, 1×10 5 cells were plated onto 6-well plates each well and treated as indicated above. 24 h after treatment, a wound was created by scraping the cell monolayer with 1 ml a pipette tip manually and the medium were replaced with fresh DMEM containing 1% FBS. At 0, 24, or 48 h, cells were washed with PBS, and images were captured under the microscope. Cell migration was estimated by wound healing rate (%) = [1-(wound area at Tt/wound area at T0)]×100.

Transwell assay
Migration assay was performed using Transwell cell culture systems (Corning, USA). Briefly, 2×10 5 cells per well were plated onto 24-well plates and cultured for 24 h, after treated for 48 h as described above, cells were suspended in DMEM at the density of 1×10 6 cells/ml and added to the upper chamber (100 μl/each) while 600 μl medium (conditioned medium: DMEM containing 10% FBS=1:1) was added into the lower chamber, the conditioned medium was the medium from normal cell supernatant cultured for 24 h. After being incubated at 37 °C for 24 h, the cells on the top membrane surface of upper chamber were removed and the infiltrating cells on the bottom surface were fixed in 10% formaldehyde for 30 s, stained with 5% crystal violet for 30 min and observed under a microscope. Cell numbers were counted from five random visions.

Matrigel-based transwell assay
Cell invasion abilities were detected by matrigel-based transwell assay. Briefly, 2×10 5 cells per well were plated onto 24-well plates and cultured for 24 h, after treated for 48 h as described above, cells were suspended in DMEM at the density of 1×10 6 cells/ml. Before treatments, 100 μl matrigel (BD Biosciences, USA) (5 mg/ml) was added into upper chamber and incubated with 300 μl DMEM for 15 min at room temperature. Cells were suspended in DMEM at the density of 1×10 6 cells/ml and added to the upper chamber (100 μl/each) while 600 μl medium was added into the lower chamber. After incubation at 37 °C for 24 h, the cells on the top membrane surface of upper chamber were carefully removed and cells on the bottom surface were fixed in 10% formaldehyde for 30 s, stained with 5% crystal violet for 30 min and observed under a microscope. Infiltrating cell numbers were counted from five random visions.

Cell apoptosis assay
Cell apoptotic rate was detected by Annexin V-FITC/Propidium Iodide (PI) followed by flow cytometry (FCM). In brief, 3×10 5 cells were plated onto 6-well plates per well and incubated for 24 h. Cells were collected after being treated for 48 h, centrifuged at 1,000 rpm for 5 min, washed with PBS, and then suspended in 195 μl 1×Annexin V-FITC binding buffer and 5 μl Annexin V-FITC (Sigma-Aldrich, USA). After 10 min incubation at room temperature protected from light, the cells were centrifuged at 1,000 rpm for 5 min, re-suspended in 190 μl 1×Annexin V-FITC binding buffer, and then incubated with 10 μl PI protected from light. Finally, cells were analyzed using Flow Cytometer (BD Biosciences, USA).

Statistical analysis
Statistical analyses were performed using SPSS20.0 software. All data was shown as mean ± SD. Paired t-test was used to analyze the difference of RACK1 expression between paired tissues. Significance between multiple groups was evaluated by one-way analysis of variance (ANOVA) followed by a Dunnett post hoc test. For immunohistochemical staining, Kruskal Wallis test was used to analyze the difference of RACK1 expression among three different tissues, Kruskal-Wallis one-way ANOVA followed by all pairwise method was employed to further explore the difference of RACK1 expression between groups. Kendall correlation and Spearman's rank correlation were used to analyze the correlation between RACK1 expression and clinicopathological factors. P values are based on a two-tailed statistical analysis. Statistical significance was determined at the level of P< 0.05. All assays were performed in triplicate times independently.

RACK1 was highly expressed in cell lines and melanoma patients
To understand whether RACK1 was related to the tumorigenesis of melanoma, We first examined the expression of RACK1 in melanoma cell lines (A375 and A875) and the normal skin cell line HaCaT, both mRNA and protein levels of RACK1 in A375 and A875 cells were higher than that in HaCaT (P<0.05) ( Fig. 1A and B). The mRNA and protein levels of RACK1 were also checked in tumor tissues from patients with melanoma and the adjacent non-tumor skin tissues, RT-qPCR and Western blot showed that both mRNA and protein levels of RACK1 was higher in melanoma tissues compared with their peripheral non-tumor skin tissues (P<0.05) (Fig. 1C and D).

RACK1 expression was correlated with clinical characteristics in melanoma patients
The expression of RACK1 was explored by immunohistochemical staining in total of 107 tissue samples which including 67 cases of malignant melanoma tissues, 23 cases of normal skin tissues and 17 cases of benign tissues. RACK1 was significantly high expressed in malignant melanoma tissues, compared with that in normal skin tissues and benign tissues ( Table 1, Fig. 1E-F). More specifically, 62.69% (42/67) of the melanoma tissues showed strong or moderate staining (+++/++) and 37.31% (25/67) showed weak staining (-/+) of RACK-1, while most of control normal skin and benign tissues showed weak staining (-/+), 91.30% (21/23) and 100% (17/17), respectively. Moreover, RACK1 expression was strongly related with the TNM stage of melanoma (P=0.009) (Fig. 1F and Table 2). These results confirmed that RACK is upregulated in melanoma tissues and suggested that RACK1 might contribute to the tumorigenesis and progression of melanoma.

RACK1 expression was downregulated by siRNA in melanoma cells
To further examine the role of RACK1 in melanoma cells, we designed siRNA (si-RACK1) to downregulate endogenous RACK1 levels in A375 and A875 cells. The interference and knockdown efficiency was confirmed by RT-qPCR ( Fig. 2A and B), Western blot analysis (Fig. 2C and D) and fluorescence microscopy (Fig. 2E). All the above data proved that compared with si-NC treated or untreated cells, RACK1 expression was significantly inhibited by si-RACK1 in both A375 and A875 cells (Fig. 2).

The growth of melanoma cells was inhibited by RACK1 downregulation
MTT assay was utilized in order to investigate the inhibitory effects of RACK1 downregulation on the growth of melanoma cells. The result showed that compared with si-NC treated or untreated cells, si-RACK1 clearly inhibited the growth of A375 and A875 cells at 48 h and 72 h (P<0.05) (Fig. 3A and B).

The migration and invasion of melanoma cells was inhibited by RACK1 downregulation
Both wound-healing and transwell assay were used to observe the effect of RACK1 downregulation on melanoma cell migration. Matrigel-based transwell assay was utilized to evaluate the influence of RACK1 depletion on melanoma cell invasion. Compared with that of si-NC treated or untreated cells, si-RACK1 treatment significantly decreased both migration (P<0.05, Fig. 3C and D) and invasion (P<0.05, Fig. 3E) of A375 and A875 cells.

The apoptosis of melanoma cells was induced by RACK1 downregulation
Annexin-V-FITC/PI followed by FCM was performed to evaluate the effect of RACK1 downregulation on melanoma cell apoptosis. Compared with that of si-NC treated or untreated cells, si-RACK1 treatment significantly increased apoptosis of A375 and A875 cells (P<0.05) (Fig. 4).

Discussion
Melanoma is a highly malignant tumor with limited treatment options [1]. Despite much research, the mechanisms of the occurrence and development of melanoma remains unclear. Surgery, radiotherapy and chemotherapy have long been the main methods for melanoma treatment, but these methods also have some problems, such as severe trauma, obvious side effects and low patient tolerance [31]. With the recent process in treatment, immune therapies and targeted therapies are two main treatment approaches for patients with advanced melanoma, but both still have limitations, and not all patients experience sustained responses [32]. RACK1 was originally indentified as a key anchor for protein kinase C [11], and has been shown to be involved in numerous different biological processes. In recent years, RACK1 has been reported as an oncogene in many human cancers [13]. But it is interesting to note that the role of RACK1 in different tumors is not the same and sometimes even the opposite. Cao et al. demonstrated that RACK1 was high expressed in breast cancer and was a prognostic factor that promoted breast carcinoma metastasis by interacting with RhoA and activating the RhoA/Rho kinase pathway [33]. RACK1 has also been found highly expressed in human HCC, which contributed to in vitro chemoresistance and in vivo tumor growth and survival of HCC [34]. In non-small-cell lung cancer, RACK1 served as an oncogene and silence of RACK1 resulted in inhibition of tumor growth and metastasis through the sonic hedgehog signaling pathway [35]. Highly expression of RACK1 was also found in prostate cancer [14], colorectal cancer [24,25] and nasopharyngeal carcinoma [36]. But in gastric cancer and pancreatic ductal adenocarcinoma, things have changed. Deng et al. demonstrated that RACK1 was decreased in gastric cancer patients and associated with tumor infiltration depth and poor differentiation, indicated that RACK1 suppressed gastric tumorigenesis [37]. The tumor suppressor role of RACK1 in gastric cancer was also supported by Chen et al, indicated that low expression of RACK1 was correlated with invasion and metastasis phenotype as well as 5-year survival in clinical cases, suggesting RACK1 decrease in gastric cancer links epigenetics to interleukin 8 (IL8) to promote tumor metastasis [38]. Additionally, low RACK1 expression was involved in pancreatic cancer growth and metastasis, and significantly correlated with metastasis, invasion of nerves as well as TNM stage; especially 3-year survival rate of pancreatic ductal adenocarcinoma patients with high RACK1 expression was significantly higher than those patients with low ones [39]. These studies have elucidated that the function of RACK1 is very complex and varies in different cancers. In this study, we reported that RACK1 was highly expressed in melanoma tissues compared with matched nontumorous skin tissues. The results were also supported by tissue microarray staining data: RACK1 expression in tumor tissues was significantly higher than that in benign lesions and normal skin tissues. Moreover, high levels of RACK1 in melanoma tissues were highly correlated with TNM stage (P=0.009), strongly predicted that RACK1 expression was associated with the progression of melanoma.
Evidences indicate that RACK1 participating in multiple cell functions. Silencing of RACK1 resulted in inhibition of tumor growth of non-small-cell lung cancer cells [35], induced cell apoptosis and inhibited cell proliferation of HCC and glioma cells [40,41]; RACK1 has also been found to be engaged in the regulation of cell migration and invasion, two important initial steps in cell metastasis, because it served as a molecular bridge linking the integrin effector focal adhesion kinase to the recruitment of a cAMP-degrading phosphodiesterase in the "direction-sensing" pathway [42], such phenomenon was confirmed in various tumor types, such as prostate cancer cells, epithelial ovarian cancer cells, esophageal squamous cell carcinoma cells, glioma cells, colorectal cancer cells, OSCC cells and nasopharyngeal carcinoma cells [14-17, 24, 41, 43, 44].
To understand what cellular processes are modulated by RACK1 in melanoma, we investigated the effect of RACK1 downregulation on the functions of A375 and A875 cells. In the present study, RACK1 knockdown resulted in inhibition of cell proliferation and promotion of cell apoptosis of A375 and A875 cells; in vitro study also showed that the migration and invasion of melanoma cells were inhibited. It is worth noting that, the significantly correlation of RACK1 overexpression with metastasis of melanoma patients has not been found in our study, which may be due to tumor heterogeneity and the limited samples used here, it is necessary to further study in a larger sample size.
In conclusion, the present work suggests that RACK1 may be a factor affecting the prognosis of melanoma, and the strategy of RNAi-mediated RACK1 silencing may be an effective method for melanoma treatment.