Background
Colorectal cancer is one of the most common cancers in the world, and is associated with a high death rate [
1]. Moreover, an apparent increase in the incidence of this cancer has been reported in many Asian countries [
2]. In spite of major advances in screening and treatment, the overall survival rates are still low in patients diagnosed in late stages of the disease. Distant metastasis causes most of the cancer-related morbidity and mortality after initial treatment. However, targeted treatment, such as anti-epidermal growth factor receptor (EGFR) and anti-vascular endothelial growth factor (VEGF) therapies, have had a relatively minor effect on the survival of metastatic colorectal cancer patients [
3]. Unraveling the mechanism of tumor progression and further discovery of novel prognostic markers for prediction and treatment evaluation is urgently required.
RAB GTPases, a large family of Ras small GTPases, play crucial roles in normal human physiology by regulating membrane identity and vesicle trafficking, including budding, sorting, uncoating, motility, tethering, and fusion [
4]. Loss of RAB GTPase activity is known to cause many inherited disorders [
5]. Neurons, which are connected through synapses, are vulnerable to dysfunction of RAB-regulated membrane trafficking. RAB GTPases have been reported to be involved in the pathogenesis of Parkinson’s disease and Huntington’s disease [
6‐
8]. RABs also have a role in the pathogenesis of type II diabetes through regulation of glucose transporter GLUT4 translocation [
9]. In addition, RABs also participate in phagocytosis of many pathogens, such as intracellular bacteria and viruses, and are crucial mediators of the innate immune response against infection [
10,
11].
Although the role of the Ras proto-oncogene in tumorigenesis has been well studied, the importance of RAB GTPases in cancer remains largely unknown. Among all the RABs, endocytic RABs, such as RAB5, RAB21, and RAB25, are the most extensively studied. The well-characterized example is RAB25, which modulates the movement of integrin-recycling vesicles [
12]. Several lines of evidence have indicated that RAB25 has a large impact on epithelial cell transformation, tumor motility and the invasive ability of several epithelial cancers [
13‐
15]. Overexpression of RAB25 is associated with aggressive behavior in breast cancer and ovarian cancer [
16,
17]. However, an opposite effect has also been reported, in which RAB25 acts as a tumor suppressor in colon cancer [
14]. RAB5, which is essential for the fusion of early endosomes, is another intensively studied endocytic RAB in cancer. RAB5 regulates cell survival and migration through caspase-8-mediated signal transduction [
18]. However, several studies have noted that RAB5 expression appears to be decreased during cell migration [
19‐
21]. Both overexpression and downregulation of RAB5 have been reported in different cancers [
22,
23].
Ras-related protein 3C (RAB3C), a secretory RAB, participates in the modulation of secretory vesicle exocytosis [
5]. The secretory RABs, which include RAB3, RAB26, RAB27, and RAB37, have effects on carcinogenesis similar to those of endocytic RABs. Recent studies have shown that RAB27A and RAB27B are associated with invasiveness and metastasis in cancer [
24,
25]. However, there has been far less research on the correlation between cancer and RAB3. RAB3 consists of 4 isoforms, RAB3A, RAB3B, RAB3C, and RAB3D. The estimated protein expression from the Genecard website indicates that RAB3A and RAB3B are predominantly expressed in neural systems and neuroendocrine cells, whereas RAB3D is primarily present in nonneural tissues [
26‐
28]. RAB3C is normally expressed in peripheral blood mononuclear cells and platelets, and in nervous system, colon, ovary and seminal vesicles. However, the function of RAB3C is relatively unclear, and an association between RAB3C and cancer has yet to be determined. In addition, in recent years, increased attention has been paid to the role of RAB-regulated exosome secretion in the progression of various cancers, including colon cancer [
29,
30]. Therefore, in this study, we investigated the role of RAB3C in colon carcinogenesis. Our immunohistochemistry analysis results showed that high RAB3C expression was significantly correlated with poor prognosis and more frequent distant metastasis in clinical colorectal cancer patients. Increases in migration and invasion ability in vitro and the metastasis-promoting ability in vivo were found after RAB3C overexpression in colon cancer cells. The dose-dependent decrease in the migration-enhancing ability of RAB3C-overexpressing cell-conditioned medium after IL-6 blockade further confirmed the results of both RNA microarray and proteomics analyses, which showed that the IL-6-STAT3 signaling axis is the top ranking activated pathway. Collectively, RAB3C upregulation facilitates colorectal cancer metastasis by promoting IL-6 secretion and recruiting members of the STAT3-related pathway. Therefore, targeting the RAB3C-IL-6-STAT3 axis by using therapeutics, such as Ruxolitinib, might be a useful strategy to combat metastatic colon cancers.
Methods
Patients
In total, 215 patients diagnosed with colorectal adenocarcinoma at the Taipei Municipal Wan Fang Hospital of Taiwan from 1998 to 2005 were included in this study. Patients who received preoperative chemotherapy or radiation therapy or who received incomplete surgical resection were excluded. All cases were staged according to the 7th version of the cancer staging manual of the American Joint Committee on Cancer, and the histological cancer type was classified according to the World Health Organization classification. Follow-up data were available in all cases, and the last clinical follow-up time was January 2011. Overall survival (OS) and disease-free survival (DFS) were defined as the interval from surgery to death from any cause and recurrence or distant metastasis or death, respectively.
Tissue microarray construction and immunohistochemistry staining
Paraffin-embedded tissues used to generate tissue microarrays were collected from Taipei Medical University Hospital with IRB approval (TMU-IRB 99049). Written informed consent was obtained from each patient included in the study. Three representative 1-mm-diameter cores from each tumor taken from the formalin-fixed paraffin-embedded tissues were selected on the basis of morphology typical of the diagnosis. Assessable cores were obtained in a total of 215 cases. Moreover, paired normal mucosal tissues were also obtained in 62 of the 215 cases. The histopathological diagnoses of all samples were reviewed and confirmed by two pathologists via hematoxylin- and eosin-stained slides. Immunohistochemistry staining was performed on serial 5-μm-thick tissue sections cut from the tissue microarray (TMA) by using an automated immunostainer (Ventana Discovery XT autostainer, Ventana Medical Systems, Tucson, AZ). Briefly, sections were dewaxed in a 60 °C oven, deparaffinized in xylene, and rehydrated in graded alcohol. Antigens were retrieved by heat-induced antigen retrieval for 30 min with TRIS-EDTA buffer. Slides were stained with a polyclonal rabbit anti-human RAB3A antibody (15029–1-AP, 1:200; Proteintech, Chicago, USA), a polyclonal rabbit anti-human RAB3B antibody (GTX104360, 1:100; GeneTex, Taipei, Taiwan), a polyclonal rabbit anti-human RAB3C antibody (GTX108610, 1:500; GeneTex, Taipei, Taiwan), a polyclonal rabbit anti-human RAB3D antibody (12320–1-AP, 1:50; Proteintech, Chicago, USA), a polyclonal rabbit anti-human RAB26 antibody (GTX118872, 1:100; GeneTex, Taipei, Taiwan), a polyclonal rabbit anti-human RAB27A antibody (GTX109180, 1:750; GeneTex, Taipei, Taiwan), a polyclonal rabbit anti-human RAB27B antibody (13412–1-AP, 1:200; Proteintech, Chicago, USA), and a polyclonal rabbit anti-human RAB37 antibody (13051–1-AP, 1:100; Proteintech, Chicago, USA). The sections were subsequently counterstained with hematoxylin, dehydrated, and mounted.
TMA immunohistochemistry interpretation
The IHC staining assessment was independently conducted by 2 pathologists who were blinded to patient outcome. Only cytoplasmic expression of tumor cells in the cores were evaluated. Both the immunoreactivity intensity and the percentage were recorded. The intensity of staining was scored using a four-tier scale and defined as follows: 0, no staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining. The extent of staining was scored by the percentage of positive cells (0–100%). The final IHC scores (0–300) were obtained by multiplying the staining intensity score by the percentage of positive cells. All cases were divided into two groups according to the final IHC scores. High IHC expression level was defined as a score greater than or equal to 150, and a score less than 150 was defined as low expression.
Cell culture
Eight human colon cancer cell lines of which three cell lines (CX-1, DLD-1, H3347) were maintained in RPMI 1640 medium and two cell lines (HCT116 and HT-29) were maintained in McCoy’s 5A modified medium (Sigma, St. Louis, MO, USA). Mediums were all supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), penicillin (100 unit/ml), and streptomycin (100 μg/ml). Cells were incubated in 95% air, 5% CO2 humidified atmosphere at 37 °C. SW48, SW480, and SW620 cells were cultured in Leibovitz L-15 medium (Sigma, St. Louis, MO, USA) and incubated in CO2 free incubator.
Western blot analysis
The cells were lysed at 4 °C in RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts (30 μg) of protein were separated electrophoretically using SDS-polyacrylamide gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). After being blocked with 5% non-fat milk, the membrane was incubated with specific antibodies (RAB3C: GTX108610, 1:5000, GeneTex, Taipei, Taiwan; RAB27A: GTX109180, 1:5000, GeneTex, Taipei, Taiwan; RAB3B: GTX108610, 1:5000; GeneTex, Taipei, Taiwan; RAB26: GTX118872, 1:5000; GeneTex, Taipei, Taiwan; IL-6: GTX110527, 1:1000, GeneTex, Taipei, Taiwan; STAT3: #4904, 1:1000, Cell Signaling, USA; phospho-STAT3: #9145, 1:1000, Cell Signaling, USA) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The blots were visualized using an ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).
Virus production and Infection
Full length RAB3C cDNAs were amplified from the MGC gene bank (Open Biosystem Inc., from Dr. Michael Hsiao’s library) by using PCR. The cDNAs were first cloned into a pENTR1A vector (Gateway pENTR 1A Dual Selection Vector), then subcloned into pLenti6.3/V5-DEST. Target cells were seeded at the appropriate density in a 6-well dish 24 h prior to infection. On the day of infection, the growth medium was removed, and 1500 μl of medium containing lentivirus and polybrene (final concentration 8 μg/ml) was added to each well of the 6-well plate. The plate was then centrifuged for 1 h at 37 °C and 1200 g. Subsequently, the plate was incubated for 24 h. After incubation, the medium was removed, and fresh medium containing blasticidin was added. Then, 72 h after infection, the cells were further split, and the selection was continued until all of the control cells were dead.
Migration and invasion assay
Migration and invasion abilities of cells were evaluated by transwell assay (Millipore, Bedford, MA, USA). For invasion assay, transwells were pre-coated with 35 μl of 3X diluted matrix matrigel (Bd Biosciences Pharmingen, San Diego, CA, USA) for 30 min. The upper chamber of the device were added with 2 × 105 cells in serum-free culture medium, and the lower chamber was filled with 10% FBS culture medium. After indicated hours of incubation, the remaining cells on the upper surface of the filter were carefully removed with a cotton swab. The filter was then fixed, stained and photographed. Migrated or invaded cells were quantified by counting the cells in three random fields per filter.
Animal study
All animal experiments were conducted in accordance with a protocol approved by the Academia Sinica Institutional Animal Care and Utilization Committee. Age-matched male NSG mice (6–8 weeks of age) were used. To evaluate metastasis status, 1 × 106 cells were resuspended in 0.1 ml of PBS and injected into the lateral tail vein (n = 6). Metastatic lung nodules were counted and were further confirmed via H&E staining under a microscope.
cDNA microarray and data analysis
Total RNA extracted from cells with a A260/280 ratio greater than 1.9 was used in the Affymetrix cDNA microarray analysis. In the analysis, hybridization was performed with Affymetrix human U133 2.0 plus arrays, and the chips were scanned with an Affymetrix GeneChip scanner 3000. Then, Affymetrix DAT files were processed by an Affymetrix Gene Chip Operating System (GCOS) to generate CEL files. The raw intensities in the CEL files were normalized by robust multi-chip analysis, and a fold-change analysis was performed using GeneSpring GX11 (Agilent Technologies).
Proteomics
A non-labeling quantification method was used to analyze our proteomic data. Briefly, 20 μg of protein from each sample was loaded on SDS-PAGE gels for separation. After electrophoresis, the protein was detected with Coomassie blue staining. Each lane of the SDS-PAGE gels was cut into 10 pieces, with a similar amount of protein in each piece. Then, every piece was processed by in-gel digestion with trypsin to produce a large number of peptide fragments. These fragments were detected and measured by LTQ-FT (Thermo) at the proteomics core facility at the Genomics Research Center, Academia Sinica. Data from 10 pieces of the same original sample were combined, and the protein expression was calculated by using MaxQuant (version: 1.3.0.5) and analyzed with Perseus (version: 1.3.0.4).
In silico analysis
Gene expression levels were normalized as log2 values in GeneSpring software (Agilent Technologies, Palo Alto, CA, USA). Genes that were up or downregulated with a greater than 2.0-fold change in the RAB3C overexpression group compared with the vector control group were collected. We further performed computational simulation by using Ingenuity Pathway Analysis (IPA; QIAGEN, Valencia, CA, USA) online tools to predict potential upstream regulators and canonical pathways. Pathway analysis was performed according to the genes and proteins with over 2.0-fold change in expression and an activation z-score of over 2.0 compared with vector control cells identified from the microarray and proteomics data, respectively.
Statistical analysis
Statistical analysis was performed with SPSS 20 software (SPSS, Chicago, Illinois, USA). A paired t-test was performed to compare the RAB3C IHC expression in cancer tissue and the corresponding normal mucosal tissue. The associations between clinicopathological categorical variables and RAB3C IHC expression were assessed by Pearson’s chi-square test. Survival rates were analyzed by using the Kaplan-Meier method and were compared with a log-rank test. Univariate and multivariate analysis were performed using Cox proportional hazards regression analysis without and with an adjustment for RAB3C IHC expression level and various clinicopathological parameters. For all clinical analyses, a P value of <0.05 was considered statistically significant. Student’s t-test was also used to compare the results of migration and invasion assays and in vivo metastasis experiments. The P values with the following levels were considered significant: *P < 0.05, **P < 0.01, and ***P < 0.001.
Discussion
In this study, the high expression of RAB3C in colorectal cancer tissue compared with that in normal colonic mucosal tissue provided strong evidence allowing us to determine its value as a prognostic indicator. Survival analyses showed that patients with high RAB3C expression had poor overall and disease-free survival, and RAB3C overexpression remained an independent prognostic factor in multivariate analyses. Moreover, high RAB3C expression was significantly correlated with distant metastasis. RAB3C overexpression was also confirmed to increase the migration and invasion ability of colon cancer cells and the number of metastatic nodules in animal models. Knowledge-based pathway analysis using RNA microarray and proteomics data analysis revealed that the IL-6 pathway is the major signaling pathway involved in RAB3C’s effects. The gradient decrease in the migration ability induced by RAB3C-overexpressing cell-conditioned medium by blocking of IL-6 further indicated that promotion of metastasis by RAB3C depends on IL-6 secretion. Together, these results indicated that the RAB3C protein plays a critical role in tumor progression, invasion, and metastasis through IL-6 exocytosis.
The majority of research related to RAB3 has focused on its normal physiological functions, whereas relatively little is known about the role of RAB3 in tumorigenesis. Among 4 highly homologous isoforms of RAB3, their subcellular targets and functional roles have been proposed to be distinct because of differences in their N- and C-terminal domains [
31,
32]. RAB3B, a key exocytosis regulator in anterior pituitary cells, has been demonstrated by immunohistochemistry staining to be overexpressed in pituitary adenoma [
33,
34]. RAB3D, which is predominantly expressed in non-neuronal cells such as adipocytes and various exocrine glands, has recently been studied in breast cancer. However, there was no correlation between tumor progression and the presence of endogenous RAB3D mRNA and protein [
24]. The metastasis-promoting ability of RAB3C in colorectal cancer in the present study underscored the importance of conducting more research to elucidate whether other RAB3 isoforms and other exocytic RABs also participate in and coordinately regulate exocytosis, thereby leading to tumor metastasis.
RAB3 has been found to regulate the final steps of exocytosis and function as a gate-keeper of late stage exocytosis [
35]. Exocytosis is a critical factor in the adaptation of cancer cells to the challenging environment encountered during invasion and metastasis. Cancer cell exocytosis plays an important role in liberating growth factors into the microenvironment, thus facilitating invasive the growth of tumors. The importance of RABs in this process has been illustrated by two recent studies of RAB27 in breast cancer. Overexpression of RAB27A has been shown to enhance tumor invasion and metastasis in breast cancer cell lines through secretion of insulin-like factor-II (IGF-II), which in turn modulates many important tumor progression markers including p16, vascular endothelial growth factor, cathepsin D, cyclin D1, matrix metalloproteinase-9, and urokinase-type plasminogen activator [
25]. In another study, heat-shock protein 90α has been identified in RAB27B-secretory vesicles as a key pro-invasive growth regulator inducing activation of matrix metalloproteinase-2 in breast cancer [
24]. Moreover, this study has also revealed a correlation between RAB27B and poor differentiation and lymph node metastasis in ER-positive breast cancer.
Our research is the first study focused on the role and the function of RAB3C in cancer. In the present study, we found that IL-6 secretion is the major mechanism by which RAB3C induces cancer metastasis. IL-6 has been reported to induce tumor progression, especially metastasis, in various cancer types and is also considered to be a potential therapeutic target [
36,
37]. In colon cancer, IL-6 participates in almost every step of cancer progression, including tumor initiation, proliferation, migration, and angiogenesis [
38], and IL-6 expression has been confirmed to be correlated with poor prognosis [
39]. IL-6 is generally known to be secreted by tumor-associated fibroblasts and to create an environmental niche for cancer progression [
38,
40]. However, increasing evidence shows that tumor cell-secreted IL-6 also promotes tumorigenesis through autocrine regulation [
41,
42]. In our study, we found that IL-6 secreted by colon cancer cells modulates tumor metastasis. Our study is the first to reveal the relationship between exocytic RABs and cytokine secretion, and it further solidifies the role of RAB-regulated IL-6 autocrine signaling in cancer progression.
In addition, secretory RABs control exosome secretion, thus facilitating angiogenesis, degradation of the extracellular matrix, and creation of an immune-privileged environment for cancer cells [
43,
44]. Cancer progression markers, including molecules related to metastasis processes and signaling transduction and some lipid raft-associated proteins, have been isolated from metastatic colon cancer-derived exosomes [
45]. In addition, the level of circulating exosomes has also been reported to be an indicator of colon cancer prognosis [
30]. Exosomes also affect chemoresistance and chemosensitivity by modulating drug efflux mechanisms against cytotoxic drugs such as cisplatin and microtubule stability targeted by drugs such as taxanes [
46,
47]. The strong effects of RAB3C expression on disease-free survival and tumor recurrence in the present study may be attributed to treatment resistance modulated by RAB3C. However, whether and how RAB3C-regulated exocytosis has a direct effect on chemoresistance needs further exploration. Furthermore, recent research on blocking exosome liberation by interfering with exocytic RABs also provided new insights in studying chemoresistance mechanisms [
44].
In conclusion, increased RAB3C expression is correlated with poor prognosis and distant metastasis in colorectal cancer patients and regulates exocytosis and IL-6 secretion. Moreover, its further activation of the JAK2-STAT3 signaling pathway may be essential for tumor invasiveness and metastasis. Our study not only suggests a new direction for studies focused on deciphering the relationship between exocytic RABs and cancer progression but also reveals that the RAB3C-IL6-STAT3 axis may serve as a target for prognostic prediction and future therapeutic intervention with drugs such as Ruxolitinib.