The online version of this article (doi:10.1186/s12943-017-0687-7) contains supplementary material, which is available to authorized users.
RAB GTPases are important in the regulation of membrane trafficking and cell movement. Recently, exocytic RABs have received increasing attention in cancer research. However, the functional roles of exocytic RABs in colorectal carcinogenesis remain to be elucidated.
Immunohistochemistry analysis of a microarray containing 215 colorectal adenocarcinoma tissues was used to identify the association between exocytic RABs and patient prognosis. Complementary functional RAB3C overexpression and knockdown experiments were performed. The molecular mechanism of RAB3C in inducing colon cancer cell metastasis was determined.
High RAB3C expression in patients was found to be significantly associated with advanced pathological stage, distant metastasis and poor prognosis. Multivariate analyses showed that high RAB3C expression was an independent prognostic marker in overall (P = 0.001) and disease-free survival (P < 0.001). Furthermore, our experimental results showed an increase in the migration and invasion ability of RAB3C-overexpressing colon cancer cells and increased metastatic nodules in a mouse metastasis model. The effect of RAB3C-overexpressing cell-conditioned medium was found to significantly promote the migration ability of parental colon cancer cells, thus suggesting that the promotion of migration is exocytosis dependent. Upregulation of other exocytic RABs was also seen in RAB3C-overexpressing cells. Through microarray and proteomics analyses, increased production of multiple cytokines was observed in RAB3C-overexpressing cell lines, and the IL-6 pathway was the top pathway whose members exhibited gene expression changes after RAB3C overexpression, according to Ingenuity Pathway Analysis. Blocking IL-6 with IL-6 antibody treatment or IL-6 knockdown significantly inhibited the migration potential of RAB3C-overexpressing colon cancer cells. In addition, IL-6 was found to induce STAT3 phosphorylation in RAB3C-overexpressing colon cancer cells, thus promoting migration. Ruxolitinib, a JAK2 inhibitor, was found to significantly inhibit RAB3C-induced colon cancer cell migration.
Our study revealed that RAB3C overexpression promotes tumor metastasis and is associated with poor prognosis in colorectal cancer, through modulating the ability of cancer cells to release IL-6 through exocytosis and activate the JAK2-STAT3 signaling pathway. These results further suggest that inhibition of STAT3 phosphorylation in the RAB3C-IL-6-STAT3 axis by using Ruxolitinib may be a new therapeutic strategy to combat metastatic colon cancers.
Additional file 1: Table S1. Correlation of clinicopathological features of colorectal cancer patients and the RAB3C tumor expression. (DOCX 2201 kb)12943_2017_687_MOESM1_ESM.docx
Additional file 2: Figure S1. High RAB3C expression is an independent indicator in colorectal cancer patients. (A) the box plot shows that higher RAB3C expression was correlated with a poor overall survival rate in patients in the GSE17536, (n = 177) from the SurvExpress database (P = 0.015). (B) Heatmap indicates RABs family mRNA level correlated with grade and pathological stage in the clinicopathological analysis by the Oncomine online tool. (TIFF 32000 kb)12943_2017_687_MOESM2_ESM.tif
Additional file 3: Figure S2. RAB3C overexpression increases exocytosis of colon cancer cells and promotes metastasis through IL-6 secretion. (A) The significant effect of RAB3C overexpressing cell-conditioned medium on the migration ability of parental colon cancer cells indicate that the metastasis-promoting role of RAB3C is exocytosis dependent. (B) Relative IL-6 activity in conditioned medium of CX-1 cells and SW48 cells with or without the exogenous RAB3C gene. The data were the average of three independent experiments and are presented as the mean ± SEM. The significance of the difference was analyzed using the nonparametric Mann-Whitney U test. (TIFF 28902 kb)12943_2017_687_MOESM3_ESM.tif
Goldenring JR, Shen KR, Vaughan HD, Modlin IM. Identification of a small GTP-binding protein, Rab25, expressed in the gastrointestinal mucosa, kidney, and lung. J Biol Chem. 1993;268:18419–22. PubMed
Li Y, Meng X, Feng H, Zhang G, Liu C, Li P. Over-expression of the RAB5 gene in human lung adenocarcinoma cells with high metastatic potential. Chin Med Sci J. 1999;14:96–101. PubMed
Millar AL, Pavios NJ, Xu J, Zheng MH. Rab3D: a regulator of exocytosis in non-neuronal cells. Histol Histopathol. 2002;17:929–36. PubMed
Chung YC, Chaen YL, Hsu CP. Clinical significance of tissue expression of interleukin-6 in colorectal carcinoma. Anticancer Res. 2006;26:3905–11. PubMed
Nagasaki T, Hara M, Nakanishi H, Takahashi H, Sato M, Takeyama H. Interleukin-6 released by colon cancer-associated fibroblasts is critical for tumour angiogenesis: anti-interleukin-6 receptor antibody suppressed angiogenesis and inhibited tumour-stroma interaction. Br J Cancer. 2014;110:469–78. CrossRefPubMed
Sansone P, Storci G, Tavolari S, Guarnieri T, Giovannini C, Taffurelli M, Ceccarelli C, Santini D, Paterini P, Marcu KB, et al. IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland. J Clin Invest. 2007;117:3988–4002. CrossRefPubMedPubMedCentral
Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, Moita CF, Schauer K, Hume AN, Freitas RP, et al: Rab27a and Rab27b control different steps of the exosome secretion pathway . Nat Cell Biol 2010, 12 :19–30; sup pp 11-13.
Ji H, Greening DW, Barnes TW, Lim JW, Tauro BJ, Rai A, Xu R, Adda C, Mathivanan S, Zhao W, et al. Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components. Proteomics. 2013;13:1672–86. CrossRefPubMed
Shedden K, Xie XT, Chandaroy P, Chang YT, Rosania GR. Expulsion of small molecules in vesicles shed by cancer cells: association with gene expression and chemosensitivity profiles. Cancer Res. 2003;63:4331–7. PubMed
- Secretory RAB GTPase 3C modulates IL6-STAT3 pathway to promote colon cancer metastasis and is associated with poor prognosis
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