The online version of this article (doi:10.1186/1476-4598-11-62) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
VM conceived the study, made substantial contributions to the conception and design of the study, and drafted the manuscript. BG participated in the design of the study, generated rab31 overexpressing cell lines, isolated cell clones, carried out cell-based in vitro assays, participated in the characterization of antibodies and in the in vivo experiment, and has been substantially involved in drafting the manuscript. SSö generated rab31(-mutant) overexpressing cell lines and carried out cell-based in vitro assays. SSch produced and purified the rab31 immunogen and was involved in the characterization of antibodies by Western blot and microtiter plate-based assays. BS performed the experimental in vivo metastasis assay. TK participated in the characterization of antibodies and carried out the immunoassay experiments. TL and MK were involved in the design and coordination of the study, participated in the characterization of antibodies and its application in immunoassays and immunohistochemistry, and have been substantially involved in drafting the manuscript. AK supervised the in vivo experiment and has been involved in drafting and critically revising the manuscript. GB and MS participated in the design of the study and have been involved in drafting and critically revising the manuscript. All authors have read and approved the final version of the manuscript.
Rab proteins constitute a large family of monomeric GTP-binding proteins that regulate intracellular vesicle transport. Several Rab proteins, including rab31, have been shown to affect cancer progression and are related with prognosis in various types of cancer including breast cancer. Recently, the gene encoding rab31 was found to be overexpressed in estrogen receptor-positive breast cancer tissue. In a previous study we found a significant association of high rab31 mRNA expression with poor prognosis in node-negative breast cancer patients. In the present study, we aimed to investigate the impact of rab31 (over)-expression on important aspects of tumor progression in vitro and in vivo.
Breast cancer cells displaying low (MDA-MB-231) or no (CAMA-1) endogenous rab31 expression were stably transfected with a rab31 expression plasmid. Batch-transfected cells as well as selected cell clones, expressing different levels of rab31 protein, were analyzed with regard to proliferation, cell adhesion, the invasive capacity of tumor cells, and in vivo in a xenograft tumor model. Polyclonal antibodies directed to recombinantly expressed rab31 were generated and protein expression analyzed by immunohistochemistry, Western blot analysis, and a newly developed sensitive ELISA.
Elevated rab31 protein levels were associated with enhanced proliferation of breast cancer cells. Interestingly, weak to moderate overexpression of rab31 in cell lines with no detectable endogenous rab31 expression was already sufficient to elicit distinct effects on cell proliferation. By contrast, increased expression of rab31 in breast cancer cells led to reduced adhesion towards several extracellular matrix proteins and decreased invasive capacity through MatrigelTM. Again, the rab31-mediated effects on cell adhesion and invasion were dose-dependent. Finally, in a xenograft mouse model, we observed a significantly impaired metastatic dissemination of rab31 overexpressing MDA-MB-231 breast cancer cells to the lung.
Overexpression of rab31 in breast cancer cells leads to a switch from an invasive to a proliferative phenotype as indicated by an increased cell proliferation, reduced adhesion and invasion in vitro, and a reduced capacity to form lung metastases in vivo.
Additional file 1: Figure S1. Analysis of MDA-MB-231 cell transfectants for rab31 expression by immunofluorescence. After overnight growth on fibronectin-coated glass slides, the cell monolayers were fixed and permeabilized with PBS containing 4% (w/v) paraformaldehyde, 0.025% (w/v) saponin. After incubation, the rabbit antibody directed to rab31 was detected with Alexa488-labeled goat anti-rabbit IgG (Sigma-Aldrich) and fluorescence signal intensity visualized by confocal laser scanning microscopy (CLSM). Typical fluorescent images (lower panel) together with the corresponding differential interference contrast images (upper panel) are depicted. In immunocytochemistry, a often pronounced perinuclear staining is visible. There are no apparent morphological differences between vector control cells and cells overexpressing rab31. (PDF 541 KB)12943_2012_1042_MOESM1_ESM.pdf
Additional file 2: Figure S2. The constitutively active rab31-mutant Q64L, but not the inactive form rab31-ΔCC, induces cell growth and reduces cell adhesion, when overexpressed in MDA-MB-231 breast cancer cells. (A) Overexpression of rab31 mutants in breast cancer cells. MDA-MB-231 cells were stably transfected with rab31 expression plasmids encoding either the constitutively active mutant Q64L, the inactive form rab31-ΔCC, or the empty vector pRcRSV only. Western blot analysis demonstrated low endogenous rab31 expression in vector control cells, whereas the cells overexpressing either of the rab31 mutants display strongly elevated, but similar rab31-reactive protein levels. (B) Overexpression of rab31-Q64L, but not rab31-ΔCC, enhances cell growth of breast cancer cells. Stably transfected MDA-MB-231 cells were seeded in triplicate onto 24-well plates, detached with 0.1% EDTA-solution after 24 and 96 h of cultivation and counted with a Neubauer hemocytometer under trypan blue exclusion. Cell number at 24 h was set to 100%; increase in cell number was expressed relative to the 24 h value in %. Mean values (± SEM) of seven independent experiments are depicted. Statistically significant differences (p ≤ 0.01) are indicated by an asterisk. (C) Overexpression of rab31-Q64L, but not rab31-ΔCC, reduces the adhesive capacity of breast cancer cells. Stably transfected MDA-MB-231 cells were seeded on collagen type IV-coated 96-well plates. After 2 h of cell cultivation, the number of adherent cells was monitored by the hexosaminidase activity assay as described in the Methods section. Seven independent experiments were performed in triplicates each. The results are given in % relative to the cell number of adherent vector-transfected control cells. Whisker box plots indicate the 25th and 75th percentile, the vertical bars indicate the 10th and 90th percentile. The median value of seven experiments is indicated by a bar within the box. Statistically significant differences (p < 0.01) are indicated by an asterisk. Similar effects were observed with the extracellular matrix proteins collagen type I, laminin, vitronectin, and fibronectin (data not shown). (PDF 410 KB)12943_2012_1042_MOESM2_ESM.pdf
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