Direct targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization

Abstract

Although the role of miR-200s in regulating E-cadherin expression and epithelial-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here, we use clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. MiR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin-dependent epithelial traits and Sec23a-mediated tumor cell secretome.

Comparative microRNA Profiling of Prostate Carcinomas with Increasing Tumor Stage by Deep Sequencing

“…Our observation that miR-200b and miR-200c first increase in their expression with the stage of malignancy but then decrease with progression to a metastasizing tumor would be in line with their proposition that there is a shift in SEC23A levels from nonmetastatic to metastatic tumors (21). In addition, Kong and colleagues showed that the miR-200 family regulates epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells which are characteristic features of metastatic cells (32).…”

“…2 and 3). miRNAs are short, noncoding RNAs of approximately [19][20][21][22][23][24][25] nucleotides that bind preferentially to specific sequences in the 3 0 -untranslated region (3 0 UTR) of mRNAs, resulting in either translational repression or mRNA degradation. Interaction of the miRNAs with their mRNA target ultimately leads to reduced protein synthesis via association with the Argonaute (Ago-) containing RNAinduced silencing complex (for a review, see ref.…”

Global secretome analysis identifies novel mediators of bone metastasis

“…All peptide digests were desalted using homemade C18 STAGE tips [67] and diluted in 10 µl of 0.1% acetic acid for MS analysis. For quantitative (SILAC-based) mass spectrometry preparation, samples were treated with DTT and iodoacetamide and subjected to in-solution trypsin digest overnight prior to desalting and subsequent SCX fractionation as described previously [68].…”

“…For quantitative (SILAC-based) analyses, mass spectrometry was performed as previously described [68]. Briefly, capillary nano LC-MS/MS of each SCX fraction was performed using an LTQ-Oribitrap hybrid mass spectromteter interfaced with an Eksigent nano HPLC system.…”

“…Peptide identifications were accepted at greater than 95% probability as specified by the Peptide Prophet algorithm [69] or better than 0.01 peptide probability by the Sequest algorithm. False discovery rates were estimated to be 1% by searching a reverse database as previously described [70] Quantitative (SILAC-based) mass spectrometry was performed as previously described [68,71]. Briefly, Arg-10 and Lys-8 labeled peptides were quantified using area under extracted ion chromatograms (XICs).…”