Anti-microRNA-1976 as a Novel Approach to Enhance Chemosensitivity in XAF1+ Pancreatic and Liver Cancer.

The current cancer treatments using chemoagents are not satisfactory in terms of outcomes and prognosis. Chemoagent treatments result in cell death or arrest, but the accompanying cellular responses are not well-studied. Exosomes, which are extracellular vesicles secreted by living cells, might mediate cellular responses through microRNAs. We found that miR-1976 was highly enriched in exosomes secreted after chemoagent treatment. We developed a novel approach for in situ mRNA target screening and discovered several miR-1976-specific mRNA targets, including the proapoptotic gene XAF1, which was targeted by miR-1976 and which suppressed chemoagent-induced cell apoptosis. Increased RPS6KA1 gene transcription was associated with the increase in its intronic pre-miR-1976 expression. Blockade of miR-1976 could enhance chemosensitivities of hepatoma and pancreatic cancer cells in an XAF1-dependent manner, as evidenced by increased levels of cell apoptosis, reduced IC50 in cell toxicity assays, and suppressed tumor growth in animal xenograft experiments in vivo. We propose that intracellular levels of miR-1976 determine chemosensitivity, and its blockade could be a novel strategy and potential therapeutic application in cancer treatment.

Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin ß4 axis.

The detachment of tumor cells from extracellular matrix and survival under anchorage-independence were recognized as the initial step of tumor metastasis. Previously we had demonstrated that anchorage-independence altered gene expressions and showed characteristics of cell invasiveness loss, enhanced chemosensitivity, and enhanced subcutaneous tumor formation. However, whether it affected histological phenotypes in tumor tissues remained unclear. Melanoma metastases were generated in nude mice using adherent or suspended melanoma cells. Examination of melanoma metastases revealed histological features of extensive vascular structures in adherent cell-derived tumors, while not seen in suspended cell-derived tumors. Quantitative proteomic analysis at adherent, suspended, and re-attached melanoma cells suggested that aminopeptidase N was potentially downregulated upon cell suspension or reattachment. Downregulation of aminopeptidase N by gene-specific shRNAs showed reduced cell invasiveness and enhanced subcutaneous tumor formation that was consistent with previous observations. Experiments by suppression or overexpression of aminopeptidase N expression demonstrated that aminopeptidase N regulated syndecan-1 and integrin ß4 expression through PKCδ pathway. Histological analysis at melanoma metastases further suggested that CD31+/aminopeptidase N+/syndecan-1+/integrin ß4+ phenotypes were associated with vascular structures. In summary, we suggested the expression axis of aminopeptidase N/syndecan-1/integrin ß4 in melanoma cells was suppressed by detachment stress, which diminished vascular phenotypes of melanoma metastases.

Anchorage independency promoted tumor malignancy of melanoma cells under reattachment through elevated interleukin-8 and CXC chemokine receptor 1 expression.

Metastasis of melanoma cells during the recurrence or the late stage of melanoma has been characterized as the dissemination of tumor cells under anchorage independency. The secreted interleukin-8 (IL-8) and its conical receptors from melanoma cells have been associated with melanoma malignancy. However, their correlations with melanoma cells under anchorage independency were unclear. Suspension of adherent melanoma cells generated the suspended melanoma cell model of anoikis resistance. The in-vivo xenograft experiment, in-vitro cell proliferation/migration assay, microarray, and bioinformatics analysis were used to compare the malignancy and gene expression profiling in adherent and suspended melanoma cells. PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and kinase inhibition assay were adapted to validate the expression and regulation of IL-8 and CXCR1/2. Suspended melanoma cells were anoikis resistant and showed elevated malignancy in vivo and in vitro. Gene expression profiling of adherent and suspended melanoma cells showed extensive alteration associated with cell survival/death, cell signaling, and regulation of gene expression. Microarray and bioinformatics analysis on gene set enrichment analysis further showed elevated IL-8 expression in suspended melanoma cells. The upregulation of IL-8 and the effect on chemotaxis were mediated by MEK/ERK activation upon cell suspension. Change in JNK phosphorylation induced CXCR1 downregulation under cell suspension, but upregulation by cell reattachment. We suggest the possible roles of elevated IL-8 secretion and change in CXCR expression contributing toward elevated melanoma malignancy upon reattachment from cell suspension. We show that the suspension of melanoma cells is critical in promoting melanoma malignancy in vivo and in vitro.