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.

Detachment stress mediated bioenergetic switch of malignant melanoma cells into anti-Warburg phenotype.

One of the biological features of cancer cells is their aerobic glycolysis by extensive glucose fermentation to harvest energy, so called Warburg effect. Melanoma is one of the most aggressive human cancers with poor prognosis and high mortality for its high metastatic ability. During the metastatic process, the metastatic tumor cells should survive under detachment stress. However, whether the detachment stress could affect the tumor phenotype is worthy to investigate. We had established the cell model of human melanoma cells under detachment stress, which mimicked circulating melanoma. It had been demonstrated that the detachment stress altered melanoma cell activities, malignancy, and drug sensitivity. In this study, we found that adherent melanoma cells were more sensitive to glucose depletion. Gene expression profiling altered expressions of transporters associated with glucose metabolism. In addition, detachment stress reduced lactate secretion owing to the reduced MCT4 and GLUT1 expressions, the altered glycolytic and respiratory capacities, and the increased superoxide production. Detachment stress also increases the sensitivity of melanoma cells toward the blockade of electron transport chains. Investigation of the change in glucose metabolism of melanoma cells under detachment stress would be critical to provide a novel molecular mechanism to develop potential therapeutics.

Drug-selected population in melanoma A2058 cells as melanoma stem-like cells retained angiogenic features - the potential roles of heparan-sulfate binding ANGPTL4 protein.

Malignant cancer may contain highly heterogeneous populations of cells, including stem-like cells which were resistant to chemotherapy agents, radiation, mechanical stress, and immune surveillance. The characterization of these specific subpopulations might be critical to develop novel strategy to remove malignant tumors. We selected and enriched small population of human melanoma A2058 cells by repetitive selection cycles (selection, restoration, and amplification). These subpopulation of melanoma cells persisted the characteristics of slower cell proliferation, enhanced drug-resistance, elevated percentage of side population as analyzed by Hoechst33342 exclusion, in vitro sphere formation, and in vivo xenograft tumor formation by small amount of tumor cells. The selected populations would be melanoma stem-like cells with high expression of stem cell markers and altered kinase activation. Microarray and bioinformatics analysis highlighted the high expression of angiopoietin-like 4 protein in drug-selected melanoma stem-like cells. Further validation by specific shRNA demonstrated the role of angiopoietin-like 4 protein in drug-selected subpopulation associated with enhanced drug-resistance, sphere formation, reduced kinase activation, in vitro tube-forming ability correlated with heparan-sulfate proteoglycans. Our finding would be applicable to explore the mechanism of melanoma stemness and use angiopoietin-like 4 as potential biomarkers to identify melanoma stem-like cells.

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.

Role of syndecan-1 and exogenous heparin in hepatoma sphere formation.

Glycosaminoglycan-modified proteoglycans play important roles in many cell activities, including cell differentiation and stem cell development. Tumor sphere formation ability is one of properties in cancer stem cells (CSCs). The correlation between CSC markers and proteoglycan remains to be clarified. Upon hepatoma sphere formation, expression of CSC markers CD13, CD90, CD133, and CD44, as well the syndecan family protein syndecan-1 (SDC1), increased as analyzed by PCR. Further examination by suppression of CD13 expression showed downregulation of SDC1 and CD44 gene expression, whereas suppression of SDC1 gene expression downregulated CD13 and CD44 gene expression. Suppression of SDC1 gene expression also suppressed sphere development, as analyzed by a novel sphereocrit assay to quantify the level of sphere formation. The heparin disaccharide components, but not those of chondroitin disaccharide, changed with hepatoma sphere development, revealing the increased levels of N-sulfation and 2-O-sulfation. These explained the inhibition of hepatoma sphere formation by exogenous heparin. In conclusion, we found that SDC1 affected CSC marker CD13 and CD44 expression. SDC1 proteoglycan and heparin components changed and affected hepatoma sphere development. Application of heparin mimics in reduction of hepatoma stem cells might be possible.

Mining of potential microRNAs with clinical correlation - regulation of syndecan-1 expression by miR-122-5p altered mobility of breast cancer cells and possible correlation with liver injury.

MicroRNAs are small noncoding RNAs acting as novel biomarkers of various diseases and potential regulators of protein expression and functions. Syndecan-1 is the heparan sulfate proteoglycan associated with malignancy of various cancers, including breast cancer. In this study, we proposed a experimental workflow to investigate potential microRNAs that regulate SDC1 expression and affect breast cancer cell mobility. MicroRNA candidates were selected from available Gene Expression Omnibus datasets on breast malignancy. Further in silico duplex hybridization and multiplex PCR approach were used to screen potential microRNAs. Analysis showed increased syndecan-1 expression but decreased miR-122-5p level upon breast malignancy. Western blot and in vitro luciferase assay confirmed the targeting of 3'-untranslated region of syndecan-1 and suppression of syndecan-1 expression by miR-122-5p. The suppression of syndecan-1 expression by miR-122-5p or shRNAs against syndecan-1 increased breast cancer cell mobility; while overexpression of syndecan-1 inhibited cell mobility. In further, miR-122-5p was enriched in liver cell-derived exosomes that was able to suppress syndecan-1 expression and increase cell mobility in breast cancer cells. In conclusion, our results suggested the downregulation of SDC1 by miR-122-5p or liver-cell-derived exosomes would enhance breast cancer cell mobility. Metastasis or mobility of breast cancer cells might be affected by circulating miR-122-5p and not directly correlated with progression of breast cancer.

Enhanced chemosensitization of anoikis-resistant melanoma cells through syndecan-2 upregulation upon anchorage independency.

Syndecan family proteins are heparan sulfate proteoglycans, which involved in various cellular activities and associating with metastatic potential and chemosensitivity of tumor cells. Melanoma is one of malignant tumors with poor prognosis upon metastasis. Previously, we had shown that melanoma cells remained survived under cell detachment, which was similar to the initial steps of tumor metastasis. Downregulation of syndecan-1 and upregulation of syndecan-2 in melanoma A375 cells were observed by different suspension conditions. Specific gene alterations also increased melanoma malignancy under anchorage independency. Thus, we would like to investigate in further the role of specific gene alteration, so that it could be used to develop novel strategy to treat melanoma. In this paper, we found that syndecan-2 expression level as well the kinase phosphorylation levels increased upon anchorage independency. The pathway to regulate syndecan-2 expression shifted from PKCα/ß-dependent under adhesion into PKCδ-dependent under cell suspension. Manipulation of syndecan-2 expression showed that PI3K and ERK phosphorylation as well the migratory ability increased with increased syndecan-2 expression level. In addition, suspended melanoma cells were more sensitive to chemoagents, which correlated with syndecan-2 overexpression, PI3K and ERK activations, serum level, and the presence of glycosaminoglycans. In conclusion, we showed upregulation of syndecan-2 in anoikis-resistant melanoma cells enhanced chemosensitivity through PI3K and ERK activation. This observation would support and refine the strategy of adjuvant chemotherapy to overcome metastatic melanoma.

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.

Loss of cell invasiveness through PKC-mediated syndecan-1 downregulation in melanoma cells under anchorage independency.

Anchorage-independent survival is one of the key features for malignant tumor cells. Whether specific gene alterations contributed by anchorage independency would further affect metastatic phenotypes of melanoma cells was unclear. We adapted suspension culture of melanoma cells to establish anchorage independency. The suspended melanoma cells lost their invasive abilities in vitro. Specific loss of laminin-binding ability in suspended melanoma cells was observed, which was correlated with downregulation of syndecan-1 as revealed by microarray and validated by qPCR and Western blot. Modulation of syndecan-1 expression level affected laminin binding, transwell migration and matrix metalloproteinase-2 secretion in melanoma cells. SDC1 expression and transwell migration were correlated with activity or level of protein kinase Cδ as evidence by specific inhibitors and shRNA transfection. In this study, we compared metastatic phenotypes and gene expressions of adherent and suspended melanoma cells. The anchorage independency led to protein kinase Cδ-mediated syndecan-1 downregulation, which contributed to loss of laminin-binding ability, reduced metalloproteinase-2 secretion and loss of invasiveness.