Stem cell marker (Nanog) and Stat-3 signaling promote MicroRNA-21 expression and chemoresistance in hyaluronan/CD44-activated head and neck squamous cell carcinoma cells

Abstract

MicroRNAs are often associated with the pathogenesis of many cancers including Head and Neck Squamous Cell Carcinoma (HNSCC). In particular, microRNA-21 (miR-21) appears to play a critical role in tumor cell survival, chemoresistance and HNSCC progression. In this study we investigated matrix hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with the stem cell markers, Nanog and Stat-3, in HNSCC cells (HSC-3 cells). Our results indicate that HA binding to CD44 promotes Nanog-Stat-3 (also tyrosine phosphorylated Stat-3) complex formation, nuclear translocation and transcriptional activation. Further analyses reveal that miR-21 is controlled by an upstream promoter containing Stat-3 binding site(s), while chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-21 expression by HA/CD44 signaling is Nanog/Stat-3-dependent in HNSCC cells. This process results in a decrease of a tumor suppressor protein (PDCD4), and an upregulation of inhibitors of the apoptosis family of proteins (IAPs) as well as chemoresistance in HSC-3 cells. Treatment of HSC-3 cells with Nanog- and/or Stat-3-specific small interfering RNAs (siRNAs) effectively blocks HA-mediated Nanog-Stat-3 signaling events, abrogates miR-21 production and increases PDCD4 expression. Subsequently, this Nanog-Stat-3 signaling inhibition causes downregulation of survival protein (IAP) expression and enhancement of chemosensitivity. To further evaluate the role of miR-21 in tumor cell-specific functions, HSC-3 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and block its target functions. Our results demonstrate that anti-miR-21 inhibitor not only upregulates PDCD4 expression, but also decreases IAP expression and enhances chemosensitivity in HA-treated HNSCC cells. Together, these findings indicate that the HA-induced CD44 interaction with Nanog and Stat-3 plays a pivotal role in miR-21 production leading to PDCD4 reduction, IAP upregulation and chemoresistance in HNSCC cells. This novel Nanog/Stat-3 signaling pathway-specific mechanism involved in miR-21 production is significant for the formation of future intervention strategies in the treatment of HA/CD44-activated HNSCC.

Up-regulation of Histone Methyltransferase, DOT1L, by Matrix Hyaluronan Promotes MicroRNA-10 Expression Leading to Tumor Cell Invasion and Chemoresistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma

“…Cisplatin was obtained from Sigma. The preparation of HA (ϳ500,000 -700,000-dalton polymers) used in these experiments was described previously (9,10).…”

“…It is commonly expressed as various isoforms generated by alternative mRNA splicing of variant exons inserted into an extracellular membrane-proximal site (8). Most importantly, expression of certain CD44 variant (CD44v) isoforms (especially, CD44v3) is known to be associated with HNSCC progression (6,9,10).…”

“…In particular, certain miRNAs appear to play a critical role in various CSC functions and tumor progression (9,10). However, very little is known concerning the regulation of these miRNAs and their function in head and neck cancer.…”

Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2

“…MSTO is a human malignant mesothelioma cell-line that highly expresses EGFR (11). These cells have been cultured previously using general medium with 10% FBS (12)(13)(14). The present study aimed to examine how STK1 or STK2 affected the cell growth of human gingival fibroblasts (HGF-1), CA9-22, HSC-3 and MSTO by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.…”

Effect of radiotherapy on expression of hyaluronan and EGFR and presence of mast cells in squamous cell carcinoma of the head and neck

“…When deregulated, EGFR aids in the growth and survival of cancer cells, and is therefore an important target in cancer therapy using monoclonal antibodies (10). EGFR-mediated pathways are involved in the HA/CD44 promotion of chemoresistance in SCCHN (11).…”