Abstract
MicroRNAs (miRs) are short non-coding single stranded RNAs regulating the translation of target mRNAs in normal and cancer cells in which they are frequently dysregulated promoting tumor progression. Cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC), identified by aldehyde-dehydrogenase expression (ALDH), are a cell subset within the tumor cell population that takes part in the genesis and progression of cancer. The relevance of epithelial-mesenchymal transition (EMT) has recently been recognized for tumor development and metastasis. Several studies have illustrated that miRs regulate EMT of CSC. CSC from 8 HNSCC lines, 4 of which are human papillomavirus (HPV)‑positive, were enriched by spheroid culture (spheroid-derived cells, SDC) and compared to their parental monolayer-derived cells (MDC) to analyze expression patterns of miR‑34a, CSC-related transcription factors (CSC-TFs: Sox2, Nanog, Oct3/4) and EMT-related TFs (EMT-TFs: Twist, Snail1, Snail2) by RT-qPCR. Flow cytometry was used to quantify and enrich ALDH+ CSCs. Transfection of miR‑34a mimics was used to evaluate its regulatory potential for CSC marker profiles as well as CSC- and EMT-TFs expression in HNSCC-SDC. Invasive, colony-forming and clonogenic capability of the miR‑34a mimics transfected SDC after sorting for ALDH+ and ALDH- cells was assessed by Matrigel invasion, clonogenicity and spheroid formation assay, respectively. miR‑34a expression levels were significantly downregulated in the majority of SDC derived from HNSCC-lines as compared to parental MDC (-1.6-16.4-fold). For EMT- and CSC-related TF expression, all HNSCC-derived SDC showed a significantly increased level compared to parental MDC (≤36.8-fold). Significantly increased expression of ALDH was found in SDC (2-3-fold). Compared to the HPV+, the HPV- group showed a significantly higher mean expression level of EMT-TFs, CSCs-TFs and ALDH (30.3 v.s. 12.8%). Transfection of miR‑34a mimics significantly reduced the EMT- and CSC-related TF expression level in UM-SCC9 (HPV-) and UM-SCC47 (HPV+) SDC. Simultaneously, the ALDH expression was reduced significantly (1.5-2-fold) and the invasive capacity (≤30%) and clonogenicity of HNSCC-SDC was also inhibited by transfection of miR‑34a mimics compared to controls. Restoration of miR‑34a significantly inhibited the capability for EMT formation of CSC-phenotype and functionally reduced clonogenic and invasive capacity in HNSCC cell lines. Therapeutic modulation of miR‑34a in HNSCC and CSCs may reduce the rate of metastasis and recurrence of tumors after therapy.
Highlights
Head and neck squamous cell carcinoma (HNSCC) with >600,000 newly diagnosed cases per year is the sixth most common cancer in the world [1]
We found that the expression of miR‐34a was consistently and significantly downregulated in SDC compared to the parental monolayer-derived cells (MDC) in all tested HNSCC cell lines (1.61-16.37-fold, P
Spheroid enriched cancer stem (like) cells (CSCs) can be derived from a panel of different solid malignancies such as HNSCC [10], melanoma [46], breast cancer [47] and gliosarcoma [14]
Summary
Head and neck squamous cell carcinoma (HNSCC) with >600,000 newly diagnosed cases per year is the sixth most common cancer in the world [1]. In developed countries the average 5-year survival rate is ~50% [2]. Local and distant recurrence is common in HNSCC and during the past years rates of survival have only marginally improved [3]. According to the cancer stem cell (CSC) theory, tumor formation, relapse after therapy and the development of metastases is thought to involve CSC in different phenotypic and functional states. This particular subset of cells was named cancer stem (like) cells (CSCs), similar to that conceptualised in normal tissue as stem cells [4].
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