Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas

Abstract

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44Lin and CD44Lin subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44Lin compared to CD44Lin cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

A perspective on challenges and opportunities in characterizing oral cancer stem cells

“…The primary reason for testing CD44 in HNSC was that it had been previously used successfully to mark CSCs in another epithelial tissue, e.g., breast tumors (3, 4). There was also evidence from immunohistochemistry of head and neck tumor sections that CD44 stained only the basal or poorly differentiated tumor cells, and its staining pattern was mutually exclusive to that of involucrin, a well-known terminal differentiation marker in these epithelia (5,21). Although this has been corroborated by more recent studies, ambiguities have persisted (22).…”

“…CD44 is widely expressed on the surface of hematopoietic cells, and head and neck cancers are typically wellinfiltrated by immune cells. In fact, studies that have reported FACS profiles of cells from primary patient HNSCs have estimated the percentage of mature lineage marker-positive or Lin + cells (includes hematopoietic and stromal cell populations) to be as high as 50-60% in many tumors (21,24). There are two consequences to this observation-1.…”

“…In addition to this, some specific cell-cell adhesion molecules have also been implicated. A transcriptomics analysis of CD44 + Linand CD44 -Lincells derived from primary OSCCs identified significantly lowered expression of the cellcell adhesion molecule, protocadherin 18 (PCDH18) in the CD44 + Linsubpopulation (21). Interestingly, lower PCDH18 expression correlated with poorer overall survival in The Cancer Genome Atlas (TCGA)-HNSC data highlighting it as a potential negative prognostic factor in this cancer.…”

“…A key protein that maintains the proliferative potential of cells in the basal layer of squamous epithelia is TP63, wherein it controls the expression of basal markers (e.g., keratins 5/14 (K5/14)) amongst other genes (37,38). Immunohistochemical analysis of TP63 and CD44 indicated that these proteins are co-expressed in undifferentiated, basal layer-like cells in HNSC tissue sections (21,39). Conversely, terminal differentiation markers such as involucrin and TGM1 (transglutaminase1) are not expressed by these CD44 + cells.…”

Targeting cancer stem cells in squamous cell carcinoma

“…For example, BE-43547A2, which belongs to the naturally occurring cyclic depsipeptides, can selectively target pancreatic CSCs 178 . Researcher have also found several naturally derived compounds that can ablate CSCs in melanoma more effectively 177 . In addition, curcumin, a natural product extracted from Curcuma longa plants, can suppress stemness of HNSCC CSCs through inhibition of RXRα 178 …”