CD44 expression in oro-pharyngeal carcinoma tissues and cell lines.

Expression of CD44, a transmembrane hyaluronan-binding glycoprotein, is variably considered to have prognostic significance for different cancers, including oral squamous cell carcinoma. Although unclear at present, tissue-specific expression of particular isoforms of CD44 might underlie the different outcomes in currently available studies. We mined public transcriptomics databases for gene expression data on CD44, and analyzed normal, immortalized and tumour-derived human cell lines for splice variants of CD44 at both the transcript and protein levels. Bioinformatics readouts, from a total of more than 15,000 analyses, implied an increased CD44 expression in head and neck cancer, including increased expression levels relative to many normal and tumor tissue types. Also, meta-analysis of over 260 cell lines and over 4,000 tissue specimens of diverse origins indicated lower CD44 expression levels in cell lines compared to tissue. With minor exceptions, reverse transcribed polymerase chain reaction identified expression of the four main isoforms of CD44 in normal oral keratinocytes, transformed lines termed DT and HaCaT, and a series of paired primary and metastasis-derived cell lines from oral or pharyngeal carcinomas termed HN4/HN12, HN22/HN8 and HN30/HN31. Immunocytochemistry, Western blotting and flow cytometric assessments all confirmed the isoform expression pattern at the protein level. Overall, bioinformatic processing of large numbers of global gene expression analyses demonstrated elevated CD44 expression in head and neck cancer relative to other cancer types, and that the application of standard cell culture protocols might decrease CD44 expression. Additionally, the results show that the many variant CD44 exons are not fundamentally deregulated in a diverse range of cultured normal and transformed keratinocyte lines.

Variant CD44 expression by human fibroblasts.

CD44 is a cell surface glycoprotein with roles in tumour invasion and metastasis. CD44 is variably spliced from ten variant exons and mis-splicing is a biomarker for detection of colon, urothelial and other carcinomas. Fibroblasts are normally considered to lack variant exons and thus should not generate false-positive signals. Transcription of variant exons by fibroblasts was investigated by exon-specific reverse transcription-polymerase chain reaction (RT-PCR) for variant exons v2-v10 using normal primary fibroblasts, immortalized and experimentally transformed fibroblasts. Flow cytometry, immunocytochemistry and Western blotting were used to determine expression. All types of fibroblasts, including normal primary culture fibroblasts, transcribed low levels of variant exon mRNA. Expression could not be detected by blotting or immunocytochemistry but flow cytometry revealed minor expression of some exons by all three types of cultured fibroblast. Fibroblasts do transcribe and express small amounts of variant exon CD44. This may need to be considered when using exon splicing as a biomarker for malignancy in clinical samples containing connective tissue.

Transcription and expression of CD44 variant exons by oro-pharyngeal squamous cell carcinomas.

The CD44 cell surface hyaluronan binding protein has many isoforms formed by alternative splicing and glycosylation, and some variants have been associated with a malignant phenotype, invasion and metastasis. CD44 splicing patterns and intron 9 retention in 24 oro-pharyngeal squamous cell carcinomas (OSCC) were investigated by RT-PCR. Transcription efficiency and quantity were determined in 10 carcinomas and normal control mucosa by real-time PCR using dual-labeled fluorescent Taqman probes. Most of the carcinomas, regardless of grade, showed one major transcript including exons v2-v10, similar to that expressed by normal keratinocytes. In addition, most carcinomas expressed a variety of truncated transcripts of contiguous variant exons. Real-time PCR revealed that carcinomas showed both over-expression and down-regulation in transcription compared to normal keratinocytes, but this change was independent of the state of differentiation or the sub-site of biopsy. Intron 9 was not retained in normal keratinocytes or carcinomas. Overall, the results indicate that OSCC, like normal keratinocytes, constitutively express all variant exons and that the missplicing seen in other malignancies does not appear to occur. OSCC do not reproducibly over-express CD44 and show a wide range of transcription levels. CD44 expression does not appear to be fundamentally deranged in OSCC and is unlikely to be of value in early diagnosis, or as a prognostic marker.