Differential expression of CD44 isoforms in head and neck squamous cell carcinoma

Abstract

Problem: CD44 is a trans-membrane molecule comprising multiple isoforms. These isoforms result from alternative splicing of a variant exon region. Several isoforms occur normally while others, termed CD44 variants (CD44v), are expressed in tumors. Specific isoforms CD44v3–10 and CD44 standard exist in normal keratinocytes. Isoform CD44v3 has been identified in tumor tissue. We hypothesize that total CD44 RNA transcripts are differentially expressed in head and neck squamous cell carcinoma (HNSCC) tissues compared to normal controls and that isoform CD44v3 is overexpressed in tumor tissue. Methods: Five samples of microdissected mucosa from uvulopalatopharyngoplasty patients and 5 samples of HNSCC tissue from various sites were examined. We performed RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR) using primers that amplify the alternative splicing region followed by gel electrophoresis. We performed quantitative PCR using a junction primer strategy that only amplifies isoform CD44v3 and western blot analysis using an antibody to total CD44. Results: RT-PCR results suggest that at least 3 of 5 tumors and only 1 of 5 normals contain more than 3 CD44 isoforms. The 3 isoforms seen consistently in both normal and tumor tissues correspond to specific isoforms CD44 standard, CD44v3, and CD44v3–10 by base-pair analysis. Quantitative PCR demonstrated that 4 of 5 tumor samples showed greater expression of isoform CD44v3 than all the normal samples. Western blot suggests that there are CD44 isoforms present in tumor that are not present or expressed at low levels in normals. Conclusion: These results support the hypothesis that isoforms of CD44 are differentially expressed in HNSCC compared to normals. We plan to study more tissues at the transcript and protein level and characterize additional specific isoforms using cloning and sequencing. Significance: Understanding CD44 isoform expression in HNSCC tissue compared to normal upper aerodigestive tract mucosa may establish CD44 as a tumor marker. Support: This work was supported by funding from the Flight Attendant Medical Research Institute and the Sylvester Comprehensive Cancer Center.