Abstract 758: CD44 induces P-gp expression through hyaluronic acid binding and transcriptional activation

Abstract

Abstract The main causes of treatment failure and mortality in cancer are metastases and the development of drug resistance. CD44 and P-gp are two membrane proteins well-known determinants of metastases and drug resistance respectively. We and others have shown that CD44 induces P-gp expression in cancer cells. We also showed that there is a physical interaction between these two proteins as they immunoprecipitate and co-localize in the cell membrane. Hyaluronan (HA) is a well known ligand for CD44, involved in tumor cell signaling and the development of malignant properties. Others have shown that HA is necessary for the CD44 induction of P-gp expression and drug resistance. However, it is not known whether HA binding to CD44 is indeed necessary for this induction. To answer this question, we generated different mutants of the CD44 HA binding domains (CD44 HABD). We transfected CD44 HABD mutants as well as CD44 wt into ovarian cancer cells (TOV112D) and human embryonic kidney cell lines (HEK 293). Unexpectedly, we found that P-gp expression was induced both in CD44 wt and CD44 HABD mutants. However, the P-gp induction was significantly less in the CD44 HABD mutants. We tested whether the P-gp induced by the CD44-HABD mutants was functional by testing for drug sensitivity in an MTT assay. We observed that cells transfected with CD44 HABD mutants became drug sensitive as compared to CD44 wt transfected cells even when the whole HA binding domain was deleted. We then determined if deletion of the CD44 HABD domain had an effect on the physical interaction between CD44 and P-gp. By co-immunoprecipitation experiments we showed that CD44 and P-gp were physically interacting even in CD44 HABD mutants. These results indicate the existence of an additional mechanism for P-gp induction through CD44 that is independent of HA binding. Previously, we showed that the intracytoplasmic domain of CD44 (CD44-ICD) is transported into the nucleus where it binds to DNA promoters and is involved in the transcriptional regulation of various genes. Therefore, we investigated whether CD44 was involved in transcriptional regulation of P-gp as the additional mechanism of Pgp upregulation. Co-transfection of CD44 or CD44-HABD mutants with luciferase driven MDR1 promoter showed increased luciferase activity in both CD44wt and CD44-HABD mutants. However, this MDR1 promoter does not have the CD44 DNA binding consensus sequence. Therefore, CD44-ICD could be inducing other genes that in turn activate the MDR1 gene. We conclude that although HA binding to CD44 is important for the induction of P-gp, CD44 transcriptional activation of MDR1 promoter also plays a role. Furthermore, we show that CD44 transcriptional activation of MDR1 is independent of HA binding. These results further the understanding to the present knowledge of CD44 involvement in drug resistance and uncovers new mechanisms involved in this process that are HA-independent. Citation Format: Swayamjot Kaur, Kyle Murphy, Karl Miletti, Abhilash Ravindranath, Lorna Rodriguez-Rodriguez. CD44 induces P-gp expression through hyaluronic acid binding and transcriptional activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 758. doi:10.1158/1538-7445.AM2014-758