Elucidation of the Role of CD44 Receptor and its Intracellular Domain in the Progression of Prostate Cancer

Abstract

Prostate cancer (PCa) is the second leading cause of cancer-related death in men in the United States partially due to metastatic spread to secondary sites in the bone, brain, lymph nodes and visceral organs. Here, we examine the mechanisms that can facilitate PCa progression/metastasis. CD44 is a multifunctional receptor that functions in tumorigenesis and cancer progression. Recent studies point to CD44 cleavage product; CD44 Intracellular Domain (ICD) as the fragment responsible for transcription of metastasis-related genes (MRGs). Using knockdown and overexpression strategy, we 1) determine the effect of androgen receptor (AR) and CD44 expression on stemness characteristics of PCa cells (PC3), 2) characterize CD44-ICD interaction with RUNX2 in PC3 cells and 3) determine the specificity of interaction of CD44-ICD sequences with RUNX2 and its contribution to PCa progression. We show SOX2 expression is high at mRNA and protein levels of bone metastatic PC3 cells that are AR-negative. Additionally, CD44 regulates the expression of SOX2 and knockdown of SOX2, or CD44 resulted in decrease of cell migration which was reversed with the re-expression of AR. To examine the contribution of CD44-ICD to the tumorigenic potential of PCa cells, we characterized CD44-ICD expression in PCa cells then determined its interaction with RUNX2, a transcription factor also known to promote tumorigenesis. We show the interaction of CD44-ICD/RUNX2 in the nucleus and overexpression of RUNX2 resulted in increased cell migration and tumorsphere formation via the upregulation of MRGs. Consistently, generation and overexpression of CD44-ICD-FL and CD44-ICD carboxyl-terminal deletion constructs resulted in increased interaction with RUNX2 in the nucleus with greater specificity for interacting with constructs: FL and D1 to D3. Likewise, these constructs show an increase in the expression of OPN and MMP-9 genes. However, CD44-ICD-FL, as well as D1 to D3 constructs, shows more specificity to the promoter of MMP-9 in CHIP assay analyses. This also corresponds with the expression levels of MMP-9 protein. These findings not only suggest the specificity of CD44-ICD function towards the expression of MMP-9 but also metastasis relevant events including migration and tumorigenesis. These also implicate CD44-ICD as a novel therapeutic target in cancer cells that express CD44.