Validation of Novel Transcriptional Targets that Underpin CD44-promoted breast cancer cell invasion

Abstract

Introduction: Breast cancer (BC) is the most common cancer worldwide, and metastasis is its worst aspect and the first cause of death. Metastasis is a multistep process, where an invasion is a recurring event. The process of BC cell invasion involves three major factors, including cell adhesion molecules (CAM), proteinases and Growth factors.CD44, a family of CAM proteins and the hyaluronic acid (HA) cell surface receptor, acts as cell differentiation, cell migration/invasion and apoptosis regulator. Rationale: We have previously established a tetracycline (Tet)-OFF-regulated expression system, both in vitro and in vivo (Hill et al, 2006). As a complementary approach, the highly metastatic MDA-MB-231 BC cells expressing high levels of endogenous CD44s (the standard form of CD44), was cultured in the presence and absence of 50 µg/ml of HA. RNA samples were isolated from both cell experimental models, and microarray analysis (12K CHIP from Affymetrix) was applied. More than 200 CD44s transcriptional target genes were identified and were sub-divided into groups of genes based on their function: cell motility, cytoskeletal organization, ability to degrade ECM, and cell survival. Hypothesis: Among these 200 identified genes, we selected seven genes (ICAP-1, KYNU, AHR, SIRT1, SRSF8, PRAD1, and SOD2) and hypothesized that based on evidence from literature, these genes are potential novel targets of CD44-downstream signaling mediating BC cell invasion. Specific Aims: Pursuant to this goal, we proposed the following objectives: 1- Structural validation of ICAP-1, KYNU, AHR, SIRT1, SRSF8, PRAD1 and SOD2 as novel transcriptional targets of CD44/HA-downstream signaling at both RNA and Protein level using reverse transcription polymerase chain reaction (RT-PCR) and Western Blot respectively. 2-Functional validation of ICAP-1, KYNU, AHR, SIRT1, SRSF8, PRAD1and SOD2 as novel transcriptional targets that underpin CD44-promoted BC cell migration using wound healing assay after the transfection with siRNA.Innovation/Consclusion: This study validated seven transcriptional targets of CD44/HA-downstream signaling promoting BC cell invasion. Ongoing experiments aim to dissect the signaling pathways that link CD44 activation by HA to the transcription of these seven genes.