Involvement of CD44 during tumorigenic transformation of pre-cancerous human uroepithelial cells

Abstract

Bladder cancer is a common disease in man aged over 50, making it the fourth most prevalent cancer in men. Superficial transition cell carcinoma contributes to 70% of clinical cases and is typically treated by transurethral resection (TUR) with or without Bacille Calmette-Guerin (BCG) treatment. However, effective treatment is hampered by the high cancer recurrence rate and around 30% of the patients may even progress to a high grade cancer making it the most expensive cancer to be treated. This study proposes that the high recurrence rate may be due to the presence of sensitized cell - urothelial cells that accumulated certain degree of genetic alternations but phenotypically similar to normal cells. Therefore, they can escape from the surgery removal and stem future tumor formations, i.e. cancer recurrence. However, little is known about the existence and properties of the sensitized cell. Identification and characterization this cell type will be crucial to develop means for recurrent prediction, monitoring and prevention. Information presented by this study is not only important for understanding the cancer development process but also useful for prediction of bladder cancer recurrences. An in vitro tumorigenic model involving two human uroepithelial cell lines at two different statuses, namely HUC-PC (pre-cancerous) and HUC-1 (normal) was used to study the transformation process. HUC-PC mimics the sensitized cell remained in bladder lining after TUR and tumorigenic transformation was triggered by the exposure of bladder carcinogen, 4-aminobiphenyl (ABP). Due to the fact that HUC-PC is sensitized to ABP and will transform to cancer state while HUC-1 will not, we can then target different molecules for their involvement in cancer development by comparing the molecular changes between two statuses of cell line. The success of transformation process was confirmed by functional assays and cancer markers. In this study, HUC-PC was treated with tobacco carcinogen ABP at concentrations 200μM for 24hr. After 6 weeks of incubation, the tumorigenicity of cells was evaluated via functional assays. The results showed that the transformed HUC-PC displayed neoplastic transformation phenotype, as marked by their induced proliferation rate and invasion ability. In addition, bladder markers, survivin and telomerase, were also found highly expressed in the transformed HUC-PC, which further confirmed the success of the transformation process. During transformation, our target CD44 and its partner HA were quantified for evaluating their involvement in the process. Flow cytometry and RT-qPCR analyses showed that the transformed HUC-PC expressed increased levels of CD44 in the first week after exposure to 4-ABP. The elevated level of CD44 mRNA expression was great contributed by the CD44s instead of CD44v6 (exon v6 containing CD44 isoforms). In parallel, an increase in HA was found after the CD44 induction and maintained at a high level throughout the transformation process. Based on the above findings, we demonstrated that in vitro ABP exposure could neoplastically transform the pre-cancerous HUC-PC and suggested CD44 is involved in the initiation step of cancer development, possibly by interacting with HA.