Abstract 3675: The role of the mitotic spindle protein RHAMM in early stage bladder malignancy.
Authors: Stone Randolph , Sabichi Anita , Gill Jennifer , Adegboyega Patrick , Kleiner Heather , Davis Nathan , Meyers Shari , Clifford John 
Published: August 2014

Abstract
Purpose:
To determine if RHAMM is involved in early steps in bladder tumorigenesis.
Hypothesis:
RHAMM has pro-malignant function in bladder cancer and suppression of this protein in tumorigenic bladder derived cells is expected to reduce malignant characteristics.
Results:
We have successfully infected three human bladder cancer cell lines (UMUC-13, T24, and T24M) with shRNA that reduces the expression of RHAMM. Quantitative RT-PCR was performed on RNA samples from knockdown (RH5 and RH7 lentiviral clones) and non-target siRNA infected (NT) cells. We demonstrated that the RH5, but not the RH7 virus reduced message levels. RHAMM protein expression was reduced by almost 100% for RH5, and by 60% for RH7, as early as 72 hours post infection, the reduced expression for the RH5 infected cells is maintained even after a freeze/thaw passage.
A 96-well format MTT viability assay was used to indirectly assess the proliferation rates of the UMUC13, T24 and T24M cell lines infected with NT or RH5. RH5 drastically reduced the viability of T24 and T24M cells. Microscopic observation revealed an increase in apoptotic cells in the knockdown cultures. We observed an increase in c-PARP expression in the RH5-infected U13 and T24 cells, but not in the T24M cells. This was unexpected in the case of the T24M knockdown cells since viability, as determined by the MTT assay, was lower suggesting an alternate mechanism for the reduced viability.
Another possible explanation for the apparent differences in viability between NT and knockdown cells could be differences in their adhesive properties. This was examined by measuring absorbance using the MTT assay after short time periods (2 and 4 hrs after plating). Our results indicate RH5 increased the adhesion of UMUC-13 cells but, decreased it in T24 and T24M cells. These changes were not large (10-20% difference) but statistically significant. Scratch wound assays were also performed and indicated reduced migration with the RH5 cells.
Other groups have observed a G2/M block in RHAMM deficient mouse embryo fibroblasts isolated from RHAMM null mice. A FACS cell cycle experiment was performed assessing DNA content via propridium iodide staining. Our results were not consistent with a G2/M block, however we did observe increased quantities of sub-G1 particles in the RH5 knockdown T24 and T24M cell lines.
Conclusion:
We conclude that RHAMM is a potential marker of bladder premalignancy that could also have a very early etiological function in bladder tumorigenesis. In addition, our siRNA data suggests the possibility of targeting RHAMM for treating premalignancy or even malignancy. This work could advance our understanding of the process of bladder cancer development and will help validate RHAMM as a potential biomarker for bladder premalignancy.
[The project described was supported by Grant Number F31CA153934 from the National Cancer Institute.]
Citation Format: Randolph Stone, Anita Sabichi, Jennifer Gill, Patrick Adegboyega, Heather Kleiner, Nathan Davis, Shari Meyers, John Clifford. The role of the mitotic spindle protein RHAMM in early stage bladder malignancy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3675. doi:10.1158/1538-7445.AM2013-3675