Abstract 3073: Rab25 expression, loss and therapeutic implications in human breast cancer
Authors: Cheng JiMing , Sunkara Bharath , Ayyagari Vijayalakshmi , Rao Krishna A. 
Published: February 2011

Abstract
Introduction: Our previous work has shown that the loss of Rab25 occurs in estrogen receptor (ER) and progesterone receptor (PR) negative breast cancer cell lines and human tumor samples and seems to correlate with an active ras pathway. We have expressed Rab25 in several hormonally insensitive breast cancer cell lines and have demonstrated marked reduction in tumor growth. Histologically, the tumor cells appear smaller, and angiogenesis is significantly reduced. We have investigated the mechanism by which Rab25 exerts its anti-tumorigenic effect. Loss of Rab25 cooperates with mutant Ras to promote tumorigenesis in primary human mammary epithelial cells (HMEC). HIF1-alpha levels are markedly increased with loss of Rab25 if Ras is overexpressed. Rab25 is expressed in both luminal and basal mammary epithelium at similar levels. The loss of rab25 enhances VEGF-A secretion and VEGFR1 expression. Monoclonal antibodies can be potentially used to target these pathways which appear to be active in Rab25 negative tumors.
Materials and Methods:
Cell culture - MDA-MB-231 cells were grown in DME medium with 10% FBS, and Rao-3 cell line and HMEC were grown in DFCI-1 medium. Cells were transduced with retroviral supernatant and selected. Expression of RAB25 in transduced cells was determined by RT-PCR and western blot. Silencing of Rab25-Rab-25 was silenced in RAO-2 cells by pENTRTM/H1/TO siRNA system (Invitrogen, Carlsbad, CA). Our shRNA sequences are designed using the BLOCK-iTTM RNAi Designer. The most effective silencer of Rab25 was selected on the basis of testing 3 sequence pairs. The double-stranded oligos were inserted into the pENTR/H1/TO vector and transfected into cells. Silencing of Rab25 in transduced cells was confirmed by RT-PCR and Western blot. qPCR analysis- Expression of HIF1-alpha was quantified using real-time PCR with fluorescence detection. Western blot- Western blot was performed on cell extracts for rab25 and ras. Immunohistochemistry - CK5/6 served as a basal maker, and CK18 served as a luminal maker for co-staining with Rab25 in human mammary gland. In vivo tumor formation assay − 5 × 106 cells in 0.1 ml PBS were injected into the left mammary fat pad of five week-old female nude mice. Mice were monitored for the following 7 weeks for tumor volume. Therapeutic assays- Cell lines were incubated with anti-VEGF-A, anti-VEGFR1, or both, and cell numbers were tabulated over 5 days.
Results:
1) Rab25 loss cooperated with Ras to promote tumorigenesis in several donors.
2) HIF1α levels rose significantly with loss of Rab25 and Ras co-expression.
3) Rab25 is expressed in both basal and luminal normal mammary epithelium.
4) Both anti-VEGF-A and anti-VEGFR1 antibodies have therapeutic implications for Rab25 negative breast cancer cell lines.
Conclusions: Rab25 is a tumor suppressor that antagonizes Ras and may be part of an important therapeutic pathway. Its loss may serve as therapeutic marker for certain types of breast cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3073.