Genomic and proteomic approaches identify Rab25 as a potential prognostic marker for luminal B breast tumors
Authors: Shreya Mitra, Gordon B. Mills, Kwai Wa Cheng
Journal: Clinical Cancer Research (2010)

Abstract Estrogen receptor (ER) +ve luminal B subtype of breast tumors initially respond well to tamoxifen treatment. However, over longer periods of time, their frequent recurrence contributes to the overall high mortality rate of hormone receptor +ve breast cancer patients. Recently, with the development of PARP inhibitors, there is a potential line of treatment for the triple negative subtype. But a huge dearth exists in therapeutically targeting luminal B tumors since these tumors are also insensitive to chemotherapy. Our current research addresses this urgent need by utilizing a systems biology approach to identify potential therapeutic targets and diagnostic markers for luminal B subtype of breast cancer. The endocytic pathway, as demonstrated by our laboratory is a frequent target of genomic aberrations in cancer, and plays a critical role in maintenance of cellular polarity, stem cell function, bioenergetics, proliferation, motility, invasion, metastasis, apoptosis and autophagy. Derailed endocytosis is therefore a hallmark of cancer. The Rab GTPases along with their effectors are critical regulators of this endocytic machinery and can have a huge impact on the cellular itinerary of growth factor as well as integrin receptor signaling. A broad spectrum analysis of distribution of all the Rab family members across 52 breast cancer cell lines reflected a dichotomous distribution of Rab25, with higher mRNA and protein expression in ER+ cell lines. Rab 25 is of immense interest to us because of its unique substitution of leucine for glutamine at position 71 which typically decreases the intrinsic GTPase activity, resulting in a dominant, constitutively active protein, akin to a transforming RAS mutation. Here we evaluate the role of Rab25 as a valuable predictive and prognostic marker for ER+ breast cancers, with critical implication in disease management and predicting response to therapy. Using high throughput data analysis from publicly available mRNA expression datasets, we report that Rab25 and its effector Rab coupling protein (RCP) are frequently coamplified and coordinately elevated in ER +ve breast cancers. Specifically in the luminal B subtypes, they are significantly associated with a markedly worsened outcome. Compatible with Rab25 and RCP being drivers of the amplicons, their respective mRNA levels are highly correlated across multiple tumor sets, most prominently in luminal B tumors (Soritiou p&amp;lt;0.0015; Pawitan p&amp;lt;0.01). Patients with low RNA levels of both Rab25 and RCP have a very good outcome, those with high level of either Rab25 or RCP have an intermediate outcome and patients with coordinate elevation of Rab25 and RCP mRNA have a bad outcome. Additionally, using our Reverse Phase Protein Array (RPPA) technology, we found that at protein level Rab25 and RCP correlate with poorer outcome in luminal B tumors. In vitro, using MCF7 cell line, which is a prototype of luminal B subtype, we observed an altered EGFR stability and signaling in Rab25 overexpressing stable clones which could be rescued with shRNA knock-down of Rab25, confirming specificity of Rab25 action. In MCF7 cells overexpressing Rab25, we found higher proliferation index as well as increased cell migration and invasion potentials. When injected as xenografts in mice, Rab25 overexpressing cells formed larger tumors than their wildtype controls, strongly suggesting a role for Rab25 in promoting tumor progression in luminal B tumors. The proposed studies will have a marked impact on our understanding of the mechanisms by which Rab25 and RCP contribute to breast cancer aggressiveness, and also of the therapeutic and clinical potential of Rab25 and the Rab25: RCP complex as a target and marker of the behavior of breast tumors.