Bladder cancer is the fifth most prevalent cancer in the USA. The gold standard therapy for the majority of bladder cancers is instillation of an attenuated Mycobacterium Bovis, Bacillus Calmette-Gu?rin (BCG), into the bladder, with 50% of patients positively responding to this treatment. The internalization of BCG by bladder cancer cells has long been suspected as playing a significant role in BCG treatment efficacy, however how BCG is internalized into bladder cancer cells remains undefined. Furthermore, while previous studies have explored the mechanism of action of BCG treatment on bladder cancer cells, none have addressed a role for the internalization of BCG in mediating biological responses. A subset of bladder cancers contain oncogenic Ras mutations, which among a myriad of other effects, induces elevated levels of macropinocytosis, a process previously implicated in Mycobacteria internalization in lung epithelial cells. The central hypothesis of this proposal is that BCG is internalized by oncogenic Ras-mediated macropinocytosis in bladder cancer cells and this process is essential for BCG-induced responses in bladder cancer cells. This hypothesis will be examined in a panel of bladder cancer cell lines, half of which contain oncogenic Ras mutation, through the following specific aims:
Specific Aim 1 : To determine the role of oncogenic Ras-mediated macropinocytosis in BCG internalization. Oncogenic Ras-mediated macropinocytosis might be required for the internalization of BCG in bladder cancer cells and the ensuing BCG-induced anti-tumor response. The extent of BCG internalization in a panel of bladder cancer cell lines positive or negative for oncogenic Ras will be quantified by a combination of microscopy and microbiological assays.
Specific Aim 2 : To define the role of oncogenic Ras-mediated macropinocytosis in BCG-induced responses. Oncogenic Ras-mediated macropinocytosis might be required for BCG-induced responses in bladder cancer cells. Macropinocytosed BCG could survive within macropinosomes by blocking macropinosome-lysosome fusion resulting in the induction of a cellular stress response which would have deleterious effects on cell fitness and/or affect host immunity. Alternatively, macropinocytosed BCG might be rapidly degraded and mediate antigen presentation. The role of oncogenic Ras-mediated macropinocytosis in BCG-induced responses will be tested through a combination of microbiological assays, microscopy experiments, as well as Western Blot and FACS analysis in bladder cancer cell lines positive or negative for oncogenic Ras treated without or with the macropinocytosis inhibitor EIPA in order to: a. Delineate the fate of macropinocytosed BCG. b. Elucidate the biological consequences that arise from the macropinocytosis of BCG.
Amongst non-muscle invasive bladder cancer patients; 50-70% respond positively to the gold standard treatment; Mycobacterium Bovis Bacillus Calmette-Gu rin (BCG) intravesical therapy; yet its mechanism of action remains elusive. A better understanding of how BCG treatment works and which patients might benefit from this treatment should result from uncovering how BCG is internalized into bladder cancer cells; as the internalization of BCG by bladder cancer cells is believed to be an important determinant of BCG treatment efficacy. This study seeks to uncover a previously unrecognized role for oncogenic Ras-mediated macropinocytosis; a process previously implicated in Mycobacteria internalization; in BCG-induced responses in bladder cancer cells; which has the potential to shed light on the mechanism of action of BCG treatment.
|Commisso, Cosimo; Flinn, Rory J; Bar-Sagi, Dafna (2014) Determining the macropinocytic index of cells through a quantitative image-based assay. Nat Protoc 9:182-92|