Abstract
Background
Colorectal cancer is the 3rd most common cancer in the UK, with around 40,000 new cases diagnosed annually. CRC patients have a 5-year overall survival rate of 50% cases) are associated with poor prognosis; this mutation has proven to be an important predictive factor for response to EGFR targeted therapies. Failure to target the RAS oncogene has resulted in a concentrated effort to discover targets within the downstream components of this pathway. In this study, we evaluate the roles of the small GTPases, RalA and RalB, as novel targets in RAS mutant (MT) CRC. The RALGDS/RAL pathway constitutes a RAS effector pathway and mediates cell survival, proliferation and tumorigenesis. RalB in particular contributes to cell survival through TBK1 signalling.
Methods
We used an siRNA-based approach silencing RALA and RALB both individually and simultaneously in a panel of KRASMT and WT cells with and without the addition of the MEKi AZD6244 (Selumetinib). Knockdown efficiency and subsequent signalling events were assessed by western blotting. Flow cytometry and MTT assays were used to measure cell death and cell viability respectively. Connectivity mapping using data from microarray experiments was used to identify drugs mimicking the phenotype observed with siRALB, subsequently leading to the investigation of a TBK1 inhibitor which is currently ongoing.
Results
We found that silencing RALB but not RALA, led to the greatest amount of cell death in RASMT but not WT CRC cells. In addition, a significant increase in cell death was observed when RALB silencing was combined with MEK inhibition. Cell death was found to be mediated by Caspase 8 and involved an upregulation of death receptor 5 (DR5). Furthermore, TBK1 inhibition was found to mimic the phenotype observed with siRALB.
Conclusions
RalB but not RalA, is associated with cell survival and may contribute to drug resistance in RASMT CRC. Thus, the development of novel RalB-specific therapies may lead to new treatment strategies for RAS MT CRC.
Colorectal cancer is the 3rd most common cancer in the UK, with around 40,000 new cases diagnosed annually. CRC patients have a 5-year overall survival rate of 50% cases) are associated with poor prognosis; this mutation has proven to be an important predictive factor for response to EGFR targeted therapies. Failure to target the RAS oncogene has resulted in a concentrated effort to discover targets within the downstream components of this pathway. In this study, we evaluate the roles of the small GTPases, RalA and RalB, as novel targets in RAS mutant (MT) CRC. The RALGDS/RAL pathway constitutes a RAS effector pathway and mediates cell survival, proliferation and tumorigenesis. RalB in particular contributes to cell survival through TBK1 signalling.
Methods
We used an siRNA-based approach silencing RALA and RALB both individually and simultaneously in a panel of KRASMT and WT cells with and without the addition of the MEKi AZD6244 (Selumetinib). Knockdown efficiency and subsequent signalling events were assessed by western blotting. Flow cytometry and MTT assays were used to measure cell death and cell viability respectively. Connectivity mapping using data from microarray experiments was used to identify drugs mimicking the phenotype observed with siRALB, subsequently leading to the investigation of a TBK1 inhibitor which is currently ongoing.
Results
We found that silencing RALB but not RALA, led to the greatest amount of cell death in RASMT but not WT CRC cells. In addition, a significant increase in cell death was observed when RALB silencing was combined with MEK inhibition. Cell death was found to be mediated by Caspase 8 and involved an upregulation of death receptor 5 (DR5). Furthermore, TBK1 inhibition was found to mimic the phenotype observed with siRALB.
Conclusions
RalB but not RalA, is associated with cell survival and may contribute to drug resistance in RASMT CRC. Thus, the development of novel RalB-specific therapies may lead to new treatment strategies for RAS MT CRC.
| Original language | English |
|---|---|
| Pages (from-to) | v573 |
| Journal | Annals of Oncology |
| Volume | 28 |
| Issue number | supply_5 |
| DOIs | |
| Publication status | Published - 18 Sept 2017 |
Keywords
- CRC
- ras GTPase-Activating Proteins/chemistry
