GP5d

RALB is Required for the Survival of KRASMT CRC Cells

RAS gene mutations are common in CRC, resulting in poor survival and challenging treatment due to activated downstream pathways. RALA and RALB, RAS-like proteins, have roles in tumorigenesis, but their specific functions in apoptosis, especially for KRASMT CRC, remain unclear. Khawaja et al. aimed to unravel the role of RALB GTPase in apoptosis regulation, particularly its interaction with TRAIL Death Receptor 5 (DR5) in KRASMT CRC.

Khawaja's lab previously showed that RALA, not RALB, influences the migration of KRASMT CRC cells. They investigated if RALA and RALB have similar roles in cell survival (Fig. 1A). Silencing RALA slightly reduced cell viability, while siRALB significantly affected it, corroborated by additional siRNA sequences against RALB (Fig. S1A). Overexpressing WT-RALB or active G23V-RALB notably enhanced colony formation in KRASMT CRC cells (Fig. 1A). siRALB induced cell death only in KRASMT cells, evident by PARP cleavage and Caspase-3/7 activation (Fig. 1B, C; Fig. S1A–C), unlike siRALA (Fig. 1B, C; Fig. S1D). KRASMT cancers resist MEK inhibition (MEKi). They hypothesized RALB might influence MEKi resistance in KRASMT CRC. Combining RALB siRNA with MEKi AZD6244 increased cell death in HCT116 cells, shown by PARP cleavage and Caspase activity (Fig. 1B; Fig. S1B), but not in KRASMT cells (Fig. 1B; Fig. S1C). In KRASMT cell lines (SW620, GP5D, LoVo), AZD6244 added to siRALB didn't increase cell death beyond siRALB alone (Fig. 1C; Fig. S1D). AZD6244 with siRALA showed no effect. No changes in pERK1/2 levels were observed. Hence, RALB, not RALA, regulates survival in KRASMT but not KRASMT CRC cells.

Fig. 1. RASMT CRC cells are dependent on RALB for survival (Khawaja H, Campbell A, et al., 2020).

RRSP Inhibits Proliferation and Perk Activation in CRC Cell Lines

Ras oncoproteins, such as KRAS, play a crucial role in cell signaling related to proliferation and survival. Mutations in RAS genes are present in around 30% of human cancers and significantly drive tumorigenesis, making RAS a prime target for cancer therapy.

To examine the impact of RAS processing on downstream signaling, Stubbs et al. analyzed four KRAS mutant CRC cell lines (HCT-116, SW1463, SW620, GP5d) with different KRAS mutations (Fig. 2A). Due to varying expression of the anthrax toxin receptor, they used a potent RRSP chimeric toxin (RRSP-DTB) linked to the diphtheria toxin's translocation fragment. RRSP-DTB binds to the human HB-EGF receptor, is endocytosed, and translocates into the cytosol. The HB-EGF receptor expression was consistent across CRC cell lines. They treated cells with increasing doses of RRSP-DTB or inactive RRSP*-DTB, monitoring growth inhibition. HCT-116 cells were most affected, showing the lowest IC50 (Fig. 2B), confirming high susceptibility to RRSP. Inactive RRSP*-DTB had no effect, verifying that RAS processing caused the sensitivity (Fig. 2B). SW1463 cells were also sensitive but had a 12-fold higher IC50 than HCT-116 (Fig. 2C). SW620 showed about 40% growth inhibition with RRSP-DTB at 10 nM, reflecting prior categorization of growth inhibition response (Fig. 2D, E). GP5d was similarly less susceptible but showed some growth inhibition. RRSP cleaved at least 80% of RAS across all lines and reduced ERK phosphorylation compared to RRSP*-DTB samples (Fig. 2F-H). There was variability in uncleaved RAS detection, likely due to compensatory RAS upregulation, especially in HCT-116 (Fig. 2F).

Fig. 2. RRSP-DTB growth inhibition in CRC cell lines (Stubbs CK, Biancucci M, et al., 2021).