The RAL Small G Proteins Are Clinically Relevant Targets in Triple Negative Breast Cancer

Abstract

Breast cancer (BC) is the most frequent cancer and second-leading cause of cancer deaths in women in the United States. While RAS mutations are infrequent in BC, triple-negative (TN) and HER2-positive (HER2+) BC both exhibit increased RAS activity. Here, we tested the RAS effectors RALA and RALB, which are overexpressed in BC, as tractable molecular targets in these subtypes. While analysis of the breast cancer patient sample data suggests that the RALs are associated with poor outcome in both TNBC and HER2+ BC, our in vivo and in vitro experimental findings revealed the RALs to be essential in only the TNBC cell lines. While testing the response of the BC cell lines to the RAL inhibitors RBC8 and BQU57, we observed no correlation between drug efficacy and cell line dependency on RAL expression for survival, suggesting that these compounds kill via off-target effects. Finally, we report the discovery of a new small molecule inhibitor, OSURALi, which exhibits strong RAL binding, effectively inhibits RAL activation, and is significantly more toxic to RAL-dependent TNBC cells than RAL-independent HER2+ and normal cell lines. These results support the RALs as viable molecular targets in TNBC and the further investigation of OSURALi as a therapeutic agent.

Keywords: BQU57; HER2+; OSURALi; RALA; RALB; RAS; RBC8; TNBC; breast cancer; chemotherapy.

Figure 1

RALA is associated with poor outcomes in multiple breast cancer subtypes. (A) Kaplan–Meier analysis grouping BC patients within the luminal B, HER2+, and basal BC subtypes. Grouping was accomplished by segregation between high (upper 50th percentile) vs. low expression (lower 50th percentile) groups for RALA (upper panel) and RALB (lower panel) expression with relapse–free survival as the measured outcome. Data are from KM Plotter, p < 0.05. (B) RALA overexpression, but not RALB overexpression, predicts patient response to HER2 therapy. Five–year relapse free survival data were from ROC Plotter, *, p < 0.05. (C) Kaplan–Meier analysis of HER2+ BC patient overall survival (OS, p = 0.0090) as segregated by RALA H–score (upper 50th percentile vs. lower 50th percentile) based on patient sample immunostaining with representative images for samples with low and high RALA staining.

Figure 2

Depletion of RALA or RALB reduces MDA-MB-468 tumor growth and is associated with changes in the tumor microenvironment. (A) Western blots displaying RALA and RALB expression in the MDA-MB-468 shRNA control (shCTRL), shRALA, and shRALB cells. ImageJ was used for quantification. (B,C) Effects of stable knockdown of RALA or RALB in MDA-MB-468 cells on 2D growth as measured by MTT ((B), n = 4), and 3D growth as measured by GILA ((C), n = 4) *, p < 0.05. (D) Stable knockdown of RALA or RALB had no effect on MDA-MB-468 cell migration (left: representative images, right: composite mean values, n = 2). (E) Comparison of tumor growth following orthotopic mammary fat pad injection of MDA-MB-468 shCtrl (n = 20), shRALA (n = 20), or shRALB (n = 20). Results were combined from three independent experiments. (F–H) MDA-MB-468 shCTRL (n = 9), shRALA (n = 10), and shRALB (n = 10) tumors IHC stained for (F) Ki-67, (G) CC3, or (H) CD31. ROIs were determined on each image separating the tumor from stroma before color deconvolution to extract DAB staining. A signal threshold (equivalent for each image) was then applied to the samples before measurement of the ROIs was performed to measure the % area of target staining in each region. Three representative photos from each sample were separately analyzed, and the mean values were used for comparisons among groups. *, p < 0.05. (I) MDA-MB-468 tumors stained with Masson’s Trichrome, denoted by white arrows, to analyze the collagen deposition in shCTRL (n = 7), shRALA (n = 8), or shRALB (n = 9) MDA-MB-468 tumors. *, p < 0.05. (J,K) Graphs summarize the relative amounts of secreted proteins detected in the conditioned media from the MDA-MB-468 shCTRL, shRALA, and shRALB cultures.

Figure 3

Loss of RALA or RALB does not negatively impact SKBR3 tumor growth or viability. (A) Western blots displaying RALA and RALB expression in the SKBR3 shRNA control (shCTRL), shRALA, and shRALB cells. ImageJ was used for quantification. (B) Comparison of tumor growth following orthotopic mammary fat pad injection of SKBR3 shCTRL (n = 10), shRALA (n = 9), or shRALB (n = 10). Results were from a single experiment. (C–E) Effects of stable knockdown of RALA or RALB in SKBR3 cells on 2D growth as measured by MTT ((C), n = 5), 3D growth as measured by GILA ((D), n = 5), and cell migration ((E), n = 2). *, p < 0.05.

Figure 4

RAL depletion impairs the viability of TNBC but not HER2+ breast cancer cell lines. (A) Quantification of 2D growth over 3 days as measured by the MTT assay across a panel of BC cell lines grouped by molecular subtype. Cells were treated with the indicated pairs of siRNA sequences targeting RALA and RALB for 2 days prior to plating. n = 3–12. *, p < 0.05. (B) Quantification of 3D growth over 5 days as measured by GILA across a panel of BC cell lines grouped by molecular subtype. Cells were treated with the indicated pairs of siRNA sequences targeting RALA and RALB for 2 days prior to plating. n = 3–12. *, p < 0.05.

Figure 5

RAL activity is elevated in TNBC compared to HER2+ breast cancer cell lines. (A) Western blot of GTP-RALA, GTP-RALB, and total RALA and RALB expression in the TNBC and HER2 overexpressing cell lines. (B) Quantification of RALA activation in the TNBC and HER2 cell lines. Quantification was performed by averaging the normalized GTP-RALA/(t)RALA values in TNBC (MDA-MB-231, BT549, MDA-MB-468) or HER2 (SKBR3, BT474, MDA-MB-453) per experiment (n = 2). (C) Quantification of RALB expression by TNBC or HER2. Quantification was performed by averaging the normalized (t)RALB/GAPDH values in the TNBC (MDA-MB-231, BT549, MDA-MB-468) or HER2 (SKBR3, BT474, MDA-MB-453) lines from each experiment (n = 2). (D) Gene expression analysis was performed by qPCR to determine the levels of RAL GEFs (RALGDS, RCC2, RGL1, RGL2, RGL3, RALGPS1, and RALGPS2) and RAL GAPs (RALGAPA1, RALGAPA2, and RALGAPB) within a panel of BC cell lines normalized to MDA-MB-231 (n = 2–3). Four out of the seven RAL GEFs (RALGDS, RGL1, RGL2, and RGL3) were RAS activated RALGEFS. Expression above 1 indicates an increase in gene expression and expression below 1 indicates a decrease in gene expression compared to MDA-MB-231.

Figure 6

RBC8 and BQU57 treatment inhibits the in vitro viability in a panel of BC lines in a non-RAL dependent manner. (A) Relative viability of BC cells in 3D culture following treatment with the indicated concentrations of RBC8 for 5 days as measured by GILA (n = 3). (B) Relative viability of BC cells in 3D culture following treatment with the indicated concentrations of BQU57 for 5 days as measured by GILA (n = 3–4).

Figure 7

OSURALi inhibits RAL activation and is cytotoxic to RAL–dependent TNBC cell lines. (A) Response curve for OSURALi interaction with recombinant RALA. (B) Western blot (top) and quantitation (bottom, n = 4) of the inhibition of RALA and RALB activity by BQU57, RBC8, and OSURALi. MDA–MB–468 cells were pre–incubated with 50 μM of RAL inhibitor for 1 h prior to the stimulation of RAL activity by EGF. The novel RAL inhibitor OSURALi decreased the 3D culture viability to a greater degree in TNBC relative to the HER2+ BC lines. ImageJ was used for quantitation. *, p < 0.05. (C) Relative 3D viability of cells following treatment with varying concentrations of OSURALi for 5 days as measured by GILA (n = 3). (D) Viability data from Figure 7D displayed at selected concentrations to enable comparison by subtype. (E) Relative 3D (GILA) viability of HMEC–1 cells treated with increasing doses of OSURALi for 5 days (n = 2).

Figure 8

Proposed model of RAL-dependent and -independent signaling pathways through RAS. In TNBC, signaling through EGFR leads to the activation of RALs where they function to increase tumor growth. Conversely, in HER2+ BC, signaling through HER2 does not utilize RAL signaling and instead influences BC growth through the other RAS effectors. OSURALi will inhibit RAL activity, reducing TNBC, but not HER2+ BC proliferation. Figure was produced using Biorender.