Abstract PD9-04: Context-dependent tumor suppressor role of RHOA in breast cancer through cooperation with CDH1 loss

Abstract Background: RHOA is a small GTPase that regulates actin cytoskeleton dynamics, playing key roles in various solid tumors, such as in diffuse gastric cancer (DGC). DGC and invasive lobular carcinoma (ILC) of the breast share biologic and phenotypic features, including E-cadherin (CDH1) deficiency, and a characteristic single-cell invasive pattern due to impaired cell-cell adhesion. In breast cancer (BC), the prevalence, spectrum and functional impact of RHOA mutations remain to be determined. Here, we sought to characterize the RHOA mutational landscape in BC compared to gastric cancer (GC), assess its association with CDH1 mutations, and investigate its functional impact in BC cell models. Methods: We conducted a re-analysis of targeted sequencing data from 8,939 BCs (n=4,182 primary, n=4,757 metastatic) and from 1,378 GC (n=1,044 primary; n=334 metastatic). RHOA mutational frequency, spectra and co-occurrence with other genomic alterations were assessed. The functional impact of RHOA alterations was investigated in the MDA-MB-134-VI ILC cells with RHOA knockdown (RHOA-KD) and inducible CDH1 overexpression. Results: RHOA alterations were rare in BC overall (0.76%) but significantly enriched in ILC, occurring in 2.7% of cases (29/1,058), compared to 0.25% (15/5,940) in invasive ductal carcinoma of no special type (IDC-NST; P<0.001). In BC, the most common RHOA mutations were the dominant negative loss-of-function (LOF) G17 hotspot mutation (61%), followed by E40 (10%), L22 and R5 (8%, each), L57 (6%), and G2 and Y42 (3%, each) mutations. In contrast, in GC, gain-of function (GOF) Y42 mutations, associated with altered effector binding and signaling activity, were the most frequent ones (32%), followed by R5 (15%), G62 (14%), L57 (11%), and G17 (11%) mutations. In BC, RHOA mutations frequently co-occurred with CDH1 (54%) and PIK3CA (49%) mutations, significantly more often than in GC (CDH1, 21%, P<0.001; PIK3CA, 10%, P<0.001). Compared to RHOA-wild-type BCs, RHOA-mutant BCs were also significantly enriched for mutations in chromatin remodeling genes such as CBFB (23.7% vs. 4%, [MOU1] P<0.001) and KMT2C (22.7% vs. 8%, P=0.0003), as well as in transcription factors including FOXP1 (9.3% vs. 1.7%, P<0.0001) and TBX3 (16% vs. 5.8%, P=0.001). In addition, RHOA-mutant GCs had a significantly lower KRAS mutation rate (4.2% vs. 12.4%, P=0.014) compared to RHOA-wild-type GCs. We assessed the functional impact of co-occurring RHOA and CDH1 alterations using the MDA-MB-134-VI ILC BC cells with and without CDH1 overexpression. These analyses showed that RHOA knockdown increased cell migration, invasion, anoikis resistance and proliferation, with these effects being markedly amplified in CDH1-deficient cells. Conclusions: RHOA mutations, while rare in BC overall, are enriched in ILC and frequently co-occur with CDH1 and PIK3CA mutations. Our data further suggest lineage specific differences in the role of RHOA in oncogenesis between BC and GC, with RHOA GOF mutations prevailing in GC, and RHOA LOF mutations in BC. Through functional studies we demonstrate that RHOA inactivation synergizes with CDH1 deficiency to promote oncogenic phenotypes in BC. Taken together, these findings support a context dependent tumor suppressor role of RHOA in BC, particularly in ILC, through its cooperation with CDH1 loss. Citation Format: H. Dopeso, L. Ferrando, L. Gusain, S. Farahani, S. Shen, A. Mamtani, G. Joergensen, M. Repetto, C. Schwartz, D. Ross, H. Wen, E. Brogi, H. Zhang, A. Lee, S. Oesterreich, B. Weigelt, F. Pareja. Context-dependent tumor suppressor role of RHOA in breast cancer through cooperation with CDH1 loss [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD9-04.