Targeting the Hippo Pathway in Breast Cancer: A Proteomic Analysis of Yes-Associated Protein Inhibition

Abstract

The dysregulation of the Hippo signaling pathway leads to the aberrant activation of oncogenic YAP and TAZ, driving tumor progression. In breast cancer, this disruption promotes proliferation and metastasis. This study investigates the effects of CA3, a selective YAP inhibitor, on the proteome of triple-negative breast cancer MDA-MB-231 and luminal-A-like MCF7 cells. Proteomic changes were analyzed via nano-LC-MS/MS, while cytotoxicity, apoptosis, and autophagy were assessed through WST-1 assays, flow cytometry, and Western blot analyses. Bioinformatics tools were employed to identify enriched pathways. MDA-MB-231 cells exhibited an increased expression of DNA repair proteins (p < 0.05), indicating a compensatory response to maintain genomic stability. In contrast, MCF7 cells showed a downregulation of DNA repair factors (p < 0.005). Additionally, metabolic reprogramming was apparent in MCF7 cells (p < 0.001). Apoptosis assays revealed a rise in cell death, while cell cycle analysis indicated pronounced G1-phase arrest in MDA-MB-231 cells (p < 0.01). Moreover, autophagic suppression was particularly evident in MCF7 cells. This study, for the first time, provides evidence that breast cancer subtypes exhibit distinct dependencies on YAP-driven pathways, revealing potential therapeutic vulnerabilities. Targeting Hippo signaling alongside DNA repair in triple-negative breast cancer or combining YAP inhibition with metabolic blockade in luminal breast cancer holds significant potential to enhance treatment efficacy.

Keywords: CA3 (CIL56); DNA repair; apoptosis; autophagy; breast cancer; hippo pathway; metabolic reprogramming; proteomics; yes-associated protein (YAP).

Figure 1

(a) The effects of CA3 on the viability of MDA-MB231, MCF7, and MCF10A cells. (NC: negative control, * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001). (b) Representative optical microscopy images showing morphological changes in MDA-MB-231 and MCF7 cells after treatment with CA3 at their respective IC₅₀ concentrations, compared to control.

Figure 2

Representative Western blot images and relative expression levels of YAP in MDA-MB231 and MCF7 cells after 48 h of CA3 treatment. CA3 was applied at its IC₅₀ concentration: 0.5 μM for MDA-MB-231 and 1 μM for MCF7 (NC: negative control, * p < 0.0005, ** p < 0.0001).

Figure 3

Pathway enrichment analysis visualized using bubble plots for CA3-treated (a) MDA-MB231 and (b) MCF7 cells based on data from Gene Ontology (GO); BP: biological function, MF: molecular function, KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 4

KEGG pathway enrichment analysis using ExpressAnalyst for MDA-MB-231 (a) and MCF7 (b) cells. Each colored node represents a distinct functional pathway category (e.g., proteasome, ribosome, DNA repair, metabolism), as labeled directly on the graph. Small gray nodes indicate individual proteins associated with these pathways. Proteins marked with a red star (LIG1, POLD3, and PARP1) are selected for validation. (c) Western blot analysis of these proteins, with corresponding quantifications presented in bar graphs (NC: negative control, * p < 0.0005, ** p < 0.0001).

Figure 5

The effects of CA3 treatment for 48 h on apoptosis and cell cycle distribution in MDA-MB-231 and MCF7 cells. (a) Representative flow cytometry dot plots of Annexin V/PI staining. The percentage of cells in each quadrant (LC: live cell, EA: early apoptosis, LA: late apoptosis, and DC: death cell) is indicated. (b) Statistical analysis of early, late, and total apoptosis rates based on Annexin V/PI staining from three independent experiments. (c) Representative flow cytometric histograms showing DNA content profiles of cells stained with PI for cell cycle analysis. The proportions of cells in G1, S, and G2/M phases are indicated. (d) Statistical analysis of cell cycle distribution in G1, S, and G2/M phases. The percentages were calculated based on PI-stained DNA content using flow cytometry. Data represent mean values from three independent experiments (ns: not significant,* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 6

Representative Western blot images and relative expression levels of Beclin1 and LC3 protein expression in CA-3-treated MDA-MB231 and MCF7 cells (NC: negative control, ** p < 0.0001).