GRB2 promotes brain metastasis in HER2-positive breast cancer by regulating the Ras/MAPK pathway

Abstract

Brain metastasis is emerging as the most serious concern for breast cancer patients. HER2-positive breast cancer is more prone to undergo brain metastasis than other subtypes; notably, there has been little improvement in the treatment of brain metastasis .Our study confirmed the relevance of HER2 status to brain metastasis risk via clinical data analysis and revealed that exerts GRB2 tumorigenic effects by regulating the Ras/MAPK pathway in vivo and in vitro. Both an in situ injection model and a direct cerebral injection model were used to explore the ability of GRB2 to promote the brain metastasis. Results indicated that HER2- positive is a risk factor for brain metastasis according to clinical data. GRB2 enhances proliferation, migration, and invasion while suppressing apoptosis in HER2-positive breast cancer cells in vitro, primarily by regulating phosphorylation and alternative splicing of key proteins within the Ras/MAPK pathway. Notably, tumor cells were able to cross the blood‒brain barrier in both models assessed in this study. Thus, GRB2 is an oncogenic factor that contributes to the malignancy of HER2-positive breast cancer, GRB2 and HER2 can synergistically promote tumor cell penetration of the blood‒brain barrier and induce metastasis.

Keywords: Brain metastasis; GRB2; HER2-positive breast cancer; Ras/MAPK pathway.

Fig. 1

Association between GRB2/HER2 expression and prognosis in breast cancer. (A) Time to Brain Metastasis (TTBM) in breast cancer patients stratified by ER and HER2 Status. P-value was calculated by Mantel-Cox. **** denotes a P-value < 0.0001. (B) Overall Survival After Brain Metastasis (OSBM) in breast cancer patients stratified by ER and HER2 Status. (C) Overall Survival (OS) in breast cancer patients stratified by ER and HER2 Status. (D) GRB2 is a differentially expressed gene associated with brain metastasis in HER2-Positive breast cancer: Evidence from GSE43837. (E) Box plots illustrating differences in GRB2 expression levels between normal breast tissue(N) and breast cancer tissues(T). P-value < 0.001. (F) Overall Survival of breast cancer patients stratified by low or high GRB2 protein expression (lighter lines represent 95% confidence intervals). (G) Scatter plot of the correlation between GRB2 and HER2 expression. (H) Bubble plot of enriched pathways identified by GO: BP analysis. (I) Venn diagram illustrating the overlap between the MAPK pathway and the ERBB pathway. (J) Protein-protein interaction network analysis of key proteins.

Fig. 2

Results of cell proliferation, apoptosis, migration, and invasion across cell Lines. A-B. Expression levels of GRB2 and HER2.* denotes a P-value < 0.05. C-D. Differences in cell proliferation and apoptosis across experimental groups. E. Differences in cell migration and invasion capabilities.* denotes a P-value < 0.05.***denotes a P-value < 0.01. F. Microscopic examination of transwell migration and invasion assays. Blue staining indicates migrated or invaded cells, with a higher cell number reflecting stronger migration or invasion capability. G. Differential expression of proteins in the Ras/MAPK pathway by western blot. β-actin serves as the reference protein band, and the darkness of the target band indicates high expression of the target protein in this sample.

Fig. 3

GRB2 promotes HER2-positive tumor cells to cross the blood brain barrier via the Ras/MAPK pathway in vivo. (A) In situ injection model (model 1). (B) Fluorescence intensity analysis using an in vivo animal imaging system.areas of high fluorescence intensity indicate active tumor sites or metastases. (C) Measurement of ex vivo tumor size and characteristics. (D) Results of Hematoxylin & Eosin (H&E) staining (×400), TUNEL assay, and Immunohistochemistry (IHC). H&E staining: Blue-purple represents nuclear staining, highlighting cell nuclei for morphological analysis. TUNEL assay: Yellow staining indicates apoptotic cells, reflecting DNA fragmentation during apoptosis. Immunohistochemistry: Brown represents a moderate positive signal for the target protein, while yellow indicates a weak positive signal, demonstrating varying levels of target protein expression. (E) H&E staining (×400) of Metastatic sites in the Brain, Lung, and Liver. (F) H-Score evaluation of GRB2 and HER2 protein expression levels. (G) Expression levels of key proteins in the Ras/MAPK pathway by western blot. (H) Western blot analysis of key proteins in the Ras/MAPK pathway.

Fig. 4

GRB2 promotes HER2-positive tumor cells to cross the blood brain barrier and metastasize in a retrograde manner in vivo. (A) Direct cerebral injection model (Model 2). (B) H&E Staining (×400) of metastatic sites in the Brain, Lung, and Liver.

Fig. 5

Overlap analysis of RNA sequencing and fRIP-Seq results. (A) Heatmap visualization of GRB2 knockdown-induced up- or down-regulation of 5 Ras/MAPK pathway-related genes. (B) Heatmap reveals 16 Ras/MAPK signaling pathway-related genes with alternative splicing changes in GRB2 knockdown cells. (C) Venn diagram shows 1 overlapped gene between 11 Ras/MAPK pathway-related DEGs and 3196 GRB2-bound peaks genes from fRIP-seq data. (D) Venn diagram shows 1 overlapped gene between 16 Ras/MAPK pathway-related RASE and 40,709 GRB2-bound peaks genes from fRIP-seq data. (E) GRB2-binding peak genes of MESD. (F) GRB2-binding peak genes of KITLG.