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The scaffolding adapter GAB2 maps to a region (11q13-14) commonly amplified in human breast cancer, and is overexpressed in breast cancer cell lines and primary tumors, but its functional role in mammary carcinogenesis has remained unexplored. We found that overexpression of GAB2 (Grb2-associated binding protein 2) increases proliferation of MCF10A mammary cells in three-dimensional culture. Coexpression of GAB2 with antiapoptotic oncogenes causes lumenal filling, whereas coexpression with Neu (also known as ErbB2 and HER2) results in an invasive phenotype. These effects of GAB2 are mediated by hyperactivation of the Shp2-Erk pathway. Furthermore, overexpression of Gab2 potentiates, whereas deficiency of Gab2 ameliorates, Neu-evoked breast carcinogenesis in mice. Finally, GAB2 is amplified in some GAB2-overexpressing human breast tumors. Our data suggest that GAB2 may be a key gene within an 11q13 amplicon in human breast cancer and propose a role for overexpression of GAB2 in mammary carcinogenesis. Agents that target GAB2 or GAB2-dependent pathways may be useful for treating breast tumors that overexpress GAB2 or HER2 or both., Author(s): Mohamed Bentires-Alj (corresponding author) [1, 2]; Susana G Gil [1]; Richard Chan [1]; Zhigang C Wang [3]; Yongping Wang [1]; Naoko Imanaka [1]; Lyndsay N Harris [4]; Andrea Richardson [...]

Author(s): Haihua Gu (corresponding author) [1]; Kan Saito [2]; Lori D. Klaman [1]; Junqing Shen [1]; Tony Fleming [2]; YongPing Wang [1]; Joanne C. Pratt [3]; Guosheng Lin [1]; Bing [...]

Metastasis is responsible for the death of most breast cancer patients. Robo1 has been implicated as a tumor suppressor for various cancers including breast cancer. However, it is not well understood how Robo1 expression is regulated during tumorigenesis. In this study, we uncovered that the transmembrane proline rich γ-carboxyglutamic acid protein 4 (PRRG4) promotes breast cancer metastasis by downregulating Robo1. Analysis of mRNA expression data in The Cancer Genome Atlas and immunohistochemistry assay on breast tumor samples showed that PRRG4 expression was higher in breast tumors than in normal breast tissues. Experiments with PRRG4 knockdown and overexpression revealed that PRRG4 promoted migration and invasion of breast cancer cells, and enhanced metastasis in an experimental metastasis model. Mechanistically, we found that PRRG4 via its LPSY and PPPY motifs recruited the E3 ubiquitin ligase NEDD4, which induced ubiquitination and degradation of Robo1, thus contributing to migration and invasion of breast cancer cells. In addition, PRRG4 interacted with and enhanced protein tyrosine kinase Src and FAK activation. Overall, our data support a model that PRRG4 via NEDD4 downregulates the Robo1, resulting in the activation of Src and FAK and promoting breast cancer metastasis. PRRG4 may be a novel target for treating metastatic breast cancer.

Androgen receptor (AR) is widely expressed in different subtypes of breast cancer (BC). However, it is unclear how AR functions in HER2 positive (+) BC. Knockdown of AR with shRNAs and a new generation anti-androgen drug, Enzalutamide, were used to explore the involvement of AR on the growth of HER2 + BC cells (HCC1954 and SKBR3). AR shRNA or Enzalutamide inhibited the growth of SKBR3 cells at a similar extend compared to trastuzumab, an approved HER2 targeted drug. Combining Enzalutamide with trastuzumab further decreased the growth of HCC1954 and SKBR3 cells compared with single agent alone in vitro. Biochemical analysis revealed that inhibiting AR resulted in decreased HER2 phosphorylation and activation of Erk and Akt, without affecting the HER2 and HER3 expression. The in vivo efficacy of Enzalutamide was further tested using the HCC1954 xenograft model. Enzalutamide impaired the growth of HCC1954 tumor at a level comparable to that by trastuzumab. Enzalutamide decreased Ki67 staining and increased activated caspase3 staining compared with vehicle control in HCC1954 tumors. Our results indicate AR plays an important role in promoting the growth of HER2 + BC by cross-talking with the HER2 signaling. AR drug may be used as an alternative second line therapy for treating HER2 + BC.

Transformation of fibroblasts by V-SEA involves activation of the ERK and phosphatidylinositol 3-kinase (PI3K) pathways. Effector proteins that are key mediators of the ERK and PI3K pathways, namely Grb2, the tyrosine phosphatase, SHP2 and PI3K, interact with the two phosphotyrosines found in the bidentate motif in the carboxy-terminal region of V-SEA. Genetic analysis demonstrated that while Y557 was a primary binding site and thus activator of the PI3K-Akt pathway, Y564 also contributed to the activation of this pathway. Y564 was located within a Grb2-binding motif, this raised the possibility that a protein that associated with Grb2 might be important for this PI3K activation. The scaffolding proteins Gab1 and/or Gab2 were candidates for this role. In this report, we demonstrate that V-SEA preferentially interacts with Gab2. Furthermore by using Gab2 null fibroblasts, we demonstrate that Gab2 is essential for fibroblast transformation by V-SEA. Using mutant forms of Gab2, we show that activation of the PI3K-Akt pathway via Gab2 is required for V-SEA-induced transformation. However, efficient fibroblast transformation also requires the SHP2 interaction site on Gab2.

Grb2-associated binder 2 (Gab2) is a recently identified member of the Gab/Daughter of sevenless family of docking proteins, which localize, amplify and integrate signaling pathways activated by various receptors including receptor tyrosine kinases (RTKs). To date, Gab2 signaling has been primarily investigated in hematopoietic cells. Here we report marked overexpression of Gab2 in a subset of breast cancer cell lines relative to normal breast epithelial strains and a trend for increased Gab2 expression in estrogen receptor (ER)-positive lines. Overexpression relative to normal ductal epithelium was also observed in some primary breast cancers. In MCF-7 breast cancer cells Gab2 was markedly tyrosine phosphorylated in response to heregulin and also following EGF, insulin or bFGF administration, indicating that a variety of RTKs implicated in breast cancer development or progression couple to this docking protein. In hormone-responsive breast cancer cells, GAB2 mRNA and protein expression were induced by estradiol in a manner sensitive to the pure anti-estrogen ICI 182780, indicating that this regulation is mediated via the ER. Gab2 therefore represents a novel link between steroid and growth factor signaling in breast cancer, and when overexpressed, may modulate the sensitivity of breast cancer cells to these important growth regulators.