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Mucin 1 (MUC1) has been regarded as an ideal target for cancer treatment, since it is overexpressed in a variety of different cancers including the majority of breast cancer. However, there are still no approved monoclonal antibody drugs targeting MUC1. In this study, we generated a humanized MUC1 (HzMUC1) antibody from our previously developed MUC1 mouse monoclonal antibody that only recognizes MUC1 on the surface of tumor cells. Furthermore, an antibody-drug conjugate (ADC) was generated by conjugating HzMUC1 with monomethyl auristatin (MMAE), and the efficacy of HzMUC1-MMAE on the MUC1-positive HER2+ breast cancer in vitro and in 'Xenograft' model was tested. Results from western blot analysis and immunoprecipitation revealed that the HzMUC1 antibody did not recognize cell-free MUC1-N in sera from breast cancer patients. Confocal microscopy analysis showed that HzMUC1 antibody bound to MUC1 on the surface of breast cancer cells. Results from mapping experiments suggested that HzMUC1 may recognize an epitope present in the interaction region between MUC1-N and MUC1-C. Results from colony formation assay and flow cytometry demonstrated that HzMUC1-MMAE significantly inhibited cell growth by inducing G2/M cell cycle arrest and apoptosis in trastuzumab-resistant HER2-positive breast cancer cells. Meanwhile, HzMUC1-MMAE significantly reduced the growth of HCC1954 xenograft tumors by inhibiting cell proliferation and enhancing cell death. In conclusion, our results indicate that HzMUC1-ADC is a novel therapeutic drug that can overcome trastuzumab resistance of breast cancer. HzMUC1-ADC should also be an effective therapeutic drug for the treatment of different MUC1-positive cancers in clinic., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although endocrine therapies targeting estrogen receptor α (ERα) are effective in managing ER positive (+) breast cancer, many patients have primary resistance or develop resistance to endocrine therapies. In addition, ER+ breast cancer with PIK3CA activating mutations and 11q13-14 amplification have poor survival with unclear mechanism. We uncovered that higher expression of deubiquitinase USP35, located in 11q14.1, was associated with ER+ breast cancer and poor survival. Estrogen enhanced USP35 protein levels by downregulating USP35-targeting miRNA-140-3p and miRNA-26a-5p. USP35 promoted the growth of ER+ breast cancer in vitro and in vivo, and reduced the sensitivity of ER+ breast cancer cells to endocrine therapies such as tamoxifen and fulvestrant. Mechanistically, USP35 enhanced ERα stability by interacting and deubiquitinating ERα, and transcriptional activity of ERα by interacting with ERα in DNA regions containing estrogen response element. In addition, AKT, a key effector of PI3K, phosphorylated USP35 at Serine613, which promoted USP35 nuclear translocation, ERα transcriptional activity, and the growth of ER+ breast cancer cells. Our data indicate that USP35 and ERα form a positive feedback loop in promoting the growth of ER+ breast cancer. USP35 may be a treatment target for ER+ breast cancer with endocrine resistance or with PIK3CA mutations or hyperactivation of the PI3K pathway.

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.

Cholesterol is a risk factor for breast cancer. However, it is still unclear whether the cholesterol biosynthesis pathway plays any significant role in breast carcinogenesis. 24-Dehydrocholesterol reductase (DHCR24) is a key enzyme in the cholesterol synthesis pathway. Although DHCR24 is reported to have different functions in different cancers, it is not clear whether DHCR24 is involved in breast cancer. In this study, we found that DHCR24 expression was higher in breast cancer especially in luminal and HER2 positive breast cancer tissues compared with normal breast. Changes in DHCR24 expression altered cellular cholesterol content without affecting the adherent growth of breast cancer cells. However, DHCR24 knockdown reduced whereas DHCR24 overexpression enhanced breast cancer stem-like cell populations such as mammosphere and aldehyde dehydrogenase positive cell numbers. In addition, DHCR24 overexpression increased the expression of the Hedgehog pathway-regulated genes. Treating DHCR24 overexpressing breast cancer cell lines with the Hedgehog pathway inhibitor GANT61 blocked DHCR24-induced mammosphere growth and increased mRNA levels of the Hedgehog regulated genes. Furthermore, expression of a constitutively activated mutant of Smoothened, a key hedgehog signal transducer, rescued the decreases in mammosphere growth and Hedgehog regulated gene expression induced by knockdown of DHCR24. These results indicate that DHCR24 promotes the growth of breast cancer stem-like cells in part through enhancing the Hedgehog signaling pathway. Our data suggest that cholesterol contribute to breast carcinogenesis by enhancing Hedgehog signaling and cancer stem-like cell populations. Enzymes including DHCR24 involved in cholesterol biosynthesis should be considered as potential treatment targets for breast cancer., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

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.

ErbB family of the receptor protein-tyrosine kinase plays an important role in the progression of human cancers including breast cancer. Finding protein-tyrosine phosphatase (PTPs) that can specifically regulate the function of ErbB should help design novel therapies for treatment. By performing a small interfering RNA screen against 43 human PTPs, we find that knockdown of protein-tyrosine phosphatase PTPN9 significantly increases ErbB2 tyrosyl phosphorylation in the SKBR3 breast cancer cell line. In addition, knockdown of PTPN9 expression also enhances tyrosyl phosphorylation of the ErbB1/epidermal growth factor receptor (EGFR) in the MDA-MB-231 breast cancer cell line. Conversely, increasing expression of PTPN9 wild type (WT) inhibits tyrosyl phosphorylation of ErbB2 and EGFR. To test whether ErbB2 and EGFR are substrates of PTPN9, PTPN9 WT, and a substrate trapping mutant (PTPN9 DA) are overexpressed in SKBR3 and MDA-MB-231 cells. Compared with vector control, expression of PTPN9 WT significantly inhibits whereas expression of PTPN9 DA dramatically enhances tyrosyl phosphorylation of ErbB2 and EGFR, respectively. In contrast, expression of PTPN9 WT or DA mutant does not affect tyrosyl phosphorylation of ErbB3 and Shc. Importantly, coimmunoprecipitation and glutathione S-transferase fusion protein pulldown experiments show that tyrosol-phosphorylated ErbB2 or EGFR is preferentially associated with PTPN9 DA compared with PTPN9 WT, indicating that ErbB2 and EGFR are substrates of PTPN9. Furthermore, PTPN9 WT expression specifically impairs EGF-induced STAT3 and STAT5 activation, and inhibits the cell growth in soft agar. Last, PTPN9 WT expression also reduces invasion and MMP2 expression of MDA-MB-231 cells. Our data suggest PTPN9 as a negative regulator of breast cancer cells by targeting ErbB2 and EGFR and inhibiting STAT activation.

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.

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.