Interindividual variation contributes to differential PCB 126 induced gene expression in primary breast epithelial cells and tissues.

PCB 126 is a pervasive, dioxin-like chemical pollutant which can activate the aryl hydrocarbon receptor (AhR). Despite being banned from the market, PCB 126 can be detected in breast milk to this day. The extent to which interindividual variation impacts the adverse responses to this chemical in the breast tissue remains unclear. This study aimed to investigate the impact of 3 nM PCB 126 on gene expression in a panel of genetically diverse benign human breast epithelial cell (HBEC) cultures and patient derived breast tissues. Six patient derived HBEC cultures were treated with 3 nM PCB 126. RNAseq was used to interrogate the impact of exposure on differential gene expression. Gene expression changes from the top critical pathways were confirmed via qRT-PCR in a larger panel of benign patient derived HBEC cultures, as well as in patient-derived breast tissue explant cultures. RNAseq analysis of HBEC cultures revealed a signature of 144 genes significantly altered by 3 nM PCB 126 treatment. Confirmation of 8 targets using a panel of 12 HBEC cultures and commercially available breast cell lines demonstrated that while the induction of canonical downstream target gene, CYP1A1, was consistent across our primary HBECs, other genes including AREG, S100A8, IL1A, IL1B, MMP7, and CCL28 exhibited significant variability across individuals. The dependence on the activity of the aryl hydrocarbon receptor was confirmed using inhibitors. PCB 126 can induce significant and consistent changes in gene expression associated with xenobiotic metabolism in benign breast epithelial cells. Although the induction of most genes was reliant on the AhR, significant variability was noted between genes and individuals. These data suggest that there is a bifurcation of the pathway following AhR activation that contributes to the variation in interindividual responses.

HER2 Protein Overexpression and Gene Amplification in Tubo-Ovarian High-grade Serous Carcinomas.

Most tubo-ovarian high-grade serous carcinomas (TO-HGSC) are diagnosed in advanced stages. Although the majority of patients achieve initial remission with cytoreductive surgery and chemotherapy, mortality rate remains high due to recurrent/progressive disease. The addition of trastuzumab to carboplatin-paclitaxel improved progression-free survival of patients with human epidermal growth factor receptor 2 (HER2)-positive uterine serous carcinoma. After this encouraging result of transtuzumab in HER2-positive uterine serous carcinoma, we aimed to determine the frequency of HER2 overexpression/amplification in TO-HGSC and reveal the utility of 2018 ASCO/CAP HER2 testing guideline in breast cancer for TO-HGSC. For 100 cases, HER2 protein expression was assessed by immunohistochemistry and scored from 0 to 3+ according to 2018 ASCO/CAP HER2 testing guideline. HER2 gene amplification was assessed by florescence in situ hybridization for all the 2+ and 3+ cases as well as 5 of the 0/1+ cases. Among 100 cases, immunohistochemistry scores were 0/1+ in 81 cases, 2+ in 18 cases and 3+ in 1 case. By florescence in situ hybridization, the only 3+ case and 1 of the 2+ cases were HER2-amplified and all 5 of the 0/1+ cases were HER2 nonamplified. Subclonal HER2 overexpression/amplification was identified in 1 of the neoadjuvant cases comprising <10% of the entire tumor. In summary, HER2 overexpression/amplification was found in 2% of TO-HGSC. The 2018 ASCO/CAP HER2 testing guideline in breast cancer can be utilized for TO-HGSC. Future studies are needed to explore HER2-targeted therapies in TO-HGSC and expand the patient population who may benefit from HER2-targeted therapies such as patients with activating mutations in HER2 gene without overexpression/amplification.

Inhibitory activity of YKL-40 in mammary epithelial cell differentiation and polarization induced by lactogenic hormones: a role in mammary tissue involution.

We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells.

GATA3 inhibits breast cancer metastasis through the reversal of epithelial-mesenchymal transition.

GATA3, a transcription factor that regulates T lymphocyte differentiation and maturation, is exclusively expressed in early stage well differentiated breast cancers but not in advanced invasive cancers. However, little is understood regarding its activity and the mechanisms underlying this differential expression in cancers. Here, we employed GATA3-positive, non-invasive (MCF-7) and GATA3-negative, invasive (MDA-MB-231) breast cancer cells to define its role in the transformation between these two distinct phenotypes. Ectopic expression of GATA3 in MDA-MB-231 cells led to a cuboidal-like epithelial phenotype and reduced cell invasive activity. These cells also increased E-cadherin expression but decreased levels of vimentin, N-cadherin, and MMP-9. Further, MDA-MB-231 cells expressing GATA3 grew smaller primary tumors without metastasis compared with larger metastatic tumors derived from control MDA-MB-231 cells in xenografted mice. GATA3 was found to induce E-cadherin expression through binding GATA-like motifs located in the E-cadherin promoter. Blockade of GATA3 using small interfering RNA gene knockdown in MCF-7 cells triggered fibroblastic transformation and cell invasion, resulting in distant metastasis. Studies of human breast cancer showed that GATA3 expression correlated with elevated E-cadherin levels, ER expression, and long disease-free survival. These data suggest that GATA3 drives invasive breast cancer cells to undergo the reversal of epithelial-mesenchymal transition, leading to the suppression of cancer metastasis.