Non-cancerous Mammary Epithelial Cells Research Articles

Abstract 28: Chemoprevention strategies to target abnormal imprinting in high-risk African American women who do or do not carry a BRCA1 mutation

Abstract Background: Genomic imprinting is an inherited form of parent-of-origin-specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Methods/Results: Here we show that KCNK9 has imprinted expression in breast tissue and provide the first identification of the KCNK9 DMR and DMR-associated open chromatin. Our studies show that loss of KCNK9 regulatory methylation and imprinted expression occurs in 75% of triple-negative breast cancer (TNBC) and loss of methylation occurs in non-cancerous tissue from high-risk women. Functional studies show that TASK3 protein regulates mitochondrial membrane potential and apoptosis-sensitivity. In live TNBC cells and non-cancerous mammary epithelial cells from high-risk women, loss of KCNK9 DMR methylation predicts increased mitochondrial membrane potential (p=0.007). African American women have a significantly higher frequency of loss of KCNK9 imprinting than do risk-matched Caucasian women (p=0.005). African American women with BRCA1 mutation have the highest frequency of loss (p<0.0001). Clinical Prevention: This is the first identification of the KCNK9 DMR and demonstration that loss of methylation and overexpression of TASK3 increases mitochondrial membrane potential and promotes apoptosis resistance. Low-toxicity inhibitors of TASK3 are in clinical use and can be readily re-purposed to provide cost effective chemoprevention. These inhibitors include K+ inhibitors (developed for cardiac indications) and Schwann Pepper. Our studies identify TASK3 as a “drugable”, low-toxicity target that can be readily employed as a novel target for prevention and treatment of TNBC, particularly in high-risk African American women. Citation Format: Victoria Seewaldt, David Skaar, Eric Dietze, Shraddha Desai, Randy Jirtle. Chemoprevention strategies to target abnormal imprinting in high-risk African American women who do or do not carry a BRCA1 mutation. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 28. doi:10.1158/1538-7445.CANSUSC14-28

Abstract C65: Loss of imprinting of KCNK9 in African American women with triple-negative breast cancer

Abstract Background: It has been long suspected that prenatal nutrition and environmental toxins play a role in promoting breast cancer. Genomic imprinting is an inherited form of parent-of-origin-specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. Recent studies show that exposure to the environmental plastic bis-phenol A and vitamin D deficiencies promote loss of normal imprinting. African American (AA) women are known to have vitamin D deficiency at a significantly higher frequency than women of Northern European descent. Here we investigated a mechanism that may explain the higher rate of aggressive triple-negative breast cancers (TNBC) in AA women. Methods and Results: KCNK9 encodes TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. Our studies have identified a differentially methylated region (DMR) for KCNK9 and shown monoallelic expression of KCNK9 in breast tissue. Using patient samples, we show loss of imprinted expression in tumor cells, loss of KCNK9 regulatory methylation in 75% of TNBC samples, and loss of methylation in non-cancerous tissue from high-risk AA women. In live TNBC cells and non-cancerous mammary epithelial cells from high-risk women, loss of KCNK9 DMR methylation correlates with increased mitochondrial membrane potential. Significance: This is the first identification of the KCNK9 DMR and demonstration that loss of methylation and overexpression of TASK3 increases mitochondrial membrane potential and promotes apoptosis resistance. These novel findings regarding KCNK9 loss of methylation and its disruption in a subset of tumors may lead to new approaches in diagnosis and targeted therapy of breast cancer. Citation Format: Shraddha Desai, David Skaar, Eric Dietze, Adrian Ambrose, Randy Jirtle, Victoria Seewaldt. Loss of imprinting of KCNK9 in African American women with triple-negative breast cancer. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C65. doi:10.1158/1538-7755.DISP13-C65

Abstract 3219: Synergistic effect between Ganoderma lucidum (Reishi) and lapatinib in HER2+ inflammatory breast cancer cells

Abstract Inflammatory Breast Cancer (IBC) is the most lethal and rare form of breast cancer. It is characterized by rapid progression, local and distant metastases, younger age of onset, and lower overall survival compared with other breast cancers. Although IBC, like non-IBC breast cancers, is a heterogeneous disease and can occur as any of the five molecular breast cancer subtypes, they are most commonly either triple negative; or ER-, PR- and HER2+ overexpressed. There is no specific IBC treatment; thus, it seems feasible to find a therapeutic for this deadly disease. Our published data demonstrates that Ganoderma lucidum (Reishi) inhibits the viability of the triple negative IBC SUM149 cell line, but not of MCF10A noncancerous mammary epithelial cells. Consequently, in this study, we aimed to investigate Reishi effects in the viability of the ER-, PR-, HER2+ IBC cells lines SUM190 and KPL4. Also we studied the effects of Reishi on IBC cells when treated in complement with the HER2 Tyrosine Kinase Inhibitor, lapatinib (Tykerb®). Our hypothesis is that Reishi chemosensitizes IBC cells to lapatinib therapy. Herein, SUM190 and KPL-4 cells were treated with increasing concentrations of lapatinib and/or Reishi for 24 or 72h. Our results demonstrate that SUM190 and KPL-4 cells are sensitive to Reishi treatment. The combination of lapatinib plus Reishi reduced cell viability of SUM190 cells to 70% when treated with a concentration of 0.5mg/mL Reishi for 24h. Also, our data shows a reduction in cell viability of 94% in KPL-4 cells treated with lapatinib plus Reishi for 72h. We are currently directing our efforts to continue studying the contribution of Reishi in IBC cells when treated with lapatinib after 24 or 72h. Our results provide evidence that Reishi inhibits cancer cell viability and chemosensitizes IBC cells to lapatinib therapy, highlighting its anti-IBC-therapeutic potential. We thank Dr. Kurebayashi (Kawasaki Medical School, Japan) for providing KPL-4 cells. This project was sponsored by NIH/NCI #1F31CA174307 to ISA, Title V PPOHA US Department of Education #P031M105050 to UCC, NIH/RCMI #G12MD007583 to UCC, NIH/INBRE #5P20GM103475 to UPR/UCC and a research donation from the Commonwealth of Puerto Rico to UCC's University Center of Integral and Complementary Medicine (CUMIC)/MMM. Citation Format: Yismeilin Feliz-Mosquea, Ivette Suárez-Arroyo, Luis A. Cubano, Michelle M. Martínez-Montemayor. Synergistic effect between Ganoderma lucidum (Reishi) and lapatinib in HER2+ inflammatory breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3219. doi:10.1158/1538-7445.AM2014-3219

Differential expression of bitter taste receptors in non-cancerous breast epithelial and breast cancer cells

The human bitter taste receptors (T2Rs) are chemosensory receptors that belong to the G protein-coupled receptor superfamily. T2Rs are present on the surface of oral and many extra-oral cells. In humans 25 T2Rs are present, and these are activated by hundreds of chemical molecules of diverse structure. Previous studies have shown that many bitter compounds including chloroquine, quinidine, bitter melon extract and cucurbitacins B and E inhibit tumor growth and induce apoptosis in cancer cells. However, the existence of T2Rs in cancer cell is not yet elucidated. In this report using quantitative (q)-PCR and flow cytometry, we characterized the expression of T2R1, T2R4, T2R10, T2R38 and T2R49 in the highly metastatic breast cancer cell line MDA-MB-231, poorly metastatic cell line MCF-7, and non-cancerous mammary epithelial cell line MCF-10A. Among the 5 T2Rs analyzed by qPCR and flow cytometry, T2R4 is expressed at 40–70% in mammary epithelial cells in comparison to commonly used breast cancer marker proteins, estrogen receptor and E-cadherin. Interestingly, the expression of T2R4 was downregulated in breast cancer cells. An increase in intracellular calcium mobilization was observed after the application of bitter agonists, quinine, dextromethorphan, and phenylthiocarbamide that are specific for some of the 5 T2Rs. This suggests that the endogenous T2Rs expressed in these cells are functional. Taken together, our novel findings suggest that T2Rs are differentially expressed in mammary epithelial cells, with some T2Rs downregulated in breast cancer cells.

Breast stromal fibroblasts from histologically normal surgical margins are pro‐carcinogenic

There is evidence that normal breast stromal fibroblasts (NBFs) suppress tumour growth, while cancer-associated fibroblasts (CAFs) promote tumourigenesis through functional interactions with tumour cells. Little is known about the biology and the carcinogenic potential of stromal fibroblasts present in histologically normal surgical margins (TCFs). Therefore, we first undertook gene expression analysis on five CAF/TCF pairs from breast cancer patients and three NBF samples (derived from mammoplasties). This comparative analysis revealed variation in gene expression between these three categories of cells, with a TCF-specific gene expression profile. This variability was higher in TCFs than in their paired CAFs and also NBFs. Cytokine arrays show that TCFs have a specific secretory cytokine profile. In addition, stromal fibroblasts from surgical margins expressed high levels of α-SMA and SDF-1 and exhibited higher migratory/invasiveness abilities. Indirect co-culture showed that TCF cells enhance the proliferation of non-cancerous mammary epithelial cells and the epithelial-to-mesenchymal transition of breast cancer cells. Moreover, TCF and CAF cells increased the level of PCNA, MMP-2 and the phosphorylated/activated form of Akt in normal breast luminal fibroblasts in a paracrine manner. Furthermore, TCFs were able to promote the formation and growth of humanized orthotopic breast tumours in nude mice. Interestingly, these TCF phenotypes and the extent of their effects were intermediate between those of NBFs and CAFs. Together, these results indicate that stromal fibroblasts located in non-cancerous tissues exhibit a tumour-promoting phenotype, indicating that their presence post-surgery may play important roles in cancer recurrence. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 1992: Ganoderma lucidum (Reishi) induces autophagy in inflammatory breast cancer by regulation of the mTOR signaling pathway

Abstract The control of cell growth by the mammalian target of rapamycin (mTOR) signaling pathway and autophagy are relevant to disease, as altered regulation of either pathway may result in tumorigenesis. Autophagy is a regulated pathway involving the lysosomal degradation of cytoplasmic components and organelles. Activation of the human autophagy-initiation kinase (ULK1) is directly controlled by mTOR and is a critical part of the autophagy machinery. In this study we test the autophagy inducing effects of the medicinal mushroom Ganoderma lucidum (Reishi) in the patient derived inflammatory breast cancer (IBC) cell line SUM-149 and in the non-cancerous mammary epithelial cell line MCF10A. IBC is a rare, lethal and aggressive form of locally advanced breast cancer that has a poor prognosis and no targeted therapy. Herein, we demonstrate that Reishi modulates the expression of autophagy related molecules depending on treatment time. At 2h post-Reishi treatment SUM-149 cells show that ULK1, Atg5, Atg12, Beclin1 and the microtubule-associated protein 1 light chain 3 (LC3) are α1.2 fold upregulated. Furthermore, autophagosomal structures were identified in SUM-149 but not in MCF10A Reishi treated cells. At 4h post-Reishi Atg5 and Atg12 (required for phagophore formation) are ∼1.5 fold upregulated, while at 6h LC3 expression (involved in autophagosome formation) is ∼2 fold upregulated compared to vehicle. We also show that autophagy induction in SUM-149 cells is mediated via downregulation of the PI3K/mTOR signaling pathway. Reishi downregulates PI3K (p85 and p110), mTOR phosphorylation and reduces the expression of Raptor, which is the regulatory subunit of mTORC1, the complex that directly regulates proteins involved in autophagy. Rictor, the regulatory subunit of the mTORC2 complex, which inhibits autophagy indirectly through Akt is also downregulated by Reishi. We demonstrate that Reishi has great potential to be used as an anti-IBC-therapeutic. This project was sponsored by Title V PPOHA grant number P031M105050 from the US Dept of Education to UCC, and NIH/RCMI 2G12RR003035 to UCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1992. doi:1538-7445.AM2012-1992

Ganoderma lucidum (Reishi) Inhibits Cancer Cell Growth and Expression of Key Molecules in Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the lymphatic system and absence of a palpable tumor mass. Different from other metastatic breast cancer cells, IBC cells invade by forming tumor spheroids that retain E-cadherin-based cell–cell adhesions. Herein we describe the potential of the medicinal mushroom Ganoderma lucidum (Reishi) as an attractive candidate for anti-IBC therapy. Reishi contains biological compounds that are cytotoxic against cancer cells. We report the effects of Reishi on viability, apoptosis, invasion, and its mechanism of action in IBC cells (SUM-149). Results show that Reishi selectively inhibits cancer cell viability although it does not affect the viability of noncancerous mammary epithelial cells. Apoptosis induction is consistent with decreased cell viability. Reishi inhibits cell invasion and disrupts the cell spheroids that are characteristic of the IBC invasive pathology. Reishi decreases the expression of genes involved in cancer cell survival and proliferation (BCL-2, TERT, PDGFB), and invasion and metastasis (MMP-9), whereas it increases the expression of IL8. Reishi reduces BCL-2, BCL-XL, E-cadherin, eIF4G, p120-catenin, and c-Myc protein expression and gelatinase activity. These findings suggest that Reishi is an effective anti-IBC therapeutic.

Mitochondria of highly metastatic breast cancer cell line MDA-MB-231 exhibits increased autophagic properties

Autophagy is a cellular housekeeping process that removes damaged or unwanted cellular components and recycles them to build new constituents. It is essential for tumor growth under adverse environment. Mitochondria play an important role in the formation of autophagosome and its subsequent docking and fusion with lysosome. To understand the contribution of mitochondria to the regulation of homeostatic autophagy in cancer cells, we used the transmitochondrial cytoplasmic hybrid (cybrid) model. Cybrid system allowed us to compare mitochondria from different cell types including highly metastatic breast cancer cell line MDA-MB-231 (c231), less metastatic breast cancer cell lines: MDA-MB-436 (c436) and MDA-MB-468 (c468), as well as non-cancerous mammary epithelial cell MCF-10A (c10A) in a defined nuclear background. The c231 exhibited lower LC3-II levels but higher ratio of LC3-II/LC3-I than c436, c468 and c10A. In addition, c231 displayed more punctate LC3-positive cells and had lower levels of sequestosome 1 (p62/SQSTM1) than other cybrids. These suggested that mitochondria could contribute to the increased autophagy and autophagic flux in metastatic cancer. This increased autophagy was found to be non-selective autophagy instead of selective mitophagy since LC3 puncta in c231 did not co-localize with mitochondria labeled by Mitotracker red or Tomm 20. The promotion of mitochondrial permeability transition (MPT) in c231 also contributed to increased autophagy. Block of MPT by the inhibition of low-conductance stage of MPT pores resulted in a decrease of LC3 puncta in c231. These results suggested that mitochondria from highly metastatic breast cancer cell line MDA-MB-231 can promote homeostatic autophagy of cancer through opening low-conductance MPT pores.

Paclitaxel resistance in untransformed human mammary epithelial cells is associated with an aneuploidy-prone phenotype

Despite its increasing clinical use, almost no data are currently available about paclitaxel effects on non-cancerous mammary epithelial cells. We have previously established paclitaxel-resistant sub-cell lines (paclitaxel-surviving populations, PSPs; n=20), and sensitive controls (control clones, CCs; n=10), from the untransformed human mammary epithelial cell line HME1. In this study, we aimed to establish whether paclitaxel resistance was associated with a modified sensitivity to paclitaxel-induced aneuploidy. For this purpose, we analysed basal and paclitaxel-induced chromosome missegregation, apoptosis and aberrant spindle multipolarisation as well as microtubular network composition for each subline. PSP sublines showed higher basal and paclitaxel-induced chromosome missegregation than the CC sublines. This phenomenon was associated with resistance to paclitaxel-induced apoptosis. No significant difference in paclitaxel-induced spindle pole abnormalities between CC and PSP sublines was found. Besides, we showed that a majority of PSPs display a constitutively disrupted microtubular network composition due to aberrant tubulin expression and post-translational modifications. These results clearly indicate that paclitaxel resistance in untransformed human mammary epithelial cells is related to an increased susceptibility to acquire aneuploidy in response to this agent. The consequences of these paclitaxel-associated alterations could be deleterious as they can potentially trigger tumorigenesis.

Steroid regulation of epithelial sodium channels (ENaC) in mammary epithelial cells

The epithelial sodium channel (ENaC) is important in lactating mammary cells for regulating the Na+ content of milk, while in cancerous mammary cells ENaC may play a role in oncogenesis. The goal of this study is to determine how steroids and/or the serum-and glucocorticoid induced kinase (SGK1), affect ENaC expression in mammary epithelial cells (MEC). Using real-time PCR we determined ENaC (α, β, γ ) mRNA induction in response to steroid hormones in 3 MEC lines. Under basal conditions, mouse HC11 MEC expressed α and γ ENaC mRNA; these levels were 10-fold and 2-fold lower than those in cortical collecting duct cells. In HC11, dexamethasone (dex) (1μM, 24h) induced ENaC mRNA levels 3-fold and γ ENaC mRNA levels 12-fold (βENaC was too low for detection). Next we determined the effects of changes in SGK1 expression/activity on ENaC mRNA levels. We previously reported that in CCD cells SGK1 upregulates α and βENaC mRNA levels. In contrast, in HC11 cells, SGK1 most significantly increased γ ENaC mRNA as compared to cells expressing dominant negative SGK1. This suggests that the regulation of ENaC mRNA levels by SGK1 is cell specific. We also tested the effect of dex in two human MEC lines. In MCF10A, dex induced all three subunits: α(5-fold), β(18-fold), and γ (15-fold). In the cancerous T47D, both the β and γ subunit were induced by dex (2- and 10-fold), while surprisingly αENaC was unchanged. In summary, these data reveal that MEC express ENaC subunits, which are regulated by steroid hormones in a subunit and cell-specific manner. ENaC gene expression in the cancerous MEC is regulated differently than in non-cancerous MEC.