Epigenetic reprogramming of epithelial mesenchymal transition in triple negative breast cancer cells with DNA methyltransferase and histone deacetylase inhibitors.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive neoplasia with no effective therapy. Our laboratory has developed a unique TNBC cell model presenting epithelial mesenchymal transition (EMT) a process known to be important for tumor progression and metastasis. There is increasing evidence showing that epigenetic mechanisms are involved in the activation of EMT. The objective of this study is to epigenetically reverse the process of EMT in TNBC by using DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi). METHODS: We evaluated the antitumor effect of three DNMTi and six HDACi using our TNBC cell model by MTT assay, migration and invasion assay, three dimensional culture, and colony formation assay. We then performed the combined treatment both in vitro and in vivo using the most potent DNMTi and HDACi, and tested the combined treatment in a panel of breast cancer cell lines. We investigated changes of EMT markers and potential signaling pathways associated with the antitumor effects. RESULTS: We showed that DNMTi and HDACi can reprogram highly aggressive TNBC cells that have undergone EMT to a less aggressive phenotype. SGI-110 and MS275 are superior to other seven compounds being tested. The combination of SGI with MS275 exerts a greater effect than single agent alone in inhibiting cell proliferation, motility, colony formation, and stemness of cancer cells. We also demonstrated that MS275 and the combination of SGI with MS275 exert in vivo antitumor effect. We revealed that the combined treatment synergistically reverses EMT through inhibiting EpCAM cleavage and WNT signaling, suppressing mutant p53, ZEB1, and EZH2, and inducing E-cadherin, apoptosis, as well as histone H3 tri-methylation. CONCLUSIONS: Our study showed that DNMTi and HDACi exert antitumor activity in TNBC cells partially by epigenetically reprograming EMT. Our findings strongly suggest that TNBC is sensitive to epigenetic therapies. Therefore, we propose a new strategy to treat TNBC by using the combination of SGI-110 with MS275, which exerts superior antitumor effects by simultaneously targeting multiple pathways.

Aspirin abrogates impairment of mammary gland differentiation induced by early in life second-hand smoke in mice.

Epidemiological studies show that there is limited evidence that tobacco smoking causes breast cancer in humans. In rodents, many tobacco smoke chemicals cause mammary gland tumors. This study evaluated the mammary gland differentiation in mice exposed to environmental cigarette smoke (ECS), using 3R4F Kentucky reference cigarettes, starting after birth and continuing daily for 10 weeks (total particulate exposure 95 mg/m3; CO 610 ppm). We also analyzed the effects of oral administration of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin (1600 mg/kg) or naproxen (320 mg/kg), on mammary gland differentiation, either in unexposed or ECS-exposed mice. The ECS exposure caused delay of mammary glands development. We speculate that this delay may result from aryl hydrocarbon receptor (AHR) signaling activation, which has an antiestrogenic effect and crosstalk to the estrogen metabolism pathway. Similarly, naproxen impaired gland differentiation in unexposed and ECS-exposed mice, while aspirin hindered its development only in unexposed mice. The lack of differentiation induced by the NSAIDs could be explained by their antiestrogenic effect through inhibition of aldo-keto reductases. In ECS-exposed animals, aspirin induced intense lobular formation, which could indicate that aspirin is counteracting the AHR signaling induced by ECS. Based on the differentiation induced by aspirin in ECS-exposed animals, we postulate that these mice would be less susceptible to mammary carcinogenesis. Our results suggest that exposure to smoke at an early age impairs the development of the mammary gland, thus resulting in a longer period of susceptibility and increased risk of breast cancer. However, addition of aspirin can abrogate this effect.

Development and characterization of two human triple-negative breast cancer cell lines with highly tumorigenic and metastatic capabilities.

Triple-negative breast cancer (TNBC) is a group of cancer with high diversity, limited therapies, and poor prognosis. TNBC cell lines and animal models provide effective tools for studies and drug discovery. Here, we report the development of two TNBC cell lines (XtMCF and LmMCF) based on our existing cell model that consists of normal breast epithelial cell line MCF10F, estradiol-transformed cells trMCF, and Boyden chamber-selected tumorigenic cells bsMCF. The XtMCF and LmMCF cell line were derived from xenograft and lung metastasis of bsMCF cells, respectively. The bsMCF, XtMCF, and LmMCF cells have undergone epithelial-mesenchymal transition (EMT), exhibiting a mesenchymal-like feature. In vivo studies showed XtMCF and LmMCF cells were highly tumorigenic and metastatic. The injection of 5 × 10(4) cells to CB17/SCID mice mammary fat pad produced xenografts in 9/9 mice and tumors reached 10 millimeters in diameter in 5 weeks. The injection of 1 × 10(6) XtMCF or 8 × 10(4) LmMCF cells into the mice tail vein was sufficient to form extensive lung metastases in 4 weeks. The two new cell lines exhibited CD44(+) /CD49f(+) and CD44(+) /EpCAM(+) cancer stem cell (CSC) characteristics, and the EGF-like domain of EpCAM was cleaved off. Together with the normal and early transformed counterparts, herein we provide a complete cancer model for the study of initiation, evolution, and identification of new therapeutics for TNBC. The finding that EGF-like domain of EpCAM was cleaved off in cells which have undergone EMT suggests this cleavage may be involved in the EMT process and the cancer stem cell properties of these cells.

Oxidized derivative of docosahexaenoic acid preferentially inhibit cell proliferation in triple negative over luminal breast cancer cells.

Omega-3 polyunsaturated fatty acids (PUFAs) exert an anticancer effect by affecting multiple cellular mechanisms leading to inhibition of proliferation and induction of apoptosis. It is well known that breast cancer comprises distinct molecular subtypes which differ in their responsiveness to therapeutic and preventive agents. We tested the hypothesis that n-3FA may preferentially affect triple-negative breast cancer cells for which no targeted intervention is presently available. The in vitro antiproliferative effects of n-3 PUFA docosahexaenoic acid (DHA) and its metabolite, 4-OH-DHA as well as its putative metabolite 4-OXO-DHA, were tested in five triple-negative human basal breast cell lines at different stages of transformation (MCF-10F, trMCF, bsMCF, MDA-MB-231, and BT-549) and three luminal breast cancer cell lines (MCF-7, T-47D, and SK-BR-3). Cell proliferation was measured with the tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. DHA and its oxidized derivatives significantly inhibited cell proliferation (20-90% reduction) of both basal and luminal breast cancer cell lines. The inhibitory effect was more pronounced on triple-negative basal breast cancer cell lines as compared to luminal breast cancer cell lines after 4-OXO-DHA treatment. Our data provide novel information regarding the preferential antitumor effect of oxidized derivatives of DHA on basal type breast cancer.

Expression and DNA methylation changes in human breast epithelial cells after bisphenol A exposure.

It has been suggested that xenoestrogens, a group of agents termed endocrine disruptors, may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. We previously demonstrated that the xenoestrogen, bisphenol A (BPA), was able to induce the transformation in vitro of human breast epithelial cells. The normal-like human breast epithelial cell line, MCF-10F, formed tubules in collagen (3-D cultures), although after treatment with BPA (10-5 M and 10-6 M BPA) the cells produced less tubules (73% and 80%, respectively) and some spherical masses (27% and 20%, respectively). In the present study, expression and DNA methylation analyses were performed in these cells after exposure to BPA. These cells showed an increased expression of BRCA1, BRCA2, BARD1, CtIP, RAD51 and BRCC3, all of which are genes involved in DNA repair, as well as the downregulation of PDCD5 and BCL2L11 (BIM), both of which are involved in apoptosis. Furthermore, DNA methylation analysis showed that the BPA exposure induced the hypermethylation of BCL2L11, PARD6G, FOXP1 and SFRS11, as well as the hypomethylation of NUP98 and CtIP (RBBP8). Our results indicate that normal human breast epithelial cells exposed to BPA have increased expressions of genes involved in DNA repair in order to overcome the DNA damage induced by this chemical. These results suggest that the breast tissue of women with BRCA1 or BRCA2 mutations could be more susceptible to the effects of BPA.