Non-malignant Breast Epithelial Cell Line Research Articles

Effectiveness of hypofractionated and normofractionated radiotherapy in a triple‐negative breast cancer model

Breast cancer (BC) is one of the most diagnosed malignant carcinomas in women with a triple-negative breast cancer (TNBC) phenotype being correlated with poorer prognosis. Fractionated radiotherapy (RT) is a central component of breast cancer management, especially after breast conserving surgery and is increasingly important for TNBC subtype prognosis. In recent years, moderately hypofractionated radiation schedules are established as a standard of care, but many professionals remain skeptical and are concerned about their efficiency and side effects. In the present study, two different triple-negative breast cancer cell lines, a non-malignant breast epithelial cell line and fibroblasts, were irradiated daily under normofractionated and hypofractionated schedules to evaluate the impact of different irradiation regimens on radiation-induced cell-biological effects. During the series of radiotherapy, proliferation, growth rate, double-strand DNA break-repair (DDR), cellular senescence, and cell survival were measured. Investigated normal and cancer cells differed in their responses and receptivity to different irradiation regimens, indicating cell line/cell type specificity of the effect. At the end of both therapy concepts, normal and malignant cells reach almost the same endpoint of cell count and proliferation inhibition, confirming the clinical observations in the follow-up at the cellular level. These result in cell lines closely replicating the irradiation schedules in clinical practice and, to some extent, contributing to the understanding of growth rate or remission of tumors and the development of fibrosis.

Cytotoxic Activity of Carrageenan on Malignant MCF-7 Breast Cancer and the non-Malignant SVCT Breast Epithelial Cell Lines

Objective: Recent studies have shown that sulfated polysaccharides obtained from marine algae have many biological and physiological activities. Kappa-carrageenan, a sulfated polysaccharide obtained from red marine algae, is known to have anti-proliferative effects in various cancer cell lines. In this study, the cytotoxic effect of kappa-carrageenan on two different cell lines, namely the malignant MCF-7 breast cancer cell line and the non-malignant SVCT breast epithelial cell lines, was investigated in an in vitro model. Materials & Methods: Three different concentrations of carrageenan were prepared for each cell in its own medium, with an initial dose of 1000 µg/ml (Dilution I: 1000 µg/ml; Dilution II: 250 µg/ml; Dilution III: 62.5 µg/ml). Cells were incubated with carrageenan at the doses set for each experimental group. Cells incubated in a carrageenan-free medium comprised the control group. Results: To measure cell viability, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) analysis was performed. Cell morphology was investigated by the acridine orange (AO)/propidium iodide (PI) fluorescent staining method. The present results indicated that carrageenan caused cytotoxic effects on both malignant and non-malignant cell lines. Conclusion: Considering that the different phenotypic features of the subtypes of the MCF-7 cell line may affect cell viability and cell proliferation, cell selection should be performed very carefully in cytotoxicity studies. We suggest that using the MCF-7 cell line for cytotoxicity experiments needs to contemplate this important phenomenon for further experimental setups.

Expression analysis and function of mitochondrial genome-encoded microRNAs

MicroRNAs (miRNAs) play a significant role in nuclear and mitochondrial anterograde and retrograde signaling. Most of the miRNAs found inside mitochondria are encoded in the nuclear genome, with a few mitochondrial genome-encoded non-coding RNAs having been reported. In this study, we have identified 13 mitochondrial genome-encoded microRNAs (mitomiRs), which were differentially expressed in breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231), non-malignant breast epithelial cell line (MCF-10A), and normal and breast cancer tissue specimens. We found that mitochondrial DNA (mtDNA) depletion and inhibition of mitochondrial transcription led to reduced expression of mitomiRs in breast cancer cells. MitomiRs physically interacted with Ago2, an RNA-induced silencing complex (RISC) protein, in the cytoplasm and inside mitochondria. MitomiRs regulate the expression of both nuclear and mitochondrial transcripts in breast cancer cells. We showed that mitomiR-5 targets the PPARGC1A gene and regulates mtDNA copy number in breast cancer cells. MitomiRs identified in the present study may be a promising tool for expression and functional analysis in patients with a defective mitochondrial phenotype, including cancer and metabolic syndromes. This article has an associated First Person interview with the first author of the paper.

Abstract P140: Chromomycin A5, a marine-derived antibiotic, targets the oncogenic TBX2 in breast cancer

Abstract Breast cancer (BC) continues to pose a major health burden. In 2020 alone, over 2 million people were diagnosed with BC globally and of these 684 996 people succumbed to the disease. Cancer cell drug resistance and the notion of transcription factors as undruggable targets has slowed progress for novel BC therapies. The transcription factor, TBX2, is commonly over-expressed in breast cancer with various hormone receptor statuses where it functions as a powerful pro-proliferative factor by directly repressing p21, PTEN, CST-6, and NDGR1 expression. Furthermore, TBX2 mediates Cisplatin drug resistance by inducing an S-phase cell cycle arrest allowing for cisplatin-induced DNA damage repair and breast cancer cell survival. Targeting TBX2 with DNA-damaging chemotherapies therefore emerges as a promising anti-cancer strategy to treat BC. To this end, we performed a reverse affinity screen to fish for marine-derived compounds with DNA-damaging- and TBX2 binding- activity and identified the antibiotic Chromomycin A5 (CMA5) as a promising agent. This project confirms the binding affinity of CMA5 for TBX2 by thermophoresis and fully characterizes the in vitro anticancer activity of CMA5 in TBX2-driven BC cells. Short-term and long-term selective cytotoxicity of CMA5 were investigated in two TBX2-driven BC cell lines (MCF-7 and T47D) and a non-malignant breast epithelial cell line (MCF-12A) using MTT and clonogenic assays. Our results show that CMA5 exhibited potent (IC50 ≤6.5 nM) and selective (SI ~ 3.3 ) cytotoxicity against BC cells. Importantly, CMA5 inhibited TBX2 protein levels and co-treatment of the BC cells with CMA5 and the proteasome 26S inhibitor MG132 rescued TBX2 levels. These results show that CMA5 targets TBX2 for protein degradation. Furthermore, whereas knocking down TBX2 using shRNA blocked the cytotoxicity of CMA5 (IC50 increased by 5-fold), ectopically over-expressing TBX2 increased the cytotoxic activity of CMA5 (IC50 decreased by 1.2-fold). These results suggest that CMA5 cytotoxicity is dependent, in part, on the presence of TBX2. To further explore the significance of TBX2 inhibition by CMA5 we investigated the impact of CMA5 on expression levels of the TBX2 target genes p21, PTEN, CST-6 and NDGR1 by quantitative real-time PCR analyses. The results revealed that all four genes were upregulated in the presence of CMA5 treatment which suggested that CMA5 inhibits BC cell viability and survival by targeting TBX2 and thus releasing its ability to repress these tumor suppressors. To elucidate the mechanism(s) underpinning the cytotoxic activity of CMA5 we investigated its effects on several markers of DNA damage (yH2AX expression) and apoptosis (cleaved caspase 3, 7, 8, 9 and PARP) by western blotting and immunocytochemistry. Our results show that CMA5 induced DNA damage and killed cells, in part, by both the intrinsic and extrinsic apoptotic programmed cell death pathways. Overall, this study identifies CMA5 as a promising anti-breast cancer agent because of its capability to induce DNA damage and to inhibit the oncogenic TBX2. Citation Format: Claire Bellis, Suparna Chakraborty, Mihlali Mlaza, Bianca Del Bianco Sahm, Paula Rezende Teixeira, Leticia Costa-Lotufo, Sharon Prince. Chromomycin A5, a marine-derived antibiotic, targets the oncogenic TBX2 in breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P140.

Breast-Specific Epigenetic Regulation of DeltaNp73 and Its Role in DNA-Damage-Response of BRCA1-Mutated Human Mammary Epithelial Cells.

The function of BRCA1/2 proteins is essential for maintaining genomic integrity in all cell types. However, why women who carry deleterious germline mutations in BRCA face an extremely high risk of developing breast and ovarian cancers specifically has remained an enigma. We propose that breast-specific epigenetic modifications, which regulate tissue differentiation, could team up with BRCA deficiency and affect tissue susceptibility to cancer. In earlier work, we compared genome-wide methylation profiles of various normal epithelial tissues and identified breast-specific methylated gene promoter regions. Here, we focused on deltaNp73, the truncated isoform of p73, which possesses antiapoptotic and pro-oncogenic functions. We showed that the promoter of deltaNp73 is unmethylated in normal human breast epithelium and methylated in various other normal epithelial tissues and cell types. Accordingly, deltaNp73 was markedly induced by DNA damage in human mammary epithelial cells (HMECs) but not in other epithelial cell types. Moreover, the induction of deltaNp73 protected HMECs from DNA damage-induced cell death, and this effect was more substantial in HMECs from BRCA1 mutation carriers. Notably, when BRCA1 was knocked down in MCF10A, a non-malignant breast epithelial cell line, both deltaNp73 induction and its protective effect from cell death were augmented upon DNA damage. Interestingly, deltaNp73 induction also resulted in inhibition of BRCA1 and BRCA2 expression following DNA damage. In conclusion, breast-specific induction of deltaNp73 promotes survival of BRCA1-deficient mammary epithelial cells upon DNA damage. This might result in the accumulation of genomic alterations and allow the outgrowth of breast cancers. These findings indicate deltaNp73 as a potential modifier of breast cancer susceptibility in BRCA1 mutation carriers and may stimulate novel strategies of prevention and treatment for these high-risk women.

Berberine and Emodin abrogates breast cancer growth and facilitates apoptosis through inactivation of SIK3-induced mTOR and Akt signaling pathway

Salt-inducible kinases 3 (SIK3) belong to the AMPK-related family of kinases, which have been implicated in the regulation of cell metabolism, cell polarity remodelling, and epithelial–mesenchymal transition. Elevated SIK3 expressions in breast cancer cells are shown to contribute to tumorigenesis; however, the underlying mechanism remains to be elucidated. In this study, we demonstrate that SIK3 expression is upregulated and concurrently high expression of SIK3 is associated with poor survival in breast cancer. Specifically, SIK3 knockdown revealed that SIK3 is required for the mTOR/Akt signaling pathway and proliferation of breast cancer cells. Furthermore, our findings showed that Emodin (EMO) combined with Berberine (BBR) significantly inhibited SIK3 activity, leading to reduced cell growth, increased cell cycle arrest and apoptosis in breast cancer cells, but not in non-malignant breast epithelial cell line. Mechanistic studies further reveal that EMO and BBR in combined treatment inhibited SIK3-potentiated mTOR-mediated aerobic glycolysis and cell growth in breast cancer cells. Moreover, combination treatments attenuate Akt signaling, thereby inducing G0/G1 phase cell cycle arrest and apoptosis of breast cancer cells in a SIK3-dependent manner. CRISPR/Cas9 or siRNA-mediated SIK3 knockout/knockdown showed an opposite trend in both the luminal and basal-like breast cancer. Collectively, our findings reveal that combination of EMO and BBR attenuates SIK3-driven tumor growth in breast cancer, and thus, EMO and BBR might be a novel SIK3 inhibitor explored into the prevention of breast cancer.

Abstract P6-09-01: RAD51B loss-of-function variants confer susceptibility to hereditary breast and ovarian cancers and result in tumors with genomic features of homologous recombination repair defects

Abstract Introduction: Germline pathogenic variants in genes in the homologous recombination (HR) pathway such as BRCA1, BRCA2, ATM, PALB2, RAD51C and RAD51D confer an increased risk of breast and ovarian cancers. Screening for germline variants in these cancer susceptibility genes is critical as prophylactic measures and monitoring can be performed in these individuals to minimize their risk of developing breast and/or ovarian cancers. More recently, it has been recognized that cancers arising in carriers of germline pathogenic (P)/ likely pathogenic (LP) variants in HR genes often show a homologous recombination (HR) deficient (HRD) phenotype and can be targeted with platinum agents or PARP inhibitors. RAD51B is a RAD51 paralog that binds to RAD51C and functions as a heterodimer in the HR pathway. Whilst RAD51B germline variants have been reported in isolated cases of breast and ovarian cancers, it is unclear as to whether RAD51B P/LP germline variants would confer predisposition to these cancer types. Materials and Methods: We screened 9,287 consecutive unselected cancer patients who consented for both tumor and germline testing using the MSK-IMPACT platform for the presence of RAD51B germline truncating variants. Selected RAD51B germline mutant breast cancers identified by MSK-IMPACT were subjected to whole-exome sequencing (WES) analysis to determine the dominant mutational signatures using DeconstructSigs. Functional assays were performed to ascertain the impact of RAD51B loss on HR DNA repair and PARP inhibitor sensitivity using genome editing methods in non-malignant breast epithelial cell lines. Results: Out of the 9,287 cancer patients, we detected likely pathogenic loss of function RAD51B germline variants in 11 patients (0.12%), which was similar to the frequency detected in the gnomAD database (0.09%). All female carriers of RAD51B loss of function variants (n=8) had breast or ovarian cancers (8/1,619 breast or ovarian cancers, 0.5%). The observed carrier frequencies of germline truncating variants in RAD51B was statistically enriched in breast and ovarian cancer patients compared to individuals in the gnomAD database (0.5% vs 0.09% P=0.0003; odds ratio = 5.06 (95% CI: 2.1-10.3)). Although segregation studies were not available, 9/11 of the RAD51B germline loss of function variant carriers had a personal or family history of breast or ovarian cancers. All five breast and ovarian cancers from RAD51B mutation carriers investigated by WES were found to harbor RAD51B bi-allelic inactivation through loss of heterozygosity of the RAD51B wild-type allele. In addition, these five cases were found to display genomic features of HRD including high large-scale state transition scores and a dominant mutational signature 3. CRISPR/Cas9 genome editing of RAD51B in a non-malignant breast epithelial cell model revealed that RAD51B deficient cells display PARP inhibitor sensitivity similar to that reported in BRCA1 and BRCA2 deficient cell lines. Conclusion: RAD51B loss-of-function germline variants confer susceptibility to breast and ovarian cancer development. Breast and ovarian cancers occurring in the context of RAD51B germline mutations harbor bi-allelic inactivation of RAD51B through loss-of-heterozygosity of the wild-type allele, genomic features of HRD and are likely sensitive to PARP inhibition. Albeit rarely germline mutated, RAD51B should be considered as an addition to clinical germline testing panels for hereditary breast and ovarian cancer syndrome patients. <!–EndFragment→ Citation Format: Diana SReis-FilhoReis-Filho Mandelker, Semanti Mukherjee, Jeremy Setton, Pier Selenica, Yelena Kemel, Ozge Ceyhan-Birsoy, Margaret Sheehan, Kaitlyn Tkachuk, David N Brown, Simon Powell, Britta Weigelt, Mark E Robson, Nadeem Riaz, Kenneth Offit, Jorge S Reis-Filho. RAD51B loss-of-function variants confer susceptibility to hereditary breast and ovarian cancers and result in tumors with genomic features of homologous recombination repair defects [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-09-01.

First evidence of SGPL1 expression in the cell membrane silencing the extracellular S1P siren in mammary epithelial cells

The bioactive lipid sphingosine-1-phosphate (S1P) is a main regulator of cell survival, proliferation, motility, and platelet aggregation, and it is essential for angiogenesis and lymphocyte trafficking. In that S1P acts as a second messenger intra- and extracellularly, it might promote cancer progression. The main cause is found in the high S1P concentration in the blood, which encourage cancer cells to migrate through the endothelial barrier into the blood vessels. The irreversible degradation of S1P is solely caused by the sphingosine-1-phosphate lyase (SGPL1). SGPL1 overexpression reduces cancer cell migration and therefore silences the endogenous S1P siren, which promotes cancer cell attraction—the main reason for metastasis. Since our previous metabolomics studies revealed an increased SGPL1 activity in association with successful breast cancer cell treatment in vitro, we further investigated expression and localization of SGPL1. Expression analyses confirmed a very low SGPL1 expression in all breast cancer samples, regardless of their subtype. Additionally, we were able to prove a novel SGPL expression in the cytoplasm membrane of non-tumorigenic breast cells by fusing three independent methods. The general SGPL1 downregulation and the loss of the plasma membrane expression resulted in S1P dependent stimulation of migration in the breast cancer cell lines MCF-7 and BT-20. Not only S1P stimulated migration could be repressed by overexpressing the natural SGPL1 variant not but also more general migratory activity was significantly reduced. Here, for the first time, we report on the SGPL1 plasma membrane location in human, non-malignant breast epithelial cell lines silencing the extracellular S1P siren in vitro, and thereby regulating pivotal cellular functions. Loss of this plasma membrane distribution as well as low SGPL1 expression levels could be a potential prognostic marker and a viable target for therapy. Therefore, the precise role of SGPL1 for cancer treatment should be evaluated.

Abstract 831: TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM

Abstract Transforming growth factor β (TGFβ) is a well-documented tumor suppressor; a poorly studied aspect of TGFβ biology is its control of genomic stability. Our prior work showed that TGFβ compromises ATM (ataxia telangiectasia mutated) kinase activity, which mediates recognition and repair of DNA damage. Consequently, inhibiting TGFβ following ionizing radiation increases clonogenic death in vitro and tumor control in vivo in breast, brain, and lung cancer preclinical models. As yet unknown is how TGFβ controls ATM kinase. We recently determined that TGFβ regulates mammary lineage commitment by post-transcriptional control of BRCA1 mRNA and protein by its suppression of miR-182 (Martinez-Ruiz et al., Science Signaling, in press). miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors in cancer cell lines (10.1016/j.molcel.2010.12.005) Here we used two cell culture models to investigate the effect of TGFβ regulation of BRCA1 and miR-182 on DNA damage response. Spontaneously immortalized Tgfb1 heterozygote and wild type fibroblasts from primary mammary epithelial preparations were synchronized in S-phase and exposed to UV radiation before fixation to detect unrepaired DNA damage by quantitative image analysis of 53BP1 foci. Consistent with compromised BRCA1 function, more than twice as many cells with 53BP1 foci were evident in Tgfb1 heterozygote fibroblasts compared to wild type cells (p<0.01). The second model consisted of non-malignant human breast epithelial cell line, MCF10A, stably transduced with a scrambled or miR-182 antagomir, grown under serum free conditions and treated with a small molecule inhibitor of TGFβ type I receptor kinase, LY364937. FOXO3 is a target of miR-182 and thus also regulated by TGFβ. and FOXO3 association with ATM is required for its kinase activity (10.1038/ncb1709). As reported, FOXO3 and ATM co-immunoprecipitated, but was eliminated by TGFβ inhibition and this effect was lost in MCF10A cells expressing a miR-182 antagomir. The impaired DNA repair phenotype induced by TGFβ inhibition can be fully rescued by antagonizing miR-182. These studies support TGFβ as stringent regulator of the DNA damage response via suppression miR-182, which directly targets BRCA1 and indirectly affects ATM activity via FOXO3. We predict that cancers that have lost TGFβ signaling capacity will be genomically unstable due to defective DNA damage control, which is consistent with our studies showing that TGFβ inhibition sensitize cancers to radiation therapy. Citation Format: Qi Liu, Haydeliz Martinez-Ruiz, John Murnane, Simon N. Powell, Mary Helen Barcellos-Hoff. TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 831. doi:10.1158/1538-7445.AM2017-831

Critical role of SIK3 in mediating high salt and IL-17 synergy leading to breast cancer cell proliferation.

Chronic inflammation is a well-known precursor for cancer development and proliferation. We have recently demonstrated that high salt (NaCl) synergizes with sub-effective interleukin (IL)-17 to induce breast cancer cell proliferation. However, the exact molecular mechanisms mediating this effect are unclear. In our current study, we adopted a phosphoproteomic-based approach to identify salt modulated kinase-proteome specific molecular targets. The phosphoprotemics based binary comparison between heavy labelled MCF-7 cells treated with high salt (Δ0.05 M NaCl) and light labelled MCF-7 cells cultured under basal conditions demonstrated an enhanced phosphorylation of Serine-493 of SIK3 protein. The mRNA transcript and protein expression analysis of SIK3 in MCF-7 cells demonstrated a synergistic enhancement following co-treatment with high salt and sub-effective IL-17 (0.1 ng/mL), as compared to either treatments alone. A similar increase in SIK3 expression was observed in other breast cancer cell lines, MDA-MB-231, BT20, and AU565, while non-malignant breast epithelial cell line, MCF10A, did not induce SIK3 expression under similar conditions. Biochemical studies revealed mTORC2 acted as upstream mediator of SIK3 phosphorylation. Importantly, cell cycle analysis by flow cytometry demonstrated SIK3 induced G0/G1-phase release mediated cell proliferation, while SIK3 silencing abolished this effect. Also, SIK3 induced pro-inflammatory arginine metabolism, as evidenced by upregulation of the enzymes iNOS and ASS-1, along with downregulation of anti-inflammatory enzymes, arginase-1 and ornithine decarboxylase. Furthermore, gelatin zymography analysis has demonstrated that SIK3 induced expression of tumor metastatic CXCR4 through MMP-9 activation. Taken together, our data suggests a critical role of SIK3 in mediating three important hallmarks of cancer namely, cell proliferation, inflammation and metastasis. These studies provide a mechanistic basis for the future utilization of SIK3 as a key drug discovery target to improve breast cancer therapy.

Abstract P1-05-03: The genomic landscape of breast metaplastic carcinoma

Abstract Introduction: Metaplastic breast carcinoma (MBC) is a rare histologic type of triple-negative breast cancer (TNBC), characterized by the presence of cells displaying squamous and/or mesenchymal differentiation. The transcriptomic profiles of MBCs have been reported to vary according to the type of metaplastic elements. The somatic genetic alterations that underpin this breast cancer subtype remain to be fully characterized. Here we sought to define the genomic landscape of MBCs, whether different subtypes of MBC would be driven by distinct constellations of genetic alterations, and to investigate functionally the impact of mutations affecting WNT pathway genes using non-malignant breast epithelial cells. Methods: Thirty-five MBCs were retrieved from the pathology department of the authors' institutions and classified into the MBC histologic subtypes. All but one of the MBCs were of triple-negative phenotype. DNA was extracted from microdissected tumor-normal pairs and subjected to whole-exome sequencing. Somatic genetic alterations were identified using state-of-the-art bioinformatics algorithms. The genomic profiles of MBCs were compared to those of 69 common type TNBCs from The Cancer Genome Atlas. Overall mutation rates were compared using the Mann Whitney U test, and the frequency of mutations in each gene was compared using Fisher's exact test. RNA was extracted from a subset of MBCs and subjected to WNT signaling pathway activation analysis with the RT2 Profiler PCR Array. Triple-negative non-malignant breast epithelial cells (MCF10A and MCF12A) and cancer cell lines were utilized for 2D and 3D functional studies. Results: Whole-exome analysis revealed that MBCs displayed a median of 103 (15-344) somatic mutations, which did not differ from the median number of somatic mutations in common type TNBCs (76, range 14-233). The most frequent recurrently mutated cancer genes included TP53 (69%) and PIK3CA (29%). MBCs more frequently harbored mutations in PI3K pathway genes than common type TNBCs (57% vs 22%, P<0.05), including mutations affecting PIK3CA (29% vs 7%), PIK3R1 (11% vs 0) and PTEN (11% vs 1%). MBCs also more frequently harbored mutations affecting WNT signaling pathway genes (46% vs 26%, P<0.05), including AXIN1 (6% vs 1%), WNT5A (6% vs 0) and APC (3% vs 0). MBC subtype analysis revealed that PIK3CA mutations were only detected in non-chondroid MBCs (53% vs 0), CHERP mutations were only found in chondroid MBCs (25% vs 0), whereas USP5 mutations only found in squamous MBCs (33% vs 0). MBCs with somatic mutations in WNT pathway genes had significantly higher WNT pathway activation than MBCs lacking mutations in these genes (P=0.0244). Consistent with the mesenchymal phenotype frequently exhibited by MBCs, in vitro experiments provided functional evidence that aberrant WNT pathway activation induces an epithelial-to-mesenchymal transition (EMT) phenotype, with downregulation of epithelial markers and upregulation of EMT transcriptional inducers. Conclusions: MBCs are significantly enriched for mutations affecting PI3K and WNT pathways, highlighting the importance of the dysregulation of the WNT pathway in MBC carcinogenesis. Moreover, our findings suggest that specific mutations are significantly associated with distinct histologic subtypes of MBCs. Citation Format: Geyer FC, Ng CK, Piscuoglio S, Wen YH, Wen H-C, Pareja F, Eberle CA, Burke KA, Lim RS, Natrajan R, Mariani O, Brogi E, Norton L, Vincent-Salomon A, Weigelt B, Reis-Filho JS. The genomic landscape of breast metaplastic carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-03.

MicroRNA-130b targets PTEN to mediate drug resistance and proliferation of breast cancer cells via the PI3K/Akt signaling pathway

Multidrug resistance (MDR) correlates with treatment failure and poor prognosis among breast cancer patients. This study was aimed to investigate the possible mechanism by which microRNA-130b-3p (miR-130b) mediates the chemoresistance and proliferation of breast cancer. MiR-130b was found to be up-regulated in tumor tissues versus adjacent tissues of breast cancer, as well as in adriamycin (ADR) resistant breast cancer cell line (MCF-7/ADR) versus its parental line (MCF-7) and the non-malignant breast epithelial cell line (MCF-10A), demonstrating its crucial relevance for breast cancer biology. We identified that PTEN was a direct target of miR-130b and inversely correlated with miR-130b expression in breast cancer. Moreover, over-expression of miR-130b promoted drug resistance, proliferation and decreased apoptosis of MCF-7 cells, while suppression of miR-130b enhanced drug cytotoxicity and apoptosis, as well as reduced proliferation of MCF-7/ADR cells in vitro and in vivo. Particularly, miR-130b mediated the activity of phosphoinositide-3 kinase (PI3K)/Akt signaling pathway as well as the chemoresistance and proliferation of breast cancer cell lines, which was partially blocked following knockdown of PTEN. Altogether, miR-130b targets PTEN to induce MDR, proliferation, and apoptosis via PI3K/Akt signaling pathway. This provides a novel promising candidate for breast cancer therapy.

Epigenetic changes in cancer by Raman imaging, fluorescence imaging, AFM and scanning near-field optical microscopy (SNOM). Acetylation in normal and human cancer breast cells MCF10A, MCF7 and MDA-MB-231.

This paper examines epigenetic changes in breast cancer by Raman imaging, fluorescence imaging, AFM and SNOM and discusses how they contribute to different aspects of tumourigenesis in malignant human breast epithelial cell lines MCF7 and MDA-MB-231 compared with non-malignant MCF10A cell lines. The paper focuses on information that can be extracted from Raman microscopy and Raman imaging for the biological material of nucleoli contained within the cell nucleus and lipid droplets within the cell cytoplasm. The biochemical composition of the nuclei and lipid droplets in the non-malignant and malignant human breast epithelial cell lines has been monitored. The potential of Raman microspectroscopy to monitor acetylation processes and a prognostic value of Raman biomarkers in breast cancer have been discussed.

Anti-breast cancer effects of live, heat-killed and cytoplasmic fractions of Enterococcus faecalis and Staphylococcus hominis isolated from human breast milk.

Development of tumour that is resistant to chemotherapeutics and synthetic drugs, coupled with their life-threatening side effects and the adverse effects of surgery and hormone therapies, led to increased research on probiotics' anticancer potentials. The current study investigated the potential of live, heat-killed cells (HKC) and the cytoplasmic fractions (CF) of Enterococcus faecalis and Staphylococcus hominis as anti-breast cancer agents. MCF-7 cell line was treated with 25, 50, 100 and 200 μg/mL each of live, HKC and CF of the bacteria; and cytotoxicity was evaluated for 24, 48 and 72 h using MTT assay. The morphological features of the treated cells were examined by fluorescence microscopy. The stage of cell cycle arrest and apoptosis were quantified by flow cytometry. The bacterial effect on non-malignant breast epithelial cell line, MCF-10A, was assessed using MTT assay for 24, 48 and 72 h. All the three forms of the bacteria caused a significant decrease in MCF-7 (up to 33.29%) cell proliferation in concentration- and time-dependent manner. Morphological features of apoptosis like cell death, cell shrinkage and membrane blebbing were observed. Flow cytometry analyses suggested that about 34.60% of treated MCF-7 was undergoing apoptosis. A strong anti-proliferative activity was efficiently induced through sub-G1 accumulation (up to 83.17%) in treated MCF-7 and decreased number in the G0/G1 phase (74.39%). MCF-10A cells treated with both bacteria showed no significant difference with the untreated (>90% viability). These bacteria can be used as good alternative nutraceutical with promising therapeutic indexes for breast cancer because of their non-cytotoxic effects to normal cells.

The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-β-Induced Epithelial to Mesenchymal Transition.

The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.

Abstract 1084: An EMT-like gene expression profile differentiates helical from kinase domain PIK3CA mutations

Abstract A recent study of the mutation landscape of >3000 cancers across 12 major cancer types from the Cancer Genome Atlas (TCGA) program revealed PIK3CA as the second most commonly mutated gene, occurring at >10% frequency across most cancer types, with mutations occurring mostly in the PIK3CA kinase (39% overall, 52% for breast cancer) and helical (45% overall, 39% for breast cancer) domains and their most frequent hotspot mutations being H1047R and E545K, respectively. Limited preclinical evidence has suggested that mutations affecting the kinase and helical domains can produce different phenotypic consequences; and our recent analysis of the TCGA data indicates that helical domain mutations, relative to kinase domain mutations, are preferentially associated with pathway/PARADIGM activity features enriched for cell motility mechanisms (e.g. RHO GTPases) and loss of gap junctions, suggesting that helical domain mutations might serve to enhance cell invasiveness and metastatic motility. To validate the functional differences predicted by these two classes of PIK3CA mutations, we turned to the non-malignant breast epithelial cell line MCF10A bearing targeted pathogenic knock-ins of H1047R or E545K at the PIK3CA locus; and we compared RNA-seq expression profiles from the wildtype (wt) and knockin isogenic clones. RSEM was used to estimate gene and isoform abundance; and EBseq was used to perform differential expression analysis. In a three-group comparison, over 2500 differentially expressed genes were identified as differentiating wt cells and the PIK3CA mutated MCF10A clones, with the greatest transcriptional changes (2043 genes) observed between either kinase or helical domain mutations and wt cells. Functional enrichment analysis (DAVID) revealed enrichment of 17 pathway categories among these 2043 genes, representing 3 major functional groups, the most prominent being protein translation. Only 409 genes were differentially expressed across all three groups, suggesting the transcriptome effect differentiating E545K from H2047R bearing MCF10A clones was less pronounced. In a direct comparison of the kinase and helical domain clones, >900 differentially expressed genes were identified that showed enrichment in 64 pathways representing altered cell morphology, motility, apoptosis and phosphorylation, but without enrichment in protein translation function. In particular, MCF10A E545K clones showed significant loss of expression in 19 tight junction-associated genes linked to oncogenic EMT (e.g. OCLN, CLDN-7, TJP1, MYL9). Thus, consistent with our pathway conclusions drawn from the TCGA clinical dataset, helical domain mutations appear to confer an EMT-like expression phenotype beyond the effects of both helical and kinases domain mutations on protein translation. Citation Format: Christina Yau, Joe Garay, Stephen Benz, Joe Gray, Ben Park, Christopher C. Benz. An EMT-like gene expression profile differentiates helical from kinase domain PIK3CA mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1084. doi:10.1158/1538-7445.AM2015-1084

Synergistic anticancer effects of a bioactive subfraction of Strobilanthes crispus and tamoxifen on MCF-7 and MDA-MB-231 human breast cancer cell lines.

BackgroundDevelopment of tumour resistance to chemotherapeutic drugs and concerns over their toxic effects has led to the increased use of medicinal herbs or natural products by cancer patients. Strobilanthes crispus is a traditional remedy for many ailments including cancer. Its purported anticancer effects have led to the commercialization of the plant leaves as medicinal herbal tea, although the scientific basis for its use has not been established. We previously reported that a bioactive subfraction of Strobilanthes crispus leaves (SCS) exhibit potent cytotoxic activity against human breast cancer cell lines. The current study investigates the effect of this subfraction on cell death activities induced by the antiestrogen drug, tamoxifen, in estrogen receptor-responsive and nonresponsive breast cancer cells.MethodsCytotoxic activity of SCS and tamoxifen in MCF-7 and MDA-MB-231 human breast cancer cells was determined using lactate dehydrogenase release assay and synergism was evaluated using the CalcuSyn software. Apoptosis was quantified by flow cytometry following Annexin V and propidium iodide staining. Cells were also stained with JC-1 dye to determine changes in the mitochondrial membrane potential. Fluorescence imaging using FAM-FLICA assay detects caspase-8 and caspase-9 activities. DNA damage in the non-malignant breast epithelial cell line, MCF-10A, was evaluated using Comet assay.ResultsThe combined SCS and tamoxifen treatment displayed strong synergistic inhibition of MCF-7 and MDA-MB-231 cell growth at low doses of the antiestrogen. SCS further promoted the tamoxifen-induced apoptosis that was associated with modulation of mitochondrial membrane potential and activation of caspase-8 and caspase-9, suggesting the involvement of intrinsic and extrinsic signaling pathways. Interestingly, the non-malignant MCF-10A cells displayed no cytotoxicity or DNA damage when treated with either SCS or SCS-tamoxifen combination.ConclusionsThe combined use of SCS and lower tamoxifen dose could potentially reduce the side effects/toxicity of the drug. However, further studies are needed to determine the effectiveness and safety of the combination treatment in vivo.

Suppressive role of miR-502-5p in breast cancer via downregulation of TRAF2

TRAF2 promotes cancer cell survival, proliferation and metastasis through the NF-κB pathway by directly interacting with various TNF recepors. However, the molecular mechanism of TRAF2 dysregulation in breast cancer remains to be elucidated. In the present study, miR-502-5p was predicted as a potential regulator of TRAF2. miR-502-5p was significantly downregulated in breast cancer tissues when compared to the level in paired normal breast tissues. The breast cancer cell lines including MCF-7 and MDA-MB-231 expressed a lower level of miR-502-5p when compared to the level in the non-malignant breast epithelial cell line MCF-10A. In vitro, miR-502-5p enhanced early apoptosis and inhibited proliferation of breast cancer cells. Luciferase reporter assay results showed that miR-502-5p could bind to the 3'-untranslated region of the TRAF2 gene, thus, exerting an inhibitory effect on TRAF2. Furthermore, silencing of TRAF2 exhibited effects similar to those of exogenous miR‑502-5p, while overexpression of TRAF2 partially abrogated miR-502-5p-mediated suppression in breast cancer cells. In conclusion, miR-502-5p may act as a tumor-suppressor gene by targeting oncogenic TRAF2 in breast cancer and, therefore, may be a potential diagnostic and anticancer therapeutic marker for breast cancer.

Borrelidin has limited anti‐cancer effects in bcl‐2 overexpressing breast cancer and leukemia cells and reveals toxicity in non‐malignant breast epithelial cells

Clinically effective anti-cancer drugs have to tread a narrow line between selective cytotoxicity on tumor cells and tolerable adverse effects against healthy tissues. This causes the failure of many potential cancer drugs in advanced clinical trials, hence signifying the importance of a comprehensive initial estimate of the cytotoxicity of prospective anti-cancer drugs in preclinical studies. In this study, the cytotoxicity of borrelidin, a macrolide antibiotic with a high cytotoxic selectivity for proliferating endothelial cells and leukemia cells, was tested on malignant and non-malignant breast cells. Highly metastatic breast cancer cell lines (MDA-MB-231 and MDA-MB-435) showed promising results and exhibited good sensitivity to borrelidin at low nanomolar concentrations, but borrelidin was cytotoxic to a non-malignant breast epithelial cell line (MCF10A) as well. Furthermore, although a high sensitivity of endothelial cells (human umbilical vein endothelial cells; HUVEC) and individual leukemia cell lines (Jurkat and IM9) to borrelidin was confirmed in this study, another leukemia cell line (HL60) and an immortalized endothelial cell line (EA.hy926) displayed a significantly decreased sensitivity. Reduced sensitivity to borrelidin was associated with elevated bcl-2 expression in these cell lines. In conclusion, the results presented show that borrelidin displays high and selective cytotoxicity against subgroups of cancer cells and endothelial cells, but, owing to its non-specific toxicity to non-malignant cells, its clinical application might be restricted because of likely adverse effects and limited efficacy in bcl2-overexpressing cancer cells.

ATM Suppresses SATB1-Induced Malignant Progression in Breast Epithelial Cells

SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.