Malignant Breast Epithelial Cells Research Articles

Drug repositioning for immunotherapy in breast cancer using single-cell analysis.

Immunomodulatory peptides, while exhibiting potential antimicrobial, antifungal, and/or antiviral properties, can play a role in stimulating or suppressing the immune system, especially in pathological conditions like breast cancer (BC). Thus, deregulation of these peptides may serve as an immunotherapeutic strategy to enhance the immune response. In this meta-analysis, we utilized single-cell RNA sequencing data and known therapeutic peptides to investigate the deregulation of these peptides in malignant versus normal human breast epithelial cells. We corroborated our findings at the chromatin level using ATAC-seq. Additionally, we assessed the protein levels in various BC cell lines. Moreover, our in-house drug repositioning approach was employed to identify potential drugs that could positively impact the relapse-free survival of BC patients. Considering significantly deregulated therapeutic peptides and their role in BC pathology, our approach aims to downregulate B2M and SLPI, while upregulating PIGR, DEFB1, LTF, CLU, S100A7, and SCGB2A1 in BC epithelial cells through our drug repositioning pipeline. Leveraging the LINCS L1000 database, we propose BRD-A06641369 for B2M downregulation and ST-4070043 and BRD-K97926541 for SLPI downregulation without negatively affecting the MHC complex as a significantly correlated pathway with these two genes. Furthermore, we have compiled a comprehensive list of drugs for the upregulation of other selected immunomodulatory peptides. Employing an immunotherapeutic approach by integrating our drug repositioning pipeline with single-cell analysis, we proposed potential drugs and drug targets to fortify the immune system against BC.

Mitochondrial DNA methylation is a predictor of immunotherapy response and prognosis in breast cancer: scRNA-seq and bulk-seq data insights.

Alterations in Mitochondrial DNA methylation (MTDM) exist in many tumors, but their role in breast cancer (BC) development remains unclear. We analyzed BC patient data by combining scRNA-seq and bulk sequencing. Weighted co-expression network analysis (WGCNA) of TCGA data identified mitochondrial DNA methylation (MTDM)-associated genes in BC. COX regression and LASSO regression were used to build prognostic models. The biological function of MTDM was assessed using various methods, such as signaling pathway enrichment analysis, copynumber karyotyping analysis, and quantitative analysis of the cell proliferation rate. We also evaluated MTDM-mediated alterations in the immune microenvironment using immune microenvironment, microsatellite instability, mutation, unsupervised clustering, malignant cell subtype differentiation, immune cell subtype differentiation, and cell-communication signature analyses. Finally, we performed cellular experiments to validate the role of the MTDM-associated prognostic gene NCAPD3 in BC. In this study, MTDM-associated prognostic models divided BC patients into high/low MTDM groups in TCGA/GEO datasets. The difference in survival time between the two groups was statistically significant (P<0.001). We found that high MTDM status was positively correlated with tumor cell proliferation. We analyzed the immune microenvironment and found that low-MTDM group had higher immune checkpoint gene expression/immune cell infiltration, which could lead to potential benefits from immunotherapy. In contrast, the high MTDM group had higher proliferation rates and levels of CD8+T cell exhaustion, which may be related to the secretion of GDF15 by malignant breast epithelial cells with a high MTDM status. Cellular experiments validated the role of the MTDM-associated prognostic gene NCAPD3 (the gene most positively correlated with epithelial malignant cell proliferation in the model) in BC. Knockdown of NCAPD3 significantly reduced the activity and proliferation of MDA-MB-231 and BCAP-37 cells, and significantly reduced their migration ability of BCAP-37 cell line. This study presented a holistic evaluation of the multifaceted roles of MTDM in BC. The analysis of MTDM levels not only enables the prediction of response to immunotherapy but also serves as an accurate prognostic indicator for patients with BC. These insightful discoveries provide novel perspectives on tumor immunity and have the potentially to revolutionize the diagnosis and treatment of BC.

GP5 regulates epithelial-mesenchymal transition in breast cancer via the PI3K/AKT signaling pathway.

Recent evidences have shown that glycoprotein V (GP5) protein, which is initially considered as an important adhesion molecule unique to the megakaryocyte line, was also specifically expressed in malignant human breast epithelial cells. However, its expression level and function are not clear. This study aimed to reveal the abnormal expression of GP5 in breast cancer (BC), research the associations between the GP5 abnormal expression and BC progression, and explore the molecular mechanism of GP5 in BC. Immunohistochemistry, Western blot (WB), and quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays were used to determine the expression patterns of GP5 in BC tissues and cells. The expression profiles of GP5 in the Cancer Genome Atlas databases were analyzed by UALCAN. The GP5 knockdown and over-expression BC cell lines were constructed and confirmed by RT-PCR and WB. Transcriptome sequencing and KEGG database were performed to screen cellular processes and signal pathways. Phosphatidylinositol 3-kinase (PI3K)/AKT pathway was verified by RT-qPCR, and epithelial-mesenchymal transition (EMT) was confirmed by WB. The results indicated GP5 was highly expressed in BC tissues and might play an important role as a cancer-promoting gene in BC. The high expression of GP5 was significantly associated with higher nuclear grade, higher TNM stage, and human epidermal growth factor receptor 2 (HER2) negativity. GP5 may promote the proliferation, invasion, and metastasis of BC cells by activating PI3K/AKT signaling pathway to up-regulate the EMT. This study provides a new idea that GP5 was expected to become a potential molecular target for early BC clinic diagnosis and treatment.

Acacetin and Pinostrobin Inhibit Malignant Breast Epithelial Cell Adhesion and Focal Adhesion Formation to Attenuate Cell Migration.

Naturally occurring flavonoids, such as acacetin and pinostrobin, disrupt a wide range of processes during tumor progression, such as cell proliferation, apoptosis, and angiogenesis. Although the antiproliferative and antiapoptotic effects of acacetin and pinostrobin have been studied using various cell lines, relatively little is known about the effects of acacetin and pinostrobin on cancer cell migration and metastasis. For instance, it is unclear whether acacetin or pinostrobin have any effect on breast cancer cell migration or adhesion. In this study, we assessed the effects of acacetin and pinostrobin on malignant MDA-MB-231 and T47D breast epithelial cells and non-tumorigenic MCF10A breast epithelial cells. Our results demonstrate that both acacetin and pinostrobin selectively inhibit the migration of both MDA-MB-231 and T47D cells in a dose-dependent manner while exhibiting blunted effects on MCF10A cells. Interestingly, neither compound had an effect on cell proliferation in any of the 3 cell lines. Furthermore, both acacetin and pinostrobin inhibit MDA-MB-231 and T47D cell adhesion, cell spreading, and focal adhesion formation, but have no significant effect on MCF10A cells. Collectively, these results suggest that both acacetin and pinostrobin selectively inhibit malignant breast epithelial cell migration through attenuation of cell adhesion and focal adhesion formation. These findings indicate that both acacetin and pinostrobin may serve as potential therapeutic options to target breast tumor cell migration during late-stage tumor progression.

KISS1/KISS1R SIGNAL SYSTEM AND ITS ROLE IN THE CARCINOGENESIS

The KISS1 / KISS1R signaling system can serve as a regulator of metastasis of tumors and is a potential prognostic marker of tumor processes. The action of kisspeptin10 on the Era-negative non - malignant breast epithelial cells or KISS1R expression in these cells can induce passage to the mesenchymal phenotype and to stimulate the invasiveness. The level of expression of KISS1 in remote breast cancer metastases is lower than in the primary tumor: methylation of the KISS1 promoter may be one of the reasons for the decrease of the expression of mRNA and KISS1 protein in the cells of breast cancer metastases in the brain. The clinical significance of KISS1 lies in the prediction of involvement in the neoplastic process in the lymphnodes. Features of expression of KISS1 / KISS1R in Era-positive tumors give hope for the emergence of new approaches to the treatment of these tumors. The level of KISS1 expression can serve as a molecular marker predicting the quality of tumor response to Tamoxifen therapy, especially in postmenopausal women.

GPER-induced signaling is essential for the survival of breast cancer stem cells.

G protein‐coupled estrogen receptor‐1 (GPER), a member of the G protein‐coupled receptor (GPCR) superfamily, mediates estrogen‐induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non‐BCSCs of three patient‐derived xenografts of ER−/PR+ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER‐mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD‐Ser118 to sustain BCSC characteristics. Transfection with a dominant‐negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs.

Visco-Node-Pore Sensing: A Microfluidic Rheology Platform to Characterize Viscoelastic Properties of Epithelial Cells

SummaryViscoelastic properties of cells provide valuable information regarding biological or clinically relevant cellular characteristics. Here, we introduce a new, electronic-based, microfluidic platform—visco-node-pore sensing (visco-NPS)—which quantifies cellular viscoelastic properties under periodic deformation. We measure the storage (G′) and loss (G″) moduli (i.e., elasticity and viscosity, respectively) of cells. By applying a wide range of deformation frequencies, our platform quantifies the frequency dependence of viscoelastic properties. G′ and G″ measurements show that the viscoelastic properties of malignant breast epithelial cells (MCF-7) are distinctly different from those of non-malignant breast epithelial cells (MCF-10A). With its sensitivity, visco-NPS can dissect the individual contributions of different cytoskeletal components to whole-cell mechanical properties. Moreover, visco-NPS can quantify the mechanical transitions of cells as they traverse the cell cycle or are initiated into an epithelial-mesenchymal transition. Visco-NPS identifies viscoelastic characteristics of cell populations, providing a biophysical understanding of cellular behavior and a potential for clinical applications.

Abstract P6-09-12: Using multiphoton laser scanning microscopy to assess neoadjuvant therapy outcome in core needle biopsies: A novel methodology

Abstract Background: Over-expression of Human Epidermal Growth Factor receptor-2 (HER2) in breast cancer is associated with an aggressive clinical course and poor prognosis. Targeting HER2 over-expression has been shown to be a remarkably effective therapeutic modality in the metastatic, adjuvant and neoadjuvant setting and the pathologic response to neoadjuvant treatment in HER2-positive breast cancer has been shown to be an excellent surrogate for a good outcome. The stromal tumor microenvironment is implicated in fostering tumor growth, facilitating cell migration and ultimately resulting in metastatic disease. Specifically, the collagenous extracellular matrix, which includes fibrillar collagen, has been suggested to play a role in the migration of malignant breast epithelial cells within the surrounding stroma. We have developed a novel methodology which uses an intrinsic optical signature to quantitatively evaluate fibrillar collagen (Burke et al. BMC Cancer 15 (2015): 929). Here, we evaluate the ability of this quantitative methodology to predict the pathologic response after neoadjuvant HER2-targeted treatment as assessed by the Residual Cancer Burden score/class (RCB). This quantitative evaluation in pre-treatment biopsies is then correlated with the pathologic response to treatment in the post-therapy resection. Material and Methods: Clinical pathologic variables for 29 cases of HER2-positive breast cancer that had undergone neoadjuvant chemotherapy plus HER2-targeted therapy were retrieved from the medical record database at URMC, including the post-treatment RCB score and ER/PR/HER2 status. Second harmonic generation (SHG) is an intrinsic optical signal produced by fibrillar collagen. To quantify collagen microstructure in the pre-treatment core biopsy, we used SHG imaging to determine the average forward to backward-light scattering ratio (F/B). The F/B ratio is sensitive to structural properties of collagen fibers. Results: Logistic regression was used to assess the association between F/B and the binary response variable RCB class (0/1 or 2/3). A likelihood ratio test was used to calculate the p-value to test whether the regression coefficient for F/B was zero (i.e. no effect) in the tumor-stromal interface. The average F/B ratio at the leading edge of the tumor stratified by RCB class is shown in Table 1. When evaluated in the bulk of the tumor tissue, F/B was not correlated with RCB status; however, when evaluated at the leading edge of the tumor stromal interface, F/B was significantly correlated with RCB status (p=0.035). Table 1:RCB class and average F/BRCB class (n)Average F/B ± SEM0/1 (19)16.95 ± 1.062/3 (10)12.32 ± 1.84 Conclusions: We have previously shown that the measurement of F/B in the primary tumor after resection is an independent prognostic indicator of metastasis-free survival in breast cancer. Our results in the current study furthers these observations and suggests that the evaluations of the microstructure of collagen fibers by F/B measurement from the pre-treatment biopsy, specifically at the leading edge of the tumor-stroma interface, may be useful for predicting pathologic response to trastuzumab-based neoadjuvant therapy. Further studies in a larger patient cohort are warranted. Citation Format: Desa DE, Turner BM, Buscaglia B, Hill RL, Strawderman RL, Hicks DG, Brown EB. Using multiphoton laser scanning microscopy to assess neoadjuvant therapy outcome in core needle biopsies: A novel methodology [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-12.

Abstract 37: Pulmonary pre-metastatic modifications induced by breast cancer

Abstract Breast cancer can be highly curable at early stages, while the survival rate plummets with the occurrence of metastasis. Thus, studies of molecular mechanisms that allow cancer cells to leave the primary tumor and ultimately establish colonies at distant sites are of crucial importance. First proposed by Stephen Paget in 1889, the “seed and soil” hypothesis suggests that the secondary organ microenvironment undergoes constant modification induced by the primary tumor through circulating chemokines. This leads to the migration of activated bone marrow derived cells to secondary organs, increased matrix metalloproteases (MMPs), and extracellular matrix (ECM) modifications in the secondary organ, collectively termed the pre-metastatic niche, which provides desirable docking sites for cancer cells. Our previous studies have shown persistent changes in the collagenous stroma and proteoglycans in pre-metastatic lungs of mice growing metastatic breast tumors, whose extent directly correlated with the malignancy of the primary tumor [1]. Elevated proteolytic MMP activity was also detected in these pre-metastatic lungs [2]. Based on these findings, we have continued our pre-metastatic niche studies by building an appropriate cell culture model, which consists of lung fibroblasts that were exposed to cell culture media conditioned by differentially malignant human breast epithelial cells. Cell lines used for fibroblast conditioning were nonmalignant MCF-12A breast epithelial cells (control), non-metastatic MCF-7 breast cancer cells, and highly metastatic, triple-negative MDA-MB-231 breast cancer cells. We focused our studies on ECM- and MMP-related changes, which we analyzed using qRT-PCR and immunoblotting. qRT-PCR analysis revealed that metastatic MDA-MB-231 cells induced an increase in the ECM protein Col14A1 as well as MMP-2 and MMP-14 in lung fibroblast as compared to induction by MCF-12A control. Immunoblotting analyses of lung fibroblasts showed that breast cancer cells induced increases in MMP expression in lung fibroblasts. Even higher MMP levels in fibroblasts were induced by metastatic MDA-MB-231 cells as compared to non-metastatic MCF-7 cells. Breast cancer cells also induced decreased expression levels of secreted MMPs in lung fibroblasts, most likely due to an enhanced release of these MMPs. This decrease was most significantly induced by the metastatic cell line. In conclusion, we have observed elevated levels in ECM molecules and MMPs in lung fibroblasts upon exposure of conditioned media from breast cancer cells. Our findings point towards an important role of lung fibroblasts in the evolution of the pre-metastatic niche in modifying the ECM, and their ability to directly respond to factors released from primary breast cancer cells with which they are not in direct contact. [1] Paidi SK et al. Cancer Res. 2017; 77:247-56. [2] Rizwan et al. Npj Breast Cancer 2015; 1: 15017. Citation Format: Ruoqing Cai, Caitlin M. Tressler, Menglin Cheng, Kanchan Sonkar, Santosh K. Paidi, Ishan Barman, Kristine Glunde. Pulmonary pre-metastatic modifications induced by breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 37.

Glucose insult elicits hyperactivation of cancer stem cells through miR-424\u2013cdc42\u2013prdm14 signalling axis

Background:Meta-analysis shows that women with diabetes have a 20% increased risk of breast cancer and also an increased risk for distant metastasis and mortality. The molecular mechanisms for distant metastasis and mortality in breast cancer patients with diabetes are not very well understood.Methods:We compared the effect of physiological (5 mM) and diabetic (10 mM) levels of glucose on malignant breast epithelial cell invasion and stemness capabilities. We performed microRNA array to determine the dysregulated microRNAs in hyperglycaemic conditions and performed functional and molecular analysis of the gene targets.Results:Hyperglycaemia leads to hyperactivation of cancer stem cell pool and enhances invasive ability of breast cancer cells. MiR-424 seems to be a key regulator of cancer cell stemness and invasion. Knockdown of miR-424 in cancer cells under euglycaemic conditions leads to enhanced invasion and stem cell activity, whereas ectopic expression of miR-424 in cancer cells under hyperglycaemic conditions results in suppressed invasion and stem cell activity. Cdc42, a target of miR-424, influences cancer stem cell activity by positively regulating prdm14 through activation of pak1 (p-21-activated kinase 1) and stat5.Conclusions:Our findings establish miR-424→cdc42→prdm14 axis as a key molecular signalling cascade that might influence breast cancer progression in diabetic patients through hyperactivation of cancer stem cells.

TGF-β-Dependent Growth Arrest and Cell Migration in Benign and Malignant Breast Epithelial Cells Are Antagonistically Controlled by Rac1 and Rac1b.

Despite improvements in diagnosis and treatment, breast cancer is still the most common cancer type among non-smoking females. TGF-β can inhibit breast cancer development by inducing cell cycle arrest in both, cancer cells and, as part of a senescence program in normal human mammary epithelial cells (HMEC). Moreover, TGF-β also drives cell migration and invasion, in part through the small GTPases Rac1 and Rac1b. Depletion of Rac1b or Rac1 and Rac1b in MDA-MB-231 or MDA-MB-435s breast cancer cells by RNA interference enhanced or suppressed, respectively, TGF-β1-induced migration/invasion. Rac1b depletion in MDA-MB-231 cells also increased TGF-β-induced p21WAF1 expression and ERK1/2 phosphorylation. Senescent HMEC (P15/P16), when compared to their non-senescent counterparts (P11/P12), presented with dramatically increased migratory activity. These effects were paralleled by elevated expression of genes associated with TGF-β signaling and metastasis, downregulated Rac1b, and upregulated Rac1. Our data suggest that acquisition of a motile phenotype in HMEC resulted from enhanced autocrine TGF-β signaling, invasion/metastasis-associated gene expression, and a shift in the ratio of antimigratory Rac1b to promigratory Rac1. We conclude that although enhanced TGF-β signaling is considered antioncogenic in HMEC by suppressing oncogene-induced transformation, this occurs at the expense of a higher migration and invasion potential.

1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase

Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies, we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)₂D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)₂D (10 nM) for 5 and 7 days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)₂D treatment for 5 days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)₂D-mediated regulation and hypothesized that 1,25(OH)₂D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was down-regulated with 1,25(OH)₂D treatment at 2 (73%±6 relative to vehicle) and 5 (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at 5 days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)₂D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)₂D regulates lipid metabolism in malignant breast epithelial cells.

Exosomes in Human Breast Milk Promote EMT.

Pregnancy increases breast cancer risk for all women for at least 5 years after parturition. During weaning and involution, the breast microenvironment becomes tumor promotional. Exosomes provide cell-cell communication during physiologic processes such as lactation, but also in breast cancer. We determined whether molecules in milk exosomes from healthy lactating women modulate the development and progression of breast cancer. Thirteen nursing women provided three (transitional, mature, and wean) milk samples. Exosomes were extracted and MCF7 and MCF10A breast cells labeled. The expression of six proteins linked to breast cancer was measured. On the basis of the findings, TGFβ2 concentration in exosome samples was measured, breast cells incubated with the exosomes and effect (epithelial-mesenchymal transition, EMT) on EMT-related proteins [E-cadherin, α-smooth muscle actin (α-SMA), filamentous (F)-actin and vimentin] measured. Human milk exosomes entered benign and malignant breast cells. The greatest change in wean milk protein was in TGFβ2 (P = 0.01). Exosomes with a high (but not low) level of TGFβ2 led to EMT in both cancer and benign cells, based on (i) change in cell morphology, actin cytoskeleton, and loss of cell-cell junction structure and (ii) increased α-SMA and vimentin and decreased E-cadherin. TGFβ2 is significantly upregulated in breast milk exosomes during weaning/early involution. Breast milk exosomes containing high levels of TGFβ2 induce changes in both benign and malignant breast epithelial cells, consistent with the development and progression of breast cancer, suggesting a role for high TGFβ2-expressing breast milk exosomes in influencing breast cancer risk. Clin Cancer Res; 22(17); 4517-24. ©2016 AACR.

Expression of tropomyosin 2 gene isoforms in human breast cancer cell lines.

In humans, four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) are known to produce a multitude of isoforms via alternate splicing and/or using alternate promoters. Expression of tropomyosin has been shown to be modulated at both the transcription and the translational levels. Tropomyosins are known to make up some of the stress fibers of human epithelial cells and differences in their expression has been demonstrated in malignant breast epithelial cell lines compared to 'normal' breast cell lines. We have recently reported the expression of four novel TPM1 isoforms (TPM1λ, TPM1µ, TPM1ν, and TPM1ξ) from human malignant tumor breast cell lines that are not expressed in adult and fetal cardiac tissue. Also, we evaluated their expression in relation to the stress fiber formation. In this study, nine malignant breast epithelial cell lines and three 'normal' breast cell lines were examined for stress fiber formation and expression of tropomyosin 2 (TPM2) isoform-specific RNAs and proteins. Stress fiber formation was assessed by immunofluorescence using Leica AF6000 Deconvolution microscope. Stress fiber formation was strong (++++) in the 'normal' cell lines and varied among the malignant cell lines (negative to +++). No new TPM2 gene RNA isoforms were identified, and TPM2β was the most frequently expressed TPM2 RNA and protein isoform. Stress fiber formation positively correlated with TPM2β RNA or protein expression at high, statistically significant degrees. Previously, we had shown that TPM1δ and TPM1λ positively and inversely, respectively, correlated with stress fiber formation. The most powerful predictor of stress fiber formation was the combination of TPM2β RNA, TPM1δ RNA, and the inverse of TPM1λ RNA expression. Our results suggest that the increased expression of TPM1λ and the decreased expression of TPM1δ RNA and TPM2β may lead to decreased stress fiber formation and malignant transformation in human breast epithelial cells.

Histone deacetylase 9 regulates breast cancer cell proliferation and the response to histone deacetylase inhibitors.

Histone lysine acetylation is an epigenetic mark regulated by histone acetyltransferases and histone deacetylases (HDAC) which plays an important role in tumorigenesis. In this study, we observed a strong overexpression of class IIa HDAC9, at the mRNA and protein levels, in the most aggressive human breast cancer cell lines (i.e. in basal breast cancer cells vs luminal ones or in malignant vs begnin MCF10A breast epithelial cell lines). HDAC9 overexpression was associated with higher rates of gene transcription and increased epigenetic marks on the HDAC9 promoter. Ectopic expression of HDAC9 in MCF7 luminal breast cancer cells led to an increase in cell proliferation and to a decrease in apoptosis. These effects were associated with a deregulated expression of several genes controlled by HDAC inhibitors such as CDKN1A, BAX and TNFRSF10A. Inversely, knock-down of HDAC9 expression in MDA-MB436 basal breast cancer cells reduced cell proliferation. Moreover, high HDAC9 expression decreased the efficacy of HDAC inhibitors to reduce cell proliferation and to regulate CDKN1A gene expression. Interestingly, the gene encoding the transcription factor SOX9 was identified by a global transcriptomic approach as an HDAC9 target gene. In stably transfected MCF7 cells, SOX9 silencing significantly decreased HDAC9 mitogenic activity. Finally, in a large panel of breast cancer biopsies, HDAC9 expression was significantly increased in tumors of the basal subtype, correlated with SOX9 expression and associated with poor prognosis. Altogether, these results indicate that HDAC9 is a key factor involved in mammary carcinogenesis and in the response to HDAC inhibitors.

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.

Cancer stem cells and early stage basal-like breast cancer.

Ductal carcinoma in situ (DCIS) is a category of early stage, non-invasive breast tumor defined by the intraductal proliferation of malignant breast epithelial cells. DCIS is a heterogeneous disease composed of multiple molecular subtypes including luminal, HER2 and basal-like types, which are characterized by immunohistochemical analyses and gene expression profiling. Following surgical and radiation therapies, patients with luminal-type, estrogen receptor-positive DCIS breast tumors can benefit from adjuvant endocrine-based treatment. However, there are no available targeted therapies for patients with basal-like DCIS (BL-DCIS) tumors due to their frequent lack of endocrine receptors and HER2 amplification, rendering them potentially susceptible to recurrence. Moreover, multiple lines of evidence suggest that DCIS is a non-obligate precursor of invasive breast carcinoma. This raises the possibility that targeting precursor BL-DCIS is a promising strategy to prevent BL-DCIS patients from the development of invasive basal-like breast cancer. An accumulating body of evidence demonstrates the existence of cancer stem-like cells (CSCs) in BL-DCIS, which potentially determine the features of BL-DCIS and their ability to progress into invasive cancer. This review encompasses the current knowledge in regard to the characteristics of BL-DCIS, identification of CSCs, and their biological properties in BL-DCIS. We summarize recently discovered relevant molecular signaling alterations that promote the generation of CSCs in BL-DCIS and the progression of BL-DCIS to invasive breast cancer, as well as the influence of the tissue microenvironment on CSCs and the invasive transition. Finally, we discuss the translational implications of these findings for the prognosis and prevention of BL-DCIS relapse and progression.

The nicotinic acetylcholine receptor-mediated reciprocal effects of the tobacco nitrosamine NNK and SLURP-1 on human mammary epithelial cells

Recent research has demonstrated that the nicotinergic signaling network of mammary epithelium can both mediate the physiological control of normal breast epithelial cells (BECs) and exhibit tumor-promoting effects on malignant BECs. Therefore, mammary nicotinic acetylcholine (ACh) receptors (nAChRs) may become a specific target for novel anti-breast cancer therapies. Toward this goal, we investigated the difference in the ACh receptor repertoires between normal and malignant BECs, determined effects of nicotinic ligands on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-dependent activation of ERK1/2 and tumorigenic transformation of MCF10A cells, and characterized reciprocal effects of NNK and SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor related protein-1)-1 on the expression of nAChR subunits and several oncogenes and tumor-suppressing genes in BECs. Both the non-malignant MCF10A and malignant MCF7 breast cells expressed α3, α5, α7, α9, α10, β1, β2, γ, δ and ε nAChR subunits and M1, M3, M4 and M5 muscarinic receptor subtypes. The malignancy was associated with expression of α1, α4 and β4 nAChR subunits and M2 subtype. Malignant transformation of BECs was also associated with overexpression of α7-, and α9-made nAChRs. NNK upregulated ERK1/2 phosphorylation, stimulated expression of the gene encoding the tumor-promoter HGF, downregulated expression of the tumor suppressor gene CDKN2A, and induced tumorigenic transformation of MCF10A cells. Compared to the canonical nAChR antagonists, SLURP-1 showed the highest ability to abolish the nAChR-mediated effects of NNK in both cell-signaling and cell-transformation assays and reversed many effects of NNK on gene expression. SLURP-1 also markedly upregulated the tumor suppressor genes CDKN2B, RUNX3 and TP73. Altogether, the obtained results provided new insight into the molecular mechanisms of nAChR-mediated oncogenic effects of NNK on BECs and demonstrated the ability to abolish or reverse these effects by SLURP-1.

MicroRNA-125a influences breast cancer stem cells by targeting leukemia inhibitory factor receptor which regulates the Hippo signaling pathway.

Cancer stem cells (CSC) are the main driving force behind cancer initiation and progression. The molecular mechanisms that regulate CSC properties are poorly understood. MicroRNAs (miRNAs) play a significant role in normal and cancer tissues. Here, we show that miRNA-125a indirectly regulates TAZ, an effector molecule in the Hippo pathway, through the leukemia inhibitory factor receptor (LIFR). The miR-125a→LIFR axis affected the homeostasis of nonmalignant and malignant breast epithelial stem cells through the Hippo signaling pathway. Inhibition of miR-125a in breast cancer cells led to a significant reduction in the CSC pool. In contrast, enhanced expression of miR-125a in nonmalignant breast epithelial cells resulted in significant expansion of the stem cell pool. Gain of function and loss of function of LIFR directly correlated with the inhibition and overexpression of miR-125a, respectively. Modulation of miR-125a led to a change in the activity of TAZ and its subcellular localization. We further demonstrated that miR-125a influenced stem cells by regulating Hippo signaling through LIFR in human primary breast cancer cells confirming the data obtained from established cell lines. We suggest that miR-125a could be a potential target against CSCs that maybe used along with the existing conventional therapies.

Expression of Tropomyosin 1 Gene Isoforms in Human Breast Cancer Cell Lines.

Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1γ and TPM1δ were the dominant transcripts of TPM1, there was no clear evidence for TPM1δ protein expression. Four novel human TPM1 gene RNA isoforms were discovered (λ, μ, ν, and ξ), which were not identified in adult and fetal human cardiac tissues. TPM1λ was the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1γ, λ, and ν protein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1γ RNA expression but significantly and inversely correlated with TPM1δ and TPM1λ expression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study.