Human Mammary Epithelial Cell Line Research Articles

PRRX2 as a novel TGF‐β‐induced factor enhances invasion and migration in mammary epithelial cell and correlates with poor prognosis in breast cancer

TGF-β and cancer progression share a multifaceted relationship. Despite the knowledge of TGF-β biology in the development of cancer, several factors that mediate the cancer-promoting role of TGF-β continue to be identified. This study aimed to identify and characterise novel factors potentially related to TGF-β-mediated tumour aggression in breast cells. We treated the human mammary epithelial cell line MCF10A with TGF-β and identified TGF-β-dependent upregulation of PRRX2, the gene encoding paired-related homeobox 2 transcription factor. Overexpression of PRRX2 enhanced migration, invasion and anchorage-independent growth of MCF10A cells and induced partial epithelial mesenchymal transition (EMT), as determined by partial fibroblastoid morphology of cells, upregulation of EMT markers and partially disrupted acinar structure in a three-dimensional culture. We further identified PLAT, the gene encoding tissue-type plasminogen activator (tPA), as the highest differentially expressed gene in PRRX2-overexpressing MCF10A cells, and demonstrated direct binding and transactivation of the PLAT promoter by PRRX2. Furthermore, PLAT knockdown inhibited PRRX2-mediated enhanced migration and invasion, suggesting that tPA may mediate PRRX2-induced migration and invasion. Finally, the significant correlation of PRRX2 expression with poor survival in 118 primary breast tumour samples (P = 0.027) and the increased PRRX2 expression in metaplastic breast carcinoma samples, which is pathogenetically related to EMT, validated the biological importance of PRRX2-enhanced migration and invasion and PRRX2-induced EMT. Thus, our data suggest that upregulation of PRRX2 may be a mechanism contributing to TGF-β-induced invasion and EMT in breast cancer. © 2016 Wiley Periodicals, Inc.

Rad51c- and Trp53-double-mutant mouse model reveals common features of homologous recombination-deficient breast cancers.

Almost half of all hereditary breast cancers (BCs) are associated with germ-line mutations in homologous recombination (HR) genes. However, the tumor phenotypes associated with different HR genes vary, making it difficult to define the role of HR in BC predisposition. To distinguish between HR-dependent and -independent features of BCs, we generated a mouse model in which an essential HR gene, Rad51c, is knocked-out specifically in epidermal tissues. Rad51c is one of the key mediators of HR and a well-known BC predisposition gene. Here, we demonstrate that deletion of Rad51c invariably requires inactivation of the Trp53 tumor suppressor (TP53 in humans) to produce mammary carcinomas in 63% of female mice. Nonetheless, loss of Rad51c shortens the latency of Trp53-deficient mouse tumors from 11 to 6 months. Remarkably, the histopathological features of Rad51c-deficient mammary carcinomas, such as expression of hormone receptors and luminal epithelial markers, faithfully recapitulate the histopathology of human RAD51C-mutated BCs. Similar to other BC models, Rad51c/p53 double-mutant mouse mammary tumors also reveal a propensity for genomic instability, but lack the focal amplification of the Met locus or distinct mutational signatures reported for other HR genes. Using the human mammary epithelial cell line MCF10A, we show that deletion of TP53 can rescue RAD51C-deficient cells from radiation-induced cellular senescence, whereas it exacerbates their centrosome amplification and nuclear abnormalities. Altogether, our data indicate that a trend for genomic instability and inactivation of Trp53 are common features of HR-mediated BCs, whereas histopathology and somatic mutation patterns are specific for different HR genes.

Polymeric nanocapsules prevent oxidation of core-loaded molecules: evidence based on the effects of docosahexaenoic acid and neuroprostane on breast cancer cells proliferation

BackgroundNanocapsules, as a delivery system, are able to target drugs and other biologically sensitive molecules to specific cells or organs. This system has been intensively investigated as a way to protect bioactives drugs from inactivation upon interaction with the body and to ensure the release to the target. However, the mechanism of improved activity of the nanoencapsulated molecules is far from being understood at the cellular and subcellular levels. Epidemiological studies suggest that dietary polyunsaturated fatty acids (PUFA) can reduce the morbidity and mortality from breast cancer. This influence could be modulated by the oxidative status of the diet and it has been suggested that the anti-proliferative properties of docosahexaenoic acid (DHA) are enhanced by pro-oxidant agents.MethodsThe effect of encapsulation of PUFA on breast cancer cell proliferation in different oxidative medium was evaluated in vitro. We compared the proliferation of the human breast cancer cell line MDA-MB-231 and of the non-cancer human mammary epithelial cell line MCF-10A in different experimental conditions.ResultsDHA possessed anti-proliferative properties that were prevented by alpha-tocopherol (an antioxidant) and enhanced by the pro-oxidant hydrogen peroxide that confirms that DHA has to be oxidized to exert its anti-proliferative properties. We also evaluated the anti-proliferative effects of the 4(RS)-4-F4t-neuroprostane, a bioactive, non-enzymatic oxygenated metabolite of DHA known to play a major role in the prevention of cardiovascular diseases. DHA-loaded nanocapsules was less potent than non-encapsulated DHA while co-encapsulation of DHA with H2O2 maintained the inhibition of proliferation. The nanocapsules slightly improves the anti-proliferative effect in the case of 4(RS)-4-F4t-neuroprostane that is more hydrophilic than DHA.ConclusionOverall, our findings suggest that the sensitivity of tumor cell lines to DHA involves oxidized metabolites. They also indicate that neuroprostane is a metabolite participating in the growth reducing effect of DHA, but it is not the sole. These results also suggest that NC seek to enhance the stability against degradation, enhance cellular availability, and control the release of bioactive fatty acids following their lipophilicities.

Filamin A (FLNA) modulates chemosensitivity to docetaxel in triple-negative breast cancer through the MAPK/ERK pathway.

A previous RNA interference (RNAi) screen identified filamin A (FLNA) as a potential biomarker to predict chemosensitivity in triple-negative breast cancer (TNBC). However, its ability to modulate chemosensitivity and the underlying mechanism has not been investigated. Genetic manipulation of FLNA expression has been performed in an immortalized noncancerous human mammary epithelial cell line and four TNBC cell lines to investigate its effect on chemosensitivity. Western blot analysis was performed to identify the potential signaling pathway involved. Xenograft mouse model was used to examine the in vivo role of FLNA in modulating chemosensitivity. Overexpression of FLNA conferred chemoresistance to docetaxel in noncancerous human mammary epithelial cells. Knockdown of FLNA sensitized four TNBC cell lines, MDA-MB-231, HCC38, Htb126, and HCC1937 to docetaxel which was reversed by reconstituted FLNA expression. Decreased FLNA expression correlated with decreased activation of ERK. Constitutive activation of ERK2 reversed siFLNA-induced chemosensitization. Inhibition of MEK1 recapitulates the effect of FLNA knockdown. MDA-MB-231 xenograft with FLNA knockdown showed enhanced response to docetaxel compared with control xenograft with increased apoptosis. FLNA can function as a modulator of chemosensitivity to docetaxel in TNBC cells through regulation of the MAPK/ERK pathway both in vitro and in vivo. FLNA may serve as a novel therapeutic target for improvement of chemotherapy efficacy in TNBC.

Secreted factors derived from obese visceral adipose tissue regulate the expression of breast malignant transformation genes.

Obese adipose tissue, especially the visceral depot, exhibits altered production of several molecules that could have a role on the initiation/promotion of breast cancer development. The aim of this work was to evaluate the effect of excess adipose tissue and its secreted factors on the expression of genes involved in the early steps of tumor promotion on the mammary gland. Carcinogenesis-related gene expression was evaluated in mammary gland tissue from female diet-induced obese (DIO) Sprague-Dawley rats and circulating leukocytes isolated from a group of breast cancer diagnosed and non-diagnosed obese women and compared with their normal weight counterparts. In addition, the human non-tumoral mammary epithelial cell line MCF10A was treated in vitro with the visceral (retroperitoneal adipose tissue (RPAT)) or subcutaneous adipose tissue (SAT) secretome and with rising concentrations of the lipid peroxidation by-product 4-hydroxynonenal (4-HNE). DIO rats were classified as susceptible to DIO (DIO-S) or partially resistant to DIO (DIO-R) according to the maximum fat mass gain of the lean group as a cut-off. As compared with lean and DIO-R, the DIO-S group showed a higher fat mass and lower lean mass. The anatomical characteristic of DIO-S was correlated with differential expression of cellular proliferation (ALDH3A1 and MYC) and antioxidant and DNA protection (GSTM2, SIRT1), and tumor suppression (TP53, PTEN, TGFB1) genes. Remarkably, this carcinogenesis-related gene expression pattern was reproduced in MCF10A treated with the RPAT secretome from DIO-S rats and with the lipid peroxidation by-product 4-HNE. Moreover, this pattern was also detected in leukocytes from obese women compared with normal weight women without evidence of breast cancer. Lipid peroxides secreted by the obese visceral adipose tissue could be among the relevant factors that promote changes involved in the early steps of tumor development in mammary gland. These changes can be detected even before histological alterations and in circulating leukocytes.

Abstract 4040: Transcription factor Snail mediates EMT by altering vesicular trafficking protein Rab25

Abstract Epithelial cells maintain intercellular communication directly by cell-cell, cell-ECM interaction and indirectly by paracrine secretion of extracellular messengers either as free molecules or via exosomes. The vesicular trafficking machinery is an integral part of such communication, paralleling digital cables that transmit the information bytes to the correct hub. During the process of EMT, derailing of the trafficking machinery leads to a systemic breakdown of intra and inter-cellular communication pathways. The purpose of the current study is to elucidate the functional role of Rab25, a key member of the vesicular trafficking machinery in mediating EMT-MET processes during breast cancer progression. Aberrant endocytosis is a new hallmark of cancer. Rab GTPases are the primary regulators of endocytosis. Many members of this large family are implicated in a wide array of cancers including breast cancer. Specifically, Rab25, a GTPase associated with the recycling endosomes, is deregulated in most epithelial cancers. Rab25 appears to function both as a tumor suppressor and an oncogene in a context dependent manner. We found that Rab25 is lost in Claudin Low tumors as well as in representative cell lines such as the MDA MB231. Rab25 is a robust and sufficient marker of breast cancers and cell lines with an increased EMT signature. Additionally, in immortalized human mammary epithelial cell lines (HMLE), induction of EMT by overexpression of Snail1 completely obliterates Rab25 protein. Exogenous re-expression of Rab25 dramatically rescues these cells from Snail driven invasion through MatrigelTM. However this Snail dependent regulation is unique only to TN and Claudin Low tumors and does not hold true for hormone receptor positive tumors. Further, analysis of RNA Seq data from a MMTV mouse model showed that tumors with low CDH1/Claudins expression and enriched in EMT markers also lost Rab25 transcripts. Snail mediated transcriptional repression targets recycling endosomal proteins such as Rab25 to decrease delivery of junction proteins that facilitate cell-cell communication to the appropriate compartment. In parallel, it potentially enhances paracrine regulation by secreted exosomes. Overall, our study shows presence and absence of Rab25 markedly alters cell-cell communication, which is essential for maintenance of epithelial features of cells. Rab25 could be a prognostic marker and a therapeutic target for a subset of patients with increased risk of metastatic disease. Note: This abstract was not presented at the meeting. Citation Format: Shreya Mitra, Lorenzo Federico, Tapashree Roysarkar, Sendurai Mani, Gordon B. Mills. Transcription factor Snail mediates EMT by altering vesicular trafficking protein Rab25. [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 4040. doi:10.1158/1538-7445.AM2015-4040

Abstract 3562: Protein palmitoylation in breast cancer: DHHC5 palmitoyltransferase as a potential therapeutic target

Abstract Protein S-palmitoylation is a widespread and dynamic post-translational modification that regulates protein-membrane interactions, protein-protein interactions, and protein stability. S-palmitoylation is catalyzed by members of a family of 23 palmitoyl acyl transferases, termed the DHHC family due to the presence of a common catalytic motif. The role of protein palmitoylation in cancer is largely unexplored. In our previous study, we have shown that DHHC5 palmitoyltransferase supports the growth of non-small cell lung cancer. To further investigate the role of DHHC5 in cancer, we expanded our study to include breast cancer cell lines. Stable knockdowns of DHHC5 were undertaken in 8 breast cancer cell lines (including 4 “triple-negative” breast cancer lines) as well as in two normal human mammary epithelial cell lines (HMECs) using lentiviral shRNAs. The protein levels of DHHC5 were analyzed by immunoblotting, which all showed efficient knockdown. Cell proliferation and anchorage-dependent/ independent colony formation assays indicated that DHHC5 knockdown led to cell growth arrest and decreased cell clonogenicity in most of the tested breast cancer cell lines in vitro, but not in the HMECs. BT-549, SkBr3 and T-47D cell lines were particularly dependent on DHHC5 expression, and rescue experiments involving ectopic plasmid-driven DHHC5 and its catalytic mutant DHHS expression are in progress. In vivo tumor formation assays will be carried out by injecting MCF-7 and T-47D control cells and DHHC5 stable knockdown cells as well as rescued cells subcutaneously into NOD/SCID mice. Tumor xenograft formation will be measured. As DHHC5 function is needed for optimal breast cancer cell growth, it will be important to identify physiologically relevant substrates in order to develop inhibitors of DHHC5 function. Future plans include microarray and protein kinase array analysis to identify possible signaling pathways in which DHHC5 may be involved, as well as palmitoyl-protein profiling of control and knockdown DHHC5 cell lines by mass spectrometry. Citation Format: Hui Tian, Xiaoxia Qi, Kenneth Huffman, John Minna, Sandra L. Hofmann. Protein palmitoylation in breast cancer: DHHC5 palmitoyltransferase as a potential therapeutic target. [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 3562. doi:10.1158/1538-7445.AM2015-3562

Abstract LB-207: ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell type-specific manner

Abstract [Introduction] Despite apparent resection of tumors, breast cancer patients often suffer relapse years after due to remnant dormant tumor cells. Nevertheless, the molecular mechanism regulating dormancy of breast cancer cell is still unclear. On the other hand, relapsed tumor is generally believed to be derived from cancer stem cells retaining normal stem cell property, which can undergo quiescence. ΔNp63α, an N-terminally truncated isoform of p51/p63 protein was shown to play crucial roles in the maintenance of stem cells within mammary epithelium. Furthermore, both tumor suppressive and oncogenic roles of ΔNp63α have been reported in mammary carcinogenesis. Thus, we investigated the role of ΔNp63α in the regulation of normal and malignant breast cells. [Method] MCF10A, normal human mammary epithelial cells and breast cancer cell lines of different subtypes were introduced with doxycycline-inducible constructs of ΔNp63α. These cells were subjected to cell cycle analysis, western blot, BrdU-Ki-67 immunostaining. Stemness was determined by mammosphere formation assay combined with flow cytometry analysis of cell surface markers. Drug response was studied using MTT assay. Also luminal breast cancer cell line, MCF7, was subjected to combined mRNA-miRNA microarray analysis. [Result] The induction of ΔNp63α in MCF7 luminal breast cancer cell line led the cells to acquire phenotype typical to quiescent luminal progenitor-like cells. Furthermore, the BMP and Wnt signaling pathways emerged as regulators of ΔNp63α-induced quiescence by microarray analysis. Interestingly, these quiescent cells exhibited down-regulation of BRCA1-dependent DNA repair pathways making them more sensitive to DNA damaging drug, Doxorubicin, but more resistant to anti-mitotic drug, Paclitaxel. Conversely, induction of ΔNp63α in MCF10A normal basal cells resulted in increased cell proliferation and expansion of mammary stem-like cells. ΔNp63α expression also led to the increase of cells expressing breast cancer stem cell markers in both MCF7 and MCF10A cells. However, induction of ΔNp63α affected neither cell proliferation nor the increase of mammary stem- or luminal progenitor-like cells in more aggressive luminal (T47D) and basal (MDA-MB-231) breast cancer cells containing mutant p53. This result interestingly suggested a cell type-specific function of ΔNp63α. We also identified a microRNA network that might regulate quiescence in ΔNp63α-expressing breast cancer cells which will be presented at the meeting in detail. [Conclusion] Our results suggest that the role of ΔNp63α in regulating quiescence, stem or progenitor activity in normal and breast cancer cells is cell type-specific. Given the heterogeneity in the cellular origin of breast cancer subtypes, ΔNp63α-directed chemotherapeutic strategy might be instructive to target therapeutically resistant breast cancer cells. Citation Format: Md. Ruhul Amin, Yuiko Morita-Fujimura, Shuntaro Ikawa. ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell type-specific manner. [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 LB-207. doi:10.1158/1538-7445.AM2015-LB-207

Abstract 3488: BPM 31510 has broad utility as both a single agent and in combination with standard-of-care chemotherapy in triple-negative breast cancer

Abstract Deregulated metabolism is recognized as a ‘hallmark of cancer’ although our understanding of the interplay between metabolic pathway activity and responses to standard-of-care chemotherapy remains limited. In vitro studies have demonstrated that BPM 31510, a Coenzyme Q10-containing proprietary formulation, alters both glycolysis and mitochondrial OXPHOS in cancer cells. Building on the clinical observation that triple-negative breast cancer (TNBC) is distinctly glycolytic compared to other breast sub-types, we examined the effects of BPM 31510 alone and in combination with standard-of-care chemotherapy in a murine xenograft model of TNBC (MDA-MB231). Tumor-bearing mice were treated with cycles of the standard ‘TAC regimen’ (5mg/kg docetaxel and 1 mg/kg doxorubicin for 3 days followed by 35 mg/kg cyclophosphamide) with or without BPM 31510 every 3 weeks. As expected, chemotherapy treatment extended survival. Notably, BPM 31510 was more effective as a single agent compared to traditional chemotherapy and extended survival by more than 6 weeks. The combination of BPM 31510 with chemotherapy further enhanced survival over either treatment regimen alone, and this was associated with attenuated tumor growth over the duration of the study. Analysis of a subset of tumors after 2 cycles of treatment confirmed a decrease in tumor weight in all treatment conditions compared to control and a significant synergistic effect of combined therapy (p = 0.02). Importantly, changes were not observed in the weight of other organs from the same mice, suggesting lack of systemic toxicity. Evidence of decreased proliferation (Ki67 staining) and enhanced caspase activity were also observed in tumors by immunohistochemical analysis. Assessment of the BPM 31510 IC50 in vitro using a panel of primary human mammary epithelial cells (HMEC), non-tumorigenic (MCF12A), and breast cancer cell lines demonstrated selective cytotoxicity in cancer cells compared to primary and non-tumorigenic cells. Moreover, MDA-MB231 was the most sensitive to BPM 31510-induced cell death in the breast cancer cell panel that was representative of multiple breast tumor sub-types. Taken together, these data demonstrate that the metabolic modulating agent BPM 31510 has broad utility as both a single agent and in combination with standard-of-care in breast cancer including distinctly glycolytic TNBC for which prognosis is poor and few therapeutic options exist. Citation Format: Niven R. Narain, Anne R. Diers, Arleide Lee, Rakibou Ouro-Djobo, Vivek K. Vishnudas, Ely Benaim, Joaquin J. Jimenez, Rangaprasad Sarangarajan. BPM 31510 has broad utility as both a single agent and in combination with standard-of-care chemotherapy in triple-negative breast cancer. [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 3488. doi:10.1158/1538-7445.AM2015-3488

Differential Effects of Glycyrrhiza Species on Genotoxic Estrogen Metabolism: Licochalcone A Downregulates P450 1B1, whereas Isoliquiritigenin Stimulates It.

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 μg/mL), and LigC (1 μM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 μM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 μM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 μg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women's health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.

Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells.

Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture), three-dimensional (3D) “on-top” Matrigel, 3D “cell-embedded” Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D) culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

Abstract A28: Butterfly effect in cancer: How a PI3-kinase mutation remodels the cell

Abstract PI 3-kinase is a frequently mutated oncogene. Along with the phosphatase PTEN, numerous receptor tyrosine kinases, and Akt, the PI 3-kinase pathway is among the most frequently activated in cancer. The H1047R mutation of PIK3CA is a representative gain-of-function mutation in the PI 3-kinase pathway and is transforming in both chicken fibroblasts and mouse embryonic fibroblasts. Recently, a knock-in of this mutation has been made in the normal human mammary epithelial cell line, MCF-10A. Here, H1047R confers growth factor independence, but does enable the knock-in cells to grow in soft agar or to form xenografts in mice. We have investigated this knock-in cell line and the parental MCF-10A by both deep sequencing and proteomics to determine the transcriptional and translational changes induced by PIK3CA-H1047R. We can confirm that the only coding mutation introduced into the MCF-10A cell line is PIK3CA-H1047R. Surprisingly, this single base mutation induces changes in the expression of thousands of transcripts and proteins. Despite rapid proliferation and growth factor independence, H1047R also induces a decrease in cell cycle genes including Cyclin A2 and Cyclin D2. The decrease in these genes may be the cause of an observed decrease in S and G2 phase cells as compared with the parental MCF-10A cell line. The parental cells overexpress MYC, however this overexpression is lost upon H1047R expression. Inhibition of PI 3-kinase with small molecule compounds that are selective for p110α or affect all isoforms of p110 as well as inhibition of mTOR do not restore the expression of c-MYC. This suggests that long-term activation of the PI 3-kinase pathway can result in changes to the gene expression landscape which no longer depend upon the PI 3-kinase pathway. Finally, H104R causes changes in 3D cell culture morphology. Both MCF-10A and the H1047R knock-in form acinar structures with hollow lumen when grown on reduced growth factor Matrigel substrates. However, if these cultures are treated with IGF1, the MCF-10A cells retain their acinar structures while, H1047R form branching tube-like structures which invade into Matrigel. The formation of these structures can be inhibited by the introduction of GDC-0941, a pan-PI3K inhibitor. IGF1 has no effect upon cell proliferation or migration in 2D cell culture systems. In summary, the H1047R mutation of PIK3CA while possessing a single catalytic activity can lead to a diverse range of phenotypes beyond growth factor independence. This work was supported by the National Institutes of Health under award numbers R01 CA078230 and R21 AG039716 (JRH and PKV), P30 NS057096 and P41 RR011823 (LL and JRY). This is abstract 24060 of The Scripps Research Institute. Citation Format: Jonathan R. Hart, Yaoyang Zhang, Lujian Liao, Lisa Du, John R. Yates, III, Peter K. Vogt. Butterfly effect in cancer: How a PI3-kinase mutation remodels the cell. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A28.

Linear mRNA amplification approach for RNAseq from limited amount of RNA

Whole-transcriptome evaluation by next-generation sequencing (NGS) has been widely applied in the investigation of diverse transcriptional scenarios. In many clinical situations, including needle biopsy samples or laser microdissected cells, limited amounts of RNA are usually available for the assessment of the whole transcriptome. Here, we describe an mRNA amplification protocol based on in vitro T7 transcription for transcriptome evaluation by NGS. Initially, we performed RNAseq from two human mammary epithelial cell lines and evaluated several aspects of the transcriptomes generated by linear amplification of Poly (A)+ mRNA species, including transcript representation, variability and abundance. Our protocol showed to be efficient with respect to full-length transcript coverage and quantitative expression levels. We then evaluated the applicability of using this protocol in a more realistic research scenario, analyzing tumor tissue samples microdissected by laser capture. In order to increase the quantification power of the libraries only the 3′ end of transcripts were sequenced. We found highly reproducible RNAseq data among amplified tumor samples, with a median Spearman's correlation of 80%, strongly suggesting that the amplification step and library protocol preparation lead to a consistent transcriptional profile. Altogether, we established a robust protocol for assessing the polyadenylated transcriptome derived from limited amounts of total RNA that is applicable to all NGS platforms.

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16INK4A, or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

Inhibitory mechanism of FAT4 gene expression in response to actin dynamics during Src-induced carcinogenesis.

Oncogenic transformation is characterized by morphological changes resulting from alterations in actin dynamics and adhesive activities. Emerging evidence suggests that the protocadherin FAT4 acts as a tumor suppressor in humans, and reduced FAT4 gene expression has been reported in breast and lung cancers and melanoma. However, the mechanism controlling FAT4 gene expression is poorly understood. In this study, we show that transient activation of the Src oncoprotein represses FAT4 mRNA expression through actin depolymerization in the immortalized normal human mammary epithelial cell line MCF-10A. Src activation causes actin depolymerization via the MEK/Erk/Cofilin cascade. The MEK inhibitor U0126 blocks the inhibitory effect of Src on FAT4 mRNA expression and Src-induced actin depolymerization. To determine whether actin dynamics act on the regulation of FAT4 mRNA expression, we treated MCF-10A cells with the ROCK inhibitor Y-27632. Y-27632 treatment decreased FAT4 mRNA expression. This suppressive effect was blocked by siRNA-mediated knockdown of Cofilin1. Furthermore, simultaneous administration of Latrunculin A (an actin depolymerizing agent), Y-27632, and Cofilin1 siRNA to the cells resulted in a marked reduction of FAT4 mRNA expression. Intriguingly, we also found that FAT4 mRNA expression was reduced under both low cell density and low stiffness conditions, which suggests that mechanotransduction affects FAT4 mRNA expression. Additionally, we show that siRNA-mediated FAT4 knockdown induced the activity of the Hippo effector YAP/TAZ in MCF-10A cells. Taken together, our results reveal a novel inhibitory mechanism of FAT4 gene expression through actin depolymerization during Src-induced carcinogenesis in human breast cells.

Abstract A42: Exogenous normal mammary epithelial mitochondria increase the drug sensitivity of human breast cancer MCF-7 cells

Abstract The mitochondrial dysfunction of cancer cells leads to the suppression of glucose oxidation, increased glycolysis, proliferated advantage, resistance to apoptosis and drug resistance. We hypothesize that the introduction of normal mitochondria into cancer cells will restore the mitochondrial function of cancer cells and increase drug sensitivity. We stained the mitochondria of the normal human mammary epithelial cell line MCF-12A with JC-1 dye, and found orange fluorescence (JC-1 aggregates in mitochondria due to high mitochondrial membrane potential) in the MCF-12A cells. We then isolated the JC-1 stained-mitochondria of the MCF-12A cells and co-cultured them with the cells of human breast cancer MCF-7 cell line. At 30 minutes after co-culture of MCF-7 cells with the isolated JC-1-stained mitochondria, the orange fluorescence was observed in the MCF-7 cells. The fluorescence continued to increase for at least 6 hours and was still detected at 24 hours. This demonstrated that exogenous normal mitochondria of MCF-12A can enter into MCF-7 breast cancer cells. In order to determine whether mitochondria of normal MCF-12A cells improve the sensitivity of breast cancer cells to chemotherapeutic drugs, we co-cultured MCF-7 cells for 24 hours with normal mitochondria of MCF-12A, aspirated off the media and treated the MCF-7 cells with the drugs doxorubicin, abraxane or carboplatin. We found that MCF-7 cells, treated with the normal mitochondria of MCF-12A, were more sensitive to doxorubicin, abraxane and carboplatin than non-treated MCF-7 cells. We also have RT-PCR results demonstrating that uptake of normal mitochondria by cancer cells down-regulates the expression of membrane glucose transporter GLUT3 in MCF-7 cells. In conclusion, we suggest that the introduction of normal cell mitochondria into cancer cells could restore cancer cell mitochondrial function and significantly impact cancer therapy. Citation Format: Xian-Peng Jiang, Jeff Philips, Robert L. Elliott, Jonathan F. Head. Exogenous normal mammary epithelial mitochondria increase the drug sensitivity of human breast cancer MCF-7 cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A42.

Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling.

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT (TertHMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached TertHMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.

Photoluminescent graphene quantum dots for in vivo imaging of apoptotic cells.

Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL(-1) over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications.

Serotonin regulates β-casein expression via 5-HT7 receptors in human mammary epithelial MCF-12A cells.

We previously reported that serotonin (5-hydroxytryptamine; 5-HT) suppresses β-casein expression, a differentiation marker in mammary epithelial cells, via inhibition of the signal transducer and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial cell line, MCF-12A. In this study, we investigated the expression pattern of the different 5-HT receptor subtypes in MCF-12A cells, and identified the receptors involved in 5-HT-mediated suppression of β-casein protein expression. β-Casein mRNA expression was inhibited by 30 µM 5-HT in a time-dependent manner. Treatment with 30 µM 5-HT for 72 h decreased β-casein protein levels and STAT5 phosphorylation (pSTAT5). The cells expressed four 5-HT receptors subtypes (5-HTR1D, 2B, 3A, and 7) at the mRNA and protein level, and their expression was elevated by prolactin (PRL) treatment. Additionally, the mRNA levels of 5-HTR1D and 5-HTR7 were significantly higher than the other 5-HT receptors in the cells. Tryptophan hydroxylase 1 mRNA was detectable in the cells in the absence of PRL, and PRL treatment significantly increased its expression. β-Casein and pSTAT5/STAT5 levels in the cells co-treated with 5-HT and a selective 5-HTR1D inhibitor, BRL15572, were equal to those observed in cells treated with 5-HT alone. However, in the cells co-treated with 5-HT and a selective 5-HTR7 inhibitor, SB269970, β-casein and pSTAT5/STAT5 levels increased in a SB269970 concentration-dependent manner. In conclusion, we showed that 5-HT regulates β-casein expression via 5-HTR7 in MCF-12A human mammary epithelial cells.

Effect of oncogene activating mutations and kinase inhibitors on amino acid metabolism of human isogenic breast cancer cells.

We investigated the changes in amino acid (AA) metabolism induced in MCF10A, a human mammary epithelial cell line, by the sequential knock-in of K-Ras and PI3K mutant oncogenes. Differentially regulated genes associated to AA pathways were identified on comparing gene expression patterns in the isogenic cell lines. Additionally, we monitored the changes in the levels of AAs and transcripts in the cell lines treated with kinase inhibitors (REGO: a multi-kinase inhibitor, PI3K-i: a PI3K inhibitor, and MEK-i: a MEK inhibitor). In total, 19 AAs and 58 AA-associated transcripts were found to be differentially regulated by oncogene knock-in and by drug treatment. In particular, the multi-kinase and MEK inhibitor affected pathways in K-Ras mutant cells, whereas the PI3K inhibitor showed a major impact in the K-Ras/PI3K double mutant cells. These findings may indicate the dependency of AA metabolism on the oncogene mutation pattern in human cancer. Thus, future therapy might include combinations of kinase inhibitors and drug targeting enzymes of AA pathways.