Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model.

BACKGROUND: Triple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking. METHODS: We use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining. RESULTS: TU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres. CONCLUSIONS: Here we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture.

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix. CONCLUSIONS: Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers.

INTRODUCTION: The steady increase in the incidence of obesity among adults has been paralleled with higher levels of obesity-associated breast cancer. While recent studies have suggested that adipose stromal/stem cells (ASCs) isolated from obese women enhance tumorigenicity, the mechanism(s) by which this occurs remains undefined. Evidence suggests that increased adiposity results in increased leptin secretion from adipose tissue, which has been shown to increased cancer cell proliferation. Previously, our group demonstrated that ASCs isolated from obese women (obASCs) also express higher levels of leptin relative to ASCs isolated from lean women (lnASCs) and that this obASC-derived leptin may account for enhanced breast cancer cell growth. The current study investigates the impact of inhibiting leptin expression in lnASCs and obASCs on breast cancer cell (BCC) growth and progression. METHODS: Estrogen receptor positive (ER+) BCCs were co-cultured with leptin shRNA lnASCs or leptin shRNA obASCs and changes in the proliferation, migration, invasion, and gene expression of BCCs were investigated. To assess the direct impact of leptin inhibition in obASCs on BCC proliferation, MCF7 cells were injected alone or mixed with control shRNA obASCs or leptin shRNA obASCs into SCID/beige mice. RESULTS: ER+ BCCs were responsive to obASCs during direct co-culture, whereas lnASCs were unable to increase ER(+) BCC growth. shRNA silencing of leptin in obASCs negated the enhanced proliferative effects of obASC on BCCs following direct co-culture. BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction. Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver. These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs. CONCLUSION: This study provides mechanistic insight as to how obesity enhances the proliferation and metastasis of breast cancer cells; specifically, obASC-derived leptin contributes to the aggressiveness of breast cancer in obese women.

Elevated expression of long intergenic non-coding RNA HOTAIR in a basal-like variant of MCF-7 breast cancer cells.

Epigenetic regulation of gene expression is critical to phenotypic maintenance and transition of human breast cancer cells. HOX antisense intergenic RNA (HOTAIR) is a long intergenic non-coding RNA that epigenetically represses gene expression via recruitment of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase. Elevated expression of HOTAIR promotes progression of breast cancer. In the current study we examined the expression and function of HOTAIR in MCF-7-TNR cells, a derivative of the luminal-like breast cancer cell line MCF-7 that acquired resistance to TNF-α-induced cell death. The expression of HOTAIR, markers of the luminal-like and basal-like subtypes, and growth were compared between MCF-7 and MCF-7-TNR cells. These variables were further assessed upon inhibition of HOTAIR, EZH2, p38 MAPK, and SRC kinase in MCF-7-TNR cells. When compared with MCF-7 cells, MCF-7-TNR cells exhibited an increase in the expression of HOTAIR, which correlated with characteristics of a luminal-like to basal-like transition as evidenced by dysregulated gene expression and accelerated growth. MCF-7-TNR cells exhibited reduced suppressive histone H3 lysine27 trimethylation on the HOTAIR promoter. Inhibition of HOTAIR and EZH2 attenuated the luminal-like to basal-like transition in terms of gene expression and growth in MCF-7-TNR cells. Inhibition of p38 and SRC diminished HOTAIR expression and the basal-like phenotype in MCF-7-TNR cells. HOTAIR was robustly expressed in the native basal-like breast cancer cells and inhibition of HOTAIR reduced the basal-like gene expression and growth. Our findings suggest HOTAIR-mediated regulation of gene expression and growth associated with the basal-like phenotype of breast cancer cells.

microRNA regulation of mammalian target of rapamycin expression and activity controls estrogen receptor function and RAD001 sensitivity.

BACKGROUND: The AKT/mammalian target of rapamycin (mTOR) signaling pathway is regulated by 17α-estradiol (E2) signaling and mediates E2-induced proliferation and progesterone receptor (PgR) expression in breast cancer. METHODS AND RESULTS: Here we use deep sequencing analysis of previously published data from The Cancer Genome Atlas to demonstrate that expression of a key component of mTOR signaling, rapamycin-insensitive companion of mTOR (Rictor), positively correlated with an estrogen receptor-α positive (ERα+) breast tumor signature. Through increased microRNA-155 (miR-155) expression in the ERα+ breast cancer cells we demonstrate repression of Rictor enhanced activation of mTOR complex 1 (mTORC1) signaling with both qPCR and western blot. miR-155-mediated mTOR signaling resulted in deregulated ERα signaling both in cultured cells in vitro and in xenografts in vivo in addition to repressed PgR expression and activity. Furthermore we observed that miR-155 enhanced mTORC1 signaling (observed through western blot for increased phosphorylation on mTOR S2448) and induced inhibition of mTORC2 signaling (evident through repressed Rictor and tuberous sclerosis 1 (TSC1) gene expression). mTORC1 induced deregulation of E2 signaling was confirmed using qPCR and the mTORC1-specific inhibitor RAD001. Co-treatment of MCF7 breast cancer cells stably overexpressing miR-155 with RAD001 and E2 restored E2-induced PgR gene expression. RAD001 treatment of SCID/CB17 mice inhibited E2-induced tumorigenesis of the MCF7 miR-155 overexpressing cell line. Finally we demonstrated a strong positive correlation between Rictor and PgR expression and a negative correlation with Raptor expression in Luminal B breast cancer samples, a breast cancer histological subtype known for having an altered ERα-signaling pathway. CONCLUSIONS: miRNA mediated alterations in mTOR and ERα signaling establishes a new mechanism for altered estrogen responses independent of growth factor stimulation.

Suppression of triple-negative breast cancer metastasis by pan-DAC inhibitor panobinostat via inhibition of ZEB family of EMT master regulators.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial-to-mesenchymal transition (EMT) is a key contributor in the metastatic process. We previously showed the pan-deacetylase inhibitor LBH589 induces CDH1 expression in TNBC cells, suggesting regulation of EMT. The purpose of this study was to examine the effects of LBH589 on the metastatic qualities of TNBC cells and the role of EMT in this process. A panel of breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549), drugged with LBH589, was examined for changes in cell morphology, migration, and invasion in vitro. The effect on in vivo metastasis was examined using immunofluorescent staining of lung sections. EMT gene expression profiling was used to determine LBH589-induced changes in TNBC cells. ZEB overexpression studies were conducted to validate requirement of ZEB in LBH589-mediated proliferation and tumorigenesis. Our results indicate a reversal of EMT by LBH589 as demonstrated by altered morphology and altered gene expression in TNBC. LBH589 was shown to be a more potent inhibitor of EMT than other HDAC inhibitors, SAHA and TMP269. Additionally, we found that LBH589 inhibits metastasis of MDA-MB-231 cells in vivo. These effects of LBH589 were mediated in part by inhibition of ZEB, as overexpression of ZEB1 or ZEB2 mitigated the effects of LBH589 on MDA-MB-231 EMT-associated gene expression, migration, invasion, CDH1 expression, and tumorigenesis. These data indicate therapeutic potential of LBH589 in targeting EMT and metastasis of TNBC.

Preferential star strand biogenesis of pre-miR-24-2 targets PKC-alpha and suppresses cell survival in MCF-7 breast cancer cells.

microRNAs (miRNA) are regulators of cellular pathways and alterations of normal miRNA expression levels have been shown to increase tumorigenesis. miR-24 has been demonstrated as having both tumor suppressive and oncogenic properties depending on cell context. Here, we demonstrate a possible role for pre-miR-24-2 as a tumor suppressor in the MCF-7 breast cancer cell line through the preferential processing of mature miR-24-2* over miR-24. Specifically, we show that the ectopic expression of miR-24-2* in MCF-7 breast cancer cells results in a suppression of cellular survival both in vivo and in vitro. Notably, the overexpression of miR-24-2* results in a dampening of cell survival through the targeted suppression of PKCα. In addition, a similar biological change is observed in vivo where MCF-7 cells overexpressing pre-miR-24-2 have decreased tumorigenicity and tumor incidence. Taken together our data demonstrate that when overexpressed biogenesis of the pre-miR-24-2 favors miR-24-2* in the MCF-7 breast cancer cell line and suggests a tumor suppressive role for miR-24-2* observed through the inhibition of PKCα-mediated cellular survival.

A new method for stranded whole transcriptome RNA-seq.

This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).

Obesity associated alterations in the biology of adipose stem cells mediate enhanced tumorigenesis by estrogen dependent pathways.

INTRODUCTION: Obesity has been associated with increased incidence and mortality of breast cancer. While the precise correlation between obesity and breast cancer remains to be determined, recent studies suggest that adipose tissue and adipose stem cells (ASCs) influence breast cancer tumorigenesis and tumor progression. METHODS: Breast cancer cells lines were co-cultured with ASCs (n = 24), categorized based on tissue site of origin and body mass index (BMI), and assessed for enhanced proliferation, alterations in gene expression profile with PCR arrays, and enhanced tumorigenesis in immunocompromised mice. The gene expression profile of ASCs was assess with PCR arrays and qRT-PCR and confirmed with Western blot analysis. Inhibitory studies were conducted by delivering estrogen antagonist ICI182,780, leptin neutralizing antibody, or aromatase inhibitor letrozole and assessing breast cancer cell proliferation. To assess the role of leptin in human breast cancers, Oncomine and Kaplan Meier plot analyses were conducted. RESULTS: ASCs derived from the abdominal subcutaneous adipose tissue of obese subjects (BMI > 30) enhanced breast cancer cell proliferation in vitro and tumorigenicity in vivo. These findings were correlated with changes in the gene expression profile of breast cancer cells after co-culturing with ASCs, particularly in estrogen receptor-alpha (ESR1) and progesterone receptor (PGR) expression. Analysis of the gene expression profile of the four groups of ASCs revealed obesity induced alterations in several key genes, including leptin (LEP). Blocking estrogen signaling with ICI182,780, leptin neutralizing antibody, or letrozole diminished the impact of ASCs derived from obese subjects. Women diagnosed with estrogen receptor/progesterone receptor positive (ER+/PR+) breast cancers that also expressed high levels of leptin had poorer prognosis than women with low leptin expression. CONCLUSION: ASCs isolated from the abdomen of obese subjects demonstrated increased expression of leptin, through estrogen stimulation, which increased breast cancer cell proliferation. The results from this study demonstrate that abdominal obesity induces significant changes in the biological properties of ASCs and that these alterations enhance ER+/PR+ breast cancer tumorigenesis through estrogen dependent pathways.

Single Live Cell Imaging of Multidrug Resistance Using Silver Ultrasmall Nanoparticles as Biosensing Probes in Triple-Negative Breast Cancer Cells.

Silver ultrasmall nanoparticles (Ag UNPs) (size < 5 nm) were used as biosensing probes to analyze the efflux kinetics contributing to multidrug resistance (MDR) in single live triple-negative breast cancer (TNBC) cells by using dark-field optical microscopy to follow their size-dependent localized surface plasmon resonance. TNBC cells lack expression of estrogen (ER-), progesterone (PR-), and human epidermal growth factor 2 (HER2-) receptors and are more likely to acquire resistance to anticancer drugs due to their ability to transport harmful substances outside the cell. The TNBC cells displayed greater nuclear and cytoplasmic efflux, resulting in less toxicity of Ag UNPs in a concentration-independent manner. In contrast, more Ag UNPs and an increase in cytotoxic effects were observed in the receptor-positive breast cancer cells that have receptors for ER+, PR+, and HER2+ and are known to better respond to anticancer therapies. Ag UNPs accumulated in receptor-positive breast cancer cells in a time-and concentration-dependent mode and caused decreased cellular growth, whereas the TNBC cells due to the efflux were able to continue to grow. The TNBC cells demonstrated a marked increase in survival due to their ability to have MDR determined by efflux of Ag UNPs outside the nucleus and the cytoplasm of the cells. Further evaluation of the nuclear efflux kinetics of TNBC cells with Ag UNPs as biosensing probes is critical to gain a better understanding of MDR and potential for enhancement of cancer drug delivery.

Targeting triple-negative breast cancer cells with the histone deacetylase inhibitor panobinostat.

INTRODUCTION: Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo. METHODS: TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology. RESULTS: Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype. CONCLUSIONS: This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.

Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration.

INTRODUCTION: Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam). METHODS: We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions. RESULTS: Quantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility. CONCLUSIONS: Our data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity.

Effects of SDF-1-CXCR4 signaling on microRNA expression and tumorigenesis in estrogen receptor-alpha (ER-α)-positive breast cancer cells.

The majority of breast cancer cases ultimately become unresponsive to endocrine therapies, and this progression of breast cancer from hormone-responsive to hormone-independent represents an area in need of further research. Additionally, hormone-independent carcinomas are characterized as being more aggressive and metastatic, key features of more advanced disease. Having previously shown the ability of the stromal-cell derived factor-1 (SDF-1)-CXCR4 signaling axis to promote primary tumorigenesis and hormone independence by overexpressing CXCR4 in MCF-7 cells, in this study we further examined the role of SDF-1/CXCR4 in the endogenously CXCR4-positive, estrogen receptor α (ER-α)-positive breast carcinoma cell line, MDA-MB-361. In addition to regulating estrogen-induced and hormone-independent tumor growth, CXCR4 signaling stimulated the epithelial-to-mesenchymal transition, evidenced by decreased CDH1 expression following SDF-1 treatment. Furthermore, inhibition of CXCR4 with the small molecule inhibitor AMD3100 induced CDH1 gene expression and inhibited CDH2 gene expression in MDA-MB-361 cells. Further, exogenous SDF-1 treatment induced ER-α-phosphorylation in both MDA-MB-361 and MCF-7-CXCR4 cells, demonstrating ligand-independent activation of ER-α through CXCR4 crosstalk. qPCR microRNA array analyses of the MDA-MB-361 and MCF-7-CXCR4 cell lines revealed changes in microRNA expression profiles induced by SDF-1, consistent with a more advanced disease phenotype and further supporting our hypothesis that the SDF-1/CXCR4 signaling axis drives ER-α-positive breast cancer cells to a hormone independent and more aggressive phenotype. In this first demonstration of SDF-1-CXCR4-induced microRNAs in breast cancer, we suggest that this signaling axis may promote tumorigenesis via microRNA regulation. These findings represent future potential therapeutic targets for the treatment of hormone-independent and endocrine-resistant breast cancer.

Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells.

Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demonstrate the application of a phenotypic screening approach using a small molecule inhibitor library to identify potential CXCR4-mediated signaling pathways. The present study demonstrated a new application of the Published Kinase Inhibitor Set (PKIS), a library of small molecule inhibitors from diverse chemotype series with varying levels of selectivity, in a phenotypic medium-throughput screen to identify potential mechanisms to pursue. Crystal violet staining and brightfield microscopy were employed to evaluate relative cell survival and changes to cell morphology in the screens. 'Hits' or lead active compounds in the first screen were PKIS inhibitors that reversed mesenchymal morphologies in CXCR4-activated breast cancer cells without the COOH-terminal domain (MCF-7-CXCR4-ΔCTD) and in the phenotypically mesenchymal triple-negative breast cancer cells (MDA-MB-231, BT-549 and MDA-MB-157), used as positive controls. In a following screen, the phenotypic and cell viability screen was used with a positive control that was both morphologically mesenchymal and had acquired fulvestrant resistance. Compounds within the same chemotype series were identified that exhibited biological activity in the screens, the 'active' inhibitors, were compared with inactive compounds. Relative kinase activity was obtained using published datasets to discover candidate kinase targets responsible for CXCR4 activity. MAP4K4 and MINK reversed both the mesenchymal and drug-resistant phenotypes, NEK9 and DYRK2 only reversed the mesenchymal morphology, and kinases, including ROS, LCK, HCK and LTK, altered the fulvestrant-resistant phenotype. Oligoarray experiments revealed pathways affected in CXCR4-activated cells, and these pathways were compared with the present screening approach to validate our screening tool. The oligoarray approach identified the integrin-mediated, ephrin B-related, RhoA, RAC1 and ErbB signaling pathways to be upregulated in MCF-7-CXCR4-ΔCTD cells, with ephrin B signaling also identified in the PKIS phenotypic screen. The present screening tool may be used to discover potential mechanisms of targeted signaling pathways in solid cancers.

Effects of human mesenchymal stem cells on ER-positive human breast carcinoma cells mediated through ER-SDF-1/CXCR4 crosstalk.

BACKGROUND: Adult human mesenchymal stem cells (hMSC) have been shown to home to sites of carcinoma and affect biological processes, including tumour growth and metastasis. Previous findings have been conflicting and a clear understanding of the effects of hMSCs on cancer remains to be established. Therefore, we set out to investigate the impact of hMSCs on the oestrogen receptor positive, hormone-dependent breast carcinoma cell line MCF-7. RESULTS: In this study, we show the effects of hMSCs on cancer cells are mediated through a secreted factor(s) which are enhanced by cancer cell-hMSC contact/communication. In addition to enhanced proliferation when in co-culture with hMSCs, MCF-7 cells were found to have increased migration potential in vitro. Inhibition of ER signalling by the pure anti-oestrogen ICI 182,780 decreased the effect of hMSCs on MCF-7 cell proliferation and migration supporting a role for ER signalling in the hMSC/MCF-7 cell interaction. Additionally, hMSCs have been shown to secrete a wide variety of growth factors and chemokines including stromal cell-derived factor-1 (SDF-1). This coupled with the knowledge that SDF-1 is an ER-mediated gene linked with hormone-independence and metastasis led to the investigation of the SDF-1/CXCR4 signalling axis in hMSC-MCF-7 cell interaction. Experiments revealed an increase in SDF-1 gene expression both in vivo and in vitro when MCF-7 cells were cultured with hMSCs. SDF-1 treatment of MCF-7 cells alone increased proliferation to just below that seen with hMSC co-culture. Additionally, blocking SDF-1 signalling using a CXCR4-specific inhibitor decreased hMSC induced proliferation and migration of MCF-7. However, the combined treatment of ICI and AMD3100 reduced MCF-7 cell proliferation and migration below control levels, indicating targeting both the ER and CXCR4 pathways is effective in decreasing the hMSCs induction of MCF-7 cell proliferation and migration. CONCLUSIONS: The sum of these data reveals the relationship between tumour microenvironment and tumour growth and progression. Better understanding of the mechanisms involved in this tumour stroma cell interaction may provide novel targets for the development of treatment strategies for oestrogen receptor positive, hormone-independent, and endocrine-resistant breast carcinoma.

Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence.

Adult human mesenchymal stem cells (hMSCs) have been shown to home to sites of breast cancer and integrate into the tumor stroma. We demonstrate here the effect of hMSCs on primary breast tumor growth and the progression of these tumors to hormone independence. Co-injection of bone marrow-derived hMSCs enhances primary tumor growth of the estrogen receptor-positive, hormone-dependent breast carcinoma cell line MCF-7 in the presence or absence of estrogen in SCID/beige mice. We also show hormone-independent growth of MCF-7 cells when co-injected with hMSCs. These effects were found in conjunction with increased immunohistochemical staining of the progesterone receptor in the MCF-7/hMSC tumors as compared to MCF-7 control tumors. This increase in PgR expression indicates a link between MCF-7 cells and MSCs through ER-mediated signaling. Taken together, our data reveal the relationship between tumor microenvironment and tumor growth and the progression to hormone independence. This tumor stroma-cell interaction may provide a novel target for the treatment of estrogen receptor-positive, hormone-independent, and endocrine-resistant breast carcinoma.

Resveratrol analogues surprisingly effective against triple­negative breast cancer, independent of ERα.

Resveratrol, a plant­derived stilbene compound, has exhibited anticancerous properties, including breast cancer. Stilbenes have a molecular structure highly similar to estrogen and have the ability to bind estrogen receptors and regulate activity. Numerous studies have demonstrated the effectiveness of resveratrol in estrogen receptor­positive (ER­positive) subtypes of breast cancer, yet the effects in ER­negative subtypes, including triple­negative breast cancer (TNBC), have been limited. In the present study, resveratrol and 28 analogues were tested on a panel of ER­positive and TNBC cell lines to determine effects on cell viability. Several compounds exhibited significant impacts on cell viability and suggested changes in cell morphology, with high potency of select compounds compared to resveratrol observed in a dose­dependent manner. Due to the lack of estrogen receptors in TNBC and the estrogenic nature of stilbenes, regulation of breast cancer­associated cellular pathways was assessed for five analogues shown to significantly inhibit cell viability. Top regulated pathways included apoptosis (confirmed by caspase assay) and DNA damage repair. Overall, our results indicated several resveratrol analogues to be active in ER­negative phenotypes, acting through an ER receptor­independent manner, supporting further investigation into their mechanism of action and use as potential chemotherapeutics in higher­risk breast cancer cases.

Argonaute 2 Expression Correlates with a Luminal B Breast Cancer Subtype and Induces Estrogen Receptor Alpha Isoform Variation.

Estrogen receptor alpha (ERα) signaling pathways are frequently disrupted in breast cancer and contribute to disease progression. ERα signaling is multifaceted and many ERα regulators have been identified including transcription factors and growth factor pathways. More recently, microRNAs (miRNAs) are shown to deregulate ERα activity in breast carcinomas, with alterations in both ERα and miRNA expression correlating to cancer progression. In this study, we show that a high expression of Argonaute 2 (AGO2), a translation regulatory protein and mediator of miRNA function, correlates with the luminal B breast cancer subtype. We further demonstrate that a high expression of AGO2 in ERα+ tumors correlates with a poor clinical outcome. MCF-7 breast cancer cells overexpressing AGO2 (MCF7-AGO2) altered ERα downstream signaling and selective ERα variant expression. Enhanced ERα-36, a 36 kDa ERα isoform, protein and gene expression was observed in vitro. Through quantitative polymerase chain reaction (qPCR), we demonstrate decreased basal expression of the full-length ERα and progesterone receptor genes, in addition to loss of estrogen stimulated gene expression in vitro. Despite the loss, MCF-7-AGO2 cells demonstrated increased estrogen stimulated tumorigenesis in vivo. Together with our clinical findings on AGO2 expression and the luminal B subtype, we suggest that AGO2 is a regulator of altered ERα signaling in breast tumors.

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer.

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype.