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1.  Preferential Star Strand Biogenesis of Pre-miR-24-2 Targets PKC-alpha and Suppresses Cell Survival in MCF-7 Breast Cancer Cells 
Molecular carcinogenesis  2012;53(1):38-48.
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. Additionally, 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.
doi:10.1002/mc.21946
PMCID: PMC4030540  PMID: 22911661
miRNA maturation; PKCα cellular survival; breast cancer; strand preference; miR-24-2*
2.  miR-155 induced transcriptome changes in the MCF-7 breast cancer cell line leads to enhanced mitogen activated protein kinase signaling 
Genes & Cancer  2014;5(9-10):353-364.
A single microRNA (miRNA) has the potential to regulate thousands of genes and thus govern multiple signaling pathways at once. miR-155 is an oncogenic miRNA which regulates many cellular pathways, designating it as a multifaceted regulator of proliferation, chemo-resistance, and apoptosis. While many singular targeted effects of miR-155 have been defined and an oncogenic role has been attributed to miR-155 expression, the global effect of miR-155 on the cellular transcriptomes of an ER+ breast cancer cell line has yet to be determined. Here we demonstrate that miR-155 expression increases tumorigenesis in vivo and we determine miR-155 mediated transcriptome changes through next generation sequencing analysis. miR-155 expression alters many signaling pathways, with the chief altered pathway being the MAPK signaling cascade and miR-155 induces shortening of target mRNA 3′UTRs and alternative isoform expression of MAPK related genes. In addition there is an observed increase in protein phosphorylation of components of MAPK signaling including ERK1/2 and AP-1 complex members (Fra-1 and c-Fos) as well as elevated gene expression of MAPK regulated genes Zeb1, Snail, Plaur, and SerpinE1.
PMCID: PMC4209600  PMID: 25352952
microRNA-155; breast cancer; MAPK; p38; 3′UTR; RNA-seq
3.  Antiestrogenic Activity of Flavonoid Phytochemicals Mediated via the c-Jun N-terminal Protein Kinase Pathway 
Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling independent of direct receptor binding. Here we demonstrate both chalcone and flavone function as cell type-specific selective ER modulators. In MCF-7 breast carcinoma cells chalcone and flavone suppress ERα activity through stimulation of the stress-activated members of the mitogen-activated protein kinase (MAPK) family: c-Jun N-terminal kinase (JNK)1 and JNK2. The use of dominant-negative mutants of JNK1 or JNK2 in stable transfected cells established that the antiestrogenic effects of chalcone and flavone required intact JNK signaling. We further show that constitutive activation of the JNK pathway partially suppresses estrogen (E2)-mediated gene expression in breast, but not endometrial carcinoma cells. Our results demonstrate a role for stress-activated MAPKs in the cell type-specific regulation of ERα function.
doi:10.1016/j.jsbmb.2012.05.004
PMCID: PMC4083692  PMID: 22634477
flavonoids; phytoestrogens; estrogen receptor; mitogen-activated protein kinase; antiestrogens; c-Jun N-terminal kinase (JNK)
4.  Design, Synthesis, and Osteogenic Activity of Daidzein Analogs on Human Mesenchymal Stem Cells 
ACS Medicinal Chemistry Letters  2013;5(2):143-148.
Osteoporosis is caused by an overstimulation of osteoclast activity and the destruction of the bone extracellular matrix. Without the normal architecture, osteoblast cells are unable to rebuild phenotypically normal bone. Hormone replacement therapy with estrogen has been effective in increasing osteoblast activity but also has resulted in the increased incidence of breast and uterine cancer. In this study we designed and synthesized a series of daidzein analogs to investigate their osteogenic induction potentials. Human bone marrow derived mesenchymal stem cells (MSCs) from three different donors were treated with daidzein analogs and demonstrated enhanced osteogenesis when compared to daidzein treatment. The enhanced osteogenic potential of these daidzein analogs resulted in increased osterix (Sp7), alkaline phosphatase (ALP), osteopontin (OPN), and insulin-like growth factor 1 (IGF-1), which are osteogenic transcription factors that regulate the maturation of osteogenic progenitor cells into mature osteoblast cells.
doi:10.1021/ml400397k
PMCID: PMC4027770  PMID: 24900787
Daidzein analogs; mesenchymal stem cells; BMSCs; osteogenesis
5.  A New Method for Stranded Whole Transcriptome RNA-seq 
Methods (San Diego, Calif.)  2013;63(2):126-134.
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).
doi:10.1016/j.ymeth.2013.03.023
PMCID: PMC3739992  PMID: 23557989
RNA-seq; transcriptome; duplex-specific nuclease; gene expression1
6.  Effects of the Endocrine-Disrupting Chemical DDT on Self-Renewal and Differentiation of Human Mesenchymal Stem Cells 
Background: Although the global use of the endocrine-disrupting chemical DDT has decreased, its persistence in the environment has resulted in continued human exposure. Accumulating evidence suggests that DDT exposure has long-term adverse effects on development, yet the impact on growth and differentiation of adult stem cells remains unclear.
Objectives: Human mesenchymal stem cells (MSCs) exposed to DDT were used to evaluate the impact on stem cell biology.
Methods: We assessed DDT-treated MSCs for self-renewal, proliferation, and differentiation potential. Whole genome RNA sequencing was performed to assess gene expression in DDT-treated MSCs.
Results: MSCs exposed to DDT formed fewer colonies, suggesting a reduction in self-renewal potential. DDT enhanced both adipogenic and osteogenic differentiation, which was confirmed by increased mRNA expression of glucose transporter type 4 (GLUT4), lipoprotein lipase (LpL), peroxisome proliferator-activated receptor gamma (PPARγ), leptin, osteonectin, core binding factor 1 (CBFA1), and FBJ murine osteosarcoma viral oncogene homolog (c-Fos). Expression of factors in DDT-treated cells was similar to that in estrogen-treated MSCs, suggesting that DDT may function via the estrogen receptor (ER)-mediated pathway. The coadministration of ICI 182,780 blocked the effects of DDT. RNA sequencing revealed 121 genes and noncoding RNAs to be differentially expressed in DDT-treated MSCs compared with controls cells.
Conclusion: Human MSCs provide a powerful biological system to investigate and identify the molecular mechanisms underlying the effects of environmental agents on stem cells and human health. MSCs exposed to DDT demonstrated profound alterations in self-renewal, proliferation, differentiation, and gene expression, which may partially explain the homeostatic imbalance and increased cancer incidence among those exposed to long-term EDCs.
Citation: Strong AL, Shi Z, Strong MJ, Miller DF, Rusch DB, Buechlein AM, Flemington EK, McLachlan JA, Nephew KP, Burow ME, Bunnell BA. 2015. Effects of the endocrine-disrupting chemical DDT on self-renewal and differentiation of human mesenchymal stem cells. Environ Health Perspect 123:42–48; http://dx.doi.org/10.1289/ehp.1408188
doi:10.1289/ehp.1408188
PMCID: PMC4286277  PMID: 25014179
7.  Inhibition of p38-MAPK alters SRC coactivation and estrogen receptor phosphorylation 
Cancer Biology & Therapy  2012;13(11):1026-1033.
The p38 mitogen activated protein kinase pathway (MAPK) is known to promote cell survival, endocrine therapy resistance and hormone independent breast cancer cell proliferation. Therefore, we utilized the novel p38 inhibitor RWJ67657 to investigate the relevance of targeting this pathway in the ER+ breast cancer cell line MCF-7. Our results show that RWJ67657 inhibits both basal and estrogen stimulated phosphorylation of p38α, resulting in decreased activation of the downstream p38α targets hsp27 and MAPAPK. Furthermore, inhibition of p38α by RWJ67657 blocks clonogenic survival of MCF-7 cells with little effect on non-cancerous breast epithelial cells. Even though p38α is known to phosphorylate ERα at residue within ER’s hinge region at Thr311, resulting in increased ERα transcriptional activation, our results suggest RWJ67657 inhibits the p38α-induced activation of ER by targeting both the AF-1 and AF-2 activation domains within ERα. We further show that RWJ67657 decreases the transcriptional activity of the ER coactivators SRC-1, SRC-2 and SRC-3. Taken together, our results strongly suggest that in addition to phosphorylating Thr311 within ERα, p38α indirectly activates the ER by phosphorylation and stimulation of the known ERα coactivators, SRC-1, -2 and-3. Overall, our data underscore the therapeutic potential of targeting the p38 MAPK pathway in the treatment of ER+ breast cancer.
doi:10.4161/cbt.20992
PMCID: PMC3461809  PMID: 22825349
p38; mitogen-activated protein kinase; estrogen receptor; breast cancer; SRC; drug discovery
8.  microRNA regulation of mammalian target of rapamycin expression and activity controls estrogen receptor function and RAD001 sensitivity 
Molecular Cancer  2014;13(1):229.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-229) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-229
PMCID: PMC4203920  PMID: 25283550
miR-155; mTOR; breast cancer; miRNA; Estrogen receptor
9.  Regulation of ERα-mediated transcription of Bcl-2 by PI3K-AKT crosstalk: Implications for breast cancer cell survival 
International journal of oncology  2010;37(3):541-550.
Both estrogen, through the estrogen receptor (ER), and growth factors, through the phosphatidylinositol-3-kinase (PI3K)-AKT pathway, have been shown to independently promote cell survival. Here, we investigated the role of ER/PI3K-AKT crosstalk in the regulation of cell survival in MCF-7 breast carcinoma cells. The ER inhibitor ICI 182,780 was used to determine the requirement of the ER for estrogen in the suppression of tumor necrosis factor-α (TNFα) induced apoptosis. Gene reporter assays and Western blot analyses were used to determine the involvement of the pro-survival factor Bcl-2 and the coactivator GRIP1 in this survival crosstalk. We demonstrated that an intact ER signaling pathway was required for estrogen to suppress apoptosis induced by TNFα. Our gene reporter assays revealed that ERα, not ERβ, was targeted by AKT, resulting in transcriptional potentiation of the full-length Bcl-2 promoter, ultimately leading to increased Bcl-2 protein levels. AKT targeted both activation function (AF) domains of the ERα for maximal induction of Bcl-2 reporter activity, although the AF-II domain was predominately targeted. In addition, AKT also caused an upregulation of GRIP1 protein levels. Finally, AKT and GRIP1 cooperated to increase Bcl-2 protein expression to a greater level than either factor alone. Collectively, our study suggests a role for ER/PI3K-AKT crosstalk in cell survival and documents the ability of AKT to regulate Bcl-2 expression via differential activation of ERα and ERβ as well as regulation of GRIP1.
PMCID: PMC3613138  PMID: 20664923
estrogen receptor; breast cancer; AKT; cell signaling; cell survival
10.  Inhibition of p38 mitogen-activated protein kinase alters microRNA expression and reverses epithelial-to-mesenchymal transition 
International Journal of Oncology  2013;42(4):1139-1150.
Acquired chemoresistance and epithelial-to-mesenchymal transition (EMT) are hallmarks of cancer progression and of increasing clinical relevance. We investigated the role of miRNA and p38 mitogen-activated protein kinase (MAPK) signaling in the progression of breast cancer to a drug-resistant and mesenchymal phenotype. We demonstrate that acquired death receptor resistance results in increased hormone-independent tumorigenesis compared to hormone-sensitive parental cells. Utilizing global miRNA gene expression profiling, we identified miRNA alterations associated with the development of death receptor resistance and EMT progression. We further investigated the role of p38 MAPK in this process, showing dose-dependent inactivation of p38 by its inhibitor RWJ67657 and decreased downstream ATF and NF-κB signaling. Pharmacological inhibition of p38 also decreased chemoresistant cancer tumor growth in xenograft animal models. Interestingly, inhibition of p38 partially reversed the EMT changes found in this cell system, as illustrated by decreased gene expression of the EMT markers Twist, Snail, Slug and ZEB and protein and mRNA levels of Twist, a known EMT promoter, concomitant with decreased N-cadherin protein. RWJ67657 treatment also altered the expression of several miRNAs known to promote therapeutic resistance, including miR-200, miR-303, miR-302, miR-199 and miR-328. Taken together, our results demonstrate the roles of multiple microRNAs and p38 signaling in the progression of cancer and demonstrate the therapeutic potential of targeting the p38 MAPK pathway for reversing EMT in an advanced tumor phenotype.
doi:10.3892/ijo.2013.1814
PMCID: PMC3622654  PMID: 23403951
p38 mitogen-activated protein kinase; epithelial-tomesenchymal transition; breast cancer; drug discovery
11.  Effects of SDF-1-CXCR4 signaling on microRNA expression and tumorigenesis in estrogen receptor-alpha (ER-α)-positive breast cancer cells 
Experimental Cell Research  2011;317(18):2573-2581.
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.
doi:10.1016/j.yexcr.2011.08.016
PMCID: PMC3334320  PMID: 21906588
SDF-1; CXCR4; microRNA; breast carcinoma; hormone independence; AMD3100
12.  Molecular effects of soy phytoalexin glyceollins in human prostate cancer cells LNCaP 
Molecular carcinogenesis  2009;48(9):862-871.
Glyceollins are soy-derived phytoalexins that have been proposed to be candidate cancer preventive compounds. The effect of the glyceollins on prostate cancer is unknown. The present study examined the molecular effects of soy phytoalexin, glyceollins, on human prostate cancer cell LNCaP to further elucidate its potential effects on prostate cancer prevention. We found that the glyceollins inhibited LNCaP cell growth similar to that of the soy isoflavone genistein. The growth inhibitory effects of the glyceollins appeared to be due to an inhibition of G1/S progression and correlated with an up-regulation of cyclin-dependent kinase inhibitor 1 A and B mRNA and protein levels. By contrast, genistein only up-regulates cyclin-dependent kinase inhibitor 1A. In addition, glyceollin treatments led to down-regulated mRNA levels for androgen responsive genes. In contrast to genistein, this effect of glyceollins on androgen responsive genes appeared to be mediated through modulation of an estrogen- but not androgen-mediated pathway. Hence, the glyceollins exerted multiple effects on LNCaP cells that may be considered cancer preventive and the mechanisms of action appeared to be different from other soy-derived phytochemicals.
doi:10.1002/mc.20532
PMCID: PMC4034473  PMID: 19263441
androgen; estrogen; cancer prevention; cell cycle; gene expression
13.  Dual inhibition of sphingosine kinase isoforms ablates TNF-induced drug resistance 
Oncology reports  2012;27(6):1779-1786.
Recent research has demonstrated that aberrant sphingolipid signaling is an important mechanism of chemo-resistance in solid tumors. Sphingosine kinase (Sphk), the primary enzyme metabolizing the sphingolipid ceramide into sphingosine-1-phosphate (S1P), is a primary mediator of breast cancer promotion, survival and chemoresistance. However, to date the mechanism of Sphk-mediated drug resistance is poorly understood. Using the dual sphingosine kinase isozyme inhibitor, SKI-II (4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol), we explored the effects of sphingosine kinase inhibition on multi-drug-resistant breast cancer cells. We demonstrate that SKI-II alters endogenous sphingolipid signaling and decreases cancer proliferation, survival and viability. Furthermore, pharmacological inhibition of Sphk1/2 induced intrinsic apoptosis in these cells through modulation of the NF-κB pathway. SKI-II decreases NF-κB transcriptional activity through altered phosphorylation of the p65 subunit. Taken together, these results suggest that Sphk may be a promising therapeutic target in chemoresistant cancers.
doi:10.3892/or.2012.1743
PMCID: PMC4028227  PMID: 22469881
sphingolipids; chemoresistance; sphingosine kinase; NF-κB; breast cancer; ceramide; experimental therapeutics; sphingosine-1-phosphate
14.  Pharmacology and anti-tumor activity of RWJ67657, a novel inhibitor of p38 mitogen activated protein kinase 
Endocrine therapy resistance is a primary cause of clinical breast cancer treatment failure. The p38 mitogen activated protein kinase (MAPK) signaling pathway is known to promote ligand independent tumor growth and resistance to endocrine therapy. In this study, we investigated the therapeutic potential of the p38 inhibitor RWJ67657 in the treatment of tamoxifen resistant MDA-MB-361 cells. RWJ67657 dose-dependently decreased both basal and stimulated activation of p38 MAPK signaling in this drug resistant cell system. Decreased activation of p38 by RWJ67657 resulted in inhibition of the downstream p38 targets hsp27 and MAPKAPK. Diminished p38 signaling resulted in inhibition of p38-medated gene transcription. Furthermore, pharmacological inhibition of p38 by RWJ67657 decreased biological effects of p38, including ER-mediated gene expression and clonogenic survival in a dose-dependent manner. Animal studies revealed significantly decreased p38 signaling in vivo following exposure to RWJ67657. Treatment with the inhibitor markedly decreased phosphorylation of p38 in MDA-MB-361 tumors, leading to decreased transcription of both Fra-1 and progesterone receptor. Utilizing well-established xenograft tumor models, we demonstrated that RWJ67657 exhibits potent anti-tumor properties. Treatment with RWJ67657 markedly decreased tamoxifen resistant tumor growth, both in the presence and absence of estrogen. Taken together, our findings demonstrate the therapeutic potential of targeting the p38-MAPK signaling cascade in the treatment of endocrine resistant breast cancer.
PMCID: PMC3410584  PMID: 22860234
p38; mitogen-activated protein kinase; endocrine resistance; breast cancer; drug discovery; cancer biology; hormone independence; kinase inhibitors; estrogen receptor; gene transcription
15.  Pharmacological inhibition of sphingosine kinase isoforms alters estrogen receptor signaling in human breast cancer 
Recently, crosstalk between sphingolipid signaling pathways and steroid hormones has been illuminated as a possible therapeutic target. Sphingosine kinase (SK), the key enzyme metabolizing pro-apoptotic ceramide to pro-survival sphingosine-1-phosphate (S1P), is a promising therapeutic target for solid tumor cancers. In this study, we examined the ability of pharmacological inhibition of S1P formation to block estrogen signaling as a targeted breast cancer therapy. We found that the Sphk1/2 selective inhibitor (SK inhibitor (SKI))-II, blocked breast cancer viability, clonogenic survival and proliferation. Furthermore, SKI-II dose-dependently decreased estrogen-stimulated estrogen response element transcriptional activity and diminished mRNA levels of the estrogen receptor (ER)-regulated genes progesterone receptor and steroid derived factor-1. This inhibitor binds the ER directly in the antagonist ligand-binding domain. Taken together, our results suggest that SKIs have the ability to act as novel ER signaling inhibitors in breast carcinoma.
doi:10.1530/JME-10-0116
PMCID: PMC4007162  PMID: 21321095
16.  The microRNA expression associated with morphogenesis of breast cancer cells in three-dimensional organotypic culture 
Oncology reports  2012;28(1):117-126.
Three-dimensional organotypic culture using reconstituted basement membrane matrix Matrigel (rBM 3-D) is an indispensable tool to characterize morphogenesis of mammary epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix (ECM). microRNAs (miRNAs) are a novel class of oncogenes and tumor suppressors. The majority of our current knowledge of miRNA expression and function in cancer cells is derived from monolayer 2-D culture on plastic substratum, which lacks consideration of the influence of ECM-mediated morphogenesis on miRNAs. In the present study, we compared the expression of miRNAs in rBM 3-D and 2-D cultures of the non-invasive MCF-7 and the invasive MDA-MB231 cells. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures within each cell type. Moreover, rBM 3-D culture exhibited greater discrimination in miRNA profiles between MCF-7 and MDA-MB231 cells than 2-D culture. The disparate miRNA profiles correlated with distinct mass morphogenesis of MCF-7 and invasive stellate morphogenesis of MDA-MB231 cells in rBM 3-D culture. Supplementation of the tumor promoting type I collagen in rBM 3-D culture substantially altered the miRNA signature of mass morphologenesis of MCF-7 cells in rBM 3-D culture. Overexpression of the differentially expressed miR-200 family member miR429 in MDA-MB231 cells attenuated their invasive stellate morphogenesis in rBM 3-D culture. In summary, we provide the first miRNA signatures of morphogenesis of human breast cancer cells in rBM 3-D culture and warrant further utilization of rBM 3-D culture in investigation of miRNAs in breast cancer.
doi:10.3892/or.2012.1764
PMCID: PMC3991116  PMID: 22576799
microRNA; three-dimensional organotypic culture; extra-cellular matrix; breast cancer; morphogenesis
17.  The histone deacetylase inhibitor trichostatin A alters microRNA expression profiles in apoptosis-resistant breast cancer cells 
Oncology reports  2011;27(1):10-16.
The development of drug resistance represents a major complication in the effective treatment of breast cancer. Epigenetic therapy, through the use of histone deacetylase inhibitors (HDACi) or demethylation agents, is an emerging area of therapeutic targeting in a number of ontological entities, particularly in the setting of aggressive therapy-resistant disease. Using the well-described HDAC inhibitor trichostatin A (TSA) we demonstrate the suppression of in vitro clonogenicity in the previously described apoptosis-resistant MCF-7TN-R breast carcinoma cell line. Additionally, recent work has demonstrated that these agents can alter the expression profile of microRNA signatures in malignant cells. Using an unbiased microRNA microarray analysis, changes in miRNA expression of MCF-7TN-R cells treated with TSA for 24 h were analyzed. We observed significant up-regulation of 22 miRNAs and down-regulation of 10 miRNAs in response to TSA treatment. Our results demonstrate that the HDACi, TSA, exerts anticancer activity in the apoptosis-resistant MCF-7TN-R breast carcinoma cell line. This activity is correlated with TSA alteration of microRNA expression profiles indicative of a less aggressive phenotype.
doi:10.3892/or.2011.1488
PMCID: PMC3982613  PMID: 21971930
microRNA; trichostatin A; histone deacetylase; MCF-7; breast cancer; drug resistance
18.  Cytokine Receptor CXCR4 Mediates Estrogen-Independent Tumorigenesis, Metastasis, and Resistance to Endocrine Therapy in Human Breast Cancer 
Cancer research  2010;71(2):603-613.
Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1–CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1–CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1–mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.
doi:10.1158/0008-5472.CAN-10-3185
PMCID: PMC3140407  PMID: 21123450
19.  Sphingosine Kinase Isoforms as a Therapeutic Target in Endocrine Therapy Resistant Luminal and Basal-A Breast Cancer 
Sphingosine kinase signaling has become of increasing interest as a cancer target in recent years. Two sphingosine kinase inhibitors, SKI-II and ABC294640, are promising as potential breast cancer therapies. However, evidence for their therapeutic properties in specific breast cancer subtypes is currently lacking. In this study, we characterize these drugs in luminal, endocrine resistant (MDA-MB-361) and Basal-A, triple negative (MDA-MB-468) breast cancer cells and compare them with previously published data in other breast cancer cell models. Both SKI-II and ABC294640 demonstrated greater efficacy in Basal-A compared to luminal breast cancer. ABC294640, in particular, induced apoptosis and blocked proliferation both in vitro and in vivo in this triple negative breast cancer system. Furthermore, Sphk expression promotes survival and endocrine therapy resistance in previously sensitive breast cancer cells. Taken together, these results characterize sphingosine kinase inhibitors across breast cancer cell systems and demonstrate their therapeutic potential as anti-cancer agents.
doi:10.1258/ebm.2012.012028
PMCID: PMC3954577  PMID: 22859737
sphingolipids; chemoresistance; sphingosine kinase; breast cancer; ceramide; experimental therapeutics; sphingosine-1-phosphate
20.  MEK5/ERK5 Pathway: The first fifteen years 
Biochimica et biophysica acta  2011;1825(1):37-48.
While conventional MAP kinase pathways are one of the most highly studied signal transduction molecules, less is known about the MEK5 signaling pathway. This pathway has been shown to play a role in normal cell growth cycles, survival and differentiation. This MEK5 pathway is also believed to mediate the effects of a number of oncogenes. MEK5 is the upstream activator of ERK5 in many epithelial cells. Activation of the MEK-MAPK pathway is a frequent event in malignant tumor formation and contributes to chemoresistance and anti-apoptotic signaling. This pathway may be involved in a number of more aggressive, metastatic varieties of cancer due to its role in cell survival, proliferation and EMT transitioning. Further study of this pathway may lead to new prognostic factors and new drug targets to combat more aggressive forms of cancer.
doi:10.1016/j.bbcan.2011.10.002
PMCID: PMC3954580  PMID: 22020294
mitogen-activated protein kinase; big-mitogen activated protein kinase; Erk5; cellular signaling; epithelial-to-mesenchymal transition; kinase inhibitors
21.  Discovery of a Series of Thiazole Derivatives as Novel Inhibitors of Metastatic Cancer Cell Migration and Invasion 
ACS medicinal chemistry letters  2013;4(2):191-196.
Effective inhibitors of cancer cell migration and invasion can potentially lead to clinical applications as therapy to block tumor metastasis, the primary cause of death in cancer patients. To this end we have designed and synthesized a series of thiazole derivatives that showed potent efficacy against cell migration and invasion in metastatic cancer cells. The most effective compound, 5k, was found to have an IC50 value of 176 nM in the dose-dependent transwell migration assays in MDA-MB-231cells. At the dose of 10 μM, 5k also blocked about 80% of migration in HeLa and A549 cells and 60% of invasion of MDA-MB-231 cells. Importantly, the majority of the derivatives exhibited no apparent cytotoxicity in the clonogenic assays. The low to negligible inhibition of cell proliferation is a desirable property of these anti-migration derivatives because they hold promise of low toxicity to healthy cells as potential therapeutic agents. Mechanistic studies analyzing the actin cytoskeleton by microscopy demonstrate that compound 5k substantially reduced cellular f-actin, and prevented localization of fascin to actin-rich membrane protrusions. These results suggest that the anti-migration activity may result from impaired actin structures in protrusions that are necessary to drive migration.
doi:10.1021/ml300322n
PMCID: PMC3601768  PMID: 23526571
Thiazole derivatives; synthesis; anti-migration; anti-invasion; f-actin; fascin
22.  Discovery of a Series of Thiazole Derivatives as Novel Inhibitors of Metastatic Cancer Cell Migration and Invasion 
ACS Medicinal Chemistry Letters  2013;4(2):191-196.
Effective inhibitors of cancer cell migration and invasion can potentially lead to clinical applications as a therapy to block tumor metastasis, the primary cause of death in cancer patients. To this end, we have designed and synthesized a series of thiazole derivatives that showed potent efficacy against cell migration and invasion in metastatic cancer cells. The most effective compound, 5k, was found to have an IC50 value of 176 nM in the dose-dependent transwell migration assays in MDA-MB-231cells. At a dose of 10 μM, 5k also blocked about 80% of migration in HeLa and A549 cells and 60% of invasion of MDA-MB-231 cells. Importantly, the majority of the derivatives exhibited no apparent cytotoxicity in the clonogenic assays. The low to negligible inhibition of cell proliferation is a desirable property of these antimigration derivatives because they hold promise of low toxicity to healthy cells as potential therapeutic agents. Mechanistic studies analyzing the actin cytoskeleton by microscopy demonstrate that compound 5k substantially reduced cellular f-actin and prevented localization of fascin to actin-rich membrane protrusions. These results suggest that the antimigration activity may result from impaired actin structures in protrusions that are necessary to drive migration.
doi:10.1021/ml300322n
PMCID: PMC3601768  PMID: 23526571
thiazole derivatives; synthesis; antimigration; anti-invasion; f-actin; fascin
23.  Obesity associated alterations in the biology of adipose stem cells mediate enhanced tumorigenesis by estrogen dependent pathways 
Breast Cancer Research : BCR  2013;15(5):R102.
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.
doi:10.1186/bcr3569
PMCID: PMC3978929  PMID: 24176089
24.  Glyceollin-elicited soy protein consumption induces distinct transcriptional effects compared to standard soy protein1 
Glyceollins are stress-induced compounds in soybeans with bioactive properties distinct from parent soy isoflavones. The goals of this study were to evaluate effects of dietary glyceollin-enriched and standard soy protein isolates and identify candidate target pathways of glyceollins on transcriptional profiles within mammary gland tissue. Thirty female postmenopausal cynomolgus monkeys were randomized to diets containing one of three protein sources for 3 weeks: (1) control casein / lactalbumin (C/L); (2) standard soy protein containing 194 mg/day isoflavones (SOY); and (3) glyceollin-enriched soy protein containing 189 mg/day isoflavones + 134 mg/day glyceollins (GLY). All diets contained a physiologic dose of estradiol (E2) (1 mg/day). All doses are expressed in human equivalents scaled by caloric intake. Relative to the control C/L diet, the GLY diet resulted in greater numbers of differentially regulated genes which showed minimal overlap with those of SOY. Effects of GLY related primarily to pathways involved in lipid and carbohydrate metabolism, including peroxisome proliferator-activated receptor (PPAR)-gamma and AMP-activated protein kinase (AMPK) signaling, adipocytokine expression, triglyceride synthesis, and lipase activity. Notable genes upregulated by the GLY diet included PPAR-gamma, adiponectin, leptin, lipin 1, and lipoprotein lipase. The GLY diet also resulted in lower serum total cholesterol, specifically non-high-density lipoprotein cholesterol, and increased serum triglycerides compared to the C/L diet. No effects of GLY or SOY were seen on serum insulin, adipocytokines, or vascular and bone turnover markers. These preliminary findings suggest that glyceollin-enriched soy protein has divergent effects from standard soy with some specificity for adipocyte activity and nutrient metabolism.
doi:10.1021/jf2034863
PMCID: PMC3750717  PMID: 22126086
glyceollin; soy; isoflavone; estrogen receptor; metabolism
25.  MEK5/ERK5 Signaling Suppresses Estrogen Receptor Expression and Promotes Hormone-Independent Tumorigenesis 
PLoS ONE  2013;8(8):e69291.
Endocrine resistance and metastatic progression are primary causes of treatment failure in breast cancer. While mitogen activated protein kinases (MAPKs) are known to promote ligand-independent cell growth, the role of the MEK5-ERK5 pathway in the progression of clinical breast carcinoma remains poorly understood. Here, we demonstrated increased ERK5 activation in 30 of 39 (76.9%) clinical tumor samples, as well as across breast cancer cell systems. Overexpression of MEK5 in MCF-7 cells promoted both hormone-dependent and hormone-independent tumorigenesis in vitro and in vivo and conferred endocrine therapy resistance to previously sensitive breast cancer cells. Expression of MEK5 suppressed estrogen receptor (ER)α, but not ER-β protein levels, and abrogated downstream estrogen response element (ERE) transcriptional activity and ER-mediated gene transcription. Global gene expression changes associated with upregulation of MEK5 included increased activation of ER-α independent growth signaling pathways and promotion of epithelial-to-mesenchymal transition (EMT) markers. Taken together, our findings show that the MEK5-ERK5 pathway mediates progression to an ER(−), mesenchymal and endocrine therapy resistant phenotype. Given the need for new clinical therapeutic targets, our results demonstrate the therapeutic potential of targeting the MEK5-ERK5 pathway in breast cancer.
doi:10.1371/journal.pone.0069291
PMCID: PMC3739787  PMID: 23950888

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