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ABSTRACT: Src kinase is elevated in breast tumors that are ER/PR negative and do not overexpress HER2, but clinical trials with Src inhibitors have shown little activity. The present study evaluated preclinical efficacy of a novel peptidomimetic compound, KX-01 (KX2-391), that exhibits dual action as an Src and pretubulin inhibitor. KX-01 was evaluated as a single-agent and in combination with paclitaxel in MDA-MB-231, MDA-MB-157, and MDA-MB-468 human ER/PR/HER2-negative breast cancer cells. Treatments were evaluated by growth/apoptosis, isobologram analysis, migration/invasion assays, tumor xenograft volume, metastasis, and measurement of Src, focal adhesion kinase (FAK), microtubules, Ki67, and microvessel density. KX-01 inhibited cell growth in vitro and in combination with paclitaxel resulted in synergistic growth inhibition. KX-01 resulted in a dose-dependent inhibition of MDA-MB-231 and MDA-MB-157 tumor xenografts (1 and 5 mg/kg, twice daily). KX-01 inhibited activity of Src and downstream mediator FAK in tumors that was coincident with reduced proliferation and angiogenesis and increased apoptosis. KX01 also resulted in microtubule disruption in tumors. Combination of KX-01 with paclitaxel resulted in significant regression of MDA-MB-231 tumors and reduced metastasis to mouse lung and liver. KX-01 is a potently active Src/pretubulin inhibitor that inhibits breast tumor growth and metastasis. As ER/PR/HER2-negative patients are candidates for paclitaxel therapy, combination with KX-01 may potentiate antitumor efficacy in management of this aggressive breast cancer subtype. Mol Cancer Ther; 11(9); 1936-47. ©2012 AACR.
Molecular Cancer Therapeutics 07/2012; 11(9):1936-47. · 5.23 Impact Factor
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ABSTRACT: Estrogen receptor α (ERα) activity is regulated by phosphorylation at several sites. Recently several antibodies specific
for individual phosphorylated sites within ERα have became available. Such antibodies potentially provide invaluable tools
to gain insight into the relevance in vivo of phosphorylated ERα in human breast tumors. However, validation of these antibodies
for immunohistochemistry in particular is necessary in the first instance. In this study we have investigated the usefulness
of several antibodies generated to specific phosphorylated sites within ERα for immunohistochemistry of formalin-fixed, paraffin-embedded
human breast cancer biopsy samples. As well, these data demonstrate for the first time, the detection of multiple phosphorylated
ERα forms in breast cancer (P-S104/106-ERα, P-S118-ERα, P-S167-ERα, P-S282-ERα, P-S294-ERα, P-T311-ERα, and P-S559-ERα) suggesting
the possibility that profiling of phosphorylated ERα isoforms might be useful in selecting subgroups of breast cancer patients
that would benefit from endocrine therapy.
Breast Cancer Research and Treatment 04/2012; 118(3):443-453. · 4.43 Impact Factor
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ABSTRACT: Src, a non-receptor tyrosine kinase is elevated in cancer with expression and activity correlated with cell proliferation, adhesion, survival, motility, metastasis and angiogenesis. There is limited data on Src expression and subcellular localization in breast cancer and no information about expression in racial/ethnic groups.
The present study evaluated Src expression, activity, and subcellular localization in triple negative breast cancer (TNBC) and ERα positive breast cancer (ER+BC), cancer tissue and adjacent normal epithelial ducts, and Caucasian and African American cases. 79 paraffin embedded breast carcinoma cases were obtained from Tulane University Hospital between 2007-2009. 39 cases represented TNBC (33-African Americans, 4-Caucasians, 2-unknowns) and 40 cases represented ER+BC (21-African Americans, 16-Caucasians, 3-unknowns). Immunohistochemistry was used to measure staining distribution and intensity of total Src and activated phospho-SrcY416 (p-Y416Src) in carcinoma tissue and adjacent normal mammary ducts. In TNBC and ER+BC, total Src was significantly higher in cancer compared to adjacent normal ducts (P<0.0001) in both cell membrane and cytoplasm. In membranes, p-Y416Src was elevated in cancer compared to normal ducts. Total Src in the tumor cytoplasm was significantly higher in TNBC compared to ER+BC (P = 0.0028); conversely, p-Y416Src in the tumor cell membranes was higher in TNBC compared to ER+BC (P = 0.0106). Comparison between African American (n = 21) and Caucasian ER+BC (n = 16) revealed no significant difference in expression and localization of total Src and p-Y416Src. TNBC cases positive for lymph node metastasis showed elevated membrane p-Y416Src compared to lymph node negative TNBC (P = 0.027).
Total Src and p-Y416Src were expressed higher in cancer compared to adjacent normal ducts. Cytoplasmic total Src and membrane p-Y416Src were significantly higher in TNBC compared to ER+BC. TNBC cases with lymph node metastasis showed elevated membrane p-Y416Src. Taken together, Src was elevated in the membrane and cytoplasm of more aggressive TNBC.
PLoS ONE 01/2012; 7(3):e33017. · 4.09 Impact Factor
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ABSTRACT: The study of breast cancer metastasis depends on the use of established breast cancer cell lines that do not accurately represent the heterogeneity and complexity of human breast tumors. A tumor model was developed using primary breast tumor-initiating cells isolated from patient core biopsies that would more accurately reflect human breast cancer metastasis.
Tumorspheres were isolated under serum-free culture conditions from core biopsies collected from five patients with clinical diagnosis of invasive ductal carcinoma (IDC). Isolated tumorspheres were transplanted into the mammary fat pad of NUDE mice to establish tumorigenicity in vivo. Tumors and metastatic lesions were analyzed by hematoxylin and eosin (H+E) staining and immunohistochemistry (IHC).
Tumorspheres were successfully isolated from all patient core biopsies, independent of the estrogen receptor α (ERα)/progesterone receptor (PR)/Her2/neu status or tumor grade. Each tumorsphere was estimated to contain 50-100 cells. Transplantation of 50 tumorspheres (1-5 × 103 cells) in combination with Matrigel into the mammary fat pad of NUDE mice resulted in small, palpable tumors that were sustained up to 12 months post-injection. Tumors were serially transplanted three times by re-isolation of tumorspheres from the tumors and injection into the mammary fat pad of NUDE mice. At 3 months post-injection, micrometastases to the lung, liver, kidneys, brain and femur were detected by measuring content of human chromosome 17. Visible macrometastases were detected in the lung, liver and kidneys by 6 months post-injection. Primary tumors variably expressed cytokeratins, Her2/neu, cytoplasmic E-cadherin, nuclear β catenin and fibronectin but were negative for ERα and vimentin. In lung and liver metastases, variable redistribution of E-cadherin and β catenin to the membrane of tumor cells was observed. ERα was re-expressed in lung metastatic cells in two of five samples.
Tumorspheres isolated under defined culture conditions from patient core biopsies were tumorigenic when transplanted into the mammary fat pad of NUDE mice, and metastasized to multiple mouse organs. Micrometastases in mouse organs demonstrated a dormancy period prior to outgrowth of macrometastases. The development of macrometastases with organ-specific phenotypic distinctions provides a superior model for the investigation of organ-specific effects on metastatic cancer cell survival and growth.
BMC Cancer 01/2012; 12:10. · 3.01 Impact Factor
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ABSTRACT: BACKGROUND: Disseminated tumor cells (DTCs) in the bone marrow may exist in a dormant state for extended periods of time, maintaining the ability to proliferate upon activation, engraft at new sites, and form detectable metastases. However, understanding of the behavior and biology of dormant breast cancer cells in the bone marrow niche remains limited, as well as their potential involvement in tumor recurrence and metastasis. Therefore, the purpose of this study was to investigate the tumorigenicity and metastatic potential of dormant disseminated breast cancer cells (prior to activation) in the bone marrow. METHODOLOGY/PRINCIPAL FINDINGS: Total bone marrow, isolated from mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice. As a negative control, bone marrow isolated from non-injected mice was injected into the mammary fat pad of NUDE mice. The resultant tumors were analyzed by immunohistochemistry for expression of epithelial and mesenchymal markers. Mouse lungs, livers, and kidneys were analyzed by H+E staining to detect metastases. The injection of bone marrow isolated from mice previously injected with tumorspheres into the mammary fat pad, resulted in large tumor formation in the mammary fat pad 2 months post-injection. However, the injection of bone marrow isolated from non-injected mice did not result in tumor formation in the mammary fat pad. The DTC-derived tumors exhibited accelerated development of metastatic lesions within the lung, liver and kidney. The resultant tumors and the majority of metastatic lesions within the lung and liver exhibited a mesenchymal-like phenotype. CONCLUSIONS/SIGNIFICANCE: Dormant DTCs within the bone marrow are highly malignant upon injection into the mammary fat pad, with the accelerated development of metastatic lesions within the lung, liver and kidney. These results suggest the acquisition of a more aggressive phenotype of DTCs during metastatic latency within the bone marrow microenvironment.
PLoS ONE 01/2012; 7(11):e47587. · 4.09 Impact Factor
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ABSTRACT: Nuclear receptors (NR) impact a myriad of physiological processes including homeostasis, reproduction, development, and metabolism. NRs are regulated by post-translational modifications (PTM) that markedly impact receptor function. Recent studies have identified NR PTMs that are involved in the onset and progression of human diseases, including cancer. The majority of evidence linking NR PTMs with disease has been demonstrated for phosphorylation, acetylation and sumoylation of androgen receptor (AR), estrogen receptor α (ERα), glucocorticoid receptor (GR) and peroxisome proliferator activated receptor γ (PPARγ). Phosphorylation of AR has been associated with hormone refractory prostate cancer and decreased disease-specific survival. AR acetylation and sumoylation increased growth of prostate cancer tumor models. AR phosphorylation reduced the toxicity of the expanded polyglutamine AR in Kennedy's Disease as a consequence of reduced ligand binding. A comprehensive evaluation of ERα phosphorylation in breast cancer revealed several sites associated with better clinical outcome to tamoxifen therapy, whereas other phosphorylation sites were associated with poorer clinical outcome. ERα acetylation and sumoylation may also have predictive value for breast cancer. GR phosphorylation and acetylation impact GR responsiveness to glucocorticoids that are used as anti-inflammatory drugs. PPARγ phosphorylation can regulate the balance between growth and differentiation in adipose tissue that is linked to obesity and insulin resistance. Sumoylation of PPARγ is linked to repression of inflammatory genes important in patients with inflammatory diseases. NR PTMs provide an additional measure of NR function that can be used as both biomarkers of disease progression, and predictive markers for patient response to NR-directed treatments.
Nuclear Receptor Signaling 01/2012; 10:e001.
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ABSTRACT: A major challenge in breast cancer therapy is the lack of an effective therapeutic option for a particularly aggressive subtype of breast cancer, triple-negative breast cancer. Here we provide the first preclinical evidence that a second-generation selenium compound, methylseleninic acid, significantly enhances the anticancer efficacy of paclitaxel in triple-negative breast cancer. Through combination-index value calculation, we demonstrated that methylseleninic acid synergistically enhanced the growth inhibitory effect of paclitaxel in triple-negative breast cancer cells. The synergism was attributable to more pronounced induction of caspase-mediated apoptosis, arrest of cell cycle progression at the G2/M checkpoint, and inhibition of cell proliferation. Treatment of SCID mice bearing MDA-MB-231 triple-negative breast cancer xenografts for four weeks with methylseleninic acid (4.5 mg/kg/day, orally) and paclitaxel (10 mg/kg/week, through intraperitoneal injection) resulted in a more pronounced inhibition of tumor growth compared with either agent alone. The attenuated tumor growth correlated with a decrease in tumor cell proliferation and an induction of apoptosis. The in vivo study also indicated the safety of using methylseleninic acid in the combination regime. Our findings thus provide strong justification for the further development of methylseleninic acid and paclitaxel combination therapy for the treatment of triple-negative breast cancer.
PLoS ONE 01/2012; 7(2):e31539. · 4.09 Impact Factor
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ABSTRACT: Phosphorylation of estrogen receptor α (ERα) is important for receptor function, although the role of specific ERα phosphorylation sites in ERα-mediated transcription remains to be fully evaluated. Transcriptional activation by ERα involves dynamic, coordinate interactions with coregulators at promoter enhancer elements to effect gene expression. To determine whether ERα phosphorylation affects recruitment of unique protein complexes at gene-specific promoters, changes in ERα Ser118 phosphorylation were assessed for effects on receptor and coregulator recruitment and transcription of ERα-regulated genes. Chromatin immunoprecipitation assays to measure promoter association found a 17β-estradiol (E2)-dependent recruitment of ERα at 150 min to ERα-regulated promoters, whereas ERα phosphorylated at Ser118 was dissociated from promoters after E2 treatment. Mutation of Ser118 to alanine (S118A) altered unliganded and ligand-induced association of ERα and p160 coregulators with ERα target promoters when compared with wild-type (WT)-ERα transfection. S118A and WT-ERα exhibited a similar level of recruitment to the estrogen response element-driven pS2 promoter and induced pS2 mRNA after E2 treatment. Although WT-ERα was recruited to c-myc and cyclin D1 promoters after E2 treatment and induced mRNA expression, S118A exhibited reduced interaction with c-myc and cyclin D1 promoters, and E2 did not induce c-myc and cyclin D1 mRNA. In addition, S118A resulted in increased recruitment of steroid receptor coactivator-1, glucocorticoid receptor interacting protein-1, and activated in breast cancer-1 to pS2, c-myc, and cyclin D1 irrespective of the presence of E2. Together, these data indicate that site specific phosphorylation of ERα directs gene-specific recruitment of ERα and transcriptional coregulators to ERα target gene promoters.
Endocrinology 06/2011; 152(6):2517-26. · 4.46 Impact Factor
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ABSTRACT: KX-01 is the first clinical Src inhibitor of the novel peptidomimetic class that targets the peptide substrate site of Src providing more specificity toward Src kinase. The present study was designed to evaluate the effects of KX-01 as a single agent and in combination with tamoxifen (TAM) on cell growth and apoptosis of ERα positive breast cancer in vitro and in vivo. Flow cytometry demonstrated that KX-01 induced cell cycle arrest in G2/M phase. Immunofluorescent staining for mitotic phase markers and TUNEL staining indicated that cells had arrested in the mitotic phase and mitotic arrested cells were undergoing apoptosis. KX-01 induced nuclear accumulation of cyclin B1, and activation of CDK1, MPM2, and Cdc25C that is required for progression past the G2/M checkpoint. Apoptosis resulted from activation of caspases 6, 7, 8, and 9. Combinational index analysis revealed that combinations of KX-01 with TAM resulted in synergistic growth inhibition of breast cancer cell lines. KX-01 combined with TAM resulted in decreased ERα phosphorylation at Src-regulated phosphorylation sites serines 118 and 167 that were associated with reduced ERα transcriptional activity. Orally administered KX-01 resulted in a dose dependent growth inhibition of MCF-7 tumor xenografts, and in combination with TAM exhibited synergistic growth inhibition. Immunohistochemical analysis revealed that combinational treatment reduced angiogenesis, and ERα signaling in tumors compared to either drug alone that may underlie the synergistic tumor growth inhibition. Combinations of KX-01 with endocrine therapy present a promising new strategy for clinical management of ERα positive breast cancer.
Breast Cancer Research and Treatment 04/2011; 132(2):391-409. · 4.43 Impact Factor
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ABSTRACT: NR4A nuclear receptors are a diverse group of orphan nuclear receptors with critical roles in regulating cell proliferation and cell differentiation. The ortholog of the NR4A nuclear receptor in Caenorhabditis elegans, NHR-6, also has a role in cell proliferation and cell differentiation during organogenesis of the spermatheca. Here we show that NHR-6 is able to bind the canonical NR4A monomer response element and can transactivate from this site in mammalian HEK293 cells. Using a functional GFP-tagged NHR-6 fusion, we also demonstrate that NHR-6 is nuclear localized during development of the spermatheca. Mutation of the DNA-binding domain of NHR-6 abolishes its activity in genetic rescue assays, demonstrating a requirement for the DNA-binding domain. This study represents the first genetic demonstration of an in vivo requirement for an NR4A nuclear receptor DNA-binding domain in a whole organism.
genesis 08/2010; 48(8):485-91. · 2.53 Impact Factor
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Shuang Liu,
Yanfeng Qi,
Yubin Ge,
Tamika Duplessis, Brian G Rowan,
Clement Ip,
Helen Cheng,
Paul S Rennie,
Izumi Horikawa,
Arthur J Lustig,
Qun Yu,
Haitao Zhang,
Yan Dong
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ABSTRACT: As the mainstay treatment for advanced prostate cancer, androgen deprivation therapy (ADT) targets the action of androgen receptor (AR) by reducing androgen level and/or by using anti-androgen to compete with androgens for binding to AR. Albeit effective in extending survival, ADT is associated with dose-limiting toxicity and the development of castration-resistant prostate cancer (CRPC) after prolonged use. Because CRPC is lethal and incurable, developing effective strategies to enhance the efficacy of ADT and circumvent resistance becomes an urgent task. Continuous AR signaling constitutes one major mechanism underlying the development of CRPC. The present study showed that methylseleninic acid (MSA), an agent that effectively reduces AR abundance, could enhance the cancer-killing efficacy of the anti-androgen bicalutamide in androgen-dependent and CRPC cells. We found that the combination of MSA and bicalutamide produced a robust downregulation of prostate-specific antigen and a recently identified AR target, telomerase, and its catalytic subunit, human telomerase reverse transcriptase. The downregulation of hTERT occurs mainly at the transcriptional level, and reduced AR occupancy of the promoter contributes to downregulation. Furthermore, apoptosis induction by the two agents is significantly mitigated by the restoration of hTERT. Our findings thus indicate that MSA in combination with anti-androgen could represent a viable approach to improve the therapeutic outcome of ADT. Given the critical role of hTERT/telomerase downregulation in mediating the combination effect and the fact that hTERT/telomerase could be measured in blood and urine, hTERT/telomerase could serve as an ideal tumor-specific biomarker to monitor the efficacy of the combination therapy noninvasively.
Molecular Cancer Therapeutics 07/2010; 9(7):2016-25. · 5.23 Impact Factor
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ABSTRACT: Estrogen receptor (ER)alpha activity is regulated by phosphorylation at several sites. Recently several antibodies specific for individual phosphorylated sites within ERalpha have became available. Validation and use of these antibodies suggests that several forms of phosphorylated ERalpha can be detected in multiple ER+ human breast tumor samples, thus providing relevance for investigating the regulation and function of phosphorylated ERalpha in human breast cancer. Generally, the phosphorylated ERalpha isoforms are associated with parameters that suggest that they are markers of an intact estrogen dependent signaling pathway and better clinical outcome with respect to tamoxifen therapy. Profiling of phosphorylated ERalpha may provide better biomarkers of endocrine therapy response over and above those currently available.
The Journal of steroid biochemistry and molecular biology 04/2009; 114(1-2):90-5. · 2.66 Impact Factor
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ABSTRACT: Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein-protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP(2), a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET(2) energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET(2) assay as it is used to examine the physical interaction between the nuclear receptor ERalpha and the transcriptional coactivator SRC-1. Description of methods include selection of donor and acceptor combinations, fusion construct generation and validation, cell culture and transfection, individual fluorescence and luminescence detection, BRET(2) detection, and data analysis.
Methods in molecular biology (Clifton, N.J.) 01/2009; 590:253-63.
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ABSTRACT: In recent years, evidence has emerged supporting the hypothesis that cancer is a stem cell disease. The cancer stem cell field was led by the discovery of leukemia stem cells (Tan, B.T., Park, C.Y., Ailles, L.E., and Weissman, I.L. (2006) The cancer stem cell hypothesis: a work in progress. Laboratory Investigation. 86, 1203-1207), and within the past few years cancer stem cells have been isolated from a number of solid tumor including those of breast and brain cancer among others (Al-Hajj M., Wicha M.S., Benito-Hernandez A., Morrison, S.J., and Clarke, M.F. (2003) Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 100, 3983-3988; Singh, S.K., Clarke, I.D., Terasaki, M., Bonn, V.E., Hawkins, C., Squire, J., and Dirks, P.B. (2003) Identification of a Cancer Stem Cell in Human Brain Tumors. Cancer Research. 63, 5821-5828). Cancer stem cells exhibit far different properties than established cells lines such as relative quiescence, multidrug resistance, and multipotency (Clarke, M.F., Dick, J.E., Dirks, P.B., Eaves, C.J., Jamieson, C.H.M., Jones, D.L., Visvader, J., Weissman, I.L., and Wahl, G.M. (2006) Cancer Stem Cells-Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Research. 66, 9339-9344). In addition, our laboratory has demonstrated that breast cancer stem cells exhibit a strong metastatic phenotype when passaged in mice. Since stem cells exhibit these somewhat unique properties, it will be important for endocrinologists to evaluate hormonal action in these precursor cells for a more thorough understanding of cancer biology and development of more effective treatment modalities. A relatively easy and low cost method was developed to isolate breast cancer stem cells from primary needle biopsies taken from patients diagnosed with primary invasive ductal carcinoma during the routine care of patients with consent and IRB approval. Fresh needle biopsies (2-3 biopsies at 2 cm in length) were enzymatically dissociated in a collagenase (300 U/ml)/hyaluronidase (100 U/ml) solution followed by sequential filtration. Single cell suspensions were cultured on ultra low attachment plastic flasks in defined medium and formed non-adherent tumorspheres. The tumorspheres exhibited surface marker expression of CD44(+)/CD24(low/-)/ESA(+), previously defined as a "breast cancer stem cell" phenotype by Al Hajj et al. (Al-Hajj M., Wicha M.S., Benito-Hernandez A., Morrison, S.J., and Clarke, M.F. (2003) Prospective identification of tumorigenic breast cancer cells.
Methods in molecular biology (Clifton, N.J.) 01/2009; 590:363-75.
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ABSTRACT: Estrogen receptor alpha (ERalpha) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERalpha phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERalpha phosphorylation sites exist, COS-1 cells expressing human ERalpha were labeled with [32P]H3PO4 in vivo and ERalpha tryptic phosphopeptides were isolated to identify phosphorylation sites.
Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERalpha in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERalpha as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERalpha at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor.
These novel ERalpha phosphorylation sites represent new means for modulation of ERalpha activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor.
BMC Biochemistry 01/2009; 10:36. · 1.99 Impact Factor
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ABSTRACT: Tamoxifen has efficacy as a breast cancer therapy and chemoprevention agent. However, toxicity and resistance to tamoxifen limit its clinical application. There is an urgent need to develop compounds that may be combined with tamoxifen to improve efficacy and overcome toxicity and resistance. We showed previously that the organoselenium compound methylseleninic acid (MSA) increased the growth-inhibitory effect of tamoxifen and reversed tamoxifen resistance in breast cancer cells. In this study, we examined the mechanism for induction of apoptosis by MSA combined with tamoxifen in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells. 4-hydroxytamoxifen (TAM; 10(-7) mol/L) alone resulted in cell cycle arrest but no apoptosis, whereas MSA alone (10 micromol/L) induced apoptosis in tamoxifen-sensitive cells. Combination of MSA with TAM resulted in a synergistic apoptosis in both tamoxifen-sensitive and tamoxifen-resistant breast cancer cells compared with either agent alone. MSA and MSA combined with TAM induced apoptosis through the intrinsic, mitochondrial apoptotic pathway. MSA induced a sequential activation of caspase-9 and then caspase-8. These results indicate that the growth inhibition synergy and reversal of tamoxifen resistance by combination of selenium with tamoxifen occurs via a tamoxifen-induced cell cycle arrest, allowing more cells to enter the intrinsic apoptotic pathway elicited by selenium.
Molecular Cancer Therapeutics 10/2008; 7(9):3056-63. · 5.23 Impact Factor
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ABSTRACT: Inhibition of protein kinase A (PKA) promotes estrogen-dependent growth of MCF7 breast cancer cells, although the mechanisms by which PKA regulates estrogen receptor (ER) function remain unclear. In this study elevation of cAMP by forskolin/3-isobutyl-1-methylxanthine (F/I) suppressed estradiol-dependent MCF7 and T47D breast cancer cell growth but not tamoxifen-resistant MCF7-LCC2 cells. Although F/I induced ligand independent activation of ERalpha, F/I also decreased estradiol-dependent reporter gene transcription. Overexpression of PKA or PKA inhibitor (PKI) demonstrated that F/I effects on repression of estradiol action occurred through the PKA pathway. 8CPT-2Me-cAMP, a selective inducer of non-PKA signaling, did not alter ER-dependent transcription. In contrast to F/I effects on reporter genes, F/I exhibited gene-specific effects on endogenous, ER-regulated genes. F/I enhanced estradiol induction of pS2 and cMyc but repressed estradiol induction of cyclin D1 mRNA and protein in MCF7 cells. To explore likely mechanisms by which F/I regulated ER, experiments examined estradiol binding, Hsp90 interaction, promoter recruitment, and ERalpha phosphorylation. F/I decreased estradiol binding and increased Hsp90 association with ERalpha. Chromatin immunoprecipitation revealed that F/I recruited ERalpha to both pS2 and cMyc promoters at earlier times than estradiol, and F/I shifted estradiol recruitment of ERalpha to earlier time points. F/I induced a unique ERalpha phosphorylation profile (increase in serine 305 and decrease in serine 118 phosphorylation) that was distinct from estradiol and estradiol + F/I. Taken together, F/I signaling through PKA selectively regulates estradiol-dependent genes in breast cancer, which is associated with reduced ligand binding and changes in promoter interaction and ERalpha phosphorylation.
Molecular Endocrinology 03/2007; 21(2):439-56. · 4.54 Impact Factor
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ABSTRACT: Tamoxifen is currently used as adjuvant therapy for estrogen receptor (ER) positive breast cancer patients and as a chemopreventative agent. Although ER is a predictive marker for tamoxifen response, ER status fails to predict tamoxifen response in a significant number of patients highlighting the need to identify new pathways for tamoxifen sensitivity/resistance. To identify novel proteins induced by tamoxifen in breast cancer cells sensitive to tamoxifen growth inhibition, two-dimensional (2D) gel electrophoresis was used to profile proteins in T47D breast cancer cells. Six proteins were identified that were differentially regulated by 17beta-estradiol, 4-hydroxytamoxifen and the pure antagonist acolbifene (EM-652); calreticulin, synapse associated protein 1 (SYAP1), CD2 antigen binding protein 2 (CD2BP2), nucleosome assembly protein 1 like 1 (NAP1L1), d-3-phosphoglycerate dehydrogenase (3-PHGDH) and pyridoxine 5' phosphate oxidase (PNPO). At the mRNA level, these ligands differentially regulated expression of mRNAs encoding the identified proteins in T47D and MCF7 cells but had no effect on mRNA in ERalpha-negative MDA-MB-231 breast cancer cells. These novel SERM-regulated proteins may participate in new or existing pathways for sensitivity or resistance to SERMs.
Steroids 12/2006; 71(11-12):966-78. · 2.83 Impact Factor
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Daniel E Frigo,
Aninda Basu,
Erica N Nierth-Simpson,
Christopher B Weldon,
Christine M Dugan,
Steven Elliott,
Bridgette M Collins-Burow,
Virgilio A Salvo,
Yun Zhu,
Lilia I Melnik,
Gabriela N Lopez,
Peter J Kushner,
Tyler J Curiel, Brian G Rowan,
John A McLachlan,
Matthew E Burow
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ABSTRACT: Nuclear hormone receptors, such as the estrogen receptors (ERs), are regulated by specific kinase signaling pathways. Here, we demonstrate that the p38 MAPK stimulates both ERalpha- and ERbeta-mediated transcription in MCF-7 breast carcinoma, Ishikawa endometrial adenocarcinoma, and human embryonic kidney 293 cells. Inhibition of this potentiation using the p38 inhibitor, RWJ67657, blocked estrogen-mediated transcription and proliferation. Activated ERs promote gene expression in part through the recruitment of the p160 class of coactivators. Because no direct p38 phosphorylation sites have been determined on either ERalpha or beta, we hypothesized that p38 could target the p160 class of coactivators. We show for the first time using pharmacological and molecular techniques that the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) is phosphorylated and potentiated by the p38 MAPK signaling cascade in vitro and in vivo. S736 was identified as a necessary site for p38 induction of GRIP1 transcriptional activation. The C terminus of GRIP1 was also demonstrated to contain a p38-responsive region. Taken together, these results indicate that p38 stimulates ER-mediated transcription by targeting the GRIP1 coactivator.
Molecular Endocrinology 06/2006; 20(5):971-83. · 4.54 Impact Factor
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ABSTRACT: Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.
Nuclear Receptor Signaling 02/2006; 4:e021.
