Re-Expression of ERα and AR in Receptor Negative Endocrine Cancers via GSK3 Inhibition.

DNA methylation, catalyzed by DNA methyltransferase (DNMT), is a well-characterized epigenetic modification in cancer cells. In particular, promoter hypermethylation of AR and ESR1 results in loss of expression on Androgen Receptor (AR) and Estrogen Receptor (ER), respectively, and is associated with a hormone refractory state. We now report that Glycogen Synthase Kinase 3 (GSK3) phosphorylates DNMT1 at S714, which is localized to a 62 amino acid region referred to as auto-inhibitory linker, which functions to occlude the DNA from the active site of DNMT1 to prevent the methylation of unmethylated DNA. Molecular Dynamics simulation indicates that phosphorylation at S714 resulted in conformational rearrangement of the autoinhibitory domain that inactivated its ability to block the methylation of unmethylated DNA and resulted in enhanced DNA binding. Treatment with a novel and more selective inhibitor of GSK3 resulted in decreased methylation of the promoter region of genes encoding the Androgen Receptor (AR) and Estrogen Receptor alpha (ERa) and re-expression of the AR and ERa in AR negative prostate cancer and ER negative breast cancer cells, respectively. As a result, concurrent treatment with the GSK3 inhibitor resulted in responsiveness of AR negative prostate cancer and ER negative breast cancer cells to inhibitors of the AR or ER, respectively, in in vitro and in vivo experimental models.

Downregulation of Dihydrotestosterone and Estradiol Levels by HEXIM1.

We have previously reported that hexamethylene bis-acetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound estrogen receptor α (ERα) and the androgen receptor (AR) by disrupting the interaction between these receptors and positive transcriptional elongation factor b (P-TEFb) and attenuating RNA polymerase II (RNAPII) phosphorylation at serine 2. Functional consequences of the inhibition of transcriptional activity of ERα and AR by HEXIM1 include the inhibition of ERα- and AR-dependent gene expression, respectively, and the resulting attenuation of breast cancer (BCa) and prostate cancer (PCa) cell proliferation and growth. In our present study, we determined that HEXIM1 inhibited AKR1C3 expression in BCa and PCa cells. AKR1C3, also known as 17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 5, is a key enzyme involved in the synthesis of 17ß-estradiol (E2) and 5-dihydrotestosterone (DHT). Downregulation of AKR1C3 by HEXIM1 influenced E2 and DHT production, estrogen- and androgen-dependent gene expression, and cell proliferation. Our studies indicate that HEXIM1 has the unique ability to inhibit both the transcriptional activity of the ER and AR and the synthesis of the endogenous ligands of these receptors.

A structure and function relationship study to identify the impact of the R721G mutation in the human mitochondrial lon protease.

Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The Km values for the intrinsic as well as protein-stimulated ATPase were increased whereas the kcat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings.

Non-epigenetic induction of HEXIM1 by DNMT1 inhibitors and functional relevance.

We have been studying the role of Hexamethylene bisacetamide (HMBA) Induced Protein 1 (HEXIM1) as a tumor suppressor whose expression is decreased in breast and prostate cancer. The anti-cancer actions of HEXIM1 in melanomas and AML have been reported by other groups. Previous studies have shown that 5-Aza-2'deoxycytidine (5-AzadC), a DNMT1 inhibitor, induces re-expression of tumor suppressor genes by removing/erasing methylation marks from their promoters. Our studies highlighted another mechanism wherein 5-AzadC induced DNA damage, which then resulted in enhanced occupancy of NF-ĸB, P-TEFb, and serine 2 phosphorylated RNA Polymerase II on the HEXIM1 gene. As a consequence, 5-AzadC induced HEXIM1 expression in prostate cancer cell lines and triple negative breast cancers. 5-AzadC-induced DNA damage enhanced P-TEFb occupancy via a mechanism that involved activation of ATR and ATM and induction of NF-ĸB recruitment to the HEXIM1 promoter. Downregulation of NF-ĸB attenuated 5-AzadC-induced HEXIM1 expression in prostate and breast cancer cells. The functional relevance of 5-AzadC-induced HEXIM1 expression is revealed by studies showing the HEXIM1 is required for the induction of apoptosis. Collectively, our findings support a non-epigenetic mechanism for 5-AzadC-induced re-expression of HEXIM1 protein, and may contribute to the clinical efficacy of 5-AzadC.

Inhibition of the histone demethylase, KDM5B, directly induces re-expression of tumor suppressor protein HEXIM1 in cancer cells.

BACKGROUND: The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS: We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS: We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION: HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

Phosphorylation of the histone demethylase KDM5B and regulation of the phenotype of triple negative breast cancer.

Epigenetic modifications are known to play critical roles in the expression of genes related to differentiation and dedifferentiation. Histone lysine demethylase KDM5B (PLU-1) catalyzes the demethylation of histone H3 on Lys 4 (H3K4), which results in the repression of gene expression. KDM5B is involved in regulation of luminal and basal cell specific gene expression in breast cancers. However, the mechanisms by which KDM5B is regulated in breast cancer, in particular in response to post-translational signals is not well-defined. Here, we demonstrate that KDM5B is phosphorylated at Ser1456 by the cyclin-dependent kinase 1 (CDK1). Phosphorylation of KDM5B at Ser1456 attenuated the occupancy of KDM5B on the promoters of pluripotency genes. Moreover, KDM5B inhibited the expression of pluripotency genes, SOX2 and NANOG, and decreased the stem cell population in triple-negative breast cancer cell lines (TNBC). We previously reported that the tumor suppressor HEXIM1 is a mediator of KDM5B recruitment to its target genes, and HEXIM1 is required for the inhibition of nuclear hormone receptor activity by KDM5B. Similarly, HEXIM1 is required for regulation of pluripotency genes by KDM5B.

Anticancer effect of nor-wogonin (5, 7, 8-trihydroxyflavone) on human triple-negative breast cancer cells via downregulation of TAK1, NF-κB, and STAT3.

BACKGROUND: Nor-wogonin, a polyhydroxy flavone, has been shown to possess antitumor activity. However, the mechanisms responsible for its antitumor activity are poorly studied. Herein, we investigated the mechanisms of nor-wogonin actions in triple-negative breast cancer (TNBC) cells. METHODS: Effects of nor-wogonin on cell proliferation and viability of four TNBC cell lines (MDA-MB-231, BT-549, HCC70, and HCC1806) and two non-tumorigenic breast cell lines (MCF-10A and AG11132) were assessed by BrdU incorporation assays and trypan blue dye exclusion tests. Cell cycle and apoptosis analyses were carried out by flow cytometry. Protein expression was analyzed by immunoblotting. RESULTS: Nor-wogonin significantly inhibited the growth and decreased the viability of TNBC cells; however, it exhibited no or minimal effects in non-tumorigenic breast cells. Nor-wogonin (40 µM) was a more potent anti-proliferative and cytotoxic agent than wogonin (100 µM) and wogonoside (100 µM), which are structurally related to nor-wogonin. The antitumor effects of nor-wogonin can be attributed to cell cycle arrest via reduction of the expression of cyclin D1, cyclin B1, and CDK1. Furthermore, nor-wogonin induced mitochondrial apoptosis, (as evidenced by the increase in % of cells that are apoptotic), decreases in the mitochondrial membrane potential (ΔΨm), increases in Bax/Bcl-2 ratio, and caspase-3 cleavage. Moreover, nor-wogonin attenuated the expression of the nuclear factor kappa-B and activation of signal transducer and activator of transcription 3 pathways, which can be correlated with suppression of transforming growth factor-ß-activated kinase 1 in TNBC cells. CONCLUSION: These results showed that nor-wogonin might be a potential multi-target agent for TNBC treatment.

New quinoline/chalcone hybrids as anti-cancer agents: Design, synthesis, and evaluations of cytotoxicity and PI3K inhibitory activity.

A series of quinoline-chalcone hybrids was designed as potential anti-cancer agents, synthesized and evaluated. Different cytotoxic assays revealed that compounds experienced promising activity. Compounds 9i and 9j were the most potent against all the cell lines tested with IC50 = 1.91-5.29 µM against A549 and K-562 cells. Mechanistically, 9i and 9j induced G2/M cell cycle arrest and apoptosis in both A549 and K562 cells. Moreover, all PI3K isoforms were inhibited non selectively with IC50s of 52-473 nM when tested against the two mentioned compounds with 9i being most potent against PI3K-γ (IC50 = 52 nM). Docking of 9i and 9j showed a possible formation of H-bonding with essential valine residues in the active site of PI3K-γ isoform. Meanwhile, Western blotting analysis revealed that 9i and 9j inhibited the phosphorylation of PI3K, Akt, mTOR, as well as GSK-3ß in both A549 and K562 cells, suggesting the correlation of blocking PI3K/Akt/mTOR pathway with the above antitumor activities. Together, our findings support the antitumor potential of quinoline-chalcone derivatives for NSCLC and CML by inhibiting the PI3K/Akt/mTOR pathway.

1,3,4-oxadiazole/chalcone hybrids: Design, synthesis, and inhibition of leukemia cell growth and EGFR, Src, IL-6 and STAT3 activities.

A new series of 1,3,4-oxadiazole/chalcone hybrids was designed, synthesized, identified with different spectroscopic techniques and biologically evaluated as inhibitors of EGFR, Src, and IL-6. The synthesized compounds showed promising anticancer activity, particularly against leukemia, with 8v being the most potent. The synthesized compounds exhibited strong to moderate cytotoxic activities against K-562, KG-1a, and Jurkat leukemia cell lines in MTT assays. Compound 8v showed the strongest cytotoxic activity with IC50 of 1.95 µM, 2.36 µM and 3.45 µM against K-562, Jurkat and KG-1a leukemia cell lines, respectively. Moreover; the synthesized compounds inhibited EGFR, Src, and IL-6. Compound 8v was most effective at inhibiting EGFR (IC50 = 0.24 µM), Src (IC50 = 0.96 µM), and IL-6 (% of control = 20%). Additionally, most of the compounds decreased STAT3 activation.

Simultaneous determination of dihydrotestosterone and its metabolites in mouse sera by LC-MS/MS with chemical derivatization.

Androgens play a vital role in prostate cancer development, and their elimination and blockade are essential in the disease management. DHT is the key ligand for androgen receptor (AR) in the prostate. It is locally synthesized from testosterone. In the prostate, DHT is predominantly metabolized to α-diol and ß-diol. Recent studies indicate that impaired DHT catabolism is associated with prostate cancer, signifying the necessity of a sensitive quantitative method for the determination of DHT and its metabolites. In this work, an LC-MS/MS method for the simultaneous quantification of DHT and its metabolites was developed and validated. Steroid-free sera were prepared and used for the preparation of sera calibrators and quality controls (QCs). DHT and its metabolites along with their respective stable heavy isotope labeled analytes representing internal standards were first extracted with methyl tertiary-butyl ether (MTBE) and derivatized with picolinic acid (PA). The derivatized analytes were then extracted again with MTBE, dried under nitrogen and reconstituted in the mobile phase (80% methanol and 0.2% formic acid in water). Baseline chromatographic separation of the derivatized analytes was achieved isocratically on XTerra C18 column (2.1 × 100 mm) using the mobile phase at a flow rate of 0.25 mL/min. Quantitation was performed using multiple-reaction-monitoring mode with positive electrospray ionization. The method has calibration ranges from 0.0500 ng/mL to 50.0 ng/mL for DHT and its two metabolites with acceptable assay precision, accuracy, recovery, and matrix factor. It was applied to the determination of DHT and its metabolites in an animal study.

Induction of HEXIM1 activities by HMBA derivative 4a1: Functional consequences and mechanism.

We have been studying the role of Hexamethylene bisacetamide (HMBA) Induced Protein 1 (HEXIM1) as a tumor suppressor whose expression is decreased in tamoxifen resistant and metastatic breast cancer. HMBA was considered the most potent and specific inducer for HMBA inducible protein 1 (HEXIM1) prior to our studies. Moreover, the ability of HMBA to induce differentiation is advantageous for its therapeutic use when compared to cytotoxic agents. However, HMBA induced HEXIM1 expression required at mM concentrations and induced dose limiting toxicity, thrombocytopenia. Thus we structurally optimized HMBA and identified a more potent inducer of HEXIM1 expression, 4a1. The studies reported herein tested the ability of 4a1 to induce HEXIM1 activities using a combination of biochemical, cell phenotypic, and in vivo assays. 4a1 induced breast cell differentiation, including the stem cell fraction in triple negative breast cancer cells. Clinically relevant HEXIM1 activities that are also induced by 4a1 include enhancement of the inhibitory effects of tamoxifen and inhibition of breast tumor metastasis. We also provide mechanistic basis for the phenotypic effects of 4a1. Our results support the potential of an unsymmetrical HMBA derivative, such as 4a1, as lead compound for further drug development.

Mitochondrial complex I defect and increased fatty acid oxidation enhance protein lysine acetylation in the diabetic heart.

AIMS: Cardiomyopathy is a major complication of diabetes. Our study was aimed to identify the sites of mitochondrial dysfunction and delineate its consequences on mitochondrial metabolism in a model of type 1 diabetes. METHODS AND RESULTS: Diabetes was induced by streptozotocin injection to male Lewis rats. We found a decrease in mitochondrial biogenesis pathway and electron transport chain complex assembly that targets Complex I. Oxidation of Complex II and long-chain fatty acid substrates support the electron leak and superoxide production. Mitochondrial defects do not limit fatty acid oxidation as the heart's preferred energy source indicating that the diabetic heart has a significant reserve in Complex I- and II-supported ATP production. Both mitochondrial fatty acid oxidation and Complex I defect are responsible for increased protein lysine acetylation despite an unchanged amount of the NAD(+)-dependent mitochondrial deacetylase sirt3. We quantitatively analysed mitochondrial lysine acetylation post-translational modifications and identified that the extent of lysine acetylation on 54 sites in 22 mitochondrial proteins is higher in diabetes compared with the same sites in the control. The increased lysine acetylation of the mitochondrial trifunctional protein subunit α may be responsible for the increased fatty acid oxidation in the diabetic heart. CONCLUSION: We identified the specific defective sites in the electron transport chain responsible for the decreased mitochondrial oxidative phosphorylation in the diabetic heart. Mitochondrial protein lysine acetylation is the common consequence of both increased fatty acid oxidation and mitochondrial Complex I defect, and may be responsible for the metabolic inflexibility of the diabetic heart.

Downregulation of hPMC2 imparts chemotherapeutic sensitivity to alkylating agents in breast cancer cells.

Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2) has a role in the repair of estrogen-induced abasic sites. Our present study provides evidence that downregulation of hPMC2 in MDA-MB-231 and MDA-MB-468 breast cancer cells treated with temozolomide (TMZ) decreases cell survival. This increased sensitivity to TMZ is associated with an increase in number of apurinic/apyrimidinic (AP) sites in the DNA. We also show that treatment with another alkylating agent, BCNU, results in an increase in AP sites and decrease in cell survival. Quantification of western blot analyses and immunofluorescence experiments reveal that treatment of hPMC2 downregulated cells with TMZ results in an increase in γ-H2AX levels, suggesting an increase in double strand DNA breaks. The enhancement of DNA double strand breaks in TMZ treated cells upon downregulation of hPCM2 is also revealed by the comet assay. Overall, we provide evidence that downregulation of hPMC2 in breast cancer cells increases cytotoxicity of alkylating agents, representing a novel mechanism of treatment for breast cancer. Our data thus has important clinical implications in the management of breast cancer and brings forth potentially new therapeutic strategies.

HEXIM1 plays a critical role in the inhibition of the androgen receptor by anti-androgens.

We show that HEXIM1 (hexamethylene bis-acetamide inducible 1) functions as an AR (androgen receptor) co-repressor as it physically interacts with the AR and is required for the ability of anti-androgens to inhibit androgen-induced target gene expression and cell proliferation. Oncomine™ database and IHC (immunohistochemistry) analyses of human prostate tissues revealed that expression of HEXIM1 mRNA and protein are down-regulated during the development and progression of prostate cancer. Enforced down-regulation of HEXIM1 in parental hormone-dependent LNCaP cells results in resistance to the inhibitory action of anti-androgens. Conversely, ectopic expression of HEXIM1 in the CRPC (castration-resistant prostate cancer) cell line, C4-2, enhances their sensitivity to the repressive effects of the anti-androgen bicalutamide. Novel insight into the mechanistic basis for HEXIM1 inhibition of AR activity is provided by the present studies showing that HEXIM1 induces expression of the histone demethylase KDM5B (lysine-specific demethylase 5B) and inhibits histone methylation, resulting in the inhibition of FOXA1 (forkhead box A1) licensing activity. This is a new mechanism of action attributed to HEXIM1, and distinct from what has been reported so far to be involved in HEXIM1 regulation of other nuclear hormone receptors, including the oestrogen receptor.

Lead optimization of HMBA to develop potent HEXIM1 inducers.

The potency of a series of Hexamethylene bis-acetamide (HMBA) derivatives inducing Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) was determined in LNCaP prostate cancer cells. Several compounds with unsymmetrical structures showed significantly improved activity. Distinct from HMBA, these analogs have increased hydrophobicity and can improve the short half-life of HMBA, which is one of the factors that have limited the application of HMBA in clinics. The unsymmetrical scaffolds of the new analogs provide the basis for further lead optimization of the compounds using combinatorial chemistry strategy.

HEXIM1 down-regulates hypoxia-inducible factor-1α protein stability.

We have previously reported on the inhibition of HIF-1α (hypoxia-inducible factor α)-regulated pathways by HEXIM1 [HMBA (hexamethylene-bis-acetamide)-inducible protein 1]. Disruption of HEXIM1 activity in a knock-in mouse model expressing a mutant HEXIM1 protein resulted in increased susceptibility to the development of mammary tumours, partly by up-regulation of VEGF (vascular endothelial growth factor) expression, HIF-1α expression and aberrant vascularization. We now report on the mechanistic basis for HEXIM1 regulation of HIF-1α. We observed direct interaction between HIF-1α and HEXIM1, and HEXIM1 up-regulated hydroxylation of HIF-1α, resulting in the induction of the interaction of HIF-1α with pVHL (von Hippel-Lindau protein) and ubiquitination of HIF-1α. The up-regulation of hydroxylation involves HEXIM1-mediated induction of PHD3 (prolyl hydroxylase 3) expression and interaction of PHD3 with HIF-1α. Acetylation of HIF-1α has been proposed to result in increased interaction of HIF-1α with pVHL and induced pVHL-mediated ubiquitination, which leads to the proteasomal degradation of HIF-1α. HEXIM1 also attenuated the interaction of HIF-1α with HDAC1 (histone deacetylase 1), resulting in acetylation of HIF-1α. The consequence of HEXIM1 down-regulation of HIF-1α protein expression is attenuated expression of HIF-1α target genes in addition to VEGF and inhibition of HIF-1α-regulated cell invasion.

Inducible re-expression of HEXIM1 causes physiological cardiac hypertrophy in the adult mouse.

AIMS: The transcription factor hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) regulates myocardial vascularization and growth during cardiogenesis. Our aim was to determine whether HEXIM1 also has a beneficial role in modulating vascularization, myocardial growth, and function within the adult heart. METHODS AND RESULTS: To achieve our objective, we created and investigated a mouse line wherein HEXIM1 was re-expressed in adult cardiomyocytes to levels found in the foetal heart. Our findings support a beneficial role for HEXIM1 through increased vascularization, myocardial growth, and increased ejection fraction within the adult heart. HEXIM1 re-expression induces angiogenesis, that is, essential for physiological hypertrophy and maintenance of cardiac function. The ability of HEXIM1 to co-ordinate processes associated with physiological hypertrophy may be attributed to HEXIM1 regulation of other transcription factors (HIF-1-α, c-Myc, GATA4, and PPAR-α) that, in turn, control many genes involved in myocardial vascularization, growth, and metabolism. Moreover, the mechanism for HEXIM1-induced physiological hypertrophy appears to be distinct from that involving the PI3K/AKT pathway. CONCLUSION: HEXIM1 re-expression results in the induction of angiogenesis that allows for the co-ordination of tissue growth and angiogenesis during physiological hypertrophy.

Induction of quinone reductase by tamoxifen or DPN protects against mammary tumorigenesis.

We have previously shown that estrogen receptor ß (ERß)-mediated up-regulation of quinone reductase (QR) is involved in the protection against estrogen-induced mammary tumorigenesis. Our present study provides evidence that the ERß agonist, 2,3-bis-(4-hydroxy-phenyl)-propionitrile (DPN), and the selective estrogen receptor modulator tamoxifen (Tam), inhibit estrogen-induced DNA damage and mammary tumorigenesis in the aromatase transgenic (Arom) mouse model. We also show that either DPN or Tam treatment increases QR levels and results in a decrease in ductal hyperplasia, proliferation, oxidative DNA damage (ODD), and an increase in apoptosis. To corroborate the role of QR, we provide additional evidence in triple transgenic MMTV/QR/Arom mice, wherein the QR expression is induced in the mammary glands via doxycycline, causing a decrease in ductal hyperplasia and ODD. Overall, we provide evidence that up-regulation of QR through induction by Tam or DPN can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a novel mechanism of prevention against breast cancer. Thus, our data have important clinical implications in the management of breast cancer; our findings bring forth potentially new therapeutic strategies involving ERß agonists.

TARGETING THE GENOTOXIC EFFECTS OF ESTROGENS.

Our studies indicate that expression of antioxidative stress enzymes is upregulated by Selective Estrogen Receptor Modulators (SERMs) in breast epithelial cell lines, providing protection against the genotoxic effects of estrogens and against estrogen-induced mammary tumorigenesis. This upregulation of antioxidative stress enzymes requires Estrogen Receptor beta (ERß) and human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2). Further studies indicate that hPMC2 has a functional exonuclease domain that is required for upregulation of antioxidative stress enzymes by SERMs and repair of estrogen-induced abasic sites.

Determination of hexamethylene bisacetamide, an antineoplastic compound, in mouse and human plasma by LC-MS/MS.

Hexamethylene bisacetamide (HMBA) is a polar compound which has recently been discovered to have antineoplastic activity by up-regulating the expression of an endogenous antiproliferative breast cancer protein, HEXIM1 (hexamethylene bisacetamide inducible protein 1) in vivo. HMBA has been shown in the past to induce terminal differentiation in multiple leukemia types at a concentration of 2-5mM, but its phase I and II clinical trials were largely unsuccessful due to serious side effects (notably, thrombocytopenia) with dose escalation. In this work, a sensitive and simple LC-MS/MS method for direct determination of HMBA in mouse and human plasma is described. Plasma samples were prepared by deproteinization with acetonitrile. Separation was achieved on a Waters Atlantis(®) T3 (2.1 mm × 50 mm, 3 µm) column with retention times of 2.2 and 3.7 min for HMBA and 7MBA (internal standard), respectively. The quantitation was carried out by tandem mass spectrometry using positive MRM mode. The linear range of the method was 0.500-100 ng/mL in both mouse and human plasma with injection volume of 5 µL. This method has been validated in accordance with the US Food and Drug Administration (FDA) guidelines for bioanalytical method development and applied to the determination of HMBA concentrations in FVB mice over time after a single dose of HMBA in saline (0.9% NaCl) at 10mg/kg.