Estrogen-dependent Cell Proliferation Research Articles

Young Shoots of Red Beet and the Root at Full Maturity Inhibit Proliferation and Induce Apoptosis in Breast Cancer Cell Lines.

Modern medicine is struggling with the problem of fully effective treatment of neoplastic diseases despite deploying innovative chemotherapeutic agents. Therefore, undertaking cancer-prevention measures, such as proper eating habits, should be strongly recommended. The present research aimed to compare the effects of juice from young shoots of beetroot compared to juice from root at full maturity on human breast cancer and normal cells. The juice from young shoots, both in the native and digested form, was most often a significantly stronger inhibitor of the proliferation of both analyzed breast cancer cell lines (MCF-7 and MDA-MB-231), compared to the native and digested juice from red beetroot. Regardless of juice type, a significantly greater reduction was most often shown in the proliferation of estrogen-dependent cells (MCF-7 line) than of estrogen-independent cells (MDA-MB-231 line). All analyzed types of beetroot juice and, in particular, the ones from young shoots and the root subjected to digestion and absorption, exerted an antiproliferative and apoptotic effect (pinpointing the internal apoptosis pathway) on the cells of both cancer lines studied. There is a need to continue the research to comprehensively investigate the factors responsible for both these effects.

Cytotoxicity, anti-tumor effects and structure-activity relationships of nickel and palladium S,C,S pincer complexes against double and triple-positive and triple-negative breast cancer (TNBC) cells

Triple-Negative Breast Cancer (TNBC) is a highly aggressive form of breast cancer. The high rate of metastasis associated with TNBC is attributed to its multidrug resistance, making the treatment of this metastatic condition difficult. The development of metal-based antitumor agents was launched with the discovery of cisplatin, followed by the development of related antitumor drugs such as carboplatin and oxaliplatin. Yet, the severe side effects of this approach represent a limitation for its clinical use. The current search for new metal-based antitumor agents possessing less severe side effects than these platinum-based complexes has focused on various complexes of nickel and palladium, the group 10 congeners of platinum. In this work, we have prepared a series of SCS-type pincer complexes of nickel and palladium featuring a stable meta-phenylene central moiety and two chelating but labile thioamide donor moieties at the peripheries of the ligand. We have demonstrated that the complexes in question, namely L1NiCl, L1NiBr, L1PdCl, L2PdCl, and L3PdCl, are active on the proliferation of estrogen-dependent breast tumor cells (MCF-7 and MC4L2) and triple-negative breast cancer (4 T1). Among the complexes studied, the palladium derivatives were found to be much safer anticancer agents than nickel counterparts; these were thus selected for further investigations for their effects on tumor cell adhesion and migration as well. The results of our studies show that palladium complexes are effective for inhibiting TNBC 4 T1 cells adhesion and migration. Finally, the HOMO and LUMO analysis was used to determine the reactivity and charge transfer within the compounds.

PGRMC1 can trigger estrogen-dependent proliferation of breast cancer cells: estradiol vs. equilin vs. ethinylestradiol

Objective: Previous studies have shown that progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer tissue can predict a worse prognosis for breast cancer patients. Moreover, we demonstrated that PGRMC1 can increase the proliferation of progestogens. However, the role of PGRMC1 in terms of estrogen-induced proliferation and comparing different estrogens is still unclear.Methods: Non-transfected and PGRMC1-transfected T-47D cells were stimulated with estradiol (E2), with equilin (EQ), or with ethinylestradiol (EE) at 1, 10, and 100 nmol/l. Increase of proliferation was compared with a control (without estrogens) and with the estrogen-induced stimulation in empty vector cells vs. PGRMC1-transfected cells.Results: The empty vector cells showed significant proliferation (12–15%) with all three estrogens only at the highest concentration, with no relevant differences between the estrogens. PGRMC1-transfected cells showed about three-fold higher proliferation (29–66%), whereby E2 elicited the strongest and EE the lowest proliferating effects, significantly lower compared to E2 and also compared to EQ. No significant differences were seen between E2 and EQ.Conclusions: PGRMC1 increases strongly the estrogen-dependent breast cell proliferation. The proliferating effects of EE may be lower compared to E2 and EQ. This could have importance in comparing hormone therapy and contraception. Thus, PGRMC1 not only could predict the risk using progestogens but also of different estrogens.

Abstract 3458: Neurotensin increases breast cancer proliferation through activation of AMPK

Abstract Introduction. Neurotensin (NT), a thirteen amino acid peptide predominantly localized to enteroendocrine cells of the small intestine, is released after fat ingestion. While its major role involves regulating lipid metabolism and storage, NT, acting through its high-affinity receptor (NTR1), stimulates the growth and progression of a variety of NTR1-positive cancers. Proneurotensin—a stable precursor produced in equimolar amounts as NT—has been associated with breast cancer development in women, and prior work has implicated the NT/NTR1 axis in breast cancer progression; however, very little is known about the underlying NT signaling pathways that stimulate breast cancer growth. The purpose of this study is to elucidate mechanisms by which NT leads to increased proliferation and aggression of breast cancer—with particular emphasis placed on the role of AMP-activated protein kinase (AMPK). Methods. MCF-7 (estrogen-dependent) and MDA-MB-231 (triple negative) are breast cancer cell lines that express NTR1. (i) Proliferation assays were performed by treating both cell lines with NT (0 or 100 nM) in serum-free media for 24, 48, and 72 h and then counting the viable cells. (ii) Next, to assess signaling pathways mediating the effects of NT, both cell lines were treated with NT (0 or 100 nM) in serum-free media for 15 min, 30 min, 1 h, 6 h, 12 h, and 24 h; immunoblotting was performed for phosphorylated and total forms of AMPK and its downstream effector acetyl CoA carboxylase (ACC). Results. (i) Increased proliferation of MCF-7 cells treated with NT (100 nM) was observed after serum-starvation for 48 h. While MDA-MB-231 cells treated with NT demonstrated improved survival after serum-starvation for 48 h, the effects were not significantly different. (ii) NT induced activation of AMPK and ACC in MCF-7 cells after treatment for 6 and 12 h, but these effects were not observed in MDA-MB-231 cells. Conclusions. Our findings suggest a possible linkage between NT and increased proliferation of estrogen-dependent breast cancer cells through activation of AMPK and its downstream effector proteins. These effects were minimal in NTR1-expressing triple negative breast cancer cells, suggesting that the molecular classification of the tumor plays an important role in NT signaling and downstream effects. Further delineation of the differential effects of NT in specific breast cancer phenotypes has the potential to identify novel therapeutic targets in the treatment of this disease. Citation Format: Jeremy Johnson, Jing Li, B. Mark Evers. Neurotensin increases breast cancer proliferation through activation of AMPK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3458.

A ferutinin analogue with enhanced potency and selectivity against ER-positive breast cancer cells in vitro.

Estrogen is considered a risk factor for breast cancer since it promotes breast-cell proliferation. The jaesckeanadiol-3-p-hydroxyphenylpropanoate, a hemi-synthetic analogue of the natural phytoestrogen ferutinin (jaesckeanadiol-p-hydroxybenzoate), is designed to be devoid of estrogenic activity. This analogue induces a cytotoxic effect 30 times higher than that of ferutinin towards MCF-7 breast cancer cell line. We compared these two compounds with respect to their effect on proliferation, cell cycle distribution and cancer stem-like cells in the MCF-7 cell line. Treatment with ferutinin (30 μM) and its analogue (1 μM) produced significant accumulation of cells at the pre G0/G1 cell cycle phase and triggered apoptosis. Importantly, this compound retains its anti-proliferative activity against breast cancer stem/progenitor cells that are naturally insensitive to ferutinin at the same dose. These results position ferutinin analogue as an effective compound inhibiting the proliferation of estrogen-dependent breast cancer cells and consistently targeting their stem-like cells.

The regulation of Hh/Gli1 signaling cascade involves Gsk3\u03b2- mediated mechanism in estrogen-derived endometrial hyperplasia

The present study was undertaken to explore the functional involvement of Hh signaling and its regulatory mechanism in endometrial hyperplasia. Differential expression of Hh signaling molecules i.e., Ihh, Shh, Gli1 or Gsk3β was observed in endometrial hyperplasial (EH) cells as compared to normal endometrial cells. Estradiol induced the expression of Hh signaling molecules and attenuated the expression of Gsk3β whereas anti-estrogen (K1) or progestin (MPA) suppressed these effects in EH cells. Cyclopamine treatment or Gli1 siRNA knockdown suppressed the growth of EH cells and reduced the expression of proliferative markers. Estradiol also induced the nuclear translocation of Gli1 which was suppressed by both MPA and K1 in EH cells. While exploring non-canonical mechanism, LY-294002 (Gsk3β activator) caused a decrease in Gli1 expression indicating the involvement of Gsk3β in Gli1 regulation. Further, Gsk3β silencing promoted the expression and nuclear translocation of Gli1 demonstrating that Gsk3β serves as a negative kinase regulator of Gli1 in EH cells. Similar attenuation of Hh signaling molecules was observed in rats with uterine hyperplasia undergoing anti-estrogen treatment. The study suggested that Hh/Gli1 cascade (canonical pathway) as well as Gsk3β-Gli1 crosstalk (non-canonical pathway) play crucial role in estrogen-dependent cell proliferation in endometrial hyperplasia.

Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents

The safety, including the endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (≥10,000 μg/L or 59 μM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-KBluc cells, which was blocked by the estrogen antagonist ICI 182,780. Commercial GBH formulations or their adjuvants alone did not exhibit estrogenic effects in either assay. Transcriptomics analysis of MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced cell proliferation but did not overlap with an ERα gene expression biomarker. Calculation of glyphosate binding energy to ERα predicts a weak and unstable interaction (−4.10 kcal mol−1) compared to estradiol (−25.79 kcal mol−1), which suggests that activation of this receptor by glyphosate is via a ligand-independent mechanism. Induction of ERE-luc expression by the PKA signalling activator IBMX shows that ERE-luc is responsive to ligand-independent activation, suggesting a possible mechanism of glyphosate-mediated activation. Our study reveals that glyphosate, but not other components present in GBHs, can activate ERα in vitro, albeit at relatively high concentrations.

Aryl hydrocarbon receptor induced intratumoral aromatase in breast cancer.

Aryl hydrocarbon receptor (AhR) inhibits estrogen receptor (ER) pathway, which may suppress estrogen-dependent cell proliferation. However, the correlation between AhR stimulation and intratumoral estrogen synthesis, especially through aromatase, has not been reported to date. In the present study, we examined this correlation in breast cancer cells. We examined AhR and aromatase immunoreactivity in 29 patients with invasive ductal carcinoma. We performed in vitro studies using three breast carcinoma cell lines, MCF-7, T47D, and MDA-MB-231. AhR stimulation induced the mRNA expression of the aromatase gene in vitro in three breast carcinoma cell lines, and increased estrogen synthesis in MCF-7 cell line. Results of microarray analysis showed that AhR-induced aromatase expression was associated with BRCA1 induction. Analysis of patients with breast cancer showed a significant positive correlation between intratumoral AhR and aromatase status. We also compared the effects of AhR stimulation on the induction of intratumoral estrogen synthesis and inhibition of the ER signaling pathway, because AhR exerts contradictory effects on estrogen action in breast carcinoma cells. AhR-induced aromatase expression persisted for a significantly longer duration than AhR-induced ER pathway inhibition. Moreover, breast carcinoma cells treated with an AhR agonist tended to show earlier cell proliferation after removing the agonist than cells not treated with the AhR agonist. The results of the present study suggest that AhR stimulates estrogen-dependent progression of breast carcinoma by inducing aromatase expression under some conditions. These results provide new insights on the possible roles of environmental toxins in breast cancer development.

Differences in the action of lower and higher chlorinated polychlorinated naphthalene (PCN) congeners on estrogen dependent breast cancer cell line viability and apoptosis, and its correlation with Ahr and CYP1A1 expression.

There are data showing that exposition to PCNs mixture increased incidence of gastrointestinal and respiratory neoplasms, but data regarding incidence of hormone-dependent cancer so far not shown. The objective was to determine if exposure to single lower and higher chlorinated PCN congeners is associated with altered proliferation and apoptosis of estrogen dependent breast cancer cells, and whether such effects are related to induction of AhR and CYP1A1 protein expression. MCF-7 cells were exposed to PCN 34, 39, 42, 46, 48, 52, 53, 54, 66, 67, 70, 71, 73 and 74 at concentrations of 100-10,000pg/ml. We evaluated the action of these PCN congeners on cell proliferation, DNA fragmentation and caspase-8,-9 activity. AhR and CYP1A1 protein expression and CYP1A1 activity was evaluated at a concentration of 1000pg/ml. An opposite action of tri- to tetraCNs than of penta-to heptaCNs on cell proliferation and apoptosis was evident. Tetra PCNs increased cell proliferation, but had no effect on DNA fragmentation nor caspase activity. Fast induction of CYP1A1 protein expression under the influence of lower chlorinated PCNs suggests faster metabolism and a possible stimulatory action of locally formed metabolites on cell proliferation. None of the higher chlorinated PCNs affected cell proliferation but all higher chlorinated PCNs increased caspase-8 activity, and hexa PCNs also increased caspase-9 activity. The rapid activation of the Ah receptor and CYP1A1 protein expression by higher chlorinated PCNs point to their toxicity; however, it is not sufficient for potential carcinogenicity. Action of lower chlorinated naphthalenes metabolites should be explored.

Estrogenic Activity of Mineral Oil Aromatic Hydrocarbons Used in Printing Inks.

The majority of printing inks are based on mineral oils (MOs) which contain complex mixtures of saturated and aromatic hydrocarbons. Consumer exposure to these oils occurs either through direct skin contacts or, more frequently, as a result of MO migration into the contents of food packaging that was made from recycled newspaper. Despite this ubiquitous and frequent exposure little is known about the potential toxicological effects, particularly with regard to the aromatic MO fractions. From a toxicological point of view the huge amount of alkylated and unsubstituted compounds therein is reason for concern as they can harbor genotoxicants as well as potential endocrine disruptors. The aim of this study was to assess both the genotoxic and estrogenic potential of MOs used in printing inks. Mineral oils with various aromatic hydrocarbon contents were tested using a battery of in vitro assays selected to address various endpoints such as estrogen-dependent cell proliferation, activation of estrogen receptor α or transcriptional induction of estrogenic target genes. In addition, the comet assay has been applied to test for genotoxicity. Out of 15 MOs tested, 10 were found to potentially act as xenoestrogens. For most of the oils the effects were clearly triggered by constituents of the aromatic hydrocarbon fraction. From 5 oils tested in the comet assay, 2 showed slight genotoxicity. Altogether it appears that MOs used in printing inks are potential endocrine disruptors and should thus be assessed carefully to what extent they might contribute to the total estrogenic burden in humans.

Steroid Sulfatase Inhibitor Reduces Proliferation of Ishikawa Endometrial Cancer Cells in Co-Culture Systems

Objectives: Estrogens significantly contribute toward the growth and development of endometrial cancers. Two principal pathways have been implicated in the final steps of estrogen synthesis: the steroid sulfatase (STS) and aromatase pathways. In this study, we aimed to evaluate the possible effects of tumor-stromal interactions on local estrogen biosynthesis in endometrial cancer. We also assessed the biological effects of inhibitors of steroid sulfatase and aromatase in the co-culture system compared with usual monocultures. Methods/Materials: We isolated stromal cells from endometrial cancer patients to examine local biosynthesis of estrogens and tumor-stromal interactions. Next we examined the effects of steroid sulfatase inhibitor and aromatase inhibitor in monoculture of endometrial cancer cell line (Ishikawa) and in a co-culture system involving an Ishikawa cells and stromal cells. Results: Estrogen receptor and steroid sulfatase mRNA levels in cancer cells were significantly higher in the co-cultures compared with the monocultures of endometrial cancer cells. Estradiol and androstenediol concentrations were also significantly higher in the co-cultured cells. Proliferation of the cancer cells was significantly increased through the steroid sulfatase pathway, which metabolizes androgens, estrone sulfate, and estradiol sulfate as its substrates. However, its proliferation was significantly decreased by the treatment of steroid sulfatase or aromatase inhibitors. The significant growth inhibition by the steroid sulfatase and aromatase inhibitors were also observed in the co-culture system. Conclusions: We evaluated the effects of STS inhibitor and aromatase inhibitors on the proliferation of estrogen-dependent endometrial cancer cells. Considering that intratumoral estrogen metabolism plays an important role, our co-culture systems provide an environment similar to that of the tumor in living patients in terms of metabolism and synthesis of intratumoral estrogens. The results of this study may aid in achieving improved clinical responses from patients treated with STS inhibitors.

Liganded ERα Stimulates the E3 Ubiquitin Ligase Activity of UBE3C to Facilitate Cell Proliferation.

Estrogen receptor (ER)α is a well-characterized ligand-dependent transcription factor. However, the global picture of its nongenomic functions remains to be illustrated. Here, we demonstrate a novel function of ERα during mitosis that facilitates estrogen-dependent cell proliferation. An E3 ubiquitin ligase, UBE3C, was identified in an ERα complex from estrogen-treated MCF-7 breast cancer cells arrested at mitosis. UBE3C interacts with ERα during mitosis in an estrogen-dependent manner. In vitro, estrogen dramatically stimulates the E3 activity of UBE3C in the presence of ERα. This effect was inhibited by the estrogen antagonist tamoxifen. Importantly, estrogen enhances the ubiquitination of cyclin B1 (CCNB1) and destabilizes CCNB1 during mitosis in a manner dependent on endogenous UBE3C. ERα, UBE3C, and CCNB1 colocalize in prophase nuclei and at metaphase spindles before CCNB1 is degraded in anaphase. Depletion of UBE3C attenuates estrogen-dependent cell proliferation without affecting the transactivation function of ERα. Collectively, these results demonstrate a novel ligand-dependent action of ERα that stimulates the activity of an E3 ligase. The mitotic role of estrogen may contribute to its effects on proliferation in addition to its roles in target gene expression.

Peroxisome Proliferator-Activated Receptor as a Therapeutic Target in Human Breast Cancer

Objective: Peroxisome proliferator-activated receptor γ (PPARγ) is aberrantly expressed in most breast tumors, suggesting the potential of agents that target this receptor in treatment and chemoprevention of breast cancer. However, whether PPARγ leads to the promotion or reduction of tumor formation has remained controversial and is further complicated by the ability of its available thiazolidinedione (TZD) ligands to activate another PPAR subtype, PPARδ. Method: To examine the role of each receptor in breast cancer biology, we performed a systematic evaluation of PPARγ and PPARδ agonists on the growth of human estrogen receptor (ER)-positive and -negative breast cancer cells. Results: In this manner we found that TZD-activated PPARγ was highly effective in suppressing the proliferation of estrogen-dependent cancer cells. Activated PPARδ, however, displayed growth-enhancing effects independent of estradiol and regardless of the ER status of the cells. Strikingly, in ER-negative cancer cells expressing a favorable PPARδ/γ ratio, TZDs promoted growth in a PPAR γ-independent manner by direct activation of PPARδ. A screen for ligands with increased receptor selectivity compared to TZDs revealed that GW7845 functioned as a full agonist of PPARγ, yet this agent lacked the ability to activate PPARδ and elicit its associated mitogenic effects. Conclusion: These studies indicate that selective PPAR γ modulators (SPPARγMs) that lack agonist activity on PPARδ may be clinically useful in future cancer treatment and chemoprevention.

Abstract LB-294: Growth factor genes, interaction with hormone therapy use and breast cancer risk in the California Teachers Study

Abstract Background: Growth factor and angiogenesis pathway genes are important candidate genes in breast carcinogenesis. Breast cell proliferation is modified by exogenous hormone therapy (HT) use, particularly estrogen plus progestin therapy (EPT). Estrogen-dependent cell proliferation requires activation of growth factor pathways, such as IGF-1, and potential interactions between a few FGFR2 single nucleotide polymorphisms (SNPs) and EPT have been reported. Further, angiogenesis genes such as VEGF and EGR3 were shown to be induced by steroid hormones. Therefore, we investigated SNPs in growth factor and angiogenesis genes and their interaction with HT in relation to breast cancer risk. Methods: We analyzed data from a breast cancer case-control study nested within the California Teachers Study. We genotyped 202 tagging SNPs in 12 growth factor and angiogenesis genes in 2746 non-Hispanic white women: 1351 cases and 1395 controls. We used conditional logistic regression models to estimate odds ratios (ORs) per minor allele and 95% confidence intervals (CI), and we repeated the analyses separately by HT use at cohort enrollment. P values were corrected for multiple testing using the Bonferroni method (Padj). Results: The most significant associations were observed for rs6899540 in VEGFA and 3 other VEGFA SNPs that are not in linkage disequilibrium with rs6899540. After Bonferroni correction within this gene, only rs6899540, located 4kb distal of the 3’end of the gene, remained statistically significant: OR per C allele=1.27 (95% CI=1.10-1.47), P=0.001, Padj=0.020. Additional SNPs nominally associated with breast cancer risk included rs2072454 in EGFR, rs4970503 in RPS6KA1, and rs9580185 in FGF9. Analyses among postmenopausal EPT users identified nominal associations with 5 VEGFR3 SNPs including rs6889643 and rs2290983 (ORs per risk allele ranging from 1.28 to 1.46; P-values range 0.008-0.015). Although none of these associations were statistically significant after Bonferroni correction, P values for interaction with EPT use (vs. never-HT use) were 0.03 for both rs6889643 and rs2290983. Among ET users, rs308441 in FGF2 was significantly associated with breast cancer risk (OR=1.69, Padj=0.019). Conclusions: Our results suggest that SNPs in growth factor and VEGF pathways may be associated with breast cancer risk, and these associations may be modified by ET or EPT use. Of particular interest are potential gene-HT interactions for SNPs in VEGFA, a key player in angiogenesis, and FGF2, a gene involved in VEGF signaling. Our results provide a strong rationale for further exploring these pathways and potential gene-environment interactions in pooled analyses. Citation Format: Eunjung Lee, Fredrick Schumacher, Susan L. Neuhausen, Hoda Anton Culver, Argyrios Ziogas, David Van Den Berg, Leslie Bernstein, Giske Ursin. Growth factor genes, interaction with hormone therapy use and breast cancer risk in the California Teachers Study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-294. doi:10.1158/1538-7445.AM2014-LB-294

MicroRNA-206 overexpression inhibits cellular proliferation and invasion of estrogen receptor α-positive ovarian cancer cells

The expression levels of estrogen receptor (ER α) are closely associated with estrogen-dependent growth, invasion and response to endocrine therapy in ERα-positive ovarian cancer. However, the underlying regulatory mechanisms remain to be fully understood. Previous studies have demonstrated that ERα is a direct target of microRNA (miR)-206. miR-206 has been found to be an important tumor suppressor in several cancer types, including ovarian, gastric and laryngeal cancer. However, the specific role of miR-206 in ovarian cancer remains unclear. The aim of the present study was to investigate the role of miR-206 in ER-a positive ovarian cancer in vitro. The present study demonstrated that miR-206 is significantly downregulated in ERα-positive but not ERα‑negative ovarian cancer tissues, compared with normal ovarian epithelium tissue. It was also found that the expression of miR-206 was decreased in ERα-positive ovarian cancer cell lines, CAOV-3 and BG-1, compared with normal ovarian epithelium tissues. This suggests that miR-206 may play a role in ERα-positive ovarian cancer cells via an estrogen-dependent mechanism. Further analysis revealed that 17β-E2 treatment significantly promoted cellular proliferation and invasion of estrogen-dependent CAOV-3 and BG-1 cells, which could be reversed by the introduction of miR-206 mimics. In conclusion, the present study suggests that miR-206 has an inhibitory role in estrogen-dependent ovarian cancer cells. Thus, miR-206 may be a promising candidate for the endocrine therapy of ERα-positive ovarian cancer.

Estrogen-dependent sushi domain containing 3 regulates cytoskeleton organization and migration in breast cancer cells.

Aromatase inhibitors (AI) are the standard endocrine therapy for postmenopausal breast cancer; however, currently used biomarkers, i.e., estrogen receptor-alpha/progesterone receptor (ERα/PR), predict only slightly more than half of the potential responders to AI treatment. To identify novel markers of AI responsiveness, a genome-wide microarray analysis was performed using primary breast tumor samples from 50 postmenopausal women (PMW) who later developed metastatic breast cancer. Sushi domain containing 3 (SUSD3) was significantly differentially expressed gene, with 3.38-fold higher mRNA levels in AI-responsive breast tumors versus non-responders (p<0.001). SUSD3 was highly expressed in ERα-positive breast tumors and treatment with estradiol increased SUSD3 expression in ERα-positive breast cancer cells. Treatment with an antiestrogen or ERα knockdown abolished basal and estradiol-dependent SUSD3 expression. Recruitment of ERα upstream of the transcription start site of SUSD3 was demonstrated by chromatin immunoprecipitation (ChIP)-PCR. Flow cytometric analysis of SUSD3 knockdown cells revealed blunted estradiol effects on progression into S and M phases. SUSD3 was localized to the plasma membrane of breast cancer cells. SUSD3 knockdown decreased the appearance of actin-rich protrusions, stress fibers and large basal focal adhesions, while increasing the presence of cortical actin concomitant with a decrease in Rho and FAK activity. SUSD3-deficient cells demonstrated diminished cell spreading, cell-cell adhesion, and motility. In conclusion, SUSD3 is a novel promoter of estrogen-dependent cell proliferation and regulator of cell-cell and cell-substrate interactions and migration in breast cancer. It may serve as a novel predictor of response to endocrine therapy and potential therapeutic target.

Carboxyl-Terminal Modulator Protein Positively Regulates Akt Phosphorylation and Acts as an Oncogenic Driver in Breast Cancer

Akt activation has been implicated broadly in tumorigenesis, but the basis for its dysregulation in cancer cells is incompletely understood. In this study, we sought to clarify a regulatory role for the Akt-binding carboxy-terminal modulator protein (CTMP), which has been controversial. In evaluating CTMP expression in paired normal-tumor specimens of 198 patients with breast cancer, we found that CTMP was upregulated in breast tumors, where it was associated with poor patient survival. Notably, CTMP expression also correlated positively with Akt phosphorylation in breast cancer clinical specimens and cell lines. Furthermore, ectopic expression of CTMP promoted cell proliferation and enhanced the tumorigenic properties of estrogen-dependent breast cancer cells. This effect was correlated with increased sensitivity to insulin-induced Akt phosphorylation, which is mediated primarily by the phosphoinositide 3-kinase-Akt pathway. In contrast, short hairpin RNA-mediated silencing of endogenous CTMP decreased the proliferation of estrogen-dependent or estrogen-independent breast cancer cells. Mechanistic investigations defined the N-terminal domain of CTMP at amino acids 1 to 64 as responsible for Akt binding. Taken together, our results firmly corroborate the concept that CTMP promotes Akt phosphorylation and functions as an oncogenic molecule in breast cancer.

Synthesis of Naturally Occurring Norlignan (±)-Nyasol

The norlignans including nyasol 1, hinokiresinol 2, agatharesinol 3, sequirin C 4 and sugiresinol 5 have received widespread interest as pharmaceutically valuable compounds, mainly because of their various clinically important biological activities: e.g. antiplasmodial activity, antifungal activity, anticancer activity, antimalarial activity, anti-oomycete activity, and estrogen receptor binding activity. Nyasol is one of the naturally occurring norlignans, which constitute abundant classes of phenylpropanoids. Nyasol has been isolated from various species of plants (Asparagus africanus, Anemarrhena asphodeloides, Asparagus officinalis, and Asparagus cochinchinensis). Nyasol and hinokiresinol are geometrical isomers and the confusion concerning the nomenclature of the two compounds nyasol and hinokiresinol was settled by Oketch-Rabah et al., who agreed that the Z-isomer should be named nyasol and the Eisomer hinokiresinol. In particular, nyasol shows various biological activities. For example, nyasol stimulated the proliferation of estrogendependent T47D breast cancer cells, and their stimulatory effects were blocked by an estrogen antagonist. In addition, nyasol showed binding affinity for the bovine uterine estrogen receptor, but (+)-nyasol was found to bind approximately seven times more strongly than (−)-nyasol. It was reported that (−)-nyasol increases hexobarbital sleeping time in mice. Nyasol was also reported to possess antifungal activity and inhibit testosterone 5α-reductase. Furthermore, nyasol was found to exhibit antioxidant and antiatherogenic activities. These results have encouraged us to complete adaptable and scalable total synthesis of these classes of natural products for further pharmacological study. So far, hinokiresinol derivatives have been easily prepared from chalcones by using reduction and dehydration. However, few successful synthetic procedures for nyasol have been reported other than the recent report by the Hoveyda group, which limits a scalable synthesis of nyasol. Quan et al. also reported an approach to synthesis of di-O-methyl ethers of (+)-nyasol and related norlignans, however, they might fail deprotection of the methyl ethers to obtain the final nyasol since there

Abstract 1706: Estrogen induces stearoyl-CoA desaturase expression in human MCF-7 breast carcinoma cells.

Abstract Approximately 95% of breast cancers are estrogen-dependant. Estrogen plays a crucial role in many cellular processes in estrogen-sensitive breast cancer such as carcinogenesis, production of growth factors, angiogenesis and growth stimulation. To sustain cell growth, many cancer cells exhibit an altered metabolism that is characterized by increased glycolysis and lipogenesis. We observed for the first time an up regulation of stearoyl-CoA desaturase-1 (SCD1) in response to estrogen in the hormone-dependent MCF7 human breast carcinoma cell line. SCD1 is a key enzyme in the production of monounsaturated fatty acids that are essential for membrane biogenesis. Using estrogen-starved MCF7 cells, treatment with 2 nM of estrogen induced cell proliferation, increased SCD-1 gene expression measured by qPCR (2.6±0.1 fold increase, p&amp;lt;0.05) and SCD-1 protein expression (7.2 fold increase) determined by immunoblot analysis, compared to control cells. Estrogen-induced SCD1 expression was accompanied by important changes in cellular fatty acid compositions with significant increases in the monounsaturated fatty acid content in membrane phospholipids and in SCD-1 product/substrate ratios with a 4.3±0.03 fold increase in the 16:1n-7/16:0 ratio and a 1.9±0.01 fold increase in the 18:1n-9/18:0 ratio compared to untreated cells (both p&amp;lt;0.05). Treatment of estrogen-stimulated cells with a selective SCD1 inhibitor inhibited estrogen-induced cell proliferation and prevented the estrogen-induced changes in membrane lipid composition. Overall, these results show for the first time that estrogen induces SCD-1 expression in breast carcinoma cells and suggests a crucial role for SCD1 in estrogen dependent cancer cell proliferation. SCD1 may therefore represent a potential therapeutic target for estrogen-dependent cancer. (This work was supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program). Citation Format: Anissa Belkaid, Rodney J. Ouellette, Marc E. Surette. Estrogen induces stearoyl-CoA desaturase expression in human MCF-7 breast carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1706. doi:10.1158/1538-7445.AM2013-1706

Abstract LB-21: Growth factor genes and change in mammographic density after quitting estrogen and progestin hormone therapy in the California Teachers Study.

Abstract Mammographic density (MD), a measure of the amount of epithelium and stroma in the breast, is a strong risk factor for breast cancer. Estrogen and progestin therapy (EPT) in postmenopausal women increases women's breast cancer risk. Although MD increases following EPT use and decreases following EPT cessation, substantial inter-individual variation in EPT-related MD changes is observed. Recent findings suggest that lifestyle factors do not explain much of the variation in the MD decrease following EPT cessation, suggesting that genetic factors could be major determinants. However, few studies have investigated genetic predictors of EPT-associated MD change. EPT increases breast cell proliferation, and activation of growth factor signaling pathways, such as epidermal growth factor and insulin-like growth factor 1 (IGF-1), may be required for estrogen-dependent cell proliferation. Therefore, we hypothesized that polymorphisms in growth factor genes are associated with EPT-related MD change. Using data from a mammogram substudy of the California Teachers Study, we investigated the association between 196 single nucleotide polymorphisms (SNPs) in 13 growth factor genes and MD changes after quitting EPT use. We studied genotypes and longitudinal MD data obtained before and after quitting EPT in 284 non-Hispanic white women. The median time interval between the two mammograms was 4 years (range: 1-9 years). We determined the MD (as percent MD) using a previously validated computer-assisted program. We used linear regression models to estimate the difference in MD between the ‘on-EPT’ and ‘off-EPT’ mammograms, adjusting for age and body mass index (BMI) at on-EPT mammogram, time interval and BMI change between the two mammograms, and baseline MD. After adjusting for multiple testing within each gene, we observed statistically significant associations for rs1983210 in INHA (P values adjusted for multiple correlated tests within a gene (Padj) = 0.021) and rs35539615 in IGFBP1/3 (Padj = 0.042) in relation to change in MD. The estimated differences in MD change per minor allele of these SNPs were -1.80% and 1.79%, respectively. In addition, rs2278200 in INHA showed some evidence of association with borderline statistical significance (Padj =0.053). Functional significance of these SNPs in regulating expression levels of INHA and IGFBP1/3 is not known. INHA encodes the alpha subunit of inhibins A and B, which are involved in cell differentiation and proliferation, and have been found to be expressed in the breast tissue. IGFBP1 and IGFBP3 are important regulators of bioavailability of IGF-1, which has been shown to stimulate proliferation of breast cancer cell lines in synergy with estrogen. While our findings require replication, our results suggest that genetic variation in these growth factor genes may influence MD change following EPT cessation. Citation Format: Eunjung Lee, Fred Schumacher, Anna H. Wu, Leslie Bernstein, Giske Ursin. Growth factor genes and change in mammographic density after quitting estrogen and progestin hormone therapy in the California Teachers Study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-21. doi:10.1158/1538-7445.AM2013-LB-21 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.