Mammary Cell Proliferation Research Articles

5α-Dihydrotestosterone regulates the expression of L-type calcium channels and calcium-binding protein regucalcin in human breast cancer cells with suppression of cell growth.

Androgens have been associated with the development of normal breast, and their role in mammary gland carcinogenesis has also been described. Several studies reported that androgens inhibit breast cancer cell growth, whereas others linked their action with the modulation of calcium (Ca(2+)) pumps, Ca(2+) channels and Ca(2+)-binding proteins. Also, it is known that deregulated Ca(2+) homeostasis has been implicated in the pathophysiology of breast. The L-type Ca(2+) channels (LTCCs) were found to be up-regulated in colon, colorectal and prostate cancer, but their presence in breast tissues remains uncharacterized. On the other hand, regucalcin (RGN) is a Ca(2+)-binding protein involved in the control of mammary gland cell proliferation, which has been identified as an androgen target gene in distinct tissues except breast. This study aimed to confirm the expression and activity of LTCCs in human breast cancer cells and investigate the effect of androgens in regulating the expression of α1C subunit (Cav1.2) of LTCCs and Ca(2+)-binding protein RGN. PCR, Western blot, immunofluorescence and electrophysiological experiments demonstrated the expression and activity of Cav1.2 subunit in MCF-7 cells. The MCF-7 cells were treated with 1, 10 or 100nM of 5α-dihydrotestosterone (DHT) for 24-72h. The obtained results showed that 1nM DHT up-regulated the expression of Cav1.2 subunit while diminishing RGN protein levels, which was underpinned by reduced cell viability. These findings first confirmed the presence of LTCCs in breast cancer cells and opened new perspectives for the development of therapeutic approaches targeting Ca(2+) signaling.

Abstract LB-207: ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell type-specific manner

Abstract [Introduction] Despite apparent resection of tumors, breast cancer patients often suffer relapse years after due to remnant dormant tumor cells. Nevertheless, the molecular mechanism regulating dormancy of breast cancer cell is still unclear. On the other hand, relapsed tumor is generally believed to be derived from cancer stem cells retaining normal stem cell property, which can undergo quiescence. ΔNp63α, an N-terminally truncated isoform of p51/p63 protein was shown to play crucial roles in the maintenance of stem cells within mammary epithelium. Furthermore, both tumor suppressive and oncogenic roles of ΔNp63α have been reported in mammary carcinogenesis. Thus, we investigated the role of ΔNp63α in the regulation of normal and malignant breast cells. [Method] MCF10A, normal human mammary epithelial cells and breast cancer cell lines of different subtypes were introduced with doxycycline-inducible constructs of ΔNp63α. These cells were subjected to cell cycle analysis, western blot, BrdU-Ki-67 immunostaining. Stemness was determined by mammosphere formation assay combined with flow cytometry analysis of cell surface markers. Drug response was studied using MTT assay. Also luminal breast cancer cell line, MCF7, was subjected to combined mRNA-miRNA microarray analysis. [Result] The induction of ΔNp63α in MCF7 luminal breast cancer cell line led the cells to acquire phenotype typical to quiescent luminal progenitor-like cells. Furthermore, the BMP and Wnt signaling pathways emerged as regulators of ΔNp63α-induced quiescence by microarray analysis. Interestingly, these quiescent cells exhibited down-regulation of BRCA1-dependent DNA repair pathways making them more sensitive to DNA damaging drug, Doxorubicin, but more resistant to anti-mitotic drug, Paclitaxel. Conversely, induction of ΔNp63α in MCF10A normal basal cells resulted in increased cell proliferation and expansion of mammary stem-like cells. ΔNp63α expression also led to the increase of cells expressing breast cancer stem cell markers in both MCF7 and MCF10A cells. However, induction of ΔNp63α affected neither cell proliferation nor the increase of mammary stem- or luminal progenitor-like cells in more aggressive luminal (T47D) and basal (MDA-MB-231) breast cancer cells containing mutant p53. This result interestingly suggested a cell type-specific function of ΔNp63α. We also identified a microRNA network that might regulate quiescence in ΔNp63α-expressing breast cancer cells which will be presented at the meeting in detail. [Conclusion] Our results suggest that the role of ΔNp63α in regulating quiescence, stem or progenitor activity in normal and breast cancer cells is cell type-specific. Given the heterogeneity in the cellular origin of breast cancer subtypes, ΔNp63α-directed chemotherapeutic strategy might be instructive to target therapeutically resistant breast cancer cells. Citation Format: Md. Ruhul Amin, Yuiko Morita-Fujimura, Shuntaro Ikawa. ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell type-specific manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-207. doi:10.1158/1538-7445.AM2015-LB-207

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

Impact of diethylhexyl phthalate on gene expression and development of mammary glands of pregnant mouse.

The widely used diethylhexyl phthalate (DEHP) is a known endocrine disruptor that causes persistent alterations in the structure and function of female reproductive system, including ovaries, uterus and oviducts. To explore the molecular mechanism of the effect of DEHP on the development of mammary glands, we investigated the cell cycle, growth, proliferation and gene expression of mammary gland cells of pregnant mice exposed to DEHP. It was demonstrated, for the first time, that the mammary gland cells of pregnant mice treated with DEHP for 0.5-3.5days post-coitum had increased proliferation, growth rate and number of cells in the G2/S phase. The expression of cell proliferation-related genes was significantly altered after short time and low-dose DEHP treatment of mammary gland cells in vivo and in vitro. These findings showed adverse effects of DEHP on mammary gland cells in pregnant mice.

Anti-proliferative effects of γ-tocotrienol are associated with suppression of c-Myc expression in mammary tumour cells.

Aberrant c-Myc activity plays a central role in cancer transformation. γ-tocotrienol is a member of the vitamin E family that displays potent anti-cancer activity. Here, studies were conducted to determine the role of c-Myc in mediating anti-proliferative effects of γ-tocotrienol in mammary cancer cells. Treatment effects on mouse +SA and human MCF-7 mammary cancer cell proliferation were determined by MTT assay and Ki-67 staining. Protein expression was determined by western blot analysis. Immunofluorescence staining and qRT-PCR were used to characterize cellular c-Myc and MYC levels respectively. Anti-proliferative effects of γ-tocotrienol were associated with reduction in total c-Myc and phosphorylated-c-Myc-serine 62, and increase in phosphorylated-c-Myc-threonine 58 levels. γ-tocotrienol also reduced PI3K/Akt/mTOR and Ras/MEK/Erk mitogenic signalling, cyclin D1 and cyclin-dependent kinase 4 levels, and increased p27 levels. However, γ-tocotrienol had no effect on MYC mRNA levels. γ-tocotrienol also increased levels of FBW7 (E3 ligase that initiates ubiquitination of c-Myc), but had no effect on serine/threonine phosphatase PP2A or isomerase Pin 1 levels. Combined treatment with GSK3α/β inhibitor LiCl or proteasome inhibitor MG132 blocked γ-tocotrienol-induced reductions in c-Myc. These findings indicate that anti-proliferative effects of γ-tocotrienol are associated with reduction in c-Myc that results from increase in GSK-3α/β-dependent ubiquitination and degradation, rather than from reduction in c-Myc synthesis in +SA and MCF-7 mammary cancer cells.

The potential in breast tissue engineering for the combined effects of linoleic acid and tamoxifen

Literature to date has strongly suggested that linoleic acid (LA) enhances mammary cancer cell proliferation when used in the range of 0·5–20 μg/ml. In contrast, the current in vitro studies show that deoxyribonucleic acid (DNA) levels of estrogen receptor-positive (ER+) MCF7 breast cancer cells were diminished following treatment with 30 μg/ml (100 μM) LA. MCF7 cells, when treated for 6 days with LA, consumed significantly less glucose than untreated controls (P-value<0·01). MFC7 cells treated with 30 μg/ml LA and 10 μM tamoxifen had even lower levels of DNA and lower glucose uptake. The same combination treatment of tamoxifen and LA was applied to a noncancerous mammary cell line, MCF10A, and did not have a significant effect on MCF10A DNA concentrations. Linoleic acid and its byproducts are important to cell proliferation and tissue reconstruction and, in combination with tamoxifen, can provide a uniquely different approach to soft tissue engineering. That is, LA and tamoxifen-loaded scaffolds may provide a viable reconstructive approach for patients undergoing lumpectomy and mastectomy by encouraging natural breast healing while limiting the ability of ER+ cancer cells to regrow. This approach could allow the suppression of estrogen-dependent mammary carcinomas while allowing natural cell proliferation after a mastectomy or lumpectomy.

Abstract P4-05-01: Genetic analysis of the role of Brca1 in suppression of basal-like breast cancer

Abstract BRCA1 mutation carriers are predisposed to developing basal-like breast cancers with high metastasis and poor prognosis. However, the mechanism underlying the function of BRCA1 in suppressing basal-like breast cancer remains unclear. We previously reported that deletion of p18Ink4c (p18), an inhibitor of CDK4 and CDK6, functionally inactivates the RB pathway, stimulates mammary luminal stem cell proliferation, and leads to spontaneous luminal tumor development whereas germline mutation of Brca1 alters the fate of luminal cells and causes luminal-to-basal mammary tumor transformation. We report here that disruption of Brca1 by both germline and epithelium-specific mutation of Brca1 in p18 deficient mice activates epithelial-to-mesenchymal transition (EMT) and induces basal-like breast tumors. Introduction of BRCA1 suppresses expression of EMT-inducing transcription factors (EMT-TFs) and inhibits EMT in mammary tumor cells. Knockdown of BRCA1 in human luminal cancer cells activates EMT and increases tumor formation potential. Consistently, EMT features are inversely correlated with BRCA1 in human breast cancers. These findings provide genetic evidence suggesting that BRCA1 suppresses EMT and inhibits basal-like tumor development. Citation Format: Xin-Hai Pei, Feng Bai, Ho Lam Chan, Alexandria Scott, Matthew D Smith, Cheng Fan, Jason I Herschkowitz, Charles M Perou, Alan S Livingstone, David J Robbins, Anthony J Capobianco. Genetic analysis of the role of Brca1 in suppression of basal-like breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-01.

Investigating the role of surface micro/nano structure in cell adhesion behavior of superhydrophobic polypropylene/nanosilica surfaces

The main aim of the current study was to investigate the effects of different topographical features on the biological performance of polypropylene (PP)/silica coatings. To this end, a novel method including combined use of nanoparticles and non-solvent was used for preparation of superhydrophobic PP coatings. The proposed method led to a much more homogeneous appearance with a better adhesion to the glass substrate. Moreover, a notable reduction was observed in the required contents of nanoparticles (100–20wt% with respect to the polymer) and non-solvent (35.5–9vol%) for achieving superhydrophobicity. Surface composition and morphology of the coatings were also investigated via X-ray photoelectron spectroscopy and scanning electron microscopy. Based on both qualitative and quantitative evaluations, it was found that the superhydrophobic coatings with only nano-scale roughness strongly prevented adhesion and proliferation of 4T1 mouse mammary tumor cells as compared to the superhydrophobic surfaces with micro-scale structure. Such results demonstrate that the cell behavior could be controlled onto the polymer and nanocomposite-based surfaces via tuning the surface micro/nano structure.

Downregulation of the KLF4 transcription factor inhibits the proliferation and migration of canine mammary tumor cells

Canine mammary tumor (CMT) is the most common neoplasm in female dogs, and over 50% of CMTs are diagnosed as malignant. Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and is associated with cell proliferation, differentiation, migration, and apoptosis. Although the role of KLF4 is still controversial in various human cancers, KLF4 has been identified as an oncogene in human breast cancer. Moreover, high KLF4 expression is correlated with an aggressive phenotype in CMT. Therefore, investigating the function of KLF4 may help better understand the pathogenesis of CMT.In this study, partial sequences of canine KLF4 and KLF4 expression were identified in various normal canine tissues, as well as CMT cells and Madin–Darby canine kidney (MDCK) cells. Kenpaullone, a small molecule inhibitor of KLF4, downregulated KLF4 expression in CMT cells and reduced CMT cell proliferation, migration, and colony formation in soft agar. Kenpaullone treatment induced S and G2/M phase arrest in CMT and MDCK cells, and induced death in CMT cells, but not in MDCK cells. It was concluded that KLF4 is expressed in various normal canine tissues, and downregulation of KLF4 inhibited CMT cell proliferation and migration, and induced cell death. The results of this study suggest that KLF4 may represent a suitable therapeutic target for CMT therapy.

Encapsulation of curcumin in diblock copolymer micelles for cancer therapy.

Application of nanoparticles has recently promising results for water insoluble agents like curcumin. In this study, we synthesized polymeric nanoparticle-curcumin (PNPC) and then showed its efficiency, drug loading, stability, and safety. Therapeutic effects of PNPC were also assessed on two cell lines and in an animal model of breast cancer. PNPC remarkably suppressed mammary and hepatocellular carcinoma cells proliferation (P < 0.05). Under the dosing procedure, PNPC was safe at 31.25 mg/kg and lower doses. Higher doses demonstrated minimal hepatocellular and renal toxicity in paraclinical and histopathological examinations. Tumor take rate in PNPC-treated group was 37.5% compared with 87.5% in control (P < 0.05). Average tumor size and weight were significantly lower in PNPC group than control (P < 0.05). PNPC increased proapoptotic Bax protein expression (P < 0.05). Antiapoptotic Bcl-2 protein expression, however, was lower in PNPC-treated animals than the control ones (P < 0.05). In addition, proliferative and angiogenic parameters were statistically decreased in PNPC-treated animals (P < 0.05). These results highlight the suppressing role for PNPC in in vitro and in vivo tumor growth models. Our findings provide credible evidence for superior biocompatibility of the polymeric nanocarrier in pharmacological arena together with an excellent tumor-suppressing response.

The effects of milking frequency in early lactation on milk yield, mammary cell turnover, and secretory activity in grazing dairy cows

In dairy cows, short-term changes of milking frequency in early lactation have been shown to produce an immediate and a long-term effect on milk yield in stall-fed cows. The effect is controlled locally within mammary glands and could be a function of either secretory mammary epithelial cell number or activity. To resolve this and determine its applicability in other feed management systems, a unilateral milking frequency experiment was conducted with udder halves of 17 multiparous, pasture-fed dairy cows milked either 4 times (4×) or once a day (1×) for 14d from 5±2d in milk. Mean half-udder milk yield during the treatment period was higher from the 4× compared with 1× udder halves and continued to be higher until 200d in milk once returned to twice a day milking. Mammary biopsies were obtained on d 14 of treatment from both udder halves of 10 cows. Proliferation of mammary cells was higher in 4× udder halves compared with 1×, whereas no difference in apoptosis levels was detected. Abundance of αS1-casein, β-casein, α-lactalbumin, and β-lactoglobulin mRNA was higher in tissue samples from 4× udder halves compared with 1×, whereas lactoferrin mRNA abundance was lower in 4× udder halves. In summary, change in milking frequency during early lactation affects proliferation of mammary cells as well as expression of the major milk protein genes, which both contribute to the observed changes in milk yield during and after unilateral milking frequency treatment.

Evaluation of melatonin treatment in primary culture of canine mammary tumors.

Mammary neoplasias are the most common tumors observed in female dogs. Identification of these tumors is valuable in order to identify beneficial therapeutic agents as alternative treatments for this tumor type. Oral administration of melatonin appears to exert an oncostatic effect on mammary neoplasia and may have a possible mechanism of action through its interaction with estrogen receptors on epithelial cells. Hence, we analyzed the potential therapeutic value of melatonin in tumors that are estrogen-dependent or -independent, and established a relationship of its action with the expression of the melatonin receptors MT1 and MT2. Furthermore, we analyzed the rate of cell proliferation and apoptosis after treatment with melatonin. Cell cultures were performed using 10 canine mammary tumor fragments and were divided into estrogen receptor (ER)-positive and ER-negative tumors. The results showed that both ER-positive and ER-negative tumors had decreased cell viability and proliferation after treatment with melatonin (p<0.05), although treatment was more effective in the ER-positive tumors. Analysis of the relative expression of the MT1 and MT2 genes by quantitative PCR was performed and the data were compared with the expression of ER in 24 canine mammary tumors and the cellular response to melatonin in 10 samples. MT1 was overexpressed in ER-positive tumors (p<0.05), whereas MT2 was not expressed. Furthermore, melatonin treatment in ER-positive tumors showed an efficient oncostatic effect by inhibiting cell viability and proliferation and inducing apoptosis. These results suggest that melatonin decreased neoplastic mammary cell proliferation and viability and induced apoptosis, with greater efficacy in ER-positive tumors that have a high expression of melatonin receptor MT1. This is a strong evidence for the use of melatonin as a therapeutic agent for estrogen-dependent canine mammary tumors.

Centchroman suppresses breast cancer metastasis by reversing epithelial–mesenchymal transition via downregulation of HER2/ERK1/2/MMP-9 signaling

Metastatic spread during carcinogenesis worsens disease prognosis and accelerates the cancer progression. Therefore, newer therapeutic options with higher specificity toward metastatic cancer are required. Centchroman (CC), a female oral contraceptive, has previously been reported to possess antiproliferative and proapoptotic activities in human breast cancer cells. Here, we investigated the effect of CC-treatment against breast cancer metastasis and associated molecular mechanism using in vitro and in vivo models. CC significantly inhibited the proliferation of human and mouse mammary cancer cells. CC-treatment also inhibited migration and invasion capacities of highly metastatic MDA-MB-231 and 4T1 cells, at sub-IC50 concentrations. Inhibition of cell migration and invasion was found to be associated with the reversal of epithelial-to-mesenchymal transition (EMT) as observed by the upregulation of epithelial markers and downregulation of mesenchymal markers as well as decreased activities of matrix metalloproteinases. Experimental EMT induced by exposure to TGFβ/TNFα in nontumorigenic human mammary epithelial MCF10A cells was also reversed by CC as evidenced by morphological changes and modulation in the expression levels of EMT-markers. CC-mediated inhibition of cellular migration was, at least partially, mediated through inhibition of ERK1/2 signaling, which was further validated by using MEK1/2 inhibitor (PD0325901). Furthermore, CC-treatment resulted in suppression of tumor growth and lung metastasis in 4T1-syngeneic mouse model. Collectively, our findings suggest that CC-treatment at higher doses specifically induces cellular apoptosis and inhibits cellular proliferation; whereas at lower doses, it inhibits cellular migration and invasion. Therefore, CC could further be developed as an effective drug candidate against metastatic breast cancer.

Ovariectomy in young prepubertal dairy heifers causes complete suppression of mammary progesterone receptors

Mammary growth and development depends on ovarian steroids and particularly interaction of estrogen and progesterone with their intracellular receptors. The objectives of this study were to determine the effect of ovariectomy on the expression of protein and messenger RNA for estrogen receptor-alpha (ESR1) and progesterone receptor (PGR) and their relation to mammary ductal development and cell proliferation. Prepubertal Holstein heifers 2, 3, or 4 mo of age were randomly assigned to one of 2 treatments, ovariectomized (OVX; n = 8) or sham operated (INT; n = 12). Mammary parenchymal (PAR) tissue samples were harvested 30 d after surgery. Localization and quantitation of ESR1 and PGR in PAR were determined by immunohistochemistry and quantitative multispectral imaging. Relative messenger RNA expression of ESR1 and PGR in PAR was measured by quantitative real time polymerase chain reaction. We observed the complete absence of PGR-positive epithelial cell nuclei and reduced PGR transcript abundance in mammary parenchyma of OVX heifers. The percent of epithelial cells expressing ESR1 did not differ by treatment but was decreased with age. However, average intensity of ESR1 expression per cell was reduced in OVX heifers. The abundance of Ki67 labeled epithelial cells and stromal cells was reduced after ovariectomy. These data suggest that reduced mammary development after ovariectomy may be mediated by loss of PGR expression and reduced ESR1 expression in positive cells. A presumptive relationship with ovarian-derived circulating estradiol remains unresolved, but data suggest other ovarian-derived agents may play a role. Use of specific antagonists to manipulate expression or action of PGR and ESR1 receptors should provide direct evidence for roles of these receptors in prepubertal bovine mammary development.

BRCA1 suppresses epithelial-to-mesenchymal transition and stem cell dedifferentiation during mammary and tumor development.

BRCA1 mutation carriers are predisposed to developing basal-like breast cancers with high metastasis and poor prognosis. Yet, how BRCA1 suppresses formation of basal-like breast cancers is still obscure. Deletion of p18(Ink4c) (p18), an inhibitor of CDK4 and CDK6, functionally inactivates the RB pathway, stimulates mammary luminal stem cell (LSC) proliferation, and leads to spontaneous luminal tumor development. Alternately, germline mutation of Brca1 shifts the fate of luminal cells to cause luminal-to-basal mammary tumor transformation. Here, we report that disrupting Brca1 by either germline or epithelium-specific mutation in p18-deficient mice activates epithelial-to-mesenchymal transition (EMT) and induces dedifferentiation of LSCs, which associate closely with expansion of basal and cancer stem cells and formation of basal-like tumors. Mechanistically, BRCA1 bound to the TWIST promoter, suppressing its activity and inhibiting EMT in mammary tumor cells. In human luminal cancer cells, BRCA1 silencing was sufficient to activate TWIST and EMT and increase tumor formation. In parallel, TWIST expression and EMT features correlated inversely with BRCA1 expression in human breast cancers. Together, our findings showed that BRCA1 suppressed TWIST and EMT, inhibited LSC dedifferentiation, and repressed expansion of basal stem cells and basal-like tumors. Thus, our work offers the first genetic evidence that Brca1 directly suppresses EMT and LSC dedifferentiation during breast tumorigenesis.

Adolescent meat intake and breast cancer risk.

The breast is particularly vulnerable to carcinogenic influences during adolescence due to rapid proliferation of mammary cells and lack of terminal differentiation. We investigated consumption of adolescent red meat and other protein sources in relation to breast cancer risk in the Nurses' Health Study II cohort. We followed prospectively 44,231 women aged 33-52 years who, in 1998, completed a detailed questionnaire about diet during adolescence. Relative risks (RR) and 95% confidence intervals (95%CI) were estimated using Cox proportional hazard regression. We documented 1132 breast cancer cases during 13-year follow-up. In multivariable Cox regression models with major breast cancer risk factors adjustment, greater consumption of total red meat in adolescence was significantly associated with higher premenopausal breast cancer risk (highest vs. lowest quintiles, RR, 1.43; 95%CI, 1.05-1.94; Ptrend = 0.007), but not postmenopausal breast cancer. Adolescent intake of poultry was associated with lower risk of breast cancer overall (RR, 0.76; 95%CI, 0.60-0.97; for each serving/day). Adolescent intakes of iron, heme iron, fish, eggs, legumes and nuts were not associated with breast cancer. Replacement of one serving/day of total red meat with one serving of combination of poultry, fish, legumes, and nuts was associated with a 15% lower risk of breast cancer overall (RR, 0.85; 95%CI, 0.74-0.96) and a 23% lower risk of premenopausal breast cancer (RR, 0.77; 95%CI, 0.64-0.92). In conclusion, higher consumption of red meat during adolescence was associated with premenopausal breast cancer. Substituting other dietary protein sources for red meat in adolescent diet may decrease premenopausal breast cancer risk.

Abstract 76: Liver receptor homologue-1 increases incidence of DMBA-induced mammary tumors

Abstract Breast cancer is one of the most common malignancies globally. It accounts for approximately 15% of cancer-related deaths in Australian women. The orphan nuclear receptor liver receptor homolog-1 (LRH-1) promotes increased cell proliferation, motility and invasion in breast cancer cell lines. Additionally, high LRH-1 expression in human breast cancers is positively associated with estrogen receptor alpha status and aromatase activity. However, the role of LRH-1 in tumour growth is not well understood. Therefore, we aimed to generate a doxycycline (dox)-inducible mammary epithelial specific LRH-1 knock-in mouse in order to define the role of LRH-1 in mammary epithelial proliferation in vivo. In addition, the Dimethylbenz(a)anthracene (DMBA) induced mammary tumour model was utilized with the LRH-1 transgenic mice to determine the role LRH-1 plays in promoting mammary carcinogenesis. Given clear in vitro roles for LRH-1 in breast cancer cell proliferation, we hypothesise LRH-1 stimulates mouse mammary epithelial cell proliferation and promotes DMBA-mediated mammary tumorigenesis. We demonstrate increase in proliferation markers Ki-67, PCNA and PH3 immunoreactivity in luminal epithelial cells of dox-treated animals indicating that LRH-1 plays a role in mammary cell proliferation in vivo. In DMBA treated animals, we observed a five-fold increase of dense pre-neoplastic epithelial foci (p=0.0267), and a four-fold increase in microscopic lesions in dox-treated animals (p=0.0003). We also demonstrated that LRH-1 over-expression significantly reduced breast tumour-free survival in the transgenic DMBA model (no dox n=11; dox n=12, Mantel-Cox test p=0.0375). Tumour penetrance in DMBA animals not treated with dox was 9% (out of eleven animals) versus 25.0% (out of twelve animals) in the dox treated cohort. LRH-1 is known synergise with β-catenin to induce cyclin D1/E1 mediated cell proliferation in vitro. We analysed transcript levels of cyclin D1/E1 and show a significant increase in dox treated animals with or without DMBA. Finally, we demonstrate an increase in the oncogene β-catenin nuclear localisation in the dox treated animals with or without DMBA. Taken together, these data suggest that LRH-1 increases incidence of DMBA-induced mammary tumours. Further analyses on mechanism(s) mediated by LRH-1 are warranted to fully understand its role in mammary tumorigenesis. Note: This abstract was not presented at the meeting. Citation Format: Kyren A. Lazarus, Kristy Brown, Morag Young, Jason Cain, Samantha Jayasekara, Rhiannon Coulson, Neil Watkins, Colin Clyne, Ashwini Chand. Liver receptor homologue-1 increases incidence of DMBA-induced mammary tumors. [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 76. doi:10.1158/1538-7445.AM2014-76

Abstract 2151: Evaluation of a citrus flavonoid as a chemopreventive agent against breast cancer

Abstract Naringenin is a flavonone found in citrus fruits and tomatoes that may have anti-tumor properties. Previously, our lab discovered that naringenin induced the activity of AMP-activated protein kinase (AMPK) and reduced phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP) in MCF-7 human breast cancer cells. These properties have been linked with anti-tumor actions of drugs such as metformin and mTOR inhibitors. Based on these findings, we tested the ability of naringenin (0 uM - 200 uM) to modulate growth of MCF-7 human breast cancer cells and E0771 murine mammary tumor cells. In both models for cancer cell growth, naringenin dose-dependently decreased live cell number in a time-dependent manner. Furthermore, results of flow cytometry and propidium iodide staining assays showed accumulation of apoptotic cells after naringenin treatment. Naringenin also decreased expression of cyclin D1 and elevated expression of pro-apoptotic protein Bax suggesting that naringenin may inhibit the proliferation of mammary tumor cells by inducing cell cycle arrest and promoting apoptosis. To test for dose-dependent accumulation of naringenin in mammary gland tissue, female mice were fed varying doses of naringenin (control, 0%; low dose,1.0 wt%; or high dose, 3.0 wt% naringenin) in a high saturated fat diet for 11 weeks. Body weights and cumulative food intake were not different between diet groups. Dietary naringenin resulted in dose-responsive accumulation in mammary gland. Relative accumulation of naringenin in tissues was greatest in liver (low dose, 7.61 ± 1.14 umol/kg tissue and high dose, 37.04 ± 8.33 umol/kg tissue) followed by mammary gland and muscle which had a similar magnitude of naringenin accumulation (low dose ∼ 0.4 umol/kg tissue and high dose naringenin, 2.5 umol/kg tissue) of accumulation. Naringenin and monoglucuronide naringenin were two of the main forms of accumulated flavonoid in mammary gland raising the possibility that naringenin and/or metabolites are the active form of flavonoid responsible for anti-tumor activity against mammary cancer. Evaluating the species of naringenin metabolites in mammary gland that exert pro-apoptotic actions will be important to evaluate in order to understand the potential of naringenin-rich foods to contribute to prevention of breast cancer in women. Citation Format: Jia-Yu Ke, Min Tian, Kara L. Kliewer, Steve J. Schwartz, Ken M. Reidl, Shin-yu Tsai, Lisa D. Yee, Martha A. Belury. Evaluation of a citrus flavonoid as a chemopreventive agent against breast cancer. [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 2151. doi:10.1158/1538-7445.AM2014-2151

Abstract 246: Mammary cancer preventive activity of an extract of withania somnifera roots

Abstract Mammary cancer preventive activity of an alcoholic extract of Withania somnifera (WS) roots was examined in female ErbB2+/- (MMTV/Neu) mice. The WS root extract was mixed with feed at a concentration of 750 mg/kg of diet. Mice treated with the root extract developed fewer mammary tumors relative to mice in the untreated group; after 310 days, mice in the treated group had an average of 1.62 tumors, and mice in the control group had 2.48, a reduction of 33% (p=0.1). Representative samples of the mammary tumors were examined histologically after staining with hematoxylin and eosin (H&amp;E) and for proliferation (Ki67 and PCNA). For mammary tumors of control mice, sections stained with H&amp;E showed high mitotic activity, whereas in tumors from treated mice, there was low mitotic activity. Labeling indices for Ki67 and PCNA markers in cancers of treated mice were 35% and 30% lower, respectively, than those of the control group. Expression of the cytokine CCL2 was 50% lower in cancers of the treated group. These results indicate that the root extract of WS reduces mammary tumor formation and tumor cell proliferation. Supported by grant U54 CA118948. Citation Format: kamel F. Khazal, Donald Hill, Clinton J. Grubbs. Mammary cancer preventive activity of an extract of withania somnifera roots. [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 246. doi:10.1158/1538-7445.AM2014-246

Abstract 1439: Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21)

Abstract Caloric Restriction (CR) is known to be the only model to consistently increase the lifespan and postpone age-related diseases in all species. CR increases glycogenolysis, lipolysis with fatty acid and ketone body utilization and accelerates protein catabolism. We previously reported that CR induces metabolic and signaling changes that affect the tumor microenvironment preventing the growth of 4T1 mammary tumors and their metastases. 4T1 is a highly metastatic mouse breast cancer model which resembles breast cancer in patients. Obesity causes subclinical inflammation in adipose tissue that contributes to insulin resistance and cancer progression. Chronic inflammation predisposes to different forms of cancer and diabetes, and is correlated with increased risk of breast cancer. CR reduces insulin resistance, adiposity and inflammation and the anti-diabetic drug metformin (MET) is a CR mimetic agent. In this study, we evaluated whether the combination of MET with the anti-obesity drug orlistat (OR) enhances the CR mimetic and anti-cancer properties of MET on 4T1 mammary tumor cells. Female 8 week-old BALB/c mice received: vehicle, MET (in drinking water, 3mg/ml), OR i.p. injection (240mg/kg/day) or MET + OR. After 3 weeks on drugs, 4T1 cells (105) were injected into mice and drugs continued till the end of the experiment. Treated-mice had a reduced adiposity (p&amp;lt;0.05) without any signs of toxicity. At 30 days, MET + OR co-treatment reduced tumor volume (p&amp;lt;0.01) and displayed a range of cellular alterations such as decreased proliferative index (p&amp;lt;0.05); increased apoptotic rate (p&amp;lt;0.01) and altered intra-tumor collagen deposit. MET + OR treatment reduced total vessel length (p&amp;lt;0.01) and the number of spontaneous lung metastases (p&amp;lt;0.01). In addition, combinations of MET (1-2.5mM) + OR (2.5-5µM) decreased in vitro 4T1 mammary cell proliferation (p&amp;lt;0.01), adhesion (p&amp;lt;0.05) and migration (p&amp;lt;0.01) compared to vehicle and drugs alone. Interestingly, we found that levels of fibroblast growth factor 21 (FGF21), an endocrine factor that regulates glucose and lipid metabolism, were significantly increased with MET + OR treatment (p&amp;lt;0.05). It has been shown that increased circulating levels of FGF21 correlated with decreased obesity and increased sensitivity to insulin; but up to date the role of FGF21 in tumor progression was not elucidated. We found that treatment of 4T1 cells with MET + OR increased the secretion of FGF21 (p&amp;lt;0.05) measured by ELISA assays. To elucidate the role of FGF21; we initially demonstrated that addition of recombinant protein FGF21 (0-10nM) did not change 4T1 cell proliferation. In contrast, the reduced cell proliferation and adhesion induced by MET + OR, were abolished by the pre- and co-treatment with anti-FGF21 neutralizing antibody; indicating that FGF21 may play an important role. Our results provide a new rationale basis for the use of MET plus OR as cancer therapy. Citation Format: Shobika Sivaram, Erdene Baljinnyam, Kousaku Iwatsubo, Lydia I. Puricelli, Mariana S. De Lorenzo. Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21). [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 1439. doi:10.1158/1538-7445.AM2014-1439