Spontaneous Mammary Tumorigenesis Research Articles

Inactivation of the Tumor Suppressor CYLD Sensitizes Mice to Breast Cancer Development.

Breast cancer is a leading cause of cancer-related deaths among women. Down-regulation of the tumor suppressor gene Cyld in breast cancer has been linked to a poor prognosis. This study investigated the role of Cyld in breast cancer using conditional mutant mouse models carrying a Cyld mutation, which inactivates the deubiquitinating activity of its protein product CYLD in mammary epithelial cells. We examined the potential of CYLD inactivation to induce mammary tumors spontaneously or modify the susceptibility of mice to mammary tumorigenesis by DMBA treatment or ErbB2 over-expression. CYLD inactivation significantly increased susceptibility to breast cancer induced by either DMBA treatment or ErbB2 over-expression. Moreover, while CYLD inactivation alone did not lead to spontaneous mammary tumorigenesis, it did contribute to the formation of multifocal hyperplastic lesions in virgin mice of predominantly FVB/NJ background. Our study demonstrates the tumor enhancing potential of CYLD inactivation in mammary tumorigenesis in vivo and establishes novel relevant mouse models that can be exploited for developing prognostic and therapeutic protocols.

Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models.

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment.

Transgenic mouse models of breast cancer.

Transgenic breast cancer mouse models are critical tools for preclinical studies of human breast cancer. Genetic editing of the murine mammary gland allows for modeling of abnormal genetic events frequently found in human breast cancers. Genetically engineered mouse models (GEMMs) of breast cancer employ tissue-specific genetic manipulation for tumorigenic induction within the mammary tissue. Under the transcriptional control of mammary-specific promoters, transgenic mouse models can simulate spontaneous mammary tumorigenesis by expressing one or more putative oncogenes, such as MYC, HRAS, and PIK3CA. Alternatively, the Cre-Lox system allows for tissue-specific deletion of tumor suppressors, such as p53, Rb1, and Brca1, or specific knock-in of putative oncogenes. Thus, GEMMs can be designed to implement one or more genetic events to induce mammary tumorigenesis. Features of GEMMs, such as age of transgene expression, breeding quality, tumor latency, histopathological characteristics, and propensity for local and distant metastasis, are variable and strain-dependent. This review aims to summarize currently available transgenic breast cancer mouse models that undergo spontaneous mammary tumorigenesis upon genetic manipulation, their varying characteristics, and their individual genetic manipulations that model aberrant signaling events observed in human breast cancers.

Abstract A48: Perinatal DDT exposure shortens latency of mouse mammary tumorigenesis

Abstract The contribution of exposure to the persistent, lipophilic pesticide dichlorodiphenyltrichloroethane (DDT) to breast cancer risk is controversial. A recent report by the International Agency for Research on Cancer (IARC) concluded that no consistent association exists between DDT exposure and breast cancer, yet stressed that there was a lack of existing evidence to allow for the determination of the effects of early-life exposure to DDT. It has been hypothesized that exposure to DDT during critical periods of breast development may increase breast cancer risk. We hypothesized that perinatal DDT exposure would result in shortened latency of spontaneous mammary tumorigenesis and accelerated growth of mammary tumors and increase the incidence of lung metastasis. To test the effect on mammary tumor early progression and growth as well as lung metastasis, C57BL/6J female mice, pregnant as a result of mating with B6.FVB-Tg(MMTV-PyVT)634Mul/LellJ male mice, were administered DDT from gestational day 11.5 to postnatal day 5 to produce circulating levels of DDT in dams within the prenatal range associated with breast cancer in adult daughters. This perinatal DDT exposure significantly accelerated the development of palpable mammary tumors (hazard ratio = 3.7 (95% confidence interval 2.0-6.8; p<0.0001). We also aimed to study possible mechanisms for DDT increasing mammary cancer risk, including alterations in puberty tempo and the presence of breast cancer risk factors associated with impaired metabolism. We found that mice exposed to DDT perinatally had increased terminal end buds in their mammary glands late in puberty (postnatal day 63, p<0.05). Tumor-free mice exposed to DDT perinatally also had significantly elevated circulating glucose levels (p<0.05). The risk of mammary cancer associated with perinatal DDT exposure observed in mice closely mirrors that observed in women previously. Our findings raise the possibility that perinatal DDT exposure may increase risk of breast cancer through its effects on pubertal mammary gland development and/or on glucose homeostasis. Future research to further evaluate whether these effects are mechanisms by which perinatal DDT exposure accelerates breast tumorigenesis is highly warranted. Citation Format: Tomoko Ishikawa, Jason Tong, Alexander Borowsky, Robert Cardiff, Michele La Merrill. Perinatal DDT exposure shortens latency of mouse mammary tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A48.

Abstract 5290: Loss-of-Irf5 in mammary epithelial cells drives spontaneous mammary tumorigenesis by regulating EMT

Abstract Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors. In human breast cancer (BC), IRF5 expression is lost in ~ 60% of ductal carcinoma in situs (DCISs) and ~90% of invasive ductal carcinomas (IDCs). BCs expressing the lower quartile of IRF5 transcript expression were found to significantly correlate with poor prognosis for recurrence-free survival. To examine how loss-of-Irf5 promotes breast tumor initiation and progression, we generated Irf5 knockout (Irf5-/-) mice on a BALB/C background that are susceptible to spontaneous mammary tumorigenesis. At 1 year-old, female nulliparous Irf5-/- mice (n=21) had a >3-fold increase in the incidence of spontaneous mammary tumorigenesis as compared to Irf5+/+ littermate mice (WT, n=25). As early as 6 months old, we detected dramatic differences in the presence of atypical ductal hyperplasia, and at 9 months old, ~50% of Irf5-/- mice had ADH while only 17% of WT littermates had detectable ADH (n=12/group). Immunofluorescence (IF) staining showed expansion of the luminal epithelial compartment in Irf5-/- mice with ADH and/or mammary tumors. Further, proliferation of Irf5-/- luminal epithelial cells was significantly increased over WT mice (15 week-old, n=3/group, 2-way ANOVA, Genotype factor F (1, 4) = 0.04493), supporting a loss of cell growth control in this compartment. To identify the molecular mechanism(s) driving enhanced mammary tumorigenesis, we performed RNA-seq on FACS-sorted luminal and basal epithelial cells from 9 month-old Irf5-/- and Irf5+/+ littermate mice (n=3 mice/group). Gene Set Enrichment Analysis (GSEA) identified signatures that were either positively or negatively regulated in Irf5-/- luminal and basal epithelial cells as compared to WT littermates. We found that epithelial to mesenchymal transition (EMT)-transcription factors were significantly enriched in both Irf5-/- luminal (p=0.046) and basal (p=0.004) epithelial cells. A diverse array of signaling pathways and molecules that participate in EMT induction were also found enriched in Irf5-/- cells, including Wnt/β-catenin (Luminal p=0.021; basal p=0.294), Sonic hedgehog (Shh) (luminal p=0.849), Gli1 (Irf5-/- vs. WT, luminal 3.56 vs. 1.35; basal 10.04 vs. 8.88) and PI3K signaling (luminal p=0.941; basal p=0.970). Findings suggest that loss-of-Irf5 promotes EMT through one or more of these pathways, thus contributing to the striking increase in spontaneous mammary tumorigenesis that occurs in Irf5-/- mice. Results from this study are expected to identify mechanisms and pathways that may be therapeutically targeted in patients with IRF5-negative BC. Note: This abstract was not presented at the meeting. Citation Format: Su Song, Dan Li, Kyle Kampta, Betsy Barnes. Loss-of-Irf5 in mammary epithelial cells drives spontaneous mammary tumorigenesis by regulating EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5290.

Discriminating the earliest stages of mammary carcinoma using myoepithelial and proliferative markers.

Mammographic screening has led to increased detection of breast cancer at a pre-invasive state, hence modelling the earliest stages of breast cancer invasion is important in defining candidate biomarkers to predict risk of relapse. Discrimination of pre-invasive from invasive lesions is critically important for such studies. Myoepithelial cells are the barrier between epithelial cells and the surrounding stroma in the breast ductal system. A number of myoepithelial immunohistochemistry markers have been identified and validated in human tissue for use by pathologists as diagnostic tools to distinguish in situ carcinoma from invasive breast cancer. However, robust myoepithelial markers for mouse mammary tissue have been largely under-utilised. Here, we investigated the utility of the myoepithelial markers smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), cytokeratin-14 (CK14) and p63 to discriminate mammary intraepithelial neoplasia (MIN) from invasive disease in the C57BL/6J MMTV-PyMT transgenic model of mammary carcinoma. We identified that SMMHC and CK14 are retained in early in situ neoplasia and are appropriate markers for distinguishing MIN from invasive disease in this model. Additionally, the proliferation marker Ki67 is a superior marker for differentiating between normal and hyperplastic ducts, prior to the development of MIN. Based on this, we developed a scoring matrix for discriminating normal, hyperplasia, MIN and invasive lesions in this spontaneous mammary tumorigenesis model. This study demonstrates heterogeneous expression of myoepithelial proteins throughout tumour development, and highlights the need to characterise the most appropriate markers in other models of early breast cancer to allow accurate classification of disease state.

Energy Balance Modulation Impacts Epigenetic Reprogramming, ERα and ERβ Expression, and Mammary Tumor Development in MMTV-neu Transgenic Mice.

The association between obesity and breast cancer risk and prognosis is well established in estrogen receptor (ER)-positive disease but less clear in HER2-positive disease. Here, we report preclinical evidence suggesting weight maintenance through calorie restriction (CR) may limit risk of HER2-positive breast cancer. In female MMTV-HER2/neu transgenic mice, we found that ERα and ERβ expression, mammary tumorigenesis, and survival are energy balance dependent in association with epigenetic reprogramming. Mice were randomized to receive a CR, overweight-inducing, or diet-induced obesity regimen (n = 27/group). Subsets of mice (n = 4/group/time point) were euthanized after 1, 3, and 5 months to characterize diet-dependent metabolic, transcriptional, and epigenetic perturbations. Remaining mice were followed up to 22 months. Relative to the overweight and diet-induced obesity regimens, CR decreased body weight, adiposity, and serum metabolic hormones as expected and also elicited an increase in mammary ERα and ERβ expression. Increased DNA methylation accompanied this pattern, particularly at CpG dinucleotides located within binding or flanking regions for the transcriptional regulator CCCTC-binding factor of ESR1 and ESR2, consistent with sustained transcriptional activation of ERα and ERβ. Mammary expression of the DNA methylation enzyme DNMT1 was stable in CR mice but increased over time in overweight and diet-induced obesity mice, suggesting CR obviates epigenetic alterations concurrent with chronic excess energy intake. In the survival study, CR elicited a significant suppression in spontaneous mammary tumorigenesis. Overall, our findings suggest a mechanistic rationale to prevent or reverse excess body weight as a strategy to reduce HER2-positive breast cancer risk. Cancer Res; 77(9); 2500-11. ©2017 AACR.

Abstract P6-08-08: GATA3 inhibits breast basal-like tumorigenesis

Abstract BACKGROUND: Breast cancer can be broadly categorized into two groups depending on the cell type affected. Luminal-type tumors are typically estrogen receptor (ER) positive that are associated with better survival and respond to hormone therapies whereas basal-like tumors are ER negative, more aggressive, and associated with a poor prognosis. GATA3 is a transcription factor well studied for its role as a master regulator of cellular differentiation and stem cell self renewal. Loss of Gata3 in mouse mammary glands blocks luminal cell differentiation and induces growth defects, and low levels of GATA3 are associated with basal-like and metastatic human breast cancers with epithelial-to-mesenchymal transition (EMT). Importantly, luminal cells have been shown to be the origin of some basal-like breast cancers. Due to the proliferation defects caused by GATA3 deficiency, it remains elusive how loss of function of GATA3 contributes to breast cancers development and progression. METHODS: We previously demonstrated that p18Ink4c (p18), a cell cycle inhibitor, is a downstream target of GATA3 in regulating mammary luminal cell proliferation and loss of p18 leads to luminal type tumorigenesis. To test the role of Gata3 deficiency in tumorigenesis, we generated p18-/-;Gata3+/- mice. Mammary gland development and tumorigenesis were characterized in vivo using a panel of cellular and molecular assays. Results were further confirmed in vitro with well established cell lines. RESULTS: Loss of p18 rescued mammary growth defects caused by Gata3 heterozygosity. Gata3 heterozygosity impaired luminal, but promoted basal gene expression in mammary epithelial cells. Gata3 heterozygosity in p18 null mice accelerated spontaneous mammary tumorigenesis, reducing the average latency of tumor onset. More importantly, Gata3 heterozygosity transformed the luminal type tumors of p18 null mice into heterogeneous basal-like breast cancers with activated EMT. Conversely, reintroduction of GATA3 inhibited tumor growth and reduced expression of EMT markers in basal-like tumor xenografts. We discovered that expression of GATA3 and Vimentin, an EMT marker, is inversely related in human breast cancers. CONCLUSION: Our data indicates that GATA3 promotes luminal but suppresses basal cell differentiation in the mammary gland and in tumor development. Mechanisms underlying the role of GATA3 in suppressing basal-like tumor development are under investigation.BACKGROUND: Breast cancer can be broadly categorized into two groups depending on the cell type affected. Luminal-type tumors are typically estrogen receptor (ER) positive that are associated with better survival and respond to hormone therapies whereas basal-like tumors are ER negative, more aggressive, and associated with a poor prognosis. GATA3 is a transcription factor well studied for its role as a master regulator of cellular differentiation and stem cell self renewal. Loss of Gata3 in mouse mammary glands blocks luminal cell differentiation and induces growth defects, and low levels of GATA3 are associated with basal-like and metastatic human breast cancers with epithelial-to-mesenchymal transition (EMT). Importantly, luminal cells have been shown to be the origin of some basal-like breast cancers. Due to the proliferation defects caused by GATA3 deficiency, it remains elusive how loss of function of GATA3 contributes to breast cancers development and progression. METHODS: We previously demonstrated that p18Ink4c (p18), a cell cycle inhibitor, is a downstream target of GATA3 in regulating mammary luminal cell proliferation and loss of p18 leads to luminal type tumorigenesis. To test the role of Gata3 deficiency in tumorigenesis, we generated p18-/-;Gata3+/- mice. Mammary gland development and tumorigenesis were characterized in vivo using a panel of cellular and molecular assays. Results were further confirmed in vitro with well established cell lines. RESULTS: Loss of p18 rescued mammary growth defects caused by Gata3 heterozygosity. Gata3 heterozygosity impaired luminal, but promoted basal gene expression in mammary epithelial cells. Gata3 heterozygosity in p18 null mice accelerated spontaneous mammary tumorigenesis, reducing the average latency of tumor onset. More importantly, Gata3 heterozygosity transformed the luminal type tumors of p18 null mice into heterogeneous basal-like breast cancers with activated EMT. Conversely, reintroduction of GATA3 inhibited tumor growth and reduced expression of EMT markers in basal-like tumor xenografts. We discovered that expression of GATA3 and Vimentin, an EMT marker, is inversely related in human breast cancers. CONCLUSION: Our data indicates that GATA3 promotes luminal but suppresses basal cell differentiation in the mammary gland and in tumor development. Mechanisms underlying the role of GATA3 in suppressing basal-like tumor development are under investigation. Citation Format: Pei X-H, Chan HL, Liu S, Scott A, Pimentel E, Slingerland J, Robbins D, Capobianco A, Bai F. GATA3 inhibits breast basal-like tumorigenesis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-08.

Abstract 3677: Loss of IRF5 induces spontaneous murine mammary tumorigenesis

Abstract Despite significant research efforts, breast cancer is still the most common malignancy in women and the most common cause of cancer death worldwide. A major challenge in breast cancer treatment is disease heterogeneity that is contributed by many factors including intrinsic cell factors, microenvironment, angiogenesis and tumor-specific immune responses. Interferon regulatory factor 5 (IRF5) is a transcription factor that controls inflammatory and immune responses. Analysis of over 3,000 human breast cancer tissues revealed that high expression of IRF5 correlates with increased survival and lower incidence of metastasis, whereas the lower quartile of IRF5 expression is a significant marker of poor prognosis for recurrence-free survival. These data support a tumor suppressor role for IRF5. To examine how loss of IRF5 expression may play a role in spontaneous mammary tumorigenesis, we generated Irf5 knockout (ko) mice in a BALB/C background. In mammary glands harvested from one-year-old female virgin ko mice (n = 15), we found an increased incidence (16%) of mammary carcinoma in situ as compared to their wild-type littermates (4%; n = 18). Of interest, the number of tumor bearing mice was significantly higher (38%) in retired ko female breeders (n = 8); however, there were no abnormalities detected in one-year-old male ko virgins or breeders (n = 6). Cellular and molecular analysis of female ko and wild-type littermates revealed that Irf5 deficiency enhanced mammary epithelial cell proliferation as indicated by an increased number of Ki67 expressing cells, and resulted in abnormal hyperbranching, which may contribute to mammary tumorigenesis. Irf5 ko breeders also showed impaired mammary gland involution after weaning, which has been shown to facilitate mammary tumor formation in humans. Taken together, these findings demonstrate that IRF5 is a gender-specific tumor suppressor in breast cancer. In this ongoing study, we are further investigating the intrinsic and extrinsic (microenvironment) role of IRF5 in mammary tumor initiation and progression. Citation Format: Dan Li, Xiaohui Bi, Corey Goyeneche, Amy Pitler, Betsy Barnes. Loss of IRF5 induces spontaneous murine mammary tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3677.

Tumoral Vitamin D Synthesis by CYP27B1 1-α-Hydroxylase Delays Mammary Tumor Progression in the PyMT-MMTV Mouse Model and Its Action Involves NF-κB Modulation.

Biologically active vitamin D (1,25-dihydroxycholecalciferol or 1,25(OH)2D) is synthetized from inactive prohormone 25-hydroxycholecalciferol (25(OH)D) by the enzyme CYP27B1 1-α-hydroxylase in kidney and several extrarenal tissues including breast. Although the development of breast cancer has been linked to inadequate vitamin D status, the importance of bioactive vitamin D production within tumors themselves is not fully understood. To investigate the role of tumoral vitamin D production in mammary epithelial cell progression to breast cancer, we conducted a Cre-loxP-mediated Cyp27b1 gene ablation in the mammary epithelium of the polyoma middle T antigen-mouse mammary tumor virus (PyMT-MMTV) mouse breast cancer model. Targeted ablation of Cyp27b1 was accompanied by significant acceleration in initiation of spontaneous mammary tumorigenesis. In vivo, cell proliferation, angiogenesis, cell cycle progression, and survival markers were up-regulated in tumors by Cyp27b1 ablation, and apoptosis was decreased. AK thymoma (AKT) phosphorylation and expression of several components of nuclear factor κB (NF-κB), integrin, and signal transducer and activator of transcription 3 (STAT3) signaling pathways were increased in Cyp27b1-ablated tumors compared with nonablated controls. In vitro, 1,25(OH)2D treatment induced a strong antiproliferative action on tumor cells from both ablated and nonablated mice, accompanied by rapid disappearance of NF-κB p65 from the nucleus and segregation in the cytoplasm. In contrast, treatment with the metabolic precursor 25(OH)D was only effective against cells from nonablated mice. 25(OH)D did not inhibit growth of Cyp27b1-ablated cells, and their nuclear NF-κB p65 remained abundant. Our findings demonstrate that in-tumor CYP27B1 1-α-hydroxylase activity plays a crucial role in controlling early oncogene-mediated mammary carcinogenesis events, at least in part by modulating tumoral cell NF-κB p65 nuclear translocation.

MDA-7/IL-24 functions as a tumor suppressor gene in vivo in transgenic mouse models of breast cancer.

Melanoma differentiation associated gene-7/Interleukin-24 (MDA-7/IL-24) is a novel member of the IL-10 gene family that selectively induces apoptosis and toxic autophagy in a broad spectrum of human cancers, including breast cancer, without harming normal cells or tissues. The ability to investigate the critical events underlying cancer initiation and progression, as well as the capacity to test the efficacy of novel therapeutics, has been significantly advanced by the development of genetically engineered mice (GEMs) that accurately recapitulate specific human cancers. We utilized three transgenic mouse models to better comprehend the in vivo role of MDA-7/IL-24 in breast cancer. Using the MMTV-PyMT spontaneous mammary tumor model, we confirmed that exogenously introducing MDA-7/IL-24 using a Cancer Terminator Virus caused a reduction in tumor burden and also produced an antitumor "bystander" effect. Next we performed xenograft studies in a newly created MMTV-MDA-7 transgenic model that over-expresses MDA-7/IL-24 in the mammary glands during pregnancy and lactation, and found that MDA-7/IL-24 overexpression delayed tumor growth following orthotopic injection of a murine PDX tumor cell line (mPDX) derived from a tumor formed in an MMTV-PyMT mouse. We also crossed the MMTV-MDA-7 line to MMTV-Erbb2 transgenic mice and found that MDA-7/IL-24 overexpression delayed the onset of mammary tumor development in this model of spontaneous mammary tumorigenesis as well. Finally, we assessed the role of MDA-7/IL-24 in immune regulation, which can potentially contribute to tumor suppression in vivo. Our findings provide further direct in vivo evidence for the role of MDA-7/IL-24 in tumor suppression in breast cancer in immune-competent transgenic mice.

Pttg1/securin is required for the branching morphogenesis of the mammary gland and suppresses mammary tumorigenesis

Pituitary tumor transforming gene 1 (Pttg1) encodes the mammalian securin, which is an inhibitor of separase (a protease required for the separation of sister chromatids in mitosis and meiosis). PTTG1 is overexpressed in a number of human cancers and has been suggested to be an oncogene. However, we found that, in Pttg1-mutant females, the mammary epithelial cells showed increased proliferation and precocious branching morphogenesis. In accord with these phenotypic changes, progesterone receptor, cyclin D1, and Mmp2 were up-regulated whereas p21 (Cdkn1a) was down-regulated. These molecular changes provide explanation for the observed developmental defects, and suggest that Pttg1 is a tumor suppressor. Indeed, mice lacking Pttg1 developed spontaneous mammary tumors. Furthermore, in human breast tumors, PTTG1 protein levels were down-regulated and the reduction was significantly correlated with the tumor grade.

Dietary supplemented 2-mercaptoethanol prevents spontaneous and delays virally-induced murine mammary tumorigenesis

There seems to be little doubt that organosulfur compounds have enormous benefits for biological processes, especially those of diseases like cancer. The preliminary results herein define a cancer model in which benefits/mechanisms of multitudes of xenobiotic and nature’s organosulfurs could easily be compared. Mice from three strains with a high incidence for naturally occurring tumors were treated daily with 2-mercaptoethanol (2-Me) starting at weaning. The 100% tumor incidence of undefined etiology in untreated BXSB-Yaa+ males was completely prevented by 2-Me. In contrast, 2-Me treatment of female and male C3H.OL and C3H.OH congenic strains, did not change the 100% tumor incidence due to milk-borne retrovirus, MMTV(S), but did: (1) delay the appearance of tumors by 42%; (2) increase longevity 56%; and (3) increase longevity, post-tumor detection, 95%. The addition of these results to the increasingly impressive anti-cancer benefits of simple xenobiotic organosulfurs raise the question: Can they be adapted for use as a preventive modality for human cancer?

Abstract 174: Calorie restriction decreases spontaneous mammary tumorigenesis and upregulates ER-β in MMTV-neu transgenic mice.

Abstract Obesity increases postmenopausal breast cancer risk and increases mortality in pre- and postmenopausal women. While the majority of breast cancers in obese women are estrogen receptor (ERα)-positive, ERα-negative tumors confer a much worse prognosis. The mechanism by which obesity affects ER-negative breast cancer risk and prognosis is not clear, and strategies for offsetting the negative effects of obesity are urgently needed. In this study, we utilized the MMTV-neu mouse model of luminal-type breast cancer to test the hypothesis that energy balance modulation, through diet-induced obesity (DIO) or calorie restriction (CR) regimens, alters mammary tumor development and progression through regulation of ER in the mammary epithelium. MMTV-neu mice form spontaneous mammary tumors that progress from an ERα-positive hyperplasia to aggressive ERα-negative ductal adenocarcinomas. Female MMTV-neu transgenic mice and non-transgenic host strain (FVB) mice (6-8 weeks old; n=90/genotype) were randomized (30/group) to receive: control diet (modified AIN-76A); a 30% CR regimen (isonutrient); or a DIO regimen. A subset of mice (n=4 per group) was killed at 1, 3, and 5 months following diet initiation, and tissues were collected for analysis; remaining animals were followed for a 60-week survival study. We found that, relative to control diet, the DIO regimen significantly increased body weight, percent body fat (p<0.0001), and obesity-associated serum hormones and growth factors (IGF-I, insulin, leptin, and estradiol; p<0.01 for all) in both MMTV-neu and FVB controls. Conversely, CR significantly decreased body weight, percent body fat (p<0.0001) and decreased serum hormones/ growth factors, while increasing adiponectin (each at p<0.01). Gene expression (qRT-PCR) and protein expression (immunohistochemistry) analysis revealed that loss of mammary ERα expression, known to occur by 8 weeks of age in control MMTV-neu mice, was accelerated by DIO and delayed by CR. Additionally, we found that CR (relative to control diet) significantly increased mammary ERβ expression (p<0.0001) and delayed the onset of hyperplasia (p<0.001) in both MMTV-neu and FVB mice. Importantly, we found that after 60 weeks of feeding, CR significantly increased tumor-free survival of MMTV-neu mice (p=0.01). CR tumor samples showed a decrease in vascularity and presence of mitotic figures. In conclusion, dietary energy balance modulation impacts spontaneous MMTV-neu mammary tumor development and ERα and ERβ levels in normal and tumor tissue. In addition, increased mammary ERβ expression may represent a novel mechanism underlying the anticancer effects of CR. Citation Format: Sarah M. Dunlap, Nikki A. Ford, Laura W. Bowers, Emily L. Rossi, Samantha Miller, Lucia J. Chiao, Powel H. Brown, Stephen D. Hursting. Calorie restriction decreases spontaneous mammary tumorigenesis and upregulates ER-β in MMTV-neu transgenic mice. [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 174. doi:10.1158/1538-7445.AM2013-174

The manner in which calories are restricted impacts mammary tumor cancer prevention

Although treatments for breast cancer have improved and long-term survival after diagnosis is now common, prevention of the disease is the ultimate goal. Weight loss or weight maintenance is one approach that has been recommended to reduce the risk of breast cancer, particularly for peri/postmenopausal women. This approach is supported by decades of data indicating that calorie restriction prevents spontaneous and chemically induced mammary tumor development in rodents. In most cases, calorie restriction was implemented by a consistent daily reduction of calories, i.e. chronic calorie restriction (CCR). There have also been several studies where periods of reduced caloric intake were followed by periods of refeeding, i.e. intermittent calorie restriction (ICR), resulting in the prevention of spontaneous mammary tumorigenesis. In most of the early studies, there were no direct comparisons of CCR to ICR. One study using moderate calorie restriction in a chemically induced breast cancer rat model found a slight increase in mammary tumor incidence compared with ad libitum fed and CCR rats. However, recently, it has been demonstrated in several transgenic mouse models of breast cancer that ICR consistently provided a greater degree of protection than CCR. This review will provide a detailed comparison of ICR and CCR for breast cancer prevention. It will also examine potential mechanisms of action that may include periods of reduced IGF-I and leptin as well as an increase in the adiponectin:leptin ratio. Application of this approach to at-risk women may provide an approach to lower the risk of breast cancer in overweight/obese women.

Targeted production of proprotein convertase PC1 enhances mammary development and tumorigenesis in transgenic miceThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Elevated production of proprotein convertases (PCs), proteolytic enzymes that posttranslationally modify the biological activities of diverse groups of cellular proteins, is a common occurrence in human breast carcinomas. A transgenic mouse model was developed to gain insight into the significance of PC production in breast development and neoplasia. Mammary epithelium-specific and early expression of PC1 was targeted by the use of the mouse mammary tumor virus promoter/enhancer. Whole-mount examinations revealed that the mammary glands of 83-day-old virgin PC1 transgenic mice exhibited an accelerated lobuloalveolar development compared with that of age-matched wild-type mice (p < 0.001). This phenotypic change was accompanied by extensive alterations in gene expression assessed by gene expression microarray analyses. Pathway analysis of PC1-induced alterations in gene expression has revealed possible mechanism of action of PC1 in the mammary gland. PC1 expression alone, however, did not promote spontaneous mammary tumorigenesis in the transgenic mice. PC1 transgene expression resulted in a significantly higher incidence (p = 0.008) and accelerated growth (p = 0.023) of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas. The present study therefore shows that PC1 expression can promote normal and neoplastic mammary development and growth and suggests that proprotein convertases may be important etiological factors in human breast neoplasia.

Effects of bitter melon (Momordica charantia l.) or ginger rhizome (Zingiber offifinale rosc) on spontaneous mammary tumorigenesis in SHN mice.

The effects of chronic treatment with hot water extract of Bitter Melon (Momordica charantia L.) or Ginger Rhizome (Zingiber offifinale Rosc) on spontaneous mammary tumorigenesis were examined in SHN virgin mice. In mice given free access to extract of Bitter Melon (0.5%) or Ginger (0.125%) in drinking water, the development of mammary tumors was significantly inhibited. Furthermore, the former inhibited uterine adenomyosis with a common pathological background to mammary tumors and the latterinhibited mammary tumor growth. While the mechanism of the effects of these natural products remains to be clarified, there were no adverse effects of chronic treatment with these agents as estimated from body weight, food and water intake and various plasma component levels as well as external appearance. Thus, these natural products, popular in Japan as foodstuffs, also appear to have a health benefit.

Effects of hydroxyapatite in combination with far-infrared rays on spontaneous mammary tumorigenesis in SHN mice.

We have found that the administration of a diet containing 5% hydroxyapatite (HAP) derived from pig and cattle bones, and exposure to far-infrared rays (FIR) markedly inhibited spontaneous mammary tumorigenesis in SHN mice. Thus, the effect of combined treatment with HAP and FIR on mammary tumorigenesis was examined. The significant inhibition of tumor development by individual treatment with HAP or FIR was not enhanced by combined treatment; instead, the decrease in the inhibitory effect of HAP with age was ameliorated. Associated with this, life span was elongated and a decline in ovarian function was prevented by HAP plus FIR. Normal and preneoplastic growth of mammary glands and plasma component levels were not significantly affected by any treatment. The findings indicate that HAP and FIR have characteristics common to most natural products; in combination with other agents, they have little additive effect, when each is highly active.

Validity of mouse mammary tumour models for human breast cancer: Comparative pathology

In March 1999, a panel of distinguished pathologists was convened by the U.S. National Institutes of Health Breast Cancer Think Tank to develop a classification of breast lesions based on their examination of 39 models of Genetically Engineered Mice (GEM) associated mouse mammary cancer (Cardiff et al., 2000). The meeting, in Annapolis, Maryland, resulted in a published summary report from the Pathology Panel (Cardiff et al., 2000). The Annapolis consensus report, developed from the Panel's deliberations, pointed out that the mammary lesions of GEM were different from most (spontaneous) mouse mammary tumors and could be divided into three distinct categories: (1) lesions that resemble those found in spontaneous mouse mammary tumorigenesis, (2) lesions that have a unique “signature” tumor phenotype that was specific for the transgene, and (3) lesions that resemble those found in human breast diseases (Cardiff et al., 2000). This review emphasizes the proposed nomenclature and the differences between the models and human breast cancer with the intention of stimulating discussion and the development of new models. Microsc. Res. Tech. 52:224–230, 2001. © 2001 Wiley-Liss, Inc.

Hypomethylation of an exon I estrogen receptor CpG island in spontaneous and carcinogen-induced mammary tumorigenesis in the rat

Loss of methylation at a CpG island in exon I of the rat ER gene was observed in 48% of the spontaneous mammary tumors in old female Wistar rats and 22% of the contralateral normal mammary tissues. The majority of the methylation losses were total. Similarly, 50% of 7,12-dimethylbenz[ a]anthracene (DMBA)-induced mammary tumors in young Sprague–Dawley rats exhibited a partial or total loss of methylation at this site, while all normal mammary tissues in young rats were fully methylated. Loss of ER methylation also increased with age in normal mammary tissues of tumor-free rats approaching 12.5% in middle-aged and 43% in old rats. In addition, 66% of mammary glands obtained from young rats that are subsequently at an increased risk to develop breast cancer due to manipulation of in utero dietary fat intake, exhibited methylation loss while no methylation changes were observed in rats at no increased risk for breast cancer. Therefore, the loss of ER methylation is more extensive in mammary glands of rats at high than low breast cancer risk, in old than young, and in mammary tumors than in normal tissues. The data suggest that hypomethylation of a growth-associated ER gene may be a common event in mammary tumorigenesis in the rat and may be of predictive value as a marker of increased breast cancer risk in aged individuals.