Splenic Elemental Composition of Breast Cancer-Suffering Rats Supplemented with Pomegranate Seed Oil and Bitter Melon Extract.

The aim of this study was to investigate how dietary modifications with pomegranate seed oil (PSO) and bitter melon aqueous extract (BME) affect mineral content in the spleen of rats both under normal physiological conditions and with coexisting mammary tumorigenesis. The diet of Sprague-Dawley female rats was supplemented either with PSO or with BME, or with a combination for 21 weeks. A chemical carcinogen (7,12-dimethylbenz[a]anthracene) was applied intragastrically to induce mammary tumors. In the spleen of rats, the selected elements were determined with a quadrupole mass spectrometer with inductively coupled plasma ionization (ICP-MS). ANOVA was used to evaluate differences in elemental composition among experimental groups. Multivariate statistical methods were used to discover whether some subtle dependencies exist between experimental factors and thus influence the element content. Experimental factors affected the splenic levels of macroelements, except for potassium. Both diet modification and the cancerogenic process resulted in significant changes in the content of Fe, Se, Co, Cr, Ni, Al, Sr, Pb, Cd, B, and Tl in rat spleen. Chemometric analysis revealed the greatest impact of the ongoing carcinogenic process on the mineral composition of the spleen. The obtained results may contribute to a better understanding of peripheral immune organ functioning, especially during the neoplastic process, and thus may help develop anticancer prevention and treatment strategies.

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

BACKGROUND/AIM: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

USP22 overexpression fails to augment tumor formation in MMTV-ERBB2 mice but loss of function impacts MMTV promoter activity.

The Ubiquitin Specific Peptidase 22 (USP22), a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) histone modifying complex, is overexpressed in multiple human cancers, but how USP22 impacts tumorigenesis is not clear. We reported previously that Usp22 loss in mice impacts execution of several signaling pathways driven by growth factor receptors such as erythroblastic oncogene B b2 (ERBB2). To determine whether changes in USP22 expression affects ERBB2-driven tumorigenesis, we introduced conditional overexpression or deletion alleles of Usp22 into mice bearing the Mouse mammary tumor virus-Neu-Ires-Cre (MMTV-NIC) transgene, which drives both rat ERBB2/NEU expression and Cre recombinase activity from the MMTV promoter resulting in mammary tumor formation. We found that USP22 overexpression in mammary glands did not further enhance primary tumorigenesis in MMTV-NIC female mice, but increased lung metastases were observed. However, deletion of Usp22 significantly decreased tumor burden and increased survival of MMTV-NIC mice. These effects were associated with markedly decreased levels of both Erbb2 mRNA and protein, indicating Usp22 loss impacts MMTV promoter activity. Usp22 loss had no impact on ERBB2 expression in other settings, including MCF10A cells bearing a Cytomegalovirus (CMV)-driven ERBB2 transgene or in human epidermal growth factor receptor 2 (HER2)+ human SKBR3 and HCC1953 cells. Decreased activity of the MMTV promoter in MMTV-NIC mice correlated with decreased expression of known regulatory factors, including the glucocorticoid receptor (GR), the progesterone receptor (PR), and the chromatin remodeling factor Brahma-related gene-1 (BRG1). Together our findings indicate that increased expression of USP22 does not augment the activity of an activated ERBB2/NEU transgene but impacts of Usp22 loss on tumorigenesis cannot be assessed in this model due to unexpected effects on MMTV-driven Erbb2/Neu expression.

Body weight gain and control: beneficial effect of extra virgin olive oil versus corn oil in an experimental model of mammary cancer.

Obesity is a known risk factor for breast cancer, the most common malignancy among women worldwide. We have previously described different effects of high-fat diets on mammary experimental carcinogenesis. In this work, we analyzed the animal growth data obtained in six experimental assays, in healthy and carcinogen-induced rats undergoing different dietary interventions. The animals were fed with three experimental diets administered at different periods of development: a control low-fat diet, and two isocaloric high-fat diets (rich in corn oil or in extravirgin olive oil -EVOO-). Weekly weight throughout the development of 818 animals have been compiled and reanalyzed using adjusted mathematical models. Molecular mechanisms have been investigated: ethanolamides in small intestine, neuropeptides controlling satiety in hypothalamus, and proteins controlling lipid metabolism in adipose and mammary tissues. The results indicated that the effect of diets depended on type of lipid, timing of intervention and health status. The high corn oil diet, but not the high EVOO diet, increased body weight and mass, especially if administered from weaning, in healthy animals and in those that received a moderate dose of carcinogen. The potential protective effect of EVOO on weight maintenance may be related to anorexigenic neuropeptides such as oxytocin and lipolysis/deposition balance in adipose tissue (increasing phospho-PKA, HSL, MGL and decreasing FAS). In animals with cancer, body weight gain was related to the severity of the disease. Taken together, our results suggest that EVOO has a beneficial effect on body weight maintenance in both health and cancer.

Insulin receptor loss impairs mammary tumorigenesis in mice.

Breast cancer (BC) prognosis and outcome are adversely affected by obesity. Hyperinsulinemia, common in the obese state, is associated with higher risk of death and recurrence in BC. Up to 80% of BCs overexpress the insulin receptor (INSR), which correlates with worse prognosis. INSR's role in mammary tumorigenesis was tested by generating MMTV-driven polyoma middle T (PyMT) and ErbB2/Her2 BC mouse models, respectively, with coordinate mammary epithelium-restricted deletion of INSR. In both models, deletion of either one or both copies of INSR leads to a marked delay in tumor onset and burden. Longitudinal phenotypic characterization of mouse tumors and cells reveals that INSR deletion affects tumor initiation, not progression and metastasis. INSR upholds a bioenergetic phenotype in non-transformed mammary epithelial cells, independent of its kinase activity. Similarity of phenotypes elicited by deletion of one or both copies of INSR suggest a dose-dependent threshold for INSR impact on mammary tumorigenesis.

Environmental cadmium exposure facilitates mammary tumorigenesis via reprogramming gut microbiota-mediated glutamine metabolism in MMTV-Erbb2 mice.

Cadmium (Cd) is a heavy metal that has been widely reported to be linked to the onset and progression of breast cancer (BC). However, the mechanism of Cd-induced mammary tumorigenesis remains elusive. In our study, a transgenic mouse model that spontaneously develops tumors through overexpression of wild-type Erbb2 (MMTV-Erbb2) was constructed to investigate the effects of Cd exposure on BC tumorigenesis. The results showed that oral exposure to 3.6 mg/L Cd for 23 weeks dramatically accelerated tumor appearance and growth, increased Ki67 density and enhanced focal necrosis and neovascularization in the tumor tissue of MMTV-Erbb2 mice. Notably, Cd exposure enhanced glutamine (Gln) metabolism in tumor tissue, and 6-diazo-5-oxo-l-norleucine (DON), a Gln metabolism antagonist, inhibited Cd-induced breast carcinogenesis. Then our metagenomic sequencing and mass spectrometry-based metabolomics confirmed that Cd exposure disturbed gut microbiota homeostasis, especially Helicobacter and Campylobacter abundance remodeling, which altered the gut metabolic homeostasis of Gln. Moreover, intratumoral Gln metabolism profoundly increased under Cd-elevated gut permeability. Importantly, depletion of microbiota with an antibiotic cocktail (AbX) treatment led to a significant delay in the appearance of palpable tumors, inhibition of tumor growth, decrease in tumor weight, reduction in Ki67 expression and low-grade pathology in Cd-exposed MMTV-Erbb2 mice. Also, transplantation of Cd-modulated microbiota decreased tumor latency, accelerated tumor growth, increased tumor weight, upregulated Ki67 expression and exacerbated neovascularization as well as focal necrosis in MMTV-Erbb2 mice. In summary, Cd exposure induced gut microbiota dysbiosis, elevated gut permeability and increased intratumoral Gln metabolism, leading to the promotion of mammary tumorigenesis. This study provides novel insights into environmental Cd exposure-mediated carcinogenesis.

The Role of Cancer and Somatic Stem Cells in the Anti-Inflammatory and Antitumor Effects of Aconitum baicalense Extract on Experimental Breast Cancer.

We studied the effects of the extract of the terrestrial part of Aconitum baicalense in BALB/c female mice at the early stages after the injection of N-methyl-N-nitrosourea (MNU). The extract reduced inflammatory activity and tumor growth in the mammary gland. The antitumor and anti-inflammatory effects of the extract are based on the inhibition of cancer stem cells, hematopoietic stem cells, and hematopoietic progenitor cells that promote inflammation. The extract of A. baicalense disrupted the recruitment of epithelial progenitor cells and angiogenesis precursors to the mammary gland preventing neovascularization and transformation of epithelial cells into tumor cells.

Anticancer role of mango (Mangifera indica L.) peel and seed kernel extracts against 7,12- dimethylbenz[a]anthracene-induced mammary carcinogenesis in female rats.

Breast cancer is the second leading cause of cancer death among women. The present study is an effort to reveal the antiproliferative and antioxidant actions of mango seed kernel extract (KE), peel extract (PE), and their combination (KEPE) on mammary tumors induced by 7,12 dimethylbenz[a]anthracene (DMBA). Seven groups of adult female Sprague-Dawley rats were prepared, including C: (control), DMBA: (rats were administered with DMBA), (DMBA-KE), (DMBA-PE), and (DMBA-KEPE): rats were administered with DMBA and then treated with KE, PE, and (both KE and PE), respectively, (KE) and (PE): rats were administered with KE and PE, separately. The study focused on the assessment of markers of endocrine derangement [serum 17-ß estradiol (E2)], apoptosis [caspase-3 and deoxyribonucleic acid fragmentation (DNAF)], and oxidative stress [lipid peroxidation and antioxidants (glutathione, glutathione-S-transferase, glutathione reductase, glutathione peroxidase, and superoxide dismutase)]. Histopathological examination and immunohistochemical expression of caspase-3 and estrogen receptor-α (ER-α) in mammary gland tissues (MGTs) were determined, as well as the characterization of mango extracts. The results showed that DMBA administration induced mammary tumors by increasing cell proliferation and evading apoptosis. In addition, DMBA administration caused oxidative stress by the production of reactive oxygen species, which increased lipid peroxidation and decreased cellular antioxidants, allowing cancer to progress. In contrast, treatment with DMBA-KE, DMBA-PE, or DMBA-KEPE diminished mammary tumors induced by DMBA, where they reduced oxidative stress via increased antioxidant parameters including reduced glutathione, superoxide dismutase, total glutathione peroxidase, glutathione reductase, and glutathione S-transferase. Also, different treatments decreased proliferation through the reduction of E2, and ER-α expression levels. However, these treatments increased the apoptosis of unwanted cells as they increased caspase-3 activity and DNAF. All these changes led to the prevention of breast injuries and the reduction of mammary tumors. This demonstrates that the contents of mango extracts, especially phenolics and flavonoids, have an important role in mammary tumor treatment through their potential antioxidant, antiproliferative, proapoptotic, and anti-estrogenic effects. KE and PE administration for 4 weeks had no adverse effects. Conclusion: Each of KE, PE, and KEPE has a therapeutic effect against DMBA-induced mammary tumors via induction of apoptosis and reduction of each of the OS, proliferation, and estrogenic effects. So, they can play an important role in the pharmacological tole.

Protective Effects of Pelargonidin against DMBA-Induced Mammary Tumorigenesis in BALB/c Mice through Reduced Oxidative Stress and Lipid Anomalies.

Among luminal types of breast cancers, ER + breast cancer is the most frequently diagnosed cancer globally. ER + breast cancer is commonly treated with SERM drugs that block ER to prevent ER-mediated cancerous growth. Our previous computational screening found pelargonidin (PG) can inhibit ER-signaling with potent bioactivity and satisfactory toxicological features. The present study explored the anti-tumoral prospect of PG against DMBA-induced ER + murine mammary carcinogenesis. The female BALB/c mice were divided into control (A) and DMBA-exposed groups. Following tumor appearance, the DMBA-exposed group was divided into five groups: tumor control, PG-treated (Groups P25, P50, and P100), and tamoxifen-treated (TAM). The results indicated that PG-treatment dose-dependently reduced the mean tumor volume, reinstated body weight loss, and enhanced the percentage survival of tumor-bearing mice. In addition, we recorded a significant reduction in LPO, total cholesterol, and triglycerides and a surge in the activity of antioxidases and phase II detoxifying enzymes in PG-treated animals. PG also dose-dependently increased the serum level of unbound estradiol, an indicator of competitive ER binding by an ER agonist/antagonist. These data suggest that pelargonidin has potent anticancer potential against the animal model of ER + breast cancer that matches the efficiency of tamoxifen with conceivably fewer side effects.

Global Down-regulation of Gene Expression Induced by Mouse Mammary Tumor Virus (MMTV) in Normal Mammary Epithelial Cells.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes breast cancer in mice. The mouse mammary epithelial cells are the most permissive cells for MMTV, expressing the highest levels of virus upon infection and being the ones later transformed by the virus due to repeated rounds of infection/superinfection and integration, leading eventually to mammary tumors. The aim of this study was to identify genes and molecular pathways dysregulated by MMTV expression in mammary epithelial cells. Towards this end, mRNAseq was performed on normal mouse mammary epithelial cells stably expressing MMTV, and expression of host genes was analyzed compared with cells in its absence. The identified differentially expressed genes (DEGs) were grouped on the basis of gene ontology and relevant molecular pathways. Bioinformatics analysis identified 12 hub genes, of which 4 were up-regulated (Angp2, Ccl2, Icam, and Myc) and 8 were down-regulated (Acta2, Cd34, Col1a1, Col1a2, Cxcl12, Eln, Igf1, and Itgam) upon MMTV expression. Further screening of these DEGs showed their involvement in many diseases, especially in breast cancer progression when compared with available data. Gene Set Enrichment Analysis (GSEA) identified 31 molecular pathways dysregulated upon MMTV expression, amongst which the PI3-AKT-mTOR was observed to be the central pathway down-regulated by MMTV. Many of the DEGs and 6 of the 12 hub genes identified in this study showed expression profile similar to that observed in the PyMT mouse model of breast cancer, especially during tumor progression. Interestingly, a global down-regulation of gene expression was observed, where nearly 74% of the DEGs in HC11 cells were repressed by MMTV expression, an observation similar to what was observed in the PyMT mouse model during tumor progression, from hyperplasia to adenoma to early and late carcinomas. Comparison of our results with the Wnt1 mouse model revealed further insights into how MMTV expression could lead to activation of the Wnt1 pathway independent of insertional mutagenesis. Thus, the key pathways, DEGs, and hub genes identified in this study can provide important clues to elucidate the molecular mechanisms involved in MMTV replication, escape from cellular anti-viral response, and potential to cause cell transformation. These data also validate the use of the MMTV-infected HC11 cells as an important model to study early transcriptional changes that could lead to mammary cell transformation.

PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

PD-1 monoclonal antibodies enhance the cryoablation-induced antitumor immune response: a breast cancer murine model research.

BACKGROUND: It has been demonstrated that cryoablation (Cryo) causes specific T-cell immune responses in the body; however, it is not sufficient to prevent tumor recurrence and metastasis. In this report, we evaluated changes in the tumor immune microenvironment (TIME) in distant tumor tissues after Cryo and investigated the immunosuppressive mechanisms that limit the efficacy of Cryo. METHODS: Bilateral mammary tumor models were established in mice, and we first observed the dynamic changes in immune cells and cytokines at different time points after Cryo. Then, we confirmed that the upregulation of PD-1 and PD-L1 signaling in the contralateral tumor tissue was closely related to the immunosuppressive state in the TIME at the later stage after Cryo. Finally, we also evaluated the synergistic antitumor effects of Cryo combined with PD-1 monoclonal antibody (mAb) in the treatment of breast cancer (BC) mouse. RESULTS: We found that Cryo can stimulate the body's immune response, but it also induces immunosuppression. The elevated PD-1/PD-L1 expression in distant tumor tissues at the later stage after Cryo was closely related to the immunosuppressive state in the TIME but also created the conditions for Cryo combined with PD-1 mAb for BC mouse treatment. Cryo + PD-1 mAb could improve the immunosuppressive state of tumors and enhance the Cryo-induced immune response, thus exerting a synergistic antitumor effect. CONCLUSIONS: The PD-1/PD-L1 axis plays an important role in suppressing Cryo-induced antitumor immune responses. This study provides a theoretical basis for Cryo combined with PD-1 mAb therapy in clinical BC patients.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection.

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

Rank ectopic expression in the presence of Neu and MMTV oncogenes alters mammary epithelial cell populations and their tumorigenic potential.

Determination of the mammary epithelial cell that serves as the cell of origin for breast cancer is key to understand tumor heterogeneity and clinical management. In this study, we aimed to decipher whether Rank expression in the presence of PyMT and Neu oncogenes might affect the cell of origin of mammary gland tumors. We observed that Rank expression in PyMT+/- and Neu+/- mammary glands alters the basal and luminal mammary cell populations already in preneoplasic tissue, which may interfere with the tumor cell of origin restricting their tumorigenesis ability upon transplantation assays. In spite of this, Rank expression eventually promotes tumor aggressiveness once tumorigenesis is established.

Herbal Honey Preparations of Curcuma Xanthorriza and Black Cumin Protect against Carcinogenesis through Antioxidant and Immunomodulatory Activities in Sprague Dawley (SD) Rats Induced with Dimethylbenz(a)anthracene.

BACKGROUND: Traditionally, Curcuma xanthorriza (CX), black cumin seed (BC), and honey have been used by the Indonesian people as medicinal ingredients to treat various health symptoms. CX extracts and BC have been proven in the laboratory as chemopreventive agents, antioxidants, and immunomodulators. In this study, we developed CX extract, BC oil, and honey into herbal honey preparations (CXBCH) and hypothesized that the preparations show chemopreventive activity. The purpose of the study was to determine the CXBCH potential as chemopreventive, antioxidant, and immunomodulatory. METHOD: In this experimental laboratory research, antioxidant, immunomodulatory, and cytotoxic activities were tested on human mammary cancer cell lines (T47D cells) while the chemopreventive activity of the CXBCH preparations on Sprague Dawley (SD) rats induced with dimethylbenzene(a)anthracene (DMBA). RESULTS: CXBCH preparations demonstrated immunomodulatory, antioxidant, and cytotoxic activities in T47D, Hela, and HTB-183 cells and in DMBA-induced SD rats, as the preparations inhibited tumor nodule formation, increased the number of CD4, CD8 and CD4CD25 cells, and glutathione-S-transferase (GST) activity, and decreased serum NO levels. CONCLUSIONS: CXBCH preparations display chemopreventive, antioxidant, and immunomodulatory properties.

Feeding a High-Fat Diet for a Limited Duration Increases Cancer Incidence in a Breast Cancer Model.

High-fat intake by young Asian women impacts the risk of breast cancer. Understanding the underlying molecular mechanisms may be essential for disease prevention in Asia as well as globally. We aimed to examine the effects of corn oil- and animal fat-based high-fat diets (32.9 and 31.4%, respectively, of fat energy ratio as compared to 12.3% in the standard diet) on mammary carcinogenesis and alterations in gene expression and epigenetic statuses in the mammary gland during the growth stages in a rat model. An increased incidence of carcinomas was observed after the cessation of high-fat feeding. In addition, rapid tumor growth and elevations in Celsr2 expression, which may be a result of DNA hypomethylation patterns in the 3' untranslated region of the gene were noted in the animal fat group. In the human breast carcinoma cell line MCF7, a marginal decrease in cell viability was observed following the knockdown of Celsr2, suggesting that the animal fat-associated risk of cancer is partly due to the deregulation of mammary cell proliferation via non-metabolic gene functions. The present results will contribute to the development of strategies for controlling the food-associated risk of breast cancer, particularly in younger age groups.

Adolescent- and adult-initiated alcohol exposure in mice differentially promotes tumorigenesis and metastasis of breast cancer.

BACKGROUND: Alcohol exposure increases the risk of breast cancer. Alcohol consumption by adolescents is a serious social and public health issue. This study investigated the impact of adolescent alcohol consumption on mammary tumorigenesis and progression and compared it to that of adult alcohol exposure in animal models. METHODS: Female adolescent (5 weeks) and adult (8 weeks) MMTV-Wnt1 mice were exposed to alcohol either chronically or acutely. For chronic alcohol exposure, animals were fed a liquid diet containing 6.7% ethanol for 23 weeks. For acute exposure, animals were treated with ethanol (2.5 g/kg, 25% w/v) via intraperitoneal (IP) injection for 15 days. RESULTS: In control animals, the tumor latency was 18.5 to 22 weeks. Both chronic and acute alcohol exposure in adolescent mice significantly shortened the tumor latency to 9.5 and 8.4 weeks, respectively. However, adult-initiated alcohol exposure had little effect on the tumor latency. Both adolescent- and adult-initiated alcohol exposure significantly increased lung metastasis. Adolescent-initiated alcohol exposure but not adult-initiated alcohol exposure increased the breast cancer stem cell population. Adolescent-initiated alcohol exposure significantly altered the proliferation of mammary epithelial cells, ductal growth, and the formation of terminal end buds in the mammary glands. Adolescent-initiated alcohol exposure but not adult-initiated alcohol exposure increased estradiol levels in the blood. Acute adolescent alcohol exposure also significantly increased blood progesterone levels. Furthermore, adolescent-initiated alcohol exposure activated PAK1 and p38γ MAPK, critical regulators of mammary tumorigenesis and aggressiveness, respectively, while adult-initiated alcohol exposure activated only p38γ MAPK. In addition, both adolescent and adult alcohol exposure significantly decreased the levels of a prognostic marker miR200b. CONCLUSIONS: Adolescent-initiated alcohol exposure enhanced both tumorigenesis and aggressiveness of mammary tumors, while adult-initiated alcohol exposure mainly promoted tumor metastasis. Thus, adolescent mice were more sensitive than adult mice in response to alcohol-induced tumor promotion.

Mammary Tumor Growth and Proliferation Are Dependent on Growth Hormone in Female SV40 C3(1) T-Antigen Mice.

Female SV40 C3(1) T-antigen (C3(1)/TAg) transgenic mice develop mammary tumors that are molecularly similar to human basal-like breast cancers with 100% incidence at 16 weeks of age. To determine the requirement for growth hormone (GH) signaling in these tumors, genetic crosses were used to create cohorts of female mice that were homozygous for a floxed growth hormone receptor (Ghr) gene and carried one copy each of the Rosa-Cre-ERT2 transgene and the C3(1)/TAg transgene (Ghrflox/flox; Rosa-Cre-ERT2; C3(1)/TAg+/0 mice). When the largest mammary tumor reached 200 mm3, mice were treated with tamoxifen to delete Ghr or with vehicle as a control. An additional group of Ghrflox/flox; C3(1)/TAg+/0 mice were also treated with tamoxifen when the largest mammary tumor reached 200 mm3 as a control for the effects of tamoxifen. After 3 weeks, tumors in mice in which Ghr was deleted began to shrink while vehicle and tamoxifen treatment control mouse tumors continued to grow. Pathological analysis of tumors revealed similar growth patterns and varying levels of necrosis throughout all groups. A decrease in cancer cell proliferation in Ghr-/- tumors relative to controls was observed as measured by Ki67 immunohistochemistry labeling index. These data suggest that even established C3(1)/TAg mammary tumors are dependent on the GH/IGF-1 axis.