Riluzole Suppresses Growth and Enhances Response to Endocrine Therapy in ER+ Breast Cancer.

Background: Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a significant clinical problem. Riluzole is FDA-approved for the treatment of amyotrophic lateral sclerosis. A benzothiazole-based glutamate release inhibitor with several context-dependent mechanism(s) of action, riluzole has shown antitumor activity in multiple malignancies, including melanoma, glioblastoma, and breast cancer. We previously reported that the acquisition of tamoxifen resistance in a cellular model of invasive lobular breast cancer is accompanied by the upregulation of GRM mRNA expression and growth inhibition by riluzole. Methods: We tested the ability of riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated invasive lobular breast cancer patient-derived xenograft model HCI-013EI. Results: Single-agent riluzole suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing a histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). Riluzole induced apoptosis and ferroptosis and reduced phosphorylation of multiple prosurvival signaling molecules, including Akt/mTOR, CREB, and Fak/Src family kinases. Riluzole, in combination with either fulvestrant or 4-hydroxytamoxifen, additively suppressed ER+ breast cancer cell growth in vitro. Single-agent riluzole significantly inhibited HCI-013EI patient-derived xenograft growth in vivo, and the combination of riluzole plus fulvestrant significantly reduced proliferation in ex vivo primary breast tumor explant cultures. Conclusion: Riluzole may offer therapeutic benefits in diverse ER+ breast cancers, including lobular breast cancer.

Environmentally Induced Sperm RNAs Transmit Cancer Susceptibility to Offspring in a Mouse Model.

DNA sequence accounts for the majority of disease heritability, including cancer. Yet, not all familial cancer cases can be explained by genetic factors. It is becoming clear that environmentally induced epigenetic inheritance occurs and that the progeny's traits can be shaped by parental environmental experiences. In humans, epidemiological studies have implicated environmental toxicants, such as the pesticide DDT, in intergenerational cancer development, including breast and childhood tumors. Here, we show that the female progeny of males exposed to DDT in the pre-conception period have higher susceptibility to developing aggressive tumors in mouse models of breast cancer. Sperm of DDT-exposed males exhibited distinct patterns of small non-coding RNAs, with an increase in miRNAs and a specific surge in miRNA-10b levels. Remarkably, embryonic injection of the entire sperm RNA load of DDT-exposed males, or synthetic miRNA-10b, recapitulated the tumor phenotypes observed in DDT offspring. Mechanistically, miR-10b injection altered the transcriptional profile in early embryos with enrichment of genes associated with cell differentiation, tissue and immune system development. In adult DDT-derived progeny, transcriptional and protein analysis of mammary tumors revealed alterations in stromal and in immune system compartments. Our findings reveal a causal role for sperm RNAs in environmentally induced inheritance of cancer predisposition and, if confirmed in humans, this could help partially explain some of the "missing heritability" of breast, and other, malignancies.

Systemic alterations play a dominant role in epigenetic predisposition to breast cancer in offspring of obese fathers and is transmitted to a second generation.

We previously showed that environmentally-induced epigenetic inheritance of cancer occurs in rodent models. For instance, we reported that paternal consumption of an obesity-inducing diet (OID) increased breast cancer susceptibility in the offspring (F1). Nevertheless, it is still unclear whether programming of breast cancer in daughters is due to systemic alterations or mammary epithelium-specific factors and whether the breast cancer predisposition in F1 progeny can be transmitted to subsequent generations. In this study, we show that mammary glands from F1 control (CO) female offspring exhibit enhanced growth when transplanted into OID females compared to CO mammary glands transplanted into CO females. Similarly, carcinogen-induced mammary tumors from F1 CO female offspring transplanted into OID females has a higher proliferation/apoptosis rate. Further, we show that granddaughters (F2) from the OID grand-paternal germline have accelerated tumor growth compared to CO granddaughters. This between-generation transmission of cancer predisposition is associated with changes in sperm tRNA fragments in OID males. Our findings indicate that systemic and mammary stromal alterations are significant contributors to programming of mammary development and likely cancer predisposition in OID daughters. Our data also show that breast cancer predisposition is transmitted to subsequent generations and may explain some familial cancers, if confirmed in humans.

Theaphenon E prevents fatty liver disease and increases CD4+ T cell survival in mice fed a high-fat diet.

BACKGROUND & AIMS: Obesity is a major cause of non-alcoholic fatty liver disease (NAFLD). NAFLD is an epidemic affecting nearly 34% of the adult population in the US. As a chronic inflammatory disease, NAFLD influences the immune system by dysregulating T-cell activity. Remedies for the adverse effects on the immune system are urgently needed. We studied Theaphenon E (TE), a standardized formulation of green tea extract, on the adverse effects of NAFLD in C57BL/6J mice fed a high fat diet (HFD). METHODS: Mice received HFD, low fat diet (LFD) or HFD+2% TE for 35 weeks. Hepatic lipid accumulation, cell proliferation, apoptosis and CD4+T lymphocytes were measured throughout the bioassay. The hepatic composition of fatty acids was determined. The effects of epigallocatechin gallate (EGCG) metabolites on lipid accumulation in mouse and primary human liver cells were studied. RESULTS: Unlike mice receiving HFD, mice on HFD+2% TE maintained normal liver to body weight ratios with low levels of alanine and aspartate aminotransferase (ALT and AST). Hepatic lipid accumulation was observed in HFD mice, accompanied by increased proliferation, reduced apoptosis and loss of CD4+ T lymphocytes. TE significantly inhibited lipid accumulation, decreased proliferation, induced apoptosis and increased CD4+ T cell survival in HFD mice. It was found that the EGCG metabolite EGC-M3 reduced lipid accumulation in mouse and human hepatocytes. Linoleic acid showed the largest increase (2.5-fold) in livers of mice on a HFD and this increase was significantly suppressed by TE. CONCLUSIONS: Livers of HFD-fed mice showed lipid accumulation, increased proliferation, reduced apoptosis, elevated linoleic acid and loss of CD4+ T cells. TE effectively ameliorated all of these adverse effects.

Parental obesity programs pancreatic cancer development in offspring.

Epidemiological studies suggest that timing of obesity onset - and underlying metabolic dysfunction - is important in determining pancreatic cancer rates: early and young adult abdominal overweight/obesity is more strongly associated with this cancer than obesity that develops later in life. Parental obesity and overweight are associated with metabolic dysfunction and obesity in their children. Here, we evaluated the impact of parental overweight on offspring's susceptibility of pancreatic cancer using the P48Cre/+/KrasG12D/+ mouse model. Male mice were fed an obesity-inducing diet (OID) before conception and mated with females raised on a control diet (CO) to generate the offspring. In a separate experiment, pregnant dams were fed CO or OID throughout gestation. The resulting OID offspring from the maternal (OID-m) or paternal lineage (OID-p) were used to study body weight, metabolic parameters and pancreatic cancer development and for molecular analysis. Parental obesity increased offspring's body weight at birth, weaning and in adulthood compared to CO, with gender- and genotype-specific differences. OID-p and OID-m offspring showed metabolic disorder and accelerated development of high-grade PanIN/PDAC. OID offspring also had higher rates of acinar-to-ductal reprogramming assessed by CPA1+/SOX9+-positive pancreatic cells. Levels of Tenascin C (TNC), an ECM glycoprotein shown to suppress apoptosis, were elevated in OID offspring, particularly females. In line with that, OID offspring displayed increased collagen content and decreased apoptosis in pancreatic lesions compared to CO. An ancestral history of obesity through either the paternal or maternal lineages increases offspring's susceptibility to pancreatic cancer development.

Paternal malnutrition programs breast cancer risk and tumor metabolism in offspring.

BACKGROUND: While many studies have shown that maternal factors in pregnancy affect the cancer risk for offspring, few studies have investigated the impact of paternal exposures on their progeny's risk of this disease. Population studies generally show a U-shaped association between birthweight and breast cancer risk, with both high and low birthweight increasing the risk compared with average birthweight. Here, we investigated whether paternal malnutrition would modulate the birthweight and later breast cancer risk of daughters. METHODS: Male mice were fed AIN93G-based diets containing either 17.7% (control) or 8.9% (low-protein (LP)) energy from protein from 3 to 10 weeks of age. Males on either group were mated to females raised on a control diet. Female offspring from control and LP fathers were treated with 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary carcinogenesis. Mature sperm from fathers and mammary tissue and tumors from female offspring were used for epigenetic and other molecular analyses. RESULTS: We found that paternal malnutrition reduces the birthweight of daughters and leads to epigenetic and metabolic reprogramming of their mammary tissue and tumors. Daughters of LP fathers have higher rates of mammary cancer, with tumors arising earlier and growing faster than in controls. The energy sensor, the AMP-activated protein kinase (AMPK) pathway, is suppressed in both mammary glands and tumors of LP daughters, with consequent activation of mammalian target of rapamycin (mTOR) signaling. Furthermore, LP mammary tumors show altered amino-acid metabolism with increased glutamine utilization. These changes are linked to alterations in noncoding RNAs regulating those pathways in mammary glands and tumors. Importantly, we detect alterations in some of the same microRNAs/target genes found in our animal model in breast tumors of women from populations where low birthweight is prevalent. CONCLUSIONS: Our study suggests that ancestral paternal malnutrition plays a role in programming offspring cancer risk and phenotype by likely providing a metabolic advantage to cancer cells.

Prevention of Lipid Peroxidation-derived Cyclic DNA Adduct and Mutation in High-Fat Diet-induced Hepatocarcinogenesis by Theaphenon E.

Obesity is associated with cancer risk and its link with liver cancer is particularly strong. Obesity causes non-alcoholic fatty liver disease (NAFLD) that could progress to hepatocellular carcinoma (HCC). Chronic inflammation likely plays a key role. We carried out a bioassay in the high-fat diet (HFD)-fed C57BL/6J mice to provide insight into the mechanisms of obesity-related HCC by studying γ-OHPdG, a mutagenic DNA adduct derived from lipid peroxidation. In an 80-week bioassay, mice received a low-fat diet (LFD), high-fat diet (HFD), and HFD with 2% Theaphenon E (TE) (HFD+TE). HFD mice developed a 42% incidence of HCC and LFD mice a 16%. Remarkably, TE, a standardized green tea extract formulation, completely blocked HCC in HFD mice with a 0% incidence. γ-OHPdG measured in the hepatic DNA of mice fed HFD and HFD+TE showed its levels increased during the early stages of NAFLD in HFD mice and the increases were significantly suppressed by TE, correlating with the tumor data. Whole-exome sequencing showed an increased mutation load in the liver tumors of HFD mice with G>A and G>T as the predominant mutations, consistent with the report that γ-OHPdG induces G>A and G>T. Furthermore, the mutation loads were significantly reduced in HFD+TE mice, particularly G>T, the most common mutation in human HCC. These results demonstrate in a relevant model of obesity-induced HCC that γ-OHPdG formation during fatty liver disease may be an initiating event for accumulated mutations that leads to HCC and this process can be effectively inhibited by TE. Cancer Prev Res; 11(10); 665-76. ©2018 AACR.

Maternal intake of high n-6 polyunsaturated fatty acid diet during pregnancy causes transgenerational increase in mammary cancer risk in mice.

BACKGROUND: Maternal and paternal high-fat (HF) diet intake before and/or during pregnancy increases mammary cancer risk in several preclinical models. We studied if maternal consumption of a HF diet that began at a time when the fetal primordial germ cells travel to the genital ridge and start differentiating into germ cells would result in a transgenerational inheritance of increased mammary cancer risk. METHODS: Pregnant C57BL/6NTac mouse dams were fed either a control AIN93G or isocaloric HF diet composed of corn oil high in n-6 polyunsaturated fatty acids between gestational days 10 and 20. Offspring in subsequent F1-F3 generations were fed only the control diet. RESULTS: Mammary tumor incidence induced by 7,12-dimethylbenz[a]anthracene was significantly higher in F1 (p < 0.016) and F3 generation offspring of HF diet-fed dams (p < 0.040) than in the control offspring. Further, tumor latency was significantly shorter (p < 0.028) and burden higher (p < 0.027) in F1 generation HF offspring, and similar trends were seen in F3 generation HF offspring. RNA sequencing was done on normal mammary glands to identify signaling differences that may predispose to increased breast cancer risk by maternal HF intake. Analysis revealed 1587 and 4423 differentially expressed genes between HF and control offspring in F1 and F3 generations, respectively, of which 48 genes were similarly altered in both generations. Quantitative real-time polymerase chain reaction analysis validated 13 chosen up- and downregulated genes in F3 HF offspring, but only downregulated genes in F1 HF offspring. Ingenuity Pathway Analysis identified upregulation of Notch signaling as a key alteration in HF offspring. Further, knowledge-fused differential dependency network analysis identified ten node genes that in the HF offspring were uniquely connected to genes linked to increased cancer risk (ANKEF1, IGFBP6, SEMA5B), increased resistance to cancer treatments (SLC26A3), poor prognosis (ID4, JAM3, TBX2), and impaired anticancer immunity (EGR3, ZBP1). CONCLUSIONS: We conclude that maternal HF diet intake during pregnancy induces a transgenerational increase in offspring mammary cancer risk in mice. The mechanisms of inheritance in the F3 generation may be different from the F1 generation because significantly more changes were seen in the transcriptome.

Effects of In Utero Exposure to Ethinyl Estradiol on Tamoxifen Resistance and Breast Cancer Recurrence in a Preclinical Model.

Background: Responses to endocrine therapies vary among patients with estrogen receptor (ER+) breast cancer. We studied whether in utero exposure to endocrine-disrupting compounds might explain these variations. Methods: We describe a novel ER+ breast cancer model to study de novo and acquired tamoxifen (TAM) resistance. Pregnant Sprague Dawley rats were exposed to 0 or 0.1 ppm ethinyl estradiol (EE2), and the response of 9,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors to 15 mg/kg TAM, with (n = 17 tumors in the controls and n = 20 tumors in EE2 offspring) or without 1.2 g/kg valproic acid and 5 mg/kg hydralazine (n = 24 tumors in the controls and n = 32 tumors in EE2 offspring) in the female offspring, was assessed. One-sided Chi2 tests were used to calculate P values. Comparisons of differentially expressed genes between mammary tumors in in utero EE2-exposed and control rats, and between anti-estrogen-resistant LCC9 and -sensitive LCC1 human breast cancer cells, were also performed. Results: In our preclinical model, 54.2% of mammary tumors in the control rats exhibited a complete response to TAM, of which 23.1% acquired resistance with continued anti-estrogen treatment and recurred. Mammary tumors in the EE2 offspring were statistically significantly less likely to respond to TAM (P = .047) and recur (P = .007). In the EE2 offspring, but not in controls, adding valproic acid and hydralazine to TAM prevented recurrence (P < .001). Three downregulated and hypermethylated genes (KLF4, LGALS3, MICB) and one upregulated gene (ETV4) were identified in EE2 tumors and LCC9 breast cancer cells, and valproic acid and hydralazine normalized the altered expression of all four genes. Conclusions: Resistance to TAM may be preprogrammed by in utero exposure to high estrogen levels and mediated through reversible epigenetic alterations in genes associated with epithelial-mesenchymal transition and tumor immune responses.

Endoplasmic Reticulum Stress Protein GRP78 Modulates Lipid Metabolism to Control Drug Sensitivity and Antitumor Immunity in Breast Cancer.

The unfolded protein response is an endoplasmic reticulum stress pathway mediated by the protein chaperone glucose regulated-protein 78 (GRP78). Metabolic analysis of breast cancer cells shows that GRP78 silencing increases the intracellular concentrations of essential polyunsaturated fats, including linoleic acid. Accumulation of fatty acids is due to an inhibition of mitochondrial fatty acid transport, resulting in a reduction of fatty acid oxidation. These data suggest a novel role of GRP78-mediating cellular metabolism. We validated the effect of GRP78-regulated metabolite changes by treating tumor-bearing mice with tamoxifen and/or linoleic acid. Tumors treated with linoleic acid plus tamoxifen exhibited reduced tumor area and tumor weight. Inhibition of either GRP78 or linoleic acid treatment increased MCP-1 serum levels, decreased CD47 expression, and increased macrophage infiltration, suggesting a novel role for GRP78 in regulating innate immunity. GRP78 control of fatty acid oxidation may represent a new homeostatic function for GRP78. Cancer Res; 76(19); 5657-70. ©2016 AACR.

Paternal overweight is associated with increased breast cancer risk in daughters in a mouse model.

While many studies have shown that maternal weight and nutrition in pregnancy affects offspring's breast cancer risk, no studies have investigated the impact of paternal body weight on daughters' risk of this disease. Here, we show that diet-induced paternal overweight around the time of conception can epigenetically reprogram father's germ-line and modulate their daughters' birth weight and likelihood of developing breast cancer, using a mouse model. Increased body weight was associated with changes in the miRNA expression profile in paternal sperm. Daughters of overweight fathers had higher rates of carcinogen-induced mammary tumors which were associated with delayed mammary gland development and alterations in mammary miRNA expression. The hypoxia signaling pathway, targeted by miRNAs down-regulated in daughters of overweight fathers, was activated in their mammary tissues and tumors. This study provides evidence that paternal peri-conceptional body weight may affect daughters' mammary development and breast cancer risk and warrants further studies in other animal models and humans.

Hydroxychloroquine inhibits autophagy to potentiate antiestrogen responsiveness in ER+ breast cancer.

PURPOSE: Estrogen receptor-α (ERα)-targeted therapies including tamoxifen (TAM) or Faslodex (ICI) are used to treat ER(+) breast cancers. Up to 50% of tumors will acquire resistance to these interventions. Autophagy has been implicated as a major driver of antiestrogen resistance. We have explored the ability of hydroxychloroquine (HCQ), which inhibits autophagy, to affect antiestrogen responsiveness. EXPERIMENTAL DESIGN: TAM-resistant MCF7-RR and ICI-resistant/TAM cross-resistant LCC9 ER(+) breast cancer cells were injected into mammary fat pads of female athymic mice and treated with TAM and/or ICI in combination with oral low-dose HCQ. RESULTS: We show that HCQ can increase antiestrogen responsiveness in MCF7-RR and LCC9 cells and tumors, likely through the inhibition of autophagy. However, the combination of ICI+HCQ was less effective than HCQ alone in vivo, unlike the TAM+HCQ combination. Antiestrogen treatment stimulated angiogenesis in tumors but did not prevent HCQ effectiveness. The lower efficacy of ICI+HCQ was associated with ICI effects on cell-mediated immunity within the tumor microenvironment. The mouse chemokine KC (CXCL1) and IFNγ were differentially regulated by both TAM and ICI treatments, suggesting a possible effect on macrophage development/activity. Consistent with these observations, TAM+HCQ treatment increased tumor CD68(+) cells infiltration, whereas ICI and ICI+HCQ reduced peripheral tumor macrophage content. Moreover, macrophage elimination of breast cancer target cells in vitro was reduced following exposure to ICI. CONCLUSION: HCQ restores antiestrogen sensitivity to resistant tumors. Moreover, the beneficial combination of TAM+HCQ suggests a positive outcome for ongoing neoadjuvant clinical trials using this combination for the treatment of ER(+) ductal carcinoma in situ lesions.

High-fat or ethinyl-oestradiol intake during pregnancy increases mammary cancer risk in several generations of offspring.

Maternal exposures to environmental factors during pregnancy influence the risk of many chronic adult-onset diseases in the offspring. Here we investigate whether feeding pregnant rats a high-fat (HF)- or ethinyl-oestradiol (EE2)-supplemented diet affects carcinogen-induced mammary cancer risk in daughters, granddaughters and great-granddaughters. We show that mammary tumourigenesis is higher in daughters and granddaughters of HF rat dams and in daughters and great-granddaughters of EE2 rat dams. Outcross experiments suggest that the increase in mammary cancer risk is transmitted to HF granddaughters equally through the female or male germ lines, but it is only transmitted to EE2 granddaughters through the female germ line. The effects of maternal EE2 exposure on offspring's mammary cancer risk are associated with changes in the DNA methylation machinery and methylation patterns in mammary tissue of all three EE2 generations. We conclude that dietary and oestrogenic exposures in pregnancy increase breast cancer risk in multiple generations of offspring, possibly through epigenetic means.

Changes in mammary gland morphology and breast cancer risk in rats.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription. In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.