Mammary Tumorigenesis Research Articles

Abstract 2561: Tumorigenic and metabolomic impacts of adipose-derived monocyte chemotactic protein-1 in a model of obesity-enhanced, male breast cancer

Abstract Breast cancer is a rare but aggressive disease in men. Approximately, 90% of all breast cancer in men are invasive carcinomas, 25% of which have distant metastasis at the time of clinical presentation. Obesity is a risk factor for breast cancer. Monocyte chemotactic protein-1 (MCP-1) is an adipose-derived cytokine whose concentrations are elevated by obesity. This study tested the hypothesis that adipose-derived MCP-1 contributes to male breast cancer. In a 2x2 design, male MMTV-PyMT mice with or without adipose specific Mcp-1 knockdown [designated as Mcp-1-/- or wild-type (WT)] were fed the standard AIN93G diet or an obesogenic, high-fat diet (HFD) for 25 weeks. The AIN93G diet and HFD contained 16% and 45% of energy from soybean oil, respectively. Mcp-1-/- mice had lower adipose Mcp-1 expression than WT mice. Adipose Mcp-1 deficiency reduced plasma concentrations of MCP-1 in mice fed the HFD. Mice fed the HFD had a greater tumor progression rate than mice fed the AIN93G diet (4679% vs 2430%), regardless of genotype. Mcp-1-/- mice, compared to WT mice, had a longer tumor latency (25.2 weeks vs 18.0 weeks) and lower tumor incidence (19% vs 56%), tumor progression rate (2317% vs 4792%), and tumor weights (0.23 g vs 0.64 g). Metabolomic analysis of plasma identified 87 metabolites, 56 of which differed among the four groups, including 24 that differed between the diets and 22 that differed between the genotypes. Sparse partial least squares-discriminant analysis identified 10 major metabolites that were altered the most among the four groups. These included amino acids (alanine, isoleucine, leucine, phenylalanine, threonine, and valine) and carbohydrate metabolites (fumaric acid, glucuronic acid, hexuronic acid, and malic acid). The integrated pathway analysis of the 87 identified metabolites showed that protein biosynthesis and carbohydrate metabolism pathways are the most disturbed in MMTV-PyMT mice. In conclusion, adipose-derived MCP-1 contributes to mammary tumorigenesis and alters metabolism in male MMTV-PyMT mice. Citation Format: Lin Yan, Sneha Sundaram, Bret M. Rust, Matthew J. Picklo, Michael R. Bukowski. Tumorigenic and metabolomic impacts of adipose-derived monocyte chemotactic protein-1 in a model of obesity-enhanced, male breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2561.

Abstract 2673: The cytoplasmic adaptor FRS2beta fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis

Abstract It is widely held that pro-inflammatory changes often precede the onset of breast cancer. However, the underlying molecular mechanisms are largely unknown. Here, we demonstrate that FRS2β, a membrane-linked adaptor protein expressed in a small subset of luminal epithelial cells, triggers the inflammatory changes in precancerous mammary tissues and is responsible for the onset of the disease. FRS2β-deficiency in a mouse mammary tumor virus (MMTV)-ErbB2 genetic background markedly attenuated mammary tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2β-deficient precancerous mammary tissue niche. we observed that the co-localization of FRS2β and the NEMO subunit of the IκB kinase (IKK) complex on early endosomes led to activation of nuclear factor-κB (NFκB). Moreover, the expression of numerous cytokines, including CXC chemokine ligand (CXCL)12, was elevated owing to the activation of NFκB. CXCL12 may in turn activate NFκB in an autocrine and paracrine loop, leading to the inflammatory changes that promote tumorigenesis. The elucidation of the FRS2β-dependent NFκB-activation pathway uncovers a hitherto unknown molecular link between the tissue inflammation and the onset of breast cancer. Citation Format: Noriko Gotoh, Yasuto Takeuchi, Natsuko Kimura, Takehiko Murayama, Yukino Machida, Daisuke Iejima, Tatsunori Nishimura, Mizuki Yamamoto, Jun-ichito Inoue, Kouichi Akashi, Hideyuki Saya, Masahiko Kuroda, Issay Kitabayashi, Arinobu Tojo. The cytoplasmic adaptor FRS2beta fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2673.

Abstract LB229: Environmentally-induced epigeneticinheritance of cancer: Paternal exposure to pesticides induces intergenerational programming of breast cancer predisposition via sperm small RNAs

Abstract Background: DNA sequence accounts for the majority of disease heritability, including cancer. However, it is becoming clear that environmentally-induced epigenetic inheritance of disease can also occur. Epidemiological studies have shown that parental exposure to the pesticide DDT and other environmental toxicants is associated with increased tumor development, particularly breast cancer, in offspring. Here, we tested whether paternal exposure to DDT could lead to intergenerational increase in breast cancer development using a mouse model. We also investigated the mechanisms of intergenerational transmission.Methods: Male mice (c57bl/6) were exposed to DDT or to a control-vehicle (CO) solution and used for sperm collection or mating with unexposed females to produce the DDT or CO daughters. In another experiment, normal mouse embryos (zygote stage) were injected with purified sperm RNAs or synthetic miRNA-10b and implanted into surrogate mothers to produce RNA-injected offspring. DDT daughters or RNA injected females were used to study breast cancer development (carcinogen-induced or orthotopic models) and metabolic parameters. Paternal sperm was used for RNA-seq analysis and miRNA expression levels. Results: Pre-conception paternal DDT exposure altered the sperm small non-coding RNA load, with an increase in miRNAs and a specific surge in miRNA-10b levels. DDT offspring weighed less at birth and at weaning, but became overweight and showed metabolic dysfunction in adulthood compared to CO. DDT daughters also showed increased mammary tumorigenesis, developing more aggressive tumors that grew faster than in CO. This tumor phenotype was linked to suppression of the AMPK energy sensing pathway and mTOR activation in mammary tissues. Remarkably, embryonic injection of purified DDT sperm RNA or synthetic miRNA-10b recapitulated the mammary gland and tumor phenotypes observed in DDT daughters. Conclusions: To our knowledge, this is the first report of an association between paternal DDT exposure and breast cancer in offspring. Paternal DDT-induced programming of breast cancer development in daughters is mechanistically linked to sperm RNAs and, more specifically, miRNA-10b. The impact of DDT and other endocrine disrupting chemicals on sperm and programming of breast and other cancers in offspring needs to be evaluated in humans. Citation Format: Raquel Santana Da Cruz, Hong Cao, Camile C. Fontelles, Apsra Nasir, Alexandra K. Gonsiewski, M. Idalia Cruz, Lu Jin, Sonia de Assis. Environmentally-induced epigeneticinheritance of cancer: Paternal exposure to pesticides induces intergenerational programming of breast cancer predisposition via sperm small RNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB229.

Pregnancy Inhibits Mammary Carcinogenesis by Persistently Altering the Hypothalamic-Pituitary Axis.

Simple SummaryBreast cancer is one of the most frequently diagnosed cancers and it is the second leading cause of cancer-related death in women. Early first full-term pregnancy has been known to reduce the life-time risk of breast cancer. The actual mechanism by which pregnancy reduces the life-time risk of breast cancer is not well understood. It is well established that hormones are vital for a successful full-term pregnancy and they can also influence the risk of breast cancer. The emphasis has been placed mainly on the ovarian hormones estrogen and progesterone. It is also known that hypothalamic and pituitary hormones can impact the breast. In this study, we investigated how pregnancy alters the hypothalamic/pituitary hormones and what effect these hormonal alterations have on the risk of breast cancer development. Our results demonstrate that pregnancy persistently alters the hypothalamic–pituitary hormonal axis leading to the reduction of breast cancer risk.Early full-term pregnancy is known to reduce the lifetime risk of breast cancer. Although the phenomenon of parity-induced protection is well-established, the physiological mechanisms involved in this protection are not clear. Earlier reports have shown that pregnancy results in alterations of hormone levels. How pregnancy affects hypothalamic hormones and how the change, if any, influences breast cancer is not well understood. Seven-week-old female Lewis rats were given N-methyl-N-nitrosourea. Two weeks post carcinogen exposure, a set of females were housed with males to generate the parous rats and another set of rats served as the nulliparous controls. Mammary tumorigenesis was assessed for 9 months. Hypothalamic and pituitary levels of hormones were measured at various timepoints. Further, animals were also challenged with growth hormone and prolactin secretagogues to test the effect of pregnancy on the hypothalamic–pituitary hormonal axis. Persistent alterations in the level of growth hormone-releasing hormone, thyrotropin releasing hormone, dopamine, and somatostatin in the hypothalamus of parous animals was observed. Further, we also observed that pregnancy had a significant effect on the pituitary gland and its response to growth hormone and prolactin secretagogues. Our studies using the rodent model system demonstrate that pregnancy could be reducing the risk of breast cancer by persistently altering the hypothalamic–pituitary axis, which could have implications for breast cancers in humans as well.

Single Nucleotide Polymorphism in Prolactin Gene Is Associated With Clinical Aggressiveness and Outcome of Canine Mammary Malignant Tumors.

Prolactin (PRL) is a key hormone involved in canine mammary development and tumorigenesis. In this study, the influence of a single nucleotide polymorphism (SNP) in the PRL gene (rs23932236) on the clinicopathological parameters and survival of dogs with canine mammary tumors (CMTs) was investigated. A total of 206 female dogs with spontaneous mammary tumors were enrolled in this study and circulating blood cells were genotyped. This specific SNP was associated with larger size (>3 cm diameter) for malignant tumors (P = .036), tumors with infiltrative/invasive growth pattern (P = .010), vascular invasion (P = .006), and lymph node metastasis (P = .004). Carriers of the variant allele had a shorter overall survival compared to the wild-type population with an overall survival of 18.7 months and 22.7 months, respectively (P = .004). These findings suggest that SNP rs23932236 of canine PRL gene may be used as an indicator for the development of clinically aggressive forms of CMTs.

Cumin Prevents 17β-Estradiol-Associated Breast Cancer in ACI Rats.

Breast cancer (BC) is a leading cause of cancer deaths in women in less developed countries and the second leading cause of cancer death in women in the U.S. In this study, we report the inhibition of E2-mediated mammary tumorigenesis by Cuminum cyminum (cumin) administered via the diet as cumin powder, as well as dried ethanolic extract. Groups of female ACI rats were given either an AIN-93M diet or a diet supplemented with cumin powder (5% and 7.5%, w/w) or dried ethanolic cumin extract (1%, w/w), and then challenged with subcutaneous E2 silastic implants (1.2 cm; 9 mg). The first appearance of a palpable mammary tumor was significantly delayed by both the cumin powder and extract. At the end of the study, the tumor incidence was 96% in the control group, whereas only 55% and 45% animals had palpable tumors in the cumin powder and extract groups, respectively. Significant reductions in tumor volume (660 ± 122 vs. 138 ± 49 and 75 ± 46 mm3) and tumor multiplicity (4.21 ± 0.43 vs. 1.16 ± 0.26 and 0.9 ± 0.29 tumors/animal) were also observed by the cumin powder and cumin extract groups, respectively. The cumin powder diet intervention dose- and time-dependently offset E2-related pituitary growth, and reduced the levels of circulating prolactin and the levels of PCNA in the mammary tissues. Mechanistically, the cumin powder diet resulted in a significant reversal of E2-associated modulation in ERα, CYP1A1 and CYP1B1. Further, the cumin powder diet reversed the expression levels of miRNAs (miR-182, miR-375, miR-127 and miR-206) that were highly modulated by E2 treatment. We analyzed the composition of the extract by GC/MS and established cymene and cuminaldehyde as major components, and further detected no signs of gross or systemic toxicity. Thus, cumin bioactives can significantly delay and prevent E2-mediated mammary tumorigenesis in a safe and effective manner, and warrant continued efforts to develop these clinically translatable spice bioactives as chemopreventives and therapeutics against BC.

Fish oil supplementation increases expression of mammary tumor apoptosis mediators and reduces inflammation in an obesity-associated HER-2 breast cancer model

Obesity is associated with inflammation and has been shown to increase breast cancer severity. The objective of this study was to examine the effect of fish oil (FO) supplementation in obesity-associated mammary tumorigenesis in the MMTV-neu(ndl)-YD5 mouse model of human epidermal growth factor receptor-2 positive BC. Female mice were fed one of three diets for 16 weeks: i) high fat diet [HF, % kacl: 41.2% lard, 18.7% corn oil (CO)], ii) an isocaloric HF plus menhaden FO diet (HF+FO, % kcal: 41.2 lard, 13.4% CO, 5.3% FO), iii) low fat diet (LF, % kcal: 4.7% lard, 6% CO). HF mice had increased body weight, visceral adipose weight and serum hormone concentrations (increased leptin and resistin; decreased adiponectin) versus LF, which was attenuated in the HF+FO group versus HF (P<.05). Compared to HF, tumor onset was delayed in HF+FO and LF mice (P<0.05). Compared to HF, HF+FO reduced mammary tumor multiplicity (-27%), tumor weight (-46%) and total tumor volume (-50%) (P<0.05). Additionally, HF+FO reduced mammary tumor multiplicity (-33%), tumor weight (-39%) and total tumor volume (-60%) versus LF. HF+FO improved mammary tumor apoptosis status with increased expression of pro-apoptotic Bad and decreased expression of anti-apoptotic Bcl-xLmediators versus HF (P<0.05). Additionally, HF+FO decreased tumor protein expression of activated Akt, NFκB p65 and STAT3, versus HF (P<0.05). Tumor mRNA expression of inflammatory mediators TNFα, IL-6 and leptin were reduced in HF+FO, whereas IL-10 expression was increased compared to HF (P<0.05). Collectively these results demonstrate the efficacy of FO supplementation for improving obesity-associated breast cancer outcomes.

Oxytocin receptor induces mammary tumorigenesis through prolactin/p-STAT5 pathway

Oxytocin receptor (OXTR) is involved in social behaviors, thermoregulation, and milk ejection, yet little is known about its role in breast cancer. To investigate the role of OXTR in mammary gland development and tumorigenesis, a transgenic mouse model of OXTR overexpression (++Oxtr) was used. Overexpression of OXTR-induced progressive mammary hyperplasia, unexpected milk production, and tumorigenesis in females. OXTR-induced mammary tumors showed ERBB2 upregulation and mixed histological subtypes with predomination of papillary and medullary carcinomas. OXTR overexpression led to an activation of prolactin (PRL)/p-STAT5 pathway and created a microenvironment that promotes mammary-specific tumorigenesis. PRL inhibitor bromocriptine (Br) could mitigate OXTR-driven mammary tumor growth. The study demonstrates Oxtr is an oncogene and a potential drug target for HER2-type breast cancer.

IL-33-Induced Transcriptional Activation of LPIN1 Accelerates Breast Tumorigenesis.

Simple SummaryLPIN1 is a protein that exhibits dual functions as a phosphatidic acid phosphatase enzyme in the regulation of triglyceride and glycerophospholipid metabolism and a transcriptional coregulator. Recent advances in understanding suggest that LPIN1 frequently observed in various human cancer cell lines controls the main cellular processes in cancer progression. Therefore, there is considerable interest in the underlying mechanism regulating LPIN1 expression. We have determined for the first time that IL-33 regulates LPIN1 expression by recruiting c-Jun to the LPIN1 promoter. Consistent with these observations, IL-33 levels positively correlate with LPIN1 expression in human breast cancer. Our findings point to a critical role of IL-33-induced LPIN1 expression via COT/JNK1/2 pathway in promoting epithelial transformation and breast tumorigenesis.Phospholipids are crucial materials that are not only required for cell membrane construction but also play significant roles as signaling molecules. LPIN1 is an enzyme that displays phosphatidate phosphatase activity in the triglyceride and phospholipid synthesis pathway. Recent studies have shown that overexpression of LPIN1 is involved in breast tumorigenesis, but the underlying mechanism regulating LPIN1 expression has not been elucidated yet. In the present study, we showed that the IL-33-induced COT-JNK1/2 signaling pathway regulates LPIN1 mRNA and protein expression by recruiting c-Jun to the LPIN1 promoter in breast cancer cells. IL-33 dose-dependently and time-dependently increased LPIN1 mRNA and protein expression. Moreover, IL-33 promoted colony formation and mammary tumorigenesis via induction of LPIN1 expression, while inhibition of LPIN1 disturbed IL-33-induced cell proliferation and mammary tumorigenesis. IL-33-driven LPIN1 expression was mediated by the COT-JNK1/2 signaling pathway, and inhibition of COT or JNK1/2 reduced LPIN1 expression. COT-JNK1/2-mediated IL-33 signaling activated c-Jun and promoted its binding to the promoter region of LPIN1 to induce LPIN1 expression. These findings demonstrated the regulatory mechanism of LPIN1 transcription by the IL-33-induced COT/JNK1/2 pathway for the first time, providing a potential mechanism underlying the upregulation of LPIN1 in cancer.

IRS4 promotes the progression of non-small cell lung cancer and confers resistance to EGFR-TKI through the activation of PI3K/Akt and Ras-MAPK pathways

IRS4 is a member of the insulin receptor substrate (IRS) protein family. It acts as a cytoplasmic adaptor protein, integrating and transmitting signals from receptor protein tyrosine kinases to the intracellular environment. IRS4 can induce mammary tumorigenesis and is usually overexpressed in non-small cell lung cancer (NSCLC). However, little is known about the role of IRS4 in the development and progression of lung cancer. In this study, we show that IRS4 knockout suppresses the proliferation, colony formation, migration, and invasion of A549 lung cancer cells, as well as tumor growth in a nude mouse xenograft model. In contrast, stable expression of IRS4 showed the opposite effects. As expected, IRS4 was found to activate the PI3K/Akt and Ras-MAPK pathways, and we also showed that IRS4 depletion significantly enhanced the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI)-resistant cells to gefitinib. Taken together, these results show that IRS4 promotes NSCLC progression and may represent a potential therapeutic target for EGFR-TKI-resistant NSCLC.

Maternal fish oil consumption has a negative impact on mammary gland tumorigenesis in C3(1) Tag mice offspring.

Omega-3 fatty acids have been shown to reduce the incidence and slow the growth of mammary gland cancer in rodent models. Since exposure to dietary components during the critical developmental times of gestation and lactation may alter risk for mammary gland cancer in females, we tested whether exposure to increased levels of long-chain omega-3 fatty acids from fish oils would be preventive or promotional to mammary gland cancer in the offspring. Normal SV129 female mice were fed AIN 76 diets containing either 10% corn oil (control, 50% omega 6, n-6) or 5% of an omega-3 (n-3) fatty acid concentrate (fish oil 60% n-3) + 5% canola oil (10% n-3 + 20% n-6). Females were then mated with C(3)1 TAg transgenic mice. At weaning (3weeks), pups were randomized to either the corn (C) or fish oil (F) diet, 15-17 mice per group. Four experimental groups were generated: FF, FC, CF and CC. Tumor incidence and multiplicity were assessed at the following time points 120, 130 and 140days of age. A panel of genes encoding signal transduction proteins were analyzed in mammary glands at 130days. Mice never exposed to fish oil (CC group) had a significantly higher incidence and multiplicity of mammary gland tumors than mice exposed to fish oil throughout life (FF group). Mice exposed to fish oil during a portion of life (CF or FC) had intermediate tumor incidences and multiplicities. Results also indicate that maternal consumption of fish oil increased the expression of genes associated with immune system activation (Ccl20, Cd5, Il2, Lef1, Lta). Adequate omega-3 fatty acids in the maternal diet may reduce the risk for mammary gland cancer in the offspring. If humans make dietary change by consuming more omega-3 fat instead of corn oil with 0% omega 3 fat, breast cancer may be reduced in the next generation.

Autophagy inhibition perturbs ERBB2 trafficking and abolishes tumorigenesis in ERBB2-driven breast cancer

ABSTRACT Macroautophagy/autophagy modulation is increasingly recognized as a potential strategy for cancer therapy. Using a recently developed Rb1cc1 mutant knockin mice model, we have taken a rigorous genetic approach to assess the role of both its autophagy and non-canonical functions in an ERBB2-driven BrCA model. We found that autophagy abrogation virtually abolishes mammary tumorigenesis in the ERBB2-driven model, exhibiting stronger inhibitory effects than in our previous studies using PyMT and brca1-null mouse models. Mechanistically, autophagy inhibition perturbs ERBB2 intracellular trafficking and triggers its release via small extracellular vesicles. Our results demonstrate a new mechanism for autophagy to promote tumorigenesis in ERBB2-driven BrCA and could supplement current strategies for anti-ERBB2 therapy.

Time-resolved single-cell analysis of Brca1 associated mammary tumourigenesis reveals aberrant differentiation of luminal progenitors

It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.

Prenatal polycyclic aromatic hydrocarbons, altered ERα pathway-related methylation and expression, and mammary epithelial cell proliferation in offspring and grandoffspring adult mice

BackgroundAirborne polycyclic aromatic hydrocarbons (PAH) possess carcinogenic and endocrine disrupting properties linked to mammary tumorigenesis. These effects may be initiated during a prenatal period of susceptibility to PAH activation of the aryl hydrocarbon receptor (Ahr) and through downstream effects on estrogen receptor (Er) α. PurposeWe hypothesized prenatal airborne PAH exposure induces sustained effects in female adult wild type BALB/cByj mice detected in the offspring (F1) and grandoffspring (F2) generation. We hypothesized these effects would include altered expression and epigenetic regulation of Erα and altered expression of aryl hydrocarbon receptor repressor (Ahrr, Ahrr/aryl hydrocarbon receptor nuclear translocator (Arnt), and breast cancer type 1 susceptibility (Brca1). Further, we hypothesized that PAH would induce precancerous outcomes such as epithelial cell proliferation and epithelial cell hyperplasia in mammary glands of adult female offspring and grandoffspring. ResultsPrenatal ambient PAH exposure lowered Erα mRNA expression (F1 and F2: p<0.001 for each) and induced methylation in the Erα promoter in mammary tissue in offspring and grandoffspring mice on postnatal day (PND) 60. Prenatal PAH lowered Brca1 mRNA (F1: p=0.002, F2: p=0.02); Erα mRNA was correlated with Brca1 (F1: r=0.42, p=0.02; F2: r=0.53, p=0.005). Prenatal PAH lowered Ahrr (F1: p=0.03, F2: p=0.009) and raised Arnt mRNA expression (F1: p=0.01, F2: p=0.03). Alterations in Erα mRNA (F2: p<0.0001) and Ahrr (F2: p=0.02) in the grandoffspring mice also occured by PND 28, and similarly occurred in the dam on postpartum day (PPD) 28. Finally, prenatal PAH was associated with higher mammary epithelial cell proliferation in the offspring (p=0.02), but not grandoffspring mice, without differences in the frequency of mammary cell hyperplasia. These results did not differ after adjustment by each candidate gene expression level. ConclusionsPrenatal PAH exposure induces DNA methylation and alters gene expression in the Erα-mediated pathway across generations, and suggests that functional outcomes such as mammary cell proliferation also may occur in offspring as a result.

Functional cooperation between co-amplified genes promotes aggressive phenotypes of HER2-positive breast cancer

SUMMARYMED1 (mediator subunit 1)co-amplifies with HER2, but its role in HER2-driven mammary tumorigenesis is still unknown. Here, we generate MED1 mammary-specific overexpression mice and cross them with mouse mammary tumor virus (MMTV)-HER2 mice. We observe significantly promoted onset, growth, metastasis, and multiplicity of HER2 tumors by MED1 overexpression. Further studies reveal critical roles for MED1 in epithelial-mesenchymal transition, cancer stem cell formation, and response to anti-HER2 therapy. Mechanistically, RNA sequencing (RNA-seq) transcriptome analyses and clinical sample correlation studies identify Jab1, a component of the COP9 signalosome complex, as the key direct target gene of MED1 contributing to these processes. Further studies reveal that Jab1 can also reciprocally regulate the stability and transcriptional activity of MED1. Together, our findings support a functional cooperation between these co-amplified genes in HER2+ mammary tumorigenesis and their potential usage as therapeutic targets for the treatment of HER2+ breast cancers.

Abstract PD11-08: Low dose endoxifen represses mouse mammary tumorigenesis: A preclinical study of monotherapy and combination with ulipristal acetate

Abstract Background: Endoxifen (ENX), a major active metabolite of the selective estrogen receptor (ER) modulator tamoxifen (TAM), has been shown to inhibit the growth of ER+ cancer. However, it has not been evaluated for prevention of mammary cancer. We report here the cancer prevention efficacy of ENX as monotherapy and in combination with ulipristal acetate (UPA), a selective progesterone receptor (PR) modulator. We compare these to TAM using the SV40 C3 T-antigen (C3-TAg) mouse, a well-documented mammary cancer prevention model. Methods: C3-TAg female virgin mice aged 7-8 weeks were randomized to no-treatment control or to drug treatment groups (TAM, ENX, UPA and ENX+UPA). Drug pellets were subcutaneously implanted on backside of mice. The doses of drug pellet were 12.5mg/30d for TAM, 9mg/90d for ENX, 10mg/30d for UPA, and combination of ENX (9mg/90d) + UPA (10mg/30d). The dose of ENX was equivalent to the exposure resulting from 20 mg/day, the lowest dose used in a recent Phase I human trial. Pellets were replaced every 30 days or 90 days. Mammary tumor formation was monitored twice weekly by palpation. Tumor latency, multiplicity, and tumor volume were recorded; animals are euthanized at 23 weeks of age, or earlier if tumors reach &amp;gt; 1 cm2. Tumors and mammary glands were formalin fixed and paraffin embedded for histology evaluation by a rodent pathologist. The primary endpoint was a reduction in tumor incidence in drug treated versus control groups. Secondary endpoints included prolongation of latency, reduction in tumor multiplicity, and tumor burden. Statistical significance between groups was calculated with Wilcoxon log-rank test for % tumor-free survival and Mann Whitney test for tumor multiplicity and burden.Results: All mice in the control group developed tumors by 18 weeks of age. Tumor-free survival % of ENX, UPA, and ENX+UPA treated groups were significantly higher than control animals, with the greatest increase in the ENX group (p=0.02). TAM had no effect on tumor-free survival (p=0.32). Median tumor latencies were similar in three treated groups: ENX (112 days), UPA (114 days), and ENX+UPA (111 days) groups, and were significantly delayed compared to the control or TAM group (100 days in both) (p&amp;lt;0.05). Median tumor multiplicity (invasive adenocarcinoma) per animal was significantly lower in ENX group compared to control group (3 vs. 7, p=0.02). UPA and TAM did not significantly reduce tumor multiplicity (4 for TAM and 6 for UPA). We observed non-significant 32% and 23% reduction in median tumor weight of ENX+UPA group and ENX group, respectively compared to control group. Histology evaluation for ENX+UPA group and pathologic size of invasive carcinoma is ongoing, along with the expression of Ki67, ERα and PR of mammary tumors and mammary gland by immunohistochemistry.Conclusions: TAM showed no significant effect on delaying tumorigenesis, consistent with a previous study. However, low dose of ENX (equivalent to 24 mg/day in human) effectively repressed tumor development and growth than TAM or UPA treatment. Our data suggest that ENX is promising for prevention of ER+ mammary tumorigenesis and warrant a dose optimization study for improving efficacy. Citation Format: Oukseub Lee, Minhua Wang, Maarten C. Bosland, Omid Hosseini, Irene Helenowski, Seema A. Khan. Low dose endoxifen represses mouse mammary tumorigenesis: A preclinical study of monotherapy and combination with ulipristal acetate [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD11-08.

Abstract PS17-31: Investigating the estrogen receptor Y537S mutation in transgenic models of luminal B breast cancer

Abstract Breast cancer is the most prevalent cancer in women and over two-thirds of cases express the estrogen receptor (ER). Significantly, metastatic ER positive breast cancer is the leading cause of breast cancer mortality. Despite the success of endocrine therapy to treat ER-positive breast cancers, resistance to treatment is a major problem and relapse commonly occurs though patients initially respond well to treatment. The ER Y537S mutant represents one of the most common gain-of-function mutations detected in the metastatic biopsies of patients with ER-positive breast cancer. Previous in-vitro analyses have shown that the ER Y537S mutant exhibits enhanced ER transcriptional activity by adopting a ligand-independent agonist confirmation. In our study, we seek to provide a deeper understanding of the role of this mutant by modeling the human ER Y537S mutant (ER Y541S in mice). Our lab has generated a Cre-inducible knock-in mouse model expressing the activating point mutation driven by the endogenous ER promoter, and it has been shown that when expressed ubiquitously the mutated ER leads to dramatic developmental effects. In order to further our observations in the context of endocrine resistant metastatic breast cancer, we characterize mamary tumour growth of the ER Y541S mutant in combination with luminal B models of mammary tumourigenesis. Consistent with the notion that these ER mutations occur seldom in primary tumours, preliminary data shows that tumour onset and tumour outgrowth is unaffected by the activating point mutation. When analysing tumours from both ER mutant and wildtype ER control animals, it was found that tumours with mutant ER display more basal-like cytokeratins at the RNA level via RNA sequencing and at the protein level via immunohistochemistry. This expression pattern is often associated with poorer prognosis in the clinic. Through ongoing analyses, we also seek to better understand our model through tumour regression studies (doxycycline withdrawal), treatment with tamoxifen and blocking the main source of estrogens via ovariotomies. By studying the ER Y537S mutation in models of luminal B breast cancer, we hope to investigate the factors leading to tumour recurrence and provide a deeper understanding of potential escape pathways to therapy. Citation Format: Gabriella Johnson, Joshua Roccamo, Cynthia Lavoie, Dongmei Zuo, Virginie Sanguin-Gendreau, Adrian Lee, Zheqi Li, Steffi Oesterreich, William Muller. Investigating the estrogen receptor Y537S mutation in transgenic models of luminal B breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-31.

Abstract PS19-17: The role of myoglobin in breast cancer

Abstract Breast cancer today is typically seen as malignancy with longer survival due to early-detection diagnostics, yet the cancer remains by far the most common tumor type in adult females. Recently, our group co-discovered myoglobin (MB) to be expressed in luminal cells of healthy and cancerous breast epithelia of human and mice. In human patients, MB positivity emerged as hallmark of the luminal subtype and was directly correlated with estrogen receptor alpha positivity and hence a significantly better prognosis. Cancerous cells’ MB also appears to interact with the known tumor suppressor p53. However, if and how myoglobin itself restricts mammary tumorigenesis is completely unclear. To understand how MB exerts its alleged tumor-suppressive effects and if it impacts response of mammary tumors to chemo- and/or radiotherapy, we examine the molecular role of MB in breast tumor formation and progression through a) in vitro studies (MCF7 breast cancer BrCa cell clones; wildtype (wt) controls versus CRISPR/Cas9-engineered clones of a MB and/or p53 knockout (MBko/p53ko), b) in vivo mouse models to obtain spontaneously forming breast tumors, with or without MB, in a p53 deficient background. Comparing MCF7 BrCa MBko and p53ko clones vs. their corresponding wildtype counterpart supports our hypothesis of a feed forward loop between MB and p53 proteins, besides interfering with estrogen receptor expression. In addition to that, loss of MB was noticed to exert p53-independent upregulation on cell cyclins with more S phase cell population. That was reflected by a significant increase in survival of the MBko cells, in presence or absence of p53. Hypoxic MBko cells exhibited a partial epithelial to mesenchymal transition and hence a more migratory phenotype, relative to MBwt controls. Additionally, MB seems to stimulate apoptosis of the normoxic cancer cells. On the other hand, murine tumors were found to phenocopy human BrCa in the extent of their MB expression. Interestingly, MB-devoid tumors showed a significantly higher proliferation index and fat accumulation. Further studies will look into the clinical significance of that. In conclusion, MB seems to occupy unknown roles in cancer cells beyond or different from its classical O2 storage/transport functions. Unraveling these novel roles in governing tumor development and virulence will hopefully provide innovative strategies for future breast cancer interventions. Citation Format: Mostafa A. Aboouf, Julia Armbruster, Franco Guscetti, Markus Thiersch, Max Gassmann, Thomas A. Gorr. The role of myoglobin in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-17.

RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors.

Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop-prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.

Time-restricted feeding normalizes hyperinsulinemia to inhibit breast cancer in obese postmenopausal mouse models

Accumulating evidence indicates that obesity with its associated metabolic dysregulation, including hyperinsulinemia and aberrant circadian rhythms, increases the risk for a variety of cancers including postmenopausal breast cancer. Caloric restriction can ameliorate the harmful metabolic effects of obesity and inhibit cancer progression but is difficult to implement and maintain outside of the clinic. In this study, we aim to test a time-restricted feeding (TRF) approach on mouse models of obesity-driven postmenopausal breast cancer. We show that TRF abrogates the obesity-enhanced mammary tumor growth in two orthotopic models in the absence of calorie restriction or weight loss. TRF also reduces breast cancer metastasis to the lung. Furthermore, TRF delays tumor initiation in a transgenic model of mammary tumorigenesis prior to the onset of obesity. Notably, TRF increases whole-body insulin sensitivity, reduces hyperinsulinemia, restores diurnal gene expression rhythms in the tumor, and attenuates tumor growth and insulin signaling. Importantly, inhibition of insulin secretion with diazoxide mimics TRF whereas artificial elevation of insulin through insulin pumps implantation reverses the effect of TRF, suggesting that TRF acts through modulating hyperinsulinemia. Our data suggest that TRF is likely to be effective in breast cancer prevention and therapy.