Mammary Tumorigenesis Research Articles

Targeting AU-rich element-mediated mRNA decay with a truncated active form of the zinc-finger protein TIS11b/BRF1 impairs major hallmarks of mammary tumorigenesis.

Altered expression of regulatory RNA-binding proteins (RBPs) in cancer leads to abnormal expression of mRNAs encoding many factors involved in cancer hallmarks. While conventional anticancer therapies usually target one pathway at a time, targeting key RBP would affect multiple genes and thus overcome drug resistance. Among the Tristetraprolin family of RBP, TIS11b/BRF1/ZFP36L1 mediates mRNA decay through binding to Adenylate/Uridylate (AU-rich elements) in mRNA 3'-untranslated region and recruitment of mRNA degradation enzymes. Here, we show that TIS11b is markedly underexpressed in three breast cancer cell lines, as well as in breast tumor samples. We hypothesized that restoring intracellular TIS11b levels could impair cancer cell phenotypic traits. We thus generated a derivative of TIS11b called R9-ZnCS334D, by combining N-terminal domain deletion, serine-to-aspartate substitution at position 334 to enhance the function of the protein and fusion to the cell-penetrating peptide polyarginine R9. R9-ZnCS334D not only blunted secretion of vascular endothelial growth factor (VEGF) but also inhibited proliferation, migration, invasion, and anchorage-independent growth of murine 4T1 or human MDA-MB-231 breast cancer cells. Moreover, R9-ZnCS334D prevented endothelial cell organization into vessel-like structures, suggesting that it could potentially target various cell types within the tumor microenvironment. In vivo, injection of R9-ZnCS334D in 4T1 tumors impaired tumor growth, decreased tumor hypoxia, and expression of the epithelial-to-mesenchymal transition (EMT) markers Snail, Vimentin, and N-cadherin. R9-ZnCS334D also hindered the expression of chemokines and proteins involved in cancer-related inflammation and invasion including Fractalkine (CX3CL1), SDF-1 (CXCL12), MCP-1 (CCL2), NOV (CCN3), and Pentraxin-3 (PTX3). Collectively, our data indicate that R9-ZnCS334D counteracts multiple traits of breast cancer cell aggressiveness and suggest that this novel protein could serve as the basis for innovative multi-target therapies in cancer.

Optimized Bexarotene Aerosol Formulation Inhibits Major Subtypes of Lung Cancer in Mice.

Bexarotene has shown inhibition of lung and mammary gland tumorigenesis in preclinical models and in clinical trials. The main side effects of orally administered bexarotene are hypertriglyceridemia and hypercholesterolemia. We previously demonstrated that aerosolized bexarotene administered by nasal inhalation has potent chemopreventive activity in a lung adenoma preclinical model without causing hypertriglyceridemia. To facilitate its future clinical translation, we modified the formula of the aerosolized bexarotene with a clinically relevant solvent system. This optimized aerosolized bexarotene formulation was tested against lung squamous cell carcinoma mouse model and lung adenocarcinoma mouse model and showed significant chemopreventive effect. This new formula did not cause visible signs of toxicity and did not increase plasma triglycerides or cholesterol. This aerosolized bexarotene was evenly distributed to the mouse lung parenchyma, and it modulated the microenvironment in vivo by increasing the tumor-infiltrating T cell population. RNA sequencing of the lung cancer cell lines demonstrated that multiple pathways are altered by bexarotene. For the first time, these studies demonstrate a new, clinically relevant aerosolized bexarotene formulation that exhibits preventive efficacy against the major subtypes of lung cancer. This approach could be a major advancement in lung cancer prevention for high risk populations, including former and present smokers.

Author Correction: The Rho GTPase Rnd1 suppresses mammary tumorigenesis and EMT by restraining Ras-MAPK signalling.

In the version of this Article originally published the same blot was inadvertently presented as both p-Rb and Cyclin A in Fig. 2a. This blot corresponds to the p-Rb panel, as can be seen in the unprocessed version of these blots in Supplementary Fig. 9. The corrected version of the panel is shown below, together with a completely uncropped image of both blots. In addition, in the 'Viral transduction' section of the Methods, the pLKO.1 plasmids encoding short hairpin RNAs targeting human Rnd1 were incorrectly listed as clones TRCN0000018338 and TRCN0000039977. The correct clone numbers are TRCN0000047434 and TRCN0000047435.

Abstract ES12-2: Link between BRCA1 and Pol II pausing in mammary gland development and tumorigenesis

Abstract Breast cancer risk is significantly elevated for women who carry germ-line mutations in the tumor suppressor gene BRCA1. BRCA1-asociated breast tumors are basal-like, yet they originate from luminal progenitor cells. These precancerous cells of origin are defective in luminal differentiation in BRCA1 mutation carriers before any clinical evidence of cancer. While BRCA1 is best known for its functions in double strand break DNA repair and response to DNA replication stress, it is unclear whether loss of these ubiquitously important functions of BRCA1 is sufficient to account for lineage-specific breast tumor development in BRCA1 mutation-carrying women. Filling this longstanding intellectual disconnect could inform more effective risk assessment and disease prevention. Recent discoveries from others and us challenge the prevailing DNA repair/replication-centric paradigm. First, in vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Using breast tissue of cancer-free BRCA1 mutation-carrying women, we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5' end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1 knockout mouse mammary epithelium. Of note, this BRCA1/COBRA1 interaction occurs in a DSB repair-independent manner. Based on these findings, we suggest that as part of its tumor suppressor activity, BRCA1 promotes luminal epithelial differentiation by mitigating Pol II pausing-dependent transcriptional stress at luminal fate genes. We further propose that accumulation of transcriptional stress in BRCA1-deficient luminal cells redirects luminal transcription and alters luminal homeostasis, which ultimately contributes to lineage-specific BRCA1-associated tumorigenesis. Citation Format: Li R. Link between BRCA1 and Pol II pausing in mammary gland development and tumorigenesis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES12-2.

Abstract P5-05-15: Pregnancy inhibits mammary carcinogenesis by persistently altering the hypothalamic-pituitary axis

Abstract Pregnancy, carried to term at an early age, is probably the best natural protection against breast cancer development. The relative life-long breast cancer risk for women that give birth to their first child before the age of 20 years is approximately half that of nulliparous women. In contrast, if a woman undergoes her first full-term pregnancy after the age of 35, her risk for breast cancer is increased even more than nulliparous women. In the current generation many women are career oriented and have children later in life. Universally the average age at first birth is on the rise. It is critical to understand the underlying mechanism of this protective effect of pregnancy against breast cancer to develop novel prevention strategies to reduce the risk of breast cancer without women having to undergo pregnancy early in life. Earlier, we and others have demonstrated that post-pregnancy there were persistent changes in circulating levels of hormones. In order to understand the significance of these systemic changes we determined alterations in the hypothalamic-pituitary axis in parous rats. In particular, we examined the static and dynamic alterations in the hypothalamic-pituitary axis in response to pregnancy. Seven weeks old female Lewis rats were injected with the chemical carcinogen N-methyl-N-nitrosourea (MNU) at a dose of 50mg/kg body weight intraperitoneally. Two weeks post-carcinogen treatment these rats were housed with a male rat. On the observation of the vaginal plug the male was removed from the cage. Once the rats gave birth they nursed the pups for three weeks and were weaned after that period. Mammary tumorigenesis was monitored through weekly palpation for a period of nine months. A subset of rats at 6, 12 and 24 weeks post-weaning were used to study static and dynamic changes in the level of hormones. We investigated the static alterations in the hypothalamic-pituitary axis in response to pregnancy by measuring the levels of thyrotropin releasing hormone (TRH), growth hormone releasing hormone (GHRH), somatostatin (SS), dopamine (DA), growth hormone (GH) and prolactin (PRL). Next we investigated if the dynamic alterations in the hypothalamic-pituitary axis in response to pregnancy. Control and parous animals were subjected to secretogogue treatments (Growth Hormone Related Peptide 6 for GH and Perphenazine for PRL) and the levels of GH and PRL were measured. We also isolated the pituitary and treated them with the secretogogues and measured the levels of GH and PRL. Our data demonstrated that pregnancy resulted in persistent static and dynamic alterations in circulating levels of hormones. Parous rats response to the secretogogues was severely blunted compared to the control nulliparous rats. The levels of TRH, GHRH, GH and PRL were significantly lowered in parous rats while DA and SS levels were higher in nulliparous rats. As expected mammary carcinogenesis was significantly inhibited in parous rats. Overall, these preliminary results suggest that pregnancy induces persistent changes in hypothalamic-pituitary axis, which results in a lowered hormonal promotion environment resulting in inhibition of mammary carcinogenesis. Citation Format: Subramani R, Galvez A, Pedroza D, Lakshmanaswamy R. Pregnancy inhibits mammary carcinogenesis by persistently altering the hypothalamic-pituitary axis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-15.

Abstract PD5-06: The immunomodulatory potential of denosumab in breast cancer: results from D-BEYOND, a window of opportunity trial evaluating a RANK-ligand (RANKL) inhibitor and its biological effects in young pre-menopausal women diagnosed with early breast cancer

Abstract Background Breast cancer (BC) in young women has unique biology and poor prognosis. Previous reports suggest that they often express RANKL, which was also shown to play a role in mammary tumorigenesis and various immune processes. Here, we present the primary results of D-BEYOND, a window study investigating the biological activity of the RANKL inhibitor; denosumab in pre-menopausal BC patients. Methods D-BEYOND is a prospective, phase Iia, single-arm, multicenter study assessing the effect of denosumab on BC biology in premenopausal women with early BC (NCT01864798). Patients received two subcutaneous injections of denosumab (120mg), one week apart, followed by breast surgery. The primary endpoint was geometric mean change in tumor Ki67 assessed by immunohistochemistry (IHC). Blood, tumor and normal adjacent breast tissue were collected pre- and post-treatment. Serum levels of RANKL, OPG and CTX were assessed by ELISA. RNA was extracted from fresh-frozen tissue and RNAseq was performed. DESeq2 was used for differential expression analysis, GAGE was used for pathway analysis and CIBERSORT was used to infer immune cell subsets between pre- and post-treatment. Ki67, CD4/Foxp3 and CD4/CD8 IHC were performed on FFPE tissue to further assess the immune microenvironment. The percentage of TILs was independently evaluated by two pathologists on H&E slides. Pre- and post-treatment values were compared using a paired t-test. Results A total of 27 patients were enrolled in the study between October 2013 and July 2016. The median age was 45 years (range 35-51 years). Tumors of 21 patients were hormone receptor positive (77.8%), 4 were HER2 positive (14.8%) and 2 were triple negative (7.4%). No serious adverse events were reported, the most frequent non-serious adverse event being arthralgia (14.8%). After treatment, serum levels of CTX and RANKL decreased in all patients (P < 0.001) whereas OPG increased in 76.9% of patients (P = 0.009, 95% CI 0.56-0.91). There was no significant reduction of Ki67 values from baseline (geometric mean [GM] change after treatment; 0.98, 95% CI 0.76-1.26; P = 0.90). Twenty-four pre- and post-treatment tumor pairs were available for RNAseq, IHC and TILs evaluation. There was a significant increase in the percentage of stromal TILs after treatment (GM change of 1.75, 95% CI 1.28–2.39; P = 0.001). 1084 differentially expressed genes were identified and pathway analysis revealed enrichment of several immune processes. CIBERSORT revealed an enrichment of CD8+ T cells (GM change 1.72, 95% CI 1.19–2.48; P = 0.006) and a decrease of Treg cells (0.71, 95% CI 0.52–0.98, P = 0.040). These results were confirmed by IHC of CD8+ and CD4+/Foxp3+ cells (GM change 1.59, 95% CI 1.14–2.21; P = 0.008 and 0.63, 95% CI 0.49–0.83, P = 0.001, respectively). Conclusion Short course of denosumab did not reduce tumor proliferation rate. However, it induced a significant increase in TILs and CD8 cytotoxic T cells, while Treg infiltration decreased. These findings suggest an immunomodulatory role for denosumab in young breast cancer and that its use in combination could boost immunotherapy efficacy. Citation Format: Nguyen B, Marion M, Salgado R, Venet D, Vuylsteke P, Polastro L, Wieldiers H, Simon P, Lindeman G, Larsimont D, Van den Eynden G, Velghe C, Rothe F, Garaud S, Michiels S, Willard-Gallo K, Azim Jr HA, Loi S, Piccart M, Sotiriou C. The immunomodulatory potential of denosumab in breast cancer: results from D-BEYOND, a window of opportunity trial evaluating a RANK-ligand (RANKL) inhibitor and its biological effects in young pre-menopausal women diagnosed with early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-06.

Abstract OT2-09-01: Pilot study of denosumab in BRCA1/2 mutation carriers scheduling for risk-reducing salpingo-oophorectomy

Abstract Background: Denosumab is a monoclonal antibody that inhibits RANKL and is approved for the prevention of fractures in patients with osteoporosis or bone metastases. The RANKL signaling pathway is also involved in BRCA1-associated mammary tumorigenesis via a progesterone-induced paracrine effect of RANKL on luminal progenitor cells. Pre-clinical studies have demonstrated that RANKL inhibition resulted in reduced proliferation of mammary tumors. Early findings from an ongoing pre-surgical study demonstrated that denosumab treatment resulted in decreased Ki67 proliferation index in benign breast tissue. Based on these data, denosumab is being pursued as a potential preventive agent for breast cancer in BRCA1 mutation carriers. While promising, the effect of RANKL inhibition on gynecologic tissues such as the ovaries and fallopian tubes, in which progesterone has a protective effect, is unknown. Trial design: We will conduct a multicenter, open-label randomized pilot study of presurgical administration of denosumab versus no treatment in premenopausal women with BRCA1/2 mutations undergoing risk-reducing salpingo-oophorectomy (RRSO). A total of 60 women will be randomized 1:1 to Arm 1) 3-4 doses of 120 mg denosumab subcutaneously every 4 weeks or Arm 2) No treatment. Participants will be stratified by 1) BRCA1 versus BRCA2 mutation status and 2) Use of hormonal contraceptives within the past 3 months (yes/no). Assuming a 10% unevaluable rate, we expect to have 54 evaluable participants (27 per arm). Eligibility criteria: 1) Premenopausal women (defined as < 3 months since last menstrual period OR serum follicle-stimulating hormone (FSH) < 20 mIU/mL), age > 18 years; 2) Documented germline pathogenic mutation or likely pathogenic variant in the BRCA1 or BRCA2 gene; 3) Plan for RRSO with or without hysterectomy; 4) ECOG performance status ≤ 1 (Karnofsky ≥ 70%); 5) Normal organ and marrow function; 6) Negative pregnancy test and use of adequate contraception; 7) Willingness to take supplemental oral calcium and vitamin D3; 8) Dental examination within 6 months of enrollment and no evidence of active dental issues; 9) Ability to understand and willingness to provide informed consent. Specific aims: Our primary objective is to compare the effect of denosumab to no treatment on Ki67 expression in the fimbrial end of the fallopian tube. Secondary objectives are to assess Ki67 in ovary and endometrium; cleaved caspase-3, RANK/RANKL, ER/PR, CD44, and STAT3/pSTAT3 expression in fallopian tube, ovary, and endometrium; gene expression profiling in the fallopian tube and ovary; serum markers (progesterone, estradiol, C-terminal telopeptide) and denosumab levels; and toxicity. Statistical methods: The primary endpoint is post-treatment Ki67 expression in the fimbrial end of the fallopian tube in the denosumab arm compared to the no treatment arm. Assuming a standard deviation of 5.0%, we will have 82% power to detect a 4.0% absolute difference (or effect size of 0.8) in Ki67 proliferation index between the denosumab and no treatment groups by applying a 2-sample t-test at a 0.05 significance level. Target accrual: 60 participants, to be activated in Summer 2018. Citation Format: Trivedi MS, Samimi G, Wright JD, Holcomb K, Garber JE, Horowitz NS, Arber N, Friedman E, Wenham RM, House M, Parnes H, Lee JJ, Abutaseh S, Vornik LA, Heckman-Stoddard BM, Brown PH, Crew KD. Pilot study of denosumab in BRCA1/2 mutation carriers scheduling for risk-reducing salpingo-oophorectomy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT2-09-01.

Multi-targeting chemoprevention of Chinese herb formula Yanghe Huayan decoction on experimentally induced mammary tumorigenesis

BackgroundDevelopment of safe and effective chemopreventive agents is a winning strategy in reducing the morbidity and mortality of breast cancer. The current study was to investigate the mechanism-based chemopreventive potential of a Chinese herb formula Yanghe Huayan (YHHY) Decoction on the classical 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary carcinogenesis model.MethodsFemale Sprague-Dawley rats at 42 days of age were orally administered with a human equivalent dose of YHHY Decoction at 0.02 ml/g (10 mg/ml) once daily, starting 1 wk. before and 4 wks following DMBA treatment. Mammary tumor occurrence was monitored every day. The length of time before palpable tumor is examined is defined as tumor-free survival time. High performance liquid chromatography (HPLC) analyses were adopted to identify major chemical compositions of the decoction. Following bioinformatics data mining and experimental analyses were performed to demonstrate the underlying mechanism of action.ResultsDMBA animals receiving YHHY Decoction exhibited a significant delay (P = 0.014) and in some animals prevention (P = 0.046) of tumor occurrence without obvious toxicity. Oncogenic myc activation was significantly suppressed in the DMBA-induced rats by the YHHY treatment. Eight major chemical compositions of the decoction were identified and were shown to interfere with multiple tumorigenic pathways simultaneously in the mammary tumors, including inducing tumor apoptosis and up-regulating pro-apoptotic protein Bax and down-regulating anti-apoptotic protein Bcl-2; suppressing abnormal cell proliferation and the MAPK/ERK, PI3K/AKT signalings; blocking neo-angiogenesis and the VEGF/KDR signaling, and inhibiting oxidative stress in the mammary tumors.ConclusionThe multi-components and multi-targeting properties of the YHHY Decoction support its use as a potent chemopreventive drug in breast cancer.

A miRNA-HERC4 pathway promotes breast tumorigenesis by inactivating tumor suppressor LATS1

sThe E3 ligase HERC4 is overexpressed in human breast cancer and its expression levels correlated with the prognosis of breast cancer patients. However, the roles of HERC4 in mammary tumorigenesis remain unclear. Here we demonstrate that the knockdown of HERC4 in human breast cancer cells dramatically suppressed their proliferation, survival, migration, and tumor growth in vivo, while the overexpression of HERC4 promoted their aggressive tumorigenic activities. HERC4 is a new E3 ligase for the tumor suppressor LATS1 and destabilizes LATS1 by promoting the ubiquitination of LATS1. miRNA-136-5p and miRNA-1285-5p, expression of which is decreased in human breast cancers and is inversely correlated with the prognosis of breast cancer patients, are directly involved in suppressing the expression of HERC4. In summary, we discover a miRNA-HERC4-LATS1 pathway that plays important roles in the pathogenesis of breast cancer and represents new therapeutic targets for human breast cancer.

Comparative oncogenomics identifies combinations of driver genes and drug targets in BRCA1-mutated breast cancer

BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy.

Dynamic role of the codon 72 p53 single-nucleotide polymorphism in mammary tumorigenesis in a humanized mouse model

Female breast cancer (BrCa) is the most common noncutaneous cancer among women in the United States. Human epidemiological studies reveal that a p53 single-nucleotide polymorphism (SNP) at codon 72, encoding proline (P72) or arginine (R72), is associated with differential risk of several cancers, including BrCa. However, the molecular mechanisms by which these variants affect mammary tumorigenesis remain unresolved. To investigate the effects of this polymorphism on susceptibility to mammary cancer, we used a humanized p53 mouse model, homozygous for either P72 or R72. Our studies revealed that R72 mice had a significantly higher mammary tumor incidence and reduced latency in both DMBA-induced and MMTV-Erbb2/Neu mouse mammary tumor models compared to P72 mice. Analyses showed that susceptible mammary glands from E-R72 (R72 x MMTV-Erbb2/Neu) mice developed a senescence-associated secretory phenotype (SASP) with influx of proinflammatory macrophages, ultimately resulting in chronic, protumorigenic inflammation. Mammary tumors arising in E-R72 mice also had an increased influx of tumor-associated macrophages, contributing to angiogenesis and elevated tumor growth rates. These results demonstrate that the p53 R72 variant increased susceptibility to mammary tumorigenesis through chronic inflammation.

Star-related lipid transfer protein 10 (STARD10): a novel key player in alcohol-induced breast cancer progression

BackgroundEthanol abuse promotes breast cancer development, metastasis and recurrence stimulating mammary tumorigenesis by mechanisms that remain unclear. Normally, 35% of breast cancer is Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2)-positive that predisposes to poor prognosis and relapse, while ethanol drinking leads to invasion of their ERBB2 positive cells triggering the phosphorylation status of mitogen-activated protein kinase. StAR-related lipid transfer protein 10 (STARD10) is a lipid transporter of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); changes on membrane composition of PC and PE occur before the morphological tumorigenic events. Interestingly, STARD10 has been described to be highly expressed in 35–40% of ERBB2-positive breast cancers. In this study, we demonstrate that ethanol administration promotes STARD10 and ERBB2 expression that is significantly associated with increased cell malignancy and aggressiveness.Material and methodsWe investigated the effect of ethanol on STARD10-ERBB2 cross-talk in breast cancer cells, MMTV-neu transgenic mice and in clinical ERBB2-positive breast cancer specimens with Western Blotting and Real-time PCR. We also examined the effects of their knockdown and overexpression on transient transfected breast cancer cells using promoter activity, MTT, cell migration, calcium and membrane fluidity assays in vitro.ResultsEthanol administration induces STARD10 and ERBB2 expression in vitro and in vivo. ERBB2 overexpression causes an increase in STARD10 expression, while overexpression of ERBB2’s downstream targets, p65, c-MYC, c-FOS or c-JUN induces STARD10 promoter activity, correlative of enhanced ERBB2 function. Ethanol and STARD10-mediated cellular membrane fluidity and intracellular calcium concentration impact ERBB2 signaling pathway as evaluated by enhanced p65 nuclear translocation and binding to both ERBB2 and STARD10 promoters.ConclusionOur finding proved that STARD10 and ERBB2 positively regulate each other’s expression and function. Taken together, our data demonstrate that ethanol can modulate ERBB2’s function in breast cancer via a novel interplay with STARD10.

Biochemical studies evaluating the chemopreventive potential of brucine in chemically induced mammary carcinogenesis of rats

The present study was aimed to investigate the dose dependent chemopreventive activity of brucine against 7, 12-dimethylbenz (a) anthracene induced mammary gland tumorigenesis in rats. The mammary tumor was induced by a single dose of DMBA (25 mg/rat) injected subcutaneously near the mammary gland. We observed reduced body weight and increase in tumor incidence, the total number of tumors, and tumor volume in DMBA alone injected rats and also observed decreased antioxidant status (SOD, CAT, GPX, and GSH) and increased lipid peroxidation (TBARS and LOOH) in plasma and mammary tissues. Increased levels of CYP450, Cyt-b5 and decreased levels of phase II (GST and GR) biotransformation enzymes were noticed in the liver and mammary tissues. Further, increased levels of lipid profile (TC, TG, PL, and FFA) and lipoprotein (LDL and VLDL) were noticed. Whereas, decrease in the levels of HDL in plasma and decreased levels of PL and FFA in mammary tissues were observed. Oral administration of brucine in different doses (2, 4 and 8 mg/kg bw) inhibited the tumor incidence and restored the levels of biochemical markers near to normal in dose responsive manner. Biochemical findings are supported by histopathological studies. The results suggest that brucine at a dose of 8 mg/kg bw shows more significant chemopreventive activity in DMBA-induced mammary carcinogenesis.

Mouse Models of Overexpression Reveal Distinct Oncogenic Roles for Different Type I Protein Arginine Methyltransferases.

Protein arginine methyltransferases (PRMT) are generally not mutated in diseased states, but they are overexpressed in a number of cancers, including breast cancer. To address the possible roles of PRMT overexpression in mammary gland tumorigenesis, we generated Cre-activated PRMT1, CARM1, and PRMT6 overexpression mouse models. These three enzymes are the primary type I PRMTs and are responsible for the majority of the asymmetric arginine methylation deposited in the cells. Using either a keratin 5-Cre recombinase (K5-Cre) cross or an MMTV-NIC mouse, we investigated the impact of PRMT overexpression alone or in the context of a HER2-driven model of breast cancer, respectively. The overexpression of all three PRMTs induced hyper-branching of the mammary glands and increased Ki-67 staining. When combined with the MMTV-NIC model, these in vivo experiments provided the first genetic evidence implicating elevated levels of these three PRMTs in mammary gland tumorigenesis, albeit with variable degrees of tumor promotion and latency. In addition, these mouse models provided valuable tools for exploring the biological roles and molecular mechanisms of PRMT overexpression in the mammary gland. For example, transcriptome analysis of purified mammary epithelial cells isolated from bigenic NIC-PRMT1 Tg and NIC-PRMT6 Tg mice revealed a deregulated PI3K-AKT pathway. In the future, these PRMT Tg lines can be leveraged to investigate the roles of arginine methylation in other tissues and tumor model systems using different tissue-specific Cre crosses, and they can also be used for testing the in vivo efficacy of small molecule inhibitors that target these PRMT. SIGNIFICANCE: These findings establish Cre-activated mouse models of three different arginine methyltransferases, PRMT1, CARM1, and PRMT6, which are overexpressed in human cancers, providing a valuable tool for the study of PRMT function in tumorigenesis.See related commentary by Watson and Bitler, p. 3.

Emerging Role of Novel Biomarkers of Ly6 Gene Family in Pan Cancer.

Stem cell antigen-1 (Sca-1) is the first identified member of mouse Ly6 gene family. We discovered that Sca-1 disrupts TGFβ signaling and enhances mammary tumorigenesis in a DMBA-induced mammary tumor model. Sca-1 gene is lost during evolution in humans. Human Ly6 genes Ly6D, LyE, LyH, and LyK on human chromosome 8q24.3 genes are syntenic to the mouse chromosome 15 where Sca-1 is located. We found that Ly6D, E, H, and K are upregulated in human cancer compared to normal tissue and that the increased expression of these genes are associated with poor prognosis of multiple types of human cancer. Several other groups have indicated increased expression of Ly6 genes in human cancer. Here we described the relevance of expression of human Ly6D, LyE, LyH, and LyK in functioning of normal tissues and tumor progression.

Microfluidic MeDIP-seq for low-input methylomic analysis of mammary tumorigenesis in mice.

Studies of dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, we developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ∼100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled us to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using our data, we identified differentially methylated regions and their associated genes in different periods of cancer development. Our results showed that unique methylomic features were presented in various tumor developmental stages.

Global transcriptional analysis identifies a novel role for SOX4 in tumor-induced angiogenesis.

The expression of the transcription factor SOX4 is increased in many human cancers, however, the pro-oncogenic capacity of SOX4 can vary greatly depending on the type of tumor. Both the contextual nature and the mechanisms underlying the pro-oncogenic SOX4 response remain unexplored. Here, we demonstrate that in mammary tumorigenesis, the SOX4 transcriptional network is dictated by the epigenome and is enriched for pro-angiogenic processes. We show that SOX4 directly regulates endothelin-1 (ET-1) expression and can thereby promote tumor-induced angiogenesis both in vitro and in vivo. Furthermore, in breast tumors, SOX4 expression correlates with blood vessel density and size, and predicts poor-prognosis in patients with breast cancer. Our data provide novel mechanistic insights into context-dependent SOX4 target gene selection, and uncover a novel pro-oncogenic role for this transcription factor in promoting tumor-induced angiogenesis. These findings establish a key role for SOX4 in promoting metastasis through exploiting diverse pro-tumorigenic pathways.

Loss of Limb-Bud-and-Heart (LBH) attenuates mammary hyperplasia and tumor development in MMTV-Wnt1 transgenic mice

WNT/β-catenin signaling plays pivotal roles in mammary development and tumorigenesis; and aberrant activation of this pathway is frequently observed in human breast cancer, correlating with poor outcome. However, the mechanisms underlying WNT-driven mammary tumorigenesis remain incompletely understood. Here, we used mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice, which develop aggressive mammary adenocarcinomas, to examine whether Limb-Bud-and-Heart (LBH) - a WNT/β-catenin target transcription co-factor overexpressed in human triple-negative breast cancers with WNT pathway hyperactivation, contributes to WNT-induced tumorigenesis. We found LBH is specifically overexpressed in basal epithelial tumor cells of MMTV-Wnt1 mammary tumors reminiscent of its basal cell-restricted expression in the normal postnatal mammary gland. To determine the role of LBH in mammary tumorigenesis, we crossed MMTV-Wnt1 mice with basal epithelial-specific Keratin 14/K14-Cre;LbhloxP knockout mice. Mammary glands from virgin LBH-deficient MMTV-Wnt1 mice exhibited reduced hyperplasia, cell proliferation and increased apoptosis. Importantly, LBH inactivation in mammary epithelium significantly delayed tumor onset in MMTV-Wnt1 transgenic mice, with a median tumor-free survival of 32.5 weeks compared to 22.5 weeks in control LBH wild type MMTV-Wnt1 mice (p < 0.05). This data provides the first evidence that LBH plays an essential role in WNT-induced mammary tumorigenesis by promoting hyperplastic growth and tumor formation.

Deletion of the murine ortholog of the 8q24 gene desert has anti-cancer effects in transgenic mammary cancer models

BackgroundThe gene desert on human chromosomal band 8q24 harbors multiple genetic variants associated with common cancers, including breast cancer. The locus, including the gene desert and its flanking genes, MYC, PVT1 and FAM84B, is also frequently amplified in human breast cancer. We generated a megadeletion (MD) mouse model lacking 430-Kb of sequence orthologous to the breast cancer-associated region in the gene desert. The goals were to examine the effect of the deletion on mammary cancer development and on transcript level regulation of the candidate genes within the locus.MethodsThe MD allele was engineered using the MICER system in embryonic stem cells and bred onto 3 well-characterized transgenic models for breast cancer, namely MMTV-PyVT, MMTV-neu and C3(1)-TAg. Mammary tumor growth, latency, multiplicity and metastasis were compared between homozygous MD and wild type mice carrying the transgenes. A reciprocal mammary gland transplantation assay was conducted to distinguish mammary cell-autonomous from non-mammary cell-autonomous anti-cancer effects. Gene expression analysis was done using quantitative real-time PCR. Chromatin interactions were evaluated by 3C. Gene-specific patient outcome data were analysed using the METABRIC and TCGA data sets through the cBioPortal website.ResultsMice homozygous for the MD allele are viable, fertile, lactate sufficiently to nourish their pups, but maintain a 10% lower body weight mainly due to decreased adiposity. The deletion interferes with mammary tumorigenesis in mouse models for luminal and basal breast cancer. In the MMTV-PyVT model the mammary cancer-reducing effects of the allele are mammary cell-autonomous. We found organ-specific effects on transcript level regulation, with Myc and Fam84b being downregulated in mammary gland, prostate and mammary tumor samples. Through analysis using the METABRIC and TCGA datasets, we provide evidence that MYC and FAM84B are frequently co-amplified in breast cancer, but in contrast with MYC, FAM84B is frequently overexpressed in the luminal subtype, whereas MYC activity affect basal breast cancer outcomes.ConclusionDeletion of a breast cancer-associated non-protein coding region affects mammary cancer development in 3 transgenic mouse models. We propose Myc as a candidate susceptibility gene, regulated by the gene desert locus, and a potential role for Fam84b in modifying breast cancer development.

Conditional knockout of SHP2 in ErbB2 transgenic mice or inhibition in HER2-amplified breast cancer cell lines blocks oncogene expression and tumorigenesis.

Overexpression of the human epidermal growth factor receptor 2 (HER2) is the cause of HER2-positive breast cancer (BC). Although HER2-inactivating therapies have benefited BC patients, development of resistance and disease recurrence have been the major clinical problems, pointing to a need for alternative therapeutic strategies. For that to happen, proteins that play critical roles in the biology of HER2-induced tumorigenesis have to be identified and characterized. Here, we show that the Src homology phosphotyrosyl phosphatase 2 (Shp2) encoded by the Ptpn11 gene is a requisite for ErbB2-induced tumorigenesis. We report that conditional knockout of Shp2 alleles in the ErbB2 BC model mice abrogates mammary tumorigenesis by blocking the expression of the ErbB2 transgene. We also show that inhibition of SHP2 encoded by the PTPN11 gene in the HER2-amplified BC cells induces a normal-like cellular phenotype and suppresses tumorigenesis and metastasis by blocking HER2 overexpression. These findings demonstrate that ErbB2-induced tumors in mice or xenograft tumors induced by transplantation of HER2-amplified BC cells are vulnerable to SHP2 inhibition since it abrogates the expression of the very oncogene that causes of the disease. This report paves the way for developing SHP2-targeting therapies for BC treatment in the future.