Incidence Of Mammary Tumors Research Articles

Elevated EDAR signalling promotes mammary gland tumourigenesis with squamous metaplasia

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong β-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes β-catenin. Deletion of this exon yields unconstrained β-catenin signalling activity. We also demonstrate that β-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which β-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways.

Coiled-Coil Domain: Uncoiling Tumor Suppression by BRCA1.

The coiled-coil domain of BRCA1 is essential for its interaction with partner and localizer of BRCA2 (PALB2). In mice, loss of this interaction is known to result in Fanconi anemia-associated phenotypes. In a study published in this issue of Cancer Research, Pulver and colleagues from the Jonkers lab have generated a mouse model with a leucine to proline change in codon 1363 in the coiled-coil domain of BRCA1 (Brca1LP ), which disrupts its binding with PALB2. Unlike the previously reported viable coiled-coil defective mice, homozygous Brca1LP/LP mutant mice die during embryogenesis. The authors examined the role of the BRCA1/PALB2 interaction on mammary tumorigenesis and reported increased incidence of mammary tumors that are carcinosarcomas or sarcomatoids, unlike the adenocarcinomas that are characteristic mammary tumor types associated with loss of Brca1 and Trp53 in mice. The findings reveal the relevance of the coiled-coil domain in mammary tumor suppression by BRCA1.See related article by Pulver et al., p. 6171.

Inhibition of Antiestrogen-Promoted Pro-Survival Autophagy and Tamoxifen Resistance in Breast Cancer through Vitamin D Receptor.

We determined how vitamin D receptor (VDR) is linked to disease outcome in estrogen receptor-positive (ER+) breast cancer patients treated with tamoxifen (TAM). Breast cancer patients (n = 581) in four different datasets were divided into those expressing higher (above median) and lower levels of VDR in pretreatment ER+ tumors. Across all datasets, TAM-treated patients with higher pretreatment tumor VDR expression exhibited significantly longer recurrence-free survival. Ingenuity pathway analysis identified autophagy and unfolded protein response (UPR) as top differentially expressed pathways between high and low VDR-expressing ER+ cancers. Activation of VDR with vitamin D (VitD), either calcitriol or its synthetic analog EB1089, sensitized MCF-7-derived, antiestrogen-resistant LCC9 human breast cancer cells to TAM, and attenuated increased UPR and pro-survival autophagy. Silencing of VDR blocked these effects through the IRE1α-JNK pathway. Further, silencing of VDR impaired sensitivity to TAM in antiestrogen-responsive LCC1 cells, and prevented the effects of calcitriol and EB1089 on UPR and autophagy. In a preclinical mouse model, dietary VitD supplementation induced VDR activation and reduced carcinogen-induced ER+ mammary tumor incidence. In addition, IRE1α-JNK signaling was downregulated and survival autophagy was inhibited in mammary tumors of VitD-supplemented mice. Thus, activation of VDR is predictive of reduced risk of breast cancer recurrence in ER+ patients, possibly by inhibiting antiestrogen-promoted pro-survival autophagy.

Current invasive and non-invasive biomarkers in canine mammary tumors

In bitches, the incidence of mammary tumors is determined at %50 of all neoplasms. Most of the cases are malignant and tend to metastasize. Because of this, early diagnosis has an important value. Due to the molecular and clinical similarities of canine mammary tumors to human breast cancers, human breast cancer markers are also detectable in cases of canine mammary tumors. Generally, biomarkers provide information about criteria such as clinical diagnosis, early diagnosis, prognosis, and determination of the treatment protocol. When choosing the appropriate biomarker, it should be taken into account that it is easy to apply, has a positive correlation with tumor specificity and malignancy, and whether gives precise information about prognosis. Due to the heterogeneous nature of canine mammary tumors, an ideal biomarker has not determined yet. However, new studies have revealed the existence of many biomarkers. In this review it is given to qualified and current biomarkers can be used in dogs, including invasive and non-invasive mammary tumor biomarkers.

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.

Attenuated TGFB signalling in macrophages decreases susceptibility to DMBA-induced mammary cancer in mice

BackgroundTransforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates mammary gland development and cancer progression through endocrine, paracrine and autocrine mechanisms. TGFB1 also plays roles in tumour development and progression, and its increased expression is associated with an increased breast cancer risk. Macrophages are key target cells for TGFB1 action, also playing crucial roles in tumourigenesis. However, the precise role of TGFB-regulated macrophages in the mammary gland is unclear. This study investigated the effect of attenuated TGFB signalling in macrophages on mammary gland development and mammary cancer susceptibility in mice.MethodsA transgenic mouse model was generated, wherein a dominant negative TGFB receptor is activated in macrophages, in turn attenuating the TGFB signalling pathway specifically in the macrophage population. The mammary glands were assessed for morphological changes through wholemount and H&E analysis, and the abundance and phenotype of macrophages were analysed through immunohistochemistry. Another cohort of mice received carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), and tumour development was monitored weekly. Human non-neoplastic breast tissue was also immunohistochemically assessed for latent TGFB1 and macrophage marker CD68.ResultsAttenuation of TGFB signalling resulted in an increase in the percentage of alveolar epithelium in the mammary gland at dioestrus and an increase in macrophage abundance. The phenotype of macrophages was also altered, with inflammatory macrophage markers iNOS and CCR7 increased by 110% and 40%, respectively. A significant decrease in DMBA-induced mammary tumour incidence and prolonged tumour-free survival in mice with attenuated TGFB signalling were observed. In human non-neoplastic breast tissue, there was a significant inverse relationship between latent TGFB1 protein and CD68-positive macrophages.ConclusionsTGFB acts on macrophage populations in the mammary gland to reduce their abundance and dampen the inflammatory phenotype. TGFB signalling in macrophages increases mammary cancer susceptibility potentially through suppression of immune surveillance activities of macrophages.

Prolactin synergizes with canonical Wnt signals to drive development of ER+ mammary tumors via activation of the Notch pathway.

Prolactin (PRL) cooperates with other factors to orchestrate mammary development and lactation, and is epidemiologically linked to higher risk for breast cancer. However, how PRL collaborates with oncogenes to foster tumorigenesis and influence breast cancer phenotype is not well understood. To understand its interactions with canonical Wnt signals, which elevate mammary stem cell activity, we crossed heterozygous NRL-PRL mice with ApcMin/+ mice and treated pubertal females with a single dose of mutagen. PRL in the context of ApcMin/+ fueled a dramatic increase in tumor incidence in nulliparous mice, compared to ApcMin/+ alone. Although carcinomas in both NRL-PRL/ApcMin/+ and ApcMin/+ females acquired a mutation in the remaining wildtype Apc allele and expressed abundant β-catenin, PRL-promoted tumors displayed higher levels of Notch-driven target genes and Notch-dependent cancer stem cell activity, compared to β-catenin-driven activity in ApcMin/+ tumors. This PRL-induced shift to dominant Notch signals was evident in preneoplastic epithelial hyperplasias at 120 days of age. In NRL-PRL/ApcMin/+ females, rapidly proliferating hyperplasias, characterized by β-catenin at cell junctions and high NOTCH1 expression, contrasted with slower growing lesions with nuclear β-catenin in ApcMin/+ females. These studies demonstrate that PRL can powerfully modulate the incidence and phenotype of mammary tumors, shedding light on mechanisms whereby PRL elevates risk of breast cancer.

COX-2 promotes mammary adipose tissue inflammation, local estrogen biosynthesis, and carcinogenesis in high-sugar/fat diet treated mice.

Obesity is a major risk factor for breast cancer, especially in post-menopausal women. In the breast tissue of obese women, cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production has been correlated with inflammation and local estrogen biosynthesis via aromatase. Using a mouse model of 7,12-dimethylbenz[a]anthracene/medroxyprogesterone-acetate (DMBA/MPA)-induced carcinogenesis, we demonstrated that an obesogenic diet promotes mammary tissue inflammation and local estrogen production, and accelerates mammary tumor formation in a COX-2-dependent manner. High-sugar/fat (HSF) diet augmented the levels of the pro-inflammatory mediators MCP-1, IL-6, COX-2, and PGE2 in mammary tissue, and this was accompanied by crown-like structures of breast (CLS-B) formation and aromatase/estrogen upregulation. Treatment with a COX-2 selective inhibitor, etoricoxib, decreased PGE2, IL-6, MCP-1, and CLS-B formation as well as reduced aromatase protein and estrogen levels in the mammary tissue of mice fed a HSF diet. Etoricoxib-treated mice showed increased latency and decreased incidence of mammary tumors, which resulted in prolonged animal survival when compared to HSF diet alone. Inhibition of tumor angiogenesis also seemed to account for the prolonged survival of COX-2 inhibitor-treated animals. In conclusion, obesogenic diet-induced COX-2 is sufficient to trigger inflammation, local estrogen biosynthesis, and mammary tumorigenesis.

Characterization of weaning-induced breast involution in women: implications for young women\u2019s breast cancer

In rodents, weaning-induced mammary gland involution supports increased mammary tumor incidence, growth, and progression to metastasis. Further, the protumor attributes of gland involution are COX-2 dependent and mitigated by short-duration non-steroidal anti-inflammatory drugs (NSAIDs), suggesting a potential prevention strategy. However, the transition from lactation to postweaning breast involution has not been rigorously evaluated in healthy women. Here we queried breast biopsies from healthy women (n = 112) obtained at nulliparity, lactation, and multiple postweaning time points using quantitative immunohistochemistry. We found that mammary remodeling programs observed in rodents are mirrored in the human breast. Specifically, lactation associates with the expansion of large, secretory mammary lobules and weaning associates with lobule loss concurrent with epithelial cell death and stromal hallmarks of wound healing, including COX-2 upregulation. Altogether, our data demonstrate that weaning-induced breast involution occurs rapidly, concurrent with protumor-like attributes, and is a potential target for NSAID-based breast cancer prevention.

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer.

SummaryThe receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl+ cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.

Abstract 3685: Sustained Ret expression during mammary gland post-lactation induces premature involution and enhances cancer potential

Abstract Loss of normal development is a hallmark of cancer. Thus, understanding the mechanisms of tissue-specific developmental regulation and the changes that occur during tumorigenesis may provide insights of both diagnostic and therapeutic importance. In breast cancer, several members of the receptor tyrosine kinases (RTK) family that are well known to promote aggressive breast cancers also have roles in normal breast. We found that Ret, a RTK member, is normally expressed in the mouse glands in lactation. We determined that inhibition of Ret activity in vivo does not alter lactation, however impacts in the transition to involution. Involution is the period with high inflammation which returns the lactating mammary gland to a quiescent state after weaning. Involution has been well described as a post-lactation stage that drives cancer progression. Ret is overexpressed in about 40% of human breast tumors. Previously, using a doxycycline-inducible transgenic mouse model (Ret/MTB) we determined that chronic expression of Ret is oncogenic in the mammary epithelium. However, the stage of development at which Ret expression results in increase mammary tumor incidence has not been identified. To address this, we used the Ret/MTB system, to conditionally overexpress Ret during discrete periods of mammary gland development. We found that Ret is required for efficient transition to involution. We determined that the induction of Ret in Ret/MTB females promotes the expression of factors that drives involution, including premature Stat3 activation. RNA-seq data in Ret-overexpressing glands is supporting these findings, which were confirmed by several techniques. In addition, sustained expression of Ret during post-lactation enhances cancer potential showing a significant increase in pre-neoplastic lesions, defective milk recycling and disrupting Stat3 signaling. These results demonstrate that Ret deregulation increases cancer potential in post-lactation and might be considered as a prognostic marker for post-partum breast cancer. Citation Format: Sabrina A. Vallone, Martín García Solá, Robert D. Cardiff, Roberto P. Meiss, Lewis A. Chodosh, Carolina Shere-Levy, Edith C C. Kordon, Nancy E. Hynes, Albana Gattelli. Sustained Ret expression during mammary gland post-lactation induces premature involution and enhances cancer potential [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3685.

Isolated Soy Protein Promotes Mammary Tumor Development Induced by the Type I Insulin-like Growth Factor Receptor in Transgenic Mice

Studies suggest consuming soy may protect women from breast cancer. In this study, lifetime exposure to 20%, 5% and 1% ISP in MTB-IGFIR mice (mammary-specific expression of IGF-IR) were evaluated to determine whether ISP could protect against mammary tumorigenesis. MTB-IGFIR mice fed ISP diets displayed increased mammary tumor incidence and reduced tumor latency compared to mice fed 20% casein. To evaluate whether a diet containing a less refined form of soy could protect against mammary tumor development MTB-IGFIR mice were fed Teklad 2018 (contains soybean meal). MTB-IGFIR mice fed the Teklad 2018 diet were completely protected against mammary tumor development. To determine whether dietary ISP was sufficient to induce mammary tumorigenesis, MTB-IGFIR mice were fed Teklad 2018ISP (soybean meal of Teklad 2018 was replaced with an equivalent amount of ISP). Only two of 10 MTB-IGFIR mice fed Teklad 2018ISP developed mammary tumors. This study demonstrates the complex interaction between soy and other dietary components in modifying mammary tumor development.

Abstract A03: Preconception paternal DDT exposure and programming of metabolic dysfunction and breast cancer in offspring

Abstract The pesticide dichlorodiphenyltrichloroethane or DDT was widely used in agriculture or to combat insect-borne diseases until the 1970s in the United States and the 1980s in Europe. It continues to be used in developing countries to combat malaria or illegally to protect crops. Because of its prolonged half-life, DDT is a persistent environmental pollutant that still can detected in the American population, particularly in minorities and recent immigrants. Epidemiologic studies have shown that early-life (in utero and early childhood) exposure to DDT is associated with a four-fold increase in breast cancer risk in women, and these findings are supported by animal studies. While maternal DDT exposure in pregnancy has been linked to breast cancer in offspring, the effects of paternal DDT exposure on their progeny’s breast cancer development have not been investigated. Environmental exposures can induce epigenetic changes in paternal sperm and affect the risk of disease in their offspring. Here, we evaluated the effects of preconception exposure to DDT on the male sperm epigenome and programming of offspring’s susceptibility to breast cancer, using a mouse model. Male mice were exposed to DDT (1.7mg/kg body weight) by oral gavage in the preconception window. DDT and control-vehicle (CO) males were then used for sperm collection or housed with female mice, with free access to a standard chow diet, for three days. Pregnancy onset was assessed by the presence of a vaginal plug. The weight and number of pups per litter were determined two days after birth. Pups were weaned at 21 days of age and fed a standard chow diet for the extent of the study. Offspring were weighed weekly and used to study metabolic parameters. Mammary tumors were induced by administration of 15mg of medroxyprogesterone to six-week-old female offspring, followed by oral administration of 1mg 7,12-dimethylbenz[a]anthracene once a week for 3 weeks. RNA extracted from sperm CO and DDT males was used for small RNA-seq analysis. Preconception exposure to DDT altered small noncoding RNAs (e.g., miRNAs and tRNAs) in paternal sperm. In line with that, offspring of DDT-exposed fathers had a significant decrease in birth and weaning weights compared to controls. Further, DDT offspring showed metabolic dysfunction. Compared to CO, female offspring of DDT fathers had significantly accelerated breast tumor growth and a nonsignificant increase in mammary tumor incidence and shorter tumor latency. These tumor phenotypes were linked to mammary tissue inhibition of AMPK, a central metabolic sensor, and increased mTOR activity. In conclusion, our findings support a role for DDT exposure from the paternal lineage in metabolic dysfunction and increased breast cancer development in the offspring. Defining the mechanism by which paternal exposures to EDC such as DDT can promote breast cancer development in offspring is critical to identifying early detection/preventive approaches or treatments for this malignancy in a subset of women. Citation Format: Raquel Santana da Cruz, Hong Cao, Camile Fontelles, Apsra Nasir, Volkan Tekam, M. Idalia Cruz, Lu Jin, Sonia de Assis. Preconception paternal DDT exposure and programming of metabolic dysfunction and breast cancer in offspring [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A03.

Insights from the in silico structural, functional and phylogenetic characterization of canine lysyl oxidase protein

BackgroundLysyl oxidase is an extracellular regulatory enzyme with an imperative role in interlinking of collagen and elastin by oxidizing lysine residues. Lysyl oxidase has been implicated in incidence of mammary tumors in bitches. Therefore, it becomes significant to study the structural and functional features of this enzyme for a better understanding of its molecular mechanisms. ResultsThe detailed computational investigation of the canine lysyl oxidase protein was analyzed in silico with respect to its physicochemical properties, secondary and tertiary structure predictions and functional analysis using standard bioinformatic tools. Lysyl oxidase is a flexible protein with an average molecular weight of around 46 kDa, unstable, hydrophilic, and extracellular (secretory) in nature. Twelve cysteine residues and a disulfide bridge were also found. Secondary structure analysis shows that most of the protein has predominant coiled configuration. A putative copper-binding region signature was predicted. The phylogenetic relationship of canine lysyl oxidase with a vast range of mammalian species indicates that the protein was very well conserved throughout the course of evolution. Top 10 interacting proteins were identified using STRING v10.0 analysis, elastin being the closest interacting protein. Functional analysis by InterproScan predicted protein’s biological role in oxidation-reduction process. ConclusionUnderstanding the structural and functional properties of the protein will facilitate a better understanding of its mechanism of enzyme action. Further, the predicted 3D model will serve as a cornerstone for further understanding towards the tumorigenesis potential of the protein.

Abstract B27: Modeling the impact of miR-200s on mammary tumor initiation and progression in vitro and in vivo

Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that regulate mRNA translation through inhibiting the translation of mRNAs into proteins or through degrading mRNAs. Each miRNA potentially targets hundreds of mRNAs, and thus an individual miRNA can regulate entire gene networks. The miR-200 family consists of 5 miRNAs (miR-141, miR-200a, miR-200b, miR-200c, miR-429) organized into two clusters: the miR-200c/141 cluster and the miR-200b/200a/429 cluster. The miR-200s regulate a variety of cellular functions, with the best characterized being their ability to target mesenchymal transcription factors and thus maintain an epithelial phenotype. Given that miR-200s maintain an epithelial phenotype, we evaluated their role in mesenchymal mammary tumors. All five miR-200s were expressed at significantly lower levels in the murine mesenchymal mammary tumor cell line RJ423 and the human mesenchymal breast cancer cell line MDA-MB-231 compared to epithelial tumor cell lines. To evaluate the function of miR-200s, the miR-200c/141 and miR-200b/200a/429 clusters were re-expressed in the mesenchymal mammary tumors. Re-expression of the miR-200c/141 cluster partially restored an epithelial phenotype in MDA-MB-231 cells while the miR-200b/200a/429 cluster partially restored an epithelial phenotype in RJ423 cells. Re-expression of the miR-200c/141 cluster in MDA-MB-231 cells and re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed tumor growth following intramammary injection and altered the tumor microenvironment. To identify genes regulated by miR-200 expression, RNA sequencing was performed on control and miR-200 re-expressing cells grown in 2D culture and following intramammary injection. Interestingly, the in vivo environment dramatically altered the gene repertoire regulated by the miR-200s. Hierarchical clustering revealed that alterations in gene expression induced by miR-200 re-expression in vitro poorly correlated with the alterations in gene expression induced by miR-200 in vivo. Since miR-200 re-expression significantly decreased tumor growth in vivo, a transgenic model was used to determine whether miR-200s could also impact tumor initiation. Using doxycycline-inducible miR-200 expression in combination with the mammary tumor oncogene, IGF-IR, we found that overexpression of the miR-200b/200a/429 cluster dramatically reduced mammary tumor incidence (100% vs. 19%). These data show the potent impact miR-200s have on mammary tumorigenesis and that gene expression profiling of 2D cultures is a poor predictor of genes regulated by miR-200s in vivo. Citation Format: Katrina L. Watson, Kaitlyn Simpson, Majesta Roth, Courtney Martin, Garret Conquer-Van Heumen, Roger A. Moorehead. Modeling the impact of miR-200s on mammary tumor initiation and progression in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B27.

Increased insulin receptor binding and increased IGF-1 receptor binding are linked with increased growth of L6hIR cell xenografts in vivo

Insulin analogue X10 has a higher mitogenic potency than native human insulin in vitro and supra-pharmacological doses of insulin X10 increased the incidence of mammary tumours in rats. Compared to native human insulin, insulin X10 has increased binding affinity to the insulin receptor and the IGF-1 receptor, but it is not known whether either or both characteristics are important for stimulation of cell proliferation in vivo. The aim of this study was to explore how increased binding affinity to the insulin receptor or the IGF-1 receptor contributes to stimulation of cell proliferation in vivo. A mouse xenograft model was established with rat L6 myoblast cells transfected with the human insulin receptor (L6hIR cells) and effects of supra-pharmacological doses of native human insulin, insulin X10 or novel insulin analogues with increased binding affinity to either the insulin receptor or the IGF-1 receptor were examined. Treatment with insulin X10 and insulin analogues with increased binding affinity to either the insulin receptor or the IGF-1 receptor increased growth of L6hIR cell xenografts significantly compared to native human insulin. Thus, increased binding affinity to the insulin receptor and the IGF-1 receptor are each independently linked to increased growth of L6hIR cell xenografts in vivo.

Reversal of increased mammary tumorigenesis by valproic acid and hydralazine in offspring of dams fed high fat diet during pregnancy

Maternal or paternal high fat (HF) diet can modify the epigenome in germ cells and fetal somatic cells leading to an increased susceptibility among female offspring of multiple generations to develop breast cancer. We determined if combined treatment with broad spectrum DNA methyltransferase (DNMT) inhibitor hydralazine and histone deacetylase (HDAC) inhibitor valproic acid (VPA) will reverse this increased risk. C57BL/6 mouse dams were fed either a corn oil-based HF or control diet during pregnancy. Starting at age 7 weeks, female offspring were administered 3 doses of 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. After last dose, offspring started receiving VPA/hydralazine administered via drinking water: no adverse health effects were detected. VPA/hydralazine reduced mammary tumor multiplicity and lengthened tumor latency in HF offspring when compared with non-treated HF offspring. The drug combination inhibited DNMT3a protein levels and increased expression of the tumor suppressor gene Cdkn2a/p16 in mammary tumors of HF offspring. In control mice not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden, and elevated expression of the unfolded protein response and autophagy genes, including HIF-1α, NFkB, PERK, and SQSTM1/p62. Expression of these genes was already upregulated in HF offspring prior to VPA/hydralazine treatment. These findings suggest that breast cancer prevention strategies with HDAC/DNMT inhibitors need to be individually tailored.

Arsenite and cadmium promote the development of mammary tumors.

Previous studies demonstrate that the heavy metal cadmium and the metalloid arsenite activate estrogen receptor-alpha in breast cancer cells by forming a high-affinity complex with the ligand-binding domain of the receptor and that environmentally relevant doses of cadmium have estrogen-like activity in vivo. The present study showed that in estrogen-receptor positive cells, arsenite and cadmium increased the global expression of estrogen-responsive genes and that an environmentally relevant dose of arsenite also had estrogen-like activity in vivo. Similar to estrogens, exposure of ovariectomized animals to arsenite induced the expression of the progesterone receptor, GREB1, and c-fos in the mammary gland and the expression of complement C3, c-fos, and cyclin D1 in the uterus and the increase was blocked by the antiestrogen ICI-182,780. When virgin female animals were fed a diet, that mimics exposure to either arsenite or cadmium, and challenged with the chemical carcinogen dimethylbenzanthracene, there was an increase in the incidence of mammary tumors and a decrease in the time to tumor onset, but no difference in the total number of tumors, tumor multiplicity, or total tumor volume. Together with published results, these data showed that environmentally relevant amounts of arsenite and cadmium had estrogen-like activity in vivo and promoted mammary tumorigenesis.

Carcinogenic potential of fluorinated estrogens in mammary tumorigenesis

Fluorination preventing metabolic hydroxylation of 17β-estradiol (E2) was applied to investigate the mechanisms underlying estrogen-induced carcinogenesis. Either 2-fluoro-17β-estradiol (2-FE2) or 4-fluoro-17β-estradiol (4-FE2) was administered subcutaneously for 52 weeks to August Copenhagen Irish (ACI) rats, the preferred animal model for human breast cancer. 4-FE2 induced frequent mammary tumors whereas 2-FE2 did not. The cumulative incidence of mammary tumors in rats treated with 4-FE2 was comparable to that observed with E2. The carcinogenic results were supported by histological examination of mammary glands of fluorinated estrogen-treated ACI rats. To evaluate the estrogenic potential of the fluorinated estrogens, 2-FE2 or 4-FE2 was administrated subcutaneously to ovariectomized rats. Both 4-FE2 and 2-FE2 showed high uterotrophic potency. Our results indicate that estrogenic potential may not be the sole factor driving mammary tumorigenesis. Since fluorination inhibits metabolic hydroxylation of E2 at the substituted position, the carcinogenic effect may occur through the metabolic activation of 2-hydroxylated E2, in combination with the compound’s estrogenic potency.

Rodent Models Assessing Mammary Tumor Prevention by Soy or Soy Isoflavones.

While epidemiological studies performed in Asian countries generally show that high levels of dietary soy are associated with reduced breast cancer risk, studies in Western countries have typically failed to show this correlation. In an attempt to model the preventative actions of soy on mammary tumor development, rodent models have been employed. Thirty-four studies were identified that evaluated the impact of soy products or purified soy isoflavones on mammary tumor initiation (studies evaluating established mammary tumors or mammary tumor cell lines were not included) and these studies were separated into mammary tumors induced by chemical carcinogens or transgenic expression of oncogenes based on the timing of soy administration. Regardless of when soy-based diets or purified isoflavones were administered, no consistent protective effects were observed in either carcinogen-induced or oncogene-induced mammary tumors. While some studies demonstrated that soy or purified isoflavones could reduce mammary tumor incidence, other studies showed either no effect or tumor promoting effects of soy products or isoflavones. Most importantly, only five studies found a decrease in mammary tumor incidence and six studies observed a decrease in tumor multiplicity, two relevant measures of the tumor preventative effects of soy or isoflavones. The variable outcomes of the studies examined were not completely surprising given that few studies employed the same experimental design. Future studies should be carefully designed to more accurately emulate soy consumption observed in Asian cultures including lifetime exposure to less refined soy products and potentially the incorporation of multigenerational feeding studies.