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.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5109-8) contains supplementary material, which is available to authorized users.
Pharmacologic inhibitors of poly(ADP-ribose) polymerase (PARP) putatively enhance radiation toxicity in cancer cells. Although there is considerable information on the molecular interactions of PARP and BRCA1- and BRCA2-deficient cancers, very little is known of the PARP inhibition effect upon cancers proficient in DNA double-strand break repair after ionizing radiation or after stalled replication forks. In this work, we investigate whether PARP inhibition by ABT-888 (veliparib) augments death-provoking effects of ionizing radiation, or of the topoisomerase I poison topotecan, within uterine cervix cancers cells harboring an unfettered, overactive ribonucleotide reductase facilitating DNA double-strand break repair and contrast these findings with ovarian cancer cells whose regulation of ribonucleotide reductase is relatively intact. Cell lethality of a radiation-ABT-888 combination is radiation and drug dose dependent. Data particularly highlight an enhanced topotecan-ABT-888 cytotoxicity, and corresponds to an increased number of unrepaired DNA double-strand breaks. Overall, our findings support enhanced radiochemotherapy toxicity in cancers proficient in DNA double-strand break repair when PARP is inhibited by ABT-888.
Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27 + quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b + mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b -/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b -/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b +/+ and Cdkn1b -/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk.
With the rising incidence of breast cancer cases in the population, it is imperative to understand mechanisms of susceptibility. Genome-wide association studies (GWAS) have identified many variants associated with breast cancer susceptibility. These loci have low penetrance and are common in the population. Some of the alleles are associated with specific subtypes of breast cancer and/or associate specifically within ancestral populations. Variants in the TOX3/LOC643714 locus on human chromosomal band 16q12.1 are strongly associated with risk to estrogen receptor-positive (ER+) breast cancer in cohorts of women of European ancestry. Remarkably, replication of the association of these same variants with breast cancer risk in women of African American ancestry failed, but other variants in a more distal area of the locus were found to be associated with risk. The correlated polymorphisms comprising these variants exist in noncoding regions, providing the possibility that these polymorphisms alter gene expression. The most likely causal gene, TOX3, encodes a transcription factor that has previously been found to regulate estrogen receptor alpha (ERα)-responsive genes in breast cancer cells. We hypothesize that noncoding breast cancer-associated polymorphisms on 16q12.1 regulate TOX3, subsequently modifying risk of developing ER+ breast cancer. To genetically dissect this locus, we implemented the CRISPR-Cas9 genetic engineering system in the rat model organism. The rat is the preferred rodent model for ER+ breast cancer. We engineered an allelic series of mutations generating rats with deletions in the portion of the Tox3 locus orthologous to the human risk-associated regions. We obtained viable mutants across all genotypes, despite partial embryonic lethality in homozygous Tox3 knockouts. The mutants in the allelic series display variable levels of Tox3 downregulation in the mammary gland, suggesting the presence of multiple Tox3-regulatory elements. Mutants showing altered Tox3 expression also show significant effects on mammary gland development, implicating a role for Tox3 in mammary stem/progenitor cell biology potentially through ERα gene regulation. In accordance with the association of the human risk-increasing allele with lower TOX3 transcript level, preliminary data from mammary carcinogenesis experiments in our rat model indicate that lower Tox3 levels result in increased mammary carcinoma multiplicity. This result suggests that Tox3 is a breast cancer susceptibility gene. Interestingly, Tox3 knockout rats present an obesity phenotype, male and female sterility, and a behavioral phenotype (increased anxiety), indicating pleiotropic effects of TOX3 loss-of-function mutations. Ongoing studies are focused on Tox3 in rat mammary gland biology and carcinogenesis to elucidate the mechanism of Tox3 regulation in susceptibility to ER+ breast cancer. Understanding mechanisms of susceptibility genes will ultimately lead to innovative strategies aimed at preventing breast cancer. Citation Format: Lauren B. Shunkwiler, Royal Pipaliya, Cody C. Ashy, Benjamine Van Peel, Yang Zhao, Jan Guz, Alexander Awgulewitsch, Michael J. Kern, Bart M.G. Smits. An allelic series of rat mutations reveal a role of TOX3 in mammary gland development, obesity, and breast cancer susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B29.
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