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Breast cancer is a very heterogeneous disease at clinical, histological, and molecular levels. It is the leading cause of cancer-related deaths among women. Breast cancer is manageable if diagnosed early at a localized stage, but late diagnosis of metastatic disease has a very low patient survival rate. Further, limited treatment options, insufficient prognostic and diagnostic markers, misdiagnosis and drug resistance pose a greater problem for patient survival and clinical outcome. Consequently, there is a great need to explore newer and more effective diagnostic, prognostic and therapeutic options for managing breast cancer. It is now a well-known fact that along with genetic changes, epigenetic modifications play an important role in the origin and pathogenesis of breast cancer. Universal involvement of epigenetic modifications in breast cancer development makes them useful for diagnosis, prognosis, and follow-up purposes. Further, the reversibility of epigenetic changes makes them attractive targets for breast cancer therapy. Therefore, in this chapter, we will discuss current knowledge on epigenetic involvement in the development of breast cancer and epi drugs as treatment options for breast cancer management.

The risk of cancer development in offspring due to carcinogen exposure during pregnancy is a serious issue. In this study, we explored the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and microRNA-21 (miR-21) in transplacental lung tumorigenesis and its prevention by dietary compound inositol hexaphosphate (IP6) in F1 mice. Balb/c mice were exposed to the N-ethyl-N-nitrosourea (ENU) intraperitoneally on the 17th day of gestation. After weaning, half of the litters were fed with oral 2% IP6. At the end of 30, 120, or 240 days, we did not observe any effect on fetal viability or weight between ENU-exposed and non-exposed litters and the same was true of IP6. Altered expressions of the PI3K/Akt pathway were observed in F1 mice. Further, miR-21 expressions were found to be modulated at the respective time as well, along with the activation of matrix metalloproteinase (MMP-9) and vascular endothelial growth factor expression. Akt activation also enhanced the expression of cyclin D1, cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-κBp50), and mammalian target of rapamycin (mTOR). IP6-fed F1 mice showed reduced tumorigenesis along with reduced expression of the PI3K/Akt pathway miR-21 and downstream targets. The PI3K/Akt pathway and miR-21 are involved in transplacental lung tumorigenesis, whereas IP6 seemed to affect lung tumorigenesis by suppressing the expression of the PI3K/Akt pathway in F1 mice.

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Skin cancer is among the most common cancers worldwide and identifiable molecular changes for early and late stage of skin tumorigenesis can suggest the better targets for its control. In this study, we investigated the status of K-Ras-PI3K-AKTpathway followed by NF-κB, cyclin D1, MMP-9 and regulatory micro RNA during 7, 12-dimethylbenz[a]anthracene (DMBA) induced mouse skin tumorigenesis and its prevention by butyric acid (BA), nicotinamide (NA) and calcium glucarate (CAG), individually or in combination with respect to time. DMBA upregulated the K-Ras, PI3K, Akt, NF-κB, cyclin D1 and MMP-9, but downregulated the PTEN in a time dependent manner. DMBA also reduced the levels of micoRNA let-7a but induced the levels of miR-21 and miR-20a as a function of time. BA, NA and CAG were found to prevent DMBA induced changes, but they were most effective when used together in a combination. Reduced let-7a and miR-211 were correlated with the overexpression of K-Ras and MMP-9. Overexpression of miR-21 and miR-20a was correlated with the down regulation of PTEN and overexpression of Cyclin D1. Collectively, the enhanced chemopreventive potential of natural compound in combination via regulation of K-Ras-PI3K-AKTpathway along with regulatory micro RNAs provide a newer and effective mean for cancer management.

Acrylamide, a probable human carcinogen, is ubiquitously present in the human environment, with sources including heated starchy foods, coffee and cigarette smoke. Humans are also exposed to acrylamide occupationally. Acrylamide is genotoxic, inducing gene mutations and chromosomal aberrations in various experimental settings. Covalent haemoglobin adducts were reported in acrylamide-exposed humans and DNA adducts in experimental systems. The carcinogenicity of acrylamide has been attributed to the effects of glycidamide, its reactive and mutagenic metabolite capable of inducing rodent tumors at various anatomical sites. In order to characterize the pre-mutagenic DNA lesions and global mutation spectra induced by acrylamide and glycidamide, we combined DNA-adduct and whole-exome sequencing analyses in an established exposure-clonal immortalization system based on mouse embryonic fibroblasts. Sequencing and computational analysis revealed a unique mutational signature of glycidamide, characterized by predominant T:A>A:T transversions, followed by T:A>C:G and C:G>A:T mutations exhibiting specific trinucleotide contexts and significant transcription strand bias. Computational interrogation of human cancer genome sequencing data indicated that a combination of the glycidamide signature and an experimental benzo[a]pyrene signature are nearly equivalent to the COSMIC tobacco-smoking related signature 4 in lung adenocarcinomas and squamous cell carcinomas. We found a more variable relationship between the glycidamide‐ and benzo[a]pyrene-signatures and COSMIC signature 4 in liver cancer, indicating more complex exposures in the liver. Our study demonstrates that the controlled experimental characterization of specific genetic damage associated with glycidamide exposure facilitates identifying corresponding patterns in cancer genome data, thereby underscoring how mutation signature laboratory experimentation contributes to the elucidation of cancer causation.A 40-word summaryInnovative experimental approaches identify a novel mutational signature of glycidamide, a metabolite of the probable human carcinogen acrylamide. The results may elucidate the cancer risks associated with exposure to acrylamide, commonly found in tobacco smoke, thermally processed foods and beverages.

We explored the basis of the combinatorial chemopreventive effect of butyric acid (BA), nicotinamide (NA) and calcium glucarate (CAG) on mouse skin exposed to 7,12-dimethylbenz(a)anthracene (DMBA). We studied the effects of topical application of DMBA in the presence or absence of BA, NA and CAG on the regulators of apoptosis. DMBA treatment suppressed Bax, Bax/Bcl-2 ratio, release of cyt c, Apaf1, caspase-9, -3 mediated apoptosis. Downregulation of p21 and upregulation of Bcl-2, mut p53 were also observed in only DMBA treated mice. Simultaneous application of BA, NA and CAG induced a mitochondria-mediated apoptosis, characterized by a rise in the Bax, Bax/Bcl-2 ratio, release of cyt c, upregulation of Apaf1 with down-stream activation of caspase-9, -3. Furthermore treatment with BA, NA and CAG demonstrated an upregulation of p21 and downregulation of Bcl-2, mut p53. But this effect was enhanced in the presence of all the three compounds together in combination. Chemoprevention by a combination of BA, NA and CAG by inducing the apoptosis, the natural cell death, suggest the importance of the potential combinational strategies capable of preventing skin tumor development.

In the present study, we showed the correlation of EZH2, SUV39H1 or G9a expression and histone modifications with the urethane induced mouse lung tumorigenesis in the presence or absence of antitumor agent, inositol hexaphosphate (IP6). Tumorigenesis and the molecular events involved therein were studied at 1, 4, 12 or 36 weeks after the exposure. There were no tumors at 1 or 4 weeks but tumors started appearing at 12 weeks and grew further till 36 weeks after urethane exposure. Among the molecular events, upregulation of EZH2 and SUV39H1 expressions appeared to be time dependent, but G9a expression was altered significantly only at later stages of 12 or 36 weeks. Alteration in miR-138 expression supports the upregulation of its target, EZH2. H3K9me2, H3K27me3 or H4K20me3 was found to be altered at 12 or 36 weeks. However, ChIP analysis of p16 and MLH1 promoters showed their binding with H3K9me2 and H3K27me3 which was maximum at 36 weeks. Thus, histone modification and their interactions with gene promoter resulted in the reduced expression of p16 and MLH1. IP6 prevented the incidence and the size of urethane induced lung tumors. IP6 also prevented the urethane induced alterations in EZH2, SUV39H1, G9a expressions and histone modifications. Our results suggest that the alterations in the histone modification pathways involving EZH2 and SUV39H1 expressions are among the early events in urethane induced mouse lung tumorigenesis and could be exploited for cancer control.

Gastric and esophageal adenocarcinomas (G/EAC) exhibit a characteristic pattern of somatic variants, known as signature 17. Signature 17 associates with high mutation burden, elevated neoantigen levels and a potential sensitivity to cell cycle inhibitors. Signature 17 correlates with poor survival, and is increased in cancer patients with a history of the common pre-malignant lesion called Barrett’s esophagus. We recently showed that oxidative damage during DNA replication is a likely source of signature 17; however this hypothesis remains to be validated. Intriguingly, we observe signature 17 arising in clonally expanded primary mouse cells (Hupki MEF) and in mouse models of liver cancer. Here, we aim to identify the mechanisms underlying signature 17 by mining TCGA and ICGC data, as well as generating genome-wide molecular profiles of relevant experimental cell systems. Mutational signatures were extracted from exome and whole-genome sequencing (WGS) data of over 460 G/EAC tumors. Pathway analysis of RNA-seq data from matching samples was performed using NIH DAVID. Exposure to signature 17 was next assessed with regards to clinico-pathologic data. Lastly, we compared the asymmetry of signature 17 mutations around replication origins and early/late replicating regions in WGS data from MEF cells and G/EAC cancers. Comparing the transcriptomes of signature 17-high and -low tumors implicated cell proliferation, regulation of apoptosis, DNA damage response and repair, oxidoreductase activity and gastric acid secretion as relevant pathways. We found positive associations between signature 17 intensity and Barrett’s esophagus history, poorer survival, and microsatellite instability. MEF WGS data revealed signature 17 enrichment in late-replicating genomic regions and on the lagging strand, analogous to observations in human tumors. Importantly, in all cultured MEF clones as well as in most EAC genomes, signature 17 co-occurred with signature 18 which is likely linked to oxidative damage of DNA, supporting the hypothesis that inflammation-driven oxidative DNA damage leads to these human cancers. The replication strand and timing asymmetries were significant in both signature 17 and signature 18 in EAC genomes, while the Hupki MEF clones exhibited significant strand asymmetry in signature 17 but not in signature 18, and showed significant correlation with replication timing in signature 18. Collectively, our results elucidate new features of a modifiable mutagenic activity underlying signature 17 in cancers of the esophagus, stomach, and in model systems. We have also identified novel clinical and molecular associations with signature 17. Investigations using the reported innovative cell-based models will help to identify the precise mechanistic basis of signature 17, with potential implications for primary and secondary cancer prevention and personalized therapy. Citation Format: Marketa Tomkova, Claire Renard, Lara Urban, Souad Kolli, Maude Ardin, Manuraj Pandey, Maria Zhivagui, Hana Huskova, Magali Olivier, Hiroyuki Marusawa, Benjamin Schuster-Böckler, Jiri Zavadil. Deciphering the causes of the COSMIC mutational signature 17 by combining pan-cancer data with experimental mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4661.

Here we have shown the alteration of transcription factors STAT3, NF-κB and downstream associated molecules much before the appearance of lung tumor and their response to antitumor agent, inositol hexaphosphate. Histological examination revealed the pathophysiology of the lung tissues and the onset or progression of tumor from 4 or 9 to 24 weeks in terms of tumor volume and the number. Over expression of NF-κB (p50/Rel A), COX-2, STAT3, pSTAT3 (Tyr 705), IL-6 and cyclin D1 also progressed from the time of no tumor to the time of tumor appearance and was reduced in mice drinking 2%IP6. We suggest that the alterations of STAT3, NF-κB and downstream associated molecules are critical in the development of lung tumors and can be exploited as possible mechanisms after the exposure. Status of these altered genes before the tumor development suggests their possible use as targets for the tumor control in the predisposed conditions.

Pan-cancer analyses of tumor genomes reveal mutational signatures characteristic of particular etiologic factors. The signatures are extracted mathematically from mixed patterns typically observed by tumor sequencing. However, the components of signatures originating from complex carcinogen mixtures have not been studied in detail. Lung, head and neck and liver tumors of tobacco smokers exhibit COSMIC signature 4 marked by predominant G>N mutations, involving mainly G:C>T:A transversions with transcription strand bias, consistent with the mutagenic effects of benzo[a]pyrene (B[a]P). Additionally, A>N mutations (strand-biased A:T>T:A transversions and A:T>G:C transitions) are also prominently present, yet their origins are less understood. By using exposure-coupled clonal immortalization of human and mouse primary cells and deep sequencing, we were able to dissect ‘clean' mutational signatures of tobacco smoke carcinogens B[a]P and glycidamide (GA), a key reactive metabolite of acrylamide (ACR). Whole-genome sequencing of multiple clones derived from primary B[a]P-treated human mammary epithelial cells identified a robust mutational signature marked by strand-biased G>N mutations and increased GG>TT dinucleotides, while no apparent enrichment of A:T>T:A mutations was observed. Next, in ACR and GA-treated primary mouse embryonic fibroblasts, we established by the LC-MS/MS DNA adduct analysis that ACR exerts its mutagenic effects exclusively via GA. We then extracted from 15 treated clones the exome-scale mutational signature of GA, marked by predominant A:T>T:A transversions followed by A:T>G:C transitions and G:C>T:A transversions, all showing transcription strand bias. Similarity analysis involving known primary-cancer and experimental mutational signatures indicated that the GA mutational signature was novel. A more in-depth comparison with mutation patterns from lung adenocarcinomas of heavy smokers revealed that the GA signature, including its strand bias features, matched closely with and may thus account for the A>N mutation component of the tobacco smoking-derived signature 4. Thus, mutational signatures generated in controlled experimental settings may explain particular sub-features of cancer signatures arising from co-exposures to multiple carcinogens. Furthermore, the use of innovative in vitro systems, characterized by biological barrier bypass to mimic early steps of cell transformation, can provide revealing insights into the molecular links between mutagenesis and carcinogenesis. Funding: INCa-INSERM Plan Cancer 2015; NIH/NIEHS 1R03ES025023-01A1 Citation Format: Manuraj Pandey, Maria Zhivagui, Mona I. Churchwell, Alvin W. Ng, Liacine Bouaoun, Vincent Cahais, Martha R. Stampfer, Magali Olivier, Zdenko Herceg, Ewy Mathé, Steven G. Rozen, Frederick A. Beland, Michael Korenjak, Jiri Zavadil. Deciphering components of mutational signatures arising from carcinogen co-exposures: A genome-scale experimental approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3088.

Introduction Based on cancer genome sequencing, almost 600 genes have been implicated in cancer development to date, and recently, chromatin structural changes have emerged as a key mechanism for translating exogenous cues, such as carcinogen exposures, to the deregulation of biological processes implicated in tumorigenesis. However, the functional relationship between genetic and epigenetic alterations in the context of early events of carcinogen-driven cell transformation is not well understood. Material and methods We employ an in vitro carcinogen exposure system using primary human mammary epithelial cells (HMEC), which utilises a biological barrier bypass to mimic early steps of cell transformation, to introduce genetic and epigenetic alterations and to investigate their functional interplay during cell immortalization. A temporal, integrated (epi)genomic analysis is applied to benzo[a]pyrene (B[a]P)-exposed, immortalised HMEC, involving whole genome sequencing (WGS), analyses of chromatin structure (ChIP-seq, ATAC-seq), and patterns of gene regulation (DNA methylome 850K array, RNA-seq). Results and discussions WGS of sequentially derived post-stasis and immortalised clones, as well as tumorigenic clones (generated by TP53 insertional mutagenesis of post-stasis lines), identified a prominent B[a]P mutational signature across all clones. While all sequenced clones had comparable SBS counts, we observed a gradual increase of structural variants, most notably copy number variations, from post-stasis to immortal and tumorigenic clones. Reproducibly distinct methylome patterns also correlated closely with the individual phenotypic stages of cell transformation. Chromatin accessibility analysis further showed changes characteristic to individual as well as multiple exposed clones, such as in the breast cancer tumour suppressor gene BRCA2 or the promoter region of PCDH18, a gene frequently inactivated by promoter methylation in colorectal cancer. Cell type-matched histone modification and RNA expression patterns are being established to enable a thorough integrated analysis of genetic and epigenetic determinants of carcinogen-driven HMEC transformation. Conclusion We propose that in-depth, integrated (epi)genomic analysis of this innovative model system can reveal novel molecular determinants of carcinogen-driven cellular immortalization, thus improving our understanding of cancer causes, development and progression. Funding INCa-INSERM Plan Cancer 2015 Grant.

Epigenetic changes are correlated with tumor development showing aberrations in DNA methylation and histone modifications. To find the early changes, we evaluated the epigenetic events from early to late stage of the urethane induced lung tumor development in mouse model and tried to correlate the molecular events with the progression of tumor. We addressed the hypothesis by examining the tumor development, status of DNMTs, HDACs and MBDs, DNA methylation and expression of microRNA-29b during 1 to 36 weeks after urethane exposure that included the period before and after the tumor appearance. Tumors did not appear after 1 or 4 weeks but well defined tumors appeared after 12 weeks and larger tumors appeared at 36 weeks which was prevented by IP6. DNMT1, DNMT3a and DNMT3b were upregulated after urethane exposure at the time of no tumor till the tumor developed and showed its upregulated functional activity. DNMTs are shown to be the targets of microRNA-29b and we showed that microRNA-29b was downregulated in the line of DNMT upregulation. HDAC, the histone modifier, also showed progressive upregulation. Periodic increase in methyl binding proteins, MBD2, supported the expression of gene silencing pathways in terms of the downregulation of tumor suppressor genes, p16 and MLH1. All these molecular alterations were protected in the presence of IP6. Our results showed that the key steps of epigenetics, DNMTs, mir29b, and HDAC1, are altered both before and after the development of tumors.

Mechanisms of anticancer effects of inositol hexaphosphate are not fully understood. Epigenetic changes are the early changes in tumorigenesis. DNA methyl transferases, methyl CpG binding proteins, methyl CpG DNA binding domain protein, and histone deacetylases are the major molecules involved in epigenetics. We have shown the effects of IP6 at the molecular level in mouse lungs before the tumor is developed. After 3 mo of ENU exposure, there was no tumor formation, but there was hyperplasia and lymphocytic infiltration in the lungs. Inflammation and DNA damage repair enzymes COX-2 and MLH1 appear to be upregulated, whereas tumor suppressor gene p16 was downregulated by ENU. On the other hand, ENU exposure more or less upregulated the epigenetic events such as the expressions of DNMT1, MeCP2, MBD1, and HDAC1. This alteration was reduced by IP6 administration. Results were supported by modulation of global DNA methylation and the modulation of promoter CpG methylation of p16, MLH1, and COX-2 genes. Hence, this study indicates the possible role of epigenetics at the early stage of tumor development and in the regulation of gene expression by IP6 before the onset of ENU-induced lung tumors.