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Supplementary Figure 2 from FOXO3 Encodes a Carcinogen-Activated Transcription Factor Frequently Deleted in Early-Stage Lung Adenocarcinoma

Supplementary Figure 4 from FOXO3 Encodes a Carcinogen-Activated Transcription Factor Frequently Deleted in Early-Stage Lung Adenocarcinoma

Supplementary Figure 3 from FOXO3 Encodes a Carcinogen-Activated Transcription Factor Frequently Deleted in Early-Stage Lung Adenocarcinoma

Supplementary Figure 1 from FOXO3 Encodes a Carcinogen-Activated Transcription Factor Frequently Deleted in Early-Stage Lung Adenocarcinoma

The FOXO family of transcription factors elicits cell cycle arrest, apoptosis, and resistance to various physiologic and pathologic stresses relevant to sporadic cancer, such as DNA damage and oxidative stress. Although implicated as tumor suppressors, FOXO genetic inactivation has not been observed in human cancer. In an investigation of the two major types of non–small cell lung cancer, here, we identify the FOXO3 gene as a novel target of deletion in human lung adenocarcinoma (LAC). Biallelic or homozygous deletion (HD) of FOXO3 was detected in 8 of 33 (24.2%) mostly early-stage LAC of smokers. Another 60.6% of these tumors had losses of FOXO3 not reaching the level of HD (hereafter referred to as sub-HD). In contrast, no HD of FOXO3 was observed in 19 lung squamous cell carcinoma. Consistent with the deletion of FOXO3 were corresponding decreases in its mRNA and protein levels in LAC. The potential role of FOXO3 loss in LAC was also investigated. The carcinogen (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) is strongly implicated as a cause of human lung cancer. Here, we show that FOXO3a is functionally activated and augments the level of caspase-dependent apoptosis in cells exposed to this DNA-damaging carcinogen. These results implicate FOXO3 as a suppressor of LAC carcinogenesis, a role frequently lost through gene deletion. Cancer Res; 70(15); 6205–15. ©2010 AACR.

Many women are affected by vulvodynia, but medical therapies to date have proven ineffective. We performed a pilot study using gel-based proteomics to develop a map of proteins present in vaginal/vestibular secretions and identify proteins that could be considered for future evaluation as potential therapeutic targets.We collected vestibular fluid from 4 controls and 4 patients with vulvodynia by placing a cotton swab in the vestibule and extracting the absorbed proteins. The proteins underwent 2-dimensional difference gel electrophoresis and mass spectrometry to develop a protein map. Immunohistochemistry was used to validate proteomic findings.A map was constructed of 32 of the more abundant proteins in vestibular fluid and their levels compared in control subjects and vulvodynia patients. Among these were annexin A1, interleukin 1 receptor antagonist, protein S100 A9, and a number of antiproteases and proteases. Many of these proteins differed by at least 50% between groups, but only annexin A1, one of the protease inhibitors, and immunoglobulin G κ chain were significantly different. The results with annexin A1 were validated by similar findings with immunohistochemistry.The findings of this pilot study demonstrate a set of vestibule mucosa proteins that differ significantly-either increasing or decreasing-in vulvodynia patients compared with controls, and several others that exhibited greater than 1.5-fold change but did not reach statistical significance. This study constitutes a proof-of-principle that an open, unbiased proteomic approach can identify molecular participants in vulvodynia, some of which had not been identified to date by hypothesis-driven studies.

Alveolar macrophages (AMs) are differentially regulated by human surfactant protein-A1 (SP-A1) or SP-A2. However, AMs are very heterogeneous and differences are difficult to characterize in intact cells. Using the Toponome Imaging System (TIS), an imaging technique that uses sequential immunostaining to identify patterns of biomarker expression or combinatorial molecular phenotypes (CMPs), we studied individual single cells and identified subgroups of AMs (n = 168) from SP-A–KO mice and mice expressing either SP-A1 or SP-A2. The effects, as shown by CMPs, of SP-A1 and SP-A2 on AMs were significant and differed. SP-A1 AMs were the most diverse and shared the fewest CMPs with KO and SP-A2. Clustering analysis of each group showed 3 clusters where the CMP-based phenotype was distinct in each cluster. Moreover, a clustering analysis of all 168 AMs revealed 10 clusters, many dominated by 1 group. Some CMP overlap among groups was observed with SP-A2 AMs sharing the most CMPs and SP-A1 AMs the fewest. The CMP-based patterns identified here provide a basis for understanding not only AMs’ diversity, but also most importantly, the molecular basis for the diversity of functional differences in mouse models where the impact of genetics of innate immune molecules on AMs has been studied.

Annexins and S100 proteins form two large families of Ca2+-binding proteins. They are quite different both structurally and functionally, with S100 proteins being small (10–12 kDa) acidic regulatory proteins from the EF-hand superfamily of Ca2+-binding proteins, and with annexins being at least three-fold larger (329 ± 12 versus 98 ± 7 residues) and using non-EF-hand-based mechanism for calcium binding. Members of both families have multiple biological roles, being able to bind to a large cohort of partners and possessing a multitude of functions. Furthermore, annexins and S100 proteins can interact with each other in either a Ca2+-dependent or Ca2+-independent manner, forming functional annexin-S100 complexes. Such functional polymorphism and binding indiscrimination are rather unexpected, since structural information is available for many annexins and S100 proteins, which therefore are considered as ordered proteins that should follow the classical “one protein–one structure–one function” model. On the other hand, the ability to be engaged in a wide range of interactions with multiple, often unrelated, binding partners and possess multiple functions represent characteristic features of intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs); i.e., functional proteins or protein regions lacking unique tertiary structures. The aim of this paper is to provide an overview of the functional roles of human annexins and S100 proteins, and to use the protein intrinsic disorder perspective to explain their exceptional multifunctionality and binding promiscuity.

BackgroundWe used the Toponome Imaging System (TIS) to identify “patterns of marker expression”, referred to here as combinatorial molecular phenotypes (CMPs) in alveolar macrophages (AM) in response to the innate immune molecule, SP-A1.MethodsWe compared 114 AM from male SP-A deficient mice. One group (n = 3) was treated with exogenous human surfactant protein A1 (hSP-A1) and the other with vehicle (n = 3). AM obtained by bronchoalveolar lavage were plated onto slides and analyzed using TIS to study the AM toponome, the spatial network of proteins within intact cells. With TIS, each slide is sequentially immunostained with multiple FITC-conjugated antibodies. Images are analyzed pixel-by-pixel identifying all of the proteins within each pixel, which are then designated as CMPs. CMPs represent organized protein clusters postulated to contribute to specific functions.Results1) We compared identical CMPs in KO and SP-A1 cells and found them to differ significantly (p = 0.0007). Similarities between pairs of markers in the two populations also differed significantly (p ConclusionsAM diversity is far more extensive than originally thought. The increased diversity of SP-A1-treated mice points to the possibility that SP-A1 enhances or activates several pathways in the AM to better prepare it for its innate immune functions and other functions shown previously to be affected by SP-A treatment. Future studies may identify key protein(s) responsible for CMP integrity and consequently for a given function, and target it for therapeutic purposes.

Goodson, William H. et al., © The Author 2015. Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/ mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology., We gratefully acknowledge the support of the National Institute of Health-National Institute of Environmental Health Sciences (NIEHS) conference grant travel support (R13ES023276); Glenn Rice, Office of Research and Development, United States Environmental Protection Agency, Cincinnati, OH, USA also deserves thanks for his thoughtful feedback and inputs on the manuscript; William H.Goodson III was supported by the California Breast Cancer Research Program, Clarence Heller Foundation and California Pacific Medical Center Foundation; Abdul M.Ali would like to acknowledge the financial support of the University of Sultan Zainal Abidin, Malaysia; Ahmed Lasfar was supported by an award from the Rutgers Cancer Institute of New Jersey; Ann-Karin Olsen and Gunnar Brunborg were supported by the Research Council of Norway (RCN) through its Centres of Excellence funding scheme (223268/F50), Amancio Carnero’s lab was supported by grants from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0306-2012); Matilde E. Lleonart was supported by a trienal project grant PI12/01104 and by project CP03/00101 for personal support. Amaya Azqueta would like to thank the Ministerio de Educacion y Ciencia (‘Juande la Cierva’ programme, 2009) of the Spanish Government for personal support; Amedeo Amedei was supported by the Italian Ministry of University and Research (2009FZZ4XM_002), and the University of Florence (ex60%2012); Andrew R.Collins was supported by the University of Oslo; Annamaria Colacci was supported by the Emilia-Romagna Region - Project ‘Supersite’ in Italy; Carolyn Baglole was supported by a salary award from the Fonds de recherche du Quebec-Sante (FRQ-S); Chiara Mondello’s laboratory is supported by Fondazione Cariplo in Milan, Italy (grant n. 2011-0370); Christian C.Naus holds a Canada Research Chair; Clement Yedjou was supported by a grant from the National Institutes of Health (NIH-NIMHD grant no. G12MD007581); Daniel C.Koch is supported by the Burroughs Wellcome Fund Postdoctoral Enrichment Award and the Tumor Biology Training grant: NIH T32CA09151; Dean W. Felsher would like to acknowledge the support of United States Department of Health and Human Services, NIH grants (R01 CA170378 PQ22, R01 CA184384, U54 CA149145, U54 CA151459, P50 CA114747 and R21 CA169964); Emilio Rojas would like to thank CONACyT support 152473, Ezio Laconi was supported by AIRC (Italian Association for Cancer Research, grant no. IG 14640) and by the Sardinian Regional Government (RAS); Eun-Yi Moon was supported by grants from the Public Problem-Solving Program (NRF-015M3C8A6A06014500) and Nuclear R&D Program (#2013M2B2A9A03051296 and 2010-0018545) through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science and Technology (MEST) in Korea; Fahd Al-Mulla was supported by the Kuwait Foundation for the Advancement of Sciences (2011-1302-06); Ferdinando Chiaradonna is supported by SysBioNet, a grant for the Italian Roadmap of European Strategy Forum on Research Infrastructures (ESFRI) and by AIRC (Associazione Italiana Ricerca sul Cancro; IG 15364); Francis L.Martin acknowledges funding from Rosemere Cancer Foundation; he also thanks Lancashire Teaching Hospitals NHS trust and the patients who have facilitated the studies he has undertaken over the course of the last 10 years; Gary S.Goldberg would like to acknowledge the support of the New Jersey Health Foundation; Gloria M.Calaf was supported by Fondo Nacional de Ciencia y Tecnología (FONDECYT), Ministerio de Educación de Chile (MINEDUC), Universidad de Tarapacá (UTA); Gudrun Koppen was supported by the Flemish Institute for Technological Research (VITO), Belgium; Hemad Yasaei was supported from a triennial project grant (Strategic Award) from the National Centre for the Replacement, Refinement and Reduction (NC3Rs) of animals in research (NC.K500045.1 and G0800697); Hiroshi Kondoh was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Science and Technology Agency and by JST, CREST; Hsue-Yin Hsu was supported by the Ministry of Science and Technology of Taiwan (NSC93-2314-B-320-006 and NSC94-2314-B-320-002); Hyun Ho Park was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) of the Ministry of Education, Science and Technology (2012R1A2A2A01010870) and a grant from the Korea Healthcare Technology R&D project, Ministry of Health and Welfare, Republic of Korea (HI13C1449); Igor Koturbash is supported by the UAMS/NIH Clinical and Translational Science Award (UL1TR000039 and KL2TR000063) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000; Jan Vondráček acknowledges funding from the Czech Science Foundation (13-07711S); Jesse Roman thanks the NIH for their support (CA116812), John Pierce Wise Sr. and Sandra S.Wise were supported by National Institute of Environmental Health Sciences (ES016893 to J.P.W.) and the Maine Center for Toxicology and Environmental Health; Jonathan Whitfield acknowledges support from the FERO Foundation in Barcelona, Spain; Joseph Christopher is funded by Cancer Research UK and the International Journal of Experimental Pathology; Julia Kravchenko is supported by a philanthropic donation by Fred and Alice Stanback; Jun Sun is supported by a Swim Across America Cancer Research Award; Karine A.Cohen-Solal is supported by a research scholar grant from the American Cancer Society (116683-RSG-09-087-01-TBE); Laetitia Gonzalez received a postdoctoral fellowship from the Fund for Scientific Research–Flanders (FWO-Vlaanderen) and support by an InterUniversity Attraction Pole grant (IAP-P7-07); Laura Soucek is supported by grant #CP10/00656 from the Miguel Servet Research Contract Program and acknowledges support from the FERO Foundation in Barcelona, Spain; Liang-Tzung Lin was supported by funding from the Taipei Medical University (TMU101-AE3-Y19); Linda Gulliver is supported by a Genesis Oncology Trust (NZ) Professional Development Grant, and the Faculty of Medicine, University of Otago, Dunedin, New Zealand; Louis Vermeulen is supported by a Fellowship of the Dutch Cancer Society (KWF, UVA2011-4969) and a grant from the AICR (14–1164); Mahara Valverde would like to thank CONACyT support 153781; Masoud H. Manjili was supported by the office of the Assistant Secretary of Defense for Health Affairs (USA) through the Breast Cancer Research Program under Award No. W81XWH-14-1-0087 Neetu Singh was supported by grant #SR/FT/LS-063/2008 from the Department of Science and Technology, Government of India; Nicole Kleinstreuer is supported by NIEHS contracts (N01-ES 35504 and HHSN27320140003C); P.K. Krishnakumar is supported by the Funding (No. T.K. 11-0629) of King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia; Paola A.Marignani is supported by the Dalhousie Medical Research Foundation, The Beatrice Hunter Cancer Institute and CIHR and the Nova Scotia Lung Association; Paul Dent is the holder of the Universal Inc.Chair in Signal Transduction Research and is supported with funds from PHS grants from the NIH (R01-CA141704, R01-CA150214, R01-DK52825 and R01-CA61774); Petr Heneberg was supported by the Charles University in Prague projects UNCE 204015 and PRVOUK P31/2012, and by the Czech Science Foundation projects P301/12/1686 and 15-03834Y; Po Sing Leung was supported by the Health and Medical Research Fund of Food and Health Bureau, Hong Kong Special Administrative Region, Ref. No: 10110021; Qiang Cheng was supported in part by grant NSF IIS-1218712; R. Brooks Robey is supported by the United States Department of Veterans Affairs; Rabindra Roy was supported by United States Public Health Service Grants (RO1 CA92306, RO1 CA92306-S1 and RO1 CA113447); Rafaela Andrade-Vieira is supported by the Beatrice Hunter Cancer Research Institute and the Nova Scotia Health Research Foundation, Renza Vento was partially funded by European Regional Development Fund, European Territorial Cooperation 2007–13 (CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–13) and grants from the Italian Ministry of Education, University and Research (MIUR) ex-60%, 2007; Riccardo Di Fiore was a recipient of fellowship granted by European Regional Development Fund, European Territorial Cooperation 2007–2013 (CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–2013); Rita Dornetshuber-Fleiss was supported by the Austrian Science Fund (FWF, project number T 451-B18) and the Johanna Mahlke, geb.-Obermann-Stiftung; Roberta Palorini is supported by a SysBioNet fellowship; Roslida Abd Hamid is supported by the Ministry of Education, Malaysia-Exploratory Research Grant Scheme-Project no: ERGS/1-2013/5527165; Sabine A.S.Langie is the beneficiary of a postdoctoral grant from the AXA Research Fund and the Cefic-LRI Innovative Science Award 2013; Sakina Eltom is supported by NIH grant SC1CA153326; Samira A.Brooks was supported by National Research Service Award (T32 ES007126) from the National Institute of Environmental Health Sciences and the HHMI Translational Medicine Fellowship; Sandra Ryeom was supported by The Garrett B. Smith Foundation and the TedDriven Foundation; Thierry Massfelder was supported by the Institut National de la Santé et de la Recherche Médicale INSERM and Université de Strasbourg; Thomas Sanderson is supported by the Canadian Institutes of Health Research (CIHR; MOP-115019), the Natural Sciences and Engineering Council of Canada (NSERC; 313313) and the California Breast Cancer Research Program (CBCRP; 17UB-8703); Tiziana Guarnieri is supported by a grant from Fundamental Oriented Research (RFO) to the Alma Mater Studiorum University of Bologna, Bologna, Italy and thanks the Fondazione Cassa di Risparmio di Bologna and the Fondazione Banca del Monte di Bologna e Ravenna for supporting the Center for Applied Biomedical Research, S.Orsola-Malpighi University Hospital, Bologna, Italy; W.Kimryn Rathmell is supported by the V Foundation for Cancer Research and the American Cancer Society; William K.Decker was supported in part by grant RP110545 from the Cancer Prevention Research Institute of Texas; William H.Bisson was supported with funding from the NIH P30 ES000210; Yon Rojanasakul was supported with NIH grant R01-ES022968; Zhenbang Chen is supported by NIH grants (MD004038, CA163069 and MD007593); Zhiwei Hu is grateful for the grant support from an institutional start-up fund from The Ohio State University College of Medicine and The OSU James Comprehensive Cancer Center (OSUCCC) and a Seed Award from the OSUCCC Translational Therapeutics Program.

Naheliegend und dennoch oft unerwartet erweisen sich die Fuse, Grundlage unserer aufrechten Haltung, als Ursache chronischer Beschwerden des Bewegungsapparats. Betrachtet man die Fuse naher, findet man haufig Hinweise, mit denen rezidivierende Schmerzen von Fus bis Kopf zu erklaren sind − und dann auch erfolgreich behandelt werden konnen. Die Autorin zeigt exemplarisch anhand von Fallen aus ihrer Praxis, welche korperlichen Beschwerden des Bewegungsapparats ihren Ursprung am Fus haben konnen und mittels zielgerichteter Behandlung kurativ therapiert werden konnen.

Studies of the decades-long latent stages of breast carcinogenesis have been limited to when hyperplastic lesions are already present. Investigations of earlier stages of breast cancer (BC) latency have been stymied by the lack of fiducial biomarkers needed to identify where in histologically normal tissues progression toward a BC might be taking place. Recent evidence suggests that a marker of chronic oxidative stress (OxS), protein adducts of 4-hydroxy-2-nonenal (4HNE), can meet this need. Specifically: (1) 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells were found to be prevalent in normal (reduction mammoplasty) tissues of most women (including many teenagers) studied, representative of those living in the United States’ high risk-posing environment and: (2) marked (>1.5-fold) differences were identified between tissues of healthy young women with many vs. few 4HNE+ ME cells in the relative levels of transcripts for 42 of the 84 OxS-associated genes represented in SABioscience Oxidative-Stress/Oxidative-Defense PCR array. Herein we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to identify molecular changes associated with 4HNE adducts in basal and luminal ME cells in terminal ductal units (TDLU), which are the cells of origin of BC, and associated intralobular and interlobular stroma, known contributors to carcinogenesis. Multivariate analysis-derived wavenumbers differentiated 4HNE+ and 4HNE− cells in each of the anatomical compartments. Specifically, principal component and linear discriminant analyses of mid-infrared spectra obtained from these cells revealed unambiguous, statistically highly significant differences in the “biochemical fingerprint” of 4HNE+ vs. 4HNE− luminal and basal ME cells, as well as between associated intralobular and interlobular stroma. These findings demonstrate further SR-FTIR microspectroscopy’s ability to identify molecular changes associated with altered physiological and/or pathophysiological states, in this case with a state of chronic OxS that provides a pro-carcinogenic microenvironment.

Problem Our knowledge of the innate host defenses in the vagina, a site where these defenses are essential to protecting the host upper reproductive tract from invasion by pathogens, is as yet rudimentary. Specifically, little is known about the pattern-recognition component of vaginal innate immunity, the relationship of pattern-recognition molecules to known cytokine levels, and the role of gonadal hormones in their regulation. Method of study We measured levels of Surfactant Protein-A (SP-A), a prototypic innate pattern-recognition protein, in vaginal fluid (VF) and correlated them with levels of IL-1β and IL-8, two cytokines known to be present in VF. Assays were carried out on VF collected over three consecutive cycles from ten healthy naturally cycling women who were sampled at three specific time points in the menstrual cycle. The three time points were chosen to enable correlation with distinct hormonal states. Results Both SP-A and cytokines levels were highest 5–6 days after menses (P

Survival of mice after Klebsiella pneumoniae infection and phagocytosis by alveolar macrophages (AMs), in the presence or absence of ozone (O(3)) exposure prior to infection, is sex dependent. The objective of this work was to study the role of gonadal hormones, 5α-dihydrotestosterone (DHT) and 17β-estradiol (E(2)), on mouse survival after filtered air (FA) or O(3) exposure. Gonadectomized female (G×F) and male (G×M) mice implanted with control or hormone pellets (DHT in G×F, or E(2) in G×M), exposed to O(3) (2 ppm, 3h) or FA, and infected with K. pneumoniae were monitored for survival. Survival in G×F was identical after FA or O(3) exposure; in G×M O(3) exposure resulted in lower survival compared to FA. In O(3)-exposed females, gonadectomy resulted in increased survival compared to intact females or to G×M+E(2). A similar effect was observed in G×F+DHT. The combined negative effect of oxidative stress and hormone on survival was higher for E(2). Gonadectomy eliminated (females) or minimized (males) the previously observed sex differences in survival in response to oxidative stress, and hormone treatment restored them. These findings indicate that gonadal hormones and/or oxidative stress have a significant effect on mouse survival.

The FOXO family of transcription factors elicits cell cycle arrest, apoptosis, and resistance to various physiologic and pathologic stresses relevant to sporadic cancer, such as DNA damage and oxidative stress. Although implicated as tumor suppressors, FOXO genetic inactivation has not been observed in human cancer. In an investigation of the two major types of non–small cell lung cancer, here, we identify the FOXO3 gene as a novel target of deletion in human lung adenocarcinoma (LAC). Biallelic or homozygous deletion (HD) of FOXO3 was detected in 8 of 33 (24.2%) mostly early-stage LAC of smokers. Another 60.6% of these tumors had losses of FOXO3 not reaching the level of HD (hereafter referred to as sub-HD). In contrast, no HD of FOXO3 was observed in 19 lung squamous cell carcinoma. Consistent with the deletion of FOXO3 were corresponding decreases in its mRNA and protein levels in LAC. The potential role of FOXO3 loss in LAC was also investigated. The carcinogen (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) is strongly implicated as a cause of human lung cancer. Here, we show that FOXO3a is functionally activated and augments the level of caspase-dependent apoptosis in cells exposed to this DNA-damaging carcinogen. These results implicate FOXO3 as a suppressor of LAC carcinogenesis, a role frequently lost through gene deletion. Cancer Res; 70(15); 6205–15. ©2010 AACR.

Environmental contributions to cancer development are widely accepted, but only a fraction of all pertinent exposures have probably been identified. Traditional toxicological approaches to the problem have largely focused on the effects of individual agents at singular endpoints. As such, they have incompletely addressed both the pro-carcinogenic contributions of environmentally relevant low-dose chemical mixtures and the fact that exposures can influence multiple cancer-associated endpoints over varying timescales. Of these endpoints, dysregulated metabolism is one of the most common and recognizable features of cancer, but its specific roles in exposure-associated cancer development remain poorly understood. Most studies have focused on discrete aspects of cancer metabolism and have incompletely considered both its dynamic integrated nature and the complex controlling influences of substrate availability, external trophic signals and environmental conditions. Emerging high throughput approaches to environmental risk assessment also do not directly address the metabolic causes or consequences of changes in gene expression. As such, there is a compelling need to establish common or complementary frameworks for further exploration that experimentally and conceptually consider the gestalt of cancer metabolism and its causal relationships to both carcinogenesis and the development of other cancer hallmarks. A literature review to identify environmentally relevant exposures unambiguously linked to both cancer development and dysregulated metabolism suggests major gaps in our understanding of exposure-associated carcinogenesis and metabolic reprogramming. Although limited evidence exists to support primary causal roles for metabolism in carcinogenesis, the universality of altered cancer metabolism underscores its fundamental biological importance, and multiple pleiomorphic, even dichotomous, roles for metabolism in promoting, antagonizing or otherwise enabling the development and selection of cancer are suggested.

Background: Our previous studies in pre-invasive mammary tumors revealed that estrogen receptor negative cell clusters (ER NCC) overlying focally disrupted myoepithelial (ME) cell layers showed a significantly higher rate of genetic abnormalities and cell proliferation than adjacent cells without ME cell layer disruptions. A subset of these ER NCC, however, completely lacked expression of Ki-67, a most commonly used marker for cell proliferation. The purpose of this study was to further elucidate the immunohistochemical and morphological profiles of these ER NCC. Methods: Fifteen cases with such ER NCC were selected from our previous studies and assessed with a panel of commonly used biomarkers for cell proliferation, tumor progression, and normal stem cells. Results: Immunohistochemically, in addition to Ki-67 and ER, these ER NCC completely lacked expression of all other proliferation and progression related markers that were distinctly expressed in adjacent cells within the same duct but overlying the non-disrupted ME cell layer. These ER NCC also lacked expression of all normal stem cell-related markers tested. These cell clusters, however, showed a higher and atypical expression of c-erb-B2, compared to their adjacent counterparts. Morphologically, these ER NCC were generally arranged as triangle shaped structures penetrating into the stroma, similar to micro-invasive lesions. About 15% of these ER NCC appeared to directly spread into blood vessel-like structures. These ER NCC and their possible derivatives within the stroma and blood vessels-like structures shared the same morphologic and immunohistochemical features. No comparable ER positive cell clusters were identified in any of the cases. Conclusions: These findings suggest that these ER NCC and their possible derivatives are likely regulated by yet to be defined molecules and mechanisms, and they are unlikely to respond to currently available anti-mitotic agents.

Objective: Retinoic acid plays an essential role in epithelial differentiation, and retinoid homeostasis is disrupted in cancers of epithelial origin. The goal of this study was to determine whether hRoDH-4, an enzyme that can catalyze the first and rate-limiting step in retinoic acid biosynthesis, is expressed in normal endometrium and, if so, whether its expression is altered in endometrial cancer. Study design: Proliferative, secretory, hyperplastic, and neoplastic endometria were examined by immunocytochemistry for hRoDH-4 protein and by reverse transcriptase-polymerase chain reaction for the hRoDH-4 transcript. Results: In proliferative and secretory glandular epithelia, immunoreactive hRoDH-4 was uniformly pres-ent. In endometrial cancers, hRoDH-4 immunoreactivity was markedly reduced in many neoplastic epithelial cells. Expression of hRoDH-4 in normal and neoplastic endometrium was confirmed by findings on reverse transcriptase-polymerase chain reaction. Conclusion: These findings are consistent with the hypothesis that altered expression of enzymes essential for in situ retinoic acid biosynthesis is an important phenotypic change associated with the development of endometrial cancer. (Am J Obstet Gynecol 2002;186:675-83.)

Advances in vitamin A research in 1999 and 2000 have improved the understanding the molecular processes through which beta-carotene and other provitamin A carotenoids are converted to vitamin A, the roles of cellular retinoid-binding proteins that serve as retinoid chaperones during metabolism, the regulation of retinoid transport, and the nature and regulation of several enzymes required for the absorption, storage, activation, and inactivation or degradation of retinoids. Not only has a clearer picture emerged of specific molecular processes, but it is also becoming evident that whole-body retinoid homeostasis is facilitated by close communication among organs due to the rapid interorgan recirculation of retinoids, and by the "autoregulation" by retinoic acid of several enzymes and retinoid-binding proteins that mediate retinoid homeostasis.

The involvement of estrogens in carcinogenic processes within estrogen-responsive tissues has been recognized for a number of years. Classically, mitogenicity associated with estrogen receptor-mediated cellular events was believed to be the mechanism by which estrogens contributed to carcinogenesis. Recently, the possibility that estrogens might contribute directly to mutagenesis resulting from DNA damage has been investigated. That damage is apparently a result of the formation of catechol estrogens that can be further oxidized to semiquinones and quinones. Those molecules represent reactive oxygen species and electrophilic molecules that can form depurinating DNA adducts, thus having the potential to result in permanent nucleotide mutation. Conjugation of parent estrogens to sulfate and glucuronide moieties; of catechol estrogens to methyl, sulfate, and glucuronide conjugates; and of catechol estrogen quinones to glutathione conjugates all represent potential "detoxification" reactions that may protect the cell from estrogen-mediated mitogenicity and mutagenesis. In this chapter, the biochemistry and molecular genetics of those conjugative reaction pathways are discussed. When applicable, the involvement of specific enzymatic isoforms is presented. Finally, the activity of many of these conjugative biotransformation reactions is subject to large interindividual variation--often due to the presence of common nucleotide polymorphisms within the genes encoding those enzymes. Functionally significant genetic polymorphisms that might contribute to variable conjugation of estrogens and catechol estrogens are also discussed.

Catechol-O-methyltransferase (COMT) plays both a regulatory and protective role in catechol homeostasis. It contributes to the regulation of tissue levels of catecholamines and catecholestrogens (CEs) and, by blocking oxidative metabolism of catechols, prevents endogenous and exogenous catechols from becoming a source of potentially mutagenic electrophiles. Evidence implicating CEs in carcinogenesis, in particular in the hamster kidney model of estrogen-induced cancer, has focused attention on the protective role of COMT in estrogen target tissues. We have previously reported that treating hamsters with estrogens causes translocation of COMT to nuclei of epithelial cells in the renal cortex, the site of CE biosynthesis and where the cancers arise. This finding suggested that nuclear COMT may be a marker of a threat to the genome by catechols, including CEs. It is postulated that CEs play a role in the genesis of breast cancer by contributing to a state of chronic oxidative stress that is presumed to underlie the high incidence of this disease in the United States. Therefore, here we used immunocytochemistry to re-examine human breast parenchyma for nuclear COMT. In addition to confirming previous reports of cytoplasmic COMT in mammary epithelial cells, we identified nuclear COMT in foci of mammary epithelial cells in histologically normal breast tissue of virtually all control (macromastia) and cancer patients and in breast cancer cells. There was no correlation between tissue histology and the numbers of cells with nuclear COMT, the size of foci containing such cells, or intensity of nuclear COMT immunostaining. The focal nature of the phenomenon suggests that nuclear COMT does not serve a housekeeping function but that it reflects a protective response to an increased local catechol load, presumably of CEs and, as such, that it may be a characteristic of the population of women studied who share the same major risk factor for developing breast cancer, that of living in the industrialized West.

Catecholestrogens are postulated to contribute to carcinogenesis by causing DNA damage mediated by reactive oxygen species generated during redox cycling between catechol and quinone estrogens, and by quinone estrogens that can form depurinating adducts. The above hypothesis is based principally on studies of the cancers that develop in renal cortex of hamsters treated with primary estrogens: Hamster kidney can catalyze 2- and 4-hydroxylation of estrogens and support their redox cycling, and the kidneys of estradiol-treated hamsters show evidence of oxidative cellular and DNA damage. Here we used immunocytochemisty to test the postulate that catechol-O-methyltransferase (COMT), the enzyme that can prevent oxidation of catecholestrogens to their quinone derivatives, would be induced in renal cortex of hamsters treated with estradiol or ethinyl estradiol. In kidneys of control hamsters, COMT was localized in cytoplasm of epithelial cells of proximal convoluted tubules, predominantly in the juxtamedullary region where the estrogen-induced cancers arise. After 2- or 4-weeks of treatment with either estrogen, COMT was seen in epithelial cells of proximal convoluted tubules throughout the cortex, and many cells also showed intense nuclear COMT immunoreactivity. Estradiol-induced renal cancers were negative for COMT, but were surrounded by tubules with intense cytoplasmic and nuclear immunostaining. The nucleus-associated COMT was shown by immunoblot analysis to be the soluble form of the enzyme. Using reverse transcription-polymerase chain reaction amplification, hamster kidney COMT was shown to lack the putative nuclear localization signal sequence present in human COMT. A second phase II enzyme, CuZn-superoxide dismutase (CuZnSOD), was shown by immunocytochemistry to remain extranuclear in proximal convoluted tubules of estrogen-treated hamsters, which indicates entry of COMT into the nucleus to be selective. The findings are consistent with the catechol/quinone estrogen hypothesis of estrogen-induced cancer, while the translocation of the enzyme to the nucleus following estrogen treatment suggests a response to a threat to the genome by electrophilic products of catechols.

The expression of aromatase by breast cancer cells and the role of locally produced estrogen in the stimulation of tumor growth has been controversial. The present study was performed to determine the site of aromatization in human breast cancers, using both immunocytochemistry and in situ hybridization. The functional significance of locally produced estrogens on growth of the tumor was addressed by measuring aromatase activity and a marker of proliferation (PCNA score). In addition, histocultures of some tumors were carried out to investigate whether testosterone aromatization could stimulate tumor proliferation. Of the 19 tumors investigated, 10 (52.6%) showed significant immunoreactivity to antiaromatase antibody in the cytoplasm of tumor epithelial cells and in surrounding stromal cells. The presence of aromatase mRNA detected by ISH was also located in tumor epithelial cells and stromal cell, and the pattern of expression was the same as with immunocytochemistry. In the ten tumors that showed immunoreaction to aromatase, the average aromatase activity measured in cryosections was 286.5 +/- 18.6 (SE) fmol estrogen/mg protein.h, whereas in nine tumors with weak aromatase immunoreaction, the enzyme activity was 154.7 +/- 19.3 (SE) fmol estrogen/mg protein-h (P < 0.05). The mean PCNA score was 33.8 +/- 5.1 (SE)% in strongly stained tumors and 20.8 +/- 2.0 (SE)% in weakly stained tumors (P < 0.05). Aromatase activity level and PCNA score were significantly correlated. In histoculture of four tumors, estradiol increased the incorporation of [3H]-thymidine into DNA. In two of these tumors, aromatase activity was high and [3H]-thymidine incorporation into DNA was also stimulated by testosterone. In the other two tumors that had low aromatase activity, no such stimulation occurred with testosterone. The results indicate that aromatase is expressed mainly in tumor epithelial cells and that sufficient amounts of estrogen are synthesized by the tumor to produce a proliferative response. It is concluded that estrogen synthesis by cancer cells could play a important role in promoting growth in a significant proportion of breast tumors.

In the previous communication we described a histochemical method for measuring soluble guanylate cyclase (sGC) activity in sections of rat liver. In theory, this method could be used to assess nitric oxide synthase (NOS) activity by the increased sGC activity induced by the additional presence of the substrates for NOS activity. We found that this was correct provided that the concentration of the colloid stabilizer in the reaction medium was decreased to just below the concentration required to fully stabilize the guanylate cyclase activity in the sections. This was related to the fact that the site of NOS activity was different from that of the sGC activity in the hepatocytes, so that the NO generated had to diffuse from the Kupffer cells to the hepatocytes as could occur only in partially unstabilized sections. Optimal concentrations of arginine and of NADPH have been determined for demonstrating NOS activity; the increased reaction was shown to be largely inhibited by methyl-arginine.

Guanylate cyclase liberates pyrophosphate from guanosine triphosphate (GTP). In studies published previously, this phosphate is trapped by lead ions even though it is known that free lead ions inactivate a considerable proportion of this enzymatic activity. To overcome the damaging effects of fixation, this study used fresh cryostat sections stabilized with a sufficient concentration of a collagen-derived polypeptide to ensure no measurable loss of guanylate cyclase activity. To avoid the damaging influence of free lead ions, we used a hidden metal capture reagent, i.e., a complex of lead ammonium citrate/acetate that does not react with GTP but which rapidly forms a precipitate with the pyrophosphate liberated by the enzyme. The lead precipitate is then converted into the colored sulfide which is measured in individual cells by microdensitometry. This system was used to measure guanylate cyclase activity in individual cells in unfixed sections of rat liver.

Current knowledge of changes in the mammary epithelium relevant to breast carcinogenesis is limited to when histological changes are already present because of a lack of biomarkers needed to identify where such molecular changes might be ongoing earlier during the decades-long latent stages of breast carcinogenesis. Breast reduction tissues from young women and teenagers, representative of the USA's high breast cancer incidence population, were studied using immunocytochemistry and a targeted PCR array in order to learn whether a marker of chronic oxidative stress [protein adducts of 4-hydroxy-2-nonenal (4HNE)] can identify where molecular changes relevant to carcinogenesis might be taking place prior to any histological changes. 4HNE-immunopositive (4HNE+) mammary epithelial cell-clusters were identified in breast tissue sections from most women and from many teenagers (ages 14–30 y) and, in tissues from women ages 17–27 y with many vs. few 4HNE+ cells, the expression of 30 of 84 oxidative stress associated genes represented in SA Bioscience RT2 Oxidative Stress and Antioxidant PCR array was decreased and only one was increased > 2-fold. This is in contrast to increased expression of many of these genes known to be elicited by acute oxidative stress. The findings validate using 4HNE-adducts to identify where molecular changes of potential relevance to carcinogenesis are taking place in histologically normal mammary epithelium and highlight differences between responses to acute vs. chronic oxidative stress. We posit that the altered gene expression in 4HNE+ tissues identified reflects adaptive responses to chronic oxidative stress that enable some cells to evade mechanisms that have evolved to prevent propagation of cells with oxidatively-damaged DNA and to accrue heritable changes needed to establish a cancer.

Catecholestrogens (CE) are products of aromatic hydroxylation at C−2 or C−4 of primary estrogens and their synthetic steroidal agonists. CE are generated in a number of target organs where they may act as. local mediators of estrogen action. They have also been implicated in carcinogenesis, since CE are substrates for redox cycling and hence, a potential source of free radicals. Biogenesis of CE can occur via two mechanisms, one monooxygenase and the other peroxidative. These two mechanisms have different implications for estrogen action because of differences in their co−factor requirement and in the amounts of 2− and 4−hydroxylated estrogens they generate. This review focuses on the phase I enzymes, the cytochromes P450 that catalyze 2− and 4−hydroxylation of estrogens, on the distinctive properties of 2− and 4−OHCE and on the phase II enzymes responsible for their inactivation.

Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways.

Intracerebroventricular (i.c.v.) administration of N G - monomethyl- l -arginine monoacetate (NMMA; 500 μg; 402 mM) and G - nitro- l -arginine methyl ester (NAME; 270 μg; 200 mM), inhibitors of nitric oxide synthase, enhanced the rise in oxytocin but not vasopressin levels in plasma of conscious rats following 24 h of water deprivation. This effect of NMMA occurred by 10 min after administration, reached its peak at 15 min and decreased by 20 min. Daily administration of lower doses (50 μg and 0.5 μ g 5 μ l , i.c.v.) of another inhibitor of nitric oxide synthase, N G - nitro- l -arginine , just before and after 24 h of water deprivation and in control animals treated similarly were without effect on either vasopression or oxytocin levels. Nitric oxide, therefore, attenuates preferentially the release of oxytocin during dehydration.

Characterization of enzymes mediating the formation of catecholestrogens (CE) by hamster kidney is of importance because of the proposed role of CE in renal cancer induced in this species by estrogens. We have reexamined the potential of hamster kidney to convert estradiol (E2) to 2- and 4-hydroxylated CE because of recent evidence of the limitations of assays used in previous studies, in particular in measuring 4-hydroxylation of estrogens. Under conditions optimized for NADPH-dependent activity, hamster kidney microsomes exhibited high levels of both E2-2- and E2-4-hydroxylase activities. Evidence that the two activities depend on different forms of cytochrome P-450 was obtained by the demonstration that 2- and 4-hydroxylation of E2 were affected differentially 1) by chronic treatment of hamsters with E2 and 2) by fadrozole hydrochloride, a selective cytochrome P-450 inhibitor. NADPH-dependent 2-hydroxylation of E2 from control and E2-treated hamsters, measured by a direct product isolation assay, was 1 order of magnitude higher (apparent maximum velocity, 24-32 and 6-12.5 pmol/mg protein.min in control and E2-treated hamsters, respectively) than that reported previously using radioenzymatic assays. NADPH-dependent 4-hydroxylation of E2 in controls approached and in E2-treated hamsters exceeded 2-hydroxylation of E2 (apparent maximum velocity, 17-21 and 7.5-19 pmol/mg protein.min in control and E2-treated hamsters, respectively). Thus, estrogen treatment reversed the ratios of NADPH-dependent E2-2-/4-hydroxylase activities by causing a much greater decline in 2- than 4-hydroxylation of E2 (P less than 0.007, by analysis of variance). Fadrozole hydrochloride caused a marked dose-dependent decrease in 2-hydroxylation of E2, in contrast to a small nondose-dependent inhibition of 4-hydroxylation. Under conditions optimized for peroxidatic organic hydroperoxide-dependent activity, hamster kidney microsomes generated 2- and 4-hydroxylated CE in similar amounts. The amounts of the two CE and, consequently, the ratios remained unaffected by estrogen treatment (1:0.9 and 1:1.0 in control and E2-treated hamsters, respectively). Thus, this study establishes that CE can be generated in the same tissue by three different pathways, i.e. NADPH-dependent E2-2-hydroxylase, NADPH-dependent E2-4-hydroxylase, and organic hydroperoxide-dependent E2-2/4-hydroxylase activities. We also show that these three activities can be regulated differentially and are, thus, probably mediated by different forms of cytochrome P-450. In hamster kidney, the potential to generate 4-hydroxylated CE metabolites with distinct properties could be a factor in this tissue's vulnerability to estrogen-induced carcinogenesis.

We previously developed a highly aggressive cell line from heart metastases of 4T1 breast carcinoma (designated 4THM), which produced liver metastases (designated 4TLM). In this study, gene array analysis (GAEA) compared gene expression profiles in 4TLM with profiles in 4T1 and 4THM primary tumors. GAEA demonstrated that 4T1 and 4THM tumors differed in about 250 genes. Over 1,000 genes, however, were expressed differently in 4TLM compared with primary tumors. A cohort of 16 genes showed significantly decreased expression in 4THM tumors, which decreased even further in 4TLM. Many of these genes have been implicated in breast cancer, and many are involved in cell adhesion and junctional complexes. Expression of multiple tight and adherence junction proteins was either downregulated or disappeared in 4TLM; downregulation of claudin 4, claudin 7 and gamma-catenin was confirmed by quantitative polymerase chain reaction, immunoblot, and immunocytochemical (ICC) analyses. At the protein level, intact ZO-1 was also observed in 4T1 tumors, but was not expressed in 4THM or 4TLM tumors. ICC demonstrated expression of gamma-catenin at the plasma membrane with 4T1 tumors, whereas staining appeared to be nuclear/perinuclear in 4THM tumors. Claudin 7 staining was also seen in monocyte/pmacrophage-like cells in liver around metastatic lesions by ICC, and it appeared that larger 4TLM tumors apparently reexpressed claudin 7 RNA and protein. Our results demonstrate that decreased or abnormal expression of a number of cell adhesion/junctional proteins, including claudin 4, 7, ZO-1 and gamma-catenin, correlates with liver metastases, and that cell adhesion molecules in the microenvironment are also altered.

Rat fetuses from mothers stressed chronically by immobilization and high intensity illumination beginning on day 14 of gestation have higher than normal levels of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in Leydig cells on day 17 of gestation and lower than normal levels on days 18 and 19. Plasma testosterone titers in normal and stressed male fetuses closely parallel the activity of 3 beta HSD in fetal Leydig cells. In the present study quantitative cytochemistry was used to determine whether the stress-induced alterations in 3 beta HSD activity could be prevented by treating the mother with naltrexone, an opioid receptor blocker, before each stress session. Naltrexone normalized 3 beta HSD activity on days 18 and 19 of gestation, suggesting that the stress-induced suppression involves the endogenous opioid system. In contrast, naltrexone did not prevent the elevation in enzyme activity seen on day 17 in stressed fetuses. The persistence of a stress-induced surge on day 17, in spite of naltrexone therapy, suggests that some nonopioid mechanism is operational at that time.

Knowledge of latent stages of breast carcinogenesis is limited to when histological changes are already present due to a lack of; a) cytochemical marker(s) needed to identify loci in histologically normal breast tissues where procarcinogenic conditions exist and; b) microscopic systems needed to geno- and phenotype cells in such loci. In 2 recent publications we presented evidence that protein adducts of 4-hydroxy-2-nonenal (4HNE), markers of a procarcinogenic state of chronic oxidative stress (OxS), might provide the needed fiducial marker. Specifically, a) we documented the presence of 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells in terminal ductal units (TDLUs) in breast tissues of healthy teenagers and young women and their association with altered expression of OxS-related mRNAs and; b) presented Fourier-transform infrared microspectroscopic evidence of differences in biomolecular profile of ME cells and associated intra- and interlobular stromal cells in 4HNE+ vs. 4HNE- TDLUs, i.e., of cells of origin of BCs and cells in their supportive stromal microenvironment. Here we present further evidence of molecular changes in breast tissues of young women (ages 17-30 y) associated with 4HNE adducts. A comparison of the transcriptome of breast tissues with many 4HNE+ vs. few 4HNE+ TDLUs (controls) by Illumina 24,000 gene platform and SABioscience 84 gene Cell Death PCR Array revealed reprogramming of many homeostatic mechanism in 4HNE+ TDLUs. Notably, a) DASL platform identified transcripts for 395 genes that differed (p = 2 fold between 4HNE+ vs. 4HNE- tissues. The findings support using 4HNE adducts to identify in histologically normal breast tissues loci harboring cells that might initiate a cancer. Our findings fit an evolutionary model of carcinogenesis and reflect adaptive responses to stressors that serve to preserve tissue integrity, e.g., by developing resistance to OxS and to apoptosis, responses however that over time can lead to the emergence of a cancer. Our recently acquired Toponome Imaging System (TIS) enables identifying such responses to chronic OxS implied by the presence of 4HNE adducts. TIS is the first high throughput fluorescence robotic microscopic system that can provide a map, a toponome, of up to 100 proteins at 40nm functional resolution in individual cells in a tissue section. In ongoing experiments we are using TIS and antibodies against up to 21 proteins to test the postulate that 4HNE+ TDLUs are enriched with cells with an apoptosis resistant phenotype, a postulate supported by our transcriptional data and predicted by the evolutionary model of carcinogenesis. Citation Format: Judith Weisz, Debra Shearer, Anna C. Salzberg. Using reduction mammoplasty tissues from healthy young women living in USA's high breast cancer (BC) risk posing environment to open up to study the “black box” of latent stages of breast carcinogenesis; a key to develop strategies for BC [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4558. doi:10.1158/1538-7445.AM2015-4558