Tumor Metastasis: Molecular Insights and Evolving Paradigms

Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing Mena, a Tie2hi/Vegfhi macrophage and an endothelial cell, creates a micro-anatomic doorway called “tumor microenvironment of metastasis” (TMEM). TMEM are responsible for cancer cell intravasation and dissemination to distant sites. The density of TMEM doorways is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that TMEM doorways create increased localized vascular permeability which cancer cells utilize to intravasate, the precise molecular mechanisms relating TMEM-doorway function and intravasation has not been elucidated. Active TMEM doorways are found in pre-invasive and invasive ductal breast carcinoma as well as in metastatic foci in lymph nodes and lungs, indicating that TMEM-mediated cancer cell dissemination occurs not only at the primary tumor site but also at metastatic sites, which may perpetuate metastatic dissemination even after removal of the primary tumor. Thus it is essential to understand the exact molecular mechanism of TMEM-doorway function so that specific targeted therapies can be developed to intercept systemic cancer cell dissemination. We outline here the exact molecular mechanism of TMEM-doorway functions. TMEM doorway endothelial cell-secreted Ang2 (a Tie2 ligand) stimulates VEGF expression and production by the Tie2hi TMEM macrophage. Subsequently, the TMEM doorway tumor cell- secreted CSF1 stimulates local secretion of VEGF from the Tie2hi TMEM macrophages, leading to dissociation of endothelial adherens and tight junctions near TMEM and cancer cell intravasation. In addition, we show that acute blockage of CSF1R and Tie2-Ang2 signaling by inhibitors and blocking antibodies both in vitro and in mammary tumors leads to decreased macrophage VEGF production and secretion, decreased tumor cell trans-endothelial migration, and decreased TMEM-dependent vascular permeability, tumor cell dissemination and circulating tumor cells. This is the first description of the molecular mechanisms regulating TMEM doorway function and thus represents a major step in defining new biomarkers and targets for the treatment of metastatic tumors. Citation Format: Chinmay Surve, Allison S. Harney, Mary Chen, Yarong Wang, Xianjun Ye, Yu Lin, Ved Sharma, Richard Stanley, Maja H. Oktay, John S. Condeelis. Regulation of breast tumor metastasis by the dynamic interaction between the TMEM doorway macrophage, tumor and endothelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-10.

Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Metastases, rather than the primary tumors from which these malignant growths are spawned, are culpable for greater than 90 % of human cancer-associated mortality. Metastases arise through the completion of a series of cell-biological events - collectively termed "the invasion-metastasis cascade" - which involve the dissemination of tumor cells to distant organ sites and their subsequent adaptation to these foreign microenvironments. Importantly, a number of endogenous mechanisms exist that serve to prevent metastatic progression. These safeguards must be overcome by incipient metastatic tumor cells in order for them to generate detectable metastases. Here, I highlight four endogenous mechanisms that protect against the development of metastatic disease in breast carcinomas. I discuss how the expression of these genes are dampened during malignant progression, the downstream responses they orchestrate, and clinical opportunities to therapeutically target these mechanisms. Indeed, one potentially effective strategy for the remediation of metastatic disease involves the reactivation of endogenous anti-metastasis mechanisms. Therefore, knowledge regarding endogenous anti-metastasis mechanisms may both further our comprehension of the basic etiology of metastasis and also guide the treatment of human tumors.

Making the Case for DCC and UNC5C as Tumor-Suppressor Genes in the Colon

Primary tumor versus metastasis: new experimental models for studies on cancer cell homing and metastasis in melanoma

Cancer metastasis is a complex and multistep process whereby cancer cells escape the confines of the primary site to establish a new residency at distant sites. This multistep process is also known as the invasion-metastasis cascade. The biological and molecular mechanisms that control the invasion-metastasis cascade, which ultimately leads to the spread of cancer cells into distant sites, remain poorly understood. Kindlin-2 (K2) belongs to the 4.1-ezrin-ridixin-moesin (FERM) domain family of proteins, which interact with the cytoplasmic tails of β-integrin subunits, leading to the activation of extensive biological functions. These biological functions include cell migration, differentiation, cancer initiation, development, and invasion. In this review, we will discuss the various molecular signaling pathways that are regulated by K2 during the invasion-metastasis cascade of cancer tumors. These signaling pathways include TGFβ, Wnt/β-Catenin, Hedgehog, p53 and senescence, and cancer stem cell (CSC) maintenance. We will also discuss the molecular signaling pathways that regulate K2 function both at the transcriptional and the posttranslational levels. Finally, we will consider molecular mechanisms to specifically target K2 as novel therapeutic options for cancer treatment.

BackgroundGenetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood.ResultsWe investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations.ConclusionsOur results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of somaclonal variation in rice, which is concomitant with epigenetic alterations. Perturbed expression by tissue culture of a set of 41 genes encoding for enzymes involved in DNA repair and DNA methylation is associated with both genetic and epigenetic alterations. There exist fundamental differences among distinct genotypes, pure-lines, hybrids and tetraploids, in propensities of generating both genetic and epigenetic alterations under the tissue culture condition. Parent-of-origin has a conspicuous effect on the alteration frequencies.

Barrett's esophagus (BE) is described by the transformation of the normal squamous epithelium into metaplastic columnar epithelium, driven by chronic gastroesophageal reflux disease (GERD). BE is a recognized premalignant condition and the main precursor to esophageal adenocarcinoma (EAC). Understanding the molecular mechanisms underlying BE carcinogenesis is crucial for improving prevention, surveillance, and treatment strategies. This narrative review examines the molecular abnormalities associated with the progression of BE to EAC. This study highlights inflammatory, genetic, epigenetic, and chromosomal alterations, emphasizing key pathways and biomarkers. BE progression follows a multistep process involving dysplasia and genetic alterations such as TP53 and CDKN2A (p16) mutations, chromosomal instability, and dysregulation of pathways like PI3K/AKT/mTOR. Epigenetic alterations, including aberrant microRNA expression or DNA methylation, further contribute to this progression. These molecular changes are stage-specific, with some alterations occurring early in BE during the transition to high-grade dysplasia or EAC. Innovations in chemoprevention, such as combining proton pump inhibitors and aspirin, and the potential of antireflux surgery to halt disease progression are promising. Incorporating molecular biomarkers into surveillance strategies and advancing precision medicine may enable earlier detection and personalized treatments. BE is the primary preneoplastic condition for EAC. A deeper understanding of its molecular transformation can enhance surveillance protocols, optimize the management of gastroesophageal reflux inflammation, and refine prevention and therapeutic strategies, ultimately contributing to a reduction in the global burden of EAC.

Biological Markers in Oral Squamous Cell Carcinoma

Our understanding of cancer as a disease process has evolved tremendously over the centuries, culminating in the late 20th century with the discovery of oncogenes and tumor suppressor genes and subsequent understanding of carcinogenesis as it is depicted in the classic hallmarks of cancer paper by Hanahan and Weinberg [1]. Genetic and epigenetic alterations have been increasingly identified in many diseases, including a wide variety of neoplasms. As more of these alterations are being discovered, their significance in some diseases remains still obscure, while they have become diagnostic, prognostic, and predictive genetic signatures for others. It is becoming clear that a given genetic alteration and associated molecular changes involving particular pathways in the neoplastic cell may not necessarily be specific for that particular type of cancer. Rather, such a genetic alteration represents a more general abnormality involved in the neoplastic transformation of a variety of cancers in different organs. For instance, mutations in BRAF can be seen in unrelated cancers such as melanoma, colorectal and lung carcinomas [2, 3], brain tumors [4], and hematolymphoid malignancies [5]. This paves the way to potentially identifying which of these alterations a cancer has, rather than the classical diagnostic approach of which organ it originates from or what the histologic type is, essentially redesigning the cancer taxonomy. This disease or organ-agnostic type of approach is also the mainstay of a “personalized” approach to cancer treatment. Some of these alterations are also used as diagnostic aids in differential diagnostic settings, such as IDH-1 R132H identification by immunohistochemistry or the identification of other IDH-1 or IDH-2 mutations in diffuse gliomas, in contrast to well-circumscribed gliomas or reactive gliosis [6]. An increasingly growing number of these alterations are now the subject of targeted therapies especially in the form of small molecule kinase inhibitors. They can also provide significant prognostic (such as FLT-3 mutation in acute myelogenous leukemia) and predictive information, further blurring the boundaries between diagnosis and treatment, as well as between basic and clinical sciences. It is not enough anymore for pathologists to provide only diagnosis but also an array of molecular markers that facilitate the discussion about prognosis for given cancer and potential therapeutic options. Of paramount importance are the explosion of knowledge in molecular biology and its clinical application in the form of molecular diagnostics, involving high-technology testing. Altogether, we have a better understanding of how such alterations operate in the process of oncogenesis, which in turn helps us better diagnose and treat neoplasms based on these alterations. These discoveries have also influenced the pharmaceutical and biotechnological fields, resulting in development of additional treatment options for cancer patients: O6-methylguanine DNA methyltransferase (MGMT) gene methylation status in glioblastoma and response to alkylating agents [7], KIT mutations in gastrointestinal stromal tumor (GIST) and response to imatinib [8], ALK gene rearrangements in ALK-positive nonsmall cell lung carcinoma and response to crizotinib [9], and EGFR mutations in nonsmall cell lung carcinoma and response to gefitinib [10] are a few examples of how genetic alterations, identified by molecular diagnostic testing, can impact treatment decisions. Despite this enormous success over the last 50 years since the discovery of DNA double helix and the discovery of the first human oncogene, there are still a lot of questions in regard to the optimal way of molecular testing, distinguishing between passenger and driver mutations in a tumor, dealing with the vast intra- and intertumor heterogeneity, and introducing other nongenetic molecular markers such as proteins (proteomics) and metabolites (metabolomics). In this special issue, we present a variety of manuscripts that report technical, basic, and clinical research, molecular biology, and diagnostic and therapeutic aspects of neoplasia, as well as reviews of these subjects. The topics are not limited to a particular organ, system, or type of neoplasia. The manuscripts emphasize the importance of molecular markers in various aspects of neoplasia in an attempt to provide the reader with an up-to-date source of current research on molecular markers in cancer.

Mesenchymal stem cells (MSCs) are multipotent stem cells that reside in various parts of the body like adipose tissue, bone marrow and umbilical cord with an ability to differentiate into chondrocytes, adipocytes and osteocytes. Rigorous research has helped us understand that MSCs home to wound sites and this homing mechanism has been used in the treatment of many inflammatory diseases. It is now emerging that MSCs are an important component of the tumor microenvironment (TME) and contribute to tumor plasticity Plasticity in tumor cells refers to the ability to undergo molecular and phenotypic changes due to environmental cues or genetic alterations. . MSCs are one of the key players within the TME and can either inhibit or promote tumor cell growth by distinct types of cellular interaction. These multifunctional cells can reorganize the tumor stroma : It refers to the supportive tissue consisting of connective tissue or blood vessels around an organ or tumor. which, in turn, can trigger changes in metastatic behavior and promote dedifferentiation to develop cancer stem-like cells. On the contrary, MSCs have been proposed as ideal candidates as drug delivery agents in treatment of various cancers. The double-edged role of MSCs in tumor has made it difficult to pinpoint whether MSCs promote or inhibit tumor growth and progression. During cancer progression, tumor cells undergo molecular and phenotypic changes as a result of microenvironmental cues, genetic and epigenetic alterations and treatment-imposed selective pressures which contribute to tumor heterogeneity and therapy resistance. So, understanding the mechanisms underlying the tumor plasticity may deliver new strategies for targeting cancer metastasis and resistance to therapy. Accordingly, this review focuses on diverse mechanisms of interaction between MSCs and cancer cells with emphasis on different types of intercellular communication affecting tumor progression and metastasis.

The American Association for Cancer Research has been the citadel for communicating research on chemical carcinogens for over a century. It therefore seems appropriate that a review of chemical carcinogenesis inaugurates a series of articles highlighting advances in understanding, treating, and preventing cancer.At the dawn of the 20th century, we had recognized that chemicals cause cancer, but we had not yet identified individual cancer-causing molecules, nor did we know their cellular targets. We clearly understood that carcinogenesis, at the cellular level, was predominantly an irreversible process. What we lacked was knowledge of the mechanisms by which chemicals cause cancer and the molecular changes that characterize tumor progression.We now are early in a century in which cancer is being investigated at the molecular level, and we have developed technologies that afford unprecedented power to delineate and manipulate altered pathways in cancer cells. Can we harness new insights and technologies to prevent or obliterate human cancers or delay their progression? Can we identify individuals who have a particularly high susceptibility to specific environmental carcinogens?The history of chemical carcinogenesis is punctuated by key epidemiologic observations and animal experiments that identified cancer-causing chemicals and that led to increasingly insightful experiments to establish molecular mechanisms and to reduction of human exposure. In 1914, Boveri (1) made key observations of chromosomal changes, including aneuploidy. His analysis of mitosis in frog cells and his extrapolation to human cancer is an early example of a basic research finding generating an important hypothesis (the somatic mutation hypothesis). The first experimental induction of cancer in rabbits exposed to coal tar was performed in Japan by Yamagiwa and Ichikawa (2) and was a confirmation of Pott's epidemiologic observation of scrotal cancer in chimney sweeps in the previous century (Fig. 1; ref. 3). Because coal tar is a complex mixture of chemicals, a search for specific chemical carcinogens was undertaken. British chemists, including Kennaway (4), took on this challenge and identified polycyclic aromatic hydrocarbons, for example, benzopryene, which was shown to be carcinogenic in mouse skin by Cook, Hewett, and Hieger in 1933 (5). The fact that benzopyrene and many other carcinogens were polyaromatic hydrocarbons lead the Millers (6) to postulate and verify that many chemical carcinogens required activation to electrophiles to form covalent adducts with cellular macromolecules. This in turn prompted Conney and the Millers (7) to identify microsomal enzymes (P450s) that activated many drugs and chemical carcinogens.The discovery of DNA as the genetic material by Avery, MacLeod, and McCarthy (8) and the description of the structure of DNA by Watson and Crick (9) indicated that DNA was the cellular target for activated chemical carcinogens and that mutations were key to understanding mechanisms of cancer. This led to defining the structure of the principal adducts in DNA by benzo(a)pyrene (10) and aflatoxin B1 (11). The concepts developed in investigating mechanisms of chemical carcinogenesis also led to discoveries that are relevant to other human conditions in addition to cancer, including atherosclerosis, cirrhosis, and aging.Global epidemiologic studies have indentified environmental and occupational chemicals as potential carcinogens. The most definitive epidemiologic studies have been those in which a small group is exposed to an inordinately large amount of a specific chemical, such as aniline dyes.Figure 1 illustrates exposure of individuals to residues from fossil fuel in chimneys, to tobacco smoke, and to fungi containing aflatoxin, and the identification of the responsible carcinogen(s). Active smoking and exposure to second-hand smoke are among the major causes of cancer mortality worldwide. Even after causative chemicals are identified, however, measurement of accumulated exposure of individuals in different environments remains an important challenge.The fact that genetic changes in individual cancer cells are essentially irreversible and that malignant changes are transmitted from one generation of cells to another strongly points to DNA as the critical cellular target modified by tobacco smoke and environmental chemicals. DNA damage by chemicals occurs randomly; the phenotypes of associated carcinogenic changes are determined by selection.Cancers caused by environmental agents frequently occur in tissues with the greatest surface exposure to the agents: lung, gastrointestinal tract, and skin. Recently, the study of chemical carcinogenesis has merged with studies on the molecular changes in cancer cells, thus generating biological markers to assess altered metabolic pathways and providing new targets for therapy. Although these are exciting areas, they may be peripheral to attacking the primary causes of the most common human cancers. As we catalog more and more mutations in cancer cells and more and more changes in transcription regulation, it becomes increasingly apparent that we need to understand what generates these changes. The fact that chemicals cause random changes in our genome immediately implies that our efforts need to be directed to quantifying these changes, reducing exposure, and developing approaches to chemoprevention.Chemical carcinogens cause genetic and epigenetic alterations in susceptible cells imparting a selective growth advantage; these cells can undergo clonal expansion, become genomically unstable, and become transformed into neoplastic cells. This classic view of carcinogenesis has its origin in experimental animal studies conducted in the mid 20th century. The first stage of carcinogenesis, tumor initiation, involves exposure of normal cells to chemical or physical carcinogens. These carcinogens cause genetic damage to DNA and other cellular macromolecules that provide initiated cells with both an altered responsiveness to their microenvironment and a proliferative advantage relative to the surrounding normal cells.Early in the field of chemical carcinogenesis, investigators recognized that perturbation of the normal microenvironment by physical means, such as wounding of mouse skin or partial hepatectomy in rodents (12, 13) or chemical agents, such as exposure of the mouse skin to certain phorbol esters (14), can drive clonal expansion of the initiated cells toward cancer. In the second stage, tumor promotion results in proliferation of the initiated cells to a greater extent than normal cells and enhances the probability of additional genetic damage, including endogenous mutations that accumulate in the expanding population. This classic view of two-stage carcinogenesis (14) has been conceptually important but also an oversimplification of our increasing understanding of the multiplicity of biological processes that are deregulated in cancer. In addition, an active debate continues on the relative contribution of procarcinogenic endogenous mechanisms—for example, free-radical–induced DNA damage (15), DNA depurination (16), DNA polymerase infidelity (17), and deamination of 5-methylycytosine (18)—compared with exposure to exogenous environmental carcinogens (19). The enhancement of carcinogens by epigenetic mechanisms such as halogenated organic chemicals and phytoestrogens (20), as well as the extrapolation of results from animal bioassays for identifying carcinogens to human cancer risk assessment, are also difficult to quantify (21). As discussed below, this debate is not merely an academic one, in that societal and regulatory decisions critical to public health are at issue. The identification of chemical carcinogens in the environment and occupational settings [benzo(a)pyrene and tobacco-specific nitrosamines in cigarette smoke, aflatoxin B1 (AFB1) residues from fossil fuel, vinyl chloride, and benzene] has led to regulations that have reduced the incidence of cancer.A timeline of selected experimental advances in chemical carcinogenesis that have important implications is presented in Fig. 2. First, the selected advances reflect the judgment of the authors and consultants, and remain to be modified by the readers, and, ultimately, by history. Second, the timeline shows the progression of results; an important observation generates new hypotheses that are tested by experiments with increasing mechanistic focus. Third, the timeline is punctuated with three important molecular discoveries (DNA structure, DNA sequence, and the PCR) that refocused experiments in chemical carcinogenesis (9, 22, 23). Fourth, many technological advances have allowed conceptual ideas to be experimentally tested, including the sensitive detection of chemical carcinogens by high-pressure liquid chromatography (24) and mass spectrometry (25), detection of DNA adducts by postlabeling (26) and by specific antibodies (27), transcriptional profiling by arrays (28, 29), and quantitation of mutagenicity of carcinogens using bacterial genetics (19).In the first half of the 20th century, the experimental focus was on identifying chemical carcinogens in complex mixtures, and on determining their metabolism and cellular targets. With the recognition that genes are encoded in DNA (9) and that DNA is transferred from one cellular generation to the next (30), research rapidly focused on the interaction of activated chemical carcinogens with DNA and on mutations that result from DNA alterations as well as the identification of key mutated (31) or deregulated genes including oncogenes and tumor suppressor genes (32). Underlying these studies was the expectation that delineation of mutated genes would identify them as specific targets for chemotherapy. The expectation that targeting individual mutated or rearranged gene products would be efficacious for cancer treatment has thus far been verified in only a limited number of situations, such as the use of imatinib for chronic myelogenous leukemia (33).The experimental landmarks highlighted in Fig. 2 frequently generated new experiments, and this progression has foretold some of our key concepts on the mechanisms of chemical carcinogenesis. An overriding concept has emerged that links DNA damage by reactive chemicals, the production of mutations by unrepaired DNA adducts, and the selection of cells harboring mutated genes that characterize the malignant phenotype. Studies on arylhydroxylamines provided a paradigm for tracing the metabolism of carcinogens to chemically reactive electrophiles that covalently bind to DNA. 2-Acetylaminofluorene (AAF) is metabolically activated by liver microsomal mixed–function oxygenases to N-hydroxy- and then to N-sulf oxy-AAF, a strong electrophile that forms covalent adducts with guanine moieties in DNA (34). AAF is not mutagenic in bacterial assays, whereas N-hydroxy-AAF is highly carcinogenic (34). N-hydroxy-AAF is rendered inactive by the formation of a glucuronide in the liver that is transported to the bladder and excreted (35). Unfortunately, it is subjected to acid hydrolysis in the bladder to yield active N-hydroxy-AAF, which is associated with human bladder cancer. Thus, the activation and detoxification of a chemical carcinogen in specific cells or tissues can be a major factor in determining tissue and host specificity.The testing of certain concepts in chemical carcinogenesis awaited the development of new technologies. For example, the concept of somatic mutations in cancer (1, 36) preceded by 40 years the establishment of DNA as the genetic material (8) and by 63 years the development of DNA sequencing methods (23) that directly showed clonal mutations in human cancer cells. Also, the mutator phenotype hypothesis formulated in 1974 (17) has been only recently experimentally verified (37).Many hypotheses are still under active investigation. These include the potential importance of carcinogen-protein interactions (38), carcinogen-induced reversion to stem cell–like phenotypes (39), inherited changes in gene expression (40, 41), direct action of nongenotoxic chemicals (42), and targeted interactions of carcinogens with specific genes such as TP53 (43–45). Other concepts focus on carcinogenesis mediated by RNA damage (46), RNA-templated DNA repair (47), specific metastasis genes (48, 49), and sequential clonal lineage pathways in cancer (50, 51).Emerging hypothesis such as anticarcinogens (52), overlapping pathways to malignancy (53), coordinated changes in gene expression (54), epigenetic silencing by chemical carcinogens (40, 55, 56), and oncogene addiction (57) are just beginning to be explored. Finally, there are concepts for which quantitation is lacking, yet have stood the test of time based on their inherent significance; these include the importance of anaerobic metabolism by tumors (58, 59) and the initiation of tumorigenesis by the generation of oxygen-reactive species (15).Although establishing DNA as the genetic material provided a structure that faithfully can be duplicated during each cell division, it rapidly became apparent that DNA was also subject to direct modification by X-rays (60), alkylating agents (61), and by an increasing number of environmental chemicals (62, 63). Changes in DNA by many chemical carcinogens are indirect; they first require activation by P-450 aryl hydroxylases into electrophiles to form covalent adducts with DNA and with other cellular macromolecules (64, 65). Many normally generated reactive molecules that are intermediates in metabolism modify many cellular molecules including DNA and therefore are mutagens and carcinogens. However, not all mutagens seem to be carcinogens. What was unanticipated was the magnitude of DNA modification by normal cellular processes in the absence of exposure to environmental mutagens (66, 67).The lability of DNA in an aqueous environment was first quantified by Lindahl and Nyberg, who measured the rates of depurination (16) and deamination (18) in solution under different conditions and extrapolated these results to those predicted to be present in human cells. They calculated that each normal cell could undergo >10,000 DNA damaging events per day. Endogenously generated modifications of DNA include methylation by S-adenosylmethione, modification by lipid peroxidation products, chlorination, glycosylation, oxidation, and nitrosylation (66–71). Reactive oxygen and nitrogen species are particularly relevant because the activated species are generated by host cells, and the process of resynthesis results in the replacement of >50,000 nucleotides per cell per day (68). To maintain our genomes, we have evolved a network of DNA repair pathways to excise altered residues from DNA (Fig. 3). A major consideration is the relative contribution of environmental and endogenous DNA damage to carcinogenesis. DNA damage by environmental agents would have to be extensive and exceed that produced by normal endogenous reactive chemicals to be a major contributor to mutations and cancer. This consideration underlines the difficulty in extrapolating risk of exposure to that which would occur at very low doses of carcinogens.Human cells possess an armamentarium of mechanisms for DNA repair that counter the extensiveness of DNA damage caused both by endogenous and environmental chemicals. These mechanisms include base excision repair (BER) that removes products of alkylation and oxidation (72–74); nucleotide excision repair (NER) that excises oligonucleotide segments containing larger adducts (75); mismatch repair that scans DNA immediately after polymerization for misincorporation by DNA polymerases (76); and oxidative demethylation (77), transcription-coupled repair (TCR) that preferentially repairs lesions that block transcription (78); double-strand break repair and recombination that avoids errors by copying the opposite DNA strand (79); as well as mechanisms for the repair of cross-links between strands (80, 81) that yet need to be established.Most DNA lesions are subject to repair by more than one pathway. As a result, only a minute fraction of DNA lesions escapes correction are present at the time of DNA replication and can direct the incorporation of noncomplementary nucleotides resulting in mutation (Fig. 3). Unrepaired DNA lesions initiate mutagenesis by stalling DNA replication forks or are copied over by error-prone trans-lesion DNA polymerases (82–84). Alternatively, incomplete DNA repair can result in the accumulation of mutations and mutagenic lesions, such as abasic sites (85).Damage to DNA by chemical carcinogens activates checkpoint signaling pathways leading to cell cycle arrest and allows time for DNA repair processes. In the absence of repair, cells can use special DNA polymerases that copy past DNA adducts (86, 87), or undergo apoptosis by signaling the recruitment of immunologic and inflammatory host defense mechanisms. The demonstration that each methylcholanthrene-induced tumor has a unique antigenic signature provided one of the earliest glimpses into the stochastic nature of cellular responses to carcinogens (88). The immunologic and inflammatory responses facilitate not only engulfment and clearance of damaged cells but also the resulting generation of reactive oxygen (89) and nitrogen radicals (90) that further damage cellular DNA.The concept that chronic inflammation can result in cancer is supported by Virchow's (91) histologic observation of inflammatory lymphocytes infiltrating tumors. Inflammation accompanying the "painting" of coal tar was described by Japanese pathologists in the earliest experimental study of chemical carcinogenesis (2). The classic tumor promoter, croton oil, and its most active ingredient, 12-O-tetradecanoylphorbol-13-acetate, are potent inflammatory agents. In addition to studies of "two-stage" skin carcinogenesis, other animal models have shown the synergistic interaction of chemical carcinogens with proinflammatory agents; for example, respiratory infection with influenza virus synergistically increases the lung cancer response in rats to a carcinogenic N-nitrosamine (92).Chronic inflammation can have a strong inherited basis, e.g. hemochromatosis, or can be acquired from infection by viruses, bacteria, or parasites or be associated with metabolic or physical conditions (93). Obesity has been considered to be a chronic inflammatory condition associated with multiple types of human cancer (94); gastric acid reflux causes chronic inflammation and can progress to Barrett's-associated esophageal adenocarcinoma (95); and colitis can progress to colon cancer (96, 97). Recent advances have begun to uncover the underlying mechanisms of the association between chronic inflammation and cancer.The identification of specific genes by allelic replacements and "knockouts" has facilitated the delineation of complex immune response networks that govern cellular responses to chemical carcinogens. The innate immune system is the first line of defense against pathogenic microorganisms and toxins and responds by generating free radicals, inflammatory cytokines, and the activation of the complement cascade (93, 98). In addition to reactive oxygen species, the past two decades have shown the significance of nitrogen-based free radicals, including nitric oxide and its derivatives (90, 93). The concentration and length of exposure can determine the seemingly paradoxical procarcinogenic and anticarcinogenic activities of free As be discussed in another in the chronic activation of the innate immune system is procarcinogenic and immune system is anticarcinogenic there is a to from an individual is exposed to a carcinogen to the detection of a For most there is an in cancer incidence as a of that tumor progression in a series of sequential This process has been most in colon cancer, with the progression from to to and to metastasis of cancers at different from to a sequential of mutations and genome mutations in DNA activation of of on and of This concept of sequential mutations has been by new including the of somatic mutations in and colon cancers and the demonstration that only a small fraction of colon cancers the three most frequently identified mutations this may identify potential not cancers a mutator a more stochastic cancer cell in a tumor of different and yet only a small of cells preferentially during to random mutations that a selective advantage for this concept is the demonstration that the of mutations in human cancers is greater than that in normal tissues in cell and adenocarcinoma of the colon The genetic of cancer cells produced by mutator mutations increases the that a tumor many cells to and is with the of of research in chemical carcinogenesis have provided a for the analysis of adducts and somatic mutations in as of carcinogen exposure. A paradigm for between of carcinogens exposure and a cancer risk is shown in Fig. a is a example of an environmental chemical carcinogen that has been using this a polycyclic aromatic (53), an aromatic and a tobacco-specific N-nitrosamine are other key epidemiologic studies a association between exposure and the incidence of studies of in multiple animal species, chemical and analysis of the identification of DNA adducts, and of mutagenic the for and to as a human carcinogen from these experimental animal and studies were then and to assess exposure and biological in studies conducted in of high exposure and high incidence of such as and The were and to the of the that is a human The between and was further by the association between exposure and a specific mutation in the nucleotide of of the tumor suppressor gene in In from and The a synergistic interaction between exposure or and of virus infection in the risk of was remain to be For example, the molecular of the synergistic interaction between and is still the and oxidative of to the gene incorporation in the genome of their of by advances in molecular are and they increasingly are being to understanding the interaction of chemical carcinogens with cellular and of DNA has facilitated the identification of specific genes mutated in human cancers. including mass to carcinogen with unprecedented and spectrometry is being with mutagenesis to specific alterations in DNA of the human genome and the identification of DNA enzymes the field of molecular in on individual susceptibility to carcinogens. analysis of carcinogen-induced alterations in the expression of both and the are that can molecules of carcinogens in cells, random mutations in individual cells, analysis of the of molecules and and and genetic to delineate complex pathways in cells. Underlying this progress in understanding chemical carcinogenesis is a cascade of advances in molecular that it to quantify DNA damage by chemical agents, and changes in gene the structure of DNA and the cell including carcinogenesis. in detection of DNA damage, including postlabeling of DNA (27), and mass spectrometry (25), have allowed the detection of a altered base in nucleotides using human DNA. This can be to DNA or RNA in a cell in cell including and it to assess changes in RNA and expression during carcinogenesis. these technologies it increasingly to pathways in cancer cells from to to to have made in identifying chemical carcinogens and their mechanisms of We have increasingly focused on DNA as a the fact at the cellular level, cancer is an inherited a cancer, a cancer. The efforts to chemicals as potential or human carcinogens are not but in most are in The need to identify chemical carcinogens in of human exposure and epidemiologic is on mechanistic and knowledge of and among animal species is a For example, the of in the by a not to be relevant to carcinogenesis, is initiated by epidemiologic verified by animal experiments, and by mechanistic and studies The between carcinogen exposure and the induction of cancer continues to be a of and public debate The of a is a in the of public health that to be as mechanistic accumulate in the field of chemical carcinogenesis has a history of that of cancer cancer risk assessment, public health and and occupational causes of cancer. The concepts of interactions and in the molecular of human cancer risk were generated by the of chemical carcinogenesis, cellular and molecular and cancer genetic in DNA repair and enzymes are of an inherited of in cancer susceptibility of the of cancer risk and detection are based on the knowledge of chemical carcinogenesis, including adducts, somatic and mutation carcinogen exposure and DNA with interactions can have synergistic for example, and in carcinogenesis. models of chemical carcinogenesis to a critical in the field of cancer and in our understanding the mechanisms of cancer and the of in in the field of chemical carcinogenesis remain to be stem cells mutated by chemical carcinogens and become of human chemical carcinogens epigenetic changes during These and other many to be formulated by to investigators in chemical carcinogenesis our understanding of carcinogenesis, and, as a result, cancer and potential of were Cancer and and by The Research of the Cancer for Cancer Research of of this were in by the of This therefore be in with to this selection of the major events in this review of the field are the primary of the with the of the The authors for many of which are of importance to the field of chemical carcinogenesis. We on this subject We for Fig. of chimney and for Fig. and of smoking and aflatoxin, and and for their critical

Metastases, rather than the primary tumors from which these malignant growths are spawned, are culpable for greater than 90 % of human cancer-associated mortality. Metastases arise through the completion of a series of cell-biological events - collectively termed "the invasion-metastasis cascade" - which involve the dissemination of tumor cells to distant organ sites and their subsequent adaptation to these foreign microenvironments. Importantly, a number of endogenous mechanisms exist that serve to prevent metastatic progression. These safeguards must be overcome by incipient metastatic tumor cells in order for them to generate detectable metastases. Here, I highlight four endogenous mechanisms that protect against the development of metastatic disease in breast carcinomas. I discuss how the expression of these genes are dampened during malignant progression, the downstream responses they orchestrate, and clinical opportunities to therapeutically target these mechanisms. Indeed, one potentially effective strategy for the remediation of metastatic disease involves the reactivation of endogenous anti-metastasis mechanisms. Therefore, knowledge regarding endogenous anti-metastasis mechanisms may both further our comprehension of the basic etiology of metastasis and also guide the treatment of human tumors.

Epigenetic alterations in regulatory genes, genetic factors, and genomic instability, which cause breast cancer, can also contribute to disease resistance. HORMAD , which encode proteins containing HORMA domains and are involved in homologous recombination, have important roles in cancer emergence and progression. In this study, we uncovered putative breast cancer therapeutic targets by examining HORMAD1 and HORMAD2 genetic and epigenetic alterations. mRNA levels of HORMAD1 and HORMAD2 in breast cancer samples and normal breast tissues, as well as mRNA levels in normal, breast cancer, and metastatic breast cancer samples, were analyzed using TNMplot. Prognostic value, genetic alterations, epigenetic alterations, genetic variations, ROC plots, functional prediction, and immune infiltration of HORMAD1 and HORMAD2 were conducted with KMPlotter, cBioportal, methsurv, ClinVar, ROC Plotter, PredictSNP, PANTHER, and TIMER 2.0, respectively. Both HORMAD1 and HORMAD2 mRNA levels were lower in breast cancer samples, and lower in metastatic breast cancer samples. Patients expressing higher HORMAD1 and HORMAD2 levels had favorable overall survival (OS) rates than the opposite groups. HORMAD1 and HORMAD2 gene amplifications and deletions were also observed. Pathway enrichment analyses showed that Wnt signaling alterations contributed to cell proliferation. Increased DNA methylation levels were identified in HORMAD2 when compared with HORMAD1 in patients. Two 1021C>T (Q334) and 430A>G (T144A) variants of HORMAD1 were shown to have clinical significance in patients. Also, functional prediction mutant analysis of HORMAD1 confirmed that S287F exerted a deleterious effect on amino acid impact, however, further investigations are warranted. Receiver operating characteristic (ROC) plot data indicated a significant correlation between HORMAD2 levels and anti-human epidermal growth factor receptor 2 (HER2) sensitivity. Genetic and epigenetic changes in HORMAD1 and HORMAD2 genes may be used as indicators and targets for overcoming breast cancer resistance and limiting metastasis in breast cancer cells via Wnt targeting. Further research is required to verify our findings.

Recent evidence suggests that genetic and epigenetic mechanisms might be associated with acquired resistance to cancer therapies. The aim of this study was to assess the association of genome-wide genetic and epigenetic alterations with the response to anti-HER2 agents in HER2-positive breast cancer patients. PubMed was screened for articles published until March 2021 on observational studies investigating the association of genome-wide genetic and epigenetic alterations, measured in breast cancer tissues or blood, with the response to targeted treatment in HER2-positive breast cancer patients. Sixteen studies were included in the review along with ours, in which we compared the genome-wide DNA methylation pattern in breast tumor tissues of patients who acquired resistance to treatment (case group, n = 6) to that of patients who did not develop resistance (control group, n = 6). Among genes identified as differentially methylated between the breast cancer tissue of cases and controls, one of them, PRKACA, was also reported as differentially expressed in two studies included in the review. Although included studies were heterogeneous in terms of methodology and study population, our review suggests that genes of the PI3K pathway may play an important role in developing resistance to anti-HER2 agents in breast cancer patients. Genome-wide genetic and epigenetic alterations measured in breast cancer tissue or blood might be promising markers of resistance to anti-HER2 agents in HER2-positive breast cancer patients. Further studies are needed to confirm these data.