Resolving DNA Damage: Epigenetic Regulation of DNA Repair.

Epigenetic research has rapidly evolved into a dynamic field of genome biology. Chromatin regulation has been proved to be an essential aspect for all genomic processes, including DNA repair. Chromatin structure is modified by enzymes and factors that deposit, erase, and interact with epigenetic marks such as DNA and histone modifications, as well as by complexes that remodel nucleosomes. In this review we discuss recent advances on how the chromatin state is modulated during this multi-step process of damage recognition, signaling, and repair. Moreover, we examine how chromatin is regulated when different pathways of DNA repair are utilized. Furthermore, we review additional modes of regulation of DNA repair, such as through the role of global and localized chromatin states in maintaining expression of DNA repair genes, as well as through the activity of epigenetic enzymes on non-nucleosome substrates. Finally, we discuss current and future applications of the mechanistic interplays between chromatin regulation and DNA repair in the context cancer treatment.

Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.

Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression.

Laser Bioprinting of Cells Using UV and Visible Wavelengths: A Comparative DNA Damage Study.

Laser-based techniques for printing cells onto different substrates with high precision and resolution present unique opportunities for contributing to a wide range of biomedical applications, including tissue engineering. In this study, laser-induced forward transfer (LIFT) printing was employed to rapidly and accurately deposit patterns of cancer cells in a non-contact manner, using two different wavelengths, 532 and 355 nm. To evaluate the effect of LIFT on the printed cells, their growth and DNA damage profiles were assessed and evaluated quantitatively over several days. The damaging effect of LIFT-printing was thoroughly investigated, for the first time at a single cell level, by counting individual double strand breaks (DSB). Overall, we found that LIFT was able to safely print patterns of breast cancer cells with high viability with little or no heat or shear damage to the cells, as indicated by unperturbed growth and negligible gross DNA damage.

Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions.

DNA damage checkpoint genes, such as p53, are frequently mutated in human cancer, but the selective pressure for their inactivation remains elusive. We analysed a panel of human lung hyperplasias, all of which retained wild-type p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and Chk2 phosphorylation, p53 accumulation, focal staining of p53 binding protein 1 (53BP1) and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). We propose that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability and selective pressure for p53 mutations.

Monitoring of Active Notch Signaling in Mouse Bladder Urothelium.

Notch signaling plays a crucial role in differentiation and homeostasis in a wide variety of epithelia. The tumor suppressor role of Notch in bladder urothelium is well accepted as the inactivation of this pathway due to damaging mutations in its components is associated with neoplastic transformation. Monitoring Notch signaling is therefore critical to understand how the deregulation of cell-cell communication can lead to differentiation loss and carcinogenesis. In this chapter, we provide a method to visualize active Notch signaling by the detection of the nuclear levels of Notch intracellular domain in mouse urothelium. The technique outlined below is characterized by high sensitivity and specificity and has been successfully applied to human tumor specimens. In this context, this technique could be used to characterize the molecular profile of Notch-deficient tumors and analyze the clonal expansion dynamics and the heterogeneity patterns of Notch inactivation.

Parametric Study of Jet/Droplet Formation Process during LIFT Printing of Living Cell-Laden Bioink.

Bioprinting offers great potential for the fabrication of three-dimensional living tissues by the precise layer-by-layer printing of biological materials, including living cells and cell-laden hydrogels. The laser-induced forward transfer (LIFT) of cell-laden bioinks is one of the most promising laser-printing technologies enabling biofabrication. However, for it to be a viable bioprinting technology, bioink printability must be carefully examined. In this study, we used a time-resolved imaging system to study the cell-laden bioink droplet formation process in terms of the droplet size, velocity, and traveling distance. For this purpose, the bioinks were prepared using breast cancer cells with different cell concentrations to evaluate the effect of the cell concentration on the droplet formation process and the survival of the cells after printing. These bioinks were compared with cell-free bioinks under the same printing conditions to understand the effect of the particle physical properties on the droplet formation procedure. The morphology of the printed droplets indicated that it is possible to print uniform droplets for a wide range of cell concentrations. Overall, it is concluded that the laser fluence and the distance of the donor-receiver substrates play an important role in the printing impingement type; consequently, a careful adjustment of these parameters can lead to high-quality printing.

Mitonuclear Interactions in the Maintenance of Mitochondrial Integrity.

In eukaryotic cells, mitochondria originated in an α-proteobacterial endosymbiont. Although these organelles harbor their own genome, the large majority of genes, originally encoded in the endosymbiont, were either lost or transferred to the nucleus. As a consequence, mitochondria have become semi-autonomous and most of their processes require the import of nuclear-encoded components to be functional. Therefore, the mitochondrial-specific translation has evolved to be coordinated by mitonuclear interactions to respond to the energetic demands of the cell, acquiring unique and mosaic features. However, mitochondrial-DNA-encoded genes are essential for the assembly of the respiratory chain complexes. Impaired mitochondrial function due to oxidative damage and mutations has been associated with numerous human pathologies, the aging process, and cancer. In this review, we highlight the unique features of mitochondrial protein synthesis and provide a comprehensive insight into the mitonuclear crosstalk and its co-evolution, as well as the vulnerabilities of the animal mitochondrial genome.

Mutant p53 enhances nuclear factor kappaB activation by tumor necrosis factor alpha in cancer cells.

Mutations in the p53 tumor suppressor are very frequent in human cancer. Often, such mutations lead to the constitutive overproduction of mutant p53 proteins, which may exert a cancer-promoting gain of function. We now report that cancer-associated mutant p53 can augment the induction of nuclear factor kappaB (NFkappaB) transcriptional activity in response to the cytokine tumor necrosis factor alpha (TNFalpha). Conversely, down-regulation of endogenous mutant p53 sensitizes cancer cells to the apoptotic effects of TNFalpha. Analysis of human head and neck tumors and lung tumors reveals a close correlation between the presence of abundant mutant p53 proteins and the constitutive activation of NFkappaB. Together, these findings suggest that p53 mutations may promote cancer progression by augmenting NFkappaB activation in the context of chronic inflammation.

LSD1/KDM1A, a Gate-Keeper of Cancer Stemness and a Promising Therapeutic Target.

A new exciting area in cancer research is the study of cancer stem cells (CSCs) and the translational implications for putative epigenetic therapies targeted against them. Accumulating evidence of the effects of epigenetic modulating agents has revealed their dramatic consequences on cellular reprogramming and, particularly, reversing cancer stemness characteristics, such as self-renewal and chemoresistance. Lysine specific demethylase 1 (LSD1/KDM1A) plays a well-established role in the normal hematopoietic and neuronal stem cells. Overexpression of LSD1 has been documented in a variety of cancers, where the enzyme is, usually, associated with the more aggressive types of the disease. Interestingly, recent studies have implicated LSD1 in the regulation of the pool of CSCs in different leukemias and solid tumors. However, the precise mechanisms that LSD1 uses to mediate its effects on cancer stemness are largely unknown. Herein, we review the literature on LSD1's role in normal and cancer stem cells, highlighting the analogies of its mode of action in the two biological settings. Given its potential as a pharmacological target, we, also, discuss current advances in the design of novel therapeutic regimes in cancer that incorporate LSD1 inhibitors, as well as their future perspectives.

The Histone Demethylase LSD1/ΚDM1A Mediates Chemoresistance in Breast Cancer via Regulation of a Stem Cell Program.

Breast cancer is the leading cause of cancer death in the female population, despite advances in diagnosis and treatment. The highly heterogeneous nature of the disease represents a major obstacle to successful therapy and results in a significant number of patients developing drug resistance and, eventually, suffering from tumor relapse. Cancer stem cells (CSCs) are a small subset of tumor cells characterized by self-renewal, increased tumor-initiation capacity, and resistance to conventional therapies. As such, they have been implicated in the etiology of tumor recurrence and have emerged as promising targets for the development of novel therapies. Here, we show that the histone demethylase lysine-specific demethylase 1 (LSD1) plays an important role in the chemoresistance of breast cancer cells. Our data, from a series of in vitro and in vivo assays, advocate for LSD1 being critical in maintaining a pool of tumor-initiating cells that may contribute to the development of drug resistance. Combinatory administration of LSD1 inhibitors and anti-cancer drugs is more efficacious than monotherapy alone in eliminating all tumor cells in a 3D spheroid system. In conclusion, we provide compelling evidence that LSD1 is a key regulator of breast cancer stemness and a potential target for the design of future combination therapies.

Changes in end products of nitric oxide in urine and induction of nitric oxide synthase expression in urinary bladder during intravesical instillations of IFN-alpha2b.

Interferon-alpha2b (IFN-alpha2b) is being used intravesically for preventing recurrence and progression of superficial transitional cell carcinoma of the bladder. However, its mechanism of action when instilled intravesically is not yet elucidated. We monitored end products of nitric oxide (NO) in urine in 12 bladder cancer patients undergoing intravesical instillations of IFN-alpha2b. Urine end products of NO levels rose gradually after each instillation, reaching a peak value after the third instillation. Although the patients continued their treatment for 5 more weeks, no further alteration was observed. Inducible nitric oxide synthase (iNOS) expression was immunohistochemically evaluated in urinary bladder biopsy specimens before and after IFN-alpha2b instillations. It was shown that IFN-alpha2b induced urothelial iNOS expression, with subsequent oxidative stress. The peroxynitrite (ONOO-) formed from the combination of NO with superoxide (O2-) provides important clues in the role of ONOO- as a causative factor in the antineoplastic action of IFN-alpha2b.

Distinct expression patterns of the transcription factor E2F-1 in relation to tumour growth parameters in common human carcinomas.

E2F-1 is a pivotal transcription factor that integrates signals from a variety of G1/S phase regulators and modulates diverse cellular functions, such as DNA synthesis, repair, mitosis, and apoptosis. Its role in cellular proliferation and apoptosis, as depicted from experimental models and limited reports in human malignancies, remains a matter of debate. Recently, in non-small cell lung cancer, it was observed that E2F-1 overexpression was associated with tumour growth, implying an 'oncogenic' effect. To clarify further the role of E2F-1 in carcinogenesis, the investigation was expanded in four of the most common human malignancies by examining its expression status and putative impact on tumour kinetics. These issues were addressed by immunohistochemical and molecular means in 52 breast carcinomas, 42 prostate adenocarcinomas, 58 colon adenocarcinomas, and 77 superficial bladder transitional cell carcinomas (TCCs). The following results were found: (i). in breast carcinomas, E2F-1 expression correlated with proliferation (p < 0.001) and growth index (p = 0.001); (ii). in prostate adenocarcinomas, absence of E2F-1 was noted, in contrast to its expression in normal and hyperplastic glands; (iii). in colon adenocarcinomas, E2F-1 expression was inversely related to growth index (p = 0.001), being expressed in lesions with increased apoptosis (p = 0.001) and low proliferation (p < 0.001); and (iv) in superficial TCCs, E2F-1 expression correlated with proliferation (p = 0.002). Taken together, these results suggest that E2F-1 has a growth-promoting effect in breast carcinomas and superficial TCC, whereas the opposite seems to be the case for colon and prostate cancer. To interpret the above findings, the status of the pRb and p53 tumour suppressor pathways, which are known to affect E2F-1 activity, was further investigated. The results suggest that the actions of E2F-1 are mainly dependent on the functionality of these pathways. Nevertheless, the data also imply that p53-independent pathways may play a nodal role in the function of E2F-1 in colon cancer.

Overexpression of the replication licensing regulators hCdt1 and hCdc6 characterizes a subset of non-small-cell lung carcinomas: synergistic effect with mutant p53 on tumor growth and chromosomal instability--evidence of E2F-1 transcriptional control over hCdt1.

Replication licensing ensures once per cell cycle replication and is essential for genome stability. Overexpression of two key licensing factors, Cdc6 and Cdt1, leads to overreplication and chromosomal instability (CIN) in lower eukaryotes and recently in human cell lines. In this report, we analyzed hCdt1, hCdc6, and hGeminin, the hCdt1 inhibitor expression, in a series of non-small-cell lung carcinomas, and investigated for putative relations with G(1)/S phase regulators, tumor kinetics, and ploidy. This is the first study of these fundamental licensing elements in primary human lung carcinomas. We herein demonstrate elevated levels (more than fourfold) of hCdt1 and hCdc6 in 43% and 50% of neoplasms, respectively, whereas aberrant expression of hGeminin was observed in 49% of cases (underexpression, 12%; overexpression, 37%). hCdt1 expression positively correlated with hCdc6 and E2F-1 levels (P = 0.001 and P = 0.048, respectively). Supportive of the observed link between E2F-1 and hCdt1, we provide evidence that E2F-1 up-regulates the hCdt1 promoter in cultured mammalian cells. Interestingly, hGeminin overexpression was statistically related to increased hCdt1 levels (P = 0.025). Regarding the kinetic and ploidy status of hCdt1- and/or hCdc6-overexpressing tumors, p53-mutant cases exhibited significantly increased tumor growth values (Growth Index; GI) and aneuploidy/CIN compared to those bearing intact p53 (P = 0.008 for GI, P = 0.001 for CIN). The significance of these results was underscored by the fact that the latter parameters were independent of p53 within the hCdt1-hCdc6 normally expressing cases. Cumulatively, the above suggest a synergistic effect between hCdt1-hCdc6 overexpression and mutant-p53 over tumor growth and CIN in non-small-cell lung carcinomas.