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DNA helicases, known for their fundamentally important roles in genomic stability, are high profile players in cancer. Not only are there monogenic helicase disorders with a strong disposition to cancer, it is well appreciated that helicase variants are associated with specific cancers (e.g., breast cancer). Flipping the coin, DNA helicases are frequently overexpressed in cancerous tissues and reduction in helicase gene expression results in reduced proliferation and growth capacity, as well as DNA damage induction and apoptosis of cancer cells. The seminal roles of helicases in the DNA damage and replication stress responses, as well as DNA repair pathways, validate their vital importance in cancer biology and suggest their potential values as targets in anti-cancer therapy. In recent years, many laboratories have characterized the specialized roles of helicase to resolve transcription-replication conflicts, maintain telomeres, mediate cell cycle checkpoints, remodel stalled replication forks, and regulate transcription. In vivo models, particularly mice, have been used to interrogate helicase function and serve as a bridge for preclinical studies that may lead to novel therapeutic approaches. In this review, we will summarize our current knowledge of DNA helicases and their roles in cancer, emphasizing the latest developments.

DNA helicases have risen to the forefront as genome caretakers. Their prominent roles in chromosomal stability is demonstrated by the linkage of mutations in helicase genes to hereditary disorders with defects in DNA repair, the replication stress response, and/or transcriptional activation. Conversely, accumulating evidence suggests that in cancer cells DNA helicases have a network of pathway interactions such that co-deficiency of certain helicases and their genetically interacting proteins result in synthetic lethality (SL). Such genetic interactions may potentially be exploited for cancer therapies. We will discuss the roles of RECQ DNA helicases in cancer, emphasizing some of the more recent developments in synthetic lethality.

Timely recruitment of DNA damage response proteins to sites of genomic structural lesions is very important for signaling mechanisms to activate appropriate cell cycle checkpoints but also repair the altered DNA sequence to suppress mutagenesis. The eukaryotic cell is characterized by a complex cadre of players and pathways to ensure genomic stability in the face of replication stress or outright genomic insult by endogenous metabolites or environmental agents. Among the key performers are molecular motor DNA unwinding enzymes known as helicases that sense genomic perturbations and separate structured DNA strands so that replacement of a damaged base or sugar-phosphate backbone lesion can occur efficiently. Mutations in the BLM gene encoding the DNA helicase BLM leads to a rare chromosomal instability disorder known as Bloom's syndrome. In a recent paper by the Sengupta lab, BLM's role in the correction of double-strand breaks (DSB), a particularly dangerous form of DNA damage, was investigated. Adding to the complexity, BLM appears to be a key ringmaster of DSB repair as it acts both positively and negatively to regulate correction pathways of high or low fidelity. The FANCJ DNA helicase, mutated in another chromosomal instability disorder known as Fanconi Anemia, is an important player that likely coordinates with BLM in the balancing act. Further studies to dissect the roles of DNA helicases like FANCJ and BLM in DSB repair are warranted.

DNA helicases represent a specialized class of enzymes that play crucial roles in the DNA damage response. Using the energy of nucleoside triphosphate binding and hydrolysis, helicases behave as molecular motors capable of efficiently disrupting the many noncovalent hydrogen bonds that stabilize DNA molecules with secondary structure. In addition to their importance in DNA damage sensing and signaling, DNA helicases facilitate specific steps in DNA repair mechanisms that require polynucleotide tract unwinding or resolution. Because they play fundamental roles in the DNA damage response and DNA repair, defects in helicases disrupt cellular homeostasis. Thus, helicase deficiency or inhibition may result in reduced cell proliferation and survival, apoptosis, DNA damage induction, defective localization of repair proteins to sites of genomic DNA damage, chromosomal instability, and defective DNA repair pathways such as homologous recombination of double-strand breaks. In this chapter, we will describe step-by-step protocols to assay the functional importance of human DNA repair helicases in genome stability and cellular homeostasis.

Understanding the molecular dynamics of DNA replication in vivo has been a formidable challenge requiring the development of advanced technologies. Over the past 50 years or so, studies involving DNA autoradiography in bacterial cells have led to sophisticated DNA tract analyses in human cells to characterize replication dynamics at the single-molecule level. Our own lab has used DNA fiber analysis to characterize replication in helicase-deficient human cells. This work led us to propose a model in which the human DNA helicase RECQ1 acts as a governor of the single-stranded DNA binding protein RPA and regulates its bioavailability for DNA synthesis. We have also used the DNA fiber approach to investigate the interactive role of DDX11 helicase with a replication fork protection protein (Timeless) in human cells when they are under pharmacologically induced stress. In this methods chapter, we present a step-by-step protocol for the single-molecule DNA fiber assay. We describe experimental designs to study replication stress and staining patterns from pulse-chase labeling experiments to address the dynamics of replication forks in stressed cells.

Upregulation of ADAM-12, a novel member of the multifunctional ADAM family of proteins is linked to cancer, arthritis and cardiac hypertrophy. Basal expression of ADAM-12 is very low in adult tissues but rises markedly in response to certain physiological cues, such as during pregnancy in the placenta, during development in neonatal skeletal muscle and bone and in regenerating muscle. Studies on ADAM-12 regulation have identified a highly conserved negative regulatory element (NRE) at the 5′-UTR of human ADAM-12 gene, which acts as a transcriptional repressor. The NRE contains a stretch of dinucleotide-repeat sequence that is able to adopt a Z-DNA conformation both in vitro and in vivo and interacts with hZ α ADAR1 , a bona fide Z-DNA-binding protein. Substitution of the dinucleotide-repeat-element with a non-Z-DNA-forming sequence inhibited NRE function. We have detected a NRE DNA-binding protein activity in several tissues where ADAM-12 expression is low while no such activity was seen in the placenta where ADAM-12 expression is high. These observations suggest that interaction of these proteins with ADAM-12 NRE is critical for transcriptional repression of ADAM-12. We also show that the Z-DNA forming transcriptional repressor element, by interacting with these putative Z-DNA-binding proteins, is involved in the maintenance of constitutive low-level expression of human ADAM-12. Together these results provide a foundation for therapeutic down-regulation of ADAM-12 in cancer, arthritis and cardiac hypertrophy.

Objective We aimed to investigate whether advanced non-enzymatic glycation of the extracellular matrix (ECM) protein, fibronectin, impacts its normal integrin-mediated interaction with arteriolar vascular smooth muscle cells (VSMC). Methods Atomic force microscopy (AFM) was performed on cultured VSMC from rat cremaster arterioles to study native and glycated fibronectin (FN and gFN) interactions with cellular integrins. AFM probes were functionalized with FN or gFN or with native or glycated albumin (gAlb) as controls. Results VSMC showed increased adhesion probability to gFN (72.9 ± 3.5%) compared to native FN (63.0 ± 1.6%). VSMCs similarly showed increased probability of adhesion (63.8 ± 1.7%) to gAlb compared to native Alb (40.1 ± 4.7%). Adhesion of native FN to VSMC was α5 and β1 integrin-dependent whereas adhesion of gFN to VSMC was integrin-independent. The RAGE-selective inhibitor, FPS-ZM1, blocked gFN (and gAlb) adhesion suggesting that adhesion of glycated proteins was RAGE-dependent. Interaction of FN with VSMC was not altered by soluble gFN while soluble native FN did not inhibit adhesion of gFN to VSMC. In contrast, gAlb inhibited adhesion of gFN to VSMC in a concentration-dependent manner. Conclusions Glycation of FN shifts the nature of cellular adhesion from integrin- to RAGE-dependent mechanisms. This article is protected by copyright. All rights reserved.

A disintegrin and metalloprotease domain-containing protein 12 (ADAM-12) is upregulated in many human cancers and promotes cancer metastasis. Increased urinary level of ADAM-12 in breast and bladder cancers correlates with disease progression. However, the mechanism of its induction in cancer remains less understood. Previously, we reported a Z-DNA–forming negative regulatory element (NRE) in ADAM-12 that functions as a transcriptional suppressor to maintain a low-level expression of ADAM-12 in most normal cells. We now report here that overexpression of ADAM-12 in triple-negative MDA-MB-231 breast cancer cells and breast cancer tumors is likely due to a marked loss of this Z-DNA–mediated transcriptional suppression function. We show that Z-DNA suppressor operates by interaction with methyl-CpG-binding protein, MeCP2, a prominent epigenetic regulator, and two members of the nuclear factor 1 family of transcription factors, NF1C and NF1X. While this tripartite interaction is highly prevalent in normal breast epithelial cells, both in vitro and in vivo, it is significantly lower in breast cancer cells. Western blot analysis has revealed significant differences in the levels of these 3 proteins between normal mammary epithelial and breast cancer cells. Furthermore, we show, by NRE mutation analysis, that interaction of these proteins with the NRE is necessary for effective suppressor function. Our findings unveil a new epigenetic regulatory process in which Z-DNA/MeCP2/NF1 interaction leads to transcriptional suppression, loss of which results in ADAM-12 overexpression in breast cancer cells. Cancer Res; 73(2); 736–44. ©2012 AACR.

Modification of proteins by non-enzymatic glycation occurs in diabetes mellitus, aging and pathological states including renal failure. Glycation reactions span early reversible reactions to irreversibly modified species termed advanced glycation endproducts (AGEs). Long-lived extracellular matrix (ECM) proteins are particularly susceptible to AGE modification. As ECM proteins are known to regulate vascular function through binding with integrins, AGE formation may modify normal interactions between matrix and vascular smooth muscle cells (VSMCs) ultimately impacting vasoregulation. The purpose of this study was to selectively investigate the adhesion between either non-glycated or glycated fibronectin (FN) with VSMCs using Atomic Force Microscopy (AFM). AFM probes were functionalized with FN or glycated FN and cyclically interacted with isolated VSMCs using a force mode nano-indentation protocol. Adhesion was quantified as the rupture force required to disrupt adhesion bonds the formed during contact of the AFM probe with the cell and also as the probability of an adhesive interaction occurring. AFM probes coated with FN alone required 78.6 ± 19.9 pN (N=5) to disrupt adhesive bonds whereas glycated FN required 49.9 ± 5.7 pN (N=5). Binding probability was, however, significantly increased for AFM probes coated with glycated FN vs FN alone, 72.9 ± 3.5 % vs 63.0 ± 1.6 %, (N=5) respectively. Selectivity for binding to alpha5 integrin was investigated by adding a solubilized function-blocking antibody to the cell bath. Anti-alpha5 antibody significantly reduced binding to FN (14.27%) but had no effect on glycated FN. These results establish that glycation of ECM proteins can significantly affect their adhesive interaction with VSM and integrins and this could in turn lead to altered cell-integrin based regulation of VSM function. (Supp by P01 HL095486 and RO1 HL92241)

Angiogenesis plays a significant role in cancer by providing increased blood supply to the affected tissues and thus bringing in growth factors, cytokines, and various nutrients for tumor growth. VEGF is the most prominent angiogenic agent that is markedly induced in cancer. Induction of VEGF has been widely studied but as cancer cells are quite adept at acquiring new alternative processes to circumvent surrounding environmental pressures, our understanding of the molecular mechanisms regulating VEGF expression in cancer, especially in triple-negative breast cancer cells, remains incomplete. Here, we present evidence of a novel mode of VEGF induction in triple-negative MDA-MB-231 breast cancer cells that is regulated by serum amyloid A activating factor 1 (SAF-1) transcription factor. Inhibition of SAF-1 by antisense short hairpin RNA profoundly reduces VEGF expression along with reduction in endothelial cell proliferation and migration. By both in vitro and in vivo molecular studies, we show that the effect of SAF-1 is mediated through its direct interaction with the VEGF promoter. In correlation, DNA-binding activity of SAF-1 is found to be significantly higher in MDA-MB-231 breast cancer cells. Examination of several breast cancer samples further revealed that SAF-1 is overexpressed in clinical breast cancer tissues. Taken together, these findings reveal that SAF-1 is a hitherto unrecognized participant in inducing VEGF expression in triple-negative breast cancer cells, an aggressive form of breast cancer that currently lacks effective treatment options. Suppression of SAF-1 activity in these cells can inhibit VEGF expression, providing a possible new method to control angiogenesis. Mol Cancer Res; 9(8); 1030–41. ©2011 AACR.

A disintegrin and metalloproteinase-12 (ADAM-12), a member of multifunctional family of proteins, is upregulated in many cancers, including breast, lung, liver, prostate, gastric, and bladder. The multidomain structure, composed of a prodomain, a metalloproteinase, disintegrin-like, epidermal growth factor–like, cysteine-rich and transmembrane domains, and a cytoplasmic tail, allows ADAM-12 to promote matrix degradation, cell-cell adhesion, and intracellular signaling capacities and thereby to play a critical role in cancer growth and metastasis. Despite ample evidence linking increased ADAM-12 expression with cancer, the mechanisms controlling its upregulation are still unknown. In the present study, transforming growth factor-β1 (TGF-β1) is shown to increase ADAM-12 mRNA expression in MDA-MB-231 breast carcinoma cells. We have identified a promoter element responsible for TGF-β1–mediated ADAM-12 induction. We show interaction of NF-κB with ADAM-12 promoter and that high level of NF-κB activity in breast carcinoma cells results in the upregulation of ADAM-12 expression. Site-directed mutagenesis of the NF-κB element in ADAM-12 promoter and inhibition of NF-κB activity by Bay-11-7085 and MG-132 significantly reduced TGF-β1–mediated increase of ADAM-12 promoter-driven gene expression. Transfection of cells with a dominant-negative mutant form of IκBα (IκBαΔN), which inhibits activation of NF-κB, significantly reduced transcription from ADAM-12 promoter-reporter in TGF-β1–stimulated MDA-MB-231 cancer cells. In correlation, overexpression of NF-κB induced ADAM-12 expression in a dose-dependent manner. DNA-binding and ChIP assays indicated that p65 subunit of NF-κB binds to ADAM-12 promoter. Together, our study identified a cellular mechanism for induction of ADAM-12, which involves NF-κB and its activation by TGF-β1. Mol Cancer Res; 8(9); 1261–70. ©2010 AACR.