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AraC-FdUMP[10] (CF10) is a second-generation polymeric fluoropyrimidine that targets both thymidylate synthase (TS), the target of 5-fluorouracil (5-FU), and DNA topoisomerase 1 (Top1), the target of irinotecan, two drugs that are key components of FOLFIRNOX, a standard-of-care regimen for pancreatic ductal adenocarcinoma (PDAC). We demonstrated that F10 and CF10 are potent inhibitors of PDAC cell survival (in multiple cell lines including patient-derived lines) with IC 50 s in the nanomolar range and are nearly 1,000-fold more potent than 5-FU. The increased potency of CF10 relative to 5-FU correlated with enhanced TS inhibition and strong Top1 cleavage complex formation. Furthermore, CF10 displayed single-agent activity in PDAC murine xenografts without inducing weight loss. Through a focused drug synergy screen, we identified that combining CF10 with targeting the DNA repair enzyme, poly (ADP-ribose) glycohydrolase, induces substantial DNA damage and apoptosis. This work moves CF10 closer to a clinical trial for the treatment of PDAC. IMPLICATIONS: CF10 is a promising polymeric fluoropyrimidine with dual mechanisms of action (i.e., TS and Top1 inhibition) for the treatment of PDAC and synergizes with targeting of DNA repair. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/4/565/F1.large.jpg., (©2021 American Association for Cancer Research.)

Background: The prevalence of cognitive dysfunction increases in elderly due to cardiovascular disease related risk factors in rural communities like West Texas., Objective: The purpose of this study was to find risk factors of cardiovascular disease (CVD) related to cognitive dysfunction and their impact on elderly adults in rural West Texans., Methods: Statistical methods such as Pearson's chi-squared and a multinomial logistic regression were utilized to analyze data. We used SPSS software to detect and understand the nature of the risk factors., Results: A summary of statistics was obtained by using Pearson's chi-squared test for categorical variables. CVD, diabetes mellitus, and depression were significantly associated with cognitive dysfunction for both males and females ( p  = 0.0001), whereas anxiety was found to be significantly associated with cognitive dysfunction for females ( p  = 0.0001). Age group and race/ethnicity were significantly associated with cognitive dysfunction for both males and females ( p  = 0.0001). By performing a multinomial logistic regression method and controlling for confounders, the significant risk factors ( p  <  0.05)- age (65- 84 years), diabetes, and memory loss for age-associated cognitive impairment; diabetes for cognitive impairment no dementia; age (65- 84, ≥85 years), CVD, diabetes, depression, memory loss, non-Hispanic Whites, and Black/African-Americans for mild cognitive impairment; and age, memory loss, non-Hispanic Whites, Black/African-Americans, and male gender were found for dementia., Conclusion: CVD related risk factors in developing cognitive dysfunction exist and integrating such risk variables may guide relevant policy interventions to reduce Alzheimer's incidence or dementia in rural communities in West Texans., Competing Interests: The authors declare that there is no conflict of interests., (© 2021 – The authors. Published by IOS Press.)

Chemotherapy regimens that include 5-fluorouracil (5-FU) are central to colorectal cancer treatment; however, risk/benefit concerns limit 5-FU's use, necessitating development of improved fluoropyrimidine (FP) drugs. In our study, we evaluated a second-generation nanoscale FP polymer, CF10, for improved antitumor activity. CF10 was more potent than the prototype FP polymer F10 and much more potent than 5-FU in multiple colorectal cancer cell lines including HCT-116, LS174T, SW480, and T84D. CF10 displayed improved stability to exonuclease degradation relative to F10 and reduced susceptibility to thymidine antagonism due to extension of the polymer with arabinosyl cytidine. In colorectal cancer cells, CF10 strongly inhibited thymidylate synthase (TS), induced Top1 cleavage complex formation and caused replication stress, while similar concentrations of 5-FU were ineffective. CF10 was well tolerated in vivo and invoked a reduced inflammatory response relative to 5-FU. Blood chemistry parameters in CF10-treated mice were within normal limits. In vivo , CF10 displayed antitumor activity in several colorectal cancer flank tumor models including HCT-116, HT-29, and CT-26. CF10's antitumor activity was associated with increased plasma levels of FP deoxynucleotide metabolites relative to 5-FU. CF10 significantly reduced tumor growth and improved survival (84.5 days vs. 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116- luc colorectal cancer model that spontaneously metastasized to liver. Improved survival in the orthotopic model correlated with localization of a fluorescent CF10 conjugate to tumor. Together, our preclinical data support an early-phase clinical trial of CF10 for treatment of colorectal cancer., (©2020 American Association for Cancer Research.)

Objectives: Breast cancer is the second highest female mortality rate in Texas for all races and ethnicities, except for Hispanics. Interestingly, Hale County is a rural underserved county in West Texas which experiences a lower rate of cancer, has higher age-adjusted mortality rates (26.2/100 000), on average, compared to all of Texas (23.1/100 000). The purpose of this study was to determine the relationship between sociodemographic variables and breast cancer outcomes in underserved Hale County which contributed to the highest mortality rate in Texas., Methods: Hale County breast cancer data (1995-2014) were obtained from the Texas Cancer Registry. Statistical methods independent samples t-test, Kaplan-Meier curve, and Cox proportional hazard were used to describe the significant relationship between survival time, sociodemographic, and prognostic variables., Results: Women with breast cancer in Hale County were more likely to be White non-Hispanics (n = 266, 65.5%) and had the highest longevity (2753.6 ± 2073.5 days). White Hispanics experienced the worst survival (2369.6 ± 2060.2 days) and were more likely to develop a serious grade of cancer. Significant relationships were found between the stage of cancer and insurance status with survival time for both White non-Hispanics and White Hispanics ( P < .001). Patients in grades II and III were found to be significantly ( P < .01) associated with breast cancer death, and grades II and III which had around five-fold and eleven-fold increased risk of death, respectively, compared with the referent group, grade I., Conclusion: Determining the impact of sociodemographic variables on breast cancer outcome is essential to addressing issues of geographic disparities and integrating such variables may guide relevant policy interventions to reduce breast cancer's incidence in rural underserved communities in West Texans.

Stem cells are a sub population of cell types that form the foundation of our body, and have the potential to replicate, replenish and repair limitlessly to maintain the tissue and organ homeostasis. Increased lifetime and frequent replication set them vulnerable for both exogenous and endogenous agents-induced DNA damage compared to normal cells. To counter these damages and preserve genetic information, stem cells have evolved with various DNA damage response and repair mechanisms. Furthermore, upon experiencing irreparable DNA damage, stem cells mostly prefer early senescence or apoptosis to avoid the accumulation of damages. However, the failure of these mechanisms leads to various diseases, including cancer. Especially, given the importance of stem cells in early development, DNA repair deficiency in stem cells leads to various disabilities like developmental delay, premature aging, sensitivity to DNA damaging agents, degenerative diseases, etc. In this review, we have summarized the recent update about how DNA repair mechanisms are regulated in stem cells and their association with disease progression and pathogenesis., (Copyright © 2019. Published by Elsevier B.V.)

Phosphofurin acidic cluster sorting protein-1 (PACS-1) is a multifunctional membrane traffic regulator that plays important roles in organ homeostasis and disease. In this study, we elucidate a novel nuclear function for PACS-1 in maintaining chromosomal integrity. PACS-1 progressively accumulates in the nucleus during cell cycle progression, where it interacts with class I histone deacetylases 2 and 3 (HDAC2 and HDAC3) to regulate chromatin dynamics by maintaining the acetylation status of histones. PACS-1 knockdown results in the proteasome-mediated degradation of HDAC2 and HDAC3, compromised chromatin maturation, as indicated by elevated levels of histones H3K9 and H4K16 acetylation, and, consequently, increased replication stress-induced DNA damage and genomic instability.

Background: Breast cancer remains as one of the most lethal types of cancer in women. Among various subtypes, triple-negative breast cancer (TNBC) is the most aggressive and hard to treat type of breast cancer. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and disease recurrence., Methods: We evaluated the mechanism of prexasertib-induced regulation of homologous recombination (HR) proteins using 20S proteasome inhibitors and RT-PCR. HR efficiency and DNA damages were evaluated using Dr-GFP and comet assays. DNA morphology and DNA repair focus studies were analyzed using immunofluorescence. UALCAN portal was used to evaluate the expression of RAD51 and survival probability based on tumor stage, subtype, and race in breast cancer patients., Results: Our results show that prexasertib treatment promotes both post-translational and transcriptional mediated regulation of BRCA1 and RAD51 proteins. Additionally, prexasertib-treated TNBC cells revealed over 55% reduction in HR efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) and thus should synergize with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of prexasertib and PARPi olaparib increased DNA strand breaks, γH2AX foci, and nuclear disintegration relative to single-agent treatment. Further, the prexasertib and olaparib combination was synergistic in multiple TNBC cell lines, as indicated by combination index (CI) values. Analysis of TCGA data revealed elevated RAD51 expression in breast tumors compared to normal breast tissues, especially in TNBC subtype. Interestingly, there was a discrepancy in RAD51 expression in racial groups, with African-American and Asian breast cancer patients showing elevated RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African-American and Asian TNBC patients show decreased survival., Conclusions: Based on these data, RAD51 could be a biomarker for aggressive TNBC and for racial disparity in breast cancer. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, the in vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved outcomes and reduced racial disparity in TNBC.

We are developing the fluoropyrimidine polymer F10 to overcome limitations of 5-fluorouracil (5-FU) that result from inefficient metabolism to 5-fluoro-2'-deoxyuridine-5'-mono- and tri-phosphate, the deoxyribonucleotide metabolites that are responsible for 5-FU's anticancer activity. F10 is much more cytotoxic than 5-FU to colorectal cancer (CRC) cells; however, the mechanism of enhanced F10 cytotoxicity remains incompletely characterized. Using DNA fiber analysis, we establish that F10 decreases replication fork velocity and causes replication fork collapse, while 1000-fold excess of 5-FU is required to achieve similar endpoints. Treatment of HCT-116 cells with F10 results in Chk1 phosphorylation and activation of intra-S-phase checkpoint. Combining F10 with pharmacological inhibition of Chk1 with either PF-477736 or prexasertib in CRC cells enhanced DNA damage relative to single-agent treatment as assessed by γH2AX intensity and COMET assay. PF-477736 or prexasertib co-treatment also inhibited upregulation of Rad51 levels in response to F10, resulting in reduced homologous repair. siRNA knockdown of Chk1 also increased F10-induced DNA damage assessed and sensitized CRC cells to F10. However, Chk1 knockdown did not inhibit Rad51 upregulation by F10, indicating that the scaffolding activity of Chk1 imparts activity in DNA repair distinct from Chk1 enzymatic activity. Our results indicate that F10 is cytotoxic to CRC cells in part through DNA damage subsequent to replication fork collapse. F10 is ~1000-fold more potent than 5-FU at inducing replication-mediated DNA damage which correlates with the increased overall potency of F10 relative to 5-FU. F10 efficacy can be enhanced by pharmacological inhibition of Chk1., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Mortality in ovarian cancer is predominantly due to acquired chemoresistance and tumor recurrence. UBIQUITIN CONJUGATING ENZYME E2 or RAD6 expression increases in cell lines and patient tumors in response to platinum-based chemotherapy and promotes both activation of DNA damage response pathways and expression of stemness genes and a stem cell-like phenotype driving ovarian cancer chemoresistance.

A series of novel N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)piperidine-4-carboxamide derivatives 10(a-f), 12(a-c) and 14(a-c) were synthesized and characterized by FTIR, 1 H-NMR, mass spectral and elemental analysis. The efficacy of these derivatives to inhibit in vivo angiogenesis was evaluated using chick chorioallantoic membrane (CAM) model and their DNA cleavage abilities were evaluated after incubating with calf thymus DNA followed by gel electrophoresis. These novel piperidine analogues efficiently blocked the formation of blood vessels in vivo in CAM model and exhibited differential migration and band intensities in DNA binding/cleavage assays. Among the tested compounds 10a, 10b, 10c, 12b, 14b and 14c showed significant anti-angiogenic and DNA cleavage activities compared to their respective controls and the other derivatives used in this study. These observations suggest that the presence of electron donating and withdrawing groups at positions 2, 3 and 4 of the phenyl ring of the side chain may determine their potency and as anticancer agents by exerting both anti-angiogenic and cytotoxic effects .

The normal female reproductive hormone estrogen has been linked with increased risk of breast and many other forms of cancer. This is largely due to metabolic conversion of estrogens into highly reactive catechol estrogen quinones which can interact with DNA and cause a variety of DNA adducts and lesions. Detection and analysis of these adducts and their associated cellular responses involve complex chemical, enzymatic, and LC-MS based methods, which are both laborious and require specialized expertise and instrumentation. Herein, we show that using a biotin-labeled estradiol allows immunodetection of estrogen-induced DNA adducts by slot blot and single-cell molecular combing and proximity ligation assays. The biotinylated and unlabeled estradiols induced similar levels of DNA single and double strand breaks as measured by comet assays. Using biotinylated estrogen, we further show that estrogens are able to activate the Fanconi anemia-BRCA tumor suppressor pathway and cause DNA strand breaks and oxidatively modified DNA bases as well as gross chromosomal aberrations. Utilization of biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and associated DNA damage, and to track estrogen adduct-induced cellular responses and carcinogenic mechanisms in cultured cells. The techniques presented here allow simple and rapid detection and quantitation of estrogen adducts by slot blot as well as direct visualization on the DNA strand and could pave the way for developing new treatments to protect the genome from the effects of reactive estrogen metabolites. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Resistance to current chemotherapeutic or radiation-based cancer treatment strategies is a serious concern. Cancer stem cells (CSCs) are typically able to evade treatment and establish a recurrent tumor or metastasis, and it is these that lead to the majority of cancer deaths. Therefore, a major current goal is to develop treatment strategies that eliminate the resistant CSCs as well as the bulk tumor cells in order to achieve complete disease clearance. Aldehyde dehydrogenases (ALDHs) are important for maintenance and differentiation of stem cells as well as normal development. There is expanding evidence that ALDH expression increases in response to therapy and promotes chemoresistance and survival mechanisms in CSCs. This perspective will discuss a paper by Cojoc and colleagues recently published in Cancer Research, that indicates ALDHs play a key role in resistance to radiation therapy and tumor recurrence in prostate cancer. The authors suggest that ALDHs are a potential therapeutic target for treatment prostate cancer patients to limit radiation resistance and disease recurrence. The findings are consistent with work from other cancers showing ALDHs are major contributors of CSC signaling and resistance to anti-cancer treatments. This perspective will address representative work concerning the validity of ALDH and the associated retinoic acid signaling pathway as chemotherapeutic targets for prostate as well as other cancers., Competing Interests: The authors have no conflicts of interest to declare.

Series of 4-amino-6-(arylamino)-1,3,5-triazine-2-carbohydrazides (3a-e) and N'-phenyl-4,6-bis(arylamino)-1,3,5-triazine-2-carbohydrazides (6a-e), for ease of readership, we will abbreviate our compound names as 'new triazines', have been synthesized, based on the previously reported Rad6B-inhibitory diamino-triazinylmethyl benzoate anticancer agents TZ9 and 4-amino-N'-phenyl-6-(arylamino)-1,3,5-triazine-2-carbohydrazides. Synthesis of the target compounds was readily accomplished in two steps from either bis-aryl/aryl biguanides via reaction of phenylhydrazine or hydrazinehydrate with key 4-amino-6-bis(arylamino)/(arylamino)-1,3,5-triazine-2-carboxylate intermediates. These new triazine derivatives were evaluated for their abilities to inhibit Rad6B ubiquitin conjugation and in vitro anticancer activity against several human cancer cell lines: ovarian (OV90 and A2780), lung (H1299 and A549), breast (MCF-7 and MDA-MB231) and colon (HT29) cancer cells by MTS assays. All the 10 new triazines exhibited superior Rad6B inhibitory activities in comparison to selective Rad6 inhibitor TZ9 that was reported previously. Similarly, new triazines also showed better IC50 values in survival assays of various tumor cell lines. Particularly, new triazines 6a-c, exhibited lower IC50 (3.3-22 μM) values compared to TZ9., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB.

Increased body weight and metabolic disorder including insulin resistance, type 2 diabetes and cardiovascular complications together constitute metabolic syndrome. The pathogenesis of metabolic syndrome involves multitude of factors. A number of studies however indicate, with some conformity, that oxidative stress along with chronic inflammatory condition pave the way for the development of metabolic diseases. Oxidative stress, a state of lost balance between the oxidative and anti-oxidative systems of the cells and tissues, results in the over production of oxidative free radicals and reactive oxygen species (ROS). Excessive ROS generated could attack the cellular proteins, lipids and nucleic acids leading to cellular dysfunction including loss of energy metabolism, altered cell signalling and cell cycle control, genetic mutations, altered cellular transport mechanisms and overall decreased biological activity, immune activation and inflammation. In addition, nutritional stress such as that caused by high fat high carbohydrate diet also promotes oxidative stress as evident by increased lipid peroxidation products, protein carbonylation, and decreased antioxidant system and reduced glutathione (GSH) levels. These changes lead to initiation of pathogenic milieu and development of several chronic diseases. Studies suggest that in obese person oxidative stress and chronic inflammation are the important underlying factors that lead to development of pathologies such as carcinogenesis, obesity, diabetes, and cardiovascular diseases through altered cellular and nuclear mechanisms, including impaired DNA damage repair and cell cycle regulation. Here we discuss the aspects of metabolic disorders-induced oxidative stress in major pathological conditions and strategies for their prevention and therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ovarian cancer is the fifth most deadly cancer in women in the United States and despite advances in surgical and chemotherapeutic treatments survival rates have not significantly improved in decades. The poor prognosis for ovarian cancer patients is largely due to the extremely high (80%) recurrence rate of ovarian cancer and because the recurrent tumors are often resistant to the widely utilized platinum-based chemotherapeutic drugs. In this study, expression of Rad6, an E2 ubiquitin-conjugating enzyme, was found to strongly correlate with ovarian cancer progression. Furthermore, in ovarian cancer cells Rad6 was found to stabilize β-catenin promoting stem cell-related characteristics, including expression of stem cell markers and anchorage-independent growth. Cancer stem cells can promote chemoresistance, tumor recurrence and metastasis, all of which are limiting factors in treating ovarian cancer. Thus it is significant that Rad6 overexpression led to increased resistance to the chemotherapeutic drug carboplatin and correlated with tumor cell invasion. These findings show the importance of Rad6 in ovarian cancer and emphasize the need for further studies of Rad6 as a potential target for the treatment of ovarian cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of GLIs must be tightly regulated as they modulate target genes which control tissue patterning, stem cell maintenance, and differentiation during development. However, dysregulation or mutations in HH signaling leads to genomic instability (GI) and various cancers, for example, germline mutation in PTCH1 lead to Gorlin syndrome, a condition where patients develop numerous basal cell carcinomas and rarely rhabdomyosarcoma (RMS). Activating mutations in SMO have also been recognized in sporadic cases of medulloblastoma and SMO is overexpressed in many other cancers. Recently, studies in several human cancers have shown that GLI1 expression is independent from HH ligand and canonical intracellular signaling through PTCH and SMO. In fact, this aberrantly regulated GLI1 has been linked to several non-canonical oncogenic growth signals such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor β (TGFβ), wingless-type MMTV integration site family (WNT) and β-catenin. Recent studies from our lab and other independent studies demonstrate that aberrantly expressed GLI1 influences the integrity of several DNA damage response and repair signals, and if altered, these networks can contribute to GI and impact tumor response to chemo- and radiation therapies. Furthermore, the ineffectiveness of SMO inhibitors in clinical studies argues for the development of GLI1-specific inhibitors in order to develop effective therapeutic modalities to treat these tumors. In this review, we focus on summarizing current understanding of the molecular, biochemical and cellular basis for aberrant GLI1 expression and discuss GLI1-mediated HH signaling on DNA damage responses, carcinogenesis and chemoresistance.

Fanconi anemia (FA) is a rare genome instability syndrome with progressive bone marrow failure and cancer susceptibility. FANCJ is one of 17 genes mutated in FA-patients, comprises a DNA helicase that is vital for properly maintaining genomic stability and is known to function in the FA-BRCA DNA repair pathway. While exact role(s) of FANCJ in this repair process is yet to be determined, it is known to interact with primary effector FANCD2. However, FANCJ is not required for FANCD2 activation but is important for its ability to fully respond to DNA damage. In this report, we determined that transient depletion of FANCJ adversely affects stability of FANCD2 and its co-regulator FANCI in multiple cell lines. Loss of FANCJ does not significantly alter cell cycle progression or FANCD2 transcription. However, in the absence of FANCJ, the majority of FANCD2 is degraded by both the proteasome and Caspase-3 dependent mechanism. FANCJ is capable of complexing with and stabilizing FANCD2 even in the absence of a functional helicase domain. Furthermore, our data demonstrate that FANCJ is important for FANCD2 stability and proper activation of DNA damage responses to replication blocks induced by hydroxyurea.

Allyl isothiocyanate (AITC), a constituent of many cruciferous vegetables exhibits significant anticancer activities in many cancer models. Our studies provide novel insights into AITC-induced anticancer mechanisms in human A549 and H1299 non-small cell lung cancer (NSCLC) cells. AITC exposure induced replication stress in NSCLC cells as evidenced by γH2AX and FANCD2 foci, ATM/ATR-mediated checkpoint responses and S and G2/M cell cycle arrest. Furthermore, AITC-induced FANCD2 foci displayed co-localization with BrdU foci, indicating stalled or collapsed replication forks in these cells. Although PITC (phenyl isothiocyanate) exhibited concentration-dependent cytotoxic effects, treatment was less effective compared to AITC. Previously, agents that induce cell cycle arrest in S and G2/M phases were shown to sensitize tumor cells to radiation. Similar to these observations, combination therapy involving AITC followed by radiation treatment exhibited increased DDR and cell killing in NSCLC cells compared to single agent treatment. Combination index (CI) analysis revealed synergistic effects at multiple doses of AITC and radiation, resulting in CI values of less than 0.7 at Fa of 0.5 (50% reduction in survival). Collectively, these studies identify an important anticancer mechanism displayed by AITC, and suggest that the combination of AITC and radiation could be an effective therapy for NSCLC.

Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors.

Aberrant expression of hedgehog molecules, particularly Gli1, is common in cancers of many tissues and is responsible for their aggressive behavior and chemoresistance. Here we demonstrate a novel and tumor-specific role for aberrant Gli1 in the regulation of the S-phase checkpoint that suppresses replication stress and resistance to chemotherapy. Inhibition of Gli1 in tumor cells induced replication stress-mediated DNA damage response, attenuated their clonogenic potential, abrogated camptothecin (CPT)-induced Chk1 phosphorylation, and potentiated its cytotoxicity. However, in normal fibroblasts, Gli1 siRNAs showed no significant changes in CPT-induced Chk1 phosphorylation. Further analysis of ataxia telangiectasia and Rad3-related protein (ATR)/Chk1 signaling cascade genes in tumor cells revealed an unexpected mechanism whereby Gli1 regulates ATR-mediated Chk1 phosphorylation by transcriptional regulation of the BH3-only protein Bid. Consistent with its role in DNA damage response, Bid down-regulation in tumor cells abolished CPT-induced Chk1 phosphorylation and sensitized them to CPT. Correspondingly, Gli1 inhibition affected the expression of Bid and the association of replication protein A (RPA) with the ATR- interacting protein (ATRIP)-ATR complex, and this compromised the S-phase checkpoint. Conversely, complementation of Bid in Gli1-deficient cells restored CPT-induced Chk1 phosphorylation. An in silico analysis of the Bid promoter identified a putative Gli1 binding site, and further studies using luciferase reporter assays confirmed Gli1-dependent promoter activity. Collectively, our studies established a novel connection between aberrant Gli1 and Bid in the survival of tumor cells and their response to chemotherapy, at least in part, by regulating the S-phase checkpoint. Importantly, our data suggest a novel drug combination of Gli1 and Top1 inhibitors as an effective therapeutic strategy in treating tumors that expresses Gli1., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: Aldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance., Methods: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators., Results: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells., Conclusion: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1-dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage-independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη-PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance.

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.

The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad 18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability.

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

In eukaryotes, DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a conserved mechanism of transient DNA strand breakage, rotation, and religation. The unusual architecture of the monomeric human enzyme comprises a conserved protein clamp, which is tightly wrapped about duplex DNA, and an extended coiled-coil linker domain that appropriately positions the C-terminal active site tyrosine domain against the Top1 core to form the catalytic pocket. A structurally undefined N-terminal domain, dispensable for enzyme activity, mediates protein-protein interactions. Previously, reversible disulfide bonds were designed to assess whether locking the Top1 clamp around duplex DNA would restrict DNA strand rotation within the covalent Top1-DNA intermediate. The active site proximal disulfide bond in full-length Top1-clamp(534) restricted DNA rotation (Woo, M. H., Losasso, C., Guo, H., Pattarello, L., Benedetti, P., and Bjornsti, M. A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 13767-13772), whereas the more distal disulfide bond of the N-terminally truncated Topo70-clamp(499) did not (Carey, J. F., Schultz, S. J., Sisson, L., Fazzio, T. G., and Champoux, J. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5640-5645). To assess the contribution of the N-terminal domain to the dynamics of Top1 clamping of DNA, the same disulfide bonds were engineered into full-length Top1 and truncated Topo70, and the activities of these proteins were assessed in vitro and in yeast. Here we report that the N terminus impacts the opening and closing of the Top1 protein clamp. We also show that the architecture of yeast and human Top1 is conserved in so far as cysteine substitutions of the corresponding residues suffice to lock the Top1-clamp. However, the composition of the divergent N-terminal/linker domains impacts Top1-clamp activity and stability in vivo.

Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils. Topotecan is thought to stabilize a covalent topoisomerase-DNA complex, rendering it an obstacle to DNA replication forks. Here we use single-molecule nanomanipulation to monitor the dynamics of human topoisomerase I in the presence of topotecan. This allowed us to detect the binding and unbinding of an individual topotecan molecule in real time and to quantify the drug-induced trapping of topoisomerase on DNA. Unexpectedly, our findings also show that topotecan significantly hinders topoisomerase-mediated DNA uncoiling, with a more pronounced effect on the removal of positive (overwound) versus negative supercoils. In vivo experiments in the budding yeast verified the resulting prediction that positive supercoils would accumulate during transcription and replication as a consequence of camptothecin poisoning of topoisomerase I. Positive supercoils, however, were not induced by drug treatment of cells expressing a catalytically active, camptothecin-resistant topoisomerase I mutant. This combination of single-molecule and in vivo data suggests a cytotoxic mechanism for camptothecins, in which the accumulation of positive supercoils ahead of the replication machinery induces potentially lethal DNA lesions.

DNA topoisomerase I (Top1) is a nuclear enzyme that plays a crucial role in the removal of DNA supercoiling associated with replication and transcription. It is also the target of the anticancer agent, camptothecin (CPT). Top1 contains eight cysteines, including two vicinal residues (504 and 505), which are highly conserved across species. In this study, we show that thiol-reactive compounds such as N-ethylmaleimide and phenylarsine oxide can impair Top1 catalytic activity. We demonstrate that in contrast to CPT, which inhibits Top1-catalyzed religation, thiolation of Top1 inhibited the DNA cleavage step of the reaction. This inhibition was more pronounced when Top1 was preincubated with the thiol-reactive compound and could be reversed in the presence of dithiothreitol. We also established that phenylarsine oxide-mediated inhibition of Top1 cleavage involved the two vicinal cysteines 504 and 505, as this effect was suppressed when cysteines were mutated to alanines. Interestingly, mutation of Cys-505 also altered Top1 sensitivity to CPT, even in the context of the double Cys-504 to Cys-505 mutant, which relaxed supercoiled DNA with a comparable efficiency to that of wild-type Top1. This indicates that cysteine 505, which is located in the lower Lip domain of human Top1, is critical for optimal poisoning of the enzyme by CPT and its analogs. Altogether, our results suggest that conserved vicinal cysteines 504 and 505 of human Top1 play a critical role in enzyme catalytic activity and are the target of thiol-reactive compounds, which may be developed as efficient Top1 catalytic inhibitors.

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala(653) to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti P. (2003) J. Biol. Chem. 278, 43268-43275). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p.