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Supplemental Figures S1-4. Supplementary Figure S1: eRapa prevents DMBA/TPA-induced dermal neoplasia and malignant degeneration in WT mice. Supplementary Figure S2: eRapa does not suppress mTORC1 signaling in whole skin. Supplementary Figure S3: eRapa is not a calorie restriction mimetic. Supplementary Figure S4: eRapa prevents DMBA/TPA-induced dermal neoplasia in T cell deficient mice.

Mutation of a single copy of the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer. ApcMin/+ mice exhibit multiple intestinal neoplasia (MIN) that causes anemia and death from bleeding by 6 months. Mechanistic target of rapamycin complex 1 (mTORC1) inhibitors were shown to improve ApcMin/+ mouse survival when administered by oral gavage or added directly to the chow, but these mice still died from neoplasia well short of a natural life span. The National Institute of Aging Intervention Testing Program showed that enterically targeted rapamycin (eRapa) extended life span for wild-type genetically heterogeneous mice in part by inhibiting age-associated cancer. We hypothesized that eRapa would be effective in preventing neoplasia and extend survival of ApcMin/+ mice. We show that eRapa improved survival of ApcMin/+ mice in a dose-dependent manner. Remarkably, and in contrast to previous reports, most of the ApcMin/+ mice fed 42 parts per million eRapa lived beyond the median life span reported for wild-type syngeneic mice. Furthermore, chronic eRapa did not cause detrimental immune effects in mouse models of cancer, infection, or autoimmunity, thus assuaging concerns that chronic rapamycin treatment suppresses immunity. Our studies suggest that a novel formulation (enteric targeting) of a well-known and widely used drug (rapamycin) can dramatically improve its efficacy in targeted settings. eRapa or other mTORC1 inhibitors could serve as effective cancer preventatives for people with FAP without suppressing the immune system, thus reducing the dependency on surgery as standard therapy. Cancer Prev Res; 7(1); 169–78. ©2013 AACR.

MOV file - 3205K, Three ApcMIn/+ 42 ppm eRapa-fed female mice at 902 days of age.

AVI file - 9806K, A 42 ppm-fed ApcMin/+ female at 1087 days of age.

Supplementary Table 1 from Cisplatin Depletes TREX2 and Causes Robertsonian Translocations as Seen in TREX2 Knockout Cells

Cisplatin, an anticancer drug, forms DNA interstrand cross-links (ICL) that interfere with replication, whereas TREX2 is a 3′→5′ exonuclease that removes 3′ mismatched nucleotides and promotes cellular proliferation. Here, we show that TREX2 is depleted in human cells derived from cancer after exposure to cisplatin but not other genotoxins including another cross-linking agent, mitomycin C (MMC), indicating a potential role for TREX2 depletion in cisplatin-induced cytotoxicity. To better understand TREX2 cellular function, we deleted TREX2 in mouse embryonic stem (ES) cells by gene targeting and find these cells exhibit reduced proliferation and gross chromosomal rearrangements including Robertsonian translocations (RbT). Quite interestingly, ES cells exposed to cisplatin also exhibit RbTs. By contrast, RbTs are not observed for ES cells exposed to MMC, indicating that RbTs are not caused by ICLs but instead TREX2 depletion by either cisplatin exposure or mutation. Taken together, our results show that cisplatin depletes TREX2 and causes genomic instability that is similarly observed in TREX2-mutant cells. Thus, cisplatin has two potential cytotoxic activities: (a) the generation of ICLs and (b) the depletion of TREX2. [Cancer Res 2007;67(19):9077–83]

MICRO: Rapamycin extends life and health span in wild type mice suggesting prevention of cancer. We show rapamycin extends survival in colon cancer prone mice and that crypt cells are primary targets of mTOR inhibition that mediate tumor prevention or delay. STRUCTURED: We previously showed that lifelong rapamycin treatment of short lived Apc(Min/+) mice, a model for familial adenomatous polyposis (FAP), resulted in a normal lifespan. Apc(Min/+) mice develop colon polyps with a low frequency but can be converted to a colon cancer model by dextran sodium sulfate (DSS) treatments (Apc(Min/+)-DSS model). We asked, what effect would pretreatment of Apc(Min/+) mice with chronic rapamycin prior to DSS exposure have on survival and colonic neoplasia? Forty-two ppm eRapa diet exacerbated the temporary weight loss associated with DSS treatment in both sexes. However, our survival studies showed that chronic rapamycin treatment significantly extended lifespan of Apc(Min/+)-DSS mice (both sexes) by reductions in colon neoplasia and prevention of anemia. Rapamycin also had prophylactic effects on colon neoplasia induced by azoxymethane (AOM) and DSS in C57BL/6 males and females. Immunoblot assays showed the expected inhibition of mTORC1 and effectors (S6K→rpS6 and S6K→eEF2K→eEF2) in colon by lifelong rapamycin treatments. To address the question of cell types affected by chronic enteric rapamycin treatment, immunohistochemistry analyses demonstrated that crypt cells had a prominent reduction in rpS6 phosphorylation and increase in eEF2 phosphorylation relative controls. These data indicate that enteric rapamycin prevents or delays colon neoplasia in Apc(Min/+)+DSS mice through inhibition of mTORC1 in the crypt cells.

Background: ApcMin/+ mice model familial adenomatous polyposis (FAP), a disease that causes numerous colon polyps leading to colorectal cancer. We previously showed that chronic treatment of ApcMin/+ females with the anti-aging drug, rapamycin, restored a normal lifespan through reduced polyposis and anemia prevention. Lifespan extension by chronic rapamycin in wildtype UM-HET3 mice is sex-dependent with females gaining the most benefit. Whether ApcMin/+ mice have a similar sex-dependent response to chronic mTOR inhibition is not known. Methods: To address this knowledge gap and gain deeper insight into how chronic mTOR inhibition prevents intestinal polyposis, we compared male and female ApcMin/+ mice responses to chronic treatment with a rapamycin-containing diet. Animals were fed a diet containing either 42 ppm microencapsulate rapamycin or empty capsules, one group was used to determine lifespan and a second group with similar treatment was harvested at 16 weeks of age for cross-sectional studies. Results: We found that the survival of males is greater than females in this setting (P

BACKGROUND: Apc(Min/+) mice model familial adenomatous polyposis (FAP), a disease that causes numerous colon polyps leading to colorectal cancer. We previously showed that chronic treatment of Apc(Min/+) females with the anti-aging drug, rapamycin, restored a normal lifespan through reduced polyposis and anemia prevention. Lifespan extension by chronic rapamycin in wildtype UM-HET3 mice is sex-dependent with females gaining the most benefit. Whether Apc(Min/+) mice have a similar sex-dependent response to chronic mTOR inhibition is not known. METHODS: To address this knowledge gap and gain deeper insight into how chronic mTOR inhibition prevents intestinal polyposis, we compared male and female Apc(Min/+) mice responses to chronic treatment with a rapamycin-containing diet. Animals were fed a diet containing either 42 ppm microencapsulate rapamycin or empty capsules, one group was used to determine lifespan and a second group with similar treatment was harvested at 16 weeks of age for cross-sectional studies. RESULTS: We found that the survival of males is greater than females in this setting (P < 0.0197). To explore the potential basis for this difference we analyzed factors affected by chronic rapamycin. Immunoblot assays showed that males and females exhibited approximately the same level of mTORC1 inhibition using phosphorylation of ribosomal protein S6 (rpS6) as an indirect measure. Immunohistochemistry assays of rpS6 phosphorylation showed that rapamycin reduction of mTORC1 activity was on the same level, with the most prominent difference being in intestinal crypt Paneth cells in both sexes. Chronic rapamycin also reduced crypt depths in both male and female Apc(Min/+) mice (P < 0.0001), consistent with reduced crypt epithelial cell proliferation. Finally, chronic rapamycin prevented anemia equally in males and females. CONCLUSIONS: In males and females, these findings link rapamycin-mediated intestinal polyposis prevention with mTORC1 inhibition in Paneth cells and concomitant reduced epithelial cell proliferation.

Simple Summary Glioblastoma (GBM) is one of the most aggressive tumor types with no effective treatment options. To create new routes for therapy, it is necessary to continue mapping new pathways contributing to gliomagenesis. In this regard, there is growing evidence that RNA binding proteins (RBPs) are major contributors to expression alterations affecting genes in signaling pathways critical to GBM growth and response to therapy. We have established Musashi1 (Msi1) as a main player in GBM and medulloblastoma and as a marker of clinical outcome and response to therapy. Our genomic and functional analyses established that Msi1 directly and indirectly regulates the expression of a network of genes, promoting cell cycle progression and DNA replication. Ultimately, Msi1 impact on this network has important consequences in tumor initiation, growth and response to therapy. Abstract RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in their levels are often observed in tumors with numerous oncogenic RBPs identified in recent years. Musashi1 (Msi1) is an RBP and stem cell gene that controls the balance between self-renewal and differentiation. High Msi1 levels have been observed in multiple tumors including glioblastoma and are often associated with poor patient outcomes and tumor growth. A comprehensive genomic analysis identified a network of cell cycle/division and DNA replication genes and established these processes as Msi1’s core regulatory functions in glioblastoma. Msi1 controls this gene network via two mechanisms: direct interaction and indirect regulation mediated by the transcription factors E2F2 and E2F8. Moreover, glioblastoma lines with Msi1 knockout (KO) displayed increased sensitivity to cell cycle and DNA replication inhibitors. Our results suggest that a drug combination strategy (Msi1 + cell cycle/DNA replication inhibitors) could be a viable route to treat glioblastoma.

SUMMARY During the repeated cycles of damage and repair in many muscle disorders, including Duchenne muscular dystrophy (DMD), the muscle stem cell (MuSC) pool becomes less efficient at responding to and repairing damage. The underlying mechanism of such stem cell dysfunction is not fully known. Here, we demonstrate that the distinct early telomere shortening of diseased MuSCs in both mice and young DMD patients is associated with aberrant NF-κB activation. We find that prolonged NF-κB activation in MuSCs in chronic injuries leads to shortened telomeres and Ku80 dysregulation and results in severe skeletal muscle defects. Our studies provide evidence of a role for NF-κB in regulating stem-cell-specific telomere length, independently of cell replication, and could be a congruent mechanism that is applicable to additional tissues and/or diseases characterized by systemic chronic inflammation., In brief Tichy et al. reveal a role for NF-κB signaling in regulating telomere length in muscle stem cells (MuSCs) after chronic injuries. Persistent activation of NF-κB leads to shortened telomeres, Ku80 dysregulation, and muscle defects. The findings link stem cell dysfunction and NF-κB-dependent telomere shortening in Duchenne muscular dystrophy., Graphical Abstract

Acarbose blocks the digestion of complex carbohydrates, and the NIA Intervention Testing Program (ITP) found that it improved survival when fed to mice. Yet, we do not know if lifespan extension was caused by its effect on metabolism with regard to the soma or cancer suppression. Cancer caused death for ~80% of ITP mice. The ITP found rapamycin, an inhibitor to the pro‐growth mTORC1 (mechanistic target of rapamycin complex 1) pathway, improved survival and it suppressed tumors in Apc+/Min mice providing a plausible rationale to ask if acarbose had a similar effect. Apc+/Min is a mouse model prone to intestinal polyposis and a mimic of familial adenomatous polyposis in people. Polyp‐associated anemia contributed to their death. To address this knowledge gap, we fed two doses of acarbose to Apc+/Min mice. Acarbose improved median survival at both doses. A cross‐sectional analysis was performed next. At both doses, ACA fed mice exhibited reduced intestinal crypt depth, weight loss despite increased food consumption and reduced postprandial blood glucose and plasma insulin, indicative of improved insulin sensitivity. Dose‐independent and dose‐dependent compensatory liver responses were observed for AMPK and mTORC1 activities, respectively. Only mice fed the high dose diet exhibited reductions in tumor number with higher hematocrits. Because low‐dose acarbose improved lifespan but failed to reduced tumors, its effects seem to be independent of cancer. These data implicate the importance of improved carbohydrate metabolism on survival., Acarbose, a commonly used drug to treat diabetes, extends lifespan in a Apc Min/+ cancer‐prone mice

Homologous recombination (HR) repairs DNA double strand breaks (DSBs) and stabilizes replication forks (RFs). RAD51 is the recombinase for the HR pathway. To preserve genomic integrity, RAD51 forms a filament on the 3″ end of a DSB and on a single-stranded DNA (ssDNA) gap. But unregulated HR results in undesirable chromosomal rearrangements. This review describes the multiple mechanisms that regulate HR with a focus on those mechanisms that promote and contain RAD51 filaments to limit chromosomal rearrangements. If any of these pathways break down and HR becomes unregulated then disease, primarily cancer, can result.

Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus-Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer.

SUMMARY DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance., Graphical Abstract, In Brief Ko et al. find that TREX2’s exonuclease activity causes mutations, RF stalls, and strand degradation in cells expressing RAD51K133A. RAD18 deletion and PCNA overexpression reduce mutations, implicating DNA damage tolerance. Deleting TREX2 in PARP1-deficient or FANCB mutant cells increases strand degradation that is rescued by TREX2H188A, showing that TREX2’s catalytic activity is unimportant.

ERCC1 (excision repair cross complementing‐group 1) is a mammalian endonuclease that incises the damaged strand of DNA during nucleotide excision repair and interstrand cross‐link repair. Ercc1−/Δ mice, carrying one null and one hypomorphic Ercc1 allele, have been widely used to study aging due to accelerated aging phenotypes in numerous organs and their shortened lifespan. Ercc1−/Δ mice display combined features of human progeroid and cancer‐prone syndromes. Although several studies report cellular senescence and apoptosis associated with the premature aging of Ercc1−/Δ mice, the link between these two processes and their physiological relevance in the phenotypes of Ercc1−/Δ mice are incompletely understood. Here, we show that ERCC1 depletion, both in cultured human fibroblasts and the skin of Ercc1−/Δ mice, initially induces cellular senescence and, importantly, increased expression of several SASP (senescence‐associated secretory phenotype) factors. Cellular senescence induced by ERCC1 deficiency was dependent on activity of the p53 tumor‐suppressor protein. In turn, TNFα secreted by senescent cells induced apoptosis, not only in neighboring ERCC1‐deficient nonsenescent cells, but also cell autonomously in the senescent cells themselves. In addition, expression of the stem cell markers p63 and Lgr6 was significantly decreased in Ercc1−/Δ mouse skin, where the apoptotic cells are localized, compared to age‐matched wild‐type skin, possibly due to the apoptosis of stem cells. These data suggest that ERCC1‐depleted cells become susceptible to apoptosis via TNFα secreted from neighboring senescent cells. We speculate that parts of the premature aging phenotypes and shortened health‐ or lifespan may be due to stem cell depletion through apoptosis promoted by senescent cells., ERCC1‐depleted cells become susceptible to apoptosis via TNFalpha secreted from neighboring senescent cells. Part of the premature aging phenotypes in ERCC1‐deficient mice may be due to stem cell depletion through apoptosis promoted by cellular senescence.

// Mi Young Son 1 , Chu-Xia Deng 2 , Jan H. Hoeijmarkers 3 , Vivienne I. Rebel 4, 5, 6, 7, 8 , Paul Hasty 1, 5, 6 1 Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 2 Faculty of Health Sciences, University of Macau, Macau SAR China 3 Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, The Netherlands 4 Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 5 The Cancer Therapy Research Center, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 6 The Barshop Center of Aging, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 7 Greehey Children’s Cancer Research Center, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 8 Current address: BioAffinity, San Antonio, Texas, USA Correspondence to: Paul Hasty, email: hastye@uthscsa.edu Keywords: Fanconi anemia, double strand break repair, replication fork maintenance, type 1 topoisomerase Received: April 18, 2016 Accepted: May 22, 2016 Published: June 20, 2016 ABSTRACT Benzene is a common environmental toxin and its metabolite, 1-4-Benzoquinone (BQ) causes hematopoietic cancers like myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). BQ has not been comprehensively assessed for its impact on genome maintenance, limiting our understanding of the true health risks associated with benzene exposure and our ability to identify people with increased sensitivity to this genotoxin. Here we analyze the impact BQ exposure has on wild type and DNA repair-defective mouse embryonic stem (ES) cells and wild type human cells. We find that double strand break (DSB) repair and replication fork maintenance pathways including homologous recombination (HR) and Fanconi anemia (FA) suppress BQ toxicity. BQ-induced damage efficiently stalls replication forks, yet poorly induces ATR/DNA-PK CS responses. Furthermore, the pattern of BQ-induced γH2AX and 53BP1foci is consistent with the formation of poly(ADP-ribose) polymerase 1 (PARP1)-stabilized regressed replication forks. At a biochemical level, BQ inhibited topoisomerase 1 (topo1)-mediated DNA ligation and nicking in vitro ; thus providing mechanism for the cellular phenotype. These data are consistent with a model that proposes BQ interferes with type I topoisomerase’s ability to maintain replication fork restart and progression leading to chromosomal instability that has the potential to cause hematopoietic cancers like MDS and AML.

DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.

The mechanistic (or mammalian) target of rapamycin, mTOR, is a highly conserved PI3K-related kinase that regulates cell growth, proliferation, survival, transcription, and protein synthesis. It forms two different complexes, mTOR complex-1 (mTORC1) and -2 (mTORC2). Rapamycin is an allosteric inhibitor specific to mTORC1 and extends life and health span with chronic use. Previous research from our lab shows that an enteric release formulation of rapamycin, eRapa, enhances the lifespan of mice mutated for adenomatous polyposis coli (Apc) tumor suppressor (ApcMin/+ mice), which normally develop intestinal cancer. These mice model familial adenomatous polyposis (FAP) in humans. eRapa restored a normal (and in 60% of the mice longer) life span in female ApcMin/+ mice. Mutations in Apc, a part of the Wnt/β-catenin pathway, accelerate the initiation of the adeno-carcinoma in FAP, resulting in numerous colorectal polyps at a young age, and if left untreated these polyps progress to colorectal cancer.eRapa treatment prevents polyposis in ApcMin/+ mice but the precise mechanism of action remains to be determined. We used a cross sectional and survival approach to test the preventive effects of eRapa and gain mechanistic insights. Four-week-old male and female animals were put on a diet containing either 42 ppm of eRapa or empty microcapsules. A subset of the animals was sacrificed to harvest tissue for analysis at 16 weeks of age and the rest were allowed to live out their lifespans.eRapa prevented polyposis in the small intestine of ApcMin/+ mice with only a few to no tumors in both males and females. In addition to adenomas in the intestine, ApcMin/+ mice also suffer from severe anemia. Hematocrits from the animals showed anemia in the control mice, however, eRapa treated animals did not have anemia. There was a reduction in phosphorylation of ribosomal protein S6 in the small intestine tissue, showing suppression of mTORC1 activity with eRapa. Histological analysis revealed that this suppression occurred in the Paneth cells of the small intestinal crypt. The Paneth cells, along with the stem cells at the base of the crypt form a niche and signaling between this niche maintains homeostasis in the crypt. Although the polyps are believed to originate from stem cells, our results suggest involvement of Paneth cells in tumor prevention perhaps by signaling changes in the niche. These are novel effects of rapamycin and help define its mechanisms of cancer prevention. Citation Format: Manish Parihar, Sherry G. Dodds, Paul Hasty, Zelton Dave Sharp. mTORC1 suppression in Paneth cells prevents tumors in a mouse model of intestinal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 671.

Yeast Rad1–Rad10 (XPF–ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1–Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1–Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1–Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1–Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1–Rad10 removes ICL via NER, whereas in S/G2, Rad1–Rad10 facilitates NER-independent replication-coupled ICLR., The yeast Rad1–Rad10 complex has multiple roles in DNA damage repair. Here the authors uncover mutants that uncouple the roles in UV excision repair and non-NER functions.

Cancer prevention is a cost-effective alternative to treatment. In mice, the mTOR inhibitor rapamycin prevents distinct spontaneous, noninflammatory cancers, making it a candidate broad-spectrum cancer prevention agent. We now show that oral microencapsulated rapamycin (eRapa) prevents skin cancer in dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) carcinogen-induced, inflammation-driven carcinogenesis. eRapa given before DMBA/TPA exposure significantly increased tumor latency, reduced papilloma prevalence and numbers, and completely inhibited malignant degeneration into squamous cell carcinoma. Rapamycin is primarily an mTORC1-specific inhibitor, but eRapa did not reduce mTORC1 signaling in skin or papillomas, and did not reduce important proinflammatory factors in this model, including p-Stat3, IL17A, IL23, IL12, IL1β, IL6, or TNFα. In support of lack of mTORC1 inhibition, eRapa did not reduce numbers or proliferation of CD45−CD34+CD49fmid skin cancer initiating stem cells in vivo and marginally reduced epidermal hyperplasia. Interestingly, eRapa reduced DMBA/TPA-induced skin DNA damage and the hras codon 61 mutation that specifically drives carcinogenesis in this model, suggesting reduction of DNA damage as a cancer prevention mechanism. In support, cancer prevention and DNA damage reduction effects were lost when eRapa was given after DMBA-induced DNA damage in vivo. eRapa afforded picomolar concentrations of rapamycin in skin of DMBA/TPA-exposed mice, concentrations that also reduced DMBA-induced DNA damage in mouse and human fibroblasts in vitro. Thus, we have identified DNA damage reduction as a novel mechanism by which rapamycin can prevent cancer, which could lay the foundation for its use as a cancer prevention agent in selected human populations. Cancer Prev Res; 8(5); 400–9. ©2015 AACR.

Hematopoietic stem cells (HSCs) are responsible for the continuous regeneration of all types of blood cells, including themselves. To ensure the functional and genomic integrity of blood tissue, a network of regulatory pathways tightly controls the proliferative status of HSCs. Nevertheless, normal HSC aging is associated with a noticeable decline in regenerative potential and possible changes in other functions. Myelodysplastic syndrome (MDS) is an age-associated hematopoietic malignancy, characterized by abnormal blood cell maturation and a high propensity for leukemic transformation. It is furthermore thought to originate in a HSC and to be associated with the accrual of multiple genetic and epigenetic aberrations. This raises the question whether MDS is, in part, related to an inability to adequately cope with DNA damage. Here we discuss the various components of the cellular response to DNA damage. For each component, we evaluate related studies that may shed light on a potential relationship between MDS development and aberrant DNA damage response/repair.

Fanconi anemia (FA) patients exhibit bone marrow failure, developmental defects and cancer. The FA pathway maintains chromosomal stability in concert with replication fork maintenance and DNA double strand break (DSB) repair pathways including RAD51-mediated homologous recombination (HR). RAD51 is a recombinase that maintains replication forks and repairs DSBs, but also rearranges chromosomes. Two RecQ helicases, RECQL5 and Bloom syndrome mutated (BLM) suppress HR through nonredundant mechanisms. Here we test the impact deletion of RECQL5 and BLM has on mouse embryonic stem (ES) cells deleted for FANCB, a member of the FA core complex. We show that RECQL5, but not BLM, conferred resistance to mitomycin C (MMC, an interstrand crosslinker) and camptothecin (CPT, a type 1 topoisomerase inhibitor) in FANCB-defective cells. RECQL5 suppressed, while BLM caused, breaks and radials in FANCB-deleted cells exposed to CPT or MMC, respectively. RECQL5 protected the nascent replication strand from MRE11-mediated degradation and restarted stressed replication forks in a manner additive to FANCB. By contrast BLM restarted, but did not protect, replication forks in a manner epistatic to FANCB. RECQL5 also lowered RAD51 levels in FANCB-deleted cells at stressed replication sites implicating a rearrangement avoidance mechanism. Thus, RECQL5 and BLM impact FANCB-defective cells differently in response to replication stress with relevance to chemotherapeutic regimes.

Proline-, glutamic acid- and leucine-rich protein-1 (PELP1) is a scaffolding oncogenic protein that functions as a coregulator for a number of nuclear receptors. p53 is an important transcription factor and tumor suppressor that has a critical role in DNA damage response (DDR) including cell cycle arrest, repair or apoptosis. In this study, we found an unexpected role for PELP1 in modulating p53-mediated DDR. PELP1 is phosphorylated at Serine1033 by various DDR kinases like ataxia-telangiectasia mutated, ataxia telangiectasia and Rad3-related or DNAPKc and this phosphorylation of PELP1 is important for p53 coactivation functions. PELP1-depleted p53 (wild-type) breast cancer cells were less sensitive to various genotoxic agents including etoposide, camptothecin or γ-radiation. PELP1 interacts with p53, functions as p53-coactivator and is required for optimal activation of p53 target genes under genomic stress. Overall, these studies established a new role of PELP1 in DDRs and these findings will have future implications in our understanding of PELP1's role in cancer progression.

Background: Arsenic trioxide is currently used to treat acute promyelocytic leukemia and is known as a cytotoxic agent. Arsenic is also known as a carcinogen. However, a cytoprotective effect of low-dose arsenic (LDA) has also been observed in some studies. Our previous studies have found that pretreatment with LDA before DNA damaging agents such as chemotherapy and radiation therapy protects normal cells without impairing the cancer killing effects. We have demonstrated that intact p53 function is required in this process, and therefore, LDA does not protect cancer cells as most of them have mutated or dysfunctional p53 pathway. We have also reported that LDA helps protect the telomere from enhanced erosion by DNA damaging agents in ConA-activated normal human lymphocytes. As enhanced telomere shortening is associated with genomic instability, we decided to investigate the effect of LDA in genomic integrity. Methods: Mouse embryonic stem cells (mESCs) with a mutator phenotype in DNA repair pathways (MSH2-/-, Rad51KA and Ku70null) and their wildtype counterparts were utilized for cell viability studies and HPRT minigene mutation assay. Cell viability under different doses of LDA (0-1000nM) and radiation (0-4Gy) was firstly examined by colony formation assay. DNA damage and DNA repair capacity were also measured by comet assay in MSH2-/- mESCs. TailDNA% was analyzed by OpenComet and used to describe the comet feature. Results: Arsenic had little effect on the cell viability at doses ≤200nM in all tested cell lines. In MSH2-/- mESCs, there was no significant difference between the tailDNA% of cells treated with 200nM LDA (11.41±0.52%) and the control (10.82±0.49%) immediately after radiation. However, LDA treatment significantly decreased the tailDNA% 30 minutes after radiation (4.68±0.35% vs. 7.29±0.51%, Student’s test P Discussion: Our data suggest that brief use of LDA enhances DNA repair activities and provides protection from radiation-induced mutations in mESCs. The inability to correctly repair DNA damage from chemotherapy and radiation therapy has been attributed to the development of therapy related secondary malignancy. Helping maintain genomic integrity with LDA during chemotherapy and radiation therapy may have a role in reducing therapy related secondary malignancy. Further work is in progress to test the potential use of LDA as a genome protector in vivo. Citation Format: Hang Su, Meijun Long, Mi Young Son, Teresa C Marple, Paul Hasty, Chul Soo Ha. An unexpected effect of low-dose arsenic on genomic integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3509.

Replication fork maintenance pathways preserve chromosomes, but their faulty application at nonallelic repeats could generate rearrangements causing cancer, genomic disorders and speciation. Potential causal mechanisms are homologous recombination and error-free postreplication repair (EF-PRR). Homologous recombination repairs damage-induced DNA double-strand breaks (DSBs) and single-ended DSBs within replication. To facilitate homologous recombination, the recombinase RAD51 and mediator BRCA2 form a filament on the 3' DNA strand at a break to enable annealing to the complementary sister chromatid while the RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to prevent recombination. Homologous recombination also stabilizes and restarts replication forks without a DSB. EF-PRR bypasses DNA incongruities that impede replication by ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6-RAD18 and UBC13-MMS2-RAD5 ubiquitin ligase complexes. Some components are common to both homologous recombination and EF-PRR such as RAD51 and RAD18. Here we delineate two pathways that spontaneously fuse inverted repeats to generate unstable chromosomal rearrangements in wild-type mouse embryonic stem (ES) cells. Gamma-radiation induced a BLM-regulated pathway that selectively fused identical, but not mismatched, repeats. By contrast, ultraviolet light induced a RAD18-dependent pathway that efficiently fused mismatched repeats. Furthermore, TREX2 (a 3'→5' exonuclease) suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly separating these pathways. TREX2 associated with UBC13 and enhanced PCNA ubiquitination in response to ultraviolet light, consistent with it being a novel member of EF-PRR. RAD18 and TREX2 also suppressed replication fork stalling in response to nucleotide depletion. Interestingly, replication fork stalling induced fusion for identical and mismatched repeats, implicating faulty replication as a causal mechanism for both pathways.

Myelodysplastic syndrome (MDS) is considered a hematopoietic stem cell disease that is characterized by abnormal hematopoietic differentiation and a high propensity to develop acute myeloid leukemia. It is mostly associated with advanced age, but also with prior cancer therapy and inherited syndromes related to abnormalities in DNA repair. Recent technologic advances have led to the identification of a myriad of frequently occurring genomic perturbations associated with MDS. These observations suggest that MDS and its progression to acute myeloid leukemia is a genomic instability disorder, resulting from a stepwise accumulation of genetic abnormalities. The notion is now emerging that the underlying mechanism of this disease could be a defect in one or more pathways that are involved in responding to or repairing damaged DNA. In this review, we discuss these pathways in relationship to a large number of studies performed with MDS patient samples and MDS mouse models. Moreover, in view of our current understanding of how DNA damage response and repair pathways are affected by age in hematopoietic stem cells, we also explore how this might relate to MDS development.

Steady-state levels of spontaneous DNA damage, the by-product of normal metabolism and environmental exposure, are controlled by DNA repair pathways. Incomplete repair or an age-related increase in damage production and/or decline in repair could lead to an accumulation of DNA damage, increasing mutation rate, affecting transcription, and/or activating programmed cell death or senescence. These consequences of DNA damage metabolism are highly conserved, and the accumulation of lesions in the DNA of the genome could therefore provide a universal cause of aging. An important corollary of this hypothesis is that defects in DNA repair cause both premature aging and accelerated DNA damage accumulation. While the former has been well-documented, the reliable quantification of the various lesions thought to accumulate in DNA during aging has been a challenge. Here, we quantified inhibition of long-distance PCR as a measure of DNA damage in liver and brain of both normal and prematurely aging, DNA repair defective mice. The results indicate a marginal, but statistically significant, increase in spontaneous DNA damage with age in normal mouse liver but not in brain. Increased levels of DNA damage were not observed in the DNA repair defective mice. We also show that oxidative lesions do not increase with age. These results indicate that neither normal nor premature aging is accompanied by a dramatic increase in DNA damage. This suggests that factors other than DNA damage per se, for example, cellular responses to DNA damage, are responsible for the aging phenotype in mice.

Many genotoxins are found in the environment from synthetic to natural, yet very few have been studied in depth. This means we fail to understand many molecules that damage DNA, we do not understand the type of damage they cause and the repair pathways required to correct their lesions. It is surprising so little is known about the vast majority of genotoxins since they have potential to cause disease from developmental defects to cancer to degenerative ailments. By contrast, some of these molecules have commercial and medical potential and some can be weaponized. Therefore, we need a systematic method to efficiently generate a genotoxic profile for these agents. A genotoxic profile would include the type of damage the genotoxin causes, the pathways used to repair the damage and the resultant mutations if repair fails. Mouse embryonic stem (ES) cells are well suited for identifying pathways and mutations. Mouse ES cells are genetically tractable and many DNA repair mutant cells are available. ES cells have a high mitotic index and form colonies so experiments can be completed quickly and easily. Furthermore, ES cells have robust DNA repair pathways to minimize genetic mutations at a particularly vulnerable time in life, early development when a mutation in a single cell could ultimately contribute to a large fraction of the individual. After an initial screen, other types of cells and mouse models can be used to complement the analysis. This review discusses the merging field of genotoxic screens in mouse ES cells that can be used to discover and study potential genotoxic activity for chemicals commonly found in our environment.

Chronic treatment of mice with an enterically released formulation of rapamycin (eRapa) extends median and maximum life span, partly by attenuating cancer. The mechanistic basis of this response is not known. To gain a better understanding of these in vivo effects, we used a defined preclinical model of neuroendocrine cancer, Rb1+/− mice. Previous results showed that diet restriction (DR) had minimal or no effect on the lifespan of Rb1+/− mice, suggesting that the beneficial response to DR is dependent on pRb1. Since long-term eRapa treatment may at least partially mimic chronic DR in lifespan extension, we predicted that it would have a minimal effect in Rb1+/− mice. Beginning at 9 weeks of age until death, we fed Rb1+/− mice a diet without or with eRapa at 14 mg/kg food, which results in an approximate dose of 2.24 mg/kg body weight per day, and yielded rapamycin blood levels of about 4 ng/ml. Surprisingly, we found that eRapa dramatically extended life span of both female and male Rb1+/− mice, and slowed the appearance and growth of pituitary and decreased the incidence of thyroid tumors commonly observed in these mice. In this model, eRapa appears to act differently than DR, suggesting diverse mechanisms of action on survival and anti-tumor effects. In particular the beneficial effects of rapamycin did not depend on the dose of Rb1.

p53 is a transcriptional regulator that responds to cellular stresses to suppress oncogenesis, but some of these responses can have unintended consequences that influence non-cancer-related aging processes. The impact of these consequences is not well understood—partly due to the many complex processes that influence p53 function and partly due to the vast array of processes that p53 affects. p53 has the potential to both accelerate and hinder cellular aging processes, which would likely have antithetical biological outcomes with regard to organismal aging. To accelerate aging, p53 induces apoptosis or cell cycle arrest as a prerequisite to cellular senescence; both can impair the mobilization of stem and progenitor cell populations. To suppress aging, p53 inhibits unregulated proliferation pathways that could lead to cellular senescence and a senescence-associated secretory phenotype (SASP), which creates a pro-inflammatory and degenerative tissue milieu. A review of mouse models supports both possibilities, highlighting the complexity of the p53 influence over organismal aging. A deeper knowledge of how p53 integrates and is integrated with various biological processes will improve our understanding of its influence over the aging process.

Rapamycin inhibits mechanistic (or mammalian) target of rapamycin (mTOR) that promotes protein production in cells by facilitating ribosome biogenesis (RiBi) and eIF4E-mediated 5’cap mRNA translation. Chronic treatment with encapsulated rapamycin (eRapa) extended health and life span for wild-type and cancer-prone mice. Yet, the long-term consequences of chronic eRapa treatment are not known at the organ level. Here, we report our observations of chronic eRapa treatment on mTORC1 signaling and RiBi in mouse colon and visceral adipose. As expected, chronic eRapa treatment decreased detection of phosphorylated mTORC1/S6K substrate, ribosomal protein (rpS6) in colon and fat. However, in colon, contrary to expectations, there was an upregulation of 18S rRNA and some ribosomal protein genes (RPGs) suggesting increased RiBi. Among RPGs, eRapa increases rpl22l1 mRNA but not its paralog rpl22 . Furthermore, there was an increase in the cap-binding protein, eIF4E relative to its repressor 4E-BP1 suggesting increased translation. By comparison, in fat, there was a decrease in the level of 18S rRNA (opposite to colon), while overall mRNAs encoding ribosomal protein genes appeared to increase, including rpl22 , but not rpl22l1 (opposite to colon). In fat, there was a decrease in eIF4E relative to actin (opposite to colon) but also an increase in the eIF4E/4E-BP1 ratio likely due to reductions in 4E-BP1 at our lower eRapa dose (similar to colon). Thus, in contrast to predictions of decreased protein production seen in cell-based studies, we provide evidence that colon from chronically treated mice exhibited an adaptive ‘pseudo-anabolic’ state, which is only partially present in fat, which might relate to differing tissue levels of rapamycin, cell-type-specific responses, and/or strain differences. Keywords : mTORC1; rapamycin; translation; ribosome biogenesis (Published: 27 May 2016) To access the supplementary material for this article, please see Supplementary files in the column to the right (under ‘Article Tools’). Citation: Pathobiology of Aging & Age-related Diseases 2016, 6 : 31688 - http://dx.doi.org/10.3402/pba.v6.31688

mTOR and aging appear to have co-evolved in eukaryotes, suggesting that cancer would be inexorably linked to these fundamental aspects of life. We argue this is a perilous linkage, although there is an opportunity to suppress cancer and aging as Shakespeare said of red kites attacking chickens “as one fell swoop,” which up to now might have seemed fantastical. We review current knowledge concerning the role of mTOR in both aging and cancer and proof of concept results indicating that rapamycin, or similar inhibitors, should be considered for this “down to earth” application. We also discuss the immunosuppression controversy regarding chronic rapamycin use in longevity and cancer prevention and argue that this putative caveat is not supported by available evidence. If we are going to get serious about a looming economic burden of the aging population and their associated diseases (like cancer), we might need to consider approaches that prevent or treat more than one disease at a time.

Genome maintenance is considered a prime longevity assurance mechanism as apparent from many progeroid human syndromes that are caused by genome maintenance defects. The ERCC1 protein is involved in three genome maintenance systems: nucleotide excision repair, interstrand cross-link repair, and homologous recombination. Here we describe in-life and post-mortem observations for a hypomorphic Ercc1 variant, Ercc1 −/Δ7, which is hemizygous for a single truncated Ercc1 allele, encoding a protein lacking the last seven amino acids. Ercc1 −/Δ7 mice were much smaller and median life span was markedly reduced compared to wild-type siblings: 20 and 118 weeks, respectively. Multiple signs and symptoms of aging were found to occur at an accelerated rate in the Ercc1 −/Δ7 mice as compared to wild-type controls, including a decline in weight of both whole body and various organs, numerous histopathological lesions, and immune parameters. Together they define a segmental progeroid phenotype of the Ercc1 −/Δ7 mouse model.

Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor and money. Here we describe a one-step Cre-mediated knockin system in mouse embryonic stem (ES) cells that positions the TA and inducible expression cassette to a single location. Using this system, we show doxycycline-dependent regulation of eGFP at the DNA topoisomerase 3β (Top3β) promoter. Since Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.