Telomere dysfunction has been strongly implicated in the initiation of genomic instability and is suspected to be an early event in the carcinogenesis of human solid tumors. Recent findings have established the presence of telomere fusions in human breast and prostate malignancies; however, the onset of this genomic instability mechanism during progression of other solid cancers is not well understood. Herein, we explored telomere dynamics in patient-derived epithelial ovarian cancers (OC), a malignancy characterized by multiple distinct subtypes, extensive molecular heterogeneity, and widespread genomic instability. We discovered a high frequency of telomere fusions in ovarian tumor tissues; however, limited telomere fusions were detected in normal adjacent tissues or benign ovarian samples. In addition, we found relatively high levels of both telomerase activity and hTERT expression, along with anaphase bridges in tumor tissues, which were notably absent in adjacent normal ovarian tissues and benign lesions. These results suggest that telomere dysfunction may occur early in ovarian carcinogenesis and, importantly, that it may play a critical role in the initiation and progression of the disease. Recognizing telomere dysfunction as a pervasive feature of this heterogeneous malignancy may facilitate the future development of novel diagnostic tools and improved methods of disease monitoring and treatment.

Background:Even before the onset of age-related diseases, obesity might be a contributing factor to the cumulative burden of oxidative stress and chronic inflammation throughout the life course. Obesity may therefore contribute to accelerated shortening of telomeres. Consequently, obese persons are more likely to have shorter telomeres, but the association between body mass index (BMI) and leukocyte telomere length (TL) might differ across the life span and between ethnicities and sexes. Objective:A collaborative cross-sectional meta-analysis of observational studies was conducted to investigate the associations between BMI and TL across the life span. Design:Eighty-seven distinct study samples were included in the meta-analysis capturing data from 146,114 individuals. Study-specific age- and sex-adjusted regression coefficients were combined by using a random-effects model in which absolute [base pairs (bp)] and relative telomere to single-copy gene ratio (T/S ratio) TLs were regressed against BMI. Stratified analysis was performed by 3 age categories ("young": 18-60 y; "middle": 61-75 y; and "old": >75 y), sex, and ethnicity. Results:Each unit increase in BMI corresponded to a -3.99 bp (95% CI: -5.17, -2.81 bp) difference in TL in the total pooled sample; among young adults, each unit increase in BMI corresponded to a -7.67 bp (95% CI: -10.03, -5.31 bp) difference. Each unit increase in BMI corresponded to a -1.58 × 10(-3) unit T/S ratio (0.16% decrease; 95% CI: -2.14 × 10(-3), -1.01 × 10(-3)) difference in age- and sex-adjusted relative TL in the total pooled sample; among young adults, each unit increase in BMI corresponded to a -2.58 × 10(-3) unit T/S ratio (0.26% decrease; 95% CI: -3.92 × 10(-3), -1.25 × 10(-3)). The associations were predominantly for the white pooled population. No sex differences were observed. Conclusions:A higher BMI is associated with shorter telomeres, especially in younger individuals. The presently observed difference is not negligible. Meta-analyses of longitudinal studies evaluating change in body weight alongside change in TL are warranted.

Constitutive activation of the pro-survival transcription factor NF-κB has been associated with resistance to both chemotherapy and radiation therapy in many human cancers, including prostate cancer. Our lab and others have demonstrated that the natural product parthenolide can inhibit NF-κB activity and sensitize PC-3 prostate cancers cells to X-rays in vitro; however, parthenolide has poor bioavailability in vivo and therefore has little clinical utility in this regard. We show here that treatment of PC-3 and DU145 human prostate cancer cells with dimethylaminoparthenolide (DMAPT), a parthenolide derivative with increased bioavailability, inhibits constitutive and radiation-induced NF-κB binding activity and slows prostate cancer cell growth. We also show that DMAPT increases single and fractionated X-ray-induced killing of prostate cancer cells through inhibition of DNA double strand break repair and also that DMAPT-induced radiosensitization is, at least partially, dependent upon the alteration of intracellular thiol reduction-oxidation chemistry. Finally, we demonstrate that the treatment of PC-3 prostate tumor xenografts with oral DMAPT in addition to radiation therapy significantly decreases tumor growth and results in significantly smaller tumor volumes compared to xenografts treated with either DMAPT or radiation therapy alone, suggesting that DMAPT might have a potential clinical role as a radiosensitizing agent in the treatment of prostate cancer.

Telomere length homeostasis, critical for chromosomal integrity and genome stability, is controlled by intricate molecular regulatory machinery that includes epigenetic modifications. Here, we examine site-specific and spatiotemporal alteration of the subtelomeric methylation of CpG islands using optogenetic tools to understand the epigenetic regulatory mechanisms of telomere length maintenance. Human DNA methyltransferase3A (DNMT3A) were assembled selectively at chromosome ends by fusion to cryptochrome 2 protein (CRY2) and its interacting complement, the basic helix loop helix protein-1 (CIB1). CIB1 was fused to the telomere-associated protein telomere repeat binding factor-1 (TRF1), which localized the protein complex DNMT3A-CRY2 at telomeric regions upon excitation by blue-light monitored by single-molecule fluorescence analyses. Increased methylation was achieved selectively at subtelomeric CpG sites on the six examined chromosome ends specifically after blue-light activation, which resulted in progressive increase in telomere length over three generations of HeLa cell replications. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin modifying enzymes and for loci-specific targeting to regulate the epigenetic pathways at telomeres and other selected genomic regions of interest.

The transcription factor nuclear factor-kappaB (NF- κ B) is constitutively active in several cancers and is a target of therapeutic development. We recently developed dimethylaminoparthenolide (DMAPT), a clinical grade water-soluble analog of parthenolide, as a potent inhibitor of NF- κ B and demonstrated in vitro and in vivo anti-tumor activities in multiple cancers. In this study, we show DMAPT is an epigenetic modulator functioning in an NF- κ B-dependent and -independent manner. DMAPT-mediated NF- κ B inhibition resulted in elevated histone H3K36 trimethylation (H3K36me3), which could be recapitulated through genetic ablation of the p65 subunit of NF- κ B or inhibitor-of-kappaB alpha super-repressor overexpression. DMAPT treatment and p65 ablation increased the levels of H3K36 trimethylases NSD1 (KMT3B) and SETD2 (KMT3A), suggesting that NF- κ B directly represses their expression and that lower H3K36me3 is an epigenetic marker of constitutive NF- κ B activity. Overexpression of a constitutively active p65 subunit of NF- κ B reduced NSD1 and H3K36me3 levels. NSD1 is essential for DMAPT-induced expression of pro-apoptotic BIM, indicating a functional link between epigenetic modification and gene expression. Interestingly, we observed enhanced H4K20 trimethylation and induction of H4K20 trimethylase KMT5C in DMAPT-treated cells independent of NF- κ B inhibition. These results add KMT5C to the list NF- κ B-independent epigenetic targets of parthenolide, which include previously described histone deacetylase 1 (HDAC-1) and DNA methyltransferase 1. As NSD1 and SETD2 are known tumor suppressors and loss of H4K20 trimethylation is an early event in cancer progression, which contributes to genomic instability, we propose DMAPT as a potent pharmacologic agent that can reverse NF- κ B-dependent and -independent cancer-specific epigenetic abnormalities.

A critical function of the telomere is to disguise chromosome ends from cellular recognition as double strand breaks, thereby preventing aberrant chromosome fusion events. Such chromosome end-to-end fusions are known to initiate genomic instability via breakage-fusion-bridge cycles. Telomere dysfunction and other forms of genomic assault likely result in misregulation of genes involved in growth control, cell death, and senescence pathways, lowering the threshold to malignancy and likely drive disease progression. Shortened telomeres and anaphase bridges have been reported in a wide variety of early precursor and malignant cancer lesions including those of the prostate. These findings are being extended using methods for the analysis of telomere fusions (decisive genetic markers for telomere dysfunction) specifically within human tissue DNA. Here we report that benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (PIN), and prostate cancer (PCa) prostate lesions all contain similarly high frequencies of telomere fusions and anaphase bridges. Tumor-adjacent, histologically normal prostate tissue generally did not contain telomere fusions or anaphase bridges as compared to matched PCa tissues. However, we found relatively high levels of telomerase activity in this histologically normal tumor-adjacent tissue that was reduced but closely correlated with telomerase levels in corresponding PCa samples. Thus, we present evidence of high levels of telomere dysfunction in BPH, an established early precursor (PIN) and prostate cancer lesions but not generally in tumor adjacent normal tissue. Our results suggest that telomere dysfunction may be a common gateway event leading to genomic instability in prostate tumorigenesis. .

The mission of IUPUI breast cancer signature center is to address prevention, early detection, and treatment of breast cancer through translational projects, supportive cores, and synergistic programs. This poster details our efforts improve resources for breast cancer research and efforts to develop multi-PI investigator proposals. The Signature Center has developed two web resources: the Breast Cancer Prognostics Database (PROGgene) to study prognostic implications of genes of interest in publically available breast cancer databases and PROGmiR, a microRNA database. The PROGgene can be used to study overall, recurrence free and metastasis free survival in large patient series. PROGmiR allows investigators to study the prognostic importance of microRNAs. Both PROGgene and PROGmiR have recently been published and accessed by investigators from >10 countries. The signature center has also devoted considerable efforts in developing tumor tissue resource. Tissue Bank includes a total sample of N = 600 cases with 30% non-Caucasian cases. Currently 460 cases have been assembled into a Tissue Microarray with clinical and follow up data. Expression pattern of AP2γ, a potential marker of breast cancer progression, has been analyzed in a TMA with ~170 cases. The breast cancer signature center has funded four pilot projects and projects for the fourth round of funding are currently under review. Drs. Clark Wells received funding for the project " Histologic Analysis of the Protein Levels of Amot130, AmotL1 and YAP in Normal, Hyperplastic and Invasive Breast Cancer Tissues " , which resulted in a publication in PNAS. Dr. David Gilley and his group received funding for the project: " Luminal mammary progenitors are a unique site of telomere dysfunction " , which was published in Stem Cell Reports. In the third project, Dr. Theresa Guise is investigating the mechanisms of cancer-associated muscular dysfunction with a future plan for a clinical trial. Drs. Ken Nephew and Milan Radovich received funding to obtain preliminary results for a multi-PI R01 or P01, which will explore genomics and epigenomics of breast cancer using clinical trial materials. Progress made by the signature center was integral in our request to Vera Bradley Foundation for Breast Cancer. This foundation has recently committed $15 million for the breast cancer program, which will be used to develop three themes of research with a focus on personalized therapies to improve outcome in breast cancer patients.

Telomeres are essential for genomic integrity, but little is known about their regulation in the normal human mammary gland. We now demonstrate that a phenotypically defined cell population enriched in luminal progenitors (LPs) is characterized by unusually short telomeres independently of donor age. Furthermore, we find that multiple DNA damage response proteins colocalize with telomeres in >95% of LPs but in <5% of basal cells. Paradoxically, 25% of LPs are still capable of exhibiting robust clonogenic activity in vitro. This may be partially explained by the elevated telomerase activity that was also seen only in LPs. Interestingly, this potential telomere salvage mechanism declines with age. Our findings thus reveal marked differences in the telomere biology of different subsets of primitive normal human mammary cells. The chronically dysfunctional telomeres unique to LPs have potentially important implications for normal mammary tissue homeostasis as well as the development of certain breast cancers.

The Indiana Center for Breast Cancer Research (ICBCR) was funded under the IUPUI Signature Center Initiative in 2010. Its mission is to address the full range of prevention, early detection, and treatment of breast cancer through translational projects, supportive cores, and synergistic programs. This poster details our efforts to date towards applying for a National Cancer Institute Specialized Program of Research Excellence (SPORE) in January 2013. The proposed IU Breast Cancer SPORE will include 4-5 individual research projects, 3 cores, developmental research and career development programs. The SPORE Biostatistics and Bioinformatics core has developed the Breast Cancer Prognostics Database (BCDB), an online tool to study prognostic implications of genes of interest in publically available breast cancer databases. The BCDB can be used to study overall, recurrence free and metastasis free survival in large patient series. Supporting the SPORE Biospecimen/Pathology core, the IU Breast Cancer Tissue Bank includes a total sample of N = 500 cases with 30% non-Caucasian cases from Wishard Memorial Hospital. Currently there are N = 333 cases with tissue microarray data and complete clinical data with an additional 200 cases pending tissue confirmation. Dr. Clark D. Wells together with S. Badve and G. Sandusky are collaborating on the project: “Histologic Analysis of the Protein Levels of Amot130, AmotL1 and YAP in Normal, Hyperplastic and Invasive Breast Cancer Tissues”, a candidate SPORE individual research project. This project is investigating localized protein expression in paraffin-embedded tissues to associate expression levels with disease subtype and patient outcome. Dr. David P. Gilley together with N. Kannan, N. Huda, L. Tu, R. Droumeva, R. Brinkman, J. Emerman, S. Abe, and C. Eaves, are collaborating on the project: “Luminal mammary progenitors are a unique site of telomere dysfunction”, a candidate SPORE developmental research project. This project is investigating the relationship between telomere dysfunction and breast cancer tumorigenesis. These SPORE projects and cores were discussed at the IUSCC Breast Cancer Program retreat held on 1/13/12. Two additional planning meetings were held on 1/5 and 2/23. A timeline was generated to include final project selection in April, internal review in June, external review in August-September, and draft completion by 12/1, to meet the 1/20/13 NIH receipt deadline.

Several lines of evidence suggest that the telomere-associated protein TRF2 plays critical roles in the DNA damage response. TRF2 is rapidly and transiently phosphorylated by an ATM-dependent pathway in response to DNA damage and this DNA damage-induced phosphoryation is essential for the DNA-PK-dependent pathway of DNA double-strand break repair (DSB). However, the type of DNA damage that induces TRF2 localization to the damage sites, the requirement for DNA damage-induced phosphorylation of TRF2 for its recruitment, as well as the detailed kinetics of TRF2 accumulation at DNA damage sites have not been fully investigated. In order to address these questions, we used an ultrafast femtosecond multiphoton laser and a continuous wave 405-nm single photon laser to induce DNA damage at defined nuclear locations. Our results showed that DNA damage produced by a femtosecond multiphoton laser was sufficient for localization of TRF2 to these DNA damage sites. We also demonstrate that ectopically expressed TRF2 was recruited to DNA lesions created by a 405-nm laser. Our data suggest that ATM and DNA-PKcs kinases are not required for TRF2 localization to DNA damage sites. Furthermore, we found that phosphorylation of TRF2 at residue T188 was not essential for its recruitment to laser-induced DNA damage sites. Thus, we provide further evidence that a protein known to function in telomere maintenance, TRF2, is recruited to sites of DNA damage and plays critical roles in the DNA damage response.

Poly‐ADP ribose polymerase (PARP) inhibitors have shown promise in the treatment of human malignancies characterized by deficiencies in the DNA damage repair proteins BRCA1 and BRCA2 and preclinical studies have demonstrated the potential effectiveness of PARP inhibitors in targeting ataxia‐telangiectasia mutated (ATM)‐deficient tumours. Here, we show that mantle cell lymphoma (MCL) cells deficient in both ATM and p53 are more sensitive to the PARP inhibitor olaparib than cells lacking ATM function alone. In ATM‐deficient MCL cells, olaparib induced DNA‐PK‐dependent phosphorylation and stabilization of p53 as well as expression of p53‐responsive cell cycle checkpoint regulators, and inhibition of DNA‐PK reduced the toxicity of olaparib in ATM‐deficient MCL cells. Thus, both DNA‐PK and p53 regulate the response of ATM‐deficient MCL cells to olaparib. In addition, small molecule inhibition of both ATM and PARP was cytotoxic in normal human fibroblasts with disruption of p53, implying that the combination of ATM and PARP inhibitors may have utility in targeting p53‐deficient malignancies.

Several lines of evidence suggest that defects in telomere maintenance play a significant role in the initiation of genomic instability during carcinogenesis. Although the general concept of defective telomere maintenance initiating genomic instability has been acknowledged, there remains a critical gap in the direct evidence of telomere dysfunction in human solid tumors. To address this topic, we devised a multiplex PCR-based assay, termed TAR (telomere-associated repeat) fusion PCR, to detect and analyze chromosome end-to-end associations (telomere fusions) within human breast tumor tissue. Using TAR fusion PCR, we found that human breast lesions, but not normal breast tissues from healthy volunteers, contained telomere fusions. Telomere fusions were detected at similar frequencies during early ductal carcinoma in situ and in the later invasive ductal carcinoma stage. Our results provide direct evidence that telomere fusions are present in human breast tumor tissue and suggest that telomere dysfunction may be an important component of the genomic instability observed in this cancer. Development of this robust method that allows identification of these genetic aberrations (telomere fusions) is anticipated to be a valuable tool for dissecting mechanisms of telomere dysfunction.

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human chromosome 3, via microcell-mediated chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT. To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated chromosome 3 regions produced by a novel chromosomal engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT. We found a telomerase repressor region located within a 7-Mb interval on chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements.

We investigated the efficacy and mechanism of dimethylaminoparthenolide (DMAPT), an NF-κB inhibitor, to sensitize human lung cancer cells to X-ray killing in vitro and in vivo. We tested whether DMAPT increased the effectiveness of single and fractionated X-ray treatment through inhibition of NF-κB and/or DNA double-strand break (DSB) repair. Treatment with DMAPT decreased plating efficiency, inhibited constitutive and radiation-induced NF-κB binding activity, and enhanced radiation-induced cell killing by dose modification factors of 1.8 and 1.4 in vitro. X-ray fractionation demonstrated that DMAPT inhibited split-dose recovery/repair, and neutral DNA comet assays confirmed that DMAPT altered the fast and slow components of X-ray-induced DNA DSB repair. Knockdown of the NF-κB family member p65 by siRNA increased radiation sensitivity and completely inhibited split-dose recovery in a manner very similar to DMAPT treatment. The data suggest a link between inhibition of NF-κB and inhibition of DSB repair by DMAPT that leads to enhancement of X-ray-induced cell killing in vitro in non-small-cell lung cancer cells. Studies of A549 tumor xenografts in nude mice demonstrated that DMAPT enhanced X-ray-induced tumor growth delay in vivo.

PURPOSE:To test the hypothesis that the robust expansion of bovine corneal endothelial cells (BCECs) in vitro is due to the presence of individual endothelial cells with various levels of proliferative potential.METHODS:BCECs and bovine vascular endothelial cells (ECs) derived from aorta, coronary artery, and pulmonary artery were cultivated in optimized medium. These cell populations were confirmed by morphologic features, functional assays, and gene expression profiles. Moreover, ECs were plated in a single-cell clonogenic assay to evaluate colony-forming ability.RESULTS:Both corneal and vascular ECs were confirmed to be pure populations of endothelium uncontaminated with hematopoietic cells. A complete hierarchy of endothelial colony-forming cells (ECFCs) was identified in BCECs by a single-cell clonogenic assay. The distribution of the various types of ECFCs was similar to the control ECs removed from the systemic vessels.CONCLUSIONS:Cultured BCECs display clonal proliferative properties similar to those of vascular ECs.