Fbxw7 Deletion Accelerates KrasG12D-Driven Pancreatic Tumorigenesis via Yap Accumulation

AbstractPancreatic cancers driven by KRAS mutations require additional mutations for tumor progression. The tumor suppressor FBXW7 is altered in pancreatic cancers, but its contribution to pancreatic tumorigenesis is unknown. To determine potential cooperation between Kras mutation and Fbxw7 inactivation in pancreatic tumorigenesis, we generated P48-Cre;LSL-KrasG12D;Fbxw7fl/fl (KFCfl/fl) compound mice. We found that KFCfl/fl mice displayed accelerated tumorigenesis: all mice succumbed to pancreatic ductal adenocarcinoma (PDA) by 40 days of age, with PDA onset occurring by 2 weeks of age. PDA in KFCfl/fl mice was preceded by earlier onset of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions, and associated with chromosomal instability and the accumulation of Fbxw7 substrates Yes-associated protein (Yap), c-Myc, and Notch. Using KFCfl/fl and FBXW7-deficient human pancreatic cancer cells, we found that Yap silencing attenuated growth promotion by Fbxw7 deletion. Our data demonstrate that Fbxw7 is a potent suppressor of KrasG12D-induced pancreatic tumorigenesis due, at least in part, to negative regulation of Yap

Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

AbstractIn order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells

Sperm protein 17 is a novel marker for predicting cisplatin response in esophageal squamous cancer cell lines

Expression of sperm protein 17 (Sp17) mRNA has been reported in various malignancies. In an earlier study, we reported the upregulation of Sp17 transcripts in primary esophageal squamous cell carcinomas (ESCCs) using differential display and detected Sp17 transcripts in 86% of ESCCs by RT-PCR, whereas no transcripts were detected in the paired normal esophageal tissues. Herein we hypothesized that Sp17 might be used as a marker for detecting the response of anticancer therapies in ESCCs. Our results indicated that Sp17 protein levels in esophageal squamous cancer cell lines decreased in response to treatment with ( i ) the HSP90 activity inhibitor geldanamycin, ( ii ) the tyrosine kinase inhibitor erlotinib and ( iii ) cisplatin (chemotherapeutic agent commonly used in management of ESCC). In contrast, the Sp17 levels did not decrease in response to radiation therapy and treatment with the chemotherapeutic agent, gemcitabine. Further investigations showed that cisplatin induced decrease in Sp17 levels was due to transcriptional inhibition and cisplatin-resistant cell lines did not show this decrease in Sp17 levels in response to cisplatin treatment. In addition, we also carried our mass spectophotometric analysis to identify the binding partners of Sp17 to characterize its possible involvement in esophageal tumorigenesis and chemoresistance.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64923/1/24828_ftp.pd

CONCORDE: A phase I platform study of novel agents in combination with conventional radiotherapy in non-small-cell lung cancer

Lung cancer is the leading cause of cancer mortality worldwide and most patients are unsuitable for ‘gold standard’ treatment, which is concurrent chemoradiotherapy. CONCORDE is a platform study seeking to establish the toxicity profiles of multiple novel radiosensitisers targeting DNA repair proteins in patients treated with sequential chemoradiotherapy. Time-to-event continual reassessment will facilitate efficient dose-finding. Abbreviations ATM: Ataxia telangiectasia mutated ATR: Ataxia telangiectasia and Rad3 related cfDNA: Cell-free DNA CRT: Chemoradiotherapy CT: Computed tomography CTCAE: Common terminology criteria for adverse events CTRad: Clinical and Translational Radiotherapy Research Working Group DDRi: DNA damage response inhibitor DLT: Dose limiting toxicity DNA: Deoxyribonucleic acid DNA-PK: DNA-dependent protein kinase ECOG: Eastern Cooperative Oncology Group EORTC: European Organisation for Research and Treatment of Cancer ICRU: International Commission on Radiation Units and Measurements IMPs: Investigational medicinal products LA: Locally advanced MRC: Medical Research Council NCRI: National Cancer Research Institute NSCLC: Non-small cell lung cancer PARP: Poly (ADP-ribose) polymerase PET: Positron emission tomography PFS: Progression free survival PROMs: Patient-reported outcome measures RECIST: Response evaluation criteria in solid tumours RP2D: Recommended phase II dose RT: Radiotherapy SACT: Systemic anti-cancer therapy SRC: Safety review committee TiTE-CRM: Time to event continual reassessment method TNM: Tumour node metastasi

Checkpoint kinase inhibitor AZD7762 strongly sensitises urothelial carcinoma cells to gemcitabine

Background: More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer. Methods: Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by ?H2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs. Results: Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762. Conclusions: AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy

DNA repair: the culprit for tumor-initiating cell survival?

The existence of “tumor-initiating cells” (TICs) has been a topic of heated debate for the last few years within the field of cancer biology. Their continuous characterization in a variety of solid tumors has led to an abundance of evidence supporting their existence. TICs are believed to be responsible for resistance against conventional treatment regimes of chemotherapy and radiation, ultimately leading to metastasis and patient demise. This review summarizes DNA repair mechanism(s) and their role in the maintenance and regulation of stem cells. There is evidence supporting the hypothesis that TICs, similar to embryonic stem (ES) cells and hematopoietic stem cells (HSCs), display an increase in their ability to survive genotoxic stress and injury. Mechanistically, the ability of ES cells, HSCs and TICs to survive under stressful conditions can be attributed to an increase in the efficiency at which these cells undergo DNA repair. Furthermore, the data presented in this review summarize the results found by our lab and others demonstrating that TICs have an increase in their genomic stability, which can allow for TIC survival under conditions such as anticancer treatments, while the bulk population of tumor cells dies. We believe that these data will greatly impact the development and design of future therapies being engineered to target and eradicate this highly aggressive cancer cell population

Checkpoint Signaling, Base Excision Repair, and PARP Promote Survival of Colon Cancer Cells Treated with 5-Fluorodeoxyuridine but Not 5-Fluorouracil

The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin-induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor

Mechanistic studies on the induction of fluoropyrimidine resistance by E. colidUTPase expression, wild typep53 induction or thymidylate synthase overexpression in human cells.

One limitation of fluoropyrimidine chemotherapy is the sensitivity of normal host tissues to these drugs. The development of techniques required for the selective induction of genes in vivo has led to the consideration of gene therapy-directed host protection strategies designed to enhance chemotherapy by allowing the administration of previously intolerable doses of drug. This dissertation presents an evaluation of the relative abilities of three genes: E. coli dUTPase (dutE), wt-p53 and thymidylate synthase (TS) to confer resistance in TS inhibitors in vitro. Previous studies indicated dUTPase has a role in determining the sensitivity of a cell to TS inhibition. We tested the hypothesis that induction of dutE expression could protect human cells from TS inhibitors. Although dutE expression reduced drug-induced DNA damage 5 fold in HT29 cells, protection from cytotoxicity was only 2 fold, and dutE did not protect HuTu80 cells from the cytotoxic effects of 5-fluorodeoxyuridine (FdUrd). These findings indicate dutE would not be a good gene for induction of fluoropyrimidine resistance in vivo. We also compared the relative abilities of two forms of TS, human (hTS) and E. coli (eTS), to protect human cells from FdUrd. E. coli TS was expressed at slightly higher levels (2-4 fold) than hTS in human cells, and FdUrd sensitivity in eTS-expressing cells was correspondingly lower. Cells expressing eTS were much more resistant to FdUrd when leucovorin (LV) was also present. While LV potentiated FdUrd-induced cytotoxicity in hTS-expressing cells about 10 fold, it had no effect in eTS-expressing cells. Cells expressing eTS were also relatively more resistant to AG337-induced cytotoxicity. These results suggest that eTS would be the gene of choice for a liver-directed host protection with either of these drugs. Finally, we examined the ability of TS-overexpressing cells to protect cells with normal TS content from the cytotoxic effects of TS inhibitors. Cells expressing eTS were twice as effective as hTS-overexpressing cells at protecting neighboring lacZ cells from FdUrd and AG337-induced cytotoxicity, further suggesting that eTS may be more effective than hTS at conferring fluoropyrimidine resistance in vivo.Ph.D.Biological SciencesHealth and Environmental SciencesMolecular biologyPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131068/2/9825318.pd