bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1731: Decreased cyclin B1 expression contributes to mitochondrial apoptosis in medulloblastoma.
Authors: Dasari Venkata Ramesh , Asuthkar Swapna , Nalla Arun Kumar , Rao Jasti S.
Published: August 2014

Medulloblastoma, the most common malignant pediatric brain tumor, remains difficult to cure. Previously, we have shown that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest in medulloblastoma cells. In this study, we observed that downregulation of MMP-9 in nude mice D283 xenografts downregulated the expression of cyclin B1. Increasing evidence indicates that the deregulation of cyclin B1 is involved in neoplastic transformation, suggesting that inhibition of cyclin B1 could be an attractive strategy for anti-tumor therapy. To determine the effect of downregulation of cyclin B1, we treated medulloblastoma cells with nocodazole, which is an anti-neoplastic agent. Nocodazole-treated cells have undergone G2/M cell-cycle arrest because of the mitotic spindle arrest. Prolonged arrest of cells in mitosis due to nocodazole treatment typically results in cell death by apoptosis. Hence, we confirmed the induction of apoptosis in medulloblastoma cells by TUNEL analysis. Immunoblot analysis of cell cycle checkpoint proteins Chk1, Chk2 and cyclin B1 revealed that they were downregulated in combination treatments of shMMP-9 with radiation. Further, apoptotic proteins BAD, BAX and BAK were highly upregulated, which shows the involvement of mitochondria-mediated apoptosis in our treatments. In vivo analysis of BAD molecules in D283 nude mice xenografts confirmed that downregulation of cyclin B1 induces BAD-mediated apoptotic death in medulloblastoma tumors treated with either shMMP-9 alone or in combination with radiation. With this study, we demonstrate that downregulation of cyclin B1 is an effective and specific approach to induce apoptosis for effective anti-tumor therapy against medulloblastoma.

Citation Format: Venkata Ramesh Dasari, Swapna Asuthkar, Arun Kumar Nalla, Jasti S. Rao. Decreased cyclin B1 expression contributes to mitochondrial apoptosis in medulloblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1731. doi:10.1158/1538-7445.AM2013-1731
Abstract 1732: PI3K inhibition potentiates ABT-737 induced apoptosis in colorectal cancer cells independent of MCL1 downregulation or AKT/mTOR inhibition.
Authors: Potter Danielle S. , Kelly Paul J. , Denneny Olive A. , Dive Caroline , Morrow Christopher J.
Published: August 2014

Background

Colorectal cancer (CRC) is the third most common cancer and is a leading cause of morbidity worldwide. The five year survival rate of patients with advanced or metastatic CRC is less than 5%, identifying the need for new and improved therapies in the treatment of advanced and metastatic CRC. Phosphoinositide 3-kinase (PI3K) signaling has been implicated in several hallmarks of cancer, such as sustained proliferation, resisting cell death and invasion, and is frequently deregulated in CRC via a mutation of effectors such as PIK3CA, PIK3R1 and PTEN, making it an attractive therapeutic drug target. Despite this, inhibition of PI3K signaling does not cause cell death in CRC cells. Furthermore, CRC cells are relatively resistant to the BH3 mimetic ABT-737, which directly targets the BCL2 family of proteins inducing apoptosis via the mitochondrial pathway. Therefore, we investigated whether combining PI3K inhibitors and ABT-737 in CRC cells might synergise to induce apoptosis.

Results

PI3K inhibition enhanced ABT-737 induced apoptosis by 2.3-4.5 fold and reduced expression levels of Mcl-1, an established resistance biomarker for ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis a further 1.4-2.4 fold in CRC cells with siRNA depleted Mcl-1 suggesting contributory mechanisms additional to Mcl-1 down regulation. PI3K inhibition increased levels of Bcl-xL bound BimEL independently of Mcl-1 depletion. Further investigations are being carried out to determine whether this is the cause of increase ABT-737 sensitivity. ABT-737-induced apoptosis was unaffected by inhibition of PI3K downstream effectors AKT and mTOR. A siRNA library screen has been conducted to identify proteins downstream of PI3K which affect ABT-737 sensitivity and the results will be reported.

Conclusions

These data reveal a PI3K inhibition enhanced, ABT-737 driven apoptosis that occurs via Mcl-1, AKT and mTOR independent mechanism(s) consistent with increased association of BimEL and Bcl-xL. Further work is ongoing to elucidate the molecular mechanisms that underpin enhanced response to this BH-3 mimetic in CRC.

Citation Format: Danielle S. Potter, Paul J. Kelly, Olive A. Denneny, Caroline Dive, Christopher J. Morrow. PI3K inhibition potentiates ABT-737 induced apoptosis in colorectal cancer cells independent of MCL1 downregulation or AKT/mTOR inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1732. doi:10.1158/1538-7445.AM2013-1732
Abstract 1739: Unexpected complexity in Mcl-1 phosphorylation in response to microtubule inhibitors.
Authors: Chu Rong , Chambers Timothy C.
Published: August 2014

Microtubule inhibitors promote phosphorylation and degradation of Mcl-1, and the loss of expression of this key anti-apoptotic Bcl-2 family member appears to be critical for apoptosis induction. While several sites and kinases have been implicated, a phosphorylation/degradation-resistant mutant has not been definitively described. Contrary to previous reports suggesting that Thr92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a Thr92Ala Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type following vinblastine-treatment. Similarly, when Mcl-1 with alanine replacements of five putative major sites (S64, T70A, T92, S121, T163) was expressed, it was phosphorylated and degraded like the wild-type. These results suggested redundancy or the existence of additional regulatory sites. To analyze Mcl-1 phosphorylation in more detail, cell extracts, made using a novel extraction solution to improve resolution, were subjected to 2D-PAGE/immunoblotting. Extracts from asynchronous control cells showed one major and two minor, more acidic, spots, the latter sensitive to acid phosphatase treatment. Extracts from vinblastine-treated cells showed up to 9 distinct spots all but two of which were eliminated after acid phosphatase treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to microtubule inhibitors and the existence of many more sites than previously suspected. Identification of the additional sites of phosphorylation, the kinase(s) responsible, and their role in regulation of Mcl-1 degradation are ongoing.

Supported by NIH CA109821.

Citation Format: Rong Chu, Timothy C. Chambers. Unexpected complexity in Mcl-1 phosphorylation in response to microtubule inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2013-1739
Abstract 1741: Mitotic catastrophe and inhibition of growth of Castration Resistant Prostate Cancers by a dietary agent Withaferin-A.
Authors: Vadodkar Aditi Shirish , Suman Suman , Roy Ram Vinod , Damodaran Chendil
Published: August 2014

Cell cycle deregulation has been strongly associated with the pathogenesis of prostate cancer. It has been well documented that an increased expression of cell cycle regulatory proteins (cyclin D1-25%, cyclin A-35% and cyclin B-75%) have been reported in castration resistant prostate cancer (CRPC) cells, when compared to normal prostate tissue. There are several cell cycle regulators undergoing clinical trials targeting either G0/G1 or G2/M phase of cell cycle to inhibit the growth of many cancers including prostate cancer. We previously published that Withaferin-A (WA); a natural compound effectively suppresses CRPC growth both in vitro and in vivo. Hence, in this study we intend to determine the cell-cycle regulatory potential of this agent on CRPC cells. WA caused irreversible G2/M arrest in both CRPC cell lines (PC-3 and C4-2B) till 72 hours . Phosphorylation of cdc2 by Wee1 and Myt1 is an important step to maintain an inactive state of cdc2 that lead to G2/M cell cycle arrest. Additionally decreased levels of phosphorylated Wee-1 and Myt1 followed by increase in the phosphorylation status of cdc2 (Tyr15 and Thr14) supported the G2/M phase of cell cycle arrest in both CRPC cell lines. Further checkpoint kinase assay showed increased activity in WA treated CRPC cells suggesting activation of CDC25C that activates cyclinB/cdc2 complex .We also observed down regulation of several cyclin family proteins in both CRPC cells. Altogether, these results conclude d that WA inhibits CRPC cell growth by induction of G2/M cell cycle arrest, highlighting the potential of WA could be a therapeutic agent for CRPC. Currently, we are investigating the effect of WA on cell cycle markers using both xenograft and TRAMP models. The outcome of our study may enable bringing WA to the mainstream of medicine for prevention and/or therapy for CRPC.

Citation Format: Aditi Shirish Vadodkar, Suman Suman, Ram Vinod Roy, Chendil Damodaran. Mitotic catastrophe and inhibition of growth of Castration Resistant Prostate Cancers by a dietary agent Withaferin-A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1741. doi:10.1158/1538-7445.AM2013-1741
Abstract 1742: Potential roles of histone deacetylase 11 in cell cycle regulation and cancer.
Authors: Srikumar Thejal , Telles Elphine , Seto Edward
Published: August 2014

Histone deacetylases (HDACs) are enzymes that deacetylate lysine residues on histone and non-histone proteins. They regulate various cellular functions including transcriptional regulation, cell proliferation, apoptosis, and cellular metabolism. Broad spectrum HDAC inhibitors, including Vorinostat and Romidepsin, have been approved for cancer therapy, but their use is limited due to toxicity and side effects. A deeper understanding of the function of individual histone deacetylases could lead to the development of more specific and potentially more effective anti-cancer agents. In this context, Histone Deacetylase 11 (HDAC11) could be a potential target for anticancer therapeutics. HDAC11 is the most recently discovered Histone Deacetylase and, therefore, the least characterized among the 18 HDACs. Its expression is altered in various cancers and recent studies have suggested that HDAC11 might inhibit the activity and function of the DNA replication licensing factor Cdt1. To examine whether HDAC11 controls Cdt1-mediated cell cycle regulation, HDAC11 was overexpressed in human U87 glioblastoma cells. We found that overexpression of HDAC11 represses Cdt1 expression levels. Most importantly, we observed that HDAC11 overexpression promotes earlier cell entry into the G2/M phase of the cell cycle than non-HDAC11-overexpressed cells. Finally, we discovered that HDAC11 levels change throughout the cell cycle in a manner that is contrary to the Cdt1 expression levels. These results indicate that HDAC11 negatively regulates Cdt1, and uncover an important and previously unknown role of this enzyme in cell cycle regulation. Together, our findings suggest that development of drugs that target HDAC11 might be a viable strategy for overcoming cancers where this protein is overexpressed.

Citation Format: Thejal Srikumar, Elphine Telles, Edward Seto. Potential roles of histone deacetylase 11 in cell cycle regulation and cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1742. doi:10.1158/1538-7445.AM2013-1742
Abstract 1748: Cyclin D1 harboring T286I mutation promotes oncogenic activation in endometrial cancer.
Authors: Ikeda Yuji , Oda Katsutoshi , Koso Takahiro , Miyasaka Aki , Kashiyama Tomoko , Hiraike-Wada Osamu , Maeda Daichi , Kawana Kei , Nakagawa Shunsuke , Tetsu Osamu , Kikuchi Yoshihiro , Fujii Tomoyuki , Yano Tetsu , Kozuma Shiro
Published: August 2014

Introduction. Cyclin D1 is an important regulator of cell cycle progression. Phosphorylation of cyclin D1 at Thr-286 by GSK3beta triggers nuclear export and its cytoplasmic proteolysis via the 26S proteasome. Cyclin D1 overexpression is a common event in various types of human cancers; however,mutations of CCND1 (encoding cyclin D1) have been only reported in endometrial cancer and esophageal cancer. We previously reported T286I mutations of CCND1 in 2 endometrial carcinomas (2.3%). The objective of this study was to identify the functions of T286I mutant of cyclin D1 in endometrial cancer. Materials and methods. T286I mutant cyclin D1 (CD1-T286I) plasmid was generated using wild-type cyclin D1 (CD1-WT) plasmid via site-directed mutagenesis technique. pcDNA3 empty plasmid was used as a control (CD1-Ct). Using these plasmids, the functions of mutant CD1-T286I were analyzed by luciferase assays, immunofluorescence, western blotting and colony formation assays in HEK293T (kidney) and HEC-50B (endometrial cancer) cells, both of which do not possess CCND1 mutations. Statistical analysis was performed by student-T test and p<0.05 was considered statistically significant. Result. Immunofluorescence and western blotting showed predominant accumulation of exogenous CD1-T286I in nucleus, whereas expression of exogenous CD1-WT and endogenous cyclin D1 were observed diffusely in cytoplasm and nucleus. Luciferase assay revealed that pRB1 expression was partially decreased by introduction of CD1-WT and that the pRB1 expression was further decreased by introduction of CD1-T286I (p=0.002). In colony formation assays, introduction of CD1-T286I significantly increased the colony number, compared with CD1-WT (p=0.007) and CD1-Ct. Conclusion. Stabilization and accumulation of mutant cyclin D1 (T286I) in the nucleus might overcome regulation by pRb and induce cell proliferation in certain endometrial cancers. Phosphorylation-dependent nuclear export of cyclin D1 at Thr286 might be critical for prevention of aberrant cell proliferation in human cells.

Citation Format: Yuji Ikeda, Katsutoshi Oda, Takahiro Koso, Aki Miyasaka, Tomoko Kashiyama, Osamu Hiraike-Wada, Daichi Maeda, Kei Kawana, Shunsuke Nakagawa, Osamu Tetsu, Yoshihiro Kikuchi, Tomoyuki Fujii, Tetsu Yano, Shiro Kozuma. Cyclin D1 harboring T286I mutation promotes oncogenic activation in endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2013-1748
Abstract 1751: The E2F activators signal centrosome amplification in breast cancer cells through centrosome regulatory molecules.
Authors: Lee Mi-Young , Saavedra Harold Ivan
Published: August 2014

Based on the elevated frequencies of centrosome amplification in premalignant breast lesions and in breast tumors, centrosome amplification is proposed to play a role in breast tumorigenesis. The coordination between cell and centrosome cycles is of paramount importance to maintain genomic stability and to the prevention of mammary tumorigenesis. Thus, both cell cycle regulators and molecules that regulate centrosome duplication work together to prevent abnormal centrosome duplication. E2Fs are transcription factors that regulate various cellular processes, such as DNA replication, DNA repair, cell proliferation and apoptosis; their role on centrosomal and mitotic regulation is understood poorly. We hypothesize that the E2F activators (E2F1, E2F2, E2F3a) play key roles in regulating centrosome amplification in HER2 positive breast cancer cells. We found that the E2Fs activators are deregulated or overexpressed in HER2 positive breast cancer cells displaying centrosome amplification, since serum withdrawal does not downregulate the E2F activators (which occurs in control MCF10A cells upon serum-withdrawal). Upregulated E2Fs correlated with the transcriptional overexpression of cell cycle and centrosome regulatory targets, including cylin D, cyclin E, Nek2 and Plk4. Knockdown of E2Fs in HER2 positive cells decreased centrosome amplification without affecting the cell cycle, or entry into S phase. The chromatin immunoprecipitation (ChIP) assay showed binding of E2F1, E2F2 and E2F3 into the promoter of Nek2 and between exons 1 and 2 of Plk4. In two out of three of the Her2+ breast cancer cell lines displaying centrosome amplification, extinguishing E2F expression leads to downregulation of Nek2 and cyclin D1 -potent inducers of centrosome amplification-. In conclusion, centrosome amplification in Her2+ breast cancer cells is dependent on the E2Fs activators and is mediated by molecules that play critical roles in centrosome duplication. We have identified a novel mechanism where E2F-generated centrosome amplification may contribute to breast carcinogenesis.

Citation Format: Mi-Young Lee, Harold Ivan Saavedra. The E2F activators signal centrosome amplification in breast cancer cells through centrosome regulatory molecules. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1751. doi:10.1158/1538-7445.AM2013-1751
Abstract 1753: KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro.
Authors: Rauhala Hanna E. , Laurila Eeva , Vuorinen Elisa , Savinainen Kimmo , Kallioniemi Anne
Published: August 2014

Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for the early detection of the disease and a shortage of effective therapies. We have previously shown that karyopherin alpha 7 (KPNA7) is frequently amplified and overexpressed in pancreatic cancer. KPNA7 is a recently characterized member of the alpha karyopherin family of nuclear import receptors. The nuclear import machinery is composed of a complex network of proteins required for the appropriate transport of proteins from the cytoplasm to the nucleus. Abnormal function of this machinery leads to incorrect localization of nuclear proteins and disturbances in cellular homeostasis, which may subsequently contribute to the development of various diseases including cancer. Indeed, previous evidence has linked members of the karyopherin alpha family, especially KPNA2, to cancer pathogenesis.

In this study we demonstrate that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines as well as in some other cancer types. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a striking reduction in cell growth due to the induction of the cell cycle regulator p21 and subsequent G1 arrest of the cell cycle. In both of these cell lines KPNA7 silencing also resulted in decreased anchorage independent growth, and additionally in the AsPC-1 cells reduced cell migration and invasion. Furthermore, in the Hs700T cells KPNA7 silencing resulted in dramatic morphological change where the cells acquired fibroblast-like shape. This phenotypic change was not explained by induction of EMT or senescence.

In conclusion, our data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role of KPNA7 in human cancer. Moreover, the lack of KPNA7 expression in normal adult tissues highlights it as a potential novel therapeutic target for cancer. Further studies are needed to identify the cargo proteins transported by KPNA7. These studies are likely to reveal important new information on nuclear transport and may highlight the molecular mechanisms involved in KPNA7 mediated phenotypes in cancer cells.

Citation Format: Hanna E. Rauhala, Eeva Laurila, Elisa Vuorinen, Kimmo Savinainen, Anne Kallioniemi. KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1753. doi:10.1158/1538-7445.AM2013-1753
Abstract 1755: Invasive cancer cells are mostly in the G0/G1 phase of the cell cycle.
Authors: Yano Shuya , Mii Sumiyuki , Tome Yasunori , Hiroshima Yukihiko , Uehara Fuminari , Miwa Shinji , Fujiwara Toshiyoshi , Hoffman Robert M.
Published: August 2014

Invasive cancer cells are a critical target for chemotherapy. However, the cell cycle phase of invasive cancer cells is poorly understood. Cancer cells were cultured in 3D Gelfoam® culture where they have in vivo-like cell cycle kinetics. Cancer cell-cycle kinetics were visualized using a fluorescence ubiquitination cell cycle indicator (FUCCI), whereby quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). Real-time imaging demonstrated that cancer cells in the G0/G1 phase are invasive and disseminate in Gelfoam histoculture. Moreover, in vivo, FUCCI-expressing cancer cells were in G0/G1 phase when they metastasized to the liver. Disseminated cancer cells in the peritoneal cavity were also in G0/G1 phase. Most drugs currently in use target cells in S/G2/M are therefore may not be able to target invasive cancer cells. Color-coded cell-cycle imaging of cancer cell invasion should provide a novel platform for the rapid screening of candidate therapeutic agents against quiescent, invasive cancer cells.

Citation Format: Shuya Yano, Sumiyuki Mii, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Toshiyoshi Fujiwara, Robert M. Hoffman. Invasive cancer cells are mostly in the G0/G1 phase of the cell cycle. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1755. doi:10.1158/1538-7445.AM2013-1755
Abstract 1758: Cancer cell location in tumor determines cell cycle position and dormancy.
Authors: Yano Shuya , Tome Yasunori , Digman Michelle , Momiyama Masashi , Suetsugu Atsushi , Gratton Enrico , Hoffman Robert M.
Published: August 2014

The phase of the cell cycle can determine whether a cancer cell can respond to most drugs. The fluorescence ubiquitination cell cycle indicator (FUCCI) was used to image the cell cycle phase within tumors. In nascent tumors in nude mice, approximately 30% of the cells in the center of the tumor were in G0/G1 and 70% in S/G2/M. In contrast, approximately 90% of cancer cells in the center of an established tumor were in G0/G1 phase. Similarly, approximately 75% of cancer cells far from (>100 μm) tumor vessels were in G0/G1. Although 60-70% of cancer cells near the surface of an established tumor (< 100 μm) or near blood vessels were in S/G2/M, these cells are a small minority of an established tumor. Moreover, tumors that are stable in size for more than six months in nude mice consisted of mostly G0/G1 cells, suggesting they are dormant. The results presented here have profound implications for cancer therapy, since most drugs currently in use target cells in S/G2/M, which are a minor fraction in an established tumor. With the developments reported here, drugs that target non-dividing and dormant cancer cells can be readily identified.

Citation Format: Shuya Yano, Yasunori Tome, Michelle Digman, Masashi Momiyama, Atsushi Suetsugu, Enrico Gratton, Robert M. Hoffman. Cancer cell location in tumor determines cell cycle position and dormancy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1758. doi:10.1158/1538-7445.AM2013-1758
Abstract 1765: Recurrent fusion of NTRK1 in glioblastoma multiforme.
Authors: Kim Jinkuk , Cho Hee-Jin , Cho Gye-Hyun , Ko Young-Hyeh , Paik Soonmyung , Nam Do-Hyun
Published: August 2014

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on many types of solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBM, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA). From the analysis, we identified recurrent fusion of neurotrophic tyrosine kinase receptor type 1 (NTRK1, also called TrkA) to the genes highly expressed in neuronal tissues, neurofascin (NFASC) and brevican (BCAN). NTRK1 is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lack NTRK1 expression, the fusion-positive GBMs express fusion transcripts in high abundance that retain NTRK1 kinase domain in frame. Analyses of the matching Exome-Seq data indicated that NFASC-NTRK1 fusion occurred through DNA-level rearrangement, whereas BCAN-NTRK1 fusion presumably occurred at RNA-level. As analogous NTRK1-fusions with thyroid-expressed genes were previously shown to drive thyroid cancer, NTRK1-fusion in GBM might have a similar role. Consistent with this hypothesis, the GBMs that harbor the NTRK1-fusion showed elevated NGF/NTRK1 pathway activity. About 1% of GBM patients are estimated to have NTRK1-fusion; these patients might benefit from potential NTRK1 inhibition therapies.

Citation Format: Jinkuk Kim, Hee-Jin Cho, Gye-Hyun Cho, Young-Hyeh Ko, Soonmyung Paik, Do-Hyun Nam. Recurrent fusion of NTRK1 in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1765. doi:10.1158/1538-7445.AM2013-1765

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1766: Investigating Chk1 biology with selective oral Chk1 inhibitor.
Authors: Wang Zhenxiong
Published: August 2014

Chk1 inhibitor as a chemo-potentiator in cancer therapy was proposed more than 10 years ago. Most of the first generation i.v. dosing chk1 inhibitors were terminated in clinical trials recently. The second generation oral chk1 inhibitors were just launched by several companies. However, many questions in Chk1 biology remain unclear, which is important to guide Pfizer to develop oral chk1 inhibitor in the future.

Chk1 biology team investigated preclinical scheduling, biomarker, combination strategy, TGI, primary indication and patient selection with chk1 tool compounds. Biomarker is critical in evaluating POM in clinical trial phase I. Significant target modulations and dose responses of biomarkers p-Chk1 345, r-H2AX, cleaved-PARP were observed in both in vitro and in vivo studies, suggesting their potential application in clinical setting. The scheduling study indicated its importance in chk1 project. Both administration of chk1 inhibitor soon after chemotherapy and sustained inhibition of chk1 robustly improved efficacy in Miapaca2 pancreatic cancer model. There was no clear patient selection strategy for Chk1 clinical trials in the past. To investigate the primary indication and patient selection, 6 indications were indentified according to clinical trial results and human tumor tissue genomics, more than 100 cancer cell lines were screened with either chk1 inhibitor alone or in combination with SOC. 75%(12/16) cell lines in mesothelioma and 44% (4/9) in endometrial carcinoma demonstrated significant synergy in the combination treatment. Next generation sequencing and SiRNA library screening in DNA damage repair genes (DDR) will be used to clarify the mechanisms.

In summary, we have identified treatment schedule and potential new indications for Chk1 inhibitor, which could significantly increase efficacy, lower toxicity, and improve probability of success in clinical trials.

Citation Format: Zhenxiong Wang. Investigating Chk1 biology with selective oral Chk1 inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1766. doi:10.1158/1538-7445.AM2013-1766
Abstract 1768: The oncogene Mdmx promotes genomic instability independent of p53.
Authors: Melo Alexia N. , Bouska Alyssa , Arrate Maria P. , Eischen Christine M.
Published: August 2014

Mdmx is upregulated in many human cancers. As a member of the Mdm2 protein family, Mdmx functions similar to Mdm2 as a negative regulator of the tumor suppressor p53. With the ability to form heterodimers with Mdm2, p53 regulation by Mdmx predictably relies on Mdm2. However, while the p53- and Mdm2-dependent functions of Mdmx understandably contribute to cancer, Mdmx has also displayed p53- and Mdm2-independent functions in tumor development that are less well characterized. Therefore, we investigated novel avenues in which Mdmx contributes to tumorigenesis. We demonstrate increased expression of Mdmx results in an inhibition in double-strand DNA break repair and a delay in DNA damage signaling that was independent of p53 and surprisingly, independent of Mdm2. We identified a novel association between Mdmx and a key mediator of DNA repair, the Mre11-Rad50-Nbs1 complex. We also detected increased genomic instability and promotion of in vitro transformation as a result of Mdmx overexpression that was also independent of p53 and Mdm2. Our data show that Mdmx interacts with the Mre11-Rad50-Nbs1 complex and delays DNA break repair, which leads to genome instability and cellular transformation. This novel function of Mdmx provides insight into an alternative mechanism by which Mdmx contributes to tumor formation and possibly tumor progression independent of its interaction with Mdm2 or its negative regulation of p53. These data also highlight the potential for novel therapeutic intervention strategies that capitalize on this newly identified function of Mdmx.

Citation Format: Alexia N. Melo, Alyssa Bouska, Maria P. Arrate, Christine M. Eischen. The oncogene Mdmx promotes genomic instability independent of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1768. doi:10.1158/1538-7445.AM2013-1768
Abstract 1771: Extrachromosomal microDNAs in normal vertebrate tissues.
Authors: Dillon Laura , Kumar Pankaj , Shibata Yoshiyuki , Dutta Anindya
Published: August 2014

MicroDNAs are small (<400 base pairs in length) single- or double-stranded extrachromosomal circular DNAs that are derived by excision from tens of thousands of sites within the mammalian genome (Science, 2012, 336:86). Thus far, microDNAs have been detected in several mouse tissues, mouse and human cell lines, and both normal and malignant cells. Here, we examine the microDNAs across eight tissue types (brain, heart, lung, liver, thymus, spleen, kidney, and skeletal muscle) from normal adult mice and in chicken cell lines. We observed microDNAs in all tissue types, originating from thousands of unique genomic loci. As was previously described, these microDNAs are small circles (100 to 400 base pairs), possess a high GC content and are enriched in genic regions. The quantity and loci-of-origin of the microDNAs are compared between tissue types to ascertain tissue-specific differences that may be correlated with amount of cell proliferation and expression of specific genes. Previously, we suggested that the release of microDNAs leaves behind microdeletions within the source genomic loci, resulting in somatically mosaic cells. Our results confirm this possibility by demonstrating that microDNAs are universally present across cell and tissue types in vertebrates. In addition, short (2-15 bp) direct repeats that usually flank the genomic loci yielding microDNAs lead us to hypothesize that homologous-recombination or microhomology-mediated-repair may be involved in microDNA generation. A survey of microDNAs in cell lines defective in many of these repair pathways is being carried out to verify this hypothesis.

Citation Format: Laura Dillon, Pankaj Kumar, Yoshiyuki Shibata, Anindya Dutta. Extrachromosomal microDNAs in normal vertebrate tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1771. doi:10.1158/1538-7445.AM2013-1771
Abstract 1772: p53 promotes repair of heterochromatin DNA by regulating JMJD2b and SUV39H1 expression.
Authors: Zheng Hong , Chen Jiandong
Published: August 2014

Constitutive heterochromatin is important for maintaining chromosome stability but also delays the repair of DNA double strand breaks (DSB). DSB repair in complex mammalian genomes involves a fast phase (2-6 hrs) where most of the breaks are rapidly repaired, and a slow phase (up to 24 hrs) where the remaining damages in heterochromatin are repaired. We found that p53 deficiency delays the slow phase DNA repair after ionizing irradiation. P53 deficiency prevents down regulation of histone H3K9 trimethylation at pericentric heterochromatin after DNA damage. Moreover, p53 directly induces expression of the H3 K9 demethylase JMJD2b through promoter binding. P53 activation also indirectly down regulates expression of the H3 K9 methytransferase SUV39H1. Depletion of JMJD2b or sustained expression of SUV39H1 delays the repair of heterochromatin DNA and reduces clonogenic survival after ionizing irradiation. The results suggest that by regulating JMJD2b and SUV39H1 expression, p53 not only controls transcription but also promotes heterochromatin relaxation to accelerate a rate-limiting step in the repair of complex genomes.

Citation Format: Hong Zheng, Jiandong Chen. p53 promotes repair of heterochromatin DNA by regulating JMJD2b and SUV39H1 expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1772. doi:10.1158/1538-7445.AM2013-1772
Abstract 1773: Nucleotide-resolution genomic breakpoint analysis of TMPRSS2-ERG rearrangements in prostate cancer by target-capture next-generation sequencing.
Authors: Weier Christopher , Haffner Michael , Mosbruger Timothy , Esopi David , Hicks Jessica , Zheng Qizhi , Isaacs William B. , De Marzo Angelo M. , Nelson William G. , Yegnasubramanian Srinivasan
Published: August 2014

TMPRSS2-ERG translocations occur in approximately 50% of prostate cancers and therefore represent one of the most frequently observed structural rearrangements in all cancers. However, little is known about the genomic architecture of such rearrangements. We therefore designed and optimized a pipeline involving target-capture of TMPRSS2 and ERG genomic sequences coupled with paired-end next generation sequencing to resolve genomic rearrangement breakpoints in TMPRSS2 and ERG at nucleotide resolution in a large series of primary prostate cancer specimens (n = 83). This strategy showed >90% sensitivity and specificity in identifying TMPRSS2-ERG rearrangements, and allowed identification of intra- and inter-chromosomal rearrangements involving TMPRSS2 and ERG with known and novel fusion partners. Our results indicate that rearrangement breakpoints show strong clustering in specific intronic regions of TMPRSS2 and ERG. The observed TMPRSS2-ERG rearrangements often exhibited complex chromosomal architecture associated with several intra- and inter-chromosomal rearrangements. Nucleotide resolution analysis of breakpoint junctions revealed that the majority of TMPRSS2 and ERG rearrangements (∼88%) occurred at or near regions of microhomology or involved insertions of one or more base pairs. This architecture implicates nonhomologous end joining (NHEJ) and microhomology mediated end joining (MMEJ) pathways in the generation of such rearrangements. These analyses have provided important insights into the molecular mechanisms involved in generating prostate cancer-specific recurrent rearrangements.

Citation Format: Christopher Weier, Michael Haffner, Timothy Mosbruger, David Esopi, Jessica Hicks, Qizhi Zheng, William B. Isaacs, Angelo M. De Marzo, William G. Nelson, Srinivasan Yegnasubramanian. Nucleotide-resolution genomic breakpoint analysis of TMPRSS2-ERG rearrangements in prostate cancer by target-capture next-generation sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1773. doi:10.1158/1538-7445.AM2013-1773
Abstract 1774: Emerging role of Smad7 in DNA damage response pathway as a positive regulator.
Authors: Park Sujin , Kang Jin Muk , Kim Staci Jakyong , Kim Hyojung , Kim Seong-Jin
Published: August 2014

The cellular response to DNA damage is essential in maintaining genomic stability. As DNA damage accumulates, the occurrence rate of cancer is significantly enhanced. Smad7 is known as an inhibitory Smad which negatively regulates TGF-β-induced signaling pathway. Smad7 is predominantly localized in the nucleus even without TGF-β stimulation. However, TGF-β-independent Smad7 function in the nucleus is still not clear. Here, we report Smad7 regulates DNA damage signaling in response to double-strand break induced by radiomimetic drug, neocarzinostatin (NCS). Smad7 overexpression accelerates DNA repair by enhancing phosphorylation of ATM and γH2AX, major DNA damage response signaling, after DNA damage. In Smad7−/− MEF cell, loss of Smad7 leads to failure of ATM phosphorylation and nuclear foci formation at the site of DNA damage upon NCS treatment. We show that Smad7 interacts with activated ATM/MRN complex and this interaction is significantly increased after DNA damage. Additionally, Smad7 co-localizes with pATM, MRN complex and γH2AX at the sites of DNA damage. Taken together, our data suggests that Smad7 plays a critical role in DNA damage response pathway through interaction with activated ATM. In this study, we reveal the de novo function of Smad7 in the nucleus.

Citation Format: Sujin Park, Jin Muk Kang, Staci Jakyong Kim, Hyojung Kim, Seong-Jin Kim. Emerging role of Smad7 in DNA damage response pathway as a positive regulator. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1774. doi:10.1158/1538-7445.AM2013-1774
Abstract 1777: Genome-wide identification of replication-associated breakpoints.
Authors: Tsai Yu-Chen , Shastri Nishita , Smith Kevin D. , Schug Jonathan , Casellas Rafael , Brown Eric J.
Published: August 2014

Previous studies have defined sequences that are difficult to replicate and, consequently, are more vulnerable to replication-associated breaks and recombination events. However, many of these sequences have been identified through indirect and biased approaches. To identify genomic sequences that contribute to replication-associated breakpoints, we have performed genome-wide screens to determine the location, sequence, and frequency of replication perturbations within the mammalian genome. To convert these transient pause sites in S phase into longer-lived intermediates, asynchronous ATR-inhibited cells were treated with a low dose of polymerase inhibitor (0.2 μM aphidicolin) to promote selective replication fork collapse at difficult-to-replicate sequences. Affected regions were then isolated by chromatin immunoprecipitation of RPA (RPA-ChIP) and by a recently developed novel DNA breakpoint isolation method. Following deep sequencing, these sites were mapped within 1-5 kb regions and sequences at the center of these peaks were binned to quantify the overall frequency of replication fork collapse at such sites. Surprisingly, sequences that have long been thought to be among the most difficult to replicate, such as CGG repeats, were overshadowed by distinct repetitive sequences that are relatively uncharacterized. These sites were greater than 50-fold enriched over background, and were more common as replication-troubled regions than other well characterized sequences (e.g. triplet repeats, fragile sites, etc). In total, over 100 independently confirmed sites were identified, with most sequences observed in multiple locations across the genome. Notably, common fragile sites, which have been speculated to harbor such replication slow zones, were not observed to be particularly enriched in RPA-ChIP samples, consistent with recent observations that these sites are fragile for reasons other than impeded DNA replication rates. These findings and others indicating a larger role of chromatin structure in common fragile site breakage will be discussed.

Citation Format: Yu-Chen Tsai, Nishita Shastri, Kevin D. Smith, Jonathan Schug, Rafael Casellas, Eric J. Brown. Genome-wide identification of replication-associated breakpoints. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1777. doi:10.1158/1538-7445.AM2013-1777

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1779: BRCA2 interacts with an essential replication factor, MCM10.
Authors: Alcivar Allen L. , Ma Jianglin , Xia Bing
Published: August 2014

The BRCA2 (Breast Cancer 2, early onset) gene is implicated in a variety of familial cancers. Loss of BRCA2 function results in severe defects in DNA double-strand break repair, DNA damage-induced checkpoint response and the stability of stalled DNA replication forks, all of which are important for genomic stability. About 50% of BRCA2 is associated with PALB2 (partner and localizer of BRCA2), which anchors BRCA2 onto chromatin and directs its recruitment to DNA damage sites. By tandem affinity purification of PALB2 we identified MCM10, an essential DNA replication factor, as a component of the PALB2/BRCA2 complex. MCM10 promotes initiation of DNA replication through binding to MCM2-7 helicase complex and recruiting the initial DNA polymerase, pol α, to replication origins.

Interestingly, we found that MCM10 binds to BRCA2 instead of PALB2 in the complex. Cells depleted of BRCA2 or MCM10 showed similar instability of stalled replication forks, indicating possible cooperation between the two proteins in fork stabilization following replication stress. Moreover, DNA damage-induced CHK1 activation is markedly reduced in both BRCA2- and MCM10-depleted cells even though RPA (replication protein A) is hyperphosphorylated. Our findings suggest that BRCA2 checkpoint function may be mediated through MCM10 and their interaction may be important for stalled replication fork stability. Domain mapping experiments showed that an evolutionarily conserved coiled-coil motif in the N-terminus of MCM10 which is required for BRCA2 binding and that BRCA2 has at least two MCM10-binding sites, both in its central region. BRCA2 and MCM10 mutants lacking these interaction domains will be used to further test the functional relevance of their interaction.

Citation Format: Allen L. Alcivar, Jianglin Ma, Bing Xia. BRCA2 interacts with an essential replication factor, MCM10. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1779. doi:10.1158/1538-7445.AM2013-1779
Abstract 1780: The MAD1 1673 GA polymorphism alters the function of the mitotic splindle assembly checkpoint and is asociated with a worse response to induction chemotherapy and sensitivity to treatment in patients with advanced epithelial ovarian cancer.
Authors: Santibañez Miguel , Gallardo Dolores , Morales Flavia , Lopez Alejandro , Prada Diddier , Mendoza Julia , Castro Clementina , de Leon David Cantu , Oñate Luis F. , Perez Delia , Mohar Alejandro , Herrera Luis A.
Published: August 2014

Introduction: MAD1, a protein of the mitotic spindle assembly checkpoint (SAC), recognizes MAD2 through two leucine zippers, transporting and activating MAD2, which promotes a metaphase arrest signal. A SNP of Mad1 was found to affect the SAC function that could be involved in a bad response to therapeutic agents that alter the dynamics of microtubules. Objective: To investigate the relationship of the polymorphism Mad1 1673 GA (rs1801368) with the efficiency of the SAC and the generation of aneuploidies, and with the therapeutic response of patients with ovarian cancer. Methods: The polymorphism was evaluated in 144 healthy individuals and 91 patients. The mitotic arrest and the presence of errors in segregation were analyzed in cultured human lymphocytes treated with nocodazol and paclitaxel. Errors in segregation were also evaluated in 27 biopsies of patients. Results: Allele frequencies in healthy individuals were G: 50, A: 50, while in the patients, they were G: 38%, A: 62% (p<0.05). The percentage of cells with mitotic arrest was higher in GG cells (p<0.05). The frequency of micronuclei and nondisjunction events increased in AA cells (p<0.05). Tumors from polymorphic patients had a higher percentage of aneuploid cells (p<0.05). The GG patients showed a higher biochemical response, optimal cytoreduction and sensitivity to the treatment. There were no differences in progression-free or overall survival between both groups. Conclusions: the polymorphism Mad1 1673 GA affects the SAC functionality, increasing the frequency of aneuploid cells. This polymorphism modifies the response to agents that alter the dynamics of microtubules in patients with ovarian cancer.

Citation Format: Miguel Santibañez, Dolores Gallardo, Flavia Morales, Alejandro Lopez, Diddier Prada, Julia Mendoza, Clementina Castro, David Cantu de Leon, Luis F. Oñate, Delia Perez, Alejandro Mohar, Luis A. Herrera. The MAD1 1673 GA polymorphism alters the function of the mitotic splindle assembly checkpoint and is asociated with a worse response to induction chemotherapy and sensitivity to treatment in patients with advanced epithelial ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1780. doi:10.1158/1538-7445.AM2013-1780