bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2768: Global DNA hypomethylation is an important feature of melanoma
Authors: Micevic Goran , Theodosakis Nicholas , Taube Janis M. , Bosenberg Marcus W. , Rodic Nemanja
Published: July 2016

Aberrant DNA methylation occurs nearly universally in cancer. Paradoxically, changes in DNA methylation are bi-directional: there is a focal increase in DNA methylation (hypermethylation), and a global overall decrease in DNA methylation (hypomethylation). Focal hypermethylation plays well described roles in silencing tumor suppressor genes in many malignancies, including melanoma. However, the functional role of global hypomethylation in tumorigenesis is relatively poorly understood. Melanoma is the deadliest form of skin cancer, accounting for approximately 74,000 new cases and 10,000 deaths in the United States in 2015. Changes in DNA methylation are also arguably one of the most common features of melanoma. Similar to other malignancies, most studies have focused on focal hypermethylation, identifying genomic loci differentially methylated between melanocytes and melanoma cells. However, global changes in DNA methylation have not been well described thus far. In this study, we describe global changes in DNA methylation in two melanoma clinical cohorts (JHH cohort, n = 13 nevi and n = 39 melanomas; NCI cohort n = 31 nevi and n = 97 melanomas) relative to benign nevi using a semi-quantitative immunohistochemistry based assay. We report a global decrease in DNA methylation levels in melanoma relative to benign melanocytic nevi consistent between both cohorts (p < 0.001, Student's t-test). Furthermore, we studied the functional effect of increasing the level of global DNA methylation in a panel of five early passage melanoma cell lines. Increases in global DNA methylation suppressed proliferation of melanoma cells (p <0.05, Student's t-test), and increased the proportion of apoptotic cells (p <0.01, Student's t-test). Global DNA hypomethylation is thus an important epigenetic feature of melanoma that may regulate proliferative capacity, and merits further investigation.

Citation Format: Goran Micevic, Nicholas Theodosakis, Janis M. Taube, Marcus W. Bosenberg, Nemanja Rodic. Global DNA hypomethylation is an important feature of melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2768.
Abstract 2769: Response to 5FU therapy in colorectal cancer is affected by EZH2-mediated BclX splicing
Authors: Benderska Natalya , Gazouli Maria , Aravantinos Gerasimos , Hartmann Arndt , Schneider-Stock Regine
Published: July 2016

Background and Aims: The chromatin modifier EZH2 is remarkably up-regulated in many cancer types including colon cancer. High levels of EZH2 have been correlated with tumor metastasis and regulation of tumor-initiating cell subpopulation. Since, DZNep, an EZH2 inhibitor, can inhibit the growth capability of colon cancer by inducing apoptosis; down-regulation of EZH2 seems to be a promising therapeutic approach. Our preliminary data showed that EZH2 levels were remarkably increased in blood samples from metastatic colorectal cancer patients who did not respond to the combination therapy with bevacizumab-irinotecan-5FU in comparison to patients who were responders and developed a stable disease. Many apoptosis-related proteins possess a dual function (pro- and anti-apoptotic) due to alternative splicing. Therefore we aimed to find possible apoptosis-associated EZH2 targets to explain these interesting findings.

Methods: HCT116 colon cancer cells were used for DZNep and 5FU treatment and its efficiency was verified by Western blot analysis. In vivo splicing assay was performed using BclX minigene construct. The mRNA expression of EZH2, caspase 2, Death-associated protein kinase (DAPK) and BclX isoforms was assessed by quantitative real-time PCR in cells (BclX also in blood of patients) and normalized to the GAPDH. Cell viability was examined by Crystal Violet staining.

Results: Western Blot for caspase 3 cleavage indicated increased apoptotic levels after DZNep treatment. Combined DZNep and 5FU treatment of cells led to a synergistic effect in reducing cell viability. DZNep treatment specifically promoted pro-apoptotic BclXs isoform, but did not affect the shift of pro-apoptotic isoforms of caspase 2 and DAPK. Using a BclX minigene transfection we confirmed this effect exogenously. The ratio BclXs/Xl was significantly altered by overexpression of different splicing factors such as ASF/SF2, Sam68, after DZnep treatment. Moreover, patients who did not respond to the combination therapy had the highest EZH2 and BclXl isoform levels.

Conclusion: Our data indicate a new interesting link between epigenetic regulation and the splicing machinery. Detailed investigation of EZH2-mediated regulation of BclXs/Xl ratio could have the potential for prediction of 5FU-based therapy response in colon cancer patients.

Citation Format: Natalya Benderska, Maria Gazouli, Gerasimos Aravantinos, Arndt Hartmann, Regine Schneider-Stock. Response to 5FU therapy in colorectal cancer is affected by EZH2-mediated BclX splicing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2769.
Abstract 2785: The role of heat shock factors for cancer stem cell regulation
Authors: Thakkar Ankita , Wang Bin , Ince Tan A.
Published: July 2016

Introduction: Heat shock factor family is a group of transcription factors that can bind specifically to heat shock elements (HSE) within the promoter region of heat shock protein (HSP). The HSF pathway activation helps cells survive under stress by preventing protein misfolding and aggregation. Three heat shock factors (HSF1, HSF2 and HSF4) have been identified in humans, among them, HSF1, which is believed to be the master regulator of cellular response to stress, is by far the best-characterized member of the HSF family.

We first identified that high HSF1 is associated with high tumor grade and poor survival in multiple cancer models including breast, ovarian, lung and colon cancers. In addition, our recent data indicate that HSF1 drives a distinct transcription program that is unrelated to heat shock response in malignant tumors, and this HSF1 “non-heat shock related transcription program” correlates with poor outcome in breast cancer patients. Moreover, we confirmed that HSF1 drives the Cancer Stem Cell (CSC) phenotype in multiple breast cancer cell lines and facilitate chemo-treatment resistance by increasing CSC frequency in breast cancer cells.

Previous studies have shown that HSF2 and HSF4 may play a role in cancer progression either on their own or through interaction with HSF1. HSF2 forms hetero-trimers with HSF1 and facilitate its function in response to heat shock. Inactivation of HSF4 in p53 null mouse models causes cell senescence and suppression of tumorigenesis, with great similarities to HSF1-/- p53 null mice.

Results: In an attempt to investigate how HSF1 is activated to regulate CSC phenotype, we constructed phospho HSF1 at three different sites. We observed that there is no correlation of phospho activation of HSF1 with CSC phenotype. In follow-up work to the studies above, we found that both HSF2 and HSF4 are also significantly up-regulated in CSCs, suggesting that they may cooperate with HSF1 in regulating CSCs. Moreover, we discovered that HSF4 is over-expressed in breast cancers compared to normal mammary gland, and over-expression of HSF4 induces tumorsphere formation in breast cancer cells.

Conclusion: These observations indicated that in order to gain a complete understanding of the role of the HSFs in cancer it is now necessary to study all three HSFs together.

Citation Format: Ankita Thakkar, Bin Wang, Tan A. Ince. The role of heat shock factors for cancer stem cell regulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2785.
Abstract 2795: MARVELD1 regulates hypoxia-induced oxidative stress response and tumor growth
Authors: Shi Ming , Yao Yuanfei , Li Yu
Published: July 2016

Nuclear factor MARVELD1 (MARVEL domain-containing 1) is a potential tumor suppressor, which negatively regulates tumor cell proliferation. It is widely expressed in human normal tissues, but down-regulated in multiple primary carcinomas. In this study, we show that MARVELD1 acts as tumor suppressor via its ability to inhibit reative oxygen species (ROS) in response to hypoxic condition. Data showed that overexpression of MARVELD1 in uterine cancer cells significantly suppressed tumor growth, while MARVELD1 silencing promoted colony formation of tumor cells in hypoxic environment. Furthermore, we found that ROS production in hypoxia-treated cancer cells was elevated by MARVELD1 silencing. Mitochondria function assay showed that compared to RL952/vector cells, a decrease in oxygen consumption and derived mitochondrial ATP production was observed in hypoxia-treated RL952/MARVELD1 cells. Moreover, considering that ROS that induced by hypoxia promoted tumor cell proliferation, our data further indicated ROS generation that inhibited by MARVELD1 alleviated the growth of uterine tumor cells. Taken together, MARVELD1 regulated hypoxia-induced oxidative stress response and cell growth through inhibiting ROS generation in uterine cancer, suggesting that targeting MARVELD1 might be a novel strategy to cancer therapy.

Keywords: MARVELD1, Hypoxia, Oxidative stress, ROS

Citation Format: Ming Shi, Yuanfei Yao, Yu Li. MARVELD1 regulates hypoxia-induced oxidative stress response and tumor growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2795.
Abstract 2798: Enhanced HIF1α inhibition through dual inhibition of CDK and HSP90
Authors: Zhao Shuai , Dicker David T. , El-Deiry Wafik S.
Published: July 2016

A prevalent characteristic of solid tumors is the presence of hypoxic areas. Intratumoral hypoxia plays a well-known role in chemotherapy and radiotherapy resistance and is associated with poor prognosis as well as enhanced metastasis. Hypoxia-inducible factor 1α (HIF1α) is a major mediator of the cellular response to hypoxia, which promotes malignant proliferation and progression in cancers. HIF-1α expression is increased in a variety of tumors but this is not restricted to hypoxic regions. Understanding and targeting the mechanism of cancer-related HIF1α stabilization may help to improve current cancer treatments. We have previously shown that cyclin-dependent kinase 1 (CDK1) stabilizes HIF1α through direct phosphorylation of its Ser668 residue in a Von Hippel-Lindau (VHL)-independent manner both under hypoxia and at G2/M under normoxia (Warfel et al., Cell Cycle, 2013). Another previously acknowledged VHL-independent HIF1α stabilizer is the heat shock protein 90 (HSP90) that has been correlated with adverse prognosis and used as a therapeutic target in cancer. We investigated potential crosstalk between CDK1-mediated and HSP90-mediated HIF1α stabilization and the potential for therapeutic targeting. We found that under hypoxia, the interaction between HSP90 and HIF1α proteins is impaired by CDK1 inhibition in HCT116 colon cancer cells. Moreover, the level of HIF1α protein is increased by heat shock (40°C) treatment (as compared to 37°C), which can be reversed by the treatment with the HSP90 inhibitor, geldanamycin. Interestingly, administration of the CDK1 inhibitor, Ro-3306, is also able to decrease heat shock-induced HIF1α. Our results suggest that CDK1 activity may contribute to HSP90-mediated HIF1α stabilization. On the other hand, HIF1α level is decreased by HSP90 inhibition under hypoxia, which can be further reduced by the combination of HSP90 inhibition and CDK1 inhibition or knockdown. The joint inhibitory effect of CDK1 and HSP90 is observed in other cancer cell lines. In addition, we evaluated the combinational treatment of CDK4/HSP90 inhibition and found it robustly suppresses HIF1α expression in glioblastoma cell lines (i.e. T98G and SNB19). Ongoing studies are examining the biological function of CDK/HSP90 dual inhibition on cancer cell invasiveness as well as effects on cell viability. Our findings provide a rationale for targeting HIF1α through a novel combination of CDK and HSP90 inhibitors, a therapeutic strategy with clinical implications.

Citation Format: Shuai Zhao, David T. Dicker, Wafik S. El-Deiry. Enhanced HIF1α inhibition through dual inhibition of CDK and HSP90. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2798.
Abstract 2800: Target RNA sequencing analysis of hypoxia induced expression of stem cell genes in primary cultures of prostate cancer cells
Authors: Yin Hong , Greer Adam H. , Mills Glenn
Published: July 2016

Hypoxia plays important regulatory roles in expanding the tumor stem cell population. In prostate cancer, hypoxia is a feature of poor prognosis. The association of prostate cancer stem cells with hypoxia has been investigated. The altered stem cell gene expression in prostate cancer cells under the hypoxic condition has been documented. The majority of the investigations were performed with established prostate cancer cell lines. However, little is known about the expression of stem cell genes in primary cultures of prostate cancer. In this study, we examined the hypoxia-regulated expression of stem cell genes with primary cultures of human prostate cancer. Cells were isolated from three patient's prostate cancer samples. Isolated cells were cultured in prostate epithelium culture medium CnT-52. After 48 hours of exposure to 1% oxygen, RNA was extracted and quantified. The library was prepared with Illumina's TruSeq Targeted RNA Expression Stem Cell Panel. The sequencing was performed on Miseq platform with MiSeq Reagent Kits v2 (300cycles). FastQ files were generated by Miseq Reporter and analyzed further by Truseg Target RNA v1.0(Illumina), Cufflinks Assembly &DE(Illumina), and Biomedical Genomics Workstation(Qiagen).The results showed that of the 100 tested stem cell genes, seven genes were significantly upregulated(FDR p-value<0.05). They are BMP1, BMP2, FZD7, JUND, NOTCH1, NOTCH3, and PPARD. Three genes, PSEN2, SDAD1, and SOX2, were significantly down-regulated. To validate sequencing results, we examined NOTCH1 and NOTCH3 expression with RT-PCR. Compared to cells under normoxic condition, hypoxia induced expression of NOTCH1 increased by 3.7, 3.9 and 3.1 fold, respectively among three hypoxic cultures, while the expression of NOTCH3 increased by 8.1, 5.9, and 7.5 fold, respectively. However, the investigation of hypoxia affected expression of stem cell genes in LNCaP, an established prostate cell line, showed that the significant decreased expression of BMP1, FZD7, PPARD, and NOTCH3 was seen under hypoxic condition(FDR p-value <0.05). RT-PCR analysis showed that expression of NOTCH3 in hypoxic LNCaP, PC3, and DU145 cells was decreased by 7.7, 6.1, and 1.2 fold, respectively. The expression profile of stem cell genes in response to hypoxia was different between primary cultures of prostate cancer cells and established prostate cancer cell lines. The activation of BMP-NOTCH signaling could be one character of stemness of prostate cancer in response to hypoxia.

Citation Format: Hong Yin, Adam H. Greer, Glenn Mills. Target RNA sequencing analysis of hypoxia induced expression of stem cell genes in primary cultures of prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2800.
Abstract 2801: Arginylation as a novel regulator of prostate cancer progression
Authors: Birnbaum Michael , Kumar Akhilesh , Zhang Fangliang
Published: July 2016

Post-translational arginylation, mediated by Arginyltransferase (Ate1), is an important protein modification involved in stress response yet remains poorly studied. Our lab recently reported that a loss of Ate1 is sufficient to induce tumorigenesis and loss of contact inhibition in fibroblasts. However, the role of Ate1 in cancer initiation and progression is not clear. This study examines the effects of Ate1 loss in prostate cancer models of tumorigenesis and progression. We first show that Ate1 is required for normal cellular responses to several types of stress present in cancer. Preliminary data in fibroblasts shows that Ate1 is essential for cell death following oxidative stress, hypoxia, UV and gamma radiation, and apoptosis-inducing drugs. These results inspired further tests to examine if cancer progression may be directly stimulated by a loss of Ate1 through increased cell survival and invasiveness. To study the effects of lost Ate1, we characterize established prostate cancer cell lines with Ate1 stably knocked down. In PC-3 prostate cancer cells, a reduction of Ate1 corresponded with increased invasiveness in the Boyden Chamber invasion assay, and reduced H2O2-induced cell death. In LnCaP prostate cancer cells, reduced Ate1 induced significantly higher anchorage-independent growth during a soft-agar growth assay. In conclusion, our data suggests that the loss of Ate1 in prostate cancer serves to stimulate cancer progression, and that Ate1 protein expression levels may predict malignancy.

Citation Format: Michael Birnbaum, Akhilesh Kumar, Fangliang Zhang. Arginylation as a novel regulator of prostate cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2801.
Abstract 2807: Coordinated upregulation of peroxiredoxins in breast cancer
Authors: Muhlbauer Jillian , Phelan Shelley A.
Published: July 2016

Peroxiredoxins (Prdxs) are a group of thiol-specific antioxidant proteins that are involved in many cellular processes, including growth, proliferation, differentiation, and the stress response. The family includes six proteins in humans, which show differences in substrate specificity, tissue expression, and cellar localization and distribution. Cancer cells commonly exhibit aberrantly regulated expression of antioxidants, which can support cancer cell survival in the face of elevated levels of reactive oxygen species. Breast cancers exhibit significantly elevated Prdx levels of Prdx. We previously reported overexpression of many Prdx proteins in the MCF-7 breast cancer line, and further demonstrated their importance in cell survival and doxorubicin-resistance in this line. To further investigate the misregulation of these proteins in human breast cancer, we compared Prdx levels between breast tumor and adjacent normal breast tissue from 20 breast cancer patients. We showed that most patients exhibited elevated levels of several Prdx proteins in the tumor tissue. We have gone on to analyze the promoters of these genes in an effort to identify potential mechanisms of coordinate regulation, as well as downstream targets that may mediate their role in cell survival. Together, we feel that this family of proteins may be useful biomarkers, and also targets, in breast cancer biology.

Citation Format: Jillian Muhlbauer, Shelley A. Phelan. Coordinated upregulation of peroxiredoxins in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2807.
Abstract 2812: Targeting reactive oxygen species in human cancer cell growth and metastasis in nude mouse
Authors: Inufusa Haruhiko
Published: July 2016

[Purposes of the study] Reactive oxygen species (ROS) are associated with clinical condition, and closely involved in cancer growth and metastasis. Twendee X is a composition consisting of Vitamin C, Glutamine, Cystine, Riboflavin, Succinic acid, Fumaric acid, Coenzyme Q10, and Niacin. Twendee X strongly reduces oxygen radicals or ROS in vitro and in vivo (World Intellectual Property Organization WO/2013/072441 COMPOSITION FOR PROTECTION AGAINST CELL-DAMAGING EFFECTS). Effects of Twendee X on human colon cancer cell lines growth and metastasis in nude mouse was examined.

[Experimental procedures] Human colon cancer cell line RPMI4788 human gastric cancer cell line MKN45 was used in this study. Ten nude mice (BALB/C nu-nu, 4wks, Male) per each group of experiments were used, and repeated twice. Two cell lines were inoculated in subcutaneous of nude mouse, and tumor growth monitored for days. Lung metastasis was counted weeks after injection RPMI4788 cells into the tale vein of nude mouse. Twendee X was added to the free drink water around 37mg/kg/day by calculation. Serum of tumor bearing nude mouse was sampled just before sacrifice. Free Calpe Diem (Diacron International, Italy) was used to measure ROS related marker d-ROMs test (detect hydrogen peroxide). Natural Killer cell (NK) activity also measured in tumor bearing nude mouse.

[Data] Tumor growth of RPMI4788 and MKN45 was reduced 25% and 40% respectively by Twendee X administration. Metastatic nodule of RPMI4788 was reduced to 1/7 by Twendee X administration. Increase value of d-ROMs test reduced to 60% compare to the control by Twendee X administration. NK activity of RPMI4788 tumor bearing nude mouse was reduced to 28% compare to the normal mouse, and increased to 56% by Twendee X administration.

[Conclusion] Twendee X has significant effects of reducing tumor growth and inhibition metastasis. Hydrogen peroxide level (d-ROMs test) of tumor bearing nude mouse in serum was significantly reduced. ROS effects on immune cell activity, and Twendee X recovered suppressed NK activity. Thus, Twendee X effects on ROS inhibition and suppressed RPMI 4788 tumor growth and metastasis to the lung of nude mouse. Metastatic nodules receiving larger blood flow compare to the tumor in the back of mouse, thus means anti-tumor immune cells in blood may easier attack cancer cells.

Citation Format: Haruhiko Inufusa. Targeting reactive oxygen species in human cancer cell growth and metastasis in nude mouse. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2812.
Abstract 2814: Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro
Authors: Nelson Garret B. , Thai Sheau-Fung Y. , Jones Carlton P. , Barbee Audrey , Killius Micaela , Ross Jeffrey A.
Published: July 2016

A series of six titanium dioxide and two cerium oxide engineered nanomaterials were assessed for their ability to induce cytotoxicity, reactive oxygen species (ROS), various types of DNA damage, and transcriptional changes in human respiratory BEAS-2B cells exposed in vitro at several concentrations for 72 hours. Only limited cytotoxicity was observed at concentrations up to 300 μg/ml for all of the nanomaterials. Small increases in 8-oxo-deoxyguanosine were induced by some of the nanomaterials, but did not achieve statistical significance. No increases in ethenoadenosine or ethenocytidine were detected by ELISA assays for any of the tested nanomaterials. Several of the nanomaterials exhibited concentration related increases in levels of apurinic/apyrimidinic sites, endogenous DNA adducts measured by 32P-postlabeling, lipid peroxidation, and ROS. Consistent with these findings, several of the nanomaterials also affected expression of genes involved in p53, ATM, and mismatch repair pathways. Integrin signaling pathways were also altered by a majority of the nanomaterials tested. There was general agreement between activity in DNA damage assays and extent of pathway transcriptional alteration. One out of the cerium oxide nanomaterials tested did not induce a high enough incidence of differentially expressed genes relative to controls to allow analysis at the pathway level, and also elicited the lowest response in multiple DNA damage assays. Taken together, these data are consistent with the contribution of DNA damage induced by reactive oxygen species as mediators of potentially adverse biological effects following exposure to engineered titanium and cerium oxide nanomaterials, and suggests the utility of short term in vitro tests to predict relative potencies of these particles.

This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.

Citation Format: Garret B. Nelson, Sheau-Fung Y. Thai, Carlton P. Jones, Audrey Barbee, Micaela Killius, Jeffrey A. Ross. Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2814.
Abstract 2823: Identification of highly selective inhibitors of cyclin-dependent kinase 12
Authors: Choidas Axel , Schultz-Fademrecht Carsten , Degeenhardt Carsten , Habenberger Peter , Koch Uwe , Eickhoff Jan E. , Greifenberg Ann Kathrin , Nussbaumer Peter , Geyer Matthias , Klebl Bert M.
Published: July 2016

CDK12 and CDK13 belong to the cyclin-dependent kinase (CDK) family, which includes at least 20 different human CDKs and CDK-like enzymes. One subclass of CDKs (e.g. CDK7, CDK8, CDK9, CDK12 and CDK13) preferentially regulates transcription by phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII), whereas another subgroup of CDKs plays a pivotal role in controlling cell cycle progression (e.g. CDK1, CDK2, CDK4, CDK6 and CDK7). Both groups - cell cycle as well as transcriptional CDKs - have been reported to be involved in cancer development and progression. Recent reports indicated CDK12 to be involved in DNA damage repair mechanisms and linked its mutation to high-grade serous ovarian carcinoma rendering CDK12 a promising target for drug development. However, the pivotal role of individual CDKs and their complex regulation requests for highly selective inhibitors to avoid unwanted adverse effects. Non-specific first-generation CDK-inhibitors cause toxicity in vivo issues. They showed little to no therapeutic window in clinical trials and thus shifted the focus to the optimization of selective small molecule CDK inhibitors.

To identify selective small molecular weight inhibitors of CDK12 and CDK13 we employed a rationale drug discovery approach based on a focused kinase library screen. Screening of slightly over 16.000 compounds identified several hit compound classes. Medicinal chemistry based optimization yielded nanomolar CDK12 inhibitors. The selectivity of these improved CDK12 inhibitors is excellent, when tested against 12 CDK family members. Such selective CDK12 inhibitors have also shown promising physicochemical properties, indicating lead- and drug-likeness. Although, potency on the target still needs to be improved, the frontrunners from our selective CDK12 inhibitor family have shown cellular inhibition of CDK12 substrate phosphorylation such as those of RNA polymerase II. Currently, potency of these CDK12 inhibitors is being improved by using structure-guided medicinal chemistry-based optimization. The resulting lead candidates will then be tested in in vivo cancer models.

Citation Format: Axel Choidas, Carsten Schultz-Fademrecht, Carsten Degeenhardt, Peter Habenberger, Uwe Koch, Jan E. Eickhoff, Ann Kathrin Greifenberg, Peter Nussbaumer, Matthias Geyer, Bert M. Klebl. Identification of highly selective inhibitors of cyclin-dependent kinase 12. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2823.
Abstract 2829: Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures
Authors: Boehnke Karsten , Calsina Bruna , Amat Joaquín , Hermoso Ana , Torres Raquel , Reinhard Christoph , Velasco Juan A. , Iversen Philip W. , De Dios Alfonso , Buchanan Sean , Beckmann Richard P. , Schumacher Dirk , Regenbrecht Christian RA , Yaspo Marie-Laure , Lehrach Hans , Lallena María José
Published: July 2016

Proper patient-tailoring strategy and the validation of novel therapeutic targets remain enormous challenges during drug discovery processes. Patient-derived three-dimensional organoid cell culture models possess great potential to associate compound sensitivity and disease complexity in order to provide a key missing link between compound screening and clinical trials. Abemaciclib is a reversible, ATP competitive, selective inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing advanced clinical testing.

In this study, we established and characterized three-dimensional organoid cultures from primary colorectal cancer patients and validated their use as drug sensitivity models. We aimed to explore the antitumor activity of abemaciclib in colon cancer organoid cultures by assessing markers for cell viability, proliferation, cell cycle, senescence and apoptosis. Single cell suspension of patient-derived samples were precultured for four days to allow for complete morphogenesis of three-dimensional organoid structures. Subsequently, the cultures were treated for at least two population doubling times and analyzed by luminescent cell viability, immunohistochemistry and flow cytometry assays.

Our data suggest that abemaciclib treatment decreased the cell viability of patient-derived colorectal cancer organoid cultures characterized by G1 cell cycle arrest and reduced Ki-67-positive cells. Furthermore, treated cultures showed elevated levels of reactive oxygen species and increased markers for early and late apoptosis. In summary, complex organoid models have the potential to further evaluate the antitumor activity of abemaciclib in various tumor types by enabling mechanistic studies in a patient-specific preclinical setting.

Citation Format: Karsten Boehnke, Bruna Calsina, Joaquín Amat, Ana Hermoso, Raquel Torres, Christoph Reinhard, Juan A. Velasco, Philip W. Iversen, Alfonso De Dios, Sean Buchanan, Richard P. Beckmann, Dirk Schumacher, Christian RA Regenbrecht, Marie-Laure Yaspo, Hans Lehrach, María José Lallena. Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2829.
Abstract 2833: Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins
Authors: Zhang Shuling , Gary Joy M. , Simmons John K. , Xu Jinfei , Gamache Benjamin J. , Zhang Ke , Watson Nicholas , Kovalchuk Alexander L. , Michalowski Aleksandra M. , Chen Jin-Qiu , Herrmann Michelle A. , Thaiwong Tuddow , Kiupel Matti , Dubois Wendy , Testa Joseph R. , Mock Beverly A.
Published: July 2016

The AKT/mTOR pathway is frequently hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with T-lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) were crossed to mice with genetically reduced mTOR expression (knock-down, KD). Mice with genetic reduction of mTOR had increased survival by 10 weeks relative to wild type mTOR mice, though both developed thymic pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Similarly, when mTOR wild type Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex, survival was also increased. Gene expression profiling of thymic lymphomas from the mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T-cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. The combination of a mTOR inhibitor (rapamycin) and a CDK4/6 inhibitor (PD-0332991, Palbociclib) synergistically decreased the overall viability and signaling downstream of drug targets in mouse lymphoma cells and in human T-ALL/LBL cell lines. This combination was also evaluated in mice using a disseminated leukemia model. In vivo treatment with this combination not only reduced tumor size by inhibiting tumor cell proliferation and arresting tumor cell cycle, but also increased overall survival. We are currently validating upstream regulators of Cdk6 as well as downstream targets in the pre-T LBL tumors from the mTOR deficient mice.

Citation Format: Shuling Zhang, Joy M. Gary, John K. Simmons, Jinfei Xu, Benjamin J. Gamache, Ke Zhang, Nicholas Watson, Alexander L. Kovalchuk, Aleksandra M. Michalowski, Jin-Qiu Chen, Michelle A. Herrmann, Tuddow Thaiwong, Matti Kiupel, Wendy Dubois, Joseph R. Testa, Beverly A. Mock. Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2833.
Abstract 2835: Voruciclib, a clinical stage oral CDK inhibitor, sensitizes triple negative breast cancer xenografts to proteasome inhibition
Authors: Dey Joyoti , Casalini Joseph , Ditzler Sally , Biery Matt , Merrell Angela , Thirstrup Derek , Grenley Marc , Klinghoffer Richard
Published: July 2016

Triple negative breast cancer (TNBC) is a highly heterogeneous disease, notoriously challenging to treat with standard chemotherapy options, and therefore is an area of intense focus for discovery of novel effective combination therapies. Here we used a previously described technology platform called CIVO, which enables assessment of multiple drugs and drug combinations simultaneously in living tumors, to identify drug combinations that result in synergistic anti-tumor activity in the HCC1187 model of TNBC. Our study focused on agents that combine with Voruciclib, a novel clinical stage oral CDK inhibitor with potent activity (<100 nM) against CDKs 1, 4, and 9. Among the drug combinations investigated, robust localized anti-tumor activity as measured by cleaved caspase 3 (CC3) positive apoptotic cells, was observed upon combined tumor exposure to Voruciclib and the proteasome inhibitor Bortezomib. In contrast, exposure to either Voruciclib or Bortezomib as single agents showed little anti-tumor activity. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies where tumor regression was induced by the Voruciclib/Bortezomib combination, but no significant impact on tumor progression was observed in xenografted subjects treated with either single agent. The ability of TNBC cells to withstand stressors such as chemotherapy may be due in part to activation of adaptive survival pathways including the unfolded protein (UPR) and endoplasmic reticulum (ER) stress responses. As observed in previous reports, exposure of HCC1187 cells to Bortezomib alone led to an increase in two markers of the cytoprotective arm of the UPR/ER stress pathway: XBP-1s and GRP-78/BIP. Consistent with the possibility that Voruciclib impedes the cytoprotective UPR/ER stress response induced by Bortezomib, exposure to the drug combination substantially reduced protein expression of both XBP-1s and GRP-78 with concomitant induction of cPARP. Voruciclib also neutralized upregulation of these same proteins by the classic ER stress inducing agent Tunicamycin. These studies provide a foundation for further investigation of breast cancer relevant anti-cancer agents that induce UPR/ER stress responses in combination with Voruciclib for treating TNBC patients.

Citation Format: Joyoti Dey, Joseph Casalini, Sally Ditzler, Matt Biery, Angela Merrell, Derek Thirstrup, Marc Grenley, Richard Klinghoffer. Voruciclib, a clinical stage oral CDK inhibitor, sensitizes triple negative breast cancer xenografts to proteasome inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2835.
Abstract 2838: The role of cyclin D1-associated kinase activity in the regulation of autophagy and senescence in gastric cancer cells
Authors: Vargas Leandro , Bravo Sindy , Valenzuela Claudio , Brown Nelson E.
Published: July 2016

Introduction: Cyclin D1 regulates cell cycle progression through its ability to bind and activate cyclin-dependent kinases 4 and 6 (CDK4/6). Recent work carried out in mammary epithelial cells (MECs) propagated in vitro has revealed that the functional status of cyclin D1 modulates a balance between senescence and autophagy, two processes commonly disrupted in human cancers. Thus, in contrast to the commonly held assumption that autophagy is an effector process of senescence, inhibition of autophagy was accompanied by an induction or exacerbation of senescence in MECs deficient in cyclin D1-associated kinase activity, a reflection of the pro-survival function of autophagy in these cells. Materials and methods: In the present study, we set out to confirm these findings in gastric cancer cell lines (AGS, MKN-45 and KATO III) that may differ in their ability to induce senescence. To this end, we first treated AGS (cyclin D1+; pRB+; p53+) cells with Palbociclib (a specific inhibitor of cyclin D-CDK4/6 complexes), Spautin-1 (a specific inhibitor of autophagy), or a combination of these drugs, and assessed the effect of the treatment on the ability of these cells to proliferate or undergo senescence. Results and discussion: Unlike what was previously reported, our results suggest that autophagy is not required for the implementation of the senescent phenotype in AGS cells treated with Palbociclib. Nonetheless, senescent cells in which autophagy was disrupted were larger and often displayed multiple nuclei, suggesting the existence of different senescence programs depending on autophagy function.

Citation Format: Leandro Vargas, Sindy Bravo, Claudio Valenzuela, Nelson E. Brown. The role of cyclin D1-associated kinase activity in the regulation of autophagy and senescence in gastric cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2838.
Abstract 2851: EGFR-TERT cooperation in the development of EGFR-TKI treatment induced pulmonary fibrosis
Authors: Wei Rongrong , Liu Wanqing
Published: July 2016

Background: Previous studies have shown that epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment in lung cancer can lead to the development of pulmonary fibrosis (PF). In other studies, it has been shown that reduced telomerase reverse transcriptase (TERT) function increases susceptibility to PF. In this work, we hypothesize that TERT is associated with the pathogenesis of EGFR-TKI induced PF via important signaling pathways.

Methods: HCC827 cells (EGFR L858R) was treated with gefitinib and TERT expression level was detected following the treatment. To investigate the molecular changes following gefitinib treatment, we also performed a 84-TF qPCR- microarray and a qPCR microarray for 84-key secreted proteins of cytokines and chemokines. The level of interleukin-6(IL-6) was further measured with ELISA in cell medium following the gefitinib treatment. Fibrogenic markers were detected after co-culturing HCC827 with lung fibroblast IMR-90 cells.

Results: TERT gene expression was significantly decreased over 90% after gefitinib treatment. Early growth response protein 1(EGR-1) and E2F transcription factor 1(E2F1) were significantly down-regulated as major TFs that are responsive to gefitinib treatment. Both EGR-1 and E2F1 have been shown to regulate TERT expression in previous studies. In addition, TFs including JUN and most REL members involved in the NF-κB pathway, were significantly up-regulated after gefitinib treatment, which are known to lead to the activation of the senescence-associated secretory phenotype (SASP) pathway. IL-6 mRNA as well as protein secretion were both significantly up-regulated after gefitinib treatment. After co-culturing HCC827 and IMR-90 for 7 days and treating with gefitinib, the growth of IMR-90 cells were increased compare to the control group. Fibrogenic markers, e.g. COL1A1 (Collagen, Type I, Alpha 1), α-SMA (alpha smooth muscle actin), FSP-1(Fibroblast-specific protein 1), etc. were all up-regulated.

Conclusion: Our data suggest that EGFR inhibition leads to lung epithelial cell senescence via inhibiting TERT function and activating of the SASP pathway, which further activates fibroblasts and induces fribrogenesis. Continued studies are warranted to further understand the molecular signaling in this mechanism.

Citation Format: Rongrong Wei, Wanqing Liu. EGFR-TERT cooperation in the development of EGFR-TKI treatment induced pulmonary fibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2851.
Abstract 2856: Inhibition of glycolysis sensitizes cancer cells to metformin-induced anoikis
Authors: Yi Yong , Xiao Zhi-Xiong Jim
Published: July 2016

Metformin is a widely used medicine to treat type 2 diabetes. Clinical studies have shown that metformin can reduce cancer incidence, yet the molecular mechanism of which is not totally understood. The p53-related p63 plays a critical role in regulation of cell proliferation, apoptosis, differentiation and development. We and others have shown that the major isform of p63 proteins, ΔNp63α, is important in regulation of squamous carcinoma cell proliferation, survival and cancer metastasis (Bergholz et al.,Oncogene 33:212-224, 2014;Li et al.,CDDis,2013, e943; doi:10.10384). In this study, we show that metformin can induce instability of ΔNp63α protein, leading to subsequent anoikis. In addition, glucose restriction or inhibition of glycolysis significantly sensitizes cells to metformin. Furthermore, we show that integrin β1 is involved in this process. Taken together, this study reveals a novel molecular mechanism by which metformin inhibits squamous carcinoma formation in vitro and in vivo.

Citation Format: Yong Yi, Zhi-Xiong Jim Xiao. Inhibition of glycolysis sensitizes cancer cells to metformin-induced anoikis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2856.
Abstract 2859: Chloroquine and vitamin D3 modulate proliferation in early stage breast cancer models
Authors: Espina Virginia A. , Yeon Solomon , VanHouten Joshua N. , Wysolmerski John , Liotta Lance A.
Published: July 2016

The defining feature of premalignant mammary lesions is proliferation of cells within the intraductal niche. Survival in this stressful duct microenvironment requires sophisticated mechanisms to avoid apoptosis caused by hypoxia, nutrient deprivation, metabolic acidosis, and intracellular calcium overload. Two major survival mechanisms of breast ductal carcinoma cells (DCIS) are 1) the upregulation of autophagy and 2) the enhanced export of calcium via plasma membrane calcium-ATPase isoform 2 (PMCA2) efflux pumps. We hypothesize that calcium efflux, through PMCA2, and activation of autophagic catabolism are critical interdependent mechanisms for the survival of pre-invasive human breast neoplastic cells.

To assess the proteomic response to autophagy inhibition and/or elevated intracellular calcium, 41 signal transduction proteins/post-translationally modified proteins related to proliferation, autophagy, and calcium pathways were quantified by reverse phase protein microarrays (RPPA) in time course studies of T-47D metastatic ductal breast cancer cell line, and MCF-10A non-tumorigenic breast cell line, both of which express low basal levels of PMCA2, and in human primary DCIS organoid cultures. Cell lines were treated with chloroquine (50μM), or 1,25 dihydroxyvitamin D3 (Calcitriol) (50nM or 100nM), or chloroquine + Calcitriol, and harvested at periodic intervals over 197 hours. Stoichiometric time course studies (24 hours) were evaluated for Calcitriol as a single agent in both T-47D and MCF-10A cell lines. Cell morphology, adhesion, proliferation and autophagic flux in the DCIS organoids were assessed by Immunohistochemistry and RPPA.

Calcitriol alone did not increase proliferation based on PCNA expression within DCIS ducts. Chloroquine markedly increased Cleaved PARP (Chi Sq p = 0.004) and CAMKinase II (p = 0.0068). Chloroquine (50μM) plus Calcitriol (100nM) increased E-Cadherin (p = 0.0379) and phosphoPAK1/2 (p = 0.0163), while PMCA2 levels were not significantly different between treatment groups.

Autophagy inhibition and calcium efflux may be exploited therapeutically by co-administration of chloroquine and Calcitrol, to inhibit lysosomal catabolism and increase intracellular calcium levels, respectively, switching them from autophagic survival into apoptotic death.

Citation Format: Virginia A. Espina, Solomon Yeon, Joshua N. VanHouten, John Wysolmerski, Lance A. Liotta. Chloroquine and vitamin D3 modulate proliferation in early stage breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2859.