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GZ17-6.02 is undergoing clinical evaluation in solid tumors and lymphoma. The present studies were performed to define its biology in renal carcinoma cells and to determine whether it interacted with axitinib to enhance tumor cell killing. GZ17-6.02 interacted in an arithmetically greater than additive fashion with axitinib to kill kidney cancer cells. GZ17-6.02 and axitinib cooperated to inactivate ERBB2, c-MET, c-KIT, c-SRC, the AMPK, STAT3, STAT5 and eIF2α and to activate PERK, ULK1 and ATG13. The drugs interacted to increase the expression of FAS-L and to decrease the levels of MCL1, BCL-XL, and HDACs 1-3. The drugs as single agents inactivated the Hippo pathway. GZ17-6.02 and axitinib interacted to enhance autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5, eIF2α, toxic BH3 domain proteins or CD95/FADD significantly reduced drug combination lethality. GZ17-6.02 and axitinib increased the expression of BAK, BIM, Beclin1 and ATG5, effects blocked by knock down of eIF2α. The drugs increased phosphorylation of ULK1 S757 and ATG13 S318 and decreased the phosphorylation of mTORC1 and mTORC2, effects blocked by knock down of AMPKα. Knock down of Beclin1 or ATG5 prevented the drug combination reducing expression of HDACs 1-3 and from enhancing the expression of MHCA. Knock down of HDACs 1-3 enhanced MHCA expression. We conclude that GZ17-6.02 and axitinib interact to kill requiring ER stress signaling, autophagy and death receptor signaling. Autophagic degradation of HDACs played a key role in enhancing MHCA expression and of a potential improved response to checkpoint inhibitory immunotherapy.

GZ17-6.02 is presently undergoing clinical evaluation in solid tumors and lymphoma. The present studies were performed to define its biology in estrogen receptor positive breast cancer cells and to determine whether it interacted with palbociclib to enhance tumor cell killing. GZ17-6.02 interacted in an additive fashion with palbociclib to kill ER+ breast cancer cells. GZ17-6.02 and palbociclib cooperated to inactivate mTOR and AKT and to activate ULK1 and PERK. The drugs interacted to increase the expression of FAS-L and BAX, and to decrease the levels of MCL1, the estrogen receptor, and HDACs 1–3. Palbociclib activated ERBB3, an effect blocked by GZ17-6.02. GZ17-6.02 and palbociclib interacted to increase the expression of multiple toxic BH3 domain proteins and to reduce MCL1 and BCL-XL expression. Knock down of FAS-L reduced the lethality of [GZ17-6.02 + palbociclib]. GZ17-6.02 and palbociclib interacted to enhance autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5, BAG3, eIF2α, toxic BH3 domain proteins or CD95 significantly reduced drug combination lethality. GZ17-6.02 and palbociclib increased the expression of Beclin1 and ATG5, effects blocked by knock down of eIF2α. The drugs also increased the phosphorylation of the AMPK and ATG13, effects blocked by knock down of ATM. Knock down of ATM or the AMPK, or expression of activated mTOR significantly reduced the abilities of GZ17-6.02 and palbociclib to enhance autophagosome formation and autophagic flux.

This paper aims to systematically review the major findings from meta-analyses comparing different treatment options for hepatocellular carcinoma (HCC). A total of 153 relevant papers were searched via the PubMed, EMBASE, and Cochrane library databases. They were classified according to the mainstay treatment modalities (i.e., liver transplantation, surgical resection, radiofrequency ablation, transarterial embolization or chemoembolization, sorafenib, and others). The primary outcome data, such as overall survival, diseases-free survival or recurrence-free survival, progression-free survival, and safety, were summarized. The recommendations and uncertainties regarding the treatment of HCC were also proposed.

// Qing Xie 1, * , Shuai Wang 2, * , Yue Zhao 1 , Zhenchao Zhang 2 , Chuan Qin 1 , Xianjun Yang 1 1 Tumor Signaling and Transduction Lab, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang City, Henan Province, 453003, PR China 2 Department of Human Parasitology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang City, Henan Province, 453003, PR China * These authors have contributed equally to this work Correspondence to: Qing Xie, email: hnxxqingxie@126.com Keywords: BCSCs, miR-519d, cisplatin, resistance, MCL-1 Received: December 28, 2016 Accepted: January 25, 2017 Published: February 28, 2017 ABSTRACT Cancer stem cells are considered as the cell population which is responsible for chemoresistance and treatment failure in breast cancer patients. Therefore, it is urgent to explore the mechanism by which cancer stem cells survive under the treatment of chemotherapeutic drugs such as cisplatin. In this paper, we demonstrated significant decrease of miR-519d in breast cancer stem cells by quantitative RT-PCR analysis. Furthermore, we found the enforced expression of miR-519d in T-47D-cancer stem cells significantly increased their sensitivity to cisplatin through the apoptosis pathway. In addition, the gene of MCL-1, which is a member of pro-apoptotic Bcl-2 family, was found to be the target of miR-519d in T-47D-cancer stem cells. Our date demonstrated that enforced miR-519d expression enhanced the cisplatin-induced apoptosis through the MCL-1 dependent mitochondria pathway in breast cancer stem cells. Taken together, the present study suggests that miR-519d reduces chemoresistance in breast cancer stem cells, and understanding of miR-519d may be helpful for increasing the efficacy of chemotherapy.

// Artur Kadurin 1, 2, 3, 4 , Alexander Aliper 2 , Andrey Kazennov 2, 7 , Polina Mamoshina 2, 5 , Quentin Vanhaelen 2 , Kuzma Khrabrov 1 , Alex Zhavoronkov 2, 6, 7 1 Search Department, Mail.Ru Group Ltd., Moscow, Russia 2 Pharmaceutical Artificial Intelligence Department, Insilico Medicine, Inc., Emerging Technology Centers, Johns Hopkins University at Eastern, Baltimore, Maryland, USA 3 Big Data and Text Analysis Laboratory, Kazan Federal University, Kazan, Republic of Tatarstan, Russia 4 St. Petersburg Department of V.A. Steklov Institute of Mathematics of the Russian Academy of Sciences, Petersburg, Russia 5 Department of Computer Science, University of Oxford, Oxford, UK 6 The Biogerontology Research Foundation, Trevissome Park, Truro TR4 8UN, UK 7 Moscow Institute of Physics and Technology, Dolgoprudny, Russia Correspondence to: Alex Zhavoronkov, email: alex@insilicomedicine.com Keywords: generative adversarian networks, adversarial autoencoder, deep learning, drug discovery, artificial intelligence Received: June 14, 2016 Accepted: November 24, 2016 Published: December 22, 2016 ABSTRACT Recent advances in deep learning and specifically in generative adversarial networks have demonstrated surprising results in generating new images and videos upon request even using natural language as input. In this paper we present the first application of generative adversarial autoencoders (AAE) for generating novel molecular fingerprints with a defined set of parameters. We developed a 7-layer AAE architecture with the latent middle layer serving as a discriminator. As an input and output the AAE uses a vector of binary fingerprints and concentration of the molecule. In the latent layer we also introduced a neuron responsible for growth inhibition percentage, which when negative indicates the reduction in the number of tumor cells after the treatment. To train the AAE we used the NCI-60 cell line assay data for 6252 compounds profiled on MCF-7 cell line. The output of the AAE was used to screen 72 million compounds in PubChem and select candidate molecules with potential anti-cancer properties. This approach is a proof of concept of an artificially-intelligent drug discovery engine, where AAEs are used to generate new molecular fingerprints with the desired molecular properties.

// Nicolle M. Linnerth-Petrik 1 , Lisa A. Santry 1 , Roger Moorehead 2 , Manfred Jucker 3 , Sarah K. Wootton 1, * , Jim Petrik 2, * 1 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada 2 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada 3 Center of Experimental Medicine, Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Hamburg, Germany * These authors have contributed equally to this work Correspondence to: Jim Petrik, email: jpetrik@uoguelph.ca Keywords: ovarian cancer, Akt isoforms, Akt inhibitors, tumor development Received: March 21, 2016 Accepted: July 27, 2016 Published: August 11, 2016 ABSTRACT Ovarian cancer remains a significant therapeutic problem and novel, effective therapies are needed. Akt is a serine-threonine kinase that is overexpressed in numerous cancers, including ovarian. Mammalian cells express three Akt isoforms which are encoded by distinct genes. Although there are several Akt inhibitors in clinical trials, most indiscriminately target all isoforms. Current in vitro data and animal knockout experiments suggest that the Akt isoforms may have divergent roles. In this paper, we determined the isoform-specific functions of Akt in ovarian cancer cell proliferation in vitro and in ovarian cancer progression in vivo. For in vitro experiments, murine and human ovarian cancer cells were treated with Akt inhibitors and cell viability was assessed. We used two different in vivo approaches to identify the roles of Akt isoforms in ovarian cancer progression and their influence on the primary tumor and tumor microenvironment. In one experiment, wild-type C57Bl6 mice were orthotopically injected with ID8 cells with stable knockdown of Akt isoforms. In a separate experiment, mice null for Akt 1-3 were orthotopically injected with WT ID8 cells ( Figure 1 ). Our data show that inhibition of Akt1 significantly reduced ovarian cancer cell proliferation and inhibited tumor progression in vivo . Conversely, disruption of Akt2 increased tumor growth. Inhibition of Akt3 had an intermediate phenotype, but also increased growth of ovarian cancer cells. These data suggest that there is minimal redundancy between the Akt isoforms in ovarian cancer progression. These findings have important implications in the design of Akt inhibitors for the effective treatment of ovarian cancer.

// Giada Poli 1, * , Giulia Cantini 1, * , Roberta Armignacco 1, * , Rossella Fucci 1 , Raffaella Santi 2 , Letizia Canu 1 , Gabriella Nesi 2 , Massimo Mannelli 1 , Michaela Luconi 1 1 Endocrinology Unit, Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence Italy 2 Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Florence Italy * These authors contributed equally to this work Correspondence to: Michaela Luconi, email: michaela.luconi@unifi.it Keywords: metformin, H295R, IGF-1R, apoptosis, tumor proliferation Received: March 24, 2016 Accepted: June 09, 2016 Published: July 06, 2016 ABSTRACT Adrenocortical carcinoma (ACC) is a rare heterogeneous malignancy with poor prognosis. Since radical surgery is the only available treatment, more specific and effective drugs are urgently required. The anti-diabetic drug metformin has been associated with a decreased cancer prevalence and mortality in several solid tumors, prompting its possible use for ACC treatment. This paper evaluates the in vitro and in vivo anti-cancer effects of metformin using the ACC cell model H295R. Metformin treatment significantly reduces cell viability and proliferation in a dose- and time-dependent manner and associates with a significant inhibition of ERK1/2 and mTOR phosphorylation/activation, as well as with stimulation of AMPK activity. Metformin also triggers the apoptotic pathway, shown by the decreased expression of Bcl-2 and HSP27, HSP60 and HSP70, and enhanced membrane exposure of annexin V, resulting in activation of caspase-3 apoptotic effector. Metformin interferes with the proliferative autocrine loop of IGF2/IGF-1R, which supports adrenal cancer growth. Finally, in the ACC xenograft mouse model, obtained by subcutaneous injection of H295R cells, metformin intraperitoneal administration inhibits tumor growth, confirmed by the significant reduction of Ki67%. Our data suggest that metformin inhibits H295R cell growth both in vitro and in vivo . Further preclinical studies are necessary to validate the potential anti-cancer effect of metformin in patients affected by ACC.

// Peter E. Ghaly 1 , Rabab M. Abou El-Magd 2, 4 , Cassandra D. M. Churchill 1 , Jack A. Tuszynski 2, 3 , F. G. West 1 1 Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada 2 Department of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada 3 Department of Physics, University of Alberta, Edmonton, AB T6G 2E1, Canada 4 Genetic Engineering and Biotechnology Institute, City of Scientific Research and Technological Application, New Borg El-Arab City, Alexandria, 21934, Egypt Correspondence to: F. G. West, email: frederick.west@ualberta.ca Jack A. Tuszynski, email: jackt@ualberta.ca Keywords: noscapine, tubulin, microtubules, fluorescence quenching, docking Received: October 28, 2015 Accepted: April 10, 2016 Published: May 26, 2016 ABSTRACT Noscapine, a naturally occurring opium alkaloid, is a widely used antitussive medication. Noscapine has low toxicity and recently it was also found to possess cytotoxic activity which led to the development of many noscapine analogues. In this paper we report on the synthesis and testing of a novel noscapine analogue. Cytotoxicity was assessed by MTT colorimetric assay using SKBR-3 and paclitaxel-resistant SKBR-3 breast cancer cell lines using different concentrations for both noscapine and the novel compound. Microtubule polymerization assay was used to determine the effect of the new compound on microtubules. To compare the binding affinity of noscapine and the novel compound to tubulin, we have done a fluorescence quenching assay. Finally, in silico methods using docking calculations were used to illustrate the binding mode of the new compound to α,β-tubulin. Our cytotoxicity results show that the new compound is more cytotoxic than noscapine on both SKBR-3 cell lines. This was confirmed by the stronger binding affinity of the new compound, compared to noscapine, to tubulin. Surprisingly, our new compound was found to have strong microtubule-destabilizing properties, while noscapine is shown to slightly stabilize microtubules. Our calculation indicated that the new compound has more binding affinity to the colchicine-binding site than to the noscapine site. This novel compound has a more potent cytotoxic effect on cancer cell lines than its parent, noscapine, and hence should be of interest as a potential anti-cancer drug.

// Shogo Hosogoe 1 , Shingo Hatakeyama 1 , Ayumu Kusaka 1 , Itsuto Hamano 1 , Yoshimi Tanaka 1 , Kazuhisa Hagiwara 1 , Hideaki Hirai 2 , Satoko Morohashi 2 , Hiroshi Kijima 2 , Hayato Yamamoto 1 , Yuki Tobisawa 1 , Tohru Yoneyama 3 , Takahiro Yoneyama 1 , Yasuhiro Hashimoto 3 , Takuya Koie 1 and Chikara Ohyama 1, 3 1 Department of Urology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan 2 Department of Pathology and Bioscience, Hirosaki University Graduate School of Medicine, Hirosaki, Japan 3 Department of Advanced Transplant and Regenerative Medicine, Hirosaki University Graduate School of Medicine, Hirosaki, Japan Correspondence to: Shingo Hatakeyama, email: shingoh@hirosaki-u.ac.jp Keywords: renal cell carcinoma, presurgical therapy, axitinib, radiological response Received: March 29, 2017 Accepted: May 04, 2017 Published: May 17, 2017 ABSTRACT Background and Objective: A quantitative tumor response evaluation to molecular-targeting agents in advanced renal cell carcinoma (RCC) is debatable. We aimed to evaluate the relationship between radiologic tumor response and pathological response in patients with advanced RCC who underwent presurgical therapy. Results: Of 34 patients, 31 underwent scheduled radical nephrectomy. Presurgical therapy agents included axitinib ( n = 26), everolimus ( n = 3), sunitinib ( n = 1), and axitinib followed by temsirolimus ( n = 1). The major presurgical treatment-related adverse event was grade 2 or 3 hypertension (44%). The median radiologic tumor response by RECIST, Choi, and CMER were −19%, −24%, and −49%, respectively. Among the radiologic tumor response tests, CMER showed a higher association with tumor necrosis in surgical specimens than others. Ki67/MIB1 status was significantly decreased in surgical specimens than in biopsy specimens. The magnitude of the slope of the regression line associated with the tumor necrosis percentage was greater in CMER than in Choi and RECIST. Materials and Methods: Between March 2012 and December 2016, we prospectively enrolled 34 locally advanced and/or metastatic RCC who underwent presurgical molecular-targeting therapy followed by radical nephrectomy. Primary endpoint was comparison of radiologic tumor response among Response Evaluation Criteria in Solid Tumors (RECIST), Choi, and contrast media enhancement reduction (CMER). Secondary endpoint included pathological downstaging, treatment related adverse events, postoperative complications, Ki67/MIB1 status, and tumor necrosis. Conclusions: CMER may predict tumor response after presurgical molecular-targeting therapy. Larger prospective studies are needed to develop an optimal tumor response evaluation for molecular-targeting therapy.

// Ravyn M. Thompson 1 , Dominik Dytfeld 2 , Leticia Reyes 1 , Reeder M. Robinson 1 , Brittany Smith 1 , Yefim Manevich 1 , Andrzej Jakubowiak 3 , Mieczyslaw Komarnicki 2 , Anna Przybylowicz-Chalecka 2 , Tomasz Szczepaniak 2 , Amit K. Mitra 5 , Brian G. Van Ness 5 , Magdalena Luczak 4 , Nathan G. Dolloff 1, * 1 Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA 2 Karol Marcinkowski University of Medical Sciences, Poznan, Poland 3 University of Chicago, Chicago, IL, USA 4 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland 5 University of Minnesota, Minneapolis, MN, USA * These authors contributed equally to this work Correspondence to: Nathan G. Dolloff, email: dolloffn@musc.edu Keywords: multiple myeloma, proteasome inhibitor, glutaminase, carfilzomib Received: December 08, 2016 Accepted: March 01, 2017 Published: March 16, 2017 ABSTRACT Curative responses in the treatment of multiple myeloma (MM) are limited by the emergence of therapeutic resistance. To address this problem, we set out to identify druggable mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib), which are cornerstone agents in the treatment of MM. In isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. PI resistant cells exhibited increased mitochondrial respiration driven by glutamine as the principle fuel source. To target glutamine-induced respiration in PI resistant cells, we utilized the glutaminase-1 inhibitor, CB-839. CB-839 inhibited mitochondrial respiration and was more cytotoxic in PI resistant cells as a single agent. Furthermore, we found that CB-839 synergistically enhanced the activity of multiple PIs with the most dramatic synergy being observed with carfilzomib (Crflz), which was confirmed in a panel of genetically diverse PI sensitive and resistant MM cells. Mechanistically, CB-839 enhanced Crflz-induced ER stress and apoptosis, characterized by a robust induction of ATF4 and CHOP and the activation of caspases. Our findings suggest that the acquisition of PI resistance involves adaptations in cellular bioenergetics, supporting the combination of CB-839 with Crflz for the treatment of refractory MM.

// Tetsuo Mashima 1 , Yoko Taneda 1, 2 , Myung-Kyu Jang 1, 2 , Anna Mizutani 1 , Yukiko Muramatsu 1 , Haruka Yoshida 1 , Ayana Sato 1, 2 , Noritaka Tanaka 1, 3 , Yoshikazu Sugimoto 3 and Hiroyuki Seimiya 1, 2 1 Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan 2 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan 3 Division of Chemotherapy, Faculty of Pharmacy, Keio University, Tokyo, Japan Correspondence to: Hiroyuki Seimiya, email: hseimiya@jfcr.or.jp Keywords: tankyrase, Wnt, colorectal cancer, resistance, mTOR Received: August 26, 2016 Accepted: May 03, 2017 Published: May 24, 2017 ABSTRACT Activation of Wnt/β-catenin signaling is essential for colorectal carcinogenesis. Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, is a positive regulator of the Wnt/β-catenin signaling. Accordingly, tankyrase inhibitors are under preclinical development for colorectal cancer (CRC) therapy. However, Wnt-driven colorectal cancer cells are not equally sensitive to tankyrase inhibitors, and cellular factors that affect tankyrase inhibitor sensitivity remain elusive. Here, we established a tankyrase inhibitor-resistant cell line, 320-IWR, from Wnt/β-catenin-dependent CRC COLO-320DM cells. 320-IWR cells exhibited resistance to tankyrase inhibitors, IWR-1 and G007-LK, but remained sensitive to a PARP-1/2 inhibitor, olaparib, and several anti-CRC agents. In 320-IWR cells, nuclear localization of active β-catenin was decreased and expression of β-catenin target genes was constitutively repressed, suggesting that these cells repressed the Wnt/β-catenin signaling and were dependent on alternative proliferation pathways. 320-IWR cells exhibited upregulated mTOR signaling and were more sensitive to mTOR inhibition than the parental cells. Importantly, mTOR inhibition reversed resistance to tankyrase inhibitors and potentiated their anti-proliferative effects in 320-IWR cells as well as in CRC cell lines in which the mTOR pathway was intrinsically activated. These results indicate that mTOR signaling confers resistance to tankyrase inhibitors in CRC cells and suggest that the combination of tankyrase and mTOR inhibitors would be a useful therapeutic approach for a subset of CRCs.

// Long Chen 1, 2, * , Lei Shi 3, * , Wenhua Wang 2, * and Youxin Zhou 1 1 Department of Neurosurgery, The First Affiliated Hospital of Soochow University, Suzhou 215006, P. R. China 2 Department of Neurosurgery, Traditional Chinese Medicine Hospital of Kunshan, Affiliated Nanjing University of Traditional Chinese Medicine, Suzhou 215300, P. R. China 3 Department of Neurosurgery, The First People’s Hospital of Kunshan Affiliated with Jiangsu University, Suzhou 215300, P. R. China * These authors have contributed equally to this work Correspondence to: Lei Shi, email: sl1012002322@126.com Youxin Zhou, email: zhouyxyq2008@sohu.com Keywords: glioma stem cells, demethoxycurcumin, reactive oxygen species, caspase-3, ABCG2 Received: February 20, 2017 Accepted: April 06, 2017 Published: May 19, 2017 ABSTRACT We analyzed the role of ABCG2, a drug transporter, in determining the sensitivity of glioma stem cells (GSCs) to demethoxycurcumin (DMC). We first demonstrated that ABCG2 is more highly expressed in GSCs than primary astrocytes. Modulation of ABCG2 levels in GSCs by transfection of ABCG2 shRNA or a lentiviral vector encoding ABCG2 revealed an inverse relation between ABCG2 levels and DMC-induced GSC growth inhibition. Suppressing ABCG2 increased DMC-induced apoptosis and G0/G1 cell cycle arrest in GSCs. It also increased levels reactive oxygen species (ROS) in GSCs treated with DMC, resulting in increased cytochrome C and caspase-3 activity. When GSCs transfected with ABCG2 shRNA or overexpressing ABCG2 were xenografted and the tumor-bearing, immunodeficient mice were treated with DMC, ABCG2 expression suppressed the tumor proliferation rate (T/C %). These findings demonstrate that ABCG2 expression is critical for DMC resistance in GSCs and is a potential therapeutic target for GBM.

// Yongmei Liu 1, * , Yan Zhang 1, * , Li Zhang 2 , Bin Liu 3 , Yongsheng Wang 1 , Xiaojuan Zhou 1 , Yanying Li 1 , Qian Zhao 4 , Youling Gong 1 , Lin Zhou 1 , Jiang Zhu 1 , Zhenyu Ding 1 , Jin Wang 1 , Feng Peng 1 , Meijuan Huang 1 , Lu Li 1 , Li Ren 1 and You Lu 1 1 Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China 2 Department of Medical Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, China 3 Pulmonary Tumor Ward, Sichuan Cancer Hospital, Chengdu, China 4 West China School of Medicine, Sichuan University, Chengdu, China * These authors contributed equally to this work Correspondence to: You Lu, email: radyoulu@hotmail.com Keywords: squamous cell carcinoma, EGFR mutation, epidermal growth factor receptor-tyrosine kinase inhibitor Received: February 14, 2017 Accepted: May 01, 2017 Published: May 17, 2017 ABSTRACT Epidermal growth factor receptor (EGFR) mutations are common in lung adenocarcinoma (ADC) but rare in squamous cell carcinoma (SQC). The efficacy of EGFR-tyrosine kinase inhibitors (EGFR-TKIs) for SQC with EGFR mutations is unclear. The aim of this study was to evaluate the efficacy of EGFR-TKIs for these patients. We performed a retrospective matched-pair case-control study from 3 cancer centers, including 44 SQC and 44 ADC patients with EGFR mutation who were treated with EGFR-TKI. Subsequently, we performed a pooled analysis on the efficacy of EGFR-TKIs for EGFR-mutant SQC in 115 patients, including 71 patients selected from 25 published reports. In our multicenter study, EGFR-mutant SQC and ADC patients had similar objective response rate (ORR) (43.2% vs. 54.5%, p = 0.290), but SQC patients had lower disease control rate (DCR) (71.3% vs. 100%, p = 0.001), significant shorter median progression free survival (PFS) (5.1 vs. 13.0 months, p = 0.000) and median overall survival (OS) (17.2 vs. 23.6 months, p = 0.027). In pooled analysis, the ORR, DCR, PFS and OS of SQC patients were 39.1%, 71.3%, 5.6 months and 15.0 months, respectively. Performance status was the only independent predictor of PFS and erlotinib treatment was associated with a better survival. In conclusion, EGFR-TKI was less effective in EGFR-mutant SQC than in ADC but still has clinical benefit for SQC patients. Further study is need to evaluate the using of EGFR-TKIs in these SQC patients.

// Di Zheng 1, * , Min Hu 2, * , Yu Bai 2 , Xuehua Zhu 2 , Xuesong Lu 3 , Chunyan Wu 4 , Jiying Wang 1 , Li Liu 1 , Zheng Wang 3 , Jian Ni 1 , Zhenfan Yang 2 and Jianfang Xu 1 1 Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School, Shanghai, China 2 IMED Asia, AstraZeneca, Shanghai, China 3 Research and Development Information, AstraZeneca, Shanghai, China 4 Department of Pathology, Shanghai Pulmonary Hospital, Tongji University Medical School, Shanghai, China * These authors contributed equally to this work Correspondence to: Jianfang Xu, email: xujianfang63@aliyun.com Zhenfan Yang, email: Pamela.Yang@astrazeneca.com Keywords: EGFR, NSCLC, osimertinib, drug resistance, G796D Received: March 08, 2017 Accepted: May 04, 2017 Published: May 16, 2017 ABSTRACT Osimertinib is an effective third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved in multiple countries and regions for patients with EGFR T790M mutation-positive non-small cell lung cancer (NSCLC). Despite impressive initial tumor responses, development of drug resistance ultimately limits the benefit of this compound. Mechanisms of resistance to osimertinib are just beginning to emerge, such as EGFR C797S and L718Q mutations, BRAF V600E and PIK3CA E545K mutations, as well as ERBB2 and MET amplification. However, a comprehensive view is still missing. In this study, we presented the first case of Chinese NSCLC patient who developed resistance to osimertinib, and discovered de novo EGFR G796D mutation as a potential mechanism. Our findings provided insights into mechanisms of resistance to osimertinib and highlighted tumor heterogeneity and clonal evolution during the development of drug resistance.

Chemotherapy is the major choice for the cancer treatment of early and advanced stages. However, intrinsic or acquired drug resistance significantly restricts the clinical efficacy of chemotherapy. It is critical to develop novel approaches to detect and overcome drug resistance. In this study, we demonstrated that accelerated glycolysis played a pivotal role in both intrinsic and acquired cisplatin-resistance of gastric cancer cells. The metabolic reprogramming of cisplatin-resistant cells was characterized by increased glycolysis dependence. Inhibition of glycolysis with glucose starvation or 2-Deoxy-D-glucose (2-DG) treatment significantly reversed drug resistance. By proteomic screening, we found the increased expression of the glycolytic enzyme Enolase 1 (ENO1) in cisplatin-resistant gastric cancer cells. Depletion of ENO1 by siRNA significantly reduced glycolysis and reversed drug resistance. Moreover, the increased expression of ENO1 was attributed to the down-regulation of ENO1-targeting miR-22, rather than activated gene transcriptional or prolonged protein stability. Finally, the elevated levels of ENO1 proteins were associated with the shorter overall survival of gastric cancer patients. In conclusion, ENO1 is a novel biomarker to predict drug resistance and overall prognosis in gastric cancer. Targeting ENO1 by chemical inhibitors or up-regulating miR-22 could be valuable to overcome drug resistance.

// Loredana Urso 1 , Ilaria Cavallari 2, * , Micol Silic-Benussi 2, * , Lorena Biasini 2 , Giulia Zago 3 , Fiorella Calabrese 4 , Pier Franco Conte 1, 3 , Vincenzo Ciminale 1, 2 and Giulia Pasello 3 1 Department of Surgery, Oncology and Gastroenterology, University of Padova, 35128, Padova, Italy 2 Immunology and Molecular Oncology Unit, Veneto Institute of Oncology, IRCCS, 35128, Padova, Italy 3 Medical Oncology Unit 2, Veneto Institute of Oncology, IRCCS, 35128, Padova, Italy 4 Department of Cardio-Thoracic and Vascular Sciences, University of Padova, 35128, Padova, Italy * These authors contributed equally to this work Correspondence to: Vincenzo Ciminale, email: v.ciminale@unipd.it Keywords: mesothelioma, MDM2, p53, RG7112, TRAIL Received: March 10, 2017 Accepted: April 24, 2017 Published: May 11, 2017 ABSTRACT Malignant Pleural Mesothelioma (MPM) is a chemoresistant tumor characterized by low rate of p53 mutation and upregulation of Murine Double Minute 2 (MDM2), suggesting that it may be effectively targeted using MDM2 inhibitors. In the present study, we investigated the anticancer activity of the MDM2 inhibitors Nutlin 3a ( in vitro ) and RG7112 ( in vivo ), as single agents or in combination with rhTRAIL. In vitro studies were performed using MPM cell lines derived from epithelioid (ZL55, M14K), biphasic (MSTO211H) and sarcomatoid (ZL34) MPMs. In vivo studies were conducted on a sarcomatoid MPM mouse model. In all the cell lines tested (with the exception of ZL55, which carries a biallelic loss-of-function mutation of p53), Nutlin 3a enhanced p21, MDM2 and DR5 expression, and decreased survivin expression. These changes were associated to cell cycle arrest but not to a significant induction of apoptosis. A synergistic pro-apoptotic effect was obtained through the association of rhTRAIL in all the cell lines harboring functional p53. This synergistic interaction of MDM2 inhibitor and TRAIL agonist was confirmed using a mouse preclinical model. Our results suggest that the combined targeting of MDM2 and TRAIL might provide a novel therapeutic option for treatment of MPM patients, particularly in the case of sarcomatoid MPM with MDM2 overexpression and functional inactivation of wild-type p53.

// Baoan Hong 1 , Yong Yang 2 , Sheng Guo 3 , Shayiremu Duoerkun 4 , Xiaohu Deng 5 , Dawei Chen 3 , Shijun Yu 3 , Wubin Qian 3 , Qixiang Li 3 , Qing Li 6 , Kan Gong 1 and Ning Zhang 2 1 Department of Urology, Peking University First Hospital, Institute of Urology, Peking University, Beijing, P.R. China 2 Department of Urology, Beijing Cancer Hospital, Beijing Institute for Cancer Research, Beijing, P.R. China 3 Division of Translational Oncology, Crown Bioscience, Taicang, Jiangsu, P.R. China 4 Department of Urology, Central Hospital of HaMi Region, Xinjiang, P.R. China 5 Department of Urology, People’s Hospital of Kelamayi, Xinjiang, P.R. China 6 Center for Cellular & Structural Biology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, P.R. China Correspondence to: Ning Zhang, email: niru7429@126.com Kan Gong, email: gongkan2013@163.com Keywords: clear cell renal cell carcinoma, molecular heterogeneity, targeted therapy, patient-derived xenograft model, precision medicine Received: December 08, 2016 Accepted: April 26, 2017 Published: May 10, 2017 ABSTRACT Inter- and intra-tumour molecular heterogeneity is increasingly recognized in clear cell renal cell carcinoma (ccRCC). It may partially explain the diversity of responses to targeted therapies and the various clinical outcomes. In this study, a 56-year-old male ccRCC patient with multiple metastases received radical nephrectomy and resection of the metastatic tumour in chest wall. The surgical specimens were implanted into nude mice to establish patient-derived xenograft (PDX) models with KI2367 model derived from the primary tumour and KI2368 model from the metastastic tumour. The two modles were treated with Sorafenib, Sunitinib, Axitinib, combined Sorafenib/Sunitinib, or alternating therapy of Sorafenib and Sunitinib. Significant anti-tumour activity was found in KI2367 treated with Sorafenib/Sunitinib monotherapy, combined Sorafenib/Sunitinib, and alternating therapy of Sorafenib/Sunitinib (P 5-fold higher in KI2367 than in KI2368 and the expression levels of 994 genes were > 5-fold higher in KI2368 than in KI2367. These results suggest the presence of intra-tumour molecular heterogeneity in this patient. This heterogeneity may influence the response to targeted therapies. Multiple biopsy, liquid biopsy and genomic analysis of intra- tumour molecular heterogeneity may help guide a more precise and effective plan in selecting targeted therapies for ccRCC patients.

For patients with advanced hepatocellular carcinoma (HCC), sorafenib is the only systemic treatment recommended by international guidelines. We recently reported that HCC patients with a low MoRAL (model to predict tumor recurrence after LDLT) score (≤ 314.8) have excellent treatment outcomes after living-donor liver transplantation (LDLT), even though they are beyond the Milan criteria. In the present study, we investigated whether LDLT offers a better treatment outcome than sorafenib for patients with HCC beyond the Milan criteria according to the MoRAL score. A retrospective cohort study of 325 consecutive patients who were treated with either LDLT (n = 122) or sorafenib (n = 203) for HCC beyond the Milan criteria from 2005 to 2014 at a tertiary hospital was performed. The primary and secondary endpoints were overall survival (OS) and time-to-progression. When baseline characteristics were balanced using inverse probability weighting, OS was significantly longer in the LDLT group than in the sorafenib group (5-year OS rate, 71.9% vs. 4.9%; HR=0.1; P < 0.001). The LDLT group exhibited a significantly lower risk of tumor progression (5-year recurrence rate, 34.7% vs. 96%; HR=0.14; P < 0.001) than the sorafenib group. The increase in OS with LDLT was predominantly among patients with a low MoRAL score (5-year OS rate, 81.1% vs. 5.8%; HR=0.06; P < 0.001) compared with those with a high MoRAL score (5-year OS rate, 28.3% vs. 4.3%; HR = 0.42; P = 0.047). Patients with a low MoRAL score and without extrahepatic metastasis or hepatic vein invasion might be good candidates for LDLT instead of sorafenib treatment if there is a willing living related donor.

// Gordon W. Rewcastle 1, 2 , Sharada Kolekar 3 , Christina M. Buchanan 2, 3 , Swarna A. Gamage 1 , Anna C. Giddens 1 , Kit Y. Tsang 1 , Jackie D. Kendall 1, 2 , Ripudaman Singh 1 , Woo-Jeong Lee 3 , Greg C. Smith 4 , Weiping Han 5 , David J. Matthews 6 , William A. Denny 1, 2 , Peter R. Shepherd 2, 3 and Stephen M.F. Jamieson 1, 2, 7 1 Auckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand 2 Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand 3 Department of Molecular Medicine and Pathology, School of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand 4 Department of Pharmacology, School of Medical Sciences, The University of New South Wales, Sydney, Australia 5 Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore 6 PharmIntuition, San Francisco, CA, USA 7 Department of Pharmacology and Clinical Pharmacology, School of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand Correspondence to: William A. Denny, email: b.denny@auckland.ac.nz Keywords: SN32976, phosphatidylinositol 3-kinase, pan PI3K inhibitor, PI3Kα-preferential, kinase selectivity Received: January 31, 2017 Accepted: April 14, 2017 Published: May 09, 2017 ABSTRACT Multiple therapeutic agents have been developed to target the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is frequently dysregulated in cancer promoting tumor growth and survival. These include pan PI3K inhibitors, which target class Ia PI3K isoforms and have largely shown limited single agent activity with narrow therapeutic windows in clinical trials. Here, we characterize SN32976, a novel pan PI3K inhibitor, for its biochemical potency against PI3K isoforms and mTOR, kinase selectivity, cellular activity, pharmacokinetics, pharmacodynamics and antitumor efficacy relative to five clinically-evaluated pan PI3K inhibitors: buparlisib, dactolisib, pictilisib, omipalisib and ZSTK474. SN32976 potently inhibited PI3K isoforms and mTOR, displaying preferential activity for PI3Kα and sparing of PI3Kδ relative to the other inhibitors, while showing less off-target activity than the clinical inhibitors in a panel of 442 kinases. The major metabolites of SN32976 were also potent PI3K inhibitors with similar selectivity for PI3Kα as the parent compound. SN32976 compared favorably with the clinically-evaluated PI3K inhibitors in cellular assays, inhibiting pAKT expression and cell proliferation at nM concentrations, and in animal models, inducing a greater extent and duration of pAKT inhibition in tumors than pictilisib, dactolisib and omipalisib at similarly tolerated dose levels and inhibiting tumor growth to a greater extent than dactolisib and ZSTK474 and with similar efficacy to pictilisib and omipalisib. These results suggest that SN32976 is a promising clinical candidate for cancer therapy with enhanced kinase selectivity and preferential inhibition of PI3Kα compared to first generation pan PI3K inhibitors, while retaining comparable anticancer activity.

// Simona Salati 1 , Valentina Salvestrini 2 , Chiara Carretta 1 , Elena Genovese 1 , Sebastiano Rontauroli 1 , Roberta Zini 1 , Chiara Rossi 1 , Samantha Ruberti 1 , Elisa Bianchi 1 , Greta Barbieri 1 , Antonio Curti 2 , Fausto Castagnetti 2 , Gabriele Gugliotta 2 , Gianantonio Rosti 2 , Micaela Bergamaschi 3 , Agostino Tafuri 4 , Enrico Tagliafico 5 , Roberto Lemoli 3 and Rossella Manfredini 1 1 Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy 2 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. and A. Seragnoli”, University of Bologna, S.Orsola-Malpighi Hospital, Bologna, Italy 3 Department of Internal Medicine (DiMI), Clinic of Hematology, University of Genoa, IRCCS Azienda Ospedaliera Universitaria S. Martino-IST, Genoa, Italy 4 Department of Clinical and Molecular Medicine, Clinic of Hematology, University “La Sapienza”, Rome, Italy 5 Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy Correspondence to: Rossella Manfredini, email: rossella.manfredini@unimore.it Simona Salati, email: simona.salati@unimore.it Keywords: chronic myeloid leukemia, leukemic stem cells, tyrosine kinase inhibitors, microRNAs, apoptosis Received: January 10, 2017 Accepted: April 24, 2017 Published: May 08, 2017 ABSTRACT The development of Imatinib mesylate (IM), which targets the oncogenic BCR-ABL fusion protein, has greatly improved the outcome of Chronic Myeloid Leukemia (CML) patients. However, BCR-ABL–positive progenitors can be detected in CML patients in complete cytogenetic response. Several evidence suggests that CML stem cells are intrinsically resistant to Tyrosine Kinase Inhibitors (TKI), and therefore they represent the most likely candidate responsible for disease relapse. In this work, we investigated the microRNA (miRNA) expression profile of different subpopulations of CML Leukemic Stem Cells (LSCs): Lin-CD34+CD38- and Lin-CD34-CD38- cells. These cell fractions have been previously shown to be endowed with TKI intrinsic resistance. Our analysis identified 33 common deregulated miRNAs in CML LSCs. Among those, 8 miRNAs were deregulated in CML independently from BCR-ABL kinase activity and therefore are likely to be involved in the BCR-ABL-independent resistance to TKI that characterizes CML LSCs. In particular, the up-regulation of miR-29a-3p and miR-660-5p observed in CML LSCs, led to the down-regulation of their respective targets TET2 and EPAS1 and conferred TKI-resistance to CML LSCs in vitro . On the other hand, miR-494-3p down-regulation in CML LSCs, leading to c-MYC up-regulation, was able to decrease TKI-induced apoptosis. These results demonstrate that aberrant miRNA expression in CML LSCs could contribute to the intrinsic TKI-resistance observed in these cell populations, and support the development of novel therapies aimed at targeting aberrantly regulated miRNAs or their targets in order to effectively eradicate CML LSCs.

// Pei-Ming Yang 1, 3 , Chia-Jung Chou 3 , Ssu-Hsueh Tseng 1 and Chien-Fu Hung 1, 2 1 Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 2 Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 3 Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Correspondence to: Chien-Fu Hung, email: chung2@jhmi.edu Keywords: cell cycle, cervical cancer, drug repurposing, flavonoid, interferon Received: July 07, 2016 Accepted: April 05, 2017 Published: May 02, 2017 ABSTRACT The clinical management and treatment of cervical cancer, one of the most commonly diagnosed cancers and a leading cause of cancer-related female death, remains a huge challenge for researchers and health professionals. Cervical cancer can be categorized into two major subtypes: common squamous cell carcinoma (SCC) and adenocarcinoma (AC). Although it is a relatively rare histological subtype of cervical cancer, there has been a steady increase in the incidences of AC. Therefore, new strategies to treat cervical cancer are urgently needed. In this study, the potential uses of IFNγ-based therapy for cervical cancer were evaluated using bioinformatics approaches. Gene expression profiling identified that cell cycle dysregulation was a major hallmark of cervical cancer including SCC and AC subtypes, and was associated with poor clinical outcomes for cervical cancer patients. In silico and in vitro experimental analyses demonstrated that IFNγ treatment could reverse the cervical cancer hallmark and induce cell cycle arrest and apoptosis. Furthermore, we demonstrated that apigenin could enhance the anticancer activity of IFNγ in a HeLa cervical AC cell line by targeting cyclin-dependent kinase 1. Taken together, the present study suggests the selective therapeutic potential of IFNγ alone or in combination with apigenin for managing cervical SCC and AC.

Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the signaling pathway status, share the common parameter which is an increase in reactive oxygen species (ROS) and undergo metabolic reprogramming. CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism. They took up twice as much glutamine and were highly sensitive to glutamine deprivation. Glutamine is hydrolyzed to glutamate for glutathione synthesis, an essential factor to abrogate high ROS via xCT antiporter. Thus, blocking glutamate flux using riluzole (an amyotropic lateral sclerosis approved drug) can selectively kill CR cells in vitro and in vivo. However, we discovered here that glutathione suppression is not the primary pathway in eradicating the CR cells. Riluzole can lead to further decrease in NAD(+) (nicotinamide adenine dinucleotide) and lactate dehydrogenase-A (LDHA) expressions which in turn further heightened oxidative stress in CR cells. LDHA knocked-down cells became hypersensitive to riluzole treatments and possessed increased levels of ROS. Addition of NAD(+) re-stabilized LDHA and reversed riluzole induced cell death. Thus far, no drugs are available which could overcome cisplatin resistance or kill cisplatin resistant cells. CR cells possess high levels of ROS and undergo metabolic reprogramming. These metabolic adaptations can be exploited and targeted by riluzole. Riluzole may serve as a dual-targeting agent by suppression LDHA and blocking xCT antiporter. Repurposing of riluzole should be considered for future treatment of cisplatin resistant lung cancer patients., Department of Veterans Affairs, BLR&D Career Development Award-2; NIH/NCI; Woman Cancer Association of the University of Miami Award

The insulin growth factor-I receptor (IGF1R) signaling is a key mechanism for osteosarcoma (OS) cell proliferation. GSK1904529A is a novel small molecule IGF1R kinase inhibitor. Its activity against OS cells was tested. In both established OS cell lines (Saos-2 and MG-63) and primary human OS cells, treatment with GSK1904529A (at nM concentrations) significantly inhibited cell proliferation. At the molecular level, GSK1904529A almost completely blocked IGF1R activation in OS cells, and inhibited downstream AKT-ERK activation. IGF1R silence by targeted shRNA also inhibited AKT-ERK activation and Saos-2 cell proliferation. Significantly, GSK1904529A was unable to further inhibit proliferation of IGF1R-silenced Saos-2 cells. In vivo, GSK1904529A administration orally inhibited Saos-2 tumor growth in nude mice. Together, these results suggest that targeting IGF1R by GSK1904529A inhibits OS cell growth in vitro and in vivo.

// Tharcisio Citrangulo Tortelli Junior 1 , Lyris Martins Franco de Godoy 2, 4, 6 , Gustavo Antonio de Souza 2, 5, 6 , Diego Bonatto 3 , Andreia Hanada Otake 1 , Renata de Freitas Saito 1 , Jose Cesar Rosa 2, 6 , Lewis Joel Greene 2, 6 and Roger Chammas 1 1 Centro de Investigacao Translacional em Oncologia (LIM24), Departamento de Radiologia e Oncologia, Faculdade de Medicina da Universidade de Sao Paulo and Instituto do Câncer do Estado de Sao Paulo, Sao Paulo, SP, Brazil 2 Departamento de Biologia Celular, Molecular e Bioagentes Patogenicos, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil 3 Centro de Biotecnologia, Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil 4 Instituto Carlos Chagas, Fiocruz Parana, PR, Brazil 5 Instituto do Cerebro, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil 6 Centro de Terapia Celular, Fundacao Hemocentro de Ribeirao Preto, Ribeirao Preto, SP, Brazil Correspondence to: Roger Chammas, email: rchammas@usp.br Lewis Joel Greene, email: ljgreene@fmrp.usp.br Keywords: melanoma, prohibitin, mitochondria, cisplatin, tunicamycin Received: December 23, 2016 Accepted: April 19, 2017 Published: May 11, 2017 ABSTRACT Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy.

// Cavan P. Bailey 1 , Tulin Budak-Alpdogan 3 , Christopher T. Sauter 1 , Michelle M. Panis 1 , Cihangir Buyukgoz 1 , Emily K. Jeng 2 , Hing C. Wong 2 , Neal Flomenberg 1 and Onder Alpdogan 1 1 Department of Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 2 Altor BioScience Corporation, Miramar, FL, USA 3 Department of Hematology and Oncology, MD Anderson Cancer Center at Cooper, Camden, NJ, USA Correspondence to: Onder Alpdogan, email: onder.alpdogan@jefferson.edu Keywords: stem cell transplantation, interleukin-15, cytokine therapy, graft-versus-tumor activity, animal models Received: August 15, 2016 Accepted: April 28, 2017 Published: May 15, 2017 ABSTRACT Interleukin-15 (IL-15) is a potent cytokine that increases CD8 + T and NK cell numbers and function in experimental models. However, obstacles remain in using IL-15 therapeutically, specifically its low potency and short in vivo half-life. To help overcome this, a new IL-15 superagonist complex comprised of an IL-15N72D mutation and IL-15RαSu/Fc fusion (IL-15SA, also known as ALT-803) was developed. IL-15SA exhibits a significantly longer serum half-life and increased in vivo activity against various tumors. Herein, we evaluated the effects of IL-15SA in recipients of allogeneic hematopoietic stem cell transplantation. Weekly administration of IL-15SA to transplant recipients significantly increased the number of CD8 + T cells (specifically CD44 + memory/activated phenotype) and NK cells. Intracellular IFN-γ and TNF-α secretion by CD8 + T cells increased in the IL-15SA-treated group. IL-15SA also upregulated NKG2D expression on CD8 + T cells. Moreover, IL-15SA enhanced proliferation and cytokine secretion of adoptively transferred CFSE-labeled T cells in syngeneic and allogeneic models by specifically stimulating the slowly proliferative and nonproliferative cells into actively proliferating cells. We then evaluated IL-15SA’s effects on anti-tumor activity against murine mastocytoma (P815) and murine B cell lymphoma (A20). IL-15SA enhanced graft-versus-tumor (GVT) activity in these tumors following T cell infusion. Interestingly, IL-15 SA administration provided GVT activity against A20 lymphoma cells in the murine donor leukocyte infusion (DLI) model without increasing graft versus host disease. In conclusion, IL-15SA could be a highly potent T- cell lymphoid growth factor and novel immunotherapeutic agent to complement stem cell transplantation and adoptive immunotherapy.

A pancreatic ductal adenocarcinoma (PDAC), obtained from a patient, was grown orthotopically in the pancreatic tail of nude mice to establish a patient-derived orthotopic (PDOX) model. Seven weeks after implantation, PDOX nude mice were divided into the following groups: untreated control (n = 7); gemcitabine (100 mg/kg, i.p., once a week for 2 weeks, n = 7); cobimetinib (5 mg/kg, p.o., 14 consecutive days, n = 7); trametinib (0.3 mg/kg, p.o., 14 consecutive days, n = 7); trabectedin (0.15 mg/kg, i.v., once a week for 2 weeks, n = 7); temozolomide (25 mg/kg, p.o., 14 consecutive days, n = 7); carfilzomib (2 mg/kg, i.v., twice a week for 2 weeks, n = 7); bortezomib (1 mg/kg, i.v., twice a week for 2 weeks, n = 7); MK-1775 (20 mg/kg, p.o., 14 consecutive days, n = 7); BEZ-235 (45 mg/kg, p.o., 14 consecutive days, n = 7); vorinostat (50 mg/kg, i.p., 14 consecutive days, n = 7). Only the MEK inhibitors, cobimetinib and trametinib, regressed tumor growth, and they were more significantly effective than other therapies (p < 0.0001, respectively), thereby demonstrating the precision of the PDOX models of PDAC and its potential for individualizing pancreatic-cancer therapy.

Prolactinomas are the most prevalent functional pituitary adenomas. The preferred treatments for prolactinomas are dopamine agonists (DAs) such as bromocriptine (BRC), but DAs still have the challenges of tumor recurrence and drug resistance. This study demonstrates that the synergy of function and mechanism between artesunate (ART) and BRC inhibits prolactinoma cell growth in vitro. We found that low-dose ART combined with BRC synergistically inhibited the growth of GH3 and MMQ cell lines, caused cell death, attenuated cell migration and invasion, and suppressed the expression of extracellular prolactin. The induction of apoptosis after co-treatment was confirmed by immunofluorescent staining, assessment of caspase-3 protein expression, and flow cytometry. Expression of miR-200c, a carcinogenic factor in pituitary adenoma, was reduced following co-treatment with ART and BRC. This was accompanied by increased expression of the antitumor factor Pten. Transfection experiments with miR-200c analogs and inhibitors confirmed that miR-200c expression was inversely associated with Pten expression. We suggest that ART and BRC used in combination exert synergistic apoptotic and antitumor effects by suppressing miR-200c and stimulating Pten expression.

// Huifang Peng 1, * , Hongwei Jin 2, * , Huiqin Zhuo 3, 4 and Heqing Huang 1, 5, 6 1 State Key Laboratory of Stress Cell Biology, School of Life Science, Xiamen University, Xiamen, Fujian 361004, China 2 Xiamen Center of Clinical Laboratory, Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China 3 Department of Gastrointestinal Surgery, Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China 4 Institute of Gastrointestinal Oncology, Medical College of Xiamen University, Xiamen University, Xiamen, Fujian 361004, China 5 State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen, Fujian 361004, China 6 The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry & Chemical Engineering, Xiamen University, Xiamen, Fujian 361004, China * These authors have contributed equally to this work Correspondence to: Heqing Huang, email: hqhuang@xmu.edu.cn Huiqin Zhuo, email: zhuohuiqin@xmu.edu.cn Keywords: cisplatin, transferrin, targeted drug delivery, ovarian cancer, antitumor treatment Received: May 12, 2016 Accepted: April 02, 2017 Published: April 21, 2017 ABSTRACT Cisplatin is a widely used anticancer drug, while non-targeted delivery, development of drug resistance, and serious side effects significantly limit its clinical use. In order to improve the tumor-targeting properties of cisplatin, transferrin (Tf) was employed as a carrier to transfer cisplatin into cancer cells via transferrin receptor 1 (TfR1) mediated endocytosis. The binding ability of cisplatin and Tf could be improved by pretreating Tf with 10% ethanol, and the binding number of cisplatin for each Tf molecule could reach to 40 without structural or functional impairment of Tf. The Tf-cisplatin complex could be delivered into human ovarian carcinoma cells high efficiently. In tumor-bearing nude-mice model, the Tf-cisplatin complex inhibited tumor growth in vivo more effectively than free cisplatin, with less toxicity in other tissues. Tumor targeting efficiency of the Tf-cisplatin complex was supported by in vivo and ex vivo imaging and platinum residues detected in each ex vivo organ. These data suggested that Tf-cisplatin was more effective and less drug-resistance than cisplatin, with targeting to tumor cells. Therefore, Tf-mediated delivery of cisplatin is a potential strategy for targeted delivery into tumor cells.

// Jung-Suk Choi 1 , Seol Kim 2 , Edward Motea 3 and Anthony Berdis 1, 2, 4, 5 1 Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA 2 Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA 3 Departments of Radiation Oncology and Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, TX 75390, USA 4 Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH 44115, USA 5 Case Comprehensive Cancer Center, Cleveland, OH 44106, USA Correspondence to: Anthony Berdis, email: a.berdis@csuohio.edu Keywords: DNA damage, DNA polymerization, chemotherapy, nucleoside analogs, leukemia Received: February 24, 2017 Accepted: April 11, 2017 Published: April 19, 2017 ABSTRACT Anti-cancer agents exert therapeutic effects by damaging DNA. Unfortunately, DNA polymerases can effectively replicate the formed DNA lesions to cause drug resistance and create more aggressive cancers. To understand this process at the cellular level, we developed an artificial nucleoside that visualizes the replication of damaged DNA to identify cells that acquire drug resistance through this mechanism. Visualization is achieved using "click" chemistry to covalently attach azide-containing fluorophores to the ethynyl group present on the nucleoside analog after its incorporation opposite damaged DNA. Flow cytometry and microscopy techniques demonstrate that the extent of nucleotide incorporation into genomic DNA is enhanced by treatment with DNA damaging agents. In addition, this nucleoside analog inhibits translesion DNA synthesis and synergizes the therapeutic activity of certain anti-cancer agents such as temozolomide. The combined diagnostic and therapeutic activities of this synthetic nucleoside analog represent a new paradigm in personalized medicine.

// Miriam S. Butler 1, *, # , Mani Roshan-Moniri 1, *, # , Michael Hsing 1, *, # , Desmond Lau 2, *, # , Ari Kim 1 , Paul Yen 1 , Marta Mroczek 1 , Mannan Nouri 1 , Scott Lien 1 , Peter Axerio-Cilies 1 , Kush Dalal 1 , Clement Yau 1 , Fariba Ghaidi 1 , Yubin Guo 1 , Takeshi Yamazaki 1 , Sam Lawn 1 , Martin E. Gleave 1 , Cheryl Y. Gregory-Evans 3 , Lawrence P. McIntosh 2, * , Michael E. Cox 1, * , Paul S. Rennie 1, * and Artem Cherkasov 1, * 1 Vancouver Prostate Centre and the Department of Urologic Sciences, University of British Columbia, Vancouver, BC V6H 3Z6, Canada 2 Department of Biochemistry and Molecular Biology, Department of Chemistry, Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada 3 Department of Ophthalmology and Visual Sciences, Eye Care Centre, University of British Columbia, Vancouver, BC V5Z 3N9, Canada * These authors contributed equally to this work # Co-first authors Correspondence to: Michael E. Cox, email: mcox@prostatecentre.com Artem Cherkasov, email: acherkasov@prostatecentre.com Keywords: prostate cancer, ERG, rational drug design, small molecule inhibitor, TMPRSS2-ERG Received: July 29, 2016 Accepted: April 04, 2017 Published: April 15, 2017 ABSTRACT Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.

// Craig T. Wallington-Beddoe 1,2,3 , Melissa K. Bennett 1,2,3 , Kate Vandyke 2,3,4 , Lorena Davies 1,2 , Julia R. Zebol 1,2 , Paul A.B. Moretti 1,2 , Melissa R. Pitman 1,2 , Duncan R. Hewett 3,4 , Andrew C.W. Zannettino 1,2,3,4,* and Stuart M. Pitson 1,2,3,* 1 Center for Cancer Biology, University of South Australia, Adelaide, Australia 2 SA Pathology, Adelaide, Australia 3 School of Medicine, University of Adelaide, Australia 4 South Australian Health and Medical Research Institute, Adelaide, Australia * Co-senior authorship of this article Correspondence to: Andrew C.W. Zannettino, email: // Stuart M. Pitson, email: // Keywords : myeloma, endoplasmic reticulum, proteasome inhibitor, sphingosine kinase Received : March 29, 2017 Accepted : April 04, 2017 Published : April 14, 2017 Abstract The proteasome inhibitor bortezomib has proven to be invaluable in the treatment of myeloma. By exploiting the inherent high immunoglobulin protein production of malignant plasma cells, bortezomib induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in myeloma cell death. In most cases, however, the disease remains incurable highlighting the need for new therapeutic targets. Sphingosine kinase 2 (SK2) has been proposed as one such therapeutic target for myeloma. Our observations that bortezomib and SK2 inhibitors independently elicited induction of ER stress and the UPR prompted us to examine potential synergy between these agents in myeloma. Targeting SK2 synergistically contributed to ER stress and UPR activation induced by bortezomib, as evidenced by activation of the IRE1 pathway and stress kinases JNK and p38MAPK, thereby resulting in potent synergistic myeloma apoptosis in vitro . The combination of bortezomib and SK2 inhibition also exhibited strong in vivo synergy and favourable effects on bone disease. Therefore, our studies suggest that perturbations of sphingolipid signalling can synergistically enhance the effects seen with proteasome inhibition, highlighting the potential for the combination of these two modes of increasing ER stress to be formally evaluated in clinical trials for the treatment of myeloma patients.

Increased glycolysis and overexpression of glucose transporters (GLUTs) are physiological characteristics of human malignancies. Based on the so-called Warburg effect, 18flurodeoxyglucose-positron emission tomography (FDG-PET) has successfully developed as clinical modality for the diagnosis and staging of many cancers. To leverage this glucose transporter mediated metabolic disparity between normal and malignant cells, in the current report, we focus on the fluorine substituted series of glucose, mannose and galactose-conjugated (trans-R,R-cyclohexane-1,2-diamine)-2-flouromalonato-platinum(II) complexes for a comprehensive evaluation on their selective tumor targeting. Besides highly improved water solubility, these sugar-conjugates presented improved cytotoxicity than oxaliplatin in glucose tranporters (GLUTs) overexpressing cancer cell lines and exhibited no cross-resistance to cisplatin. For the highly water soluble glucose-conjugated complex (5a), two novel in vivo assessments were conducted and the results revealed that 5a was more efficacious at a lower equitoxic dose (70% MTD) than oxaliplatin (100% MTD) in HT29 xenograft model, and it was significantly more potent than oxaliplatin in leukemia-bearing DBA/2 mice as well even at equimolar dose levels (18% vs 90% MTD). GLUT inhibitor mediated cell viability analysis, GLUT1 knockdown cell line-based cytotoxicity evaluation, and platinum accumulation study demonstrated that the cellular uptake of the sugar-conjugates was regulated by GLUT1. The higher intrinsic DNA reactivity of the sugar-conjugates was confirmed by kinetic study of platinum(II)-guanosine adduct formation. The mechanistic origin of the antitumor effect of the fluorine complexes was found to be forming the bifunctional Pt-guanine-guanine (Pt-GG) intrastrand cross-links with DNA. The results provide a rationale for Warburg effect targeted anticancer drug design.

// Rong Wang 1, * , Chengmeng Jin 1, * and Xun Hu 1 1 Cancer Institute, A Key Laboratory For Cancer Prevention & Intervention, Ministry of Education of the People’s Republic of China, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China * These authors have contributed equally to this work Correspondence to: Xun Hu, email: huxun@zju.edu.cn Keywords: heterogeneity, cancer, drug-response Received: November 04, 2016 Accepted: March 07, 2017 Published: April 12, 2017 ABSTRACT One cancer cell line is believed to be composed of numerous clones with different drug sensitivity. We sought to investigate the difference of drug-response pattern in clones from a cell line or from a single cell. We showed that 22 clones derived from 4T1 cells were drastically different from each other with respect to drug-response pattern against 11 anticancer drugs and expression profile of 19 genes associated with drug resistance or sensitivity. Similar results were obtained using daughter clones derived from a single 4T1 cell. Each daughter clone showed distinct drug-response pattern and gene expression profile. Similar results were also obtained using Bcap37 cells. We conclude that a single cancer cell can rapidly produce a population of cells with high heterogeneity of drug response and the acquisition of drug-response heterogeneity is random.

// Liyan Ji 1, 2, * , Simin Yang 1, * , Shasha Li 1 , Shan Liu 1 , Shunan Tang 1 , Zhongqiu Liu 2 , Xiangbao Meng 1 , Siwang Yu 1 1 Department of Chemical Biology, Peking University School of Pharmaceutical Sciences, Beijing 100191, China 2 International Institute for Translational Chinese Medicine, Guangzhou Traditional Chinese Medicine University, Guangzhou 510006, China * These authors contributed equally to this work Correspondence to: Siwang Yu, email: swang_yu@bjmu.edu.cn Xiangbao Meng, email: xbmeng@bjmu.edu.cn Keywords: triazolonaphthalimide, DNA binding, topoisomerase 2, DNA replication, cell cycle arrest Received: June 29, 2016 Accepted: March 27, 2017 Published: April 08, 2017 ABSTRACT DNA and DNA-associated processes have been classes of the most important targets of chemotherapeutic drugs. As classic DNA intercalators and topoisomerase inhibitors, naphthalimides have been extensively investigated as potential anti-cancer drugs. We recently synthesized a novel series of triazolonaphthalimides with excellent anti-cancer activities. In the present study, one of the most potent triazolonaphthalimides, LSS-11, was investigated. LSS-11 bound to DNA in vitro and in cell mainly by minor groove binding and significantly increased the stability of DNA, which could be fundamental for the biological activities of LSS-11. In addition to inhibiting DNA topoisomerase II-catalyzed decatenation of knotted circulated DNA, LSS-11 dramatically inhibited DNA replication mediated by polymerase chain reaction and isothermal helicase-dependent amplification, as well as the expression of luciferase driven by a minimal TA promoter in cell . Furthermore, LSS-11 exhibited strong cytotoxicity in selected human colon cancer cell lines by inducing cell cycle arrest and apoptosis, which was accompanied by DNA damage response. Finally, LSS-11 potently inhibited the growth of S180 murine sarcoma and SW480 human colorectal cancer xenografts in vivo without significant major toxicities. These results suggest that LSS-11 deserves further research and development as a novel anti-cancer agent, and provided new understandings of mechanisms by which LSS-11 inhibited multiple DNA-associated processes and tumor growth.

// Chencheng Xie 1 , Matthew J. Freeman 1 , Huarui Lu 1 , Xiaohong Wang 1 , Colleen L. Forster 2 , Aaron L. Sarver 3 and Timothy C. Hallstrom 1 1 Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA 2 BioNet, Academic Health Center, University of Minnesota, Minneapolis, MN, USA 3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA Correspondence to: Timothy C. Hallstrom, email: // Keywords : retinoblastoma, AKT, NVP-BEZ235, carboplatin, topotecan Received : October 19, 2016 Accepted : March 10, 2017 Published : April 08, 2017 Abstract Retinoblastoma is a pediatric cancer of the retina most often caused by inactivation of the retinoblastoma ( RB1) tumor suppressor gene. We previously showed that Rb1 loss cooperates with either co-activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, or co-deleting Pten, to initiate retinoblastoma tumors in mice. The objectives of this study were to determine if the AKT pathway is activated in human retinoblastomas and the extent that anti-PI3K therapy induces apoptosis in retinoblastoma cells, alone or in combination with the DNA damaging drugs carboplatin and topotecan. Serial sections from human retinoblastoma tissue microarrays containing 27 tumors were stained with antibodies specific to p-AKT, Ki-67, forkhead box O1 (p-FOXO1), and ribosomal protein S6 (p-S6) using immunohistochemistry and each tumor sample scored for intensity. Human retinoblastoma tumors displayed significant correlation between p-AKT intensity with highly proliferative tumors ( p = 0.008) that were also highly positive for p-FOXO1 ( p = 0.002). Treatment with BEZ235, a dual PI3K/mTOR inhibitor, reduced phosphorylation levels of the AKT targets p-FOXO and p-S6 and effectively induced apoptosis the Y79 and Weri-1 human retinoblastoma cell lines and in vivo in our retinoblastoma mouse model. Long-term treatment with BEZ235 in vivo using our retinoblastoma-bearing mice induced apoptosis but did not significantly extend the lifespan of the mice. We then co-administered BEZ235 with topotecan and carboplatin chemotherapeutics in vivo , which more effectively induced apoptosis of retinoblastoma, but not normal retinal cells than either treatment alone. Our study has increased the variety of potentially effective targeted treatments that can be considered for human retinoblastoma.

We established two patient derived tumor cells (PDCs) from right and left pulmonary metastatic lesions respectively of a patient with giant cell tumor. At that time, patient-derived tumor cells from right and left surgical specimens were collected and cultured. High-throughput screening (HTS) for 24 drugs was conducted with a micropillar/microwell chip platform using giant cell tumor PDCs. Using 6 doses per drug in 6 replicates for giant cell tumor PDCs, the dose response curves and corresponding IC50 values were calculated from the scanned images using the S+ Chip Analyzer. A sensitive response was more significantly achieved for AZD4547 (FGFR2 inhibitor) in giant cell tumor PDCs originated from the right pulmonary nodule under the micropillar/microwell chip platform using 3D culture. This sensitivity was consistent with the target expression patterns of giant cell tumor PDCs (FGFR2-IIIC mRNA expression in giant cell tumor PDCs originated from the right pulmonary nodule was increased significantly as compared to those originated from left). However, in a conventional 2D cultured MTT assay, there was no difference for IC50 values of AZD4547 between giant cell tumor PDCs originated from right and left pulmonary nodules. An HTS platform based on 3D culture on micropillar/microwell chips and PDC models could be applied as a useful preclinical tool to evaluate the intrapatient tumor/response heterogeneity. This platform based on 3D culture might reflect far better the relation between the tumor-biology and the matched targeted agent as compared to a conventional 2D cultured MTT assay.

IF 5.168; International audience; HLA-A*0201/DRB1*0101 transgenic mice (A2/DR1 mice) have been developed to study the immunogenicity of tumor antigen-derived T cell epitopes. To extend the use and application of this mouse model in the field of antitumor immunotherapy, we described a tumor cell line generated from a naturally occurring tumor in A2/DR1 mouse named SARC-L1. Histological and genes signature analysis supported the sarcoma origin of this cell line. While SARC-L1 tumor cells lack HLA-DRB1*0101 expression, a very low expression of HLA-A*0201 molecules was found on these cells. Furthermore they also weakly but constitutively expressed the programmed death-ligand 1 (PD-L1). Interestingly both HLA-A*0201 and PD-L1 expressions can be increased on SARC-L1 after IFN-γ exposure in vitro. We also obtained two genetically modified cell lines highly expressing either HLA-A*0201 or both HLA-A*0201/ HLA-DRB1*0101 molecules referred as SARC-A2 and SARC-A2DR1 respectively. All the SARC-L1-derived cell lines induced aggressive subcutaneous tumors in A2DR1 mice in vivo. The analysis of SARC-L1 tumor microenvironment revealed a strong infiltration by T cells expressing inhibitory receptors such as PD-1 and TIM-3. Finally, we found that SARC-L1 is sensitive to several drugs commonly used to treat sarcoma and also susceptible to anti-PD-L1 monoclonal antibody therapy in vivo. Collectively, we described a novel syngeneic tumor model A2/DR1 mice that could be used as preclinical tool for the evaluation of antitumor immunotherapies.

// Tomasz Rzymski 1, * , Michal Mikula 2, * , Eliza Żylkiewicz 1 , Agnieszka Dreas 1 , Katarzyna Wiklik 1 , Aniela Golas 1 , Katarzyna Wojcik 1 , Magdalena Masiejczyk 1 , Anna Wrobel 1 , Izabela Dolata 1 , Agata Kitlinska 1 , Malgorzata Statkiewicz 2 , Urszula Kuklinska 2 , Krzysztof Goryca 2 , Łukasz Sapala 1 , Aleksandra Grochowska 3 , Aleksandra Cabaj 2, 4 , Malgorzata Szajewska-Skuta 1 , Ewelina Gabor-Worwa 1 , Katarzyna Kucwaj 1 , Arkadiusz Bialas 1 , Adam Radzimierski 1 , Michal Combik 1 , Jakub Woyciechowski 1 , Maciej Mikulski 1 , Renata Windak 1 , Jerzy Ostrowski 2, 3 and Krzysztof Brzozka 1 1 R&D Department, Selvita S.A., Krakow, Poland 2 Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center, Warsaw, Poland 3 Department of Gastroenterology, Hepatology and Clinical Oncology, Medical Center for Postgraduate Education, Warsaw, Poland 4 Laboratory of Bioinformatics, Nencki Institute of Experimental Biology, Warsaw, Poland * These authors have contributed equally to this work Correspondence to: Krzysztof Brzozka, email: krzysztof.brzozka@selvita.com Keywords: CDK8 mediator, kinase inhibitor, STAT5, AML, leukemia stem cells Received: August 04, 2016 Accepted: March 09, 2017 Published: April 04, 2017 ABSTRACT Inhibition of oncogenic transcriptional programs is a promising therapeutic strategy. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. X-ray crystallography has shown SEL120-34A to be a type I inhibitor forming halogen bonds with the protein’s hinge region and hydrophobic complementarities within its front pocket. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 in cancer cells in vitro . Consistently, regulation of STATs- and NUP98-HOXA9- dependent transcription has been observed as a dominant mechanism of action in vivo . Treatment with the compound resulted in a differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. In vivo efficacy in xenotransplanted AML models correlated with significant repression of STAT5 S726. Favorable pharmacokinetics, confirmed safety and in vivo efficacy provide a rationale for the further clinical development of SEL120-34A as a personalized therapeutic approach in AML.

Inhibition of Brd4 by JQ1 treatment showed potential in the treatment of glioma, however, some cases showed low sensitivity of JQ1. In addition, the pre-clinical analysis showed its limitation by demonstrating that transient treatment with JQ1 leads to aggressive tumor development. Thus, an improved understanding of the mechanisms underlying JQ1 is urgently required to design strategies to improve its efficiency, as well as overcome its limitation. HEXIM1 has been confirmed to have an important role in regulating JQ1 sensitivity. In our study, ubenimex, a classical anti-cancer drug showed potential in regulating the JQ1 sensitivity of glioma cells using the WST-1 proliferation assay. Further studies demonstrated that ubenimex inhibited autophagy and downregulated the autophagic degradation of HEXIM1. The role of HEXIM1 in regulating JQ1 sensitivity was verified by the HEXIM1 knockdown. Since ubenimex was verified as an Akt inhibitor, we further studied the role of Akt inhibition in regulating JQ1 sensitivity and migration of glioma cells. Data showed that ubenimex improved the efficiency of JQ1 treatment and suppressed migration both in the in vitro and in vivo xenografts models. The Akt agonist attenuated these effects, pointing to the role of Akt inhibition in JQ1 sensitivity and suppressed migration. Our findings suggest the potential of ubenimex adjuvant treatment to enhance JQ1 efficiency and attenuate parts of its side effect (enhancing tumor aggressive) by regulating the autophagic degradation of HEXIM1 and Akt inhibition.

// Zhijie Wang 1, 6 , Sarina Piha-Paul 1 , Filip Janku 1 , Vivek Subbiah 1 , Naiyi Shi 1 , Jing Gong 1 , Chetna Wathoo 2 , Kenna Shaw 2 , Kenneth Hess 3 , Russell Broaddus 4 , Aung Naing 1 , David Hong 1 , Apostolia M. Tsimberidou 1 , Daniel Karp 1 , James Yao 5 , Funda Meric-Bernstam 1 and Siqing Fu 1 1 Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Institute of Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 6 National Cancer Center/Cancer Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China Correspondence to: Siqing Fu, email: siqingfu@mdanderson.org Keywords: KRAS, TP53, chronic inflammation, phase I trial, gene aberration-related therapy Received: October 15, 2016 Accepted: March 16, 2017 Published: April 05, 2017 ABSTRACT Purpose: Genetic alterations such as activating KRAS and/or inactivating TP53 are thought to be the most common drivers to tumorigenesis. Therefore, we assessed phase I cancer patients with KRAS+ / TP53+ mutations. Results: Approximately 8% of patients referred to phase I clinical trials harbored concurrent KRAS and TP53 mutations. Patients who received a phase I trial therapy (n = 57) had a median OS of 12 months, compared with 4.6 months in those who were not treated (n = 106; p = 0.003). KRAS G13 and TP53 R273 mutations were associated with poor overall survival (OS), while antiangiogenesis and gene aberration-related therapies were associated with prolonged OS. A prognostic model using neutrophilia, thrombocytosis, hypoalbuminemia, body mass index

// Pierre Vandenberghe 1 , Perrine Hague 1 , Steven C. Hockman 2 , Vincent C. Manganiello 2, ** , Pieter Demetter 3 , Christophe Erneux 4, * and Jean-Marie Vanderwinden 1, * 1 Laboratory of Neurophysiology, Faculty of Medicine, Universite Libre de Bruxelles, Brussels, Belgium 2 Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA 3 Department of Pathology, Erasme University Hospital, Universite Libre de Bruxelles, Brussels, Belgium 4 IRIBHM, Faculty of Medicine, Universite Libre de Bruxelles, Brussels, Belgium * These authors have contributed equally to this work ** deceased Correspondence to: Christophe Erneux, email: cerneux@ulb.ac.be Jean-Marie Vanderwinden, email: jmvdwin@ulb.ac.be Keywords: transgenic mice, cilostazol, tissue array, KIT, cancer Received: August 15, 2016 Accepted: March 25, 2017 Published: April 10, 2017 ABSTRACT We previously identified phosphodiesterase 3A (PDE3A) as a marker for interstitial cells of Cajal (ICC) in adult mouse gut. However, PDE3A expression and function during gut development and in ICC-derived gastrointestinal stromal tumors (GIST) remained unknown. Here we found that PDE3A was expressed throughout ICC development and that ICC density was halved in PDE3A-deficient mice. In the human imatinib-sensitive GIST882 cell line, the PDE3 inhibitor cilostazol halved cell viability (IC 50 0.35 μM) and this effect synergized with imatinib (Chou-Talalay’s CI 50 0.15). Recently the compound 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP was found to be cytotoxic selectively for cells expressing both PDE3A and Schlafen12 (SLFN12) (de Waal L et al. Nat Chem Bio 2016), identifying a new, non-catalytic, role for PDE3A. 108 out of 117 (92%) of our human GIST samples displayed both PDE3A and SLFN12 immunoreactivity. GIST882 cells express both PDE3A and SLFN12 and DNMDP decreased their viability by 90%. Our results suggest a role for PDE3A during ICC development and open novel perspectives for PDE3A in targeted GIST therapy, on one hand by the synergism between imatinib and cilostazol, a PDE3 inhibitor already in clinical use for other indications, and, on the other hand, by the neomorphic, druggable, PDE3A-SLFN12 cytotoxic interplay.

// Markus Wallner 1, 2, * , Mounir Khafaga 1, * , Ewald Kolesnik 1 , Aris Vafiadis 1 , Gerold Schwantzer 3 , Deborah M. Eaton 2 , Pero Curcic 4 , Martin Kostenberger 5 , Igor Knez 4 , Peter P. Rainer 1 , Martin Pichler 6 , Burkert Pieske 1, 7, 8 and Dirk Von Lewinski 1 1 Division of Cardiology, Department of Internal Medicine, Medical University of Graz, 8036 Graz, Austria 2 Cardiovascular Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, 19140 PA, United States of America 3 Institute for Medical Informatics, Statistics, and Documentation, Medical University of Graz, 8036 Graz, Austria 4 Division of Cardiac Surgery, Department of Surgery, Medical University of Graz, 8036 Graz, Austria 5 Department of Pediatric Cardiology, Medical University of Graz, 8036 Graz, Austria 6 Division of Clinical Oncology, Department of Internal Medicine, Medical University of Graz, 8036 Graz, Austria 7 Department of Internal Medicine and Cardiology, Campus Virchow Klinikum, Charite University Medicine, Berlin, 13353 Berlin, Germany 8 Department of Internal Medicine and Cardiology, German Heart Center, Berlin, 13353 Berlin, Germany * These authors contributed equally to this work Correspondence to: Ewald Kolesnik, email: ewald.kolesnik@medunigraz.at Keywords: prostate cancer, hormone therapy, cardiac disease models, heart failure, istaroxime Received: March 06, 2017 Accepted: April 14, 2017 Published: April 30, 2017 ABSTRACT The current gold standard for prostate cancer treatment is androgen deprivation therapy and antiandrogenic agents. However, adverse cardiovascular events including heart failure can limit therapeutic use. Istaroxime, which combines Na + -K + -ATPase (NKA) inhibition with sarco/endoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) stimulation, has recently shown promising anti-neoplastic effects in prostate cancer (PC) models and may also improve cardiac function. Considering the promising anticancer effects of istaroxime, we aimed to assess its functional effects on human myocardium. Results: Istaroxime and strophanthidin elicited dose-dependent positive inotropic effects with a decline in developed force at supraphysiological concentrations in human atrial, nonfailing, and failing ventricular (ToF) myocardium. Diastolic force and RT50% did not change after exposure to both drugs. The maximal developed force in our in-vitro model of heart failure (ToF) was significantly higher after istaroxime administration. Such a difference did not occur in atrial or nonfailing ventricular trabeculae and was not applicable to the diastolic force. Materials and Methods: Human atrial and ventricular trabeculae were isolated from nonfailing hearts and hearts of infants with tetralogy of Fallot (ToF), which were used as an in-vitro model of heart failure. The samples were electrically stimulated and treated with increasing concentrations of istaroxime and strophanthidin (10 nM–1 μM). Systolic and diastolic force development and relaxation parameters (RT50%) were analyzed. Conclusions: Combined NKA inhibition/SERCA2a stimulation increases contractility in atrial, nonfailing, and failing myocardium. Considering that heart failure is a potential side effect of current PC treatments, especially in elderly patients, istaroxime might combine beneficial cardiac and anti-cancer properties.

// Cheng-Yu Yang 1, 6 , Chih-Kung Lin 3 , Chang-Huei Tsao 7, 8 , Cheng-Chih Hsieh 5 , Gu-Jiun Lin 4 , Kuo-Hsing Ma 4 , Yi-Shing Shieh 6 , Huey-Kang Sytwu 1, 7 and Yuan-Wu Chen 1, 2, 6 1 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 2 Department of Oral and Maxillofacial Surgery, Tri-Service General Hospital, Taipei, Taiwan 3 Division of Anatomic Pathology, Taipei Tzu Chi Hospital, Taipei, Taiwan 4 Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan 5 Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan 6 School of Dentistry, National Defense Medical Center, Taipei, Taiwan 7 Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan 8 Department of Medical Research, Tri-Service General Hospital, Taipei, Taiwan Correspondence to: Yuan-Wu Chen, email: h6183@yahoo.com.tw Keywords: patient-derived tumor xenograft, LSD1, melatonin, oral cancer, cell proliferation Received: June 16, 2016 Accepted: March 08, 2017 Published: April 04, 2017 ABSTRACT Aberrant activation of histone lysine-specific demethylase (LSD1) increases tumorigenicity; hence, LSD1 is considered a therapeutic target for various human cancers. Although melatonin, an endogenously produced molecule, may defend against various cancers, the precise mechanism involved in its anti-oral cancer effect remains unclear. Patient-derived tumor xenograft (PDTX) models are preclinical models that can more accurately reflect human tumor biology compared with cell line xenograft models. Here, we evaluated the anticancer activity of melatonin by using LSD1-overexpressing oral cancer PDTX models. By assessing oral squamous cell carcinoma (OSCC) tissue arrays through immunohistochemistry, we examined whether aberrant LSD1 overexpression in OSCC is associated with poor prognosis. We also evaluated the action mechanism of melatonin against OSCC with lymphatic metastases by using the PDTX models. Our results indicated that melatonin, at pharmacological concentrations, significantly suppresses cell proliferation in a dose- and time-dependent manner. The observed suppression of proliferation was accompanied by the melatonin-mediated inhibition of LSD1 in oral cancer PDTXs and oral cancer cell lines. In conclusion, we determined that the beneficial effects of melatonin in reducing oral cancer cell proliferation are associated with reduced LSD1 expression in vivo and in vitro .

// Zih-Yin Lai 1 , Hsin-Yueh Yeo 1 , Ya-Tse Chen 1 , Kuo-Ming Chang 2 , Tze-Chien Chen 3 , Yung-Jen Chuang 1 and Shing-Jyh Chang 4 1 Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan (R.O.C.) 2 Department of Pathology, Hsinchu MacKay Memorial Hospital, Hsinchu 30071, Taiwan (R.O.C.) 3 Department of Obstetrics and Gynecology, MacKay Memorial Hospital, Taipei 10449, Taiwan (R.O.C.) 4 Department of Obstetrics and Gynecology, Hsinchu MacKay Memorial Hospital, Hsinchu 30071, Taiwan (R.O.C.) Correspondence to: Yung-Jen Chuang, email: yjchuang@life.nthu.edu.tw Shing-Jyh Chang, email: justine3@ms8.hinet.net Keywords: neuroendocrine cervical carcinoma, combination therapy, genotoxic drug, PI3K inhibitor, BEZ235 Received: February 14, 2017 Accepted: April 12, 2017 Published: April 21, 2017 ABSTRACT Background: Neuroendocrine cervical carcinoma (NECC) is a rare and aggressive subtype of cervical cancer. To date, no NECC cell-based model is available, which hinders the development of new therapeutic strategies for NECC. In this study, we derived a new NECC cell line from an ex vivo biopsy and used it to explore novel drug combination approach for NECC. Results: The stable HM-1 cell line displayed high expression levels of the neuroendocrine marker, synaptophysin. HM-1 cell transplantation could induce tumor growth in nude mice. As expected, the combination of etoposide and cisplatin synergistically inhibited HM-1 cell proliferation. Strikingly, when etoposide and cisplatin were combined with PI3K inhibitor BEZ235, the growth of HM-1 cells was significantly reduced. Taken together, the data implied the combination of etoposide and cisplatin with BEZ235 not only inhibited HM-1 cell proliferation but also increased cell apoptosis. Materials and Methods: A NECC tissue sample from a 75-year-old female patient was processed to derive a primary cell line annotated as HM-1. The features of HM-1 were analyzed to establish its characteristic profile. Next, HM-1 was treated with PI3K inhibitors, BKM120 and/or BEZ235, in combination with two well-known genotoxic drugs, etoposide and/or cisplatin, to evaluate which combination could serve as a more effective treatment approach. Their inhibiting effects on HM-1 were evaluated by cell viability, apoptosis, and target kinase expression. Conclusions: The newly established NECC cell line HM-1 could serve as a cell-based model for NECC research. The synergistic drug combination of PI3K inhibitor with genotoxic drugs might become a potential new treatment strategy against NECC.

// Jun An 1, * , Ying Xue 2, * , Meijun Long 3, * , Ge Zhang 2 , Junhang Zhang 1 and Hang Su 4 1 Department of Cardiothoracic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China 2 Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China 3 Breast Cancer Center and Department of Thyroid and Breast Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China 4 Department of Radiation Oncology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA * These authors have contributed equally to this work Correspondence to: Junhang Zhang, email: zhangjh33@mail.sysu.edu.cn Hang Su, email: suh3@uthscsa.edu Keywords: CCR2 antagonist, MMP-9, non-small cell lung cancer, viability, motility and invasion Received: September 23, 2016 Accepted: March 09, 2017 Published: April 05, 2017 ABSTRACT Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, which is the leading cancer killer in the world. Despite the recent advances in its diagnosis and therapy, the prognosis of NSCLC patients remains very poor, mainly due to the development of drug resistance and metastasis. Both the chemokine network and the matrix metalloproteinase (MMP) system play important roles in cancer cell metastasis. The disruption of CCL2/CCR2 chemokine signaling has been shown to suppress cancer cellviability and metastasis. CCL2-neutralizing antibodies, which have shown promising therapeutic efficacy in several cancer models, are not widely used due to technical issues. CCR2 antagonism has thus become an alternative method for cancer treatment. However, the effect of CCR2 antagonists on NSCLC progression remains poorly understood. Here, we investigated the effect of CCR2 antagonist (CAS445479-97-0) on the proliferation, migration and invasion of human lung adenocarcinoma A549 cells by using WST-1 cell viability assay, transwell migration assay, wound healing scratch assay and Matrigel invasion assay. We demonstrated that CCL2 treatment promoted A549 cell viability, motility and invasion by upregulating MMP-9 expression and that this induction was significantly suppressed by CAS 445479-97-0. Taken together, our data suggested that the CCR2 antagonist would be a potential drug for treating CCR2-positive NSCLC patients.

// Hui-Er Gao 1, * , Yue Sun 1, * , Ya-Hui Ding 2 , Jing Long 2 , Xiao-Lei Liu 1 , Ming Yang 1 , Qing Ji 1 , Ying-Hui Li 1 , Yue Chen 2 , Quan Zhang 2 , Ying-Dai Gao 1 1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, P. R. China 2 State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin 300353, P. R. China * These authors contributed equally to this work Correspondence to: Quan Zhang, email: zhangquan612@163.com Ying-Dai Gao, email: ydgao@ihcams.ac.cn Keywords: CPPTL, acute myeloid leukemia, ROS, JNK pathway Received: February 23, 2017 Accepted: April 03, 2017 Published: April 17, 2017 ABSTRACT Drug resistance and human leukocyte antigen (HLA) matching limit conventional treatment of acute myeloid leukemia (AML). Although several small molecule drugs are clinically used, single drug administration is not sufficient to cure AML, which has a high molecular diversity. Metabolic homeostasis plays a key role in determining cellular fate. Appropriate levels of reactive oxygen species (ROS) maintain the redox system balance, and excessive amounts of ROS cause oxidative damage, thus providing a strategy to eliminate cancer cells. CPPTL is a novel analogue of parthenolide that exhibited significant cytotoxicity to AML cells in vitro and induced apoptosis in a dose-dependent manner. Additionally, CPPTL’s prodrug DMA-CPPTL decreased the burden of AML engraftment and prolonged survival in a mouse model administered human primary AML cells in vivo . CPPTL induced apoptosis of AML cells by stimulating ROS production, and accumulation of ROS then activated the JNK pathway, thereby promoting mitochondrial damage. These results demonstrated that CPPTL effectively eradicated AML cells in vitro and in vivo and suggested that CPPTL may be a novel candidate for auxiliary AML therapy.

Isocitrate dehydrogenase (IDH) plays an indispensable role in the tricarboxylic acid cycle, and IDH mutations are present in nearly 75% of glioma and 20% of acute myeloid leukemia. One IDH1R132H inhibitor (clomifene citrate) was found by virtual screening method, which can selectively suppress mutant enzyme activities in vitro and in vivo with a dose-dependent manner. The molecular docking indicated that clomifene occupied the allosteric site of the mutant IDH1. Enzymatic kinetics also demonstrated that clomifene inhibited mutant enzyme in a non-competitive manner. Moreover, knockdown of mutant IDH1 in HT1080 cells decreased the sensitivity to clomifene. In vivo studies indicated that clomifene significantly suppressed the tumor growth of HT1080-bearing CB-17/Icr-scid mice with oral administration of 100 mg/kg and 50 mg/kg per day. In short, our findings highlight clomifene may have clinical potential in tumor therapies as a safe and effective inhibitor of mutant IDH1.

Extracellular signal-regulated kinase 8 (ERK8), also known as mitogen-activated protein kinase 15 (MAPK15), is the most recently identified protein kinase of the ERK family members and yet the least has been studied so far. Here, we report that ERK8 is highly expressed in several human lung cancer cell lines and is positively correlated with their sensitivities to the anti-cancer drug arsenic trioxide (As2O3). As2O3 at physiologically relevant concentrations (5-20 μM) potently stimulates the phosphorylation of ERK8 at Thr175 and Tyr177 within the TEY motif in the kinase domain, leading to its activation. Interestingly, activated ERK8 interacts and directly phosphorylates IkappaBalpha (IκBα) at Ser32 and Ser36, resulting in IκBα degradation. This in turn promotes nuclear factor-kappaB (NF-κB) p65 nuclear translocation and chromatin-binding, as well as the subsequent induction and activation of proteins involved in apoptosis. We also show that stable short-hairpin RNA-specific knockdown of endogenous ERK8 or inhibition of NF-κB activity by NF-κB inhibitor in high ERK8 expressing lung cancer H1299 cells blunted the As2O3-induced NF-κB activation and cytotoxicity towards these cells, indicating the critical role of ERK8 and NF-κB in mediating the As2O3 effects. Taken together, our findings suggest for the first time a regulatory paradigm of NF-κB activation by ERK8 upon As2O3 treatment in human lung cancer cells; and implicate a potential therapeutic advantage of As2O3 that might gain more selective killing of cancer cells with high ERK8 expression.

// Yan-Feng Huang 1 , Da-Jian Zhu 2 , Xiao-Wu Chen 3 , Qi-Kang Chen 2 , Zhen-Tao Luo 3 , Chang-Chun Liu 3 , Guo-Xin Wang 3 , Wei-Jie Zhang 3 and Nv-Zhu Liao 2 1 Department of Traditional Chinese Medicine, Shunde Hospital of Southern Medical University, Guangdong 528300, China 2 Department of Gastrointestinal Surgery, Shunde Women and Children’s Hospital Affiliated to Jinan University, Guangdong 528300, China 3 Department of Gastrointestinal Surgery, Shunde Hospital of Southern Medical University, Guangdong 528300, China Correspondence to: Da-Jian Zhu, email: zhudajian2016@126.com Keywords: curcumin, colorectal cancer, irinotecan, ROS, ER stress Received: May 13, 2016 Accepted: February 27, 2017 Published: April 04, 2017 ABSTRACT Although initially effective against metastatic colorectal cancer (CRC), irinotecan-based chemotherapy leads to resistance and adverse toxicity. Curcumin is well known for its anti-cancer effects in many cancers, including CRC. Here, we describe reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress as important mechanisms by which curcumin enhances irinotecan’s effects on CRC cells. CRC cell lines were treated with curcumin and/or irinotecan for 24 h, and then evaluated using cell proliferation assays, cell apoptosis assays, cell cycle analysis, intracellular Ca 2+ measurements, ROS measurements and immunoblotting for key ER stress-related proteins. We found that cell viability was inhibited and apoptosis was increased, accompanied by ROS generation and ER stress activation in CRC cells treated with curcumin alone or in combination with irinotecan. Blocking ROS production attenuated the expression of two markers of ER stress: binding of immunoglobulin protein (BIP) and CCAAT/enhancer-binding protein homologous protein (CHOP). Blocking CHOP expression using RNA interference also inhibited ROS generation. These results demonstrated that curcumin could enhance the effects of irinotecan on CRC cells by inhibiting cell viability and inducing cell cycle arrest and apoptosis, and that these effects may be mediated, in part, by ROS generation and activation of the ER stress pathway.

// Mengdan Li 1, * , Lihong Chen 2, * , Yiping Qu 1 , Fang Sui 1 , Qi Yang 1, 3 , Meiju Ji 3 , Bingyin Shi 1, 3 , Mingwei Chen 4 , Peng Hou 1, 3 1 Department of Endocrinology, The First Affiliated Hospital of Xi’an Jiaotong University School of Medicine, Xi’an 710061, P.R. China 2 Department of Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, P.R. China 3 Key Laboratory for Tumor Precision Medicine of Shaanxi Province, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, P.R. China 4 Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, P.R. China * These authors contributed equally to this work Correspondence to: Peng Hou, email: phou@xjtu.edu.cn Keywords: gallbladder carcinoma (GBC), targeted massively parallel sequencing, MAP kinase pathways, Wnt/β-catenin pathway, NF-κB pathway Received: February 22, 2017 Accepted: March 21, 2017 Published: March 31, 2017 ABSTRACT The aim of this study was to profile somatic mutation spectrum in gallbladder cancers (GBCs), and determine the role of MAP kinase pathway in GBC by a series of in vitro and in vivo studies. We performed targeted massively parallel sequencing of DNA isolated from GBCs and matched blood from 14 GBC patients to search for mutations in 504 genes commonly altered in human cancers. We identified recurrent mutations enriched in several major signaling pathways including MAP kinase, Wnt/β-catenin and NF-κB pathways. Immunohistochemistry analysis further validated overactivation of MAP kinase and Wnt pathways in a panel of GBC samples. By treating GBC cells with MEK inhibitor trametinib, we found that trametinib not only dramatically inhibited the activity of MAPK/ERK pathway, but also blocked the Wnt/β-catenin signaling through decreasing β-catenin expression or suppressing nucleus translocation of β-catenin. Moreover, trametinib inhibited the proliferation of GBC cell in a dose- and time-dependent manner, induced GBC cell apoptosis, and inhibited GBC cell migration and invasion. Growth of xenograft tumors derived from GBC cell line NOZ in nude mice was also significantly inhibited by trametinib. Our data highlight the critical role of MAP kinase pathways in GBC pathogenesis, and may represent therapeutic targets for this cancer.