bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 263: Effects of the sodium glucose cotransporter 2 inhibitor tofogliflozin on hepatoma cell lines and liver tumorigenesis
Authors: Shirakami Yohei , Obara Koki , Kubota Masaya , Sakai Hiroyasu , Tanaka Takuji , Shimizu Masahito , Seishima Mitsuru
Published: August 2018

Obesity, diabetes mellitus, and their related metabolic abnormalities are associated with increased risk of hepatocellular carcinoma (HCC). Anti-diabetic agents sodium glucose cotransporter (SGLT)-2 inhibitors are expected to ameliorate the abnormalities associated with metabolic syndrome including non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of the SGLT2 inhibitor tofogliflozin on the development of NAFLD-related liver tumorigenesis in C57BL/KsJ-+Leprdb/+Leprdb (db/db) obese and diabetic mice. The direct effects of tofogliflozin on human hepatoma cell lines were also evaluated. Male db/db mice were administered diethylnitrosamine-containing water for two weeks and were then treated with tofogliflozin throughout the experiment. Tofogliflozin was kindly provided by Kowa Co., Ltd. and the chemical structure will be disclosed at the time of presentation at the meeting. In mice treated with tofogliflozin, the development of hepatic pre-neoplastic lesions, foci of cellular alterations, was markedly suppressed, and hepatic steatosis and inflammation significantly reduced, as evaluated using the NAFLD activity score (NAS), in comparison to those in the control mice. Serum levels of glucose and free fatty acid and mRNA expression levels of pro-inflammatory markers in the liver were reduced by tofogliflozin treatment. Conversely, the proliferation of SGLT2 protein-expressing hepatoma cells was not inhibited and the insulin signaling in those cells was not altered by this agent. These findings suggest that tofogliflozin suppressed the early phase of obesity- and NAFLD-related liver carcinogenesis by attenuating chronic inflammation and steatosis in the liver, while the agent had no significant direct effect on the proliferation of hepatoma cells. Therefore, SGLT2 inhibitors may have a chemopreventive effect on obesity-related HCC.

Citation Format: Yohei Shirakami, Koki Obara, Masaya Kubota, Hiroyasu Sakai, Takuji Tanaka, Masahito Shimizu, Mitsuru Seishima. Effects of the sodium glucose cotransporter 2 inhibitor tofogliflozin on hepatoma cell lines and liver tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 263.
Abstract 2639: Prognostic significance of non-small cell lung cancer and response to EGFR-TKI of EGFR mutated lung cancer besed on PD-L1 expression
Authors: Kobayashi Kenichi , Seike Masahiro , Noro Rintaro , Hemmi Shinobu , Gemma Akihiko
Published: August 2018

Background: Recent clinical trials have shown that immune checkpoint blockades that target either PD-1 or PD-L1 yield remarkable responses in a subgroup of non-small cell lung cancer (NSCLC) patients.

Materials and Methods: We retrospectively examined, by immunohistochemical analysis, 211 NSCLC patients who had undergone complete surgical resection. Using 32 independent samples, we also evaluated PD-L1 expression in 32 NSCLC patients with epidermal growth factor receptor (EGFR) mutations treated by EGFR-tyrosine kinase inhibitors (EGFR-TKIs).

Results: Overall survival of PD-L1 positive surgical NSCLC patients with stages I-III and stage I disease were significantly shorter than those of PD-L1 negative NSCLC patients (P <0.01 and P = 0.02, respectively). In 86 squamous cell carcinoma (SQ) cases, PD-L1 positive patients showed significantly poorer survival than PD-L1 negative patients (P = 0.01). In stage I NSCLC patients, gender and PD-L1 expression were found to be independent predictors of death after multivariate analysis (Hazard ratio [HR] 3.57, P <0.01 and HR 2.05, P = 0.04, respectively). In SQ patients, p-stage and PD-L1 expression were independent predictors of death (HR 2.53, P <0.01 and HR 2.26, P = 0.02, respectively). Response to EGFR-TKIs, progression-free survival and overall survival were not significantly different between PD-L1 positive and negative NSCLC patients with EGFR mutations.

Conclusion: PD-L1 positive expression in a tumor was a significant independent predictor of poor outcome in patients with lung SQ and in stage I NSCLC patients. Based on PD-L1 expression, the clinical response to EGFR-TKIs and prognosis did not differ in EGFR-mutated NSCLC patients.

Citation Format: Kenichi Kobayashi, Masahiro Seike, Rintaro Noro, Shinobu Hemmi, Akihiko Gemma. Prognostic significance of non-small cell lung cancer and response to EGFR-TKI of EGFR mutated lung cancer besed on PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2639.
Abstract 2642: CBLC is a potential biomarker for the susceptibility to paclitaxel in lung adenocarcinoma
Authors: Lin Yi-Ping , Hong Shiao-Ya , Kao Yu-Rung , Wu Cheng-Wen
Published: August 2018

CBL family proteins have been reported as E3 ubiquitin ligases of receptor tyrosine kinases. CBLC is the most divergent member of CBL family in human due to lack of the distal part of C terminus found in other two proteins, CBL and CBLB. By data mining from TCGA database, CBLC expression was frequently upregulated in lung adenocarcinoma. However, very little is known about the tumorigenic functions of CBLC compared to other CBL family proteins. Although patients with lung adenocarcinoma harboring EGFR mutations can be treated with tyrosine kinase inhibitors (TKIs), there is no good target therapy for treating lung adenocarcinoma patients with EGFR wild-type or TKIs resistance. Here, this study is to investigate if CBLC can be a good target for treatment to benefit these patients. In lung adenocarcinoma cells, CBLC knockdown significantly decreased cell viability, while CBLC overexpression increased the ability to form colonies in soft agar. Flow cytometry analyses revealed that CBLC depletion decreased the cell population of G2/M phase and increased cell apoptosis. Our study also showed that CBLC knockdown in EGFR wild-type cells increased the drug resistance to paclitaxel, which is widely used as a first-line chemotherapy drug in lung adenocarcinoma patients with EGFR wild-type or TKI resistance. Taken together, these findings indicate that CBLC might play a role in promoting cell mitosis and could be a potential biomarker of paclitaxel susceptibility for patients with EGFR wild-type lung adenocarcinoma.

Citation Format: Yi-Ping Lin, Shiao-Ya Hong, Yu-Rung Kao, Cheng-Wen Wu. CBLC is a potential biomarker for the susceptibility to paclitaxel in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2642.
Abstract 266: Disulfiram overcomes docetaxel resistance in prostate cancer by targeting the neddylation pathway and down-regulation of Skp2 expression
Authors: Song Liankun , Yu Bin , Liu Hong-Min , Zi Xiaolin
Published: August 2018

Disulfiram (DSF), an inhibitor of aldehyde dehydrogenase and a dithiocarbamate chelator of copper, has been clinically used for treatment of alcohol dependence for decades. A population-based case control study has suggested that the use of DSF is associated with reduced risk of prostate cancer. In addition, copper is accumulated in prostate cancer and can act synergistically with DSF to inhibit the growth of prostate cancer cells. These accumulating data suggested that DSF is a very promising agent to be repurposed for prostate cancer treatment and prevention. However, the anti-cancer mechanisms of DSF currently remain largely unknown. Here we have shown that DSF treatment decreases the neddylation levels of NEDD8-conjugating enzyme UBE2M and Cullin-1 leading to down-regulation of Skp2 expression in prostate cancer DU145 cells. Further co-immunoprecipitation experiment revealed that DSF disrupted the formation of the defective in Cul neddylation 1 protein (DCN1) and UBE2M complex. DSF synergistically enhanced the growth inhibitory effect of docetaxel on prostate cancer cell lines and overcame the drug resistance. Taken together, these data may provide proof of concept for rationally designed repurpose of DSF to treat docetaxel-resistant prostate cancer by involvement of the neddylation pathway.

Citation Format: Liankun Song, Bin Yu, Hong-Min Liu, Xiaolin Zi. Disulfiram overcomes docetaxel resistance in prostate cancer by targeting the neddylation pathway and down-regulation of Skp2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 266.
Abstract 2716: Development of 4-1BB agonists: Ligand fusion shows greater anti-tumor activity than antibodies in vivo
Authors: Tam Eric , Craig Galina , Ghassemifar Sara , Koshkaryev Alexander , Movassaghian Sara , Cosgrove Cormac , Sampson James , Drummond Daryl , Raue Andreas
Published: August 2018

Members of the tumor necrosis factor (TNF) superfamily of costimulatory receptors have emerged as promising targets for modulation of tumor infiltrating lymphocytes. Among these, 4-1BB (CD137) has a prominent role in promoting survival and expansion of cytotoxic CD8 T cells as well as activation of CD4 T cells, NK cells, and antigen presenting cells. The clinical relevance of 4-1BB agonism is currently being evaluated for two antibodies: urelumab (IgG4) and utomilumab (IgG2). However, it has been established that for the trimeric TNF receptor family, the homotrimeric ligands are superior activators compared to bivalent agonistic antibodies. Here we describe a prototype 4-1BB agonist consisting of three human 4-1BB ligands linked together as a single polypeptide chain and fused to the C-terminus of human IgG1 Fc (STIM-41BBL). Compared to a clinically relevant human 4-1BB antibody, STIM-41BBL shows increased activation of 4-1BB in a NF-kappa B reporter cell line. STIM-4-1BBL also shows augmented cytokine release compared to the antibody in CD8 T cells. Furthermore, we have developed a mouse STIM-41BBL and like the human counterpart, the ligand fusion induced greater cytokine release than an agonistic mouse 4-1BB antibody (IgG2a) in CD8 T cells. We have also introduced mutations (D265A/N297A) to the mouse 4-1BB antibody to make it effectorless in order to better approximate the clinical 4-1BB antibodies. Subsequently, we demonstrate that the murine STIM-41BBL had greater anti-tumor activity than the 4-1BB antibody in a MC38 tumor model. These results support the continued development of a 4-1BB ligand based therapeutic and suggests the greater potential of a STIM-41BBL fusion to a targeting antibody for more precise activation of immune system.

Citation Format: Eric Tam, Galina Craig, Sara Ghassemifar, Alexander Koshkaryev, Sara Movassaghian, Cormac Cosgrove, James Sampson, Daryl Drummond, Andreas Raue. Development of 4-1BB agonists: Ligand fusion shows greater anti-tumor activity than antibodies in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2716.
Abstract 2751: Regorafenib attenuates IFN-γ-induced PD-L1 expression and enhances antitumor immunity
Authors: Zhu Xiao-Feng
Published: August 2018

Immune checkpoint blocked therapy (ICB) targeting the PD-1/PD-L1 axis induce durable tumor regressions in a sizeable minority of cancer patients. To enhance the efficacy of existing immunotherapies, we implemented a screen of kinase inhibitors capable of increasing the activity of T cells suppressed by PD-L1. Here, we identified regorafenib, which was approved to treat patients with advanced gastrointestinal stromal tumors and metastatic colorectal cancer, as a potent agent significantly attenuating IFN-γ-induced PD-L1 expression without affecting MHC-I expression, contributing to enhanced anti-tumor effects with ICB in vitro and in vivo, due in part to inhibit JAK1/2-STAT1 signaling and the activation of ERK by suppressing the tyrosine kinase receptor RET-Src axis. Additionally, our analysis of GEO database and TCGA database revealed that RET and Src co-high expression was an independent unfavorable prognosis factor in both melanoma patients with or without ICB via inhibiting anti-tumor immune response. Further, gene set enrichment analysis (GSEA) demonstrated, compared to RET and Src co-high expression group, anti-tumor immune response related gene signatures as prominent modules in other group. Collectively, our data unveiled a new mechanism of reducing IFN-γ-induced PD-L1 expression and provided a rationale to explore a new combination of immunotherapies and regorafenib in melanoma treatment.

Citation Format: Xiao-Feng Zhu. Regorafenib attenuates IFN-γ-induced PD-L1 expression and enhances antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2751.
Abstract 2783: Empowering therapeutic monoclonal antibodies with IFN-alpha for cancer immunotherapy
Authors: Guo Jun , Xiao Yu , Lyer Ramesh B. , Lake Marc R. , Ladror Uri S. , Lu Xin , Pappano Bill , Harlan John E. , Tomlinson Medha J. , Bukofzer Gail T. , Donawho Cherrie K. , Shoemaker Alexander R. , Huang Tzu-Hsuan
Published: August 2018

Type 1 IFNs stimulates secretion of IP-10 (CXCL10) which is a critical chemokine to recruit effector T cells to the tumor microenvironment and IP-10 knockout mice exhibit a phenotype with compromised effector T cell generation and trafficking. Type 1 IFNs also induces MHC class 1 upregulation on tumor cells which can enhance anti-tumor CD8 T cell effector response in the tumor microenvironment. Although type 1 IFNs show great promise in potentiating anti-tumor immune response, systemic delivery of type 1 IFNs is associated with toxicity thereby limiting clinical application. In this study, we fused tumor targeting antibodies with IFN-alpha and showed that the fusion proteins can be produced with high yields and purity. IFN fusions selectively induced IP-10 secretion from antigen positive tumor cells, which was critical in recruiting the effector T cells to the tumor microenvironment. Further, we establish a real time in vitro antigen specific CTL killing assay using IncuCyte Zoom and tested the anti-tumor efficacy of anti-PDL1-IFN-alpha fusion using this system. We found anti-PDL1-IFN-alpha as low as 10 pM exhibits potent activity in potentiating OT1 CD8 T cells killing against OVA expressing tumor cells, while control IFN fusion did not exhibit any activity in the same experiment. Furthermore, IFN-alpha fusion antibody was well tolerated in vivo and demonstrated anti-tumor efficacy in an anti-PD-L1 resistant syngeneic mouse tumor model. Our data supports the hypothesis of targeting type 1 IFN to the tumor microenvironment may enhance effector T cell responses in non-inflamed tumors.

Citation Format: Jun Guo, Yu Xiao, Ramesh B. Lyer, Marc R. Lake, Uri S. Ladror, Xin Lu, Bill Pappano, John E. Harlan, Medha J. Tomlinson, Gail T. Bukofzer, Cherrie K. Donawho, Alexander R. Shoemaker, Tzu-Hsuan Huang. Empowering therapeutic monoclonal antibodies with IFN-alpha for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2783.
Abstract 2829: Targeting the DNA damage response and DNA-PK signaling via small molecule Ku inhibitors
Authors: Turchi John J. , Gavande Navnath S. , VanderVere-Carozza Pamela S. , Vernon Tyler , Pawelczak Katherine S.
Published: August 2018

Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} The DNA dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role as a primary sensor in the non-homologous end joining (NHEJ) DNA double strand break (DSB) repair pathway. Various anti-cancer therapeutic strategies mediate their cytotoxic effects by inducing DSBs, including ionizing radiation (IR), and clinical outcomes are directly related to the repair of DNA damage. Modulating the pathway responsible for repairing DSBs will have a profound impact on the efficacy of DNA damaging agents in the clinic. To date, development of inhibitors for DNA-PK has focused on targeting the active site with ATP mimetics. We have taken the novel and innovative approach to inhibiting DNA-PK via blocking the Ku 70/80 heterodimer interaction with DNA, a necessary step in DNA-PK activation. Exploiting this unique mechanism of kinase activation, we have identified a series of highly potent and specific DNA-PK inhibitors that impart their inhibitory activity via disruption of the binding of Ku protein to DNA ends. This novel approach affords significant advantages to current approaches in kinase inhibition. Novel derivatives of our initial hit inhibit DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing agents like etoposide and bleomycin. Data demonstrate that the cellular effects observed are a function of Ku inhibition and that this novel class of DNA-PK inhibitors can be further developed as anti-cancer therapeutics that can be used as an adjuvant to, or concomitant with radiotherapy and other cancer therapies that induce DNA damage.

Citation Format: John J. Turchi, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Tyler Vernon, Katherine S. Pawelczak. Targeting the DNA damage response and DNA-PK signaling via small molecule Ku inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2829.
Abstract 2832: Novel therapy for oral squamous cell carcinoma using non-thermal atmospheric pressure biocompatible plasma activated water
Authors: Han Ihn , Song In Seok , Li Ying , Kang Min Ho , Ryu Jae Jun , Choi Eun Ha
Published: August 2018

Non-thermal atmospheric pressure biocompatible plasma (NBP) has been reported that was possible to induce apoptosis on various cancer cells including lung, ovary, brain, liver, breast, and pancreas cancer. We developed NBP device, which has been reported to have cytotoxicity on cancer cells induce DNA damage and apoptosis. In particular,reactive oxygen and/or nitrogen species (RONS) were generated by plasma device to give oxidative stress and depolarization of mitochondria membrane in cancer cells. Now we developed a microwave plasma jet system, which could generate nitric oxide (NO) and NO-plasma activated water (NO-PAW). Previously, we found that NO-PAW could induce apoptosis and accumulate ROS in human cervical cancer cells (1). In this study we investigated the efficacy of NO-PAW on oral squamous cancer cells (OSCC), a common malignant tumor of the head and neck (2, 3). We observed that cytotoxicity and apoptotic stimulation by NO-PAW were significantly increased. Also, expression level of caspase and PARP in OSCC was increased after treatment by NO-PAW. However, the apoptosis effect was not observed in human gingival fibroblasts (as normal human cells). Additionally, we found that NO-PAW inhibited cancer progression in vivo. In conclusion, NO-PAW could suppress the progress of OSCC by inducing cell apoptosis. This indicated NO-PAW as a novel tool for OSCC therapy, and probably will be also effective for other oral cancer.

Acknowledgments: This work was supported by the Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF- 2016K1A4A3914113) funded by the Ministry of Science, ICT, and Future Planning (MSIP) of the Korean Government for EH. Choi, I. Han. The Korea government, and the Basic Science Research Program through the NRF of Korea, funded by the Ministry of Education (NRF-2015R1C1A2A01054137) for I. Han.

References:

1. Y. Li, M.H. Kang, H.S. Uhm, G.J. Lee, E.H. Choi, I. Han. Sci Rep 2017;7:1.

2. E. Majchrzak, B. Szybiak, A. Wegner, P. Pienkowski, J. Pazdrowski, L. Luczewski, M. Sowka, P. Golusinski, J. Malicki, W. Golusinski. Radiol Oncol 2014;48:1.

3. B. Wang, S. Zhang, K. Yue, X.D. Wang. Chin J Cancer 2013;32:614.

Citation Format: Ihn Han, In Seok Song, Ying Li, Min Ho Kang, Jae Jun Ryu, Eun Ha Choi. Novel therapy for oral squamous cell carcinoma using non-thermal atmospheric pressure biocompatible plasma activated water [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2832.
Abstract 3158: DLL3 regulates migration and invasion of small cell lung cancer
Authors: Furuta Megumi , Sakakibara Jun Konishi , Shoji Tetsuaki , Takashima Yuta , Kikuchi Hajime , Kikuchi Eiki , Kikuchi Junko , Kinoshita Ichiro , Akita Hirotoshi Dosaka , Nishimura Masaharu
Published: August 2018

Background: Delta-like protein 3 (DLL3) is a ligand of Notch signaling, which is reported to be a tumor suppressor in small cell lung cancer (SCLC). Previous studies suggest that DLL3 might be associated with neuroendocrine tumorigenesis through inhibition of Notch signaling unlike other activating ligands. Moreover, DLL3 was highly expressed in SCLC, but not in normal lung tissue. However, little is known about function of DLL3 in SCLC. In this study, we examine the effect of DLL3 in tumorigenesis of SCLC.

Methods: The mRNA expression levels and proteins of DLL3, Notch receptors (Notch1, Notch2, Notch3 and Notch4), Hes1 and EMT markers (E-cadherin, Snail and Vimentin) were examined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot in 9 SCLC cell lines (SBC-3, SBC-5, MS-1, RERF-LC-MA, H69, H82, H209, H529 and H1688). We used small interfering RNA (siRNA) to down-regulate the expression of DLL3 in SCLC cell lines. Anchorage-dependent and anchorage-independent cell growth was measured by MTT assay and migration and invasion were assessed by transwell assay.

Results: The mRNA of DLL3 was expressed in all of 9 SCLC cell lines. The expression of DLL3 was especially higher in H82, H69, H209, H529 and H1688 cells. Notch1 protein was expressed in SBC-3, SBC-5, MS-1 and H82. Based on DLL3 expression analysis data, we used H82 and H69 in the following experiments. The suppression of DLL3 by siRNA resulted in the moderate inhibition of cell growth of H82 cells in both anchorage-dependent and anchorage-independent cell proliferation. The depletion of DLL3 prevented migration and invasion in the two cell lines. The expressions of Notch1 and Notch target gene, Hes1 were downregulated by DLL3 knockdown in both SCLC cells. Because Notch pathway was reported to regulate EMT, we evaluated the EMT markers when DLL3 was inhibited in SCLC cells. Snail was downregulated in DLL3 knockdown SCLC cells, while protein expression levels of E-cadherin and Vimentin were not changed in these DLL3 knockdown cells compared to control cells.

Conclusions: DLL3 promotes the migration and invasion in SCLC cells by modulating Notch1 and Snail.

Citation Format: Megumi Furuta, Jun Konishi Sakakibara, Tetsuaki Shoji, Yuta Takashima, Hajime Kikuchi, Eiki Kikuchi, Junko Kikuchi, Ichiro Kinoshita, Hirotoshi Dosaka Akita, Masaharu Nishimura. DLL3 regulates migration and invasion of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3158.
Abstract 3159: Rac1 and Cdc42 as drivers in ovarian cancer metastasis
Authors: Rivera Melanie , Dominguez Dayna , Pauken Christine , Romero Elsa , Kenney S. Ray , Shi Yang , Lee Ji-Hyun , Gillette Jennifer , Hudson Laurie G. , Wandinger-Ness Angela
Published: August 2018

Five-year ovarian cancer patient survival has not improved for decades, largely due to disease recurrence. Therefore, drivers of relapse need to be identified. We first reported that Rac1 and Cdc42 GTPases are highly overexpressed in serous ovarian cancer using immunohistochemistry. To correlate GTPase expression with survival outcomes, we analyzed gene expression data in The Cancer Genome Atlas, revealing that ovarian cancer patients with the highest RAC1 RNA levels had significantly worse age-adjusted survival than those with low RAC1. Quantitative analyses of Rac1b, a constitutively active Rac1 splice variant, show dramatic overexpression in tumor epithelia relative to stroma and normal epithelia. Since high Rac1 and Cdc42 levels drive cancer cell adhesion, invasion and metastasis, we used multiple assays to assess the dependence of ovarian cancer cell behaviors on Rac1 and Cdc42. Using Matrigel invasion chambers, treatment of multiple ovarian cancer cell lines with specific Rac1 and Cdc42 inhibitors resulted in significantly decreased cellular invasion. Conversely, with overexpression of Rac1, ovarian cancer cells demonstrated increased invasion. Furthermore, IP injection of ovarian cancer cells overexpressing Rac1 resulted in increased adhesion, invasion and metastasis in the omentum and peritoneum in vivo. These data support Rac1 and Cdc42 as important drivers of invasive and metastatic ovarian cancer and as novel high-value therapeutic targets with potential impact in reducing disease metastasis.

Citation Format: Melanie Rivera, Dayna Dominguez, Christine Pauken, Elsa Romero, S. Ray Kenney, Yang Shi, Ji-Hyun Lee, Jennifer Gillette, Laurie G. Hudson, Angela Wandinger-Ness. Rac1 and Cdc42 as drivers in ovarian cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3159.
Abstract 320: Frequency and patient characteristics of homologous recombination deficiency in metastatic cutaneous melanoma
Authors: Kim Kevin B. , Millis Sherri Z. , Ross Jeffrey , Gay Laurie M. , Vosoughi Elham , Moretto John , Leong Stanley P. , Singer Mark I. , Parrett Brian M. , Minor David R. , Kashani-Sabet Mohammed
Published: August 2018

Purpose: In various cancer types, cells with a homologous recombination (HR) deficiency have been shown to be sensitive to PARP inhibitors. We investigated the frequency of HR pathway gene mutations in melanoma.Experimental Design: Next-generation sequencing analysis that included the entire coding sequence of 315 cancer-related genes was performed on formalin-fixed, paraffin-embedded melanoma samples. Clinical and pathologic characteristics of 62 patients with advanced melanoma were reviewed, and compared with a dataset of 1,986 melanoma patients with available molecular profiling data.Results: Nineteen (30.6%) patients had ≥1 functional HR mutation. The characteristics of patients with HR-deficient melanoma were: median age 69, male predominance (79%) and primary head and neck melanoma (53%). TMB was high in 12 (67%) patients and low in 1 (6%) patient. The most commonly mutated HR-associated gene was ARID2 (13%), followed by ARID1A, ATM, BRCA1, and FANCA (3% each). Among the 19 patients with HR mutation(s), concurrent NF1, NRAS, V600 BRAF, and KIT mutations were found in 7 (37%), 6 (32%), 5 (26%) and 1 (5%) patients, respectively. Presence of HR pathway mutation was associated with absence of ulceration at the primary site, high TMB and clinical response to checkpoint immunotherapy. A larger dataset analyzed by Foundation Medicine, Inc. (n=1,986) showed a similar frequency and pattern of HR mutations. Conclusions: HR pathway gene mutations are frequently observed in advanced melanoma. Melanomas with these alterations may represent a unique subset of patients who are more likely to benefit from checkpoint blockade, and also may be targeted with PARP inhibitors.

Clinical and pathologic characteristics of patients with genetic HR mutationsHR mutation (n=19)Non HR mutation (n=43)p-valueUlceration statusn=16*n=33*0.0050Present2 (12.5%)18 (54.5%)0.0114Absent14 (87.5%)15 (45.5%)0.0479Tumor mutation burdenn=18*n=38*0.0114Low1 (5.6%)17 (44.7%)Intermediate5 (27.8%)8 (21.1%)High12 (66.7%)13 (34.2%)Response to anti-PD-1 antibody inhibitors (+/- ipilimumab)n=12*n=23*0.0479Response10 (83.3%)10 (43.5%)Early Progression1 (8.3%)10 (43.5%)Stable disease1 (8.3%)3 (13.0%)

Citation Format: Kevin B. Kim, Sherri Z. Millis, Jeffrey Ross, Laurie M. Gay, Elham Vosoughi, John Moretto, Stanley P. Leong, Mark I. Singer, Brian M. Parrett, David R. Minor, Mohammed Kashani-Sabet. Frequency and patient characteristics of homologous recombination deficiency in metastatic cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 320.
Abstract 3216: H2AX is a novel prognostic marker of breast cancer
Authors: Katsuta Eriko , Ouchi Mutsuko , Ouchi Toru , Takabe Kazuaki
Published: August 2018

INTRODUCTION: Phosphorylation of histone variant H2AX, termed gamma-H2AX, is well known for its role in DNA damage repair. On the other hand, clinical relevance of expression of H2AX in breast cancer remains to be elucidated. Previously we found that H2AX determines clonogenic cell survival under metabolic oxidative stress and cellular levels of Reactive Oxygen Species (ROS). In this study, we investigated the impact of H2AX expression on breast cancer patients' survival, as well as underlying mechanisms.

METHODS: Breast cancer patients in The Cancer Genome Atlas (TCGA) dataset were classified as either high or low expression of H2AX. Overall Survival (OS) and Disease-Free Survival (DFS) as well as Gene Set Enrichment were compared between these two groups. Gene expression of each cell lines was analyzed by RNA-seq. Radiation sensitivity was quantified using cells from patient-derived xenograft (PDX). Gene expression level after neoadjuvant chemo-radio therapy was compared using publically available database (GSE25113).

RESULTS: H2AX expression level is higher in triple negative breast cancers (TNBC) than non-TNBC (p<0.001) and in HER2 positive tumors than negative tumors (p=0.048) as well as advanced Stage tumors (Stage I vs Stage II/III/IV, p=0.029). H2AX high expressing tumors showed significantly worse OS (p=0.007) as well as DFS (p=0.001) and this was the case only in advanced Stage tumors (Stage I vs Stage II/III/IV, p=0.029). There were more H2AX high tumors with advancement of Stage (Stage I: 2.8%, Stage II: 5.5%, Stage III/IV 7.4%). In agreement, breast cancer cell line clones that develop metastasis expressed higher H2AX compared from their parental cells (4T1 vs 4T1.2; p<0.001, MDA-MB-231 vs LM2-4; p=0.042). Gene Set Enrichment Analysis (GSEA) demonstrated that not only DNA repair (p<0.001) and ROS related gene set (p=0.011), but also Myc target related gene sets (p=0.002) as well as UV response related gene set (p<0.001) were significantly enriched in H2AX high patients. We also demonstrated that H2AX high expressing cells from PDX showed significantly higher sensitivity to radiation therapy with higher ROS production. Indeed, tumors that demonstrated pathological completely response (pCR) after neoadjuvant chemo-radiation therapy had a trend to have higher H2AX levels compared to non-pCR tumors (p=0.093).

CONCLUSIONS: Advanced Stage breast cancers that express high levels of H2AX associated with worse OS and DFS, but showed higher sensitivity to radiation. H2AX can be a prognostic biomarker for recurrent tumor that may be sensitive to radiation therapy.

Citation Format: Eriko Katsuta, Mutsuko Ouchi, Toru Ouchi, Kazuaki Takabe. H2AX is a novel prognostic marker of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3216.
Abstract 3293: Solid tumors have frequent mutation, copy number variation and differential mRNA expression of GPCRs: Are such GPCRs functional oncogenes
Authors: Sriram Krishna , Moyung Kevin , Corriden Ross , Insel Paul
Published: August 2018

G protein-coupled receptors (GPCRs), the largest family of membrane receptors, regulate numerous cellular functions and are drug targets for many disorders but not generally in cancer. We analyzed The Cancer Genome Atlas (TCGA) for GPCR mutations, copy number variation (CNV) and mRNA expression in 20 categories of solid tumors. We found that numerous GPCRs are frequently mutated, in particular, certain adhesion GPCRs (e.g.,GPR98 and GPR112), which appear to have a higher density of mutation events than other genes of similar length. Tumors with high mutational burden show a high frequency of such GPCR mutations that may result from DNA damage. CNV (especially amplifications and single-copy deletions) of GPCRs is widespread. In certain tumor types, particular GPCRs (e. g., GPR160) show high-level amplification in >30% of tumors. However, CNV does not appear to be a major factor in determining differential expression (DE) of GPCRs in tumors.

Compared to RNA expression in normal tissues (GTEX database), most solid tumor types (including those accounting for the most annual deaths) differentially express >50 GPCRs, including 77 GPCRs with increased expression in at least one tumor type that are targets for approved drugs, providing opportunities for drug repurposing. DE of GPCRs is largely independent of tumor stage/grade, largely insensitive to specific driver mutations, and similar in primary and metastatic tumors, which both have high expression of numerous GPCRs in nearly all members within tumor populations. Cancer cells highly express most GPCRs overexpressed in tumors, making such GPCRs (especially those that are druggable) attractive candidates for further validation and functional studies. Multiple published studies provide functional data for certain such GPCRs.

These findings imply a previously unappreciated role for GPCRs in solid tumors. Overexpressed GPCRs in tumors may regulate tumor biology as “functional oncogenes” and are potentially novel biomarkers and therapeutic targets, including for approved drugs.

Citation Format: Krishna Sriram, Kevin Moyung, Ross Corriden, Paul Insel. Solid tumors have frequent mutation, copy number variation and differential mRNA expression of GPCRs: Are such GPCRs functional oncogenes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3293.
Abstract 3318: Frequent promoter hypermethylation correlates with better prognosis in pharyngeal cancer
Authors: Nakagawa Takuya , Matsusaka Keisuke , Misawa Kiyoshi , Fukuyo Masaki , Takane Kiyoko , Hanazawa Toyoyuki , Matsubara Hisahiro , Okamoto Yoshitaka , Kaneda Atsushi
Published: August 2018

The incidence of Human Papilloma Virus (HPV) associated oropharyngeal cancer is dramatically increasing in developed countries, due to mainly HPV 16. Whereas viral infection status could affect molecular subtypes in other cancers, molecular features of HPV(+) and HPV(-) OPSCCs including epigenetic aberrations are not fully clarified. We here performed genome-wide DNA methylation analysis of OPSCC. We identified 144 candidate methylation-associated silencing genes using Infinium 450k analysis of 13 OPSCC samples, 4 normal mucosal samples and two HNSCC cell lines with/without 5Aza/TSA. Re-expression of the hypermethylated genes by 5-aza-2-deoxycytidine was confirmed by RNA-seq. Among the 144 candidate genes, 9 genes were selected and their methylation status was analyzed in 70 pharyngeal SCC cases including 55 OPSCC and 15 hypopharyngeal SCC cases using pyrosequencing. HPV(+) samples showed higher methylation level within all the samples (P=0.0002, t-test) and within OPSCC (P=0.001, t-test). While HPV infection significantly correlated with positive staining of p16 in immunohistochemistry, HPV(+) as well as p16(+) status significantly correlated with better prognosis (P = 0.005 and 0.002, log-rank test). The aberrant hypermethylation of an identified marker gene also correlated with better prognosis significantly when all the cases were analyzed (P = 0.04). Interestingly, the its hypermethylation status also correlated with better prognosis within p16(-) cases (P = 0.03). Our data suggest that aberrant DNA methylation might be involved in genesis of OPSCC, that DNA methylation might associate with HPV infection, and that methylation marker might work as prognostic marker. To elucidate the correlation between DNA methylation and OPSCC including HPV(+) and HPV(-) OPSCCs, we additionally performed genome-wide DNA methylation analysis of OPSCC using Infinium 450k analysis of 83 OPSCC samples, and integrated our data with 81 OPSCC data from the Cancer Genome Atlas (TCGA). HPV(+) and HPV(-) OPSCCs were mainly classified into two epigenotypes, respectively. Analyzing the correlation with clinicopathological data, we created a new classification strategy for OPSCC.

Citation Format: Takuya Nakagawa, Keisuke Matsusaka, Kiyoshi Misawa, Masaki Fukuyo, Kiyoko Takane, Toyoyuki Hanazawa, Hisahiro Matsubara, Yoshitaka Okamoto, Atsushi Kaneda. Frequent promoter hypermethylation correlates with better prognosis in pharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3318.
Abstract 3334: CREB is required for KRAS-driven lung tumorigenesis
Authors: Lee Jong woo , Slack Frank J. , Nestler Eric J. , Herbst Roy S. , Koo Jaseok Peter
Published: August 2018

The aberrant constitutive activation of the transcription factor, cyclic-AMP response element- binding (CREB) protein, has been associated with poor prognosis and chemotherapeutic resistance in KRAS-mutated human cancers, one of which includes non-small cell lung cancer (NSCLC). However, the role of CREB in KRAS-driven lung tumorigenesis has not been fully understood. Using adenoviral delivery of Cre specifically to the mouse lung, we generated a new transgenic lung cancer mouse model driven by Kras activating mutation G12D and concomitant with Creb loss (KrasLSL-G12D/+; CrebFlox/Flox). In this study, we used this model to show that a proto-oncogenic transcription factor CREB, is required for KRAS-driven lung tumorigenesis. Loss of Creb (Creb-/-) in the mice harboring Kras-mutated lung tumors significantly suppressed tumor development, and thus, dramatically prolonged mouse survival when compared to either wild (Creb+/+) or heterozygous (Creb+/-) deletion of Creb. Mechanistically, we identified a few novel CREB target gene. These data suggest that CREB is essential for KRAS-driven lung tumorigenesis and that CREB could be a potential target for therapeutic treatment for individuals suffering from KRAS-mutant lung cancers.

Citation Format: Jong woo Lee, Frank J. Slack, Eric J. Nestler, Roy S. Herbst, Jaseok Peter Koo. CREB is required for KRAS-driven lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3334.
Abstract 3342: Truncated glioma-associated oncogene homolog 1 (tGLI1) is a novel mediator of mesenchymal subtype of glioblastoma and a novel transcriptional activator of CD44
Authors: Rimkus Tadas K.
Published: August 2018

The subpopulation of stem-like cells, or glioma stem cells (GSCs), is a key driver of tumor initiation, recurrence, and chemoresistance. Recently, patient-derived GSC lines were identified to contain two mutually exclusive subtypes termed proneural and mesenchymal. Mesenchymal GBM and GSCs were found to be more aggressive and radioresistant; however, the molecular pathways supporting mesenchymal GBM and mesenchymal GSCs are not well understood. To address this knowledge gap, we examined truncated glioma-associated oncogene homolog 1 (tGLI1) for its role in mesenchymal subtypes of GBM and GSCs. Discovered in our laboratory, tGLI1 is a novel alternatively spliced, gain-of-function variant of GLI1 zinc-finger transcription factor with an in-frame deletion of 41 codons retains all of the known functional domains of GLI1, but gains the ability to activate genes not regulated by GLI1. In this study, we report for the first time that tGLI1 is a tumor-specific transcription factor that facilitates GBM growth, enriches in the mesenchymal subtype of GBM and GSCs, and promotes mesenchymal GSCs via transcriptionally activating CD44 expression. Using an orthotopic GBM xenograft mouse model, we observed that tGLI1-overexpressing tumors grew more aggressively, with increased proliferation and angiogenesis, compared to control and GLI1-overexpressing xenografts. IHC analysis using tGLI1- and GLI1-selective antibodies we developed showed that tGLI1 was significantly expressed in GBM specimens but undetectable in normal brains whereas GLI1 was expressed in both tissues. Datamining further showed that tGLI1 activation signature (tGAS) was correlated with glioma grade, tumor angiogenesis, and poor overall survival, and that GBMs with high tGAS were enriched with mesenchymal GBM and GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared to the monolayer culture. Mesenchymal GSCs expressed more tGLI1 than proneural GSCs. Ectopic tGLI1 expression significantly enhanced the ability of GSCs to yield neurospheres in vitro and form aggressive tumors in mouse brains. Finally, we examined the ability of tGLI1 to regulate stemness genes and found that tGLI1 binds to and transactivates the promoter of the CD44 gene, a marker and mediator for mesenchymal GSCs. Collectively, these findings advanced our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSCs.

Citation Format: Tadas K. Rimkus. Truncated glioma-associated oncogene homolog 1 (tGLI1) is a novel mediator of mesenchymal subtype of glioblastoma and a novel transcriptional activator of CD44 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3342.
Abstract 3377: Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing
Authors: Loeb Lawrence A. , Loubet-Senear Kaitlyn J. , Kohrn Brendan F. , Schmitt Michael W. , Bronner Mary P. , Beckman Robert A.
Published: August 2018

The accumulation of somatic mutations is a defining hallmark of cancer. Human colorectal cancers (CRC) contain thousands of mutations that are detected at frequencies greater than 1%. Some of these are “driver mutations” that have been positively selected during tumor progression. In addition, subclonal mutations—those occurring only in a fraction of malignant cells—are randomly distributed and contribute to phenotypic and morphologic heterogeneity of cancer cells within a tumor. Cells containing specific subclonal mutations may be present prior to therapy and can be positively selected and account for the rapid emergence of therapeutic resistance. The extent of subclonal mutations in cancer, however, has been difficult to quantify, as the high error rate of next-generation sequencing precludes reliable detection of mutations present in fewer than 1-5% of cells. Here, we apply the highly accurate duplex sequencing methodology to quantify the extent of subclonal mutations in CRC. We analyzed somatic mutations in genes encoding replicative DNA polymerases (POLδ and POLε) as well as in genes reported to be frequently mutated in CRC. Duplex sequencing was carried out at depths varying from 500X-30,000X and an accuracy of 10-8. We find that CRCs with DNA repair deficits harbor an unexpectedly large complement of unique subclonal mutations; indicating that the mutational diversity of CRCs has been greatly underestimated. The frequency of subclonal mutations in normal colonic mucosa and CRC is greater than reported for clonal mutations. The burden of tumor-associated subclonal mutations does not correlate with age and the spectrum of single-nucleotide substitutions is different from that in nonmalignant cells, indicating that different mechanisms of mutation accumulation are operative in normal and CRC. Our data are consistent with a mutation rate of 1.1 x 10 per base pair, indicating that each malignant cell and its daughter encoded genomic DNA differs by more 3,000 nucleotides. The linearity of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from five different tumors, is in accord with a neutral model of tumor evolution. We calculate that the probability of mutations at any type is so high that it is likely that every possible somatic point mutation is present by the time a tumor is clinically detectable, and this could account for the high frequency by which tumors become resistant to therapeutic agents.

Citation Format: Lawrence A. Loeb, Kaitlyn J. Loubet-Senear, Brendan F. Kohrn, Michael W. Schmitt, Mary P. Bronner, Robert A. Beckman. Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3377.