bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5517: Antitumor effect of LRIG1, negative regulator of ErbB, in lung cancer harboring mutant-EGFR
Authors: Torigoe Hidejiro , Toyooka Shinichi , Sakaguchi Masakiyo , Shien Kazuhiko , Yamamoto Hiromasa , Soh Junichi , Asano Hiroaki , Tomida Shuta , Tsukuda Kazunori , Miyoshi Shinichiro
Published: July 2017

Introduction: EGFR is a member of the ErbB family of tyrosine kinase receptors (RTKs) and plays important roles in the pathogenesis of certain human cancers. In non-small cell lung cancer (NSCLC), mutations in EGFR gene are commonly known as oncogenic driver mutation and targeted for treatment. Leucine rich repeat and immunoglobulin-like domain protein-1 (LRIG1) is a cell surface protein and known as a negative regulator of ErbB family. In brain tumor, LRIG1 was reported to downregulate not only wild-type EGFR, but also EGFRvIII, mutant-type EGFR. But there has been no report about the relationship between LRIG1 and EGFR mutation in the kinase domain frequently found in lung cancer. In this study, we investigate the role of LRIG in EGFR mutant lung cancer from the view point of its effect on signal transduction and cell proliferation and invasion.

Method: We examined the LRIG1 expression levels in lung cancer cell lines and the surgically resected primary lung cancer tissues by quantitative PCR assay. We made stable clone of EGFR-mutant cell line (HCC827; exon19del E746-A750) overexpressing GFP tagged LRIG1, and compared with stable clone overexpressing GFP alone.

Results: The LRIG1 expression levels in lung cancer cell lines and the surgically resected primary lung cancer tissues were much lower than that in normal bronchial epithelial cells and their normal tissues, respectively. The introduction of LRIG1 decreased EGFR expression and their phosphorylation in EGFR-mutant cell line. LRIG1 strongly suppressed cell proliferation, invasion and migration of EGFR-mutant cell line. In addition, a phospho-RTK array revealed that LRIG1 also downregulated the other RTKs, such as HER2, HER3, MET, and IGF-1R.

Conclusion: Our findings demonstrates that LRIG1 decreases the expression and phosphorylation of mutant-EGFR protein and has strong anti-tumor effect in lung cancer harboring EGFR mutation. These findings suggest the importance of LRIG1 related studies to develop a novel strategy for EGFR mutant cancers.

Citation Format: Hidejiro Torigoe, Shinichi Toyooka, Masakiyo Sakaguchi, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Shuta Tomida, Kazunori Tsukuda, Shinichiro Miyoshi. Antitumor effect of LRIG1, negative regulator of ErbB, in lung cancer harboring mutant-EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5517. doi:10.1158/1538-7445.AM2017-5517
Abstract 5519: psiTPTE22-HERV functions as a tumor suppressor in gastric cancer and is associated with disease outcome
Authors: Liang Jessie Qiaoyi , Xu Fei , Zheng Shu , Yu Jun
Published: July 2017

Background and aim: The novel gene psiTPTE22-HERV identified by us previously is a human endogenous retrovirus (HERV)-related gene located adjacent to the gene psiTPTE22. With a high GC content around the promoter region, expression of psiTPTE22-HERV can be regulated epigentically by DNA methylation. We aimed to elucidate the clinical significance of epigenetic alteration and biological function of psiTPTE22-HERV in gastric cancer.

Methods and results: psiTPTE22-HERV was ubiquitously expressed in normal adult tissues including stomach, but was frequently silenced/down-reguated in gastric tumors and cancer cell lines as evidenced by RT-PCR. Bisulfite genomic sequencing results indicated that psiTPTE22-HERV was silenced by promoter DNA methylation, and its expression could be restored by demethylation treatments in gastric cancer cell lines. Ectopic expression of psiTPTE22-HERV significantly suppressed cell viability, clonogenicity and cell cycle progression, induced apoptosis, and inhibited migration and invasion of SGC7901 and MKN45 gastric cancer cells. In contrast, knock-down of psiTPTE22-HERV in the gastric cancer MKN1 cells significantly increased cell growth and migration ability, promoted cell cycle progress and inhibited cell apoptosis. psiTPTE22-HERV significantly suppressed subcutaneous tumorigenicity of SGC7901 cells in nude mice and metastasis (liver implantation) in tail vein injection models. Promoter methylation level of psiTPTE22-HERV was significantly higher in gastric tumors than in adjacent non-tumor tissues as revealed by bisulfite genomic sequencing (P<0.05); methylation levels in both tumors and adjacent non-tumor tissues of gastric cancer patients were significantly higher than in normal stomach tissues (both P<0.001). Multivariate Cox regression analysis demonstrated that promoter methylation of psiTPTE22-HERV in primary gastric tumors was an independent risk factor for poor survival. Kaplan-Meier survival curves showed that high-methylation of psiTPTE22-HERV promoter was significantly associated with shortened survival in gastric cancer patients from two independent cohorts (both P<0.05).

Conclusion: psiTPTE22-HERV functions as a tumor suppressor that is commonly down-regulated by promoter methylation in gastric cancer, which may serve as a prognostic biomarker for gastirc cancer patients.

Citation Format: Jessie Qiaoyi Liang, Fei Xu, Shu Zheng, Jun Yu. psiTPTE22-HERV functions as a tumor suppressor in gastric cancer and is associated with disease outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5519. doi:10.1158/1538-7445.AM2017-5519
Abstract 5535: Effect of LKB1 activity on the sensitivity to PI3K/mTOR inhibitor in non-small-cell lung cancer
Authors: Shukuya Takehito , Yamada Tadaaki , Koenig Michael J. , Rahman Mohammad A. , Amann Joseph M. , Carbone David P.
Published: July 2017

The serine-threonine liver kinase B1 (LKB1, also called STK11) is a tumor suppressor that functions as master regulator of cell growth, metabolism, survival and polarity. Germline mutation of LKB1 causes Peutz-Jeghers syndrome, which is an autosomal dominant disease characterized by mucocutaneous pigmentation and hamartomatous polyps. In non-small-cell lung cancer (NSCLC), LKB1 is frequently rendered non-functional, either through mutation or down regulation. As a tumor suppressor and a protein whose function is lost, identifying pathways that are activated with LKB1 loss may be the only way to target such tumors. Some previous studies have suggested that inhibition of mTOR, a growth promoting pathway negatively regulated by LKB1, as a potential target in LKB1 mutant NSCLC. In this study, we investigated the effect of LKB1 activity on the sensitivity to a PI3K/mTOR inhibitor using four LKB1 mutant NSCLC cell lines to which LKB1 gene was transduced and one LKB1 wild type NSCLC cell line in which LKB1 gene was knocked out by CRISPR technology. Transduction of LKB1 resulted in significant resistance to a PI3K/mTOR inhibitor in two of the four LKB1 add-back cell lines, while knocking out LKB1 in the LKB1 wild type cell line induced PI3K/mTOR inhibitor sensitivity. The mechanism behind these observed results appears to be through regulation of phosphorylation of AKT. The presence of LKB1 led to a persistent AKT phosphorylation even in the presence of the PI3K/mTOR inhibitor, both in add back cell and wild type cells. Our data suggest that the identification of LKB1 activity may be a promising biomarker for the sensitivity to PI3K/mTOR inhibition. Further investigation into the mechanism of LKB1 loss and increased sensitivity to inhibition of the PI3K and mTOR pathways is warranted.

Citation Format: Takehito Shukuya, Tadaaki Yamada, Michael J. Koenig, Mohammad A. Rahman, Joseph M. Amann, David P. Carbone. Effect of LKB1 activity on the sensitivity to PI3K/mTOR inhibitor in non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5535. doi:10.1158/1538-7445.AM2017-5535
Abstract 5536: The metastasis suppressor NME1 regulates stemness in melanoma
Authors: Wang Ying , Kaetzel David M.
Published: July 2017

Melanoma is a lethal skin cancer that is highly resistant to therapy and prone to metastasize to distant organs. The quiescence melanoma stem cells are believed to account for the resistance to therapies. Traditional chemotherapies designed to target fast-proliferative tumor cells tend to miss quiescent stem cells. The escaped quiescent stem cells may accumulate oncogenic mutation from common mutagenic sources and finally lead to tumor relapse. NME1 is a metastasis suppressor gene that inhibits the metastatic potential in melanoma and other cancers. In this study, we focus on the role of NME1 in the regulation of stemness and quiescence of melanoma stem cells. We find that the genesis and growth of stem cell spheroids derived from melanoma cell lines are abrogated after knockdown of NME1. We also find that the mRNA level of several melanoma stem cell markers are elevated when we knockdown NME1. These results suggest that NME1 regulates the survival and stemness of melanoma stem cells. In addition, we demonstrate that melanoma stem cells are heterogeneous in NME1 expression. Specifically, the NME1 expression is markedly elevated in a highly proliferative subpopulation in the spheroids that express the stem cell markers OCT4, SOX2 and SOX10. Conversely, we observe another subpopulation of these spheroids that exhibits low NME1 expression and is non-proliferative. We hypothesize that this subpopulation consists quiescent melanoma stem cells that are more resistance to therapies, and that their quiescent status is controlled by the level of NME1. We are now employing CRISPR technology to separately study potentials in tumorigenesis, metastasis, and therapy-resistance of the stem cell subpopulations.

Citation Format: Ying Wang, David M. Kaetzel. The metastasis suppressor NME1 regulates stemness in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5536. doi:10.1158/1538-7445.AM2017-5536
Abstract 5537: Loss of ITM2A, a novel tumor suppressor of ovarian cancer through G2/M cell cycle arrest, is a poor prognostic factor of epithelial ovarian cancer
Authors: Kim So Young , Lee Eun-Ju , Cho Min Ji , Rhee Hyemin
Published: July 2017

Integral membrane protein 2A (ITM2A) is a type 2 transmembrane protein of unknown function. The aim of this study was to investigate its expression pattern, clinical significance, and biological function in epithelial ovarian cancer. ITM2A expression in 35 normal, 20 adenoma, 11 borderline and 90 cancerous ovarian tissues was measured by immunohistochemistry. Clinicopathological parameters were obtained from medical records. Survival data was analyzed using Kaplan-Meier estimates and multivariate analysis using the Cox-regression method. Anti-tumor activities of ITM2A were explored by cell proliferation and colony formation assays, flow cytometry, Western blots and animal studies using ovarian cancer cell lines. Chemoresponsiveness was evaluated by measuring IC50 and confirmed by animal studies using an intraperitoneal orthotropic model. ITM2A was significantly downregulated in invasive carcinomas compared to normal, adenoma and borderline tumor tissues. ITM2A loss occurred in 45.6% (41 of 90) of invasive carcinomas and was significantly associated with FIGO stage, type II tumors, suboptimal debulking operation, recurrence and chemoresistance. ITM2A loss and higher FIGO stage were independent factors for poor prognosis. Expression of ITM2A inhibited growth and induced G2/M cell cycle arrest by attenuating cdc2, cyclin B1, cdc25c and p-cdc2 (Thr 161). In vitro and in vivo experiments showed that ITM2A expression significantly reduced the paclitaxel and carboplatin IC50 and tumor mass after paclitaxel treatment. ITM2A is a new biomarker of poor prognosis in ovarian cancer. It is a novel tumor suppressor that induces cell cycle arrest, acts as a chemosensitizer, and has therapeutic potential for ovarian cancer.

Citation Format: So Young Kim, Eun-Ju Lee, Min Ji Cho, Hyemin Rhee. Loss of ITM2A, a novel tumor suppressor of ovarian cancer through G2/M cell cycle arrest, is a poor prognostic factor of epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5537. doi:10.1158/1538-7445.AM2017-5537
Abstract 5556: Widespread protein interactome network rewiring in human cancer
Authors: Yi Song , Sahni Nidhi
Published: July 2017

In the past decade, genome and exome sequencing projects have identified thousands of genetic variants in patients across a large number of cancer types. However, the explosion of genomic information has left many fundamental questions regarding genotype-phenotype relationships unresolved. One critical challenge is to distinguish causal disease mutations from non-pathogenic polymorphisms. Even when causal mutations are identified, the functional consequence of such mutations is often elusive. Classical “one gene, one function, one disease” models can not reconcile with the complexity that different mutations of the same gene often lead to different phenotypes. The extent to which network perturbations are involved in disease malfunction and how distinct interaction perturbation patterns can distinguish cancer mutations are largely unknown. Here we report a systematic approach to investigate genetic variant-specific effects on molecular interactions at large scale across diverse human cancers. Remarkably, in comparison to non-disease polymorphisms, disease mutations are more likely to associate with interaction perturbations. A large fraction of missense disease mutations are found to cause protein interaction alterations. While some result in loss of all their interactions, many exhibit selective elimination of specific interactions (“edgetic”). Different mutations of the same gene give rise to different interaction profiles, accounting for distinct disease outcomes. Edgetic mutations perturb interactions through disrupting specific interaction interfaces, and the perturbed partners are more likely expressed in relevant disease tissue. Together, our approach is insightful in prioritizing disease-causing variants, and uncovering patient mutation-specific disease mechanisms at a base-pair resolution, a critical step towards personalized precision medicine. Furthermore, our results suggest distinct interaction perturbations as a widespread mechanism underlying genetic heterogeneity, providing a fundamental link between genotype and phenotype in cancer.

Citation Format: Song Yi, Nidhi Sahni. Widespread protein interactome network rewiring in human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5556. doi:10.1158/1538-7445.AM2017-5556
Abstract 5568: Towards decoding the interplay between glycolysis and oxidative phosphorylation in cancer
Authors: Jia Dongya , Yu Linglin , Lu Mingyang , Ben-Jacob Eshel , Ma Jianpeng , Levine Herbert , Kaipparettu Benny A. , Onuchic Jose
Published: July 2017

Abnormal metabolism is a hallmark of cancer, yet its regulatory mechanism is poorly understood. Cancer cells were considered to mostly utilize glycolysis, referred to as the Warburg effect. However recent evidence suggests that oxidative phosphorylation also plays a crucial role during cancer progression. Here we utilized a systems biology approach to decipher the regulatory principle of glycolysis and oxidative phosphorylation. Integrating information from literature, we constructed a regulatory network of genes and metabolites, from which we extracted a core circuit containing HIF-1, AMPK and ROS. Our circuit analysis showed that while normal cells have an oxidative state and a glycolytic state, cancer cells can access an additional hybrid state with both metabolic modes coexisting, due to higher ROS production and/or oncogenic activation, such as RAS, MYC and c-SRC. The anti-correlation between AMPK and HIF-1 and the association of metabolic states with oncogenes were further confirmed using TCGA patient transcriptomics data of multiple cancer types and single-cell RNA-seq data of lung adenocarcinoma. We propose that the hybrid phenotype contributes to metabolic plasticity, allowing cancer cells to adapt to various microenvironments. Using model simulations, we predicted the efficacies of several metabolic cancer therapies based on their effectiveness in reducing metabolic plasticity. Our theoretical framework of metabolism can serve as a platform to decode cancer metabolic plasticity and design cancer therapies targeting metabolism.

Citation Format: Dongya Jia, Linglin Yu, Mingyang Lu, Eshel Ben-Jacob, Jianpeng Ma, Herbert Levine, Benny A. Kaipparettu, Jose Onuchic. Towards decoding the interplay between glycolysis and oxidative phosphorylation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5568. doi:10.1158/1538-7445.AM2017-5568
Abstract 5586: PPMX-T003, a fully human anti-TfR1 antibody with potent efficacy against hematologic malignancies
Authors: Zhang Lilin , Nomura Fumiko , Aikawa Yoichi , Kurosawa Yoshikazu , Morishita Kazuhiro , Sudo Yukio
Published: July 2017

Transferrin receptor1 (TfR1) is a type II transmembrane glycoprotein that involves intracellular uptake of iron. TfR1 is ubiquitously expressed at low levels in normal cells except erythroblasts and placental trophoblasts, which express high levels of TfR1 due to the need of up taking a large amount of iron. Accumulating studies have shown elevated levels of TfR1 in both solid tumor cells, as well as hematologic malignant cells when compared to their normal counterparts. Since TfR1 is implicated in growth and survival in various cancer cells, targeting TfR1 could be attractive strategy for cancer therapeutics. To this end, we have developed a fully human antibody against TfR1, PPMX-T003, which displayed potent anti-tumor activity in vitro and in vivo. PPMX-T003 induced apoptosis or cell cycle arrest with EC50s of 3~200ng/ml in various tumor cell lines. Mechanistically, PPMX-T003 triggers apoptosis or cell growth arrest by inhibiting binding of TfR1 to its ligand transferrin and blocking iron uptake. In addition, PPMX-T003 elicits antibody dependent cellular cytotoxicity (ADCC) activity in cancer cells. Importantly, PPMX-T003 showed potent efficacy against several blood cancer xenograft models. PPMX-T003 completely eradicated established subcutaneous tumors in two acute myeloid leukemia (AML) models generated by Kasumi-1 and HL-60, at a dose of 10 mg/kg when administrated (I.V.) once a week for 4 weeks. Moreover, PPMX-T003 completely eradicated established tumors in a lymphoma xenograft model (SU-DHL-2) at a dose of 3 mg/kg. Furthermore, PPMX-T003 greatly prolonged mice survival in a disseminated leukemia model (CCRF-CEM: acute lymphoblastic leukemia cell line). The control mice engrafted with CCRF-CEM cells (n=10) developed leukemia and died within 42 days, whereas 8 of the 10 mice treated with PPMX-T003 survived 179 days until the experiment was terminated. A preliminary toxicology study in Cynomolgus monkeys with multiple doses was also carried out. Although moderate anemia was observed at the dose of 30 mg/kg, no other abnormalities were observed, indicating a tolerable safety profile. Taken together, these results indicate that PPMX-T003 could be a potent therapeutic antibody for the treatment of hematologic malignancies.

Citation Format: Lilin Zhang, Fumiko Nomura, Yoichi Aikawa, Yoshikazu Kurosawa, Kazuhiro Morishita, Yukio Sudo. PPMX-T003, a fully human anti-TfR1 antibody with potent efficacy against hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5586. doi:10.1158/1538-7445.AM2017-5586
Abstract 5604: Novel biomarkers Ly6E/K play important role in drug resistance and tumor immune escape
Authors: Hossiny Midar A. , Frazier William R. , Steiner Noriko , Kallakury Bhaskar , Kumar Rakesh , Upadhyay Geeta
Published: July 2017

Stem cell antigen Sca-1 is implicated in murine cancer stem cell biology and breast cancer models, but the role of its human homologs Ly6K and Ly6E in breast cancer are not established. Here we report increased expression of Ly6K/E in human breast cancer specimens correlates with poor overall survival, with an additional specific role for Ly6E in poor therapeutic outcomes. Increased expression of Ly6K/E also correlated with increased expression of the immune checkpoint molecules PDL1 and CTLA4, increased tumor-infiltrating T regulatory cells, and decreased natural killer (NK) cell activation. Mechanistically, Ly6K/E was required for TGFβ signaling and proliferation in breast cancer cells, where they contributed to phosphorylation of Smad1/5 and Smad2/3. Furthermore, Ly6K/E promoted cytokine-induced PDL1 expression and activation and binding of NK cells to cancer cells. Finally, we found that Ly6K/E promoted drug resistance and facilitated immune escape in this setting. Overall, our results establish a pivotal role for a Ly6K/E signaling axis involving TGFβ in breast cancer pathophysiology and drug response, and highlight this signaling axis as a compelling realm for therapeutic invention.

Citation Format: Midar A. Hossiny, William R. Frazier, Noriko Steiner, Bhaskar Kallakury, Rakesh Kumar, Geeta Upadhyay. Novel biomarkers Ly6E/K play important role in drug resistance and tumor immune escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5604. doi:10.1158/1538-7445.AM2017-5604
Abstract 5610: Quantitative cell-based bioassays to advance individual or combination immune checkpoint immunotherapy
Authors: Grailer Jamison , Stecha Pete , Garvin Denise , Hartnett Jim , Fan Frank , Cong Mei , Cheng Zhi-jie Jey
Published: July 2017

Immune checkpoint receptors play a critical role in maintaining immune homeostasis and are genetically and functionally associated with autoimmune disease, cancer and persistent viral infections. Blockade of immune checkpoints (e.g., PD-1 and CTLA-4) has emerged as a promising new approach to enhance anti-tumor immune responses. While immunotherapies directed against PD-1 and CTLA-4 are showing unprecedented efficacy in the treatment of cancer, many patients and tumor types remain refractory to these therapies. This has resulted in a broadening of immunotherapy research and development to include additional immune checkpoint receptors (e.g., LAG-3, TIGIT, CD112R) targeted individually or in combination with other immunotherapy strategies.

A major challenge in the development of biologics that target immune checkpoints is access to quantitative and reproducible functional bioassays. Existing methods rely on primary cells and measurement of complex functional endpoints. These assays are cumbersome, highly variable, and fail to yield the quality of data that is required for drug development in a quality-controlled environment. To address this need, we have developed a suite of immune cell line-based bioluminescent reporter bioassays for individual and combination immune checkpoint immunotherapy targets including PD-1 (PD-L1 or PD-L2), CTLA-4, LAG-3, TIGIT, PD-1+TIGIT and more. These assays consist of stable cell lines that express luciferase reporters driven by specific response elements under the precise control of intracellular signals mediated by the T cell receptor and immune checkpoint target(s). These mechanism of action-based bioassays are available in “thaw-and-use” format and demonstrate high specificity, sensitivity and reproducibility. The bioassays are pre-qualified according to ICH guidelines and demonstrate the performance required for use in antibody screening, potency testing and stability studies.

Citation Format: Jamison Grailer, Pete Stecha, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative cell-based bioassays to advance individual or combination immune checkpoint immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5610. doi:10.1158/1538-7445.AM2017-5610
Abstract 5615: Genomic differential expression analysis of fusion proteins incorporating the pro-apoptotic molecule Granzyme B reveals new potential targets for treatment of breast cancer
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , Alvarez-Cienfuegos Ana , Rosenblum Michael G.
Published: July 2017

Granzyme B (GrB) is a member of the serine protease family of enzymes that play a critical role in the body’s defense against viral infection and tumor development. Cytotoxic T lymphocyctes (CTL) and natural killer (NK) cells directly deliver granzymes to target cells, which induces apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. Anti-tumor efficacy studies have suggested that the targeted delivery of human Granzyme B to tumor cells has a significant potential for cancer treatment. We had previously developed a novel fusion construct composed of the VEGF121 growth factor and human GrB. The GrB/VEGF121 construct was found to be highly cytotoxic to vascular endothelial cells expressing the R2 (KDR) receptor for VEGF. In this study, we examined the mechanism of GrB/VEGF121-induced cytotoxicity against cells in culture at the genomic level. The TNBC cell line MDA-MB-231 was treated with an IC50 dose of GrB/VEGF121 for 24 hours; the cells were then harvested and the effect of GrB/VEGF121 on intracellular events was examined by extraction of mRNA followed by microarray analysis. Gene-level differential expression analysis revealed that a total of twenty genes were upregulated by over 3-fold, while 25 were downregulated 3-fold or more. These included genes involved in signal transduction, stress response, cell cycle control, hypoxia and metastasis. Particularly impacted were the Nuclear Receptors Meta pathway, NRF2 pathway, nuclear receptors in lipid metabolism and toxicity, matrix metalloproteinases, and the notch signaling pathway. Validated data will be reported following complete analysis of gene level differential expression and alternative splicing. Our data suggests that GrB/VEGF121 induces expression of genes known to be induced by VEGF alone, as well as molecules previously not associated with either Granzyme B or VEGF.This data suggests a previously unsuspected impact of the serine protease GrB on various molecular pathways within the cell and may lead to a new understanding of how these agents operate at the molecular level. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Michael G. Rosenblum. Genomic differential expression analysis of fusion proteins incorporating the pro-apoptotic molecule Granzyme B reveals new potential targets for treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5615. doi:10.1158/1538-7445.AM2017-5615
Abstract 5616: Activation of CD137/CD137L results in reduced tumor cell proliferation in human colon cancer
Authors: Grimmig Tanja , Callies Simone , Naegele Hannes , Wagner Martin , Moench Romana , Polat Buelent , Germer Christoph-Thomas , Gasser Martin , Waaga-Gasser Ana Maria
Published: July 2017

Background: CD137L is expressed on antigen presenting cells and CD137/CD137L interaction co-stimulates activation and proliferation of CD137 expressing T cells. CD137L expression has also been detected on various cancer cells. While signaling in immune cells showed both inhibiting and activating effects the impact of CD137L induction on tumor cells is poorly understood. In this study, the expression of CD137 and CD137L and the effects of CD137L activation were analyzed in human colorectal cancer (CRC).

Methods: Expression of CD137 and CD137L was examined in human colon cancer cell lines HT29, SW480 and SW620 treated with or without hypoxia using RT-qPCR, Western blot and FACS analysis. Its differentiated expression was further analyzed in human CRCs by immunofluorescent double-staining. To dissect the effects of CD137L activation on cancer cells, MTS proliferation assays were additionally performed.

Results: All three human colon cancer cell lines demonstrated high cellular (FACS) and protein (Western Blot) CD137L expression. CD137 was found only in SW480 cells at weak expression. Additionally, both CD137 and CD137L were significantly upregulated in cells treated with hypoxia. In human CRCs both CD137 and CD137L expressing tumor cells were detected by immunofluorescent double-staining. Interestingly, activation of CD137L by immobilized CD137-Fc resulted in reduced cancer cell proliferation compared to IgG1-Fc controls.

Conclusion: Our results demonstrate tumor cell mediated expression of the costimulatory molecule CD137 and its ligand CD137L in both colon cancer cell lines and human colorectal cancer. Moreover, CD137L reverse signaling was found to reduce cancer cell proliferation in vitro. Based on these data, the CD137/CD137L signaling pathway seems to present a promising target for cancer therapy in CRC.

Note: This abstract was not presented at the meeting.

Citation Format: Tanja Grimmig, Simone Callies, Hannes Naegele, Martin Wagner, Romana Moench, Buelent Polat, Christoph-Thomas Germer, Martin Gasser, Ana Maria Waaga-Gasser. Activation of CD137/CD137L results in reduced tumor cell proliferation in human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5616. doi:10.1158/1538-7445.AM2017-5616
Abstract 5625: Identification and validation of a PD-L1 specific peptide for determination of PD-L1 expression in tumors
Authors: Caldwell Charles , Johnson Cory , Hammer Richard , Kannan Raghuraman
Published: July 2017

In recent years, immunotherapy has seen a resurgence as a promising method of treating cancers. The interaction between Programmed Death Ligand 1 (PD-L1) and its receptor, Programmed Death 1 receptor (PD-1) has been of particular interest. Many different types of tumors have been shown to overexpress PD-L1, which causes host immune cells that express the PD-1 receptor and bind PD-L1 to cease killing the tumor. Patients who have tumors exhibiting high levels of PD-L1 show poor prognosis compared to those who do not. Inhibition of the PD-L1 PD-1 binding axis has been shown to restore host immunity to the tumor, and recently many new drugs such as atezolizumab and pembrolizumab have been developed to target this interaction. Current methods of PD-L1 diagnosis have shown to vary based on the antibody, detection kit brand, antigen retrieval method, and clinically strict defined methods for use by the FDA for assessment. To refine detection of PD-L1, we have identified a PD-L1 specific peptide which can be used to detect PD-L1 expressing tumors using either conventional immunohistochemistry or flow cytometry. Immunohistochemistry using this peptide was tested against the FDA-approved PD-L1 (SP263) Rabbit monoclonal primary antibody on biopsied patient tissues. Flow cytometry was performed on both cultured cell lines and patient tissues. We have shown that our PD-L1 peptide shows specific staining in the tumor regions of FFPE tissues where the SP263 kit does not stain, and we can also detect PD-L1 expression in both cell samples and patient tissues using flow cytometry.

Citation Format: Charles Caldwell, Cory Johnson, Richard Hammer, Raghuraman Kannan. Identification and validation of a PD-L1 specific peptide for determination of PD-L1 expression in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5625. doi:10.1158/1538-7445.AM2017-5625
Abstract 5626: Investigation of T cell activation by anti-human PD-1 antibodies Nivolumab, Pembrolizumab and BGB-A317 using tumor-infiltrating lymphocytes (TILs) from colorectal cancer and colorectal liver metastasis patients
Authors: Luo Lusong , Wu Xiaoran , Zhang Tong , Fu Chunyan , Zhang Yanjuan , Guo Amy , Zhou Dongping , Zhang Lianhai , Wang Kun , Xing Baocai , Ji Jiafu , Wang Lai , Li Kang
Published: July 2017

Blockade of the PD-1 pathway with anti-PD-1 antibody, such as nivolumab (nivo) and pembrolizumab (pembro), has led to remarkable clinical responses in patients with many different cancer types. In this study, we investigated the activation of tumor-infiltrating lymphocytes (TILs) from patients with colorectal cancer (CRC) and colorectal liver metastasis (CLM) by nivo, pembro and BGB-A317, a novel humanized IgG4 anti-PD-1 antibody under clinical development. BGB-A317 has a unique binding signature to PD-1 with high affinity. Additionally, it is engineered to remove Fc gamma receptor (FcγR) binding, including FcγRI, FcγRIIA, FcγRIIB and FcγRIIIA. In our studies, all three anti-PD-1 antibodies showed significant increases in IFN-γ and TILs proliferation in a 3D tumor spheroid model. In both CRC and CLM patients, BGB-A317 treatment at concentration levels of 0.1, 1 and 10 μg/mL led to higher IFN-γ production than that in nivo and pembro treated groups. The enhanced TILs function was associated with a high density of CD8+ T cells, but inversely correlated with the percentage of CD11b+ myeloid cells in CRC tumors. Of interest, BGB-A317 showed better activation of TILs in the liver metastasis tumor microenvironment (TME) where macrophages were more abundant. This observation is consistent with the hypothesis that removal of FcγR binding might offer advantage to PD-1 mAbs in tumor microenvironment enriched with macrophages.

Citation Format: Lusong Luo, Xiaoran Wu, Tong Zhang, Chunyan Fu, Yanjuan Zhang, Amy Guo, Dongping Zhou, Lianhai Zhang, Kun Wang, Baocai Xing, Jiafu Ji, Lai Wang, Kang Li. Investigation of T cell activation by anti-human PD-1 antibodies Nivolumab, Pembrolizumab and BGB-A317 using tumor-infiltrating lymphocytes (TILs) from colorectal cancer and colorectal liver metastasis patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5626. doi:10.1158/1538-7445.AM2017-5626
Abstract 563: Quantitative EMT expression score for predicting survival outcome
Authors: George Jason T. , Jolly Mohit K. , Levine Herbert
Published: July 2017

Tumor aggressiveness and subsequent metastasis still remain as major limitations to curative treatments for cancer patients. The epithelial to mesenchymal transition (EMT) is a process that occurs naturally during embryogenesis and has been linked to metastasis in many cancer types. EMT is characterized by phenotypic changes that allow for tissue extravasation and migration of cancer cells into the bloodstream. These changes may be linked to specific patterns in the gene expression signature of cells undergoing EMT. Recent theoretical efforts have predicted the existence of a stable, hybrid (E/M) phenotype which has also been observed experimentally in single cells. However, the effects of this hybrid phenotype on cancer patient survival have not been well characterized. The ability to quantify a patient’s EMT status via a simple test involving a small collection of prognostic genes would provide an important tool for clinical risk stratification and treatment in the context of many cancer types. Here, we apply iterative statistical methods to generate an EMT score based on gene expression samples. We use this score to characterize the degree of the hybrid phenotype signature present in test samples. Predictions from our model are verified against cell lines with known EMT status. Lastly, we apply our model to clinical samples in order to assess survival differences in various EMT groups. We demonstrate that in many cases, EMT status successfully classifies patients into groups with statistically significant differences in survival, which is of immediate clinical relevance.

Citation Format: Jason T. George, Mohit K. Jolly, Herbert Levine. Quantitative EMT expression score for predicting survival outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 563. doi:10.1158/1538-7445.AM2017-563
Abstract 5633: Unique modulation of estrogen-induced apoptosis with structural analogues of endoxifen in long-term estrogen-deprived (LTED) breast cancer cells
Authors: Abderrahman Balkees , Fanning Sean W. , Rincon Daniela Quintana , Greene Geoffrey , Maximov Philipp Y. , Jordan V. Craig
Published: July 2017

Endoxifen, a non-steroidal anti-estrogen is the active hydroxylated metabolite of N-desmethyltamoxifen. The structure is based on a synthetic triphenylethylene-type angular estrogen. Endoxifen is in clinical trials as a therapeutic agent to treat aromatase resistant breast cancer. Paradoxically, LTED breast cancer is known to respond to synthetic and natural estrogens through estrogen-induced apoptosis. A series of synthetic endoxifen analogs, were used to modulate estrogen-induced apoptosis in LTED breast cancer cells (MCF7:5C). X-ray crystallography was performed on endoxifen, bisphenol, and diethylstillbestrol (DES), bound to the ligand-binding domain (LBD) of the human estrogen receptor (ER). Cellular studies established that (MCF-7:5C) of estrogen-induced apoptosis was dependent on the LBD of the ER. Similarly, endoxifen blocked estrogen-induced apoptosis in a concentration and time-dependent manner. Apoptosis triggered by DES was blocked by endoxifen and was ER-dependent. X-ray crystallography of both endoxifen and DES illustrate the two extremes of estrogen-induced apoptosis, which is dependent upon the closure of helix 12 over the LBD to generate estrogenic activity. Despite the fact that bisphenol, an analog of endoxifen without the anti-estrogenic monomethylamino ethyl side chain, is an estrogen that causes breast cancer cell replication; bisphenol initially inhibits estrogen-induced apoptosis, but triggers estrogen-induced apoptosis itself after 7 days of treatment of LTED breast cancer cells. X-ray crystallography demonstrates that bisphenol can produce a unique complex with the ER:LBD, which is similar to the DES:LBD estrogenic complex. In both cases, the ligands are sealed into the LBD by helix 12. This delay in apoptosis caused by an analog of endoxifen, that is an angular estrogen, possess unique biological features. We hypothesize that in LTED cells the complex passes through an anti-estrogenic conformation to evolve into an estrogenic complex with helix 12 sealing the LBD. We describe a unique property of a ligand:receptor complex. For the first time we demonstrate that a ligand:receptor complex is slowly activated from an antagonist to an agonist complex over a period of days.

Citation Format: Balkees Abderrahman, Sean W. Fanning, Daniela Quintana Rincon, Geoffrey Greene, Philipp Y. Maximov, V. Craig Jordan. Unique modulation of estrogen-induced apoptosis with structural analogues of endoxifen in long-term estrogen-deprived (LTED) breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5633. doi:10.1158/1538-7445.AM2017-5633
Abstract 5635: Vitamin D supplementation decreases serum 27-hydroxycholesterol and expression of CYP27A1 in tumors of breast cancer patients
Authors: Going Catherine , Alexandrova Ludmila , Lau Ken , Yeh Christine , Telli Melinda , Jensen Kristin , Feldman David , Pitteri Sharon
Published: July 2017

The goal of this study was to investigate whether vitamin D regulates the conversion of cholesterol to 27-hydroxycholesterol (27HC), an endogenous selective estrogen receptor modulator (SERM) that can act as a driver of estrogen receptor positive (ER+) breast cancer, particularly in post-menopausal women with low circulating estrogen levels. The hypothesis of this study was that the active metabolite of vitamin D, calcitriol, can inhibit expression of CYP27A1, the synthesizing enzyme for 27HC, thereby decreasing the concentration of 27HC in the blood. Both 27HC and 25-hydroxyvitamin D (25OHD) were quantified by LC-MS/MS in the serum of 29 breast cancer patients, 26 of which had ER+ cancer, pre- and post-treatment with either a low dose (control) of 400 IU vitamin D or a high dose of 10,000 IU vitamin D per day during the neoadjuvant period, which ranged between 7 and 37 days. In addition to these blood samples, tissue biopsies pre-treatment and the excised tumor collected post-treatment were tested for gene expression by microarray analysis. A significant increase (p = 4.3E-5) was observed in serum 25OHD for patients who received the high dose vitamin D compared to those who received the low dose, whose 25OHD values remained stable. A greater decrease (p = 1.7E-1) was observed in serum 27HC for the high dose group compared to the low dose group, with greater decreases in 27HC on average for those with larger increases in 25OHD concentration. Gene expression measurements showed a decrease in CYP27A1 expression (p = 4.5E-1) in breast cancer tissue for the high dose group compared to the low dose group, with greater decreases in expression observed for patients on high dose vitamin D treatment for longer times. These preliminary data show good evidence that high dose vitamin D supplementation can decrease 27HC levels in the body by inhibiting expression of CYP27A1 and that the longer this treatment, the greater the effect. While larger scale studies are needed, the data in this study point to vitamin D supplementation as a possible simple method for inhibiting ER+ breast cancer growth and suggests this reduction in CYP27A1 expression as an additional pathway by which vitamin D can mitigate risk and improve outcomes for ER+ breast cancer patients.

Citation Format: Catherine Going, Ludmila Alexandrova, Ken Lau, Christine Yeh, Melinda Telli, Kristin Jensen, David Feldman, Sharon Pitteri. Vitamin D supplementation decreases serum 27-hydroxycholesterol and expression of CYP27A1 in tumors of breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5635. doi:10.1158/1538-7445.AM2017-5635
Abstract 5638: Combination use of EF2K and VPS34 inhibitors with anti-androgen against drug-resistant castration resistant prostate cancer
Authors: Yang Joy C. , Cucchiara Vito , Gao Allen C. , Evans Christopher P.
Published: July 2017

INTRODUCTION AND OBJECTIVES:

Modulating the activity of eukaryotic elongation factor 2 kinase (eEF2K) has been suggested to regulate protein elongation to block autophagy in the tumor microenvironment. Among inhibitors of eEF2K, one would also inhibit VPS34, a class III phosphatidylinositol-3 kinase, and abrogate autophage flux to impair the survival escape mechanism. We tested the eEF2K and VPS 34 inhibitors from Janssen alone or in combination with anti-androgens for their effects on proliferation of prostate cancer cell lines, especially some castration resistant PC (CRPC) lines.

MATERIALS AND METHODS:

Cell proliferation was assessed with various concentrations of the EF2K or VPS34 inhibitor on CRPC lines using WST-1 viability assay. Effect of combinations of EF2K or VPS34 inhibitor with anti-androgens abiraterone (Abi) or enzalutamide (Enza) on drug-resistant CRPC cells was further explored. Western blot analysis was performed to examine the response of key autophagic molecules, androgen receptor (AR) and variant. Real time RT-PCR (RT-qPCR) was used to elucidate the effect of EF2K or VPS34 inhibitor alone or together with anti-androgens on AR, AR variant and their downstream molecules.

RESULTS:

EF2K and VPS34 inhibitors suppressed CRPC cell growth in a dose-response manner with IC50 ranging from 1 to 5 µM. These inhibitors displayed synergy with Abi and Enza against drug-resistant CRPC cells; especially on the pair of enza + EF2K inhibitor with p = 0.02 for significant difference when compared to enza or EF2K inhibitor alone. To our surprise, no significant autophagy was induced by these two inhibitors according to the autophagy markers detected by Western blots. VPS34 inhibitor alone and when combined with Abi and Enza showed AR/variant degrading ability. This downregulation was at the expression level with significant change of AR and V7, together with their transactivation markers PSA, TMPRS2, NKx3.1 and FKBP5 detected by RT-qPCR.

CONCLUSION:

EF2K and VPS34 inhibitors when combined with anti-androgens may solicit profound inhibitory effect on drug-resistant CRPC cells. Molecular delineation demonstrated the direct target might be AR and its variants. These combinations offered a new therapeutic option for advanced PC treatments.

Citation Format: Joy C. Yang, Vito Cucchiara, Allen C. Gao, Christopher P. Evans. Combination use of EF2K and VPS34 inhibitors with anti-androgen against drug-resistant castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5638. doi:10.1158/1538-7445.AM2017-5638