bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1706: High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types
Authors: Bailey Shannon T. , Lund James , Ekram Muhammad , Yu Wanfeng , Williams Richard T. , Sun Hongye , Gulcher Jeffrey R.
Published: August 2019

Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, whole-genome sequencing (WGS) of these samples has remained challenging due to the difficult process of isolating high-quality DNA and distinguishing true variant calls from artifacts. As evidence of these challenges, only four WGS studies of FFPE samples have been published to date. Previous work has attempted to overcome these challenges by using whole-exome sequencing or smaller exon panels at high coverage depths with publications reporting success from less than half to most samples. Thus, there has not been widespread adoption of using FFPE samples for next-generation sequencing.

We developed a novel DNA extraction and library prep protocol for whole-genome sequencing of FFPE tissue samples. This method produces larger DNA fragments that can be successfully used for WGS. We previously presented results of a large esophageal cancer cohort where we sequenced over 1,000 samples in tumor-normal pairs with data equivalent to that generated from fresh-frozen tissues. We recently expanded the tumor types, ages, and sources and achieved similar results. We have assayed FFPE tissues derived from breast cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma. These samples were sourced from several different labs, which have used variations on the standard fixation protocol. Some of these FFPE samples were over 15 years old. Variant calling provides excellent results without excessive C>T mutations. The coverage uniformity was very good, and CNV calls were made with results comparable to published TCGA data. These samples have been analyzed for MSI and TMB, and the results are comparable to those using fresh tissues.

Our method makes new types of clinical studies possible utilizing FFPE samples that have been generated as part of standard hospital procedures. This method also facilitates performing studies comparing primary and recurrent cancers easier to establish and run as recurrent cancer cases can be enrolled and stored FFPE primary samples used to establish a cancer baseline.

Citation Format: Shannon T. Bailey, James Lund, Muhammad Ekram, Wanfeng Yu, Richard T. Williams, Hongye Sun, Jeffrey R. Gulcher. High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1706.
Abstract 1718: Down-regulation of p16INK4a by hYSK1 promotes cancer cell migration under hypoxic conditions
Authors: Lee Mee-Hyun , Shim Jung-Hyun , Dong Zigang , Surh Young-Joon , Choi Bu Young
Published: August 2019

The alteration expression of p16INK4a, a well-known cyclin-dependent kinase inhibitor involved in cancer cell cycle control, is unclear, especially under hypoxic conditions. To evaluate p16INK4a regulation, we performed a protein microarray analysis. Among 1,800 proteins, we identified hYSK1 as a novel protein that interacts with the tumor suppressor p16INK4a. hYSK1, a member of the Ste20 family of serine/threonine protein kinases, promotes cell migration & tumorigenesis and is activated by oxidative stress. However, the molecular mechanisms underlying the oncogenic potential of hYSK1 remain elusive. Here, we report here that hYSK1 interacts with p16INK4a under hypoxic conditions like tumors, where it negatively regulates p16INK4a, enhancing cancer cell migration. Hypoxic stimulation of hYSK1 reduces p16INK4a accumulation through p16 promoter regulation to interact with unphosporylated SP-1 & increases matrix metalloproteinase-2 (MMP-2) expression by activating the MMP-2 promoter associated with cell migration and proliferation.Conversely, knocking down hYSK1 expression activated p16INK4a expression & suppressed MMP-2 expression.Thus, hYSK1 is necessary as a trigger for inactivating p16INK4a & activating MMP-2 during tumor migration, suggesting that hYSK1 is a specific negative regulator of the tumor suppressor p16INK4a may represent a novel molecular target for reactivation of tumor suppressor genes in humans.

Citation Format: Mee-Hyun Lee, Jung-Hyun Shim, Zigang Dong, Young-Joon Surh, Bu Young Choi. Down-regulation of p16INK4a by hYSK1 promotes cancer cell migration under hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1718.
Abstract 1721: FOXO1, a downstream substrate of AKT, function as tumor suppressor in HCC carcinogenesis
Authors: Zhang Shu , Chen Xin
Published: August 2019

Hepatocellular carcinoma (HCC) represents the fifth most common tumor types in the world and the third leading cause of cancer-related death. However, the signaling pathways underlying hepatic pathogenesis are still poorly understood, hence lacking of effective targeted therapy. Activation of PI3K/AKT signaling cascade has been implicated in HCC development. It has been well-established that the mammalian target of rapamycin complex 1 (mTORC1), is a major downstream effector of AKT. The reason why mTORC1 can be consistently activated is partly due to inactive mutations of a negative element regulating mTORC1, tuberous sclerosis 2 (TSC2), found in 10% human HCCs. Beyond TSC2, forkhead Box O transcription factor 1 (FOXO1), is another AKT substrate. It has been proved that FOXO1 plays a crucial role in the insulin signal pathway and liver regeneration relying on AKT isoforms. However, the role of FOXO1 in HCC development has not been well established. Here we report, by using TCGA database the mRNA level of FOXO1 significantly decreases among the FOXO family, comparing tumor tissue to the surrounding tissue. The overall survival rate favors patients with high FOXO1 expression level. The inhibition efficacies of a pan-AKT inhibitor, in both TSC2 wide type cells and TSC2-null ones, indicating TSC2 is not the only target downstream of AKT. Protein analysis shows the activity of mTORC1 can still be inhibited as well as the phosphorylation of FOXO1 in TSC2-null cells. Therefore, we hypothesize that in the presence of TSC2, AKTi can suppress the activation of mTORC1 directly through TSC2. Otherwise, in the absence of TSC2, AKTi can still inhibit mTORC1 via activation of FOXO1. Furthermore, we overexpress the activated FOXO1 (FOXO1 AAA) in TSC2-null HCC cell lines. Cell growth are significantly prohibited, implicating FOXO1 is a tumor suppressor in HCC cells. Over expression of activated FOXO1 can also delay mTORC1 activation based HCC in AKT/Ras FVB mice model.Lesions consist mainly of clear-cell hepatocytes, owing to an increase in glycogen and fat storage. Proliferation is nearly the same within the neoplastic nodules. Activated AKT occurs both in the cytoplasm and on the membrane. Interestingly, FOXO1AAA escapes into the cytoplasm instead of remaining in the nuclei where it should stay. In a conclusion, AKT/mTORC1 can be a classic molecular pathway for HCC pathogenesis. Nevertheless, the parallel substrate of AKT, FOXO1, may also be an important factor in a TSC2 independent way.

Citation Format: Shu Zhang, Xin Chen. FOXO1, a downstream substrate of AKT, function as tumor suppressor in HCC carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1721.
Abstract 1723: Elucidation of novel therapeutic targets for acute myeloid leukemias with RUNX1-RUNX1T1 fusion
Authors: Yun Jae Won , Bae Yoon Kyung , Cho So Yeong , Kim Hee-Jin , Nam Do-Hyun , Kim Sun-Hee , Chun Sejong , Joo Kyeung Min , Lee Hye Won , Park Woong-Yang
Published: August 2019

The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1 fusion protein is difficult to target since it lacks kinase activities. Here, we used a sophisticated bioinformatic tool to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AMLs from the TCGA database, the expressions of 293 genes were significantly correlated with the expression of the RUNX1-RUNX1T1 fusion gene. Based on the 293 genes, the COX, VEGFR, PDGFR, and FGFR pathways are predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Compared with the AML cells without the fusion, the specific activations of the AML cell lines with RUNX1-RUNX1T1 fusion were confirmed by the increased phosphorylation of VEGFRs, PDGFRs, and FGFRs. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve treatment outcomes for AML patients in clinic.

Note: This abstract was not presented at the meeting.

Citation Format: Jae Won Yun, Yoon Kyung Bae, So Yeong Cho, Hee-Jin Kim, Do-Hyun Nam, Sun-Hee Kim, Sejong Chun, Kyeung Min Joo, Hye Won Lee, Woong-Yang Park. Elucidation of novel therapeutic targets for acute myeloid leukemias with RUNX1-RUNX1T1 fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1723.
Abstract 1728: Overactivation of tumor suppressor P53 in hepatocytes promotes hepatocarcinogenesis
Authors: Makino Yuki
Published: August 2019

Aim: p53 is a tumor suppressor and its dysfunction promotes carcinogenesis of several organs including the liver. Meanwhile, p53 is reported to be overactivated in non-tumorous liver tissues of patients with chronic liver diseases, presumably due to several stressful stimuli to hepatocytes, including viral infection, ethanol exposure, fat accumulation. The aim of this study is to clarify the impact of p53 overactivation on hepatocarcinogenesis.

Methods: To induce hepatocyte-specific overactivation of p53 in liver cancer models, hepatic Mdm2, a negative regulator of p53, was genetically ablated in diethylnitrosoamine (DEN)-treated mice (Alb-CreMdm2fl/fl) and liver-specific constitutive-active Kras-mutant knock-in mice (Alb-CreKrasLSL-G12DMdm2fl/fl called as KrasG12DΔMdm2 mice). To analyze the significance of p53, hepatic p53 was deleted in Mdm2-wild KrasG12D mice (Alb-CreKrasLSL-G12DMdm2+/+p53fl/fl) and KrasG12DΔMdm2 mice (Alb-CreKrasLSL-G12DMdm2fl/flp53fl/fl).

Results: Mdm2 deletion significantly elevated serum ALT levels and increased tumor penetrance in both DEN-treated mice and Kras-mutant mice, suggesting that p53 overactivation accelerated hepatocarcinogenesis. KrasG12DΔMdm2 mice upregulated hepatic expressions of pro-apoptotic molecules (p16, p21, Bax, Noxa), number of TUNEL positive hepatocytes and serum caspase-3/7 levels, indicating accelerated hepatocyte apoptosis. They also increased the number of β-galactosidase positive hepatocytes and upregulated hepatic inflammatory cytokines (Tnf, Ccl2, Il-1β), suggesting senescence-associated secretory phenotype (SASP). Expressions of hepatic progenitor cell (HPC) markers (Afp, Epcam, Ck7, Ck19, Cd90, Cd133, Dlk1) were up-regulated in their liver. HPC marker staining suggested that liver tumors in KrasG12DΔMdm2 mice may originate from HPCs. All the phenotypes including accelerated hepatocyte apoptosis, SASP and emergence of HPCs in KrasG12DΔMdm2 mice were significantly alleviated by p53 deletion. Most importantly, deletion of p53 significantly decelerated hepatocarcinogenesis in those mice, indicating that p53 overactivation was responsible for the accelerated hepatocarcinogenesis. In sharp contrast, p53 deletion significantly accelerated hepatocarcinogenesis in Mdm2-wild Kras-mutant mice, confirming the tumor suppressive role of normal level of p53 in the liver.

Conclusion: Whereas p53 deletion accelerated hepatocarcinogenesis, p53 overactivation induced liver injury and activation of HPCs, leading to the acceleration of hepatocarcinogenesis. p53 overactivation in hepatocytes might be associated with liver cancer development in patients with chronic liver diseases.

Citation Format: Yuki Makino. Overactivation of tumor suppressor P53 in hepatocytes promotes hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1728.
Abstract 1731: Functional genomics approach reveals distinct neo-morphic activities and co-driver mutations of different missense mutant TP53 that promote breast cancer heterogeneity and progression
Authors: Pal Anasuya , Pal Anasuya
Published: August 2019

Phenotypic and molecular profiling of breast cancer demonstrates a high degree of heterogeneity in the breast tumors. TP53 tumor suppressor is mutated in 30% of all breast tumors and 80% of the basal-like subtype with numerous co-existing somatic mutations in other genes. We hypothesize that tumor heterogeneity is a result of combination of neo-morphic functions of specific TP53 driver mutations and functionally important but distinct co-mutations (or ‘co-drivers’) for each type of TP53 mutation. We thus ectopically expressed 10 most common missense p53 mutant proteins found in breast cancer patients in normal-like mammary epithelial cells and examined their phenotypes associated with various hallmarks of cancer. Supporting our hypothesis, a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell invasion and cell polarity across the 10 mutant cell lines were observed compared to those of WT p53 expressing cells. Integrated analysis of ChIP-seq and RNA-seq showed distinct promoter binding profiles of different p53 mutant proteins, implying that non-canonical transcriptional activity of mutant p53 proteins may contribute to the phenotypic heterogeneity in TP53-mutated tumor. Further, enrichment and model-based pathway analyses revealed the key biological pathways associated with invasion of cells expressing different p53 mutant proteins. As a proof-of-concept for screening co-drivers of mutant p53, when PTEN was deleted in non-invasive cells expressing p53-Y234C mutant using CRISPR-Cas9 system, an increase in cell invasion was observed. A genome-wide CRISPR library-based screen on p53-Y234C and p53-R273C cells identified completely different candidate co-driver mutations that promoted cell invasion. The top candidates included several known mutated genes in breast cancer patients harboring TP53 mutations and were associated with cytoskeletal and apoptosis resistance pathways. Overall, our combined approach of molecular profiling and functional genomics screens highlighted distinct sets of genes and co-driver mutations that can lead to heterogeneous phenotypes and promote aggressive behaviors of cells with different TP53 mutant backgrounds, which can guide development of novel targeted therapies.

Citation Format: Anasuya Pal, Anasuya Pal. Functional genomics approach reveals distinct neo-morphic activities and co-driver mutations of different missense mutant TP53 that promote breast cancer heterogeneity and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1731.
Abstract 1733: Role of the SIX1 oncofetal protein in endometrial basal cell metaplasia and carcinogenesis following neonatal exposure to diethylstilbestrol
Authors: Suen Alisa A. , Jefferson Wendy N. , Wood Charles E. , Kenan Anna C. , Williams Carmen J.
Published: August 2019

Early-life exposure to estrogenic chemicals can increase cancer risk, likely by disrupting normal patterns of cellular differentiation. In a model of this process, female mice exposed to the potent synthetic estrogen diethylstilbestrol (DES) on postnatal days 1-5 develop endometrial adenobasal carcinoma as adults. Neoplastic glands are comprised of abnormal populations of basal cells (cytokeratin (CK)14+), luminal cells (CK18+), and low numbers of "mixed” basal/luminal cells (CK14+/18+), all of which express the oncofetal protein sine oculis homeobox 1 (SIX1). We hypothesized that DES-induced SIX1 expression is necessary for aberrant endometrial differentiation patterns and carcinogenesis. To test this hypothesis, a conditional knockout model was generated in which floxed Six1 was excised in the uterus using progesterone receptor (Pgr) cre. The most prominent change in DES-exposed SIX1 knockout (DES-Six1d/d) mice was the absence of basal cells in the uterine horns. There was a greater than 10-fold decrease in CK14 labeling in the uterine horns of DES-Six1d/d mice compared to DES-exposed SIX1 wildtype (DES-Six1+/+) mice as determined by quantitative image analysis. However, DES-Six1d/d mice exhibited a 42% increase in cancer incidence compared to DES-Six1+/+ mice at 6 months of age (16/18 DES-Six1d/d vs. 7/15 DES-Six1+/+). Interestingly, mixed cells were still present in DES-Six1d/d mice. These findings demonstrate that SIX1 is a cellular differentiation factor necessary for DES-induced basal cells but not mixed cell development or cancer. Furthermore, these data suggest that DES-induced SIX1 expression decreases endometrial carcinogenesis by facilitating basal cell differentiation. Studies investigating the mixed cell population as a putative cancer progenitor cell population are ongoing. Interestingly, mixed cells were present in 35% (63/181) of malignant human endometrial tissue biopsies and in 0% (0/29) of normal endometrial tissue biopsies, suggesting that the DES mouse model has significant similarities to human endometrial cancer.

Citation Format: Alisa A. Suen, Wendy N. Jefferson, Charles E. Wood, Anna C. Kenan, Carmen J. Williams. Role of the SIX1 oncofetal protein in endometrial basal cell metaplasia and carcinogenesis following neonatal exposure to diethylstilbestrol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1733.
Abstract 174: Functional characterization of ZNF750 in esophageal squamous cell carcinoma
Authors: Choi Sheyne SA , Yu Valen Zhuoyou , Ko Josephine Mun-Yee , Dai Wei , Lung Maria L.
Published: August 2019

Background: Esophageal squamous cell carcinoma (ESCC) is a lethal and aggressive malignancy that is a threat worldwide. It is of significant prevalence in regions of Asia including Iran, Korea, Japan and especially China. Epidemiological studies of ESCC reveal genetic variations as a risk-factor to ESCC incidence. Genomic profiling of ESCC primary tumors have already identified candidate genes involved in different cellular processes such as cell cycle and apoptosis. Data from our whole exome sequencing study of primary tumors and lymph node metastases also reveal mutations involved in these pathways; however, we also uncovered an interesting candidate involved in differentiation namely, ZNF750. ZNF750 has been reported to play important roles in other squamous cell carcinomas, and as a candidate tumor suppressor gene in ESCC.

Methods and Results: We exogenously upregulated ZNF750 expression by lentiviral system and used this model to investigate ZNF750 function in ESCC. By MTT 2D cell proliferation assay, we showed that in vitro cell proliferation in the ZNF750 group was hindered compared to controls. In addition, we set up 2D and 3D colony formation assays and found that the ZNF750 groups produced fewer colonies in 2D culture plates and produced significantly smaller colonies, when embedded in Matrigel. All of these findings are consistent with previous studies. We also studied the potential functional role of ZNF750 in 3D invasion/migration. Transwell permeable inserts were used to support a thick Matrigel culture with a nutrient gradient. Cells were embedded in Matrigel and moved along the gradient within the gel. After prolonged incubation (>2 weeks), location and size of the colonies were examined by confocal microscopy scanning. Cells with upregulated ZNF750 were found to exhibit greater mobility compared to the control cells. We also subjected ZNF750 and control groups to cisplatin to test for chemoresistance in ESCC. We found that ZNF750 expression increases chemoresistance, as opposed to the control cells.

Conclusion: From these results, ZNF750 expression inhibits cell proliferation, while enhancing cell mobility and drug resistance, all of which are characteristics of cells undergoing epithelial-mesenchymal transition (EMT). We, therefore, hypothesize that ZNF750 functions in ESSC beyond that of a tumor suppressor, but as a putative EMT regulator.

Acknowledgements: Research Grants Council Collaborative Research Fund grant number 106150246 and Asian Cancer Research Fund to MLL.

Citation Format: Sheyne SA Choi, Valen Zhuoyou Yu, Josephine Mun-Yee Ko, Wei Dai, Maria L. Lung. Functional characterization of ZNF750 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 174.
Abstract 175: Methyltransferase protein A regulates colorectal cancer cell proliferation and metastasis through methylation of protein B
Authors: Kang Songhwa , Kwon Sang-Mo
Published: August 2019

Post-translational modifications (PTMs) contribute to abnormal cell proliferation, adhesion, and morphology in cancer progression. Methylation is one such PTM that affects many of the protein properties and has a substantial role in tumorigenesis. Here, we identified that the methyltransferase protein A was upregulated in human colorectal cancer (CRC) and was associated with CRC proliferation, migration, and invasion in vitro. Our study showed that Protein B was a novel target of methyltransferase Protein A and that methylated Protein B was upregulated in human CRC. High expression of Protein A and B resulted in a poor prognosis of CRC; moreover, CRC proliferation, migration, and invasion were inhibited when protein A and B were unable to bind through specific residue, as predicted by in silico protein-protein docking analysis. Furthermore, our study showed that methylation of Protein B by methyltransferase protein A plays an important role in CRC proliferation and invasion via enhancement of HIF-1α stabilization. In conclusion, our data suggested that methylated Protein B may be a potentially valuable clinical prognostic marker of CRC and that interference with the binding of Protein B to methyltransferase Protein A may be a new anticancer target in CRC.

Citation Format: Songhwa Kang, Sang-Mo Kwon. Methyltransferase protein A regulates colorectal cancer cell proliferation and metastasis through methylation of protein B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 175.
Abstract 1751: The role of SIRT2 in BRCA1 regulation
Authors: Minten Elizabeth , Zhang Hui , Li Chunyang , Head PamelaSara , Yu David S.
Published: August 2019

The most deleterious type of DNA damage a cell can sustain is the DNA double-stranded break (DSB). One of the major pathways responsible for the repair of DNA DSB breaks is the homologous recombination (HR) pathway, an error-free mechanism of repair that occurs primarily in the S and G2 phases of the cell cycle when a sister chromatid is available to be used as a repair template. Impairment of HR can result in genomic instability, which can increase the risk of tumorigenesis. However, many of the mechanisms by which defects in HR lead to an increased risk for developing cancer are unknown. Mutations or misregulation of the Breast Cancer 1 (BRCA1) protein, a major player in HR and an established tumor suppressor, have been linked to an increased lifetime risk for the development of cancer in both men and women, including colon, breast, ovarian, and pancreatic cancer. Yet, how BRCA1 is regulated in HR is not well understood and thus highlights a major a gap in our understanding of how deficiencies in HR contribute to the development of cancer. Our lab has discovered that the histone deacetylase and putative human tumor suppressor, SIRT2, has a crucial role in the repair of DNA DSBs. We have found that SIRT2 interacts with and deacetylates BRCA1, both in vitro and in cells, and that depletion of SIRT2 decreases BRCA1 protein levels, thus impairing HR. Our results show SIRT2 is a novel regulator of BRCA1 and is critical for the efficient repair of DNA DSBs through HR. These findings will provide invaluable insight into how to exploit the interplay between SIRT2 and BRCA1 as a novel therapeutic approach for the prevention and treatment of cancer.

Citation Format: Elizabeth Minten, Hui Zhang, Chunyang Li, PamelaSara Head, David S. Yu. The role of SIRT2 in BRCA1 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1751.
Abstract 1758: Splicing component ISY1 interacts with APE1 and regulates base excision repair
Authors: Jaiswal Aruna , Williamson Elizabeth , Patel Bhavita , Srinivasan Gayathri , Kong Kimi , Lomelino Carrie , Narayan Satya , Hromas Robert
Published: August 2019

Cellular genome is continuously challenged by endogenous and exogenous DNA damaging agents. In the genome, base modifications are the most common form of DNA damage. Base excision DNA repair (BER) is the most important pathway for the removal of oxidized or alkylated DNA. If these base modifications continue to persist, the replisome may stall at DNA lesions, causing the replication fork to collapse, leading to genetic instability. While the main components of the BER pathway are well defined, its regulatory mechanism remains poorly understood. We report here that apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional protein interacts with the pre-mRNA splicing factor ISY1. However, this interaction is independent of DNA or RNA based upon our pull-down experimental results. ISY1 expression is induced by oxidative or DNA alkylation damage, which would then provide an immediate up-regulation of APE1 activity in vivo and enhanced BER of oxidized bases. This was supported from our results of in vitro reconstituted experiment suggesting that ISY1 can stimulate 5’-3’ endonuclease 1 activity of APE1 in both the short- and long-patch BER pathways and increase its binding to AP site DNA. The interaction between ISY1 and APE1 also establishes a connection between DNA damage repair and pre-mRNA splicing. Based upon these results, we propose that ISY1 is an important regulator of BER and enhances the ability of APE1 to efficiently recognize abasic sites in DNA. Role of ISY1 becomes important in aging tissues where decreased APE1 activity results in the accumulation of oxidative genomic damages. Therefore, ISY1 may work as an anti-aging factor. Induction of ISY1 in malignancies could constitute a mechanism to avoid apoptotic death after treatment with alkylating agents.

Citation Format: Aruna Jaiswal, Elizabeth Williamson, Bhavita Patel, Gayathri Srinivasan, Kimi Kong, Carrie Lomelino, Satya Narayan, Robert Hromas. Splicing component ISY1 interacts with APE1 and regulates base excision repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1758.
Abstract 176: Tetraspanins in oral squamous cell carcinoma
Authors: Reddi Kiran K. , Anderson Kenneth M. , Garcia-Godoy Franklin , Zhang Yanhui H.
Published: August 2019

Objectives: To (1) investigate the expression levels of several tetraspanins in oral cancer cells and tissues; (2) characterize the subpopulation of oral cancer cells with elevated CD82; and (3) investigate roles of the tetraspanins in oral cancer migration, invasion, and metastasis.

Methods: The protein expression levels of tetraspanins CD82(KAI1), CD9, CD81(TAPA), and their associate protein EWI-2(IGSF8) were quantified in normal human gingival epithelial cells (SG) and oral cancer cells (CAL27 and SCC-25 cells) using western blotting and immunofluorescence staining and imaging. CAL27 CD82 knockout cells (CD82KO) were created by CRISPR/CAS-9, sorted by Fluorescent Activated Cell Sorting, and validated by western blotting. A wound healing assay was used to study the role of CD82 in oral cancer cell migration. CD82 perturbation of the CAL27 genome and proteome were evaluated using Microarray (Affymetrix) and TRAQ/TMT Based Differential Protein Expression Analysis (Thermo, mass spectrometer).

Results: So far, we have found (1) CD82 is highly expressed in CAL27 cancer cells, but its expression is low in SG and SCC-25 cells; (2) CD82 is highly expressed in human tongue cancer tissues; (3) CD9 is highly expressed in CAL27 cells; (4) The expression levels of CD81 and IGSF8 are comparable in the three cancer cell lines; (5) CAL27 cancer cells migrate faster than normal SG cells; (6) Knockout of CD82 significantly decreases the migration rate of CAL27 cells, suggesting that CD82 promotes migration of these cancer cells.

Conclusions: CD82 has varied expression in different oral cancer cell lines. Its expression is very high in CAL27 cancer cells. Knockout of CD82 in CAL27 cells significantly inhibits migration of these cells. Our study suggests that CD82 might be used as a biomarker for a subpopulation of oral cancer cells and that CD82 could be a target for therapeutic interventions.

Acknowledgements:

Supported in part by the 2017 UTHSC CORNET Award.

Supported in part by the UTHSC College of Dentistry Alumni Endowment Fund and the Tennessee Dental Association Foundation.

Citation Format: Kiran K. Reddi, Kenneth M. Anderson, Franklin Garcia-Godoy, Yanhui H. Zhang. Tetraspanins in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 176.
Abstract 1765: In vivo modeling of RIT1 function in development and lung cancer
Authors: McSharry Maria
Published: August 2019

Ras-like in most tissues 1 (RIT1) encodes a ubiquitously expressed RAS-family small GTPase. Recently, somatic RIT1 mutations were identified in lung adenocarcinoma and myeloid malignancies, and germline RIT1 mutations were found in. RIT1 is also implicated hematopoietic malignancies and in Noonan syndrome, a “Rasopathy” affecting craniofacial development and associated with congenital heart disease. Of a variety of RIT1 mutations discovered, the most recurrent mutation observed was M90I. In this study, murine models were used to determine whether expression of RIT1M90I mutation is sufficient for tumor initiation or developmental phenotypes observed in Noonan patients.

Mice were genetically engineered at the Rosa26 locus to harbor RIT1M90I under the control of a lox-Cre system (LSL-RIT1M90I). The Cre recombinase protein was introduced via intratracheal delivery of adenovirus to the lungs. At one-year post treatment, 7 of the 10 LSL-RIT1M90I mice examined exhibited the expected genetic recombination. Tumors were observed in 3 of these mice, one of which was wild-type for p53. No visible tumors were seen in mice lacking the RIT1M90I gene or in cases where recombination was not observed, regardless of p53 status.

Mouse embryonic fibroblasts (MEFs) were infected by adenovirus to recapitulate the in vivo studies and evaluate transformation. In vitro experiments conducted in RIT1M90I MEFs demonstrated faithful recombination of the transgene upon treatment with Cre. Protein analysis of these same cells reflected higher levels of RIT1 protein in transgenic cells treated with Cre, while in all other conditions, RIT1 was undetectable. Preliminary data from an immortalization assay suggested enhanced proliferation of MEFs expressing the RIT1M90I gene compared to wild-type counterparts.

Cre recombinase was expressed body-wide in tamoxifen (4OHT) inducible mice (UBC-Cre-ERT2) LSL-RIT1M90I animals crossed to the inducible UBC-Cre-ERT2 system exhibited faithful recombination of the transgene upon treatment with 4OHT.

To determine the consequences of constitutive expression of RIT1M90I, RIT1M90I sires were crossed to dams harboring constitutively active Cre recombinase or to a constitutively active Cre SRY-box promoter (Sox2-Cre). Of the 100 pups observed, none contained both RIT1M90I and Cre recombinase (p < 0.0001), indicating embryonic lethality induced by RIT1 activation.

These data demonstrate the powerful effect of RIT1 activation on murine embryonic development and will enable future efforts on the characterization of the mechanism of RIT1-induced Noonan Syndrome and lung cancer.

Citation Format: Maria McSharry. In vivo modeling of RIT1 function in development and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1765.
Abstract 1768: Biochemical and structural analysis of the Neurofibromin (NF1) protein and a potential role for protein destabilization in Rasopathy diseases
Authors: Sherekar Mukul , Han Sae-Won , Messing Simon , Drew Matthew , Gillette William , Lorenzo Claire , McCormick Frank , Esposito Dominic
Published: August 2019

Neurofibromin is the 320 kilodalton protein product of the NF1 gene which is mutated in the Rasopathy disease Neurofibromatosis Type I. Defects in NF1 lead to abrogation of the GTPase activating activity of the protein, leading to aberrant signaling through the RAS-MAPK pathway. Very little is known about the function of the majority of the NF1 protein—to date only the GAP domain and a region containing a Sec-PH motif have detailed structural information. To better understand the role of this large protein, we have carried out a series of biochemical and biophysical studies which clearly show that full length NF1 protein exists primarily as a dimer. Data from in vivo experiments confirmed that this dimerization also occurs in cells, and presumably has biological implications. A model was generated from negative-stain EM data which suggests that NF1 monomers form both intermolecular contacts to form dimers and intramolecular contacts which stabilize the individual monomers. Deletion analysis permitted us to map these two interacting domains to different parts of the NF1 protein, and crosslinking studies coupled with mass spectrometry helped to identify regions of possible interaction. In addition, introduction of point mutations observed in patients with NF diseases caused a dramatic decrease in the stability of proteins both in vitro and in vivo, potentially arguing for a rationale for loss of NF1 activity by these single mutations

Citation Format: Mukul Sherekar, Sae-Won Han, Simon Messing, Matthew Drew, William Gillette, Claire Lorenzo, Frank McCormick, Dominic Esposito. Biochemical and structural analysis of the Neurofibromin (NF1) protein and a potential role for protein destabilization in Rasopathy diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1768.
Abstract 1780: iSCREAM - an unbiased pipeline to screen for activating kinase mutations
Authors: Chakroborty Deepankar , Kurppa Kari J. , Paatero Ilkka , Elo Laura L. , Elenius Klaus
Published: August 2019

Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites. Most of these somatic mutations are expected to be inconsequential passenger mutations that reflect the general instability of the tumors. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hot-spots within their respective genes. However, a vast majority of mutations in cancer tissues are rare and no information is currently available about their functional significance. Several lines of in vitro and in vivo clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations that could serve as predictive markers in oncology.

An unbiased iSCREAM (in vitro screen for activating mutations) platform was developed to functionally characterize thousands of variants of a kinase oncogene in a single assay. The functional genetics screen was based on expressing random variants of a cDNA encoding a tyrosine kinase in a cell line in which the activating mutations provide growth-advantage. Targeted next-generation sequencing of the cDNA inserts was used to quantitatively analyze variants that provided growth-advantage.

iSCREAM is able to identify activating mutations from a library of thousands of random mutations of a tyrosine kinase. The pipeline can also be modified to identify mutations conferring resistance to a tyrosine kinase inhibitor.

Citation Format: Deepankar Chakroborty, Kari J. Kurppa, Ilkka Paatero, Laura L. Elo, Klaus Elenius. iSCREAM - an unbiased pipeline to screen for activating kinase mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1780.
Abstract 1782: ANKRD52 inhibited tumor metastasis through dephosphorylation of PAK1 in lung adenocarcinomas
Authors: Lee Ting-Fang , Liu Yin-Pu , Lin Yen-Fan , Hsu Chong-Fang , Wu Cheng-Wen
Published: August 2019

PAK1 is an important oncogene involved in the progression of a variety of cancer types including lung cancer. Although PAK1 inhibition is shown to suppress tumor progression, specific PAK1 inhibitors are not available due to the complex structure and insufficient understanding of this protein. In an attempt to find TAZ (WW domain-containing transcription regulator 1) regulated tumor suppressor, a member of ankyrin repeat proteins, ankyrin repeat domain 52 (ANKRD52), is observed to suppress PAK1 phosphorylation. Ankyrin repeat proteins are known to function as a platform for protein-protein interactions, and ANKRD52 was shown to interact with protein phosphatase 6 (PP6). Immunohistochemistry staining in lung cancer tissue revealed prolonged disease-free survival in high ANKRD52 expressing cases. Using lung cancer cell lines, ANKRD52 knockdowns showed significantly increased cell mobility while forced expressed ANKRD52 decreased cell mobility. Using mass spectrometry analysis, p21-activated kinases (PAK1) was identified to interact with ANKRD52. Upregulated PAK1 phosphorylation was detected in the knockdowns of ANKRD52 or PP6. Our model demonstrated a novel anti-metastatic protein, ANKRD52, that regulates cell mobility through interactions with PP6 and dephosphorylation of PAK1.

Citation Format: Ting-Fang Lee, Yin-Pu Liu, Yen-Fan Lin, Chong-Fang Hsu, Cheng-Wen Wu. ANKRD52 inhibited tumor metastasis through dephosphorylation of PAK1 in lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1782.
Abstract 1783: A combination therapy of atypical protein kinase inhibitor and phosphatidylinositol-3- kinase inhibitor reduces multidrug resistance in renal cell carcinoma (RCC)
Authors: Khalid Khandker Mohammad
Published: August 2019

Renal Cell Carcinoma (RCC) is the most common type of kidney cancer (85%) of which 75% of the cases involve conventional clear cell RCC (ccRCC). Choice of treatments recommended for clear cell carcinoma and non-clear cell carcinoma are partial nephroctomy (Stage I), radical nephoroctomy (Stage II and III) and chemotherapy (stage IV). Phosphatidylinositol-3- kinase (PI3K) inhibitors are one of the most prospective candidate in the therapy of ccRCC as the PI3CA gene for p110α (a class of the catalytic sub unit of PI3K) is mutated in most of the cases. Hence, PI3K inhibitors like Alpelisib (BYL 719) can be a treatment. But current therapeutic prospects are limited to the fact that RCC is a chemotherapy-resistant cancer type that acts as a barrier for successful treatment with this inhibitor. In addition, of all the other aPKCs, PKC ί/λ and PKC ζ are reported to be frequently overexpressed in most cancer cells and to have different significant roles in tumor progression and carcinogenesis of different type of cancers. Caki-1 metastatic clear cells were treated with PKC ζ inhibitor [8-hydroxynaphthalene-1,3,6-trisulfonic acid] or ζ-stat, Alpelisib and combination of both ζ-stat and Alpelisib at different doses. The results of WST-1 shows that the combination of ζ-stat (7.5 µM) and Alpelisib (10.0 µM) has the most cytotoxicity compared to the single therapy of Alpelisib. Annexin-V/PI assay also suggested that the combination therapy caused greater percentage of apoptosis and cell death compared to the single therapy of Alpelisib. The efflux proteins, (p-glycoprotein (P-gp) and ATP-binding cassette sub-family G member 2 (ABCG2), are responsible for developing cell resistance in Caki-1 cells had reduced expression in the cells when treated with the combination treatment of Alpelisib and ζ-stat. Therefore, combination therapy of Alpelisib and an aPKC inhibitor for metastatic ccRCC can reduce expression of multi drug resistance (MDR) proteins/efflux transporter P-gp and ABCG2. Hence, this combination may be a possible treatment that might help eradicating MDR of ccRCC. Thus, a combination of Alpelisib and an aPKC inhibitor can be a two pronged approach reducing the cell proliferation and resistance.

Citation Format: Khandker Mohammad Khalid. A combination therapy of atypical protein kinase inhibitor and phosphatidylinositol-3- kinase inhibitor reduces multidrug resistance in renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1783.
Abstract 1786: Effect of DUSP2 on pancreatic cancer lymphatic dissemination
Authors: Tsai Shaw-Jenq , Wang Chu-An , Chang I-Heng
Published: August 2019

More than 90% of pancreatic cancers are originated from the exocrine ducts of the pancreas, which is called pancreatic ductal adenocarcinoma (PDAC). PDAC is notorious for the high early metastatic rate and low 5-year survival rate. Vascular endothelial growth factor C (VEGF-C), a member of the VEGF family, was first identified as the master lymphangiogenic factor in embryonic development. Overexpression of VEGF-C in cancers has been tightly linked to lymphangiogenesis and is highly associated with lymphatic invasion and metastasis; however, the underlying mechanism responsible for VEGF-C overexpression remains largely unknown. Constitutive activation of MAPK pathway was characterized as feature of pancreatic cancer. In genetic mouse model of pancreatic cancer, MAPK signaling is required to initiate and maintain the pancreatic intraepithelial neoplasia lesions. In normal cells, MAPKs mediated signaling is crucial to various cell functions and the activation can be balanced by the action of dual specificity phosphatases (DUSPs). Previously, we have demonstrated that hypoxia can suppress DUSP2 by which mediates tumor malignancy and drug resistance. Herein, we further identify that DUSP2 is a master regulator in controlling lymphangiogenesis and early lymphovascular invasion of PDAC. Expression of DUSP2 is silenced in PDAC, which contributes to aberrant production of VEGF-C. VEGF-C expression is controlled by DUSP2 at multiple levels including transcription, maturation, and extracellular vesicle-associated secretion. Our findings provide the proof of concept that blocking DUSP2/Exo-VEGF-C axis may serve as an attractive therapeutic approach to inhibit early dissemination of pancreatic cancer.

Citation Format: Shaw-Jenq Tsai, Chu-An Wang, I-Heng Chang. Effect of DUSP2 on pancreatic cancer lymphatic dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1786.
Abstract 1788: Targeting ornithine decarboxylase down-regulates multiple pathways involved in precancerous lesion formation of esophageal squamous cell cancer
Authors: Xie Yifei , Wu Qiong , Zhao Jimin , Liu Kangdong
Published: August 2019

Precancerous lesion of esophageal squamous cell cancer (ESCC) owns the characteristic of bidirectional instability, which can develop to cancer or reverse to normal epithelium by appropriate interference. Therefore, the feature of precancerous lesion provides possibility for ESCC chemoprevention. In the current study, we found that ornithine decarboxylase (ODC) protein level is higher in esophageal precancerous lesions compared with normal esophageal epithelium. Knocking down ODC suppresses cell proliferation and clone formation of ESCC. Moreover, difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreases N-nitrosomethylbenzylamine (NMBA)-induced rats’ esophageal precancerous lesions. Further molecular study indicated that ODC down-regulates p38, ERK in the MAP kinase pathway and AKT/mTOR/p70S6K pathway, which are involved in the carcinogenesis process. Overall, our results indicate that ODC may be a potential target for ESCC chemoprevention.

Citation Format: Yifei Xie, Qiong Wu, Jimin Zhao, Kangdong Liu. Targeting ornithine decarboxylase down-regulates multiple pathways involved in precancerous lesion formation of esophageal squamous cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1788.