bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3546: Prevalence of Lynch Syndrome among Middle Eastern endometrial cancer using targeted next generation sequencing
Authors: Parvathareddy Sandeep K. , Siraj Abdul K. , Bu Rong , Masoodi Tariq , Azam Saud , Haqawi Wael , Alobaisi Khadija , Alrasheed Maha , Balde Valorie , Siraj Nabil , Diaz Mark R. , Ghazwani Laila , DeVera Felisa , AlDossari Hassan , Al-Kuraya Khawla S.
Published: September 2020

Endometrial carcinoma (EC) is the second most common gynecologic cancer worldwide. Although most ECs are sporadic, 2%-5% tend to be familial, with Lynch syndrome (LS) being the most commonly associated. The purpose of this study was to identify the incidence of LS in a large cohort of Middle eastern ECs and determine the feasibility of microsatellite instability (MSI) screening by immunohistochemistry (IHC) followed by molecular screening in deficient cases. We performed screening of 436 unselected EC for MSI status using IHC, followed by Target Capture sequencing of MSI deficient cases. MLH1 methylation analysis was performed by Real-time PCR, for cases showing deficient MLH1 by IHC. Complete loss of tumor nuclear protein expression in at least one of the four MMR genes was noted in 12.2% (53/436) of EC cases with 32 cases being MLH1 deficient, 10 MSH2 deficient, five MSH6 deficient and six PMS2 deficient. Of the 53 EC cases showing loss of expression in MMR genes, germline MMR mutations were found in four cases (0.9%). Promoter methylation analysis of 32 MLH1 deficient cases showed four cases to be unmethylated (12.5%). Our current study highlights the incidence of LS among patients with EC in Saudi Arabia. Screening of all EC patients using immunohistochemistry and reflex MLH1 promoter methylation testing followed by gene sequencing is feasible and desirable.

Citation Format: Sandeep K. Parvathareddy, Abdul K. Siraj, Rong Bu, Tariq Masoodi, Saud Azam, Wael Haqawi, Khadija Alobaisi, Maha Alrasheed, Valorie Balde, Nabil Siraj, Mark R. Diaz, Laila Ghazwani, Felisa DeVera, Hassan AlDossari, Khawla S. Al-Kuraya. Prevalence of Lynch Syndrome among Middle Eastern endometrial cancer using targeted next generation sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3546.
Abstract 3584: CENPA, MYBL2, and FOXM1 are identified as key transcriptional regulators in lung adenocarcinoma using TENET 2.0
Authors: Mullen Daniel J. , Yan Chunli , Kang Diane S. , Zhou Beiyun , Borok Zea , Marconett Crystal N. , Farnham Peggy J. , Offringa Ite A. , Rhie Suhn K.
Published: September 2020

Lung cancer is the leading cause of cancer-related death in the United States, with lung adenocarcinoma being the most common subtype. Although a number of genetic alterations have been identified as potential drivers in lung adenocarcinoma, there is still a large gap in our understanding of tumor development. Epigenetic alterations have been implicated in the pathogenesis of tumors. To identify epigenetic alterations that drive lung adenocarcinoma, we used an improved version of the Tracing Enhancer Networks using Epigenetic Traits method (TENET 2.0) in primary normal lung and lung adenocarcinoma cells. We identified over 17,000 enhancers that appear activated in lung adenocarcinoma but not in normal lung and linked the activity of these enhancers to transcriptional regulators. We identified that CENPA, MYBL2, and FOXM1 are key transcriptional regulators activated in a subgroup of lung adenocarcinomas and they are linked to numerous cancer-specific enhancers. High expression of these regulators is associated with poor patient survival. In addition, we identified individual enhancers that are linked to these regulators and whose increased activities are strongly associated with poor patient survival. Knockdown experiments of MYBL2 and FOXM1 suggested that they regulate genes involved in controlling cell cycle progression and cell division. We also showed that high expression of TK1, a presumed target gene of a MYBL2-linked enhancer, is strongly associated with poor patient survival. Identification and characterization of key transcriptional regulators and associated cancer-specific enhancers in lung adenocarcinoma provides important insights into the deregulation of lung adenocarcinoma transcriptomes and novel potential targets for clinical intervention.

Citation Format: Daniel J. Mullen, Chunli Yan, Diane S. Kang, Beiyun Zhou, Zea Borok, Crystal N. Marconett, Peggy J. Farnham, Ite A. Offringa, Suhn K. Rhie. CENPA, MYBL2, and FOXM1 are identified as key transcriptional regulators in lung adenocarcinoma using TENET 2.0 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3584.
Abstract 3595: Riboprotein variant and their role in chondrogenic differentiation and osteosarcoma development
Authors: Orgebin Emilien , Ory Benjamin , Lamoureux Francois , O'Donohue Marie-Francoise , Charrier Céline , Gleizes Pierre-Emmanuel , Isidor Bertrand , Baud'huin Marc
Published: September 2020

Variants in genes encoding ribosomal proteins (RPs) have thus far been associated with Diamond-Blackfan anemia, a rare inherited bone marrow failure, and isolated congenital asplenia. We reported recently one de novo missense variant and three de novo splice variants in the RPL13 gene, which encodes ribosomal protein RPL13/eL13, in four unrelated individuals with a rare bone dysplasia causing severe short stature. The three splice variants (c.477+1G>T, c.477+1G>A, and c.477+2 T>C) result in partial intron retention, which leads to an 18-amino acid insertion. These RPL13 variants define a human ribosomopathy leading to a rare skeletal dysplasia and highlight the role of this ribosomal protein in bone development. It has been shown that patients suffering from ribosomopathies have a highly predisposition to develop cancer compared to general population. Moreover, recent studies have also revealed that mutations in factors involved in ribosome synthesis appear to be drivers of tumorigenesis in sporadic cancers.

In this study, we focused on the role of variants of the riboprotein RPL13 in the bone biology and in the development of osteosarcoma.

Mesenchymal stem cells expressing variants of RPL13 or wild type RPL13 have been cultured for chondrogenic and osteoblastic differentiation. Our results show that RPL13 variants did not modified the capacity of cells to differentiate into osteoblasts. However, the chondrogenic differentiation was increased in cells expressing variants of RPL13. The expression of sox9 and collagen type X were investigated by immunochemistry on chondrocyte micromass and revealed an increase of expression of these markers in RPL13 variants cells. We next investigated the role of variants of RPL13 in osteosarcoma cell lines. Several cell lines were transduced by lentiviral vector to produce variants of RPL13. The variants induced an increase of cell proliferation and survival. The clonogenecity and the invasion have been also studied.

Taken together, these data show that variants of a gene encoding for a riboprotein causing a ribosomopathy could have an effect on chondrogenic differentiation and on osteosarcoma development.

Citation Format: Emilien Orgebin, Benjamin Ory, Francois Lamoureux, Marie-Francoise O'Donohue, Céline Charrier, Pierre-Emmanuel Gleizes, Bertrand Isidor, Marc Baud'huin. Riboprotein variant and their role in chondrogenic differentiation and osteosarcoma development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3595.
Abstract 3608: Identification of differentially expressed genes in 4T1 mice triple-negative mammary gland cancer during the early phase of combination treatment with indomethacin and radiation
Authors: Cheng Robert Yuk Sing , Ridnour Lisa , Back Timothy , Basudhar Debashree , Anderson Stephen , McVicar Daniel , Wink David
Published: September 2020

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers. In order to develop the most effective treatment against TNBC, we tested the effects of the combination treatment with indomethacin and radiation on TNBC utilizing a mouse experimental TNBC equivalent model. Seven weeks old BALB/c mice were implanted with 4T1 mice triple-negative mammary gland tumor cells on the right hind leg one week before the treatments started. Mice were divided into four groups treated with vehicle control, 6 Gy radiation, 30 mg/L (in water) indomethacin, or combination regime of 6 Gy radiation and 30 mg/L indomethacin. 6 Gy radiation was given to mice on Day 0 only while indomethacin was given to mice every day. Mice received the combination treatment with radiation and indomethacin showed a significant tumor growth delay at the end of the treatment regime (Day 30). We next elucidated if there are changes in biological pathways during the early phase of combination therapy (Day 3). Total RNAs isolated from the primary tumors were subjected to whole genome transcriptomic analyses (RNAseq). We identified a total of 58 genes with 2-fold changes in expression and p-value <0.05 differentially expressed among the treatment groups. Strikingly, the trend in expression levels of differentially expressed genes identified from the combination treatment group was opposite to that from the other two treatment groups. Results indicate that the effect of combination treatment with indomethacin and radiation is neither additive or synergistic. In addition, pathway core analyses revealed “Cancer” as the top associated disease, and “GABA Receptor Signaling and cAMP-Mediated Signaling” as the top canonical pathways among the treatment groups. In conclusion, our findings provided a detailed snapshot of the early events occur in mice subject to either single or combination treatment with indomethacin and radiation. Data will improve our understanding of the treatment responses of TNBC. We believe further time-course and transcriptomic studies may be beneficial for the new therapeutic strategies for TNBC.

Citation Format: Robert Yuk Sing Cheng, Lisa Ridnour, Timothy Back, Debashree Basudhar, Stephen Anderson, Daniel McVicar, David Wink. Identification of differentially expressed genes in 4T1 mice triple-negative mammary gland cancer during the early phase of combination treatment with indomethacin and radiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3608.
Abstract 3616: Somatic mutation profiling of colorectal cancer by targeted next generation sequencing
Authors: El-Din Youssef Amira Salah , Moustafa Ahmed , Touny Ahmed Osama , Hassan Zeinab K. , Eldin Mohammed Mohey , Lotfy Mai M. , Nassar Auhood , sayed ola , Zekri Abdel-Rahman N.
Published: September 2020

Aim: This study aimed to sequence custom 96 genes of tumor suppressor genes and oncogenes frequently associated with colorectal cancer (CRC) to identify the frequency of the detected genetic mutations in the disease progression of CRC patients and to develop personalized therapy in those somatic mutation carriers.

Material and methods: Biopsy samples were collected from Egyptian patients classified into inflammatory bowel disease (IBD) (n=20), colonic polyp (CP) (n=38) and CRC (n=60) patients as well as subjects with chronic non-specific colitis served as a control group (n=20). The libraries were performed using Qiaseq UMI-based targeted panel and sequenced via Ion proton sequencer. The detected genetic variants with an average coverage of 500x were annotated against Cosmic and dbSNP and Clinvar databases.

Results: Analysis revealed that 52 genes harbor 128, 47 genes harbor 111, 40 genes harbor 71 and 39 genes harbor 66 somatic variants were detected in the CRC, CP, IBD and control groups; respectively. ARID1A (c.5548dupG (5%)), ATM (c.2572T>C (10%)), AXIN2 (c.1975C>T (5%)), FLCN (c.1285dupC (8%)), KRAS (c.35G>T (8%)), MSH6 (c.3261dupC (5%)), SLC9A9 (c.1765A>G (10%)), TP53 (c.1024C>T (5%)) were found to be the highly frequently detected pathogenic CRC specific variants. We also identified 29 genes harbor 43 common variants. The highly frequently common detected variants were ACVR2A (c.1310delA), ATM (c.5557G>A), BRCA1 (c.3548A>G, c.2612C>T), BRCA2 (c.1114A>C), IGF2 (c.677delG), KIT (c.1621A>C), MLH1 (c.655A>G), MLK4 (c.2223G>T, c.2182G>A), PIK3CA (c.1173A>G), PTPN12 (c.964G>A), RET (c.2071G>A), TP53 (c.121delG, c.215C>G)

Conclusion: Our data showed that our Egyptian genetic makeup is different from other population. Also, the identified somatic mutations are crucial for understanding cancer predisposition and developing personalized therapies for the Egyptian colorectal cancer patients.

Citation Format: Amira Salah El-Din Youssef, Ahmed Moustafa, Ahmed Osama Touny, Zeinab K. Hassan, Mohammed Mohey Eldin, Mai M. Lotfy, Auhood Nassar, ola sayed, Abdel-Rahman N. Zekri. Somatic mutation profiling of colorectal cancer by targeted next generation sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3616.
Abstract 3628: Multiregional sequencing reveals the heterogeneity and underlying complex evolutionary history in liver cancer metastasis
Authors: Sun Yun-Fan , Wu Pin , Wang Ze-Jian , Song Min-Fang , Zhou Kai-Qian , Zhang Li-Ye , Peng David P. , Fan Jia
Published: September 2020

Cancer metastasis is still the major cause of death, and poorly understood mechanistically. Therefore, increasing number of studies begins to study the genetic profiles of metastatic samples. However, studies from different tumor types show contradicting conclusions on evolution pattern. In this study, we focus on metastasis in hepatocellular carcinoma (HCC). We analyzed 92 primary and their matched 177 metastatic samples from a cohort of 64 HCC patients encompassing 10 distinct metastatic sites. To study the heterogeneity, we generated multi-regional exome and RNA sequencing for both primary and metastatic tumors. The inferred phylogenies showed both monoclonal (48 %) and polyclonal (52%) metastasis. The polyclonal metastasis is further divided into two subgroups: single branch-multiple waves and multiple-branch metastasis, while the majority of polyclonal evolution is derived from single branch-multiple waves metastasis (12 out of 13). In addition, we observed similar frequency of early, intermediate and late dissemination for tumor metastasis. Therefore, both the clonal and temporal heterogeneity lead to the genetic diversity in metastasis of HCC, which results in the inconsistency in driver (potentially targetable) mutations between primary and metastatic tumor samples for over 60% of patients in our cohort. To identify the potential driver genes for metastasis, we included another two cohorts including no-relapse/metastasis (n=32) and intrahepatic relapse (n=42) as a control. However, similar to published datasets, only TP53 showed a significant increase of frequency in metastatic cohort compared with other cohorts. To conclude, to our knowledge, we provided by far the largest metastatic HCC cohort of multi-regional sequencing and revealed the complex evolutionary history of HCC metastasis regarding to the clonal and temporal composition. We speculate that huge heterogeneity in HCC, especially in metastatic tumors, may be the main reason for frequent failure in targeted therapy in HCC.

Citation Format: Yun-Fan Sun, Pin Wu, Ze-Jian Wang, Min-Fang Song, Kai-Qian Zhou, Li-Ye Zhang, David P. Peng, Jia Fan. Multiregional sequencing reveals the heterogeneity and underlying complex evolutionary history in liver cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3628.
Abstract 3643: HDAC6 modulates colon cancer cell migration and invasion
Authors: Li Hui
Published: September 2020

Introduction: Non-histone protein acetylation in cancer cell regulates diverse biological functions that have been associated with tumorigenesis and cancer progression. HDAC6 is a tubulin-specific deacetylase which regulates microtubule-dependent cell movement. Here we investigated the role of HDAC6 on colon cancer progression.

Methods: Colon tumor tissues were analyzed by immunohistochemistry to measure the expression of HDAC6. Attenuation of HDAC6 function was achieved using either inhibitors (Tubastatin and ACY-738) or siRNAs. Cell migration and invasion were determined using transwell assays. CCK8 and flow cytometry were used to assess the cell proliferation and cell survival respectively. We performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. Immunoprecipitation assay was used to determine protein acetylation level and protein-protein interaction.

Results: We identified that HDAC6 was highly expressed in metastatic colon cancer than non-metastatic colon cancer, indicating that HDAC6 is related with tumor metastasis. Selective suppression of HDAC6 or knocking down of HDAC6 significantly decreased colon cancer cell migration and invasion ability in vitro, without affecting cell proliferation and cell survival. In order to further clarify the role of HDAC6 in cell motility, we employed mass spectrometry-based label-free quantitative proteomics using human colon cancer cell line. We identified 42 proteins with > 2-fold increase of the acetylation level in the absence of HDAC6. These proteins are significantly enriched in processes related to cell-cell adhesion, cell division and several signaling pathways including cAMP and FoxO signaling pathway. We selected ENAH, an actin associated protein, as a potential new substrate of HDAC6, which has been previously reported to play important roles in cytoskeleton remodeling and cell polarity establishment. Immunoprecipitation analysis showed that HDAC6 can associate with ENAH under physical conditions. Besides, ENAH showed elevated acetylation in response to HDAC6 deletion.

Conclusions: Our findings showed that inhibition of HDAC6 decreased the colon cancer cell migration and metastasis probably through regulating the acetylation level of ENAH. Our study also provided a resource of putative cytoplasmic targets of this deacetylase for further investigation. Considering that small molecule compounds targeting HDACs are currently undergoing clinical trials for the treatment of cancer, a better understanding of HDAC6-dependent cellular processes would be highly valuable for the evaluation of the clinical relevance and therapeutic efficacy of HDAC6 inhibitors.

Citation Format: Hui Li. HDAC6 modulates colon cancer cell migration and invasion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3643.
Abstract 367: The role of A20 - Cdc20 axis promotes tumorigenesis and metastasis in triple negative breast cancer
Authors: Song Chaeun
Published: September 2020

Triple negative breast cancer (TNBC) has a higher mitotic index compared to other types of breast cancer yet Ki-67 values are still being used, despite an unclear mechanism. Here we found that the gene Cell-Division Cycle 20 (CDC20), an essential mitotic factor, was highly expressed in TNBC cell lines by using a NCBI GEO database. CDC20 was also negatively correlated with TNBC metastasis and recurrence. CDC20 deficiency results in the decreasing of TNBC cancer cell growth and migration, and mitosis inhibitors blocked TNBC cancer cells growth and metastasis. Interestingly, CDC20 stability is regulated by tumor necrosis factor, alpha-induced protein 3 (TNFAIP3 /A20). CDC20 expression show positive correlation with A20 in TNBC patient samples but not HR positive or HER2 breast cancer patients. A20 deficiency also inhibits cell migration and invasion through the decreasing of CDC20 protein level. Our findings uncover a critical role of the A20-CDC20 axis in tumor growth and metastasis of TNBC and suggest A20 or CDC20 is a novel therapeutic target in TNBC.

Citation Format: Chaeun Song. The role of A20 - Cdc20 axis promotes tumorigenesis and metastasis in triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 367.
Abstract 3678: The role of SCAMP3 in EGFR trafficking and breast cancer cell response
Authors: Diaz Ariana C. Acevedo , Chea Jael Reyes- , Martinez-Montemayor Michelle M. , Suárez-Arroyo Ivette J.
Published: September 2020

Breast cancer (BC) affects approximately 1 in 8 women and is the second leading cause of cancer among women in the United States. BC tumors frequently overexpress Epidermal Growth Factor Receptor (EGFR) which contributes to BC progression and poor clinical outcomes. Studies associate the endocytosis related protein, Secretory Carrier Membrane Protein 3 (SCAMP3), with the regulation of EGFR recycling and degradation. Recently, we published that SCAMP3 is overexpressed in BC cells and tumors. Others have identified SCAMP3 as an indicator of poor prognosis in hepatocellular carcinoma. Therefore, we hypothesize that SCAMP3 promotes the trafficking of EGFR, induces EGFR signaling and BC progression. To evaluate whether SCAMP3 has a direct interaction with EGFR, we treated SUM-149 (EGFR+) and MDA-MB-468 (EGFR+++) BC cells with 100ng/mL EGF at different time points, then we evaluated the localization of both proteins by immunofluorescence. Results showed that SCAMP3 co-localizes with EGFR at the cytoplasm at early stimulation times and the perinuclear area after 30 mins in both cell lines. To evaluate if SCAMP3 has a role in BC progression, we knocked down SCAMP3 in SUM-149 cells using the CRISPR/Cas9 technique, and we assessed the effects in cell viability, colony, and sphere formation capacity and EGFR signaling. Knockdown (KO) and wild type (WT) cells were treated with the ligand at the same concentration described above for 30 min. Our results showed that SCAMP3 knockdown decreased cell viability, colony number and the area of formed spheres. The activation of ERK1/2 was also reduced in SCAMP3 KO cells. Taken together, SCAMP3 is involved in the internalization of EGFR, cell viability, proliferation, and tumor sphere formation. We conclude that SCAMP3 has an essential role in EGFR trafficking and BC progression through EGFR downstream signaling modulation.

Citation Format: Ariana C. Acevedo Diaz, Jael Reyes- Chea, Michelle M. Martinez-Montemayor, Ivette J. Suárez-Arroyo. The role of SCAMP3 in EGFR trafficking and breast cancer cell response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3678.
Abstract 368: Defining the invasive gene signature using patient derived pancreatic cancer organoids
Authors: Jeong Yea Ji , Lerner Michael G. , Ge Yuchen , Knutsdottir Hildur , Navarro-Serer Bernat , Chianchiano Peter E. , Ewald Andrew J. , Bader Joel S. , Wood Laura D.
Published: September 2020

Pancreatic cancer mortality is rising due to late detection and established metastasis. Despite the recent advances in cancer research, metastasis research has been hampered by the lack of models that recapitulate this complex process. Traditional cell culture and animal models provided insights on cancer cell migration and dissemination. But local invasion, which precedes all the events in the metastatic cascade, remains poorly understood. Patient-derived organoids (PDO) present an innovative platform in which the earliest step of metastasis can be observed and manipulated ex vivo. We developed a pancreatic cancer organoid model using surgically resected tumors from The Johns Hopkins Hospital. We previously reported that there are two invasive patterns in pancreatic cancer PDOs - Mesenchymal and Collective invasion, with one dominating pattern in every PDO culture. Here, we describe the gene expression signature associated with local invasion in pancreatic cancer by comparing the whole transcriptome of matched invasive and non-invasive organoids from the same tumor. For this study, PDOs were cultured for 7 days ex vivo and we manually collected 50 invasive and non-invasive organoids from each culture for RNA-seq analysis. To date, the invasive and non-invasive organoids from 6 PDO lines have been analyzed. The list of differentially expressed genes between invasive and non-invasive organoids includes 1,106 genes with FDR 0.001, with DACT1, DKK3 and KIF26B being most differentially expressed. Pathway analysis identifies that the invasive phenotype is associated with extracellular matrix receptor interaction, focal adhesion and PI3K-Akt signaling pathways. In conclusion, our findings revealed a distinct gene expression signature between invasive and non-invasive cells in PDOs. Interestingly, we did not find significant up or down-regulation in the classical EMT markers in our results, which suggests possible pancreatic cancer-specific metastasis programs exist. We believe our results can inform new diagnostic and prognostic tools and help identify anti-metastasis targets in pancreatic cancer.

Citation Format: Yea Ji Jeong, Michael G. Lerner, Yuchen Ge, Hildur Knutsdottir, Bernat Navarro-Serer, Peter E. Chianchiano, Andrew J. Ewald, Joel S. Bader, Laura D. Wood. Defining the invasive gene signature using patient derived pancreatic cancer organoids [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 368.
Abstract 3702: A lineage-specific lncRNA driven by Foxa1 associated super enhancer promotes tumor growth through interacting YBX1
Authors: Zhang Yin , Huang Yong-xin , Wang Dan-lan , Yan Hai-yan , Dong Yin
Published: September 2020

Lineage-specific expression of genes plays important roles in tumorigenesis. It has been suggested that lncRNAs tend to be more lineage-specifically expressed than protein-coding genes. However, the regulation and role of lineage-specific lncRNAs remains largely unknown. In this study, we reported a lncRNA DSCAM-AS1 that specifically expressed in lung adenocarcinoma and luminal breast cancer. The expression of DSCAM-AS1 was regulated by two super enhancer clusters driven by Foxa1. Foxa1 can directly bind to promoter of DSCAM-AS1. In breast cancer, DSCAM-AS1 was also transcriptionally regulated by ERα, which formed complex with Foxa1. Higher expression of DSCAM-AS1 was associated with poor poor prognosis in both lung adenocarcinoma and luminal breast cancer patients. Silencing or knock-out of DSCAM-AS1 significantly reduce the growth of lung adenocarcinoma and luminal breast cancer cells. We observed silencing or knock-out of DSCAM-AS1 could reduce the expression of Foxa1 in lung adenocarcinoma and breast cancer cells. We also found a significant decrease of ERα expression after silencing or knock-out of DSCAM-AS1 in luminal breast cancer cells. Mechanistically, we found DSCAM-AS1 could interact with YBX1 and enhance recruitment of YBX1 at the promoter region of Foxa1 and ERα. DSCAM-AS1 could form a positive feedback loop with Foxa1 and ERα. Our findings may provide another regulatory loop: Foxa1 and ERα drive expression of super enhancer associated lncRNA DSCAM-AS1 and form a positive feedback loop with it, which play important roles in maintaining cancer cell identity and function.

Citation Format: Yin Zhang, Yong-xin Huang, Dan-lan Wang, Hai-yan Yan, Yin Dong. A lineage-specific lncRNA driven by Foxa1 associated super enhancer promotes tumor growth through interacting YBX1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3702.
Abstract 3704: MicroRNA-3977 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting FoxM1
Authors: Karamil Selda , Altintas Nuray , Ozpolat Bulent
Published: September 2020

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC is associated with poor prognosis, high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC requires new therapeutic approaches. The Forkhead box protein M1 (FoxM1) is a member of forkhead superfamily transcription factors that has been associated with normal cell growth and proliferation through cell cycle transition and mitotic spindle control. FoxM1 is also identified as a master regulator of tumor metastasis. Based on analysis of pooled clinical datasets derived from online accessible databases as Breast Cancer Gene-Expression Miner v4.3 and Kaplan-Meier plotter, the expression and prognostic values of FoxM1 increase in TNBC compare with other breast cancer subtypes. Directly binding to the 3'-untranslated region (3' UTR) of FoxM1 mRNAs suppress its high expression in TNBC that leads to inhibition of cell proliferation, motility, and invasion. These results implied that FoxM1 is an essential poor prognostic factor and promising candidate target in the treatment of TNBC. We identified miR-3977 as a potential regulator of FoxM1 by using in silico prediction algorithms, and we investigated its role in TNBC. In this study, we found miR-3977 expression is significantly suppressed in TNBC cell lines and inversely correlated with FoxM1 levels. We demonstrate that miR-3977 directly bound to the 3′ UTR of FoxM1 mRNA and repressed its expression. We also find reduced miR-3977 expression is associated with shorter patient survival in TNBC based on Kaplan-Meier plotter database. Moreover, in vivo delivery of miR-3977 nanoparticles inhibited FoxM1 expression and tumor growth in TNBC xenograft mouse models. In conclusion, we provide the first evidence that miR-3977 functions as a tumor suppressor and gain of miR-3977 expression leads to decrease in FoxM1 expression and inhibits to cell growth, colony formation, invasion, and progression of TNBC. Taken together, our data suggest that miR-3977-based gene therapy is a potential therapeutic strategy in TNBC.

Citation Format: Selda Karamil, Nuray Altintas, Bulent Ozpolat. MicroRNA-3977 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting FoxM1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3704.
Abstract 371: A reporter mouse model enabling image-based evaluation of CD8+T cell dynamics in immunocompetent allografts
Authors: Zhao Bosong , Feng Dan , Zhu Haiyan , Ci Lei , Wan Zhipeng , Sun Ruilin
Published: September 2020

Immune cell recruitment to tumors is indispensable for the success of immunotherapy. In vivo bioluminescence imaging technology to visualize the location and functionality of immune cells is of great value for immuno-oncology research. Here, we describe the generation of a genetically engineered mouse model “Tluc” in whichall CD8+T cells constitutively express a novel luciferase reporter Akaluc. On the C57/BL6 background, the IRES-Akaluc-EGFP expression cassette was placed downstream of the translation stop codon of the endogenous Cd8a gene, allowing the bioluminescence imaging of CD8+T cell location and function under physiological and pathological conditions. As expected, the engineered mice showed constitutive luciferase expression in lymphoid organs such as thymus, lymph nodes and spleen under normal conditions. We next modeled the T cell dynamics in the context of tumor transplantation. In the case of subcutaneously implanted mouse cancer cells such as MC38 colon cancer cells and antigenic SIY-expressing B16 melanoma cells (but not the regular B16 cells), the reporter mice showed T cell recruitment and infiltration into the tumor mass. This apparent immune response was consistent with the simulation. Our study underscores the utilities of such reporter mice for uncovering the role of immune cells in tumor responses by revealing dynamics, interactions and distribution of various immune cells in tumor settings.

Citation Format: Bosong Zhao, Dan Feng, Haiyan Zhu, Lei Ci, Zhipeng Wan, Ruilin Sun. A reporter mouse model enabling image-based evaluation of CD8+T cell dynamics in immunocompetent allografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 371.
Abstract 3714: Role of miR-29a in pancreatic stellate cell mediated stromal remodeling in pancreatic ductal adenocarcinoma
Authors: Dey Shatovisha , Liu Sheng , Factora Tricia D. , Taleb Solaema , Riverahernandez Primavera , Udari Lata , Zhong Xiaoling , Wan Jun , Kota Janaiah
Published: September 2020

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive form of malignancy with nearly equal incidence and mortality rates in patients. The disease is characterized by dense desmoplastic tumor-associated stroma, which prevents effective therapeutic delivery to the tumor core and confers resistance to chemotherapy. The central effectors of such characteristic stroma in PDAC are the pancreatic stellate cells (PSCs), which are activated by autocrine and paracrine signaling of the tumor cells and deposit excessive amounts of extracellular matrix (ECM) proteins- the major component of the stroma. This interaction of the PSCs with the cancer cells greatly promote tumor progression and metastasis. In our previous work, we demonstrated that microRNA (miRNA) 29a plays a key role in PSC-mediated stromal reaction, and that the loss of the molecule in PSCs accelerates ECM protein accumulation and cancer growth. To understand the mechanistic regulation of miR-29a in PDAC stroma, we performed RNA-seq analysis with human PSCs (hPSCs), which identified 197 genes that differentially expressed with miR-29a overexpression at a false discovery rate less than 0.05. This included 19 downregulated and one upregulated direct miR-29a targets. Of these, expression of six key targets with the highest fold changes, namely, ADAMTS2, IGF1, E2F7, LAMC1, CLDN1 and ITGA6 by miR-29a were validated at the protein level in hPSCs. In addition, canonical pathways associated with the target genes and their network interactions were assessed, which identified the association of the key targets with pathways such as collagen synthesis, insulin signaling, TGF-β and PI3K/Akt pathways. ADAMTS2, E2F7, LAMC1 and CLDN1 expressions were significantly upregulated in PDAC patient pancreas compared to that in healthy individuals. Together, the current study presents a comprehensive understanding of miR-29a mediation in stromal remodeling, and elucidates associated pathways in regulating PSCs' function in PDAC desmoplasia and pancreatic carcinogenesis.

Citation Format: Shatovisha Dey, Sheng Liu, Tricia D. Factora, Solaema Taleb, Primavera Riverahernandez, Lata Udari, Xiaoling Zhong, Jun Wan, Janaiah Kota. Role of miR-29a in pancreatic stellate cell mediated stromal remodeling in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3714.
Abstract 3816: Elucidating the potential role of membrane complement regulatory proteins (CD46, CD55, CD59) in tumorigenesis of cervical cancer
Authors: Mo Xuetang , Leung Thomas Ho-Yin , Siu Michelle Kwan-Yee , Ngan Hextan Yuen-Sheung
Published: September 2020

Introduction: Cervical cancer is a common genital tract cancer. Although abundant screening and vaccination are beneficial for reducing the disease burden, the molecular mechanism underlying tumorigenicity and aggressiveness are not fully understood. Human papillomavirus (HPV) E6 protein was found to be responsible in the development of cervical cancer. Membrane complement regulatory proteins (mCRP), such as CD46, CD55, CD59, control complement activation on the surface of tumour cells and suggested to be important in contributing tumorigenesis in various types of cancers. In our recent study, we have reported that HPV E6 protein enriches the expression of CD55 in cervical cancer cells, which would further promote radio-resistance and cancer aggressiveness. In the present study, we would characterize the functional role of other two mCRPs CD46 and CD59 in contributing cervical carcinogenesis. We would further examine if triple knockout of CD46, CD55, CD59 would have synergistic effect on blockage of tumorigenesis in cervical cancer.

Material and method: Flow cytometry was performed to analyse the expression level of CD46, CD55 and CD59 in cervical cell lines C33A, C4-1, CaSki, SiHa, and a HPV18-E6-overexpressing stable clone (18E6-5, origin C33A cells). In order to investigate the effect on the blockage of those mCRPs, 18E6-5 clone with high CD46, CD55 and CD59 expression were chosen for in vitro functional study using and CRISPR/Cas9 knockout.

Results and discussion: The percentage of CD46(+), CD55(+) and CD59(+) cells were high in C4-1, CaSki, SiHa and 18E6-5 lines. The C33A cells demonstrate a low CD55(+) subpopulation while high CD46(+) and CD59(+) subpopulations. We found that single knockout of CD46 or CD59 supressed cell proliferation, sphere-forming, anchorage-independent cell growth capability, and sensitized cells to radiation treatment. In addition, triple knockout of CD46, CD55, CD59 remarkably inhibited those effects comparing with the effects of single and double knockout.

Conclusion: Silencing of CD46, CD55, CD59 expression abolished the tumorigenic, radio-resistant and aggressive effects in cervical cancer cells, with triple knockout displayed the highest blockage effects. Those mCRPs would be responsible for tumorigenicity, radio-resistance and cancer aggressiveness in cervical cancer.

Citation Format: Xuetang Mo, Thomas Ho-Yin Leung, Michelle Kwan-Yee Siu, Hextan Yuen-Sheung Ngan. Elucidating the potential role of membrane complement regulatory proteins (CD46, CD55, CD59) in tumorigenesis of cervical cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3816.
Abstract 3822: Genome-wide CRISPR library screen to identify synthetic lethal targets of a WEE1 degrader
Authors: Yue Yangbo , Su Hexiu
Published: September 2020

Background: WEE1 kinase is a G2/M cell cycle checkpoint protein, activated to allow DNA repair prior to cell entry to mitosis. WEE1 kinase inhibitors have witnessed encouraging early clinical results in combination with chemotherapy and radiotherapy. We previously identified a small molecule WEE1 degrader and showed selective growth inhibition in pancreatic cancer cell lines. To further study the functional mechanism of this compound and to investigate the combinational inhibition of WEE1 degradation, we performed genome-wide CRISPR library screening in pancreatic cancer cell lines harboring either wildtype K-RAS or mutant K-RAS. The identified synthetic lethal targets may lead to tractable therapeutic opportunities in combination with this compound.

Method: We conducted CRISPR screening in MIA-PaCa2 and PANC1 cell lines, harboring mutant and wild type K-RAS, respectively, with genome-wide Brunello library (2 vector system) in the presence or absence of the WEE1 degrader for 2 weeks. After harvesting the cells for deep sequencing, we analyzed the top hits whose inhibition showed synergetic cytotoxic effect with the compound.

Results: According to the sequencing result, the deletion of several candidate genes increased the sensitivity to the compound, indicating combinational potential with this WEE1 degrader. Further validation studies are in progress. We will discuss possible mechanisms underlying the synthetic lethality. The pipeline we have established, from experimental design to bioinformatics analysis, can be applied to investigate functional mechanism and combination strategy of any target therapeutic agents.

Citation Format: Yangbo Yue, Hexiu Su. Genome-wide CRISPR library screen to identify synthetic lethal targets of a WEE1 degrader [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3822.
Abstract 387: Analysis of FFPE human tumor tissues using CODEX with signal amplification
Authors: Mistry Sejal , Dakshinamoorthy Gajalakshmi , Yuan Jessica , Noordam Pieter , Kim Joseph , Lee Won-Mean , Kennedy-Darling Julia
Published: September 2020

Analysis of FFPE human tumor tissues using CODEX with signal amplification

Characterizing the complexities of the tumor microenvironment is fundamental to understanding disease mechanisms. The spatial relationships between infiltrating immune cells and the remodeling of the cellular matrix is widely recognized as a key component to defining tumor heterogeneity. Current methodologies for studying cells within the context of tissue architecture, like traditional immunofluorescence (IF) and immunohistochemistry (IHC), are limiting–allowing the assessment of only a few parameters at a time. The CODEX technology has overcome this limitation through a DNA-based labeling strategy, involving adding and removing dye-labeled oligonucleotides (reporters) across multiple cycles to oligonucleotide-labeled antibodies. In this manner, tens of markers can be analyzed on the same tissue. Additionally, CODEX interfaces with existing inverted microscopes and provides a cost-effective, fully automated platform for highly multiplexed imaging. Recently, we expanded our workflow to amplify the fluorescent signal intensities of low expressing biomarkers by combining Tyramide Signal Amplification (TSA) with CODEX. This approach is associated with key markers used to analyze the tumor microenvironment, including PD-L1 and PD-1. We demonstrated signal amplification of more than 50X compared with non-amplified signal. Here, we present the analysis of CODEX data for a panel of over 20 biomarkers on human FFPE tissue including signal amplification of PD-L1, PD-1 and foxp3. Through amplification of key markers as the final cycle of a CODEX experiment, low expression can be measured and used to identify key cell types, including Treg cells and PD-L1 positive tumor and stromal regions.

Citation Format: Sejal Mistry, Gajalakshmi Dakshinamoorthy, Jessica Yuan, Pieter Noordam, Joseph Kim, Won-Mean Lee, Julia Kennedy-Darling. Analysis of FFPE human tumor tissues using CODEX with signal amplification [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 387.
Abstract 3871: Infiltration of CD56dim NK cells regulated by MLH1 into tumor tissue of FAP patients predict better prognosis
Authors: Yang Jun , Zhang Hushan , Liu Wei Qing , Li Wenliang , Ma Shumin , Liang Lei , Kou Zhiyong , Huang Depei , Shen Xudong , Dong Jian
Published: September 2020

Backgroud

Familial adenomatous polyposis (FAP) is an autosomal dominant-inherited colorectal cancer (CRC) with poor prognosis, and is also considered as model to study the change of CRC from pre-tumor to tumor progression. To better predict and guide clinical applications of immune check point inhibitors(ICI), researchers proposed the classification system of human cancer, termed tumor immunity in the microenvironment (TIME), that four different TIME groups have been identified according to the presence of TILs and B7-H1(PD-L1) expression in tumor biopsies. Therefore, we followed and performed this theory into our research about TIME of FAP, to explore the potential of immunotherapy for FAP, and explore the cancerous mechanism from immune microenvironment.

Methods

We collected tissue samples of 40 FAP patients from May 2009 to December 2018, followed by detection of PD-L1 by using Dako PD-L1 IHC 22C3 pharmDx, MLH1, PMS2, MSH2, MSH6 were detected by IHC, and CD8+ T cells, tumor associated macrophages (TAM) and NK cells infiltration in the tumor biopsies by multiple fluorescence immunohistochemistry (mICH). Follow-up data of these 40 patients were analyzed.

Results

CD8+T cells, M1 and M2 TAM, CD56bright NK and CD56dim NK cells were found both in intratumoral region and tumor rim of FAP patients(n=40), no differences have been found between M1and M2 TAM, however, CD56dim NK increased more than CD56bright NK both in intratumoral region and tumor rim(p<0.01). Surprisingly, no positive expression of PD-L1 was detected in the tumor tissue of these FAP patients (Tumor Proportion Score, TPS<1%). Further results shown CD56dim NK mainly increased in the tissue of FAP patients(n=7) with loss of MLH1, and consistent with this, these patients shown better prognosis than patients with wild type MLH1, that is, have longer progression free survival (p<0.005).

Conclusions

For the TIME characteristics of FAP patient, Immunotherapy targeted PD-1/PD-L1 may not applicable for FAP, however, MLH1, the member of mismatch repair protein, was associated with infiltration of NK cells, especially CD56dim NK cells in the tumor, and also associated with prognosis of FAP patients. Therefore, it can be exploited as potential target of immunotherapy for FAP or even CRC.

Citation Format: Jun Yang, Hushan Zhang, Wei Qing Liu, Wenliang Li, Shumin Ma, Lei Liang, Zhiyong Kou, Depei Huang, Xudong Shen, Jian Dong. Infiltration of CD56dim NK cells regulated by MLH1 into tumor tissue of FAP patients predict better prognosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3871.