bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1026: Oncogenic alterations in FGFR3 and ERBB2 lead to ligand-independent activation of PPARG in bladder cancer
Authors: Goldstein Jonathan T. , Berger Ashton C. , Strathdee Craig A. , Meyerson Matthew
Published: August 2019

PPARG is genomically activated in muscle-invasive bladder cancer through focal PPARG gene amplification and hotspot mutations in its heterodimer partner, RXRA. However, more than half of the PPARG-activated bladder tumors and cell lines do not have identifiable somatic alterations in either PPARG or RXRA. Using a PPARG-driven reporter assay in RT112 bladder cancer cell line, we screened probe compounds to identify candidate drivers of ligand-independent PPARG activation. We found that pan-FGFR inhibitors and MEK1/2 inhibitors antagonized the PPARG-driven reporter assay with potency similar to reported values for their cognate targets (1-20 nM) and interestingly, RT112 cells carry an FGFR3-TACC3 oncogenic fusion. In addition to the expected effects of these inhibitors on phospho-MEK1/2 and phospho-ERK1/2, they also inhibited production of canonical PPARG targets, including FABP4. In a second subset of cell lines, a parallel story was also observed for the effects of ERBB2 inhibitors in ERBB2 hotspot mutant bladder cancer. Taken together, these data uncover additional mechanisms for functional activation of PPARG in bladder cancer, suggest potential resistance mechanisms for FGFR and ERBB2 inhibitors, and provide a rationale for therapeutic combinations of PPARG modulators.

Citation Format: Jonathan T. Goldstein, Ashton C. Berger, Craig A. Strathdee, Matthew Meyerson. Oncogenic alterations in FGFR3 and ERBB2 lead to ligand-independent activation of PPARG in bladder 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 1026.
Abstract 1035: PLEK2 promotes epithelial mesenchymal transition through increasing Snail1 in lung cancer cells
Authors: Liu En-Chu , Lee Tsai-Tu , Wu Shey-Lin , Lee Tai-Lin , Tsai Meng-Feng
Published: August 2019

Pleckstrin 2 (PLEK2), one of the pleckstrin homology domain proteins, have been suggested to modulate actin rearrangement and cell spreading in T lymphocytes. Recently studies also implicated that PLEK2 paly an importance role in human myeloproliferative neoplasms, melanoma, and lung cancers. PLEK2 mRNA genes were related to poor overall survival in lung adenocarcinoma patients. However, the biological functions of PLEK2 in lung cancer is not clear. We detected the expression of PLEK2 in various lung adenocarcinoma cell lines using quantitative RT-PCR methods. Molecular manipulations were performed to investigate the roles of PLEK2 in lung cancer cells. Over-expression of PLEK2 in lung cancer cells exhibited markedly promoting in the proliferation and anchorage independent growth, whereas reduced of PLEK2 was found to suppress lung cancer cell progression. This studies also indicated that PLEK2 may play an important role in the epithelial-mesenchymal transition (EMT) features, including a morphological change, increased invasive and migratory ability, reduction the epithelial marker expression and up-regulated EMT regulators. Our results suggested that PLEK2 expression induce lung cancer cell EMT phenomenon through up-regulation Snail1 expression. These results will provide information and help us to understand the roles of PLEK2 in lung cancer cells.

Citation Format: En-Chu Liu, Tsai-Tu Lee, Shey-Lin Wu, Tai-Lin Lee, Meng-Feng Tsai. PLEK2 promotes epithelial mesenchymal transition through increasing Snail1 in lung cancer cells [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 1035.
Abstract 1042: Cellular plasticity and migratory modalities in high-grade serous ovarian carcinoma
Authors: Bapat Sharmila A. , Varankar Sagar
Published: August 2019

Epithelial-mesenchymal (E-M) switch associated plasticity is crucial for the gradual destabilization of tumor tissue architecture to facilitate metastasis. Several studies towards comprehension of the metastatic cascade employ an overview of the phenotype specific markers and their association with distinct functionalities of migration, invasion, stemness, anoikis resistance, ECM remodeling, etc. Recent work on the dissection of phenotypic transitions has identified the existence of transitionary intermediate states across multiple cancers which exhibit greater plasticity as compared to the two phenotypic extremes. In a previous study from our group, we successfully identified molecular subclasses in high grade serous ovarian carcinoma (HGSC) that were associated with distinct modes of metastatic dissemination viz., collective cell migration (CCM) and EMT. Towards, elucidating contributions of distinct phenotypic states to CCM and EMT along with a comprehension of associated molecular regulators we resolved a phenotypic spectrum along the E-M axis. Identification of five phenotypic states followed by their functional characterization was associated with differential migratory capabilities that were further dissected with the aid of live cell imaging to quantitatively distinguish CCM and EMT. Enhanced visual output and extraction of three quantitative metrics viz., displacement, velocity and nearest neighbours generated an informative tool for assessing migratory modes in vitro. Furthermore, these metrics identified migratory modalities inclusive of wound closure by proliferation (passive CCM), sheet-like cell migration (active CCM) and individual cell migration (EMT). Interestingly, phenotypic status and migratory modes across the spectrum emerged as a consequence of a balance between expression and differential sub-cellular localization of two transcription factors viz. Tcf21 and Slug. Application of the novel migratory analyses in view of chemotherapeutic challenges identified the emergence of an epithelial state across the spectrum facilitated by Tcf21 mediated Slug repression and resulted in a switch from EMT to aCCM-mediated migration. Our study thus provides a comprehensive dissection of the E-M axis in HGSC in view of associated functional attributes.

Acknowledgements: DBT Indo-Finnish Collaborative Grants, DBT Indo-Australia Collaborative Grants and NCCS Intramural Grants to SAB funded this research. SSV received fellowship from CSIR.

Citation Format: Sharmila A. Bapat, Sagar Varankar. Cellular plasticity and migratory modalities in high-grade serous ovarian 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 1042.
Abstract 1045: CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development
Authors: Enuameh Metewo S. , Shu Weiguo , Newman Robert
Published: August 2019

Among women, breast cancer continues to be the most common cancer, with metastasis being the leading cause of mortality in cancer patients around the world (DIZON et al. 2016). Epithelial to mesenchymal transition (EMT) - the process by which epithelial cells shift to the mesenchymal state, has been implicated in many aspects of breast cancer tumorigenesis, metastasis and drug resistance ). The accumulation of a large body of data on the association of EMT with cancer over the years has not resulted in EMT being an active target for therapeutic development. This is due in part to the lack of appropriate in vitro models. Here we have exploited some of the basic biology of EMT, to create an advanced in vitro metastatic breast cancer reporter cell line model for use in both basic research and discovery of new EMT inhibitors.

During EMT, E-cadherin protein expression is down regulated in cancer cells in association with the loss of cell-to-cell adhesion, apico-basal polarity and a change to a spindle-shaped morphology. By installing an emerald green fluorescent protein (EmGFP) tag on the C-terminus of the e-cadherin (ECAD) gene in the epithelial BT-474 breast cancer cell line via CRISPR/Cas9 genome editing, end-point or real-time EMT status of cells can be tracked under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the BT-474 ECAD EmGFP cell line, shows that it responds to EMT induction. The subsequent EMT status of cells can be monitored in real time by observing and measuring ECAD EmGFP expression, in addition, the resulting mesenchymal cells have increased invasive capacities. Furthermore, this EMT reporter cell line shows sensitivity to the MEK1/2 inhibitor U0126 - thereby providing the basis for the use of this cell line in high-throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic breast cancer. The BT-474 ECAD EmGFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis.

References

DIZON, D. S., L. KRILOV, E. COHEN, T. GANGADHAR, P. A. GANZ et al., 2016 Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer From the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011.

HAY, E. D., 1995 An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154: 8-20.

Citation Format: Metewo S. Enuameh, Weiguo Shu, Robert Newman. CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development [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 1045.
Abstract 1048: CD44 promotes epithelial-mesenchymal transition (EMT) by regulating snail, ZEB1, and Caveolin-1 expression and predicts poor survival of ovarian cancer
Authors: Zhou Jiayi , Du Yan , Luan Baoxin , Yu Yinhua , Zhao Hongbo
Published: August 2019

CD44 is a stem cell marker which is involved in the generation of a stem cell niche and maintaining stem cell quiescence. Our data first demonstrated that CD44 knockdown by small silencing RNA abrogated both basal Snail expression and TGF-β1 induced Snail expression in HOPM and HOPM-snail cells. In addition, CD44 knockdown caused an decrease in ZEB1 expression. Further, RPPA data indicated that Caveolin-1 may be another regulative target of CD44, and western blotting analysis confirmed that CD44 knockdown caused an increase in Caveolin-1 expression. However, there was no obvious reciprocal regulation among ZEB1, Caveolin-1, and snail. Moreover, CD44 knockdown caused a decrease in cell migration, cell invasion and clone formation of HOPM and HOPM-snail cells.

We next observed the role of CD44 expression in the prognosis of ovarian cancer. Over-expression of CD44 was associated with advanced FIGO stage (P<0.05). Univariate analysis result showed that histological subtype, FIGO stage, intravascular tumor thrombus, CA125 and CD44 expression were associated with overall survival and disease-free survival of ovarian cancer patients. Multivariate analysis showed that CD44 expression was an independent prognostic factor to predict both overall survival and disease-free survival of ovarian cancer patients. Taken together, our data showed that CD44 may be crucial for EMT process by regulating Snail, ZEB1, and Caveolin-1 expression. And CD44 is a potential prognostic factor as well as a treatment target for ovarian cancer.

Citation Format: Jiayi Zhou, Yan Du, Baoxin Luan, Yinhua Yu, Hongbo Zhao. CD44 promotes epithelial-mesenchymal transition (EMT) by regulating snail, ZEB1, and Caveolin-1 expression and predicts poor survival of ovarian 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 1048.
Abstract 1054: Systematic establishment of robustness and standards in xenograft experiments and analysis
Authors:   The NCI PDXNet Consortium, Evrard Yvonne , Chuang Jeffrey H.
Published: August 2019

Patient-Derived Xenograft (PDX) models are a valuable model system for demonstrating clinical translatability in the preclinical testing of therapeutic agents. Large PDX collections such as the NCI PDXNet Consortium are a critical resource for assays of therapeutic efficacy. However, research groups vary in their procedures for experiments and assessments, limiting conclusions about the robustness of PDX results and their mapping to patient outcomes. To determine the sensitivity of PDX assays to different procedures, the PDXNet conducted a study of temozolomide drug response for PDX models derived from three patients, performed in replicate across four PDX Development and Trial Centers (PDTCs) with all sites blinded to the procedures and results of other groups. The three PDX models were chosen based on prior treatment experiments conducted by the NCI Patient Derived Models Repository that showed that one model was sensitive, one was resistant, and one had intermediate sensitivity. Each PDTC used their internally developed SOPs, which varied in their procedures for establishment of xenografts, treatment dosing and scheduling, time period of study, and response criteria. Additionally all groups followed internal sequencing procedures to generate one exome-seq and one RNA-seq per model. Drug responses were consistent across all five testing groups, with each group identically classifying the sensitive, resistant, and intermediate models. We also trained novel statistical classifiers for assessing sensitivity, resistance, and intermediate response on 105 retrospective cohorts of mice collected among the PDTCs. In addition, we compared and benchmarked exome-seq and RNA-seq analysis pipelines developed by each individual PDTC on synthetic test data. We assessed the robustness of sequencing assessement on PDX tumors by applying the optimized pipeline across the replicate samples generated across the separate PDTC groups. Overall, our results show that drug responses of PDX models are consistent and robust across different academic centers. We are developing standardized SOPs for experimental procedures, response measurement, statistical assessment of response, and sequence analysis based on these results. All SOPs will be released publicly through protocol descriptions and shared computational workflows within the NCI Cancer Genomics Cloud. We expect these PDXNet standards to improve the use of PDX and other in vivo models in advancing cancer precision medicine.

Citation Format: The NCI PDXNet Consortium, Yvonne Evrard, Jeffrey H. Chuang. Systematic establishment of robustness and standards in xenograft experiments and analysis [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 1054.
Abstract 1055: Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors
Authors: Tang Xuzhen , Yang Li , Qi Hui , Zhai Xianzhi , Wang Fuyang , Qiang Xiangnan , Xu Jie , Qin Xiaoran , Gu Qingyang , Yan Shaoyu , Ji Qunsheng
Published: August 2019

Tyrosine Kinase Inhibitor (TKI) of Epidermal Growth Factor Receptor (EGFR) is a first-line therapy for non-small cell carcinoma lung cancer patients with EGFR mutations, but patients often develop resistance to such a target therapy. Recently several PD-1 and PD-L1 inhibitors were approved for treating NSCLC, however, it remains unclear whether EGFR mutant NSCLC would benefit from PD-1/PD-L1 treatment. PD-1 response in cancer patients is closely associated with oncogenic mutation status, tumor microenvironment, PD-L1 expression and tumor mutation burden (TMB); however, the lack of pre-clinical models limited the investigation of the checkpoint inhibitors in EGFR mutant tumors. Here we use human PBMC reconstitution system for immune-humanization, and established a series of IO-Xenograft models (IO-CDX and IO-PDX) with cancer cell lines (IO-CDX) or patient-derived tissues (IO-PDX) that contain various EGFR mutations, including EGFR 19 del, L858R, T790M, and EGFR over-expression, amplification and fusion. We treated these models with either EGFR TKI (erlotinib, afatinib or osimertinib) or nivolumab, a PD-1 inhibitor. The predictive biomarkers including PD-L1 expression and TMB were analyzed by whole exom sequencing (WES) and FACS for tumor infiltrating lymphocytes (TILs), respectively. We found that 3 TKI-resistant PDX models bearing EGFR T790M mutation exhibited lower TILs than those bearing EGFR wild type or exon 19del mutation. Of the 8 IO-CDX or IO-PDX humanized models we have examined, those exhibiting higher TILs achieved better responses to nivolumab treatment. The 3 most sensitive models are EGFR amplification (wt), EGFR over-expression (wt) and EGFR exon 19del. In contrast, the EGFR T790M model from H1975 cell line was observed with hyper-progressive disease after nivolumab treatment. We will also discuss the contribution of PD-L1 expression and TMB to nivolumab in other models. In summary, these well-established IO-CDX and IO-PDX models can provide a human-resembling immune system for of immune therapeutics and combination with targeted therapies, and help to facilitate the understanding of relationship among tumor microenvironment, driver oncogenic mutations and drug response.

Citation Format: Xuzhen Tang, Li Yang, Hui Qi, Xianzhi Zhai, Fuyang Wang, Xiangnan Qiang, Jie Xu, Xiaoran Qin, Qingyang Gu, Shaoyu Yan, Qunsheng Ji. Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors [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 1055.
Abstract 1072: Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma
Authors: Rath Sanhita , Peng Sen , Hartman Lauren , Reid George , Dhruv Harshil , Glaser Shannon , Meng Fanyin , Alpini Gianfranco , Celinski Scott
Published: August 2019

Cholangiocarcinoma (CCA) is a fatal primary malignancy with limited treatment options and a dismal 5year survival rate of <10%. Poor prognosis and early recurrence after surgical treatment warrants further molecular characterization of these tumors to evaluate therapeutic sensitivities and identification of biomarkers for CCA. Although several Next Generation Sequencing (NGS) studies have revealed some targetable genomic aberrations, the lack of preclinical and translational models has severely limited the identification of clinically relevant targeted therapies for CCA. So far, very few putative molecular targets/pathways have been identified using patient-derived xenograft (PDX) models, CCA cell derived xenograft models (CDX) and CCA cell lines, only 4 of which have progressed to preclinical testing for therapeutic value. In this study we describe development and comprehensive characterization of patient derived xenograft models of CCA from freshly resected patient tissue. Patient tumor cells from 8 patients were engrafted subcutaneously in highly immunocompromised NSG (NOD.Cg-PrkdcscidIl2rgtm1Wj) mice for PDX development. Two (CCA-1 and CCA-7) of 8 (25 %) engraftments yielded successful PDX model. The PDX avatar and matched patient tumor samples were genomically characterized at the whole exome and transcriptome level to check the fidelity of PDX models. Exome sequencing analysis of PDX tumors demonstrate higher genomic aberrations as compared to their patient counterparts. Preliminary analysis of transcriptomic profiles from CCA TCGA dataset, long term established intra- and extra-hepatic CCA cell lines (Mz-ChA-1, TFK-1, SG231, CCLP-1, HuCC-T1 and HuH-28), CCA CDX model from Mz-ChA-1 cells and CCA PDX models demonstrate that CCA PDX models more faithfully recapitulate patient tumors as compared to long term established cell lines. In summary, this proof of concept study demonstrates that PDX avatars of CCA could serve as a good model system for preclinical target discovery and validation for a deadly and neglected disease such as CCA.

Citation Format: Sanhita Rath, Sen Peng, Lauren Hartman, George Reid, Harshil Dhruv, Shannon Glaser, Fanyin Meng, Gianfranco Alpini, Scott Celinski. Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma [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 1072.
Abstract 1073: Role of CD44 in the transition of ductal carcinoma in situ to invasive breast carcinoma
Authors: Liu Fang , Zhou Jun , Kundu-Roy Tanima , Wahler Joseph , So Jae Young , Lin Yong , Lou You-Rong , Barnard Nicola J. , Matsuura Isao , Suh Nanjoo
Published: August 2019

Ductal carcinoma in situ (DCIS) transition to invasive tumor is an important issue for breast cancer. The human MCF10DCIS.com cell line (abbreviated as MCF10DCIS) is derived from the human MCF10A breast epithelial cell line. The MCF10DCIS xenografts are similar to human high grade comedo DCIS. In the xenograft assay, MCF10DCIS cells form DCIS-like lesion, remain as DCIS-like lesion in the first few weeks, and then progress into invasive carcinoma. Thus, it is an excellent model to study human breast cancer transition from DCIS to invasive carcinoma. To the best of our knowledge, it is also the only cell model for DCIS transition to invasive carcinoma. We investigated the role of CD44 in breast cancer transition from DCIS to invasive carcinoma by knockdown the expression of CD44 in the MCF10DCIS xenograft model. Our results show that knockdown of CD44 expression in the MCF10DCIS cells enhances DCIS transition to invasive carcinoma. Accordingly, a previous study has shown that CD44 expression is lower in invasive human breast tumors compared with DCIS, especially in luminal A subtype. We have also observed that CD44 expression is lower in the invasive carcinomas compared with DCIS. Furthermore, we provide evidence that CD44 plays a role in maintaining the integrity of the myoepithelial layer. Our study uncovered an unexpected role of CD44 in the prevention of DCIS transition to invasive carcinoma. Our findings have important implications for breast cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Fang Liu, Jun Zhou, Tanima Kundu-Roy, Joseph Wahler, Jae Young So, Yong Lin, You-Rong Lou, Nicola J. Barnard, Isao Matsuura, Nanjoo Suh. Role of CD44 in the transition of ductal carcinoma in situ to invasive breast 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 1073.
Abstract 1089: Characterization of the immune response to lymph mode metastases
Authors: Jones Dennis , Padera Timothy
Published: August 2019

Metastasis remains the principal cause of cancer mortality. Lymph nodes are the most common site of solid tumor metastases and lymph node status is the most significant prognostic indicator for patient outcome in human solid cancer. Although antitumor T cells are generated in lymph nodes, metastatic cancer cells are able to grow in this seemingly hostile microenvironment. Further, we recently found that lymph nodes are a portal for metastatic progression to distant organs. The challenge is how to treat cancer cells that have spread to lymph nodes or distant organs in order to prevent their growth and ideally eradicate them from the body. Most cancer therapies are developed against the primary tumor growing in its native microenvironment. Our previous work has shown that classical sprouting angiogenesis (as observed in the primary tumor) does not occur in metastatic lymph nodes and is therefore not necessary for metastatic outgrowth. Here, our preliminary data show that cancer cells remodel the blood endothelium in metastatic lymph nodes. Although many blood vessels lack smooth muscle cell coverage, intravenous lectin perfusion reveals a functional blood vasculature within metastatic lymph nodes. However, we measured decreased expression of adhesion molecules on the vasculature in metastatic lymph nodes. Adoptive transfer of lymphocytes revealed impaired lymphocyte migration into mice harboring metastatic lymph nodes compared to naïve lymph nodes. Together, these findings suggest that decreased vascular adhesion molecule expression results in lymphocyte exclusion and impaired host immunosurveillance, leading to the progression of lymph node metastases. Our ongoing work seeks to inform therapeutic strategies that will enhance functional antitumor T cell infiltration into metastatic tumors and prevent their spread to distant organs.

Citation Format: Dennis Jones, Timothy Padera. Characterization of the immune response to lymph mode metastases [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 1089.
Abstract 1091: Comparison of TCR repertoires between cancer tissues and lymph nodes in colorectal cancer patients
Authors: Kiyotani Kazuma , Matsuda Tatsuo , Miyauchi Eisaku , Hsu Yu-Wen , Nagayama Satoshi , Zewde Makda , Park Jae-Hyun , Kato Taigo , Harada Makiko , Suzuki Nobuaki , Nagano Hiroaki , Hazama Shoichi , Nakamura Yusuke
Published: August 2019

Tumor draining lymph nodes (TDLNs) are located in the routes of lymphatic drainage from a primary tumor and have the highest metastasis in various types of solid tumors. TDLNs are considered as a tissue to activate the antitumor immunity, where antigen-specific effector T cells are generated and released to the blood stream. These effector T cells then infiltrate into the tumor site and attack cancer cells. However, T cell receptor (TCR) repertoire in TDLNs has not been well characterized. We performed TCR sequencing of cancer tissues, corresponding normal mucosa tissues and a total of 203 regional lymph nodes, including 67 metastasis-positive lymph nodes from 23 colorectal cancer patients with lymph node metastasis. Metastasis-positive lymph nodes showed a significantly lower TCR diversity and shared TCR clones more frequently with primary tumor tissues compared to metastasis-negative TDLNs. Hierarchical clustering and principal component analyses also supported that TCR repertoires in metastasis-positive TDLNs were more similar to primary tumor tissues than metastasis-negative TDLNs. These finding suggest that cancer-reactive T cell clones might expand in the metastasis-positive TDLNs, although we have not tested their reactivity against cancer cells. We are now examining whether the T cells in TDLNs can recognized autologous cancer cells.

Citation Format: Kazuma Kiyotani, Tatsuo Matsuda, Eisaku Miyauchi, Yu-Wen Hsu, Satoshi Nagayama, Makda Zewde, Jae-Hyun Park, Taigo Kato, Makiko Harada, Nobuaki Suzuki, Hiroaki Nagano, Shoichi Hazama, Yusuke Nakamura. Comparison of TCR repertoires between cancer tissues and lymph nodes in colorectal cancer patients [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 1091.
Abstract 1103: The effects of neo-adjuvant chemotherapy on myeloid cells in high-grade serous ovarian cancer metastases
Authors: Heath Owen M. , Maniati Eleni , Belato Chiara , Gopinathan Ganga , Lecker Laura , Lakhani Anissa , Pegrum Colin , McDermott Jacqueline , Lockley Michelle , Barton Desmond P. , Balkwill Frances
Published: August 2019

Many tumours have abundant macrophage populations. Tumour-associated macrophages (TAMs) frequently have tumour promoting roles and are associated with poor clinical outcome. We hypothesise that targeting TAMs in high-grade serous ovarian cancer (HGSOC) may improve response to chemotherapy.

We have assessed the effects of chemotherapy on TAM populations in human HGSOC obtained pre- and post-chemotherapy as well as in murine HGSOC models harbouring a relevant mutational profile. We find that chemotherapy treatment decreases TAM density within tumour areas. Furthermore, TAMs expressing markers known to associate with disease progression were decreased following chemotherapy. In vivo and in vitro we have demonstrated an up-regulation of inflammasome activation and TLR signalling in live myeloid cells following chemotherapy and have shown that macrophages are killed by chemotherapy at clinically relevant drug concentrations. These observations suggest a mechanism for TAM depletion and highlight chemotherapy induced activation of innate immunity in HGSOC.

The majority of HGSOC patients respond well to first line chemotherapy but will relapse and succumb to treatment resistant disease. We have developed a murine model of HGSOC relapse after first-line chemotherapy, which has the potential to extend translational studies into this clinically important area. We have found that TAMs are re-established in tumours at relapse, suggesting a clinically defined window of opportunity to target TAMs in HGSOC following first-line chemotherapy.

Overall, our results provide a rationale for targeted re-programming of TAMs in HGSOC after chemotherapy.

Citation Format: Owen M. Heath, Eleni Maniati, Chiara Belato, Ganga Gopinathan, Laura Lecker, Anissa Lakhani, Colin Pegrum, Jacqueline McDermott, Michelle Lockley, Desmond P. Barton, Frances Balkwill. The effects of neo-adjuvant chemotherapy on myeloid cells in high-grade serous ovarian cancer metastases [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 1103.
Abstract 1108: Intestine-specific homeobox recruits p300/CBP-associated factor and bromodomain-containing protein 4 to promote epithelial-mesenchymal transition
Authors: Wang Li-Ting , Chiou Shyh-Shin , Hsu Shih-Hsien
Published: August 2019

The epithelial-mesenchymal transition is an important process in the cancer progression, but its occurrence and the regulatory mechanism are not fully understood. Intestine specific homeobox acted both as a proto-oncogene and upstream regulator of EMT markers, by which modulates tumorigenic initiation and progression in lung cancer cells. ISX acetylated by PCAF recruits bromodomain-containing protein 4, to initiate chromatin remodeling, and up-regulated EMT downstream regulators in tumors cells. Ectopic expression of ISX was shown to enhance TWIST1, Snail1 expression by recruiting BRD4 to enhance Pol II dependent transcription, lead in to remodeling of the tumor microenvironment. In NSCLC carcinoma, increased ISX expression was noted correlating with distinct clinical metastatic features and poor prognosis. These results suggest that theISX-BRD4 axis mediates EMT signaling and exerts significant regulatory effects on tumor initiation and metastasis.

Citation Format: Li-Ting Wang, Shyh-Shin Chiou, Shih-Hsien Hsu. Intestine-specific homeobox recruits p300/CBP-associated factor and bromodomain-containing protein 4 to promote epithelial-mesenchymal transition [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 1108.
Abstract 1113: Targeting PRPK function blocks colon cancer metastasis
Authors: Zykova Tatyana , Yao Ke , Shin Seung Ho , Roh Eunmiri , Ma Weiya , Bode Ann M. , Dong Zigang
Published: August 2019

The biological functions of the p53-related protein kinase (PRPK) remain unclear. We have previously demonstrated that PRPK is phosphorylated by the T-LAK cell-originated protein kinase (TOPK) and that phosphorylated PRPK (p-PRPK) promotes colon cancer metastasis. Here, we analyzed colon adenocarcinomas from 87 patients and found that higher expression levels of p-PRPK were associated with later stages of metastatic dissemination (stage III and IV) as compared with earlier stages (stages I and II). Indeed, levels of p-PRPK were higher in metastatic versus malignant human colon adenocarcinomas. Knocking down PRPK expression attenuated colorectal liver and lung metastasis of colon cancer cells in vivo. An in vitro kinase assay indicated that active PRPK does not phosphorylate p53 directly. We found that PRPK phosphorylates survivin, a regulator of colon cancer metastasis. PRPK phosphorylates survivin at Thr34, which is important for survivin stability. Taken together, our data strongly suggest that the PRPK signaling pathway promotes colon cancer metastasis by modulating survivin stability, and that PRPK could be a new prognostic marker for the survival of colon cancer patients. In addition, we identified an FDA-approved bacteriostatic antibiotic, fusidic acid sodium salt (fusidic acid or FA) as an inhibitor of PRPK, and show that FA combined with 5-fluorouracil (5-FU) inhibited PRPK activity and colon cancer metastasis to the lung in mice. We contend that the combination of FA with 5-FU could be an alternative therapeutic strategy to traditional chemotherapy for colon cancer patients with poor prognosis.

Citation Format: Tatyana Zykova, Ke Yao, Seung Ho Shin, Eunmiri Roh, Weiya Ma, Ann M. Bode, Zigang Dong. Targeting PRPK function blocks colon cancer metastasis [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 1113.
Abstract 1114: Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases
Authors: Loreth Desiree , Münsterberg Justine , Maire Cecile , Werner Stefan , Gandrass Monja , Bernreuther Christian , Kropidlowski Jolanthe , Westphal Manfred , Steurer Stefan , Lamszus Katrin , Glatzel Markus , Pantel Klaus , Wikman Harriet
Published: August 2019

Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related death with still a 5-year survival rate of <10%. Every second patient with advanced NSCLC will develop brain metastases and in 50% of those the brain is the only site of tumor relapse (oligo-metastasis). Cancer cells need to have specific properties to access and colonize the brain microenvironment leading to the question which metastatic factors foster brain metastasis formation. Different cell adhesion molecules (CAMs) have been shown to be involved in the process of metastasis in different tumor entities. The activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein of the immunoglobulin superfamily and involved in the maintenance of cell-cell contacts and has a possible role in the “homing” of metastatic cells in distant organs. ALCAM is also involved in the leukocyte transmigration across the blood-brain barrier. Here we therefore investigated the role of ALCAM in NSCLC brain metastases formation. Our studies on patient material revealed that the ALCAM protein expression in brain metastases (n=71) is significantly increased (50.7% positive, p=0.023) compared to the primary tumor (21.7% positive, n=47). Analysis of matched pairs of primary tumors and brain metastases confirmed this observation, as in 33% a de novo ALCAM expression in the matching brain metastases could be observed. Additionally, patients with a strong ALCAM expression in either their primary tumors or brain metastases showed a significantly shortened overall survival (p=0.017 and p=0.035 respectively). In vitro analysis of the effect of ALCAM knock out in the H460 lung cancer cell line showed no effect on proliferation, migration or colony-forming behavior. However, cell adhesion was severely hampered in the knock out cells. Currently in vivo mice experiments are running. Together these findings indicate an important role for ALCAM in brain metastasis formation.

Citation Format: Desiree Loreth, Justine Münsterberg, Cecile Maire, Stefan Werner, Monja Gandrass, Christian Bernreuther, Jolanthe Kropidlowski, Manfred Westphal, Stefan Steurer, Katrin Lamszus, Markus Glatzel, Klaus Pantel, Harriet Wikman. Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases [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 1114.
Abstract 1115: NANOG-AKT signaling increases metastatic capacity via stabilizes HDAC1 protein through Chfr inactivation
Authors: Cho Eunho
Published: August 2019

Histone deacetylases (HDACs), in particular HDAC1, play an important role in tumor development and progression by modifying histone and non-histone proteins. Overexpression of HDAC1, as a cancer marker associated with poor prognosis. Nevertheless, the regulatory molecular mechanisms of HDAC1 expression remain largely unknown. Previously, we validated the correlation between the key pluripotency transcription factor NANOG and HDAC1 in immune-edited tumor cells by identifying that NANOG upregulates HDAC1 expression through its promotor occupancy. In this study, we further elucidated the mechanism that Nanog could regulate the Hdac1 protein level, as well as transcriptional level. Interestingly, we found that Nanog induces phosphorylation of an E3 ubiquitin ligase Chfr through Akt signaling, which results in the inactivation of Chfr. This decreases not only ubiquitinase activity but proteasomal degradation on Hdac1. Furthermore, accumulated Hdac1 on its own gives rise to metastatic capacity in cancer cells. Moreover, inhibition of Akt signaling is significantly effective at reversing HDAC1 mediated metastatic capacity in refractory cancer types. Taken together, we suggest a mechanistic rationale for the application of Akt inhibitors in HDAC1+ cancer treatment.

Citation Format: Eunho Cho. NANOG-AKT signaling increases metastatic capacity via stabilizes HDAC1 protein through Chfr inactivation [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 1115.
Abstract 1119: S100A4 alters mitochondrial metabolism to promote invasion and metastasis of non-small cell lung cancer cells through upregulation of NDUFS2
Authors: Liu Lili , Qi Lei , Knifley Teresa , Piecoro Dava W. , Rychahou Piotr , Liu Jinpeng , Mitov Mihail I. , Martin Jeremiah , Wang Chi , Wu Jianrong , Weiss Heidi L. , Butterfield D. Allan , Evers B. Mark , O'Connor Kathleen L. , Chen Min
Published: August 2019

It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which the metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we find that S100A4, a major metastasis-promoting protein, alters metabolic plasticity to drive tumor invasion and metastasis. Investigating how S100A4 regulates metabolism, we find that depletion of S100A4 decreases oxygen consumption rate, mitochondrial activity, and ATP production, and shifts cell metabolism to be more glycolytically active. We further identify that the 49 KD mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types (cBioPortal). Importantly, we find that knockdown of NDUFS2 inhibits mitochondrial complex I activity, reduces cellular ATP level, and decreases the invasive capacity in three-dimensional (3D) growth, thus mimicking the biological effect resulting from S100A4 knockdown in A549 cells. Likewise, silencing S100A4 and NDUFS2 dramatically decreases the rate of metastasis as well as tumor growth in vivo. Finally, we provide evidence that cells with S100A4 and NDUFS2 depletion shift metabolism toward glycolysis through upregulating hexokinases expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Together, our findings uncover a novel function of S100A4 in mitochondrial metabolism and highlight the importance of NDUFS2 in regulating the plasticity of mitochondrial metabolism by S100A4 to promote the invasion and metastasis in lung cancer.

Citation Format: Lili Liu, Lei Qi, Teresa Knifley, Dava W. Piecoro, Piotr Rychahou, Jinpeng Liu, Mihail I. Mitov, Jeremiah Martin, Chi Wang, Jianrong Wu, Heidi L. Weiss, D. Allan Butterfield, B. Mark Evers, Kathleen L. O'Connor, Min Chen. S100A4 alters mitochondrial metabolism to promote invasion and metastasis of non-small cell lung cancer cells through upregulation of NDUFS2 [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 1119.
Abstract 1123: Bcl-xL promotes metastasis via a novel nuclear function
Authors: Zhang Tiantian , Li Sha , Na Joseph , Chi Soyoung , Zhang George , Du Yi-Chieh Nancy
Published: August 2019

Cancer cells often evade apoptosis through upregulation of anti-apoptotic proteins and/or downregulation of pro-apoptotic proteins. Bcl-xL, a negative regulator of mitochondria-mediated apoptosis, is frequently overexpressed in cancer cells. Bcl-xL has long been known for its function in regulating apoptosis during embryonic development and in pathological conditions. Any role that Bcl-xL might play in tumor metastasis has been ascribed to its anti-apoptotic function; i.e. Bcl-xL may increase metastasis by lending survival advantage to the tumor cells during the course of metastasis. However, we have demonstrated that Bcl-xL’s metastatic function is independent of its canonical anti-apoptotic activity and instead requires a novel nuclear function in cancer. We found that Bcl-xL promotes migration, even when cells are defective in mitochondria-mediated apoptosis. Consistently, Bcl-xL mutants lacking anti-apoptotic activity can still promote migration, induce epithelial-mesenchymal transition (EMT), upregulate TGFβ, and increase migration and invasion of pancreatic neuroendocrine tumor cells and breast cancer cells. Importantly, our data demonstrated that forcible localization of Bcl-xL outside the nucleus impairs its metastatic function.

As a mitochondrial membrane protein in healthy cells, Bcl-xL’s transmembrane domain prevents its nuclear import. This suggests that Bcl-xL enters the nucleus by an active nuclear transport. We hypothesized that unique transporters likely exist so as to transport this normally mitochondria membrane-bound Bcl-xL protein to the nucleus. By using immunoprecipitation in combination with mass spectrometry, we identified a novel interacting partner of Bcl-xL. We are investigating whether this protein transports Bcl-xL into the nucleus to promote metastasis.

Citation Format: Tiantian Zhang, Sha Li, Joseph Na, Soyoung Chi, George Zhang, Yi-Chieh Nancy Du. Bcl-xL promotes metastasis via a novel nuclear function [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 1123.
Abstract 1126: Association of CXCR4 expression with lymph node metastasis in distal cholangiocarcinoma
Authors: Yagawa Yohsuke , Aruga Atsushi , Takeshita Nobuhiro , Higuchi Ryota , Yazawa Takehisa , Tanigawa Keishi , Yamamoto Masakazu
Published: August 2019

BACKGROUND: The standard treatment of resectable distal bile duct cancer is pancreatoduodenectomy. However, prognosis is still poor because of a high rate of lymph node (LN) metastasis, underlining the need to further clarify the mechanism of LN metastasis. Recently accumulated evidence has shown that cancer cells can take advantage of the homeostatic mechanism of lymphocyte homing to metastasize to LNs. The CXCL12-CXCR4 axis modulates lymphocyte homing by inducing continuous migration of dendritic cells and T cells that are expressing chemokine receptor CXCR4 to LNs where chemokine CXCL12 is homeostatically secreted.

OBJECTIVE: The aim of this study is to elucidate whether distal bile duct cancer cells express CXCR4 and investigate a possible association with LN metastasis.

METHODS: This study enrolled 32 patients with resectable distal bile duct cancer including 19 patients with LN metastasis and 13 without. All patients underwent pancreatoduodenectomy at our department between 2002 and 2008 and have been followed up (Median observation period: 50.5 (4-184) months). Histopathologically, 27 cases were tubular adenocarcinoma, and 5 cases were other types of cancer. Immunostaining with resected specimens was undertaken to examine the expression of CXCR4 in cancer cells and determine whether its expression level is associated with lymph node metastasis. When 10% and more of tumor cells were stained, specimens were diagnosed as positive for CXCR4. The result of staining level was visually assessed.

RESULTS: Cancer cells were stained heterogeneously by antibody for CXCR4. Normal cells of the bile duct were not stained, while damaged or desquamated cells were stained. With regard to the staining pattern, the cytoplasm and membrane were stained but the nucleus was not stained. 19 out of 32 cases were positive for CXCR4 expression (59.4%). 15 of 19 cases with LN metastasis were positive whereas 4 of 13 cases without LN metastasis were positive (p=0.0107). CXCR4 expression on cancer cells increased with advancing cancer cell undifferentiation in tubular adenocarcinoma; cancer cells were positive for CXCR4 in 0 of 5 (0%), 6 of 16 (37.5%), and 6 of 6 (100%) cases in tubular 1, tubular 2, and tubular 3 adenocarcinoma, respectively.

CONCLUSIONS: Our results indicated that the expression of CXCR4 in cancer cells may be associated with LN metastasis in distal bile duct cancer.

Citation Format: Yohsuke Yagawa, Atsushi Aruga, Nobuhiro Takeshita, Ryota Higuchi, Takehisa Yazawa, Keishi Tanigawa, Masakazu Yamamoto. Association of CXCR4 expression with lymph node metastasis in distal cholangiocarcinoma [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 1126.
Abstract 1143: The circulating tumor cell (CTC) microenvironment visualized by color coded imaging
Authors: Suetsugu Atsushi , Satake Tomoyuki , Nakamura Miki , Shimizu Masahito , Saji Shigetoyo , Moriwaki Hisataka , Hoffman Robert M.
Published: August 2019

Background/Aim: An important question is whether cancer cells circulate with stromal cells. A color-coded mouse model of metastatic lymphoma was utilized to investigate the microenvironment of CTC clusters using fluorescent-protein imaging.

Materials and Methods: EL-4 mouse lymphoma cells expressing red fluorescent protein (RFP) were injected into the spleen of transgenic C57BL6-green fluorescent protein (GFP) mice. The cellular composition of CTC clusters both in heart blood and portal blood was imaged with confocal microscopy.

Results: CTC clusters comprised 8.8% of CTCs determined by color-coded imaging. Heterotypic CTC clusters containing other types of cells were distinguishable from homotypic CTCs. Heterotypic CTC clusters comprising cancer cells and fibroblasts were more rare than homotypic ones. Heterotypic CTC clusters with fibroblasts were observed only in portal blood, not in heart blood.

Conclusion: CTCs can form clusters, which may contain fibroblast or other stromal cells that may play a role in promoting CTC metastasis.

Citation Format: Atsushi Suetsugu, Tomoyuki Satake, Miki Nakamura, Masahito Shimizu, Shigetoyo Saji, Hisataka Moriwaki, Robert M. Hoffman. The circulating tumor cell (CTC) microenvironment visualized by color coded imaging [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 1143.