bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4822: The 3'UTR of CAPZA1 functions separately and oppositely to its host gene in ESCC
Authors: Li Dan , Kang Nan , Li Zhangfu , Zhan Qimin
Published: September 2020

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and highly prevalent in China. According to our previous study on whole-genome sequencing (WGS) and whole-exome sequencing (WES) of ESCC, we identified that CAPZA1 displayed copy number losses in the ESCC sequencing cohort. CAPZA1, which modulates the dynamic assembly of actin filament and cell motility, has been reported dysregulated expression in neuroblastoma, gastric cancer, liver cancer and breast cancer. However, the functional role and the underlying mechanism of CAPZA1 in ESCC have not been elucidated. Here, we identify the expression of CAPZA1 significantly decreased in cancer tissues than in normal tissues by immunohistochemical assay. Through gain and loss of function approaches, we found that CAPZA1 suppressed the proliferation, migration and invasion of ESCC cells in vivo and in vitro. Moreover, we found that CAPZA1 promoted the expression of E-cadherin, while decreased the expression of Vimentin and Twist. Intriguingly, we demonstrated that the 3'UTR of CAPZA1 could function as an independent RNA molecule separately with a full length of 1487nt by RACE and Northern-blot assays. Furthermore, the RNA in situ hybridization assay was employed to validate that 3'UTR expression levels were significantly higher in cancer tissues than in normal tissues. Additionally, the 3'UTR promoted cell migratory and invasive capabilities, which is oppositely to its host gene CAPZA1. The 3'UTR performed oncogenic role by enhancing the combination of IGF2BPs proteins to c-myc mRNA and promoted the translation of c-myc. These findings offer a new dimension to the crosstalk between 3'UTR and its host gene, which may open new ways towards additional therapeutic strategies in the treatment of ESCC.

Citation Format: Dan Li, Nan Kang, Zhangfu Li, Qimin Zhan. The 3'UTR of CAPZA1 functions separately and oppositely to its host gene in ESCC [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 4822.
Abstract 4825: Super enhancer-associated lincRNA BCSL1 promotes the development of breast cancer
Authors: Yuan Xiaoqing , Peng Li , Chen Jianing , Cui Xiuying , Liu Yujie , Song Erwei
Published: September 2020

Long Intergenic non-coding RNAs (lincRNAs) expressed in tissue-specificity model are involved in a variety of tumor biological processes. Super enhancers (SEs) have been shown to drive the expression of genes including lincRNAs that control cell identity and the development of cancer. However, the role of super-enhancer-driven lincRNAs in luminal type breast cancer (BC) is poorly understood. Here we identified a lincRNA, BCSL1, was uniquely up-regulated in BC tumor tissues and cell lines. ChIP-sequencing and Hi-C analysis manifested that BCSL1 was directly regulated by the luminal subtype specific SEs in BC cells. Nuclear plasma separation assay and FISH test showed that BCSL1 slightly more located in the cytoplasm than the nucleus of MCF7 cells. Knocking down of BCSL1 significantly inhibited the proliferation, colony formation and apoptosis of BC cells, and the cell cycle switching from G1 stage to S stage. Overexpression of BCSL1 significantly increased their proliferation, colony formation and apoptosis. Mechanistically, RNA pulldown, Mass spectrometry and RNA-sequencing indicated BCSL1 may be involved in the development of luminal BC by participating in apoptosis-related pathways.In summary, we identified a lincRNA, BCSL1, that is uniquely increased in breast cancer. Increased expression of BCSL1 promotes the proliferation, colony formation and apoptosis of luminal breast cancer cells. BCSL1 might be developed as a biomarker of luminal breast cancer patients. This work was supported by the National Natural Science Foundation of China (81803636 and 81972658), the Natural Science Foundation of Guangdong Province (2018A0303130329).

Citation Format: Xiaoqing Yuan, Li Peng, Jianing Chen, Xiuying Cui, Yujie Liu, Erwei Song. Super enhancer-associated lincRNA BCSL1 promotes the development of 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 4825.
Abstract 4834: Circulating microRNA-762 promotes colorectal cancer proliferation and invasion by upregulating the Wnt-1/β-catenin pathway
Authors: Chen Ying-Yin , Lai Peng-Sheng , Chen Chung-Yu , Liao Hui-Fen
Published: September 2020

Background: A number of microRNAs (miRNAs) have been demonstrated to be associated with the diagnosis, progression and prognosis of colorectal cancer (CRC). However, the function of miRNA-762 (miR-762) in CRC remains unclear, and the molecular mechanisms underlying the effects of miR-762 in CRC require further investigation.

Methods: The circulating miRNAs from BALB/c mice with CRC CT26 cell implantation were assayed by microarray. Then, miR-762 mimic and inhibitor were transfected to CT26 cells for analysis of cell viability, invasion, and epithelial-mesenchymal transition (EMT), cell cycle, and regulatory molecule expression. Human subjects were included for comparison the circulating miR-762 levels in CRC patients and control donors, as well as the patients with and without distant metastasis.

Results: The screening for miRNA levels in mice with CRC cell implantation indicated that plasma miR-762 was upregulated. Transfection of miR-762 mimic to CT26 cells increased cell viability, invasion, and EMT, whereas transfection of miR-762 inhibitor decreased the above abilities. Western blot analysis showed that miR-762 mimic transfection upregulated the expression of Wnt-1 and β-catenin, as well as increased the nuclear translocation of β-catenin. Further analysis showed that serum miR-762 levels in CRC patients were higher than in control donors. Among the CRC patients (n = 20), six patients with distant metastasis showed higher serum miR-762 levels than the patients without distant metastasis.

Conclusions: Circulating miR-762 could promote CRC disease development and progression through the Wnt/β-catenin signaling. miR-762 might be used as a biomarker for CRC diagnosis and targeted therapy.

Citation Format: Ying-Yin Chen, Peng-Sheng Lai, Chung-Yu Chen, Hui-Fen Liao. Circulating microRNA-762 promotes colorectal cancer proliferation and invasion by upregulating the Wnt-1/β-catenin pathway [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 4834.
Abstract 4841: Functional genome-wide screening identifies triple-negative breast cancer cell cycle-dependent vulnerability driven by genomic gain of death effector domain-containing protein
Authors: Ni Yingjia , Schmidt Keon R. , Werner Barnes A. , Koenig Jenna K. , Guldner Ian H. , Schnepp Patricia M. , Tan Xuejuan , Jiang Lan , Host Misha , Sun Longhua , Howe Erin N. , Wu Junmin , Littlepage Laurie E. , Nakshatri Harikrishna , Zhang Siyuan
Published: September 2020

Triple-negative breast cancer (TNBC) persists as a clinical challenge, with targeted therapeutic options remaining limited for the subtype due to few targetable tumor-specific vulnerabilities. While basal-like TNBC tumors express epidermal growth factor receptor (EGFR), the target of multiple clinically efficacious therapies, TNBC showed little to no response to anti-EGFR therapy in clinical trials. Using a genome-wide synthetic lethal pooled barcoded shRNA screen, we identified Death Effector Domain-containing protein (DEDD), upregulated in over 60% of TNBC tumors, as a driver of mitogen-independent G1/S cell cycle transition in TNBC under EGFR inhibition. This cell cycle role of DEDD depends on cytoplasmic localization and is independent of nuclear DEDD, which maintains roles in apoptosis. Cytosolic DEDD promotes G1/S transition through direct interaction with Rb family proteins, encouraging their degradation. Further, DEDD interacts with heat shock 71kDa protein 8 (HSC70) to augment cyclin D1 expression and drive transition to S phase. These interactions of overexpressed cytosolic DEDD with the G1/S checkpoint mechanism present a vulnerability of TNBC to cell cycle inhibition. Frequent Rb-loss in TNBC has prevented clinical testing of CDK4/6 inhibitors in TNBC, however. Yet, we found TNBC with DEDD overexpression to be sensitive to a combination of CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo, independent of Rb status. This novel vulnerability presented by DEDD overexpression in TNBC suggests promising therapeutic potential for CDK4/6 inhibitor use in combinatorial regimens.

Citation Format: Yingjia Ni, Keon R. Schmidt, Barnes A. Werner, Jenna K. Koenig, Ian H. Guldner, Patricia M. Schnepp, Xuejuan Tan, Lan Jiang, Misha Host, Longhua Sun, Erin N. Howe, Junmin Wu, Laurie E. Littlepage, Harikrishna Nakshatri, Siyuan Zhang. Functional genome-wide screening identifies triple-negative breast cancer cell cycle-dependent vulnerability driven by genomic gain of death effector domain-containing protein [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 4841.
Abstract 4858: Oncogenic HIPPO-YAP1: in vivo target validation of YAP1 in malignant mesothelioma
Authors: Calvet Loreley , Santos Odette Dos , Jean-Baptiste Véronique , Spanakis Emmanuel , Ruffin Yvette , Sanchez Isabelle , Mestadier Jessica , Soubigou Stéphane , Feteanu Sylvie , Picard Pascale , Boumaya Chagri , Pollard Jack , Dib Colette , Sidhu Sukhvinder , Debussche Laurent , Valtingojer Iris
Published: September 2020

Yes associated protein YAP1 (YAP1) is a cofactor of gene transcription and exerts its action through association with transcription factors from the TEAD family. Activity of YAP1-TEAD is tightly regulated by the HIPPO pathway and upon activation the expression of pro-survival and anti-apoptotic genes is induced. The HIPPO pathway consists of a series of Ser/Thr kinases, which lead to the phosphorylation of YAP1, and hence to its inactivation by either sequestration in the cytoplasm, or degradation. HIPPO pathway deletions and YAP1 activation are particularly prevalent (> 60 %) in patients with malignant pleural mesothelioma. We use a malignant mesothelioma tumor cell line with a HIPPO deleted genetic background (MSTO-211H, LATS1 loss) to demonstrate, that downregulation of YAP1 leads to the inhibition of YAP1-dependent gene expression, the induction of apoptosis and the inhibition of cell growth in vitro. When these cell lines are grown as an in vivo subcutaneous xenograft in mice, downregulation of YAP1 leads to the inhibition of YAP1-dependent gene expression and eventually results in the regression of established tumors. This effect is specific to YAP1 activated mesothelioma models and is not observed in the HIPPO pathway independent HCT116 colon cancer model. We show here, that YAP1 activity alone is required for tumor maintenance in vivo in the context of HIPPO pathway genetic alterations, and thus confirm the relevance of this target for a drug discovery approach.

Citation Format: Loreley Calvet, Odette Dos Santos, Véronique Jean-Baptiste, Emmanuel Spanakis, Yvette Ruffin, Isabelle Sanchez, Jessica Mestadier, Stéphane Soubigou, Sylvie Feteanu, Pascale Picard, Chagri Boumaya, Jack Pollard, Colette Dib, Sukhvinder Sidhu, Laurent Debussche, Iris Valtingojer. Oncogenic HIPPO-YAP1: in vivo target validation of YAP1 in malignant mesothelioma [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 4858.
Abstract 4934: DCBLD2 mediated cisplatin-induced epithelial-to-mesenchymal transition and metastasis in non-small cell lung cancer
Authors: Chen Xiaosu , Lv Yajing , Huang Chongbiao , Xiang Rong
Published: September 2020

In clinical practice, we observed that many patients with solid tumors were diagnosed with early metastasis after chemotherapy, consistent with a large number of preclinical and clinical repots. However, the mechanism how chemotherapy induced tumor metastasis was seldomly reported. In this work, we found that cisplatin promoted epithelial-to-mesenchymal transition (EMT), cell motility and metastasis in non-small cell lung cancer (NSCLC). Moreover, we validated DCBLD2 was a key mediator in cisplatin-induced metastasis in NSCLC through an in vivo, genome-scale CRISPR/Cas9 activation screening. DCBLD2 was abnormally high-expressed in patients with NSCLC, in accordance with the analysis of TCGA database. The expression level of DCBLD2 was negatively correlated with the prognosis of NSCLC patients. Mechanically, DCBLD2 promoted EMT, cell migration in NSCLC cells and metastasis in a mouse xenograft models by binding to cytoskeletal proteins. In addition, we found that cisplatin played an essential role in promoting DCBLD2 expression. The ERK signaling pathway could be activated by cisplatin with phosphorylated, leading to the transcription factor FOS binding to the promoter region of DCBLD2, upregulating the expression of DCBLD2. In conclude, DCBLD2 was a contextual determinant of chemotherapy and metastatic in NSCLC and a potential target for anti-metastasis drugs.

Citation Format: Xiaosu Chen, Yajing Lv, Chongbiao Huang, Rong Xiang. DCBLD2 mediated cisplatin-induced epithelial-to-mesenchymal transition and metastasis in non-small cell lung 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 4934.
Abstract 4959: Targeting of the eukaryotic translation initiation factor 4A against breast cancer stemness
Authors: Sridharan Sangita , Robeson Megan , Bastihalli Diwakar Tukaramrao , Howard Cory , Subramaniyan Boopathi , Tilley Augustus , Raman Dayanidhi
Published: September 2020

Chemoresistance is a clinically significant issue in triple-negative breast cancer (TNBC) patients, leading to minimal residual disease. The surviving breast cancer stem cells (BCSCs) undergo multilineage differentiation and repopulate a more aggressive primary tumor leading to metastasis and subsequent mortality. Not only are BCSCs innately chemo/radioresistant but also contribute to therapy failure through interconversion between BCSCs and bulk tumor cells. Therefore, it is imperative to co-target BCSCs and bulk tumor cells simultaneously. Identifying novel molecular targets is key to eliminate both bulk tumor cells and BCSCs. The eukaryotic protein translation initiation machinery, the eIF4F complex, is a convergence point for many oncogenic signaling pathways. This complex plays a vital role in translation of many oncogenic mRNAs implicated in cell cycle, tumor progression, chemoresistance and metastasis. We have previously demonstrated that the mRNA helicase eIF4A1, of the eIF4F complex, might serve as a vulnerable node to kill TNBC cells. In the current study, we hypothesized that targeting of eIF4A1 would reduce/eliminate both bulk tumor cells and BCSCs and overcome chemoresistance. In order to evaluate the effects of targeting eIF4A1 in BCSCs, we isolated BCSCs based on aldehyde dehydrogenase activity and CD44 expression by Fluorescence Activated Cell Sorting and characterized for stemness. Through pharmacological targeting (Rocaglamide A) and genetic ablation (CRISPR-Cas9) of eIF4A1, we demonstrated that there was a statistically significant reduction in the self-renewal ability indicated by the reduction in primary and secondary mammosphere formation efficiency (MFE) of BCSCs, levels of the pluripotency transcription factors (SOX2, OCT4 and NANOG), expression of drug transporters (ABCG2, ABCB1 and ABCC1) and increased induction of apoptotic cell death. Overall, we demonstrated that targeting eIF4A1 is effective in eliminating BCSCs through reduction in the levels of stemness factors and drug transporters. This sets up a stage to employ small molecule inhibitors against eIF4A1 in co-targeting strategic therapies.

Citation Format: Sangita Sridharan, Megan Robeson, Diwakar Tukaramrao Bastihalli, Cory Howard, Boopathi Subramaniyan, Augustus Tilley, Dayanidhi Raman. Targeting of the eukaryotic translation initiation factor 4A against breast cancer stemness [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 4959.
Abstract 5017: EphA2 is a clinically relevant target for breast cancer bone metastatic disease
Authors: Brantley-Sieders Dana M.
Published: September 2020

While advances in molecular targeted therapies have increased survival for breast cancer patients, outcomes for patients with metastatic disease remain poor. EphA2 receptor tyrosine kinase (RTK) is highly expressed in breast tumor cells across multiple molecular subtypes and correlates with poor patient prognosis. As metastasis of breast cancer to bone is a major cause of morbidity and mortality in patients, we investigated the role of EphA2 in this clinically relevant phenomenon. Analysis of human patient gene expression datasets and human breast-to-bone metastasis samples revealed high level ephA2 expression in bone lesions relative to other sites of metastatic dissemination, and EphA2 positive staining on breast tumor cells in bone metastatic lesions, respectively. EphA2 loss of function in tumor cells reduced tumor-induced osteolysis in two independent intratibial tumor engraftment models (4T1 and MDA-MB-231) as well as in an intracardiac experimental metastasis model (MDA-MB-231) in vivo. We observed a significant reduction in osteoclasts in animals harboring EphA2 loss of function tumors relative to control tumors. Further in vitro analyses revealed that blocking EphA2 function resulted in defective precursor maturation into functional osteoclasts through an interleukin-6 (IL-6) dependent mechanism. Pharmacologic inhibition of EphA2 decreased breast tumor induced osteolytic disease severity in vivo. Together, these data provide pre-clinical validation of EphA2 as a clinically relevant molecular target for breast cancer bone metastatic, osteolytic disease, warranting further investigation of molecular mechanisms that link EphA2 to tumor-induced osteolysis and eventual clinical development.

Citation Format: Dana M. Brantley-Sieders. EphA2 is a clinically relevant target for breast cancer bone metastatic disease [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 5017.
Abstract 5038: Limited inhibition of multiple nodes in a driver network blocks metastasis
Authors: Yesilkanal Ali Ekrem , Yang Dongbo , Tiwari Payal , Sabino Alan U. , Lee Jiyoung , Xie Xiao-He , Sun Siqi , Dann Christopher , Steinberg Ethan , Stuhlmiller Timothy , Frankenberger Casey , Goldsmith Elizabeth , Johnson Gary L. , Ramos Alexandre F. , Rosner Marsha
Published: September 2020

High-dose, single-drug treatment is unsuccessful at suppressing metastasis due to cellular heterogeneity and feedback activation. Here we demonstrate that targeting driver network signaling capacity by limited inhibition at multiple nodes is a more effective anti-metastatic strategy. A physiological metastasis suppressor, Raf Kinase Inhibitory Protein, decreases the activity of multiple kinases in the stress-regulated MAP kinase (MAPK) network and reduces the output to metastatic transcription factor BACH1 that upregulates motility-related genes. This effect is mimicked by a low-dose four-drug cocktail (4D-MAPKi) that blocks metastatic colonization in mouse breast cancer models and increases survival. Drug treatment and network modeling show that 1) multi-kinase inhibition is required to suppress signaling output across cells with different MAPK network topologies, and 2) restricting inhibition of individual kinases prevents threshold activation of other compensatory kinase networks. This low-dose multi-drug approach to decrease signaling capacity of a driver network represents a transformative, clinically-relevant strategy for anti-metastatic treatment.

Citation Format: Ali Ekrem Yesilkanal, Dongbo Yang, Payal Tiwari, Alan U. Sabino, Jiyoung Lee, Xiao-He Xie, Siqi Sun, Christopher Dann, Ethan Steinberg, Timothy Stuhlmiller, Casey Frankenberger, Elizabeth Goldsmith, Gary L. Johnson, Alexandre F. Ramos, Marsha Rosner. Limited inhibition of multiple nodes in a driver network blocks 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 5038.
Abstract 5041: Inhibition of RUNX-CBFβ binding reduces RSPO3 expression and EMT features in breast cancer cells
Authors: Felcher Carla M. , Tocci Johanna M. , Sola Martin E. Garcia , Bushweller John H. , Castilla Lucio H. , Kordon Edith C.
Published: September 2020

We have recently identified R-spondin3 (RSPO3) as a novel key modulator of breast cancer development that may become a potential target for treatment of the basal subtype, which lacks efficient therapeutic options to date. RSPO3 is expressed in the basal stem cell-enriched compartment of mouse mammary glands whereas it is absent in the mature luminal cells. RSPO3 knockdown in mouse mammary tumor cells reduces canonical Wnt signaling pathway, epithelial-to-mesenchymal transition (EMT)-like features, migration capacity, and tumor formation in vivo. To evaluate the relevance of RSPO3 in human breast cancer, we analyzed samples from 75 patients with ductal infiltrating breast carcinomas, where we found a high prevalence of positive immunoreactivity for RSPO3 (70%). Expression data of The Cancer Genome Atlas Network from 1985 human breast carcinoma samples showed that RSPO3 is primarily expressed in Claudin-low breast cancer subtype, which is characterized for having stem cells features. In culture assays revealed that knockdown of RSPO3 expression reduced SOX2 expression and impaired mammary tumor cell ability to form mammospheres. Previous reports suggested the potential involvement of RUNX1-CBFβ on RSPO3 transcription in mammary tumor cells. Therefore, we evaluated if pharmacologic inhibition of RUNX1 activity would reduce RSPO3 expression and activity in the cancer stem cell compartment. Treatment of breast cancer cells with AI-10-104, a small molecule that inhibits CBFβ-RUNX interaction, reduced RSPO3 mRNA and protein levels, and reduced the migration ability of MDA-MB231 breast cancer cells, ability that was recovered upon treatment with recombinant RSPO3. In addition, AI-10-104 inhibited the expression of canonical Wnt pathway targets, EMT and stem-cell markers. Therefore, our results demonstrate that inhibition of RUNX-CBFβ transcriptional activity leads to a down-regulation of RSPO3 expression, constituting a new molecular target for therapeutic modulation in breast cancer treatment.

Citation Format: Carla M. Felcher, Johanna M. Tocci, Martin E. Garcia Sola, John H. Bushweller, Lucio H. Castilla, Edith C. Kordon. Inhibition of RUNX-CBFβ binding reduces RSPO3 expression and EMT features in breast cancer cells [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 5041.
Abstract 5059: Breast cancer patient-derived xenograft models for pre- and co-clinical investigation of immune based therapies
Authors: Wulf-Goldenberg Annika , Kiver Verena , Stecklum Maria , Brzezicha Bernadette , Jurmeister Philipp , Schweiger Caroline , Blohmer Jens-Uwe , Keilholz Ulrich , Hoffmann Jens
Published: September 2020

Breast cancer is a highly heterogeneous disease, with a heterogeneous prognosis. Patient-derived xenograft models (PDX) can reflect this heterogeneity and can be used to support the development of novel therapeutic strategies against breast cancer. Preclinical experiments with larger cohorts of breast cancer PDX can be used to model a clinical phase II study with new drugs or drug combinations. The predictive value of these preclinical trials is currently evaluated in co-clinical trials, where treatment effects are compared between patients and their corresponding PDX. We present results from our translational pre- and co-clinical studies in breast cancer immune therapy.

As part of the EFRE-POP Project, we established PDX by collecting breast cancer tissue samples from surgery and engrafting them on immunodeficient mice. The tissues were obtained from patients with disease progression after chemotherapy with three to four drugs. PDXs have been successfully engrafted, phenotypically characterized and drug-screened. The PDXs were tested for response to the standard of care therapies. The staining for estrogen/progesterone/androgen/Her2 receptors, Ki-67, and CK5/6 of the original tumor and the PDX were comparable. The patient tumors and the breast cancer PDX models were further analyzed for the expression of PD-L1. We identified four patients that had received PD-1 or PD-L1 antibodies after a sample (used to establish a corresponding PDX) was obtained.

To evaluate the response of our preclinical PDX models to immune checkpoint inhibitors, we engrafted mice with a human immune system either by transplanting allogeneic hematopoietic stem cells (HSC) or allogenic PBMC's from healthy donors. Besides, we were able to obtain PBMC's from the patients to set up an autologous model. The patient-derived tumors were transplanted to these humanized mice and after successful engraftment treated with the PD-1 or PD-L1 antibodies corresponding with the clinical treatment of the patient.

In summary, humanized PDX of refractory tumors have been established and the first pre-clinical evaluation of response to immune therapy provides evidence that xenografts respond in a qualitatively similar manner as the patient. This suggests a translational relevance of the described pre-clinical models.

Citation Format: Annika Wulf-Goldenberg, Verena Kiver, Maria Stecklum, Bernadette Brzezicha, Philipp Jurmeister, Caroline Schweiger, Jens-Uwe Blohmer, Ulrich Keilholz, Jens Hoffmann. Breast cancer patient-derived xenograft models for pre- and co-clinical investigation of immune based therapies [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 5059.
Abstract 506: A bespoke screening platform to study mono(ADP-ribosylation)
Authors: Wigle Tim J. , Blackwell Danielle J. , Schenkel Laurie B. , Ren Yue , Church William D. , Desai Hetvi J. , Swinger Kerren K. , Santospago Andrew G. , Majer Christina R. , Lu Alvin Z. , Niepel Mario , Perl Nicholas R. , Vasbinder Melissa M. , Keilhack Heike , Kuntz Kevin W.
Published: September 2020

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are post-translational modifications deposited on multiple amino acids by PARP enzymes using nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate. While there are approved drugs and clinical trials on-going for inhibitors of the polyPARP enzymes that deposit poly(ADP-ribose) (specifically PARP1 and PARP2 inhibitors), monoPARP enzymes that deposit mono(ADP-ribose) are only recently gaining recognition for their role in cellular stress signaling, inflammation and cancer. However, there is a lack of chemical probes to study their function in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop screening assays to enable determination of potency and selectivity of inhibitors during the hit finding and lead optimization phases. The development of enzyme assays is complicated by the fact that the substrates for the majority of the monoPARPs are unknown, and even for those with identified substrates, it is uncertain how they engage their substrates. Here we describe the development of robust high-throughput biochemical and cellular monoPARP assays that overcome the lack of knowledge around the substrates and construction of a family-wide screening panel. We highlight derivatized microplates that activate the enzymes to self-MARylate in dissociation enhanced lanthanide fluorescence assays (DELFIA), antibodies that recognize MARylation in in-cell western (ICW) and immunofluorescence (IF) assays, and NAD+-competitive molecular probes that are used to develop in vitro time-resolved fluorescence resonance energy transfer (TR-FRET) and cellular NanoLuc bioluminescence resonance energy transfer (NanoBRET) probe displacement assays. Additionally, we employ several methods to characterize inhibitor binding kinetics. These assays have been used in high-throughput screening campaigns of up to 500,000 compounds, as well as in the development of potent and selective inhibitors of multiple monoPARP enzymes including RBN012759, a tool compound for PARP14 that inhibits in vitro with an IC50 of 3 nM and in cells using with an IC50 of 9 nM, and is 300-fold selective over all other PARP enzymes.

Citation Format: Tim J. Wigle, Danielle J. Blackwell, Laurie B. Schenkel, Yue Ren, William D. Church, Hetvi J. Desai, Kerren K. Swinger, Andrew G. Santospago, Christina R. Majer, Alvin Z. Lu, Mario Niepel, Nicholas R. Perl, Melissa M. Vasbinder, Heike Keilhack, Kevin W. Kuntz. A bespoke screening platform to study mono(ADP-ribosylation) [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 506.
Abstract 5070: LIN28A-mediated repression of Let-7a is involved in resistin-induced STAT3 upregulation in breast cancer cells and supports growth and stemness properties
Authors: Deshmukh Sachin Kumar , Srivastava Sanjeev K. , Zubair Haseeb , Singh Ajay P. , Singh Seema
Published: September 2020

Breast cancer (BC) is the most frequently diagnosed malignancy and continues to be the second leading cause of cancer-related death in women in the United States. Dysregulation of Let-7 family microRNAs is observed in BC and associated with disease pathogenesis. Previously, we reported a significant increase in serum levels of resistin in BC patients as compared to the healthy individuals. Moreover, this increase was significantly greater in BC patients of African American background as compared to those of European descent. We also demonstrated that treatment of BC cells with resistin promoted their growth, aggressiveness, stemness and chemoresistance by inducing the expression and activation of STAT3. Here we present data to show that resistin induces STAT3 expression in BC cells in a LIN28A and Let-7a-dependent manner. Treatment of BC cells with resistin led to the most significant decrease in the expression of Let-7a among other Let-7 family members. Furthermore, resistin treatment also induced the expression of LIN28A at both transcript and protein levels and its silencing abrogated resistin-mediated Let-7a suppression. Let-7a restoration or LIN28A silencing abolished the resistin-induced growth, clonogenicity and sphere-forming ability of BC cells. Restoration of Let-7a suppressed the expression of several genes associated with growth, survival and stemness. Ingenuity Pathway Analysis suggested STAT3 as a putative central node associated with Let-7a-mediated gene regulation. Immunoblot analysis confirmed that Let-7a restoration dramatically suppressed basal as well as resistin-induced STAT3 expression. STAT3 regulation by Let-7a was further confirmed 3'UTR-reporter assay. Taken together, our findings demonstrate a novel resistin/LIN28A/Let-7a/STAT3 signaling axis regulating the growth and stemness phenotypes of BC cells.

Citation Format: Sachin Kumar Deshmukh, Sanjeev K. Srivastava, Haseeb Zubair, Ajay P. Singh, Seema Singh. LIN28A-mediated repression of Let-7a is involved in resistin-induced STAT3 upregulation in breast cancer cells and supports growth and stemness properties [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 5070.
Abstract 508: A novel orally available Wnt-pathway inhibitor for the treatment of metastatic cancers
Authors: Zhou Kun , Cheong Jae Eun , Alix-Panabières Catherine , Sun Lijun , Zetter Bruce R.
Published: September 2020

We have developed a novel drug compound (OBD9) which blocks growth of metastatic cancer cells both in vitro and in vivo. We now identify OBD9 as an effective Wnt-signaling inhibitor targeting TNIK (TRAF2 And NCK Interacting Kinase) and we hypothesize that OBD9 represent a novel therapeutic option for patients with tumors that have an activated Wnt signaling pathway.

To find novel drug candidates that selectively inhibit metastatic tumor cell viability, a drug screen was firstly performed and results of the screen identified members of the FDA-approved benzimidazole methylcarbamate family (e.g. mebendazole (MBZ) and albendazole (ALB)) as potential therapies for metastatic cancer. Earlier work also supports a role for this chemical family in the potential treatment of multiple cancers, but progress has been stalled by their poor water solubility and poor bioavailability for systemic delivery to disseminated tumors. We therefore synthesized a novel compound (OBD9) containing the scaffold of MBZ coupled to an oxetane group to enhance aqueous solubility to 361μM. OBD9 demonstrates significant cytotoxicity toward a variety of cancer cell types including colon, lung, and prostate cancers (IC50: 0.9-2μM). In a mouse xenograft model using highly aggressive PCMLN3 prostate cancer cells, OBD9 at 30 mg/kg significantly repressed growth of established tumors with no visible toxicity. In a mouse xenograft model of human A549 lung cancer cell line, orally delivered OBD9 also dramatically inhibited the growth of established tumors at 30 or 90μM without noticeable toxicity.

Mechanistically, we find that OBD9 treatment significantly reduces TNIK levels as early as 4 hours at 1uM via an autophagy-dependent protein degradation pathway. TNIK functions as an activator of Wnt signaling pathway via phosphorylation of the beta-catenin/TCF4 complex that regulates Wnt downstream targets. We show that OBD9 treatment inhibits colon cancer cell growth and both qPCR and Western blot data suggest that Wnt signaling downstream targets, such as TCF4, AXIN2 and cMyc, are all significantly suppressed by OBD9 via the inhibition of TNIK. Overall, our in vitro and in vivo data suggest that OBD9 potentially represents a novel therapeutic option for multiple cancers including but not limit to colon, lung and prostate cancer.

Citation Format: Kun Zhou, Jae Eun Cheong, Catherine Alix-Panabières, Lijun Sun, Bruce R. Zetter. A novel orally available Wnt-pathway inhibitor for the treatment of metastatic cancers [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 508.
Abstract 5108: Investigation of how extracellular matrix composition affects pancreatic cancer chemoresistance through the use of biomimetic models
Authors: Xiao Weikun , Eun Chae-Young , Dekalb Charlene , Zhang Kexin , Hill Reginald
Published: September 2020

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal and malignant type of cancer with few effective treatments. Despite recent advancement in knowledge of cancer treatment, median survival of PDAC patients remains within 2 years, and less than 8% patients live beyond 5 years after diagnosis. The main factor that drives PDAC lethality is the development of chemoresistance in nearly all clinical PDACs. While intrinsic factors that facilitates chemoresistance have been studied extensively, few studies have illustrated how complex and unique microenvironment surrounding the PDAC tumor affects therapeutic responses. Moreover, a physiologically relevant co-culture model of PDAC and stromal cells remained challenging. To address this issue, we have developed a Matrigel-based, orthogonally tunable 3-dimensional (3D) culture system to culture established human PDAC cell lines, cancer-associated fibroblast (CAF) cells and mouse model-derived organoids. Individual matrix parameters can be customized to mimic actual tumor matrix composition and mechanical properties. Moreover, we have optimized a co-culture medium to maintain viability of both organoid tumor and cancer associated fibroblasts. Through our 3D culture system, our data suggests that matrix-induced exosome secretion directly facilitate gemcitabine resistance in PDAC cells. By directly increasing stiffness of our 3D culture platform, we have observed that PDAC cells potentially display more invasive phenotype. Moreover, in the co-culture systems, we have seen that CAF cells cultured in different stiffness substrates affect PDAC drug response in organoid cultures. Our investigation provides insight into how matrix composition and stiffness together affect therapeutic outcome in PDAC. We believe that specific therapeutic strategies aimed at targeting the matrix microenvironment will be instrumental in order to overcome matrix-mediated chemoresistance in PDAC.

Citation Format: Weikun Xiao, Chae-Young Eun, Charlene Dekalb, Kexin Zhang, Reginald Hill. Investigation of how extracellular matrix composition affects pancreatic cancer chemoresistance through the use of biomimetic models [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 5108.
Abstract 5112: Interaction between RHOA mutated gastric cancer cells and cancer associated fibroblasts promotes tumorigenesis
Authors: Jeon Daye , Choi Yuri , Lee Seon Jin , Choi Kyung Un , Park Do Youn , Kwon Chae Hwa
Published: September 2020

Background: We previously observed that RHOA-mutated gastric cancer has desmoplastic morphological feature. Although desmoplastic reaction is known to consist mainly of cancer associated fibroblasts (CAF), the relationship cancer cells with RHOA mutation and CAF have not been reported. Thus, in this study, we investigated the interaction between RHOA-mutated gastric cancer cells and CAF and its underlying mechanism.

Methods: We established stable gastric cell lines (AGS, MKN-45) expressing RHOA wild type (WT) of mutation (G17E, T37I, E40K, Y42C) using lentiviral vector and cocultured these cells with CAF using transwell. And proliferation and migration of RHOA-mutated gastric cancer cells were examined. To understand molecular mechanism of RHOA-mutated gastric cancer cells proliferation and migration, RNA-sequencing was conducted and the results were analyzed and validated.

Results: Cocultivation of gastric cancer cells expressing RHOA WT or mutation (G17E, T37I, E40K, Y42C) with CAF increased proliferation relative to cells expressing an empty vector (EV). In addition, migration of RHOA mutant cells, except for cells with RHOA WT and Y42C, was increased by CAF relative to EV cells. Genes related to IL-17, IL-15, and IGF-1 signaling pathways were differentially expressed between RHOA WT or mutant cells and EV cells, suggesting that proliferation and migration of RHOA mutant cells promoted by CAF through these pathways. Furthermore, we identified CXCL1 and IGFBP2 as mediator of interaction between RHOA mutated gastric cancer cells and CAF.

Conclusion: In summary, we found that gastric cancer cells with RHOA mutation have tumor-promoting activity by interaction with CAF. Thus, molecules for impairing their interactions could be potential therapeutic targets in gastric cancer patients with RHOA mutation.

Citation Format: Daye Jeon, Yuri Choi, Seon Jin Lee, Kyung Un Choi, Do Youn Park, Chae Hwa Kwon. Interaction between RHOA mutated gastric cancer cells and cancer associated fibroblasts promotes tumorigenesis [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 5112.
Abstract 5130: Proteomic characterization of cytoplasmic lipid droplets in human metastatic breast cancer cells
Authors: Zembroski Alyssa S. , Andolino Chaylen , Buhman Kimberly K. , Teegarden Dorothy
Published: September 2020

One of the characteristic features of metastatic breast cancer is altered lipid metabolism, including increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. Although multiple hypotheses exist for how CLDs may promote cancer progression, how exactly CLDs contribute to breast cancer metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs are thought to regulate CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to altered lipid metabolism and breast cancer progression is unknown. Therefore, we utilized untargeted shotgun proteomics and transmission electron microscopy to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. We identified a total number of 1537 proteins in the CLD fraction isolated from MCF10CA1a cells, and these proteins were associated with a variety of cellular processes. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, proteins involved in cell-cell adhesion were the most enriched in the CLD fraction, and some of these proteins had associated terms in cell migration and signaling. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and aggressiveness, making them possible therapeutic targets for prevention or treatment of this disease.

Citation Format: Alyssa S. Zembroski, Chaylen Andolino, Kimberly K. Buhman, Dorothy Teegarden. Proteomic characterization of cytoplasmic lipid droplets in human metastatic breast cancer cells [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 5130.
Abstract 5133: Optimized RAS top-down proteomic assay reveals expanded proteoform landscape in malignant cells
Authors: DeHart Caroline , Sharma Kanika , Esposito Dominic , Maciag Anna , Nissley Dwight , McCormick Frank
Published: September 2020

Traditional proteomic methods face an inherent challenge with the RAS family of GTPases (HRAS, NRAS, KRAS4A, KRAS4B). Expressed at low endogenous abundance, these four isoforms possess considerable sequence similarity, rendering the confident proteomic linkage of a given isoform with a specific mutation or post-translational modification (PTM) a formidable proposition outside of isoform-specific model cell lines. Conversely, top-down proteomic analysis (TDP), which measures intact protein molecules, can precisely identify modified protein forms, or proteoforms, and confidently localize both mutations and PTMs on each RAS isoform present. Previously, a method combining immunoprecipitation and subsequent TDP (IP-TDP) led to the identification of eleven KRAS4B proteoforms, including four mutations and two novel PTMs, within colorectal cancer cell lines and tumor samples†. These initial results demonstrated the great potential for proteoform-resolved measurements to further illuminate the role of KRAS4B mutations and modifications in human cancer.

More recently, the NCI RAS Initiative has developed an enhanced RAS proteoform assay incorporating IP-TDP analysis on an Orbitrap Fusion Lumos mass spectrometer. By employing alternate antibodies, recombinant protein standards, and well-characterized model or malignant cell lines, we have obtained substantial improvements in both RAS proteoform detection and characterization, thus facilitating the identification and localization of low-abundance PTMs. Moreover, we have applied our optimized IP-TDP method to a diverse panel of malignant and RAS mutant cell lines with the goal of expanding our knowledge of the RAS proteoform landscape. In doing so, we have observed a wealth of novel RAS proteoforms, of far greater number and complexity than previously anticipated. We have also observed isoform-specific proteoform variations, most notably between KRAS4A and KRAS4B, and initial indications of proteoform context dependence, namely within a given mutation or tissue background. Our results further underscore the power of proteoform-resolved measurements and indicate that RAS-dependent signaling pathways in normal and cancer contexts may be far more complex than previously thought.

† I Ntai et. al. “Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification crosstalk.” Proc Natl Acad Sci U S A. 2018 Apr 17; 115(16):4140-4145.

Citation Format: Caroline DeHart, Kanika Sharma, Dominic Esposito, Anna Maciag, Dwight Nissley, Frank McCormick. Optimized RAS top-down proteomic assay reveals expanded proteoform landscape in malignant cells [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 5133.