bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1078: Sotorasib and metformin combination enhances cytotoxicity and apoptosis in KRAS mutant lung cancer cell lines
Authors: Hernandez-Pedro Norma Yanet , Barrios-Bernal Pedro , Lucio-Lozada José , Ramos-Ramirez Maritza , Arrieta Oscar
Published: April 2023

KRAS is the most frequently mutated oncogene in lung adenocarcinoma (50%) and it is associated with a poor response to antineoplastic treatments, as well as poorer survival rates. KRAS activates the MAPK pathway, promoting cell survival and increasing protein synthesis. Novel inhibitors of KRAS G12C mutation (sotorasib) have demonstrated short-lasting responses due to resistance mediated by the AKT-mTOR-P70S6K pathway. In this context, metformin is a promising candidate to avoid this inconvenience by inhibiting mTOR and P70S6K. Therefore, this project aimed to explore the effects of the combination of sotorasib and metformin on cytotoxicity, apoptosis, and the activity of the mTOR pathway in lung cancer cell lines with or without KRAS mutations.

Methods: We performed dose-effect curves to determine the IC50 concentration of sotorasib, and IC10 of metformin. Then, these doses were used to treat lung cancer cell lines A549 (KRAS G12S), H522 (without KRAS), and H23 (KRAS G12C). Cellular cytotoxicity was evaluated by MTT assay, apoptosis induction through flow cytometry with Annexin V and 7AAD, and the activity of P70S6K and AKT proteins by western blot. Main outcomes and measures: cell survival, apoptosis, expression, and phosphorylation of intermediaries belonging to the mTOR pathway.

Results: The combined treatment showed a synergic effect on cytotoxicity, increased apoptosis induction, and notable inhibition of AKT and P70S6K1 proteins, compared to sotorasib alone or to control groups in all the cell lines, but predominantly in KRAS-mutated cells (H23 and A549).

Conclusions: The combination of metformin with sotorasib showed synergic effects on cytotoxicity, increased apoptosis induction, and a remarkable inhibition of downstream proteins involved in the signaling of growth factor receptors in all tested cells. Moreover, these results suggest a sensitizing effect of metformin to sotorasib treatment in cells without KRAS mutations, such as H522.

Citation Format: Norma Yanet Hernandez-Pedro, Pedro Barrios-Bernal, José Lucio-Lozada, Maritza Ramos-Ramirez, Oscar Arrieta. Sotorasib and metformin combination enhances cytotoxicity and apoptosis in KRAS mutant lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1078.
Abstract 1089: High grade serous ovarian cancer: Detecting stemness and EMT levels in response to chemo and radiation therapy
Authors: Jung Yeonkyu , Keniston Aaron , Antonissen Ashley , Morcos Ann , Bertucci Antonella , Vazquez Marcelo , Unternaehrer Juli
Published: April 2023

High-Grade Serous Ovarian Cancer (HGSOC) is the most fatal of gynecological cancers. The recurrence rate of HGSOC after treatment is more than 80%. The available treatment options for HGSOC are limited; standard of care includes surgery combined with chemotherapy, with radiation therapy in some advanced cases. Both chemotherapy and radiation therapy are known to induce tumor cell aggressiveness. Many cancer studies have demonstrated that cancer stem cells (CSCs) and the epithelial-mesenchymal transition (EMT) promote cancer aggressiveness, invasion, metastasis, and drug resistance. Therefore, to better understand the cause of the poor prognosis in HGSOC, our objective is to scrutinize the role of CSCs and EMT in chemo- and radiation resistance. To lead our inquiry, we utilized the SORE6 reporter to identify Sox2/Oct4 expression, which indicates the stemness, and the Zeb1 3’ UTR reporter to detect EMT. Using flow cytometry, we quantified the reporter activity in 10 ovarian cancer cell types, including chemo-sensitive and chemo-resistant ones, considering both cell lines and patient-derived cells. Chemo-resistant cells are grown under cisplatin treatment. We exposed some of these ovarian cancer cell lines to 0, 1, 2, 4 and 8Gy of 250 MeV proton and 6 MeV photon beams, then analyzed the reporter activity 72 hours post-radiation. Our findings exhibit that chemo-resistant cells express higher stemness and EMT levels than chemo-sensitive cells and have higher resistance to radiation. These reporters can detect radiation-induced stemness and EMT. Furthermore, stemness and EMT levels positively correlate with the radiation dosage increment. We conclude that these reporters are an efficient surrogate for detection of stemness and EMT and can be used to test strategies for prevention of therapy-induced aggressive phenotypes.

Citation Format: Yeonkyu Jung, Aaron Keniston, Ashley Antonissen, Ann Morcos, Antonella Bertucci, Marcelo Vazquez, Juli Unternaehrer. High grade serous ovarian cancer: Detecting stemness and EMT levels in response to chemo and radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1089.
Abstract 1091: MDM2 inhibition as a novel radio-sensitizing strategy for endometrial cancer
Authors: Petty Aaron , Yard Brian , Durmaz Arda , Scott Jacob , Vargas Roberto
Published: April 2023

Introduction: As opposed to other malignancies, the majority of endometrial cancer (EC) cases are wild-type for TP53. Approximately 15% of TP53-mutated EC have heterozygous mutation, preserving a fraction of wild-type signaling. Given the demonstrated impact of p53 signaling on radiotherapy (RT) response in EC, we explored the role of MDM2 inhibition as a novel radio-sensitizing strategy for EC.

Methods: To assess the impact of MDM2 inhibition on cell viability and radiation response, we employed 3 parental EC cell lines of differing TP53 status (JHUEM2, Hec108, Hec1B), as well as monoclonal isolates of JHUEM2 after TP53 knockout. An intron-targeting approach facilitated TP53 cDNA complementation. Radiation response was determined by assessing cell viability with CellTiter-Glo and calculating area under the curve (AUC) with multiple dose points. Nutlin-3 and AMG-232 were both tested. All experiments were carried out in triplicate. Highest Single Agent (HSA) analysis was used to determine additive versus synergistic effects.

Results: Nutlin-3 and AMG-232 were both observed to have a negative impact on cell viability, dependent on TP53 context. In the wild-type TP53 cell line (JHUEM2), IC50s for Nutlin-3 and AMG-232 were observed to be 1.417 and 0.218 μM, respectively. The heterozygous TP53-mutated cell line, Hec108, had corresponding IC50s of 6.04 and 1.10 μM. The Hec1B cell line did not exhibit a response to either drug. Subsequently, IC50 curves were calculated in the presence of multiple dose points of gamma irradiation administered as one fraction. A synergistic effect was observed with Nutlin-3 and AMG-232 in JHUEM2, while only AMG-232 reached synergy in Hec108.

Conclusions: Overall, these findings nominate MDM2 inhibitors as potential radio-sensitizing strategies that would be applicable to a large proportion of EC. Given the lack of benefit from cisplatin-based radio-sensitization for recurrent EC in a recent clinical trial, this provides a novel biomarker driven strategy.

Citation Format: Aaron Petty, Brian Yard, Arda Durmaz, Jacob Scott, Roberto Vargas. MDM2 inhibition as a novel radio-sensitizing strategy for endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1091.
Abstract 1094: 3-Bromopyruvate reduces pancreatic tumor growth by damaging mitochondria and inhibiting glucose metabolism in both cell culture and mouse model
Authors: Roy Sanjit , Dukic Tijana , Bhandary Binny , Molitoris Jason , Ko Young H , Shukla Hem D
Published: April 2023

Pancreatic cancer (PC) is the deadly cancer in both men and women, with a one-year relative survival rate of 20%, and a five-year relative survival rate of only 8%. Pancreatic cancer is characterized by dense fibrotic stroma that constitutes up to 90% of the total tumor volume and contains extracellular matrix (ECM), immune cells, vasculature, and cancer-associated fibroblasts which are hallmark of immunosuppressive tumor microenvironment. In present investigation we have tested anti-cancer effect of 3-BromoPyruvate (3-BP), an inhibitor of aerobic glycolysis employing Panc-2 cancer cells, and syngeneic pancreatic cancer mouse model. Panc-2 cancer cells treated with 40 uM of 3-BP showed significant inhibition in cell proliferation and ATP production. Cells treated with 60 uM dose level, showed drastic phenotypic changes (membrane blebbing) within 2 hours of treatment. The qRT PCR data showed 3BP inhibited hexokinase II (HK2) expression at 40uM, suggesting 3-BP regulate HK2 expression at transcriptional level. We also observed that 3-BP in combination with low doses of irradiation was more effective in killing cancer cells than 3-BP alone in both 2D and 3D culture model. Treatment of C57BL/6 subcutaneous pancreatic tumor model with 10 and 20 mg/kg of 3BP also showed significant reduction of tumor growth. The Transmission Electron Microscopy of tumor tissues treated with 3-BP revealed severely damaged mitochondrial structure which might have severely affected ATP generation in cancer cell. Altogether, the results in this study demonstrated that 3-BP could be a promising anti-tumor drug against pancreatic cancer and its efficacy could be improved in combination with radiation therapy.

Citation Format: Sanjit Roy, Tijana Dukic, Binny Bhandary, Jason Molitoris, Young H Ko, Hem D Shukla. 3-Bromopyruvate reduces pancreatic tumor growth by damaging mitochondria and inhibiting glucose metabolism in both cell culture and mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1094.
Abstract 1134: Therapeutic potential of an antibody-drug conjugate directed against a tumor-specific epitope on podocalyxin
Authors: Dean Pamela M. Austin , Hernaez Diana Canals , Brassard Julyanne , Hughes Michael R. , Bell Erin M. , McNagny Kelly M. , Roskelley Calvin D.
Published: April 2023

Podocalyxin (Podxl) is a cell surface sialomucin that is frequently upregulated in tumors with high metastatic potential and its expression is associated with poor outcome in several human cancers. As such, Podxl is emerging as an important prognostic and theragnostic marker. While Podxl is expressed on normal vascular endothelia and kidney podocytes, we sought to produce a novel anti-Podxl antibody that selectively recognizes a tumor-restricted glycoepitope on the extracellular mucin domain of Podxl. The antibody we have produced, PODO447, is specific to the tumor glycoform of Podxl, demonstrated by a lack of binding to normal tissues that are known to express Podxl. In contrast, we show binding of the antibody to tumor cell lines, patient-derived cell lines, and primary tumor tissues. We have previously shown that the majority of tumors in an ovarian carcinoma array (219 cases), including 65% of the high-grade serous histotype, are positive for PODO447. Here, we further show the presence of PODO447 in tumors of the breast, urothelium, pancreas, endometrium, colon, prostate, lung (both small cell and non-small cell), and glioblastoma. To assess the therapeutic potential of our antibody as an antibody drug conjugate (ADC), we coupled PODO447 to the microtubule disruptor monomethyl auristatin E (MMAE) with an enzyme cleavable linker carbamoyl p-aminobenzyl carbamate (PABC), resulting in the ADC PODO447-Vedotin. We demonstrate promising in vitro activity of the ADC to various human tumor cell lines as well as in vivo efficacy to xenografted ovarian and pancreatic tumor lines. Our data reveals PODO447-Vedotin as a tumor-specific and highly efficacious therapeutic agent for the targeting of human tumors and as such, PODO447 exhibits potential for further development as a targeted clinical immunotherapy.

Citation Format: Pamela M. Austin Dean, Diana Canals Hernaez, Julyanne Brassard, Michael R. Hughes, Erin M. Bell, Kelly M. McNagny, Calvin D. Roskelley. Therapeutic potential of an antibody-drug conjugate directed against a tumor-specific epitope on podocalyxin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1134.
Abstract 1161: Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function
Authors: Bennett Neal K. , Nakaoka Hiroki J. , Laurent Danny , Okimoto Ross , Sei Yoshitaka , Horvai Andrew E. , Bivona Trever G. , Batsios Georgios , Viswanath Pavithra , Hoeve Johanna ten , Graeber Thomas G. , Nakamura Ken , Nakamura Jean L.
Published: April 2023

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells downregulate glycolytic mechanisms. Metabolic shifts associated with silencing mito-respiratory genes can be detected in vivo with metabolic imaging. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors.

Citation Format: Neal K. Bennett, Hiroki J. Nakaoka, Danny Laurent, Ross Okimoto, Yoshitaka Sei, Andrew E. Horvai, Trever G. Bivona, Georgios Batsios, Pavithra Viswanath, Johanna ten Hoeve, Thomas G. Graeber, Ken Nakamura, Jean L. Nakamura. Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1161.
Abstract 1166: APOBEC deaminases compete with tobacco smoking mutagenesis and affect age at onset of lung cancer
Authors: Zhang Tongwu , Sang Jian , Hoang Phuc H. , Zhao Wei , Rosenbaum Jennifer , Klimczak Leszek J. , McElderry John , Klein Alyssa , Wirth Christopher , Bergstrom Erik N. , Díaz-Gay Marcos , Vangara Raviteja , Hutchinson Amy , Lawrence Scott M. , Cole Nathan , Zhu Bin , Przytycka Teresa M. , Shi Jianxin , Caporaso Neil E. , Homer Robert , Pesatori Angela C. , Consonni Dario , Chanock Stephen J. , Wedge David C. , Gordenin Dmitry A. , Alexandrov Ludmil B. , Harris Reuben S. , Landi Maria Teresa
Published: April 2023

APOBEC enzymes are part of the innate immunity and are responsible for restricting viruses and retroelements by deaminating cytosine residues. Most solid tumors harbor different levels of somatic mutations attributed to the off-target activities of APOBEC3A (A3A) and/or APOBEC3B (A3B). However, how APOBEC3A/B interact with exogenous mutagenic processes in shaping tumor development is largely unknown. Here, by combining deep whole-genome sequencing with multi-omics profiling of 309 lung cancers from smokers with detailed tobacco smoking information, we identify two subtypes defined by low (LAS) and high (HAS) APOBEC mutagenesis. LAS are enriched for A3B-like mutagenesis and KRAS mutations, whereas HAS for A3A-like mutagenesis and TP53 mutations. Unlike APOBEC3A, APOBEC3B expression is strongly associated with an upregulation of the base excision repair pathway. Hypermutation by unrepaired A3A and tobacco smoking mutagenesis combined with TP53-induced genomic instability can trigger senescence, apoptosis, and cell regeneration, as indicated by telomere shortening, high expression of pulmonary healing signaling pathway and stemness markers in HAS. The expected association of tobacco smoking exposure with genomic/epigenomic changes are not observed in HAS, a plausible consequence of frequent cell senescence or apoptosis. HAS tumors have slower clonal expansion and older age at onset compared to LAS, particularly in heavy smokers, consistent with high proportions of newly generated, unmutated cells in HAS. These findings show how heterogeneity in mutational burden across competing mutational processes and cell types contributes to tumor development, with important clinical implications.

Citation Format: Tongwu Zhang, Jian Sang, Phuc H. Hoang, Wei Zhao, Jennifer Rosenbaum, Leszek J. Klimczak, John McElderry, Alyssa Klein, Christopher Wirth, Erik N. Bergstrom, Marcos Díaz-Gay, Raviteja Vangara, Amy Hutchinson, Scott M. Lawrence, Nathan Cole, Bin Zhu, Teresa M. Przytycka, Jianxin Shi, Neil E. Caporaso, Robert Homer, Angela C. Pesatori, Dario Consonni, Stephen J. Chanock, David C. Wedge, Dmitry A. Gordenin, Ludmil B. Alexandrov, Reuben S. Harris, Maria Teresa Landi. APOBEC deaminases compete with tobacco smoking mutagenesis and affect age at onset of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1166.
Abstract 1180: Identification of target proteins for breast cancer genetic risk loci and blood risk biomarkers in a large study by integrating genomic and proteomic data
Authors: Jia Guochong , Yang Yaohua , Ping Jie , Xu Shuai , Liu Lili , Guo Xingyi , Tao Ran , Long Jirong , Zheng Wei
Published: April 2023

Genome-wide association studies (GWAS) have identified around 200 loci associated with breast cancer risk. However, protein targets for these loci remain largely unknown. Identifying protein targets and biomarkers can improve the understanding of cancer biology and etiology and identify high-risk individuals for cancer prevention. In this study, we investigated genetically predicted levels of 1,142 circulating proteins with breast cancer risk in 133,384 cases and 113,789 controls of European ancestry included in the Breast Cancer Association Consortium (BCAC). We identified 22 blood protein biomarkers associated with the risk of overall breast cancer at a false discovery rate (FDR) <0.05, including nine proteins encoded by genes located at least 500kb away from previously reported risk variants for breast cancer. Analyses focusing on 124 encoding genes located at GWAS-identified breast cancer risk loci found 20 proteins associated with overall breast cancer risk and one protein associated with triple-negative breast cancer risk at FDR <0.05. Adjustment for the GWAS-identified risk variants significantly attenuated the association for 13 of these proteins, suggesting that these proteins may be the targets of these GWAS-identified risk loci. The identified proteins are involved in various biological processes, including glutathione conjugation, STAT5 signaling, and NF-kB signaling pathways. Our study identified novel protein targets and risk biomarkers for breast cancer risk.

Citation Format: Guochong Jia, Yaohua Yang, Jie Ping, Shuai Xu, Lili Liu, Xingyi Guo, Ran Tao, Jirong Long, Wei Zheng. Identification of target proteins for breast cancer genetic risk loci and blood risk biomarkers in a large study by integrating genomic and proteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1180.
Abstract 1251: Novel potential role of m6A-demethylase FTO (fat mass and obesity) protein in colorectal cancer
Authors: Landskron-Ramos Glauben T. , Domínguez-Beltrando Antonia L. , Zambra-Rojas Mauricio , Sanguineti Antonella , Vasquez Gonzalo , la Fuente Marjorie De , Simian Daniela , Valdebenito-Silva Silvana , Saavedra Juan , Gouet Solange , Soto-Rifo Ricardo , Sauma Daniela , Eugenin Eliseo , Abedrapo Mario , Hermoso Marcela A. , Hermoso Marcela A.
Published: April 2023

Colorectal cancer (CRC) is the one of the most frequent and deadly cancer worldwide. The regulation mediated by the N6methyladenosine (m6A) demethylase protein called FTO in CRC is quite controversial. As previously studied in obesity and food intake, currently is being linked to stemness, epithelial-mesenchymal transition, chemotherapy resistance and proliferation in colon cancer cells, however, its localization and function in the tumor microenvironment (TME) and how it connects with progression remains unclear. The aim of this study was to evaluate FTO protein in tumor and healthy corresponding tissue from 22 colorectal cancer patients with different histological TNM stages (hTNM) by immunohistochemistry and multiple immunofluorescence analysis; classifying the tumor and stromal FTO localization and associating this data with clinical and histopathological features. As for content, the frequency of FTO+ cells in stromal compartments increases in more invasive stages compared to non-tumor tissue and in tumors with low desmoplasia. Regarding to localization, FTO is mostly nuclear and present in the lamina propria cells in non-tumor tissues and in immune and fibro-immune compartments in the TME, such as T cells, cancer associated fibroblasts and macrophages. In tumor cells, the percentage of FTO+ cells increase in early carcinogenic stages (hTNM I vs healthy tissue) and in moderately differentiated tumors. In addition, nuclear FTO expression increases in normal T cells and fibroblasts with a particular pattern after exposure for 24h to a colon adenocarcinoma cell-derived conditioned media, suggesting FTO activity in the TME interaction following a paracrine signal. In conclusion, the increase of the FTO protein in both tumor and stroma from CRC suggests its participation mediating TME communication.

Citation Format: Glauben T. Landskron-Ramos, Antonia L. Domínguez-Beltrando, Mauricio Zambra-Rojas, Antonella Sanguineti, Gonzalo Vasquez, Marjorie De la Fuente, Daniela Simian, Silvana Valdebenito-Silva, Juan Saavedra, Solange Gouet, Ricardo Soto-Rifo, Daniela Sauma, Eliseo Eugenin, Mario Abedrapo, Marcela A. Hermoso, Marcela A. Hermoso. Novel potential role of m6A-demethylase FTO (fat mass and obesity) protein in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1251.
Abstract 1278: ID3 controlled dynamic Pyk2 localization is important for the mobility of cancer stem cells
Authors: Das Jayanta , Deoraj Alok , Roy Deodutta , Felty Quentin
Published: April 2023

Treatment of brain metastases remains a clinical barrier in breast cancer patients. Secondary breast tumors in the brain are reported to be enriched with breast cancer stem cell (BCSC) markers suggesting that BCSCs infiltrate the brain. Brain infiltration by BCSCs is complex involving detachment from the primary tumor, entering the blood, surviving the microvascular niche, and crossing the blood brain barrier (BBB). It is critical to understand how BCSCs move through the BBB to prevent brain metastases. Nuclear respiratory factor 1 (NRF1) transcription factor, Pyk2 non-receptor tyrosine kinase, and inhibitor of differentiation protein 3 (ID3) have been shown to contribute to the pathogenesis of cancer through inducing CSCs or increasing mobility of these cells. Recently, we showed that NRF1-induced BCSCs crossed the BBB which were guided by ID3 overexpressing endothelial stem cells. In this study, we investigated the NRF1/ID3/Pyk2 axis contributing to BCSCs mobility. NRF1 promoted migration of BCSCs and mesenchymal MDAMB231 cells through brain endothelial cells cultured with astrocytes. ID3 overexpressing endothelial cells promoted adhesion with BCSCs and mobility of BCSCs across the BBB model. In addition, ID3 controlled Pyk2 localization which is important for mobility of stem cells and cancer cells. Endothelial cells were treated with a known risk factors for breast cancer, 17β-estradiol (E2) and environmental pollutant PCB153. Treatment with PCB153 or E2 showed increased growth of BCSCs tumor spheroids and mobility of ESCs. Exosomal ID3 released from endothelial cells also supported the growth of BCSCs and provide the basis for paracrine effects by endothelial stem cells associated with breast tumors. These findings show for the first time a novel role for ID3 and Pyk2 by which endothelial stem cells support breast tumor metastases where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3/Pyk2 modifying agents to prevent the spread of breast cancer to the brain.

Citation Format: Jayanta Das, Alok Deoraj, Deodutta Roy, Quentin Felty. ID3 controlled dynamic Pyk2 localization is important for the mobility of cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1278.
Abstract 1286: UNC5B promotes EMT and metastasis of pancreatic adenocarcinomas and expresses different isoforms that impact sensitivity to Netrin-1 blockade
Authors: Harris Chris R. , Prela Orjola , Wang Lan , Casabianca Anthony , Narrow Wade , Sechrist Zachary , Withers Tracy , Shields Cory , Asad Asra , Hezel Aram , Carpizo Darren
Published: April 2023

The axonal guidance receptor UNC5B is considered to be a tumor suppressor because it promotes apoptosis in the absence of its protein ligand, NTN1. But we find that UNC5B is not a tumor suppressor of pancreatic ductal adenocarcinomas (PDAC), and instead promotes metastasis. In TCGA data, UNC5B expression associates with poor patient outcome. In vivo, pancreatic cancer metastasis was completely eliminated by knocking out UNC5B from the genetically engineered KPC mouse model of PDACs, and was reduced upon knockout of UNC5B in a splenectomy model of liver metastasis. An inhibitor of NTN1, NP137, also reduced metastasis in vivo. In vitro, knockout of UNC5B from mesenchymal pancreatic cell lines was sufficient to reduce many metastatic traits including invasiveness, EMT, and aerobic glycolysis. YAP1 is a known target of UNC5B and was overexpressed in high UNC5B expressing cell lines, which were sensitive to the YAP1 inhibitor CA3. NP137 is currently in clinical trial for several tumor types and we have been investigating which patients might respond best to this drug. Using an antibody (D9M7Z) that recognizes an epitope in UNC5B that overlaps with the caspase-3 cleavage site that is critical for UNC5B's apoptotic functions, we found two isoforms in murine and human PDACs: one isoform that contains the caspase-3-containing epitope as well as a previously-unreported isoform that to lack it. Cells expressing the caspase-3 site were much more sensitive to NP137. We also discovered genetic alterations in patient samples that induce expression of UNC5B. For instance, UNC5B was recurrently amplified in PDACs and these amplifications were highly focal to the UNC5B locus; also, UNC5B expression was increased by mutations of ARID1A, a component of the SWI/SNF transcription complex that is recurrently mutated in pancreatic adenocarcinomas. ARID1A mutations caused cell lines to become mesenchymal, but the cells returned to an epithelial phenotype upon knockout of UNC5B. In current and future clinical trials, NP137 may particularly benefit patients with tumors that harbor mutations in ARID1A or ARID1B, focal amplifications of UNC5B, and/or high expression of the caspase-3-containing isoform of UNC5B.

Citation Format: Chris R. Harris, Orjola Prela, Lan Wang, Anthony Casabianca, Wade Narrow, Zachary Sechrist, Tracy Withers, Cory Shields, Asra Asad, Aram Hezel, Darren Carpizo. UNC5B promotes EMT and metastasis of pancreatic adenocarcinomas and expresses different isoforms that impact sensitivity to Netrin-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1286.
Abstract 1316: Immune cell dysfunction is enhanced in a mouse model of obesity-associated breast cancer lung metastasis
Authors: Williams Abbey E. , Arendt Lisa M.
Published: April 2023

Obesity is known to worsen the overall prognosis of breast cancer patients. Breast cancer patients with a body mass index (BMI) of ≥30 kg/m2 have an increased risk for metastatic disease compared to patients with a BMI in the normal range. Further studies are necessary to understand the mechanisms driving increased breast cancer metastasis in obese patients, particularly to the lung. Although immune cells like neutrophils and myeloid derived suppressor cells have been studied in breast cancer metastasis of the lung, little is known how adaptive immune cells function in metastatic niche of the lung in obesity. To investigate these gaps, we fed 3-week-old female FVB/N mice either a low-fat diet (LFD) or high-fat diet (HFD) for 16 weeks to induce obesity. We then injected estrogen receptor positive (ER+) TC2 cells into the mammary fat pad. Tumors grew to 0.5 cm in diameter then were surgically removed. Lung tissue was collected 8 weeks after tumor removal to examine progression of metastatic disease, and T cell populations were quantified in metastatic lung tissue from obese and lean mice using flow cytometry. Lungs from obese mice showed a significant increase of total immune cells (CD45) and a decrease in total T cells and natural killer cells (NK) compared with controls. CD3+ cells and CD8+ T cells from obese mice expressed higher levels of programmed cell death protein-1 (PD1), a marker of T cell dysfunction. These results suggest that T cells from the lungs of obese mice may have a greater level of T cell dysfunction than controls. To further investigate how obesity alters the function of T cells, CD45+ cells were sorted from lungs from lean and obese mice with and without metastasis, and gene expression was examined using the Nanostring nCounter Immune Exhaustion panel. Immune cells from the lungs of obese non-tumor-bearing mice showed increased expression of NK exhaustion markers and increased TCR signaling. In metastatic lungs from obese mice, immune cells also showed impaired interferon and tumor necrosis factor signaling and an increase in genes associated with exhaustion in NK cells. Further analysis showed upregulated expression of genes associated with senescence in immune cells from metastatic lungs of obese mice. These data suggest that obesity may increase dysfunctional states of T cells and NK cells, leading to enhanced metastatic growth of breast cancer cells.

Citation Format: Abbey E. Williams, Lisa M. Arendt. Immune cell dysfunction is enhanced in a mouse model of obesity-associated breast cancer lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1316.
Abstract 1349: Exploring CD63 as a targetable mediator of the pro-tumorigenic tissue microenvironment in HNSCC
Authors: Dingendorf Elisabeth B. , Stoffel Michael C. , Kittel Dahlia L. , Kristiansen Glen , Lerbs Tristan
Published: April 2023

Introduction: Head and neck squamous cell carcinoma (HNSCC) is one of the ten most common cancer types worldwide - ranked on the sixth position with 560,000 new diagnoses and 270,000 deaths in 2020. The survival could be increased only slightly over the past decades and in addition the incidence of HNSCC continues to rise. In this study, we investigated the role of the tetraspanin CD63 during cancer progression.

Methods: First, we performed survival analyses with available statistics from data bases to correlate CD63 and survival. Then, we established a 20-marker spectral FACS panel to identify different fibroblast subpopulations in patient tissue. Upon receiving patient consent, fresh HNSCC tissue was received from surgical tumor resections at the University Hospital of Bonn. We mechanically and enzymatically digested and purified the tissue of ten patients to create single-cell suspensions. After staining, the cells were measured on a Sony ID7000 Spectral Analyzer and interpreted with FlowJo. To identify whether CD63 on fibroblasts affects the differentiation of macrophages in vitro, we isolated primary fibroblasts and monocytes from HNSCC patients and deleted CD63 in fibroblasts by CRISPR-Cas9. Thereafter, we co-incubated fibroblasts (without and with the CD63-Knockout) and macrophages over ten days in M-CSF containing medium, and measured the expression of M1 and M2 markers through flow cytometry.

Results: First, correlating CD63 gene expression with the patient outcome showed that CD63 correlated with a decreased survival. Then, analyzing fresh tumor specimens with the multicolor panel showed an increase of CD63 expression in different fibroblast subpopulations in the tumor specimens, e. g. CD9+ CD34+ CD63+ CD90+ fibroblasts increased more than eightfold. Investigating potential mechanisms, by which CD63 expression on fibroblasts affects tumor progression, deleting CD63 steered macrophages towards the pro-inflammatory M1 phenotype.

Conclusion: This study demonstrates that HNSCC increases CD63 expression in different fibroblast populations and that deleting CD63 on fibroblasts can steer macrophages towards the pro-inflammatory phenotype, thereby suggesting a rationale for testing CD63 as a therapeutic target.

Citation Format: Elisabeth B. Dingendorf, Michael C. Stoffel, Dahlia L. Kittel, Glen Kristiansen, Tristan Lerbs. Exploring CD63 as a targetable mediator of the pro-tumorigenic tissue microenvironment in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1349.
Abstract 1361: Astrocytes activate neuronal programming in melanoma brain metastases
Authors: Farnan Julia K. , Cavallo Maria R. , Hartsough Edward J. , Jackson Joshua G.
Published: April 2023

Melanoma Brain Metastases (MBM) pose a major challenge for treating metastatic melanoma. Up to 70% of advanced melanoma patients develop MBM, resulting in significant morbidity and mortality. Melanocytes, the originating cell type of melanomas, are derived from neural crest cells in development. Melanoma’s proclivity to metastasize to the brain has been anecdotally connected to its neural crest origins. Recently, transcriptomic analyses have demonstrated that MBM express a neuronal-like phenotype, upregulating neural crest factors such as NCAM (Neural cell adhesion molecule), L1CAM (L1 cell adhesion molecule protein), while expressing melanocytic markers like MITF (melanocyte induction transcription factor). The activation of neuronal development programming may be a key mediator in brain-metastatic growth, and thus necessitates a better understanding of how melanoma cells adapt to the brain. Astrocytes have emerged as the major host cell type that cancer cells encounter and interact with during brain metastasis formation. Typically, astrocytes regulate brain homeostasis and support neuronal function. Their response to injury, termed reactive astrocytosis, is a process defined by increased secretion of growth factors and inflammatory cytokine production. Post-mortem analyses of MBM patients reveal perilesional reactive astrocytosis. Here we demonstrate that astrocyte reactivity promotes MBM growth, using in vitro and in vivo model systems. We identify that astrocytes secrete factors that increase tumor growth and expression of neural development factors. Moreover, pharmacological inhibition of neural development pathways, like Wnt signaling, attenuate the effects of astrocyte secreted factors. We developed a brain adapted melanoma line, D4M3a BrJ, and demonstrate the time course of astrocyte reactivity in vivo in its relationship with tumor growth. Our findings provide evidence that astrocyte reactivity promotes MBM growth and activation of neural development programming, unveiling a new therapeutic avenue for targeting the brain metastatic niche.

Citation Format: Julia K. Farnan, Maria R. Cavallo, Edward J. Hartsough, Joshua G. Jackson. Astrocytes activate neuronal programming in melanoma brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1361.
Abstract 1365: The spatially resolved host-microbiome sequencing platform reveals the interaction between bacteria and cancer stem cell microinches
Authors: Kim Gyeongjun
Published: April 2023

The tumor microbiome is a unique element that appears differently for each type of cancer in a person. Currently, intratumoral host-microbiome sequencing was only done by bulk tissue analysis which means little is understood when it comes to the spatial distribution of microbiota in the heterogeneous tumor microenvironment and the localized effect of the microbiota. Breast cancer is known to be one of the cancers rich in the microbiome. We focused on cancer stem cell-like microniches related to the tumor microenvironment of triple-negative breast cancer(TNBC). We adapted SLACS(Spatially resolved Laser activated Cell Sorter) which isolates the region of interest from immunofluorescence-stained samples using a laser, providing the spatial distribution of localized microbiota in rare cancer stem cell samples. By applying SLACS

to point out the breast cancer stem cell micro-niches, we discovered that more microbes flourished in ALDH1 high/CD44 high groups rather than normal cells. We also identified the composition of the microbiota was different in cancer stem cells depending on the degree of expression of the marker. Comparing RNA-seq data of host and microbiome sequencing with functional studies, we reveal the distribution of microbiota in breast tumors and interactions between cancer stem cells and microbiota. Collectively, we found the spatial pattern of the microbiota localized in cancer stem cell microniches and how cells and bacteria interact with each other more precisely.

Citation Format: Gyeongjun Kim. The spatially resolved host-microbiome sequencing platform reveals the interaction between bacteria and cancer stem cell microinches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1365.
Abstract 1372: NUDT16L1 promotes colon cancer progression via inhibition of ferroptosis
Authors: Lin Yi-Syuan , Tsai Ya-Chuan , Li Chia-Jung , Wei Tzu-Tang , Wu Ya-Na , Wu Shang-Rung , Lin Shin-Chin , Tsai Shaw-Jenq , Lin Shih-Chieh
Published: April 2023

Ferroptosis is an iron-dependent oxidative cell death with accumulation of lipid peroxidation and abnormal morphology of mitochondria. Although previous studies have shown that ferroptosis inducers have therapeutic potentials in several treatment-naive and drug-resistant cancer types, lacking systematic analysis of the ferroptosis sensitivity in different cancer types makes it difficult to know which cancer type is suitable for clinical usage and critical regulator determining the ferroptosis sensitivity during cancer progression is still not clear. In this study, colon cancer was identified as one of the ferroptosis-insensitive cancer types by dry and wet bench analyses. Importantly, our large-scale analysis also showed that NUDT16L1 was a novel ferroptosis repressor and overexpressed in colon cancer clinical specimens. Similar findings are also observed in the chemical-induced and genetic mouse models of colon cancer. Furthermore, NUDT16L1 loss-of-function and gain-of-function cell models and its conditional knock-in and knock-out mouse models are used to prove its crucial role in the development of colon cancer. In addition, loss of NUDT16L1 induced several key ferroptosis characteristics and impairment of mitochondrial functions while its restoration attenuated those phenomena in colon cancer cells. Mechanistically, our RNA-Seq and RIP-Seq analyses shown that NUDT16L1 directly interacted and positively regulated several crucial negative regulators of ferroptosis. More interestingly, loss of NUDT16L1 not only restores the sensitivity of ferroptosis inducer but also impairs the function of mitochondria. Finally, a specific NUDT16L1 inhibitor was shown to not only repress colon cancer growth in vitro and in vivo but also induce the ferroptosis in colon cancer. In conclusion, this is the first study to demonstrate that overexpression of NUDT16L1 promotes colon cancer progression by inhibition of ferroptosis and maintenance proper function of mitochondria. Furthermore, specific NUDT16L1 inhibitor might be a promising therapeutic strategy for colon cancer patient in the future.

Citation Format: Yi-Syuan Lin, Ya-Chuan Tsai, Chia-Jung Li, Tzu-Tang Wei, Ya-Na Wu, Shang-Rung Wu, Shin-Chin Lin, Shaw-Jenq Tsai, Shih-Chieh Lin. NUDT16L1 promotes colon cancer progression via inhibition of ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1372.
Abstract 1382: Expression and intracellular translocation of cancer biomarkers in hepatocarcinoma cells induced by changes in mitochondrial metabolism
Authors: Larson Bradley R. , Gooz Monika , Maldonado Eduardo
Published: April 2023

Hepatocarcinoma is one of the most common cancer types in the U.S. with limited treatment options and poor survival rate. Recently, cancer bioenergetics emerged as a promising source of targets to develop novel anticancer agents. The identification of biomarkers is essential for screening and validation of small molecules proposed as chemotherapeutic agents. In this regard, there is a great need for high-throughput/high-content methods, which require short and reproducible sample preparation. In this project, we used the erastin-like compound X1 in the well-characterized SNU-449 hepatocarcinoma cell line. Erastin initially enhances mitochondrial function and ROS formation leading to subsequent mitochondrial dysfunction. As readout, we employed directly and indirectly labeled fluorescent antibodies to visualize and quantify expression of proliferative and antiproliferative markers Ki-67 and cytoskeleton-associated protein 4/p63 (CKAP4/p63), as well as expression and intracellular translocation of the tumor suppressor protein p53. Using the Agilent BioTek Cytation 5 cell imaging multimode reader and Agilent BioTek Gen5 microplate reader and imager software, we successfully validated these markers and the corresponding antibodies as tools to study long-term effects of xenobiotics on mitochondrial bioenergetics. The method allows for fast and reliable screening of potential drug candidates. Acknowledgement: This work was supported in part by the Chan Zuckerberg Initiative, Silicon Valley Community Foundation, and the NIH S10 OD028663 to M.G. and by the U.S. National Institutes of Health (NIH/NCI) R01 CA184456, and South Carolina Translational Research Pilot Project UL1 TR001450-SCTR to E.N.M.

Citation Format: Bradley R. Larson, Monika Gooz, Eduardo Maldonado. Expression and intracellular translocation of cancer biomarkers in hepatocarcinoma cells induced by changes in mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1382.
Abstract 1388: Non-thermal plasma-activated medium induces ferroptotic cell death by intracellular ferrous iron overload in head and neck cancer
Authors: Nguyen QuocKhanh , Oh Chan , Jung Seung-Nam , Piao Yudan , Lim Mi Ae , Kim Young Il , Jin Yanli , Kim Hae Jong , Koo Bon Seok
Published: April 2023

The notoriously high proliferation rate of cancer cells requires them to constantly deal with the balance adjustment between oxidative stress overload and upregulated protective antioxidant pathways. It puts them in a vulnerable state whenever one piece of this puzzle went out of control. Non-thermal plasma (NTP) is partially ionized gas operated at or around body temperature and has emerged as a new potential anti-cancer therapy due to its ability to release and induce ROS, RNS, and other free radicals as well as generate UV- photon and electromagnetic field; but the specific mechanism and its specificity to cancer cell are still controversial. Using transcriptomic analysis, we previously reported the link between NTP-activated media (NTPAM)and apoptosis; but the sequencing data also revealed a strong Ferroptotic response in all the 6Head and Neck cancer cell lines we checked. Ferroptosis is a newly identified oxidative-regulated cell death, characterized by the iron-dependent generation of lipid peroxidation. In this study, we found the prolonged treatment of NTPAM-induced cell death via the non-canonical ferroptosis pathway by directly increasing intracellular toxic ferrous Iron (Fe2+). We found that the excessive overexpression of HMOX1 under prolonged NTPAM-induced oxidative stress was actually responsible for the surge of intracellular Fe2+ due to the increase of heme degradation. Pharmacological inhibition or RNA silencing of HMOX1 was able to rescue 2 HNC cell lines from NTPAM-induced increased Iron concentration as well as ferroptosis cell death, both in vivo and in vitro. These results suggest NTP directly induces ferroptosis via an increase of Fe2+ concentration by triggering the over-responding of the NRF2 - HMOX1 antioxidant system. This finding could be exploited to develop future NTP-based cancer therapy as well as in combination with other Iron sensitive anti-neoplastic agents; and could be a potential method of overcoming conventional cancer treatment resistance.

Citation Format: QuocKhanh Nguyen, Chan Oh, Seung-Nam Jung, Yudan Piao, Mi Ae Lim, Young Il Kim, Yanli Jin, Hae Jong Kim, Bon Seok Koo. Non-thermal plasma-activated medium induces ferroptotic cell death by intracellular ferrous iron overload in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1388.