bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1934: Modulation of protein homeostasis networks upon Hsp70 inhibition in cancer cells
Authors: Sannino Sara , Brodsky Jeffrey L.
Published: July 2021

Cellular protein homeostasis is tightly regulated by balancing protein synthesis, folding, and degradation. Cancer cells constantly modulate protein folding and degradation efficiency to survive and proliferate without accumulating toxic misfolded proteins under acute stress conditions, such as low oxygen and energy, and exposure to chemotherapeutic agents. Many cancers rely on the unfolded protein response (UPR) to cope with stress, which increases the expression of molecular chaperones and mitigates the accumulation of misfolded proteins. Thus, it is unsurprising that the Hsp70 molecular chaperone, which serves as a master regulator of proteostasis, is upregulated in many cancers. Increased Hsp70 correlates with metastasis formation and poor patient prognosis in different cancers, like breast cancer. To define the role of Hsp70 in cancer survival and identify how cancer cells compensate for chaperone-mediated proteotoxicity, we investigated the response to a specific Hsp70 inhibitor, MAL3-101, in breast cancer cells. We discovered that breast cancer cells bin into distinct groups when subjected to Hsp70 inhibition based on their sensitivity to the compound. Moreover, we demonstrate that resistant cells have higher autophagy levels compared to more sensitive lines. Autophagy was further induced by MAL3-101 in resistant breast cancer cells, as evidenced by the accumulation of both autophagy related genes and proteins, as well as by autophagic-like structures detected by confocal microscopy. These data suggest that resistance to Hsp70 inhibition arises from autophagy induction. We then discovered that Hsp70 inhibition induces the UPR pathway, triggering apoptosis in sensitive cells. In particular, the activation of the UPR transducer, PERK, is required to induce apoptosis in sensitive cells when Hsp70 was inhibited. Overall, our work positions autophagy as a critical compensatory mechanism when molecular chaperone function is overwhelmed and misfolded proteins accumulate. Furthermore, our findings delineate a distinct role of PERK in apoptosis induction upon Hsp70 inhibition, highlighting the interplay between the PERK, Hsp70, and autophagy.

Citation Format: Sara Sannino, Jeffrey L. Brodsky. Modulation of protein homeostasis networks upon Hsp70 inhibition in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1934.
Abstract 1935: Antitumor effect mechanism of statin in PCa cells focusing on autophagy
Authors: Miyazawa Yoshiyuki , Oka Daisuke , Nakayama Hiroshi , Sekine Yoshitaka , Arai Seiji , Suzuki Kazuhiro
Published: July 2021

Antitumor effect mechanism of statin in prostate cancer cells focusing on autophagy Background:Statins have recently been studied for their proapoptotic and antimetastatic effects. However, the exact mechanisms of their anticancer actions remain unclear. We have studied the novel castration resistant prostate cancer (CRPC) treatment with statins. Using microarrays, we discovered up-regulation of ATG4B (Autophagy related 4 homolog B) in PC-3 cells after simvastatin treatment. In this study, we evaluated the effects of simvastatin focusing on androgen-independent prostate cancer cells. Methods:PC-3, LNCaP-LA, 22RV-1-LA (taht were derived from LNCaP and 22RV1 cells and cultured in 10% charcoal-stripped fetal bovine serum for 3 months), and DU145 human prostate cancer cell lines were used. Microarrays were performed using PC-3 untreated or administered with simvastatin (0 uM versus 5 uM). Proliferation, migration, and invasion were evaluated using MTS assay, Migration assay, and Invasion assay, respectively. The increases of autophagy were detected by western blot using microtubule associated protein 1 light chain 3 (LC-3) antibody and confirmed by a fluorescence microscope with staining autophagosomes by autophagy detection kit (abcam, Cambridge, UK). Results:Using microarrays, we discovered up-regulation of ATG4B in PC-3 cells after simvastatin treatment. Simvastatin inhibited the proliferation, migration, and invasion of PC-3, LNCaP-LA, 22RV1-LA and DU145 cells. The expression level of LC-3 was up-regulated by simvastatin in PC-3, LNCaP-LA, 22RV1-LA and DU145 cells. In PC-3 cells treated with statin, a significant increase in autophagosomes was confirmed by fluorescence microscopy. The expression of LC-3 and autophagosomes were increased in a concentration-dependent manner. Conclusions:It was found that statin administration enhances autophagy induction in a prostate cell line. We plan to study the combined effect with other drugs that induce autophagy. We would like to develop CRPC treatment by inducing autophagy cell death.

Citation Format: Yoshiyuki Miyazawa, Daisuke Oka, Hiroshi Nakayama, Yoshitaka Sekine, Seiji Arai, Kazuhiro Suzuki. Antitumor effect mechanism of statin in PCa cells focusing on autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1935.
Abstract 1942: Quantitative D2O proteomics reveals ATG9A-dependent autophagy substrates that are degraded independently of the LC3-lipidation machinery
Authors: McEwan Colten M. , Broadbent David G. , Kannangara Ashari R. , Poole Daniel M. , Naylor Bradley C. , Price John C. , Schmidt Jens C. , Andersen Josh L.
Published: July 2021

Autophagy is an essential catabolic process that cancer cells use to survive metabolic stress and maintain basal cellular homeostasis via degradation of dysfunctional organelles and protein aggregates. One protein with an unclear, yet essential, role in autophagy is the multi-pass transmembrane protein ATG9A. ATG9A is an apical autophagy regulator that is hypothesized to help seed the formation of nascent autophagosomes by providing membrane through its own unique ATG9A vesicles. To understand the role ATG9A plays in autophagic protein turnover, we performed whole proteome deuterium exchange and quantitative mass spectrometry to measure protein degradation rates in Wild-Type (WT) and ATG9A Knock-Out (KO) cells. This experiment revealed that ATG9A KO cells display significantly decreased degradation rates of aggregation prone proteins and autophagy adapters, such as p62/SQSTM1. In an effort to understand the relationship between ATG9A and p62/SQSTM1, we found that ATG9A associates directly with p62/SQSTM1 and mediates the basal degradation of p62/SQSTM1-associated protein aggregates. This ATG9A-mediated degradation occurs in a non-canonical way, involving a subset of ATG proteins (e.g., ATG13, ATG101), but independent of the LC3-lipidation machinery ATG5 and ATG7. Taken together, our data suggests that ATG9A controls the degradation of p62 in an ATG5/ATG7-independent manner.

Citation Format: Colten M. McEwan, David G. Broadbent, Ashari R. Kannangara, Daniel M. Poole, Bradley C. Naylor, John C. Price, Jens C. Schmidt, Josh L. Andersen. Quantitative D2O proteomics reveals ATG9A-dependent autophagy substrates that are degraded independently of the LC3-lipidation machinery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1942.
Abstract 1948: Investigation of the effect of LLLT on glioblastoma multiforme
Authors: Atluri Rohini , Afkhami Parsa , Choi Tae-Youl , Simmons Denise P.
Published: July 2021

Low-level Laser Treatment (LLLT) has a potential to alter tumor behavior such as proliferation in addition to therapeutic effects such as wound healing, inflammation reduction. Studies indicate that, different tumor cells respond distinctly to LLLT based on laser dose (J/sq. cm). GBM is an aggressive and invasive primary brain tumor which is heterogenic in nature and any treatment that could have a potential to kill or change tumor behavior or tumor microenvironment by making GBM sensitive to available treatments can help improve the survival rate. In this study, we investigated the effect of LLLT on human malignant glioma (U251) cells using He-Ne Laser (632.8 nm). The He-Ne laser parameters used for the treatment are power (14.87±0.3 mW), beam diameter (0.68 cm), and varying laser exposure times of 5 mins and 10 mins leading to variable doses of 12.28 J/sq.cm and 24.56 J/sq. cm respectively. The dose response of LLLT on U251 cells revealed that the dose 12.28 J/sq. cm lead to cell death of 17%, 24 hrs post treatment while the dose 24.56 J/sq. cm achieved cell death of 30%, 6 hrs post treatment along with some morphological changes such as vacuole formation in the heterogenic malignant glioma. Also, a biphasic response was observed post treatment with the dose of 24.56 J/sq. cm, as we observed increase in cell viability 18hrs post treatment along with neurite formation. Western analysis suggests the involvement of PARP and caspase 9 in killing LLL treated U251 cells. Thus, upon further study, LLLT could have a potential to be used as an adjunctive treatment when used with conventional treatments for malignant glioma.

Citation Format: Rohini Atluri, Parsa Afkhami, Tae-Youl Choi, Denise P. Simmons. Investigation of the effect of LLLT on glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1948.
Abstract 196: Redefining cancer subtypes using multi-omics and deep learning
Authors: Akalin Altuna , Uyar Bora , Ronen Jonathan , Franke Vedran
Published: July 2021

Cancer is a heterogeneous collection of diseases traditionally classified by the tissue of origin. The diversity of the molecular profiles of cancers has a big impact on the way patients are diagnosed and treated, how they respond to their prescribed treatments, the duration of survival after diagnosis, and factors such as remission, recurrence, or spread (metastasis) of the disease. While such diagnostic and prognostic outcomes are potentially predictable by taking a closer look into the changes of the genome, epigenome, transcriptome, proteome, and various other omics platforms, the contemporary cancer treatments still predominantly don't make the best use of such multi-omics profiling of patient samples. Therefore, multi-omics profiling of cancers holds great potential to define a molecularly coherent subtype definition of cancers in order to achieve the eventual goal of matching the best possible treatment to the subgroup of patients.

However, the current subtypes from consortiums such as TCGA have been defined by heterogeneous methods and molecular markers by different teams. A subset of these studies have not attempted to characterize molecular subtypes, but rather taken histopathologically defined subtypes as the gold standard and tried to characterize molecular features of these subtypes. Here we evaluate TCGA cancer subtypes based on the molecular profile coherence score. This novel metric combines survival statistics, pathways information, tumor purity estimates, and mutational signatures. We expect that subtypes that are patient subgroups should display molecular signature homogeneity. We evaluate TCGA subtypes from 21 cancers using these criteria and compare the subtypes with our own definition using multi-omics data in a deep learning framework. We have refined the several subtypes from multiple cancers towards more molecularly coherent patient subgroups.

Citation Format: Altuna Akalin, Bora Uyar, Jonathan Ronen, Vedran Franke. Redefining cancer subtypes using multi-omics and deep learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 196.
Abstract 1964: The mutational pattern of cell cycle checkpoints and regulators in Egyptian colorectal cancer patients
Authors: El-Din Youssef Amira Salah , Lotfy Mai M. , Nassar Auhood , Zekri Abdel-Rahman N.
Published: July 2021

Background: Uncontrolled proliferation is a hallmark of cancer cells. Many cell cycle checkpoints and regulators are altered in tumors which in turn results in the accumulation of genetic aberrations. So, this study aimed to identify the mutational profile of cell cycle checkpoints and regulators in tissue biopsies in Egyptian Colorectal Cancer patients.

Material and Methods: Sixty two fresh tissues samples were collected from Egyptian patients with CRC. The libraries were performed using Qiaseq UMI-based targeted panel.The detected genetic variants with an average coverage of 500x were annotated using Ingenuity Variant analysis (IVA) and pathway analysis was performed using Ingenuity Pathway analysis (IPA).

Results: Data analysis revealed that TP53& ATM genes; the checkpoints at G1/S and G2/M phases, were highly mutated. They harbored 12 and 9 pathogenic mutations respectively, which cause loss of functionality. The most frequently detected pathogenic variants were c.121delG (58%) in the TP53 & c.640delT (29%) in the ATM. While CHECK2; the checkpoint at G2/M phase, harbored 4 pathogenic variants; c.430delC (16%) was the most detected one. Also, two pathogenic variants were detected in the M phase checkpoint BUB1B and the most frequently detected one was c.1669delA (37%). We also detected pathogenic variants involved in the G1/S phase regulators; SMAD4 (c.1241delT (23%)), CDKN2A (c.238delG (18%)), STK11 (c.340delG (11%)), and CDH1 (c.1955delT (19%)). Other pathogenic variants involved in G2/M phase regulators were also detected; BRCA1 (c.3214delC (16%)) & EP300 (c.4408delA (19%)). Moreover, we detected 2 pathogenic variants in M phase regulator; CDC27 (c.1998delA (7%) & c.2070delA (5%)).

Conclusion: Our data showed the deregulated pattern of cell cycle checkpoints and regulators that are crucial for understanding colorectal cancer progression in Egyptian patients. Also, it may help in developing personalized targeted therapies that efficiently target the cell cycle genes mutations, thereby limits the CRC progression in those patients.

Citation Format: Amira Salah El-Din Youssef, Mai M. Lotfy, Auhood Nassar, Abdel-Rahman N. Zekri. The mutational pattern of cell cycle checkpoints and regulators in Egyptian colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1964.
Abstract 1965: Discovery of ZN-c3, a potent Wee-1 inhibitor with a differentiated pharmacologic and kinase selectivity profile
Authors: Li Jiali , Boren Brant , Huang Peter Q. , Bunker Kevin D. , Doñate Fernando , Samatar Ahmed A.
Published: July 2021

Genome instability initiated by DNA damage or DNA replication stress is a driver of tumor development. To ensure the accuracy of DNA replication, DNA damage response (DDR) mediated by various cell cycle checkpoints activate the DNA repair system. One such checkpoint system is the G2-M checkpoint which is involved in the repair and prevention of excessive DNA damage. A key regulator of the G2-M checkpoint is the Wee1 kinase which causes cell cycle arrest to prevent damaged cells from entering mitosis. Thus, inhibition of Wee1 should prevent G2-M checkpoint activation which would lead to unscheduled mitotic entry and induction of apoptosis. Small-molecule inhibitors of Wee1 are currently being tested in several tumor types. Preliminary results show promising clinical activity of AZD-1775, a potent inhibitor of Wee1, however, achieving an optimal dose and a schedule that is well-tolerated has been a challenge. Here, we describe the characterization of ZN-c3, a potent, selective and orally bioavailable small-molecule inhibitor of Wee1. ZN-c3 exhibits potent anti-proliferative activity across multiple tumor cell lines. Compared to AZD-1775, ZN-c3 has superior selectivity profile against a panel of kinases. In vivo, ZN-c3 induced tumor regression as a single agent in several tumor models including non-small cell lung cancer, ovarian cancer, and uterine sarcoma. In combination studies, ZN-c3 potentiates the antitumor efficacies of carboplatin and gemcitabine. The pharmacokinetic and efficacy profiles of ZN-c3 and AZD-1775 were compared in tumor xenograft models. ZN-c3 showed higher plasma and tumor exposure than AZD-1775, which correlated with more potent tumor growth inhibition than AZD-1775. In summary, ZN-c3, with its key differentiated pharmacologic and kinase selectivity profile, is a promising new generation of DDR inhibitor. ZN-c3 is currently in clinical development for the treatment of advanced solid cancers.

Citation Format: Jiali Li, Brant Boren, Peter Q. Huang, Kevin D. Bunker, Fernando Doñate, Ahmed A. Samatar. Discovery of ZN-c3, a potent Wee-1 inhibitor with a differentiated pharmacologic and kinase selectivity profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1965.
Abstract 1980: Non-canonical regulation and function of anti-apoptotic protein c-FLIP
Authors: Tsegaye Matyas Abel , McGeehan Kyle , Nemera Mati , Schafer Zachary T.
Published: July 2021

The metastasis of cancer cells to distant sites accounts for over 90% of cancer related deaths. In order to metastasize, cancer cells must be able to survive during extracellular matrix (ECM) detachment, namely by blocking anoikis (a caspase-dependent cell death) and restoring metabolic deficiencies. Such barriers during tumorigenesis are abrogated through activation of oncogenic signaling, and/or inhibition of tumor suppressors. Elucidating the signaling pathways involved in cancer cell metastasis is a crucial step in designing effective therapeutic interventions. Surprisingly, our investigation has revealed that cellular FLICE-like Inhibitory Protein (c-FLIP), a protein well known for its pro-survival function, instead antagonizes the viability of ECM-detached cancer cells. Additionally, we demonstrated that signaling emanating from oncogenic growth factor receptors results in the downregulation of c-FLIP expression providing additional evidence that cancer cells may benefit from diminished FLIP levels. Interestingly, this repression of c-FLIP expression by activated oncogenes is a consequence of PI3K signaling. Our study also suggests that low c-FLIP levels are beneficial in cancer cells due to capacity of c-FLIP to induce β-catenin degradation. Taken together, our study has revealed an unexpected role for c-FLIP in restricting the viability of tumor cells and raises the idea that the c-FLIP may have context-dependent pro- and anti-cell death roles during tumorigenesis.

Citation Format: Matyas Abel Tsegaye, Kyle McGeehan, Mati Nemera, Zachary T. Schafer. Non-canonical regulation and function of anti-apoptotic protein c-FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1980.
Abstract 1981: TGF-β induced PLEK2 promotes metastasis and chemoresistance in ESCC by up-regulation of LCN2
Authors: Wang Feng , Zhang Chaoqi , Liu Chengming , Cheng Hong , Sun Nan , He Jie
Published: July 2021

Background: Esophageal squamous cell carcinoma (ESCC) patients suffer from poor prognosis in general. Novel molecular markers to predict prognosis and serve as potential therapy target are much needed. Our previous research has established a comprehensive database of ESCC (GSE53625). Combined with data from TCGA, we found Pleckstrin 2 (PLEK2) predicts poor prognosis in ESCC. Meanwhile, its role in ESCC progression remains unknown.

Methods: The TCGA database and GSE53625 are used to establish the expression profiles of PLEK2 and overall survival rates. Loss-of-function assays were performed to examine the effect of PLEK2 on proliferation, migration and invasion of ESCC cells in vitro and in vivo. ChIP assay was used to detect the relationship between TGF-β and PLEK2. Western blot and quantitative real time PCR (qRT-PCR) were carried out to reveal the interrelation between PLEK2 and LCN2.

Results: PLEK2 had a higher expression in ESCCs and predicted poor overall survival (OS). PLEK2 attenuation by shRNA was sufficient to decrease ESCC cells proliferation, clonogenicity, migration and invasion in vitro and tumorigenicity and distant metastasis in vivo. Conversely, elevating PLEK2 levels performed opposite effects. Downregulation of PLEK2 led to higher proportions of apoptotic ESCC cells when induced by Cisplatin. The high expression of PLEK2 in ESCC patients also linked to shorter OS of patients who received adjuvant chemotherapy. TGF-β could increase the expression of PLEK2 by stimulation of Smad2/3 which can bind directly with the promoter sequences of PLEK2. Analyses of transcriptome profiling suggested LCN2 as the downstream of PLEK2. Overexpression LCN2 in PLEK2 stable knockdown ESCC cells can reverse the decreased migration and invasion, but not the proliferation of ESCC cells. TGF-β increased the expression of LCN2, but the effect disappeared when PLEK2 was knockdown. We also find that AKT pathway is involved in the whole regulatory processes.

Conclusion: Our results revealed the significance of PLEK2 in driving oncogenesis and metastasis in ESCC by regulating LCN2 which activates AKT pathway. We also indicated that PLEK2 was associated with chemo-resistance. Our findings indicates the potential use of PLEK2 as a prognostic biomarker and a candidate therapeutic target in ESCC.

Keywords: PLEK2; ESCC; TGF-β; chemo-resistance

Citation Format: Feng Wang, Chaoqi Zhang, Chengming Liu, Hong Cheng, Nan Sun, Jie He. TGF-β induced PLEK2 promotes metastasis and chemoresistance in ESCC by up-regulation of LCN2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1981.
Abstract 1986: p53/FBXL20 axis negatively regulates the protein stability of PR55α, a PP2A regulatory subunit
Authors: Yan Ying , Madduri Lepakshe , Brandquist Nichole , Palanivel Chitra , Zhou Sumin , Enke Charles , Ouellette Michel
Published: July 2021

PP2A holoenzymes are the majority of Ser/Thr phosphatases in human cells and each PP2A complex contains one scaffold subunit, one catalytic subunit, and one regulatory subunit. PP2A regulatory subunits, existing in four sub-families encoded by 15 genes, determine the substrate specificities and intracellular localizations of PP2A. We previously reported an essential role for PR55α, a PP2A regulatory subunit, in the support of malignant potential of pancreatic cancer cells, which includes promoting YAP-activation that supports anchorage-independent growth and metastasis. Furthermore, our data showed that PR55α level is elevated in pancreatic cancer relative to normal tissues and high PR55α levels correlate with poor patient survival. The studies in this report reveal a novel mechanism by which the p53 tumor suppressor negatively regulates PR55α protein stability. The results show that p53 inactivation by siRNA-knockdown, gene-deletion, HPV-E6 promoted degradation, or p53R175H mutant expression all results in the increase of PR55α expression. Subsequent studies demonstrate that this p53 function is mediated through its target gene FBXL20, a substrate recognition component of the SCF (Skp_Cullin_F-box) ubiquitin ligase complex that facilitates proteasomal degradation. As a result, the expression of mutant p53R175H in human pancreatic ductal cells mimics the PR55α effect, increasing c-Myc protein stability, and promoting anchorage-independent proliferation. Additionally, FBXL20 mRNA levels are significantly reduced in pancreatic cancers relative to normal control, and low FBXL20 levels correlate with poor patient survival, both of which inversely resemble the pathological effects of PR55α in pancreatic cancer. Collectively, these results define a novel p53 function in the suppression of PR55α expression.

Citation Format: Ying Yan, Lepakshe Madduri, Nichole Brandquist, Chitra Palanivel, Sumin Zhou, Charles Enke, Michel Ouellette. p53/FBXL20 axis negatively regulates the protein stability of PR55α, a PP2A regulatory subunit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1986.
Abstract 1988: Association between CEACAMs and hypoxia defines gastric cancer progression
Authors: Poirah Indrajit , Chakraborty Debashish , Dixit Pragyesh , Samal Supriya , Padhan Pratyush Kumar , Barik Alok , Singh Shivaram Prasad , Singer Bernhard B. , Bhattacharyya Asima
Published: July 2021

Background: Gastric cancer is one of the most prevalent cancers. As in many other solid tumors, hypoxia is a notable feature in gastric cancer as well. The hypoxic microenvironment promotes cancer by modulating expression of several master regulator oncoproteins. Carcinoembryonic antigen-related cell adhesion molecules or (CEACAMs) are adhesion proteins that have been associated with many epithelial tumors but their expression pattern in hypoxic gastric epithelial cells are not known. Since these membrane proteins have widespread roles in the intracellular and intercellular signaling, inflammation, angiogenesis and metastasis, we examined hypoxia-mediated regulation of CEACAM proteins.

Methods: AGS, MKN45 and primary gastric epithelial cells were subjected to hypoxia of varying degrees and the changes in the CEACAM protein profile were evaluated by immunoblotting, FACS assay and immunofluorescence microscopy. In vitro DNA-binding assay and luciferase assays were performed to assess the transcriptional regulation of CEACAMs. Overexpression and suppression studies were also performed to elucidate upstream regulations of CEACAM proteins. Gastric cancer biopsy tissues were examined from consenting patients by performing fluorescence and confocal microscopy.

Results: We found that varying degrees of hypoxia differentially modulate CEACAM expression in gastric cancer epithelial cells which in turn leads to altered proliferation and invasiveness of cancer cells. Interestingly, gastric adenocarcinoma and metastatic gastric cancer biopsies show distinct CEACAM signatures. These findings unfold further avenues highlighting the importance of CEACAM proteins as biomarkers of gastric cancer and as therapeutic targets to treat gastric cancer.

Funding: Department of Atomic Energy (DAE).

Citation Format: Indrajit Poirah, Debashish Chakraborty, Pragyesh Dixit, Supriya Samal, Pratyush Kumar Padhan, Alok Barik, Shivaram Prasad Singh, Bernhard B. Singer, Asima Bhattacharyya. Association between CEACAMs and hypoxia defines gastric cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1988.
Abstract 1993: Off-target effects of ultra-low dose dimethyl sulfoxide on targetable signaling events in lung cancer in vitro models
Authors: Baldelli Elisa , Subramanian Mahalakshmi , Alsubaie Abduljalil M. , Baglivo Sara , Hodge K A. , Bianconi Fortunato , Ludovini Vienna , Crino' Lucio , Petricoin Emanuel F. , Pierobon Mariaelena
Published: July 2021

Background: The identification of targetable alterations in cancer continues to offer novel opportunities to the drug discovery process. However, pre-clinical testing often requires solubilization of these drugs in cosolvents like dimethyl sulfoxide (DMSO). Using commercially available models commonly used for in vitro drug screening and pre-clinical testing, we explored the DMSO off-target effects on functional signaling networks, drug targets, and downstream substrates.

Materials and Methods: Eight commercially available Non-Small Cell Lung Cancer (NSCLC) cell lines were incubated with three ultra-low concentrations of DMSO (0.0008%, 0.002%, and 0.004% v/v, respectively) for 5 min, as well as 1, 6 and 24 hours. Following incubation, expression and activation levels of 183 proteins were captured using Reverse Phase Protein Array (RPPA) technology, of which 134 were kinases and downstream substrates.

Results: The DMSO effect was heterogenous across cell lines and varied based on multiple factors including: concentration, exposure time, and cell line. Of the 183 proteins measured, 88% were statistically significant at the highest DMSO concentration, followed by 81% and 67% at lower concentrations. Only 28 proteins were found statistically different in more than 5 cell lines indicating heterogenous response across models. pERK 1/2 T202/Y204 emerged as the most frequently affected node along with stress-induced response and mitogenic proteins. These cell line specific DMSO-induced alterations, may modulate response to targeted agents and chemotherapeutics.

Conclusions: Ultra-low DMSO concentrations have broad and heterogenous effects on targetable signaling proteins which are model, time, and concentration dependent. As such, off-target effects need to be carefully evaluated in the design and implementation of pre-clinical drug screening and testing.

Citation Format: Elisa Baldelli, Mahalakshmi Subramanian, Abduljalil M. Alsubaie, Sara Baglivo, K A. Hodge, Fortunato Bianconi, Vienna Ludovini, Lucio Crino', Emanuel F. Petricoin, Mariaelena Pierobon. Off-target effects of ultra-low dose dimethyl sulfoxide on targetable signaling events in lung cancer in vitro models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1993.
Abstract 2014: Identification of Smurf2 as a HIF-1α degrading E3 ubiquitin ligase
Authors: Zhao Shuai , El-Deiry Wafik S.
Published: July 2021

The major adaptive response to hypoxia involves HIF-1α which is conventionally regulated by VHL E3 ligase. Our previous experiments suggested a stabilization of HIF-1α by CDK1 and CDK4/6 that is independent of VHL, hypoxia or p53, and that CDK4/6 inhibitors destabilizes HIF-1α under normoxia and hypoxia. To further investigate the molecular mechanism of HIF-1α destabilization by CDK1 or CDK4/6 inhibitors, we performed a proteomic screen on immunoprecipitated HIF-1α from hypoxic colorectal cancer cells that were either untreated or treated with CDK1 inhibitor Ro3306 and CDK4/6 inhibitor palbociclib. Our proteomics screen identified a number of candidates that were enriched in palbociclib-treated hypoxic cells including SMAD specific E3 ubiquitin protein ligase 2 (Smurf2). In addition, we identified a HIF-1α peptide that appeared to be differentially phosphorylated after palbociclib treatment. Gene knockdown of Smurf2 increased basal expression of HIF-1α that was further increased in the presence of Ro3306 or two different CDK4/6 inhibitors, palbociclib and abemaciclib. Overexpression of Smurf2 inhibited expression of HIF-1α and enhanced HIF-1α ubiquitination in normoxia. Proteasome inhibitor MG-132 partially rescued HIF-1α expression when Smurf2 was overexpressed. Overexpression of Smurf2 is correlated with improved disease-free survival and overall survival in clear cell renal cell cancer. Our results unravel a previously unknown mechanism involving Smurf2 for HIF-1α destabilization in CDK4/6 inhibitor-treated cells, thereby shedding light on VHL-independent HIF-1α regulation.

Citation Format: Shuai Zhao, Wafik S. El-Deiry. Identification of Smurf2 as a HIF-1α degrading E3 ubiquitin ligase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2014.
Abstract 2019: Drug repositioning to induce the synthetic lethality in cancer cells with GSH-depleting therapy
Authors: Otsuki Yuji , Nagano Osamu , Saya Hideyuki
Published: July 2021

The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis. Inhibitor of the GSH synthesis including buthionine sulfoximine (BSO) and cystine-glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, thus potential anticancer agents. However, the efficacy of xCT or GSH synthesis-targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. In this study, we performed a synthetic lethal screen of a drug library to identify agents that sensitize the GSH deficiency-resistant cancer cells to the xCT inhibitor sulfasalazine (SSZ). This screen identified the oral anesthetic dyclonine (DYC) which act as a covalent inhibitor for aldehyde dehydrogenases (ALDHs). Treatment with DYC induced intracellular accumulation of the toxic aldehyde 4-hydroxynonenal (4-HNE) in a cooperative manner with SSZ. The combination of DYC and SSZ cooperatively suppressed the growth of SSZ-resistant tumors. Furthermore, we recently found that a vasodilator oxyfedrine (OXY) also possess the similar structure with dyclonine and induce cell death in BSO-resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase-2 expression as a potential biomarker for the efficacy of such combination therapy. Moreover, OXY-mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion.

Citation Format: Yuji Otsuki, Osamu Nagano, Hideyuki Saya. Drug repositioning to induce the synthetic lethality in cancer cells with GSH-depleting therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2019.
Abstract 2031: The PERK arm of the unfolded protein response induces CXCL8 and CCL20 through C/EBPδ
Authors: Poria Dipak Kumar , Sheshadri Namratha , Sharan Shikha , Sterneck Esta
Published: July 2021

Cancer cells often experience endoplasmic reticulum (ER) stress due to either activated oncogenes, microenvironmental conditions or therapeutic interventions. ER stress initiates an adaptive unfolded protein response (UPR) program, mediated by PERK, IRE1 and ATF6 transmembrane sensors, which alleviates the stress and promotes cells survival. However, if unmitigated, the UPR proceeds to initiation of cell death. Cancer cells often usurp the UPR pathways that contribute to cell survival and metastasis through intra- and intercellular mechanisms. Intracellular mechanisms of cellular adaptation are well-characterized; however, the intercellular consequences of ER stress are less understood. Here, we show that in cell lines representing melanoma, breast and prostate cancer, the transcription factor C/EBPδ (CEBPD) is an early ER stress response factor that is induced by the PERK arm of the UPR. CEBPD induction occurs in part through activation of PERK-mediated non-canonical JAK/STAT3 signaling. Transcriptional profiling in MDA-MB-435 melanoma cells revealed that C/EBPδ supports the expression of genes involved in cell intrinsic adaptations such as chaperones, components of ER associated degradation, and apoptosis inhibitors. In addition, C/EBPδ promotes expression of the inflammatory mediators CXCL8 and CCL20, which are known to modulate the tumor microenvironment through the receptors present on various host cells. Depletion of CEBPD by siRNA or inhibition of PERK kinase reduced CXCL8 and CCL20 transcripts and secreted proteins upon ER stress induction by thapsigargin. Bioinformatic analysis and the ENCODE ChIP-seq data suggest direct interaction of C/EBPδ with CXCL8 and CCL20 promoter/enhancer, indicative of direct transcriptional activation of the genes. Indeed, ectopic expression of C/EBPδ, but not a mutant lacking the transactivation domain, was sufficient to induced CXCL8 and CCL20 transcription in HEK293 cells in the absence of ER stress. Consistent with these results, conditioned media from ER-stressed MDA-MB-435 cells induced the gene expression of proinflammatory cytokines IL-6, IL-12, TNFα and IL-1RA in HL-60 promyelocytic cells while suppressing IL-10 expression. Taken together, these data show that the PERK arm of the UPR activates JAK/STAT3 pathway to induce expression of C/EBPδ and thereby enhance secretion of immunomodulators and transmit UPR-induced inflammatory signals to immune cells in the tumor microenvironment. This pathway may contribute to the overall outcome of the UPR on cancer cell survival, metastasis and response to anticancer therapies.

Citation Format: Dipak Kumar Poria, Namratha Sheshadri, Shikha Sharan, Esta Sterneck. The PERK arm of the unfolded protein response induces CXCL8 and CCL20 through C/EBPδ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2031.
Abstract 2032: Vitamin B12 augments Ca2+ dependent cytotoxicity of diethyldithiocarbamate through excessive ER-vacuolization, oxidative stress and mitochondrial depolarization in MCF-7 cells in culture
Authors: Solovieva Marina E.
Published: July 2021

Coordinated organization of intracellular organelles into functional networks is vital for cells. Mitochondrial and endoplasmic reticulum (ER) interaction in regulation of cell Ca2+ homeostasis is crucial for cell death and survival. Function of these intracellular organelles involved in two-way regulation of the status of major cell metabolites including protein, lipid exchange and intracellular Ca2+ homeostasis. Disulfiram and its derivatives recently attracted attention as a potential anticancer treatment (Kona et al., 2011). Combined use of hydroxycobalamin (vitamin B12b) and diethyldithiocarbamate (DDTC) demonstrated synergistic effect (combination index=0.25) and enhanced the cytotoxic effect of DDTC in number of transformed cell lines in culture (Solovieva et al., 2020). In the present work using MCF-7, human breast cancer cells in culture we demonstrate that combined action of DDTC and B12b resulted in induction of UPRER manifested by dilation of ER cisterns, increased level of polyubiquitinated proteins, and increased expression of CHOP. Confocal microscopy revealed disorganization of mitochondrial network and ER and treatment of these cells with B12b/DDTC and accompanied by mitochondrial depolarization (~50% after 6 h of exposure to drugs). Treatments also induced changes in morphology of mitochondrial network, and within 6 h of exposure, mitochondria demonstrated large amplitude swelling, characteristic feature of mitochondrial permeability transition pore opening. Drug exposure induced mitochondrial oxidative stress and increase in superoxide anion level (monitored with MitoSOX) and increased content of mitochondrial Ca2+ (xRhod-1) which almost doubled following treatment with B12b/DDTC. Short-time exposure of MCF-7 cells to mixture of drugs in Ca2+-free incubation media (2.5 mM EGTA) resulted in generation of significantly smaller size of vacuoles as compared with control. Similarly, pre-loading of cells with intracellular Ca2+ chelator (BAPTA, 5 µM, 30 min, 37oC) or inhibition of IP3 receptor(s) with 2-APB, delayed the drug-induced vacuolization. Taken together, our data indicate that the mechanism of B12b/DDTC-induced cytotoxicity in human MCF-7 cancer cell lines involves mitochondrial oxidative stress, activation of MPT pore and Ca2+ overload.

Support of Funding Information: Russian Science Foundation № 19-75-20145

Citation Format: Marina E. Solovieva. Vitamin B12 augments Ca2+ dependent cytotoxicity of diethyldithiocarbamate through excessive ER-vacuolization, oxidative stress and mitochondrial depolarization in MCF-7 cells in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2032.
Abstract 2036: Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications
Authors: Song Kai , Minami Jenna , Ridgley Trevor , Huang Arthur , Jayaraman Rachana , Crosson William , Salazar Jesus , Pazol Eli , Niroshi Senaratne , Nagesh Rao , Paraiso Kim , Graeber Thomas
Published: July 2021

Cancer cells display two modes of focal amplifications (FA): extrachromosomal double minutes (DMs/ecDNAs) and intrachromosomal homogenously staining regions (HSRs). Understanding the plasticity of these two modes is critical for preventing targeted therapy resistance. We developed a combined BRAF plus MEK inhibitor resistance melanoma model that bears high BRAF amplifications through both ecDNA and HSR modes, and investigated FA dynamics in the context of drug resistance plasticity. Cells harboring FAs displayed mode switching between ecDNAs and HSRs, from both de novo genetic changes and selection of pre-existing subpopulations. We found that copy number plasticity is not exclusive to ecDNAs. Single cell-derived clones with HSRs also exhibit BRAF copy number and corresponding HSR length plasticity that allows them to respond to dose reduction and recover from drug addiction. In addition, upon kinase inhibitor escalation, we observed reproducible selection for cells with BRAF kinase domain duplications residing on ecDNAs. Whole genome sequencing of sublines with different FA formats allowed fine reconstructions of BRAF amplicon and revealed conserved structures during the transitions due to drug dose manipulations. The amplification boundaries in our cell line model were found consistent with those in clinically observed cases of combined BRAF/MAPK inhibitor resistance. RNA-seq and CRISPR screening performed on parental, ecDNA and HSR sublines revealed distinct biology and vulnerabilities associated with each kind of FA. Vulnerabilities for genes involved in ecDNA maintenance in DNA repair pathways identified in our model are consistent with previous reports, such as a role for DNA-dependent protein kinase (DNA-PK/PRKDC). Our screen reveals many new targets. In sum, the karyotypic plasticity of FAs allows cancer cells to respond to drug dose challenges through a myriad of mechanisms, including increases or decreases in DMs, shortening of HSRs, and acquisition of secondary resistance mechanisms. The cellular vulnerabilities identified in our study provide roadmaps for therapeutically targeting each kind of FA mode.

Citation Format: Kai Song, Jenna Minami, Trevor Ridgley, Arthur Huang, Rachana Jayaraman, William Crosson, Jesus Salazar, Eli Pazol, Senaratne Niroshi, Rao Nagesh, Kim Paraiso, Thomas Graeber. Plasticity and vulnerability associated with extrachromosomal and intrachromosomal BRAF amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2036.
Abstract 2040: Analysis of DNA damage repair gene alterations and their association with tumor mutation burden in 1427 hepatocellular carcinoma patients
Authors: Yang Mengmei , Xie Wenzhuan , Huang Mengli , Zhu Xinhua
Published: July 2021

Background: DNA damage repair (DDR) pathway have attracted much attention because they modulate cancer risk, progression and therapeutic response. DDR deficiency sensitize the tumor to poly ADP-ribose polymerase inhibitors (PARPi). Also alterations in DDR genes are associated with higher tumor mutation burden (TMB) and better response to immune checkpoint inhibitors (ICI). Here we conduct a large-scale genomic analysis to clarify the landscape of DDR gene mutations in hepatocellular carcinoma and the correlation between individual gene mutation and tumor mutation burden.

Methods: Tumor samples from 1427 patients with advanced hepatocellular carcinoma were assayed through next-generation sequencing with 381 cancer-related genes including 30 predefined DDR genes: MRE11A, RAD50, BARD1, BLM, BRCA1, BRCA2, BRIP1, PALB2, MLH1, MSH2, MSH6, PMS2, POLD1, ATM, ATR, CHEK1, CHEK2, FANCA, FANCC, FANCD2, FANCG, FANCF, FANCL, RAD51, CUL3, ERCC1, POLE, MUTYH, and PRKDC. TMB (mutations per megabase [mut/Mb]) was calculated based on nonsilent somatic mutations in coding regions. Wilcoxon test was used for the comparisons and statistical significance was set at p = 0.05.

Results: Somatic DDR gene alterations were found in 18.8% of the HCC patients. The most common mutational types were copy number variation (30.54%), frameshift (21.89%), and nonsense (20%). The most frequently altered DDR genes are ATM (2.6%) and BRCA2 (2.2%), followed by FANCA (0.9%), PALB2 (0.8%), and MLH1 (0.7%). As to different functional pathways of DDR, the gene alteration frequency from high to low is HRR (6.1%), DS (3.2%), FA (2.5%), MMR (2.2%), BER (0.6%), NHEJ (0.6%), and NER (0.3%). The TMB of patients carrying somatic DDR gene alterations was significantly higher than those without somatic DDR gene alterations (7.26 vs 6.45 mut/MB, p<0.001). As to individual gene, alterations in RAD50, MLH1, MSH2, CHEK2, and FANCE were significantly correlated with higher tumor mutation burden while alterations in other DDR genes were not.

Conclusion: This is the largest study to clarify the landscape of DDR gene mutations in hepatocellular carcinoma. This study confirms that mutations in DDR genes are relatively common and are correlated with higher tumor mutation burden. This also indicates not all DDR genes are equal since mutations of certain individual genes were associated with higher TMB whereas others were not; further studies are needed to confirm this.

Citation Format: Mengmei Yang, Wenzhuan Xie, Mengli Huang, Xinhua Zhu. Analysis of DNA damage repair gene alterations and their association with tumor mutation burden in 1427 hepatocellular carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2040.