bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 711: p53 causes butein-mediated apoptosis of chronic myeloid leukemia cells
Authors: Lee SeoYeon , Choi Yu-Jeong , Kim Myeong-sun , Kang Sooyeon , Kim Ji Hye , Woo Sang Mi , Ko Seong-Gyu , Oh Sukjoong
Published: August 2019

Progression of chronic myeloid leukemia, marked by the oncogenic Bcr-Abl mutation, is tightly associated with an alteration of the p53 pathway. It is known that butein extracted from various plants represses cancer growth. Although the anticancer effects of butein are widely accepted, the mechanisms by which butein induces apoptosis of chronic myeloid leukemia cells remains to be elucidated. The present study demonstrated that butein-induced apoptosis was mediated by p53. KBM5 chronic myeloid leukemia (CML) cells expressing wild-type p53 were more sensitive to butein compared with p53-null K562 CML cells in terms of apoptotic cell death. In addition, butein arrested KBM5 cells at S-phase and altered the expression levels of certain cyclins and the p53-downstream targets, MDM2 and p21. In addition, while butein reduced the protein expression of MDM2 in the KBM5 and K562 cells, it resulted in proteasome-independent MDM2 degradation in p53-expressing KBM5 cells, however, not in p53-null K562 cells. Therefore, the present study suggested that p53 causes the butein-mediated apoptosis of leukemic cells.

Citation Format: SeoYeon Lee, Yu-Jeong Choi, Myeong-sun Kim, Sooyeon Kang, Ji Hye Kim, Sang Mi Woo, Seong-Gyu Ko, Sukjoong Oh. p53 causes butein-mediated apoptosis of chronic myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 711.
Abstract 73: Inhibition of ACK1 delays and overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib
Authors: Gu Jiajia , He Xia , Mahajan Nupam P. , Owonikoko Taofeek K. , Ramalingam Suresh S. , Sun Shi-Yong
Published: August 2019

The approval of osimertinib (AZD9291) as a third generation EGFR inhibitor for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (first-line) or those who have become resistant to 1st generation EGFR inhibitors through the T790M mutation (second-line) have generated tremendous benefits to these NSLCL patients. However, the unavoidable development of acquired resistance to osimertinib limits the long-term benefits of patients. Thus, effective treatment options are urgently needed. Activated Cdc42-associated kinase 1 or ACK1 (also named as TNK2) is a non-receptor tyrosine kinase with oncogenic function. Although ACK1 gene is amplified in lung squamous cell carcinoma, its involvement in lung cancer including regulation of drug resistance has largely not been investigated. We found that inhibition of ACK1 with (R)-9bMS, a specific ACK1 inhibitor, synergized with AZD9291 in inhibiting the growth of EGFR mutant NSCLC cell lines. Similar results were also generated when ACK1 is genetically inhibited through transient shRNA gene knockdown. The combination of AZD9291 and (R)-9bMS enhanced induction of apoptosis as evaluated by detecting annexin V-positive cells with flow cytometry and PARP cleavage using Western blotting. In an in vitro long-term resistance delay assay, the combination of (R)-9bMS and AZD9291 clearly prevented the emergence of AZD9291-resistance as evaluating resistant cell colony formation and growth. Beyond the combination was also effective in inhibiting the growth of EGFR mutant NSCLC cell lines with acquired resistance to AZD9291. In some resistant cell lines, the combinations induced senescence in addition to induction of apoptosis. These preliminary findings suggest that ACK1 inhibition might be a potential and innovative strategy for preventing and overcoming AZD9291 acquired resistance. Hence further in vivo and mechanistic studies are warranted. (This work was supported by the NIH/NCI R01 CA223220 and Winship lung cancer pilot award to SYS; NIH/NCI R01 CA208258, Bankhead Coley Award-6BC08 and Prostate Cancer Foundation (PCF) Challenge award-17CHAL06 to NPM).

Citation Format: Jiajia Gu, Xia He, Nupam P. Mahajan, Taofeek K. Owonikoko, Suresh S. Ramalingam, Shi-Yong Sun. Inhibition of ACK1 delays and overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 73.
Abstract 733: Characterizing the genetic profile of endometrioid ovarian carcinoma using exome sequencing
Authors: Pierson William E. , Peters Pamela , Quigley David A. , Chen Lee-may , Chapman Jocelyn S.
Published: August 2019

Objective: Endometrioid ovarian carcinoma is poorly understood at the genomic level. Prior sequencing investigations have been limited to small gene panels. In order to characterize the etiology of this disease and identify potential therapeutic targets, we sought to build an unbiased genetic profile of coding gene alterations in endometriod ovarian tumors.

Methods: We sequenced the exomes of primary endometrioid ovarian tumors obtained from our institutional gynecologic oncology tissue bank from 1996 - 2011. We analyzed somatic point mutations, copy number alterations, structural variations and mutational signatures and integrated these data to determine genetic drivers. Demographic and clinical data were collected by retrospective chart review.

Results: Seventeen primary endometrioid ovarian tumors were sequenced. Median age at surgical resection was 51 years (range 33-77 years). The majority of patients presented with early stage disease (71%), many had a history of endometriosis (47%) and 19% had a synchronous primary uterine carcinoma. Microsatellite instability (MSI) was present in 18% (3/17) tumors. All three of these tumors with MSI exhibited a high tumor mutational burden with an average of 174 mutations per megabase of DNA, and two of them had confirmed loss or inactivation of mismatch repair proteins. Approximately half of all cases (47%) had inactivation or truncating mutations in PTEN. 59% of cases (10/17) had alterations in WNT pathway genes, including 6/17 (35%) with missense mutations in the regulatory domain of beta catenin (CTNNB1). 2/17 (12%) had TP53 mutations. We identified one case with a mutation in the exonuclease domain of POLE (P286R), which also exhibited a characteristic POLE-mutant mutation signature.

Conclusion: Using whole exome sequencing, we identified that endometrioid ovarian carcinomas are commonly hypermutated and often harbor microsatellite instability. These findings highlight the importance of genetic sequencing to determine which patients may benefit from targeted immunotherapies.

Citation Format: William E. Pierson, Pamela Peters, David A. Quigley, Lee-may Chen, Jocelyn S. Chapman. Characterizing the genetic profile of endometrioid ovarian carcinoma using exome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 733.
Abstract 776: Posttransriptional control of steroid biosynthesis pathway in prostate cancer
Authors: Bhattarai Asmita , Shukla Girish C.
Published: August 2019

Prostate cancer (PCa) depends on androgens for growth and maintenance. Despite curative therapeutics of the androgen-sensitive PCa, one-third of patients relapse to a more aggressive form of the disease known as castration-resistant PCa (CRPC). Androgen signaling mediated via Androgen receptor (AR) continues to promote CRPC and therapeutic resistance. The synthesis of testosterone (T) and dihydrotestosterone (DHT) via Intratumoral steroidogenesis appears to play a crucial role in CRPC. It appears that PCa cells synthesize de-novo androgen from cholesterol from acetyl-coA via Terpenoid backbone and Steroid biosynthesis pathways. We found that several enzymes of the intracellular cholesterol synthesis pathways including HMGS1, HMGCR, SREBP1, FASN, SCARB1 are potential targets of hsa-miR-149 5p. Currently, we are characterizing miR-149-5p mediated posttranscriptional regulation of these genes and determining its potential therapeutic application in CRPC.

Citation Format: Asmita Bhattarai, Girish C. Shukla. Posttransriptional control of steroid biosynthesis pathway in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 776.
Abstract 793: The prometastatic effects of subcytotoxic doses of doxorubicin are countered by agents inactivating mitochondrial reactive oxygen species
Authors: Porporato Paolo E. , Sonveaux Pierre
Published: August 2019

Mitochondrial reactive oxygen species (mtROS) possess both tumor-promoting and tumor-suppressive activities according to their net concentration resulting for production levels and antioxidant detoxification. mtROS promote cancer cell fitness at low doses (mitohormesis), are cytotoxic at high doses and promote cancer migration, invasion and metastasis at intermediate doses. These intracellular doses vary from a model to another. Yet, we recently identified that mitochondria-targeted ROS scavengers mitoTEMPO and mitoQ prevent cancer metastasis in several in vivo models (Porporato et al. Cell Reports 2014;8:754-66). Interestingly, several anticancer drugs, among which doxorubicin, are well known to trigger mtROS production in cancer cells, but whether this response participates in cell killing or in resistance is still debated. We therefore tested whether combining mitochondria-targeted antioxidants with doxorubicin could help controlling cancer cell proliferation in vitro and tumor growth in mice. Using breast cancer cells lines MCF7 and 4T1 as models, we first found that subcytotoxic doses of doxorubicin increased cell respiration and, thereby, mitochondrial superoxide production. Downstream, mtROS activated the TGFβ pathway and induced cancer cell migration, invasion, epithelial-mesenchymal transition (EMT) and metastatic take in lungs. MitoTEMPO and mitoQ almost totally blocked these phenotypical changes. Together, our data provide a strong rationale for combining doxorubicin chemotherapy with drugs lowering mtROS production in breast cancer.

Citation Format: Paolo E. Porporato, Pierre Sonveaux. The prometastatic effects of subcytotoxic doses of doxorubicin are countered by agents inactivating mitochondrial reactive oxygen species [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 793.
Abstract 796: Progestins target mitochondrial respiration to shift metabolic phenotype in luminal breast cancer
Authors: Matthews Shawna B. , Fettig Lynsey M. , Ward Ashley V. , Jackman Matthew R. , MacLean Paul S. , Sartorius Carol A.
Published: August 2019

The dichotomous nature of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancers has confounded its use as an endocrine target. Historically, progestins were used to treat some advanced breast cancers and ongoing clinical trials are testing several PR ligands. PR generally opposes ER activity through multiple mechanisms, most notably by altering global ER transcriptional activity to suppress breast tumor growth. However, progestins can also stimulate expansion of endocrine-resistant cancer stem cells (CSCs). Thus, better understanding of potential consequences of progestins in breast cancer is necessary to gauge therapeutic efficacy. Given that ER-driven tumor growth relies on mitochondrial respiration, we evaluated whether PR modulates cell metabolism machinery as a mechanism of ER antagonism. We used functional and morphometric assays in an ER+ breast cancer cell line and patient-derived tumor models to show that progestin treatment dramatically alters mitochondrial function. Multivariate metabolomics analysis highlights a progestin-associated metabolic signature involving accumulation of multiple TCA cycle intermediates, including fumarate, malate, and succinate. Functional assays indicate that progestins impair mitochondrial oxidative reserve capacity and oxygen consumption while ablating estradiol-induced accumulation of ATP in PR+ but not PR− cells. Morphometric assessment of mitochondria via laser-scanning confocal and transmission electron microscopy indicate a shift away from reticular filamentous mitochondria towards smaller, rounder, more perinuclear mitochondria with altered membrane potential. These results suggest that PR targets mitochondrial respiration as part of a broader strategic shift from ER-driven tumor growth to PR-driven thrifty metabolic dormancy. Whether the latter is indicative of an acquired propensity for glycolytic metabolism in indolent tumor cells, CSCs, or perhaps both, is addressed in ongoing studies.

Citation Format: Shawna B. Matthews, Lynsey M. Fettig, Ashley V. Ward, Matthew R. Jackman, Paul S. MacLean, Carol A. Sartorius. Progestins target mitochondrial respiration to shift metabolic phenotype in luminal breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 796.
Abstract 805: Adaptation of mitochondrial organization to aggregation in serous ovarian cancer
Authors: Grieco Joseph P. , Edwards Stephanie , Allen Mitchell , Wang Yao , Perry Justin , Song Yipei , Swami Nathan , Brown David , Schmelz Eva
Published: August 2019

During metastasis, ovarian cancer cells exfoliate from the primary tumor and disseminate throughout the peritoneal cavity mostly as aggregates with a higher survival rate and metastatic potential. To determine how the mitochondrial morphology and dynamic expression of fusion and fission-regulating proteins contribute to spheroid survival, in this study we investigated changes in mitochondrial morphology in order to identify phenotypic alterations that are associated with changes in mitochondrial function. We compared monolayers and spheroids of murine and human benign, late-stage, and tumor-initiating cells in hypoxia using fluorescent microscopy and western blot analysis. In addition, we investigated how the mitochondrial membrane potential (Δψm) and ROS production contributed to this phenotype using the InCell analyzer with TMRM and Amplex Red dyes respectively. The data from this study suggest that the morphology and dynamic protein expression are highly altered during tumorigenesis, aggregation, and in hypoxic conditions, indicating a direct correlation of mitochondrial morphology in regulating survival, apoptosis and proliferation. The identification of these molecular events and changes that allow for the survival of metastatic cancer cells in an environment low in oxygen and nutrients may provide specific targets for interventions that would suppress successful metastatic outgrowth of disseminating cells and could potentially be used as a biomarker for treatment efficacy.

Citation Format: Joseph P. Grieco, Stephanie Edwards, Mitchell Allen, Yao Wang, Justin Perry, Yipei Song, Nathan Swami, David Brown, Eva Schmelz. Adaptation of mitochondrial organization to aggregation in serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 805.
Abstract 807: HDAC1 -dependent epigenetic loss of ATP5H confers a stem cell-like phenotype with features associated with metastatic capacity through mitochondrial reprogramming
Authors: Kim Suyeon , Song Kwon-Ho , Kim Tae-Woo
Published: August 2019

The failure of most cancer therapies is the emergence of tumor cells that are refractory to available agent. Here we report that single-modality cancer therapy triggers broad-spectrum cross-resistance to multiple clinical interventions. This multi-modality resistance arises through a core metabolic reprogramming pathway instigated by loss of the ATP synthase subunit ATP5H, which sequentially leads to accumulation of reactive oxygen species and stabilization of HIF-1α under normoxia. Furthermore, we found that NANOG/HDAC1 axis regulates epigenetic loss of ATP5H, this pathway confers tumor cells a stem cell-like phenotype with features associated with metastatic capacity. ATP5H loss in the tumor is strongly linked to failure of therapy, and poor survival in cancer patients. Integration of these biological features provides the best basis for understanding patient outcome in this setting and suggests that loss of ATP5H shapes the tumor resistance to multiple anti-tumor therapy.

Citation Format: Suyeon Kim, Kwon-Ho Song, Tae-Woo Kim. HDAC1 -dependent epigenetic loss of ATP5H confers a stem cell-like phenotype with features associated with metastatic capacity through mitochondrial reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 807.
Abstract 818: Changes in tumor suppressor methylation in SKBR3 cells during treatment with Olaparib or Doxorubicin
Authors: Sichmanova Zuzana , Ottesen Asta , Lonning Per Eystein , Berge Elisabet Ognedal , Knappskog Stian
Published: August 2019

DNA methylation is an epigenetic mechanism that can alter gene expression without modifying the DNA sequence. Reactivation of tumor suppressor genes that have been epigenetically silenced in cancer could be a potential therapeutic approach. In this study, we performed long-term treatment of the breast cancer cell line SKBR3, with Olaparib or Doxorubicin in order to assess the methylation status of tumor suppressor genes, after the cell cultures had become resistant to these drugs. Using bisulphite conversion and massive parallel sequencing we examined CpG islands in promoter regions of 283 tumor suppressor genes at several time points under the two treatment regiments. Methylation analysis was carried out for each sample using our in-house bioinformatic pipeline established in collaboration with Roche and publicly available tools. We assessed differentially methylated CpG’s for each drug, compared to control samples treated with dimethyl sulfoxide (DMSO) using the R package methylKit. Gene ontology and pathway analysis revealed hypermethylation in genes belonging to homologous recombination pathway and hypomethylation in genes responding to cellular stress and protein kinase signaling in Olaparib-treated samples. In Doxorubicin-treated samples, we identified genes involved in inter-strand cross-link repair to be hypomethylated, while other genes associated with more general cellular response to DNA damage were hypermethylated. While in Olaparib-treated samples, differential methylation seemed to be reversible and dependent on the dosage of Olaparib, methylation patterns in Doxorubicin-treated samples appeared to change in an incremental manner, as a function of exposure time to the drug.

Citation Format: Zuzana Sichmanova, Asta Ottesen, Per Eystein Lonning, Elisabet Ognedal Berge, Stian Knappskog. Changes in tumor suppressor methylation in SKBR3 cells during treatment with Olaparib or Doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 818.
Abstract 822: PureQuant Methylation Assay for the accurate quantification of immune cell populations
Authors: Pradhan Suman , Dargitz Carl , Guzman Jerry , Lakshmipathy Uma
Published: August 2019

A key challenge during the development and manufacturing of cell based immunotherapies is the reliable estimation of cell purity. Current flow cytometric methods, while effective for the detection of surface antigens such as CD8 that positively identifies Cytotoxic T lymphocytes, are not ideal for intracellular markers used to positively identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Additionally, characterization methods that utilize live cells pose a challenge in large scale GMP manufacturing environments due to complicated logistics, limited throughput and difficulty in standardization. Therefore, there is an emerging need for alternative assay methods that address some of these challenges.

Here, we introduce our newly developed PureQuant™ Assays that specifically measure the unique DNA methylation status of specific immune cell types. Assays were developed specifically for Cytotoxic T Cells, Regulatory T cells and T helper 17 cells by detecting methylation status of CD8, FoxP3 and IL17A, respectively, via qPCR of bisulfite converted genomic DNA. These assays are robust with minimal sample requirement, utilizing fresh/frozen cells or isolated gDNA. Results are represented as a percentage of the total cell population similar to flow cytometry based methods. Comparison of the two methods for surface markers such as CD8 show comparable results. Our results demonstrate that this method which combines accuracy, low sample requirement and flexibility, is an ideal measurement system for confirmation of T cell identity and purity that is critical for cell therapy research and development.

Citation Format: Suman Pradhan, Carl Dargitz, Jerry Guzman, Uma Lakshmipathy. PureQuant Methylation Assay for the accurate quantification of immune cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 822.
Abstract 824: LKB1 loss in lung adenocarcinoma leads to global hypomethylation and altered FOXA binding
Authors: Koenig Michael J. , Amann Joseph , Agana Bernice , Kaufman Jacob , Wysocki Vicki , Oakes Christopher , Carbone David P.
Published: August 2019

LKB1 is functionally lost in 30% of lung adenocarcinoma cases. There are no targeted therapies available for LKB1-loss tumors. Previous studies of LKB1-loss in a mouse model of pancreatic cancer suggested that LKB1 loss drives DNA hypermethylation and serine biogenesis. DNA methyltransferases (DNMTs) maintain CpG methylation, which suppresses gene transcription. The primary substrate for DNMTs is S-adenosyl Methionine (SAMe), and the maintenance of CpG methylation has been shown to be dependent on continued regeneration of SAMe. KRAS status is associated both with LKB1 loss and with a CpG island hypermethylation phenotype (CIMP). Using a previously published 16-gene signature for LKB1 loss, we set out to confirm whether the changes that occur in the mouse model of pancreatic cancer also occur in lung cancer. We applied our signature to samples in The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset (signature-positive samples are described as “LKB1-loss”), which allowed us to compare both gene expression data as well as methylation data from Illumina 450k microarrays. We then developed a cell-line model for LKB1-loss in order to study the mechanism by which LKB1 regulates DNA methylation.

We observed that LKB1-loss is associated with widespread demethylation of the genome in TCGA samples. Approximately 14,000 CpG sites were significantly hypomethylated in LKB1-loss tumors, while only 200 were hypermethylated (adjusted p-value cutoff = 0.05). Furthermore, Student’s t-test showed that LKB1-loss samples have significantly lower average methylation (β-values), regardless of KRAS status. Using motif analysis, we found that demethylated CpG sites are significantly enriched for FOXA1/2/3 consensus binding sites. FOXA transcription factors are well known as pioneering transcription factors that mediate demethylation. We discovered in vitro that FOXA1 localization and turnover is dependent on LKB1 expression, specifically the activity of downstream kinase SIK. Rather than showing enhanced serine biogenesis, LKB1-loss tumors express less DNMT1 and LC-MS/MS analysis shows depletion of SAMe. Finally, in vitro models of LKB1-loss show an increased sensitivity to DNMT inhibitors and FOXA1 silencing.

Together, these observations suggest that LKB1-loss lung adenocarcinoma patients have a distinct hypomethylation profile. This profile may make them uniquely sensitive to DNMT-inhibition in combination with other therapies, a hypothesis which we will test in future studies.

Citation Format: Michael J. Koenig, Joseph Amann, Bernice Agana, Jacob Kaufman, Vicki Wysocki, Christopher Oakes, David P. Carbone. LKB1 loss in lung adenocarcinoma leads to global hypomethylation and altered FOXA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 824.
Abstract 828: Accelerated epigenetic aging in bladder cancer patients
Authors: Chew Yap Ching , Guo Wei , Yang Xiaojing , Piatti Paolo , Jin Mingda , Booher Keith , Jara Benjamin , Jia Xi-Yu
Published: August 2019

Aging represents the most important risk factor for many chronic diseases including cardiovascular diseases, diabetes, and cancer, therefore understanding the mechanisms of aging is a fundamental step for designing new treatments for chronic diseases. DNA methylation is the most reliable and accurate molecular marker for aging quantification, however, genome-wide DNA methylation profiling techniques, such as reduced representative bisulfite sequencing and Illumina Bead Array that are widely used in aging research are prohibitively expensive and have poor data quality at low-read coverage sites. Here we report a robust targeted bisulfite sequencing approach, called SWARM® (Simplified Whole-panel Amplification Reaction Method), for the accurate biological age determination. SWARM™ is flexible and low cost, requires relatively low DNA starting material, allows the simultaneous amplification and sequencing of hundreds of loci, and has shown to increase sample throughput. Using the SWARM® approach, we were able to analyze the methylation level of several hundreds of age-associated loci including the published Horvath Clock sites. Gender-specific age-predictive models were built using the elastic net regression of DNA methylation levels of the loci and chronological age of urine DNA samples of over 300 healthy subjects of 18 to 88 years old. Urine samples from bladder cancer patients exhibit significant age acceleration, with an average of >10 years. In brief, our gender-specific urine DNAge® analysis is a tool for the precise biological aging quantification and can be used to address questions in aging and urinary track cancers.

Citation Format: Yap Ching Chew, Wei Guo, Xiaojing Yang, Paolo Piatti, Mingda Jin, Keith Booher, Benjamin Jara, Xi-Yu Jia. Accelerated epigenetic aging in bladder cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 828.
Abstract 845: c-MET nuclear transportation via membrane-bounded retrograde trafficking in breast cancer cells
Authors: Chen Mei-Kuang , Du Yi , Sun Linlin , Hsu Jennifer L. , Wang Yu-Han , Gao Yuan , Huang Jiaxing , Hung Mien-Chie
Published: August 2019

Reactive oxygen species (ROS) can be induced by anti-cancer treatments, such as ionizing irradiation and chemotherapy drugs. Hydrogen peroxide (H2O2) is one of the ROS species highly produced in cancer cells due to rapid proliferation or anti-cancer treatments. ROS serve as important intracellular messengers in cell growth and proliferation signaling pathways. However, ROS also causes cellular stress in cancer cells through oxidizing DNA, protein and lipids. Therefore, balancing ROS stress between signaling and damages at a tolerable level is important for cancer cell survival. c-MET is one of the receptor tyrosine kinases overexpressed in breast cancer. Full-length c-MET is known to be oncogenic for triggering signal transduction cascade from plasma membrane to upregulate cell proliferation and migration in cancer cells. Moreover, c-MET is reported to phosphorylate PARP1 under H2O2 stimulation, and the phosphorylated PARP1 resulting in PARP inhibitor resistance in breast cancer cells. However, it is not yet clear how c-MET travels from cell membrane to the nucleus. Here, we demonstrate that H2O2 induces retrograde transport of c-MET from plasma membrane into the nucleus and that knocking down either COPI or Sec61β attenuates H2O2-induced full-length nuclear c-MET accumulation. However, c-MET kinase inhibitor did not block the nuclear transport of c-MET, suggesting the transportation mechanisms is independent from kinase activity. Our results indicate that membrane-bound retrograde vesicles involve in membrane-to-nucleus transportation of c-MET in breast cancer cells. Moreover, nuclear c-MET interacts with both PARP1 and Ku proteins in breast cancer cells, suggesting the role of full-length nuclear c-MET in mediating repair of ROS-induced DNA damages.

Citation Format: Mei-Kuang Chen, Yi Du, Linlin Sun, Jennifer L. Hsu, Yu-Han Wang, Yuan Gao, Jiaxing Huang, Mien-Chie Hung. c-MET nuclear transportation via membrane-bounded retrograde trafficking in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 845.
Abstract 852: Determining the roles of ERO1-alpha and ERO1-beta in progression of multiple myeloma and drug resistance
Authors: Chen Weichih , Hazlehurst Lori
Published: August 2019

Multiple myeloma (MM) is a hematological malignancy characterized by dysregulated growth and accumulation of monoclonal plasma cells within the bone marrow. It is a treatable but incurable disease. Despite improvement in progression-free and overall survival of MM patients nearly all MM patients will relapse and become resistant to current treatments. Therefore, to find an effective therapeutic target that is applicable to MM, our laboratory analyzed a retrospective study, patients with relapsed MM who had received bortezomib therapy were enrolled in phase 2 and phase 3 clinical trial. Patients who had higher ERO1-alpha expression demonstrated significant poorer outcomes as compared to patients with lower ERO1-alpha expression. Moreover, patients with relapsed myeloma had higher expression of ERO1-alpha protein levels compared to newly diagnosed MM patients, suggesting that ERO1-alpha correlated with disease progress and poor survival of MM patients. Disulfide bond formation is critical for the folding, stability and function of newly synthesized secretory and membrane proteins in endoplasmic reticulum (ER). Endoplasmic reticulum oxidoreductase 1 (ERO1) is an essential component in this process. ERO1-alpha and ERO1-beta are two flavoprotein isomers that have been identified in mammalian cells. ERO1-alpha is widely expressed and up-regulated during oxidative stress and ERO1-beta is only expressed in select tissue and induced by unfold protein response (UPR). We found that knockdown of ERO1-alpha MM cells caused cell growth inhibition and decreased colony formation and lambda secretion. Moreover, overexpression of ERO1-alpha enhanced cell growth. Additionally, reducing the expression of ERO1-alpha resulted in upregulated ERO1-beta by transcriptional and translational levels. Reducing the expression of ERO1-beta in MM cells increased cell growth and induced resistance to current treatments, including the proteasome inhibitor (bortezomib), alkylating agent (melphalan), corticosteroids (dexamethasome) and histone deacetylase inhibitor (panobinostat). We found reducing the expression of ERO1-beta resulted in decreased expression of glucocorticoid receptor (GR). Taken together, our data indicate that ERO1 -alpha and -beta are not functionally redundant as increased alpha expression correlates with MM progression while increased beta expression correlates with increased sensitivity to chemotherapeutic agents. Future studies are aimed at delineation of mechanism underlying the divergent phenotypes that result from reducing ERO1- alpha or -beta in MM cells.

Citation Format: Weichih Chen, Lori Hazlehurst. Determining the roles of ERO1-alpha and ERO1-beta in progression of multiple myeloma and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 852.
Abstract 867: EGFR kinase domain duplication (EGFR-KDD) is activated by asymmetric intra-molecular dimerization
Authors: Du Zhenfang , Gallant Jean-Nicolas , Red-Brewer Monica , Brown Benjamin , Sheehan Jonathan , Meiler Jens , Lovly Christine M.
Published: August 2019

Recently, we identified a novel oncogenic EGFR exon 18 - 25 Kinase Domain Duplication (EGFR-KDD) in patients with lung cancer (PMID: 26286086). This novel EGFR alteration contains two tandem, in-frame EGFR TKDs. Computational modeling suggested that the linker between the two TKDs may support the formation of an asymmetric intra-molecular dimer. In this study, we sought to provide experimental support for the model that EGFR-KDD could form ligand-independent asymmetric intra-molecular dimers. We constructed point mutations (Table 1) disrupting the potential intra- (C1, N2) and inter-molecular dimer interfaces (N1 and C2) between each TKD of EGFR-KDD. We also introduced catalytically inactivating mutation into each TKD individually (Dead1 and Dead2). We stably expressed these mutants in YAMC and NR6, two cell lines lacking endogenous wild-type EGFR expression. In the absence of ligand, ‘wild-type’ EGFR-KDD was phosphorylated on known EGFR auto-phosphorylation sites, Y992, Y1068, and Y1173. However, mutations of the intra-molecular dimer interface (C1, N2) abrogated EGFR-KDD auto-phosphorylation, which is an accepted surrogate for EGFR enzymatic activity. In support of these data, expression of EGFR-KDD, but not the intra-molecular dimer interface mutants (C1, N2), supported growth in soft agar, an assay utilized in support of oncogenic phenotypes. Interestingly, phosphorylation of the dimer interface mutants (C1, N2) was rescued by ligand stimulation, suggesting the formation of EGFR-KDD multimers. Our structural modeling by Rosetta suggests that EGFR-KDD can form high order multimers with itself or with wild-type EGFR, and we observed co-precipitation of EGFR-KDD with itself and wild-type EGFR in vitro. Collectively, our findings define a previously unrecognized model of asymmetric intra-molecular dimers for EGFR, which provides important insights for the understanding of EGFR activation mechanisms and shed light on the exploration of therapeutic benefit.

Table 1:Kinase domain mutations constructed for these studiesLobeMutationFunctionsN1I706QDisruption of the 1st N-lobe interfaceDead1D837NKinase dead mutation of the 1st kinase domainC1V948RDisruption of the 1st C-lobe interfaceN2I1057QDisruption of the 2nd N-lobe interfaceDead2D1188NKinase dead mutation of the 2nd kinase domainC2V1299RDisruption of the 2nd C-lobe interface

Citation Format: Zhenfang Du, Jean-Nicolas Gallant, Monica Red-Brewer, Benjamin Brown, Jonathan Sheehan, Jens Meiler, Christine M. Lovly. EGFR kinase domain duplication (EGFR-KDD) is activated by asymmetric intra-molecular dimerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 867.
Abstract 879: Nicotine-induced oxidative stress causes epigenetic alterations during malignant transformation of human kidney epithelial cells
Authors: Chang Yuwei , Singh Kamaleshwar
Published: August 2019

Nicotine is a component of cigarette smoke and mounting evidence implicates tobacco smoking in kidney cancer development. Whether nicotine alone can cause kidney cancer is not clear. Moreover, the underlying molecular mechanisms for nicotine-induced carcinogenesis is still not well-understood. Therefore, the objective of this study was to determine if chronic exposure to nicotine results in malignant transformation of HK-2 kidney epithelial cells. The data revealed that chronic exposure to nicotine induced growth and malignant transformation in HK-2 cells. Additionally, the nicotine-exposed cells had inherently increased levels of intracellular ROS, and acquired stem cell-like sphere formation, as well as epithelial-mesenchymal-transition (EMT) changes during malignant transformation. Treatment with antioxidant NAC resulted in abrogation of EMT and stem cell-like sphere in HK-2 cells, suggesting the role of nicotine-induced ROS in these morphological changes. Additionally, epigenetic reagents 5-aza-2'-deoxycytidine and trichostatin A also diminished stem cell-like sphere in nicotine-exposed HK-2 cells. The nicotine also induced changes in the expression of epigenetic genes (DNMT3a, DNMT3b, and HMT1) and these changes were reversed by NAC treatment. This further supports the role of nicotine-induced ROS in epigenetic changes during malignant transformation of HK-2 cells. The altered levels of pAKT in low dose nicotine exposed cells were restored after NAC treatment, suggesting that nicotine-induced ROS, through regulation of AKT pathway, controls the EMT and stemness during early stages of carcinogenesis. In summary, to our knowledge, this is the first report showing that chronic exposure to nicotine induces malignant transformation in human kidney epithelial cells through ROS-mediated epigenetic modifications.

Citation Format: Yuwei Chang, Kamaleshwar Singh. Nicotine-induced oxidative stress causes epigenetic alterations during malignant transformation of human kidney epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 879.
Abstract 891: Plumbagin and atovaquone inhibit Na+/K+-ATPase through the generation of oxidative stress
Authors: Alharbi Yousef , Kapur Arvinder , Felder Mildred , Barroilhet Lisa , Pattnaik Bikash , Patankar Manish S.
Published: August 2019

Plumbagin and atovaquone are chemotoxic to ovarian, breast and other tumors. Both molecules inhibit oxidative phosphorylation causing a rapid increase in intracellular oxygen radicals and apoptosis. Oxidative stress is also known to inhibit the activity of Na+/K+-ATPase (NKA), the ion channel that maintains the membrane potential. Here, we investigate if the oxidative stress mediated by plumbagin and atovaquone also leads to inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit proliferation of three human (OVCAR-3, SKOV-3 and TYKNu) and one mouse (ID8) ovarian cancer cell lines. Using SKOV-3 and OVCAR-3 as models for ovarian cancer, we demonstrate that oxygen radical scavenger, N-acetylcysteine (NAC) attenuated the cytotoxicity of plumbagin and atovaquone. Using whole cell patch clamping we demonstrate that plumbagin and atovaquone inhibit outward and inward current flowing through NKA in SKOV-3 and OVCAR-3. Both drugs decrease cellular ATP. Providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either of the drugs for 1-2 h resulted in a significant decrease in the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA and hence the membrane potential cannot be optimally maintained. Evaluation of compounds that induce oxidative stress should therefore consider contribution of NKA inhibition to the cytotoxic activity of such agents.

Citation Format: Yousef Alharbi, Arvinder Kapur, Mildred Felder, Lisa Barroilhet, Bikash Pattnaik, Manish S. Patankar. Plumbagin and atovaquone inhibit Na+/K+-ATPase through the generation of oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 891.
Abstract 895: The identification of molecular mechanism underlying the progression of gastric cancer peritoneal dissemination by senescent fibroblasts
Authors: Yasuda Tadahito , Koiwa Mayu , Uchihara Tomoyuki , Yonemura Atsuko
Published: August 2019

Background: Secreted factors from cancer associated fibroblast (CAFs) is likely to be implicated in cancer progression, but the precise mechanism is not fully understood. It is reported that senescent cells survive and secrete some cytokines, so called senescence-associated secretory phenotype (SASP). The purpose of current study is to identify the molecular mechanism underlying the progression of gastric cancer (GC) peritoneal dissemination by CAFs showing SASP.

Methods: We examined the senescent state of CAFs activated NFkB signaling by inflammatory cytokines (iCAFs) and evaluated the expression of SASP factors. We investigated the growth ability of GC cell lines treated with iCAFs derived condition medium (CM). We transplanted GC cell lines with intraperitoneal administration to generate peritoneal metastasis mouse model and evaluated the influence of SASP factors on cancer progression.

Results: We found that the growth ability of iCAFs decreased and their expression of p16 and p15, which was senescent markers increased. And the comprehensive genomic analysis revealed that iCAFs was highly enriched the senescent related genes and SASP factors related genes. Next, we also found that iCAFs derived CM significantly increased the growth ability of GC cells. Moreover, also in peritoneal metastasis mouse model, iCAFs derived CM remarkably enhanced intraperitoneal tumors.

Conclusion: These findings suggest that SASP factors from senescent CAFs, which activated by inflammatory cytokines can promote peritoneal dissemination of GC cells.

Citation Format: Tadahito Yasuda, Mayu Koiwa, Tomoyuki Uchihara, Atsuko Yonemura. The identification of molecular mechanism underlying the progression of gastric cancer peritoneal dissemination by senescent fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 895.
Abstract 896: Knocking out the gene (SNCA) that codes for the Parkinson’s disease-related protein alpha-synuclein in SK-Mel-28 melanoma cells significantly retards tumor growth in SCID mice
Authors: Shekoohi Sahar , Rajasekaran Santhanasabapathy , Yang Shu , Nagarajan Sureshbabu , Patel Dhaval , Yu Xiuping , Witt Stephan N.
Published: August 2019

The Parkinson’s disease-associated protein alpha-synuclein (α-syn) is often highly expressed in melanoma. α-Syn appears to exert a pro-survival function in melanoma cells, possibly by maintaining autophagy homeostasis. The long-term goal of our work is to determine the mechanism by which α-syn confers survival in melanoma cells. Herein, we used CRISPR (clustered regularly interspaced short palindromic repeats) to disrupt SNCA, the gene that codes for α-syn, in the melanoma cell line SK-Mel-28. Western blotting and real-time-qPCR verified that we generated four α-syn knockout (KO) clones. Three clones showed no expression of α-syn, whereas one had about 50% less α-syn than the control cells. DNA sequencing is consistent with three of the clones having both alleles of SNCA knocked out, whereas one clone has only one allele knocked out. α-Syn KO clones were tested in a variety of assays to ascertain the function of α-syn. First, the percentage of cells with exofacial phosphatidylserine (PS) was determined by annexin V-FITC/PI staining in conjunction with fluorescence-activated cell sorting. On average, there was a statistically significant 50% increase in exofacial PS (annexin V+/PI-) in three of the four α-syn KO clones compared to control cells. Exofacial PS was increased in the α-syn KO clones without a concomitant increase in apoptotic cells (annexin V+/PI+). Second, the clones were tested for growth in vivo using SCID mice. All four clones showed a ~65% reduction in the rate of tumor growth, followed over 12 weeks. Third, immunohistochemical staining of excised tumors from the SCID mice showed that the proliferation marker Ki-67 was significantly (p< 0.0001) lower in the tumors generated from the KO clones compared to tumors from control cells. In conclusion, given that α-syn avidly binds to PS, one possibility is that α-syn is like molecular Velcro that binds to PS molecules embedded in the inner leaflet, and this binding helps ATP-driven flippases keep PS in the inner leaflet. Loss α-syn then increases exofacial PS, which is a marker of senescence. Our working model is that α-syn is an anti-senescence protein, and that the α-syn KO clones grow slower than control cells in vivo because the clones have entered a senescent state.

Citation Format: Sahar Shekoohi, Santhanasabapathy Rajasekaran, Shu Yang, Sureshbabu Nagarajan, Dhaval Patel, Xiuping Yu, Stephan N. Witt. Knocking out the gene (SNCA) that codes for the Parkinson’s disease-related protein alpha-synuclein in SK-Mel-28 melanoma cells significantly retards tumor growth in SCID mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 896.
Abstract 903: Real-time imaging of cellular senescence in living animals
Authors: Cui Lina
Published: August 2019

Senescence, a cellular stage that halts proliferation, plays a critical role in cellular processes such as tumor suppression, tumorigenesis and aging. Monitoring of senescence in real-time in living objects not only holds value in basic biological studies, it can also benefit treatments of various diseases such as cancer and age-related conditions. Our lab has developed a series of small molecule based agents for non-invasive imaging of cellular senescence in real-time. We have demonstrated their applications in tumor models receiving chemo- or radiotherapy, as well as aging mice models. These probes will be useful for treatment response monitoring, therapeutic regimen optimization, as well as imaging-guided removal of aged cells.

Citation Format: Lina Cui. Real-time imaging of cellular senescence in living animals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 903.
Abstract 904: Hypoxic attenuation of ΔB-Raf-induced senescence
Authors: Chang Soojeong , Park Hyunsung
Published: August 2019

Oncogene-induced senescence (OIS) is natural surveillance mechanisms against tumorigenesis. The in-vivo oxygen concentration around tumor microenvironment has been estimated as 3%~0.5%. We investigate the effects of physiological hypoxia (1.5% O2) and pathological hypoxia (<0.5% O2) on OIS. Expression of active ΔB-Raf in human lung fibroblast IMR90 cells triggered senescence processes such as activation of senescence associated-β-galactosidase (SA-β-gal) and senescence associated heterochromatin foci (SAHF). We found that hypoxic condition delayed onset of activation of SA-β-gal. Furthermore hypoxic condition blocked formation of heterochromatin foci which was identified as a hallmark of OIS with increase of histone methylation. Rearrangement of chromatin organization by activated ΔB-Raf was also inhibited under hypoxic condition. This study shows that tumor hypoxia assists the transformed cells to overcome an obstacle of senescence caused by oncogenes. [This study was supported by the National Research Foundation of Korea (NRF), grant 2016R1A2B4012840 and 2018R1A4A1025985]

Citation Format: Soojeong Chang, Hyunsung Park. Hypoxic attenuation of ΔB-Raf-induced senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 904.