bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1107: Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma
Authors: Campbell Nathaniel R. , Zhang Maomao , Baron Maayan , Heilmann Silja , Deforet Maxime , Ferretti Lorenza , Huang Ting-Hsiang , Levesque Mitchell P. , Yanai Itai , Xavier Joao B. , White Richard M.
Published: September 2020

Metastasis is the primary cause of cancer death, yet we know little about what determines its timing and aggressiveness. In melanoma, transcriptional profiling has revealed multiple co-existing cell states, including proliferative (PRO) versus invasive (INV) subpopulations that have been posited to represent a “go or grow” tradeoff in the etiology of metastasis. Although both of these populations are present in primary tumors and metastases, little is known about how they physically interact with each other in tumor dissemination. We use a combination of melanoma modeling in zebrafish and human validation to demonstrate that these subpopulations form spatially structured heterotypic clusters that cooperate in the seeding of metastasis. We unexpectedly found that INV cells were tightly adherent to each other and formed large tumor cell clusters. Mixing of PRO and INV cells generated heterotypic clusters, composed of a dense central core of INV cells surrounded by a rim of PRO cells. Using intravital imaging, we identified metastatic cooperation between these two subpopulations, in which the INV cells facilitated the earlier spread of the less metastatic PRO cells via collective extravasation. Mechanistically, we identified the TFAP2 family of neural crest transcription factors to be a master regulator of both clustering and the PRO/INV states. Knockout of TFAP2 from melanoma cells reduced proliferation but increased clustering and metastasis. TFAP2 loss resulted in increased expression of cell adhesion molecules, providing a mechanistic basis for cluster formation. Our data suggest a coherent framework for the co-existence of these two divergent cell populations in melanoma, in which differing cell states form heterotypic clusters that promote collective metastasis via cell-cell cooperation.

Citation Format: Nathaniel R. Campbell, Maomao Zhang, Maayan Baron, Silja Heilmann, Maxime Deforet, Lorenza Ferretti, Ting-Hsiang Huang, Mitchell P. Levesque, Itai Yanai, Joao B. Xavier, Richard M. White. Cell state diversity promotes metastasis through heterotypic cluster formation in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1107.
Abstract 112: Telomere proteins coordinate intracellular signaling to control telomere maintenance mechanism selection and metastatic recurrence
Authors: Robinson Nathaniel J. , Taylor Derek J. , Schiemann William P.
Published: September 2020

The relationship between telomere homeostasis and the development of cancer remains incompletely understood. In breast cancer (BC), these knowledge gaps are magnified by the fact that distinct telomere maintenance mechanisms (TMMs) are utilized across different disease subtypes and stages of progression. Accordingly, we have found that TMM identity in BC possesses an inherent plasticity that portends important prognostic and therapeutic insight, events regulated in part by SLX4-interacting protein (SLX4IP). To better understand how SLX4IP regulates TMM identity in BC, we employed proteomics of isolated chromatin segments (PICh) to capture the telomere proteomes of BC cells harboring differential SLX4IP expression. These analyzes revealed that SLX4IP blocks recruitment of the shelterin protein telomere repeat-binding factor 2-interacting protein (TERF2IP, also known as RAP1) to telomeres. Moreover, telomeres lacking RAP1 were able to be extended via the homologous recombination-based Alternative Lengthening of Telomeres (ALT) pathway. Mechanistically, SLX4IP disrupted the interaction between RAP1 and telomere repeat-binding factor 2 (TRF2), which facilitated the nuclear export of RAP1 and its binding to and activation of the NF-κB regulatory kinase, IKKβ. Activated IKKβ phosphorylated both NF-κB and β-catenin, thereby coordinating a transcriptional network of genes that are essential in promoting ALT. Conversely, loss of SLX4IP yielded telomeric retention of RAP1 with concomitant diminution of IKKβ activity, resulting in β-catenin-dependent stimulation of telomerase. Notably, telomerase was sufficient to drive the metastatic outgrowth of cells that were previously reliant upon ALT independent of its ability to extend telomere DNA. These findings shed new light on the functions of SLX4IP and the myriad roles of telomere proteins in influencing TMM plasticity and disease progression.

Citation Format: Nathaniel J. Robinson, Derek J. Taylor, William P. Schiemann. Telomere proteins coordinate intracellular signaling to control telomere maintenance mechanism selection and metastatic recurrence [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 112.
Abstract 1130: Cultural and socioeconomic factors affecting breast cancer screening in an Argentine city
Authors: Croce Maria Virginia , Cermignani Luciano , Rabassa Martin E.
Published: September 2020

In Argentina, there are no studies evaluating cultural and socioeconomic factors affecting breast cancer screening. Argentina presents social and economic disparities, and there is a mixture of features of both developed and developing societies. The goal of this research was to analyze cultural and social factors in advantaged and disadvantaged women of a metropolitan area in order to improve breast cancer screening by increasing the understanding of the complex society. A cross-sectional study was performed; a total of 739 women was included, being 379 of low economic power (ow group, LG) and 360 of middle economic power (MG); women were personally interviewed within three months. A structured previously validated questionnaire was employed considering socially shared values, beliefs, expectations, motivations, and emotions relevant to health behaviors, interactions with the health care system, educational level, occupation, and information about breast cancer and mammographic screening. Previous research in this area suggested that theoretical models and multivariate methods were needed in order to account for the complexity of relations among psychological, social structural, and cultural determinants of health behaviors. In this sense, an exhaustive statistical analysis was performed by multivariate analysis and logistic regression model. A Principal Component Analysis was performed employing previously selected variables; 3 factors were extracted which accounted for 35.8% of the total variance. Factor 1 was associated with fatalistic responses in association to attitudes to life and/or mammography screening while Factor 2 was related to concerns about cancer and exposition to radiation. These two factors and the covariates age, social group, education, health system, and primary doctor were use in a logistic regression model to assess mammographic adherence. Low score Factor 2 (p=0.003), MG (p=0.024) and the availability of a primary doctor (p=0.000) were associated with high mammographic adherence. Conclusions: This study highlights that mammographic screening is highly influenced by socioeconomic power, health system, and access to primary doctor. It appears that, some psychological factors related to concerns about cancer and exposition to radiation are also important. These data would be important to plan specific prevention and early diagnosis programs to be implemented by governmental entities.

Citation Format: Maria Virginia Croce, Luciano Cermignani, Martin E. Rabassa. Cultural and socioeconomic factors affecting breast cancer screening in an Argentine city [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1130.
Abstract 1134: Developing an early expression-based signature to assess interval colorectal carcinoma risk
Authors: Cheah Peh Yean , Thean Lai Fun , Lam Kuen Kuen , Liew Soo Chin , Wong Michelle , Koh Nicholas , Lo Michelle , Tan Emile , Tang Choong Leong
Published: September 2020

Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer mortality in the developed world including Singapore. Despite an aging populace which is now more compliant to having a baseline colonoscopy at age 50 or earlier, interval cancer (cancer that develops in between colonoscopies) is an unmet critical problem. The identification of early driver genes that can serve as biomarkers for CRC risk will aid in clinical management decision to counter cancer-associated mortality and morbidity. We profiled the genome-wide expression of matched mucosa (M), mucosa next to tumor (MT) and tumor (T) samples of a series of 23 patients using Affymetrix Gene Array. M samples are at least 20 cm away from the tumor and MT samples are adjoining the T samples but maintained the normal colonic crypt architecture of M. We discovered that thirty-nine dysregulated genes in MT samples compared to M samples persists until tumor formation. These early driver genes comprise not only tumor suppressors or oncogenes but included metabolic and transporter genes indicating that transformation towards malignancy requires the disruption of many pathways. Majority of these genes were down-regulated; the top three most drastically down-regulated genes include AQP8 (a water transporter), CA7 (a metabolic gene) and PHLPP2 (a tumor suppressor). Further analysis indicates that the three genes were already down-regulated in the normal appearing mucosa of a ‘healthy' individual compared to age-, gender-, ethnicity-matched (AGE-) controls seven years before recto-sigmoid cancer was diagnosed. This suggests that the dysregulation can occur several years before tumor formation and hence could possibly be used as early or pre-cancer biomarker. We have validated the down-regulation of the 3 genes in an independent series of 135 CRC samples from the same center using real-time RT-PCR assays. Correlation of the expression with clinicopathological features of the patients (gender, tumor site, age-group) and molecular subtypes (CMS 1-4) of the tumors will be ascertained to assess whether the signature is applicable to all CRC subtypes. Furthermore, the expression in matched blood samples will also be determined to explore feasibility of using these samples for assessing interval colorectal carcinoma risk.

Citation Format: Peh Yean Cheah, Lai Fun Thean, Kuen Kuen Lam, Soo Chin Liew, Michelle Wong, Nicholas Koh, Michelle Lo, Emile Tan, Choong Leong Tang. Developing an early expression-based signature to assess interval colorectal carcinoma risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1134.
Abstract 1148: Salivary auto-antibodies as diagnostic markers of oral cavity squamous cell carcinoma
Authors: Hsueh Pei Chun , Wu Chih Ching
Published: September 2020

Oral cavity squamous cell carcinoma (OSCC) is a destructive disease with its increasing new cases annually. OSCC is usually diagnosed at advanced clinical stage which leads to the poor outcomes of patients. Currently, no biomarkers provide capability for sufficient early detection of OSCC. Cancer-associated auto-antibody (auto-Ab) holds potentials as promising biomarkers for cancer detection because auto-Ab is easily detected with well-established secondary reagents. To ameliorate the detection of OSCC, we evaluated the capability of auto-Ab to 9 proteins as the OSCC biomarkers with multiplexed bead-based immunoassay, as the salivary levels of the 9 proteins are elevated in OSCC patients. The multiplexed bead-based assay has been established to simultaneously detect the salivary levels of the 9 auto-Abs and anti-p53, a well-characterized OSCC biomarker. Compared to the healthy individuals, the salivary levels for 8 of 9 auto-Abs as well as anti-p53 were significantly elevated in OSCC patients. The salivary levels of the 7 of 9 auto-Abs were significantly increased in individuals with oral potentially malignant disorder compared to the control group. In addition, the salivary levels of 8 of 9 auto-Abs as well as anti-p53 were statistically significant in early-stage OSCC patients. Moreover, the marker panel of the 9 auto-Abs can improve the detection of OSCC. Collectively, we have established the platform for detection of maker panels and the salivary auto-Abs are potential biomarkers for OSCC screening.

Citation Format: Pei Chun Hsueh, Chih Ching Wu. Salivary auto-antibodies as diagnostic markers of oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1148.
Abstract 1162: Amelioration of the pathologic changes in the hepatocyte-specific BRAF V600E mutated mice by administration of aspirin
Authors: Tanaka Hiroki , Horioka Kie , Yamamoto Masahiro , Yamazaki Kosuke , Ogawa Katsuhiro
Published: September 2020

We previously reported that the BrafV637E mutation, corresponding to the human BRAFV600E mutation, plays a pivotal role in hepatocarcinogenesis induced by diethylnitrosamine (DEN) in B6C3F1 mice (Mol Carcinog 2017;56:478-488). The livers of transgenic mice with a hepatocyte-specific human BRAFV600E mutation weighed 4.5 times greater than that of normal mice and was entirely consisted of hepatocytes resembling the DEN-induced neoplastic hepatocytes. However, these transgenic mice spontaneously died seven weeks after birth due to thrombotic microangiopathy accompanying interstitial pneumonia, glomerulonephropathy and erythrocyte dyscrasia (Cancer Sci 2019; 110:2748-2759). The hepatocytes of the transgenic mice showed thrombopoietin (TPO) overexpression which is associated with eventual megakaryocytosis and thrombocytosis, and activated platelets were deposited in various tissues including hepatic sinudoids, pulmonary septa and renal glomeruli. Particularly, platelets were sparsely adhered to the sinusoidal endothelial cells (LSEC) and densely adhered to the cell surface and incorporated into the cytoplasm of Kupffer cells in the liver sinusoids. TPO was also overexpressed in the DEN-induced hepatic tumors, and sinusoidal platelet deposition was observed in hepatic tumors of humans and mice, indicating that the hepatic microenvironment of the transgenic mice mimics that of the hepatic tumors. In the transgenic mice, Aspirin (Asp) administration prevented platelet activation, reduced the liver vs body weight ratio, decreased the platelet deposition in the liver, kidney and lung, prevented the erythrocyte dyscrasia and ameliorated the renal and pulmonary changes. In particular, Asp administration turned the neoplastic hepatocyte morphology to the normal morphology in the peripheral part of hepatic lobules, where the sinusoidal platelet deposition was diminished. The platelet factors are necessary for hepatic regeneration, and the interaction of platelets to sinusoidal cells can trigger the production of cytokines that are necessary for hepatocyte proliferation by LSEC and Kupffer cells. Therefore, the macroenvironmental changes including the TPO overproduction by the neoplastic hepatocytes, the eventual megakaryocytosis/thrombocytosis, the platelet activation, and platelet interaction with hepatic sinusoidal cells may contribute to hepatocarcinogenesis from a very early stage. Also, inhibition of platelet activation by Asp may have a preventive effect on hepatocarcinogenesis.

Citation Format: Hiroki Tanaka, Kie Horioka, Masahiro Yamamoto, Kosuke Yamazaki, Katsuhiro Ogawa. Amelioration of the pathologic changes in the hepatocyte-specific BRAF V600E mutated mice by administration of aspirin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1162.
Abstract 1175: Race informs survival disparities in head and neck cancer clinical trials
Authors: Liu Jeffrey C. , Egleston Brian , Blackman Elizabeth , Ragin Camille
Published: September 2020

Background: Survival disparities between blacks and whites in head and neck cancer are well documented, with access to care and socioeconomic status (SES) as major contributors. However, the impact of race alone on survival is much less clear. Previous attempts to evaluate the role of race have been limited by access to care/SES as major confounders. We set out to directly assess the role of race in head and neck cancer by evaluating treatment outcomes of subjects enrolled in clinical trials. In this population, access to care and socioeconomic confounders are minimized because all patients receive identical treatments.

Methods: Clinical trial data from the Radiation Therapy Oncology group (now NRG) studies were obtained by request. Seven RTOG Studies (RTOG 0129, 0234, 9003, 9111, 9501, 9512, 9703) were included for study. Studies were included if they were phase II or III trials that employed survival as an endpoint, e.g. progression free survival (PFS), disease free survival (DFS), overall survival (OS). Studies that were small (<200 patients) or did not use survival as an endpoint (e.g. side effect trial) were excluded. For each black patient enrolled in a clinical trial, a white patient matched in the same arm of that trial was used as a control. The study null-hypothesis was that there would be no race derived impact on survival and thus the chance of survival favoring either whites or blacks in each matched pair would be 50%.

Results: Across the seven trials, 468 blacks were identified and matched with 468 white study arm specific controls. Whites had better outcomes than blacks in 60% of matched pairs (p<0.001). Blacks were consistently more likely to have worse outcomes. When outcomes were collapsed by PFS/DFS, the failure rate was significantly higher in blacks (HR=1.46, p<0.001)). Failure was largely due to locoregional failure, and blacks were at higher risk (sHR =1.43, p=0.002). The development of distant metastasis within the paired cohorts was not significantly different (sHR=1.15, p=0.38), suggesting that locoregional failure was the major driver of failure.

Conclusions: In this analysis of head and neck cancer patients in clinical trials, blacks consistently had worse outcomes in comparison to study arm specific white controls. This suggests that race alone confers a worse survival independent of socioeconomic and access to care factors. Further study is needed to confirm these findings, and to explore other causes underlying this disparity.

Citation Format: Jeffrey C. Liu, Brian Egleston, Elizabeth Blackman, Camille Ragin. Race informs survival disparities in head and neck cancer clinical trials [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1175.
Abstract 1190: Comparison of cumulative incidence rates of colorectal cancer among Asians and Pacific Islanders (API) populations in Guam, Hawai'i, and the United States
Authors: Badowski Grazyna
Published: September 2020

Background: Colorectal cancer is one of 4 most common cancers on Guam, Hawai'i (HI) and the US. However, incidence rates vary between ethnic groups and location.

Objective: The objective of the study was to compare cumulative incidence rates (CIRs) among ethnic groups in Guam, HI, and the US; specifically, Chamorros in Guam, Filipinos in Guam, HI and the US, and Native Hawaiians in HI and the US. CIRs are weighted sums of age-specific incidence rates therefore they can be used for comparisons between populations. They are relatively stable and thus provide a good estimate of cumulative risk of cancer.

Methods: This study included all colorectal cancer cases (n=562) reported to the Guam Cancer Registry and all colorectal cancer cases (n=253,782) reported to the Surveillance, Epidemiology, and End Results (SEER) 9-Registry from 2000 to 2014. Cumulative incidence rates were calculated for three time periods: 2000-2004, 2005-2009, and 2010-2014 and compared by age, ethnicity and location.

Results: Differences were seen in CIRs by time-period, age, ethnicity, and location. For all three time periods: 2000-2004, 2005-2009, 2010-2014, the lifetime cumulative incidence rates (age 0-79) were highest for Hawai'i (5.7%, 5.4%, and 5.3% respectively), followed by the US (5.2%, 4.5%, and 3.7%) and Guam (4.3%, 3.3%, and 3.6%). Interestingly, there were large differences in lifetime CIRs between Filipinos in HI, Filipinos in the US and Filipinos on Guam (i.e. 6.5%, 3.7%, and 2.4% respectively in 2005-2009). The Chamorros on Guam had the rate at least 1.5 times higher than Filipinos on Guam (4.3 % vs 3.3%) and close to the rate of Native Hawaiians in HI (4.9%). Overall, there was downward trend for the lifetime CIRs over time. Only for the Filipinos in HI, the rates went up. However, early onset CIRs (age 0-49) went up across all locations and ethnic groups.

Conclusions: Disparities in colorectal cancer incidence were identified. Further study is needed to examine how exposure to risk factors and screening changes with immigration and how this variation contributes to the incidence disparities.

Citation Format: Grazyna Badowski. Comparison of cumulative incidence rates of colorectal cancer among Asians and Pacific Islanders (API) populations in Guam, Hawai'i, and the United States [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1190.
Abstract 1207: Custom next-generation sequencing primer designs for targeted sequencing of multi strain viral targets
Authors: Singh Ratnesh , Li Na , Luo Zunping , McGeachy Anna , Alcantara Antonio F. Martinez , Ewing Aren , Hyland Fiona C.
Published: September 2020

Viruses are infectious agents that can cause many diseases including cancers. A viral species is comprised of many strains each having a unique pathology. For example, over 170 types of HPV have been described, of which only 2 strains, HPV16 and HPV18, are implicated in 70% of cervical cancer cases. Accurate identification of viral strains is often critical and also facilitates research into their association with prognosis or response and personalized therapies. Currently, sequencing virus genes or genome requires complex procedures that involve purifying the virus particle and its genomes from the host genome. Whole genome sequencing is inefficient since viral genomes are orders of magnitude smaller than human genomes, and human DNA is orders of magnitude more abundant than viral DNA. In contrast, targeted next-generation sequencing allows focusing of sequencing and analysis to the region or species of interest using PCR based enrichment.

We developed a viral design pipeline that accepts a multiple sequence alignment of the viral genomes or genomic regions of interest to identify and design primer pairs in conserved regions flanking the variable regions. The presence/absence of a set of 1 or more amplicons is used to precisely differentiate the many strains of a viral species. For whole genome sequencing, the complete viral genomic sequence is used as target to design unique and highly specific amplicons covering the whole genome for enrichment of the entire virus. All primers pass through rigorous quality checks to meet our sensitivity, specificity, and primer quality standards.

We have successfully designed multi-strain Ion Ampliseq custom viral panels for HCV, HIV, CMV, Measles and Pestivirus. Some of these panels were designed with the objective of targeting a specific region on a virus genome; other panels were designed to enrich and sequence the whole virus genome. The panel primers were highly specific to the region of interest in the viral genomes and did not amplify the host genome.

In conclusion, our custom Ion AmpliSeq viral panels enable the enrichment and amplification of the unique regions of interest in the viral genomes or the whole virus genome, avoiding the massive excess of the host genome. These panels allow precise identification of the virus species and strains for many applications including research, phylogenomic classification and comparative genomics.

For Research Use Only. Not for use in diagnostic procedures

Citation Format: Ratnesh Singh, Na Li, Zunping Luo, Anna McGeachy, Antonio F. Martinez Alcantara, Aren Ewing, Fiona C. Hyland. Custom next-generation sequencing primer designs for targeted sequencing of multi strain viral targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1207.
Abstract 1209: The association between the cell cycle control pathway and pediatric germ cell tumors
Authors: Hubbard Aubrey K. , Pankratz Nathan , Kelley Spencer T. , Meredith John J. , Poynter Jenny N.
Published: September 2020

Pediatric germ cell tumors (GCTs) account for approximately 6% of childhood cancers. GCTs are heterogeneous but are grouped together due to a common cell of origin, the primordial germ cell. GCTs are predicted to have high heritability, therefore, genetic association studies are paramount in understanding GCT etiology. Genome-wide association (GWA) studies commonly ignore the aggregate effect of SNPs/genes with weaker individual associations. Pathway-based approaches provide the opportunity to interrogate joint SNP effects in pathways of interest. The cell cycle control pathway is a plausible candidate pathway given the knowledge that alterations in the the mitotic-meiotic switch may lead to genetic instability and initiate GCT development. In this analysis we evaluated a set of 94 genes associated with cell cycle control using data from a case-parent triad study of pediatric GCT. Genes involved in cell cycle control were identified using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We also included additional genes based on reports in the literature suggesting a role in cell cycle control in GCTs, including STRA832, 33, CYP26B134, CKS1B35, and E2F636. Variants within 25,000 base pairs upstream or downstream of the gene were selected for inclusion resulting in a total of 2,846 SNPs included in the analysis. Genotyping data were generated using the Illumina HumanCore Exome Array for 867 GCT cases and their unaffected parents. Preliminary results evaluating individual effects of SNPs in the pathway were conducted using the transmission disequilibrium test (TDT). We did not observe any individual SNP associations that reached genome-wide significance; however, suggestive associations were observed for SNPs located in CDKN2C (p=0.000208), MCM7 (p=0.000787), RB1 (p=0.000798), RBL2 (p=0.00250) and TP53 (p=0.00257). Replication of these findings will be conducted using data from a record linkage case-control study from neonatal biobanks in California and Michigan (N=837 cases and 1057 controls). In addition, we will utilize pathway-based approaches to jointly evaluate the effects of genes in this pathway, including Gene Set Enrichment Analysis for Genome wide association study (i-GSEA4GWAS), MetXcan for functional significance, and Family-based rare-variant association analysis (PedGene). In conclusion, our results suggest that genes within the cell-cycle control pathway play a role in the etiology of pediatric GCT.

Citation Format: Aubrey K. Hubbard, Nathan Pankratz, Spencer T. Kelley, John J. Meredith, Jenny N. Poynter. The association between the cell cycle control pathway and pediatric germ cell tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1209.
Abstract 122: Targeting forward and reverse EphB4/EFNB2 signaling by a peptide with dual functions
Authors: Xiong Chiyi , Wen Yunfei , Zhao Jun , Yin Dengke , Xu Lingyun , Chelariu-Raicu Anca , Yao Cody , Leng Xiaohong , Liu Jinsong , Chaudhari Rajan R , Zhang Shuxing , Sood Anil K , Li Chun
Published: September 2020

Introduction: EphB4 is frequently overexpressed in ovarian and other solid tumors and involved in interactions between tumor cells and tumor microenvironment, contributing to metastasis. EphB4 and its membrane-bound ligand ephrin B2 (EFNB2) mediates bi-directional signaling. Currently, no small-molecule-based, peptides with dual functional binding to EphB4 are available. Therefore, we developed an EphB4 agonist that is capable of activating the forward EphB4 signaling and interfering with the reverse EFNB2 signaling.

Methods: A high-affinity bi-directional ephrin agonist peptide (BIDEN-AP) that selectively activated EphB4 was discovered through structural modifications to the known EphB4 antagonist TNYLFSPNGPIARAW (TNYL-RAW) by surface plasma resonance and cell based assays. To improve pharmacokinetics and enhance peptide stability, BINDEN-AP peptide was conjugated to Core-Crosslinked Polymeric Micelles (CCPM). Anti-tumor and anti-angiogenic effects of BINDEN-AP peptide and its conjugate with CCPM were evaluated using orthotopic human A2789cp20-Luc ovarian cancer xenograft model, and PDX model of high grade serous ovarian carcinoma (HGSC).

Results: We found that BIDEN-AP was selectively internalized via receptor-mediated endocytosis, mediated phosphorylation of EphB4 and its downstream adaptor protein Crk1 (MAPK14), and suppressed invasion and epithelial-to-mesenchymal transition in ovarian cancer cells. BIDEN-AP also inhibited endothelial migration and tube formation by 50% (p<0.05). In vivo, BIDEN-AP and CCPM-BIDEN-AP significantly reduced tumor weight (p<0.05) and number of tumor nodules (p<0.01) of intraperitoneally inoculated A2780cp20 ovarian tumors. Moreover, CCPM-BIDEN-AP displayed greater anti-tumor potency than BIDEN-AP with regard to reduction in the number of tumor nodules (p<0.01). Both BIDEN-AP and CCPM-BIDEN-AP compromised angiogenesis as evidenced by significantly reduced microvessel density (p<0.01).

Conclusion: Given the robust anti-tumor effects of BIDEN-AP, these data could have direct relevance for further therapeutic development.

Citation Format: Chiyi Xiong, Yunfei Wen, Jun Zhao, Dengke Yin, Lingyun Xu, Anca Chelariu-Raicu, Cody Yao, Xiaohong Leng, Jinsong Liu, Rajan R Chaudhari, Shuxing Zhang, Anil K Sood, Chun Li. Targeting forward and reverse EphB4/EFNB2 signaling by a peptide with dual functions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 122.
Abstract 1221: Novel strategies to target autophagy to improve cancer therapy
Authors: Li Yuanpei , Ma Zhao , Zhang Dailn , Ramachandran Mythili , Lin Tzu-yin
Published: September 2020

In cancer research, autophagy is thought to be a double-edged sword because it can either prevent malignant transformation during cancer initiation or sustain tumor metabolism, growth and survival in cancer establishment and progression. For established tumors, especially the advanced or metastatic types, the harsh intercellular conditions activate autophagy so that tumor cells can adapt to the stressful conditions and efficiently recycle their nutrients, while also allowing them to respond to anti-tumor therapeutics. Therefore, interventions that deliberately inhibit autophagy in cancer therapy have been used to reduce cancer cell survival, overcome drug resistance and improve clinical outcomes. The vast majority of autophagy inhibitors used in these therapies are lysosomotopic alkaline agents, such as chloroquine (CQ) and hydroxychloroquine (HCQ). However, their efficacy is considered to be imperfect. New strategy to target autophagy for cancer therapy is needed. Through introducing nanotechnology into initial new drug entity design, we developed a series of new chemical entities, BAQ12-18, and all of them are potential building blocks to self-assemble into the single new-chemical-entity nanomedicines (BAQ SNNs). The selected BAQ12 NP and BAQ13 NP not only improve drug delivery and tumor-targeted biodistribution but also endow unique proton sponge effect to dramatically enhance the existing functions of lysosomal disruption and autophagy blockade, and thus generate significant therapeutic efficacy both in vitro and in vivo. The more striking finding is that BAQ13 NP can potently provide both a uniform drug delivery platform and synergistic effect to the combination drug, indicating a new-generation strategy of combination therapy. Considering their capacity to encapsulate different drugs and broad synergistic effect with various treatments, BAQ SNNs may make a tremendous contribution to the outcome of cancer treatment. To the best of our knowledge, this is the first attempt to extend nanotechnology into the design stage of new small-molecule drug entity. The seamless connection between new drug discovery and nanomedicine-based drug delivery will absolutely inspire researchers to develop increasingly advanced nanomedicines with a wide range of practical values and market for improved the therapy against challenging diseases.

Citation Format: Yuanpei Li, Zhao Ma, Dailn Zhang, Mythili Ramachandran, Tzu-yin Lin. Novel strategies to target autophagy to improve cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1221.
Abstract 1230: Critical role of cav1 in high-throughput identification of gallic acid as a novel late-stage autophagy suppresser against invasive breast cancer
Authors: Wang Neng , Zhang Fengxue , Wang Zhiyu , Zheng Yifeng , Wang Shengqi , Yang Bowen
Published: September 2020

Purpose: Development of autophagy inhibitors has important clinical significance in cancer therapies. We and others demonstrated that over-expression of Caveolin-1 (Cav1), which was the major scaffolding protein of caveolae, was associated with higher tumor stage, metastasis and worse prognosis in various carcinomas, particularly in breast cancer. Furthermore, we identified critical role of Cav1 in breast cancer metastasis via modulation of lysosomal function and autophagy. Based on the above, we screened a series of 2, 645 kinds of small-molecule drugs to find out certain cav1 inhibitors which could suppress autophagy and breast cancer metastasis.

Experimental Design: We conducted the 1st high-throughput screening based on surface plasmon resonance (SPR) analysis of cav-1 interaction with 2, 645 kinds of small-molecule drugs. Secondly, we launched the 2nd high-throughput screening based on CCK8 analysis to validate the anti-cancer potential of the selected compounds. We also carried out the final high-throughput screening to investigate their autophagic effects with high content image cytometry assay. The anti-cancer and anti-metastasis effects of the final drug candidate was investigated using invasive breast cancer cells MDA-MB-231 and BT-549, and its role on autophagy was further investigated by transfection of mRFP-GFP-LC3 adenoviral vector and western blot analysis of autophagic proteins.

Results: 2, 645 kinds of small-molecule drugs were screened based on a series of high-throughput screening. The compound gallic acid was identified as the candidate drug owing to its strong interaction and competitive inhibition with cav1, its potent anti-proliferation potential on both invasive breast cancer cells MDA-MB-231 and BT-549, as well as its potent autophagic inhibition compared with the other 2, 644 drugs. Further experimental validation revealed that gallic acid was a potent late-stage autophagy inhibitor via inhibiting autophagosome-lysosome fusion.

Conclusions: Collectively, these results demonstrated that the cav1 inhibitor gallic acid was a novel late-stage autophagy suppressor with potential clinical application for invasive breast cancer therapy.

Citation Format: Neng Wang, Fengxue Zhang, Zhiyu Wang, Yifeng Zheng, Shengqi Wang, Bowen Yang. Critical role of cav1 in high-throughput identification of gallic acid as a novel late-stage autophagy suppresser against invasive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1230.
Abstract 1231: Targeting JNK1/2 induces a potent cytostatic response in estrogen receptor expressing breast cancer cells, with induction of autophagy and senescence as measurable outcomes
Authors: Herrington Kyler , Joseph Carol , Abdulmoneim Samar , Lewis Allison , Cai Jingwen , Youngblood Hannah , Liu Yutao , Schoenlein Patricia
Published: September 2020

The c-Jun N-terminal kinases, JNK1/2 are members of the mitogen activated protein kinase (MAPK) family that can mediate inflammatory responses, cell differentiation, cell proliferation, and/or cell death. In breast cancer, JNK signaling has been associated with increased proliferation, angiogenesis, and tumor metastasis. Also, inhibition of JNK has been shown to promote tumor cytostasis, but this inhibition also attenuates the cytotoxic effects of certain chemotherapeutics, calling into question the therapeutic potential of JNK inhibitors. In this study, we hypothesized that JNK1/2 inhibition induced autophagy, as a mechanism of protection against chemotherapy- and endocrine-induced apoptosis. Autophagy is a normal physiologic process required for the elimination of damaged mitochondria and misfolded proteins that can be induced in cancer cells under certain stresses such as hypoxia and drug insult to provide protection from death. To characterize the role of JNK1/2 in autophagy, we utilized two ERα expressing human breast cancer cell lines, T47-D and MCF-7. Autophagy flux experiments were performed in which the steady state levels of the autophagy protein LC3 II were analyzed in control cells versus cells undergoing JNK1/2 inhibition with the non-reversible JNK-IN8 inhibitor. JNK-IN8 was used as a single agent or in combination with estradiol and/or antiestrogen treatment. These studies reproducibly identified JNK1/2 inhibition as a mechanism of autophagy activation. The induction of autophagy correlated with sustained inhibition of proliferation as determined by cell counts, MTT, and clonogenic assays, and induction of senescence (in T47-D cells) as determined by the expression of β-galactosidase. In contrast, induction of apoptosis was not a major outcome of JNK1/2 inhibition. These pre-clinical studies provide strong evidence that JNK1/2 is an attractive molecular target to improve the treatment of endocrine responsive breast cancer, but they also emphasize that cells are surviving by autophagy and/or senescence. Because cell survival by autophagy and/or senescence could ultimately lead to breast cancer relapse, ongoing studies are addressing approaches to effectively kill these surviving cell populations.

Citation Format: Kyler Herrington, Carol Joseph, Samar Abdulmoneim, Allison Lewis, Jingwen Cai, Hannah Youngblood, Yutao Liu, Patricia Schoenlein. Targeting JNK1/2 induces a potent cytostatic response in estrogen receptor expressing breast cancer cells, with induction of autophagy and senescence as measurable outcomes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1231.
Abstract 1236: In vitro association of chloroquine with alkylating and antimicrotubule agents in patient derived cell lines of glioblastoma
Authors: Kipper Franciele C. , Thomas Ajith , Lenz Guido
Published: September 2020

Autophagy is an important mechanism for cell survival after chemotherapy treatment. Many preclinical and clinical trials have tried to achieve tumor remission combining alkylating agents with autophagy inhibition. Chloroquine (CQ) has a long record as an autophagy inhibitor. In previous studies it was administered in vitro with mixed effects on central nervous system cell lines: while high doses of CQ were cytotoxic, low doses were neuroprotective. Preclinical studies showed that CQ could be used to treat glioblastoma, tumors derived from glial cells in the brain. In a phase I/II clinical trial combining CQ with the standard alkylating agent (temozolomide) plus radiotherapy treating patients with glioblastoma, a dose-limiting toxicity was observed at high doses. Although the authors reported that autophagy inhibition could not be achieved, they did not describe if this mechanism was involved in tumor resistance in that cohort of patients. In order to clarify if CQ can be used to block autophagy, we performed long term treatment combining low doses of CQ with chemotherapeutic agents. We used patient derived cell lines (PDCL) from glioblastoma tumors and treated them with therapeutic agents, using the reported plasma concentration of each drug. We measured acidic organelles with acridine orange incorporation 3, 5 and 7 days after addition of the treatment with flow cytometry. Additionally, 7 days after treatment surviving cells were counted. We did not observe significant increase in autophagy levels or decrease in cell number after temozolomide treatment. On the contrary, paclitaxel induced autophagy and decreased cell survival. Interestingly the combination of procarbazine, lomustine and vincristine (PCV), induced a broad range of results, ranging from high autophagy to increased cell death. We decided to combine PCV with CQ and perform long term analysis in order to evaluate the ability of cells undergoing or escaping apoptosis, senescence or mitotic catastrophe. After 5 days nuclear morphometric analysis demonstrated that out of 6 PDCL tested, only one presented an increase in senescence. The same PDCL also presented a decrease in total number of cells and increase in p62, although combining CQ with PCV was not better than PCV alone. Our results in vitro, using low concentrations of CQ corroborate the finding in clinical trials that chloroquine does not eliminate malignant cells in glioblastoma and better autophagy inhibitors need to be developed to achieve this goal.

Citation Format: Franciele C. Kipper, Ajith Thomas, Guido Lenz. In vitro association of chloroquine with alkylating and antimicrotubule agents in patient derived cell lines of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1236.
Abstract 1241: Galectin-3 and the modulation of autophagy in the pancreatic cancer microenvironment
Authors: Felix Antonio , Consonni Silvio , Pitta Maira , Carvalho Hernandes , Pereira Michelly , Rêgo Moacyr
Published: September 2020

Galectin-3, a member of the β-galactoside binding proteins family was found up- regulated in pancreatic cancer and may be affected by microenvironment factors, such as hypoxia and/or nutritional deprivation. In this study, we aimed to analyze the effects of hypoxia and nutritional deprivation on the Galectin-3 protein levels and the possible influence of this galectin on the autophagy of pancreatic cancer cells. Nutritional deprivation was performed through partial or total reduction of fetal bovine serum (FBS) or through complete removal of serum, glucose and amino acids. Hypoxia was established with 1% oxygen tension. Proteins Galectin-3, Beclin- 1 and LC3 were accessed through Western blot and immunofluorescence. Induction of LC3 stable production was performed through transfection of pEGFP-LC3 and evaluated by confocal microcopy. Degradative vacuole quantification was done by transmission electron microscopy and knockdown of Galectin-3 was performed using siRNA transfection. According to the analyzes, the most effective conditions for the induction of autophagy were partial FBS deprivation for PANC-1 (p = 0.0035) and complete FBS/amino acid/glucose deprivation for Mia PaCa 2 cells (p = 0.0362). PANC-1 cells presented higher levels of Galectin-3 when they were submitted to 24 hours of FBS deprivation and simultaneously FBS deprivation and hypoxia. Hypoxia alone caused increases in LC3 levels for Mia PaCa-2 and for PANC-1 cells. Knockdown of Galectin-3 caused decreases of LC3 levels in almost all experimental conditions for both pancreatic cell lines with exception of concomitant nutritional deprivation and hypoxia for PANC-1. These results show that the Galectin-3 and LC-3 protein are modulated by hypoxia and nutritional deprivation. Cells incubated under hypoxia presented higher levels of LC3, what arguments in favor of the pro-autophagic aspects of hypoxia. Moreover, inhibition of Galectin-3 causes reduction of LC3, which implies Galectin-3 as an important regulator of the autophagic process in pancreatic cancer cells.

Key-words: Pancreatic Ductal Adenocarcinoma, LC3, Hypoxia, Nutritional deprivation.

Citation Format: Antonio Felix, Silvio Consonni, Maira Pitta, Hernandes Carvalho, Michelly Pereira, Moacyr Rêgo. Galectin-3 and the modulation of autophagy in the pancreatic cancer microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1241.