bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1478: Endothelial-specific loss of glutaminase normalizes tumor blood vessels and inhibits tumor growth
Authors: Ngwa Verra , Edwards Deanna , Chen Jin
Published: September 2020

Abnormal tumor blood vessels with chaotic angiogenic sprouting result in spontaneous hemorrhages and increased interstitial fluid pressure in the tumor microenvironment. These defects of intra-tumoral vessels lead to poor leukocyte trafficking and drug delivery. Although there is a solid published premise for tumor blood vessel normalization, mechanisms of improved tumor vessel quality are only beginning to be investigated. Because tumor vascular endothelial cells are fast growing, they require nutrients to generate biomass that support their proliferative program. Indeed, inhibition of glucose metabolism has been shown to normalize tumor vasculature and improve drug delivery. In addition to glucose, glutamine is also essential for endothelial cell proliferation and angiogenic sprouting. Thus, we hypothesized that tumor associated endothelium may require glutamine metabolism to support their rapid proliferation and blocking glutaminolysis would normalize tumor vasculature. To test this hypothesis, we crossed glutaminase (GLS) floxed (GLS f/f) animals with CDH5 (VE-Cad)-CreER transgenic mice to induce endothelial-specific deletion of GLS upon tamoxifen administration (GLSECKO). We found that there is a decrease in tumor growth in GLSECKO animals compared to WT recipients. Loss of GLS in endothelium leads to a decrease in CD31+ angiogenic sprouts, and a better coverage of SMA+ pericytes in capillaries. We also observed an increase in tomato-lectin perfused blood vessels and a decrease in tissue hypoxia, as judged by pimonidazole stain. Together, these results suggest that endothelial-specific loss of glutaminase normalizes tumor blood vessels. We are currently investigating the effects of endothelial-specific deletion of GLS on endothelial cell metabolism, tumor metastasis, lymphocytes infiltration, and the response to chemotherapy.

Success of this project will uncover the role of endothelial glutaminase in tumor vessels. Further, chemotherapies are currently the main treatment for TNBC patients, so improvements in drug delivery will have significant clinical impact. Finally, TNBCs lacking tumor infiltrating lymphocytes (TILS) are poorly responsive to immune checkpoint inhibitors (ICIs). Investigating ways to normalize tumor blood vessels will lay foundation for developing novel therapeutics to increase TILs in immunologically cold TNBCs to increase their response to ICIs.

Citation Format: Verra Ngwa, Deanna Edwards, Jin Chen. Endothelial-specific loss of glutaminase normalizes tumor blood vessels and inhibits tumor growth [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 1478.
Abstract 1482: PIM1 promotes angiogenesis via phosphorylation and stabilization of HIF-1α
Authors: Casillas Andrea L. , Chauhan Shailender S. , Toth Rachel K. , Jensen Corbin C. , Warfel Noel A.
Published: September 2020

An essential process for the growth and dissemination of solid tumors is angiogenesis, or the formation of new blood vessels. Overexpression of Proviral Integration site for Moloney murine leukemia virus (PIM)-1, a serine-threonine kinase, has been implicated as a driver of aggressive prostate and colon cancer. PIM1 is known to promote tumor growth and survival, and we recently uncovered a role for PIM1 in promoting resistance to anti-angiogenic agents. Here, we identify a novel signaling pathway that directly links PIM to tumor angiogenesis. Immunohistochemical staining of prostate cancer tissue revealed a significant positive correlation between PIM1 and CD31 expression, a marker of endothelial cells. Using prostate cancer as a model, in vitro models of endothelial tube formation and DCE-MRI confirmed that PIM1 overexpression increases tumor angiogenesis. Gene expression analysis of PIM1 overexpressing cells showed that PIM kinases regulates the expression levels of many hypoxia inducible factor-1α (HIF-1α) target genes in normoxic conditions. In vitro, in vivo and kinase assays discovered a novel phosphorylation site within the oxygen-dependent degradation domain (ODDD) of HIF-1α that is a direct target of PIM1. Biochemical analysis demonstrates that phosphorylation of this site increases HIF-1α stability by decreasing PHD binding, hydroxylation, and subsequent proteasomal degradation of HIF-1α. Two CRISPR-generated cell lines with point mutations that mimic phosphorylation of this site were used in xenograft models of colon cancer to confirm the significance of this site to the identified PIM-HIF signaling and tumor angiogenesis. Importantly, we show that the anti-angiogenic and cytotoxic effects of PIM inhibitors are largely dependent on their ability to downregulate HIF-1α signaling. In summary, we uncovered a novel role for PIM1 in promoting tumor angiogenesis and identified a novel phosphorylation site that controls the stability of HIF-1α in normoxic and hypoxic conditions.This research yields important insight into the role of PIM signaling in solid tumors and provides preclinical evidence that to improve the translation of PIM kinase inhibitors in solid tumors.

Citation Format: Andrea L. Casillas, Shailender S. Chauhan, Rachel K. Toth, Corbin C. Jensen, Noel A. Warfel. PIM1 promotes angiogenesis via phosphorylation and stabilization of HIF-1α [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 1482.
Abstract 149: Non-small cell lung cancer (NSCLC) detection by DNA methylation profiling and computational deconvolution
Authors: Hornstein Nicholas J. , Noor Zorawar S. , Pellegrini Matteo , Garon Edward
Published: September 2020

Purpose: Early detection of pulmonary malignancies is critical to achieving positive treatment outcomes. However, due to the minimal symptom burden often associated with early-stage neoplasms, in addition to the cost and limitations of advanced imaging modalities, detection of disease is often delayed until nodal or metastatic spread has occurred. Ongoing cellular turnover releases DNA into systemic circulation where it is able to achieve a steady state level. We postulated that we could identify methylation sites specific to pulmonary Squamous Cell Carcinoma (SCC) and Adenocarcinoma (AD) and then computationally determine the presence or absence of neoplastic signatures from a heterogeneous tissue sample, such as is found in cell free DNA isolated from plasma.

Methods: We designed a computational framework for the detection of informative methylation sites based on an iterative Euclidean Distance algorithm. After generation of a reference matrix comprised of 25 cells of origin, SCC and AD (via 444 biopsy-derived methylomes), we sought informative methylation sites which had unique expression patterns among the tested cell types. To quantify the reliability of this approach, we generated synthetic In sillico “plasma” samples comprised of varying compositions of SCC, AD and whole blood methylation data which we then deconvolved using a NNLS algorithm.

Results: We identified 200 informative sites out of 390,000 tested. Based on the 200 identified informative sites, we were able to define a Euclidean Space with robust separation of individual cell types (L2>5). Pairings of CD4+/CD8+ T-Cells and SCC/AD had the closest Euclidean distances, likely related to cellular origin and biologic similarities. However, we accurately distinguished between these cell type pairs in >95% of In silico experiments. Additionally, we demonstrated accurate detection of neoplastic signature within synthetic heterogeneous mixtures ranging from .1 to 100% of neoplastic “spike-in” (PearsonR =.92, p=1.5e-42). Going forward, we hope to further define the operating characteristics of this approach In vivo and develop its capability as we look to recapitulate our results with plasma samples from patients with and without pulmonary malignancies.

Conclusion: Here, we describe a computational framework for the detection of pulmonary malignancy via methylation signatures. Using this framework we identified 200 informative methylation sites which allow for In silico deconvolution and detection of NSCLC within heterogeneously complex samples.

Citation Format: Nicholas J. Hornstein, Zorawar S. Noor, Matteo Pellegrini, Edward Garon. Non-small cell lung cancer (NSCLC) detection by DNA methylation profiling and computational deconvolution [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 149.
Abstract 150: Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis
Authors: Fujimoto Mao , Arai Eri , Tsumura Koji , Yotani Takuya , Yamada Yuriko , Takahashi Yoriko , Maeshima Akiko Miyagi , Fujimoto Hiroyuki , Yoshida Teruhiko , Kanai Yae
Published: September 2020

For diagnosis of upper urinary tract urothelial carcinoma (UTUC), retrograde ureteropyelography and ureteroscopy are invasive, and selective urine cytology has insufficient sensitivity and specificity. The aim of this study was to establish a less invasive and more accurate method for UTUC diagnosis based on genome-wide DNA methylation profiling. Single-CpG resolution genome-wide DNA methylation screening was performed using the Infinium assay, and DNA methylation quantification was verified using pyrosequencing. We analyzed 26 samples of normal control urothelial tissue (C), an initial cohort of 62 samples (31 samples of non-cancerous urothelium [N] from UTUC patients and 31 samples of the corresponding UTUCs), a validation cohort of 82 samples (41 N and 41 UTUC samples), and 14 samples of urinary bladder urothelial carcinoma (BUC). In the initial cohort, we identified 2,448 CpG sites showing significant differences in DNA methylation levels between both C and UTUC and N and UTUC, but not showing differences between C and N. Among these CpG sites, 10 were located within CpG islands or their shores and shelves included in genomic domains where DNA methylation levels are stably controlled, allowing discrimination of UTUC even from BUC. Receiver operating characteristic curve analysis for discrimination of UTUC from N in these 10 CpG and neighboring sites (37 diagnostic panels in total) yielded area under the curve values of 0.959-1.000, with a sensitivity and specificity of 86.6-100% and 93.5-100%, respectively. The reliability of the established criteria was successfully confirmed in the validation cohort. Application of our criteria to voided urine samples will ultimately allow non-invasive and reliable DNA methylation diagnosis of UTUC using an appropriate DNA methylation quantification system such as high-performance liquid chromatography.

Citation Format: Mao Fujimoto, Eri Arai, Koji Tsumura, Takuya Yotani, Yuriko Yamada, Yoriko Takahashi, Akiko Miyagi Maeshima, Hiroyuki Fujimoto, Teruhiko Yoshida, Yae Kanai. Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis [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 150.
Abstract 1500: Understanding tumor cell community and its evolution by single cell analysis in liver cancer
Authors: Ma Lichun , Hernandez Maria O. , Zhao Yongmei , Mehta Monika , Tran Bao , Kelly Michael , Rae Zachary , Hernandez Jonathan M. , Davis Jeremy L. , Martin Sean P. , Kleiner David E. , Hewitt Stephen M. , Ylaya Kris , Wood Bradford J. , Greten Tim F. , Wang Xin Wei
Published: September 2020

Tumor cell biodiversity is a major contributing factor to therapeutic failures and lethal outcomes of solid malignancies. Primary liver cancer, comprising mainly hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), is the second most lethal malignancy worldwide. To understand the biodiversity of tumor cell community in liver cancer, we performed single-cell transcriptome profiling of primary HCC and iCCA from 19 patients, who were enrolled at the NIH Clinical Center for immune checkpoint inhibition clinical trials. We found malignant cells differed within and between tumors, indicating both intratumor and intertumor heterogeneity. We developed a method to measure intratumor heterogeneity. Strikingly, the degree of intratumor diversity was associated with patient outcome where tumors with high diversity was associated with poor prognosis while tumors with low diversity was linked to good prognosis. The link between intratumor diversity and patient prognosis was also observed in bulk genomic and transcriptomic profiles for hundreds of HCC as well as iCCA patients. We found a polarization of the diverse landscapes of tumor microenvironment (TME) from low diversity and high diversity tumors. By searching upstream regulators of TME, we found an increased VEGF expression in high diversity tumors linked to diversity-related polarization of stromal and immune cells. Additionally, tumor evolutionary trajectory was uncovered by single-cell transcriptomic profiles generated from tumor biopsies collected from patients at baseline and after treatment with immune checkpoint inhibitors. Our results offer insight into the diverse ecosystem of HCC and iCCA and determine an impact of tumor cell biodiversity on patient prognosis, which may further benefit the prediction of clinical response to immune therapy.

Citation Format: Lichun Ma, Maria O. Hernandez, Yongmei Zhao, Monika Mehta, Bao Tran, Michael Kelly, Zachary Rae, Jonathan M. Hernandez, Jeremy L. Davis, Sean P. Martin, David E. Kleiner, Stephen M. Hewitt, Kris Ylaya, Bradford J. Wood, Tim F. Greten, Xin Wei Wang. Understanding tumor cell community and its evolution by single cell analysis in liver 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 1500.
Abstract 1520: RAB31 triggers oxaliplatin resistance in colorectal cancer via epithelial-mesenchymal transition
Authors: Chen Chi-Long , Chang Yu-Chan , Hsiao Michael
Published: September 2020

Colorectal tumorigenesis during Epithelial-Mesenchymal transition (EMT) is associated with cancer metastasis and drug resistance. The small G-proteins family has been implicated in the regulation of cell cytoskeleton, cell movement, and vesicle transport. Small G-proteins family is divided into five main families. The Rab protein family is a member of the Ras superfamily of small G-proteins. We analyzed all probes of the Rab family members from microarray datasets and RNA-sequencing. We further calculated the EMT scores by using an algorithm. Our results showed that the RAB31 expression levels of the three probes are positively correlated with EMT score (Pearson rho=0.72, 0.71 and 0.68, respectively). Compared with other family members, RAB31 had most significantly value in colorectal cancer cell lines. We established several two-way model with cDNA and shRNAs of RAB31, respectively. Furthermore, we observed that the expression of epithelial type marker CDH1 was inhibited, and mesenchymal type markers SNAI1 and SNAI2 were upregulated in RAB31 overexpression models. Moreover, we found that the protein level of RAB31 was correlated with several clinicopathological parameters (N status, M status) and disease-free survival in tissue arrays through immunohistochemistry staining. According to The Cancer Genome Atlas Program (TCGA) and online meta-database, high RNA level of RAB31 was consistent in colorectal cancer (HR=1.38, n=466 and HR=1.29, n=482) and correlated with poor prognosis of patients. In addition, we identified that RAB31 may associated with IC50 of oxaliplatin and EGFR trafficking. We concluded that RAB31 regulates the sensitivity of oxaliplatin, an additional chemotherapy strategy and an independent prognostic factor for colorectal cancer.

Citation Format: Chi-Long Chen, Yu-Chan Chang, Michael Hsiao. RAB31 triggers oxaliplatin resistance in colorectal cancer via epithelial-mesenchymal transition [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 1520.
Abstract 153: Epigenetic loss of NSUN5 in glioma drives a global protein synthesis depletion along with the emergence of a stress adaptive translational program
Authors: Barahona Vanessa Ortiz , Janin Maxime , Esteller Manel
Published: September 2020

One of the known hallmarks of cancer is an aberrant RNA transcriptome, and yet, it cannot always be fully due to genetic or epigenetic alterations at the transcriptional start sites of the genes. Indeed, recent evidences point out to new malignant alterations at the RNA level itself. RNA molecules have been found to show more than 100 differently chemically modified nucleotides, and such post-transcriptional modifications play a role in controlling RNA homeostasis, being all grouped under the term ‘epitranscriptome'. In our study, we reported that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate 5-methylcytosine RNA methyltransferase. As NSUN5 activity has not been formally described in human cells yet, we studied its potential targets in the human transcriptome, demonstrating its role in the methylation of the cytosine 3782 in the 28S ribosomal RNA (rRNA). Logically, we found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA, and this depletion drives an overall depletion of protein synthesis under stress conditions, analyzed by O-propargyl-puromycin (OP-Puro) and [3H] leucine incorporation into nascent proteins. However, together with the depletion of global protein synthesis, we observed the emergence of an adaptive translational program for survival under conditions of cellular stress, assessed by combining three NGS-experiments: RNA-seq, Ribo-seq and SILAC. Moreover, as one of the genes increasing its expression upon NSUN5 silencing is the stress-related enzyme NQO1, we found out that NSUN5-depleted gliomas are sensitive to bioactivatable substrates of the NQO1 enzyme. Most importantly, we observed that NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival, conferring it value as a predictor of better patient outcome for this otherwise devastating disease.

Citation Format: Vanessa Ortiz Barahona, Maxime Janin, Manel Esteller. Epigenetic loss of NSUN5 in glioma drives a global protein synthesis depletion along with the emergence of a stress adaptive translational program [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 153.
Abstract 1534: Vimentin prevents a miR-dependent negative regulation of tissue factor mRNA during epithelial-mesenchymal transitions and facilitates early metastasis
Authors: Vanwynsberghe Aline , Francart Marie-Emilie , Gilles Christine
Published: September 2020

Epithelial-Mesenchymal Transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified Tissue Factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (i) vimentin silencing accelerated TF mRNA decay after actinomycin-D treatment, reflecting TF mRNA stabilization, (ii) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (iii) TF 3'-UTR-luciferase reporter vector assays implicated the 3'-UTR of TF mRNA in vimentin-dependent TF regulation, and (iv) using different TF 3'UTR luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs.

Citation Format: Aline Vanwynsberghe, Marie-Emilie Francart, Christine Gilles. Vimentin prevents a miR-dependent negative regulation of tissue factor mRNA during epithelial-mesenchymal transitions and facilitates early metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1534.
Abstract 1536: MiRNA expression profile of exosomes derived from breast cancer cells following induction of the epithelial-to-mesenchymal transition
Authors: Showalter Christian A. , Huang Yinan , Burdick Monica M.
Published: September 2020

Breast cancer-derived exosomes are small extracellular vesicles that play a crucial role in the metastatic progression of breast cancer due to their ability to transport a variety of biologically active molecules (e.g., miRNAs) to local and distant sites in the body. The molecular cargo transported by breast cancer-derived exosomes alters the transcriptome of recipient cells at these sites to stimulate pro-metastatic events, including the epithelial-to-mesenchymal transition (EMT). Importantly, the molecular cargo loaded into exosomes can vary depending on cell state. However, it is unclear whether the expression of miRNAs in breast cancer-derived exosomes is altered following inducement of the EMT. Regulation of exosomal miRNAs following the EMT may enhance the ability of circulating tumor cells to target specific tissue sites or improve formation and maintenance of metastatic tumors compared to miRNAs packaged into exosomes secreted from cancer cells without induction of the EMT. To address this knowledge gap, we hypothesized that the EMT alters expression of miRNAs in exosomes derived from EMT-induced MDA-MB-468 breast cancer cells. To induce the EMT, MDA-MB-468 cells were treated with exogenous epidermal growth factor. MDA-MB-468 cells treated with epidermal growth factor exhibited greater mRNA expression of the mesenchymal marker Vimentin compared to non-treated MDA-MB-468 cells by qRT-PCR. Subsequently, exosomes were isolated from the conditioned cell culture medium of MDA-MB-468 cells, with and without induction of the EMT, using ExoQuick exosome isolation reagent. Molecular size analysis of isolated exosomes revealed that they were similar in size to the typical size range reported for exosomes (40 - 150nm). Moreover, a common exosomal marker protein, CD9, was enriched in the MDA-MB-468-derived exosome fractions compared to whole cell lysates by Western blot. Furthermore, next generation sequencing and bioinformatic analysis were performed to determine the miRNA expression profiles of these exosomes. Results from this study reveal expression of miRNAs in breast cancer cell-derived exosomes before and after activation of the EMT and may indicate how exosomes secreted from advanced stage breast cancer enhance metastatic tumor progression.

Citation Format: Christian A. Showalter, Yinan Huang, Monica M. Burdick. MiRNA expression profile of exosomes derived from breast cancer cells following induction of the epithelial-to-mesenchymal transition [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 1536.
Abstract 1539: NDRG1 promotes epithelial-mesenchymal transition in lung cancer cells by upregulation of SLUG expression
Authors: Liu En-Chu , Liu Hui-Chia , Chen Jeremy J. M. , Tsai Meng-Feng
Published: September 2020

N-myc down-regulated gene 1 (NDRG1) is a member of the NDRG family which belongs to α/β hydrolase superfamily, but without presenting a hydrolytic catalytic site. NDRG1 gene has been proposed that involved in cellular stress response pathways, including the heavy metal response, hypoxia response, DNA damage response, cellular differentiation, and proliferation. An oncogenic and tumor-promoting role of NDRG1 has been suggested in lung cancer and various malignant cancers. However, the biological functions and downstream molecular mechanisms of NDRG1 gene in lung cancer are still unclear. In this study, we detected the expression of NDRG1 in various lung adenocarcinoma cell lines using quantitative RT-PCR and Western blotting methods. The constitutive NDRG1-expression, and NDRG1-knockdown stable transfectants were established. Over-expression of NDRG1 in lung cancer cells exhibited markedly promoting in the proliferation and anchorage independent growth, whereas reduced of NDRG1 was found to suppress lung cancer cell progression. We also indicated that NDRG1 play an important role in the epithelial-mesenchymal transition (EMT) features, including a morphological change, increased migratory and invasive ability, reduction the epithelial marker expression and up-regulated EMT regulators. In addition, microarray assays were performed to further explore the potential molecular mechanisms of NDRG1 in lung cancer. The candidate NDRG1-regulated genes, including IL11, SOX7, TCF4, IGF2, SLUG and SLIT2, were verified by quantitative RT- PCR. Finally, we demonstrated that NDRG1 induces EMT and invasion process in lung cancer cell through up-regulation SLUG expression. These results will provide information and help us to understand the roles of NDRG1 in lung cancer cells.

Citation Format: En-Chu Liu, Hui-Chia Liu, Jeremy J. M. Chen, Meng-Feng Tsai. NDRG1 promotes epithelial-mesenchymal transition in lung cancer cells by upregulation of SLUG expression [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 1539.
Abstract 1547: LOXL1 is a novel responsible gene for peritoneal dissemination potentially via promoting EMT and motility in gastric cancer
Authors: Hu Qingjiang , Masuda Takaaki , Zaitsu Yoko , Tsuda Yasuo , Hisamatsu Yuichi , Nakashima Yuichiro , Ando Koji , Kimura Yasue , Oki Eiji , Mori Masaki , Mimori Koshi
Published: September 2020

Background. In gastric cancer (GC), peritoneal dissemination (PD) occurs frequently and is incurable. We have identified 25 PD-associated genes in GC by in silico analysis in previous research. In this study, we focused on Lysyl oxidase-like 1 (LOXL1), one of the 25 PD-associated genes, because LOXL1 is a member of the lysyl oxidase (LOX) gene family and may have the ability to promote EMT in GC.

Materials and Methods. We assessed LOXL1 expression using a public GC dataset (GSE15459) and a highly-disseminated peritoneal GC cell line (RT-qPCR). Next, we performed gene expression analysis, including gene set enrichment analysis (GSEA) with two public GC datasets (GSE15459 and TCGA). Moreover, we performed a series of in vitro experiments using GC cells. Finally, we evaluated the clinicopathological significance of LOXL1 expression using RT-qPCR and immunohistochemistry staining in the Kyushu cohort. We also performed survival analyses of LOXL1 expression in the Kyushu cohort, GSE15459, and TCGA datasets.

Results. The expression of LOXL1 was higher in GC patients with PD compared with GC patients without PD in the GSE15459 dataset. LOXL1 was also overexpressed in highly-disseminated peritoneal GC cells relative to its control cells. Moreover, LOXL1 expression positively correlated with the EMT gene set in GSEA. Additionally, LOXL1 overexpression reduced CDH1 expression, increased VIM expression, CDH2 expression, SNAI2 expression, and PLS3 expression, and promoted migration capacity in GC cells. Finally, high LOXL1 expression was associated with poorly-differentiated, lymph node metastasis, and PD in the Kyushu cohort, and was a poor prognostic factor in three GC datasets.

Conclusions. LOXL1 was a novel responsible gene for PD in GC, possibly through the activation of EMT and cell motility.

Citation Format: Qingjiang Hu, Takaaki Masuda, Yoko Zaitsu, Yasuo Tsuda, Yuichi Hisamatsu, Yuichiro Nakashima, Koji Ando, Yasue Kimura, Eiji Oki, Masaki Mori, Koshi Mimori. LOXL1 is a novel responsible gene for peritoneal dissemination potentially via promoting EMT and motility in gastric 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 1547.
Abstract 157: Pervasive promoter hypermethylation of silenced TERT alleles in human cancers
Authors: Graham Mindy K. , Esopi David , Brosnan-Cashman Jacqueline A. , Gupta Anuj , De Marzo Angelo , Nelson William G. , Heaphy Christopher M. , Meeker Alan K. , Wheelan Sarah J. , Yegnasubramanian Srinivasan
Published: September 2020

In cancers, the epigenetic regulation of telomerase expression is not fully characterized, with multiple conflicting reports. In particular, the role of DNA methylation of cytosine residues in the control of TERT expression remains unclear. Here, we have carried out the most comprehensive characterization of TERT promoter methylation using ultra-deep bisulfite sequencing spanning more than 310 CpGs within and surrounding the core TERT promoter in 96 different cell lines, including primary, immortalized, and cancer cell types, as well as in control and reference samples. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent G228A and G250A TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. Notably, in cancers, the hypermethylated alleles were associated with repressed expression while remaining unmethylated alleles were enriched with the open chromatin marks, H3K4me3 and H3K27ac. Furthermore, the hypomethylated alleles of the TERT promoter were responsible for the observed TERT expression in cancer. This association of hypomethylated alleles with active expression and hypermethylated alleles with repressed expression is particularly evident in cancers harboring TERT promoter mutations, and cancers displaying allele-specific expression. Reporter assays confirmed that DNA methylation of the TERT promoter greatly suppressed reporter expression in heterologous TERT promoter-reporter constructs. Overall, our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to the attenuation of TERT activation in cancer cells.

Citation Format: Mindy K. Graham, David Esopi, Jacqueline A. Brosnan-Cashman, Anuj Gupta, Angelo De Marzo, William G. Nelson, Christopher M. Heaphy, Alan K. Meeker, Sarah J. Wheelan, Srinivasan Yegnasubramanian. Pervasive promoter hypermethylation of silenced TERT alleles in human cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 157.
Abstract 1586: CCL18 signaling from breast tumor-associated macrophages fosters the activation of fibroblasts into a chemoresistance-inducing phenotype
Authors: Zhang Xiaoqian , Chen Jianing , Su Shicheng , Song Erwei
Published: September 2020

Carcinoma-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in tumor microenvironment critically involved in cancer progression. Our recent study identified a specific CAFs subset by two cell-surface molecules, CD10 and GPR77. We proved that CD10+GPR77+ CAFs could promote tumor formation and chemoresistance by providing a survival niche for cancer stem cells (CSCs). However, their origin and underlying activating initiation mechanism are unclear. Tumor-associated macrophages (TAMs) are the most abundant immune-related stromal cells and the important source of inflammatory cytokine. Here, we demonstrated that CCL18 secreted by TAMs could induces the activation of normal breast fibroblasts into a CD10+GPR77+ CAFs phenotype, which are not only resistant to chemotherapy themselves, but also can induce chemoresistance in cancer cells by abundantly producing IL-8 and IL-6 and enriching the population of cancer stem cells. In conclusion, our studies have identified an inflammatory signaling network in the interaction of different kind of stroma cells in tumor microenvironment and highlighted its potential values as predictive markers and therapeutic targets to abrogate tumor formation and improve chemotherapeutic efficacy.

Citation Format: Xiaoqian Zhang, Jianing Chen, Shicheng Su, Erwei Song. CCL18 signaling from breast tumor-associated macrophages fosters the activation of fibroblasts into a chemoresistance-inducing phenotype [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 1586.
Abstract 1597: The landscape of PD1-PD-L1 axis in the context of immune cells: Tumors versus stromal compartments
Authors: Lin Xiaoqian , Sulaiman Raed , Aske Jennifer C. , Meyer Paul , RojasEspaillat Luis , Starks David , De Pradip , Dey Nandini
Published: September 2020

Introduction: Immune response, tolerance, suppression, and resistance are the result of a complex dynamic interaction comprising PD1-PD-L1 axis in the context of immune cells (ICs) involving tumor cells, and anti/pro-tumor immune-cells of the stromal compartment.

Aim: Here we present the landscape of PD1-PD-L1 axis in the context of CD3, CD4, CD8, PD1, FOXP3, and IL12RB2 positive ICs in the tumor (T) and the tumor-adjacent normal (TAN) tissues from patients with ovarian cancers (OC), and lung cancers (LC).

Methods: The study was approved by Avera IRB. Surgically resected paired T and TAN tissue samples (from patients with OC and LC) were used to identify CD3 (ab16669), CD4 (ab133616), CD8 (ab85792), FOXP3 ([236A/E7], PD1 (ab137132), IL12RB2 (NBP1-85983), CD68 (Dako. #M0876), CD163 (Cell Signaling #93498) in ICs and PD-L1 (Clone 22C3; Dako) in T cells & tumor-associated macrophages (TAMs). Tonsil tissue was used for the IHC validation. Paired specimens were morphologically evaluated by H&E stain & Ki-67, cl-caspase 3. Anatomic pathological evaluation for the diagnosis of the disease was carried out by a Pathologist.

Results: Expression of CD3, CD4, CD8, PD1, and FOXP3 was heterogeneous, with each tumor presenting an individual pattern of distribution of ICs. However in most tumor samples, the distribution of ICs was of tumor-cooperating phenotypes. PD-L1 expression in tumors did not correlate with any other ICs of the tumor-microenvironment. In TAN tissues from OC, PD1-PD-L1 staining was absent. While macrophages in TAN tissue from LC were moderately positive for PD-L1. In T tissue from OC and LC, PD1-PD-L1 staining was only present in the stroma, lymphocytes, and absent in the tumor compartments while PD-L1 staining was identified only in the macrophages. Tumoricidal cells were rarely observed within the tumor compartment of T tissues from OC. The tumors cells from LC was only positive for PD-L1.

Conclusion: Using dual stain of PD1 and PD-L1, we present for the first time, the co-distribution pattern of PD1-PD-L1 axis in the context of the expression of CD3, CD4, CD8, FOXP3 T cells as well as CD68, and CD163 positive TAMs in the paired sample of a T tissue and its TAN tissue in LC and OC. The relationship between the PD1-PD-L1 axis and the expression of IL12 Receptor in the context of immune-landscape is being worked out and will be presented in the meeting.

Citation Format: Xiaoqian Lin, Raed Sulaiman, Jennifer C. Aske, Paul Meyer, Luis RojasEspaillat, David Starks, Pradip De, Nandini Dey. The landscape of PD1-PD-L1 axis in the context of immune cells: Tumors versus stromal compartments [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 1597.
Abstract 162: DNA methylation activates TP73 expression in human breast cancer tumorigenesis
Authors: Yao Zhixing , Yao David R. , Yu William , Sherif Zaki A.
Published: September 2020

Epigenetic events that cause changes in gene expression are common in human cancers. However, the connection between TP73 expression and its epigenetic functionality in tumorigenesis has not been widely explored. As a transcription factor, p73 is activated in a similar manner to p53 in response to DNA damage and regulates the expression of downstream genes involved in cell cycle arrest and apoptosis. However, there are other compounding functions of this gene that reflect its non-tumor-related characters, thus making it very difficult to assess its specific role in tumorigenesis, which results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. TP73 is the homologue of the master tumor suppressor TP53 involved in cellular responses to stress and development. The TP73 gene encodes two different proteins TAp73 and ΔNp73 and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors and is thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of TP73 inactivation in human cancers. Therefore, the role of TP73 in tumorigenesis has remained elusive to date. In this study, we determined TP73 expression in normal human mammary epithelial cells (HMEC), non-cancerous Li-Fraumeni Syndrome (LFS) skin fibroblasts, human breast cancer cell lines (MCF7 & MDA321), neuroblastoma cell lines (IMR32 & SK-N-SH) and normal skin fibroblast cell line (HS27). We also conducted an immunohistochemistry analysis of TP73 expression in human normal breast tissues, cancerous breast tissues and non-cancerous (cancer-adjacent) breast tissues. The results show that TP73 expression is evident only in cancer cell lines and breast cancer tissues. Moreover, methylation-specific PCR and bisulfite sequencing studies revealed that TP73 promoter is activated only in cancer cell lines by DNA methylation. Furthermore, ChIP assay results demonstrated that CTCF (a chromosomal networking protein CCCTC binding factor) and tumor protein p53 (TP53) bind to TP73 promoter and regulate TP73 expression. These observations demonstrate the existence of a positive correlation at the promoter site of TP73 between its expression and DNA methylation. However, TP73 expression is not involved in cell proliferation, which may prove significant for the development of future diagnostic and therapeutic applications.

Citation Format: Zhixing Yao, David R. Yao, William Yu, Zaki A. Sherif. DNA methylation activates TP73 expression in human breast cancer tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 162.
Abstract 1654: Highly multiplexed analysis of FFPE breast tissues using the codex technology
Authors: Nikulina Nadezhda , Braubach Oliver , Basak Sayantani , Gallina Maria Elena , Lee Won-Mean , Kim Joseph , Hempel Cassandra , Williams Edek , Shang Olive , Cheung Ben , Kennedy-Darling Julia
Published: September 2020

Highly multiplexed imaging has emerged as a critical tool in understanding the complexities of the tumor microenvironment. This technology enables detection of tens of markers within the same tissue specimen, thereby allowing for key cell identification, quantification and spatial localization. These measurements can in turn provide insights into disease mechanisms and effective treatment modalities. In particular, with the high incidence of breast cancer and the need for early diagnosis and precise treatment regimens, highly multiplexed imaging could improve our understanding of this disease and provide the necessary data for improvements to patient health. Here, we demonstrate the development of a breast cancer specific CODEX panel for the measurement of more than 30 specific markers. The CODEX (Co-Detection by indEXing) platform enables detection of tens of markers within the same tissue specimen through a DNA-based tagging approach, whereby antibodies are labeled with specific oligonucleotide tags (barcodes) and dye-oligonucleotides (reporters) are iteratively hybridized and dehybridized across multiple cycles. This process is completely automated through the CODEX instrument in combination with existing fluorescent microscopes.

Using FFPE Human breast cancer tissues at different stages as well as normal FFPE Human breast tissue utilizing the CODEX platform, data was generated for 30+ different biomarkers. This antibody panel was designed to detect cancer cells as well as non-malignant cells as part of tumor microenvironment to enable a better understanding of communication between the cells. The CODEX generated data was analyzed using the CODEX software suite to identify key cell types and analyze the spatial association between them. Upon analysis of data obtained, it was found that the biomarkers expression between the three samples were distinctly different. Using the CODEX technology, we were able to identify and classify key cell types and map the spatial localization of more than 20 key cell types.

Citation Format: Nadezhda Nikulina, Oliver Braubach, Sayantani Basak, Maria Elena Gallina, Won-Mean Lee, Joseph Kim, Cassandra Hempel, Edek Williams, Olive Shang, Ben Cheung, Julia Kennedy-Darling. Highly multiplexed analysis of FFPE breast tissues using the codex technology [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 1654.
Abstract 1657: Paired agent fluorescence imaging of cancer in a living mouse using targeted molecular probes with emission wavelengths of 690 and 830 nm
Authors: Spires (Schreiber) Cynthia L. , Zhai Canjia , Dempsey Janel , McGarraugh Hannah , Matthews Braden , Christmann Caroline , Smith Bradley
Published: September 2020

Targeted fluorescent probes were used for cancer imaging and assessing tumor targeting performance in a living mouse model. Untargeted probes and targeted probes (appended with cRGDfK peptide units for integrin targeting on cancer cells) were fabricated with emission wavelengths of 690 and 830 nm. Selective targeting of cancer cells was proved by series of cell microscopy experiments followed by in vivo imaging of subcutaneous tumors in living mice. The mouse imaging studies included a mock surgery that completely removed a fluorescently labeled tumor. Enhanced tumor accumulation due to probe targeting was first evaluated by conducting Single Agent Imaging (SAI) experiments that compared tumor imaging performance of a targeted probe and untargeted probe in separate mouse cohorts. Although there was imaging evidence for enhanced tumor accumulation of the targeted probe, there was moderate scatter in the data due to tumor-to-tumor variability of the vasculature structure and interstitial pressure. A subsequent Paired Agent Imaging (PAI) study co-injected a binary mixture of targeted probe (with emission at 690 nm) and untargeted probe (with emission at 830 nm) into the same tumor-burdened animal. The conclusion of the PAI experiment also indicated enhanced tumor accumulation of the targeted probe but the statistical significance was much higher, even though the experiment required a much smaller cohort of mice. The imaging data from the PAI experiment was analyzed to determine the targeted probe's Binding Potential (BP) for available integrin receptors within the tumor tissue. In addition, pixelated maps of BP within each tumor indicated a heterogenous spatial distribution of BP values. The results of this study show that PAI is a promising new way to analyze tumor heterogeneity and its eventual use in clinical applications such as targeted therapy, image guided surgery, and personalized medicine.

Citation Format: Cynthia L. Spires (Schreiber), Canjia Zhai, Janel Dempsey, Hannah McGarraugh, Braden Matthews, Caroline Christmann, Bradley Smith. Paired agent fluorescence imaging of cancer in a living mouse using targeted molecular probes with emission wavelengths of 690 and 830 nm [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 1657.
Abstract 1660: Reflectance confocal microscopy modeling of lentigo maligna
Authors: Hanlon Katharine L. , Grichnik James M.
Published: September 2020

Early melanoma detection in sun-damaged skin can be a challenge. Clinically, some solar lentigines or flat seborrheic keratoses may be relatively indistinguishable from Lentigo Maligna. Reflectance Confocal Microscopy can now be used in the clinic to evaluate these lesions in vivo and increase biopsy accuracy. Spatial relationships of cells and architecture of associated skin structures play a critical role in appropriate diagnoses. As we move toward artificial intelligence using computer diagnostic platforms, it will be important to base algorithms on data with high diagnostic yield. We have undertaken efforts to create 3D reconstructions from sequential depths of confocal images of skin lesion in vivo in order to allow for more precise definition spatial relationships. These efforts have facilitated clearer evaluation of architectural changes at the dermal epidermal junction, location of cells around adnexal structures, and extent of melanocytic dendricity and diversity within the lesion. The use of color maps for efficient identification and accurate localization of cellular and architectural features of interest is another approach to advanced evaluation of confocal image data. We anticipate that these efforts will further improve accuracy for both human and computer diagnoses from reflectance confocal images.

We appreciate generous support of the Theodora B Betz Foundation.

Citation Format: Katharine L. Hanlon, James M. Grichnik. Reflectance confocal microscopy modeling of lentigo maligna [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 1660.