bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4643: Mutant ASXL1 collaborates with HHEX to promote myeloid leukemogenesis
Authors: Asada Shuhei , Takeda Reina , Inoue Daichi , Goyama Susumu , Kitamura Toshio
Published: August 2019

An epigenetic modulator Additional sex combs-like 1 (ASXL1) is recurrently mutated in myeloid neoplasms and its mutations are associated with poor prognosis. Recently, we generated mutant Asxl1 conditional knock-in (Asxl1-MT KI) mice mimicking human ASXL1 E635RfsX15 mutation, one of the most common mutations in myeloid neoplasms (Nagase et al. JEM 2018). Retrovirus-mediated insertional mutagenesis study exhibited susceptibility of Asxl1-MT KI bone marrow cells to myeloid leukemia, and we identified Hematopoietically expressed homeobox (Hhex) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between the mutant ASXL1 and HHEX in myeloid leukemogenesis. We first performed colony-forming assay and found that forced expression of HHEX enhanced colony replating activity and blocked myeloid differentiation in bone marrow hematopoietic stem progenitor cells (HSPCs) derived from ASXL1-MT KI mice, while it showed only modest effect in normal HSPCs. The synergistic effect between the mutant ASXL1 and HHEX in blocking myeloid differentiation was also observed in human HL-60 cells. We next evaluated the role of endogenous Hhex in the mutant ASXL1-expressing cells. Depletion of endogenous Hhex using CRISPR-Cas9 system ameliorated mutant ASXL1-induced differentiation block in 32Dcl3 cells. Depletion of endogenous Hhex in murine mutant ASXL1-expressing leukemia cells [cSAM cells: cells with combined expression of SETBP1 and ASXL1 mutations (Inoue et al. Leukemia 2015), cRAM cells: cells with combined expression of RUNX1 and ASXL1 mutations (Nagase et al. JEM 2018)] also promoted differentiation and increased apoptosis. Furthermore, Hhex deletion profoundly attenuated the colonogenicity of cSAM and cRAM cells and leukemogenicity of cSAM cells. We then investigated target genes of the mutant ASXL1 and HHEX in myeloid neoplasms using public database and our previous RNA-Seq data. Among the potential target genes of the mutant ASXL1 and HHEX, we found that Myb, Etv5 and Oraov1 genes were upregulated by the mutant ASXL1 and HHEX in murine HSPCs. Conversely, Hhex depletion resulted in downregulation of these genes both in cSAM and cRAM leukemic cells. In addition, depletion of Myb, Etv5 or Oraov1 genes significantly abrogated the colonogenicity of cSAM cells. These data suggest that mutant ASXL1 and HHEX cooperatively induce myeloid leukemogenesis via dysregulating Myb, Etv5 and Oraov1.

Citation Format: Shuhei Asada, Reina Takeda, Daichi Inoue, Susumu Goyama, Toshio Kitamura. Mutant ASXL1 collaborates with HHEX to promote myeloid leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4643.
Abstract 4646: Loss of nuclear HoxA10 is associated with proliferation of testicular germ cell tumors
Authors: Chen RuiQi , Li Haolong , Li Yinan , Fazli Ladan , Gleave Martin , Nappi Lucia , Dong Xuesen
Published: August 2019

Background: HOXA10 is a key transcriptional factor that regulates testis development as reported from previous transgenic mouse models and human inherited diseases. However, whether it also plays important roles in promoting the development of testicular cancer is not well understood.

Objective: To study the expression of HOXA10 and its regulated signaling pathways in testicular cancers.

Design, Setting, and Participants: A tissue microarray was constructed with benign and cancerous testis. TCam2, NT-2, and NCCIT cell models were applied in this study.

Intervention: Immunohistochemistry and immunofluorescence were performed to measure the expression and cellular localization of HOXA10 in testicular cancer tissues and cell models. Cell proliferation and cell cycling rates were determined by BrdU incorporation and flow cytometry assays. HOXA10 transcriptomes were profiled with Ampliseq RNA-seq in testicular cancer cells. Immunoblotting assays were used to detect HOXA10-regulated signaling.

Results: HOXA10 is a nuclear protein in benign spermatocytes. Reduced nuclear expression and increased cytoplasmic expression of HOXA10 are associated with testicular cancers. These changes are consistent in both seminoma and non-seminoma. Enhanced HOXA10 expression in testicular cancer cell models inhibits cell proliferation and delays cell cycle progression through G2/M phases. These functions of HOXA10 mainly affect the TP53, cKit, STAT3, AKT and ERK signaling pathways.

Conclusions: Loss of nuclear functions of HOXA10 enhances proliferation of testicular cancer cells, suggesting that downregulation of HOXA10 transcription activity may promote the development of testicular cancers.

Citation Format: RuiQi Chen, Haolong Li, Yinan Li, Ladan Fazli, Martin Gleave, Lucia Nappi, Xuesen Dong. Loss of nuclear HoxA10 is associated with proliferation of testicular germ cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4646.
Abstract 465: Molecular classification of endometrial cancer provides complementary information but does not outperform current predictive models: The Karolinska and Bern experience
Authors: Nastic Denis , Imboden Sara , Ghaderi Mehran , Rydberg Filippa , Olsson Daniel , Mueller Michael , Rau Tilman T. , Epstein Elisabeth , Carlson Joseph
Published: August 2019

Purpose: The purpose of this study was to evaluate the performance of a proposed prognostic molecular classification of endometrial cancer in a retrospective cohort of n=604 patients. A secondary goal was to compare the performance to existing prognostic models.

Methods: All cases were analysed using 1) sequencing of the exonuclease domain of the POLE gene, 2) microsatellite instability by immunohistochemistry, and 3) TP53 mutation, as evaluated by immunohistochemistry. Additionally, complete histopathological and clinical data was available. The cases were classified using the molecular markers analyzed.

Results: The assembled cohort largely resembled the typical unselected distribution of FIGO grade and stage. Analysis of clinicopathologic variables showed that FIGO grade and stage, LVSI, tumor histotype, depth of myometrial invasion, ESMO 2013 and 2016 risk group, recurrence and survival all were significantly associated with molecular subgroups. A large number of patients showed multiple molecular aberrations (so called multiple classifiers, n=50, 8.4%). Mutation in P53 was a statistically prognostic marker for overall survival (OS) in the univariable Cox regression (p-value 0.003 for OS, and <0.001 for DSS and PFS, HRs of 1.61, 1.83 and 1.72). The POLE mutated cases showed a trend towards favorable prognosis. By contrast, histotype (endometrioid vs. non-endometrioid) was a highly significant predictor of OS, DSS and PFS (HR=2.23, p=<0.001). Both ESMO 2013 and 2016, which represent an aggregate of the various clinicopathologic factors, were highly significant in predicting outcome and especially so for the high and advanced risk groups with HRs up to 70.1 (CI 23.9-206.8) and all p-values of <0.001. In the multivariable Cox regression analysis, however, none of the molecular markers remained significant. The Harrell C-index for 2016 ESMO risk groups was 0.76. Adding molecular markers did not show improved discriminatory ability.

Conclusions: In this retrospective cohort, molecular markers provided important biological information that may be helpful in therapeutic decision making. However, the molecular markers did not outperform current prognostic models.

Citation Format: Denis Nastic, Sara Imboden, Mehran Ghaderi, Filippa Rydberg, Daniel Olsson, Michael Mueller, Tilman T. Rau, Elisabeth Epstein, Joseph Carlson. Molecular classification of endometrial cancer provides complementary information but does not outperform current predictive models: The Karolinska and Bern experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 465.
Abstract 4655: Association of a new germline variant in the MUTYH DNA glycosylase gene with colorectal adenoma transformation into malignancy
Authors: Mahasneh Amjad A. , Al-Shaheri Fawaz , Banihani Mohamemd N.
Published: August 2019

Background: Germline mutations in mutY homolog (E.coli) (MUTYH)DNA glycosylase gene have been associated with recessive inheritance of multiple s adenoma. Several studies revealed that germline mutations in this gene are ethnicity related. This study aimed to identify the germ-line mutations in MUTYH gene and determine their prevalence among Jordanian patients with colorectal adenoma.

Methods: 150 colorectal adenoma patients and 150cancer free individuals with no previous history of polyps were recruited. Sanger DNA sequencing of the MUTYH genewas carried out using 3130xL Genetic Analyzer. Sequencing results were analyzed by ChromasPro and mutational effects were predicted by online bioinformatics tools(accession number for the transcript used as reference isNG_008189.1).

Results: Two novel variants, g.87C>T and c.1264G>C, have been identified. G.87C>T was found in 60 (40%) patients and 10 (6.7%) controls.C.1264G>C was found in 90 (60%) patients and 7 (4.7%) controls. Thus, a significant association between g.87C>T and c.1264G>C variants and colorectal adenoma was found (p value for both variants was <0.0001). Moreover, the newly identified germline variant, c.1264G>C, was found to be significantly associated with colorectal adenoma transformation into malignancy (p< 0.0001).

Conclusion: The data showed high prevalence of two germline mutations in MUTYH gene among Jordanians with colorectal adenoma, which may make them as potential early biomarkers for diagnosis of colorectal adenoma.

Citation Format: Amjad A. Mahasneh, Fawaz Al-Shaheri, Mohamemd N. Banihani. Association of a new germline variant in the MUTYH DNA glycosylase gene with colorectal adenoma transformation into malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4655.
Abstract 4659: Cadmium induced malignant transformation involves activation of the Erk/MAPK pathway
Authors: Dasgupta Pritha , Kulkarni Priyanka , Bhat Nadeem S. , Gupta Ravi , Hashimoto Yutaka , Saini Sharanjot , Dar Altaf A. , Shahryari Varahram , Yamamura Soichiro , Tanaka Yuichiro , Shiina Marisa , Dahiya Rajvir , Majid Shahana
Published: August 2019

Cadmium (Cd) is a chemical pollutant of the natural and occupational environment and is reported to be associated with human carcinogenesis. The molecular mechanisms associated with Cd-induced prostate cancer remain elusive. This study provides evidence that Erk/MAPK signaling is a carcinogenic molecular fingerprint for Cd induced prostate cancer. Cd exposed RWPE1 (Cd-RWPE1) cells robustly formed tumors in nude mice. Functionally, chronic Cd exposure of RWPE1 cells promoted cell survival, proliferation and colony formation with inhibition of apoptosis. RT2 PCR array analysis of 84 genes involved in the Erk/MAPK pathway revealed induction of gene expression in Cd-RWPE1 cells compared to RWPE1. This was confirmed by individual TaqMan gene expression analysis. Gene Set Enrichment Analysis (GSEA) for differentially expressed genes in Cd-RWPE1 showed an enrichment of the Erk/MAPK pathway along with other pathways such as KEGG-ERBB, KEGG-Cell Cycle, KEGG-VEGF, KEGG-Pathways in cancer and KEGG-prostate cancer pathway. We randomly selected upregulated genes from the Erk/MAPK pathway and performed profile analysis in a prostate adenocarcinoma data set (n=534) from the TCGA/GDC data base. We observed upregulation of these genes in prostate cancer compared to normal prostate samples. Taken together, these data reveal that Erk/MAPK signaling is a major pathway involved in Cd-induced malignant transformation of normal prostate epithelial cells. Understanding the dominant oncogenic pathways involved in the malignant transformation of normal prostate epithelial cells may help develop optimal therapeutic strategies for prostate cancer.

Citation Format: Pritha Dasgupta, Priyanka Kulkarni, Nadeem S. Bhat, Ravi Gupta, Yutaka Hashimoto, Sharanjot Saini, Altaf A. Dar, Varahram Shahryari, Soichiro Yamamura, Yuichiro Tanaka, Marisa Shiina, Rajvir Dahiya, Shahana Majid. Cadmium induced malignant transformation involves activation of the Erk/MAPK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4659.
Abstract 4662: YYFZBJS ameliorates the progression of colon carcinogenesis in ApcMin/+ mice by inhibiting regulatory T-cell generation
Authors: Sui Hua , Gu Kaijuan , Ren Junze , Wen Haotian , Wang Ting , Hu Jing , Yang Liu , Ji Qing , Li Qi
Published: August 2019

Colorectal cancer (CRC) is the third most common solid tumor in the world and shows resistance to several immunotherapies, particularly immune checkpoint blockade which has therapeutic effects on many other types of cancer. T cell (Treg) represents one of the main populations of effector immune cells in antitumor immunity, has been considered as a double-edged sword during the progression of colon carcinogenesis. Our previous studies showed that traditional Chinese herbs had potential anticancer effects in improving the quality of life and therapeutic effect. However, little is known whether the mechanism of TCM formula is depended on the Treg-mediate innate immunity. Here, we used C57BL/6 ApcMin/+ mice, an animal model of human intestinal tumorigenesis, to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas, and to evaluate the effects of YYFZBJS on the progression of colon carcinogenesis in vivo and in vitro. Compared to unaffected spleen, intestinal lymphatic, and mesenteric lymph nodes (MLN), accumulated CD4+CD25+ FoxP3 positive Treg cells were reduced after YYFZBJS treatment in ApcMin/+ mice. Treatment with YYFZBJS blocked tumor initiation and progression in the transgenic mouse models (ApcMin/+) with less change of body weight and survival rates. Moreover, YYFZBJS significantly reduced the expression of IL-6, IL-10, TGF-β, and other oncogenes markers such as MMP-2, MMP-9, cyclinD1 and c-Myc in Peripheral Blood and adenomatous tissue. In conclusion, we show that the Treg is involved in CRC development and progression and we identify YYFZBJS as a new potential drug target for the treatment of CRC.

Note: This abstract was not presented at the meeting.

Citation Format: Hua Sui, Kaijuan Gu, Junze Ren, Haotian Wen, Ting Wang, Jing Hu, Liu Yang, Qing Ji, Qi Li. YYFZBJS ameliorates the progression of colon carcinogenesis in ApcMin/+ mice by inhibiting regulatory T-cell generation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4662.
Abstract 4670: The molecular mechanisms of EpCAM in regulating tumor progression in colon cancer cells
Authors: Liang Kang-Hao , Tso Hsien-Cheng , Hung Shao-Hsi , Liao Mei-Ying , Wu Han-Chung
Published: August 2019

Epithelial cell adhesion molecule (EpCAM) is highly expressed in advanced epithelial cancers and tumor-initiating cells, but its role in cancer progression remains to be elucidated. Here, we show that the extracellular domain of EpCAM (EpEX) acts through its EGF-like domain I to bind EGFR and activates downstream ERK1/2 and AKT signaling. Inhibition or shRNA knockdown of EGFR ablated EpEX-induced ERK1/2 and AKT phosphorylation in colon cancer cells. EGFR signaling, stimulated by either EpEX or EGF, then induces regulated intramembrane proteolysis of EpCAM, releasing both EpEX and EpCAM intracellular domain (EpICD). MEK inhibitor, U0126, prevented EpEX-induced EpCAM proteolysis by ADAM17 and presenilin 2 proteases. Furthermore, EGFR inhibitor, AG1478, and MEK inhibitor, U0126, both decreased the production of EpICD, which was found to be necessary for nuclear accumulation of β-catenin protein and expression of HIF1α target genes in vitro and in mouse xenograft models. In clinical samples from colorectal carcinoma patients, high levels of nuclear EpICD predicted metastasis and poor prognosis. Importantly, we also showed that EpAb2-6, an anti-EpCAM neutralizing monoclonal antibody, inhibited EpEX-activated EGFR-PI3K-AKT signaling and induced apoptotic signaling through FOXO3a activation of HTRA2 gene expression. This antibody also inhibited the nuclear translocation of EpICD and oncogenic signaling through β-catenin. Finally, in both metastatic and orthotopic animal models of colorectal cancer, EpAb2-6 therapy exhibited an antitumor effect and markedly extended the survival time of mice. Taken together, the results demonstrate that EpEX contributes to malignancy by functioning as a growth factor, which activates EpICD-mediated signaling, thereby enhancing colon cancer cell survival. Furthermore, the data indicate that EpEX can be considered as a promising target for treatment of colon cancer.

Citation Format: Kang-Hao Liang, Hsien-Cheng Tso, Shao-Hsi Hung, Mei-Ying Liao, Han-Chung Wu. The molecular mechanisms of EpCAM in regulating tumor progression in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4670.
Abstract 4677: Inhibition of 15-PGDH causes Kras-driven tumor expansion through prostaglandin E2-ALDH1 signaling in the pancreas
Authors: Ishimoto Takatsugu , Arima Kota , Uchihara Tomoyuki , Miyake Keisuke , Yonemura Atsuko , Yasuda Tadahito , Itoyama Rumi , Iwatsuki Masaaki , Baba Yoshifumi , Yoshida Naoya , Baba Hideo
Published: August 2019

Chronic inflammation has a crucial role in cancer development and the progression of various tumors, including pancreatic ductal adenocarcinoma (PDAC). The arachidonate cascade is a major inflammatory pathway that produces several metabolites, such as prostaglandin E2 (PGE2). 15-hydroxyprostaglandin dehydrogenase (15-PGDH) participates in the degradation of PGE2 and has attracted attention in recent years. Although inhibition of 15-PGDH is reported to lead to PGE2 accumulation and expand tissue stem cell fraction, the role for pancreatic tumor progression is still unknown. The aim of this study is to elucidate the regulatory mechanism and functional role of 15-PGDH for cancer stem-like cell expansion during tumor progression in pancreas. We found that 15-PGDH expression is frequently down-regulated in PDAC cells and significantly correlated with the number of infiltrating macrophages in human PDAC tissues. Moreover, direct co-culture assay revealed that macrophage derived interleukin-1 beta down-regulates 15-PGDH expression in PDAC cells. Furthermore, pharmacological blockade of 15-PGDH led to PGE2 accumulation, and promoted growth and sphere formation through the expansion of ALDH1-positive cells. We also elucidated the molecular mechanism that PGE2 accumulation by 15-PGDH inhibition increases CYP26A1 expression and subsequently depletes all-trans retinoic acid, and results in ALDH1 up-regulation. Finally, genetic ablation of 15-Pgdh promoted tumorigenesis in KrasLSL-G12D; Ptf1aCre/+ mice through the expansion of ALDH1-positive cells. Our findings highlight the role and significance of PGE2 degradation pathway for PDAC tumor progression.

Note: This abstract was not presented at the meeting.

Citation Format: Takatsugu Ishimoto, Kota Arima, Tomoyuki Uchihara, Keisuke Miyake, Atsuko Yonemura, Tadahito Yasuda, Rumi Itoyama, Masaaki Iwatsuki, Yoshifumi Baba, Naoya Yoshida, Hideo Baba. Inhibition of 15-PGDH causes Kras-driven tumor expansion through prostaglandin E2-ALDH1 signaling in the pancreas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4677.
Abstract 4680: KRAS activation in gastric adenocarcinoma stimulates epithelial-to-mesenchymal transition to cancer stem-like cells and promotes metastasis
Authors: Yoon Changhwan , Till Jacob , Cho Soo-Jeong , Chang Kevin , Lin Jian-Xian , Ryeom Sandra , Yoon Sam
Published: August 2019

BACKGROUND:

Our previous work showed that in a mouse model of gastric adenocarcinoma (GA) with loss of p53 and Cdh1 that adding oncogenic Kras (a.k.a triple conditional or Tcon mice) accelerates tumorigenesis and metastasis. The Receptor Tyrosine Kinase (RTK)-RAS signaling pathway is altered in 60% of GAs, and KRAS is amplified or mutated in 17% of GAs. There is some evidence that RTK-RAS signaling and oncogenic KRAS are important in the epithelial-to-mesenchymal transition (EMT) and maintenance of cancer stem-like cells (CSCs). We hypothesized that RTK-RAS activation in GA CSCs increases the metastatic potential of these cells.

METHODS:

The RTK-RAS pathway and KRAS were examined in nontransformed HFE-145 gastric epithelial cells, tumor-derived organoids derived from Tcon mice, human GA cell lines (AGS and KATOIII), AGS xenografts, and a tissue microarray of human GAs from 115 patients undergoing surgical resection. KRAS was inhibited using shRNA, and the RTK-RAS pathway was blocked using a MEK inhibitor PD0325901.

RESULTS:

Kras activity was much higher in GA cell lines grown as spheroids compared to GA cell lines grown monolayers. Metastasis following KRAS activation in GA cells results from stimulation of epithelial-to-mesenchymal transition (EMT) to cancer stem-like cells (CSCs). In organoids derived from our mouse model, Kras knockdown decreased spheroid formation, expression of EMT-related proteins, migration, and invasion; similar effects, as well as reversal of chemoresistance, were observed following KRAS knockdown in patient tumor-derived GA cell lines. KRAS inhibition in GA spheroid cells led to reduced flank xenograft growth, loss of the infiltrative tumor border, fewer lung metastases, and increased survival. Supporting clinical relevance, high tumor levels of CD44 (a marker of CSCs) and KRAS activation were independent predictors of worse overall survival in 115 GA patients.

CONCLUSION:

In this study, we show that the addition of oncogenic KRAS into gastric epithelial cells leads to EMT and acquisition of CSC phenotypes. Inhibition of KRAS in GA cell lines and in tumor derived organoids reverses EMT and inhibit CSC phenotypes. We conclude that metastasis following KRAS activation in GA cells likely results from stimulation of EMT and transition to CSCs.

Citation Format: Changhwan Yoon, Jacob Till, Soo-Jeong Cho, Kevin Chang, Jian-Xian Lin, Sandra Ryeom, Sam Yoon. KRAS activation in gastric adenocarcinoma stimulates epithelial-to-mesenchymal transition to cancer stem-like cells and promotes metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4680.
Abstract 4682: TGFB signaling disruption in the formation of oral pre-neoplasia
Authors: Andl Claudia D. , Means Anna L. , Lewis James S. , Rothaus Alexandra , Andl Thomas
Published: August 2019

In humans, tissue homeostasis has to be maintained for decades without significant loss of functionality. Epithelia such as the oral mucosa achieve this through the self-renewing capabilities of stem cells. These life-long residents of the oral mucosa maintain homeostasis of the epithelium and at the same time have to be able to protect themselves from the main threats to longevity, i.e., stem cell exhaustion and malignant conversion and carcinogenesis. A key pathway in mediating cell cycle arrest in oral keratinocytes is mediated by TGFB superfamily signaling. There is good evidence implicating TGFB signaling as a tumor suppressor pathway in oral cancer. We have previously shown that disruption of TGFB signaling and cell adhesion can lead to oral squamous cell carcinoma. In vitro using oral keratinocytes, we found that TGFB regulates the stem cell compartment, inducing gene expression of quiescent stem cell markers MECP2, XPC, ITGB1 and PDPN. Using multiplex immunofluorescence, we have stained a tissue microarray containing 28 cases of squamous cell carcinoma, 4 adenocarcinoma, 4 metastatic carcinoma, 6 hyperplasia, and 5 adjacent inflammatory as well as normal adjacent and normal tissues. The antibodies selected were MECP2, XPC, ITGB1 and K15 in combination with the proliferation marker ki67 to determine if TGFB signaling regulates markers of stem cell quiescence. This quiescent gene signature highlighted a basal niche for slow-cycling cells, reserve stem cells. During oral disease progression, marker expression was abandoned in favor of increased proliferation. In vivo, a mouse model in which KrasG12D expression was induced and the Smad4 gene deleted specifically in the adult epithelium showed invasive cancers in the oral cavity. These Smad4-negative tumors express Sox9, a marker of human OSCCs and a TGFB target, which we have also observed to be upregulated in human premalignant lesions. Sox9-positive areas in the mouse tumors are associated with a downregulation of quiescent oral stem cell markers Krt15 and Mecp2, same as we observed in immunostainings of human tissues. In conclusion, TGFB and SMAD4 regulate quiescent stem cells and loss of this pathways allows invasive cancers to develop in the setting of oncogene expression. Furthermore, we could show that MECP2 in combination with other markers identifies quiescent basal cells in the human oral mucosa and is lost as TGFB signaling is disrupted in tumorigenesis.

Citation Format: Claudia D. Andl, Anna L. Means, James S. Lewis, Alexandra Rothaus, Thomas Andl. TGFB signaling disruption in the formation of oral pre-neoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4682.
Abstract 4683: Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies
Authors: Wang Wei , Bhargava Dharmendra , Ye Yuan Chao , Chen Songhai
Published: August 2019

G protein coupled receptors (GPCRs) are the largest family of cell surface receptors with over 800 members. Aberrant activation of GPCRs has been implicated in cancer initiation and development. However, many of GPCRs are overexpressed in cancer cells, making it difficult to target individual GPCRs for cancer treatment. In this study, we have tested a new approach of targeting GPCRs in HER2+ breast cancer by blocking their common pathways downstream of G proteins. By mammary gland-specific expression of the pertussis toxin (PTx) catalytic subunit, a selective inhibitor that uncouples a subgroup of GPCRs from activating Gi/o proteins, we demonstrated that blocking GPCR downstream signaling delayed the onset of HER2-driven spontaneous mammary tumor formation and suppressed lung metastasis. Moreover, we showed that aberrant HER2 signaling upregulated Gi/o-GPCR expression in breast cancer cells, which in turn activated the ErbB/HER family of protein-tyrosine kinases via Src and PI3K pathways to maintain cancer stem cell tumorigenicity. Targeting Gi/o-GPCR signaling by PTx, Src or PI3K inhibitors sensitizes HER2+ breast cancer to HER2-targeted therapies. Together, our data demonstrate that targeting GPCR downstream signal pathways may represent a new approach to ablate CSCs to block tumor progression and augment HER2-targeted therapeutics.

Citation Format: Wei Wang, Dharmendra Bhargava, Yuan Chao Ye, Songhai Chen. Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4683.
Abstract 4686: Stem-like cells and bulk cells in breast cell lines express distinct combinations of Eph receptors and ephrin ligands
Authors: Lucero Mariana , Thind Jaspreet , Senaati Shayan , Jimenez Belinda , Zadeh Tanin , Kandpal Raj P.
Published: August 2019

Purpose: The maintenance of the breast cancer stem cell niche in mammary tumors has been attributed to aberrant expression of Eph receptors and ephrin ligands. The current study aims to identify Eph receptors and ephrin ligands that may contribute to the tumorigenic and invasive breast cancer phenotypes, and can potentially be used to classify tumor specific stem cells and target them for therapeutic intervention.

Methods: The bulk cells and stem-like cells from MCF10A, MCF7 and MDA-MB-231 cell lines were separated based on CD44+/CD24+ and CD44+/CD24- combinations of cell surface markers, respectively. These cell lines represent non-tumorigenic breast epithelium (MCF10A), non-invasive breast carcinoma (MCF7), and invasive triple negative breast carcinoma (MDA-MB-231). The profiles of Eph receptors and ephrin ligands were determined by qPCR. The changes in selected transcripts were confirmed by immunocytochemistry and western blot analysis.

Results: Our study revealed that Eph receptors and ephrin ligands have distinct expression signatures in CD44+/CD24- tumor-initiating cells relative to the bulk tumor cell population. Such combinatorial expression of these molecules in stem cells and bulk cells may modulate and maintain breast tumor phenotypes. The differential patterns of expression are evidenced by changes in qualitative as well as quantitative combinations of Eph receptors and ephrin ligands. The levels of EphA8 transcript were notably lower in CD44+/CD24- cells as compared to bulk MCF7 cells. However, EphA8 was significantly upregulated in stem cells as compared to bulk cells isolated from MDA-MB-231 cell line.

Conclusion: The relative abundance of Eph receptors and ephrin ligands in stem and bulk cells provides molecular signatures of stem cells specific to tumor phenotypes. The combinatorial expression of these molecules corresponds to invasiveness of breast cancer cells, which can be potentially exploited for diagnostics and therapeutics.

Citation Format: Mariana Lucero, Jaspreet Thind, Shayan Senaati, Belinda Jimenez, Tanin Zadeh, Raj P. Kandpal. Stem-like cells and bulk cells in breast cell lines express distinct combinations of Eph receptors and ephrin ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4686.
Abstract 47: Analyzing immune cell infiltration of cancer spheroids in a 3D cell culture platform
Authors: Lim Sei Hien , Kuan Chee Mun , Campisi Marco , Chiono Valeria , Pavesi Andrea
Published: August 2019

The adoption of in vitro 3D cell culture models as a bridge between conventional 2D cell culture models and the complex in vivo animal models has been increasing. A carefully designed 3D model can run biological assays with animal model-like complexities but with the simplicity and affordability of traditional cell culture. For example, traditional 2D and transwell assays of immune cell infiltration efficiency are unrealistic, as cell migration is gravity-driven. AIM 3D cell culture chips offer a more realistic immune cell infiltration model by compartmentalizing immune cells and cancer spheroids in parallel channels. The chips consist of a 3D hydrogel channel and two flanking media channels. Cancer spheroids are cultured within the hydrogel channel while immune cells are seeded in one of the media channels. The seeded immune cells are required to actively invade and seek out target cancer cells in 3D hydrogel in AIM chips before they can infiltrate the cancer spheroids. This is a process similar to immune cell infiltration in vivo. By utilizing high content confocal imaging, fluorescent-labeled immune cells that migrate into the 3D hydrogel can be visualized and quantified. This assay, in combination with adoptive T cell therapy or immune checkpoint blockade, can determine the roles of tumor infiltrated lymphocytes in immunotherapy through quantifying the live: dead ratio of cancer spheroids in the chips. This is particularly useful as a quality control tool for cellular adoptive immunotherapy where the infiltration efficiency and tumoricidal activity of engineered immune cells can be accessed in vitro. In summary, AIM 3D cell culture chips create a more physiologically relevant 3D microenvironment for visualizing immune cell infiltration of cancer spheroids.

Citation Format: Sei Hien Lim, Chee Mun Kuan, Marco Campisi, Valeria Chiono, Andrea Pavesi. Analyzing immune cell infiltration of cancer spheroids in a 3D cell culture platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 47.
Abstract 471: Tumor clonal status predicts clinical outcomes of non-small-cell lung cancer
Authors: Kim Eun Young , Lee Sanghoon , Kim Arum , Kim Tae Hee , Chang Yoon Soo
Published: August 2019

Background: Intratumoral heterogeneity is a major cause of resistance to therapeutic agents and treatment failure. In this study, we evaluated clonal status, which reflects intratumoral heterogeneity, in non-small-cell lung cancer (NSCLC) and we discussed its clinical implications.

Materials and Methods: Targeted sequencing was performed using a customized panel comprising 70 solid tumor-associated genes on samples obtained from 99 lung adenocarcinoma cases. In parallel, TCGA-LUAD cases with clinical information were extracted from the GDC data portal for validation. Clonal status was identified from the allele frequency of the mutated gene using the Maftools package.

Results: We found no significant relationships with stage between the total number of mutations or the number of variants when classified according to the variant effect predictor. There was a positive correlation between the maximum primary tumor diameter and the number of clones (σ=0.2318, P-value = 0.0339). Additionally, the number of clones increased with stage (p-value= 0.0220, Kruskal-Wallis chi-squared test). Disease-free survival (DFS) was significantly shorter among cases wherein tumors comprised two or more clones than among cases in which tumors were comprised of only one clone (P-value = 0.008, Log-rank test). A multivariate model that controlled for total number of mutations, tumor stage, sex, age, and smoking indicated that the number of clones was an independent determinant of DFS.

Conclusions: Number of clones comprising the primary lesion was positively correlated with tumor size and stage, and was an independent factor affecting clinical outcome, indicating that a description of tumor clonality may be required for next-generation sequencing.

Citation Format: Eun Young Kim, Sanghoon Lee, Arum Kim, Tae Hee Kim, Yoon Soo Chang. Tumor clonal status predicts clinical outcomes of non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 471.
Abstract 4730: A novel RAD51 inhibitor, CYT01B, shows anti-cancer activity in preclinical models of AID expressing solid tumors
Authors: Day Melinda , Maclay Tyler , Mills Kevin
Published: August 2019

AID, a DNA-directed cytidine deaminase, plays a critical role in somatic hypermutation and immunoglobulin class switching in maturing B-lymphocytes. Unlike site specific recombinases such as RAG1/2, AID is a promiscuous DNA damaging enzyme that deaminates cytidines at sites throughout the genome. AID expressing cells become critically dependent on the homologous recombination factor RAD51 to repair DNA double strand breaks that result from these off target deamination events. We have developed a novel small molecule, CYT01B, which inhibits RAD51 response to DSBs and is potent to a much greater extent in AID expressing cells. Previously, we have shown this molecule to have activity in mouse lymphoma xenograft models that constitutively express AID. Here we present new preclinical characterization data of CYT01B in solid cancer models. We first analyzed the frequency and levels of AID overexpression in solid tumor data in The Cancer Genome Atlas. Multiple solid tumor types displayed ectopic AID expression, including breast cancer, sarcoma, melanoma, pancreatic cancer, lung cancer, and head and neck cancers with about 30% of the patient samples 4-fold above the baseline expression level. We then tested CYT01B in several solid tumor derived human cell lines. We observed a correlation between AID expression and activity. Cell lines with low ectopic expression of AID had EC50 values in the low micromolar range (~2µM), while those without AID expression gave EC50 values of about 5µM and higher. Next, we examined the activity of our small molecule in 14 different PDX models with various levels of AID expression. These models included renal, head and neck, lung, pancreatic, ovarian, colorectal, and breast cancer samples. We observed a wide range of tumor growth inhibition across the different models (0% to 60%+), which tended to correspond with AID expression; the higher the AID expression the greater the observed tumor growth inhibition. Taken together, these data demonstrate that CYT01B shows anti-cancer activity in a range of solid tumor models. Overall, this provides the basis for continued development of CYT01B as an AID/RAD51 synthetic lethal therapeutic for solid cancers.

Citation Format: Melinda Day, Tyler Maclay, Kevin Mills. A novel RAD51 inhibitor, CYT01B, shows anti-cancer activity in preclinical models of AID expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4730.
Abstract 4731: Therapeutic targeting of RNA splicing through inhibition of protein arginine methylation
Authors: Fong Jia Yi , Low Diana , Pignata Luca , Kawabata Kimihito Cojin , Lee Stanley CW , Koh Cheryl , Musiani Daniele , Massignani Enrico , Wun Cheng Mun , Goy Pierre-Alexis V. , Shen Yudao , Wollmann Heike , Gay Florence PH , Luciani Genna , Barsyte Dalia , Jin Jian , Melnick Ari M. , Bonaldi Tiziana , Abdel-Wahab Omar , Guccione Ernesto
Published: August 2019

Mutations in RNA splicing factors commonly occur in myeloid leukemia and solid tumors. These mutations occur in a heterozygous manner and confer dependency on the wild-type allele. Studies have shown that splicing factor mutant cancers are vulnerable to further perturbation of splicing, by pharmacological intervention that directly targets core splicing factors. Our project identifies the use of PRMT inhibitors, as plausible alternative therapeutic strategy to treat spliceosomal mutant leukemia. The data show that splicing factor mutant leukemia exhibit greater sensitivity to the use of inhibitors against Type I PRMTs and PRMT5, in comparison to their wild-type counterparts. As the need for new therapeutic strategies in cancer treatment increases, this study identifies PRMT inhibitors as a potential therapeutic intervention against cancers with splicing factor mutations.

Citation Format: Jia Yi Fong, Diana Low, Luca Pignata, Kimihito Cojin Kawabata, Stanley CW Lee, Cheryl Koh, Daniele Musiani, Enrico Massignani, Cheng Mun Wun, Pierre-Alexis V. Goy, Yudao Shen, Heike Wollmann, Florence PH Gay, Genna Luciani, Dalia Barsyte, Jian Jin, Ari M. Melnick, Tiziana Bonaldi, Omar Abdel-Wahab, Ernesto Guccione. Therapeutic targeting of RNA splicing through inhibition of protein arginine methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4731.
Abstract 4737: Case report: Preclinical testing of NEDD8 and proteasome inhibitors for a treatment-refractory, metastatic high-grade mucinous colorectal cancer
Authors: Torchiaro Erica , Petti Consalvo , Isella Claudio , Medico Enzo
Published: August 2019

Colorectal cancer (CRC) is the third leading cause of death in the world. CRC shows variable phenotypic make-ups; among them, a particularly aggressive histological subtype is the “high grade mucinous” (HGM) adenocarcinoma, highly mucinous, prone to metastasis and typically refractory to treatments. In some cases, HGM is accompanied by a peculiar “signet ring” phenotype of cancer cells. Early stage diagnosis of signet ring HGM is rare, since clinical symptoms tend to occur late and most cases are usually detected at an advanced stage, with a poor overall survival. We previously demonstrated that a transcriptional signature of HGM displays negative prognosis and sensitivity to the NEDD-8 inhibitor pevonedistat, suggesting the involvement of neddylation- and ubiquitination-based mechanisms in these cases (Picco et al., JNCI 2017 djw209). To assess the clinical potential of colorectal cancer HGM identification and targeting by pevonedistat or other inhibitor of proteasome pathway, we recently propagated cells and PDXs from a case of early onset, metastatic CRC in a Lynch syndrome patient, refractory to standard care (FOLFOX6, FOLFIRI-Panitumumab) and, surprisingly, also to nivolumab. The tumor was highly mucinous, with signet ring undifferentiated cells. Mutational analysis on tumor tissue from the first surgery highlighted PIK3CA H1047R mutation and no mutations in KRAS, NRAS or BRAF. Surprisingly, exome analysis on two lesions from a subsequent surgery displayed a different scenario: KRAS G13D but not PIK3CA mutation. Probably these discording results suggest a strong tumor heterogeneity and evolution. Considering the failure of all previous therapies, the lack of actionable genetic alterations and the peculiarity of the phenotypic features, patient-derived models (organoids and 2D cell cultures) were derived from the two latter lesions and tested for sensitivity to the NEDD8 pathway inhibitor pevonedistat and the proteasome inhibitors bortezomib and carfilzomib. Interestingly, all models showed a strong sensitivity to both drugs, in particular to bortezomib. This is an example of a rare case of high-grade mucinous colorectal cancer where the integration of several approaches proves useful to identify possible therapeutic strategies for a patient without further standard treatment options.

Citation Format: Erica Torchiaro, Consalvo Petti, Claudio Isella, Enzo Medico. Case report: Preclinical testing of NEDD8 and proteasome inhibitors for a treatment-refractory, metastatic high-grade mucinous colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4737.
Abstract 4739: Induction of cell death by HDAC, Aromatase and MDM2 inhibitors in MCF-7 cells
Authors: Venkatesan Thiagarajan , Natarajan Umamaheswari , Radhakrishnan Vijayaraghavan , Samuel Shila , Rathinavelu Appu
Published: August 2019

The histone deacetylase (HDAC) enzymes are evolving as key enzymes in the regulation of many physiological processes, including chromatin remodeling, genome stability, DNA repair, regulation of transcription, protein secretion, cellular metabolism, cell cycle progression, differentiation, migration, and cell death. Among different regulators of HDACs, suberoylanilide hydroxamic acid (SAHA) is a potent inhibitor and it is known to cause strong growth arrest, differentiation and apoptosis of many tumor types in both in vitro and in vivo experimental models. Interestingly, treatment of MCF-7 breast cancer cells with SAHA was found to trigger necroptosis, which is a caspase independent form of cell death. Typically, stimulation of death domain receptors (DDR) by suitable ligands lead to the activation of RIP1, RIP3 and MLKL proteins which are the critical regulators of necroptosis. It appears that, during SAHA induced necroptosis elevation of p21 may be the main signal that triggers recruitment of RIP1, Phospho-RIP3 and MLKL intracellularly in MCF-7 cells. Also, our results confirmed that the apoptosis marker such as BAX and caspases are not elevated by SAHA in MCF-7 cells after 24 hrs of treatment. In addition, the fluorescence assay conducted with SYTOX Green after 10 and 50 µM necrostatin pre-treatments, confirmed that SAHA treatment activates RIP1 mediated necroptosis pathway in MCF-7 cells. The induction of p21 expression was also down-regulated significantly by 10 and 50 µM necrostatin pre-treatments, which further suggests that p21 is up-stream of the necroptosis executioners in MCF-7 cells. On the other hand, when the MCF-7 cells were treated with Letrazole and RG-7388, BAX appears to be involved in mediating cell death. Interestingly, both Letrazole and RG-7388 were not able to elevate p21 in MCF-7 cells. So far, our results suggest that SAHA can induce BAX independent necroptosis while Letrazole and RG-7388 induce cell death through elevation of BAX and BAK in MCF-7 cells. (The generous support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

Citation Format: Thiagarajan Venkatesan, Umamaheswari Natarajan, Vijayaraghavan Radhakrishnan, Shila Samuel, Appu Rathinavelu. Induction of cell death by HDAC, Aromatase and MDM2 inhibitors in MCF-7 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4739.