bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2524: MiR-29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells as well as on tumor cells
Authors: Kimura Yuki , Ohzawa Hideyuki , Saito Akira , Sata Naohiro , Kitayama Joji
Published: September 2020

Background: Peritoneal metastasis (PM) frequently occur in patients with gastric cancer with poor prognosis. Peritoneal cavity is lined with monolayer of mesothelial cells (MC) and contains various types of cells and exosomes. MC play an important role in the development of PM through mesothelial-mesenchymal transition (MMT). MiR-29 family is well known as tumor suppressor. However, the role of miR-29s in PM is not well characterized. We have previously reported that miR-29 family in exosome derived from peritoneal fluid was downregulated in patients with peritoneal metastasis of gastric cancer. In this study, we investigated the functions of miR-29s in the process of peritoneal metastasis.

Methods: Exosomes were purified from peritoneal fluids of 85 patients with gastric cancer and miR-29 levels in peritoneal exosomes were quantified with RT-PCR using TaqMan Advanced miRNA Assays. Proliferation and migration of human gastric cancer cell lines, MKN45 and NUGC, were examined with MTT and transwell assays, respectively. Human peritoneal mesothelial cells (HPMCs) were isolated by trypsinization of omental tissues obtained from patients undergoing bariatric surgery and cultured in DMEM supplemented with 20% fetal bovine serum. MiR-29b mimic and negative control were transfected by lipofection method using RNAiMAX. MMT was induced by the culture of HPMCs with 10 ng/ml TGF-β. Expressions of cytokeratin and vimentin were evaluated with flow cytometry.

Results: The expressions of miR-29a, miR-29b and miR-29c in peritoneal exosomes were all downregulated in 33 patients with PM as compared to those without PM. Transfection of miR-29b mimic significantly decreased the proliferation of MKN45 and NUGC-4 by 22~58% as compared with negative control miRNA (p<0.01). Migration was inhibited more strongly by 84~90% (p<0.01). After 48 hour-culture with TGF-β, HPMCs apparently changed the morphology from round to spindle shape. The expression level of cytokeratin in HPMCs was decreased while vimentin tended to be upregulated by TGF-β. However, transfection of miR-29b, but not control miRNA, significantly suppressed the morphological changes and reduced the expression levels of vimentin with restored expression of cytokeratin, suggesting the inhibition of MMT in HPMCs.

Conclusion: MiR-29b suppresses mesenchymal transition of MC as well as of tumor cells. Replacement of miR-29b to peritoneal cavity might be used for the treatment for peritoneal metastasis.

Citation Format: Yuki Kimura, Hideyuki Ohzawa, Akira Saito, Naohiro Sata, Joji Kitayama. MiR-29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells as well as on tumor cells [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 2524.
Abstract 2548: Pentose phosphate pathway inhibition potentiates metformin cytotoxic effects on HNSCC cells
Authors: Villaverde Marcela Solange , Gutkind Jorge Silvio
Published: September 2020

The pentose phosphate pathway (PPP) enables cancer cells to adapt to anabolic demands that require rapid DNA, RNA, and lipid synthesis and to oxidative cellular stress. Metformin can efficiently inhibit the malignant progression of oral premalignant lesions by indirectly inhibiting the mammalian target of rapamycin (mTORC1). Frequently activated in cancer cells, mTORC1 may upregulate the oxidative branch of the pentose phosphate pathway (PPP) through the sterol regulatory element binding protein (SREBP1). Our previous results showed that the combination of metformin and 6-AN (a PPP inhibitor) strongly potentiates their individual cytotoxic effects on melanoma, glioblastoma, mammary and colon carcinoma cell lines. However, the mechanism of this synergism remains unclear. In this context, the aim of this project was to determine the cell signaling events underlying the therapeutic potential of this combinatory approach, against HNSCC cell lines. Then, we study the effect of metformin alone or in combination with 6-AN on AMPK/mTOR/S6K/S6 and SREBP1 pathway. We found that the inhibition of PPP strongly potentiated metformin cytotoxic effect on 2D and 3D cultures of CAL33 and HN12, both HNSCC cell lines. Furthermore, the combination synergized to increase the activation of AMPK, concomitant with decreased phosphorylation of mTOR, decreased phosphorylation of S6K and decreased phosphorylation of S6. Interestingly, activation of SREBP1 was also synergistically decreased by this combinatory approach. We conclude that the inhibition of PPP and metformin converges to inhibit mTOR pathway. This interaction deserves to be further investigated as a potential strategy to potentiate metformin therapy.

Citation Format: Marcela Solange Villaverde, Jorge Silvio Gutkind. Pentose phosphate pathway inhibition potentiates metformin cytotoxic effects on HNSCC cells [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 2548.
Abstract 2568: Characterization of metabolic changes induced by anti-cancer drugs in MCF7 and MDA-MB-231 cell lines
Authors: Hoffman David L. , Pisano Michael
Published: September 2020

Chemotherapeutics can have profound effects on cancer cell metabolism due to either direct or indirect effects. In many instances these effects can vary based on cell type, and level of cancer type aggressiveness. Here we focus on two breast cancer cell lines MCF7, and the triple negative MDA-MB-231 cells in a panel of assays designed to study specific aspects of cellular metabolism. The cells will be screened against three chemotherapeutics which have previously been shown to have effects on cellular metabolism, paclitaxel, AG-879 and oxaliplatin, and the mitochondrial targeted antioxidant mitoquinol (MitoQ), which has been shown to exhibit anticancer properties. Assays in this panel include cell growth, mitochondrial function, oxidative stress, lysosomal function and lipid storage. Through the utilization of this panel of assays panel, we compare the different metabolic phenotypes of two breast cancer cell lines with varied severity. Additionally, we demonstrate the effects that a small subset of anti-cancer agents has across a wide range of metabolic processes

Citation Format: David L. Hoffman, Michael Pisano. Characterization of metabolic changes induced by anti-cancer drugs in MCF7 and MDA-MB-231 cell lines [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 2568.
Abstract 2579: Kras expression level regulates heparanase activity
Authors: Ju Donghong , Kosir Mary A.
Published: September 2020

Introduction: Sarcomas are locally and systemically aggressive, significantly interacting with extracellular matrix. The purpose of this study is to quantify the expression of heparanase by K-ras expressing sarcoma cells.

Methods: Balb/c3T3 and K234 (Ki-ras-transformed Balb/c 3T3) cells (ATCC) were cultured using DMEM with 5% FBS. Heparanase assays were performed with the heparan degrading enzyme assay kit (cat# MK412; Takara) on cell lysate for each cell line according manufacturer protocol. Dhmaracon on-Target Plus SMARTpool siRNA was used to knock down Kras. Total RNA was isolated with Qiagen RNeasy kit, cDNA was made using Invitrogen SuperScript™ III First-Strand Synthesis System, real time RT-PCR was done by use SYBR green Master Mix and Applied Biosystems® 7500 system, mRNA level for HPSE and PPBP was measured by RTPCR. The UCSC genome browser was used to show HPSE sequence and primer location.

Results: The heparan degrading activity of lysate from K234 cells was 20-30 times higher than from Balb/c3T3 cells. However, nine pairs of primers for mHPSE RT-PCR results show that mHPSE mRNA is expressed differently in Balb/c3T3 and K234 cell with K234 cell mHPSE mRNA level is lower than in Balb/c 3T3 cells. The mRNA sequence is the same in these two cell lines, however. Using siRNA knock down of greater than 80% Kras from K234 and Balb/c3T3 cells, the heparanase activity of lysate decreased by 78% in K234 cells, and 25% in Balb/c3T3 cells. Kras knock down also changed cell morphology in both cell lines, and changed the mRNA levels of HPSE, PPBP and some EMT markers.

Conclusion: Kras overexpressed in K234 cells is associated with increase in heparanase activity, and knock down of Kras decreases heparanase activity. However, heparanase activity does not correlate with HPSE (heparanase 1) expression level, so there is likely another mechanism(s) for Kras expression to regulate heparanase activity.

Citation Format: Donghong Ju, Mary A. Kosir. Kras expression level regulates heparanase activity [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 2579.
Abstract 2580: Contrasting functions of EPSTI1 in human oral and lung squamous cell cancer cell lines - its tumor promoting roles in oral and tumor suppressing roles in lung squamous cell carcinomas
Authors: Fan Meng Meng , Arai Makoto , Tawada Akinobu , Chiba Tetsuhiro , Fukushima Reo , Uzawa Katsuhiro , Shiiba Masashi , Kato Naoya , Tanzawa Hideki , Takiguchi Yuichi
Published: September 2020

Introduction: Epithelial-stromal interaction 1 (EPSTI1) is a gene mapped to human chromosome 13q13.3. Recent studies have shown that EPSTI1 regulates many malignant features of cancers. However, the relevance of EPSTI1 to oral squamous cell carcinomas (OSCC) and lung squamous cell carcinoma (LSCC) are not known. The present study aimed to uncover the roles and the underlying mechanisms of EPSTI1 in OSCC and LSCC.

Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assay were used to evaluate the expression of EPSTI1 in four OSCC cell lines, HSC2, HSC3, HSC3M3 and HSC4, and three LSCC cell lines, LK-2, EBC-1 and H226. In vitro experiments were carried out in parental, their shRNA EPSTI1 knockdown and EPSTI1 overexpressing cells. Cell cycle was evaluated by flow cytometry, and epithelial-to-mesenchymal transition (EMT)-related proteins were evaluated by western blot assay. Changes in expression of EPSTI1-related genes in transfected cells were confirmed by using RT2 Profiler PCR Array Human Cancer Pathway Finder.

Results: EPSTI1 was significantly up-regulated in OSCC cell lines, and significantly down-regulated in LSCC cell lines compared to their normal counterparts. Knockdown of EPSTI1 in OSCC cells and overexpression of the gene in LSCC cells suppressed cell proliferation accompanied by cell-cycle arrest in the G1 phase with up-regulation of p21 and down-regulation of CDK2 and cyclin D1. These indicate that EPSTI1 was significantly involved in tumor growth in OSCC and LSCC, but in an opposite direction. Furthermore, EPSTI1 was related to enhanced cell migration and EMT phenotype in OSCC, and it was related to suppressed cell migration and reversed EMT in LSCC. In PCR-array analyses in two OSCC and two LSCC cells, some common genes were regulated similarly in each OSCC and LSCC.

Conclusion: EPSTI1 was up-regulated and its down-regulation suppressed the malignant phenotypes, indicating oncogenic roles of the gene in OSCC. In contrast the gene was down-regulated and its up-regulation suppressed the malignant phenotypes, indicating tumor suppressive roles of the gene in LSCC. These findings suggest that EPSTI1 might be a therapeutic target for OSCC and LSCC.

Citation Format: Meng Meng Fan, Makoto Arai, Akinobu Tawada, Tetsuhiro Chiba, Reo Fukushima, Katsuhiro Uzawa, Masashi Shiiba, Naoya Kato, Hideki Tanzawa, Yuichi Takiguchi. Contrasting functions of EPSTI1 in human oral and lung squamous cell cancer cell lines - its tumor promoting roles in oral and tumor suppressing roles in lung squamous cell carcinomas [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 2580.
Abstract 2585: Tools for the discovery of KRAS pathway inhibitors
Authors: Kuznetsova Ekaterina , McCauley Lauren , Steinke-Neal Elaine , Sullivan Timothy , Primavera Louis , Ma Haiching
Published: September 2020

The small GTPase, KRAS, is a known oncogene that is frequently mutated in a large percentage of cancers and is associated with poor disease prognosis. Mutated KRAS is locked in the activated GTP bound state and facilitates enhanced Ras signaling in cancer cells. While being a desirable target, absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful.

There has been a resurgence of interest in discovery of KRAS modulators after recent identification of unique binding pockets and successful inhibition of the KRAS G12C mutant by covalent chemical modifiers. With five molecules in clinical trials, many researchers are joining the efforts to find new inhibitors. Ras-mediated signaling commonly occurs through the receptor tyrosine kinase SOS-Ras pathway. To facilitate drug discovery activities targeting this pathway, we have produced the full spectrum of pathway proteins including kinases, wild type and mutated KRAS, and the upstream guanine nucleotide exchange factor protein SOS1. We have also developed and validated a number of biochemical and biophysical assays for these targets. This poster will summarize our most recent assay development efforts for the KRAS mutants including two different assays that monitor SOS1-mediated KRAS nucleotide exchange activities. Molecules that target SOS1 can be characterized using nucleotide exchange and KRAS::SOS1 protein-protein interaction assays. Binding of molecules to KRAS and SOS1 can also be assessed with biophysical methods. These tools and assays will significantly advance the drug discovery efforts of the industry.

Citation Format: Ekaterina Kuznetsova, Lauren McCauley, Elaine Steinke-Neal, Timothy Sullivan, Louis Primavera, Haiching Ma. Tools for the discovery of KRAS pathway inhibitors [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 2585.
Abstract 2587: RAC1P29S expression in melanocytes is associated with increased DNA damage and altered cell cycle progression
Authors: Cannon Alexa C. , Chernoff Jonathan
Published: September 2020

Melanoma is the fifth most common cancer in the United States that has less than a 25% 5-year survival rate once the cancer has metastasized beyond the lymph nodes. Recent exome studies of cutaneous melanoma have identified RAC1P29S as the third most prevalent hotspot mutation in 4-9% of sun-exposed melanoma. RAC1 alteration in cancer is correlated to poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. As a member of the Ras superfamily of small GTPases, RAC1 functions as a master regulator of actin cytoskeleton dynamics and influences proliferation, cell migration, motility, and polarity. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are becoming increasingly evident, the RAC1-driven biological mechanisms contributing to melanomagenesis remain elusive. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis has identified the G2-M cell cycle phase transition as a hyperactive pathway in RAC1P29S-expressing melanocytes. RAC1P29S expression promotes increased kinase activity of the PAK-PLK1-AURORA A axis and increased transition into M-phase as indicated by FACS analysis. Interestingly, RAC1P29S-expressing melanocytes also possess increased DNA damage via γH2AX foci, suggesting a compromise in G2-M checkpoint integrity and an altered DNA damage response. Here, we assess the influence of RAC1P29S on the PAK-PLK1-AURORA A axis and its role in promoting G2-M transition. In addition, we investigate the DNA damage response and cell cycle regulation in the presence of cytotoxic agents. Collectively, these results suggest novel therapeutic pathways to target in RAC1P29S-driven melanoma.

Citation Format: Alexa C. Cannon, Jonathan Chernoff. RAC1P29S expression in melanocytes is associated with increased DNA damage and altered cell cycle progression [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 2587.
Abstract 2593: Inhibition of COX1/2-driven thromboxane A2 pathway suppresses Barrett's esophagus and esophageal adenocarcinoma development
Authors: Zhang Tianshun , Wang Qiushi , Ma Wei-Ya , Wang Keke , Chang Xiaoyu , Johnson Michele L. , Bai Ruihua , Bode Ann M. , Foster Nathan R. , Falk Gary W. , Iyer Prasad G. , Dong Zigang
Published: September 2020

Barrett's esophagus (BE), a complication of gastroesophageal reflux disease (GERD), predisposes patients to esophageal adenocarcinoma (EAC). Reliable biomarkers for early detection and discovery of potential drug targets are urgently needed for improved BE and EAC patient outcomes. In this study, COX2 and TBXAS are highly expressed in BE and EAC patients accompanied by a pronounced elevation of circulating TXA2 levels in patient biopsy samples. Acetylsalicylic acid (ASA) suppressed BE and EAC growth by targeting the TXA2 pathway in a esophagoduodenostomy mouse model of reflux. Additionally, biopsies from patients showed that ASA substantially decreased serum TXA2 levels, resulting in reduced inflammation in a window opportunity clinical trial. Overall, this study establishes the importance of the COX1/2-driven TXA2 pathway in BE and EAC pathophysiology and lays the groundwork for introducing a TXA2-targeting strategy for EAC prevention and early detection. ASA targets the TXA2 pathway and suppresses BE and EAC.

Citation Format: Tianshun Zhang, Qiushi Wang, Wei-Ya Ma, Keke Wang, Xiaoyu Chang, Michele L. Johnson, Ruihua Bai, Ann M. Bode, Nathan R. Foster, Gary W. Falk, Prasad G. Iyer, Zigang Dong. Inhibition of COX1/2-driven thromboxane A2 pathway suppresses Barrett's esophagus and esophageal adenocarcinoma development [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 2593.
Abstract 2596: Toll like receptor 4 as a potential DNA repair modulator in U87MG-GBM cells
Authors: Moretti Isabele F. , Lerario Antonio , Oba-Shinjo Sueli M. , Marie Suely K. , Lima Marina Trombetta
Published: September 2020

Toll like receptor 4 (TLR4) has a dual role in cellular fate, depending on the stimuli and microenvironment context. Its activation may promotes cell survival and proliferative phenotype via MyD88 with NFkB activation, or leads to cell death via TRIF. Glioblastoma (GBM), grade IV astrocytoma, is the most common and aggressive primary malignancy in the brain. Surgical tumor cytoreduction followed by radiotherapy and adjunct chemotherapy with Temozolomide (TMZ) have been considered the standard of care for GBM. And, in spite of several combinatory rescue therapeutic trials, the median overall survival of GBM patients continue to be approximately 15 months. In this context, new therapeutic strategies are urgent, and immune modulation has demonstrated optimistic results in several tumor types. Therefore, the aim of this study was to analyze the role of TLR4 in human astrocytomas. First, we evaluated TLR4 expression in 140 human astrocytoma of different grades of malignancy, by qRT-PCR. TLR4 molecular signaling pathways were investigated through LPS stimuli in U87MG-GBM cell line. In astrocytoma samples, TLR4 was up-regulated when compared to brain non-neoplastic samples (p<0.05, Kruskall Wallis and Dunn tests), with increased expression in Mesenchymal compare to Classical and Proneural subtypes. U87MG cells stimulation with LPS (10ug/ml) lead to NF-kB (p65) nuclear translocation after 12hr through western blot and immunofluorescence, which was unexpected as such translocation through the MyD88/TRAF6 canonical pathway occurs within 30min to 2hr after LPS stimulation. TRIF, IL1B, RIPK1/3 expressions were increased after LPS stimulation, suggesting an activation of inflammasome and ripoptosome pathways. Moreover, the combined LPS treatment with TMZ showed a higher apoptotic rate after 48hr when compared to TMZ alone. However, the transcriptomic analysis by RNASeq of U87MG cells after LPS stimulation showed an upregulation of DNA repair genes, particularly MBD4 and PLAUR. Both genes are upregulated in mesenchymal GBM subtype in the TCGA RNASeq dataset, and they present correlated expression with TLR4 (r=0.406, p=0.004 and r=0.338, p=0.019 by Spearman's test). Our results suggest that the anti-tumoral effect by TLR4 pathway activation by enhancement of tumor apoptosis might be more efficient with combined inhibition of DNA repair cascade. In fact, OSI-296 and BKM-120 have shown to decrease MBD4 expression, and increased overall survival was observed in a phase II clinical trial for solid tumor.

Citation Format: Isabele F. Moretti, Antonio Lerario, Sueli M. Oba-Shinjo, Suely K. Marie, Marina Trombetta Lima. Toll like receptor 4 as a potential DNA repair modulator in U87MG-GBM cells [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 2596.
Abstract 2601: Expression and clinicopathological association of RABs in pancreatic cancer
Authors: Anand Shashi , Zubair Haseeb , Khan Mohammad Aslam , Singh Seema , Singh Ajay P.
Published: September 2020

Rab proteins form the largest subfamily of small GTPases and play an indispensable role in the regulation of vesicle trafficking. They control a wide variety of endocytic, transcytic and exocytic transport pathways and thus play important roles in cellular growth and behavior. Altered expression and functions of Rab proteins have been reported in various malignancies and implicated in tumorigenic processes. Pancreatic cancer (PC) is a highly lethal malignancy and molecular mechanisms associated with its pathobiology are relatively less understood. Here we mined The Cancer Genome Atlas (TCGA) Database for differential expression and clinicopathological association of RAB genes in pancreatic cancer. Out of the 62 RAB genes analyzed, 5 (RAB3A, RAB26, RAB25, RAB21, and RAB22A) were found to have significant upregulation in cancer, while 5 (RAB6B, RAB8B, RABL2A, RABL2B and RAB32) exhibited significant downregulation. Racially disparate expression was also reported for some RABs. RAB3A and RAB26 had significantly higher and lower expression, respectively, in African American (AA) and Asian PC as compared to Caucasian Americans (CA) PC, whereas RAB25 was significantly higher in Asian PC than CA PC. No clear trend of altered expression was observed with increasing stage and grade, although stage- and grade-specific differences were recorded for some RABs. RAB21, RAB22A, and RAB8B showed significantly higher expression in PC from middle-aged patients (41-60 years) compared to older age groups, but no gender-associated differential expression was observed. PC from occasional drinkers had significantly higher expression of RAB21 compared to daily or weekly drinker, whereas RAB25 expression was significantly higher in social drinkers, compared to occasional ones. Expression of RABL2A was significantly reduced in PC of diabetic patients whereas RAB26 was significantly down in pancreatitis patients. Most importantly, a significant association of high expression of RAB21, RAB22A and RAB25, and low expression of RAB6B, RABL2A, and RABL2B was observed with poorer survival of PC patients. Together, our study is suggestive of potential diagnostic and prognostic significance of RABs in PC and warrants further investigations to define their functional and mechanistic significance.

Citation Format: Shashi Anand, Haseeb Zubair, Mohammad Aslam Khan, Seema Singh, Ajay P. Singh. Expression and clinicopathological association of RABs in pancreatic 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 2601.
Abstract 2611: Comparison of 3D culture conditions to develop an in vitro model for breast cancer cell emboli formation
Authors: Vitale Ariana , Sterneck Esta , Kuppusamy Balamurugan
Published: September 2020

Inflammatory breast cancer (IBC) is a rare type of breast cancer accounting for 2-4% of all breast cancer patients, but accounts for 7-10% of breast cancer related mortality. IBC is characterized by extensive skin invasion and the formation of tumor cell emboli within the breast and dermal lymphatic system. Characteristics of emboli include cell to cell adhesions mediated by E-cadherin, encoded by the gene CDH1. Studies from our laboratory show that CEBPD is expressed in IBC patient emboli and promotes emboli formation in vitro. Mechanistically we found that CEBPD promotes cell-cell adhesion within emboli at least in part by supporting the expression of E-cadherin adhesion complex proteins. In this study, we compared an in vitro model for intra-lymphatic emboli formation with conditions of tumor sphere formation to begin to address the role of fluid shear in emboli formation and emboli-specific molecular events, if any. We conducted experiments under slow rocking and non-rocking culture conditions with 4 different culture media. We found that the mechanical shear forces (achieved by low speed rocking) generated large, well-compacted emboli-like structures within 24 hours of suspension culture. In the absence of fluid shear, the cells assembled into smaller and more irregular aggregates. In agreement with this, our preliminary data suggest that E-cadherin expression is modestly higher under fluid shear conditions in addition to overall increase under 3D conditions. We are now validating the mechanisms for higher expression of emboli in 3D culture conditions and are designing experiments to address the functional relevance of the pathways for IBC emboli biology. Understanding the molecular mechanisms of emboli formation may provide valuable insights into the uniquely aggressive nature of IBC.

Citation Format: Ariana Vitale, Esta Sterneck, Balamurugan Kuppusamy. Comparison of 3D culture conditions to develop an in vitro model for breast cancer cell emboli formation [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 2611.
Abstract 264: Long noncoding RNA LNC01534 regulates cancer stem cells in colorectal cancer
Authors: Ohtsuka Masahisa , Xin Wu , Ichihara Momoko , Hirose Haruka , Takahashi Hidekazu , Matsuda Chu , Akamatsu Hiroki , Mizushima Tsunekazu , Doki Yuichiro , Yamamoto Hirofumi
Published: September 2020

Introduction The clinical importance of cancer stem cells (CSCs) has been widely reported, and clinical studies targeting CSCs are in progress. Additionally, accumulating evidence suggests that long noncoding RNAs (lncRNAs) have essential roles in cancer initiation and progression, while deregulated lncRNA expression is found in a variety of cancer types. In this study, we focused on lncRNAs regulating CSCs in colorectal cancer (CRC).

Objectives To identify the lncRNAs associated with CSCs and to evaluate their clinical relevance and function.

Materials and Methods We separated CSCs and other cells in CRC using the ornithine decarboxylase (ODC) degron system to visualize CSCs based on the correlation between low proteasome activity and CSC properties. We performed comprehensive gene analysis comparing CSCs and other cells to identify a candidate lncRNA that regulates the stemness of CRC cells. Finally, we analyzed the clinical relevance and function of the lncRNA.

Results The lncRNA LNC01534 showed high expression in CSCs compared with the level in other CRC cells. Using gene expression and clinical data from the Cancer Genome Atlas Project for CRC patients, we showed that LNC01534 was associated with overall survival in CRC. LNC01534 was also associated with overall survival and disease-free survival in 187 separate CRC samples. Functional studies revealed that the loss of LNC01534 function reduced proliferation and arrested the cell cycle in CRC cells. Furthermore, genes that underwent expression changes upon the knockdown of LNC01534 were evaluated by RNA-seq. This showed that the knockdown of LNC01534 affected the expression of genes related to endoplasmic reticulum (ER) stress and autophagy. Specifically, the expression levels of HSPA5, LC3-I, p62, and Beclin-1–genes related to ER stress and autophagy–increased upon suppressing LNC01534.

Conclusions We demonstrated the clinical and biological relevance of LNC01534 in association with CSCs, and discovered the effect of LNC01534 on ER stress and autophagy to maintain stemness in CRC.

Citation Format: Masahisa Ohtsuka, Wu Xin, Momoko Ichihara, Haruka Hirose, Hidekazu Takahashi, Chu Matsuda, Hiroki Akamatsu, Tsunekazu Mizushima, Yuichiro Doki, Hirofumi Yamamoto. Long noncoding RNA LNC01534 regulates cancer stem cells in colorectal 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 264.
Abstract 2688: Pan-cancer analysis of clonality and the timing of systemic spread in paired primary tumors and metastases
Authors: Hu Zheng , Li Zan , Ma Zhicheng , Curtis Christina
Published: September 2020

Metastasis is the primary cause of cancer-related deaths, but the natural history, clonal evolution and patterns of systemic spread are poorly understood. We analyzed exome sequencing data from 458 paired primary tumors (P) or metastasis (M) samples from 136 breast, colorectal and lung cancer patients, including both untreated (n=98) and treated (n=101) metastases. We find that treated metastases often harbored private driver gene mutations whereas untreated metastases did not, suggesting the acquisition and selection of additional mutations is not required for untreated metastases and treatment promotes clonal evolution. Additionally, the ratio of nonsynonymous versus synonymous mutations (dN/dS) showed decreased selection pressures in the metastasis relative to primary tumors. Polyclonal seeding was common in lymph node metastases (n=19/35, 54%; mostly untreated) and untreated distant metastases (n=20/70, 29%), but less frequent in treated metastases (n=9/90, 10%). Mathematical modeling shows that the low number of metastasis-private clonal mutations is consistent with early metastatic seeding, which commonly occurred several years prior to diagnosis in breast (2.4 years, range 0-3.3), lung (3.6 years, range 2.8-3.7) and colorectal (4.1 years, range 3.1-4.6) cancers. Thus, this pan-cancer analysis reveals early systemic spread in three common cancer types. Further, these data suggest that the natural course of metastasis is selectively relaxed relative to early tumor development and that metastasis-private mutations are not drivers of cancer spread but are instead associated with drug resistance.

Citation Format: Zheng Hu, Zan Li, Zhicheng Ma, Christina Curtis. Pan-cancer analysis of clonality and the timing of systemic spread in paired primary tumors and metastases [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 2688.
Abstract 2732: NLRP12 suppresses colorectal cancer progression and invasion via downregulation of the Wnt/b-catenin pathway
Authors: Khan Shahanshah , Zaki Hasan
Published: September 2020

Metastatic CRC is associated with increased mortality, underscoring the importance of understanding the molecular mechanism of CRC invasion and metastasis. We recently demonstrated that mice deficient in NLRP12, an innate immune sensor, are susceptible to CRC with increased incidence of invasive adenocarcinoma. Here, we investigated the molecular mechanism of NLRP12-mediated suppression of cancer progression and invasion using mouse models and in vitro biochemical studies. Nlrp12−/− mice treated with azoxymethane plus dextran sodium sulfate developed higher tumor burden and invasive adenocarcinoma compared to WT mice. RNA-seq analysis showed significantly higher levels of protooncogenes, matrix metalloproteinases (Mmp 3, 7,8, 10, 12), and epithelial to mesenchymal transition (EMT) markers (Vim, Fn1, Foxc2, Zeb2, Ezh2) in Nlrp12−/− tumors. To understand the underlying mechanism of increased expression of cancer promoting genes in Nlrp12−/− tumors, we analyzed activation of various cell signaling pathways. To our surprise, there was no major difference in the activation NF-κB, ERK, JNK, and STAT3, which have previously been shown to be downregulated by NLRP12, between WT and Nlrp12−/- tumors. However, we observed strikingly higher levels of β-catenin and expression of Wnt target genes (Ctnnb1, Axin2, Yap1, Tcf3, Lgr5, etc.) in Nlrp12−/− tumors. Consistently, deletion of NLRP12 in APCmin/+ mice led to higher polyp development and activation of the Wnt/β-catenin pathway. Notably, hyperactivation of the Wnt/β-catenin pathway is a hallmark of CRC and about 80% of CRC patients carry mutations in the Wnt/β-catenin pathway. To further explore the link between NLRP12 and the Wnt/β-catenin pathway, we overexpressed NLRP12 in HEK293T cells and CRC cell line MC38. NLRP12-overexpressed cells showed reduced activation of β-catenin and expression of Wnt target genes following stimulation with Wnt3a. An opposite result was observed when NLRP12 was knocked out from these cell lines. Consistently, Nlrp12-KO MC38 cells exhibited increased proliferation, colony formation, and migration. These data for the first time demonstrate that NLRP12 is an intrinsic negative regulator of the Wnt/β-catenin pathway. Overall, these findings reveal a novel CRC suppression pathway involving NLRP12 and suggest that the NLRP12/β-catenin signaling axis could be therapeutically targeted for the treatment of CRC invasion and metastasis.

Citation Format: Shahanshah Khan, Hasan Zaki. NLRP12 suppresses colorectal cancer progression and invasion via downregulation of the Wnt/b-catenin pathway [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 2732.
Abstract 2736: AKT1E17K activates focal adhesion kinase and promotes melanoma brain metastasis
Authors: Kircher David A. , Trombetti Kirby A. , Silvis Mark R. , Parkman Gennie L. , Fischer Grant M. , Angel Stephanie N. , Stehn Christopher M. , Strain Sean C. , Grossmann Allie H. , Duffy Keith L. , McMahon Martin , Davies Michael A. , Mendoza Michelle C. , VanBrocklin Matthew W. , Holmen Sheri L.
Published: September 2020

Hyper-activation of the PI3K/AKT signaling pathway occurs in most metastatic melanomas and increased PI3K/AKT pathway activity correlates with disease progression. The serine/threonine kinase, AKT, represents a major signaling hub within the pathway and consists of three highly conserved paralogs that have both distinct and overlapping functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated the ability of constitutively active E17K, E40K, or Q79K mutants of each AKT paralog to promote tumor progression and metastasis in the context of BRAFV600E expression and loss of Cdkn2a and Pten. Expression of AKT1E17K promoted highly aggressive melanomas that metastasized to the lungs and brain. This metastatic phenotype was not significantly observed in the case of other mutant AKT-positive tumors, suggesting that the AKT paralogs have distinct, non-overlapping roles in the development of melanoma metastases. AKT1E17K-positive tumors showed AKT1E17K-dependent up-regulation of multiple focal adhesion (FA) factors, which are key components of focal adhesions and established stimulators of cell motility, as well as phosphorylation of focal adhesion kinase (FAK). Ectopic expression of AKT1E17K in non-metastatic melanoma cells increased cell invasion, a phenotype abrogated by pharmacological inhibition of AKT or FAK. These findings strongly suggest that one mechanism by which AKT1 promotes melanoma metastasis is through regulation and activation of proteins involved in focal adhesions. This has important implications for the development of therapeutic strategies aimed at preventing or treating disseminated disease.

Citation Format: David A. Kircher, Kirby A. Trombetti, Mark R. Silvis, Gennie L. Parkman, Grant M. Fischer, Stephanie N. Angel, Christopher M. Stehn, Sean C. Strain, Allie H. Grossmann, Keith L. Duffy, Martin McMahon, Michael A. Davies, Michelle C. Mendoza, Matthew W. VanBrocklin, Sheri L. Holmen. AKT1E17K activates focal adhesion kinase and promotes melanoma brain 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 2736.
Abstract 2738: Role of KLF8 for drug-resistant metastasis of breast cancer
Authors: Hao Jie , Lu Heng , Mukherjee Debarati , Sun Aiqin , Yu Lin , Zhao Jihe
Published: September 2020

Metastasis and drug resistance are intertwined processes that are responsible for the vast majority of patient deaths from breast cancer. The underlying mechanisms still need to be investigated extensively. We previously demonstrated that KLF8 activates CXCR4 transcription in metastatic breast cancer. Here, we report a novel signaling mechanism that turns a localized metastasis into multiple organ metastases in response to chemotherapy. We show that KLF8 expression in breast cancer cells can be over-induced by chemotherapeutic drugs. Data from large-cohorts of patients indicates that post-chemotherapy there is a close correlation between the aberrant high levels of KLF8 and CXCR4, and this correlation is well associated with drug resistance, metastasis, and poor prognosis. To mimic this upregulation, we overexpressed KLF8 or CXCR4 in a human breast cancer cell line known to metastasize to the lungs only after i.v. injection in the nude mice. Surprisingly, we found that these KLF8 or CXCR4 overexpressing cells metastasized all over the body after orthotopically implanted. Tube formation assay, Ki67 staining, and western blotting revealed that KLF8 or CXCR4 overexpression promoted angiogenic vascular growth involving increased expression and secretion of VEGF protein. We also performed the matrigel-on-top 3-D culture to mimic the colonization step of metastasis and found that KLF8 or CXCR4 overexpression strongly enhances the formation of filopodium-like protrusions and proliferation via CXCR4 stimulation. Taken together, these results suggest that the chemo-induction of KLF8 upregulation is critical for drug resistance and systemic metastasis through enhanced tumor angiogenesis and colonization via CXCR4 over-activation. This discovery identifies the KLF8-CXCR4 signaling axis as a potentially new therapeutic target for breast cancer.

Citation Format: Jie Hao, Heng Lu, Debarati Mukherjee, Aiqin Sun, Lin Yu, Jihe Zhao. Role of KLF8 for drug-resistant metastasis of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2738.
Abstract 276: Mir-139 regulates autophagy in prostate cancer cells through Beclin-1 and mTOR signaling proteins
Authors: Benatar Tania C. , Nam Robert , Sherman Christopher , Amemiya Yutaka , Seth Arun
Published: September 2020

Background: Prostate cancer represents the second leading cause of cancer death in men and develops as a result of the accumulation of genetic and epigenetic alterations. We previously identified a panel of five miRNAs associated with biochemical recurrence and metastasis following prostatectomy from prostate cancer patients using NGS-based whole miRNome sequencing and qPCR-based validation analysis. We have shown that expression of miR-139, one of the downregulated miRNAs from this panel, was associated with improved prognosis in patients with localized prostate cancer. Furthermore, miR-139 overexpression inhibited prostate cancer cell growth through signaling pathways mediated in part through downregulation of AXL and IGF1R. Here, we further investigated the molecular mechanisms by which miR-139 could inhibit prostate cancer cell growth.

Methods: We examined miR-139 transfected PC3, DU145 and LNCaP cells by morphology as well as by cell-based assays, confocal microscopy and immunoblotting.

Results and Discussion: We found that treatment of prostate cancer cells with miR-139 resulted in phenotypic changes characteristic of autophagic cells. Conversion of the microtubule-associated protein light chain 3 (LC3-I to LC3-II) was used to monitor autophagy. We observed that the treatment of PC3 and LNCaP cells with miR-139 increased the conversion of LC3-I to LC3-II protein that was specifically inhibited by addition of miR-139 antagomir. The upregulation of LC3 II was further confirmed by confocal microscopy of PC3 cells treated with miR-139. Mammalian target of rapamycin (mTOR) and Beclin1 are two important autophagy-related molecules playing significant roles in different stages. mTOR negatively regulates autophagy machinery. Treatment of cells with miR-139 inhibited both activation and expression of mTOR. We also observed increased levels of phospho-Beclin 1 in PC3 cells treated with mir-139. Beclin 1 is a scaffold protein that assembles components for promoting or inhibiting autophagy and its phosphorylation controls autophagy. The cargo adaptor protein, p62/SQSTM1, interacts with autophagic substrates, delivering them to the autophagosome for degradation, and is degraded during autophagy. We found that during early treatment of cells with miR-139, p62 expression is inhibited, while at later stages, p62 expression accumulates, suggesting that autophagic flux may be blocked. These results suggest that miR-139 is regulating autophagy in prostate cancer cells at least in part through the mTOR and Beclin-1 proteins.

Citation Format: Tania C. Benatar, Robert Nam, Christopher Sherman, Yutaka Amemiya, Arun Seth. Mir-139 regulates autophagy in prostate cancer cells through Beclin-1 and mTOR signaling proteins [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 276.
Abstract 2778: Mapping the extracellular pH gradient of colorectal cancer after immuno-thermal ablation
Authors: Slovak Ryan J. , Coman Daniel , Ludwig Johannes M. , Kim Hyun S.
Published: September 2020

Background: Biosensor imaging of redundant deviation in shifts (BIRDS) is a non-invasive MR imaging modality that maps extracellular pH (pHe) by directly detecting paramagenetically shifted non-exchangeable protons of exogeneous agents. Previously it has been used to characterize various tumors in the rat brain and to monitor the response to loco-regional treatment of liver cancer. The purpose of this study was to investigate the feasibility of utilizing pHe mapping with BIRDS to differentiate ablated and non-ablated tumors in the setting of systemic immunotherapy of murine colorectal cancer (CRC).

Materials and Methods: Eight BALB/c mice (Charles River Laboratories, Wilmington, MA, USA) were implanted with bilateral flank injections of 0.5 × 106 CT26.WT colorectal cancer cells (CRL- 2638; ATCC, Manassas, VA). Mice were randomly assigned into 3 treatment groups; group 1 received injections of sham antibodies (InVivoMAb IgG controls), group 2 was treated with dual anti-PD-1 and anti-CTLA-4 antibodies (Clone J43 & Clone 9D9; BioXcell, West Lebanon, NH, USA), III). Group 3 received cryoablation in combination with systemic injections of dual checkpoint inhibitors. Mapping of pHe was performed between 3-10 days after the initiation of treatment and was immediately followed by sacrifice.

Results: The mean pHe of untreated, control tumors was significantly lower than the average pHe of tumors treated with immuno-thermal ablations (6.6 vs 6.96; p<0.05). Among the mice in the ablation groups, the targeted tumors demonstrated significantly less acidity on average than the distal, off-target tumors (6.96 vs 6.63; p<0.05). The average pHe of tumors in the DICB group was not significantly different than any other group (6.75 vs 6.6, 6.96 & 6.63; p>0.05).

Conclusions: BIRDS MR quantification was feasible for imaging murine CRC model. BIRDS quantification of pHe could serve as an imaging non-invasive biomarker for metabolic changes of the tumor microenvironment in a murine CRC model with Immuno-Thermal Ablation Therapy.

Significance at a GlanceTreatment GroupMean Extracellular pHSignificant DifferencesControl6.6Vs Target Ablated Tumors (p<0.05)Dual Immune Checkpoint Blockade (DICB)6.75NoneTargeted tumors in Ablated group6.96Vs Control (p<0.05) Vs Off-target Tumors (p<0.05)Off-target tumors in Ablated Group6.63Vs Ablated Tumors (p<0.05)

Citation Format: Ryan J. Slovak, Daniel Coman, Johannes M. Ludwig, Hyun S. Kim. Mapping the extracellular pH gradient of colorectal cancer after immuno-thermal ablation [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 2778.