bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2326: Differential lipid metabolism in mammary cancer cell progression
Authors: Andoline Chaylen , Pawar Parik , Teegarden Dorothy
Published: July 2021

There is an association between increased lipid accumulation and aggressive breast cancer phenotypes, and therefore poorer clinical outcomes. The mechanism by which dysregulated lipid metabolism may promote metastasis remains incompletely understood. Several potential mechanisms by which the neutral lipid storage organelles, cytoplasmic lipid droplets, may promote metastasis include utilization of fatty acids (FAs) for oxidation, protection against lipotoxcity, and production of lipid signaling mediators. We have previously demonstrated that metastatic MCF10CA1a mammary cancer cells rely on fatty acid oxidation (FAO) compared to non-metastatic MCF10A-ras cells, using etomoxir to inhibit the rate-limiting enzyme of FAO, carnitine palmitoyltransferase 1 (CPT1). Previous literature indicates that there is an increase in various lipid metabolism pathways, including FA uptake, de novo lipogenesis, and FA storage in neutral lipid. We found that there is no significant difference between palmitic acid uptake between the non-metastatic and metastatic cells. In the current study, we show that MCF10CA1a cells have significantly higher levels of carbon incorporation into palmitic and oleic FAs from 13C-acetate (25% and 20% greater), 13C-glucose (21% and 11% greater), and 13C-lactate (29% and 22% greater) respectively, compared to the MCF10A-ras cells, as detected by LC-MS/MS. In order to determine the source of FAs that contributes to the migration-dependent FAO, we assessed MCF10CA1a cell migration following inhibition of lipolysis (adipose triglyceride lipase—ATGL; ATGListatin) alone or in combination with the FAO inhibitor, etomoxir. We determined that inhibition of ATGL, the initial enzyme of triglyceride lipolysis pathway, significantly reduces MCF10CA1a cell migration (20% decrease compared to vehicle). Inhibition of FAO and lipolysis did not induce additional reduction in cell migration. We also showed that MCF10CA1a cells are more dependent on FA metabolism than the non-metastatic MCF10A-ras cells, demonstrated by MCF10CA1a cells having a greater decrease in cell viability compared to the MCF10A-ras cells given each FA metabolism inhibition (p<0.05: 22% greater decrease for FAO inhibition and 16% greater decrease for dual FAO and lipolysis inhibition, p=0.05: 17% greater decrease for FA synthesis inhibition). These data suggest that metastatic MCF10CA1a cells rely on their increased FA metabolism to survive and migrate, and specifically that the catabolism of FAs from triglyceride supports the FAO-driven migration of metastatic cancer cells.

Citation Format: Chaylen Andoline, Parik Pawar, Dorothy Teegarden. Differential lipid metabolism in mammary cancer cell progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2326.
Abstract 2345: Development of a novel diagnostic system for bile duct cancer using urinary metabolites
Authors: Amano Hizuru , Hinoki Akinari , Uchida Hiroo , Yokota Kazuki , Ishigaki Takashi , Sakairi Minoru , Abe Mayumi , Terui Yasushi , Oda Hirohisa , Tainaka Takahisa , Shirota Chiyoe , Sumida Wataru , Makita satoshi , Takimoto Aitaro , Okamoto Masamune , Yasui Akihiro , Nakagawa Yoichi
Published: July 2021

Background and Aim: Urine contains numerous small molecular metabolites that can be used as reliable, non-invasive cancer biomarkers. The aim of this study was to identify biomarkers for bile duct cancer by comprehensively analyzing urinary metabolites from patients with bile duct cancer.

Materials and Methods: This study was carried out on urine samples collected before and after tumor resection in 13 patients with bile duct cancer. Urine metabolites were extracted using liquid chromatography/mass spectrometry. Biomarker candidates were identified using the Wilcoxon rank-sum test, random forest method, and orthogonal partial least squares discriminant analysis.

Results: We developed an AI technology that efficiently searched for urine tumor markers based on the detailed medical information of each urine sample and used them to construct cancer test models. About 1500 metabolites, such as amino acids, nucleic acids, and lipids, were detected in one urine sample. Ten urine tumor marker candidates for bile duct cancer were identified.

Conclusions: We identified novel urine tumor marker candidates for bile duct cancer. We are currently investigating additional urine samples from 23 patients with bile duct cancer. The possibility of detecting and monitoring bile duct cancer using urine tumor markers has been examined.

Citation Format: Hizuru Amano, Akinari Hinoki, Hiroo Uchida, Kazuki Yokota, Takashi Ishigaki, Minoru Sakairi, Mayumi Abe, Yasushi Terui, Hirohisa Oda, Takahisa Tainaka, Chiyoe Shirota, Wataru Sumida, satoshi Makita, Aitaro Takimoto, Masamune Okamoto, Akihiro Yasui, Yoichi Nakagawa. Development of a novel diagnostic system for bile duct cancer using urinary metabolites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2345.
Abstract 2359: MicroRNA 494-5p acts as tumor suppressor in colorectal cancer by targeting JAK1
Authors: Allgayer Heike , Patil Nitin , Abdelrahim Omar , Leupold Joerg H.
Published: July 2021

Recently, we have characterized the metastatically relevant microRNA landscape by comparing microRNA (miR) expression between human colorectal metastasis and matched primary tumors and normal tissues (Mudduluru et. al. Cancer Res. 2015; PMID: 26069251). One of these, miR-494-5p, was deregulated already in primary tumors. In vitro invasion and migration (Boyden chamber and Matrigel) assays of cultured colorectal cancer cells were significantly reduced after miR-494-5p overexpression (mimic). Conversely, this was abolished by anti-miR-494-5p as an inhibitor. Overexpression of miR-494-5p in cell lines showed a significant reduction in cellular proliferation, which was reversed upon inhibitor co-transfection in a time dependent manner. In silico analysis suggested an interaction of miR-494-5p with the 3´UTR of JAK1. Accordingly, an overexpression of miR-494-5p in cultured colon cancer significantly reduced luciferase activity of a reporter plasmid containing the wild type JAK1-3'UTR, which was abolished when the miR-494-5p seed sequence was mutated to prevent miR-494-5p binding, suggesting a physical interaction and translational repression of JAK1 by miR-494-5p. Furthermore, overexpression of miR-494-5p led to a significant reduction of JAK1 expression in colorectal cancer cell lines in a time dependent manner, which was abolished by co-transfecting a specific anti-miR-494-5p as an inhibitor. Taken together, our preliminary data suggests that miR-494-5p inhibits the expression of JAK1 as a direct molecular target via specific interaction at the JAK1-3'UTR. Additionally, miR-494-5p is capable of reducing proliferation, migration, and invasion, suggesting miR-494-5p as an early tumor suppressor molecule in colorectal cancer.

Citation Format: Heike Allgayer, Nitin Patil, Omar Abdelrahim, Joerg H. Leupold. MicroRNA 494-5p acts as tumor suppressor in colorectal cancer by targeting JAK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2359.
Abstract 2360: Identifying differentially expressed miRNAs in CRC stem cell subpopulations
Authors: Stark Victoria A. , Viswanathan Vignesh , Facey Caroline O. , Opdenaker Lynn M. , Boman Bruce M.
Published: July 2021

Our research goal is to discover the molecular mechanisms behind tumor heterogeneity in CRC. We previously showed that CRCs contain multiple subpopulations of cancer stem cells (CSCs) which may explain the occurrence tumor heterogeneity and resistance to treatment. To determine how CSC sub-populations might arise, we are studying miRNA expression in CRC SCs. MicroRNAs are known to regulate SC phenotype and are found to be dysregulated in many cancers.

Hypothesis: Tumor heterogeneity results from existence of multiple CSC subpopulations that are regulated by distinct miRNAs. Accordingly, we are using bioinformatics and miRNA profiling to identify miRNAs that target SC genes in CRCs.

Indeed, our miRNA expression profiling of normal and malignant ALDH+ human colonic SCs showed that miRNA92a targets the SC gene LRIG1 and upregulation of miRNA92a leads to decreased LRIG1 expression. We also discovered that miRNA23b targets the SC gene LGR5 and miRNA23b is upregulated in ALDH+ CSCs. We have identified several other candidate miRNAs that are predicted to target CD166, ALDH1A1, BMI1, LRG5, and LRIG1 SC genes. We are currently in the process of validating whether these miRNAs contribute to emergence of specific CSC sub-populations in CRCs. Thus, identifying miRNAs that regulate CSC subpopulations should provide new strategies to modulate CSC composition in order to sensitize tumors to treatments.

Citation Format: Victoria A. Stark, Vignesh Viswanathan, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Identifying differentially expressed miRNAs in CRC stem cell subpopulations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2360.
Abstract 2365: Intracellular interaction of downstream molecular mediators of miR-1207-3p in prostate cancer cells
Authors: Ghosh Priyanka , Ogunwobi Oloronseun
Published: July 2021

Prostate cancer (PCa) is the second most common cancer diagnosed among men in the United States. Human chromosome 8q24 is the most important PCa susceptibility locus. This region contains the MYC oncogene, which is involved in early PCa initiation. Downstream to MYC is the PVT1 gene, which is often amplified in PCa. PVT1 is a non-protein coding gene that encodes six annotated microRNAs (miRNAs), including miR-1207-3p. Our laboratory has previously demonstrated miR1207-3p to be a significant modulator in PCa and revealed that miR-1207-3p is significantly underexpressed in PCa cells and histologically confirmed PCa tissues compared to normal prostatic cells and tissues. We have also shown that miR-1207-3p has tumor suppressive activity in vivo. Moreover, we discovered that miR-1207-3p directly targets fibronectin type III domain containing 1 (FNDC1), which was found to be overexpressed in PCa cells and there is a concomitant overexpression of (FN1)/androgen receptor (AR)/c-MYC. In an effort to better understand the role of FNDC1/FN1/AR/c-MYC in PCa progression we examined the interaction between FNDC1/FN1/AR/c-MYC and found direct physical interaction through coimmunoprecipitation. We also examined the spatial localization of the FNDC1/FN1/AR/c-MYC pathway by performing immunofluorescence staining in PCa cells. Single staining analysis revealed that FNDC1 and c-MYC localize to the nucleus and cytoplasm while AR localizes to the nucleus and FN1 localizes to the cytoplasm in PCa cells. However, in normal prostate epithelial cells FNDC1, AR and c-MYC localize to the nucleus whereas FN1 localize to the cytoplasm. These results suggest that the underexpression of miR1207-3p may be affecting the localization of FNDC1 to cytoplasm and thus promoting interaction between FNDC1/FN1/AR/c-MYC. Future studies include examining the effect of miR-1207 on the spatial organization and interactions of the FNDC1/FN1/AR/c-MYC pathway through immunofluorescence and co-immunoprecipitation assays. Thus, understanding this molecular interaction could reveal interesting insight into the biology of microRNA-1207-3p regulation in PCa.

Citation Format: Priyanka Ghosh, Oloronseun Ogunwobi. Intracellular interaction of downstream molecular mediators of miR-1207-3p in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2365.
Abstract 2367: Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma
Authors: Wu Xun , Cheng Yi-Shing Lisa , Matthen Methew , Yoon Angela , Schwartz Gary , Bala Shashi , Taylor Alison M. , Momen-Heravi Fatemeh
Published: July 2021

MicroRNAs (miR) are evolutionarily highly conserved non-coding RNAs that play roles in fundamental cellular functions by post-translational gene regulation. In cancer pathogenesis, miRs exert both anti-tumorigenic and pro-tumorigenic effects by virtue of miR-specific and context-dependent mechanisms. The role of chromosomal changes and mutation status on miR expression in cancer remains unknown. Here we provide evidence that chromosome arm 1p loss and P53 mutations are associated with lower levels of miR-34a, particularly in squamous cancers such as head and neck squamous cell carcinoma (HNSCC). miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET, and modulates cell proliferation. miR-34a expression is inversely correlated with MET in tumor tissue. We found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Moreover, ectopic expression of miR-34a reduces HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes and proteins involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Our work demonstrates a role of miR-34a in maintaining tumor immunity, as overexpression of miR-34a resulted in a decease in percentage of anti-inflammatory (M2-like) TAMs and PDL1+TAMs, which are reported to have pro-tumorigenic activity. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells, myeloid cells, and regulatory T cells. Our results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity, and could potentially represent a new therapeutic approach for HNSCC.

Citation Format: Xun Wu, Yi-Shing Lisa Cheng, Methew Matthen, Angela Yoon, Gary Schwartz, Shashi Bala, Alison M. Taylor, Fatemeh Momen-Heravi. Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2367.
Abstract 238: Exhaustive tumor specific antigen detection with RNAseq
Authors: Sharpnack Michael , Johnson Travis , Chalkley Robert , Han Zhi , Carbone David , Huang Kun , He Kai
Published: July 2021

Background: Tumor specific antigen (TSA) identification in human cancer predicts response to immunotherapy and provides vaccine targets for precision medicine. In addition to neoantigens from somatic coding mutations, numerous non-mutated TSAs can elicit T-cell responses but are often overlooked by current methods. We present a method that accurately and comprehensively predict TSAs from RNAseq data regardless of mutation status.

Methods: HLA-I genotypes were predicted with seq2HLA. RNAseq fastq files were translated into all possible peptides of length 8-11, and peptides with high expression in the tumor and comparatively low expression in normal were tested for their MHC-I binding potential with netMHCpan-4.0. We defined our predicted TSA by i) high expression in tumor samples, ii) low expression in normal samples, and iii) high predicted patient-specific MHC-I binding affinity.

Results: We developed a novel pipeline for TSA prediction from RNAseq that is not limited to mutation-derived TSAs. This pipeline was used to predict all possible unique peptides size 8-11 on previously published murine and human lung and lymphoma tumors then validated on matched tumor and control lung adenocarcinoma (LUAD) samples. This pipeline is able to predict TSAs in MHC-I ligand-purified proteomics data with favorable performance to existing methods. Furthermore, neoantigens predicted by exomeSeq are typically poorly expressed at the RNA level, (28% of predicted neoantigens with >0 expression, mean of 15.6 reads/sample) and a fraction of them (47/6,928, 0.68%) are expressed in matched normal samples. Finally, a set of 6 TSAs are expressed in 22/39 (56%) of LUAD tumors and represent attractive vaccine targets.

Conclusion: Direct quantification of RNAseq evidence of the potential peptidome in matched tumor and control RNAseq samples, via our novel pipeline, allows for exhaustive detection of TSAs.

Citation Format: Michael Sharpnack, Travis Johnson, Robert Chalkley, Zhi Han, David Carbone, Kun Huang, Kai He. Exhaustive tumor specific antigen detection with RNAseq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 238.
Abstract 2387: Targeting PVT1 exon 9 transcript is not pro-apoptotic but induces claudin 4 expression and inhibits migration in triple negative breast cancer cells
Authors: Levine Fayola A. , Ogunwobi Olorunseun O.
Published: July 2021

Triple negative breast cancer (TNBC) is a lethal molecular subtype of invasive breast cancer (BC) that is ER-, PR- and HER2-. Claudin low (CL) TNBC, a distinct molecular subtype of TNBC, has the worst prognosis compared to other BC subtypes. Aberrant expression of claudin proteins disrupts the function of tight junctions. Consequently, inducing epithelial-to-mesenchymal transition (EMT) in cancers, an important factor for enhanced motility and metastasis. Therefore, understanding the molecular mechanisms that modulate claudin expression in TNBC is crucial. The 8q24 genomic locus is an important cancer susceptibility locus that is associated with poor clinical outcomes in cancer due to common amplification events that occur in many malignant diseases. This locus contains the PVT1 gene, which encodes a long noncoding RNA (lncRNA) that has been implicated in multiple cancers including BC. PVT1 consists of 12 exons that are alternatively spliced to generate lncRNAs. Although previous research has implicated PVT1 as an important player in BC, the underlying molecular mechanisms of PVT1 in CL TNBC is unknown. To examine the role of PVT1 in CL TNBC, we assessed PVT1 expression in T47D (ER+), MDA MB 231 (CL) and MDA MB 468 (claudin high (CH)) BC cells. We observed that PVT1 exons 4A, 4B, and 9 are significantly overexpressed in CL MDA MB 231 and significantly underexpressed in CH MDA MB 468 in comparison to T47D cells, suggesting a protumorigenic role of PVT1 in CL TNBC. We analyzed the functional consequences of siRNA targeting of PVT1 exon 9 expression in CL TNBC cells. siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells led to a significant reduction in migration and the re-expression of claudin 4. To determine the role of PVT1 on EMT, we assessed the expression of EMT markers (vimentin, fibronectin, and E-cadherin) in MDA MB 231 cells. We observed no changes in the expression of EMT markers when PVT1 exon 9 is knocked down. However, our data show that mesenchymal markers are more highly expressed in MDA MB 231 cells in comparison to MDA MB 468 cells. Further, knockdown of PVT1 exon 9 did not induce apoptosis in MDA MB 231 cells as assessed by caspase 3 and caspase 9 expression. Taken together, our data indicate that PVT1 exon 9 regulates claudin expression and migration in CL TNBC, and may have implications for clinical outcomes in TNBC.

Citation Format: Fayola A. Levine, Olorunseun O. Ogunwobi. Targeting PVT1 exon 9 transcript is not pro-apoptotic but induces claudin 4 expression and inhibits migration in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2387.
Abstract 2389: LINC00901 regulates glucose metabolism in pancreatic cancer
Authors: Peng Wan-Xin , Yang Liu , Mo Yin-Yuan
Published: July 2021

Dysregulation of metabolism is one of the cancer hallmarks. However, little is known whether long coding RNAs (lncRNAs) play a role in the regulation of metabolism in pancreatic ductal adenocarcinoma (PDAC). In the present study, we focused on lncRNA LINC00901 because a high level of LINC00901 is associated with the poor outcome of PDAC patients. Functional studies showed that LINC00901 promotes PDAC cell proliferation and invasion, whereas knockout (KO) of LINC00901 impairs these abilities of PDAC cells. Furthermore, the xenograft animal model suggested that LINC00901 promotes tumorigenesis. To determine the underlying mechanism of LINC00901-mediated tumorigenesis, we performed RNA-seq analysis in LINC00901 overexpression or KO cells. Gene Set Enrichment Analysis (GSEA) analysis showed that gene sets involved in glucose biological processes such as ‘‘MYC targets”, “Glycolysis” and “Gluconeogenesis” are significantly enriched in the LINC00901 overexpression group, whereas these genes are down-regulated in the LINC00901 KO group. The top-scoring genes recurring in the three gene sets include key glycolysis-related genes, lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and enolase 1 (ENO1), all of which were validated by qRT-PCR and western blot. Moreover, we found that LINC00901 upregulates c-Myc by stabilizing c-Myc RNA. Together, these results suggest that LINC00901 is an oncogenic lncRNA capable of regulating glucose metabolism, leading to PDAC development. Experiments are underway to determine how LINC00901 regulates c-Myc RNA stability in pancreatic cancer cells.

Citation Format: Wan-Xin Peng, Liu Yang, Yin-Yuan Mo. LINC00901 regulates glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2389.
Abstract 2390: LncRNA PAINT promotes prostate cancer progression through modulation of genes involved in epithelial-mesenchymal transition and apoptosis
Authors: Ganapathy Kavya , Hasan Md Faqrul , Sun Jiao , Khatib Ayman , Andl Thomas , Soulakova Julia , Coppola Domenico , Zhang Wei , Chakrabarti Ratna
Published: July 2021

Long non-coding RNAs (LncRNAs) play regulatory roles in cellular processes and their aberrant expression may drive cancer progression. Here we report the function of a LncRNA PAINT (Prostate Cancer-Associated Intergenic Non-Coding Transcript) in promoting prostate cancer (PCa) progression. Upregulation of PAINT expression was noted in the advanced stage and metastatic PCa patient tissues and correlated with poor survival. Inhibition of PAINT decreased cell proliferation, S-phase progression, increased expression of apoptotic markers, and improved sensitivity to docetaxel and Aurora kinase inhibitor VX-680. Inhibition of PAINT also decreased cell migration and reduced expression of mesenchymal markers, Slug, and Vimentin. Ectopic expression of PAINT suppressed E-cadherin, increased S-phase progression, and cell migration. PAINT expression in PCa cells also induced larger colony formation and higher expression of mesenchymal markers. Transcriptome profiling of PCa cells overexpressing PAINT showed differential expression of genes involved in epithelial-mesenchymal transition (EMT), apoptosis, and drug resistance which were further validated by qRT-PCR analysis. Additionally, GO enrichment and KEGG pathway analysis revealed altered expression of multiple pathways including p53 signaling and drug metabolism pathways indicating that PAINT may promote an oncogenic environment by simultaneously regulating various biological processes in PCa. In summary, our findings establish the role of PAINT as an oncogene in PCa and indicate the clinical significance of PAINT as a diagnostic marker and a possible therapeutic target for aggressive PCa.

Citation Format: Kavya Ganapathy, Md Faqrul Hasan, Jiao Sun, Ayman Khatib, Thomas Andl, Julia Soulakova, Domenico Coppola, Wei Zhang, Ratna Chakrabarti. LncRNA PAINT promotes prostate cancer progression through modulation of genes involved in epithelial-mesenchymal transition and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2390.
Abstract 2393: The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique
Authors: Ray Roslyn M. , Lang Lu , Pokharel Sheela P. , O'Meally Denis , Scott Tristan A. , Chen Chun-Wei , Morris Kevin V.
Published: July 2021

Long non-coding RNAs (LncRNAs) have been found to play important roles in maintaining and promoting cellular proliferation in cancerous cells. One such lncRNA, LINC00963 has been found to promote cellular proliferation, migration and tumorigenesis in prostate, ovarian, breast and non-small cell lung (NSCLC) cancer. In this study, we aim to identify important functional regions of this lncRNA with a view to identify unique regions for targeted drug therapy. In order to achieve this, we sought to investigate this lncRNA in the A549 NSCLC cell line. We found that LINC00963 was highly expressed and when targeted with either siRNAs or antisense oligonucleotides, cells rapidly undergo apoptosis. Using cell death as a proxy for loss-of-function, we characterized LINC00963 at the genomic level using a CRIPSRScan approach. CRISPRScan is a novel way of determining lncRNA functional domains through the introduction of local mutations. These mutations could potentially disrupt lncRNA function, which for LINC00963 would lead to apoptosis of the cell. We created a unique lentivirus library comprised of 1784 single guide RNAs spanning the length of LINC00963 in order to precisely identify regions of functional importance. Upon transduction in a stably expressing Cas9-A549 cell line, we collected cellular fractions over time, and using high-throughput sequencing (NextSeq, Illumina), evaluated the depletion of these sgRNAs as a proxy for their importance in LINC00963 function. The identification of these key genomic regions that may render LINC00963 non-functional may offer new lncRNA target sites for anti-cancer therapies in the future. Additionally, the utilization of an in-depth CRISPR-Cas9 approach to determine important lncRNA domains provides a novel and precise way in which to evaluate lncRNA function.

Citation Format: Roslyn M. Ray, Lu Lang, Sheela P. Pokharel, Denis O'Meally, Tristan A. Scott, Chun-Wei Chen, Kevin V. Morris. The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2393.
Abstract 2397: Significance of the combination of biguanides and fatty acid β-oxidation inhibitors in triple-negative breast cancer
Authors: Park Junhyoung , Jung Kwang Hwa , Jia Dongya , Yang Sukjin , Sakthivel Dharaniya , Tiwari Abha , Onuchic José N. , Kaipparettu Benny Abraham
Published: July 2021

Compared to hormone receptor (HR) positive breast cancer (BC), basal or triple-negative BC (TNBC) suffers a poor prognosis, attributed to the limited understanding of its driver signaling pathways. Although TNBC exhibits unique characteristics, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are essential to reduce its severity. We have previously shown that metabolic reprogramming to fatty acid β-oxidation (FAO) is a dominant metabolic pathway in TNBC. We have also reported that FAO regulates c-Src, one of the frequently upregulated oncopathways in TNBC, via autophosphorylation of Src at Y419. However, single drug therapy using FAO inhibitors or Src inhibitors did not show a significant therapeutic advantage in TNBC. Biguanide such as metformin is one of the most widely administered anti-diabetic agents. Recently increasing evidence demonstrates that biguanide therapy can have an anticancer effect in various cancer types, including BC. Phenformin, a biguanide derivative similar to metformin, has greater potency than metformin. Both biguanides can suppress mitochondrial electron transport chain (ETC) function because of its inhibitory effect on ETC complex I activity. However, biguanides also lead to the activation of AMPK, which plays a vital role in insulin signaling and energy-sensing. Notably, AMPK is an upstream regulator of the FAO pathway because it can phosphorylate ACC and activate FAO. Considering the dependency of TNBC on mitochondrial FAO and the role of AMPK in the activation of FAO, our computational modeling predicted that the combined targeting of both ‘arms' of the pathway could provide a significant advantage to the anticancer effect in TNBC. Thus, we have used the combination of biguanides and FAO inhibitors to target both ETC and FAO respectively. Pharmacological and genetic approaches with multiple in vitro and in vivo analysis using TNBC cell lines and PDX models suggest that the combination of biguanides and FAO inhibitors can provide significant advantage in TNBC therapy. Further studies also show that the combination of biguanides with Src inhibitors can provide a better therapeutic option to target metastatic TNBC. We are currently evaluating these findings' translational significance as a cost-effective approach to managing non-targetable TNBCs.

Citation Format: Junhyoung Park, Kwang Hwa Jung, Dongya Jia, Sukjin Yang, Dharaniya Sakthivel, Abha Tiwari, José N. Onuchic, Benny Abraham Kaipparettu. Significance of the combination of biguanides and fatty acid β-oxidation inhibitors in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2397.
Abstract 2402: Tid1 deficiency disrupting mitochondria to drive NASH-dependent hepatocellular carcinoma progression
Authors: Chang Ching-Wen , Chen Yu-Syuan , Lo Jeng-Fan , Wang Xin Wei
Published: July 2021

Non-alcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) is a significant risk factor to drive hepatocellular carcinoma (HCC). However, a preclinical model of progressive NAFLD/NASH is largely lacking. Here, we report that mice with hepatocyte specific deletion of Tid1, encoding a mitochondrial cochaperone, tended to develop NASH-dependent HCC. Mice with hepatic Tid1 deficiency showed impairing mitochondrial function and causing fatty acid metabolic dysregulation, meanwhile; sequentially developed fatty liver, NASH, and cirrhosis/HCC in a Diethylnitrosamine (DEN) induced oxidative environment. The pathological signatures of human NASH, including cholesterol accumulation and activation of inflammatory and apoptotic signaling pathways, are also present in these mice. Clinically, low Tid1 mRNA expression was associated with unfavorable prognosis in patients with HCC. Empirically, hepatic Tid1 deficiency directly disrupts entire mitochondria that plays a key mechanism involved in the NASH-dependent HCC development. Overall, we established a new mouse model developing NASH-dependent HCC and provided a promising approach for improving the treatment.

Citation Format: Ching-Wen Chang, Yu-Syuan Chen, Jeng-Fan Lo, Xin Wei Wang. Tid1 deficiency disrupting mitochondria to drive NASH-dependent hepatocellular carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2402.
Abstract 2409: The regulation of mitochondria metabolism is correlated with gemcitabine resistance in MIA-PaCa-2 cell line
Authors: Fujiwara-Tani Rina , Kishi Shingo , Mori Shiori , Kuniyasu Hiroki
Published: July 2021

We established gemcitabine (GEM)-resistant pancreatic ductal adenocarcinoma (PDAC) cell line, MIA-G cells from MIA-PaCa-2 (MIA-P) cells by continuous low-dose GEM treatment. MIA-G cells was also resistant to 5-fluorouracil in comparison with MIA-P. Metabolic flux analysis showed that oxygen consumption rate (OCR) in MIA-G cells was higher than MIA-P cells, however, OCR was suppressed by GEM treatment only in MIA-G cells. In MIA-P cells, GEM treatment generated elevation of mitochondrial membrane potential and mitochondrial ROS, but not MIA-G cells. In MIA-G cells, expression of mitochondrial transcription factor A was also decreased. Moreover, glutamine uptake and glutamine peroxidase level were elevated in MIA-G cells. These findings suggest that suppression of OXPHOS contributes to GEM resistance through reduction of ROS production.

Citation Format: Rina Fujiwara-Tani, Shingo Kishi, Shiori Mori, Hiroki Kuniyasu. The regulation of mitochondria metabolism is correlated with gemcitabine resistance in MIA-PaCa-2 cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2409.
Abstract 2412: Decursin inhibits tumor progression in head and neck squamous cell cancer by down regulating CXCR7 expression
Authors: Joo Mina , Kim Solbi , Kim Nayoung , Lee Myung-Won , Jeon Heung Jin , Ryu Hyewon , Song Ik-Chan , Kim Jin-Man , Lee Hyo Jin
Published: July 2021

Background: CX chemokine receptor 7 (CXCR7) is frequently overexpressed in a variety of tumors and plays a significant role in tumor growth and metastasis. Thus, inhibition of CXCR7 is an important potential anticancer treatment strategy. Decursin, a derivative from Angelica gigas Nakai has been reported to have anti-tumor activity in various cancers. However, little is known about its anti-tumor activity and underlying mechanisms of action in head and neck squamous cell cancer (HNSCC).

Methods: CXCR7-overexpressing HNSCC cell lines were established in vitro. The effects of decursin on cell growth, motility, migration, and invasion were investigated using CCK-8, wound healing, and transwell assays. In addition, the mechanism by which decusin shows antitumor activity was investigated.

Results: Overexpression of CXCR7 significantly increased cell proliferation, migration, invasion, and cell motility compared with control cells. Treatment with decursin decreased membrane expression of CXCR7 in a concentration dependent manner and significantly inhibited tumor progression by suppressing cell growth, migration, invasion, and motility. Decursin was found to induce G0/G1 cell cycle arrest and regulate cell cycle-related proteins such as Cyclin A and CDK2 in HNSCC cells. In addition, decursin inhibited CXCR7-induced STAT3/c-MYC activation.

Conclusion: These results demonstrate that CXCR7 promotes cancer progression through the STAT3/c-Myc pathway in HNSCC and that decursin is a natural product that may target CXCR7 for HNSCC treatment.

Citation Format: Mina Joo, Solbi Kim, Nayoung Kim, Myung-Won Lee, Heung Jin Jeon, Hyewon Ryu, Ik-Chan Song, Jin-Man Kim, Hyo Jin Lee. Decursin inhibits tumor progression in head and neck squamous cell cancer by down regulating CXCR7 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2412.
Abstract 2415: The role of GTSF1 as a regulator of retrotransposons and its impact on carcinogenesis
Authors: Villarreal Amelia Martínez , Gantchev Jennifer , Litvinov Ivan
Published: July 2021

Hypomethylation of repetitive sequences is a common feature reported in cancer, particularly of retrotransposon elements. Retrotransposon elements can be deleterious due to their potential as insertional mutagens, so cells have deployed several control mechanisms to silence them through methylation of DNA. These methylation pathways are different between somatic and germline cells. In germline cells, the piRNA pathway is the main process responsible for silencing the retrotransposon LINE-1. GTSF1 is germline cell specific and an important part of this pathway. Along with PIWI proteins in the piRNA pathway, GTSF1 processes small RNA molecules and identifies active retrotransposons, recruiting the DNA methylation machinery to those sites. Aberrant expression of GTSF1 has been reported in cancer, particularly on lymphomas. We performed a Bayesian Analysis and Markov Chain Monte Carlo Method with GTSF1 levels of expression reported on The Cancer Genome Atlas (TCGA), which confirmed high levels of mRNA in lymphomas. Nonetheless, the role of GTSF1 in retrotransposon control in cancer and in carcinogenesis remains unknown. To address this issue, we have performed shRNA mediated knockdown in head and neck squamous cell carcinoma and cutaneous T-cell lymphoma cell lines, and we have overexpressed the cDNA of GTSF1 in immortalized keratinocytes. We evaluated the impact of the knockdown and overexpression on cell biology, retrotransposons, and genomic instability. Overall, GTSF1 expression seems to affect LINE-1 retrotransposon protein expression and function. Also, we are assessing the impact on genomic instability and methylation profiles. The aberrant expression of this gene poses an excellent clinical opportunity for targeted therapy development. Its germline cell restricted expression will potentially result in less secondary effects than current available therapies.

Citation Format: Amelia Martínez Villarreal, Jennifer Gantchev, Ivan Litvinov. The role of GTSF1 as a regulator of retrotransposons and its impact on carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2415.
Abstract 2416: Upregulation of amplified in breast cancer 1 contributes to pancreatic ductal adenocarcinoma progression and vulnerability to blockage of hedgehog activation
Authors: Li Licen , Bao Jiaolin , Wang Haitao , Lei Haipeng , Cheng Peng , Zeng Jianming , Hao Wenhui , Zhang Xu , Xu Xiaoling , Yu Chundong , Deng Chu-Xia , Chen Qiang
Published: July 2021

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and devastating cancers without effective treatments. Amplified in breast cancer 1 (AIB1) is a member of the steroid receptor coactivator family that mediates the transcriptional activities of nuclear receptors. While AIB1 is associated with the initiation and progression of multiple cancers, the mechanism by which AIB1 contributes to PDAC progression remains largely unknown. In this study, we aimed to explore the role of AIB1 in the progression of PDAC and elucidate the underlying mechanisms.

Methods: The clinical significance and mRNA level of AIB1 in PDAC were studied by database analysis. To demonstrate whether AIB1 mediates the malignant features of PDAC cells, namely, proliferation, migration, invasion, we performed real-time PCR and Western blot analysis, established xenograft models and used in vivo metastasis assay. With insights into the mechanism of AIB1, we performed RNA sequencing (Seq), ChIP-Seq, luciferase reporter assays and pull-down assays. Furthermore, we analyzed the relationship between AIB1 expression and its target expression in PDAC cells and patients and explored whether PDAC cells with high AIB1 levels are sensitive to inhibitors of its target.

Results: We found that AIB1 was significantly upregulated in PDAC and associated with its malignancy. Silencing AIB1 impaired hedgehog (Hh) activation by reducing the expression of smoothened (SMO), leading to cell cycle arrest and the inhibition of PDAC cell proliferation. In addition, AIB1, via upregulation of integrin αv (ITGAV) expression, promoted extracellular matrix (ECM) signaling, which played an important role in PDAC progression. Further studies showed that AIB1 preferably bound to AP-1 related elements and served as a coactivator for enhancing the transcriptional activity of MafB, which promoted the expression of SMO and ITGAV. PDAC cells with high AIB1 levels were sensitive to Hh signaling inhibitors, suggesting that blocking Hh activation is an effective treatment against PDAC with high AIB1 expression.

Conclusions: These findings reveal that AIB1 is a crucial oncogenic regulator associated with PDAC progression via Hh and ECM signaling and suggest potential therapeutic targets for PDAC treatment.

Citation Format: Licen Li, Jiaolin Bao, Haitao Wang, Haipeng Lei, Peng Cheng, Jianming Zeng, Wenhui Hao, Xu Zhang, Xiaoling Xu, Chundong Yu, Chu-Xia Deng, Qiang Chen. Upregulation of amplified in breast cancer 1 contributes to pancreatic ductal adenocarcinoma progression and vulnerability to blockage of hedgehog activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2416.
Abstract 2418: PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression
Authors: Miller Karina , Degan Seamus , Wang Yanqing , Ku Sheng-Yu , Goodrich David W. , Gelman Irwin H.
Published: July 2021

Background: The loss of PTEN function, one of the most frequent mutations in prostate cancer (CaP), is presumed to drive disease progression through AKT activation. However, two transgenic mouse CaP models, Pten/RbPE:-/- and Akap12-/-;RbPE:-/-, exhibited different CaP and metastasis development even though both shared Rb loss plus Akt activation, based on increased relative AKTpoS473 levels.

Experimental Procedures: We analyzed whether PI3K-p110 and/or AKT isoform reliance, using isoform-specific RNAi or small molecule inhibitors, played a role in the different CaP outcomes.

Findings: In comparison to Akap12/Rb-null high-grade prostatic intraepithelial neoplasias, Pten/Rb-null adenocarcinomas had increased protein and activation levels of AKT2 and decreased relative levels of Smad4, a known CaP metastasis suppressor. Comparison of isogenic PTEN +/- pairs of human or mouse CaP cell lines indicated that PTEN-deficiency correlated with dependence on both p110β and AKT2 for survival or oncogenic growth/invasiveness, whereas PTEN expression correlated with greater dependence on AKT1 plus p110α. Inhibition of AKT2 in PTEN-null CaP cells induced higher SMAD4 levels and decreased chemotaxis and Matrigel invasiveness. Given that PTEN likely regulates non-redundant roles of the AKT isoforms during CaP progression, we sought to characterize AKT isoform substrates by phosphoproteomics analysis. Specifically, after endogenous kinases were inactivated in mouse Pten/Rb-null adenocarcinoma cell lysates using 5'-fluorosulfonylbenzoyl-5'-adenosine (FSBA), dialyzed samples were subjected to in vitro kinase reactions using enzymatically-active, full-length baculovirus-expressed AKT1, AKT2 or AKT3, or as a negative control, a heat-inactivated mixture of all three isoforms. Tryptic peptides isolated on TiO2 beads were subjected to LC-MS/MS. Data will be shown on the role of AKT isoform-shared vs. -preferred substrates in specific parameters of CaP progression controlled by PTEN.

Conclusion: Our data suggest that in the context of RB1 loss, PTEN controls a signaling plasticity and drug sensitivity through specific pairings of PI3K-p110 and AKT isoforms.

Citation Format: Karina Miller, Seamus Degan, Yanqing Wang, Sheng-Yu Ku, David W. Goodrich, Irwin H. Gelman. PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2418.
Abstract 2422: Genetic aberrations of Anaplastic Lymphoma Kinase (ALK) are functional drivers in head and neck squamous cell carcinoma (HNSCC)
Authors: Wang Lan , Yeung Thomas Chun Kit , Ngan Hoi-Lam , Poon Peony Hiu Yan , Lui Vivian Wai Yan
Published: July 2021

Head and neck squamous carcinoma (HNSCC) is a cancer with high genetic heterogeneity. Fusions of Anaplastic Lymphoma Kinase (ALK) have been widely described as a functional driver for lung cancer. Yet, mutations and amplification of ALK are found in HNSCC as well as many other human cancers. Their biological impacts on cancer cell growth are underexplored.

Using the Cancer Genome Atlas (TCGA) HNSCC dataset (Firehose Legacy; N=510), we identified a subset of HNSCC patients harboring ALK aberrations (4%), including 0.4% cases with gene amplification and 3.5% with somatic mutations. Transcriptome analysis showed significant tumor-specific ALK upregulation in 43 adjacent normal-HNSCC tumor pairs (2-fold, p=0.001***, TCGA HNSCC Firehose Legacy). Interestingly, clinical analysis revealed that ~94% HNSCC patients with ALK amplification and mutations have advanced diseases (stage III or IV). Among the HNSCC-associated ALK aberrations, ALK amplification, as well as ALK p.R401L mutations are recurrent events. Thus, we sought to investigate their biological functions by ectopic expressions into an HNSCC cell line, FaDu, via retroviral infection. We found that overexpression of ALK WT gene (mimicking gene amplification) and ALK p.R401L significantly promoted cell proliferation vs. EGFP-control (p=0.0004***, 0.01*, respectively). Clonogenic assay also demonstrated both events to be drivers for colony survival vs. EGFP-control by 120%, 150% (p=0.02*, 0.0025**, respectively). Lastly, both events markedly increase FaDu cells invasiveness by Matrigel invasion assay by 67%, 58% (vs. EGFP-control, p<0.0001****, 0.0001****, respectively). In conclusion, ALK aberrations are functional drivers promoting HNSCC aggressiveness.

Acknowledgements: This study is funded by General Research Fund, Research Grant Council, Hong Kong Government, Hong Kong SAR (#17121616 ). VWYL also receives funding supports from General Research Fund, Research Grant Council, Hong Kong Government, Hong Kong SAR (#17121616, #14168517), Research Impact Fund (#R4015-19F, R4017-18), the Health and Medical Research Fund (HMRF#15160691) from the Health and Medical Research Fund, the Food and Health Bureau, the Government of the Hong Kong Special Administrative Region), University-Industry Collaboration Programme (UIM/329; Innovation and Technology Fund, Hong Kong Government, Hong Kong SAR), and the Hong Kong Cancer Fund, Hong Kong SAR.

Citation Format: Lan Wang, Thomas Chun Kit Yeung, Hoi-Lam Ngan, Peony Hiu Yan Poon, Vivian Wai Yan Lui. Genetic aberrations of Anaplastic Lymphoma Kinase (ALK) are functional drivers in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2422.