Patient-matched Normal Tissue Research Articles

7948 Integration Of Epigenomics And Metabolomics Reveals New Signaling In Aggressive Thyroid Cancer

Abstract Disclosure: A. Sanghi: None. S. Wu: None. T. Chou: None. L.A. Orloff: None. M. Kasowski: None. M. Snyder: None. Although genetic mutations are causal in human cancers, the contribution of chromatin changes to cancer onset and progression remains less well understood. Multi-omics profiling of human tumors can provide insight into how alterations in chromatin structure are propagated through the pathway of gene expression to result in cancer signaling. We applied multi-omics profiling of 36 human thyroid cancer primary tumors, metastases, and patient-matched normal tissue. Over 80% of the tumors harbored BRAFV600E mutations, and the remaining tumors mostly had activating mutations in the MAPK pathway. Through quantification of chromatin accessibility associated with active transcription units and global protein expression, we identify local enhancers that are highly correlated with coordinated RNA and protein expression. The enhancers are predicted to be actively bound by MAPK transcription factor, indicating they are under MAPK control. These cancer-specific enhancers associated with upregulation of lysosomal peptidase expression and dipeptide abundance in tumors. We show that upregulated dipeptides stimulate MAPK activity, and BRAF inhibition results in loss of lysosomal function in mutants. Perturbation of lysosomal peptidases and dipeptide abundance decreases MAPK activity only in mutant cells compared to wildtype. These analyses suggest that BRAF thyroid tumors integrate MAPK-driven epigenetic regulation with lysosomal metabolite signaling in a positive feedback circuit. Using local cancer-specific epigenetic features, we isolated key cancer signaling, making way for potential targets for cancer therapeutics. Presentation: 6/3/2024

281. PROTEOMIC, TRANSCRIPTOMIC AND GENOMIC ANALYSIS OF ESOPHAGEAL ADENOCARCINOMA UNCOVERS CANDIDATE THERAPEUTIC TARGETS AND CANCER-SELECTIVE POST-TRANSCRIPTIONAL REGULATION

Abstract Background Addressing the poor prognosis associated with esophageal adenocarcinoma (EAC) has been hindered by the absence of biomarkers for early disease detection and therapeutic targets. Despite extensive research on the somatic mutations associated with EAC, the impact of genomic aberrations on protein expression and cancer phenotype remains unclear. Methods We conducted a quantitative proteomic analysis using tumour tissue and patient-matched adjacent normal esophageal and gastric tissues from 23 patients undergoing resection for EAC. We further examined the relationship between transcript and protein levels using patient-matched whole transcriptome RNA sequencing and proteomic data from 7 patients and integrated these findings with data from a cohort of 264 EAC patients with RNA sequencing and 454 patients with whole-genome sequencing, as well as external published datasets. Results We quantified protein expression from 5897 genes in EAC and normal tissues. Several potential biomarkers showing selective expression in EAC, such as the transmembrane protein GPA33, were identified. We validated the EAC-specific expression of GPA33 in an external cohort of 115 patients, suggesting its utility as a diagnostic and therapeutic target. Additionally, integrated analysis of protein and RNA expression in EAC and normal tissues revealed genes with discordant protein and RNA levels, indicating post-transcriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, exhibited rare somatic mutations, suggesting post-transcriptional mechanisms driving this EAC-specific phenotype. AGMAT, as an example, was found to be overexpressed at the protein level in EAC compared to adjacent normal tissues, with a proposed EAC-selective, post-transcriptional mechanism regulating protein abundance. Conclusion Through quantitative proteomic analysis, we identified GPA33 as a candidate EAC biomarker. Integrated analysis of the proteome, transcriptome, and genome in EAC uncovered genes with tumor-selective post-transcriptional regulation of protein expression, offering potential exploitable vulnerabilities for therapeutic intervention.

Multi-Omic Analysis of Esophageal Adenocarcinoma Uncovers Candidate Therapeutic Targets and Cancer-Selective Posttranscriptional Regulation

Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype. We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from seven patients and further integrated these data with a cohort of EAC RNA-seq data (n = 264 patients), EAC whole-genome sequencing (n = 454 patients), and external published datasets. We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified, including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated protein and RNA abundance, suggesting posttranscriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation, suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be overexpressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, posttranscriptional regulation of protein expression, which may be an exploitable vulnerability.

Integrative HLA typing of tumor and adjacent normal tissue can reveal insights into the tumor immune response

BackgroundThe HLA complex is the most polymorphic region of the human genome, and its improved characterization can help us understand the genetics of human disease as well as the interplay between cancer and the immune system. The main function of HLA genes is to recognize “non-self” antigens and to present them on the cell surface to T cells, which instigate an immune response toward infected or transformed cells. While sequence variation in the antigen-binding groove of HLA may modulate the repertoire of immunogenic antigens presented to T cells, alterations in HLA expression can significantly influence the immune response to pathogens and cancer.MethodsRNA sequencing was used here to accurately genotype the HLA region and quantify and compare the level of allele-specific HLA expression in tumors and patient-matched adjacent normal tissue. The computational approach utilized in the study types classical and non-classical Class I and Class II HLA alleles from RNA-seq while simultaneously quantifying allele-specific or personalized HLA expression. The strategy also uses RNA-seq data to infer immune cell infiltration into tumors and the corresponding immune cell composition of matched normal tissue, to reveal potential insights related to T cell and NK cell interactions with tumor HLA alleles.ResultsThe genotyping method outperforms existing RNA-seq-based HLA typing tools for Class II HLA genotyping. Further, we demonstrate its potential for studying tumor-immune interactions by applying the method to tumor samples from two different subtypes of breast cancer and their matched normal breast tissue controls.ConclusionsThe integrative RNA-seq-based HLA typing approach described in the study, coupled with HLA expression analysis, neoantigen prediction and immune cell infiltration, may help increase our understanding of the interplay between a patient’s tumor and immune system; and provide further insights into the immune mechanisms that determine a positive or negative outcome following treatment with immunotherapy such as checkpoint blockade.

268. DEVELOPMENT OF A NOVEL ESOPHAGUS-ON-A-CHIP MODEL: A HIGH-FIDELITY PLATFORM FOR DIRECTING PERSONALIZED THERAPY IN ESOPHAGEAL ADENOCARCINOMA

Abstract Background 80% of patients diagnosed with esophageal adenocarcinoma (EAC) die of disease. Current standard of care for EAC is systemic cytotoxic, platinum-based neoadjuvant chemotherapy followed by resection and additional rounds of chemotherapy. Locally advanced, resectable patients often fail to respond due to intrinsic and/or acquired chemoresistance. Traditional preclinical models fail to offer alternative salvage options. To our knowledge, this is the first high-fidelity dynamic 3D platform implemented to study esophageal function, pathophysiology, and drug effects. Methods Primary tissues obtained from EAC patients at diagnostic biopsy were propagated into patient-derived organoids (PDOs). The upper (mucosal) channel of the esophagus-chip was designated for PDO-derived epithelial cells, while the lower stromal channel was designated for matched fibroblasts. Autologous cell types were exposed to a physiologically relevant flow of nutrient media and microtissues were formed. Epithelial permeability was determined by cascade blue assay. Using this model, a clinically relevant chemotherapy protocol was administered to study patient-specific tumor response. To determine microtissue development and cellular fate in response to chemotherapy, level of several epithelial/mesenchymal biomarkers were evaluated. Results Study cohort consisted of both chemo-sensitive and -resistant EAC patients. Esophagus-chip models were developed with samples from patient-matched tumor and normal tissue. Chips recapitulated morphological and histological features of their parental tissues. Epithelial barrier integrity was established within 5 days on-chip. Scanning electron microscopy revealed the presence of pleomorphic cells and mature microvilli in EAC-chips. In vitro administration of docetaxel triplet chemotherapy demonstrated efficacy similar to that observed in the clinical setting when assessed directly or indirectly. Confocal imaging revealed treatment-associated differential expression of epithelial (CK7, CK19), and mesenchymal (vimentin) markers. Conclusion 3D-organoids conserve cellular heterogeneity of source patients, whereas esophagus-on-chip expand on this diversity by adding microphysiological relevance, stromal influence & flexible complexity. This high-fidelity biomimetic platform enables clinical mimicry and can predict patient-specific chemosensitivity within a clinically relevant timeframe. This esophagus-chip system is a promising potential replacement for both inefficient preclinical animal models and existing tumor-only avatars lacking stromal and microphysiological context.

Free-Fatty Acid Receptor-4 (FFA4/GPR120) differentially regulates migration, invasion, proliferation and tumor growth of papillary renal cell carcinoma cells

Kidney cancer is among the 10 most common cancers, and renal cell carcinoma (RCC), which represent 90% of all kidney cancers, has the highest mortality rate of all genitourinary cancers. Papillary RCC (pRCC) is the second most frequent subtype of RCC and demonstrates distinct characteristics compared to other subtypes, including a high degree of metastasis and resistance to treatments against the more common clear cell RCC (ccRCC) subtype. Here, we demonstrate that the Free-Fatty Acid Receptor-4 (FFA4), a G protein-coupled receptor that is endogenously activated by medium-to-long chain free-fatty acids, is upregulated in pRCC compared to patient-matched normal kidney tissue, and that the expression of FFA4 increases with the degree of pathological grading of pRCC. Our data also show that FFA4 transcript is not expressed in ccRCC cell lines, but is expressed in the well-characterized metastatic pRCC cell line ACHN. Furthermore, we show that agonism of FFA4 with the selective agonist cpdA positively regulates ACHN cell migration and invasion in a manner dependent on PI3K/AKT/NF-κB signaling to COX-2 and MMP-9, with partial-dependence on EGFR transactivation. Our results also demonstrate that FFA4 agonism induces STAT-3-driven epithelial-mesenchymal transition, suggesting a significant role for FFA4 in pRCC metastasis. On the contrary, FFA4 agonism significantly reduces cell proliferation and tumor growth, suggesting that the receptor may have opposing effects on pRCC cell growth and migration. Together, our data demonstrate that FFA4 has significant functional roles in pRCC cells and may be an attractive target for study of pRCC and development of RCC pharmacotherapeutics.

Abstract LB357: CRISPR-Cas9 targeting of somatic mutations selectively kills cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for 3% of all cancers but is the 3rd leading cause of cancer death in the United States. This staggering discrepancy is partly because most pancreatic cancers evade early detection and patients most commonly present with metastatic disease. As cancer initiates from a single transformed cell, passenger mutations that accumulate over the course of the patient’s lifetime in that cell are then clonally propagated to the growing tumor and metastases, providing a targetable molecular signature throughout the cancer. Here we propose a CRISPR-Cas9 gene therapy aimed at these tumor-specific somatic mutations. Using whole genome sequencing of rapid autopsy tumor and normal samples from five patient cases, we identified single base substitutions present in tumor tissues and absent in patient-matched normal tissue. A subset (6.6% on average) of these mutations lead to novel protospacer adjacent motif (PAM) sites. These tumor specific PAM sites are necessary for Cas9 binding and DNA scission and are therefore selectively targetable by CRISPR-Cas9 systems. Using a hybrid capture/NGS approach, we demonstrate in patients with multiple metastases that the majority of these novel PAM sites, ~70%, are maintained among the different metastases. Using multitarget sgRNA which cut at a known number of sites in the human genome, we determined a relationship between number of CRISPR-Cas9 cuts and growth inhibition in PDAC cells. We found that 8-10 cut sites in non-coding regions are sufficient to induce >95% cancer cell death. Finally, using cocultures of fluorescently labeled PDAC cells and a multiplexed sgRNA array targeting multiple tumor specific PAMs, we demonstrate selective cell killing of the targeted cell line. Importantly, deep sequencing of patient-matched normal lymphocytes lacking the tumor-specific PAMs treated with the same sgRNA array exhibited no editing at the targeted loci. This work establishes a bioinformatic platform for the discovery of tumor-specific CRISPR targets in the context of metastatic disease and demonstrates selective killing to cancer cells while sparing the patient’s normal cells. These data establish the proof of principle for a gene therapy approach targeting metastatic disease. Citation Format: Kirsten Bowland, Selina Shiqing K. Teh, Alyza Skaist, Ralph H. Hruban, Nicholas J. Roberts, Chrisine Iacobuzio-Donahue, James R. Eshleman. CRISPR-Cas9 targeting of somatic mutations selectively kills cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB357.

Abstract LB251: Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma

Abstract Background: In cutaneous melanoma, the burden of chromothripsis is high prior to targeted therapy, and additional chromothripsis appears to be a key evolutionary mechanism by which cancer rapidly generates and accumulates highly dynamic structural variants (SVs). Blocking cancer genomic instability may prevent tumor escape from targeted therapies. Methods: We assembled three cohorts of tissues for WGS-based analysis of SVs. The first cohort consisted of patient-matched normal tissues, BRAF V600MUT melanoma tumors before MAPKi therapy and at disease progression (n=10 normal tissues; n=10 pretreatment tumors; n=17 acquired-resistant tumors; n=10 patients). The second cohort consisted of rapid autopsy melanoma tissues (n=3 normal tissues; n=12 acquired-resistant tumors; n=6 metastatic organ sites). The third cohort consisted of cutaneous PDX tumors. To study acquired MAPKi-resistance at the whole-genome level, we subjected PDXs (n=6 models; 1 BRAF MUT and 5 NRAS MUT models) to MAPKi therapy in NSG mice at doses sufficient to elicit tumor regression, and then generated acquired MAPKi-resistant tumors. In total, we used vehicle-treated tumors (n=6), acquired-resistant tumors (n=12), and patient-matched normal tissues (n=6) to generate WGS data. Results: Analysis of genomic amplicons due to intrachromosomal complex genomic rearrangements (CGRs) and extrachromosomal circular DNAs (ecDNAs) uncovered a significant (unpaired Student’s t-test, p=0.0002) association between acquired-resistant tumors and CGRs and/or ecDNAs harboring bona fide MAPKi-resistance genes and revealed copy number amplification of BRAF (range 4.5-27), NRAS (range 5-13), HRAS (range 13-16), MYC (range 12-15) and EGFR (CN 4.6-5), known to drive acquired MAPKi-resistance. Moreover, we validated a recurrent ecDNA by direct isolation and high-depth sequencing using a new approach referred to as CRISPR-CATCH. This alternative technique confirmed the circularized junctions of a 890 kb, driver ecDNA within this acquired-resistant clinical tumor sample. Additionally, resistance-specific (versus sensitivity-specific) chromothriptic single-base substitutions (SBSs) enriched for signatures of defects in base excision repair (BER) and in DNA mismatch repair (MMR) (Wilcoxon rank sum test, p=0.04 and p=0.005 respectively) in 14 of 31 resistant tumors (10 of 16 patients). Moreover, breakpoint-sequence analysis inferred non-homologous end-joining (NHEJ) as critical, and homologous recombination repair (HRR) as adjunctive, to DNA double-stranded break repair underlying CGR and ecDNA formation harboring MAPK-reactivation or MAPKi resistance-driver genes. Inhibition of DNA-PKc or PARP1/2, even only initially during MAPKi treatment, suppressed acquired MAPKi-resistance in melanoma cell lines (BRAF V600MUT n=3 and NRAS Q61MUT n=3) and blunted the expansion of ecDNA + CGR genomic spans. In vivo, DNA-PKi in combination with MAPKi forestalled resistance, reduced ecDNA and CGRs size, and suppressed the contribution of NHEJ in 5 out of 5 cutaneous melanoma PDXs analyzed. Conclusions: Our findings advance the concepts that preventing—instead of reversing—acquired resistant phenotypes may be more clinically impactful and that targeting DNA-PKCS and NHEJ lies at the center of this approach in stabilizing cancer genomes during oncogene-targeted therapies. Citation Format: Prashanthi Dharanipragada, Xiao Zhang, Sixue Liu, Shirley H. Lomeli, Aayoung Hong, Yan Wang, Zhentao Yang, Agustin Vega-Crespo, Antoni Ribas, Stergios J. Moschos, Gatien Moriceau, Roger S. Lo. Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB251.

Abstract 222: High frequency of structural variants in FFPE colorectal cancer tissue detected by targeting selected common fragile site genes and LINE transposable elements

Abstract Introduction: Structural variants (SVs) caused by chromosomal rearrangements or LINE retrotranspositions are a highly prevalent type of somatic DNA alterations in colorectal cancer (CRC). Studies about the function of SVs in tumor biology and their putative application as biomarkers are currently hampered by the need for fresh frozen tumor material to detect SVs by long-read or deep whole genome sequencing. This impedes the identification of SVs in retrospective cohorts and implementation in routine diagnostics (where only formalin-fixed paraffin-embedded (FFPE) tissue is available). In this study, we explored the applicability of FFPE-Targeted Locus Capture (FFPE-TLC) as a novel technology for targeted detection of SVs in CRC. Materials and methods: We defined the regions most frequently presenting SVs in CRC and included these in a customized capture panel. FFPE-TLC followed by targeted sequencing was performed on primary tumors of 29 metastatic CRC patients and eight patient-matched normal colon tissues. SV-specific droplet digital PCR (ddPCR) assays were designed for orthogonal validation of SVs in tumor tissue and detection of SVs in cell-free plasma circulating tumor DNA (ctDNA). Results: The regions associated with most SVs in metastatic CRC were four common fragile site (CFS) genes; MACROD2, PARK2, FHIT, WWOX, and three LINE source elements, and were included in a 2.7 Mb targeted panel. After filtering for germline variation, we identified 168 somatic SVs in 26/29 patients (90%), median SVs per patient: 3, range: 1-22. 110 SVs were found in CFS genes in 19/29 patients (66%), 12 of them presenting SVs in MACROD2. 58 SVs were caused by LINE integrations in 19/29 patients (66%), with different LINE behaviors observed: one source LINE generated 19 SVs in 13 patients, whilst another LINE caused 33 SVs in 5 patients. For selected cases, the tumor-specific origin of SVs was verified and its applicability as a plasma ctDNA biomarker demonstrated. Discussion: This study demonstrates, for the first time, the feasibility of targeted detection of SVs in CFS genes and SVs caused by LINE retrotransposition in routinely obtained FFPE tissue without prior knowledge of the breakpoint location. The high prevalence of SVs offers opportunities to detect many somatic alterations by targeting few genes and LINEs, e.g. for translation into ctDNA biomarker assays. We conclude that FFPE-TLC enables the investigation of SV mutations in retrospective cohorts and opens new avenues for mutation analysis in routine diagnostics. Citation Format: Carmen Rubio-Alarcon, Ellen Stelloo, Daan C. Vessies, Iris van ‘t Erve, Nienke Mekkes, Joost Swennenhuis, Soufyan Lakbir, Elise van Bree, Marianne Tijssen, Pien Delis-van Diemen, Mirthe Lanfermeijer, Theodora C. Linders, Daan van den Broek, Cornelis J. Punt, Jaap Heringa, Gerrit A. Meijer, Sanne Abeln, Harma Feitsma, Remond J. Fijneman. High frequency of structural variants in FFPE colorectal cancer tissue detected by targeting selected common fragile site genes and LINE transposable elements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 222.

Targeting oncogenic functions of miR-301a in head and neck squamous cell carcinoma by PI3K/PTEN and MEK/ERK pathways

Treatment of head and neck squamous cell carcinomas (HNSCC), the sixth most frequent cancer worldwide, remains challenging. miRNA dysregulation is closely linked to tumorigenesis and tumor progression, thus emerging as suitable targets for cancer treatment. Transcriptomic analysis of TCGA HNSCC dataset revealed that miR-301a expression levels significantly increased in primary tumors, as compared to patient-matched normal tissue. This prompted us to investigate its pathobiological role and potential as new therapeutic target using different preclinical HNSCC models. miR-301a overexpression in HNSCC-derived cell lines led to enhanced proliferation and invasion, whereas miR-301 inhibition reduced these effects. In vivo validation was performed using an orthotopic mouse model. Results concordantly showed that the mitotic counts, the percentage of infiltration depth and Ki67 proliferative index were significantly augmented in the subgroup of mice harboring miR-301a-overexpressing tumors. Further mechanistic characterization revealed PI3K/PTEN/AKT and MEK/ERK pathways as central signaling nodes responsible for mediating the oncogenic activity of miR-301a observed in HNSCC cells. Notably, pharmacological disruption of PI3K and ERK signals with BYL-719 and PD98059, respectively, was effective to completely revert/abolish miR-301a-promoted tumor cell growth and invasion. Altogether, these findings demonstrate that miR-301a dysregulation plays an oncogenic role in HNSCC, thus emerging as a candidate therapeutic target for this disease. Importantly, available PI3K and ERK inhibitors emerge as promising anti-tumor agents to effectively target miR-301a-mediated signal circuit hampering growth-promoting and pro-invasive functions.

Proteomic analysis of low- and high-grade human colon adenocarcinoma tissues and tissue-derived primary cell lines reveals unique biological functions of tumours and new protein biomarker candidates

BackgroundColon cancer is the third most common cancer and second highest cause of cancer deaths worldwide. The aim of the study was to find new biomarkers for diagnosis, prognosis and therapeutic drug targets for this disease.MethodsFour low-grade and four high-grade human colon adenocarcinoma tumours with patient-matched normal colon tissues were analysed. Additionally, tissue-derived primary cell lines were established from each tumour tissue. The cell lines were validated using DNA sequencing to confirm that they are a suitable in vitro model for colon adenocarcinoma based on conserved gene mutations. Label-free quantitation proteomics was performed to compare the proteomes of colon adenocarcinoma samples to normal colon samples, and of colon adenocarcinoma tissues to tissue-derived cell lines to find significantly differentially abundant proteins. The functions enriched within the differentially expressed proteins were assessed using STRING. Proteomics data was validated by Western blotting.ResultsA total of 4767 proteins were identified across all tissues, and 4711 across primary tissue-derived cell lines. Of these, 3302 proteins were detected in both the tissues and the cell lines. On average, primary cell lines shared about 70% of proteins with their parent tissue, and they retained mutations to key colon adenocarcinoma-related genes and did not diverge far genetically from their parent tissues. Colon adenocarcinoma tissues displayed upregulation of RNA processing, steroid biosynthesis and detoxification, and downregulation of cytoskeletal organisation and loss of normal muscle function. Tissue-derived cell lines exhibited increased interferon-gamma signalling and aberrant ferroptosis. Overall, 318 proteins were significantly up-regulated and 362 proteins significantly down-regulated by comparisons of high-grade with low-grade tumours and low-grade tumour with normal colon tissues from both sample types.ConclusionsThe differences exhibited between tissues and cell lines highlight the additional information that can be obtained from patient-derived primary cell lines. DNA sequencing and proteomics confirmed that these cell lines can be considered suitable in vitro models of the parent tumours. Various potential biomarkers for colon adenocarcinoma initiation and progression and drug targets were identified and discussed, including seven novel markers: ACSL4, ANK2, AMER3, EXOSC1, EXOSC6, GCLM, and TFRC.

Circular RNAs Hsa_circ_101555 and Hsa_circ_008068 as Diagnostic Biomarkers for Early-Stage Lung Adenocarcinoma.

BackgroundLung adenocarcinoma (LUAD) is a life-threatening disease worldwide with a high mortality rate. The early diagnosis of LUAD is crucial for improving subsequent treatment and prognosis. However, biomarkers for early detection remain a clinical challenge in LUAD. Here, we aimed to develop circular RNAs (circRNAs) in circulating plasma from LUAD patients as valuable diagnostic biomarkers in LUAD.MethodsCircRNA expression was determined by circRNA microarray in three pairs of LUAD tumour tissues and patient-matched normal lung tissues. Hsa_circ_101555 and hsa_circ_008068 were selected as potential biomarkers in LUAD tissues and plasma by RT–PCR, respectively. The diagnostic value was analysed by the area under the curve (AUC) and the receiver operating characteristic (ROC) test.ResultsOur results showed that 6261 circRNAs were upregulated and 7238 circRNAs were downregulated in LUAD tumour tissues compared with patient-matched normal lung tissues. Hsa_circ_101555 and hsa_circ_008068 were filtered as biomarkers for early-stage LUAD. Q-PCR results showed that hsa_circ_101555 and hsa_circ_008068 were significantly upregulated in both LUAD cancer tissues and circulating plasma. Hsa_circ_101555 and hsa_circ_008068 were positively associated with tumour differentiation, tumour size and CEA (P<0.05). The ROC analysis showed that hsa_circ_101555 and hsa_circ_008068 had a better diagnostic potential compared to the traditional biomarkers (CEA, SCC, CYFRA21-1) in the detection of early-stage LUAD.ConclusionThe circular RNAs hsa_circ_101555 and hsa_circ_008068 could serve as novel diagnostic biomarkers for early-stage LUAD.

Impact of Secretion-Active Osteoblast-Specific Factor 2 in Promoting Progression and Metastasis of Head and Neck Cancer.

Simple SummaryHead and neck cancers (HNC) exhibit poor survival due to metastases. Our study identified osteoblast-specific factor 2 (OSF-2) as overexpressed in primary tumors, lymph node metastases, and the tumor microenvironment. High OSF-2 levels correlate with metastatic disease and reduced survival of HPV-negative HNC patients. OSF-2’s active secretion signal seems to promote metastases by supporting the tumor microenvironment via the ß1 integrin-induced PI3K and Akt/PKB signaling pathway. We suggest OSF-2 as a potential biomarker and drug target to control (HPV-negative) HNC metastasis and disease management.Treatment success of head and neck cancer (HNC) is still hampered by tumor relapse due to metastases. Our study aimed to identify biomarkers by exploiting transcriptomics profiles of patient-matched metastases, primary tumors, and normal tissue mucosa as well as the TCGA HNC cohort data sets. Analyses identified osteoblast-specific factor 2 (OSF-2) as significantly overexpressed in lymph node metastases and primary tumors compared to normal tissue. High OSF-2 levels correlate with metastatic disease and reduced overall survival of predominantly HPV-negative HNC patients. No significant correlation was observed with tumor localization or therapy response. These findings were supported by the fact that OSF-2 expression was not elevated in cisplatin-resistant HNC cell lines. OSF-2 was strongly expressed in tumor-associated fibroblasts, suggesting a tumor microenvironment-promoting function. Molecular cloning and expression studies of OSF-2 variants from patients identified an evolutionary conserved bona fide protein secretion signal (1MIPFLPMFSLLLLLIVNPINA21). OSF-2 enhanced cell migration and cellular survival under stress conditions, which could be mimicked by the extracellular administration of recombinant protein. Here, OSF-2 executes its functions via ß1 integrin, resulting in the phosphorylation of PI3K and activation of the Akt/PKB signaling pathway. Collectively, we suggest OSF-2 as a potential prognostic biomarker and drug target, promoting metastases by supporting the tumor microenvironment and lymph node metastases survival rather than by enhancing primary tumor proliferation or therapy resistance.

Regulation of a Novel Splice Variant of Early Growth Response 4 (EGR4-S) by HER+ Signalling and HSF1 in Breast Cancer.

Simple SummaryEGR4 is known to play an important role in the proliferation of small cell lung cancer. Our research identified a new, shortened version of this protein (which we named EGR4-S), found in breast cancer but not detectable in normal breast tissue. Interestingly, our findings show that the EGR4-S expressed by breast cancer cells could be reduced by treating the cells with certain targeted cancer therapeutics. However, sustained, high-dose treatment led to EGR4-S becoming less responsive. In addition, we identified an inverse relationship between EGR4-S and molecular stress. When cancer cells were in conditions of increased molecular stress, reduced EGR4-S levels were associated with lower growth rate but enhanced properties associated with higher metastatic potential. Taken together, our research suggests further investigation of EGR4-S is warranted in order to determine its potential as a biomarker for differentiating tumours from normal tissue at the molecular level, as well as its possible resistance to targeted therapies.The zinc finger transcription factor EGR4 has previously been identified as having a critical role in the proliferation of small cell lung cancer. Here, we have identified a novel, shortened splice variant of this transcription factor (EGR4-S) that is regulated by Heat Shock Factor-1 (HSF1). Our findings demonstrate that the shortened variant (EGR4-S) is upregulated with high EGFR, HER2, and H-Rasv12-expressing breast cell lines, and its expression is inhibited in response to HER pathway inhibitors. Protein and mRNA analyses of HER2+ human breast tumours indicated the novel EGR4-S splice variant to be preferentially expressed in tumour tissue and not detectable in patient-matched normal tissue. Knockdown of EGR4-S in the HER2-amplified breast cancer cell line SKBR3 reduced cell growth, suggesting that EGR4-S supports the growth of HER2+ tumour cells. In addition to chemical inhibitors of the HER2 pathway, EGR4-S expression was also found to be suppressed by chemical stressors and the overexpression of HSF1. Under these conditions, reduced EGR4-S levels were associated with the observed lower cell growth rate, but the augmentation of properties associated with higher metastatic potential. Taken together, these findings identify EGR4-S as a potential biomarker for HER2 pathway activation in human tumours that is regulated by HSF1.

Single-cell transcriptomic profiling unravels the adenoma-initiation role of protein tyrosine kinases during colorectal tumorigenesis

The adenoma-carcinoma sequence is a well-accepted roadmap for the development of sporadic colorectal cancer. However, cellular heterogeneity in aberrant epithelial cells limits our understanding of carcinogenesis in colorectal tissues. Here, we performed a single-cell RNA sequencing survey of 54,788 cells from patient-matched tissue samples, including blood, normal tissue, para-cancer, polyp, and colorectal cancer. At each stage of carcinogenesis, we characterized cell types, transcriptional signatures, and differentially expressed genes of distinct cell populations. The molecular signatures of epithelial cells at normal, benign, and malignant stages were defined at the single-cell scale. Adenoma and carcinoma precursor cell populations were identified and characterized followed by validation with large cohort biopsies. Protein tyrosine kinases (PTKs) BMX and HCK were identified as potential drivers of adenoma initiation. Specific BMX and HCK upregulations were observed in adenoma precursor cell populations from normal and adenoma biopsies. Overexpression of BMX and HCK significantly promoted colorectal epithelial cell proliferation. Importantly, in the organoid culture system, BMX and HCK upregulations resulted in the formation of multilayered polyp-like buds protruding towards the organoid lumen, mimicking the pathological polyp morphology often observed in colorectal cancer. Molecular mechanism analysis revealed that upregulation of BMX or HCK activated the JAK-STAT pathway. In conclusion, our work improved the current knowledge regarding colorectal epithelial evolution during carcinogenesis at the single-cell resolution. These findings may lead to improvements in colorectal cancer diagnosis and treatment.

External Validation of the Prognostic Value of an Immune-Associated Gene Panel for Clear Cell Renal Cell Carcinomas.

Clear cell renal cell carcinomas (ccRCCs) are highly immune infiltrates, and many of them respond to immunotherapy with checkpoint inhibitors including anti-PD-L1 or anti-PD1 agents. However, the effect of immune genes on clinical outcomes in ccRCCs has not been fully studied. Here, we show in this study that an immune-associated gene panel has a prognostic value for clear cell renal cell carcinomas. We performed single-sample gene set enrichment analysis (ssGSEA) and cell type identification by estimating subsets of RNA transcripts (CIBERSORT) algorithms on patient-matched normal renal and RCC tissues to characterize two immunophenotypes and immunological characteristic subpopulations. Furthermore, LASSO Cox regression was applied to develop a novel prognosis-associated model for ccRCC patients based on an immune-gene panel. The results were verified by the Gene Expression Omnibus (GEO) dataset and coordinated with the clinicopathological characteristics of ccRCCs, along with genomic signatures. Finally, based on the above perspectives, we generated a nomogram with a high prognostic efficiency for ccRCC patients. Overall, this study offers a unique perspective that can contribute to improving the accuracy of prognosis prediction and treatment with immunotherapy.

Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models.

Simple SummaryOral cancer is often deadly and severely painful. Because this form of cancer pain cannot be adequately treated with current medications including opiates, new treatment approaches are needed. Cathepsin S, a lysosomal cysteine protease may play a role in oral cancer pain through a protease-activated receptor-2 (PAR2)-dependent mechanism. We undertook a series of experiments to define the role of cathepsin S in oral cancer pain. We compared cathepsin S activity in human oral cancer tumors versus patient-matched normal tissue; a human oral cancer cell line versus a benign dysplastic oral keratinocyte cell line; and in an orthotopic xenograft tongue cancer mouse model versus normal controls in mice. We localized cathepsin S in macrophages and carcinoma cells in human oral cancers. The injection of cathepsin S caused nociception in a mouse model while the injection of oral cancer cells in which the gene for cathepsin S is deleted generated less nociception. Our findings will lay the foundations for clinical trials of cathepsin S inhibitors for treating oral cancer pain.Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.

Abstract 473: Glycomic characterization of carbohydrate antigen-defined pancreatic cancer tissues using lectins and imaging mass spectrometry

Abstract N-glycosylation alterations in tumors are now recognized as cancer hallmarks for its role in oncogenesis, cancer signaling and metastasis. For pancreatic adenocarcinoma (PDAC), the sole FDA-approved biomarker is the carbohydrate antigen 19-9 (CA19-9). Because of CA19-9's association with pancreatitis and other gastrointestinal cancers, it is used for surveillance of disease progression rather than for diagnosis. A recently identified carbohydrate antigen, sialylated tumor-related antigen (sTRA) recognized by the TRA-1-60 mAb, is expressed in a non-overlapping subset of pancreatic cancer and is under consideration for use alongside CA19-9. To better understand these carbohydrate antigens in relation to PDAC pathogenesis, complementary N-glycan analysis approaches were applied to a cohort of 53 patient-matched tumor and normal tissue samples represented by tissue microarrays (TMA) and whole-tissue FFPE samples. The distribution of N-glycans in these tissues were defined using a matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) workflow. Over 100 individual N-glycan species were mapped to their histopathology locations. The same cohort was characterized by multi-round immunofluorescence for CA19-9 and sTRA expression as well as two lectins, PHA-E and GSL-II,. N-glycan IMS data from the TMAs were evaluated by LASSO-regularized logistic regression for the identification of N-glycan analytes with predictive value in differentiating between tumor and normal tissue. LASSO selected N-glycan masses were incorporated alongside biomarker expression data to construct a model for PDAC tissue classification. N-glycans associated with tumor tissue tended to be bi-, tri- and tetra-antennary complex structures with multople fucose, terminal sialic acids and bisecting GlcNAc. Quantitative analysis of the IMS data by LASSO regression identified 9 differentiating N-glycan masses, 7 from tumor cores and 2 from normal cores. When both IMS glycan masses and biomarker expression data for CA19-9 and sTRA were integrated into a single linear regression model, receiver operator curve (ROC) accuracy and AUC improvements, +0.172 and +0.221 respectively, were observed in comparison to models utilizing either dataset individually. Critically, this model was able to rescue cases where biomarker data was missing or insufficient so that they could be correctly classified as tumor or normal tissue. Analysis of this PDAC tissue cohort using MALDI-IMS illustrated the utility of investigating specific N-glycans and N-glycan structural classes correlated with carbohydrate PDAC biomarkers. Integration of N-glycan IMS and biomarker lectin immunostaining bolsters pancreatic cancer identification and posits expanded utility for each approach for PDAC surveillance and early identification. Citation Format: Colin McDowell, Zachary Klamer, Johnathan Hall, Luke Wisniewski, Peggi Angel, Anand Mehta, Brian Haab, Richard R. Drake. Glycomic characterization of carbohydrate antigen-defined pancreatic cancer tissues using lectins and imaging mass spectrometry [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 473.

Combining Bioinformatics and Experiments to Identify and Verify Gene Prognostic Markers in Hepatocellular Carcinoma

Background: Hepatocellular Carcinoma (HCC) is one of the highly malignant tumors threatening human health. The current research aimed to identify potential prognostic gene biomarkers for HCC. Materials and Methods: Microarray data of gene expression profiles of HCC from GEO were downloaded. After screening overlapping differentially expressed genes (DEGs) by R software. The STRING database and Cytoscape were used to identify hub genes. Cox proportional hazards regression was performed to screen the potential prognostic genes. Moreover, quantitative real-time PCR analyses were performed to detect the expression of ANLN in liver cancer cells and tissues. Finally, its possible pathways and functions were predicted using gene set enrichment analysis (GSEA). Result: A total of 566 DEGs were obtained from the overlapping analysis of three mRNA microarray dataset. Six key hub genes including RACGAP1, KIF20, DLGAP5, CDK1, BUB1B and ANLN, were associated with poor prognosis of patients with HCC. Higher expression of ANLN was associated with reduced overall survival and disease-free survival in patients with HCC. Multivariate analysis revealed that ANLN expression was an independent risk factor affecting overall survival. RT-PCR and Western blot analysis further demonstrated that ANLN expression was increased in HCC compared with patient-matched adjacent normal tissues. Notably, Gene enrichment analysis revealed that DEGs in ANLN-high patients were enriched in cell cycle, DNA duplication and p53 signaling pathway. Conclusion: The high expression of RACGAP1, KIF20, DLGAP5, CDK1, BUB1B and ANLN might be poor prognostic biomarkers in HCC patients, and may help to individualize the management of HCC.

HACE1 blocks HIF1\u03b1 accumulation under hypoxia in a RAC1 dependent manner

Uncovering the mechanisms that underpin how tumor cells adapt to microenvironmental stress is essential to better understand cancer progression. The HACE1 (HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase) gene is a tumor suppressor that inhibits the growth, invasive capacity, and metastasis of cancer cells. However, the direct regulatory pathways whereby HACE1 confers this tumor-suppressive effect remain to be fully elucidated. In this report, we establish a link between HACE1 and the major stress factor, hypoxia-inducible factor 1 alpha (HIF1α). We find that HACE1 blocks the accumulation of HIF1α during cellular hypoxia through decreased protein stability. This property is dependent on HACE1 E3 ligase activity and loss of Ras-related C3 botulinum toxin substrate 1 (RAC1), an established target of HACE1 mediated ubiquitinylation and degradation. In vivo, genetic deletion of Rac1 reversed the increased HIF1α expression observed in Hace1–/– mice in murine KRasG12D-driven lung tumors. An inverse relationship was observed between HACE1 and HIF1α levels in tumors compared to patient-matched normal kidney tissues, highlighting the potential pathophysiological significance of our findings. Together, our data uncover a previously unrecognized function for the HACE1 tumor suppressor in blocking HIF1α accumulation under hypoxia in a RAC1-dependent manner.