Development Of Adenocarcinoma Research Articles

Abstract 3939: Ror2, a novel key regulator driving cell fate decisions throughout pancreatic tumor progression

Abstract Introduction: Reprogramming of pancreas cell fate drives development of pancreatic ductal adenocarcinoma (PDAC). Acinar cells, the most probable origin of pancreatic cancer, undergo a rapid cell identity switch towards a duct-like phenotype upon KrasG12D expression and when combined with pancreatitis or the loss of acinar differentiation factors. Metaplastic and dysplastic duct-like cells are heterogeneous with a proportion acquiring features reminiscent of gastric lineages. While some gastric signatures are maintained in the classical PDAC subtype, they are eroded in the more aggressive, basal-like PDAC. Since subtype identity has a major impact on prognosis and therapeutic targetability, druggable targets that regulate cellular reprogramming in pancreatic cancer can be exploited to increase sensitivity to therapy. Methods: To elucidate mechanisms responsible for early reprogramming, pancreatic tissue of mice with conditional KrasG12D expression and loss of Pdx1 was analyzed by single-nucleus ATAC-seq. Expression of identified target genes was studied in precancerous lesions and PDAC by using multiplex RNAscope and IHC staining. Computational analyses of publicly available sequencing data were used to establish correlation to PDAC subtype identity. Genes were functionally studied in PDAC cell lines. Results: By performing snATAC-seq and RNA-seq of early transformed pancreatic tissue, we discovered that acinar cells with the combined expression of KrasG12D and loss of Pdx1 activate expression of a gastric metaplastic gene signature accompanied by elevated levels of the receptor kinase Ror2. Ror2 is also highly expressed in distinct subpopulations of metaplastic and dysplastic cells, associated with a gastric neck cell phenotype and enhanced proliferative capacity. In contrast, Ror2Low lesions are characterized by a gastric pit cell-like and a senescent phenotype. Genetic ablation of Ror2 resulted in a shift in lesion identity, enriching those with a pit cell and senescent identity. In PDAC, we found that Ror2 anti-correlates with a similar gastric pit cell phenotype that is maintained in the classical subtype PDAC but is strongly associated with the more aggressive basal-like subtype. Overexpression of ROR2 in human PDAC cell lines with a classical differentiation induced loss of the classical gene signature as well as epithelial-to-mesenchymal transition. Moreover, ROR2 enforces a strong dependency on AKT signaling, causing increased vulnerability of ROR2-expressing cells to AKT inhibition, but increased resistance to the KRAS inhibitor MRTX1133. Conclusions: We discovered Ror2 as a critical determinant of precancerous lesion as well as PDAC subtype identity. Its role in driving an aggressive PDAC phenotype that is inherently resistant to Kras inhibition suggests that inhibiting this receptor tyrosine kinase will enhance sensitivity to the new generation of targeted therapies. Citation Format: Simone Benitz, Alexander Steep, Malak Nasser, Jonathan Preall, Ujjwal Mahajan, Ian Loveless, Holly McQuithey, Erick Davis, Hui-Ju Wen, Daniel Long, Thomas Metzler, Samuel Zwernik, Daniel Salas-Escabillas, Ling Huang, Nina Steele, Ivonne Regel, Filip Bednar, Howard Crawford. Ror2, a novel key regulator driving cell fate decisions throughout pancreatic tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3939.

Abstract 3427: Serum metabolites and pancreatic ductal adenocarcinoma in two prospective cohorts

Abstract Objective: Metabolomics may provide insight to understand the etiology and potential mechanisms for pancreatic ductal adenocarcinoma (PDAC) development beyond established risk factors of smoking, diabetes, and adiposity. We extended our previous metabolomic study by including additional participants from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) with the aim to examine prospective associations between serum metabolites and incident PDAC. Method: We measured 1483 known metabolites in pre-diagnostic serum (up to 24 years) in two PDAC nested case-control studies within the PLCO (360 cases-control pairs) of mostly non-smoker men and women and Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC, 372 cases-control pairs) male smokers using liquid chromatography, high resolution/accurate mass spectrometry. Controls were incidence-density sampled and matched to cases by age, blood draw date, sex, race/ethnicity, and cohort. We used conditional logistic regression to calculate odds ratios and 95% confidence intervals for PDAC per standard deviation increase in log10-metabolite levels in each cohort and combined using fixed-effect meta-analyses and stratified by follow-up time. We also performed ElasticNet regression in each cohort to identify metabolites. We assessed associations of 72 predefined metabolic pathways with PDAC by comparing Fisher’s statistic of metabolite p-values per pathway with a null distribution based on 500,000 permutations. Results: Sixty-six metabolites were associated with PDAC at a false discovery rate (FDR)<0.05 with 26 below the Bonferroni threshold (P<3.4 × 10−5). Among the 66 metabolites, 14 and 15 were selected in ElasticNet regressions in ATBC and PLCO, respectively, with two in both cohorts. Notable findings include: fibrinopeptide B(1-9); seven modified, di- or poly-peptides; eight xenobiotics related to tobacco smoke; amino acids, carbohydrates, and TCA cycle metabolites known to be regulated by mutant KRAS (aspartate, glutamate, lactate, pyruvate, and α-ketoglutarate); four secondary bile acids and three aromatic amino acids (tryptophan, phenylalanine, and tyrosine) that were positively, while four fibrinogen cleavage peptides were inversely associated with PDAC. Among the top identified metabolites, aspartate, α-glutamyltyrosine, pyroglutamylglutamine, and Glu-Gly-Asn-Val showed time-varying associations overall (P≤0.05) with greater magnitudes within the first 5 years after blood draw. Thirteen metabolic pathways were associated with PDAC (Bonferroni P≤0.05). Conclusion: Our study suggests pre-diagnostic circulating metabolites, particularly those potentially related to subclinical disease, microbiome, and tobacco metabolism are associated with PDAC. The identified metabolites along with other known risk factors might be useful in algorithms to model disease risk and progression. Citation Format: Ting Zhang, Steven C. Moore, Sheng Fu, Kevin Wang, Demetrius Albanes, Stephanie J. Weinstein, Kai Yu, Rachael Z. Stolzenberg-Solomon. Serum metabolites and pancreatic ductal adenocarcinoma in two prospective cohorts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3427.

Abstract 3893: A multimodal spatial-omics atlas of lung precancer and progression to adenocarcinoma

Abstract Understanding the earliest changes during lung adenocarcinoma (LUAD) development can set the stage for discovery of fertile targets for disease interception, thereby mitigating the dire public health burden of LUAD. We previously identified bulk-level molecular and immunological changes that are enriched in normal-appearing tissue (NAT) in the local niche of human LUAD, as well as those that commence in adenomatous premalignant lesions (aPMLs, LUAD precursors) and that are further enriched in LUADs. Yet, in-depth understanding of the identities, states, and properties of specific cell subsets in NAT and precancer that trigger LUAD remain largely elusive due to inherent roadblocks to sampling and characterizing aPMLs that are at the center of this trajectory. Here, we aimed to map molecular profiles, states, and interactions of cell subsets that underlie initiation in lesion-adjacent NAT, and to determine how features of these cells evolve along the aPML-LUAD spectrum. We analyzed an expanding cohort of archived matching NATs, aPMLs and LUADs, including challenging small samples, from up to 17 patients. Samples were analyzed using combined high-resolution, multi-modal Visium spatial transcriptomics (ST, n=17) and proteomics (n=12), as well as single-cell RNA-sequencing of fixed cells (scFFPE-seq) from consecutive sections of the same tissues from 15 patients. We also studied a subset of samples by high-plex spatial proteomics (COMET) or subcellular spatial gene expression analysis (Xenium). In total, we sequenced more than half a million single cells comprising diverse epithelial, immune, and stromal subsets, which were concordant with lineage clusters identified by ST analysis of the same samples. Loss of alveolar differentiation was a hallmark of tumor cell-enriched areas in aPMLs and LUADs, with a more pronounced effect in the LUADs. Alveolar intermediate cells (AICs), which we have previously shown to be involved in LUAD initiation, were found in lesion-adjacent NAT, and their signature was enriched in matching aPMLs and further in LUADs. In line with these findings, trajectory analysis showed that AICs are derived from normal alveolar type 2 cells and likely progress towards tumor cells. Spatial neighborhood analysis pointed towards strong crosstalk between AICs and macrophages. We also noted heterogeneity in immune infiltration including patterns indicative of progressive features along the NAT-aPML-LUAD continuum. For example, B cell lineages were not only increased in abundance in LUADs relative to aPMLs, but they also mobilized into relatively more mature tertiary lymphoid aggregates/structures. While T regulatory cells aggregated close to LUADs, they were rather scattered in matching aPMLs. Our multimodal and spatial atlas of NAT, nearby aPML and LUAD elucidates the earliest cellular events underlying transition of NAT to LUAD and that could inform of targets for interception of this trajectory. Citation Format: Ansam Sinjab, Fuduan Peng, Yunhe Liu, Sujuan Yang, Tieling Zhou, Alejandra G. Serrano, Jiping Feng, Lorena Gomez Bolanos, Guangchun Han, Daniel Gustavo Rosen, Stephen G. Swisher, Avrum Spira, Steven M. Dubinett, Luisa M. Solis Soto, Mingyao Li, Junya Fujimoto, Jared Burks, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A multimodal spatial-omics atlas of lung precancer and progression to adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3893.

Abstract 2177: Postbiotic metabolites secreted by L. acidophilus reduce DNA damage in an experimental model of Barrett’s tumorigenesis

Abstract The origins of esophageal adenocarcinoma (EAC) are commonly attributed to the progression from gastroesophageal reflux disease (GERD) to Barrett’s Esophagus (BE) and finally to esophageal adenocarcinoma. During GERD, various types of bile induce oxidative stress and generate reactive oxygen species (ROS), resulting in DNA damage and inflammation in cells. Although there has been much research documenting the shift in the esophageal microbiome from gram-positive to gram-negative bacteria during this progression, there has also been an observed increase in the presence of Lactobacillus, a gram-positive commensal bacterium with probiotic qualities, in the esophagus during the progression from GERD to BE and eventually to EAC. Our previous research has demonstrated the ability of Lactobacillus spp. to not only colonize the esophagus under in vitro conditions of GERD, but also their anti-inflammatory and anti-genotoxic properties on esophageal cells exposed to bile. To assess the extent of reflux-induced DNA damage and oxidative stress during the progression of BE, we exposed two BE cell lines, CP-B and BAR-T, and the EAC cell line FLO-1 to ox bile or deoxycholic acid. Both bile types demonstrated significant increases in phosphorylated H2AX, as a marker for pronounced DNA damage repair. Next, we supplemented both live L. acidophilus and its postbiotic metabolites to cells exposed to ox bile and deoxycholic acid. We show a significant reduction in DNA damage as measured by pH2AX with both the live treatment and the treatment with postbiotic metabolites when compared to untreated, bile-exposed cells at various stages of BE and EAC. Ongoing experiments include assessing anti-genotoxic effects of postbiotic metabolites through DNA repair protein RAD51, reductions in oxidative stress though ROS marker 8-oxo-guanine, and anti-inflammatory effects through NFkB-associated inflammation. BE and EAC cell viability and proliferation upon bile exposure and postbiotic metabolite administration will also be assessed. This study aims to determine the supplementation with Lactobacillus postbiotic metabolites in the prevention and intervention of esophageal adenocarcinoma development. Citation Format: Divya Joshi, Claudia Andl. Postbiotic metabolites secreted by L. acidophilus reduce DNA damage in an experimental model of Barrett’s tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2177.

Abstract 3933: Comprehensive molecular characterization of mitochondrial mutational landscape across the evolution of lung adenocarcinoma

Abstract The human mitochondrial genome is prone to genetic alterations, which lead to somatic mutations, changes in mtDNA copy number and altered expression of respiratory chain subunits. This is in part due to the fact that mtDNA has a limited ability to repair itself when it is damaged. Although a buildup of somatic mutations in mtDNA has been associated with increased cancer risk, the role of mtDNA alterations in malignant transformation of lung adenocarcinoma has not been addressed. Therefore, a clear understanding of changes in mtDNA along the pathway of lung tumorigenesis is critical for identifying molecular biomarkers related to carcinogenesis and tumor progression. Like all solid tumors, lung adenocarcinoma is thought to be initiated and to progress through a series of genetic alterations, including changes in mtDNA. Lungs resected for primary adenocarcinomas often harbor minute discrete foci of cytologically atypical pneumocyte proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and ultimately fully invasive adenocarcinoma (ADA). In this study we used a unique cohort of 52 patients with matched specimens (n=133) representing different steps of disease progression (AAH, AIS, and MIA) coupled with novel ultra-deep mitochondrial sequencing (mtDNA-Seq) method to assess the mtDNA mutational landscape throughout the continuum of lung adenocarcinoma development. Utilizing a custom bioinformatics workflow, somatic mutations were detected in a subset of the premalignant non-invasive lesions, with overall higher mutational load observed in MIA and ACA specimens. Additionally, large degree of inter-patient heterogeneity were observed for mtDNA copy numbers in conjunction with the clonal histological evolution of pulmonary neoplasia. Moreover, an appreciable number of coding and non-coding mutations from regulatory regions of the mtDNA were detectable in the precursor AAH lesions and the continuum of LUAD progression. Majority of shared mutations showed heteroplasmic transition with increase in fractional abundance in ADA, compared to the AHH lesions, thereby indicating spatial clonal expansion as they progressed histologically. Here we report the first comprehensive characterization of mitochondrial mutational landscape in dynamic evolution of lung adenocarcinoma, and provide insight into the heterogeneity of clonal events associated with the progression from noninvasive to malignant disease. Citation Format: Alka Singh, Santanu Dasgupta, Ashwin L. Koppayi, Lisa Rooper, Vasudha Mishra, David Sidransky, Nishant Agrawal, Karthik Suresh, Evgeny Izumchenko. Comprehensive molecular characterization of mitochondrial mutational landscape across the evolution of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3933.

A 15-Inflammation-Related Gene Signature Predicts the Prognosis of Patients With Pancreatic Ductal Adenocarcinoma

Chronic inflammation promotes the development of pancreatic ductal adenocarcinoma (PDAC) and PDAC-related inflammatory tumor microenvironment facilitates tumor growth and metastasis. Thus, we aimed to study the association between inflammatory response and prognosis in patients with PDAC. We conducted the whole transcriptomic sequencing using tissue samples collected from patients diagnosed with PDAC (n = 106) recruited from Shandong Cancer Hospital. We first constructed a prognostic signature using 15 inflammation-related genes in The Cancer Genome Atlas (TCGA) cohort (n = 177) and further validated it in an independent International Cancer Genome Consortium (ICGC) cohort (n = 90) and our in-house cohort. PDAC patients with a higher risk score had poorer overall survival (OS) (P < 0.001; HR, 3.02; 95% CI, 1.94-4.70). The association between the prognostic signature and OS remained significant in the multivariable Cox regression adjusting for age, sex, alcohol exposure, diabetes, and stage (P < 0.001; HR, 2.91; 95% CI, 1.73-4.89). This gene signature also robustly predicted prognosis in the ICGC cohort (P = 0.01; HR, 1.94; 95% CI, 1.14-3.30) and our cohort (P < 0.001; HR, 2.40; 95% CI, 1.45-3.97). Immune subtype C3 (inflammatory) was enriched and CD8+ T cells were higher in patients with a lower risk score (P < 0.05). Furthermore, PDAC patients with higher risk scores were more sensitive to chemotherapy, immunotherapy, and PARP inhibitors (P < 0.05). In sum, we identified a novel gene signature that was associated with inflammatory response for risk stratification, prognosis prediction, and therapy guidance in PDAC patients. Future studies are warranted to validate the clinical utility of the signature.

An immune cell map of human lung adenocarcinoma development reveals an anti-tumoral role of the Tfh-dependent tertiary lymphoid structure

The immune responses during the initiation and invasion stages of human lung adenocarcinoma (LUAD) development are largely unknown. Here, we generated a single-cell RNA sequencing map to decipher the immune dynamics during human LUAD development. We found that T follicular helper (Tfh)-like cells, germinal center B cells, and dysfunctional CD8+ Tcells increase during tumor initiation/invasion and form a tertiary lymphoid structure (TLS) inside the tumor. This TLS starts with an aggregation of CD4+ Tcells and the generation of CXCL13-expressing Tfh-like cells, followed by an accumulation of B cells, and then forms a CD4+ T and B cell aggregate. TLS and its associated cells are correlated with better patient survival. InhibitingTLS formation by Tfh or B cell depletion promotes tumor growth in mouse models. The anti-tumoraleffect of the Tfh-dependent TLS is mediated through interleukin-21 (IL-21)-IL-21 receptor signaling. Our study establishes an anti-tumoral role of the Tfh-dependent TLS in the development of LUAD.

An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched withalveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Establishment of the molecular subtypes and a risk model for stomach adenocarcinoma based on genes related to reactive oxygen species

BackgroundOxidative stress promotes the development of stomach adenocarcinoma (STAD) and resistance of STAD patients to chemotherapy. This study developed a risk classification and prognostic model for STAD based on genes related to oxidative stress. MethodsUnivariate Cox regression and least absolute shrinkage and selection operator (Lasso) regression analysis were performed using transcriptome data of STAD from The Cancer Genome Atlas (TCGA) and reactive oxygen species (ROS)-related genes from Gene Set Enrichment Analysis (GSEA) website to develop a risk model. Genetic landscape, pathway characteristics and immune characteristics between the two risk groups were assessed to evaluate patients’ response to anti-tumor therapy. Further, a nomogram was created to evaluate the clinical outcomes of STAD patients. The mRNA levels of genes were detected by reverse transcription quantitative PCR (RT-qPCR). ResultsTwo ROS-related molecular subtypes (subtype C1 and C2) were classified, with subtype C2 having unfavorable prognosis, higher immune score, and greater infiltration of macrophages, myeloid-derived suppressor cells (MDSCs), mast cells, regulatory T cells, and C–C chemokine receptor (CCR). Five ROS-related genes (ASCL2, COMP, NOX1, PEG10, and VPREB3) were screened to develop a prognostic model, the robustness of which was validated in TCGA and external cohorts. RT-qPCR analysis showed that ASCL2, COMP, NOX1, and PEG10 were upregulated, while the mRNA level of VPREB3 was downregulated in gastric cancer cells. The risk score showed a negative relation to tumor mutation burden (TMB). Low-risk patients exhibited higher mutation frequencies of TTN, SYNE1, and ARID1A, higher response rate to immunotherapy and were more sensitive to 32 traditional chemotherapeutic drugs, while high-risk patients were sensitive to 13 drugs. Calibration curve and DCA confirmed the accuracy and reliability of the nomogram. ConclusionThese findings provided novel understanding on the mechanism of ROS in STAD. The current study developed a ROS-related signature to help predict the prognosis of patients suffering from STAD and to guide personalized treatment.

Helicobacter pylori CagA protein induces gastric cancer stem cell-like properties through the Akt/FOXO3a axis.

The presence of Helicobacter pylori (H. pylori) infection poses a substantial risk for the development of gastric adenocarcinoma. The primary mechanism through which H. pylori exerts its bacterial virulence is the cytotoxin CagA. This cytotoxin has the potential to induce inter-epithelial mesenchymal transition, proliferation, metastasis, and the acquisition of stem cell-like properties in gastric cancer (GC) cells infected with CagA-positive H. pylori. Cancer stem cells (CSCs) represent a distinct population of cells capable of self-renewal and generating heterogeneous tumor cells. Despite evidence showing that CagA can induce CSCs-like characteristics in GC cells, the precise mechanism through which CagA triggers the development of GC stem cells (GCSCs) remains uncertain. This study reveals that CagA-positive GC cells infected with H. pylori exhibit CSCs-like properties, such as heightened expression of CD44, a specific surface marker for CSCs, and increased ability to form tumor spheroids. Furthermore, we have observed that H. pylori activates the PI3K/Akt signaling pathway in a CagA-dependent manner, and our findings suggest that this activation is associated with the CSCs-like characteristics induced by H. pylori. The cytotoxin CagA, which is released during H. pylori infection, triggers the activation of the PI3K/Akt signaling pathway in a CagA-dependent manner. Additionally, CagA inhibits the transcription of FOXO3a and relocates it from the nucleus to the cytoplasm by activating the PI3K/Akt pathway. Furthermore, the regulatory function of the Akt/FOXO3a axis in the transformation of GC cells into a stemness state was successfully demonstrated.

Microsatellite instability in precancerous changes in the gastric mucosa

Microsatellite instability is a widely known and sought after tumor marker. Among other things, its prevalence and role in the development of gastric adenocarcinoma are being studied. However, the profile of microsatellite instability during precancerous changes in the gastric mucosa (atrophy, intestinal metaplasia, epithelial dysplasia) remains insufficiently studied. The purpose of this literature review is to assess the possibility of using microsatellite instability status as a diagnostic and predictive marker of precancerous changes and lesions of the gastric mucosa. A systematic review of publications in the PubMed database was conducted using a search query based on the combination of the terms “microsatellite instability” (“MSI”), “stomach/gastric cancer/adenocarcinoma”, “stomach/gastric dysplasia/intraepithelial neoplasia”, “stomach/gastric precancerous lesions” for the period from 1997 to 2023. Review articles were excluded from the search results. The systematic review included 11 relevant publications. Despite the lack of a uniform methodology and diversity in the study groups, all publications demonstrated an increase in the level of microsatellite instability in the range from normal (unchanged) gastric mucosa and/or its precancerous changes to gastric cancer: among precancerous changes in the gastric mucosa, the most common subject of study was intestinal metaplasia, where levels of microsatellite instability ranged from 0 to 53.3%, while its levels in gastric cancer ranged from 32.6 to 76.7%. The results of the studies included in the review may indicate a possible predictive role of microsatellite instability in precancerous changes in the gastric mucosa in relation to the risk of developing cancer.

Bioinformatics analysis identifies coagulation factor II receptor as a potential biomarker in stomach adenocarcinoma

Coagulation factor 2 thrombin receptor (F2R), a member of the G protein-coupled receptor family, plays an important role in regulating blood clotting through protein hydrolytic cleavage mediated receptor activation. However, the underlying biological mechanisms by which F2R affects the development of gastric adenocarcinoma are not fully understood. This study aimed to systematically analyze the role of F2R in gastric adenocarcinoma. Stomach adenocarcinoma (STAD)-related gene microarray data and corresponding clinicopathological information were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Differential expression genes (DEGs) associated with F2R were analyzed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), and protein–protein interaction (PPI) networks. F2R mRNA expression data were utilized to estimate stromal cell and immune cell scores in gastric cancer tissue samples, including stromal score, immune score, and ESTIMATE score, derived from single-sample enrichment studies. Analysis of TCGA and GEO databases revealed significantly higher F2R expression in STAD tissues compared to normal tissues. Patients with high F2R expression had shorter survival times than those with low F2R expression. F2R expression was significantly correlated with tumor (T) stage, node (N) stage, histological grade and pathological stage. Enrichment analysis of F2R-related genes showed that GO terms were mainly related to circulation-mediated human immune response, immunoglobulin, cell recognition and phagocytosis. KEGG analysis indicated associations to extracellular matrix (ECM) receptor interactions, neuroactive ligand-receptor interactions, the phosphoinositide-3-kinase-protein kinase B/Akt (PI3K-AKT) signaling pathway, the Wnt signaling pathway and the transforming growth factor-beta (TGF-β) signaling pathway. GSEA revealed connections to DNA replication, the Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway and oxidative phosphorylation. Drug sensitivity analysis demonstrated positive correlations between F2R and several drugs, including BEZ235, CGP-60474, Dasatinib, HG-6-64-1, Aazopanib, Rapamycin, Sunitinib and TGX221, while negative correlation with CP724714, FH535, GSK1904529A, JNK-9L, LY317615, pyrimidine, rTRAIL and Vinorelbine. Knocking down F2R in GC cell lines resulted in slowed proliferation, migration, and invasion. All statistical analyses were performed using R software (version 4.2.1) and GraphPad Prism 9.0. p < 0.05 was considered statistically significant. In conclusion, this study underscores the significance of F2R as a potential biomarker in gastric adenocarcinoma, shedding light on its molecular mechanisms in tumorigenesis. F2R holds promise for aiding in the diagnosis, prognosis, and targeted therapy of STAD.

On-chip Raman spectroscopy of live single cells for the staging of oesophageal adenocarcinoma progression.

The absence of early diagnosis contributes to oesophageal cancer being the sixth most common cause of global cancer-associated deaths, with a 5-year survival rate of < 20%. Barrett's oesophagus is the main pre-cancerous condition to adenocarcinoma development, characterised by the morphological transition of oesophageal squamous epithelium to metaplastic columnar epithelium. Early tracking and treatment of oesophageal adenocarcinoma could dramatically improve with diagnosis and monitoring of patients with Barrett's Oesophagus. Current diagnostic methods involve invasive techniques such as endoscopies and, with only a few identified biomarkers of disease progression, the detection of oesophageal adenocarcinoma is costly and challenging. In this work, single-cell Raman spectroscopy was combined with microfluidic techniques to characterise the development of oesophageal adenocarcinoma through the progression of healthy epithelial, Barrett's oesophagus and oesophageal adenocarcinoma cell lines. Principal component analysis and linear discriminant analysis were used to classify the different stages of cancer progression. with the ability to differentiate between healthy and cancerous cells with an accuracy of 97%. Whilst the approach could also separate the dysplastic stages from healthy or cancer with high accuracy-the intra-class separation was approximately 68%. Overall, these results highlight the potential for rapid and reliable diagnostic/prognostic screening of Barrett's Oesophagus patients.

TGFβ and Hippo Signaling Pathways Coordinate to Promote Acinar to Ductal Metaplasia in Human Pancreas.

Acinar-to-ductal metaplasia (ADM) serves as a precursor event in the development of pancreatic ductal adenocarcinoma (PDAC) upon constitutive environmental and genetical stress. While the role of ADM in PDAC progression has been established, the molecular mechanisms underlying human ADM remain elusive. We previously demonstrated the induction of ADM in human acinar cells through the transforming growth factor beta (TGFβ) signaling pathway. We aim to investigate the interaction between TGFβ and Hippo pathways in mediating ADM. RNA-sequencing was conducted on sorted normal primary human acinar, ductal, and AD (acinar cells that have undergone ADM) cells. ATAC-seq analysis was utilized to reveal the chromatin accessibility in these three cell types. ChIP-Seq of YAP1, SMAD4, and H3K27ac was performed to identify the gene targets of YAP1 and SMAD4. The role of YAP1/TAZ in ADM-driven cell proliferation, as well as in oncogenic KRAS driven proliferation, was assessed using sphere formation assay. AD cells have a unique transcription profile, with upregulated genes in open chromatin states in acinar cells. YAP1 and SMAD4 co-occupy the loci of ADM-related genes, including PROM1, HES1, and MMP7, co-regulating biological functions such as cell adhesion, cell migration, and inflammation. Overexpression of YAP1/TAZ promoted acinar cell proliferation but still required the TGFβ pathway. YAP1/TAZ were also crucial for TGFβ-induced sphere formation and were necessary for KRAS-induced proliferation. Our study reveals the intricate transition between acinar and AD states in human pancreatic tissues. It unveils the complex interaction between the Hippo and TGF-β pathways during ADM, highlighting the pivotal role of YAP1/TAZ and SMAD4 in PDAC initiation.

Abstract C094: Tff2 defines pancreatic TA progenitors that are protective against Kras-driven carcinogenesis

Abstract Background: Progenitor or stem cells play a pivotal role in maintaining tissue homeostasis and are often the source of tumor progression. While genuine stem cells are absent in the pancreatic exocrine organ, a variety of progenitor cells are present that contribute to normal homeostasis and regeneration process post-injury. In a previous study, we identified a pancreatic facultative progenitor (FP) that was marked by the gene doublecortin kinase like-1 (Dclk1). These Dclk1+ FP are rare, long-lived, quiescent cells that are critical for regeneration following injury. In contrast, cells marked by Trefoil factor 2 (Tff2) are active progenitors within the gastrointestinal tract, playing a crucial role in homeostasis and tumor progression. However, the role of Tff2+ cells within the pancreas remains relatively underexplored. Method &amp; Results: To explore the function of Tff2 cells, we developed a Tff2 BAC transgenic mouse line (Tff2-DTR-CreERT2), which was then crossed with report mice. Employing lineage tracing and single cell RNA sequencing, we uncovered a new type of progenitor, distinct from the FP - Tff2+ transit amplifying progenitors (TAP). Tff2+ acinar cells showed much greater proliferation (&amp;gt;100-fold) than all other acinar cells. Single cell RNA-seq of the Tff2-expressing lineage showed that one cluster (cluster 0) identified acinar cells with a low differentiated, progenitor-like state. These cells were found to contribute to tissue homeostasis, exhibiting expansion in the early stages, giving rise to more differentiated acinar cells, peaking around six months, and then gradually diminishing until they disappeared. To understand the role of Tff2 in PanIN lesions, we developed a compound mouse model: Tff2-DTR-CreERT; Mist1-CreERT; KrasG12D(TMK). When Tff2+ cells were then ablated using Diphtheria Toxin in the setting of caerulein-induced pancreatitis, the TMK mice developed significantly more PanIN lesions. Tff2-CreERT; KrasG12D mice were generally more resistant to developing advanced PanINs, but in the setting of caerulein pancreatitis, the Tff2; KrasG12D model developed more pancreatic ductal adenocarcinoma (PDAC). To explore the role of the TFF2 peptide in tumor progression, we used an adenovirus expressing TFF2 (Ad-TFF2), and introduced it through tail vein injections. We found that Ad-Tff2 helped prevent the development of advanced PanIN lesions. Conclusion: In summary, our research indicated that Tff2 cells play a significant role in maintaining pancreatic homeostasis and act in paracrine fashion to suppress the development of pancreatic adenocarcinoma. Citation Format: Feijing Wu, Zhengyu Jiang, Xiaofei Zhi, Yosuke Ochiai, Ruth A. White, Ermanno Malagola, Jin Qian, Biyun Zheng, Ruhong Tu, Hiroki Kobayashi, Quin Waterbury, Leah B. Zamechek, Timothy C. Wang. Tff2 defines pancreatic TA progenitors that are protective against Kras-driven carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C094.

Abstract B104: Epigenetic small molecule library screen to discover compounds that inhibit and reverse pancreatic acinar to ductal metaplasia

Abstract Pancreatic ductal adenocarcinoma (PDAC) development is believed to be preluded by the event of acinar to ductal metaplasia (ADM), the transdifferentiation of acinar cells into a duct-like state. It is widely accepted that either acinar or ductal cells bearing mutant Kras drive the development of PDAC. We have previously shown that we can inhibit and reverse ADM in a 3D organoid assay using primary pancreatic tissue from p48Cre/+LSL-KRASG12D/+ mice using the histone deacetylase inhibitor (HDACi), Trichostatin A (Da Silva, 2022). We further employed our 3D organoid model to perform a phenotypic and molecular screening of 144 epigenetic compounds. We identified class I HDACi, FK228, and the histone methyltransferase inhibitor, Chaetocin, as the top hits of reversing ADM without inducing cytotoxicity as assessed by calcein AM staining. Largazole homodomer, a novel HDACi derived from marine cyanobacteria, was also identified as both an inhibitor and reversal agent of ADM. These three compounds demonstrated phenotypic and molecular reversal of duct-like structures back into their acinar state, in addition to relative mRNA upregulation of acinar markers (Amy2a, Cela1, Cpa2) and downregulation of ductal markers (Krt19, Krt7, Sox9). Bulk-RNA sequencing combined with pathway analysis demonstrated pronounced inhibition of cancer signaling pathways during ADM reversal, such as TGFβ1, Vegf, TNF, IL-6, and STAT3. Moreover, RNA sequencing showed significant upregulation and downregulation of acinar and ductal/PDAC markers, respectively. Thus, a novel phenotypic small molecular screen based on organoid morphology was developed to discover compounds that inhibit and reverse pancreatic ADM in a mouse model with mutant Kras. Citation Format: Corey M. Perkins, Kalina R. Atanasova, Ranjala Ratnayake, Jinmai Jiang, Qi-Yin Chen, Thomas D. Schmittgen, Hendrik Luesch. Epigenetic small molecule library screen to discover compounds that inhibit and reverse pancreatic acinar to ductal metaplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B104.

Abstract B049: Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity

Abstract Epidemiologic studies have shown that obesity is a risk factor for more than a dozen cancers, including pancreatic ductal adenocarcinoma (PDAC). As PDAC is one of the leading causes of cancer-related death and obesity rates are rapidly increasing worldwide, it is imperative that we understand the mechanisms by which obesity promotes PDAC progression. To address this gap in knowledge, our lab combined a well-established genetic model for obesity (O: Lepob/ob) with a Kras-driven pancreatic cancer model (KC: Pdx1-Cre; KrasLSL-G12D/+) that mimics human PDAC progression. Resultant KCO mice had a drastic increase in tumor development compared to their lean counterparts, whereas induced weight loss intercepted tumor progression, showing that the tumor-promoting effects of obesity can be reversed. Bulk RNA sequencing of tumors from these mice revealed aberrantly increased expression of multiple peptide hormones expressed in insulin-producing beta (b) cells of pancreatic islets, including cholecystokinin (CCK) and amylin (Iapp). Strikingly, transgenic expression of CCK and Iapp in b-cells was sufficient to promote Kras-driven pancreatic tumorigenesis in non-obese mice. Single-cell RNA sequencing of b cells showed a trajectory of changes in gene expression with increasing obesity from wildtype to high-fat diet-fed to Lepob/ob mice. Further in silico analysis combining archetypal (AANet) with pseudotime (TrajectoryNet) methods demonstrated that b cells misexpressing hormones (CCK and Iapp) arose from a specific subtype of preexisting b cells, specifically virgin b cells, a rare, immature, regenerative population. Preliminary in vivo lineage tracing analyses in MIP-CreERT;LSL-TdTomato;Lepob/ob mice support these findings. Finally, we investigated the transcriptional networks that lead to aberrant CCK and Iapp expression in b cells. By combining Grainger causality analysis with known gene-gene relationships in the Transcriptional Regulatory Relationship Unraveled by Sentence-based Text mining (TRRUST) v2 database, we discovered a stress-induced mitogen-activated protein kinase (MAPK8)-cJun pathway that promotes pro-tumorigenic hormone expression in b cells. These combined experimental and computational methods nominate novel b cell targets to subvert the development of PDAC, an exocrine tumor. Citation Format: Cathy C. Garcia, Aarthi Venkat, Alex Tong, Sherry Agabiti, Lauren Lawres, Rebecca Cardone, Richard Kibbey, Smita Krishnaswamy, Mandar D Muzumdar. Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B049.

CEACAM5 and TROP2 define metaplastic and dysplastic transitions in human antral gastric precancerous lesions and tumors.

Mucosal gastric atrophy and intestinal metaplasia (IM) increase the risk for the development of gastric cancer (GC) as they represent a field for development of dysplasia and intestinal-type gastric adenocarcinoma. We have investigated the expression of two dysplasia markers, CEACAM5 and TROP2, in human antral IM and gastric tumors to assess their potential as molecular markers. In the normal antral mucosa, weak CEACAM5 and TROP2 expression was only observed in the foveolar epithelium, while inflamed antrum exhibited increased expression of both markers. Complete IM exhibited weak CEACAM5 expression at the apical surface, but no basolateral TROP2 expression. On the other hand, incomplete IM demonstrated high levels of both CEACAM5 and TROP2 expression. Notably, incomplete IM with dysplastic morphology (dysplastic incomplete IM) exhibited higher levels of CEACAM5 and TROP2 expression compared to incomplete IM without dysplastic features (simple incomplete IM). In addition, dysplastic incomplete IM showed diminished SOX2 and elevated CDX2 expression compared to simple incomplete IM. CEACAM5 and TROP2 positivity in incomplete IM was similar to that of gastric adenomas and GC. Significant association was found between CEACAM5 and TROP2 positivity and histology of GC. These findings support the concept that incomplete IM is more likely associated with GC development. Overall, our study provides evidence of the heterogeneity of gastric IM and the distinct expression profiles of CEACAM5 and TROP2 in dysplastic incomplete IM. Our findings support the potential use of CEACAM5 and TROP2 as molecular markers for identifying individuals with a higher risk of GC development in the context of incomplete IM.

Integrated multi-omic analysis and experiment reveals the role of endoplasmic reticulum stress in lung adenocarcinoma

BackgroundLung cancer is a highly prevalent malignancy worldwide and is associated with high mortality rates. While the involvement of endoplasmic reticulum (ER) stress in the development of lung adenocarcinoma (LUAD) has been established, the underlying mechanism remains unclear.MethodsIn this study, we utilized data from The Cancer Genome Atlas (TCGA) to identify differentially expressed endoplasmic reticulum stress-related genes (ERSRGs) between LUAD and normal tissues. We performed various bioinformatics analyses to investigate the biological functions of these ERSRGs. Using LASSO analysis and multivariate stepwise regression, we constructed a novel prognostic model based on the ERSRGs. We further validated the performance of the model using two independent datasets from the Gene Expression Omnibus (GEO). Additionally, we conducted functional enrichment analysis, immune checkpoint analysis, and immune infiltration analysis and drug sensitivity analysis of LUAD patients to explore the potential biological function of the model. Furthermore, we conducted a battery of experiments to verify the expression of ERSRGs in a real-world cohort.ResultsWe identified 106 ERSRGs associated with LUAD, which allowed us to classify LUAD patients into two subtypes based on gene expression differences. Using six prognostic genes (NUPR1, RHBDD2, VCP, BAK1, EIF2AK3, MBTPS2), we constructed a prognostic model that exhibited excellent predictive performance in the training dataset and was successfully validated in two independent external datasets. The risk score derived from this model emerged as an independent prognostic factor for LUAD. Confirmation of the linkage between this risk model and immune infiltration was affirmed through the utilization of Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The q-PCR results verified significant differences in the expression of prognostic genes between cancer and paracancer tissues. Notably, the protein expression of NUPR1, as determined by immunohistochemistry (IHC), exhibited an opposite pattern compared to the mRNA expression patterns.ConclusionThis study establishes a novel prognostic model for LUAD based on six ER stress-related genes, facilitating the prediction of LUAD prognosis. Additionally, NUPR1 was identified as a potential regulator of stress in LUAD.

Identification and validation of a 4-extracellular matrix gene signature associated with prognosis and immune infiltration in lung adenocarcinoma

BackgroundThe extracellular matrix (ECM) plays a crucial role in the development and tumor microenvironment of lung adenocarcinoma (LUAD). This study aimed to establish a risk score of ECM-related genes in LUAD and explore the association between the risk score and patient survival as well as immune cell infiltration, somatic mutations, and therapy response. MethodsGene expression data from The Cancer Genome Atlas (TGCA) and eight Gene Expression Omnibus (GEO) databases were used to analyze and identify differentially expressed genes (DEGs). Prognostic ECM-related genes were identified and utilized to formulate a prognostic signature. A nomogram was constructed using TCGA dataset and validated in two GEO datasets. Differences between high- and low-risk patients were analyzed for function enrichment, immune cell infiltration, somatic mutations, and therapy response. Finally, Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of DEGs in LUAD. ResultsA risk score based on four ECM-related genes, ANOS1, CD36, COL11A1, and HMMR, was identified as an independent prognostic factor for overall survival (OS) compared to other clinical variables. Subsequently, a nomogram incorporating the risk score and TNM staging was developed using the TCGA dataset. Internal and external validation of the nomogram, conducted through calibration plots, C-index, time-dependent receiver operating characteristics (ROC), integrated discrimination improvement (IDI), and decision curve analyses (DCA), demonstrated the excellent discriminatory ability and clinical practicability of this nomogram. The risk score correlated with the distribution of function enrichment, immune cell infiltration, and immune checkpoint expression. More somatic mutations occurred in the high-risk group. The risk score also demonstrated a favorable ability to predict immunotherapy response and drug sensitivity. ConclusionA novel signature based on four ECM-related genes is developed to help predict LUAD prognosis. This signature correlates with tumor immune microenvironment and can predict the response to different therapies in LUAD patients.