Tumor Microbiome Diversity and Composition Influence Pancreatic Cancer Outcomes.

Most patients diagnosed with resected pancreatic adenocarcinoma (PDAC) survive less than 5 years, but a minor subset survives longer. Here, we dissect the role of the tumor microbiota and the immune system in influencing long-term survival. Using 16S rRNA gene sequencing, we analyzed the tumor microbiome composition in PDAC patients with short-term survival (STS) and long-term survival (LTS). We found higher alpha-diversity in the tumor microbiome of LTS patients and identified an intra-tumoral microbiome signature (Pseudoxanthomonas-Streptomyces-Saccharopolyspora-Bacillus clausii) highly predictive of long-term survivorship in both discovery and validation cohorts. Through human-into-mice fecal microbiota transplantation (FMT) experiments from STS, LTS, or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration. Our study demonstrates that PDAC microbiome composition, which cross-talks to the gut microbiome, influences the host immune response and natural history of the disease.

Atlas of Metastatic Gastric Cancer Links Ferroptosis to Disease Progression and Immunotherapy Response.

BACKGROUND & AIMS: Metastases from gastric adenocarcinoma (GAC) lead to high morbidity and mortality. Developing innovative and effective therapies requires a comprehensive understanding of the tumor and immune biology of advanced GAC. Yet, collecting matched specimens from advanced, treatment-naïve patients with GAC poses a significant challenge, limiting the scope of current research, which has focused predominantly on localized tumors. This gap hinders deeper insight into the metastatic dynamics of GAC. METHODS: We performed in-depth single-cell transcriptome and immune profiling on 68 paired, treatment-naïve, primary metastatic tumors to delineate alterations in cancer cells and their tumor microenvironment during metastatic progression. To validate our observations, we conducted comprehensive functional studies both in vitro and in vivo, using cell lines and multiple patient-derived xenograft and novel mouse models of GAC. RESULTS: Liver and peritoneal metastases exhibited distinct properties in cancer cells and dynamics of tumor microenvironment phenotypes, supporting the notion that cancer cells and their local tumor microenvironments co-evolve at metastatic sites. Our study also revealed differential activation of cancer meta-programs across metastases. We observed evasion of cancer cell ferroptosis via GPX4 up-regulation during GAC progression. Conditional depletion of Gpx4 or pharmacologic inhibition of ferroptosis resistance significantly attenuated tumor growth and metastatic progression. In addition, ferroptosis-resensitizing treatments augmented the efficacy of chimeric antigen receptor T-cell therapy. CONCLUSIONS: This study represents the largest single-cell dataset of metastatic GACs to date. High-resolution mapping of the molecular and cellular dynamics of GAC metastasis has revealed a rationale for targeting ferroptosis defense in combination with chimeric antigen receptor T-cell therapy as a novel therapeutic strategy with potential immense clinical implications.

Protein biomarkers and alternatively methylated cell-free DNA detect early stage pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed. DESIGN: To assess circulating marker discrimination in training, testing and validation patient cohorts (total n=426 patients), plasma markers were measured among PDAC cases and patients with chronic pancreatitis, colorectal cancer (CRC), and healthy controls. Using CA19-9 as an anchor marker, measurements were made of two protein markers (TIMP1, LRG1) and cell-free DNA (cfDNA) pancreas-specific methylation at 9 loci encompassing 61 CpG sites. RESULTS: Comparative methylome analysis identified nine loci that were differentially methylated in exocrine pancreas DNA. In the training set (n=124 patients), cfDNA methylation markers distinguished PDAC from healthy and CRC controls. In the testing set of 86 early stage PDAC and 86 matched healthy controls, CA19-9 had an area under the receiver operating characteristic curve (AUC) of 0.88 (95% CI 0.83 to 0.94), which was increased by adding TIMP1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.06), LRG1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02) or exocrine pancreas-specific cfDNA methylation markers at nine loci (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02). In the validation set of 40 early stage PDAC and 40 matched healthy controls, a combined panel including CA19-9, TIMP1 and a 9-loci cfDNA methylation panel had greater discrimination (AUC 0.86, 95% CI 0.77 to 0.95) than CA19-9 alone (AUC 0.82; 95% CI 0.72 to 0.92). CONCLUSION: A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC.

Glycosylation in cancer as a source of biomarkers.

INTRODUCTION: Glycosylation, the process of glycan synthesis and attachment to target molecules, is a crucial and common post-translational modification (PTM) in mammalian cells. It affects the protein's hydrophilicity, charge, solubility, structure, localization, function, and protection from proteolysis. Aberrant glycosylation in proteins can reveal new detection and therapeutic Glyco-biomarkers, which help to improve accurate early diagnosis and personalized treatment. This review underscores the pivotal role of glycans and glycoproteins as a source of biomarkers in human diseases, particularly cancer. AREAS COVERED: This review delves into the implications of glycosylation, shedding light on its intricate roles in cancer-related cellular processes influencing biomarkers. It is underpinned by a thorough examination of literature up to June 2024 in PubMed, Scopus, and Google Scholar; concentrating on the terms: (Glycosylation[Title/Abstract]) OR (Glycan[Title/Abstract]) OR (glycoproteomics[Title/Abstract]) OR (Proteoglycans[Title/Abstract]) OR (Glycomarkers[Title/Abstract]) AND (Cancer[Title/Abstract]) AND ((Diagno*[Title/Abstract]) OR (Progno*[Title/Abstract])). EXPERT OPINION: Glyco-biomarkers enhance early cancer detection, allow early intervention, and improve patient prognoses. However, the abundance and complex dynamic glycan structure may make their scientific and clinical application difficult. This exploration of glycosylation signatures in cancer biomarkers can provide a detailed view of cancer etiology and instill hope in the potential of glycosylation to revolutionize cancer research.

Development of autoantibody signatures for common cancers.

A humoral immune response in the form of autoantibodies to tumor antigens occurs early during tumor development. Identification of antigens that induce an autoantibody response restricted to a cancer type has the potential to yield markers useful for early detection. A multitude of strategies are currently available for the discovery of tumor antigens directed autoantibodies in circulation. Each approach has advantages and limitations for comprehensive discovery of antigenic epitopes. Herein, we review established and novel strategies and methodologies and highlight potential cancer applications.

Circulating microRNA Panel for Prediction of Recurrence and Survival in Early-Stage Lung Adenocarcinoma.

Early-stage lung adenocarcinoma (LUAD) patients remain at substantial risk for recurrence and disease-related death, highlighting the unmet need of biomarkers for the assessment and identification of those in an early stage who would likely benefit from adjuvant chemotherapy. To identify circulating miRNAs useful for predicting recurrence in early-stage LUAD, we performed miRNA microarray analysis with pools of pretreatment plasma samples from patients with stage I LUAD who developed recurrence or remained recurrence-free during the follow-up period. Subsequent validation in 85 patients with stage I LUAD resulted in the development of a circulating miRNA panel comprising miR-23a-3p, miR-320c, and miR-125b-5p and yielding an area under the curve (AUC) of 0.776 in predicting recurrence. Furthermore, the three-miRNA panel yielded an AUC of 0.804, with a sensitivity of 45.8% at 95% specificity in the independent test set of 57 stage I and II LUAD patients. The miRNA panel score was a significant and independent factor for predicting disease-free survival (p < 0.001, hazard ratio [HR] = 1.64, 95% confidence interval [CI] = 1.51-4.22) and overall survival (p = 0.001, HR = 1.51, 95% CI = 1.17-1.94). This circulating miRNA panel is a useful noninvasive tool to stratify early-stage LUAD patients and determine an appropriate treatment plan with maximal efficacy.

Epithelial SOX9 drives progression and metastases of gastric adenocarcinoma by promoting immunosuppressive tumour microenvironment.

OBJECTIVE: Many cancers engage embryonic genes for rapid growth and evading the immune system. SOX9 has been upregulated in many tumours, yet the role of SOX9 in mediating immunosuppressive tumour microenvironment is unclear. Here, we aim to dissect the role of SOX9-mediated cancer stemness attributes and immunosuppressive microenvironment in advanced gastric adenocarcinoma (GAC) for novel therapeutic discoveries. METHODS: Bulk RNAseq/scRNA-seq, patient-derived cells/models and extensive functional studies were used to identify the expression and functions of SOX9 and its target genes in vitro and in vivo. Immune responses were studied in PBMCs or CD45+ immune cells cocultured with tumour cells with SOX9high or knockout and the KP-Luc2 syngeneic models were used for efficacy of combinations. RESULTS: SOX9 is one of the most upregulated SOX genes in GAC and highly expressed in primary and metastatic tissues and associated with poor prognosis. Depletion of SOX9 in patient-derived GAC cells significantly decreased cancer stemness attributes, tumour formation and metastases and consistently increased CD8+ T cell responses when cocultured with PBMCs/CD45+ cells from GAC patients. RNA sequencing identified the leukaemia inhibitory factor (LIF) as the top secreted molecule regulated by SOX9 in tumour cells and was enriched in malignant ascites and mediated SOX9-induced M2 macrophage repolarisation and inhibited T cell function. CONCLUSION: Epithelial SOX9 is critical in suppressing CD8+ T cell responses and modified macrophage function in GAC through the paracrine LIF factor. Cotargeting LIF/LIFR and CSF1R has great potential in targeting SOX9-mediated cancer stemness, T cell immunosuppression and metastases suggesting the novel combination therapy against advanced GAC.

A Comprehensive Search of Non-Canonical Proteins in Non-Small Cell Lung Cancer and Their Impact on the Immune Response.

There is substantial interest in mining neoantigens for cancer applications. Non-canonical proteins resulting from frameshift mutations have been identified as neoantigens in cancer. We investigated the landscape of non-canonical proteins in non-small cell lung cancer (NSCLC) and their induced immune response in the form of autoantibodies. A database of cryptoproteins was computationally constructed and comprised all alternate open reading frames (altORFs) and ORFs identified in pseudogenes, noncoding RNAs, and untranslated regions of mRNAs that did not align with known canonical proteins. Proteomic profiles of seventeen lung adenocarcinoma (LUAD) cell lines were searched to evaluate the occurrence of cryptoproteins. To assess the immunogenicity, immunoglobulin (Ig)-bound cryptoproteins in plasmas were profiled by mass spectrometry. The specimen set consisted of plasmas from 30 newly diagnosed NSCLC cases, pre-diagnostic plasmas from 51 NSCLC cases, and 102 control plasmas. An analysis of LUAD cell lines identified 420 cryptoproteins. Plasma Ig-bound analyses revealed 90 cryptoproteins uniquely found in cases and 14 cryptoproteins that had a fold-change >2 compared to controls. In pre-diagnostic samples, 17 Ig-bound cryptoproteins yielded an odds ratio ≥2. Eight Ig-bound cryptoproteins were elevated in both pre-diagnostic and newly diagnosed cases compared to controls. Cryptoproteins represent a class of neoantigens that induce an autoantibody response in NSCLC.

YAP1 mediates gastric adenocarcinoma peritoneal metastases that are attenuated by YAP1 inhibition.

OBJECTIVE: Peritoneal carcinomatosis (PC; malignant ascites or implants) occurs in approximately 45% of advanced gastric adenocarcinoma (GAC) patients and associated with a poor survival. The molecular events leading to PC are unknown. The yes-associated protein 1 (YAP1) oncogene has emerged in many tumour types, but its clinical significance in PC is unclear. Here, we investigated the role of YAP1 in PC and its potential as a therapeutic target. METHODS: Patient-derived PC cells, patient-derived xenograft (PDX) and patient-derived orthotopic (PDO) models were used to study the function of YAP1 in vitro and in vivo. Immunofluorescence and immunohistochemical staining, RNA sequencing (RNA-Seq) and single-cell RNA-Seq (sc-RNA-Seq) were used to elucidate the expression of YAP1 and PC cell heterogeneity. LentiCRISPR/Cas9 knockout of YAP1 and a YAP1 inhibitor were used to dissect its role in PC metastases. RESULTS: YAP1 was highly upregulated in PC tumour cells, conferred cancer stem cell (CSC) properties and appeared to be a metastatic driver. Dual staining of YAP1/EpCAM and sc-RNA-Seq revealed that PC tumour cells were highly heterogeneous, YAP1high PC cells had CSC-like properties and easily formed PDX/PDO tumours but also formed PC in mice, while genetic knockout YAP1 significantly slowed tumour growth and eliminated PC in PDO model. Additionally, pharmacologic inhibition of YAP1 specifically reduced CSC-like properties and suppressed tumour growth in YAP1high PC cells especially in combination with cytotoxics in vivo PDX model. CONCLUSIONS: YAP1 is essential for PC that is attenuated by YAP1 inhibition. Our data provide a strong rationale to target YAP1 in clinic for GAC patients with PC.

Integrated genomic profiling and modelling for risk stratification in patients with advanced oesophagogastric adenocarcinoma.

OBJECTIVE: Prognosis of patients with advanced oesophagogastric adenocarcinoma (mEGAC) is poor and molecular determinants of shorter or longer overall survivors are lacking. Our objective was to identify molecular features and develop a prognostic model by profiling the genomic features of patients with mEGAC with widely varying outcomes. DESIGN: We profiled 40 untreated mEGACs (20 shorter survivors <13 months and 20 longer survivors >36 months) with whole-exome sequencing (WES) and RNA sequencing and performed an integrated analysis of exome, transcriptome, immune profile and pathological phenotypes to identify the molecular determinants, developing an integrated model for prognosis and comparison with The Cancer Genome Atlas (TCGA) cohorts. RESULTS: KMT2C alterations were exclusively observed in shorter survivors together with high level of intratumour heterogeneity and complex clonal architectures, whereas the APOBEC mutational signatures were significantly enriched in longer survivors. Notably, the loss of heterozygosity in chromosome 4 (Chr4) was associated with shorter survival and 'cold' immune phenotype characterised by decreased B, CD8, natural killer cells and interferon-gamma responses. Unsupervised transcriptomic clustering revealed a shorter survivor subtype with distinct expression features (eg, upregulated druggable targets JAK2, MAP3K13 and MECOM). An integrated model was then built based on clinical variables and the identified molecular determinants, which significantly segregated shorter and longer survivors. All the above features and the integrated model have been validated independently in multiple TCGA cohorts. CONCLUSION: This study discovered novel molecular features prognosticating overall survival in patients with mEGAC and identified potential novel targets in shorter survivors.

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

Multiplex profiling of peritoneal metastases from gastric adenocarcinoma identified novel targets and molecular subtypes that predict treatment response.

OBJECTIVE: Peritoneal carcinomatosis (PC) occurs frequently in patients with gastric adenocarcinoma (GAC) and confers a poor prognosis. Multiplex profiling of primary GACs has been insightful but the underpinnings of PC's development/progression remain largely unknown. We characterised exome/transcriptome/immune landscapes of PC cells from patients with GAC aiming to identify novel therapeutic targets. DESIGN: We performed whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-seq) on 44 PC specimens (43 patients with PC) including an integrative analysis of WES, RNA-seq, immune profile, clinical and pathological phenotypes to dissect the molecular pathogenesis, identifying actionable targets and/or biomarkers and comparison with TCGA primary GACs. RESULTS: We identified distinct alterations in PC versus primary GACs, such as more frequent CDH1 and TAF1 mutations, 6q loss and chr19 gain. Alterations associated with aggressive PC phenotypes emerged with increased mutations in TP53, CDH1, TAF1 and KMT2C, higher level of 'clock-like' mutational signature, increase in whole-genome doublings, chromosomal instability (particularly, copy number losses), reprogrammed microenvironment, enriched cell cycle pathways, MYC activation and impaired immune response. Integrated analysis identified two main molecular subtypes: 'mesenchymal-like' and 'epithelial-like' with discriminating response to chemotherapy (31% vs 71%). Patients with the less responsive 'mesenchymal-like' subtype had high expression of immune checkpoint T-Cell Immunoglobulin And Mucin Domain-Containing Protein 3 (TIM-3), its ligand galectin-9, V-domain Ig suppressor of T cell activation (VISTA) and transforming growth factor-ß as potential therapeutic immune targets. CONCLUSIONS: We have uncovered the unique mutational landscape, copy number alteration and gene expression profile of PC cells and defined PC molecular subtypes, which correlated with PC therapy resistance/response. Novel targets and immune checkpoint proteins have been identified with a potential to be translated into clinics.

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

Human epididymis protein 4 antigen-autoantibody complexes complement cancer antigen 125 for detecting early-stage ovarian cancer.

BACKGROUND: Early detection of ovarian cancer could significantly improve patient outcomes. Cancer antigen 125 (CA 125) is elevated in sera from approximately 60% of patients with early-stage (I/II) disease. Sensitivity might be improved through the combination of CA 125 with other biomarkers. Among potential biomarkers, antigen-autoantibody (Ag-AAb) complexes have received relatively little attention. METHODS: Luminex-based immunoassays were used to measure human epididymis protein 4 (HE4), anti-HE4 autoantibody, and HE4 Ag-AAb complexes in sera from patients with early- (n = 73) and late-stage ovarian cancers (n = 49) at the time of diagnosis and from asymptomatic women with (n = 15) or without ovarian cancer (n = 212) enrolled in the Normal Risk Ovarian Cancer Screening Study. RESULTS: At 98% specificity for healthy, asymptomatic women, 7% of patients with early-stage (I/II) ovarian cancer and 4% of patients with late-stage (III/IV) disease had elevated levels of HE4 autoantibody, whereas elevated levels of HE4 Ag-AAb complexes were detected in sera from 38% of early-stage cases and 31% of late-stage cases. Complementarity was observed in receiver operating characteristic (ROC) curves between HE4 Ag-AAb complexes and CA 125 levels in early-stage ovarian cancer (P < .001). CA 125 detected 63% of cases, and a combination of CA 125 and HE4 Ag-AAb complexes detected 81%. Complementarity was also observed in ROC curves for an independent validation set with 69 early-stage patients (P = .039). HE4 Ag-AAb complexes were detected in serial preclinical serum samples from women destined to develop ovarian cancer: they correlated with CA 125 but did not provide a lead time. CONCLUSIONS: HE4 Ag-AAb complexes could complement CA 125 in detecting a higher fraction of early-stage ovarian cancers.

Novel urinary protein biomarker panel for early diagnosis of gastric cancer.

BACKGROUND: With the goal of discovering non-invasive biomarkers for early diagnosis of GC, we conducted a case-control study utilising urine samples from individuals with predominantly early GC vs. healthy control (HC). METHODS: Among urine samples from 372 patients, age- and sex-matched 282 patients were randomly divided into three groups: 18 patients in a discovery cohort; 176 patients in a training cohort and 88 patients in a validation cohort. RESULTS: Among urinary proteins identified in the comprehensive quantitative proteomics analysis, urinary levels of TFF1 (uTFF1) and ADAM12 (uADAM12) were significantly independent diagnostic biomarkers for GC, in addition to Helicobacter pylori status. A urinary biomarker panel combining uTFF1, uADAM12 and H. pylori significantly distinguished between HC and GC patients in both training and validation cohorts. On the analysis for sex-specific biomarkers, this combination panel demonstrated a good AUC of 0.858 for male GC, whereas another combination panel of uTFF1, uBARD1 and H. pylori also provided a good AUC of 0.893 for female GC. Notably, each panel could distinguish even stage I GC patients from HC patients (AUC = 0.850 for males; AUC = 0.845 for females). CONCLUSIONS: Novel urinary protein biomarker panels represent promising non-invasive biomarkers for GC, including early-stage disease.

In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has not been changed significantly during several decades. To seek new therapeutic targets for EOC, we performed an in vivo dropout screen in human tumor xenografts using a pooled shRNA library targeting thousands of druggable genes. Then, in follow-up studies, we performed a second screen using a genome-wide CRISPR/Cas9 library. These screens identified 10 high-confidence drug targets that included well-known oncogenes such as ERBB2 and RAF1, and novel oncogenes, notably KPNB1, which we investigated further. Genetic and pharmacological inhibition showed that KPNB1 exerts its antitumor effects through multiphase cell cycle arrest and apoptosis induction. Mechanistically, proteomic studies revealed that KPNB1 acts as a master regulator of cell cycle-related proteins, including p21, p27, and APC/C. Clinically, EOC patients with higher expression levels of KPNB1 showed earlier recurrence and worse prognosis than those with lower expression levels of KPNB1. Interestingly, ivermectin, a Food and Drug Administration-approved antiparasitic drug, showed KPNB1-dependent antitumor effects on EOC, serving as an alternative therapeutic toward EOC patients through drug repositioning. Last, we found that the combination of ivermectin and paclitaxel produces a stronger antitumor effect on EOC both in vitro and in vivo than either drug alone. Our studies have thus identified a combinatorial therapy for EOC, in addition to a plethora of potential drug targets.

Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers.

The earlier diagnosis of cancer is one of the keys to reducing cancer deaths in the future. Here we describe our efforts to develop a noninvasive blood test for the detection of pancreatic ductal adenocarcinoma. We combined blood tests for KRAS gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker. The cohort tested included 221 patients with resectable pancreatic ductal adenocarcinomas and 182 control patients without known cancer. KRAS mutations were detected in the plasma of 66 patients (30%), and every mutation found in the plasma was identical to that subsequently found in the patient's primary tumor (100% concordance). The use of KRAS in conjunction with four thresholded protein biomarkers increased the sensitivity to 64%. Only one of the 182 plasma samples from the control cohort was positive for any of the DNA or protein biomarkers (99.5% specificity). This combinatorial approach may prove useful for the earlier detection of many cancer types.

Testing the gene expression classification of the EMT spectrum.

The epithelial-mesenchymal transition (EMT) plays a central role in cancer metastasis and drug resistance-two persistent clinical challenges. Epithelial cells can undergo a partial or full EMT, attaining either a hybrid epithelial/mesenchymal (E/M) or mesenchymal phenotype, respectively. Recent studies have emphasized that hybrid E/M cells may be more aggressive than their mesenchymal counterparts. However, mechanisms driving hybrid E/M phenotypes remain largely elusive. Here, to better characterize the hybrid E/M phenotype (s) and tumor aggressiveness, we integrate two computational methods-(a) RACIPE-to identify the robust gene expression patterns emerging from the dynamics of a given gene regulatory network, and (b) EMT scoring metric-to calculate the probability that a given gene expression profile displays a hybrid E/M phenotype. We apply the EMT scoring metric to RACIPE-generated gene expression data generated from a core EMT regulatory network and classify the gene expression profiles into relevant categories (epithelial, hybrid E/M, mesenchymal). This categorization is broadly consistent with hierarchical clustering readouts of RACIPE-generated gene expression data. We also show how the EMT scoring metric can be used to distinguish between samples composed of exclusively hybrid E/M cells and those containing mixtures of epithelial and mesenchymal subpopulations using the RACIPE-generated gene expression data.

The BE GONE trial study protocol: a randomized crossover dietary intervention of dry beans targeting the gut microbiome of overweight and obese patients with a history of colorectal polyps or cancer.

BACKGROUND: Mouse and human studies support the promise of dry beans to improve metabolic health and to lower cancer risk. In overweight/obese patients with a history of colorectal polyps or cancer, the Beans to Enrich the Gut microbiome vs. Obesity's Negative Effects (BE GONE) trial will test whether and how an increase in the consumption of pre-cooked, canned dry beans within the context of usual diet and lifestyle can enhance the gut landscape to improve metabolic health and reduce cancer risk. METHODS/DESIGN: This randomized crossover trial is designed to characterize changes in (1) host markers spanning lipid metabolism, inflammation, and obesity-related cancer risk; (2) compositional and functional profiles of the fecal microbiome; and (3) host and microbial metabolites. With each subject serving as their own control, the trial will compare the participant's usual diet with (intervention) and without (control) dry beans. Canned, pre-cooked dry beans are provided to participants and the usual diet continually assessed and monitored. Following a 4-week run-in and equilibration period, each participant provides a total of 5 fasting blood and 6 stool samples over a total period of 16 weeks. The intervention consists of a 2-week ramp-up of dry bean intake to 1 cup/d, which is then continued for an additional 6 weeks. Intra- and inter-individual outcomes are assessed across each crossover period with consideration of the joint or modifying effects of the usual diet and baseline microbiome. DISCUSSION: The BE GONE trial is evaluating a scalable dietary prevention strategy targeting the gut microbiome of high-risk patients to mitigate the metabolic and inflammatory effects of adiposity that influence colorectal cancer risk, recurrence, and survival. The overarching scientific goal is to further elucidate interactions between diet, the gut microbiome, and host metabolism. Improved understanding of the diet-microbiota interplay and effective means to target these relationships will be key to the future of clinical and public health approaches to cancer and other major diet- and obesity-related diseases. TRIAL REGISTRATION: This protocol is registered with the U.S. National Institutes of Health trial registry, ClinicalTrials.gov, under the identifier NCT02843425. First posted July 25, 2016; last verified January 25, 2019.

Immunoproteasome deficiency is a feature of non-small cell lung cancer with a mesenchymal phenotype and is associated with a poor outcome.

The immunoproteasome plays a key role in generation of HLA peptides for T cell-mediated immunity. Integrative genomic and proteomic analysis of non-small cell lung carcinoma (NSCLC) cell lines revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. Low expression of immunoproteasome subunits in early stage NSCLC patients was associated with recurrence and metastasis. Depleted repertoire of HLA class I-bound peptides in mesenchymal cells deficient in immunoproteasome components was restored with either IFNγ or 5-aza-2'-deoxycytidine (5-aza-dC) treatment. Our findings point to a mechanism of immune evasion of cells with a mesenchymal phenotype and suggest a strategy to overcome immune evasion through induction of the immunoproteasome to increase the cellular repertoire of HLA class I-bound peptides.