miR-146a inhibits ovarian tumor growth in vivo via targeting immunosuppressive neutrophils and enhancing CD8+ T cell infiltration.

Immunotherapies have emerged as promising strategies for cancer treatment. However, existing immunotherapies have poor activity in high-grade serous ovarian cancer (HGSC) due to the immunosuppressive tumor microenvironment and the associated low tumoral CD8+ T cell (CTL) infiltration. Through multiple lines of evidence, including integrative analyses of human HGSC tumors, we have identified miR-146a as a master regulator of CTL infiltration in HGSC. Tumoral miR-146a expression is positively correlated with anti-cancer immune signatures in human HGSC tumors, and delivery of miR-146a to tumors resulted in significant reduction in tumor growth in both ID8-p53-/- and IG10 murine HGSC models. Increasing miR-146a expression in tumors improved anti-tumor immune responses by decreasing immune suppressive neutrophils and increasing CTL infiltration. Mechanistically, miR-146a targets IL-1 receptor-associated kinase 1 and tumor necrosis factor receptor-associated factor 6 adaptor molecules of the transcription factor nuclear factor κB signaling pathway in ID8-p53-/- cells and decreases production of the downstream neutrophil chemoattractant, C-X-C motif chemokine ligand 1. In addition to HGSC, tumoral miR-146a expression also correlates strongly with CTL infiltration in other cancer types including thyroid, prostate, breast, and adrenocortical cancers. Altogether, our findings highlight the ability of miR-146a to overcome immune suppression and improve CTL infiltration in tumors.

Clinically conserved genomic subtypes of gastric adenocarcinoma.

Gastric adenocarcinoma (GAC) is a lethal disease characterized by genomic and clinical heterogeneity. By integrating 8 previously established genomic signatures for GAC subtypes, we identified 6 clinically and molecularly distinct genomic consensus subtypes (CGSs). CGS1 have the poorest prognosis, very high stem cell characteristics, and high IGF1 expression, but low genomic alterations. CGS2 is enriched with canonical epithelial gene expression. CGS3 and CGS4 have high copy number alterations and low immune reactivity. However, CGS3 and CGS4 differ in that CGS3 has high HER2 activation, while CGS4 has high SALL4 and KRAS activation. CGS5 has the high mutation burden and moderately high immune reactivity that are characteristic of microsatellite instable tumors. Most CGS6 tumors are positive for Epstein Barr virus and show extremely high levels of methylation and high immune reactivity. In a systematic analysis of genomic and proteomic data, we estimated the potential response rate of each consensus subtype to standard and experimental treatments such as radiation therapy, targeted therapy, and immunotherapy. Interestingly, CGS3 was significantly associated with a benefit from chemoradiation therapy owing to its high basal level of ferroptosis. In addition, we also identified potential therapeutic targets for each consensus subtype. Thus, the consensus subtypes produced a robust classification and provide for additional characterizations for subtype-based customized interventions.

Inhibition of the interaction between Hippo/YAP and Akt signaling with ursolic acid and 3'3-diindolylmethane suppresses esophageal cancer tumorigenesis.

Hippo/YAP signaling hinders cancer progression. Inactivation of this pathway contributes to the development of esophageal cancer by activation of Akt. However, the possible interaction between Akt and Hippo/YAP pathways in esophageal cancer progression is unclear. In this study, we found that ursolic acid (UA) plus 3'3-diindolylmethane (DIM) efficiently suppressed the oncogenic Akt/Gsk-3ß signaling pathway while activating the Hippo tumor suppressor pathway in esophageal cancer cells. Moreover, the addition of the Akt inhibitor LY294002 and the PI3K inhibitor 3-methyladenine enhanced the inhibitory effects of UA plus DIM on Akt pathway activation and further stimulated the Hippo pathway, including the suppression of YAP nuclear translocation in esophageal cancer cells. Silencing YAP under UA plus DIM conditions significantly increased the activation of the tumor suppressor PTEN in esophageal cancer cells, while decreasing p-Akt activation, indicating that the Akt signaling pathway could be down-regulated in esophageal cancer cells by targeting PTEN. Furthermore, in a xenograft nude mice model, UA plus DIM treatment effectively diminished esophageal tumors by inactivating the Akt pathway and stimulating the Hippo signaling pathway. Thus, our study highlights a feedback loop between the PI3K/Akt and Hippo signaling pathways in esophageal cancer cells, implying that a low dose of UA plus DIM could serve as a promising chemotherapeutic combination strategy in the treatment of esophageal cancer.

Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

3,3'-Diindolylmethane Augments 5-Fluorouracil-InducedGrowth Suppression in Gastric Cancer Cells through Suppression of the Akt/GSK-3ß and WNT/Beta-Catenin.

Gastric cancer (GC) is one of the most lethal cancers in South Korea, and it is a cancer of concern worldwide. 5-fluorouracil (5-Fu) is commonly used as the first-line therapy for advanced GC; however, its side effects often limit the dosage range and impair patients' quality of life. Due to the limitations of current chemotherapy, new anticancer therapies are urgently needed. 3,3'-diindolylmethane (DIM) has been reported to have the ability to protect against various types of cancer. Our study aimed to elucidate the anticancer effect of DIM in GC when treated with the chemotherapeutic agent 5-Fu. In our results, combined treatment with DIM and 5-Fu resulted in higher apoptosis and lower cell proliferation than treatment with 5-Fu in SNU484 and SNU638 cell lines. Furthermore, when DIM and 5-Fu were administered together, cell invasion was diminished by mediated E-cadherin, MMP-9, and uPA; p-Akt and p-GSK-3ß levels were reduced more significantly than when 5-Fu was administered alone. Moreover, in the Wnt signaling pathway, combined treatment of DIM and 5-Fu diminished ß-catenin levels in the nucleus and inhibited cyclin D1and c-Myc protein levels. The Akt inhibitor, wortmannin, further inhibited the levels of ß-catenin and c-Myc that were inhibited by DIM and 5-Fu. Furthermore, an animal xenograft model demonstrated that DIM combined with 5-Fu considerably reduced tumor growth without any toxic effects by regulating the Akt/GSK-3ß and ß-catenin levels. Our findings suggest that DIM significantly potentiates the anticancer effects of 5-Fu by targeting the Akt/GSK-3ß and WNT/ß-catenin because the combination therapy is more effective than 5-Fu alone, thereby offering an innovative potential therapy for patients with GC.

Effects of Subnormothermic Regulated Hepatic Reperfusion on Mitochondrial and Transcriptomic Profiles in a Porcine Model.

OBJECTIVE: We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death model. SUMMARY OF BACKGROUND DATA: Donation after circulatory death represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation. METHODS: Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control- Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed. RESULTS: Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis. CONCLUSION: Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Sub- normothermic RHR has potential applicability to clinical liver transplantation.

Inhibition of colorectal cancer tumorigenesis by ursolic acid and doxorubicin is mediated by targeting the Akt signaling pathway and activating the Hippo signaling pathway.

Colorectal cancer (CRC) is one of the deadliest malignant tumors worldwide and its prevalence is increasing in South Korea. The efficacy of combined treatment with natural product­derived and chemotherapy agents including curcumin combined with 5­fluorouracil, resveratrol combined with cisplatin and epigallocatechin­3­gallate (EGCG) combined with cisplatin in preventing cancer progression and killing cancer cells has emerged. The Akt and Hippo signaling pathways serve a key role in colorectal tumor growth; however, the exact role of the crosstalk between Akt and Hippo signaling pathways in CRC remains poorly elucidated. The combined effect of UA and DOX on the cell proliferation, apoptosis, migration and cell cycle of CRC cells were investigated by performing Cell proliferation assay, a soft agar colony formation assay, flow cytometry, wound healing assay and western blotting assay. Subsequently, the expression of AKT and Hippo signaling pathway­associated proteins were also assessed by western blot assay. Moreover, a xenograft nude mouse model was constructed to verify the effects of UA and DOX on the tumorigenesis of HCT116 cell in vivo. The present study reported that ursolic acid (UA) strongly enhanced the antitumor action of doxorubicin (DOX) via blocking the Akt/glycogen synthase kinase­3ß (Gsk3ß) signaling pathway and activating tumor­suppressive Hippo signaling (mammalian Ste20­like kinase 1 and 2, salvador family WW domain containing protein 1 and MOB kinase activator 1), thereby downregulating downstream effector yes­associated protein 1 (Yap) and connective tissue growth factor (CTGF) protein expression levels in CRC cells. Furthermore, The PI3K inhibitor LY294002 further suppressed Akt activity and enhance the function of Hippo pathway­associated proteins in DOX + UA treated cells; this effect led to subsequent oncogenic Yap and CTGF inhibition following combined treatment, whereas Akt activator SC79 exerted an opposite effect in CTGF expression. In vivo, treatment with UA combined with DOX markedly suppressed the progression of CRC without any toxic effects on a xenograft mouse model by disrupting Akt signaling and activating the Hippo signaling pathway. These results demonstrated that UA and DOX treatment successfully induced Akt/Gsk3ß inactivation via Hippo signaling pathway activation to promote Yap degradation, resulting in the inhibition of colorectal tumorigenesis. In conclusion, these findings suggested that combination therapy with UA and DOX may be more effective than DOX alone. UA may be a novel anticancer strategy and could be considered for investigation as a complementary chemotherapy agent in the future.

Proteo-genomic characterization of virus-associated liver cancers reveals potential subtypes and therapeutic targets.

Primary liver cancer is a heterogeneous disease in terms of its etiology, histology, and therapeutic response. Concurrent proteomic and genomic characterization of a large set of clinical liver cancer samples can help elucidate the molecular basis of heterogeneity and thus serve as a valuable resource for personalized liver cancer treatment. In this study, we perform proteomic profiling of ~300 proteins on 259 primary liver cancer tissues with reverse-phase protein arrays, mutational analysis using whole genome sequencing and transcriptional analysis with RNA-Seq. Patients are of Japanese ethnic background and mainly HBV or HCV positive, providing insight into this important liver cancer subtype. Unsupervised classification of tumors based on protein expression profiles reveal three proteomic subclasses R1, R2, and R3. The R1 subclass is immunologically hot and demonstrated a good prognosis. R2 contains advanced proliferative tumor with TP53 mutations, high expression of VEGF receptor 2 and the worst prognosis. R3 is enriched with CTNNB1 mutations and elevated mTOR signaling pathway activity. Twenty-two proteins, including CDK1 and CDKN2A, are identified as potential prognostic markers. The proteomic classification presented in this study can help guide therapeutic decision making for liver cancer treatment.

Therapeutic Targeting of Macrophage Plasticity Remodels the Tumor-Immune Microenvironment.

SIGNIFICANCE: Comprehensive single-cell proteomics analyses of lung adenocarcinoma progression reveal the role of tumor-associated macrophages in resistance to PD-1 blockade therapy. See related commentary by Lee et al., p. 2515.

Midnolin Regulates Liver Cancer Cell Growth In Vitro and In Vivo.

Hepatocellular carcinoma (HCC) ranks worldwide as one of the most lethal cancers. In spite of the vast existing knowledge about HCC, the pathogenesis of HCC is not completely understood. Discovery of novel genes that contribute to HCC pathogenesis will provide new insights for better understanding and treating HCC. The relatively obscure gene midnolin has been studied for over two decades; however, its biological roles are largely unknown. Our study is the first to demonstrate the functional significance of midnolin in HCC/cancer: Midnolin expression correlates with poor prognosis in HCC patients, and suppression of midnolin severely inhibits tumorigenicity of HCC cells in vitro and in mice and disrupts retinoic acid/lipid metabolism in these cells.

Consensus subtypes of hepatocellular carcinoma associated with clinical outcomes and genomic phenotypes.

BACKGROUND AND AIMS: Although many studies revealed transcriptomic subtypes of HCC, concordance of the subtypes are not fully examined. We aim to examine a consensus of transcriptomic subtypes and correlate them with clinical outcomes. APPROACH AND RESULTS: By integrating 16 previously established genomic signatures for HCC subtypes, we identified five clinically and molecularly distinct consensus subtypes. STM (STeM) is characterized by high stem cell features, vascular invasion, and poor prognosis. CIN (Chromosomal INstability) has moderate stem cell features, but high genomic instability and low immune activity. IMH (IMmune High) is characterized by high immune activity. BCM (Beta-Catenin with high Male predominance) is characterized by prominent ß-catenin activation, low miRNA expression, hypomethylation, and high sensitivity to sorafenib. DLP (Differentiated and Low Proliferation) is differentiated with high hepatocyte nuclear factor 4A activity. We also developed and validated a robust predictor of consensus subtype with 100 genes and demonstrated that five subtypes were well conserved in patient-derived xenograft models and cell lines. By analyzing serum proteomic data from the same patients, we further identified potential serum biomarkers that can stratify patients into subtypes. CONCLUSIONS: Five HCC subtypes are correlated with genomic phenotypes and clinical outcomes and highly conserved in preclinical models, providing a framework for selecting the most appropriate models for preclinical studies.

Clinical Significance of Glycolytic Metabolic Activity in Hepatocellular Carcinoma.

High metabolic activity is a hallmark of cancers, including hepatocellular carcinoma (HCC). However, the molecular features of HCC with high metabolic activity contributing to clinical outcomes and the therapeutic implications of these characteristics are poorly understood. We aimed to define the features of HCC with high metabolic activity and uncover its association with response to current therapies. By integrating gene expression data from mouse liver tissues and tumor tissues from HCC patients (n = 1038), we uncovered three metabolically distinct HCC subtypes that differ in clinical outcomes and underlying molecular biology. The high metabolic subtype is characterized by poor survival, the strongest stem cell signature, high genomic instability, activation of EPCAM and SALL4, and low potential for benefitting from immunotherapy. Interestingly, immune cell analysis showed that regulatory T cells (Tregs) are highly enriched in high metabolic HCC tumors, suggesting that high metabolic activity of cancer cells may trigger activation or infiltration of Tregs, leading to cancer cells' evasion of anti-cancer immune cells. In summary, we identified clinically and metabolically distinct subtypes of HCC, potential biomarkers associated with these subtypes, and a potential mechanism of metabolism-mediated immune evasion by HCC cells.

Comprehensive Molecular Characterization of Adenocarcinoma of the Gastroesophageal Junction Between Esophageal and Gastric Adenocarcinomas.

OBJECTIVE: To investigate the molecular characteristics of AGEJ compared with EAC and gastric adenocarcinoma. SUMMARY OF BACKGROUND DATA: Classification of AGEJ based on differential molecular characteristics between EAC and gastric adenocarcinoma has been long-standing controversy but rarely conducted due to anatomical ambiguity and epidemiologic difference. METHODS: The molecular classification model with Bayesian compound covariate predictor was developed based on differential mRNA expression of EAC (N = 78) and GCFB (N = 102) from the Cancer Genome Atlas (TCGA) cohort. AGEJ/cardia (N = 48) in TCGA cohort and AGEJ/upper third GC (N = 46 pairs) in Seoul National University cohort were classified into the EAC-like or GCFB-like groups whose genomic, transcriptomic, and proteomic characteristics were compared. RESULTS: AGEJ in both cohorts was similarly classified as EAC-like (31.2%) or GCFB-like (68.8%) based on the 400-gene classifier. The GCFB-like group showed significantly activated phosphoinositide 3-kinase-AKT signaling with decreased expression of ERBB2. The EAC-like group presented significantly different alternative splicing including the skipped exon of RPS24, a significantly higher copy number amplification including ERBB2 amplification, and increased protein expression of ERBB2 and EGFR compared with GCFB-like group. High-throughput 3D drug test using independent cell lines revealed that the EAC-like group showed a significantly better response to lapatinib than the GCFB-like group (P = 0.015). CONCLUSIONS: AGEJ was the combined entity of the EAC-like and GCFB-like groups with consistently different molecular characteristics in both Seoul National University and TCGA cohorts. The EAC-like group with a high Bayesian compound covariate predictor score could be effectively targeted by dual inhibition of ERBB2 and EGFR.

Inhibition of Cyclin Dependent Kinase 4/6 Overcomes Primary Resistance to Programmed Cell Death 1 Blockade in Malignant Mesothelioma.

BACKGROUND: Despite the profound number of malignant pleural mesothelioma (MPM) patients now treated with programmed cell death 1 (PD-1) blockade, insight into the underpinnings of rational therapeutic strategies to treat resistance to checkpoint immunotherapy remains unrealized. Our objective was to develop a novel therapeutic approach to overcome primary resistance to PD-1 blockade in MPM. METHODS: We generated a transcriptome signature of resistance to PD-1 blockade in MPM patients treated with nivolumab (4 responders and 4 nonresponders). We used The Cancer Genome Atlas MPM cohort (n = 73) to determine what genomic alterations were associated with the resistance signature. We tested whether regulation of identified molecules could overcome resistance to PD-1 blockade in an immunocompetent mouse malignant mesothelioma model. RESULTS: Immunogenomic analysis by applying our anti-PD-1 resistance signature to The Cancer Genome Atlas cohort revealed that deletion of cyclin dependent kinase inhibitor 2A (CDKN2A) was highly associated with primary resistance to PD-1 blockade. Under the hypothesis that resistance to PD-1 blockade can be overcome by cyclin dependent kinase 4/6 (CDK4/6) inhibition, we tested whether CDK4/6 inhibitors could overcome resistance to PD-1 blockade in subcutaneous tumors derived from Cdkn2a-/- AB1 malignant mesothelioma cells, which were resistant to PD-1 blockade. The combination of daily oral administration of CDK4/6 inhibitors (abemaciclib or palbociclib) and intraperitoneal anti-PD-1 treatment markedly suppressed tumor growth compared with anti-PD-1 or CDK4/6 inhibitor alone. CONCLUSIONS: We identified a therapeutic target, CDK4/6, to overcome primary resistance to PD-1 blockade through comprehensive immunogenomic approaches. These data provide a rationale for undertaking clinical trials of CDK4/6 inhibitors in more than 40% of patients with MPM who demonstrate loss of CDKN2A.

An Overview of the Genomic Characterization of Hepatocellular Carcinoma.

Tumor classifications based on alterations in the genome, epigenome, or proteome have revealed distinct tumor subgroups that are associated with clinical outcomes. Several landmark studies have demonstrated that such classifications can significantly improve patient outcomes by enabling tailoring of therapy to specific alterations in cancer cells. Since cancer cells accumulate numerous alterations in many cancer-related genes, it is a daunting task to find and confirm important cancer-promoting alterations as therapeutic targets or biomarkers that can predict clinical outcomes such as survival and response to treatments. To aid further advances, we provide here an overview of the current understanding of molecular and genomic subtypes of hepatocellular carcinoma (HCC). System-level integration of data from multiple studies and development of new technical platforms for analyzing patient samples hold great promise for the discovery of new targets for treatment and correlated biomarkers, leading to personalized medicine for treatment of HCC patients.