Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis.

BACKGROUND: Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. METHODS: We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. FINDINGS: CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. INTERPRETATION: MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. FUNDING: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.

Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer.

Bromodomain and extraterminal domain (BET) family proteins are epigenetic master regulators of gene expression via recognition of acetylated histones and recruitment of transcription factors and co-activators to chromatin. Hence, BET family proteins have emerged as promising therapeutic targets in cancer. In this study, we examined the functional role of bromodomain containing 3 (BRD3), a BET family protein, in colorectal cancer (CRC). In vitro and vivo analyses using BRD3-knockdown or BRD3-overexpressing CRC cells showed that BRD3 suppressed tumor growth and cell cycle G1/S transition and induced p21 expression. Clinical analysis of CRC datasets from our hospital or The Cancer Genome Atlas revealed that BET family genes, including BRD3, were overexpressed in tumor tissues. In immunohistochemical analyses, BRD3 was observed mainly in the nucleus of CRC cells. According to single-cell RNA sequencing in untreated CRC tissues, BRD3 was highly expressed in malignant epithelial cells, and cell cycle checkpoint-related pathways were enriched in the epithelial cells with high BRD3 expression. Spatial transcriptomic and single-cell RNA sequencing analyses of CRC tissues showed that BRD3 expression was positively associated with high p21 expression. Furthermore, overexpression of BRD3 combined with knockdown of, a driver gene in the BRD family, showed strong inhibition of CRC cells in vitro. In conclusion, we demonstrated a novel tumor suppressive role of BRD3 that inhibits tumor growth by cell cycle inhibition in part via induction of p21 expression. BRD3 activation might be a novel therapeutic approach for CRC.

Case report: A rare case of triple negative breast cancer with development of acute pancreatitis due to dexamethasone during adjuvant chemotherapy.

Here, we present the case of a 42-year-old female who developed acute pancreatitis due to dexamethasone during adjuvant chemotherapy for early triple negative breast cancer (TNBC). The patient received partial mastectomy and sentinel lymph node biopsy for early TNBC (cT1N0M0, cStage I) of the left breast. Dose-dense doxorubicin plus cyclophosphamide (ddAC) was administered as the adjuvant-chemotherapy; however, epigastralgia appeared on the fifth day of the first administration. A blood test showed a remarkable increase of serum pancreatic enzyme levels and computed tomography (CT) showed the swelling of pancreas and surrounding effusion, and she was diagnosed with moderate acute pancreatitis. As she had no history of excessive alcohol consumption or complication of cholelithiasis, dyslipidemia, or pancreatic neoplasm, drug-induced pancreatitis was suspected. Dexamethasone, which was administered as an antiemetic, was the suspected drug based on the drug administration history and previous report, and dexamethasone was discontinued from the second administration of ddAC. There was subsequently no recurrence of pancreatitis with no increase in serum pancreatic enzyme levels, and it was possible to complete adjuvant-chemotherapy. Alcohol, gallstones, dyslipidemia, and drugs have been reported as causes of pancreatitis; however, steroid-induced acute pancreatitis is extremely rare. We present the first case of acute pancreatitis induced by dexamethasone as the antiemetic.

A rare case of liver regenerative and non-neoplastic lesion resembling a well-differentiated hepatocellular carcinoma.

BACKGROUND: Nodular regenerative hyperplasia (NRH) is a rare disease that presents pathologically as diffuse hepatic nodules without fibrous septa. It is believed to be caused by vasculopathy against a background of various systemic diseases, such as hematologic, autoimmune, and drug-induced diseases, with various symptoms. In spite of the recent imaging advances, various atypical cases of nodular lesions are observed in daily clinical practice. Cases that do not completely meet these criteria are referred to as -like or -similar lesions in clinical situations, making it difficult to understand their pathogenesis. We present a case in which two hepatic nodular lesions were noted and difficult to differentiate from malignancy preoperatively. The lesions were laparoscopically resected and a pathological diagnosis with non-neoplastic liver regenerative nodules resembling NRH was made. CASE PRESENTATION: A 49-year-old man with no alcohol or drug intake and no past medical history was identified as having liver tumors on screening examination without any symptoms. Contrast-enhanced computed tomography (CT) showed two hepatic tumors; approximately 2-cm tumors at S7 and S8. Gadolinium-ethoxybenzyl-diethylenetriamine-pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) revealed fat inclusions in their contents. Ethoxybenzyl (EOB) uptake was also observed during the hepatobiliary phase. Based on preoperative examinations, we suspected well-differentiated hepatocellular carcinoma (HCC) and performed laparoscopic S7/8 partial resection for these lesions. Macroscopically, the resected specimens showed a non-cirrhotic yellowish-cut surface containing brownish, ill-defined lesions with irregular borders. Microscopically, these lesions showed zonal necrosis, congestion, and aggregation of hemosiderin-laden macrophages around the central vein. In these areas, the fatty deposition of hepatocytes was lower than that in the surrounding background hepatocytes. Histopathologically, neither neoplastic nor hyperplastic lesions were observed, and he was diagnosed as regenerative hepatic change with centrilobular necrosis. CONCLUSIONS: Considering the pathological results, these lesions were thought to be a type of NRH-like lesion with possible hepatic vessel disorder. However, the lesion's cause and classification was difficult to determine. The accumulation of these regenerative changes accompanying fatty liver is needed to clarify the mechanism and its clinical significance.

Subclonal accumulation of immune escape mechanisms in microsatellite instability-high colorectal cancers.

BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.

Transducin Beta-Like 2 is a Potential Driver Gene that Adapts to Endoplasmic Reticulum Stress to Promote Tumor Growth of Lung Adenocarcinoma.

BACKGROUND: Endoplasmic reticulum (ER) stress has a close relation with cancer progression. Blocking the adaptive pathway of ER stress could be an anticancer strategy. Here, we identified an ER stress-related gene, Transducin beta-like 2 (TBL2), an ER-localized type I transmembrane protein, on increased chromosome 7q as a candidate driver gene of lung adenocarcinoma (LUAD). METHODS: The association between TBL2 mRNA expression and prognostic outcomes and clinicopathological factors was analyzed using The Cancer Genome Atlas (TCGA) datasets of LUAD and lung squamous cell carcinoma (LUSC). Localization of TBL2 in tumor tissues was observed by immunohistochemical staining. Gene set enrichment analysis (GSEA) was conducted using TCGA dataset. In vitro cell proliferation assays were performed using TBL2 knockdown LUAD cells, LUSC cells, and LUAD cells overexpressing TBL2. Apoptosis and ATF4 expression (ER stress marker) were evaluated by western blotting. RESULTS: TBL2 was overexpressed in LUAD and LUSC cells. Multivariate analysis indicated high TBL2 mRNA expression was an independent poor prognostic factor of LUAD. GSEA revealed high TBL2 expression was positively correlated to the ER stress response in LUAD. TBL2 knockdown attenuated LUAD cell proliferation under ER stress. TBL2 inhibited apoptosis in LUAD cells under ER stress. TBL2 knockdown reduced ATF4 expression under ER stress. CONCLUSIONS: TBL2 may be a novel driver gene that facilitates cell proliferation, possibly by upregulating ATF4 expression followed by adaptation to ER stress, and it is a poor prognostic biomarker of LUAD.

Trousseau's Syndrome with Advanced Neuroendocrine Carcinoma of Colon: A Case Report.

Here, we present a 69-year-old female with advanced neuroendocrine carcinoma (NEC) of colon with multiple liver, bone, and kidney metastases who developed Trousseau's syndrome. The patient received etoposide plus cisplatin (EP) as the first-line therapy; however, after single administration of EP, she developed the severe lower-limb edema and EP was considered to be intolerable. Etoposide plus carboplatin was administered as the second-line therapy and after 3 cycles of administration, the progressive disease (PD) was confirmed and 5-fluorouracil + leucovorin + irinotecan (FOLFIRI) plus ramucirumab was administered as the third-line therapy. However, PD was confirmed after 3 cycles of the therapy, and she was to receive the best supportive care and was hospitalized in our hospital. Four weeks after hospitalization, mild impaired consciousness and dysarthria were observed. Blood tests showed coagulation abnormalities including elevation of plasma fibrin/fibrinogen degradation products (FDPs) and D-dimer levels, and the diffusion-weighted image of magnetic resonance imaging (MRI) of the head showed multiple cerebral infarcts. She was diagnosed with Trousseau's syndrome due to the progression of NEC and intravenous unfractionated heparin was administered as anticoagulant therapy. After the administration of heparin, plasma FDP and D-dimer levels decreased; however, due to the progression of NEC, the patient died 6 weeks after hospitalization. This is the first report of NEC of the colon that developed Trousseau's syndrome.

Convergent genomic diversity and novel BCAA metabolism in intrahepatic cholangiocarcinoma.

BACKGROUND: Driver alterations may represent novel candidates for driver gene-guided therapy; however, intrahepatic cholangiocarcinoma (ICC) with multiple genomic aberrations makes them intractable. Therefore, the pathogenesis and metabolic changes of ICC need to be understood to develop new treatment strategies. We aimed to unravel the evolution of ICC and identify ICC-specific metabolic characteristics to investigate the metabolic pathway associated with ICC development using multiregional sampling to encompass the intra- and inter-tumoral heterogeneity. METHODS: We performed the genomic, transcriptomic, proteomic and metabolomic analysis of 39-77 ICC tumour samples and eleven normal samples. Further, we analysed their cell proliferation and viability. RESULTS: We demonstrated that intra-tumoral heterogeneity of ICCs with distinct driver genes per case exhibited neutral evolution, regardless of their tumour stage. Upregulation of BCAT1 and BCAT2 indicated the involvement of 'Val Leu Ile degradation pathway'. ICCs exhibit the accumulation of ubiquitous metabolites, such as branched-chain amino acids including valine, leucine, and isoleucine, to negatively affect cancer prognosis. We revealed that this metabolic pathway was almost ubiquitously altered in all cases with genomic diversity and might play important roles in tumour progression and overall survival. CONCLUSIONS: We propose a novel ICC onco-metabolic pathway that could enable the development of new therapeutic interventions.

Spatial and single-cell transcriptomics decipher the cellular environment containing HLA-G+ cancer cells and SPP1+ macrophages in colorectal cancer.

The cellular interactions in the tumor microenvironment of colorectal cancer (CRC) are poorly understood, hindering patient treatment. In the current study, we investigate whether events occurring at the invasion front are of particular importance for CRC treatment strategies. To this end, we analyze CRC tissues by combining spatial transcriptomics from patients with a public single-cell transcriptomic atlas to determine cell-cell interactions at the invasion front. We show that CRC cells are localized specifically at the invasion front. These cells induce human leukocyte antigen G (HLA-G) to produce secreted phosphoprotein 1 (SPP1)+ macrophages while conferring CRC cells with anti-tumor immunity, as well as proliferative and invasive properties. Taken together, these findings highlight the signaling between CRC cell populations and stromal cell populations at the cellular level.

Rab27b, a Regulator of Exosome Secretion, Is Associated With Peritoneal Metastases in Gastric Cancer.

BACKGROUND/AIM: Peritoneal metastasis (PM) of gastric cancer (GC) leads to poor clinical outcomes. Tumor-derived exosomes promote metastasis via communication between tumor cells and host cells. In this study, we investigated the effect of Rab27, which is required for exosome secretion, on the PM of GC. MATERIALS AND METHODS: We established a stable knockdown of two Rab27 homologs, Rab27a and Rab27b, in human GC cells (58As9) with a high potential of PM. We examined the level of exosome secretion from Rab27-knockdown 58As9 cells by Western blotting and the ability of Rab27b knockdown to suppress PM in 58As9 cells using a mouse xenograft model. In vitro proliferation and invasion assays were performed in the Rab27b-knockdown cells. Next, Rab27b expression was evaluated in human GC tissues by immunohistochemistry. Finally, we assessed the clinicopathological and prognostic significance of Rab27b expression by RT-qPCR in both our and other TCGA datasets of GC. RESULTS: Rab27a and Rab27b knockdown in 58As9 cells decreased the secretion of exosomes, characterized by the endocytic marker CD63. Rab27b knockdown decreased PM in vivo without affecting the in vitro proliferation or invasion ability of 58As9 cells. In human GC tissues, Rab27b was overexpressed in tumor cells. The overall and recurrence-free survival rates were significantly lower in GC patients with high compared to low Rab27b mRNA expression in our and other TCGA datasets. CONCLUSION: Rab27b expression potentially serves as a poor prognostic biomarker, possibly affecting PM via exosome secretion from GC cells.

Molecular and clinicopathological differences between depressed and protruded T2 colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) can be classified into four consensus molecular subtypes (CMS) according to genomic aberrations and gene expression profiles. CMS is expected to be useful in predicting prognosis and selecting chemotherapy regimens. However, there are still no reports on the relationship between the morphology and CMS. METHODS: This retrospective study included 55 subjects with T2 CRC undergoing surgical resection, of whom 30 had the depressed type and 25 the protruded type. In the classification of the CMS, we first defined cases with deficient mismatch repair as CMS1. And then, CMS2/3 and CMS4 were classified using an online classifier developed by Trinh et al. The staining intensity of CDX2, HTR2B, FRMD6, ZEB1, and KER and the percentage contents of CDX2, FRMD6, and KER are input into the classifier to obtain automatic output classifying the specimen as CMS2/3 or CMS4. RESULTS: According to the results yielded by the online classifier, of the 30 depressed-type cases, 15 (50%) were classified as CMS2/3 and 15 (50%) as CMS4. Of the 25 protruded-type cases, 3 (12%) were classified as CMS1 and 22 (88%) as CMS2/3. All of the T2 CRCs classified as CMS4 were depressed CRCs. More malignant pathological findings such as lymphatic invasion were associated with the depressed rather than protruded T2 CRC cases. CONCLUSIONS: Depressed-type T2 CRC had a significant association with CMS4, showing more malignant pathological findings such as lymphatic invasion than the protruded-type, which could explain the reported association between CMS4 CRC and poor prognosis.

GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein.

Colorectal cancer (CRC) is one of the most common types of cancer and a significant cause of cancer mortality worldwide. Further improvements of CRC therapeutic approaches are needed. BCL2-associated athanogene 6 (BAG6), a multifunctional scaffold protein, plays an important role in tumor progression. However, regulation of BAG6 in malignancies remains unclear. This study showed that guided entry of tail-anchored proteins factor 4 (GET4), a component of the BAG6 complex, regulates the intercellular localization of BAG6 in CRC. Furthermore, GET4 was identified as a candidate driver gene on the short arm of chromosome 7, which is often amplified in CRC, by our bioinformatics approach using the CRC dataset from The Cancer Genome Atlas. Clinicopathologic and prognostic analyses using CRC datasets showed that GET4 was overexpressed in tumor cells due to an increased DNA copy number. High GET4 expression was an independent poor prognostic factor in CRC, whereas BAG6 was mainly overexpressed in the cytoplasm of tumor cells without gene alteration. The biological significance of GET4 was examined using GET4 KO CRC cells generated with CRISPR-Cas9 technology or transfected CRC cells. In vitro and in vivo analyses showed that GET4 promoted tumor growth. It appears to facilitate cell cycle progression by cytoplasmic enrichment of BAG6-mediated p53 acetylation followed by reduced p21 expression. In conclusion, we showed that GET4 is a novel driver gene and a prognostic biomarker that promotes CRC progression by inducing the cytoplasmic transport of BAG6. GET4 could be a promising therapeutic molecular target in CRC.

Fanconi Anemia Complementation Group E, a DNA Repair-Related Gene, Is a Potential Marker of Poor Prognosis in Hepatocellular Carcinoma.

INTRODUCTION: Fanconi anemia complementation group E (FANCE) is a Fanconi anemia (FA) pathway gene that regulates DNA repair. We evaluated the clinical relevance of FANCE expression in hepatocellular carcinoma (HCC). METHODS: First, the associations between the expression of FA pathway genes including FANCE and clinical outcomes in HCC patients were analyzed in 2 independent cohorts: The Cancer Genome Atlas (TCGA, n = 373) and our patient cohort (n = 53). Localization of FANCE expression in HCC tissues was observed by immunohistochemical staining. Gene set enrichment analysis (GSEA) and gene network analysis (SiGN_BN) were conducted using the TCGA dataset. Next, an in vitro proliferation assay was performed using FANCE-knockdown HCC cell lines (HuH7 and HepG2). The association between mRNA expression of FANCE and that of DNA damage response genes in HCC was analyzed using TCGA and Cancer Cell Line Encyclopedia datasets. Finally, the association between FANCE mRNA expression and overall survival (OS) in various digestive carcinomas was analyzed using TCGA data. RESULTS: FANCE was highly expressed in HCC cells. Multivariate analysis indicated that high FANCE mRNA expression was an independent factor predicting poor OS. GSEA revealed a positive relationship between enhanced FANCE expression and E2F and MYC target gene expression in HCC tissues. FANCE knockdown attenuated the proliferation of HCC cells, as well as reduced cdc25A expression and elevated histone H3 pSer10 expression. SiGN_BN revealed that FANCE mRNA expression was positively correlated with DNA damage response genes (H2A histone family member X and checkpoint kinase 1) in HCC tissues. Significant effects of high FANCE expression on OS were observed in hepatobiliary pancreatic carcinomas, including HCC. CONCLUSIONS: FANCE may provide a potential therapeutic target and biomarker of poor prognosis in HCC, possibly by facilitating tumor proliferation, which is mediated partly by cell cycle signaling activation.

Oxysterol binding protein-like 3 (OSBPL3) is a novel driver gene that promotes tumor growth in part through R-Ras/Akt signaling in gastric cancer.

Gastric cancer (GC) is one of the most lethal malignant tumors. To improve the prognosis of GC, the identification of novel driver genes as therapeutic targets is in urgent need. Here, we aimed to identify novel driver genes and clarify their roles in gastric cancer. OSBPL3 was identified as a candidate driver gene by in silico analysis of public genomic datasets. OSBPL3 expression was analyzed by RT-qPCR and immunohistochemistry in GC cells and tissues. The biological functions and mechanisms of OSBPL3 in GC were examined in vitro and in vivo using GC cells. The association between OSBPL3 expression and clinical outcome in GC patients was also evaluated. Overexpression of OSBPL3 was detected in GC cells with OSBPL3 DNA copy number gains and promoter hypomethylation. OSBPL3-knockdown reduced GC cell growth in vitro and in vivo by inhibiting cell cycle progression. Moreover, an active Ras pull-down assay and western blotting demonstrated that OSBPL3 activates the R-Ras/Akt signaling pathway in GC cells. In a clinical analysis of two GC datasets, high OSBPL3 expression was predictive of a poor prognosis. Our findings suggest that OSBPL3 is a novel driver gene stimulating the R-Ras/Akt signaling pathway and a potential therapeutic target in GC patients.

Cancer-associated Fibroblast-derived Spondin-2 Promotes Motility of Gastric Cancer Cells.

BACKGROUND/AIM: Peritoneal dissemination (PD) occurs frequently in gastric cancer (GC) and is fatal. The interactions between tumor cells and stromal cells are critical for cancer progression. Our aim was to identify a novel PD-associated gene derived from stromal cells in GC. MATERIALS AND METHODS: Among the candidate PD-associated genes identified in our previous study, we focused on spondin-2 (SPON2), an extracellular matrix-secreted protein. Clinicopathological and prognostic analyses of SPON2 mRNA expression were performed using GC datasets. Localization of SPON2 expression was assessed by immunohistochemistry. In vitro migration assay and immunofluorescence staining were also conducted using GC cell lines. RESULTS: SPON2 was expressed in and secreted from cancer-associated fibroblasts in GC. High expression of SPON2 in tumor tissues was correlated with PD, tumor size and poor prognosis in GC. The motility of GC cells was increased by treatment with a SPON2 recombinant protein in vitro. CONCLUSION: Cancer-associated fibroblast-derived SPON2 may promote PD, in part, by facilitating GC cell motility and serve as a predictive marker for PD in GC.

Mitotic checkpoint regulator RAE1 promotes tumor growth in colorectal cancer.

Microtubules are among the most successful targets for anticancer therapy because they play important roles in cell proliferation as they constitute the mitotic spindle, which is critical for chromosome segregation during mitosis. Hence, identifying new therapeutic targets encoding proteins that regulate microtubule assembly and function specifically in cancer cells is critical. In the present study, we identified a candidate gene that promotes tumor progression, ribonucleic acid export 1 (RAE1), a mitotic checkpoint regulator, on chromosome 20q through a bioinformatics approach using datasets of colorectal cancer (CRC), including The Cancer Genome Atlas (TCGA). RAE1 was ubiquitously amplified and overexpressed in tumor cells. High expression of RAE1 in tumor tissues was positively associated with distant metastasis and was an independent poor prognostic factor in CRC. In vitro and in vivo analysis showed that RAE1 promoted tumor growth, inhibited apoptosis, and promoted cell cycle progression, possibly with a decreased proportion of multipolar spindle cells in CRC. Furthermore, RAE1 induced chemoresistance through its anti-apoptotic effect. In addition, overexpression of RAE1 and significant effects on survival were observed in various types of cancer, including CRC. In conclusion, we identified RAE1 as a novel gene that facilitates tumor growth in part by inhibiting apoptosis and promoting cell cycle progression through stabilizing spindle bipolarity and facilitating tumor growth. We suggest that it is a potential therapeutic target to overcome therapeutic resistance of CRC.

The novel driver gene ASAP2 is a potential druggable target in pancreatic cancer.

Targeting mutated oncogenes is an effective approach for treating cancer. The 4 main driver genes of pancreatic ductal adenocarcinoma (PDAC) are KRAS, TP53, CDKN2A, and SMAD4, collectively called the "big 4" of PDAC, however they remain challenging therapeutic targets. In this study, ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 (ASAP2), one of the ArfGAP family, was identified as a novel driver gene in PDAC. Clinical analysis with PDAC datasets showed that ASAP2 was overexpressed in PDAC cells based on increased DNA copy numbers, and high ASAP2 expression contributed to a poor prognosis in PDAC. The biological roles of ASAP2 were investigated using ASAP2-knockout PDAC cells generated with CRISPR-Cas9 technology or transfected PDAC cells. In vitro and in vivo analyses showed that ASAP2 promoted tumor growth by facilitating cell cycle progression through phosphorylation of epidermal growth factor receptor (EGFR). A repositioned drug targeting the ASAP2 pathway was identified using a bioinformatics approach. The gene perturbation correlation method showed that niclosamide, an antiparasitic drug, suppressed PDAC growth by inhibition of ASAP2 expression. These data show that ASAP2 is a novel druggable driver gene that activates the EGFR signaling pathway. Furthermore, niclosamide was identified as a repositioned therapeutic agent for PDAC possibly targeting ASAP2.

Potential association of LOXL1 with peritoneal dissemination in gastric cancer possibly via promotion of EMT.

BACKGROUND: Peritoneal dissemination (PD) frequently occurs in gastric cancer (GC) and is incurable. In this study, we aimed to identify novel PD-associated genes and clarify their clinical and biological significance in GC. MATERIALS AND METHODS: We identified LOXL1 as a PD-associated candidate gene by in silico analysis of GC datasets (highly disseminated peritoneal GC cell line and two freely available GC datasets, GSE15459 and TCGA). Next, we evaluated the clinical significance of LOXL1 expression using RT-qPCR and immunohistochemistry staining (IHC) in a validation cohort (Kyushu cohort). Moreover, we performed gene expression analysis, including gene set enrichment analysis (GSEA) with GSE15459 and TCGA datasets. Finally, we performed a series of in vitro experiments using GC cells. RESULTS: In silico analysis showed that LOXL1 was overexpressed in tumor tissues of GC patients with PD and in highly disseminated peritoneal GC cells, relative to that in the control GC patients and cells, respectively. High expression of LOXL1 was a poor prognostic factor in the TCGA dataset. Next, IHC showed that LOXL1 was highly expressed in GC cells. High LOXL1 mRNA expression was associated with poorly differentiated histological type, lymph node metastasis, and was an independent poor prognostic factor in the Kyushu validation cohort. Moreover, LOXL1 expression was positively correlated with the EMT (epithelial-mesenchymal transition) gene set in GSEA. Finally, LOXL1-overexpressing GC cells changed their morphology to a spindle-like form. LOXL1 overexpression reduced CDH1 expression; increased the expression of VIM, CDH2, SNAI2, and PLS3; and promoted the migration capacity of GC cells. CONCLUSIONS: LOXL1 is associated with PD in GC, possibly through the induction of EMT.

Prognostic Significance of PD-1, PD-L1 and CD8 Gene Expression Levels in Gastric Cancer.

INTRODUCTION: Anti-programmed cell death 1 (PD-1) therapies have shown promising clinical activity against gastric cancer (GC). We evaluated the clinical significance of immune-related gene expression in GC tissues to better understand the tumor immune microenvironment. METHODS: PD-1, PD-1 ligand 1 (PD-L1) and CD8 mRNA levels and clinicopathological factors, including survival, were examined by quantitative RT-PCR in 155 GC patients who underwent surgery. PD-1 and PD-L1 expression in tumor tissue from 24 GC patients was investigated by immunohistochemical analysis. RESULTS: PD-1, PD-L1 and CD8 mRNA levels were significantly lower in tumor tissue than in normal tissue (p < 0.0001, p < 0.05, and p < 0.0001). GC patients with low PD-1, PD-L1 and CD8 mRNA levels had significantly poorer overall survival (OS) than those with high PD-1, PD-L1 and CD8 mRNA levels, respectively (p < 0.001, p < 0.01 and p < 0.05). Low PD-1, PD-L1 and CD8 mRNA levels were more significantly associated with poor prognosis in undifferentiated-type GC patients than in differentiated-type GC patients (PD-1: differentiated p = 0.0071 vs. undifferentiated p = 0.0024; PD-L1: p = 0.6527 vs. p < 0.0001; CD8: p = 0.4465 vs. p < 0.05). Multivariate analysis showed that lymph node metastasis, peritoneal dissemination, distant metastasis, low PD-1 mRNA levels and low CD8 mRNA levels were independent prognostic factors for worse OS (low PD-1 mRNA level: OR 2.16, 95% CI 1.10-4.58, p < 0.05; low CD8 mRNA level: OR 2.55, 95% CI 1.12-6.90, p < 0.05). PD-1 and PD-L1 mRNA levels in immune cells were significantly associated with PD-1 and PD-L1 protein levels (both p < 0.05), respectively. CONCLUSIONS: PD-1, PD-L1 and CD8 mRNA levels may reflect antitumor immunity in GC, and low PD-1 and CD8 mRNA levels are potential predictive biomarkers for poor prognosis in GC patients who underwent surgery.