MCM6 promotes intrahepatic cholangiocarcinoma progression by upregulating E2F1 and enhancing epithelial-mesenchymal transition.

Minichromosome maintenance complex component 6 (MCM6), a member of the MCM family, plays a pivotal role in DNA replication initiation and genome duplication of proliferating cells. MCM6 is upregulated in multiple malignancies and is considered a novel diagnostic biomarker. However, the functional contributions and prognostic value of MCM6 in intrahepatic cholangiocarcinoma (ICC) remain unexplored. In this study, we investigated the molecular function of MCM6 in ICC. Data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO, GSE107943) indicated an upregulation of MCM6 in tumor tissues. Immunohistochemical analysis performed on 115 cases of ICC samples confirmed the upregulation of MCM6 and further suggested that a high level of MCM6 expression predicted shorter overall and disease-free survival in ICC patients. Functional studies suggested that MCM6 knockdown significantly suppressed cell viability, blocked cell cycle progression and inhibited metastasis, while the enhancement of MCM6 expression promoted the proliferation and migration of ICC cells both in vitro and in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) suggested that the epithelial-mesenchymal transition (EMT) and E2F1-correlated genes were enriched in ICC tissues with high MCM6 expression. Further verification indicated that MCM6 promoted the EMT of ICC cells via upregulating E2F1. In addition, E2F1 knockdown partially blocked the pro-malignant effects of MCM6 overexpression. In summary, MCM6 was found to be a novel prognostic and predictive marker for ICC. MCM6 promoted ICC progression via activation of E2F1-mediated EMT.

NUSAP1, a novel stemness-related protein, promotes early recurrence of hepatocellular carcinoma.

Early recurrence (within 2 years after resection) is the primary cause of poor outcomes among hepatocellular carcinoma (HCC) patients, and liver cancer stem cells are the main contributors to postsurgical HCC recurrence. Nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in tumor progression. We investigated the function and clinical value of NUSAP1 in early recurrence of HCC. Data from public datasets and our cohort were used to assess the association between NUSAP1 expression and early HCC recurrence. Gain- and loss-of-function experiments were carried out in vivo and in vitro. The predictive effect of NUSAP1 on early HCC recurrence was further evaluated by a validation cohort. We found that elevated NUSAP1 expression in HCC specimens was correlated with poor outcome, especially in cases with postoperative early recurrence. Functional studies indicated that NUSAP1 significantly promotes HCC progression. A postsurgical recurrence murine model further revealed that upregulated NUSAP1 dramatically increased the likelihood of HCC early recurrence. RNA sequencing data revealed that the gene sets of cancer stemness and the signal transducer and activator of transcription 3 (STAT3) pathway were enriched by NUSAP1 overexpression. Mechanistically, NUSAP1 enhanced cancer stemness through stimulating STAT3 nuclear translocation and activation through receptor of activated protein C kinase 1 (RACK1). In a validation cohort with 112 HCC patients, NUSAP1 effectively predicted HCC early recurrence. Our results indicated that NUSAP1 promotes early recurrence of HCC by sustaining cancer stemness and could serve as a valuable predictive indicator for postsurgical intervention in HCC patients.

Enhanced Expression of ARK5 in Hepatic Stellate Cell and Hepatocyte Synergistically Promote Liver Fibrosis.

AMPK-related protein kinase 5 (ARK5) is involved in a broad spectrum of physiological and cell events, and aberrant expression of ARK5 has been observed in a wide variety of solid tumors, including liver cancer. However, the role of ARK5 in liver fibrosis remains largely unexplored. We found that ARK5 expression was elevated in mouse fibrotic livers, and showed a positive correlation with the progression of liver fibrosis. ARK5 was highly expressed not only in activated hepatic stellate cells (HSCs), but also in hepatocytes. In HSCs, ARK5 prevents the degradation of transforming growth factor ß type I receptor (TßRI) and mothers against decapentaplegic homolog 4 (Smad4) proteins by inhibiting the expression of Smad ubiquitin regulatory factor 2 (Smurf2), thus maintaining the continuous transduction of the transforming growth factor ß (TGF-ß) signaling pathway, which is essential for cell activation, proliferation and survival. In hepatocytes, ARK5 induces the occurrence of epithelial-mesenchymal transition (EMT), and also promotes the secretion of inflammatory factors. Inflammatory factors, in turn, further enhance the activation of HSCs and deepen the degree of liver fibrosis. Notably, we demonstrated in a mouse model that targeting ARK5 with the selective inhibitor HTH-01-015 attenuates CCl4-induced liver fibrosis in mice. Taken together, the results indicate that ARK5 is a critical driver of liver fibrosis, and promotes liver fibrosis by synergy between HSCs and hepatocytes.

Percutaneous Gastrostomy Compared with Esophageal Stent Placement for the Treatment of Esophageal Cancer with Dysphagia.

PURPOSE: To compare the outcomes of self-expandable metal stent placement and percutaneous gastrostomy (PG) for the treatment of patients with esophageal cancer (EC) and dysphagia. MATERIALS AND METHODS: This retrospective observational study consisted of 113 patients with EC and dysphagia who underwent either stent placement (n = 47) or PG (n = 66) at a single center between June 2014 and June 2018. RESULTS: There were 63 men and 50 women, with a mean age of 76.5 years (standard deviation 4.9 years). The 2 groups had similar baseline characteristics, except that the PG group had a higher percentage of patients with cervical EC (22.7% vs 2.1%, P < .001). The PG group had better maintenance of nutritional status in terms of reduction in serum albumin level (P = .039) and weight loss (P = .041). Compared with the stent group, the PG group demonstrated a lower incidence of local severe pain (0% vs 21.3%, P < .001) and lower incidence of dislodgment of device (1.5% vs 19.1%, P = .002). The PG group demonstrated longer overall survival compared with the stent group for Stages II and III (201 vs 185 days, P = .034) and Stage IV (122 vs 86 days, P = .001). CONCLUSIONS: Compared with stent insertion, PG is associated with better maintenance of nutritional status, fewer complications, and better survival. Thus, PG may be the preferred choice for treating malnutrition in patients with EC and dysphagia.

Aurora B facilitates cholangiocarcinoma progression by stabilizing c-Myc.

BACKGROUND: Cholangiocarcinoma (CCA), a malignancy that arises from biliary epithelial cells, has a dismal prognosis, and few targeted therapies are available. Aurora B, a key mitotic regulator, has been reported to be involved in the progression of various tumors, yet its role in CCA is still unclarified. METHODS: Human CCA tissues and murine spontaneous CCA models were used to assess Aurora B expression in CCA. A loss-of-function model was constructed in CCA cells to determine the role of Aurora B in CCA progression. Subcutaneous and liver orthotopic xenograft models were used to assess the therapeutic potential of Aurora B inhibitors in CCA. RESULTS: In murine spontaneous CCA models, Aurora B was significantly upregulated. Elevated Aurora B expression was also observed in 62.3% of human specimens in our validation cohort (143 CCA specimens), and high Aurora B expression was positively correlated with pathological parameters of tumors and poor survival. Knockdown of Aurora B by siRNA and heteroduplex oligonucleotide (HDO) or an Aurora B kinase inhibitor (AZD1152) significantly suppressed CCA progression via G2/M arrest induction. An interaction between Aurora B and c-Myc was found in CCA cells. Targeting Aurora B significantly reduced this interaction and accelerated the proteasomal degradation of c-Myc, suggesting that Aurora B promoted the malignant properties of CCA by stabilizing c-Myc. Furthermore, sequential application of AZD1152 or Aurora B HDO drastically improved the efficacy of gemcitabine in CCA. CONCLUSIONS: Aurora B plays an essential role in CCA progression by modulating c-Myc stability and represents a new target for treatment and chemosensitization in CCA.

A DNA/RNA heteroduplex oligonucleotide coupling asparagine depletion restricts FGFR2 fusion-driven intrahepatic cholangiocarcinoma.

Pemigatinib, a pan-FGFR inhibitor, is approved to treat intrahepatic cholangiocarcinoma (ICC) harboring FGFR2 fusion mutations. Improving its targeting of FGFR2 fusions remains an unmet clinical need due to its pan selectivity and resistance. Here, we report a cholesterol-conjugated DNA/RNA heteroduplex oligonucleotide targeting the chimeric site in FGFR2-AHCYL1 (F-A Cho-HDO) that accumulates in ICC through endocytosis of low-density lipoprotein receptor (LDLR), which is highly expressed in both human and murine ICC. F-A Cho-HDO was determined to be a highly specific, sustainable, and well-tolerated agent for inhibiting ICC progression through posttranscriptional suppression of F-A in ICC patient-derived xenograft mouse models. Moreover, we identified an EGFR-orchestrated bypass signaling axis that partially offset the efficacy of F-A Cho-HDO. Mechanistically, EGFR-induced STAT1 upregulation promoted asparagine (Asn) synthesis through direct transcriptional upregulation of asparagine synthetase (ASNS) and dictated cell survival by preventing p53-dependent cell cycle arrest. Asn restriction with ASNase or ASNS inhibitors reduced the intracellular Asn, thereby reactivating p53 and sensitizing ICC to F-A Cho-HDO. Our findings highlight the application of genetic engineering therapies in ICC harboring FGFR2 fusions and reveal an axis of adaptation to FGFR2 inhibition that presents a rationale for the clinical evaluation of a strategy combining FGFR2 inhibitors with Asn depletion.

Serine/threonine kinase TBK1 promotes cholangiocarcinoma progression via direct regulation of ß-catenin.

Cholangiocarcinoma (CCA) is a highly heterogeneous and metastatic malignancy with a poor prognosis even after curative hepatectomy. Studies exploring its pathogenesis and identifying effective therapeutic targets are urgently needed. In this study, we found that TANK-binding kinase 1 (TBK1), a serine/threonine-protein kinase, showed a dynamic increase during the different stages of murine spontaneous CCA carcinogenesis (hyperplasia, dysplasia, and CCA). TBK1 was upregulated in human tissues, including intrahepatic (n = 182) and extrahepatic (n = 40) CCA tissues, compared with nontumor tissues, and the elevated expression of TBK1 was positively correlated with larger tumour diameter, lymph node metastasis, and advanced TNM stage. Functional studies indicated that TBK1 promoted CCA growth and metastasis both in vitro and in vivo. TBK1 directly interacts with ß-catenin, promoting its phosphorylation at the S552 site and its nuclear translocation, which further activates EMT-related transcriptional reprogramming. GSK-8612, a TBK1 inhibitor or a kinase-inactivating mutation, effectively suppresses the above processes. In addition, we found that low-density lipoprotein receptor (LDLR), which mediates the endocytosis of cholesterol, was upregulated in CCA. Therefore, we designed a cholesterol-conjugated DNA/RNA heteroduplex oligonucleotide targeting TBK1 (Cho-TBK1-HDO), which could accumulate in CCA cells via LDLR, reduce the TBK1 mRNA level and inhibit intrahepatic metastasis of CCA. Besides, in the experimental group of 182 ICC patients, high TBK1 expression combined with high nuclear ß-catenin expression predicted a worse prognosis. In summary, TBK1 might serve as a potential prognostic biomarker and therapeutic target for patients with CCA.

Downregulation of Circular RNA circPSD3 Promotes Metastasis by Modulating FBXW7 Expression in Clear Cell Renal Cell Carcinoma.

Circular RNAs (circRNAs), a novel class of noncoding RNAs, have been shown to play critical regulatory roles in clear cell renal cell carcinoma (ccRCC). Metastasis is the main contributor to the poor prognosis of patients with ccRCC. However, the role of circRNAs in ccRCC metastasis has not been fully elucidated. In this study, microarray and RNA-seq analyses revealed that circPSD3 (hsa_circ_0002111) was dramatically downregulated in ccRCC tissues compared to adjacent nontumor tissues. A qRT-PCR analysis performed on our ccRCC cohorts confirmed the downregulation of circPSD3 in ccRCC tissues and further suggested that a low level of circPSD3 expression was associated with tumor metastasis in patients with ccRCC. Based on the results of functional studies, circPSD3 significantly inhibited cell migration, invasion, and the epithelial-mesenchymal transition (EMT) in vitro and blocked pulmonary metastasis in vivo. Mechanistically, circPSD3 functioned as a competing endogenous RNA for microRNA 25-3p (miR-25-3p) to regulate F-box and WD repeat domain-containing 7 (FBXW7) expression. Further verification indicated that circPSD3 overexpression restrained an EMT-like phenotype in cells, while miR-25-3p partially rescued these effects. In summary, circPSD3 inhibits tumor metastasis by repressing the miR-25-3p/FBXW7-EMT axis and might be developed as a potential diagnostic and therapeutic target for ccRCC.

Periplaneta americana extract alleviates steatohepatitis in a mouse model by modulating HMGB1-mediated inflammatory response.

Alcoholic abuse and obesity are the most common lifestyle implications of chronic liver injury, and always act synergistically to increase the risk of mortality. Periplaneta americana has a long history of being applied in medicine, including wound healing, antitumor, antibacterial, antiviral, antifibrotic, and cardiomyocyte-protecting. Ganlong capsule (GLC), a natural prescription drug extracted from Periplaneta americana, has been widely used in HBV-related symptoms. However, the anti-steatohepatitis efficacy and mechanisms of GLC have not yet been characterized. Here, we found the protective effect of GLC on the development of hepatic steatosis, oxidative stress, and inflammation in vivo under alcohol exposure combined with a high-fat and high-cholesterol diet (HFHC). Consistently, GLC exhibited a hepatoprotective property by preventing hepatocytes from oxidative stress injury and lipid accumulation in vitro. In addition, it exerted an anti-inflammation characteristic by reducing macrophage recruitment and decreasing the expression of pro-inflammatory genes in vivo and in vitro. Mechanically, GLC serum, isolated from GLC-treated mice, reduced extracellular high-mobility group box 1 (HMGB1) of dying hepatocytes; and suppressed subsequent M1 polarization of macrophages in the co-culture system. Furthermore, GLC serum inhibited inflammatory response via suppressing the HMGB1 release and blocking the downstream TLR4/NF-kB pathway. Collectively, GLC alleviates steatohepatitis induced by alcohol consumption and obesity through inhibition of the HMGB1-mediated inflammatory cascade. GLC might be a therapeutic candidate for the treatment of steatohepatitis developed by alcohol abuse and metabolic disorders.

Surgical outcomes and survival for T4 gastric cancer extending to the transverse colon.

BACKGROUND: For the treatment of locally advanced (T4) gastric cancer, extended multi-organ resection remains controversial. This study aimed to evaluate the surgical outcomes and survival of patients with T4 gastric cancer extending to the transverse colon. METHODS: A total of 2,652 gastric cancer patients underwent surgery between December 2011 and December 2015. Data from 40 of these patients who underwent curative resection for T4 gastric cancer extending to the transverse colon were obtained. Patient characteristics, related complications, long-term survival, and prognostic factors for T4 gastric cancer were analyzed. RESULTS: Postoperative morbidity occurred in 5 (12.5%) patients. All of the patients were cured with conservative treatment. No procedure-related mortality occurred. The 1-, 3-, and 5-year overall survival (OS) rates were 75.0%, 49.2%, and 36.9%, respectively, with a median survival time of 24 months. Univariate analysis revealed tumor size (P=0.049), advanced T stage (P=0.013), and lymph node metastasis (P=0.006) to be poor prognostic factors of OS. Advanced T stage and lymph node metastasis were identified by multivariate analysis as being independent prognostic factors. Further, it was observed that lymph node metastasis grade was associated with poorer OS. CONCLUSIONS: Patients with T4 gastric cancer extending to the transverse colon might benefit from curative resection with acceptable morbidity and mortality.

[A scoring system to predict the risk of anastomotic leakage in patients with patients with rectal cancer older than 60 years].

OBJECTIVE: To establish a scoring system to predict the risk of anastomotic leakage in patients with rectal cancer older than 60 years. METHODS: The study included 995 patients (≥60 years) with rectal cancer locating 3-12 cm from the anal verge who underwent anterior resection or intersphincteric resection at the Department of General Surgery, Henan Cancer Hospital from January 2012 to December 2016. Potential risk factors for leakage were subjected to univariate analysis. Multivariate logistic regression analysis was used to identify the independent risk factors for anastomotic leakage. The scoring system was developed based on regression coefficient for each significant risk factor. One point was allocated to the risk factor with a regression coefficient ß<1, and two points were allocated to the risk factor with ß>1. The proposed scoring system was tested by the area under curve (AUC) of the receiver operating characteristic curve (ROC). RESULTS: Surgery was successfully performed in all 995 patients. The incidence of anastomotic fistula was 4.6%(46/995). Among these 46 patients, 31 recovered after conventional treatment, and 13 patients underwent transverse colostomy,and 2 died of multiple organ failure. Independent risk factors included age (ß=0.643, OR=1.902, 95%CI: 1.020-3.614, P=0.048), body mass index(BMI) (ß=1.218, OR=3.379, 95%CI: 1.607-7.105, P=0.001), albumin levels (ß=0.986, OR=2.681, 95%CI: 1.432-5.021, P=0.002), and level of anastomosis from the anal verge (ß=1.395, OR=4.034, 95%CI: 2.086-7.801, P=0.000). The scoring system was created base on coefficient ß of the independent risk factors (age≥70 years for 1, BMI≥25 kg/m2 for 2, albumin levels <35 g/L for 1, level of anastomosis from anal verge <4.0 cm for 2). All the scores were added up, and all patients were divided into the high-risk group(4-6 points, n=71) and intermediate-low-risk group(0-3 points, n=924) based on the scoring system. The incidence of anastomotic leakage in the two groups was 23.9%(17/71) and 3.1%(29/924), respectively (χ2=60.092, P=0.000). The AUC of age, BMI, albumin levels, and level of anastomosis from the anal verge were 0.598, 0.591, 0.622, and 0.635 respectively. The AUC of the scoring system was 0.656, which was higher than above parameters with a sensitivety of 0.37 and specificity of 0.94. CONCLUSIONS: The scoring system is effective and accurate for identifying a subgroup at high risk for postoperative anastomotic leakage in rectal cancer patients over 60 years old.