The Causes Behind the Survival Paradox in Non-Metastatic Colorectal Cancer Patients and its Impact on the Treatment Regimen: A Retrospective Cohort Study.

BACKGROUND: The survival paradox (ie, the prognosis of the population at earlier tumor stages is worse than that of the population at later stages) has been observed in colorectal cancer based on the American Joint Committee on Cancer Tumor-Nodes-Metastases staging system. We aimed to clarify the reason for the survival paradox and its impact on patient treatment. METHODS: We conducted a retrospective study analyzing eligible patients with colorectal cancer from the Surveillance, Epidemiology, and End Results database and Zhejiang Cancer Hospital between 2010 and 2019. Adjusting for confounders using propensity score matching allowed confirmation of the effect of staging on the survival paradox. RESULTS: Based on the Surveillance, Epidemiology, and End Results database, the subgroups with survival paradox might be IIB/C versus IIIA, IIA versus IIIA, and T4N0 (IIB/C) versus T3N1 (IIIB). After propensity score matching, stage IIB/C still had a worse prognosis than stage IIIA (5-year overall survival: 69.3% vs 78.5%, P < .001). Interestingly, the proportion of stage IIIA people receiving chemotherapy was higher than that of stage IIB/C (P < .001), and logistic regression models showed that staging was the reason for deciding whether a patient receives chemotherapy or not. These phenomena between stage IIB/C and IIIA were verified in the local database. CONCLUSION: These results suggested that the survival paradox was mainly due to underestimation of stage T4 weights or overestimation of stage N1 weights, and the low proportion of chemotherapy in patients with T4N0M0 colorectal cancer (proven to be more malignant than stage IIIA) might be related to the assignment to earlier stages, resulting in a lack of attention and poor compliance to chemotherapy in these patients.

Tanshinone IIA promotes vascular normalization and boosts Sorafenib's anti-hepatoma activity via modulating the PI3K-AKT pathway.

Introduction: Angiogenesis is an essential feature of liver cancer. Tumor hypoxia results from abnormal vessel architecture. Numerous studies have sufficiently demonstrated that Tanshinone IIA (Tan IIA) can increase blood flow and enhance microcirculation. The objectives of this study are to: 1 assess the impact of Tan IIA on tumor angiogenesis and architecture, 2 determine the impact of Tan IIA on tumor hypoxia and susceptibility to Sorafenib, and 3 clarify the relevant mechanisms. Methods: CCK8 and flow cytometry measured cell proliferation and apoptosis, respectively. Tube creation assay was used to investigate medication effects on angiogenesis and structure. Drug effects on tumor development, metastasis, and hypoxic tumor microenvironment are assessed in an orthotopic xenograft model of liver tumors. Protein expression was measured by Western blotting and immunohistochemistry. Results: Our results demonstrated that Tan IIA could not reduce tumor proliferation or enhance Sorafenib's anti-tumor effect in vitro. Nevertheless, it can prevent Sorafenib from demolishing the typical vascular structure and aid sorafenib in blocking the recruitment of vascular endothelial cells by liver cancer cells. Although Tan IIA cannot inhibit tumor growth in vivo, it can significantly boost Sorafenib's inhibitory effect on liver cancer, alleviate tumor microenvironment hypoxia, and minimize lung metastasis. This effect may be achieved by reducing HIF-1α and HIF-2α expression via the PI3K-AKT signal pathway. Discussion: Our results reveal the mechanism of Tan IIA in normalizing tumor blood vessels, provide innovative concepts and approaches to overcome chemotherapy resistance, and provide a theoretical basis for the clinical transformation and usage of Tan IIA.

PIK3C2A is a prognostic biomarker that is linked to immune infiltrates in kidney renal clear cell carcinoma.

Background: Phosphoinositide 3-kinases (PI3Ks) are lipid enzymes that regulate a wide range of intracellular functions. In contrast to Class I and Class III PI3K, which have more detailed descriptions, Class II PI3K has only recently become the focus of functional research. PIK3C2A is a classical member of the PI3Ks class II. However, the role of PIK3C2A in cancer prognosis and progression remains unknown. Methods: The expression pattern and prognostic significance of PIK3C2A in human malignancies were investigated using multiple datasets and scRNA-seq data. The PIK3C2A expression in renal clear cell carcinoma (KIRC) was then validated utilizing Western blot. The functional role of PIK3C2A in KIRC was assessed using combined function loss experiments with in vitro experiments. Furthermore, the correlation of PIK3C2A expression with tumor immunity was investigated in KIRC. The TCGA database was employed to investigate PIK3C2A functional networks. Results: Significant decrease in PIK3C2A expression in KIRC, demonstrated that it potentially influences the prognosis of diverse tumors, particularly KIRC. In addition, PIK3C2A was significantly correlated with the T stage, M stage, pathologic stage, and histologic grade of KIRC. Nomogram models were constructed and used to predict patient survival based on the results of multivariate Cox regression analysis. PIK3C2A knockdown resulted in significantly increased KIRC cell proliferation. Of note, PIK3C2A expression demonstrated a significant correlation with the infiltrating levels of primary immune cells in KIRC. Conclusion: These findings support the hypothesis that PIK3C2A is a novel biomarker for tumor progression and indicates dynamic shifts in immune infiltration in KIRC. Furthermore, aberrant PIK3C2A expression can influence the biological activity of cancer cells.

Decoding the Conformational Selective Mechanism of FGFR Isoforms: A Comparative Molecular Dynamics Simulation.

Fibroblast growth factor receptors (FGFRs) play critical roles in the regulation of cell growth, differentiation, and proliferation. Specifically, FGFR2 gene amplification has been implicated in gastric and breast cancer. Pan-FGFR inhibitors often cause large toxic side effects, and the highly conserved ATP-binding pocket in the FGFR1/2/3 isoforms poses an immense challenge in designing selective FGFR2 inhibitors. Recently, an indazole-based inhibitor has been discovered that can selectively target FGFR2. However, the detailed mechanism involved in selective inhibition remains to be clarified. To this end, we performed extensive molecular dynamics simulations of the apo and inhibitor-bound systems along with multiple analyses, including Markov state models, principal component analysis, a cross-correlation matrix, binding free energy calculation, and community network analysis. Our results indicated that inhibitor binding induced the phosphate-binding loop (P-loop) of FGFR2 to switch from the open to the closed conformation. This effect enhanced extensive hydrophobic FGFR2-inhibitor contacts, contributing to inhibitor selectivity. Moreover, the key conformational intermediate states, dynamics, and driving forces of this transformation were uncovered. Overall, these findings not only provided a structural basis for understanding the closed P-loop conformation for therapeutic potential but also shed light on the design of selective inhibitors for treating specific types of cancer.

Upregulation of HMGB1 in tumor-associated macrophages induced by tumor cell-derived lactate further promotes colorectal cancer progression.

BACKGROUND: Lactate accumulation leads to an acidic tumor microenvironment (TME), in turn promoting colorectal cancer (CRC) progression. Tumor-associated macrophages (TAMs) are the predominant cells in TME. This study aimed to reveal the regulation mechanism of CRC cell-derived lactate on TAMs and explore the mechanism underlying lactate accumulation-induced aggravation in CRC. METHODS: Cell growth and metastasis were evaluated by colony formation, Transwell, and wound healing assays. Western blot and RT-qPCR were applied to determine the protein and mRNA expression. Flow cytometry was used to analyze the polarization state and apoptotic rate of macrophages induced in THP-1 cells. The lactate in the cell supernatant was quantified using an ELISA kit. Immunofluorescence was performed to visualize the location of High Mobility Group Box 1 (HMGB1). H&E and Ki67 staining assays were used to assess tumorigenesis in nude mice bearing ectopic tumors. RESULTS: Cell growth and metastasis were promoted in the hypoxic CRC cells. The hypoxic cell supernatant stimulated the M2-type polarization of macrophages. The lactate level increased in hypoxic cancer cells. However, the inhibition of lactate using 3-hydroxy-butyrate (3-OBA) reversed the effects of hypoxia. Also, macrophages showed no promoting effect on cancer cell growth and migration in the presence of 3-OBA. HMGB1 was secreted into the extracellular space of lactate-induced macrophages, further enhancing the malignant behaviors of cancer cells. ERK, EMT, and Wnt signaling pathways were activated in cancer cells due to HMGB1 upregulation. CONCLUSIONS: The lactate metabolized by cancer cells stimulated M2 polarization and HMGB1 secretion by macrophages, aggravating the carcinogenic behaviors of cancer cells.

Computational Dissection of the Role of Trp305 in the Regulation of the Death-Associated Protein Kinase-Calmodulin Interaction.

Death-associated protein kinase 1 (DAPK1), as a calcium/calmodulin (CaM) regulated serine/threonine kinase, functions in apoptotic and autophagy pathways and represents an interesting drug target for inflammatory bowel disease and Alzheimer's disease. The crystal structure of the DAPK1 catalytic domain and the autoregulatory domain (ARD) in complex with CaM provides an understanding of CaM-dependent regulation of DAPK1 activity. However, the molecular basis of how distinct Trp305 (W305Y and W305D) mutations in the ARD modulate different DAPK1 activities remains unknown. Here, we performed multiple, µs-length molecular dynamics (MD) simulations of the DAPK1-CaM complex in three different (wild-type, W305Y, and W305D) states. MD simulations showed that the overall structural complex did not change significantly in the wild-type and W305Y systems, but underwent obvious conformational alteration in the W305D system. Dynamical cross-correlation and principal component analyses revealed that the W305D mutation enhanced the anti-correlated motions between the DAPK1 and CaM and sampled a broader distribution of conformational space relative to the wild-type and W305Y systems. Structural and energetical analyses further exhibited that CaM binding was unfavored in response to the W305D mutation, resulting in the decreased binding of CaM to the W305D mutant. Furthermore, the hydrogen bonds and salt bridges responsible for the loss of CaM binding on the interface of the DAPK1-CaM complex were identified in the W305D mutant. This result may provide insights into the key role of Trp305 in the regulation of CaM-mediated DAPK1 activity.

Patterns of Lymph Node Metastasis and Optimal Surgical Strategy in Small (≤20 mm) Gastroenteropancreatic Neuroendocrine Tumors.

Background: Regional lymph node metastasis (LNM) is crucial for planning additional lymphadenectomy, and is directly correlated with poor prognosis in gastroenteropancreatic neuroendocrine tumors (GEP-NETs). However, the patterns of LNM for small (≤20 mm) GEP-NETs remain unclear. This population-based study aimed at evaluating LNM patterns and identifying optimal surgical strategies from the standpoint of lymph node dissemination. Methods: This retrospective cohort study retrieved data from the Surveillance, Epidemiology, and End Results (SEER) 18 registries database for 17,308 patients diagnosed as having localized well-differentiated GEP-NETs ≤ 20 mm between January 1, 2004, and December 31, 2017. The patterns of LNM were characterized in 6,622 patients who underwent extended resection for adequate lymph node harvest. Results: Of 6,622 patients with localized small GEP-NETs in the current study, 2,380 (36%) presented with LNM after regional lymphadenectomy. Nodal involvement was observed in approximately 7.4%, 49.1%, 13.6%, 53.7%, 13.8%, 7.8%, and 15.4% of gastric (g-), small intestinal (si-), appendiceal (a-), colonic (c-), rectal (r-), non-functional pancreatic (nfp-), and functional pancreatic (fp-) NETs ≤ 20 mm. Patients with younger age, larger tumor size, and muscularis invasion were more likely to present with LNM. Additional lymphadenectomy conferred a significant survival advantage in NETs (≤10 mm: HR, 0.47; 95% CI, 0.33-0.66; p < 0.001; 11-20 mm: HR, 0.54; 95% CI, 0.34-0.85; p = 0.008) and fp-NETs ≤ 20 mm (HR, 0.08; 95% CI, 0.02-0.36; p = 0.001), as well as g-NETs (HR, 0.39; 95% CI, 0.16-0.96; p = 0.041) and c-NETs of 11-20 mm (HR, 0.07; 95% CI, 0.01-0.48; p = 0.007). Survival benefits of additional lymphadenectomy were not found in a-NETs, r-NETs, and nfp-NETs with a small size. Conclusions: Given the increased risk for nodal metastasis, primary tumor resection with regional lymphadenectomy is a potential optimal surgical strategy for si-NETs and fp-NETs ≤ 20 mm, as well as g-NETs and c-NETs of 11-20 mm. Local resection is an appropriate and reliable surgical approach for a-NETs, r-NETs, and nfp-NETs ≤ 20 mm.

Short-term and long-term outcomes of natural orifice specimen extraction surgeries (NOSES) in rectal cancer: a comparison study of NOSES and non-NOSES.

Background: Natural orifice specimen extraction surgeries (NOSES) have been applied to colorectal cancer (CRC). Different types of NOSES have been proposed. Traditional laparoscopic CRC surgeries (non-NOSES) have been widely adopted in clinical practice. Therefore, the safety and feasibility of NOSES could be clarified by comparing with non-NOSES. Methods: Consecutive cases who underwent NOSE or non-NOSE rectal surgeries were retrospectively collected at the Second Affiliated Hospital of Harbin Medical University between 1 January 2013 and 31 December 2018. Other inclusion criteria included patients with adenocarcinoma of the rectum within 15 cm of the anal verge, over the age of 18 and undergoing primary laparoscopic rectal resection. Patients who were lost to follow-up or had incomplete information were excluded. Basic characteristics including gender, tumor location, age, staging, treatment, and Body Mass Index (BMI) were analyzed. Short-term outcomes including comorbidities, intra-operative blood loss, hospital stay, gas exhaust time were compared between different NOSES and non-NOSES groups. Long-term outcomes including overall survival (OS) and disease-free survival (DFS) were also analyzed. Patients were followed-up during the inpatient period, at an outpatient clinic, or by phone call. Results: A total of 196 NOSES cases and 243 non-NOSES cases were included. There was a sex difference between the two groups and other factors were comparable. Cases were divided into NOSES groups [including extra-abdominal resection (EVER), specimen extraction and extra-abdominal resection (EXER), and intra-abdominal resection and specimen extraction (IREX)] and non-NOSES groups. Differences in sex (P=0.016), BMI (with mean of 22.08, 22.00, 22.53, and 23.26 kg/m2, P=0.003), and staging (P=0.008) were observed between the four groups. There was a difference in the intra-operative blood loss between NOSES and non-NOSES groups (57.05±62.78, 52.65±68.19, 36.52±43.99 vs. 76.12±90.11 mL, P=0.002), in which NOSES groups had less blood loss. Furthermore, NOSES groups showed a better post-operative gas exhaust time (54.68±37.80, 45.06±24.69, 47.91±28.93 vs. 56.94±27.69 hours, P=0.012). NOSES groups also had fewer ileostomies (17 vs. 37, P=0.003). There was no difference in the long-term DFS and OS between the two groups. Conclusions: NOSES in rectal cancer showed better short-term outcomes and had comparable long-term outcomes compared with non-NOSE surgeries.

Evaluating Solid Lung Adenocarcinoma Anaplastic Lymphoma Kinase Gene Rearrangement Using Noninvasive Radiomics Biomarkers.

PURPOSE: To develop a radiogenomics classifier to assess anaplastic lymphoma kinase (ALK) gene rearrangement status in pretreated solid lung adenocarcinoma noninvasively. MATERIALS AND METHODS: This study consisted of 140 consecutive pretreated solid lung adenocarcinoma patients with complete enhanced CT scans who were tested for both EGFR mutations and ALK status. Pre-contrast CT and standard post-contrast CT radiogenomics machine learning classifiers were designed as two separate classifiers. In each classifier, dataset was randomly split into training and independent testing group on a 7:3 ratio, accordingly subjected to a 5-fold cross-validation. After normalization, best feature subsets were selected by Pearson correlation coefficient (PCC) and analysis of variance (ANOVA) or recursive feature elimination (RFE), whereupon a radiomics classifier was built with support vector machine (SVM). The discriminating performance was assessed with the area under receiver-operating characteristic curve (AUC), accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). RESULTS: In classifier one, 98 cases were selected as training data set, 42 cases as independent testing data set. In classifier two, 87 cases were selected as training data set, 37 cases as independent testing data set. Both classifiers extracted 851 radiomics features. The top 25 pre-contrast features and top 19 post-contrast features were selected to build optimal ALK+ radiogenomics classifiers accordingly. The accuracies, AUCs, sensitivity, specificity, PPV, and NPV of pre-contrast CT classifier were 78.57%, 80.10% (CI: 0.6538-0.9222), 71.43%, 82.14%, 66.67%, and 85.19%, respectively. Those results of standard post-contrast CT classifier were 81.08%, 82.85% (CI: 0.6630-0.9567), 76.92%, 83.33%, 71.43%, and 86.96%. CONCLUSION: Solid lung adenocarcinoma ALK+ radiogenomics classifier of standard post-contrast CT radiomics biomarkers produced superior performance compared with that of pre-contrast one, suggesting that post-contrast CT radiomics should be recommended in the context of solid lung adenocarcinoma radiogenomics AI. Standard post-contrast CT machine learning radiogenomics classifier could help precisely identify solid adenocarcinoma ALK rearrangement status, which may act as a pragmatic and cost-efficient substitute for traditional invasive ALK status test.

Exosome-transferred long non-coding RNA ASMTL-AS1 contributes to malignant phenotypes in residual hepatocellular carcinoma after insufficient radiofrequency ablation.

OBJECTIVES: Long non-coding RNAs (lncRNAs) are emerging RNA regulators in cancer progression, including in hepatocellular carcinoma (HCC). Recently, insufficient radiofrequency ablation (RFA) has been reported to lead to recurrence and metastasis of residual HCC tumours. Herein, we aimed to the role of ASMTL-AS1 in residual HCC after insufficient RFA. MATERIALS AND METHODS: In vitro insufficient RFA model was simulated in Huh7 cells and subsequently named Huh7-H cells. In vitro and in vivo assays were conducted to investigate ASMTL-AS1 function in HCC. RESULTS: LncRNA ASMTL-AS1 low expressed in normal human liver was found to be highly expressed in HCC tissues and further increased in tumours after insufficient RFA. ASMTL-AS1 expression was related to stage, metastasis and prognosis in HCC. Huh7-H possessed higher ASMTL-AS1 level and more aggressive than Huh7 cells. ASMTL-AS1 contributed to the malignancy of HCC cells both in vitro and in vivo. Mechanistically, ASMTL-AS1 was trans-activated by MYC and promoted NLK expression to activate YAP signalling via sequestering miR-342-3p in HCC. Interestingly, ASMTL-AS1 could be wrapped by exosomes and then convey malignancy through NLK/YAP axis between cells even in residual HCC after insufficient RFA. CONCLUSIONS: Exosomal ASMTL-AS1 aggravates the malignancy in residual HCC after insufficient RFA via miR-342-3p/NLK/YAP signalling, opening a new road for the treatment of HCC and the prevention of recurrence or metastasis of residual HCC after insufficient RFA.

Hypoxia-induced downregulation of B-cell translocation gene 3 confers resistance to radiation therapy of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is now a major human cancer, and B-cell translocation gene 3 (BTG3) has been reported as a tumor-suppressor in CRC, but its upstream regulator has not been identified. METHODS: Endogenous expression levels of BTG3 were compared between normal colorectal cell line CCD-18Co and two CRC cell lines SW480 and HT29, as well as between CRC patient tumor and adjacent normal tissues. Analysis of BTG3 genomic region was performed which identified a putative hypoxia response element (HRE). Effects of hypoxia condition, BTG3 overexpression, and their combination on the radiation sensitivity of CRC cell lines were assessed. RESULTS: BTG3 was downregulated in CRC cell lines and patient tumor samples, via the HRE in its promoter region. Hypoxia and BTG3 overexpression could both induce radiation resistance in CRC cells. Combining hypoxia with BTG3 overexpression effectively rendered the resistance of CRC cells to radiation to a level lower than hypoxia alone and higher than normoxia alone, indicating the essential role of BTG3 in hypoxia-induced radiation resistance of CRC cells. CONCLUSION: We therefore propose a novel signaling cascade involving hypoxia/BTG3 to be a potential risk factor for CRC patients undergoing radiation therapy, which could possibly serve as therapeutic targets among CRC patients with acquired radiotherapy resistance.

Differentiation of Treatment-Related Effects from Glioma Recurrence Using Machine Learning Classifiers Based Upon Pre-and Post-Contrast T1WI and T2 FLAIR Subtraction Features: A Two-Center Study.

PURPOSE: We propose three support vector machine (SVM) classifiers, using pre-and post-contrast T2 fluid-attenuated inversion recovery (FLAIR) subtraction and/or pre-and post-contrast T1WI subtraction, to differentiate treatment-related effects (TRE) from glioma recurrence. MATERIALS AND METHODS: Fifty-six postoperative high-grade glioma patients with suspicious progression after radiotherapy and chemotherapy from two centers were studied. Pre-and post-contrast T1WI and T2 FLAIR were collected. Each pre-contrast image was voxel-wise subtracted from the co-registered post-contrast image. Dataset was randomly split into training, and testing on a 7:3 ratio, accordingly subjected to a five fold cross validation. Best feature subsets were selected by Pearson correlation coefficient and recursive feature elimination, whereupon a radiomics classifier was built with SVM. The discriminating performance was assessed with the area under receiver-operating characteristic curve (AUC), accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: In all, 186 features were extracted on each subtraction map. Top nine T1WI subtraction features, top thirteen T2 FLAIR subtraction features and top thirteen combination features were selected to build optimal SVM classifiers accordingly. The accuracies/AUCs/sensitivity/specificity/PPV/NPV of SVM based on sole T1WI subtraction were 80.00%/80.00% (CI: 0.5370-1.0000)/100%/70.00%/62.50%/100%. Those results of SVM based on sole T2 FLAIR subtraction were 86.67%/84.00% (CI: 0.5962-1.0000)/100%/80%/71.43%/100%. Those results of SVM based on both T1WI subtraction and T2 FLAIR subtraction were 93.33%/94.00% (CI: 0.7778-1.0000)/100%/90%/83.33%/100%, respectively. CONCLUSION: Pre- and post-contrast T2 FLAIR subtraction provided added value for diagnosis between recurrence and TRE. SVM based on a combination of T1WI and T2 FLAIR subtraction maps was superior to the sole use of T1WI or T2 FLAIR for differentiating TRE from recurrence. The SVM classifier based on combination of pre-and post-contrast subtraction T2 FLAIR and T1WI imaging allowed for the accurate differential diagnosis of TRE from recurrence, which is of paramount importance for treatment management of postoperative glioma patients after radiation therapy.

Insufficient radiofrequency ablation promotes the metastasis of residual hepatocellular carcinoma cells via upregulating flotillin proteins.

PURPOSE: Radiofrequency ablation (RFA) therapy has proven to be effective and feasible for early-stage hepatocellular carcinoma (HCC); however, rapid progression of residual tumor cells after RFA has been confirmed, but the molecular mechanisms of this phenomenon are poorly understood. This study evaluated the effect of the lipid raft proteins known as flotillins on the invasive and metastatic potential of residual HCC. METHODS: The human HCC cell line HCCLM3 was used to establish insufficient RFA models in vivo and in vitro. Changes in cellular morphology, soft agar colony formation, motility, metastasis, and epithelial-mesenchymal transition (EMT) markers after insufficient RFA intervention in vitro and in vivo were detected by real-time PCR, western blotting, immunohistochemistry and transwell assays. RESULTS: The results showed that flotillin-1 and flotillin-2 expression were upregulated in HCCLM3 cells following 45 °C heat treatment and in residual HCCLM3 xenografts cells after insufficient RFA. Knocking down flotillin-1 or flotillin-2 in HCCLM3 cells by shRNA significantly lowered insufficient RFA-induced tumor growth, EMT changes, and metastasis in vitro and in vivo. Furthermore, mechanism studies indicated that flotillins altered the EMT status and metastatic potential of heat-treated HCCLM3 cells by activating the Akt/Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our findings present new evidence that flotillins play a key role in the aggressive behaviors of residual cancer cells after insufficient RFA and provide new insights into the regulatory mechanism of Wnt/ß-catenin signaling.

Interferon-α Combined With Herbal Compound "Songyou Yin" Effectively Inhibits the Increased Invasiveness and Metastasis by Insufficient Radiofrequency Ablation of Hepatocellular Carcinoma in an Animal Model.

OBJECTIVE: We had previously proved that insufficient radiofrequency ablation (RFA) could enhance invasiveness and metastasis of hepatocellular carcinoma (HCC) through epithelial-mesenchymal transition (EMT), which is mediated by activating ß-catenin signaling. Thus, the aim of the present study was to demonstrate whether the combined treatment of interferon-α (IFN-α) and "Songyou Yin" (SYY) minimizes the pro-metastatic effects of insufficient RFA, as well as to explore its underlying mechanism. METHODS: Insufficient RFA was performed in an orthotopic nude mice model of HCCLM3 with high metastatic potential. The effects of IFN-α, SYY, and combined IFN-α and SYY were observed in the animal model. Tumor sizes, lung metastasis, and survival time were assessed. Immunochemistry staining, real-time polymerase chain reaction, and Western blot were used to examine gene expression related to metastasis and angiogenesis in residual cancer after insufficient RFA. RESULTS: For up to 8 weeks of treatment, the combined therapy significantly decreased the residual cancer sizes, minimized the lung metastasis rate, and prolonged the survival time of nude mice, which might be due to suppression of the EMT via ß-catenin signal blockade, in addition to attenuating angiogenesis in residual cancer after insufficient RFA. CONCLUSION: IFN-α combined with SYY significantly weakened the enhanced metastatic potential of residual cancer after insufficient RFA by attenuating EMT, which is mediated through inhibiting activation of ß-catenin. In addition, decreasing angiogenesis of residual cancer might also play a certain role.

The Rho GTPase Rnd1 inhibits epithelial-mesenchymal transition in hepatocellular carcinoma and is a favorable anti-metastasis target.

Rnd1, a member of Rho GTPases, was found to be downregulated in human malignancies and downregulation of Rnd1 promotes tumor invasion via various mechanisms. However, the role of Rnd1 in hepatocellular carcinoma (HCC) progression remains unclear. In this study, our results demonstrated that Rnd1 was downregulated in HCC cells and in human HCC tissues. Low expression of Rnd1 was associated with aggressive clinic-pathologic characteristics, such as vascular invasion, and poor prognosis in patients who underwent curative surgery for HCC. Overexpression of Rnd1-suppressed cell growth, migration, invasion, and EMT processes in vitro and in vivo. Furthermore, Rnd1 blocked HCC progression by restricting EMT process through inhibition of the Raf/MEK/ERK cascade, and this was correlated with a reduction in RhoA activity. Combination of Rnd1 overexpression with sorafenib, a Raf signaling pathway inhibitor, showed a more potent inhibition on HCC metastasis. Moreover, epigenetic inhibitors (5-Aza and SAHA) increased the expression of Rnd1, and potentiated sorafenib-induced toxicity in HCC cells. In a conclusion, Rnd1-suppressed EMT-mediated metastasis of HCC by reducing the activity of the RhoA/Raf/MEK/ERK signaling pathway, functioning as a favorable anti-metastasis target for HCC patients. Rnd1 overexpression in combination with sorafenib may result in enhanced anti-metastasis efficacy in HCC.

Correction to: miR-182-5p promotes hepatocellular carcinoma progression by repressing FOXO3a.

The original article [1] contains an error in Fig. 5a whereby the Western blot bands representing CyclinD1 have mistakenly been duplicated over the Western blot bands intended to represent SGK.

Dihydropyrimidine dehydrogenase predicts survival and response to interferon-α in hepatocellular carcinoma.

Metastasis and recurrence contribute to poor prognosis of hepatocellular carcinoma (HCC). Recently, we reported that interferon-α (IFN-α) can suppress metastasis of HCC; however, the underlying mechanism has not been fully described. In this study, we demonstrated that expression of dihydropyrimidine dehydrogenase (DPYD), a pyrimidine catabolic enzyme, was dose-dependently downregulated by IFN-α in HCC tissues from nude mice. Notably, DPYD expression was found to be significantly increased in HCC cell lines with higher metastatic potentials compared with their controls. Moreover, upregulation of DPYD in HCC cells could promote in vitro migration, invasion, and in vivo lung metastasis, and inducing changes characteristic of epithelial-mesenchymal transition (EMT). In contrast, knockdown of DPYD inhibited these processes. Mechanistically, DPYD functioned as a positive regulator of EMT in HCC by targeting the p38/NF-κB/Snail1 pathway. Clinically, tissue microarray analysis showed that high DPYD expression was positively associated with aggressive tumor characteristics, including larger tumor size, tumor recurrence, and advanced tumor node metastasis (TNM) stage, and independently correlated with poorer overall survival times after curative resection. HCC patients with low DPYD expression have better response to IFN-α therapy. Taken together, our findings elucidate that IFN-α could downregulate DPYD expression to inhibit EMT and HCC metastasis, and suggest that DPYD might be a potential prognostic biomarker and a therapeutic target for HCC.

miR-182-5p promotes hepatocellular carcinoma progression by repressing FOXO3a.

BACKGROUND: High frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown. METHODS: Real-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression. RESULTS: We showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3'-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/ß-catenin signaling by inhibiting the degradation of ß-catenin and enhancing the interaction between ß-catenin and TCF4 which was mediated by repressed FOXO3a. CONCLUSIONS: Consistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

Berberine hydrochloride counteracts enhanced IL-8 expression induced by SN 38 in AGS cells.

IL-8 over-expression could enhance cancer metastasis. In present study, berberine hydrochloride (BER) triggered proliferative inhibition and G2/M arrest in AGS cells, down-regulated protein expression of cyclin B1, Bcl-2, up-regulated expression of p21, p53 and cleaved caspase 3, but showed no effect on protein expression of CHOP, Bip, and caspase 4. BER could down-regulate the enhanced IL-8 expression through down-regulating ERK1/2 and p38 MAPK over-activation induced by SN 38. The increased IL-8 mediated adhesive ability of AGS cells to HUVECs induced by SN 38, could be reduced by BER. Thus, BER could reduce the side-effect of SN 38 in clinic.

Insufficient Radiofrequency Ablation Treated Hepatocellular Carcinoma Cells Promote Metastasis by Up-Regulation ITGB3.

Radiofrequency ablation (RFA) is one of the standards of care for early stage hepatocellular carcinoma (HCC). However, rapid progression of residual tumor after RFA has been confirmed. The aim of this study was to investigate the underlying mechanism of this phenomenon. Human HCC cell lines HCCLM3 and HepG2 were employed to establish insufficient RFA models in vivo and in vitro, respectively. The effects of insufficient RFA on metastatic potential of residual tumors were evaluated. The molecular changes after insufficient RFA were evaluated by PCR array, western blot, immunofluorescence, and immunohistochemistry. Results showed that insufficient RFA significantly promoted lung and intrahepatic residual tumor cells in vivo, and heat intervention promoted migration and invasion of hepatoma cells in vitro. PCR array revealed that the expression of integrin ß3 (ITGB3) and MMP2 were up-regulated in the residual tumors of HCCLM3 xenograft model. The up-regulation of ITGB3 was confirmed by qRT-PCR, Western blot and immunohistochemistry. Knockdown ITGB3 expression in HCCLM3 cells by shRNA significantly lowered the pro-metastatic effects of insufficient RFA. Mechanism studies indicated that ITGB3 mediated the expression of MMP2 by activing FAK/PI3K/AKT signaling pathway. The up-regulation of ITGB3 contributed to enhanced metastatic potential of residual cancer in HCCLM3 model after insufficient RFA. Targeting ITGB3 expression may further improve the clinical effects of RFA.