PTEN status determines therapeutic vulnerability to celastrol in cholangiocarcinoma.

BACKGROUND: A balanced protein homeostasis network helps cholangiocarcinoma (CCA) maintain their oncogenic growth, and disrupting proteostasis therapeutically will induce proteotoxic stress. Phosphatase and tensin homolog (PTEN) have been reported to be involved in proteostasis, and PTEN-associated pathways are commonly altered in CCA. Celastrol, a triterpene from plants, exhibits cytotoxic effects in various types of cancer. However, the underlying mechanisms remain unclear. PURPOSE: We investigated the therapeutic effect of celastrol in CCA and identified the molecular characteristics of tumors that were sensitive to celastrol. The target of celastrol was explored. We then evaluated the candidate combination therapeutic strategy to increase the effectiveness of celastrol in celastrol-insensitive CCA tumors. METHODS: Various CCA cells were categorized as either celastrol-sensitive or celastrol-insensitive based on their response to celastrol. The molecular characteristics of cells from different groups were determined by RNA-seq. PTEN status and its role in proteasome activity in CCA cells were investigated. The CMAP analysis, molecular docking, and functional assay were performed to explore the effect of celastrol on proteasome activities. The correlation between PTEN status and clinical outcomes, as well as proteasomal activity, were measured in CCA patients. The synergistic therapeutic effect of autophagy inhibitors on celastrol-insensitive CCA cells were measured. RESULTS: Diverse responses to celastrol were observed in CCA cells. PTEN expression varied among different CCA cells, and its status could impact cell sensitivity to celastrol: PTENhigh tumor cells were resistant to celastrol, while PTENlow cells were more sensitive. Celastrol induced proteasomal dysregulation in CCA cells by directly targeting PSMB5. Cells with low PTEN status transcriptionally promoted proteasome subunit expression in an AKT-dependent manner, making these cells more reliant on proteasomal activities to maintain proteostasis. This caused the PTENlow CCA cells sensitive to celastrol. A negative correlation was found between PTEN levels and the proteasome signature in CCA patients. Moreover, celastrol treatment could induce autophagy in PTENhigh CCA cells. Disrupting the autophagic pathway in PTENhigh CCA cells enhanced the cytotoxic effect of celastrol. CONCLUSION: PTEN status in CCA cells determines their sensitivity to celastrol, and autophagy inhibitors could enhance the anti-tumor effect in PTENhigh CCA.

Bortezomib in previously treated phosphatase and tension homology-deficient patients with advanced intrahepatic cholangiocarcinoma: An open-label, prospective and single-centre phase II trial.

INTRODUCTION: Intrahepatic cholangiocarcinoma (ICC) is characterized by a dismal prognosis with limited therapeutic alternatives. To explore phosphatase and tension homolog (PTEN) as a biomarker for proteasome inhibition in ICC, we conducted a phase II trial to assess the second-line efficacy of bortezomib in PTEN-deficient advanced ICC patients. METHODS: A total of 130 patients with advanced ICC in our centre were screened by PTEN immunohistochemical staining between 1 July 2017, and 31 December 2021, and 16 patients were ultimately enrolled and treated with single-agent bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11 of a 21-day cycle. The primary endpoint was the objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors v1.1. RESULTS: The median follow-up was 6.55 months (95% confidence interval [CI]: 0.7-19.9 months). Among the 16 enrolled patients, the ORR was 18.75% (3/16) and the disease control rate was 43.75% (7/16). The median progress-free survival was 2.95 months (95% CI: 2.1-5.1 months) and the median overall survival (mOS) was 7.2 months (95% CI: 0.7-21.6 months) in the intent-to-treat-patients. Treatment-related adverse events of any grade were reported in 16 patients, with thrombopenia being the most common toxicity. Patients with PTEN staining scores of 0 were more likely to benefit from bortezomib than those with staining scores > 0. CONCLUSIONS: Bortezomib yielded an encouraging objective response and a favourable OS as a second-line agent in PTEN-deficient ICC patients. Our findings suggest bortezomib as a promising therapeutic option for patients with PTEN-deficient ICC. HIGHLIGHTS: There is a limited strategy for the second-line option of intrahepatic cholangiocarcinoma (ICC). This investigator-initiated phase 2 study evaluated bortezomib in ICC patients with phosphatase and tension homology deficiency. The overall response rate was 18.75% and the overall survival was 7.2 months in the intent-to-treat cohort. These results justify further developing bortezomib in ICC patients with PTEN deficiency.

Arsenic trioxide augments immunogenic cell death and induces cGAS-STING-IFN pathway activation in hepatocellular carcinoma.

The treatment of hepatocellular carcinoma (HCC) is particularly challenging due to the inherent tumoral heterogeneity and easy resistance towards chemotherapy and immunotherapy. Arsenic trioxide (ATO) has emerged as a cytotoxic agent effective for treating solid tumors, including advanced HCC. However, its effectiveness in HCC treatment remains limited, and the underlying mechanisms are still uncertain. Therefore, this study aimed to characterize the effects and mechanisms of ATO in HCC. By evaluating the susceptibilities of human and murine HCC cell lines to ATO treatment, we discovered that HCC cells exhibited a range of sensitivity to ATO treatment, highlighting their inherent heterogeneity. A gene signature comprising 265 genes was identified to distinguish ATO-sensitive from ATO-insensitive cells. According to this signature, HCC patients have also been classified and exhibited differential features of ATO response. Our results showed that ATO treatment induced reactive oxygen species (ROS) accumulation and the activation of multiple cell death modalities, including necroptosis and ferroptosis, in ATO-sensitive HCC cells. Meanwhile, elevated tumoral immunogenicity was also observed in ATO-sensitive HCC cells. Similar effects were not observed in ATO-insensitive cells. We reported that ATO treatment induced mitochondrial injury and mtDNA release into the cytoplasm in ATO-sensitive HCC tumors. This subsequently activated the cGAS-STING-IFN axis, facilitating CD8+ T cell infiltration and activation. However, we found that the IFN pathway also induced tumoral PD-L1 expression, potentially antagonizing ATO-mediated immune attack. Additional anti-PD1 therapy promoted the anti-tumor response of ATO in ATO-sensitive HCC tumors. In summary, our data indicate that heterogeneous ATO responses exist in HCC tumors, and ATO treatment significantly induces immunogenic cell death (ICD) and activates the tumor-derived mtDNA-STING-IFN axis. These findings may offer a new perspective on the clinical treatment of HCC and warrant further study.

Clinical Application of Long-Pulsed 800-Nm Diode Laser Depilation Technology on Microtia Reconstruction in 965 Patients.

BACKGROUND: The issue of hair growth on reconstructed ears has been a matter of concern for both patients and surgeons, despite the notable progress made in microtia reconstruction technology in recent times. OBJECTIVE: This study aims to present the practical implementation of long-pulsed 800-nm diode laser depilation technology in the field of auricular reconstruction. Furthermore, it seeks to establish a comprehensive and standardized protocol for utilizing lasers in the reconstruction of microtia ears. METHODS: A total of 965 patients (comprising 1021 ears) diagnosed with congenital microtia underwent treatment using 800-nm long-pulsed diode laser depilation. The participants received 1-3 treatment sessions with intervals of 25-30 days. To assess the effectiveness of the treatment, two independent observers compared photographs and measured the reduction in terminal hair count before and after the final session. Clinical outcomes were evaluated using VAS questionnaires, and any adverse events were diligently recorded. RESULTS: The findings indicated that the utilization of the long-pulsed 800-nm diode laser was both safe and efficient in achieving hair removal during microtia ear reconstruction. As additional sessions were conducted, pain scores demonstrated a decline, while adverse reactions remained minimal. LIMITATIONS: This is a retrospective single-institution study. CONCLUSION: The application of a long-pulsed 800-nm diode laser has been proved to be a safe and effective method for removing hair during the process of microtia ear reconstruction, involving the use of a tissue expander and autologous costal cartilage. To achieve satisfactory results in hair removal, it was found necessary to repeat the shots procedure two to three times. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

PTEN deficiency facilitates gemcitabine efficacy in cancer by modulating the phosphorylation of PP2Ac and DCK.

Gemcitabine is a nucleoside analog that has been successfully used in the treatment of multiple cancers. However, intrinsic or acquired resistance reduces the chemotherapeutic potential of gemcitabine. Here, we revealed a previously unappreciated mechanism by which phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, dominates the decision-making process that is central to the regulation of gemcitabine efficacy in cholangiocarcinoma (CCA). By investigating a gemcitabine-treated CCA cohort, we found that PTEN deficiency was correlated with the improved efficacy of gemcitabine-based chemotherapy. Using cell-based drug sensitivity assays, cell line-derived xenograft, and patient-derived xenograft models, we further confirmed that PTEN deficiency or genetic-engineering down-regulation of PTEN facilitated gemcitabine efficacy both in vitro and in vivo. Mechanistically, PTEN directly binds to and dephosphorylates the C terminus of the catalytic subunit of protein phosphatase 2A (PP2Ac) to increase its enzymatic activity, which further dephosphorylates deoxycytidine kinase (DCK) at Ser74 to diminish gemcitabine efficacy. Therefore, PTEN deficiency and high phosphorylation of DCK predict a better response to gemcitabine-based chemotherapy in CCA. We speculate that the combination of PP2A inhibitor and gemcitabine in PTEN-positive tumors could avoid the resistance of gemcitabine, which would benefit a large population of patients with cancer receiving gemcitabine or other nucleoside analogs.

Adenosine 2A receptor contributes to the facilitation of post-infectious irritable bowel syndrome by γδ T cells via the PKA/CREB/NF-κB signaling pathway.

BACKGROUND: Immunological dysfunction-induced low-grade inflammation is regarded as one of the predominant pathogenetic mechanisms in post-infectious irritable bowel syndrome (PI-IBS). γδ T cells play a crucial role in innate and adaptive immunity. Adenosine receptors expressed on the surface of γδ T cells participate in intestinal inflammation and immunity regulation. AIM: To investigate the role of γδ T cell regulated by adenosine 2A receptor (A2AR) in PI-IBS. METHODS: The PI-IBS mouse model has been established with Trichinella spiralis (T. spiralis) infection. The intestinal A2AR and A2AR in γδ T cells were detected by immunohistochemistry, and the inflammatory cytokines were measured by western blot. The role of A2AR on the isolated γδ T cells, including proliferation, apoptosis, and cytokine production, were evaluated in vitro. Their A2AR expression was measured by western blot and reverse transcription polymerase chain reaction (RT-PCR). The animals were administered with A2AR agonist, or A2AR antagonist. Besides, γδ T cells were also injected back into the animals, and the parameters described above were examined, as well as the clinical features. Furthermore, the A2AR-associated signaling pathway molecules were assessed by western blot and RT-PCR. RESULTS: PI-IBS mice exhibited elevated ATP content and A2AR expression (P < 0.05), and suppression of A2AR enhanced PI-IBS clinical characteristics, indicated by the abdominal withdrawal reflex and colon transportation test. PI-IBS was associated with an increase in intestinal T cells, and cytokine levels of interleukin-1 (IL-1), IL-6, IL-17A, and interferon-α (IFN-α). Also, γδ T cells expressed A2AR in vitro and generated IL-1, IL-6, IL-17A, and IFN-α, which can be controlled by A2AR agonist and antagonist. Mechanistic studies demonstrated that the A2AR antagonist improved the function of γδ T cells through the PKA/CREB/NF-κB signaling pathway. CONCLUSION: Our results revealed that A2AR contributes to the facilitation of PI-IBS by regulating the function of γδ T cells via the PKA/CREB/NF-κB signaling pathway.

Aquaporin 1 is a prognostic marker and inhibits tumour progression through downregulation of Snail expression in intrahepatic cholangiocarcinoma.

BACKGROUND: Recently, some studies have suggested a link between AQP1 and cancer progression. AIMS: The aim of the present study was to investigate the influence of AQP1 on the clinicopathology and prognosis of intrahepatic cholangiocarcinoma (ICC) patients. METHODS: We retrospectively detected the expression of AQP1 protein in 307 patients with ICC who underwent partial hepatectomy. Western blot analysis was used to detect AQP1 protein levels in stable AQP1 overexpression and knockdown cell lines. The influence of AQP1 on the invasion and metastasis ability of ICC cells was assessed by wound-healing and Transwell assays in vitro as well as by a splenic liver metastasis model in vivo. RESULTS: Positive membranous AQP1 expression was identified in 34.2% (105/307) of the ICC specimens. Survival data revealed that positive AQP1 expression was significantly associated with favourable disease-free survival (DFS) and overall survival (OS) (p = 0.0290 and p = 0003, respectively). Moreover, high AQP1 expression inhibited the invasion and migration of ICC cells in vitro as well as inhibited liver metastasis in nude mice. Mechanistically, high AQP1 expression in ICC cells increased the levels of E-cadherin but decreased the levels of the Snail transcription factor. CONCLUSIONS: AQP1 expression is associated with a favourable prognosis in ICC patients. AQP1 inhibits ICC cell invasion, metastasis, and epithelial-mesenchymal transition (EMT) through downregulation of Snail expression.

PTEN Deficiency Facilitates Exosome Secretion and Metastasis in Cholangiocarcinoma by Impairing TFEB-mediated Lysosome Biogenesis.

BACKGROUND & AIMS: In eukaryotes, the ubiquitin-proteasome system and the autophagy-lysosome pathway are essential for maintaining cellular proteostasis and associated with cancer progression. Our previous studies have demonstrated that phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, limits proteasome abundance and determines chemosensitivity to proteasome inhibitors in cholangiocarcinoma (CCA). However, whether PTEN regulates the lysosome pathway remains unclear. METHODS: We tested the effects of PTEN on lysosome biogenesis and exosome secretion using loss- and gain-of-function strategies in CCA cell lines. Using in vitro dephosphorylation assays, we explored the regulatory mechanism between PTEN and the key regulator of lysosome biogenesis, transcription factor EB (TFEB). Using the migration assays, invasion assays, and trans-splenic liver metastasis mouse models, we evaluated the function of PTEN deficiency, TFEB-mediated lysosome biogenesis, and exosome secretion on tumor metastasis. Moreover, we investigated the clinical significance of PTEN expression and exosome secretion by retrospective analysis. RESULTS: PTEN facilitated lysosome biogenesis and acidification through its protein phosphatase activity to dephosphorylate TFEB at Ser211. Notably, PTEN deficiency increased exosome secretion by reducing lysosome-mediated degradation of multi-vesicular bodies, which further facilitated the proliferation and invasion of CCA. TFEB agonist curcumin analog C1 restrained the metastatic phenotype caused by PTEN deficiency in mouse models, and we highlighted the correlation between PTEN deficiency and exosome secretion in clinical cohorts. CONCLUSIONS: In CCA, PTEN deficiency impairs lysosome biogenesis to facilitate exosome secretion and cancer metastasis in a TFEB phosphorylation-dependent manner.

Gankyrin and TIGAR cooperatively accelerate glucose metabolism toward the PPP and TCA cycle in hepatocellular carcinoma.

Oncogene-derived metabolic reprogramming is important for anabolic growth of cancer cells, which is now considered to be not simply rely on glycolysis. Pentose phosphate pathway and tricarboxylic acid cycle also play pivotal roles in helping cancer cells to meet their anabolic and energy demands. The present work focused on gankyrin, a relatively specific oncogene in hepatocellular carcinoma (HCC), and its impact on glycolysis and mitochondrial homeostasis. Metabolomics, RNA-seq analysis, and subsequent conjoint analysis illustrated that gankyrin regulated the pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and mitochondrial function and homeostasis, which play pivotal roles in tumor development. Mechanistically, gankyrin was found to modulate HCC metabolic reprogramming via TIGAR. Gankyrin positively regulated the transcription of TIGAR through Nrf2, which bound to the antioxidant response elements (AREs) in the promoter of TIGAR. Interestingly, TIGAR feedback regulated the transcription of Nrf2 and subsequently gankyrin by promoting nuclear importation of PGC1α. The loop between gankyrin, Nrf2, and TIGAR accelerated glucose metabolism toward the PPP and TCA cycle, which provided vital building blocks, such as NADPH, ATP, and ribose of tumor and further facilitated the progression of HCC.

Upregulated adenosine 2A receptor accelerates post-infectious irritable bowel syndrome by promoting CD4+ T cells' T helper 17 polarization.

BACKGROUND: Post-infectious irritable bowel syndrome (PI-IBS) is generally regarded as a functional disease. Several recent studies have reported the involvement of low-grade inflammation and immunological dysfunction in PI-IBS. T helper 17 (Th17) polarization occurs in IBS. Adenosine and its receptors participate in intestinal inflammation and immune regulation. AIM: To investigate the role of Th17 polarization of CD4+ T cells regulated by adenosine 2A receptor (A2AR) in PI-IBS. METHODS: A PI-IBS model was established by infecting mice with Trichinella spiralis. The intestinal A2AR and CD4+ T lymphocytes were detected by immunohistochemistry, and the inflammatory cytokines were detected by enzyme-linked immunoassay. CD4+ T lymphocytes present in the animal's spleen were separated and cultured with or without A2AR agonist and antagonist. Western blotting and real-time quantitative polymerase chain reaction were performed to determine the effect of A2AR on the cells and intestinal tissue. Cytokine production was determined. The protein and mRNA levels of A2AR associated signaling pathway molecules were also evaluated. Furthermore, A2AR agonist and antagonist were injected into the mouse model and the clinical features were observed. RESULTS: The PI-IBS mouse model showed increased expression of ATP and A2AR (P < 0.05), and inhibition of A2AR improved the clinical features in PI-IBS, including the abdominal withdrawal reflex and colon transportation test (P < 0.05). The number of intestinal CD4+ T cells and interleukin-17 (IL-17) protein levels increased during PI-IBS, which was reversed by administration of the A2AR antagonist (P < 0.05). CD4+ T cells expressed A2AR and produced IL-17 in vitro, which was regulated by the A2AR agonist and antagonist. The A2AR antagonist increased the production of IL-17 by CD4+ T cells via the Janus kinase-signal transducer and activator of transcription-receptor-related orphan receptor γ signaling pathway. CONCLUSION: The results of the present study suggested that the upregulation of A2AR increases PI-IBS by promoting the Th17 polarization of CD4+ T cells.

PTEN deficiency facilitates the therapeutic vulnerability to proteasome inhibitor bortezomib in gallbladder cancer.

Gallbladder cancer (GBC) is an aggressive malignancy of biliary tract with poor prognosis. Although several studies have shown the frequency of relevant genetic alterations, there are few genetic models or translational studies that really benefit for GBC treatment in the era of precision medicine. By targeted sequencing and immunohistochemistry staining, we identified that phosphate and tension homology deleted on chromosome ten (PTEN) was frequently altered in GBC specimens, and loss of PTEN expression was independently correlated with poor survival outcomes. Further drug screening assays revealed proteasome inhibitor bortezomib as a promising agent for GBC treatment, and knockdown of PTEN increased bortezomib efficacy both in vivo and in vitro. Therapeutic evaluation of patient derived xenografts (PDXs) strongly supported the utilization of bortezomib in PTEN deficient GBC. Mechanically, functional PTEN inhibited ARE-dependent transcriptional activity, the same machinery regulating the transcription of proteasome subunits, thus PTEN suppressed proteasome activity and bortezomib sensitivity. Through siRNA screening, we identified the ARE-related transcriptional suppressor BACH1 involved in PTEN-mediated proteasome inhibition and regulated by PTEN-AKT1 axis. In summary, our study indicates that proteasome activity represents a prime therapeutic target in PTEN-deficient GBC tumors, which is worthy of further clinical validation.

PTEN status determines chemosensitivity to proteasome inhibition in cholangiocarcinoma.

Patient-derived xenografts (PDXs) and PDX-derived cells (PDCs) are useful in preclinical research. We performed a drug screening assay using PDCs and identified proteasome inhibitors as promising drugs for cholangiocarcinoma (CCA) treatment. Furthermore, we determined that phosphate and tensin homology deleted on chromosome ten (PTEN) deficiency promotes protein synthesis and proteasome subunit expression and proteolytic activity, creating a dependency on the proteasome for cancer cell growth and survival. Thus, targeting the proteasome machinery with the inhibitor bortezomib inhibited the proliferation and survival of CCA cells lacking functional PTEN. Therapeutic evaluation of PDXs, autochthonous mouse models, and patients confirmed this dependency on the proteasome. Mechanistically, we found that PTEN promoted the nuclear translocation of FOXO1, resulting in the increased expression of BACH1 and MAFF BACH1 and MAFF are transcriptional regulators that recognize the antioxidant response element, which is present in genes encoding proteasome subunits. PTEN induced the accumulation and nuclear translocation of these proteins, which directly repressed the transcription of genes encoding proteasome subunits. We revealed that the PTEN-proteasome axis is a potential target for therapy in PTEN-deficient CCA and other PTEN-deficient cancers.

Association of thyroid hormones with metabolic syndrome and its components in postmenopausal Chinese women.

To evaluate the association between thyroid hormones (TH) and metabolic syndrome (MS) in postmenopausal women (PmW), a cross-sectional study was conducted with a sample of 1000 participants of PmW (40-65 years). Thyroid stimulating hormone (TSH) and free thyroxine (fT4) were evaluated. The MS was defined using the International Diabetes Federation (IDF) ethnicity-specific definitions for Asian. Participants were classified into three groups according to the TSH reference range: high-TSH (≥4.2 mU/L), low-TSH (<0.1mU/L), and normal-TSH (0.1-4.2 mU/L) group. Serum triglycerides (TG) levels were higher in low-TSH group and high-TSH group compared with normal-TSH group (p < .05). The whole sample was stratified into <60 and ≥60 years subgroups. In the ≥60 years group, fT4 was negatively correlated with waist circumference (WC) (p = .028) and positively correlated with fasting blood glucose (FBG) (p = .043), meanwhile TSH was positively correlated with WC only in the control subjects (p = .014). No difference was found between TH and the number of MS components. It was demonstrated that serum fT4 levels were associated with FBG and WC, while TSH was associated with WC in elderly PmW without MS.

Cytoskeleton-Associated Protein 4, a Promising Biomarker for Tumor Diagnosis and Therapy.

Cytoskeleton-associated protein 4 (CKAP4) is located in the rough endoplasmic reticulum (ER) and plays an important role in stabilizing the structure of ER. Meanwhile, CKAP4 is also found to act as an activated receptor at the cell surface. The multifunction of CKAP4 was gradually discovered with growing research evidence. In addition to the involvement in various physiological events including cell proliferation, cell migration, and stabilizing the structure of ER, CKAP4 has been implicated in tumorigenesis. However, the role of CKAP4 is still controversial in tumor biology, which may be related to different signal transduction pathways mediated by binding to different ligands in various microenvironments. Interestingly, CKAP4 has been recently recognized as a serological marker of several tumors and CKAP4 is expected to be a tumor therapeutic target. Therefore, deciphering the gene status, expression regulation, functions of CKAP4 in different diseases may shed new light on CKAP4-based cancer diagnosis and therapeutic strategy. This review discusses the publications that describe CKAP4 in various diseases, especially on tumor promotion and suppression, and provides a detailed discussion on the discrepancy.

RPB5-Mediating Protein Promotes Cholangiocarcinoma Tumorigenesis and Drug Resistance by Competing With NRF2 for KEAP1 Binding.

BACKGROUND AND AIMS: Cancer cell survival depends on the balance between reactive oxygen species production and scavenging, which is regulated primarily by NRF2 during tumorigenesis. Here, we demonstrate that deletion of RBP5-mediating protein (RMP) in an autonomous mouse model of intrahepatic cholangiocarcinoma (ICC) delays tumor progression. APPROACH AND RESULTS: RMP-overexpressing tumor cells exhibited enhanced tolerance to oxidative stress and apoptosis. Mechanistically, RMP competes with NRF2 for binding to the Kelch domain of KEAP1 (Kelch-like ECH-associated protein 1) through the E**E motif, leading to decreased NRF2 degradation via ubiquitination, thus increasing NRF2 nuclear translocation and downstream transactivation of antioxidant genes. This RMP-KEAP1-NRF2 axis promotes ICC tumorigenesis, metastasis, and drug resistance. Consistent with these findings, the RMP level in human ICC is positively correlated with the protein level of NRF2 and is associated with poor prognosis. CONCLUSION: These findings reveal that RMP is involved in the oxidative stress defense program and could be exploited for targeted cancer therapies.

Six2 is negatively correlated with prognosis and facilitates epithelial-mesenchymal transition via TGF-ß/Smad signal pathway in hepatocellular carcinoma.

BACKGROUND: Increasing evidence indicates that Six2 contributes to tumorigenesis in various tumor including hepatocellular carcinoma (HCC). This study aimed to determine the role of Six2 in HCC and to elucidate the association of Six2 with clinical pathological characteristics. METHODS: The expressions of Six2 in HCC tumor, para-tumor tissue and portal vein tumor thrombus (PVTT) were detected by tissue microarray technique, immunohistochemistry, real-time RT-PCR and Western blotting. Chi-square and Kaplan-Meier analysis were used to analyze the correlation between Six2 expression and prognosis of HCC patients. Lentivirus mediated Six2 knockdown, spheroid formation assay, proliferation assay and subcutaneous tumor implantation were performed to determine the function of Six2. RESULTS: In 274 HCC samples, Six2 was strongly expressed. Kaplan-Meier analysis revealed that high expression of Six2 was correlated with a shorter overall survival (OS) and disease-free survival (DFS). Moreover, Six2 expression was associated with sex, alpha-fetoprotein, tumor size and portal vein invasion. Six2 was highly expressed in PVTT. Six2 knockdown inhibited HCC cell lines proliferation, migration, and self-renewal in vitro and in vivo. In addition, low-expression of Six2 weakened TGF-ß induced Smad4 activation and epithelial-mesenchymal transition in HCC cell lines. CONCLUSIONS: Elevated Six2 expression in HCC tumor patients was associated with negative prognosis. Upregulated Six2 promoted tumor growth and facilitated HCC metastasis via TGF-ß/Smad signal pathway.

Glypican-3-targeted precision diagnosis of hepatocellular carcinoma on clinical sections with a supramolecular 2D imaging probe.

The ability of chemical tools to effectively detect malignancy in frozen sections removed from patients during surgery is important for the timely determination of the subsequent surgical program. However, current clinical methods for tissue imaging rely on dye-based staining or antibody-based techniques, which are sluggish and complicated. Methods: Here, we have developed a 2D material-based supramolecular imaging probe for the simple, rapid yet precise diagnosis of hepatocellular carcinoma (HCC). The 2D probe is constructed through supramolecular self-assembly between a water soluble, fluorescent peptide ligand that selectively targets glypican-3 (GPC-3, a specific cell-surface biomarker for HCC) and 2D molybdenum disulfide that acts as a fluorescence quencher as well as imaging enhancer. Results: We show that the 2D imaging probe developed with minimal background fluorescence can sensitively and selectively image cells overexpressing GPC-3 over a range of control cells expressing other membrane proteins. Importantly, we demonstrate that the 2D probe is capable of rapidly (signal became readable within 1 min) imaging HCC tissues over para-carcinoma regions in frozen sections derived from HCC patients; the results are in accordance with those obtained using traditional clinical staining methods. Conclusion: Compared to conventional staining methods, which are laborious (e.g., over 30 min is needed for antibody-based immunosorbent assays) and complex (e.g., diagnosis is based on discrimination of the nucleus morphology of cancer cells from that of normal cells), our probe, with its simplicity and quickness, might become a promising candidate for tumor-section staining as well as fluorescence imaging-guided surgery.

Combination of Kras activation and PTEN deletion contributes to murine hepatopancreatic ductal malignancy.

Kras mutations are among the most common genetic abnormalities in human neoplasms, including cholangiocarcinomas, pancreatic cancer and colon cancer. PTEN has previously been associated with cholangiocarcinoma development in murine models. Here, we have established novel mouse models of neoplasms by liver-specific and biliary-pancreatic Kras activation and PTEN deletion. By liver-specific disruption of PTEN and activation of Kras in mice caused rapid development of intrahepatic biliary epithelial proliferative lesions (Intrahepatic cholangiocarcinoma, ICC), which progress through dysplasia to invasive carcinoma. In contrast, Kras activation in combination with heterozygous PTEN deletion induced mixed carcinomas of liver (both ICC and hepatocellular carcinoma, HCC), whereas Kras activation alone did not induce biliary tract neoplasm. Use of Sox9-Cre-LoxP-based approach to coordinately delete PTEN and activate Kras in the adult mouse resulted in not only development of low-grade biliary lesions (ICC and extrahepatic bile duct carcinoma, ECC) but also pancreatic carcinomas. Our data provide a functional link between PTEN gene status, hepatobiliary cell fate, and HCC, biliary carcinoma, pancreatic cancer pathogenesis, and present novel genetically engineered mouse models of PTEN loss-driven malignancy.

Fluorogenic 2D Peptidosheet Unravels CD47 as a Potential Biomarker for Profiling Hepatocellular Carcinoma and Cholangiocarcinoma Tissues.

A 2D peptidosheet unravels CD47 as a potential biomarker to image hepatocarcinoma and cholangiocarcinoma cells and tissues. Supramolecular assembly between water-soluble 2D MoS2 and a peptide probe produces the 2D peptidosheet suited for the profiling of hepatocarcinoma and cholangiocarcinoma tissues over healthy tissues on clinical specimens.

Elevated ZC3H15 increases HCC growth and predicts poor survival after surgical resection.

Zinc finger CCCH-type containing 15 (ZC3H15), also known as DRG family regulatory protein 1 (DFRP1), is a highly conserved eukaryotic protein that associates with active translation machinery. The aim of our study was to explore the clinical relevance and intrinsic functions of ZC3H15 in hepatocellular carcinoma (HCC). We constructed a cohort with 261 tumor and matched normal tissues from HCC patients. ZC3H15 protein and mRNA levels were determined using immunohistochemistry, western blot analysis, and quantitative polymerase chain reaction. ZC3H15 was highly expressed in the majority of HCC cases, and high ZC3H15 levels were significantly associated with high serum a-fetoprotein (AFP) levels (>20 ng/mL) and vascular invasion. Kaplan-Meier and Cox regression data indicated that elevated ZC3H15 was an independent predictor for HCC-specific disease-free survival (hazards ratio [HR], 1.789; 95% confidence interval [95% CI], 1.298-2.466 [P=0.0004]) and overall survival (HR, 1.613; 95% CI, 1.120-2.322 [P=0.0101]). Interaction of ZC3H15 with TRAF2 increased activation of NFκB signaling. These results suggest ZC3H15 is an independent prognostic marker in HCC patients that is clinicopathologically associated with tumor invasion and serum AFP levels.