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.

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.

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.

Hydrogen sulfide alleviates apoptosis and autophagy induced by beryllium sulfate in 16HBE cells.

Beryllium and its compounds are systemic toxicants that are widely applied in many industries. Hydrogen sulfide has been found to protect cells. The present study aimed to determine the protective mechanisms involved in hydrogen sulfide treatment of 16HBE cells following beryllium sulfate-induced injury. 16HBE cells were treated with beryllium sulfate doses ranging between 0 and 300 µM BeSO4 . Additionally, 16HBE cells were subjected to pretreatment with either a 300 µM dose of sodium hydrosulfide (a hydrogen sulfide donor) or 10 mM DL-propargylglycine (a cystathionine-γ-lyase inhibitor) for 6 hr before then being treated with 150 µM beryllium sulfate for 48 hr. This study illustrates that beryllium sulfate induces a reduction in cell viability, increases lactate dehydrogenase (LDH) release, and increases cellular apoptosis and autophagy in 16HBE cells. Interestingly, pretreating 16HBE cells with sodium hydrosulfide significantly reduced the beryllium sulfate-induced apoptosis and autophagy. Moreover, it increased the mitochondrial membrane potential and alleviated the G2/M-phase cell cycle arrest. However, pretreatment with 10 mM DL-propargylglycine promoted the opposite effects. PI3K/Akt/mTOR and Nrf2/ARE signaling pathways are also activated following pretreatment with sodium hydrosulfide. These results indicate the protection provided by hydrogen sulfide in 16HBE cells against beryllium sulfate-induced injury is associated with the inhibition of apoptosis and autophagy through the activation of the PI3K/Akt/mTOR and Nrf2/ARE signaling pathways. Therefore, hydrogen sulfide has the potential to be a promising candidate in the treatment against beryllium disease.

Measurable Residual Disease Detected by Multiparameter Flow Cytometry and Sequencing Improves Prediction of Relapse and Survival in Acute Myeloid Leukemia.

The clinically ideal time point and optimal approach for the assessment of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) are still inconclusive. We investigated the clinical value of multiparameter flow cytometry-based MRD (MFC MRD) after induction (n = 492) and two cycles of consolidation (n = 421). The latter time point was proved as a superior indicator with independent prognostic significance for both relapse-free survival (RFS, HR = 3.635, 95% CI: 2.433-5.431, P <0.001) and overall survival (OS: HR = 3.511, 95% CI: 2.191-5.626, P <0.001). Furthermore, several representative molecular MRD markers were compared with the MFC MRD. Both approaches can establish prognostic value in patients with NPM1 mutations, and FLT3, C-KIT, or N-RAS mutations involved in kinase-related signaling pathways, while the combination of both techniques further refined the risk stratification. The detection of RUNX1-RUNX1T1 fusion transcripts achieved a considerable net reclassification improvement in predicting the prognosis. Conversely, for patients with biallelic CEBPA or DNMT3A mutations, only the MFC method was recommended due to the poor prognostic discriminability in tracking mutant transcripts. In conclusion, this study demonstrated that the MFC MRD after two consolidation cycles independently predicted clinical outcomes, and the integration of MFC and molecular MRD should depend on different types of AML-related genetic lesions.

Molecular organizations and function of iron-stress-induced-A protein family in Anabaena sp. PCC 7120.

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions, and surround photosystem I (PSI) trimer with a ring formation. A cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, it is unknown how the IsiAs are associated with PSI. Here we report on molecular organizations and function of the IsiAs in this cyanobacterium. A deletion mutant of the isiA1 gene was constructed, and the four types of thylakoids were prepared from the wild-type (WT) and ΔisiA1 cells under iron-replete (+Fe) and iron-deficient (-Fe) conditions. Immunoblotting analysis exhibits a clear expression of the IsiA1 in the WT-Fe. The PSI-IsiA1 supercomplex is found in the WT-Fe, and excitation-energy transfer from IsiA1 to PSI is verified by time-resolved fluorescence analyses. Instead of the IsiA1, both IsiA2 and IsiA3 are bound to PSI monomer in the ΔisiA1-Fe. These findings provide insights into multiple-expression system of the IsiA family in this cyanobacterium.

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.

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.

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.

RMP predicts survival and adjuvant TACE response in hepatocellular carcinoma.

Adjuvant transcatheter arterial chemoembolization (TACE) protects against hepatocellular carcinoma (HCC) and is associated with reduced disease recurrence and improved outcome after surgery. However, deterioration of liver function after TACE negatively impacts the patient prognosis and limits it use as an option to prolong survival. We analyzed two independent cohorts that included a total of 510 patients with HCC who had undergone tumor resection. Immunohistochemistry assay was used to measure RPB5-mediating protein (RMP) expression and assessed their association with recurrence rate and response to therapy with adjuvant TACE. In patients with HCC, the expression of RMP in tumor is associated with age, gender, tumor size, portal venous invasion, TNM stages, BCLC stages and overall survival. Among patients with high RMP expression, adjuvant TACE after resection was associated with early recurrence. Even in the patients with small tumor size (no more than 5 cm) or no venous invasion, RMP status is associated with response to adjuvant TACE. RMP status in tumors may be a useful marker in estimating prognosis in patients with HCC and in assisting in the selection of patients who are likely to benefit from adjuvant TACE to prevent relapse.

CKAP4 inhibited growth and metastasis of hepatocellular carcinoma through regulating EGFR signaling.

CKAP4, one kind of type II trans-membrane protein, plays an important role to maintain endoplasmic reticulum structure and inhibits the proliferation of bladder cancer cells by combining its ligand anti-proliferative factor (APF). However, the biological function of CKAP4 in the progression of liver cancer has not been clearly demonstrated. In the present study, we knocked down or overexpressed CKAP4 in hepatocellular carcinoma (HCC) cells and cell proliferation, invasion, and migration capacities were investigated by CCK-8 and transwell assays. In vivo tumor model in mice was used to evaluate the role of CKAP4 on growth and metastasis of HCC. The data documented that HCC cells with high CKAP4 levels were featured by low proliferation capability as well as low invasion potential. Interestingly, we found that CKAP4 suppressed the activation of epithelial growth factor receptor (EGFR) signaling, which may partly explain the role of CKAP4 in cell biological behavior of HCC. Further study revealed that CKAP4 could associate with EGFR at basal status and the complex was reduced upon EGF stimulation, leading to release EGFR into cytoplasm. Thus, we demonstrate the novel mechanism, for the first time, expression of CKAP4 regulates progression and metastasis of HCC and it may provide therapeutic values in this tumor.

Prognostic significance of cytoskeleton-associated membrane protein 4 and its palmitoyl acyltransferase DHHC2 in hepatocellular carcinoma.

BACKGROUND: The functions of cytoskeleton-associated membrane protein 4 (CKAP4), one kind of type II transmembrane protein, are associated with the palmitoyl acyltransferase DHHC2. The objective of the current study was to investigate CKAP4/DHHC2 expression and its prognostic significance in patients with hepatocellular carcinoma (HCC). METHODS: Two independent cohorts of 416 patients with HCC were enrolled. All the patients included had defined clinicopathologic and follow-up data. Using real-time polymerase chain reaction and immunohistochemical assay, CKAP4 and DHHC2 expression were evaluated. The association between CKAP4/DHHC2 expression and HCC-specific disease-free survival and overall survival was analyzed by Kaplan-Meier curves, the log-rank test, and Multivariate Cox regression analyses. RESULTS: The data documented that CKAP4 expression was much higher in HCC tumor tissues compared with adjacent normal tissues and its expression was significantly correlated with tumor size, intrahepatic metastases, portal venous invasion, and Barcelona Clinic Liver Cancer stage of disease in 2 cohorts of patients. On survival analysis, patients with high CKAP4 expression appeared to have a favorable overall survival and a longer disease-free survival compared with those with low expression. DHHC2 expression was also examined in tissue microarray analysis by immunohistochemistry and the results demonstrated that 87.6% of the cases had low expression of DHHC2. Kaplan-Meier analysis indicated that a high level of DHHC2 expression predicted favorable overall survival and disease-free survival rates in both the training cohort and validation set. Furthermore, the combination of CKAP4 and DHHC2 was found to have a more powerful efficiency in prognosis prediction than either one alone. CONCLUSIONS: To the best of our knowledge, the current study is the first to demonstrate that the expression of CKAP4 and its palmitoyl acyltransferase DHHC2 correlates with disease progression and metastasis in patients with HCC and may provide prognostic and therapeutic value.