MCL1 inhibition: a promising approach to augment the efficacy of sorafenib in NSCLC through ferroptosis induction.

Ferroptosis, an iron-dependent form of regulated cell death, plays a crucial role in modulating the therapeutic response in non-small cell lung cancer (NSCLC) patients. Studies have identified the signal transducer and activator of transcription 3 (STAT3) and myeloid cell leukemia-1 (MCL1) as potential targets for sorafenib, which exhibits activities in inducing ferroptosis. However, the role of STAT3-MCL1 axis in sorafenib-induced ferroptosis in NSCLC is still unclear. This study provided evidence that ferroptosis is a critical driver of sorafenib-induced cell death in NSCLC, supported by the accumulation of lipid peroxidation products, indicative of oxidative stress-induced cell death. Additionally, both in vitro and in vivo experiments showed that ferroptosis contributed to a significant portion of the anti-cancer effects elicited by sorafenib in NSCLC. The noticeable accumulation of lipid peroxidation products in sorafenib-treated mice underscored the significance of ferroptosis as a contributing factor to the therapeutic response of sorafenib in NSCLC. Furthermore, we identified the involvement of the STAT3/MCL1 axis in sorafenib-induced antitumor activity in NSCLC. Mechanistically, sorafenib inhibited endogenous STAT3 activation and downregulated MCL1 protein expression, consequently unleashing the ferroptosis driver BECN1 from the BECN1-MCL1 complex. Conversely, there is an augmented association of BECN1 with the catalytic subunit of system Xc-, SLC7A11, whose activity to import cystine and alleviate lipid peroxidation is hindered upon its binding with BECN1. Notably, we found that MCL1 upregulation correlated with ferroptosis resistance in NSCLC upon sorafenib treatment. Our findings highlight the importance of sorafenib-triggered ferroptosis in NSCLC and offer a novel strategy to treat advanced NSCLC patients: by downregulating MCL1 and, in turn, predispose NSCLC cells to ferroptosis.

Comparison of Humoral Antibody Responses and Seroconversion Rates between Two Homologous ChAdOx1 nCoV-19 and mRNA-1273 Vaccination in Patients Undergoing Maintenance Hemodialysis.

BACKGROUND: Hemodialysis patients are at an increased risk of SARS-CoV-2 infection and are excluded from preauthorization COVID-19 vaccine trials; therefore, their immunogenicity is uncertain. METHODS: To compare the antibody responses to homologous ChAdOx1 and mRNA-1273 SARS-CoV-2 vaccination in hemodialysis patients, 103 age- and sex-matched hemodialysis patients with two homologous prime-boost vaccinations were recruited to detect anti-receptor-binding domain (RBD) IgG levels and seroconversion rates (SCRs) 14 days after a prime dose (PD14), before and 28 days after a boost dose (pre-BD0 and BD28). RESULTS: Both mRNA-1273 and ChAdOx1 vaccinations elicited immunogenicity in study subjects, and the former induced higher anti-RBD IgG levels than the latter. The SCRs of both groups increased over time and varied widely from 1.82% to 97.92%, and were significantly different at PD14 and pre-BD0 regardless of different thresholds. At BD28, the SCRs of the ChAdOx1 group and the mRNA-1273 group were comparable using a threshold ≥ 7.1 BAU/mL (93.96% vs. 97.92%) and a threshold ≥ 17 BAU/mL (92.73% vs. 97.92%), respectively, but they were significantly different using a threshold ≥ 20.2% of convalescent serum anti-RBD levels (52.73% vs. 95.83%). The seroconversion (≥20.2% of convalescent level) at BD28 was associated with mRNA-1273 vaccination after being adjusted for age, sex, body mass index, and the presence of solicited reactogenicity after a prime vaccination. CONCLUSION: Our prospective, observational cohort indicates that a full prime-boost mRNA-1273 vaccination is likely to provide higher immune protection in hemodialysis patients compared to ChAdOx1, and this population with a prime-boost ChAdOx1 vaccination should be prioritized for a third dose.

Targeting pathogenic macrophages by the application of SHP-1 agonists reduces inflammation and alleviates pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a progressive fibrotic disorder with no cure that is characterized by deterioration of lung function. Current FDA-approved drugs for IPF delay the decline in lung function, but neither reverse fibrosis nor significantly improve overall survival. SHP-1 deficiency results in hyperactive alveolar macrophages accumulating in the lung, which contribute to the induction of pulmonary fibrosis. Herein, we investigated whether employing a SHP-1 agonist ameliorates pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis murine model. Histological examination and micro-computed tomography images showed that SHP-1 agonist treatment alleviates bleomycin-induced pulmonary fibrosis. Reduced alveolar hemorrhage, lung inflammation, and collagen deposition, as well as enhanced alveolar space, lung capacity, and improved overall survival were observed in mice administered the SHP-1 agonist. The percentage of macrophages collected from bronchoalveolar lavage fluid and circulating monocytes in bleomycin-instilled mice were also significantly reduced by SHP-1 agonist treatment, suggesting that the SHP-1 agonist may alleviate pulmonary fibrosis by targeting macrophages and reshaping the immunofibrotic niche. In human monocyte-derived macrophages, SHP-1 agonist treatment downregulated CSF1R expression and inactivated STAT3/NFκB signaling, culminating in inhibited macrophage survival and perturbed macrophage polarization. The expression of pro-fibrotic markers (e.g., MRC1, CD200R1, and FN1) by IL4/IL13-induced M2 macrophages that rely on CSF1R signaling for their fate-determination was restricted by SHP-1 agonist treatment. While M2-derived medium promoted the expression of fibroblast-to-myofibroblast transition markers (e.g., ACTA2 and COL3A1), the application of SHP-1 agonist reversed the transition in a dose-dependent manner. Our report indicates that pharmacological activation of SHP-1 ameliorates pulmonary fibrosis via suppression of CSF1R signaling in macrophages, reduction of pathogenic macrophages, and the inhibition of fibroblast-to-myofibroblast transition. Our study thus identifies SHP-1 as a druggable target for the treatment of IPF, and suggests that the SHP-1 agonist may be developed as an anti-pulmonary fibrosis medication that both suppresses inflammation and restrains fibroblast-to-myofibroblast transition.

Evaluating the biological effectiveness of boron neutron capture therapy by using microfluidics-based pancreatic tumor spheroids.

Background: The recent success of boron neutron capture therapy (BNCT) for cancer treatment has attracted considerable attention. Because irradiated neutrons penetrate deep into solid tumor tissue, BNCT efficacy is strongly influenced by cell pathophysiology in tumors. The tumor microenvironment critically influences tumor pathophysiology, but its effects on BNCT remain unexplored. Methods: We used a pancreatic tumor as a model to develop a high-throughput 3D tumor spheroid platform for evaluating BNCT efficacy under different microenvironment conditions. We expanded our system to serve as a transwell-like device in order to investigate the influence of stromal fibroblasts in the tumor microenvironment. Results: With the use of the proposed microfluidic chip and a laboratory pipette, more than 40 spheroids with controllable diameters (standard deviation <10%) could be cultured on a chip for more than 10 days. The response to BNCT from each spheroid can be monitored in real time. By using pancreatic tumor spheroids of two different diameters, we found that large spheroids, characterized by more hypoxic microenvironments, exhibited lower BNCT susceptibility. The cells in the hypoxic region expressed the HIF1-α signal, which is crucial in many therapeutic resistance signal pathways. In addition, the heterogeneous presence of stemness markers (Oct-4, Sox-2, and CD 44) implied that the underlying BNCT resistance mechanism was sophisticated. In the presence of fibroblasts, we found an association between ß-catenin nuclear translocation and BNCT resistance; membrane contacts from fibroblasts were found to be indispensable for translocation activation. Conclusions: In summary, by means of easily accessible microfluidic engineering, we developed tumor spheroids to recapture the pathophysiological characteristics of pancreatic tumors. Our data suggest that hypoxia and fibrosis can reduce BNCT efficacy in pancreatic cancer treatment. Considering the growing requirement for drug screening in personalized medicine, our findings and the developed method are expected to improve the fundamental understanding of BNCT and facilitate broad applications of BNCT in clinical settings.

Premorbid use of selective beta-blockers improves sepsis incidence and course: Human cohort and animal model studies.

Introduction: Beta-blockers are widely prescribed to manage hypertension and cardiovascular diseases and have been suggested as an attractive therapy to improve the prognosis of sepsis. Herein, we investigated the potential benefits of premorbid selective beta-blocker use in sepsis with a real-world database and explored the underlying mechanism by in vivo and in vitro experiments. Methods: A total of 64,070 sepsis patients and 64,070 matched controls who were prescribed at least one anti-hypertensive drug for more than 300 days within 1 year were selected for the nested case-control study. Female C57BL/6 J mice and THP-1 cells stimulated with lipopolysaccharide (LPS) were used for studying systemic responses during sepsis to validate our clinical findings. Results: The risk of sepsis was lower in current selective beta-blocker users than in non-users (adjusted OR (aOR), 0.842; 95% CI, 0.755-0.939), and in recent users than in non-users (aOR, 0.773; 95% CI, 0.737-0.810). A mean daily dose of ≥0.5 DDD was associated with a lower risk of sepsis (aOR, 0.7; 95% CI, 0.676-0.725). Metoprolol, atenolol, and bisoprolol users had lower risk of sepsis than non-users. In a LPS-induced sepsis mouse model, mice pre-fed with atenolol had significantly reduced mortality. While atenolol had some mild effects on LPS-induced release of inflammatory cytokines in septic mice, it significantly reduced serum soluble PD-L1 levels. Notably, atenolol treatment reversed the negative correlation of sPD-L1 with inflammatory cytokines in septic mice. Moreover, atenolol markedly downregulated the PD-L1 expression on LPS-stimulated THP-1 monocytes/macrophages via targeting ROS-induced NF-κB and STAT3 activation. Conclusion: Atenolol pretreatment can reduce sepsis mortality in mice, and in vivo and in vitro studies of PD-L1 expression suggest a role for atenolol in the modulation of immune homeostasis. These findings may contribute to the reduced incidence of sepsis in hypertensive patients with premorbid treatment with selective beta-blockers, especially atenolol.

Comparative survival analysis of platinum-based adjuvant chemotherapy for early-stage squamous cell carcinoma and adenocarcinoma of the lung.

BACKGROUND AND PURPOSE: Although cytotoxic platinum-based adjuvant chemotherapy (pACT) has been recommended for patients with completely resected early-stage (ES) non-small-cell lung cancer (ES-NSCLC), therapeutic regimens for NSCLC have evolved in the past two decades. The study was aimed to examine the effectiveness of postoperative pACT for resected ES-NSCLC patients with squamous cell carcinoma (SCC) or adenocarcinoma (ADC) according to real-world data. METHODS AND PATIENTS: Inverse probability treatment weighting (IPTW) was used to adjust baseline characteristics between the group receiving pACT and those not receiving any treatment (observation, OBS) within 3 months after curative surgery. Cox regression models were used to compare overall survival (OS) and treatment failure-free survival (TFS) between the groups. RESULTS: Of 31,208 patients with ES-NSCLC, 4700 undergoing complete tumor resection were eligible, with a mean follow-up period of 4.5 years. The pACT (n = 2347) and OBS (n = 2353) groups were well-balanced after IPTW. The median OS differed between the pACT and OBS groups (77.2 vs. 75.5 months, adjusted hazard ratio [aHR] = 0.87, 95% confidence interval [CI] = 0.79-0.95, p = 0.003), and the 5-year survival rates were 58.2% and 55.3%, respectively (p < 0.001). In the SCC group, pACT was superior to OBS in OS (75.0 vs. 57.4 months, aHR = 0.74, 95% CI = 0.62-0.88, p = 0.001) and TFS (32.7 vs. 21.8 months, aHR = 0.74, 95% CI = 0.63-0.86, p < 0.001). Both OS and TFS did not differ between two groups in those with ADC. CONCLUSION: Real-world data indicated that pACT confers a survival benefit for resected ES-NSCLC patients with SCC but not ADC, which needs to be verified by a large sample of randomized controlled studies.

Humoral Immunogenicity and Reactogenicity of the Standard ChAdOx1 nCoV-19 Vaccination in Taiwan.

BACKGROUND: The ChAdOx1 nCoV-19 vaccine has been widely administered against SARS-CoV-2 infection; however, data regarding its immunogenicity, reactogenicity, and potential differences in responses among Asian populations remain scarce. METHODS: 270 participants without prior COVID-19 were enrolled to receive ChAdOx1 nCoV-19 vaccination with a prime-boost interval of 8-9 weeks. Their specific SARS-CoV-2 antibodies, neutralizing antibody titers (NT50), platelet counts, and D-dimer levels were analyzed before and after vaccination. RESULTS: The seroconversion rates of anti-RBD and anti-spike IgG at day 28 after a boost vaccination (BD28) were 100% and 95.19%, respectively. Anti-RBD and anti-spike IgG levels were highly correlated (r = 0.7891), which were 172.9 ± 170.4 and 179.3 ± 76.88 BAU/mL at BD28, respectively. The geometric mean concentrations (GMCs) of NT50 for all participants increased to 132.9 IU/mL (95% CI 120.0-147.1) at BD28 and were highly correlated with anti-RBD and anti-spike IgG levels (r = 0.8248 and 0.7474, respectively). Body weight index was statistically significantly associated with anti-RBD IgG levels (p = 0.035), while female recipients had higher anti-spike IgG levels (p = 0.038). The GMCs of NT50 declined with age (p = 0.0163) and were significantly different across age groups (159.7 IU/mL for 20-29 years, 99.4 IU/mL for ≥50 years, p = 0.0026). Injection-site pain, fever, and fatigue were the major reactogenicity, which were more pronounced after prime vaccination and in younger participants (<50 years). Platelet counts decreased and D-dimer levels increased after vaccination but were not clinically relevant. No serious adverse events or deaths were observed. CONCLUSION: The vaccine is well-tolerated and elicited robust humoral immunity against SARS-CoV-2 after standard prime-boost vaccination in Taiwanese recipients.

Stabilization of AURKA by the E3 ubiquitin ligase CBLC in lung adenocarcinoma.

CBL family proteins (CBL, CBLB and CBLC in mammals) are E3 ubiquitin ligases of protein tyrosine kinases. CBL mediates the lysosomal degradation of activated EGFR through K63-linked ubiquitination, while CBLC has an oncogenic function by positively regulating EGFR activation through K6 and K11-linked ubiquitination in EGFR mutant lung adenocarcinoma (LAD). Here, we used immunoprecipitation and mass spectrometry to study the CBLC interactome, and found that CBLC is also involved in cell cycle regulation by stabilizing Aurora kinase A (AURKA). CBLC interacted with the kinase domain of AURKA and positively regulated the stability of AURKA by conjugating monoubiquitination and K11/K63-linked polyubiquitination, which are protective from degrading K11/K48 polyubiquitination. CBLC depletion markedly decreased the half-life of AURKA in cycloheximide-treated LAD cells. When LAD cells were synchronized with double thymidine block at the G1/S boundary and then released into mitotic arrest, CBLC depletion delayed the accumulation and activation of AURKA and prevented cancer cells from entering mitosis. CBLC deficiency significantly delayed cell cycle progression, reduced the mitotic population, and increased apoptosis of LAD cells. Targeting CBLC inhibited tumor growth of LAD cells and enhanced their sensitivity to paclitaxel in xenograft models. Immunohistochemical staining of the tissue microarray also revealed a positive correlation between the expression of CBLC and AURKA in normal and LAD tissues, further supporting the positive regulation of AURKA expression by CBLC. In summary, these findings indicate that the oncogenic E3 ligase CBLC plays a role in mitotic entry by stabilizing AURKA via ubiquitination in LAD. This work demonstrates that targeting CBLC combined with paclitaxel might be a potential option for the treatment of LAD patients who have no available targeted therapies.

Tissue Architecture Influences the Biological Effectiveness of Boron Neutron Capture Therapy in In Vitro/In Silico Three-Dimensional Self-Assembly Cell Models of Pancreatic Cancers.

Pancreatic cancer is a leading cause of cancer death, and boron neutron capture therapy (BNCT) is one of the promising radiotherapy techniques for patients with pancreatic cancer. In this study, we evaluated the biological effectiveness of BNCT at multicellular levels using in vitro and in silico models. To recapture the phenotypic characteristic of pancreatic tumors, we developed a cell self-assembly approach with human pancreatic cancer cells Panc-1 and BxPC-3 cocultured with MRC-5 fibroblasts. On substrate with physiological stiffness, tumor cells self-assembled into 3D spheroids, and the cocultured fibroblasts further facilitated the assembly process, which recapture the influence of tumor stroma. Interestingly, after 1.2 MW neutron irradiation, lower survival rates and higher apoptosis (increasing by 4-fold for Panc-1 and 1.5-fold for BxPC-3) were observed in 3D spheroids, instead of in 2D monolayers. The unexpected low tolerance of 3D spheroids to BNCT highlights the unique characteristics of BNCT over conventional radiotherapy. The uptake of boron-containing compound boronophenylalanine (BPA) and the alteration of E-cadherin can partially contribute to the observed susceptibility. In addition to biological effects, the probability of induced α-particle exposure correlated to the multicellular organization was speculated to affect the cellular responses to BNCT. A Monte Carlo (MC) simulation was also established to further interpret the observed survival. Intracellular boron distribution in the multicellular structure and related treatment resistance were reconstructed in silico. Simulation results demonstrated that the physical architecture is one of the essential factors for biological effectiveness in BNCT, which supports our in vitro findings. In summary, we developed in vitro and in silico self-assembly 3D models to evaluate the effectiveness of BNCT on pancreatic tumors. Considering the easy-access of this 3D cell-assembly platform, this study may not only contribute to the current understanding of BNCT but is also expected to be applied to evaluate the BNCT efficacy for individualized treatment plans in the future.

Alisertib inhibits migration and invasion of EGFR-TKI resistant cells by partially reversing the epithelial-mesenchymal transition.

Epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used in the clinical treatment of non-small cell lung cancer (NSCLC) patients with EGFR mutations. Previous studies have shown that Aurora kinase A (AURKA) is overexpressed in a broad spectrum of cancer cells, which can induce epithelial-mesenchymal transition (EMT) and contribute to the occurrence of acquired EGFR-TKI resistance. However, whether the inhibition of AURKA could overcome EGFR-TKI resistance or reverse the EMT in TKI-resistant NSCLC cells remains unclear. In the current study, we established three EGFR-TKI-resistant cell lines and analyzed their expression profiles by RNA sequencing. The results revealed that the EMT pathway is significantly upregulated in the three cell lines with EGFR-TKI resistance. The phosphorylation of AURKA at Thr 288 was also upregulated, suggesting that the activation of AURKA plays an important role in the occurrence of EGFR-TKI resistance. Interestingly, the AURKA inhibitor, alisertib treatment restored the susceptibility of resistant cells to EGFR-TKIs and partially reversed the EMT process, thereby reducing migration and invasion in EGFR-TKI-resistant cells. This study provides evidence that targeting AURKA signaling pathway by alisertib may be a novel approach for overcoming EGFR-TKI resistance and for the treatment of metastatic EGFR-TKIs in NSCLC patients.

Allogeneic human umbilical cord-derived mesenchymal stem cells reduce lipopolysaccharide-induced inflammation and acute lung injury.

Acute lung injury (ALI) is the clinical disorder of acute hypoxemic respiratory deficiency and it is associated with a high mortality rate. Increased lung permeability, infiltration of inflammatory cells, secretion of inflammatory cytokines, and pulmonary edema are hallmarks of ALI. Currently, there is no effective pharmacological agent approved for ALI, and the treatment regimens available are mostly supportive. Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunomodulating potential, which therefore hold great promise for the treatment of ALI. We established an LPS-induced ALI mouse model by intratracheal injection of lipopolysaccharide (LPS). Human umbilical cord-derived MSCs (hUC-MSCs) were delivered through the tail vein to assess the effects of MSCs on relieving LPS-induced ALI. Intratracheal injection of LPS increased the infiltration of neutrophils and enhanced the expression of pro-inflammatory cytokines, such as IL-6, IL-1ß and TNF-α. Administration of hUC-MSCs decreased pathological signs of inflammation, as well as reduced ALI scores. The levels of IL-6, IL-1ß and TNF-α were also dose-dependently inhibited in the bronchoalveolar lavage fluids from damaged lung tissues. Moreover, MPO and BAX levels were decreased by the hUC-MSC treatment, suggesting hUC-MSCs may play the role in inhibiting ROS production and apoptotic death in ALI repair. These results highlight the potential of hUC-MSCs to alleviate bacterial endotoxin-induced inflammation, and may represent an effective modality for the treatment of ALI in clinical settings.

Identification of subcellular targeting sequences of Cten reveals its role in cell proliferation.

Spatial and temporal subcellular localization plays critical roles in regulating protein function. Cten (C-terminal tensin like) is a member of the tensin family. Cten recruits signaling molecules, such as DLC1, to focal adhesions, modulates homeostasis of receptor tyrosine kinases, including EGFR and c-Met, and promotes cell migration. These functions are likely controlled by Cten localization at focal adhesions and/or in the cytoplasm. In addition, Cten has been detected in the nucleus by which mechanism is unknown. To this end, we have examined the distribution of Cten in various cell lines, determined primary sequence requirements for its nuclear and focal adhesion localizations, and analyzed potential roles of nuclear Cten. Our results show that a proportion of Cten translocates to nuclei in cancer cell lines and that nuclear exporting of Cten is a CRM1-dependent process. A nuclear localization sequence and a nuclear export sequence are identified within Cten. In addition, like other tensins, Cten contains two independent focal adhesion binding sites. Although further expression of recombinant Cten showed no effect on cancer cell proliferation, silencing of Cten significantly reduced cell growth. Furthermore, expression of Cten mutants either with defective nuclear export sequence or tagged with SV40 nuclear localization sequence promoted cell growth. These results suggest that nuclear Cten contributes to cancer cell proliferation. Our findings identify a molecular mechanism for regulating Cten protein trafficking in mammalian cells and provide new insights into the dynamics of focal adhesion complexes in health and disease.

Upregulation of E3 Ubiquitin Ligase CBLC Enhances EGFR Dysregulation and Signaling in Lung Adenocarcinoma.

CBLC (CBL proto-oncogene c) belongs to the CBL protein family, which has E3 ubiquitin ligase activity toward activated receptor tyrosine kinases. CBLC is frequently upregulated in non-small cell lung cancer (NSCLC), yet very little is known about the functions of CBLC in tumorigenesis. Here we show that CBLC is an epigenetically demethylated target and its expression can be upregulated in NSCLC after treatment with the DNA methylation inhibitor 5'-azacytidine. Depletion of CBLC significantly inhibited cell viability and clonogenicity in vitro and reduced tumor growth in a xenograft model. CBLC silencing further sensitized EGFR-mutated NSCLC cells to treatment with tyrosine kinase inhibitors. Conversely, ectopic expression of CBLC enhanced the activation of EGFR and downstream ERK1/2 signaling after ligand stimulation by competing with CBL for EGFR binding. Analysis of ubiquitin linkages on activated EGFR (aEGFR) revealed that CBLC ubiquitinated and positively regulated aEGFR stability through the conjugation of polyubiquitin by K6 and K11 linkages. This CBLC-mediated polyubiquitination promoted either preferential recycling of aEGFR back to the plasma membrane or trafficking to the cell nucleus. IHC analyses revealed a positive correlation between phospho-EGFR and CBLC in lung adenocarcinoma. In summary, we demonstrate a novel mechanism by which aEGFR escapes lysosomal degradation in a CBLC/ubiquitin-dependent manner to sustain its activation. Our work identifies CBLC as a potential diagnostic biomarker and also points to its utilization as a novel therapeutic target for NSCLC therapy.Significance: This work demonstrates the role of CBLC expression as a diagnostic biomarker and potential therapeutic target in lung adenocarcinoma. Cancer Res; 78(17); 4984-96. ©2018 AACR.

Down-regulation of tensin2 enhances tumorigenicity and is associated with a variety of cancers.

Tensin family members, including tensin2 (TNS2), are present as major components of the focal adhesions. The N-terminal end of TNS2 contains a C1 region (protein kinase C conserved region 1) that is not found in other tensin members. Three isoforms of TNS2 have been identified with previous reports describing the shortest V3 isoform as lacking the C1 region. Although TNS2 is known to regulate cell proliferation and migration, its role in tumorigenicity is controversial. By gain-of-function overexpression approaches, results supporting either promotion or reduction of cancer cell tumorigenicity were reported. Here we report that the complete V3 isoform also contains the C1 region and describe the expression patterns of the three human TNS2 isoforms. By loss-of-function approaches, we show that silencing of TNS2 up-regulates the activities of Akt, Mek, and IRS1, and increases tumorigenicities in A549 and Hela cells. Using public database analyses we found that TNS2 is down-regulated in head and neck, esophageal, breast, lung, liver, and colon cancer. In addition, patients with low TNS2 expression showed poor relapse-free survival rates for breast and lung cancers. These results strongly suggest a role of tensin2 in suppressing cell transformation and reduction of tumorigenicity.

CTEN prolongs signaling by EGFR through reducing its ligand-induced degradation.

Activation of EGF receptor (EGFR) triggers signaling pathways regulating various cellular events that contribute to tissue development and function. Aberrant activation of EGFR contributes to tumor progression as well as therapeutic resistance in patients with cancer. C-terminal tensin-like (CTEN; TNS4) is a focal adhesion molecule that is a member of the tensin family. Its expression is upregulated by EGF and elevated CTEN mediates EGF-induced cell migration. In the presence of CTEN, we found that EGF treatment elevated the level of EGFR protein but not mRNA. The extended half-life of activated EGFR sustained its signaling cascades. CTEN reduced ligand-induced EGFR degradation by binding to the E3 ubiquitin ligase c-Cbl and decreasing the ubiquitination of EGFR. The Src homology 2 domain of CTEN is not only required for binding to the phosphorylated tyrosine residue at codon 774 of c-Cbl, but is also essential for the tumorigenicity observed in the presence of CTEN. Public database analyses indicated that CTEN mRNA levels are elevated in breast, colon, lung, and pancreas cancers, but not correlated with EGFR mRNA levels in these cancers. In contrast, immunohistochemistry analyses of lung cancer specimens showed that CTEN and EGFR protein levels were positively associated, in support of our finding that CTEN regulates EGFR protein levels through a posttranslational mechanism. Overall, this work defines a function for CTEN in prolonging signaling from EGFR by reducing its ligand-induced degradation.

Phylogenetic analysis, expression patterns, and transcriptional regulation of human CTEN gene.

Cten is a focal adhesion molecule and a member of the tensin family. Its expression is highly enriched in the prostate and placenta, suggesting that cten gene might be closely associated with mammalian species. Recent studies have reported that cten expression is frequently up-regulated in a variety of cancers and its levels appear to correlate with tumorigenicity. Here, we have (1) analyzed cten sequences of various species to build a phylogenetic tree, (2) examined cten mRNA levels in human and mouse tissues to establish its expression profiles, and (3) determined the promoter region of human CTEN gene in cell lines and in a mouse model to understand its transcriptional regulation. Our analyses indicate that all currently known cten genes are present in mammals. The prostate and placenta are the two most cten abundant tissues in human and mouse, meanwhile brain and lung also express low levels of cten. Results from cell culture reporter assays demonstrate that a 327-bp fragment is the shortest functional promoter. All functional promoter constructs produce 40- to 160-fold increases in luciferase reporter activities in normal prostate cells, whereas lower activities (<40-fold) are detected in non-prostatic cell lines. To evaluate CTEN promoter activity in mice and develop a new tissue specific Cre recombinase mouse model, we have established pCTEN-Cre:R26R mice by crossing R26R ß-galactosidase reporter mice with pCTEN-Cre transgenic mice, in which the 327-bp cten promoter drives the expression of Cre recombinase. X-gal analysis has shown strong ß-galactosidase activities in the prostate, brain, and few other tissues in pCTEN-Cre:R26R mice. Altogether, we have identified the promoter region of human cten gene and provided a useful tool for investigating cell lineages and generating tissue-specific knockout or knockin mice.

Phosphorylation of serine 177 of the small hepatitis delta antigen regulates viral antigenomic RNA replication by interacting with the processive RNA polymerase II.

Recent studies revealed that posttranslational modifications (e.g., phosphorylation and methylation) of the small hepatitis delta antigen (SHDAg) are required for hepatitis delta virus (HDV) replication from antigenomic to genomic RNA. The phosphorylation of SHDAg at serine 177 (Ser(177)) is involved in this step, and this residue is crucial for interaction with RNA polymerase II (RNAP II), the enzyme assumed to be responsible for antigenomic RNA replication. This study demonstrated that SHDAg dephosphorylated at Ser(177) interacted preferentially with hypophosphorylated RNAP II (RNAP IIA), which generally binds at the transcription initiation sites. In contrast, the Ser(177)-phosphorylated counterpart (pSer(177)-SHDAg) exhibited preferential binding to hyperphosphorylated RNAP II (RNAP IIO). In addition, RNAP IIO associated with pSer(177)-SHDAg was hyperphosphorylated at both the Ser(2) and Ser(5) residues of its carboxyl-terminal domain (CTD), which is a hallmark of the transcription elongation isoform. Moreover, the RNAP II CTD kinase inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) not only blocked the interaction between pSer(177)-SHDAg and RNAP IIO but also inhibited HDV antigenomic RNA replication. Our results suggest that the phosphorylation of SHDAg at Ser177 shifted its affinitytoward the RNAP IIO isoform [corrected] and thus is a switch for HDV antigenomic RNA replication from the initiation to the elongation stage.

ERK1/2-mediated phosphorylation of small hepatitis delta antigen at serine 177 enhances hepatitis delta virus antigenomic RNA replication.

The small hepatitis delta virus (HDV) antigen (SHDAg) plays an essential role in HDV RNA double-rolling-circle replication. Several posttranslational modifications (PTMs) of HDAgs, including phosphorylation, acetylation, and methylation, have been characterized. Among the PTMs, the serine 177 residue of SHDAg is a phosphorylation site, and its mutation preferentially abolishes HDV RNA replication from antigenomic RNA to genomic RNA. Using coimmunoprecipitation analysis, the cellular kinases extracellular signal-related kinases 1 and 2 (ERK1/2) are found to be associated with the Flag-tagged SHDAg mutant (Ser-177 replaced with Cys-177). In an in vitro kinase assay, serine 177 of SHDAg was phosphorylated directly by either Flag-ERK1 or Flag-ERK2. Activation of endogenous ERK1/2 by a constitutively active MEK1 (hemagglutinin-AcMEK1) increased phosphorylation of SHDAg at Ser-177; this phosphorylation was confirmed by immunoblotting using an antibody against phosphorylated S177 and mass spectrometric analysis. Interestingly, we found an increase in the HDV replication from antigenomic RNA to genomic RNA but not in that from genomic RNA to antigenomic RNA. The Ser-177 residue was critical for SHDAg interaction with RNA polymerase II (RNAPII), the enzyme proposed to regulate antigenomic RNA replication. These results demonstrate the role of ERK1/2-mediated Ser-177 phosphorylation in modulating HDV antigenomic RNA replication, possibly through RNAPII regulation. The results may shed light on the mechanisms of HDV RNA replication.