Binding of the host protein cyclophilin A (CypA) to the viral capsid exerts multiple effects on HIV-1 infection, including enhancement of reverse transcription, stabilization of the capsid, and promotion of nuclear entry. CypA can also inhibit infection of selected HIV-1 mutants by a poorly understood mechanism. Using atomic force microscopy methods, we previously showed that HIV-1 cores are highly elastic and that mutants with reduced capsid elasticity are impaired for nuclear entry and infection of nondividing cells. Here we demonstrate that binding of CypA to the capsids of such mutants inhibits their nuclear entry by further reducing the elasticity of their capsids. These effects were reversed by suppressor mutations that restored elasticity to the mutant capsids. Our results define the mechanism by which CypA controls HIV-1 nuclear entry. We hypothesize that nuclear entry involves temporal modulation of capsid elasticity by host proteins prior to and during passage through the nuclear pore.

Binding of the host protein cyclophilin A (CypA) to the HIV-1 capsid exerts a variety of effects on infection, including enhancement of reverse transcription, stabilization of the capsid, and promotion of nuclear entry. For several HIV-1 mutants, CypA binding inhibits nuclear entry by an unknown mechanism. We recently demonstrated that HIV-1 cores are elastic and that HIV-1 mutants with inelastic capsids are impaired for nuclear entry and infection of nondividing cells. Here we show that CypA prevents infection of nondividing cells by such mutants and inhibits their entry into the nucleus. CypA binding to mutant cores further reduced their elasticity in vitro, and this effect was reversed by suppressor mutations that restored nuclear entry. We suggest that HIV-1 nuclear entry involves temporal modulation of capsid elasticity by host proteins prior to and during traversal of the nuclear pore.

HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacapavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.

The viral capsid performs critical functions during HIV-1 infection and is a validated target for antiviral therapy. Previous studies have established that the proper structure and stability of the capsid are required for efficient HIV-1 reverse transcription in target cells. Moreover, it has recently been demonstrated that permeabilized virions and purified HIV-1 cores undergo efficient reverse transcription in vitro when the capsid is stabilized by addition of the host cell metabolite inositol hexakisphosphate (IP6). However, the molecular mechanism by which the capsid promotes reverse transcription is undefined. Here we show that wild type HIV-1 virions can undergo efficient reverse transcription in vitro in the absence of a membrane-permeabilizing agent. This activity, originally termed “natural endogenous reverse transcription” (NERT), depends on expression of the viral envelope glycoprotein during virus assembly and its incorporation into virions. Truncation of the gp41 cytoplasmic tail markedly reduced NERT activity, suggesting that gp41 licenses the entry of nucleotides into virions. By contrast to reverse transcription in permeabilized virions, NERT required neither the addition of IP6 nor a mature capsid, indicating that an intact viral membrane can substitute for the function of the viral capsid during reverse transcription in vitro. Collectively, these results demonstrate that the viral capsid functions as a nanoscale container for reverse transcription during HIV-1 infection.

Supplementary File containing supplementary Table 1 and five supplementary figures.

<p>Supplementary Methods</p>

PDF file - 215K, t-DARPP enhances ERBB2 expression on the cell surface Supplemental Figure 2. t-DARPP decreases binding of trastuzumab to ERBB2 receptor

PDF file - 696K, Real-time RT-PCR data

AXL receptor tyrosine kinase promotes an invasive phenotype and chemotherapy resistance in esophageal adenocarcinoma (EAC). AXL has been implicated in the regulation of autophagy, but the underlying molecular mechanism remains poorly understood. Herein, we investigate the mechanistic role of AXL in autophagy as well as metformin-induced effects on the growth and survival of EAC. We demonstrate that AXL mediates autophagic flux through activation of AMPK-ULK1 signaling in a reactive oxygen species (ROS)-dependent mechanism by glucose starvation. AXL positively regulates basal cellular ROS levels without significantly affecting mitochondrial ROS production in EAC cells. Pharmacological inhibition of cellular ROS using Trolox abrogates glucose starvation-induced AMPK signaling and autophagy. We demonstrate that AXL expression is required for metformin-induced apoptosis in EAC cells in vitro. The apoptosis induction by metformin is markedly attenuated by inhibition of autophagy through genetic silencing of Beclin1 or ATG7 autophagy mediators, thereby confirming the requirement of intact autophagy for enhancing metformin-induced apoptosis in EAC cells. Our data indicate that metformin-induced autophagy displays a pro-apoptotic function in EAC cells. We show that the metformin-induced suppression of tumor growth in vivo is highly dependent on AXL expression in a tumor xenograft mouse model of EAC. We demonstrate that AXL promotes metformin-induced apoptosis through activation of autophagy in EAC. AXL may be a valuable biomarker to identify tumors that are sensitive to metformin. Therefore, AXL expression could inform the selection of patients for future clinical trials to evaluate the therapeutic efficacy of metformin in EAC.

Background: Esophageal adenocarcinoma (EAC) is highly aggressive and characterized by poor prognosis. AXL expression has been linked to Barrett's tumorigenesis and resistance to chemotherapy, which is associated with c-ABL intracellular localization. However, the molecular and functional relationship between AXL and c-ABL and the clinical significance of the co-expression of these proteins in EAC remain unclear. Methods: We used immunohistochemical analysis (IHC) on tissue microarrays containing human EAC samples (n=53) and normal esophageal tissues (n=11) in combination with corresponding deidentified clinicopathological information to evaluate the expression and the prognostic significance of AXL and c-ABL in EAC. The data were statistically analyzed using Kruskal-Wallis, the chi-square, the Fisher's exact, and Pearson tests. The Kaplan-Meier method and Cox proportional hazards regression model were used to evaluate cancer patient survival. We used a serum deprivation EAC cell model to investigate the pro-survival function of AXL and c-ABL using cell viability, apoptosis, and lactate dehydrogenase activity assays. We performed in vitro assays, including Western blotting, quantitative real-time PCR, and translational chromatin immunoprecipitation (TrIP-Chip) to study the molecular relationship between AXL and c-ABL in EAC cells. Results: IHC analysis revealed that AXL and c-ABL were overexpressed in 55% and 66% of EAC samples, respectively, as compared to normal tissues. Co-overexpression of the two proteins was observed in 49% of EAC samples. The chi-square test indicated a significant association between AXL and c-ABL expression in the EAC samples (χ2 = 6.873, p = 0.032), and the expression of these proteins was significantly associated with EAC patient age (p < 0.001), tumor stage (p < 0.01), and lymph node status (p < 0.001). AXL and c-ABL protein expression data analysis exhibited an identical clinicopathological association profile. Additionally, we found a significant association between expression of AXL (χ2 = 16.7, p = 0.002) or c-ABL (χ2 = 13.4, p = 0.001) and survival of EAC patients. The Cox proportional hazards model and log rank test predicted a significant increase in mortality of patients with high expression of AXL [hazard ratio (HR): 2.86, 95% confidence interval (CI): 1.53 - 5.34, p = 0.003] or c-ABL [HR: 3.29, 95% CI: 1.35 - 8.03, p = 0.001] as compared to those patients with low expression of AXL or c-ABL proteins. Molecular investigations indicated that AXL positively regulates c-ABL protein expression through increased cap-dependent protein translation involving phosphorylation of EIF4E in EAC cells. Next, we investigated the functional relationship between AXL and c-ABL in EAC cells. We demonstrated that the pro-survival activity of AXL requires c-ABL expression in response to serum deprivation. Conclusion: This study highlights the importance of the co-overexpression of AXL and c-ABL proteins as a valuable prognostic biomarker and targeting these proteins could be an effective therapeutic approach in EAC or other solid tumors expressing high levels of AXL and c-ABL proteins.

Abstract Esophageal adenocarcinoma (EAC) is an aggressive malignancy with poor clinical outcome. The incidence of EAC has been rising rapidly in the past three decades. Here, we showed that apurinic/apyrimidinic endonuclease (APE1) is overexpressed in EAC cell lines, and patients' samples of dysplasia and EAC. Downregulation of APE1 or inhibition of its redox function significantly repressed invasion. Overexpression of a redox-defective mutant, C65A, abrogated the proinvasive phenotype of APE1. APE1 regulated invasion via upregulation of matrix metalloproteinase 14 (MMP-14), which subsequently activated MMP-2, leading to degradation of the extracellular matrix in a redox-dependent manner. Downregulation of APE1 or inhibition of its redox function decreased the rate of endocytosis and recycling of MMP-14 protein. APE1 interacted with ARF6, a key regulator of MMP-14 recycling, which maintained ARF6 activity in an APE1-redox–dependent manner, promoting its ability to regulate MMP-14 recycling to the cell surface. In summary, these findings identify a novel redox-sensitive APE1–ARF6–MMP-14 signaling axis that mediates cellular invasion in esophageal carcinogenesis. Significance: This study demonstrates the association between oxidative stress and the development and metastatic behavior of esophageal adenocarcinoma.

The endoplasmic reticulum stress (ERS)-induced autophagy and apoptosis are favorable for the suppression of many cancer types. Salidroside (Salid) has been proven to be capable of inducing the apoptosis of many cancer cells. However, the underlying mechanisms and whether Salid can activate the autophagic system have still not been explained thoroughly. Herein, the inhibition effect of Salid on the growth and progress of gastric cancer and the underlying mechanisms were investigated. With the SGC-7901 cells acting as the cancer model cells, we ascertained that Salid exerted a superior antagonism effect on the growth and migration of gastric cancer cells in a dose-dependent manner. Additionally, Salid exhibited strong capacity to induce cell apoptosis by the down-regulation of proliferation-related genes (Ki67 and PCNA), increase in the pro-apoptotic protein C-caspase-3, and changing the levels of other related genes. A mechanism study revealed that the levels of the ERS-related genes, such as CHOP, C-caspase-12, GADD34, and BiP, in the SGC-7901 cells dramatically changed post-treatment by Salid, indicating the involvement of ERS in Salid-inducing cell apoptosis. In addition, the increased LC3(+) autophagic vacuoles, enhanced conversion of LC3-I to LC3-II, and inhibition of the PI3K/Akt/mTOR pathway further confirmed the activation of autophagy induced by Salid. Importantly, the effect of Salid in regulating the levels of autophagy-related proteins or the signaling pathway could be markedly depressed by co-incubating with Wortmannin (Wort), an autophagy inhibitor. The final evaluation of the tumor therapy efficacy exhibited satisfactory cancer growth inhibition by Salid with negligible toxicity to normal tissues. In summary, the present work provides a comprehensive effective evaluation of Salid for treating gastric cancer. The detailed investigation of the underlying mechanisms may offer a rational reference for the future applications of Salid in clinic.

Background.Oncogenic BRAF V600E mutation occurs in ~8% of human colorectal cancers (CRC), but is highly enriched in tumors with microsatellite instability (MSI) and CIMP (includes epigenetic inactivation of MLH1, p16Ink4a).BRAF V600E constitutively activates MEK/ EKR signaling and is associated with poor prognosis in non MSI CRCs and in MSI tumors with recurrence or metastasis.In contrast to melanoma where BRAF V600E and MEK inhibitors are efficacious, selective inhibition of BRAF V600E in CRCs is ineffective due, in part, to reactivation of MEK/ERK signaling.Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in human BRAF V600E CRCs.We determined if CDK1 confers apoptosis resistance and whether its inhibition can enhance the efficacy of MEK inhibition in BRAF V600E CRC cells and tumor xenografts.Methods.BRAF (HT-29, RKO, VACO432, WiDr) or KRAS mutant (DLD1, SW620) and wild-type (DiFi) human CRC lines utilized as were isogenic RKO [A19 ( BRAF V600E /-/-), T29 (BRAF WT /-/-)] and VACO432 [parental (BRAF V600E /-), VT1 (BRAF WT /-)] CRC cells.Gene expression manipulated by siRNA or ectopic expression.Cells treated w/ the CDK1 selective inhibitor RO-3306 or dinaciclib (CDK1,2,5,9 inhibitor) ± the MEK inhibitor cobimetinib.Apoptotic signaling was analyzed by immunoblotting and quantified by Annexin V staining and flow cytometry.Long-term cell survival was evaluated by clonogenic assay.In vivo anti-tumor effects were determined in a murine flank xenograft model.Results.Multiple BRAF V600ECRC lines expressed CDK1 whose genetic/ pharmacological inhibition by RO-3306 or dinaciclib sensitized them to apoptosis shown by annexin V labeling.Cells isogenic for BRAF V600E or with ectopic BRAF V600E conferred resistance to CDK inhibitors, as did cells w/ ectopic MEK.The combination of RO-3306 or dinaciclib with cobimetinib cooperatively enhanced apoptosis and reduced clonogenic survival vs monotherapy.In a CRC xenograft model, dinaciclib + cobimetinib produced significantly greater tumor growth inhibition than did either drug alone.Analysis of xenograft tumors showed increased caspase cleavage and reduced Ki-67 staining to a greater extent with the drug combination compared to monotherapy or control.To establish CDK1 as a therapeutic target, we analyzed TCGA datasets which revealed CDK1 overexpression in human CRCs vs normal colonic tissue with an observed inverse relationship between CDK1 and p16 mRNA expression.To confirm this finding experimentally, we ectopically expressed p16Ink4a into RKO CRC cells and found reduced CDK1 protein expression.Conclusion.CDK1 is a novel mediator of apoptosis resistance in BRAF V600E CRCs whose combined targeting with CDK and MEK/ERK inhibitors potently suppressed tumor growth in a murine model.These data support the therapeutic evaluation of this combination in patients with BRAF V600E CRCs.

Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that is characterized by resistance to chemotherapy and a poor clinical outcome. The overexpression of the receptor tyrosine kinase AXL is frequently associated with unfavorable prognosis in EAC. Although it is well documented that AXL mediates cancer cell invasion as a downstream effector of epithelial-to-mesenchymal transition, the precise molecular mechanism underlying this process is not completely understood. Herein, we demonstrate for the first time that AXL mediates cell invasion through the regulation of lysosomes peripheral distribution and cathepsin B secretion in EAC cell lines. Furthermore, we show that AXL-dependent peripheral distribution of lysosomes and cell invasion are mediated by extracellular acidification, which is potentiated by AXL-induced secretion of lactate through AKT-NF-κB–dependent MCT-1 regulation. Our novel mechanistic findings support future clinical studies to evaluate the therapeutic potential of the AXL inhibitor R428 (BGB324) in highly invasive EAC.

to recurrence of symptoms.Conclusions: This is the first prospective study of cyclosporine and vedolizumab in steroid-refractory severe UC patients.We demonstrate significant effectiveness and safety of this treatment on week 10 after vedolizumab was started.Further trials are warranted.

AXL receptor tyrosine kinase is overexpressed in esophageal adenocarcinoma ( EAC ) and several other types of malignancies; hence, it may be a valuable therapeutic target. Herein, we investigated the role of AXL in regulating c‐ MYC expression and resistance to the chemotherapeutic agent epirubicin in EAC . Using in vitro EAC cell models, we found that AXL overexpression enhances epirubicin resistance in sensitive cells. Conversely, genetic knockdown or pharmacological inhibition of AXL sensitizes resistant cells to epirubicin. Notably, we showed that inhibition or knockdown of c‐ MYC markedly sensitizes AXL ‐dependent resistant cells to epirubicin, and our data demonstrated that AXL promotes epirubicin resistance through transcriptional upregulation of c‐ MYC . We showed that AXL overexpression significantly increased transcriptional activity, mRNA , and protein levels of c‐ MYC . Conversely, AXL knockdown reversed these effects. Mechanistic investigations indicated that AXL upregulates c‐ MYC expression through activation of the AKT /β‐catenin signaling pathway. Data from a tumor xenograft mouse model indicated that inhibition of AXL with R428 in combination with epirubicin synergistically suppresses tumor growth and proliferation. Our results demonstrate that AXL promotes epirubicin resistance through transcriptional upregulation of c‐ MYC in EAC . Our findings support future clinical trials to assess the therapeutic potential of R428 in epirubicin‐resistant tumors with overexpression of AXL and activation of c‐ MYC .

+mice and CRC cells and to elucidate the effects of miR-155 on WNT cascade in a context of constitutive WNT signaling activation.Methods: miR-155-5p levels were measured by quantitative real time PCR in normal mucosa and small colonic adenomas (< 5 mm) of FAP patients (n=6), in small intestinal tumors of Apc Min/+ mice (n=4) and CRC cell lines with different APC mutations.AXIN1 and AXIN2 were assayed by quantitative real time PCR in FAP patients.Cell counting, colony forming assay and cleaved CASPASE-3 staining were performed to evaluate the effect of miR-155-5p overexpression on cell survival, growth and apoptosis in the APC mutant CRC cell lines SW480 and DLD-1.The effects of miR-155-5p up-regulation alone or in combination with the canonical WNT ligand WNT3a on WNT/ β-CATENIN components were also characterized using western blotting (AXIN1, phosphoβ-CATENIN, β-CATENIN, phospho-GSK3β, GSK3β and TCF-4) and quantitative real time PCR (CYCLIND1, C-MYC, AXIN2 and TCF-1), on SW480 and DLD-1.Results: APC mutations were associated with low levels of miR-155-5p in normal mucosa and small colonic adenomas of FAP patients compared with healthy subjects and in small intestinal tumors of Apc Min/+ mice respect to the normal mucosa.miR-155-5p down-regulation was also confirmed in the APC-mutant CRC cell lines.Interestingly, mRNA levels of AXIN1 and AXIN2 resulted significantly increased in patients with FAP showing reduced expression of miR-155-5p.Induction of miR-155-5p in SW480 and DLD-1 cell lines was associated with reduced cell survival and clonogenic capability.Moreover, miR-155 transfected DLD-1 displayed increased cleaved CASPASE-3 levels.We also found that miR-155-5p acted as a critical regulator of WNT/β-CATENIN signaling in the absence of APC, affecting both TCF-4 and AXIN1 proteins and sensitizing CRC cells to WNT3a stimulation.Conclusions: In the setting of APC mutations, miR155 is decreased possibly as a compensatory mechanism to impair Wnt signaling through Axin1.We believe that targeting miR-155 could be a promising strategy for WNT signaling modulation in CRC patients harboring APC mutations.

Background: Gender differences in reflux esophagitis (RE) were generally recognized in several aspects, in which prevalence of RE was higher in male than in female.Although we previously reported that clinical parameters associated with lifestyle diseases rapidly worsened for years before RE onset in patients, little is known whether predictive factors of future RE are common by gender or not.Hence, we carried out subgroup analysis by gender to investigate and compare the predictive factors using long-term health checkup records.Methods: We used health checkup records between April 2004 and March 2014 at nine institutions in Japan.Subjects who were newly diagnosed as RE between April 2009 and March 2014 were included in the analysis as case subjects.For each case subject, two subjects who had no RE diagnosis between April 2004 and March 2014 and were matched for age, sex, and participating institutions with the corresponding case were included as control subjects.We divided these subjects into subgroups of male and female, and the time courses of clinical parameters in case group were compared with those in control group by the restricted maximum likelihood method for repeated measures or multivariate logistic analysis, appropriately.Results: Initial data were obtained from 230,056 individuals, and 2,066 case subjects (1,558 males and 508 females) and 4,132 control subjects (3,116 males and 1,016 females) were included in the analysis.Means and standard deviations of age were 53.9±9.6 (male case), 54.1±8.4 (female case), 54.0±9.7 (male control), and 53.9±8.3 (female control).Regarding the male group, the time courses of body mass index (BMI) (p<0.001),abdominal circumference (p<0.001),fasting blood sugar (p=0.009),serum triglyceride (p=0.022),high-density lipoprotein cholesterol (p=0.040),glutamate oxaloacetate transaminase (p=0.035),glutamic pyruvic transaminase (p=0.003),γ-glutamyl transpeptidase (p<0.001), and percentages with acid reflux symptoms (p=0.003) in the case group showed more rapid worsening than in the control group.On the other hand, regarding the female group, no significant differences in the time-courses of parameters were observed between case and control group.Conclusions: The male RE group displayed a more rapid worsening of the clinical parameters associated with lifestyle diseases compared with the male non-RE group similarly to the analysis for all subjects, whereas these results were not shown in the female RE group.These results suggest that the risk factors of RE differ between male and female, and different approaches according to gender should be necessary to prevent RE development.