BH3-only protein expression determines hepatocellular carcinoma response to sorafenib-based treatment

Hepatocellular carcinoma (HCC) represents a global health challenge with limited therapeutic options. Anti-angiogenic immune checkpoint inhibitor-based combination therapy has been introduced for progressed HCC, but improves survival only in a subset of HCC patients. Tyrosine-kinase inhibitors (TKI) such as sorafenib represent an alternative treatment option but have only modest efficacy. Using different HCC cell lines and HCC tissues from various patients reflecting HCC heterogeneity, we investigated whether the sorafenib response could be enhanced by combination with pro-apoptotic agents, such as TNF-related apoptosis-inducing ligand (TRAIL) or the BH3-mimetic ABT-737, which target the death receptor and mitochondrial pathway of apoptosis, respectively. We found that both agents could enhance sorafenib-induced cell death which was, however, dependent on specific BH3-only proteins. TRAIL augmented sorafenib-induced cell death only in NOXA-expressing HCC cells, whereas ABT-737 enhanced the sorafenib response also in NOXA-deficient cells. ABT-737, however, failed to augment sorafenib cytotoxicity in the absence of BIM, even when NOXA was strongly expressed. In the presence of NOXA, BIM-deficient HCC cells could be in turn strongly sensitized for cell death induction by the combination of sorafenib with TRAIL. Accordingly, HCC tissues sensitive to apoptosis induction by sorafenib and TRAIL revealed enhanced NOXA expression compared to HCC tissues resistant to this treatment combination. Thus, our results suggest that BH3-only protein expression determines the treatment response of HCC to different sorafenib-based drug combinations. Individual profiling of BH3-only protein expression might therefore assist patient stratification to certain TKI-based HCC therapies.Subject terms: Cancer, Diseases     

Sigma‐1 receptor protects against ferroptosis in hepatocellular carcinoma cells

Sigma‐1 receptor (S1R) regulates reactive oxygen species (ROS) accumulation via nuclear factor erythroid 2‐related factor 2 (NRF2), which plays a vital role in ferroptosis. Sorafenib is a strong inducer of ferroptosis but not of apoptosis. However, the mechanism of sorafenib‐induced ferroptosis in hepatocellular carcinoma (HCC) remains unclear. In this study, we found for the first time that sorafenib induced most of S1Rs away from nucleus compared to control groups in Huh‐7 cells, and ferrostatin‐1 completely blocked the translocation. S1R protein expression, but not mRNA expression, in HCC cells was significantly up‐regulated by sorafenib. Knockdown of NRF2, but not of p53 or hypoxia‐inducible factor 1‐alpha (HIF1α), markedly induced S1R mRNA expression in HCC cells. Inhibition of S1R (by RNAi or antagonists) increased sorafenib‐induced HCC cell death in vitro and in vivo. Knockdown of S1R blocked the expression of glutathione peroxidase 4 (GPX4), one of the core targets of ferroptosis, in vitro and in vivo. Iron metabolism and lipid peroxidation increased in the S1R knockdown groups treated with sorafenib compared to the control counterpart. Ferritin heavy chain 1 (FTH1) and transferrin receotor protein 1 (TFR1), both of which are critical for iron metabolism, were markedly up‐regulated in HCC cells treated with erastin and sorafenib, whereas knockdown of S1R inhibited these increases. In conclusion, we demonstrate that S1R protects HCC cells against sorafenib and subsequent ferroptosis. A better understanding of the role of S1R in ferroptosis may provide novel insight into this biological process.     

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.     

Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic "BH3-domain only" PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-β (TGF-β) through the intrinsic pathway and to tumor necrosis factor-α (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-β or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines.     

Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy

Sorafenib is the standard first-line drug for the treatment of advanced hepatocellular carcinoma (HCC), however, its therapeutic efficacy is not satisfactory due to primary or secondary resistance of HCC cells. In the present study, we identified Metaxin 1 (MTX1) as a new regulator of sorafenib resistance in HCC through genome-scale CRISPR activation (CRISPRa) screening. We found that MTX1 was frequently upregulated in HCC tissues and overexpression of MTX1 promoted HCC cell proliferation in vitro and in vivo. As well, MTX1 overexpression increased cell growth rate and decreased cell apoptosis upon sorafenib treatment. Consistently, the resistance induced by MTX1 was also observed in subcutaneous xenograft tumor model. Clinically, high expression of MTX1 was closely related with poor outcomes in HCC patients who received sorafenib treatment. Mechanistically, overexpression of MTX1 could promote HCC cell autophagy via interacting with and inhibiting CDGSH iron sulfur domain 1 (CISD1), an autophagy negative regulator. Taken together, our findings suggest that MTX1 is upregulated in HCC and contributes to sorafenib resistance via a possible mechanism involving CISD1 mediated autophagy.     

Combined anti-tumor effects of IFN-α and sorafenib on hepatocellular carcinoma in vitro and in vivo

Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide, and novel therapeutic strategies are urgently required to improve clinical outcome. Interferon-alpha (IFN-α) and sorafenib are widely used as anti-tumor agents against various malignancies. In this study, we investigated the combined effects of IFN-α and sorafenib against HCC. We demonstrated that the combination therapy synergistically suppressed HCC cellular viability, arrested cell cycle propagation and induced apoptosis in HCC cells. Further research revealed that IFN-α and sorafenib collaboratively regulated the expression levels of cell cycle-related proteins Cyclin A and Cyclin B as well as the pro-survival Bcl-2 family proteins Mcl-1, Bcl-2 and Bcl-X(L). Moreover, sorafenib inhibited IFN-α induced oncogenic signaling of STAT3, AKT and ERK but not the activation of the tumor suppressor STAT1. Xenograft experiments also confirmed the combined effects of IFN-α and sorafenib on tumor growth inhibition and apoptosis induction in vivo. In conclusion, these results provide rationale for the clinical application of IFN-α and sorafenib combination therapy in HCC treatment.     

Distinct role of calmodulin and calmodulin-dependent protein kinase-II in lipopolysaccharide and tumor necrosis factor-alpha-mediated suppression of apoptosis and antiapoptotic c-IAP2 gene expression in human monocytic cells

Exposure of phagocytic cells to bacterial endotoxin (lipopolysaccharide; LPS) or inflammatory cytokines confers antiapoptotic survival signals; however, in the absence of the appropriate stimulus, monocytes are programmed to undergo apoptosis. Macrophage survival may thus influence inflammatory and immune responses and susceptibility to microbial pathogens. Herein, we demonstrate that LPS and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), enhance monocytic cell survival through the induction of the antiapoptotic c-IAP2 gene in a human promonocytic THP-1 cell line. We also investigated the role of upstream signaling molecules including the mitogen-activated protein kinases, phosphatidylinositol 3-kinase, and the calcium signaling pathways in the regulation of c-IAP2 expression and eventual survival of monocytic cells. Our results suggest that LPS and TNF-alpha-induced c-IAP2 expression was regulated by calmodulin (CaM) through the activation of calmodulin-dependent protein kinase-II (CaMKII). In addition, CaM and CaMKII regulated c-IAP2 expression in LPSand TNF-alpha-stimulated cells through NF-kappaB activation. Moreover, the CaM/CaMKII pathway also regulated LPS- and TNF-alpha-mediated inhibition of apoptosis in these cells. Taken together, these results suggest that LPS- and TNF-alpha-induced c-IAP2 expression and its associated antiapoptotic survival signals in THP-1 cells are regulated selectively by CaM/CaMKII through NF-kappaB activation.     

Receptor-interacting Protein 1 Increases Chemoresistance by Maintaining Inhibitor of Apoptosis Protein Levels and Reducing Reactive Oxygen Species through a microRNA-146a-mediated Catalase Pathway

Although receptor-interacting protein 1 (RIP1) is well known as a key mediator in cell survival and death signaling, whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. In this report, we found that, in human lung cancer cells, knockdown of RIP1 substantially increased cytotoxicity induced by the frontline anticancer therapeutic drug cisplatin, which has been associated with robust cellular reactive oxygen species (ROS) accumulation and enhanced apoptosis. Scavenging ROS dramatically protected RIP1 knockdown cells against cisplatin-induced cytotoxicity. Furthermore, we found that, in RIP1 knockdown cells, the expression of the hydrogen peroxide-reducing enzyme catalase was dramatically reduced, which was associated with increased miR-146a expression. Inhibition of microRNA-146a restored catalase expression, suppressed ROS induction, and protected against cytotoxicity in cisplatin-treated RIP1 knockdown cells, suggesting that RIP1 maintains catalase expression to restrain ROS levels in therapy response in cancer cells. Additionally, cisplatin significantly triggered the proteasomal degradation of cellular inhibitor of apoptosis protein 1 and 2 (c-IAP1 and c-IAP2), and X-linked inhibitor of apoptosis (XIAP) in a ROS-dependent manner, and in RIP1 knockdown cells, ectopic expression of c-IAP2 attenuated cisplatin-induced cytotoxicity. Thus, our results establish a chemoresistant role for RIP1 that maintains inhibitor of apoptosis protein (IAP) expression by release of microRNA-146a-mediated catalase suppression, where intervention within this pathway may be exploited for chemosensitization.     

Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

c-IAP1 blocks TNFalpha-mediated cytotoxicity upstream of caspase-dependent and -independent mitochondrial events in human leukemic cells

Tumor necrosis factor-alpha (TNFalpha) mediates cytochrome c release from mitochondria, loss of mitochondrial membrane potential (DeltaPsim) and apoptosis in sensitive leukemic cells. In the present study, by using the human leukemic U937 cell line, we demonstrate that the cytochrome c release is caspase-8-dependent and can be blocked by an inhibitor of caspase-8, Z-Ile-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone (Z-IETD.fmk), or a pan caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD.fmk). However, TNFalpha-mediated loss of DeltaPsim was not inhibited by caspase inhibitors. The apoptotic process was blocked by either Z-IETD.fmk or Z-VAD.fmk in cells with lower DeltaPsim. U937 cells with stable transfection of the cellular inhibitor of apoptosis protein 1 (c-IAP1) are resistant to TNFalpha-induced activation of caspases, Bid cleavage, cytochrome c release and DeltaPsim collapse. In addition, both c-IAP1 and XIAP were not up-regulated upon prolonged exposure to TNFalpha. In contrast, there was a caspase-dependent cleavage of XIAP, but not c-IAP1, during treatment with TNFalpha for 7 days. These results demonstrate that c-IAP1 blocks TNFalpha signaling at a level controlling both activation of caspase-8 and a signal to cause loss of DeltaPsim. The sensitive U937 cell line failed to acquire resistance and gain a self-protecting advantage against apoptosis, upon induction of c-IAP1 expression.     

Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress

Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC.     

Interaction of heat-shock protein 90 beta isoform (HSP90 beta) with cellular inhibitor of apoptosis 1 (c-IAP1) is required for cell differentiation

Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90 beta. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90 beta isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its beta isoform as specific depletion of HSP90alpha does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90 beta both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90 beta prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.