Supplementary Figure Legend from Sorafenib-Mediated Targeting of the AAA+ ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death (original) (raw)
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2023
The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer-derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal-regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA þ ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients. Mol Cancer Ther; 11(12); 2610-20. Ó2012 AACR.
Molecular Cancer Therapeutics, 2012
The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer-derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal-regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA þ ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients. Mol Cancer Ther; 11(12); 1-11. Ó2012 AACR.
Molecular and Cellular Biology, 2007
Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2α (eIF2α) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompan...
Journal of cellular physiology, 2018
Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c-Jun-N-terminal kinase (JNK), Akt, and 5'AMP-activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3-12 hr) ER stress characterized by an increase of P-eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of P-JNK1/2/JNK1/2, P-AMPKα, P-Foxo3a, P-AKt/AKt and P-Foxo3a/Foxo3a ratios, and reduction of P-mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim , Beclin-1, Bcl-xL, Bcl-2, autophagy markers, and reduction of myeloid cell leukemia-1 (Mcl-1) expression. The progressive increase of CHOP expression, a...
Journal of neuro-oncology, 2016
Malignant gliomas are a lethal type of brain tumors that poorly respond to chemotherapeutic drugs. Several therapy resistance mechanisms have been characterized. However, the response to stress through mRNA translational control has not been evaluated for this type of tumor. A potential target would involve the alpha subunit of eukaryotic translation initiation factor (eIF2α) that leads to assembly of stress granules (SG) which are cytoplasmic granules mainly composed by RNA binding proteins and untranslated mRNAs. We assessed whether glioma cells are capable of assembling SG after exposure to different classes of chemotherapeutic agents through evaluation of the effects of interfering in this process by impairing the eIF2α signaling. C6 and U87MG cells were exposed to bortezomib, cisplatin, or etoposide. Forced expression of a dominant negative mutant of eIF2α (eIF2α(DN)) was employed to block this pathway. We observed that exposure to drugs stimulated SG assembly. This was reduced...
Regulation of the sensitivity of hepatocarcinoma cells by ORMDL3, to sorafenib by autophagy
Medical Oncology
Serum orosomucoid1-like protein 3 (ORMDL3) is a membrane protein in the endoplasmic reticulum, known to regulate many important signal transduction processes and autophagy regulation, but it is unclear whether it is involved in the intratumoral microenvironment and cancer drug resistance. Our present study found that silencing ORMDL3 increases the inhibitory effect of sorafenib on the viability and proliferation in HCC cells, and increases the sensitivity of HCC cells to sorafenib. In addition, silencing ORMDL3 can increase ROS levels by inhibiting autophagy, thereby increasing sorafenib-induced apoptosis of HCC cells. Further, our study also found that ORMDL3 silencing inhibits autophagy through the PERK-ATF4-Beclin1 pathway, thus affecting sorafenib sensitivity. The in vivo effects of sorafenib were tested by xenografting using nude mice. It showed that silencing ORMDL3 in HCC cells could increase the inhibitory effect of sorafenib on the growth of tumors. This is the first report...
By screening mouse monoclonal antibody libraries for Kelch repeats, we serendipitously identified monoclonal antibodies to eukaryotic elongation factor 2 (eEF2). Interestingly, eEF2 was highly expressed in lung adenocarcinoma (LADC), but not in the neighboring non-tumor lung tissue. Normally, eEF2 is involved in the peptidyl-tRNA translocation during protein synthesis. Overexpression of eEF2 would implicate an association with disease progression of LADC. In the present study, we investigated the prognostic significance of eEF2 in patients with LADC. Expression of eEF2 was detected by immunoblotting, immunohistochemistry and confocal immunofluorescence microscopy. Our results show that patients with high eEF2 expression had a significantly higher incidence of early tumor recurrence (67.8% vs 18.2%, P = 0.016), and a significantly worse prognosis (P < 0.001). In an in vitro study, silencing of eEF2 expression increased mitochondrial elongation, cellular autophagy and cisplatin sensitivity. Moreover, eEF2 was sumoylated in LADC cells, and eEF2 sumoylation correlated with drug resistance. These results suggest that eEF2 is an anti-apoptotic marker in LADC. However, biological function and involvement of eEF2 in the disease progression of LADC require further studies. (Cancer Sci 2011; 102: 1582-1589
FEBS Journal, 2011
A multikinase inhibitor of the Raf ⁄ mitogen-activated protein kinase kinase (MEK) ⁄ extracellular signal-regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK ⁄ ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co-treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1 ⁄ 2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl-2 family. ERK1 ⁄ 2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX-induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib-induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK ⁄ ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX-induced autophagy and thus promote cancer cell survival. The use of MEK ⁄ ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects.
OPA1 downregulation is involved in sorafenib-induced apoptosis in hepatocellular carcinoma
Laboratory Investigation, 2013
Sorafenib has been used to treat advanced hepatocellular carcinoma (HCC), but the underlying molecular mechanisms remain controversial and why some patients do not respond to this therapy is poorly understood. In this study, we show that sorafenib triggers cell growth inhibition and apoptosis in HCC cells by directly targeting the mitochondria. Treatment with sorafenib induces rapid mitochondrial fragmentation, which is associated with the deregulation of mitochondria fusion-related protein optic atrophy 1 (OPA1). Exposure of cells or isolated mitochondria to sorafenib substantially induces cytochrome c release. Our data indicate that siRNA-mediated OPA1 knockdown significantly sensitizes HCC cells to sorafenib-induced apoptosis. Furthermore, sorafenib has no apparent apoptotic toxicity to normal human primary hepatocytes. Sorafenib inhibits HCC xenograft tumor growth in vivo and murine xenograft tumor tissue analysis reveals mitochondria fusion protein. OPA1 expression levels are strongly downregulated by sorafenib treatment. Western blotting evaluation of patient HCC with matched non-tumor tissue samples demonstrates that OPA1 expression is decreased in up to 40% of HCC patients. Taken together, we have shown that sorafenib suppresses the tumorigenesis of HCC through the induction of mitochondrial injury via OPA1. Our results provide new insights into the pathogenesis of HCC and suggest that OPA1 is a novel therapeutic target in patients with HCC.