Background:Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma(HCC).However,resistance to sorafenib...Background:Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma(HCC).However,resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib.As the activation of AKT/mTOR played a pivotal role in sorafenib resistance,we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells.Methods:The sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines.The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method.Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting.Results:Sorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells,which underscores the significance of AKT activation.Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction.Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis,leading to the dephosphorylation of Ser136 in BAD protein,and increased the expression of total BAD,which contributed to the apoptosis in sorafenibresistant HCC cells.Conclusions:In this study,Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenibresistant HCC cells,via the suppression of mTORC2-AKT-BAD pathway.Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.展开更多
Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complex...Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.展开更多
The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism,cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase.However,i...The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism,cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase.However,its upstream regulatory pathways as well as whether it has additional function(s)remain largely unknown.We recently reported that Rictor contains a novel ubiquitin E3 ligase activity by forming a novel complex with Cullin-1,but not with other Cullin family members.Furthermore,we identified SGK1 as its downstream target.Interestingly,Rictor,but not Raptor or mTOR,promotes SGK1 ubiquitination.As a result,SGK1 expression is elevated in Rictor^(–/–)MEFs.We further defined that as a feedback mechanism,Rictor can be phosphorylated by multiple AGC family kinases including Akt,S6K and SGK1.Phosphorylation of Rictor at the Thr1135 site did not affect its kinase activity towards phosphorylating its conventional substrates including Akt and SGK1.On the other hand,it disrupted the interaction between Rictor and Cullin-1.Consequently,T1135E Rictor was defective in promoting SGK1 ubiquitination and destruction.This finding further expands our knowledge of Rictor’s function.Furthermore,our work also illustrates that Rictor E3 ligase activity could be governed by specific signaling kinase cascades,and that misregulation of this process might contribute to SGK overexpression which is frequently observed in various types of cancers.展开更多
基金a grant from Medical and Health Science and Technology Program of Zhejiang Province(2019RC076).
文摘Background:Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma(HCC).However,resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib.As the activation of AKT/mTOR played a pivotal role in sorafenib resistance,we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells.Methods:The sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines.The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method.Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting.Results:Sorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells,which underscores the significance of AKT activation.Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction.Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis,leading to the dephosphorylation of Ser136 in BAD protein,and increased the expression of total BAD,which contributed to the apoptosis in sorafenibresistant HCC cells.Conclusions:In this study,Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenibresistant HCC cells,via the suppression of mTORC2-AKT-BAD pathway.Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.
文摘Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.
基金supported in part by the DOD Prostate New Investigator award to W.W.NIH grant GM089763 to W.W.
文摘The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism,cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase.However,its upstream regulatory pathways as well as whether it has additional function(s)remain largely unknown.We recently reported that Rictor contains a novel ubiquitin E3 ligase activity by forming a novel complex with Cullin-1,but not with other Cullin family members.Furthermore,we identified SGK1 as its downstream target.Interestingly,Rictor,but not Raptor or mTOR,promotes SGK1 ubiquitination.As a result,SGK1 expression is elevated in Rictor^(–/–)MEFs.We further defined that as a feedback mechanism,Rictor can be phosphorylated by multiple AGC family kinases including Akt,S6K and SGK1.Phosphorylation of Rictor at the Thr1135 site did not affect its kinase activity towards phosphorylating its conventional substrates including Akt and SGK1.On the other hand,it disrupted the interaction between Rictor and Cullin-1.Consequently,T1135E Rictor was defective in promoting SGK1 ubiquitination and destruction.This finding further expands our knowledge of Rictor’s function.Furthermore,our work also illustrates that Rictor E3 ligase activity could be governed by specific signaling kinase cascades,and that misregulation of this process might contribute to SGK overexpression which is frequently observed in various types of cancers.
