Bis-benzylidine Piperidone RA190 Treatment of Hepatocellular Carcinoma via Binding RPN13 and Inhibiting NF-κB Signaling

Abstract Background According to GLOBOSCAN, hepatocellular carcinoma (HCC) claimed 782,000 lives in 2018. The tyrosine kinase inhibitor sofafenib is used to treat HCC, but new anticancer agents targeting different pathways are urgently needed to improve outcomes for patients with advanced disease. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as demonstrated by bortezomib treatment of multiple myeloma. However, resistance does emerge, and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, an ubiquitin receptor subunit of the proteasome’s 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Considerable evidence suggests that nuclear factor κB (NF-κB) signaling, which is dependent upon the proteasome, is a major driver of inflammation-associated cancers, including HCC. Methods Human HCC cell lines were treated with titrations of RA190. The time course of endoplasmic reticulum stress and NF-κB-related mechanisms by which RA190 may trigger apoptosis were assessed. The therapeutic activity of RA190 was also determined in an orthotopic HCC xenograft mouse model. Results RA190 is toxic to HCC cells and synergizes with sofafenib. RA190 triggers rapid accumulation of polyubiquitinated proteins, unresolved endoplasmic reticulum stress, and cell death via apoptosis. RA190 blocks proteasomal degradation of IκBα and consequent release of NF-κB into the nuclei of HCC cells. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth. Conclusions RA190 has therapeutic activity in a xenograft model, and with sorafenib exhibited synergetic killing of HCC cells in vitro, suggesting further exploration of such a combination treatment of HCC is warranted.

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Bis-benzylidine Piperidone RA190 Treatment of Hepatocellular Carcinoma via Binding RPN13 and Inhibiting NF-κB Signaling

10.21203/rs.2.18702/v4 ◽

2020 ◽

Author(s):

Ruey-Shyang Soong ◽

Ravi K. Anchoori ◽

Richard B. S. Roden ◽

Rou-Ling Cho ◽

Yi-Chan Chen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Proteasome Inhibitor ◽

Time Course ◽

Kinase Inhibitor ◽

Xenograft Model ◽

Therapeutic Activity ◽

Rapid Accumulation ◽

Hcc Cells

Abstract Background According to GLOBOSCAN, hepatocellular carcinoma (HCC) claimed 782,000 lives in 2018. The tyrosine kinase inhibitor sofafenib is used to treat HCC, but new anticancer agents targeting different pathways are urgently needed to improve outcomes for patients with advanced disease. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as demonstrated by bortezomib treatment of multiple myeloma. However, resistance does emerge, and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, an ubiquitin receptor subunit of the proteasome’s 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Considerable evidence suggests that nuclear factor κB (NF-κB) signaling, which is dependent upon the proteasome, is a major driver of inflammation-associated cancers, including HCC. Methods Human HCC cell lines were treated with titrations of RA190. The time course of endoplasmic reticulum stress and NF-κB-related mechanisms by which RA190 may trigger apoptosis were assessed. The therapeutic activity of RA190 was also determined in an orthotopic HCC xenograft mouse model. Results RA190 is toxic to HCC cells and synergizes with sofafenib. RA190 triggers rapid accumulation of polyubiquitinated proteins, unresolved endoplasmic reticulum stress, and cell death via apoptosis. RA190 blocks proteasomal degradation of IκBα and consequent release of NF-κB into the nuclei of HCC cells. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth. Conclusions RA190 has therapeutic activity in a xenograft model, and with sorafenib exhibited synergetic killing of HCC cells in vitro , suggesting further exploration of such a combination treatment of HCC is warranted.

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Combined Inhibition of AURKA and HSF1 Suppresses the Cell Proliferation and Promotes Cell Apoptosis of Hepatocellular Carcinoma by Activating Endoplasmic Reticulum Stress

10.21203/rs.3.rs-86073/v1 ◽

2020 ◽

Author(s):

Zetian Shen ◽

Li Yin ◽

Han Zhou ◽

Xiaoqin Ji ◽

Changchen Jiang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Western Blot ◽

Cell Apoptosis ◽

Histopathological Examination ◽

Mice Model ◽

And Migration ◽

Hcc Cells

Abstract Objective: This study aims to investigate the anti-tumor effect of combined inhibition of aurora kinase A (AURKA) and heat shock transcription factor 1 (HSF1) for hepatocellular carcinoma (HCC) and the underlying mechanism.Methods: The expression of AURKA and HSF1 in HCC tissues and cell lines were detected by Immunohistochemistry (IHC), qRT-PCR and Western blot. Then, AURKA was downregulated in HepG2 cells by lentivirus-induced RNA interfere. Cell viability, invasion, migration and apoptosis were examined by CCK8, clone formation, transwell assays and flow cytometry analysis. The expression of proteins related to cell cycle, apoptosis and endoplasmic reticulum stress (ERS) were detected by Western blot. The tumor in vivo growth was measured on nude mice model. Histopathological examination was performed with HE staining.Results: AURKA and HSF1 were highly expressed in HCC tissues and cells, as well as negative related with prognosis. Knockdown of AURKA significantly inhibited the colony formation and migration of HCC cells. Besides, AURKA inhibitor (Danusertib) significantly reduced the proliferation and migration of HCC cells, and promoted cell apoptosis. Combined inhibition of AURKA and HSF1 induced cell apoptosis of HCC cells and increased the expression of ERS-associated proteins including p-eIF2α, ATF4 and CHOP in vitro. Additionally, combined inhibition of AURKA and HSF1 displayed an excellent antitumor activity on HCC model with relative low cytotoxicity.Conclusion: Combined inhibition of AURKA and HSF1 display excellent anti-tumor effect on HCC by activating endoplasmic reticulum stress, suggesting that AURKA and HSF1 can served as potential targets for HCC treatment.

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Bis-benzylidine Piperidone RA190 Treatment of Hepatocellular Carcinoma via Binding RPN13 and Inhibiting NF-κB Signaling

10.21203/rs.2.18702/v1 ◽

2019 ◽

Author(s):

Yu-Chiau Shyu ◽

Ruey-Shyang Soong ◽

Anchoori Ravi ◽

Richard Roden ◽

Yi-Chan Chen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Anticancer Agents ◽

Proteasome Inhibitor ◽

Kinase Inhibitor ◽

Nuclear Factor Κb ◽

Time Frame ◽

Killing Effect ◽

Hcc Cells

Abstract Background Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third leading cause of cancer mortality worldwide. The development of new anticancer agents targeting different pathways is imperative to improve the advanced HCC. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as first demonstrated by the success of bortezomib treatment for multiple myeloma. However, resistance does emerge and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, a ubiquitin receptor subunit of the proteasome’s 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Methods Human HCC cell lines were treated by RA190 in vitro in different concentration and time frame. We checked the killing effect and the possible mechanisms that lead the tumor apoptosis. We also performed the orthotopic HCC animal model to show the RA190 had significant killing effect in vivo. Results We showed RA190 is also toxic to HCC cells by triggering the rapid build-up of polyubiquitinated proteins, resulting in endoplasmic reticulum stress and the induction of cell death via apoptosis. Considerable evidence suggests that nuclear factor κB (NF-κB) signal is essential for promoting inflammation-associated cancer. Here, we showed that RA190 inhibited the NF-κB pathway in HCC by preventing the degradation of IκBα via the proteasome. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth. We therefore explored combining RA190 with a tyrosine kinase inhibitor currently used to the treat HCC, Sorafenib. Conclusions RA190 and Sorafenib exhibited synergetic killing of HCC cells in vitro, suggesting further exploration of such a combination treatment of HCC is warranted.

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Bis-benzylidine Piperidone RA190 Treatment of Hepatocellular Carcinoma via Binding RPN13 and Inhibiting NF-κB Signaling

10.21203/rs.2.18702/v2 ◽

2020 ◽

Author(s):

Ruey-Shyang Soong ◽

Anchoori Ravi ◽

Richard Roden ◽

Rou-Ling Cho ◽

Yi-Chan Chen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Anticancer Agents ◽

Proteasome Inhibitor ◽

Kinase Inhibitor ◽

Nuclear Factor Κb ◽

Time Frame ◽

Killing Effect ◽

Hcc Cells

Abstract Background Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third leading cause of cancer mortality worldwide. The development of new anticancer agents targeting different pathways is imperative to improve the advanced HCC. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as first demonstrated by the success of bortezomib treatment for multiple myeloma. However, resistance does emerge and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, a ubiquitin receptor subunit of the proteasome’s 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Methods Human HCC cell lines were treated by RA190 in vitro in different concentration and time frame. We checked the killing effect and the possible mechanisms that lead the tumor apoptosis. We also performed the orthotopic HCC animal model to show the RA190 had significant killing effect in vivo . Results We showed RA190 is also toxic to HCC cells by triggering the rapid build-up of polyubiquitinated proteins, resulting in endoplasmic reticulum stress and the induction of cell death via apoptosis. Considerable evidence suggests that nuclear factor κB (NF-κB) signal is essential for promoting inflammation-associated cancer. Here, we showed that RA190 inhibited the NF-κB pathway in HCC by preventing the degradation of IκBα via the proteasome. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth. We therefore explored combining RA190 with a tyrosine kinase inhibitor currently used to the treat HCC, Sorafenib. Conclusions RA190 and Sorafenib exhibited synergetic killing of HCC cells in vitro , suggesting further exploration of such a combination treatment of HCC is warranted.

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USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis

Oncogenesis ◽

10.1038/s41389-021-00338-7 ◽

2021 ◽

Vol 10 (7) ◽

Author(s):

Ruize Gao ◽

David Buechel ◽

Ravi K. R. Kalathur ◽

Marco F. Morini ◽

Mairene Coto-Llerena ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Transcriptional Activity ◽

Kinase Inhibitor ◽

Aerobic Glycolysis ◽

Hypoxia Inducible Factor ◽

Aberrant Expression ◽

Key Enzyme ◽

Hcc Cells

AbstractUnderstanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

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