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PDF file - 2686K, The effect of bortezomib (BTZ) and ATRA on RARα expression and NBT reduction ability in HL60-vector and HL60-shRNA-STAT1 cells.

PDF file - 186K, The effect of bortezomib (BTZ) and ATRA on PML-RARα expression in NB4 cells.

PDF file - 147K

PDF file - 1344K, The effect of bortezomib (BTZ) on NF-κB pathway activity in HL60 cells.

PDF file - 1900K, Bortezomib (BTZ) synergized with ATRA to inhibit AML cell proliferation.

All-trans-retinoic acid (ATRA) has held great promise for differentiation-based therapy but reportedly downregulates retinoic acid receptor-α (RARα) in a proteasome-dependent manner, which leads to decreased acute myeloid leukemia (AML) cell differentiation efficiency. Therefore, research strategies that seek to further sensitize cells to retinoids and extend the range of retinoid-affected myeloid malignancies beyond acute promyelocytic leukemia (APL) are key investigative avenues. Here, we show that bortezomib, the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma, exhibited strong synergism with ATRA to promote HL60 and NB4 AML cell differentiation. We observed that bortezomib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes, indicative of myeloid differentiation without cell death. In addition, treatment of human leukemia HL60 xenografts with bortezomib and ATRA together did not increase bortezomib-induced progressive weight loss but resulted in significant tumor growth inhibition in addition to increased differentiation (P < 0.05). These enhanced differentiation effects were accompanied by RARα stabilization and STAT1 activation. Taken together, our study was the first to evaluate bortezomib and ATRA synergy in AML cell differentiation and to assess new opportunities for bortezomib and ATRA combination as a promising approach for future differentiation therapy. Mol Cancer Ther; 12(2); 195–206. ©2012 AACR.

Supp Figure 1. The treatment of CT-707 reduced the protein level of CTGF under hypoxia. Supp Figure 2. CT-707 elicited apoptotic cell death but not cell cycle arrest under hypoxia. Supp Figure 3. The protein level of HIF-1α under hypoxia was not influenced by YAP depletion by siRNA. Supp Figure 4. FAK inhibition was not required for the hypoxic activity of CT-707. Supp Figure 5. The semi-quantitative analyses using density measurements on the protein levels displayed in Figure 5. Supp Figure 6. The correlation of FAK, IGF-1R and YAP target genes expression with the overall survival days of liver cancer patients. Supp Figure 7. The in vivo activities of CT-707 on HepG2-, Bel-7402-xenograft and H22-allograft models. Supp Figure 8. PF-562271 treatment caused death of HepG2-xenografted nude mice but imposed less activity than CT-707.

Given that Yes-associated protein (YAP) signaling acts as a critical survival input for hypoxic cancer cells in hepatocellular carcinoma (HCC), disruption of YAP function and the maintenance of hypoxia is an attractive way to treat HCC. Utilizing a cell-based YAP-TEAD luciferase reporter assay and functional analyses, we identified CT-707, a China-FDA approved multi-kinase inhibitor under clinical trial with remarkable inhibitory activity against YAP function. CT-707 exhibited prominent cytotoxicity under hypoxia on HCC cells, which was attributable to the inhibition of YAP signaling. CT-707 arrested tumor growth in HepG2, Bel-7402, and HCC patient-derived xenografts. Mechanistically, the inhibitory activity of CT-707 on YAP signaling was due to the interruption of hypoxia-activated IGF1R. Overall, these findings not only identify CT-707 as a promising hypoxia-targeting agent against HCC, but they also unveil IGF1R as a new modulator specifically regulating hypoxia-activated YAP signaling.Significance: CT-707 may represent a novel clinical approach for patients with HCC suffering poor drug response due to intratumor hypoxia. Cancer Res; 78(14); 3995–4006. ©2018 AACR.

Materials and Methods for Supplemental Figures

Supplemental Table 1. The primer sequences for qRT-PCR assay Supplemental Table 2. The targeting sequences for siRNA Supplemental Table 3. The list of compounds for YAP-TEADs luciferase screening

Given that Yes-associated protein (YAP) signaling acts as a critical survival input for hypoxic cancer cells in hepatocellular carcinoma (HCC), disruption of YAP function and the maintenance of hypoxia is an attractive way to treat HCC. Utilizing a cell-based YAP-TEAD luciferase reporter assay and functional analyses, we identified CT-707, a China-FDA approved multi-kinase inhibitor under clinical trial with remarkable inhibitory activity against YAP function. CT-707 exhibited prominent cytotoxicity under hypoxia on HCC cells, which was attributable to the inhibition of YAP signaling. CT-707 arrested tumor growth in HepG2, Bel-7402, and HCC patient-derived xenografts. Mechanistically, the inhibitory activity of CT-707 on YAP signaling was due to the interruption of hypoxia-activated IGF1R. Overall, these findings not only identify CT-707 as a promising hypoxia-targeting agent against HCC, but they also unveil IGF1R as a new modulator specifically regulating hypoxia-activated YAP signaling. Significance: CT-707 may represent a novel clinical approach for patients with HCC suffering poor drug response due to intratumor hypoxia. Cancer Res; 78(14); 3995–4006. ©2018 AACR.

P53 is under exquisitely fine regulation and acts as a transcription factor that regulates the expression of thousands of genes that control apoptosis, necroptosis, ferrotosis, cell cycle arrest, senescence, metabolism and fertility. Regulation of p53 turnover is essential not only for the response to chemotherapeutic drugs or radiation in cancer cell but also to keep p53 activity under control in a normal cell. Moreover, .p53 is found to be overactivated in various diseases including idiopathic pulmonary fibrosis (IPF), alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). Promoting hyperactivated-p53 degradation can combat these diseases. However, the strategies that based on known proteasome-dependent degradation only can stabilize the expression level of p53 such as nutlins (inhibit the interaction between mdm2 and tumor suppressor p53). Autophagy-lysosomal degradation is the other pathway for controlling cellular protein stability. More and more researches have established that numerous proteins can be degraded through both proteasome and autophagy-lysosome pathways. However, very little is known to date about p53 turnover through autophagy-lysosome pathway. Here we discover that the activation of p53 autophagic degradation during an oral small molecule receptor tyrosine kinase inhibitor sunitinib in p53 wt cancer cells and normal cells. Mechanistically, the nucleus-to-cytoplasm shift is essential for the autophagic degradation of p53 induced by sunitinib, however, does not require p53 nuclear export signals. Indeed, p53 degradation is achieved by the nucleus-to-cytoplasm transport of its nuclear binding target HMGB1, shifting the distribution of p53 from the nucleus to the cytoplasm. Then cytoplasmic p53 directly interacts with the autophagy cargo receptor p62 to promote degradation. Importantly, inhibition of HMGB1 sensitizes cancer cell to sunitinib. Taken together, our study identified an alternative p53 protein turnover mechanism induced by sunitinib, thus not only elucidating the underlying mechanisms that limit sunitinib efficacy in cancer therapy but also opening an avenue for expanding the clinical indications of sunitinib. Note: This abstract was not presented at the meeting. Citation Format: Qiaojun He, Peihua Luo, Xiaochun Yang, Qinjie Weng, Ling Ding, Bo Yang. HMGB1 governs p53 to autophagic degradation via its nucleus-to-cytoplasm transport [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2070. doi:10.1158/1538-7445.AM2017-2070

All-Trans-Retinoic Acid (ATRA) has held great promise for differentiation-based therapy, but reportedly down-regulates retinoic acid receptor alpha (RARα) in a proteasome-dependent manner, which leads to decreased AML cell differentiation efficiency. Therefore, research strategies that seek to further sensitize cells to retinoids and extend the range of retinoid-affected myeloid malignancies beyond APL are key investigative avenues. Here, we demonstrate that bortezomib, the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma, exhibited strong synergism with ATRA to promote HL60 and NB4 AML cell differentiation. We observed that bortezomib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes indicative of myeloid differentiation, without cell death. Additionally, treatment of human leukemia HL60 xenografts with bortezomib and ATRA together did not increase bortezomib-induced progressive weight loss but resulted in significant tumor growth inhibition in addition to increased differentiation (p Citation Format: Meidan Ying, Lei Zhang, Like Zhong, Xuejing Shao, Peihua Luo, Xiaochun Yang, Bo Yang, Qiaojun He. Bortezomib sensitizes human acute myeloid leukemia cells to all-trans-retinoic acid-induced differentiation by modifying the RARα/STAT1 axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3267. doi:10.1158/1538-7445.AM2013-3267 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The Akt (Protein kinase B, PKB) kinase is a key intermediate of many cellular processes and plays a critical role in cancer promotion and progression. Akt signaling pathway has attached much attention as a promising target for the development of cancer therapeutics. It was showed previously that Isobavachalcone (IBC), a natural chalcone, inhibits the proliferation of human neuroblastoma cells by causing cell apoptosis. However, the precise mechanisms of growth suppressive effects of IBC were not clear. The purpose of this study was to investigate the inhibitory activity of IBC on Akt signaling, which is probably involved in the proliferation inhibition induced by IBC. Here, the anti-proliferative activity of IBC was further verified in six human cancer cell lines, including ovarian carcinoma cell OVCAR-8, prostate carcinoma cell PC3, colorectal carcinoma cell colo205, breast carcinoma cell MCF-7, lung carcinoma cell A549 and hepatocellular carcinoma cell HepG2. Modeling results from the Sybyl/FlexiDock program suggested that IBC potentially bond to the ATP-binding pocket of Akt, which indicated that IBC might be a potential Akt inhibitor. Studies performed using the in vitro Akt kinase assay kit confirmed that IBC could directly target and inhibit Akt kinase in an ATP-competitive manner. Results from cell-based assays also revealed that IBC significantly abated Akt phosphorylation at Ser-473 and cellular Akt kinase activity, but did not interfere with Thr-308 phosphorylation of Akt. Consistent with Akt inhibition, IBC abrogated EGF-stimulated nuclear translocation of Akt as evaluated by fluorescence confocal microscopy. Cellular phosphorylation of Akt downstream substrates, including GSK3β, mTOR, p70S6K, 4E-BP1, XIAP and Bad, was also diminished by IBC. Consequently, significant levels of apoptosis were detected in IBC-treated cancer cells. Typical morphologic features of apoptosis, including chromatin condensation, nuclear fragmentation and formation of apoptotic bodies, were observed in IBC-treated cells by DAPI staining. Meanwhile, IBC also evoked cleavage of pro-caspase-9 and pro-caspase-3, poly (ADP-ribose) polymerase (PARP) degradation, up-regulation of reactive oxygen species (ROS) production and down-regulation of the ratio between Bcl-2 and Bax. Thus, IBC-induced apoptosis was associated with caspase- and ROS-involved mitochondria pathway. Taken together, we first reported IBC inhibited cancer proliferation by abrogation of Akt signaling, providing evidence for the further application of IBC as a novel therapeutic agent for human cancer therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3595.