Discussion
Moderate and advanced stages of ESCC patients lack effective systemic treatment to eradicating tumor cells, and a new treatment alternatives are urgently needed to improve patient prognosis. Currently, a series of studies have supported the accumulation of ubiquitinated proteins causing tumor cell death (Soldatenkovet al. , 1998; Mimnaugh et al. , 2004; Itoh et al. , 2019). Therefore, inducing ubiquitinated protein accumulation has become an increasingly important treatment strategy in cancer therapy, proteasome inhibitors have been reported to have significant antitumor activity against various cancer cells through this mechanism(Sato et al. , 2012; Sato et al. , 2014; Sato et al. , 2017). The novel orally proteasome inhibitor ixazomib is currently used for treatment of multiple myeloma patients, and it has been confirmed to have killing efficiency to tumor cells (Sato et al. , 2017; Yue et al. , 2019; Harris et al. , 2020). In this study, we explore whether proteasome inhibitor ixazomib have therapeutic effects on ESCC. Our results proved that ixazomib effectively inhibits cell proliferation in ESCC cell lines and xenograft tumor model mice. In addition, we elucidate that ixazomib induces the apoptosis and suppresses proliferation in esophageal squamous cell carcinoma through activation of the c-Myc/NOXA pathway. Moreover, our results showed that combined treatment with ixazomib and cisplatin induces synergistic anti-ESCC activity. These results clearly suggest that ixazomib have potential to be used as anti-ESCC therapeutics.
The pro-apoptotic NOXA is a Bcl-2 family protein, and it is a crucial role that interacts with several apoptosis-related proteins such as Bad, Mcl-1, Bim and Puma (Morsi et al. , 2018). In addition, endoplasmic reticulum stress plays an important role in mediating tumor cell apoptosis by inducing the upregulation of NOXA (Cano-Gonzalez et al. , 2018). The c-Myc oncogene is well known and acts as a driving force to activate a variety of oncogenic signaling pathways (Yoshida, 2018). c-Myc is frequently dysregulated or overexpressed in multiple tumor cells, and its abnormal expression contributes to reprogramming cell metabolism and to maintaining the high rate of proliferation rate in cancer cells (Dejure et al. , 2017). Therefore, apoptosis in response to increased expression of c-Myc is an important endogenous protection mechanism against failure that inhibits the oncogenic properties and tumorigenesis of c-Myc (Pelengaris et al. , 2002; Nilsson et al. , 2003). Wirth M et al. using quantitative promoter-scanning chromatin immunoprecipitation revealed the binding of c-Myc to the promoters of NOXA upon proteasome inhibition (Wirth et al. , 2014). Other scholars also reported that c-Myc was a prime transcription factor to transactivate NOXA (Nikiforov et al. , 2007). A series of studies have shown that proteasome inhibitor bortezomib can induce c-Myc-dependent up-regulation of NOXA, leading to apoptosis in colorectal and pancreatic cancer cells (Yue et al. , 2019; Lankes et al. , 2020). Here, this study suggested that ixazomib-mediated upregulation the expression of NOXA and c-Myc in a time- and concentration-dependent manner, on the other hand, NOXA and c-Myc silencing using siRNA partially rescued the proliferation inhibitory effect of ixazomib in ESCC cells. These results confirm that the ixazomib inhibits the proliferation of ESCC cells by target on the c-Myc-NOXA pathway.
Bortezomib is the first proteasome inhibitor for used as a monotherapy for the treatment of multiple myeloma and mantle cell lymphoma approved by the FDA (Richardson, 2003). A series of clinical studies found that bortezomib combined with other drugs, including corticosteroids, lenalidomide and thalidomide can significantly improve the therapeutic effect (Kapoor et al. , 2012). In recent years, research has explored the potential value of bortezomib in combination with cytotoxic drugs in in solid malignancies. Bortezomib combined with platinum-based agents has achieved encouraging results in Phase I/II study in ovarian cancer and non-small-cell lung carcinomas (Ramirez et al. , 2008; Piperdi et al. , 2012; Zhao et al. , 2015). There is a pervasive belief that the synergism between two drugs was due to platinum agents can block bortezomib-induced STAT1 activation which suppresses apoptosis.(Fribley et al. , 2006; Kao et al. , 2013) Given that ixazomib, like bortezomib, is a boronate-based molecules, we explored whether ixazomib similarly enhances the anti-ESCC activity of the nonspecific cytotoxic agent (cisplatin and fluorouracil).