As a malignant tumor with high mortality, however, available treatment options for ESCC are still limited. In order to screen out more potential anti-ESCC drugs, we performed a HTS assay utilizing a library with 1469 FDA-approved therapeutic agents that are already used in clinic for treatment of various diseases. These drugs have been tested in humans, with the serum concentrations and side effects are widely known. It therefore obviates the long years of testing novel drugs for safety and stability in the human body. In the present study, romidepsin was selected out as one of the most promising one.
Romidepsin (also known as depsipeptide, FK228, FR901228 or NSC-630176), a naturally occurring HDAC inhibitor, was approved by the FDA in 2009 for the treatment of cutaneous T-cell lymphoma (CTCL) or peripheral T-cell lymphoma (PTCL) who have received at least one prior systemic therapy [9]. It specifically inhibits HDAC class Ⅰ and class Ⅱ mediated deacetylation, causing the hyperacetylation of histones and the re-expression of epigenetic silencing genes. During the past decade, romidepsin has been described to exert promising anti-tumor effects against several types of solid tumors, including ovarian cancer, breast cancer, lung cancer, pancreatic cancer, etc[13]. It has been reported that romidepsin suppresses tumor growth through inducing cell apoptosis and cell cycle arrest, regulating cell autophagy, inhibiting tumor angiogenesis, as well as modulating immune response[18]. However, only a few studies have focused on the impact of romidepsin on ESCC, moreover, its anti-ESCC activity in vivo has not been assessed so far. In this study, we observed that romidepsin strongly inhibited cell proliferation, and induced apoptosis and cycle arrest in ESCC cell lines, which was consistent with earlier research[19–21]. Furthermore, the in vivo anti-ESCC effect of romidepsin was first validated in CDX mouse models.
Although romidepsin has shown promise in cancer treatment, the underlying mechanisms responsible for its therapeutic effects remain poorly understood. Its activity depends not only on the cancer types, but also on the downstream molecules and its dose[22]. In this study, we found that romidepsin-induced ESCC inhibition was due to, at least partially, the upregulation of DDIT4. DDIT4 was first described in 2002 as a protein induced by hypoxia and DNA damage[23, 24]. Accumulating evidence suggests that DDIT4 plays a pivotal role in cell proliferation, metabolic signaling, oxidative stress, DNA damage response, and has been shown to be involved in cancer development[25, 26]. However, the alterations in DDIT4 expression and function are dependent on cancer type, since DDIT4 may play either oncogenic or tumor suppressor role in different types of cancer[27]. For example, DDIT4 overexpression was observed in lung adenocarcinoma and head and neck squamous cell carcinoma, with its upregulation showing a positive correlation with poor prognosis[28, 29]. In contrast, downregulation of DDIT4 was associated with tumor cell proliferation in HER2-positive and triple-negative breast cancer[30]. Here, for the first time, we identified that DDIT4 was significantly downregulated in ESCC cells and tissues, and high level of DDIT4 correlated with poor prognosis in ESCC patients. Additionally, recent research has demonstrated that the dysregulation of DDIT4 in cancer is mediated primarily by different transcription factors, such as p53, NF-κB, p63, and HIF-1, as well as some microRNAs and m6A demethylases [27, 31–34]. In the present study, we found that romidepsin could also activated transcription of DDIT4 through hyperacetylation of histone H3 and H4 of its promoter regions.
DDIT4 is known to negatively regulate the phosphorylation and activity of mammalian target of rapamycin complex 1 (mTORC1) through inhibit the formation of the downstream TSC1/TSC2 complex, although the precise mechanism for this has not been completely delineated[14, 27]. In mammalian cells, the main functions of mTORC1 are promoting protein synthesis, stimulating ribosome, lipid and nucleotide biosynthesis. S6K1 and 4EBP1 are two well-known targets, both of them are proteins that modulate translation initiation. Activated mTORC1 can phosphorylate S6K1 at T389 and 4EBP1 at multiple residues, leading to increased protein and nucleotide synthesis[35, 36]. To date, several chemical molecules, including baicalein, ZY0511, N-butylidenephthalide, and metformin, have been demonstrated to induce DDIT4 expression and exert anti-cancer activity through inhibiting mTORC1 pathway[14, 37–39]. Consistent with previous studies, we found here that romidepsin also could inhibit ESCC cell proliferation through upregulating DDIT4 and inhibiting its downstream mTORC1 pathway.
In recent years, PDX mouse models have emerged as a powerful tool for the development of novel therapies for early, advanced, and drug-resistant tumors. These animal models are established by transplanting fresh tumor tissue resected from human cancer into immunodeficient mice. Therefore, PDX models accurately recapitulate the pathological and molecular characteristics of corresponding individual tumors, better reflecting patient heterogeneity and clinical diversity when compared to standard CDX mouse models[40]. Using ESCC PDX models, we further verified the anti-ESCC activity of romidepsin in vivo, and compared its efficacy and toxicity with that of conventional chemotherapy, paclitaxel plus cisplatin. From the results, we confirm that romidepsin is a promising anti-ESCC agent with little or no toxicity.
In conclusion, here we describe the identification of romidepsin, an FDA-approved drug used to treat T-cell lymphoma, as the most potent growth inhibitor against ESCC cell lines in both 2D and 3D culture systems. Romidepsin significantly induced the expression of DDIT4 gene by promoting histone acetylation at its promoter region, thereby suppressed mTORC1 pathway, protein synthesis and cell proliferation (Fig. 7). Moreover, romidepsin exhibited better efficacy and safety than the conventional therapeutic drugs in ESCC PDX models. Our results suggest romidepsin as a new candidate, and DDIT4-mTORC1 pathway as a potential target for ESCC therapy.