Romidepsin Exhibits Anti-Esophageal Squamous Cell Carcinoma Activity through DDIT4-mTORC1 Pathway

Esophageal squamous cell carcinoma (ESCC) is one of the most common human malignancies globally, with high incidence and mortality rates. Given the limited current treatment options, there is an urgent need to develop new effective therapeutic drugs. In this study, researchers conducted a high-throughput drug screening (HTS) on ESCC cell lines and discovered that the histone deacetylase inhibitor Romidepsin significantly inhibits ESCC cell proliferation, induces apoptosis, and causes cell cycle arrest. The experimental results were also validated in cell line-derived xenografts (CDX) and patient-derived xenograft (PDX) mouse models of ESCC. This study was conducted by research teams from Chongqing Medical University and Central South University and was published in the journal “Cancer Gene Therapy.”

Background

Esophageal cancer (EC) is the seventh most common cancer worldwide and the sixth leading cause of cancer-related death. Based on pathological features, EC can be divided into two main subtypes: ESCC and esophageal adenocarcinoma (EAC). While the incidence of EAC is gradually increasing in developed countries, about 90% of EC cases worldwide are ESCC, with the highest incidence in Asia, East Africa, and South America. Due to the lack of early symptoms, most ESCC patients are diagnosed at mid or late stages, with a five-year survival rate of less than 20% in developed countries and less than 5% in many developing countries. Current treatments for ESCC mainly include surgical resection, radiotherapy, and chemotherapy, but only a few patients benefit. Therefore, developing new highly effective and low side-effect therapeutic drugs is urgently needed for treating ESCC.

Research Methods

The research team first used high-throughput drug screening to identify drugs with anti-ESCC activity from the FDA-approved drug compound library. The screening used various ESCC cell line models, including 2D and 3D cultures. Romidepsin was found to significantly inhibit ESCC cell proliferation, induce apoptosis, and cause cell cycle arrest.

Cell Culture and Transfection

Human ESCC cell lines (TE-1 and KYSE-150) and the immortalized normal esophageal epithelial cell line HET-1A were used in the experiments. The cells were cultured in RPMI 1640 medium with regular medium changes. For 2D culture, cells were grown in monolayers on culture plates, whereas for 3D culture, cells were seeded in low-adhesion culture plates to form spheroids. DDIT4-specific siRNA was used for related gene transfection studies.

Drug Screening

HTS was used to screen 1469 FDA-approved small molecule drugs. Cells were seeded in 96-well plates and treated with drugs for 72 hours, and cell viability was assessed by chemiluminescent intensity detection.

Drug Activity Detection

Cell viability in 2D and 3D cell lines was assessed using CCK-8 assay kit and CellTiter-Glo® 3D cell viability assay kit. The results showed that Romidepsin has time- and dose-dependent inhibitory effects on ESCC cells.

In Vivo Anti-Tumor Activity Validation

To validate the efficacy of Romidepsin in vivo, the research team established a TE-1 CDX mouse model. Mice were treated with Romidepsin or vehicle, and the results showed that Romidepsin significantly reduced tumor volume and weight without significantly affecting the body weight of the mice.

RNA Sequencing Analysis

RNA sequencing (RNA-seq) analysis showed significant differential expression of mRNA in ESCC cells treated with Romidepsin. The results indicated that the gene DDIT4 was upregulated after Romidepsin treatment. Further experiments demonstrated that this upregulation was associated with enhanced histone H3 and H4 acetylation at the DDIT4 promoter region.

Molecular Mechanism Exploration

To explore the molecular mechanism of Romidepsin, the research team conducted a series of rescue experiments. The results revealed that Romidepsin increased the expression of DDIT4, thereby inhibiting the mTORC1 pathway, reducing the phosphorylation of downstream target proteins S6K1 and 4EBP1, and subsequently decreasing protein synthesis and cell proliferation.

Efficacy and Toxicity Evaluation in PDX Model

The research team further evaluated the therapeutic effect and toxicity of Romidepsin in the ESCC PDX model. The results showed that Romidepsin had better anti-tumor effects and lower toxicity compared to traditional chemotherapy drugs (paclitaxel combined with cisplatin).

Research Results

The research results indicate that Romidepsin inhibits ESCC cell proliferation, induces apoptosis, and causes cell cycle arrest through the DDIT4-mTORC1 pathway. Additionally, Romidepsin performed better than traditional chemotherapy drugs in the PDX model, showing better efficacy and fewer side effects.

Conclusion and Prospects

In conclusion, this study identified Romidepsin as a potential anti-ESCC drug through HTS screening, revealed its molecular mechanism of anti-tumor activity through the DDIT4-mTORC1 pathway, and validated its efficacy and safety in in vivo models. Romidepsin provides a new treatment option for ESCC and offers new targets and strategies for future research.

Research Significance

This study not only provides a new drug option for the treatment of ESCC but also offers important theoretical support for the development of future drugs and treatment strategies for ESCC. By elucidating the molecular mechanism of Romidepsin, this research further lays the foundation for developing efficient and low-toxicity anti-tumor drugs.

The research team comprised scientists from the Department of Cardiothoracic Surgery and the Life Sciences Research Institute of Chongqing Medical University and the Institute of Clinical Pharmacology of Central South University. The research findings were published online in March 2024 in the journal “Cancer Gene Therapy.” The study indicates that Romidepsin promotes histone acetylation, upregulates DDIT4 expression, and inhibits the mTORC1 pathway, bringing new hope for ESCC treatment. Researchers suggest further exploration of Romidepsin’s clinical application and related clinical trials in the future.