Breast cancer is the most prevalent malignancy among women worldwide, posing a great threat to women's health (Wilkinson and Gathani 2022). For many years, surgery, chemotherapy, and adjuvant endocrine therapy have been the mainstay treatment for breast cancer (Trayes and Cokenakes 2021). Although survival rates for early-stage breast cancer have improved, therapies for metastatic and recurrent disease remain limited (Trayes and Cokenakes 2021, Liang et al. 2020, Hosonaga et al. 2020). Therefore, developing novel targeted therapies is imperative. Triptolide, a natural product derived from traditional Chinese medicine, has displayed anti-tumor activities against breast cancer and other cancers (Luo et al. 2019). However, its specific mechanisms of action are still not well elucidated. In this study, we utilized integrated bioinformatics approaches to systematically identify the potential targets of triptolide against breast cancer, providing insights into developing triptolide as an anti-cancer drug.
Firstly, based on TCGA-BC dataset, we identified 5206 targets associated with breast cancer progression through WGCNA analysis. We then analyzed the transcriptomic changes of breast cancer cells in response to triptolide treatment, and identified 2113 triptolide-responsive targets. These genes showed expression changes correlated with triptolide dosage and treatment time, suggesting their potential involvement in the anti-tumor effects of triptolide. By comparing breast cancer-related and triptolide-related targets, we identified 122 common targets as candidate therapeutic targets of triptolide. These common targets were aberrantly expressed in breast cancer and could be regulated by triptolide, thus likely mediating the anti-tumor action of triptolide.
Functional enrichment analysis results showed that these therapeutic targets were mainly enriched in tumor-related metabolic pathways, including pyruvate metabolism, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), valine, leucine and isoleucine degradation, tryptophan metabolism, lysine degradation, glyoxylate and dicarboxylate metabolism, propanoate metabolism, fatty acid degradation. It is known that tumor cells exhibit significant metabolic reprogramming, which enables them to survive and proliferate rapidly in the nutritionally deficient tumor microenvironment (Wang, Jiang and Dong 2020). Pyruvate metabolism, glycolysis/gluconeogenesis and citrate cycle (TCA cycle) are pathways closely related to tumor cell energy metabolism (Gray, Tompkins and Taylor 2014, Lu, Tan and Cai 2015). Pyruvate is a key intermediate metabolite of glucose metabolism in tumor cells (Gray et al. 2014), while glycolysis/gluconeogenesis is one of the major pathways for tumor cells to generate energy from glucose (Lu et al. 2015). Valine, leucine and isoleucine degradation, tryptophan metabolism and lysine degradation pathways are mainly involved in amino acid metabolism. Valine, leucine, and isoleucine are branched-chain amino acids (BCAAs) that can promote tumor cell proliferation through multiple pathways, including mTOR signaling, contributing to cellular metabolism, and providing substrates for protein synthesis (Nie et al. 2018). Tryptophan is an essential amino acid, and increased tryptophan metabolism is beneficial to the aggregation of immunosuppressive T cells and inhibits tumor cell apoptosis (Peyraud et al. 2022). Increased lysine metabolism can produce more acetylated histones to activate oncogenes, inhibit tumor suppressors, and promote tumorigenesis (Moreno-Yruela et al. 2022, Zhang et al. 2019, Guo et al. 2018). Glyoxylate/dicarboxylate metabolism, propanoate metabolism, and fatty acid degradation involve carbohydrate and fat metabolism. As a TCA cycle replenishment pathway, glyoxylate/dicarboxylate metabolism provides sugar skeletons for anabolism and gluconeogenesis in tumor cells (Pan et al. 2021). Propanoate metabolism produces methylmalonic acid to facilitate invasion and metastasis in breast cancer (Gomes et al. 2022). Park et al. (2021) found that sodium propionate can inhibit the proliferation of breast cancer cells and induce apoptosis by inhibiting STAT3, increasing ROS levels and activating p38. Additionally, fatty acid degradation generates acetyl-CoA through β-oxidation to fuel the TCA cycle and provide energy to tumor cells (Currie et al. 2013). Notably, some studies found triptolide affects these same metabolic pathways.For instance, triptolide inhibited head and neck cancer cell growth and metastasis by suppressing glycolysis/gluconeogenesis (Cai et al. 2021). Metabolomics analysis revealed triptolide treatment altered TCA cycle, amino acid, and fatty acid metabolism in mice (Zhao et al. 2018). However, whether triptolide exerts its anti-breast cancer effects through similar metabolic mechanisms warrants further investigation.
To elucidate connections between the 122 candidate targets and determine key therapeutic targets, we constructed a PPI network and performed topological analysis. Based on the MCC algorithm, we identified 10 key targets, including VIM, DLD, ACAT1, RABIF, ALDH2, RPS20, BIN1, TUBB6, CALM1 and PINK1. Furthermore, we analyzed their expression patterns in breast cancer versus normal breast tissues, as well as in triptolide-treated versus control breast cancer cells. The results showed that compared with normal breast tissues, RABIF was down-regulated in breast cancer tissues, while the other key targets were up-regulated in breast cancer tissues. Triptolide reversed the aberrant expression of these key targets in a time- and dose-dependent manner. Moreover, ROC analysis validated the potential of these key targets as diagnostic biomarkers and therapeutic targets for breast cancer. Notably, many studies have demonstrated the importance of these key targets in breast cancer and other cancers.
For example, VIM expression is downregulated in breast cancer cells compared to normal breast cells (Sharp et al. 2008) and associated with bone metastasis (Senga et al. 1986). The key target DLD encodes dihydrolipoamide dehydrogenase (DLD), which is a component of several multienzyme complexes, including the pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex and branched-chain α-keto acid dehydrogenase complex (Yan and Wang 2023). DLD is downregulated in breast cancer, correlating with poor prognosis (Jiang et al. 2022, Zhang et al. 2022). It has been shown that decreased expression of DLD will lead to metabolic reprogramming of immune cells (Palmieri et al. 2023). It may have the same effect in tumor cells, but still unclear. ACAT1, a lipid metabolism gene downregulated in breast cancer predicts poor prognosis when expressed at low levels (Kuldeep et al. 2023, Maldonado et al. 2021). Zhang et al. (2023) found that ACAT1 encoded Acetyl-CoA acetyltransferase 1 could inhibit breast cancer cell migration and invasion by binding to METTL3. ALDH2 (aldehyde dehydrogenase 2) is an enzyme related to Glyoxylate and dicarboxylate metabolism. Its low expression can enhance the glycolytic pathway in cells (Ma et al. 2023). ALDH2 deficiency promoted hepatocellular carcinoma through activating oncogenic signaling including JNK, STAT3, BCL-2, and TAZ (Seo et al. 2019). ALDH2 was also found to facilitate colon cancer growth and metastasis via β-catenin signaling (Wei et al. 2023). While direct evidence for ALDH2 in breast cancer is lacking, it represents a breast cancer biomarker (Han et al. 2023). Rab (Ras-related in brain) interacting factor (RABIF) is a GTPase activating protein that can activate the GTPase activity of the Rab family and participate in the regulation of intracellular membrane trafficking (Gulbranson et al. 2017). RABIF is up-regulated in breast cancer, and it can promote invasion, metastasis of breast cancer cells and induce drug resistance (Huang et al. 2021). PTEN-induced kinase 1 (PINK1) is an important kinase mainly located in mitochondria and participates in maintaining mitochondrial function. PINK1 protects against neurotoxin-induced mitochondrial damage, and its mutation or loss of function leads to ROS-mediated mitochondrial damage (Gautier, Kitada and Shen 2008). PINK1 is downregulated in most tumors, holding important prognostic value (Zhu et al. 2020). Miyahara et al. (2021) showed that knockdown of PINK1 promoted breast cancer cell growth, demonstrating the tumor suppressive role of PINK1. BIN1 (Myc box-dependent-interacting protein 1), a tumor suppressor, is down-regulated in various tumor diseases (Wang et al. 2017). Studies have shown that BIN1 inhibits MYC-mediated malignant transformation (Sakamuro et al. 1996) and induces apoptosis in cancer cells (Ge et al. 2000). Additionally, studies have found CALM1 is downregulated in hepatocellular carcinoma (Han et al. 2021) and TUBB6 is downregulated in breast cancer (Nami and Wang 2018), though their anti-breast cancer effects and mechanisms remain unclear. In summary, these key targets play significant roles in breast cancer progression and may serve as diagnostic biomarkers and therapeutic targets. Our study suggests triptolide may exert its anti-breast cancer activities by modulating these targets, but further experimental validation is needed.