Underlying mechanisms of novel cuproptosis-related dihydrolipoamide branched-chain transacylase E2 (DBT) signature in sunitinib-resistant clear-cell renal cell carcinoma

Introduction

Renal cell carcinoma (RCC) is the ninth most common primary renal malignancy globally and has a high mortality rate. RCC occurs more frequently in individuals with obesity or high blood pressure and individuals who smoke, and its incidence rate in men is twice as high as that in women [1, 2]. Several reports have noted that RCC has multiple subtypes, among which clear-cell RCC (ccRCC) is the most common, followed by papillary RCC (pRCC), chromophobe RCC (chRCC) and other rare subtypes [3, 4]. Patients generally do not show symptoms of RCC until it progresses to an advanced stage. The disease eventually reaches a locally advanced or metastatic stage in approximately one-third of patients, and tumor recurrence and distant metastasis are common after nephrectomy [5]. Although much progress has been made in treatment in recent years, treatment options remain limited and relatively ineffective. The 5-year survival rate for people with end-stage RCC is approximately 10% [6]. The recurrence and mortality rates of RCC are more than 40%—the highest rates among the 10 most common cancers in men and women—and approximately 25% of patients with RCC have distant metastasis [7]. The most common age of RCC onset is 60–70 years, and the incidence rate of RCC in men is 1.5–2 times that in women [8]. Obesity, hypertension, smoking, and chronic kidney disease (CKD) are also risk factors for RCC [9]. The 5-year survival for end-stage RCC is approximately 10% [10]. Approximately 30% of patients with RCC experience relapse after undergoing primary tumor resection [11]. Biomarkers of RCC, such as VHL, TP53/p53, KRAS, AKT, XIAP, MCL-1, TGase 2, MDM2, HIF1α, NRF2, and HIF2α, have been identified as key indicators for the diagnosis, prognosis, and treatment of RCC [12]. Despite the discovery of such indicators and the increased detection rate of RCC during clinical examinations owing to modern tools such as ultrasonography (US) and computed tomography, the incidence of RCC continues to rise. Most cases of early-stage RCC are detected by accident, and effective treatments for patients with advanced RCC have yet to be developed [13]. Many patients with RCC are only diagnosed after the tumor has metastasized, which poses a considerable challenge for effective treatment. Therefore, identifying diagnostic indicators and formulating effective treatments are necessary for predicting and managing advanced or recurrent RCC. Cuproptosis is a novel form of cell death dependent on copper ions, has been identified as a significant factor in preventing the onset and advancement of cancer, demonstrating notable effectiveness against drug-resistant tumors. However, its efficacy in treating drug-resistant renal cell carcinoma (RCC) is still uncertain. Thus, understanding the regulatory mechanisms of cuproptosis could yield valuable insights for enhancing RCC treatment strategies. In this research, we investigated the metabolic processes involving copper and the signaling pathways related to copper, focusing on identifying potential targets associated with DBT in the context of copper-induced apoptosis.

Results

Discussion

Renal eradication surgery, the main treatment for RCC, has achieved promising results because it can effectively remove primary tumors [14]. However, this procedure causes loss of kidney function, which can lead to other adverse effects, such as the emergence of CKD [2]. Patients with stage I–III RCC can undergo partial resection or nephrectomy, but relapse occurs in 33% of these patients. Chemotherapy, which is commonly used to treat other malignant tumors, is ineffective for treating RCC and is thus not recommended [14]. Conversely, patients with end-stage local RCC or metastatic RCC are treated with systemic therapy, including immune checkpoint inhibitors such as nivolumab and pembrolizumab, which target PD-1; avelumab and atezolizumab, which target PD-L1; ipilimumab, which targets CTLA-4; axitinib, sunitinib, pazopanib, and bevacizumab, which target vascular endothelial growth factor (VEGF); and the mTOR inhibitor everolimus [15]. Tyrosine kinase inhibitors (TKIs), mTOR inhibitors, and VEGF inhibitors are molecularly targeted drugs frequently used to treat RCC and have been demonstrated to improve the overall prognosis of patients with unresectable or metastatic RCC. TKIs can inhibit or even eliminate tumor growth and spread and are thus frequently used to treat multiple types of cancer [2]. Sunitinib, a TKI, is the first targeted drug for metastatic RCC treatment that has been approved by the FDA. Sunitinib and pazopanib, another TKI, are viable first-line treatments for RCC [5]. In addition to TKIs, anti-VEGF monoclonal antibodies have been demonstrated to be useful for managing advanced RCC [6]. As the development of targeted drugs has increased treatment options in recent years and these drugs are in widespread use in clinical applications, resistance to targeted drugs has also emerged and has limited their effectiveness. Therefore, identifying the mechanism of drug resistance and corresponding treatment methods is crucial.

Copper is an essential micronutrient for humans and is a key catalytic cofactor involved in many biological processes such as mitochondrial function, synthesis of biological compounds, and antioxidation. Normal physiological and biochemical processes require the copper concentration in the body to be strictly regulated and maintained within a specific range. Copper levels are balanced through several biochemical processes, including copper absorption, intracellular utilization, distribution, and excretion [16]. In adults, the tissue concentrations of copper are approximately 4–6 μg/g in the liver, 7–12 μg/g in the kidney, 3–5 μg/g in the brain, 1 μg/g in muscle, and 1000 ng/ml in plasma and serum [17]. Dietary copper is absorbed mainly in the intestines, especially the duodenum. In this process, copper transporter 1 (CTR1) absorbs copper into enterocytes, where Cu ²⁺ is subsequently reduced by the metalloreductase activity of six-transmembrane prostate epithelial antigen (STEAP), duodenal cytochrome b (DCYTB) 6, and DCYTB 7. The resulting Cu ⁺ is absorbed by intestinal cells, converted into Cu ²⁺, and released into the blood, where it is bound to ceruloplasmin, albumin, copper chaperone proteins, histidine, and macroglobulin in the blood and transported to various tissues and organs [18–20]. The ATP7A protein is responsible for transporting copper into the cells of tissues and organs. However, mutations in this protein that inhibit its ability to transfer copper cause copper-related genetic diseases or Menkes disease (copper deficiency), leading to copper imbalance, difficulty eating, seizures, muscle atrophy, weakness in the hands and feet, reduced ankle reflexes, delayed neurodevelopment, and other problems. These symptoms usually manifest in infants when they are 6–12 weeks of age [21]. Wilson disease (excess copper) is caused by different mutations of the ATP7B gene. When the ATP7B protein, which is responsible for excreting copper from the liver, is mutated, copper accumulates in tissues throughout the body, primarily in the brain and the liver, causing lipid peroxidation and mitochondrial dysfunction [22].

Copper plays a role in various molecular pathways in cancer that exert proliferative or proangiogenic effects on tumors, causing them to grow and spread [23]. Many copper-binding proteins associated with cancer have been identified, such as antioxidant-1 (Atox1), which is involved in the cell cycle, cell proliferation, angiogenesis, and vascular remodeling [24]. Moreover, lung cancer patients who have higher ATP7A expression exhibit poorer responses to cisplatin-based chemotherapy [25], and ATP7B and ATP7A are associated with resistance to cisplatin-based drugs in breast and ovarian cancer [26]. Ctr1, another copper-binding protein, is also involved in the transport of platinum-based antineoplastic drugs [27]. These findings demonstrate the key role of copper-associated proteins in cancer, their involvement in cancer progression, and the development of resistance to cisplatin-based chemotherapy drugs. Copper has also been reported to be involved in the mitogen-activated protein kinase (MAPLK) pathway and to regulate cancer cell apoptosis, survival, cell differentiation, cell proliferation, and metastasis [28].

Cuprotosis differs from well-known apoptosis and ferroptosis. Cuprotosis is a newly discovered regulated cell death that is triggered by Cu ²⁺, and it occurs when copper accumulates in cells and causes noncaspase-, apoptosis-, or reactive oxygen species (ROS)-induced cell death [29]. Copper plays an essential role in the regulation of cell proliferation, angiogenesis, and metastasis [30], and it is also involved in ROS generation [31]. Excess copper leads to proteotoxic stress and induces apoptosis through protein lipidation, mitochondrial respiration, lipidated protein aggregation, and Fe–S cluster protein destabilization [32]. Enhanced mitochondrial metabolism has been reported in melanoma, breast cancer, and leukemia, and drug-resistant cancer cells rely on the energy obtained through mitochondrial respiration [33]. Mitochondrial respiration regulates cancer proliferation, migration, and growth [34]. Cuprotosis involves copper binding to the fatty acylation group in the citric acid (TCA) cycle, and during mitochondrial respiration, the increase in fatty acylated TCA enzymes leads to the abnormal aggregation of fatty acylated proteins and the dysfunction of Fe–S-containing proteins, causing acute proteotoxic effects that trigger cell death [30]. In melanoma, liver cancer, and breast cancer, copper chelators can limit the bioavailability of copper and inhibit cancer progression and the metabolic process underlying cancer cell survival [31]. Cu ²⁺ is transported to the mitochondria through ionophores, is reduced to Cu ¹⁺ by ferredoxin (Fdx1) and combines with dihydrolipoyllysine-residue acetyltransferase (DLAT), a lipidation component in the TCA cycle. This leads to lipidated protein aggregation and the instability of Fe–S cluster proteins, resulting in proteotoxic oxidative stress that triggers cell death. Moreover, a key advantage of this program is that it is not inhibited by other well-known cell death inhibitors [30].

Branched-chain amino acids (BCAAs), such as isoleucine, leucine, and valine, are essential amino acids created by plants, bacteria, and fungi. Animals cannot synthesize BCAAs and consequently must obtain BCAAs from their diet [35, 36]. BCAAs are catabolized in the mitochondria and cytoplasm of cells, where they are first converted into branched-chain α-keto acids, 2-ketoisocaproate, 2-keto-3-methylvalerate, and 2-ketoisovalerate. Oxidative decarboxylation subsequently transforms branched-chain α-ketoacid dehydrogenase (BCKD) into branched-chain acyl-CoA esters, CO₂, and NADH. BCKD has three components: heterotetrameric (α2β2), decarboxylase E1, and dihydrolipoamide. Branched-chain transacylase E2 and FAD-dependent dihydrolipoyl dehydrogenase E3 are closely related to BCKD and play similar roles [37]. In non–small-cell lung cancer (NSCLC), BCAAs are used as nitrogen sources, and the dysfunction of the enzymes branched-chain amino acid transaminase (BCAT) 1 and BCAT2, which are responsible for BCAA usage, impairs NSCLC tumor formation [38]. Glioblastoma cells highly express BCAT1, which initiates the catabolism of BCAAs. Inhibiting BCAT1 can subsequently repress tumor growth, proliferation, and invasion [39]. Inhibiting BCAT1 also elevates αKG levels, impairing acute myeloid leukemia (AML) stem cell function [40]. In breast cancer, knockdown of BCAT1 eliminates BCAA catabolism, which inhibits mTOR-mediated mitochondrial biogenesis and function, repressing cancer proliferation [41]. Dysregulation of BCAA metabolism activates mTORC1, promoting the development and progression of hepatocellular carcinoma catabolism [42]. BCAA regulates the activity of mTOR, and BCAA metabolism is believed to contribute to cancer progression and may also participate in multiple pathways of carcinogenesis [43]. Therefore, the role of BCAA metabolism in cancer warrants further exploration.

As mentioned in the earlier text, copper ions and cuproptosis play a key role in cancer proliferation and metastasis, which suggests that copper can be used in cancer treatments. Therefore, copper chelators and copper ionophores are in high demand as targeted cancer drugs. Many copper ionophore drugs have been used to treat cancer, including 8-hydroxyquinolines (HQs), flavones, dithiocarbamates, and bis(thiosemicarbazone) ligands [44]. Although the concept of cuproptosis had not yet been proposed when copper ionophores were first developed, their tumor suppressor effects are well-documented. Disulfiram, an FDA-approved treatment for chronic alcoholism, has been demonstrated to have differential anticancer effects in numerous solid tumors and hematological malignancies [45]. The bis (thiosemicarbazonato) copper complexes glyoxalbis N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(gtsm)] and diacetylbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(atsm)] have been demonstrated to inhibit proteasome chymotrypsin-like activity that targets prostate cancer cells [46]. Elesclomol has been verified as an effective treatment for metastatic melanoma, leukemia, and various solid tumors [47], because it inhibits cancer cell growth by inducing oxidative stress and copper uptake in cells [48]. Elesclomol sensitivity is positively correlated with the degree of dependence of cancer cells on mitochondrial metabolism [47]. Disulfiram (DSF) is a drug approved by the US FDA for the treatment of alcoholism, but studies have demonstrated that DSF can be used for the treatment of melanoma by inhibiting the ubiquitin-proteasome system, reactive oxygen species, and death signaling pathways [49]. Clioquinol (CQ) has been widely used to treat skin infections, and the functionalization of CQ, namely the 8-HQ scaffold, confers substantial anticancer properties. This activity is enhanced by Cu, and CQ inhibits the growth of cancer cells through the inhibition of the proteasome, cell motility and metastasis, and generation of oxidative stress [50]. Thiosemiccarbazones (TSCs), which are chelating ligands, can be divided into single and double TSCs, with an example being 3-aminopyridine carboxaldehyde (3-AP, Triapine). TSC can chelate iron to interfere with tumor cell metabolism and proliferation [51]. Copper chelators and copper ionophores are potential cancer treatments because they can cause copper depletion or overload, perturbing copper homeostasis in tumors.

Prior research has indicated that in various human cancers, the expression of DBT (dihydrolipoamide branched chain transacylase E2) is often reduced. Specifically, in kidney renal clear cell carcinoma (KIRC), lower DBT levels are linked to more severe clinicopathological characteristics and a worse prognosis for patients. According to both univariate and multivariate Cox regression analyses, DBT can serve as an independent predictor of outcomes in KIRC patients. Experimentally, increasing DBT expression in KIRC cells through plasmid introduction has been shown to slow their growth and decrease their ability to migrate and invade. Further analysis suggests that DBT might play a role in pathways related to immunotherapy and drug metabolism. The CIBERSORT algorithm analysis implies that DBT may enhance anti-cancer immune responses in KIRC by activating M1 macrophages, mast cells, and dendritic cells, and concurrently reducing regulatory T cell activity. Lastly, DBT expression in KIRC has been found to have a strong association with immune checkpoints and the efficacy of targeted and immunotherapy drugs [52].

In our comprehensive analysis, we focused on the expression patterns of copper CRGs within a specific ccRCC cohort. Our findings revealed a significant variation in the expression of CRGs in ccRCC tumors, with this differential expression becoming more pronounced in tumors that were at an advanced stage (as depicted in Figure 1). This notable disparity in CRG expression was further corroborated by our observations in the TCGA-KIRC cohort. Here, we noticed a strong association between elevated levels of CRG expression and more advanced tumor stages, as well as higher TNM stages (illustrated in Figure 2).

Our research also included an in-depth examination of the relationship between CRG expression and patient outcomes, specifically overall survival (OS) and disease-free survival (DFS). The results of this analysis indicated a potential link between CRG expression levels and these crucial clinical outcomes, suggesting that targeting CRGs might offer a novel and effective therapeutic strategy for ccRCC. This hypothesis, however, requires further investigation, as emphasized in Figures 3, 4. We extended our research to include an analysis of CRG expression in two distinct drug resistant RCC cohorts. In this phase of our study, we discovered that two specific CRGs—DBT and GLS—exhibited significantly different expression profiles in drug-resistant RCC tumors. Notably, these differences in expression were strongly correlated with the tumors’ resistance to treatment (as shown in Figure 5). This finding adds a new dimension to our understanding of drug resistance in RCC.

In addition to these observational studies, we conducted experimental work to explore the functional implications of these findings. We observed that overexpression of DBT in RCC cells led to a noticeable decrease in cell growth, migration, and invasion. These experimental outcomes, detailed in Figure 6, underscore the potential of DBT as a target for therapeutic intervention in RCC, particularly in the context of its role in influencing tumor behavior and treatment resistance. This comprehensive approach, integrating both observational and experimental data, provides a more nuanced understanding of the role of CRGs in ccRCC and opens new avenues for research and treatment strategies.

Conclusions

Figure 7 presents a graphical abstract illustrating the research project's execution. This project aims to shed light on the impact of cuproptosis in the progression of Renal Cell Carcinoma (RCC) and to unravel the molecular mechanisms underlying drug resistance. The ultimate goal of this study is to pave the way for the development of innovative therapeutic approaches for the treatment of drug resistant RCC. This study aims to determine the significance of dihydrolipoamide branched chain transacylase E2 (DBT) in patients with Sunitinib-Resistant Clear-Cell Renal Cell Carcinoma (RCC). We found that DBT expression is reduced in various cancers, and in RCC, low DBT levels are associated with advanced disease characteristics and poor prognosis. The study examined DBT's predictive value in various cancers, highlighting its significant prognostic importance in RCC, leading to further investigations. Through comprehensive bioinformatics analysis, we explored the relationship between DBT and clinicopathological features in RCC patients. Our findings indicate a significant reduction in DBT expression in RCC, correlating with disease progression and lower survival rates. Experimental results showed that DBT overexpression inhibited RCC cell growth, migration, and invasion. Moreover, we uncovered DBT's role in the tumor immune environment, suggesting that DBT may contribute to cancer development by initiating abnormal inflammatory and immune reactions. This research enhances our understanding of RCC pathophysiology and potential molecular targets.

Materials and Methods

Supplementary Materials

Supplementary Figures

Supplementary Tables