2.1 VHL–HIF–VEGFR–mTOR signaling pathway

The VHL–HIF–VEGFR–mTOR signaling pathway plays a crucial role in the pathogenesis of renal cancer (Figure 2). The VHL gene is one of the most important tumor suppressor genes in renal cancer.⁴ Under physiological conditions, the VHL protein (pVHL) inhibits angiogenesis and tumor growth, and regulates the stability of HIFs.^{22, 23} HIF is a heterodimeric transcription factor composed of α and β subunits. The α subunit is stable under hypoxic conditions, while under normoxic conditions, it is recognized and ubiquitinated by the VHL E3 ubiquitin ligase complex and ultimately degraded by the 26S proteasome.^{4, 24}

In ccRCC, the VHL gene frequently undergoes inactivating mutations or deletions, resulting in the dysfunction of the pVHL and an inability to properly degrade HIF-α, leading to its abnormal accumulation.^{25, 26} The accumulated HIF-α combines with HIF-β to form a heterodimer, which enters the nucleus and binds to the promoter regions of genes containing hypoxia response elements, thus activating the transcription of downstream genes, including VEGF, PDGF, carbonic anhydrase IX (CAIX), and others, thereby promoting tumor angiogenesis, proliferation, and metastasis.^{22, 23} Among these, VEGF not only stimulates the proliferation of vascular endothelial cells but also mediates the self-renewal of cancer stem cells through the activation of the VEGFR-2/Janus kinase (JAK)2/signal transducer and activator of transcription 3 (STAT3) signaling axis.²⁷ Furthermore, VHL inactivation also leads to the overactivation of the mTOR, which is associated with tumor progression and a poorer prognosis in ccRCC.⁴ Moreover, in addition to regulating angiogenesis and metabolic reprogramming, the VHL–HIF pathway is also closely related to epithelial–mesenchymal transition (EMT).¹⁶ HIF-1α can activate the expression of Twist-related protein 1 (TWIST1) and regulate the Snail/Slug/ZEB1 axis, inducing the downregulation of E-cadherin and promoting the occurrence of EMT, ultimately enabling the tumor cells to acquire a mesenchymal phenotype and enhance their invasive and metastatic capabilities.^{28, 29} Simultaneously, this signaling pathway can also stimulate the secretion of inflammatory factors, such as TGF-β, further exacerbating the EMT process and tumor progression.³⁰

In summary, the VHL–HIF–VEGFR–mTOR signaling pathway plays a crucial regulatory role in the initiation and progression of renal cancer. Inactivation of VHL leads to aberrant activation of HIF, which in turn causes overexpression of downstream target genes such as VEGF, thereby promoting tumor angiogenesis and tumor cell proliferation, and accelerating tumor progression through multiple mechanisms such as EMT and inflammation. Meanwhile, the hyperactivation of the mTOR signaling pathway further exacerbates this malignant signaling cascade. Therefore, developing novel antirenal cancer therapeutic strategies targeting this pathway may be highly significant for improving the prognosis of renal cancer patients. Several small molecule inhibitors targeting HIF, VEGFR, and mTOR have entered clinical trial phases, potentially providing new hope for advanced renal cancer patients. Future studies should further elucidate the precise regulatory mechanisms of this pathway, identify new therapeutic targets, and ultimately achieve precise treatment of renal cancer.

2.2 PI3K/AKT/mTOR signaling pathway

The PI3K/AKT/mTOR signaling pathway is critically involved in the pathogenesis and progression of renal cancer. Abnormal activation of the PI3K/AKT/mTOR pathway correlates significantly with the occurrence, invasion, and metastasis of renal cancer. In-depth research on the regulatory mechanisms of the PI3K/AKT/mTOR pathway in renal cancer holds immense potential in elucidating the molecular pathological mechanisms of renal cancer and developing new therapeutic strategies and drug targets. The PI3K/AKT/mTOR pathway is an evolutionarily highly conserved signaling pathway that is critically involved in various physiological processes such as cell proliferation, differentiation, apoptosis, and metabolism. PI3K is activated by various growth factors and cytokines, which in turn catalyzes the phosphorylation of PIP2 to generate PIP3; PIP3 subsequently recruits AKT to the cell membrane and activates it. Activated AKT further phosphorylates downstream substrates such as mTOR, consequently modulating cell growth, proliferation, and survival.³¹

In RCC, abnormal activation of the PI3K/AKT/mTOR signaling pathway is highly prevalent,³¹ with extracellular signaling molecules and transmembrane receptors playing a pivotal role in initiating the activation of this pathway. In ccRCC, the rate of genetic alterations in PI3K/AKT pathway components is 27.7%, primarily encompassing changes such as gene amplification, mutation, and deletion.³¹ Inactivation of the VHL gene and activation of the HIF signaling pathway are key driving factors in the onset and progression of ccRCC, both of which are intimately interconnected with the PI3K/AKT signaling pathway, collectively forming an intricate signaling network that facilitates the occurrence and development of ccRCC. In pRCC, the rate of genetic alterations in PI3K/AKT signaling pathway-related components is 28%.³¹ Type 1 pRCC is associated with MET gene mutations, and activation of MET can further lead to the activation of the PI3K/AKT signaling pathway.³² Conversely, type 2 pRCC is associated with fumarate hydratase (FH) gene mutations.³³ chRCC is a rare subtype of RCC; however, its genetic alterations in the PI3K/AKT signaling pathway are highly significant, with approximately 32% of patients exhibiting mutations or deletions of pathway-related components.³¹ Recent evidence suggests that the VHL/HIF and PI3K/AKT signaling pathways extensively interact within a complex signaling network, thus jointly promoting the occurrence and development of ccRCC. For example, the upregulation of HIF expression caused by VHL gene inactivation can promote the expression of multiple growth factors, including epidermal growth factor (EGF), PDGF, and VEGF, potentially further activating the PI3K/AKT signaling pathway. Simultaneously, activation of mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) also promotes HIF expression, thereby forming a positive feedback loop and leading to sustained activation of the entire signaling network.³¹ Furthermore, studies have also demonstrated that VHL gene mutations can promote the progression of ccRCC through a PI3K/AKT signaling pathway-dependent mechanism involving cholesterol ester accumulation.³⁴

In RCC, the activation of the PI3K/AKT/mTOR signaling pathway can be triggered not only by extracellular signaling molecules and transmembrane receptors but also by alternative mechanisms.^{31, 35} For example, glucose deprivation can induce a distinct form of AKT phosphorylation and activation in various RCC cell lines. Furthermore, microRNAs (miRNAs), as emerging critical regulators of the PI3K/AKT pathway, are garnering significant attention. Numerous studies have demonstrated that miRNAs, including miR-148a, miR-182-5p, and miR-137, can inhibit the PI3K/AKT/mTOR pathway, consequently suppressing the proliferation, migration, and invasive capabilities of RCC cells.³⁶ miR-148a targets AKT2, inhibiting its expression and activity, subsequently suppressing the activation of downstream mTOR, ultimately leading to the inhibition of tumor cell growth. miR-182-5p indirectly inhibits AKT2 activity by targeting FLOT1, resulting in increased activity of the downstream transcription factor FOXO3a, thereby suppressing tumor cell proliferation.³⁷ miR-137 directly inhibits the PI3K/AKT pathway, leading to increased tumor cell apoptosis, thereby suppressing their growth and metastatic abilities.³⁸ In contrast, certain miRNAs, such as miR-122, have the effect of activating the PI3K/AKT/mTOR pathway. miR-122 indirectly promotes the activation of the PI3K/AKT/mTOR pathway by suppressing the expression of tumor suppressor genes, such as SPRY2, thereby relieving the inhibition of the Rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK) pathway.^{39, 40} mTOR is a critical downstream effector of the PI3K/AKT signaling pathway. Studies have demonstrated that certain miRNAs can modulate the activity of the PI3K/AKT/mTOR signaling pathway by directly targeting mTOR. miR-99a and miR-144 have been shown to directly target mTOR, suppressing its expression. Nevertheless, their precise roles in RCC remain controversial and warrant further investigation for elucidation.^{36, 41} PTEN serves as a crucial negative regulator of the PI3K/AKT/mTOR signaling pathway. Numerous studies have demonstrated that miRNAs can indirectly modulate the activity of the PI3K/AKT/mTOR signaling pathway by targeting PTEN. These studies have revealed that miRNAs, including miR-23b, miR-193a-3p, and miR-21, can directly target PTEN, suppressing its expression, thus promoting the constitutive activation of the PI3K/AKT/mTOR signaling pathway and playing an oncogenic role in the initiation and progression of RCC.³⁶ Moreover, certain miRNAs can modulate the activity of the PI3K/AKT/mTOR signaling pathway through alternative mechanisms. For instance, miR-193a-3p and miR-224 can modulate the activity of the PI3K/AKT/mTOR signaling pathway by targeting ST3GalIV.⁴² Concurrently, miR-124-3p can exert tumor-suppressive effects by targeting CAV1 and FLOT1, thus inhibiting the PI3K/AKT and Ras/MAPK signaling pathways.⁴³

Endogenous molecules, such as fibroblast activation protein-α (FAP) and cell division cycle-associated 5 (CDCA5), have been demonstrated to promote the progression of RCC by activating the PI3K/AKT signaling pathway.^{44, 45} Knockdown of FAP expression attenuates the activity of the PI3K/AKT/mTOR signaling pathway, thereby inhibiting the growth of RCC, suggesting that targeting FAP may be a potential therapeutic strategy for RCC treatment.⁴⁴ In contrast, high expression of CDCA5 upregulates the PI3K/AKT signaling pathway, thereby stimulating RCC cell proliferation and invasion.⁴⁵ Natural compounds, such as shikonin and thymoquinone (TQ), have also been shown to exert anti-RCC effects by modulating the PI3K/AKT signaling pathway.^{46, 47} Shikonin inhibits RCC cell proliferation in a dose-dependent manner and induces apoptosis by activating the Ras/MAPK and PI3K/AKT signaling pathways.⁴⁶ Similarly, TQ inhibits RCC cell migration by suppressing the activation of the prostaglandin E2 (PGE2)-mediated EP2 receptor–PI3K/AKT axis.⁴⁷

As a key downstream effector molecule of the PI3K/AKT signaling pathway, mTOR is commonly found in a state of sustained activation in RCC.⁴⁸ Substantial evidence suggests that the mTOR signaling pathway is not only closely associated with the proliferation and differentiation of tumor cells but also plays a crucial role in the maintenance of cancer stem cells, potentially contributing to drug resistance in RCC.^{48, 49} In comparison with normal tissues, mTOR activity is significantly elevated in RCC tissues, and mTOR inhibitors have demonstrated efficacy in slowing the progression of RCC.⁴⁸ Various mechanisms can lead to abnormal activation of mTOR in RCC, including PTEN functional deficiency, enhanced function of PI3K catalytic subunits, and LKB1 gene mutations.^{2, 50-55} Notably, mTOR activation primarily stems from an increase in its phosphorylation level rather than changes in protein expression, indicating that targeted inhibition of mTOR phosphorylation may be an important strategy for suppressing mTOR activity.^{55, 56}

The PI3K/AKT/mTOR signaling pathway plays a pivotal role in the initiation and progression of renal cancer. Constitutive aberrant activation of this pathway exerts both direct and indirect promoting effects on renal cancer progression. Specifically, this pathway can directly promote tumor cell proliferation, survival, and invasion; furthermore, it can also indirectly facilitate renal cancer progression by maintaining cancer stem cells and conferring drug resistance. Consequently, drugs targeting PI3K, AKT, and mTOR could offer novel insights and therapeutic approaches for renal cancer treatment. Future research should focus on elucidating the molecular mechanisms underlying PI3K/AKT/mTOR pathway activation and identifying novel therapeutic targets to improve renal cancer diagnosis and treatment.

2.3 Hippo–YAP signaling pathway

The Hippo signaling pathway is a highly conserved signal transduction pathway throughout evolution, playing a crucial role in regulating organ size, maintaining tissue homeostasis, controlling cell proliferation, apoptosis, and other essential biological processes. This pathway consists of a series of kinase cascades, including the upstream mammalian Sterile 20-like kinase 1/2 (MST1/2), large tumor suppressor 1/2 (LATS1/2) serine/threonine kinases, and the downstream transcriptional coactivators yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). When the Hippo pathway is activated, MST1/2 phosphorylates and activates LATS1/2, which in turn phosphorylates YAP/TAZ, leading to their cytoplasmic retention and degradation. Conversely, when the Hippo pathway is inactivated, nonphosphorylated YAP/TAZ translocate into the nucleus, binds to transcription factors such as the TEAD family, and promotes the transcriptional expression of downstream target genes, thereby promoting cell proliferation and inhibiting apoptosis. Accumulating evidence suggests that dysregulation of the Hippo–YAP pathway is closely associated with the occurrence and development of various tumors, including renal cancer.⁵⁷

Numerous studies have demonstrated that aberrant activation of the Hippo signaling pathway is pervasive in renal cancer tissues.⁵⁷ As an upstream regulatory gene of the Hippo pathway, mutations in the neurofibromatosis type 2 (NF2) gene can result in inactivation of the Hippo pathway and aberrant activation of YAP.⁵⁸ Alterations in the Hippo pathway and persistent activation of YAP have been identified in NF2-deficient uRCC.^{57, 59} Moreover, mechanistic studies have elucidated that silencing YAP/TAZ in NF2-deficient tumors can facilitate tumor regression. The underlying mechanism is that the absence of YAP/TAZ enhances mitochondrial respiration, diminishes reliance on glycolytic growth, and results in the accumulation of reactive oxygen species (ROS) and oxidative stress-induced cell death under nutrient-deprived conditions.^{57, 60}

The expression of the core kinases LATS1/2 in the Hippo pathway is generally downregulated in RCC.⁵⁷ Studies have demonstrated that RCC patients with high expression of LATS1/2 exhibit significantly prolonged overall survival (OS) and disease-free survival compared with those with low expression.⁶¹ Furthermore, elevated levels of methylation have been observed in the promoter region of LATS1 in RCC tissues and cell lines. Treatment of RCC cells with the DNA methylation inhibitor 5-aza-2′-deoxycytidine can demethylate LATS1, downregulate YAP expression, promote cell apoptosis, arrest the cell cycle, and inhibit cell proliferation.⁶² As key effector molecules downstream of the Hippo pathway, abnormal activation of YAP/TAZ is closely associated with the occurrence, progression, and prognosis of RCC.⁵⁷ In ccRCC patients, TAZ expression was found to be significantly upregulated, and high expression of TAZ is indicative of a poor prognosis.⁶³ Further studies have revealed that TAZ can induce the expression of NADPH oxidase 4 (Nox4) by regulating epithelial membrane protein 1 (EMP1). Nox4 is enriched in the kidney and generates ROS associated with ferroptosis; therefore, the absence of TAZ can counteract ferroptosis.^{57, 64, 65}

The Hippo–YAP pathway forms a complex cross-regulatory network with multiple key molecules and signaling pathways in the occurrence and progression of RCC. Research suggests that the overexpression of transferrin and B4GALNT1 may contribute to the occurrence and progression of ccRCC by modulating the Hippo pathway. Investigations have revealed that the downregulation of SAV1 expression, a core component of the Hippo pathway, results in the aberrant activation of YAP, which plays a crucial role in the pathogenesis of high-grade ccRCC.^{57, 66} Moreover, in mucinous tubular and spindle cell carcinoma (MTSCC), YAP1 overexpression is a critical factor driving its pathogenesis, and pertinent studies have demonstrated that the YAP inhibitor verteporfin exhibits a potential therapeutic effect on metastatic MTSCC.⁵⁷ Conversely, leukemia inhibitory factor receptor suppresses tumor metastasis by activating the Hippo pathway and downregulating YAP expression.⁵⁷ Various noncoding RNAs (ncRNAs), including TUG1 and miR-9, have also been identified as regulators of renal cancer growth and metastasis through their modulation of YAP activity. Furthermore, molecules including Claudin-2, SH3BGRL2, QKI, and REGγ/casein kinase 1ε (CK1ε) have also been demonstrated to exert critical regulatory functions in renal cancer progression through their interactions with the Hippo–YAP pathway. These research findings elucidate the intricate regulatory network of the Hippo–YAP pathway in renal cancer occurrence and progression, offering valuable insights for further elucidating the molecular mechanisms underlying renal cancer and identifying novel therapeutic targets.⁵⁷

Overall, the dysregulation of the Hippo–YAP signaling pathway plays a pivotal role in the initiation and progression of renal cancer. Upstream regulators of the Hippo pathway, such as NF2 and LATS1/2, are frequently mutated or downregulated in renal cancer tissues, resulting in pathway inactivation. Conversely, downstream effector molecules, including YAP/TAZ, are aberrantly activated, promoting renal cancer cell proliferation and survival. The Hippo–YAP pathway has an extensive cross-regulatory network with multiple critical molecules and signaling cascades, which collectively participate in regulating renal cancer progression. Comprehensive characterization of the dysregulation mechanisms of the Hippo–YAP pathway is anticipated to provide novel insights and strategies for targeted therapy of renal cancer. Interventions targeting pivotal components of this pathway, including LATS1/2 and YAP/TAZ, may serve as effective approaches for the future treatment of renal cancer.

2.4 Wnt/β-catenin signaling pathway

The WNT/β-catenin pathway is a highly conserved signal transduction pathway that plays a pivotal role in various biological processes, including embryonic development, organogenesis, and maintenance of tissue homeostasis. This pathway primarily consists of Wnt ligands, transmembrane receptors, cytoplasmic protein degradation complexes, and nuclear transcription factors. In the absence of Wnt ligand stimulation, cytoplasmic β-catenin is phosphorylated by kinases such as glycogen synthase kinase 3β (GSK3β), which subsequently leads to its ubiquitination and degradation, thus maintaining low levels of β-catenin. Upon Wnt ligand binding to transmembrane receptors, GSK3β kinase activity is suppressed, resulting in the accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus. In the nucleus, β-catenin interacts with transcription factors such as TCF/LEF, activating the transcription of downstream target genes and ultimately triggering a cascade of cell fate determination events. In addition to the canonical Wnt/β-catenin pathway, the noncanonical Wnt/Ca2+ and Wnt/planar cell polarity pathways also play crucial roles in regulating cell polarity and migration.¹⁹

In recent years, numerous studies have demonstrated that the WNT/β-catenin signaling pathway is frequently aberrantly activated during the development of various solid tumors and is closely associated with the malignant phenotype of these neoplasms.^67-69 In RCC, the key components of the Wnt/β-catenin signaling pathway are generally dysregulated.¹⁹ For instance, canonical Wnt ligands, such as Wnt1 and Wnt10A, are highly expressed in RCC tissues and are associated with tumor staging and invasiveness.^{70, 71} Conversely, noncanonical ligands, including Wnt7A and Wnt5A, are downregulated,^{72, 73} and their reduced expression may also be implicated in the development of renal cancer.¹⁹ Notably, β-catenin frequently accumulates aberrantly in RCC tissues, and its cytoplasmic levels are closely correlated with tumor diameter, staging, and prognosis.^{74, 75} Although β-catenin gene mutations are relatively uncommon in RCC, multiomics analyses have confirmed that genes associated with the WNT/β-catenin pathway generally undergo genetic and epigenetic alterations.¹⁹ In addition to Wnt ligands and receptors, antagonists such as secreted frizzled-related proteins (sFRPs), WNT inhibitory factor 1, and Dickkopf (DKK) family members are also frequently deficient in RCC tissues, further exacerbating the sustained activation of the WNT/β-catenin signaling pathway.¹⁹ The overexpression of genes such as CENPA and GBP2 can accelerate the cell cycle progression of RCC cells by activating the WNT/β-catenin pathway, thereby promoting their proliferation, migration, and invasion.^{76, 77} Moreover, the sustained activation of the WNT/β-catenin pathway can lead to chemotherapy drug tolerance in RCC cells and induce T cell differentiation into stem cell memory T-cell subpopulations, thus enhancing the tumor's immune escape capability.⁷⁸ These results suggest that the dysregulated expression of WNT/β-catenin pathway components may be a crucial factor driving the occurrence and development of RCC, and targeting this pathway could potentially serve as an effective strategy for RCC treatment.

The WNT/β-catenin pathway does not function independently during the development of RCC; instead, it is closely interconnected with multiple other signaling pathways. Research has demonstrated that the WNT/β-catenin pathway can promote carcinogenesis by modulating the expression of oncogenes and cell cycle regulators, including c-Myc and Cyclin D1.¹⁹ Moreover, the WNT/β-catenin pathway can act synergistically with the VHL–HIF pathway to augment the motility and invasive potential of RCC cells. Nonetheless, HIF-1α and HIF-2α appear to exert opposing biological effects in RCC,¹⁹ and the maintenance of intracellular homeostasis following VHL inactivation is contingent upon the delicate balance between these HIF isoforms.^{79, 80} Consequently, further investigation is necessary to elucidate the interaction patterns among the VHL, HIF, and WNT/β-catenin pathways and their influence on the RCC phenotype. Recent studies have also revealed that multiple miRNAs can function as either oncogenes or tumor suppressor genes in the progression of RCC by targeting various components of the WNT/β-catenin signaling pathway.³⁶ For instance, miR-106b-5p, miR-1260b, and miR-203a target the negative regulatory factors LZTFL1, SFRP1, DKK2, and GSK3β of the WNT/β-catenin pathway, respectively, thereby promoting RCC cell proliferation, invasion, and stem cell phenotype, and thus acting as oncogenes.^81-83 Conversely, miR-372 targets the IGF2BP1 gene, inhibiting the WNT/β-catenin pathway and exhibiting tumor suppressor properties.⁸⁴ These research findings unveil the intricate regulatory network of the WNT/β-catenin signaling pathway in the development and progression of RCC, offering crucial insights into the molecular mechanisms underlying this malignancy. Future research should focus on elucidating the crosstalk between the WNT/β-catenin signaling pathway and other signaling cascades, as well as unraveling the precise mechanisms by which various miRNAs regulate this pathway. Such endeavors will facilitate the identification of novel therapeutic targets and strategies for RCC management.

The function of noncanonical Wnt signaling pathways in RCC has not been extensively studied, and the related research is relatively limited compared with that of the Wnt/β-catenin signaling pathway.¹⁹ Receptor tyrosine kinase (RTK)-like orphan receptor 2 (RoR2) is a type of Wnt ligand receptor that is typically expressed only during embryonic development. However, it is overexpressed in ccRCC.⁸⁵ Suppressing the expression of Ror2 in RCC using shRNA or mutagenesis methods can effectively attenuate tumor growth, cell migration, and invasion promoted by Wnt/Rho signal transduction.^{85, 86} The activity of DICKKOPF-3 (Dkk-3), an antagonist of the Wnt pathway, is reduced in RCC. Restoring its expression can not only inhibit tumor cell proliferation but also promote cell apoptosis.⁸⁷

In summary, the WNT/β-catenin signaling pathway plays a pivotal role in the initiation and progression of renal cancer. On one hand, components of the Wnt/β-catenin signaling pathway, including Wnt ligands, receptors, and β-catenin, are frequently dysregulated in renal cancer tissues. Furthermore, the loss of Wnt antagonists exacerbates the constitutive activation of the pathway. On the other hand, the aberrantly activated Wnt/β-catenin signaling pathway confers multiple malignant phenotypes upon renal cancer cells, including enhanced proliferation, invasion, drug resistance, and immune evasion, by regulating downstream target genes. Moreover, this pathway synergistically promotes renal cancer progression in cooperation with other signaling pathways, such as the VHL–HIF pathway, while also being intricately regulated by ncRNAs, including miRNAs. These research findings not only enhance our understanding of renal cancer pathogenesis but also provide a theoretical basis for the development of targeted therapies against the WNT/β-catenin signaling pathway. It is anticipated that as the regulatory network and aberrant activation mechanisms of the WNT/β-catenin signaling pathway in tumors are further elucidated, novel therapeutic strategies will continue to emerge, ultimately benefiting a substantial number of renal cancer patients.

2.5 cAMP signaling pathway

cAMP is a pivotal second messenger molecule that plays a crucial role in intracellular signaling. It is primarily synthesized by membrane-bound adenylyl cyclase through the catalysis of adenosine triphosphate (ATP) and is subsequently hydrolyzed by phosphodiesterase into adenosine monophosphate (AMP). cAMP is involved in the regulation of various cellular functions, including cell growth, differentiation, apoptosis, and metabolism. cAMP primarily binds to effector molecules, such as protein kinase A (PKA) and cyclic AMP-responsive element-binding protein (CREB), thereby regulating downstream signaling pathways and gene expression.² In recent years, numerous studies have demonstrated that the cAMP signaling pathway plays a significant role in the development and progression of various tumors; however, its specific mechanisms in renal cancer have not been fully elucidated and warrant further in-depth investigation.

Studies have discovered that increased synthesis of acetylcholine (ACh) activates the cAMP/PKA pathway through its receptors, subsequently phosphorylating CREB, leading to invasive migration and proliferation of renal cancer cells.^88-90 Phosphorylated CREB can promote the invasive metastasis of renal cancer by regulating the expression of matrix metallopeptidases (MMP2 and MMP9) and EMT-related proteins.⁸⁹ Moreover, research indicates that the level of CREB phosphorylation is upregulated in ccRCC tissues and cell lines, while inhibiting CREB phosphorylation at the serine 133 site can significantly suppress the growth and metastatic activity of OS-RC-2 cells.^{88, 89} In addition to ACh, dysregulation of the cAMP signaling pathway may be associated with the development of renal cancer. An analysis of ccRCC cell lines confirmed the abnormal expression of CREB1 protein, suggesting that a posttranscriptional regulatory mechanism may contribute to CREB1 dysregulation.⁹¹ As one of the downstream effector molecules of cAMP, CREB activity is regulated by cAMP levels. Under the influence of the tumor microenvironment (TME), the cAMP signaling pathway can exert a dual effect on tumor cell growth, either promoting or inhibiting it.

In summary, the cAMP signaling pathway is implicated in the regulation of renal cancer initiation and progression through diverse mechanisms. On one hand, factors such as ACh can activate the cAMP/PKA/CREB pathway, leading to the upregulation of MMP2/9) and EMT-related molecules, thereby promoting the proliferation, invasion, and metastasis of renal cancer cells. On the other hand, the dysregulation of cAMP signaling molecules, particularly CREB, is strongly associated with renal cancer, indicating that targeting the cAMP pathway and its downstream effectors may offer novel strategies for the prevention and treatment of this malignancy. In future studies, it will be crucial to further elucidate the molecular mechanisms underlying the cAMP signaling pathway's role in regulating the initiation and progression of renal cancer, facilitating the identification of potential therapeutic targets.

2.6 HGF/c-Met signaling pathway

c-Met, a transmembrane tyrosine kinase receptor, is activated by HGF and plays a crucial role in regulating cell migration under both physiological and pathological conditions. Extensive research has shown that c-Met is implicated in the EMT process of multiple cancer types. Moreover, the interaction between c-Met and HGF can stimulate tumor cell mitosis, motility, angiogenesis, migration, and invasion.⁹² Recently, the oncogenic role of c-Met in urinary system malignancies, particularly in renal cancer, has garnered significant attention.

VHL gene mutations and hypoxic conditions can lead to the upregulation of HGF and its receptor c-Met in renal cancer.^93-95 Furthermore, HIF-1 can also regulate the expression of c-Met and VEGF under hypoxic conditions.⁹⁶ These findings suggest that the HGF/c-Met signaling pathway may contribute to the progression of renal cancer by promoting tumor angiogenesis. Consequently, c-Met has emerged as a crucial target for antiangiogenic therapy in renal cancer. Besides inducing tumor angiogenesis, c-Met can also facilitate the malignant progression of renal cancer through alternative mechanisms. Research has demonstrated that the phosphorylation level of the c-Met receptor positively correlates with tumor growth, vascularization, propensity for lung metastasis, and cell migration capacity in renal cancer.⁹⁷ Moreover, an increase in MET gene copy number is also linked to poor prognosis and distant metastasis in patients with ccRCC.⁹⁸ These findings further highlight the crucial role of c-Met in the metastatic cascade of renal cancer.

Given the crucial role of the HGF/c-Met pathway in the pathogenesis and progression of RCC, therapeutic strategies targeting this pathway have emerged as a promising area of research. For example, the combination of axitinib, a VEGF inhibitor, and crizotinib, a c-Met inhibitor, has demonstrated significant improvements in the therapeutic efficacy of RCC by simultaneously targeting these two key pathways.⁹² Furthermore, natural small-molecule compounds, including honokiol, rapamycin, and piperine, have exhibited anti-RCC effects by inhibiting c-Met activity, thus demonstrating promising potential for clinical application.⁹⁹

In summary, the HGF/c-Met signaling pathway plays a pivotal role in the initiation and progression of renal cancer. First, this pathway can directly promote renal cancer progression by facilitating tumor angiogenesis and EMT. Second, abnormalities in c-Met activity and MET gene copy number are also strongly correlated with renal cancer metastasis. Consequently, therapeutic strategies targeting the HGF/c-Met axis hold promise for providing novel treatment options for patients with advanced and metastatic renal cancer. Future research should further elucidate the molecular mechanisms underpinning the role of the HGF/c-Met pathway in renal cancer initiation and progression and explore safer and more effective c-Met inhibitors, ultimately providing innovative strategies for the precision treatment of renal cancer.

2.7 p53 signaling pathway

In the context of renal cancer, the p53 signaling pathway plays a critical role. The p53 protein, encoded by the TP53 gene, is a crucial tumor suppressor that maintains genomic integrity and inhibits tumor development. Under physiological conditions, p53 is activated in response to various stress signals, initiating downstream transcriptional programs that suppress tumor growth. Upon detection of cellular damage or stress, p53 triggers various biological processes, including cell cycle arrest, DNA repair, apoptosis, and autophagy, which maintain genomic stability and prevent malignant cell transformation.^{2, 9}

The dysregulation of the p53 signaling pathway is closely associated with the initiation and progression of RCC.⁹ Studies have demonstrated that although the mutation rate of the TP53 gene in RCC is relatively low, the loss of p53 activity remains a common characteristic of this malignancy.^{9, 100, 101} This finding suggests that alternative mechanisms may exist that impede the transmission of p53 signaling in RCC. For instance, the activation of mTOR and the inactivation of the p38MAPK-p53/p16 pathway are believed to synergistically trigger the transformation of the proximal renal tubule into RCC.¹⁰² Moreover, the interplay between p53 and the VHL gene is essential for regulating p53-mediated DNA damage response, and their dysregulation may facilitate the progression of RCC.¹⁰³ Additionally, there exists an intricate regulatory relationship between p53 and HIF-1α. Certain studies propose that HIF-1α can induce the inhibition of MDM2, thus stabilizing p53 and promoting cellular apoptosis^{104, 105}; conversely, other investigations suggest that hypoxia and HIF-1α may negatively regulate the stability and activity of p53 under specific conditions.¹⁰⁴ These findings underscore the extensive cross-talk between the p53 pathway and other critical pathways implicated in the pathogenesis and progression of RCC.

In addition to p53 itself, other components of the p53 pathway are also implicated in the pathogenesis of renal cancer. PPM1D, a transcriptional target gene of p53, can reverse DNA damage checkpoints and attenuate cell cycle arrest by dephosphorylating p53 and Chk1.¹⁰⁶ MDM2, a negative regulator of p53, promotes the degradation of p53 through ubiquitin-mediated proteasomal degradation.¹⁰⁷ In addition to MDM2, other molecules, including ARF-BP1/Mule, MdmX/Mdm4, Cop1, and Pirh2, have been identified as negative regulators of p53 signaling.^{2, 108} Alterations in these factors that regulate the stability and activity of p53 are likely to contribute to the loss of p53 function in RCC.

In summary, the p53 signaling pathway plays a pivotal role in the initiation and progression of renal cancer. On one hand, as the guardian of the genome, the loss of p53 activity renders renal cells vulnerable to malignant transformation; on the other hand, there exists significant reciprocal regulation between the p53 pathway and critical renal cancer pathways such as VHL and HIF, and the imbalance among these pathways further facilitates tumor progression. In-depth investigation of p53 pathway abnormalities not only contributes to elucidating the molecular mechanisms underlying renal cancer but also provides new insights and potential therapeutic targets for the diagnosis and treatment of this malignancy. Moving forward, it is imperative to further elucidate the mechanisms by which p53 signaling integrates various stress responses and investigate strategies for targeted regulation of the p53 pathway, thereby providing more clues for precise diagnosis and personalized treatment of renal cancer.

2.8 Ferroptosis-related signaling pathways

Ferroptosis, an emerging form of regulated cell death, is characterized by iron-mediated lipid peroxidation on the cell membrane, distinguishing it from other types of cell death, such as apoptosis and necroptosis, in terms of morphological and molecular mechanisms. The primary characteristic of ferroptotic cells is the shrinkage of mitochondrial cristae, without displaying typical apoptotic features, including chromatin condensation and apoptotic body formation. The occurrence of ferroptosis depends on three crucial factors: (1) the synthesis of polyunsaturated fatty acid phospholipids (PLs) and their iron-catalyzed peroxidation; (2) the regulation of iron metabolism, including the promotion of the Fenton reaction and serving as an essential cofactor for lipid peroxidases; and (3) the modulation of mitochondrial metabolism, as mitochondria facilitate ferroptosis through their roles in bioenergetics, biosynthesis, and ROS generation. This iron-dependent PL peroxidation process is stringently regulated by various intracellular metabolic pathways, encompassing redox balance, iron metabolism, mitochondrial activity, amino acid and lipid metabolism, and glucose metabolism. It is crucial to highlight the pivotal role of iron metabolism in the regulatory network governing ferroptosis.¹⁰⁹

Recent investigations have demonstrated that ferroptosis is suppressed during the initiation and progression of renal cancer. Elucidating the mechanisms by which RCC tumors inhibit cellular ferroptosis and developing strategies to reactivate ferroptosis may offer novel insights for RCC treatment. ccRCC, the predominant subtype of RCC, exhibits a distinct metabolic profile that is intimately associated with its sensitivity to ferroptosis. Protein disulfide-isomerase A4 (PDIA4) confers resistance to ferroptosis in ccRCC cells by upregulating ATF4/SLC7A11, whereas salinomycin exerts antitumor effects by suppressing PDIA4.¹¹⁰ Moreover, Glutathione peroxidase 4 (GPX4) plays a pivotal role in ccRCC progression, and Kruppel Like Factor 2 (KLF2) is implicated in the regulation of GPX4 expression in ccRCC. Overexpression of KLF2 suppresses tumor growth and invasion by modulating ferroptosis.¹¹¹ Acyl CoA long-chain synthetase 3 (ACSL3) regulates lipid droplet accumulation in ccRCC, a process crucial for tumor growth, and also modulates ferroptosis sensitivity in a manner dependent on the composition of exogenous fatty acids. Both ferroptosis-inducing and ferroptosis-inhibiting functions can be exploited for the treatment of clear ccRCC. The absence of AIM2, a tumor suppressor, promotes ccRCC progression and sunitinib resistance through the inhibition of ferroptosis regulated by the FOXO3a/ACSL4 axis.¹¹² Similarly, the Hippo pathway effector TAZ and iron–sulfur cluster assembly enzyme 2 (ISCA2) have been shown to induce ferroptosis in ccRCC.¹¹³ Furthermore, a prognostic model based on eight ferroptosis-related long noncoding RNAs (lncRNAs) has been developed for predicting the prognosis of ccRCC patients. The combination of URB597, a fatty acid amide hydrolase (FAAH) inhibitor, and (1S,3R)-RSL3, a ferroptosis inducer, demonstrates potent synergistic inhibition of RCC growth by inducing G1 cell cycle arrest and promoting ROS generation. This dual-targeted therapy modulates the sensitivity of RCC cells to ferroptosis.¹¹⁴

In conclusion, ferroptosis plays a pivotal role in the initiation and progression of renal cancer. The distinct metabolic profile of renal cancer is intimately associated with its sensitivity to ferroptosis, with multiple molecular mechanisms implicated in the regulation of ferroptosis in renal cancer, including PDIA4, GPX4, KLF2, ACSL3, and AIM2. Ferroptosis holds promise as a novel therapeutic target and offers potential avenues for the prevention, diagnosis, and treatment of renal cancer.

2.9 Cuproptosis-related signaling pathways

Copper is one of the essential trace elements that maintains the balance of the internal environment in the human body. As a cofactor of various enzymes, copper participates in regulating multiple physiological processes within cells, such as the function of cytochrome coxidase (COX), which requires the involvement of copper ions to complete cellular respiration.¹¹⁵ However, recent research indicates that excessive intracellular copper concentrations can trigger a unique form of cell death known as “cuproptosis.”¹¹⁶ The primary mechanism of cuproptosis involves the binding of excess copper to succinylated proteins in the tricarboxylic acid cycle, leading to abnormal aggregation of lipidation-related proteins and degradation of iron–sulfur cluster proteins, ultimately resulting in cell death due to an intracellular protein toxicity stress response.¹¹⁶ Appropriate levels of copper can induce autophagy-mediated degradation of GPX4, thereby promoting ferroptosis, indicating a crosstalk between ferroptosis and cuproptosis.¹¹⁷

ccRCC is insensitive to conventional radiotherapy and chemotherapy, which may be attributed to its resistance to cell death-related signaling pathways. Currently, various forms of cell death, such as apoptosis, necrosis, pyroptosis, ferroptosis, and autophagic cell death, have been extensively studied in ccRCC²²; however, the role of cuproptosis in the pathogenesis and progression of ccRCC remains largely unexplored.¹¹⁵ Multiple studies have demonstrated that copper levels are elevated to varying degrees in ccRCC patients, suggesting the potential involvement of cuproptosis in the pathogenesis and progression of ccRCC. For instance, Pirincci et al.¹¹⁸ reported that serum copper levels were significantly higher in ccRCC patients compared with the control group. Similarly, Panaiyadiyan et al.¹¹⁹ observed a significant elevation in blood copper concentration in renal cancer patients. Furthermore, elevated expression of the copper transporter protein CTR2 is associated with poor prognosis in ccRCC.¹²⁰

Cuproptosis may contribute to metabolic reprogramming in ccRCC by modulating the activity of crucial mitochondrial enzymes.¹¹⁵ Dysregulation of the VHL/HIF pathway and perturbations in copper homeostasis may synergistically contribute to metabolic reprogramming in ccRCC, offering a novel insight into the role of cuproptosis in the initiation and progression of ccRCC. Mitochondrial dysfunction and metabolic reprogramming are hallmark features of ccRCC.¹²¹ Deletion of the VHL gene and aberrant activation of the HIF pathway are pivotal in driving the metabolic reprogramming of ccRCC.¹¹⁵ Deletion of the VHL gene results in enhanced stability of HIF-1α and HIF-2α proteins, leading to upregulated glycolysis and consequently diminished mitochondrial respiration and energy metabolism.¹¹⁵ Subsequent investigations have revealed an intricate regulatory interplay between copper and the HIF-1 pathway.¹¹⁵ On the one hand, copper can enhance the expression and transcriptional activity of HIF-1, thereby promoting the expression of its downstream target genes, such as VEGF.^{122, 123} Conversely, HIF-1 can also negatively modulate the expression of copper transport proteins via posttranscriptional mechanisms, such as suppressing the expression of copper transporter 1A (CTR1A).¹²⁴ ATPase copper-transporting beta (ATP7B) is a copper-transporting ATPase that exhibits markedly increased expression in ccRCC, potentially serving as a compensatory mechanism in response to elevated copper levels.¹²⁵

Another important characteristic of cuproptosis is the disruption of lipoic acid metabolism and Fe–S cluster proteins.¹¹⁵ Studies have demonstrated that the use of copper ion carriers leads to the depletion of Fe–S cluster proteins in an FDX1-dependent manner, thereby triggering proteotoxic stress.¹¹⁶ In ccRCC, the expression of multiple Fe–S cluster proteins, such as FDX1, LIAS, ACO-2, and SDHB, is generally reduced and is associated with a poor prognosis.¹¹⁵ Furthermore, FDX1 knockout can suppress cuproptosis in ccRCC cells.¹²⁶ Research has also revealed that the expression and activity of key enzymes involved in lipoic acid biosynthesis, such as LIAS and LIPT, are altered in ccRCC cells, leading to the dysfunction of lipoylated proteins, which may contribute to metabolic reprogramming in ccRCC.^{115, 127} It is worth noting that the activation of the HIF pathway induced by VHL gene deletion can suppress the expression of the Fe–S protein SDHD by upregulating miR-210, suggesting that the VHL/HIF pathway is implicated in cuproptosis regulation.¹¹⁵

In summary, cuproptosis, a recently discovered form of cell death, is strongly correlated with the onset and advancement of renal cancer. Patients with renal cancer frequently exhibit elevated copper levels. Furthermore, cuproptosis-associated molecular mechanisms, including mitochondrial dysfunction, Fe–S cluster protein dysregulation, and protein persulfidation, might contribute to the metabolic reprogramming observed in renal cancer.

2.10 NF-κB signaling pathway

NF-κB is a ubiquitous transcription factor in mammalian cells, generally existing as heterodimers.¹²⁸ The canonical NF-κB pathway involves complexes formed by p50 and RelA, while the noncanonical NF-κB pathway is primarily associated with p52/RelB.¹²⁸ The NF-κB pathway is involved in the inflammatory stress response triggered by cytokines, bacterial toxins, viral products, and cell death stimuli.¹²⁹ Under physiological conditions, the NF-κB pathway regulates the innate immune system, whereas its abnormal activation may lead to pathological reactions during tumor development.¹³⁰ For example, the NF-κB pathway can promote cancer cell migration and invasion by upregulating multidrug resistance genes, proangiogenic factors, and proinflammatory cytokines, such as EGF, VEGFA, IL-8, and IL-6.¹³¹ The NF-κB subunit p50 can regulate the transition of macrophages from a protumorigenic phenotype to an M1 type, thereby inhibiting tumor growth.¹³² VHL deficiency can activate the NF-κB pathway,¹³³ which in turn induces the expression of antiapoptotic genes Bcl-xL and Bcl-2 and suppresses the expression of tumor suppressor genes (such as p53), suggesting its potential role in the development of renal cancer.^{129, 134} Increased expression of NF-κB is associated with lower survival rates in RCC patients.¹³⁵ Activation of the NF-κB and STAT3 pathways increases the infiltration of regulatory T cells (Tregs) in tumor tissues, thereby promoting the initiation, development, and metastasis of renal cancer.^{129, 136}

2.11 TGF-β signaling pathway

TGF-β is a class of evolutionarily highly conserved, multifunctional cytokines that play crucial roles in regulating various biological processes, including cell proliferation, differentiation, apoptosis, and the synthesis and degradation of extracellular matrix (ECM).^{137, 138} The TGF-β family consists of three subtypes: TGF-β1, TGF-β2, and TGF-β3, with TGF-β1 being abundantly expressed in the kidneys. TGF-β exerts its effects by binding to its specific receptors, namely type I and type II transmembrane serine/threonine kinase receptors. Upon ligand binding, the type II receptor recruits and phosphorylates the type I receptor, which in turn is activated and specifically phosphorylates the intracellular R-Smad proteins (Smad2/3).¹³⁸ Phosphorylated R-Smads form heterotrimeric complexes with Co-Smad (Smad4), translocate into the nucleus, and interact with other transcription factors, coactivators, or corepressors to regulate the transcriptional expression of downstream target genes.¹²

In adult kidneys, the TGF-β/Smad signaling pathway plays a crucial role in regulating the pathophysiological processes of glomeruli and renal tubules.^{139, 140} Numerous studies have demonstrated that aberrant activation of TGF-β1 is strongly correlated with the development of renal fibrosis. TGF-β1 can stimulate the synthesis of ECM proteins, including fibronectin and type I collagen, while suppressing the expression of MMPs. This imbalance leads to an excessive accumulation of ECM, ultimately resulting in renal interstitial fibrosis and loss of renal function.^{2, 139} Apart from its involvement in the process of renal fibrosis, the TGF-β signaling pathway is also intimately associated with the initiation and progression of renal cancer.^{12, 141} Accumulating evidence suggests that TGF-β plays a paradoxical role in the progression of renal cancer.^142-144 In the early stages of renal cancer, TGF-β acts as a tumor suppressor by inhibiting cell proliferation and promoting cell apoptosis. However, as the renal tumor advances, cancer cells progressively become resistant to TGF-β-mediated growth inhibition and apoptosis. Instead, they acquire phenotypic changes that facilitate tumor invasion and metastasis.^{12, 145-152}

Mechanistic studies have demonstrated that TGF-β promotes the malignant progression of RCC through multiple signaling pathways. TGF-β1 can promote the invasion and metastasis of RCC cells by regulating the expression of secreted protein acidic and rich in cysteine (SPARC), activating the AKT pathway, and upregulating the expression of MMP2.¹⁵³ Furthermore, TGF-β can also exert a protumorigenic effect by regulating the expression of ncRNAs. For instance, the overexpression of TGF-β1-induced long noncoding RNA-activated by TGF-β (lncRNA-ATB) and SPRY4 intronic transcript 1 (SPRY4-IT1) can promote EMT and the invasive capacity of RCC cells. Conversely, inhibiting TGF-β-induced EMT can effectively suppress the metastasis of RCC.^{154, 155} Moreover, there exists a cross-regulation between the TGF-β signaling pathway and the hypoxia pathway caused by VHL gene deletion.¹⁵² Studies have revealed that VHL gene knockout can activate the TGF-β pathway, promoting the proliferation and survival of RCC cells. Conversely, inhibiting TGF-β can attenuate the invasive capacity of ccRCC cells caused by VHL deficiency.¹² These findings suggest that targeting the TGF-β signaling pathway may provide novel therapeutic strategies for the treatment of RCC.

In summary, the TGF-β/Smad signaling pathway plays a pivotal role in the pathophysiological processes of the kidney and the progression of renal cancer. TGF-β facilitates the onset and progression of renal fibrosis by inducing the accumulation of ECM proteins. During the progression of renal cancer, TGF-β exhibits a dual role, exerting antitumor effects in the early stages, while promoting EMT, invasion, and metastasis of tumor cells in the advanced stages. Elucidating the molecular mechanisms of TGF-β in the development of kidney diseases and renal cancer will facilitate the identification of novel diagnostic biomarkers and therapeutic targets, offering new perspectives for the prevention and treatment of renal cancer.

3.1 Surgical treatment

Surgical resection is irreplaceable in treating localized RCC, making it the preferred option. Commonly used nephrectomy modalities include radical nephrectomy (RN) and partial nephrectomy (PN). RN implies the removal of the entire kidney, perirenal adipose tissue, and Gerota's fascia, maximizing the resection of the diseased area. However, RN requires a large incision, especially in open RN, leading to acute nephron loss, which increases the load on the contralateral kidney and risks postoperative acute kidney injury.¹⁶¹ PN, including nephron-sparing surgery (NSS), removes only the lesion site, preserving as many normal nephrons as possible, thus better preserving postoperative renal function and reducing cardiovascular disease risk.¹⁶² In early-stage tumor lesions, NSS presents noninferiority compared with RN. Although PN can lead to complications like hematuria, perinephric hematoma, and urinary fistulae, it is recommended for early-stage RCC patients, especially T1 and T2, when technically feasible.¹⁶³ Recent studies indicate that removing the primary tumor in metastatic RCC can benefit patients through cytoreductive nephrectomy (CN) or debulking nephrectomy.¹⁶⁴ CN works synergistically with immunotherapy by eliminating the primary tumor and removing cytokines and proteins that inhibit the immune response.¹⁶⁵ Studies comparing CN combined with IFN-α to IFN-α alone in mRCC patients show that combination therapy significantly improves median survival (13.6 vs. 7.8 months), regardless of performance status, metastasis site, and measurable disease presence.¹⁶⁶ However, with the advent of targeted therapies and immunotherapy, the benefits of CN in comprehensive mRCC treatment remain controversial for both immediate and deferred CN.¹⁶⁷ More studies are needed to confirm CN's value and role.

Minimally invasive surgery (MIS), including laparoscopic and robotic-assisted surgery, has significant OS advantages over open surgery for stage I and II RCC. MIS also has lower readmission rates (2.4 vs. 2.87%), 30-day mortality rates (0.53 vs. 0.96%), and 90-day mortality rates (1.04 vs. 1.77%).¹⁶⁸ For T3 stage RCC, the laparoscopic radical nephrectomy (LRN) group had lower estimated blood loss (100 vs. 650 mL, p < 0.001) and shorter hospital stays (4 vs. 9 days, p < 0.001) compared with the open radical nephrectomy (ORN) group. This suggests that LRN performs better than ORN in the perioperative period in the treatment of pT3a/b RCC and has no adverse effect on mid-term oncologic outcomes.¹⁶⁹ In recent years, despite an increase in the use of robot-assisted radical nephrectomy (RARN) in RCC, the advantages of RARN over LRN for RCC remain controversial. Two studies showed no substantial differences in perioperative outcomes, including operative time, bleeding, conversion rate, complications, and local recurrence rates.^{170, 171} For PN, laparoscopic and robotic-assisted surgery has significant advantages over open surgery in terms of bleeding control and shorter hospital stays.¹⁷² A meta-analysis shows that robotic-assisted PN has advantages over laparoscopic PN, including a lower conversion rate to open (p = 0.02) and radical surgery (p = 0.0006), shorter thermal ischemia time (p = 0.005), less change in postoperative estimated glomerular filtration rate (p = 0.03), and shorter hospital stays (p = 0.004); Although there were no significant differences between the two groups in terms of complications, postoperative serum creatinine changes (p = 0.65), operative time (p = 0.35), estimated blood loss (p = 0.76), and positive surgical margins (p = 0.75).¹⁷³

Despite the controversy, the value of MIS is gradually being demonstrated, particularly in controlling bleeding and shortening hospital stays. This allows patients to undergo surgical treatment while minimizing harm and complications, greatly reducing resistance and anxiety associated with open surgery.

For patients with multiple RCC tumors at increased tumor risk (e.g., bilateral kidney tumors), those who have difficulty adapting to surgery due to age or poor physical condition, and those with solitary kidneys, ablation technology offers an additional option.¹⁷⁴ the value of MIS is gradually being demonstrated, particularly in controlling bleeding and shortening hospital stays. This allows patients to undergo surgical treatment while minimizing harm and complications, greatly reducing resistance and anxiety associated with open surgery.^{175, 176} Tumor ablation techniques are minimally invasive, have shorter treatment times, are reproducible, have fewer complications, and higher success rates than traditional surgery.¹⁷⁷ The effectiveness of MWA and RFA treatments is similar for tumors ≤4 cm, while CA is the best option for larger tumors (>3 cm).^{178, 179}

3.2 Systemic treatment options

Systemic therapy for RCC mainly consists of cytokines, targeted therapies, and ICIs. Single-class drug therapy is prone to drug resistance; thus, current research focuses on combining standard clinical therapies to resist drug resistance and expand treatment options. However, even combination therapy with existing classical therapies cannot solve all problems. Effective interventions need to be developed for patients who are insensitive to existing therapies, do not have a durable response, or fail treatment.⁵ Therefore, the continuous development of new drug targets and research into new prospective therapeutic techniques remain crucial in the field of RCC treatment, which will provide more diverse therapeutic options and contribute to the solution of the drug resistance problem and the early realization of precision medicine and individualized therapy (Figure 4).

3.3 Cytokine therapy

IL-2 is a key cytokine used in RCC treatment. In 1992, high-dose IL-2 became the first cytokine approved for treating metastatic RCC.¹⁸⁰ IL-2 promotes the expansion of effector T-cells, enhances their function, and improves T-cell viability. However, it can also exert immunosuppressive effects. The mode of action differs by dose, and the effect of IL-2 treatment varies significantly with different doses. Studies have shown that low-dose IL-2 specifically activate T-reg cells and enhance their function,¹⁸¹ exerting an immunosuppressive effect to regulate chronic inflammation and autoimmune diseases.¹⁸² And high doses of IL-2 can significantly stimulate NK cell and effector T cell responses, activating immune activity and playing an antitumor role.¹⁸³ Although high-dose IL-2 significantly improves RCC efficacy, it can damage organs, causing multiple adverse events and even patient death.¹⁸⁴ This severely limits the clinical use of IL-2. Currently, several new attempts to modify IL-2 have yielded promising results. For example, a pegylated form of IL-2, NKTR-214 binds more favorably to IL-2R β/γ, improving CD8+ T cell activation and immunoreactivity. More importantly, polyethylene glycolization improves IL-2′s pharmacokinetics, eliminating the toxic reactions associated with high doses.¹⁸⁵ A study has shown that Bempegaldesleukin (NKTR-214) plus nivolumab demonstrates preliminary antitumor activity as first-line therapy in advanced ccRCC patients and is well tolerated.¹⁸⁶

Unlike IL-2, interferon-α (IFN-α) can demonstrate antitumor efficacy at low doses and inhibit tumor progression by modulating tumor immunity, inhibiting tumor angiogenesis, proliferation, and differentiation.¹⁸⁷ However, studies show that IFN-α alone does not achieve satisfactory results in RCC, but its efficacy can be significantly improved when combined with antiangiogenic drugs or mTOR inhibitors.¹⁸⁸

Other cytokines such as tumor necrosis factor-α (TNF-α) also play a role in treating advanced RCC, but their antitumor effects are unsatisfactory, and their systemic toxicity should not be ignored.¹⁸⁹ Although TNF-α mutants with lower systemic toxicity and higher efficiency can be obtained through genetic modification,¹⁹⁰ the future prospects require further observation.

Overall, cytokine therapy for advanced, metastatic RCC has achieved limited efficacy. Although high-dose IL-2 and IFN-α were once standard care for metastatic RCC, the 5-year survival rate remains low. As understanding of RCC tumor biology and pathogenesis increases, cytokine therapy is gradually being replaced by more efficient therapies.

3.4 Target therapy in RCC

RCC is closely associated with VHL mutations, which leads to an increase in the activity of its downstream the HIF and ultimately leads to elevated VEGF expression, promoting tumor angiogenesis.^{191, 192} Besides, the mTOR pathway also plays an important role in activating HIF.¹⁹³ Therefore, targeted therapeutic strategies for RCC are mainly focused on targeting the VEGF and mTOR pathways.

3.5 Immune therapy in RCC

Previous immunotherapies have focused on cytokine therapy, including IL-2,¹⁸⁰ IFN-α,¹⁸⁷ and TNF-α.¹⁸⁹ Studies on immune checkpoints have revolutionized the understanding of tumor immune escape mechanisms and improved immunotherapy. With the discovery of immune checkpoints like CTLA4, PD-1, PD-L1, and new targets like LAG-3 in tumor progression, many ICIs have shown significant antitumor effects (Table 2).²²²

3.6 Combined therapy

Recent studies have revealed a link between VHL–HIF-1–VEGF signaling-mediated tumor angiogenesis and changes in the TME. For instance, HIF-1α-induced VEGF-a promotes CD8+ T cell infiltration by regulating endothelial permeability and maintaining the effector state of CD8+ T cells.²³⁴ Inhibition of HIF1-α reduced PD-L1 expression in a mouse tumor model.²³⁵ These studies suggest the potential of combining TKIs and ICIs for RCC treatment. Indeed, combining these agents significantly improved survival compared with sunitinib alone.²³⁶ Combination therapy is now the recommended first-line treatment for advanced, metastatic RCC (Table 2).¹⁶³

Given the multitude of targeted agents and ICIs for RCC treatment, clinical studies of combination therapies have attracted much attention. For TKI and immunotherapy combination, a study showed that in previously untreated advanced RCC patients, pembrolizumab plus axitinib resulted in significantly longer PFS (15.1 vs. 11.1 months) and higher ORR (59.3 vs. 35.7%) than sunitinib.²³⁶ Similarly, another phase III trial has revealed that nivolumab plus cabozantinib was more effective than sunitinib in previously untreated advanced RCC patients, with median OS of 37.7 months in the combination group and 34.3 months in the sunitinib group. Updated median PFS was 16.6 versus 8.3 months.²³⁷

Additionally, in advanced RCC patients, durable clinical benefits were observed with nivolumab plus ipilimumab versus sunitinib at 5 years, with median OS of 55.7 versus 38.4 months and objective response of 39.3 versus 32.4%.²³⁸ An ongoing phase III b trial (NCT03873402) is testing the efficacy and safety of nivolumab plus ipilimumab versus nivolumab monotherapy in previously untreated advanced and intermediate- or low-risk RCC. Recent studies have also explored the efficacy and safety of three-drug combination therapies. In a phase III, double-blind trial, 855 previously untreated advanced ccRCC patients with intermediate or poor prognostic risk were randomly assigned to receive either 40 mg of cabozantinib per day with nivolumab and ipilimumab (experimental group) or a matching placebo with nivolumab and ipilimumab (control group). An interim analysis supported a better ORR for patients receiving triple therapy (43 vs. 36%). However, patients receiving triple therapy had higher grade ≥3 toxicity (73 vs. 41%) and more treatment interruptions (12 vs. 5%) compared with doublet therapy controls. This result supports that cabozantinib combined with nivolumab and ipilimumab is more effective and significantly prolongs PFS compared with nivolumab and ipilimumab alone in previously untreated advanced RCC patients with intermediate or poor prognostic risk. However, safety concerns remain, as grade 3 or 4 adverse events were more common in the experimental group.²³⁹

These results suggest that combination therapy is significantly superior to monotherapy, both for targeted therapy with immunotherapy and for combining different ICIs.

3.7 CAR-T therapy

RCC is infiltrated with many T cells, but these T cells do not always exert antitumor effects due to numerous inhibitory factors.²⁴⁰ Therefore, restoring T cell function in RCC is a valuable therapeutic strategy, including CAR-T cell therapy, which uses bioengineering to highly express CAR on T cells, enabling them to exert antitumor effects by interacting with tumor target antigens.²⁴¹ The striking response of CAR-T cell therapy in hematologic malignancies has drawn attention to its use in solid tumors, including RCC.²⁴² It was first validated in a phase I/II trial against carboxy-anhydrase-IX (CAIX), a gene regulated by HIF and upregulated in RCC, but no significant therapeutic response was found.²⁴³ Encouragingly, the combination with sunitinib demonstrated a favorable antitumor response in animal RCC models, suggesting that sunitinib's immunomodulatory effects may enhance the efficacy of CAR-T cell therapy.²⁴⁴

Additionally, clinical studies of CAR-T cell therapy targeting CD70, AXL, and ROR2 are in progress, and these may represent the next generation of RCC therapies. A phase I dose-escalation study (NCT04696731) is evaluating the safety, efficacy, and cell kinetics of ALLO-316 (targeting CD70) in adults with advanced or metastatic ccRCC following a lymphodepleting regimen that includes fludarabine, cyclophosphamide, and ALLO-647 (targeting CD52) to determine the phase II dose. Another phase I dose escalation and cohort expansion study (NCT04438083) is now assessing the safety and efficacy of CTX130, an allogeneic CRISPR–Cas9-engineered T cell targeting CD70, in subjects with advanced, recurrent, or refractory RCC. ROR2 and RTK AXL are highly expressed in RCC, correlate with poor prognosis, and are valuable therapeutic targets.^{86, 245} A phase I/II trial (NCT03393936) is evaluating the safety, tolerability, and antitumor activity of T-cell infusion for treating adults with relapsed and refractory stage IV metastatic RCC. ROR2-positive RCC subjects will receive CCT301-59 T-cells, while AXL-expressing RCC subjects will receive CCT301-38 T-cell therapy. These studies will help us understand the value of CAR-T cell therapy, potentially improving RCC treatment options (Table 2).

3.8 DCs vaccine

As major antigen-presenting cells, DCs activate T cells by expressing various costimulatory molecules..²⁴⁶ Thus, autologous DC vaccines can stimulate tumor-specific immune responses by exposing tumor antigens to DCs.²⁴⁷ Several clinical studies are currently validating the responsiveness and safety of DC vaccines in the treatment of RCC. For example, a phase II trial (NCT04203901) is investigating the efficacy of an autologous tumor antigen-loaded DC vaccine, CMN-001, combined with ipilimumab/nivolumab (I/N) in patients with intermediate/low-risk mRCC compared with I/N alone. The safety of a DC-tumor fusion vaccine with granulocyte macrophage colony stimulating factor is being tested in an active phase I/II trial (NCT00458536) in 38 patients with mRCC who underwent reduced-volume nephrectomy and had not received systemic therapy. And a phase II trial (NCT05127824) is evaluating the role of neoadjuvant autologous tumor vascular antigen DCs vaccine injections intradermally with cabozantinib prior to surgical resection in patients with localized RCC. These studies will soon provide definitive evidence about DC vaccine treatment for RCC (Table 2).

3.9 CDKs inhibitor

CDKs are serine/threonine kinases that promote DNA synthesis and chromosome segregation by regulating substrate phosphorylation, thereby controlling the cell cycle and participating in key processes like gene transcription, insulin secretion, glycogen synthesis, and neuronal function.^{248, 249} Consequently, CDK inhibitors have been developed to treat various diseases caused by CDK abnormalities. CDK has long been an attractive target for cancer therapy due to defective cell cycle and proliferation regulation in most tumors, leading to the development and testing of many CDK inhibitors.²⁵⁰ However, only a few CDK inhibitors have shown potential therapeutic value for RCC. Ribociclib, a CDK4/6 inhibitor, has been shown to inhibit RCC proliferation and induce apoptosis in vivo and in vitro, acting synergistically with chemotherapeutic or immunotherapeutic agents without damaging normal renal cells and fibroblasts.²⁵¹ Synergistic effects of CDK4/6 inhibitors with HIF-2α inhibitors in VHL-deficient RCC have also been identified.²⁵² The safety, tolerability, and maximum tolerated dose of the oral combination of Abemaciclib and sunitinib in patients with advanced and metastatic RCC are being evaluated in a study (NCT03905889). A phase II study (NCT05935748) is evaluating the safety, efficacy, and pharmacokinetics of the HIF-2α inhibitor NKT2152 in combination with palbociclib (doublet) and the PD-1 inhibitor sasanlimab (triplet) in subjects with advanced or metastatic ccRCC who have received prior therapy to determine the recommended dose expansion (Table 1).

3.10 HIF inhibitor

HIF-α enhances tumor cell growth and resistance to treatment by regulating the expression of downstream target genes such as VEGF and glucose transporter protein-1, improving the angiogenic and glycolytic capacity of tissues.²⁵³ Of the three HIFs, HIF-2α is considered the major RCC promoter, making it a potential therapeutic target.²⁵⁴ The first-generation HIF-2α inhibitor PT2385 demonstrated a favorable safety profile in a phase I dose-escalation trial involving patients with metastatic ccRCC who had undergone extensive pretreatment. Moreover, 52% of patients were stable, 12% had a partial response, and 2% had a complete response.²⁵⁵ Although this novel drug has promising efficacy in heavily pretreated mRCC patients, variability in drug exposure is a concern, leading to underdosing in some patients and inadequate drug exposure. The second-generation HIF-2α inhibitor belzutifan (MK-6482) is more selective. In a single-arm phase II study, 61 patients with VHL disease-associated localized RCC and pancreatic lesions were treated with 120 mg of belzutifan daily, showing activity in RCC and non-RCC neoplasms associated with VHL disease.²⁵⁶ An ongoing open-label, single-arm, phase II study (NCT03634540) demonstrates promising antitumor activity of belzutifan plus cabozantinib in patients with pretreated ccRCC.²⁵⁷

HIF-2α promotes the formation of an immunosuppressive TME through multiple pathways, such as promoting the development and proliferation of Tregs,²⁵⁸ upregulating the expression of stem cell factors, thereby inducing the secretion of TGF-β and IL-10,²⁵⁹ and HIF-2α expression correlates with an increase in the expression of PD-L1 on ccRCC tumor cells, which indirectly inhibits T cell function.²⁶⁰ These results provide a strong basis for the use of HIF-2α inhibitors in combination with checkpoint inhibitors. ccRCC preclinical data suggest that the combination of HIF-2α inhibitors and checkpoint inhibitors can inhibit tumor growth by increasing T-cell infiltration, modulating myeloid cells, and altering chemokine expression.²⁶¹ The phase III LITESPARK-022 study is assessing the role of belzutifan plus pembrolizumab in adjuvant therapy after nephrectomy (NCT05239728). More extensive combination therapy studies are underway. An open-label, randomized phase III study (NCT04736706) is evaluating the efficacy and safety of pembrolizumab in combination with belzutifan and lenvatinib, or quavonlimab (CTLA-4 antagonist) in combination with lenvatinib, compared with pembrolizumab and lenvatinib, as first-line treatment in participants with advanced ccRCC. The United States Food and Drug Administration has approved belzutifan for patients with VHL-related cancers, including RCC (Table 1).²⁶²

3.11 Antibody–drug conjugates

ADCs are a novel therapeutic approach combining monoclonal antibodies (mAbs) and cytotoxic drugs via a linker. The mAb anchors the ADC to the tumor cell surface by binding to a tumor-specific antigen (TSA), allowing the cytotoxic payload to enter the tumor cell and exert its effect.²⁶³ This approach leverages the specificity of mAbs and the potency of highly cytotoxic drugs to selectively target tumor cells and deliver the cytotoxic payload to the tumor site, potentially reducing drug-induced side effects.²⁶⁴ Consequently, ADC therapies have been approved for treating various hematologic and solid tumors, including breast cancer,²⁶⁵ gastric or gastroesophageal junction adenocarcinoma,²⁶⁶ cervical cancer,²⁶⁷ ovarian cancer,²⁶⁸ and metastatic urothelial carcinoma.²⁶⁹

Although no ADC has been approved for use in RCC, numerous clinical trials are exploring various ADCs for potential applications. CDX-014, an ADC targeting immunoglobulin mucin-1 (TIM-1), resulted in clinical benefit in 31% of patients in a study of previously treated ccRCC and pRCC, with PFS and OS of 2.7 and 12.6 months, respectively. This finding suggests that CDX-014 has a manageable toxicity profile and early signs of activity, supporting further evaluation in patients with advanced RCC and other cancers expressing TIM-1.²⁷⁰ CD70 is another potential target that is highly expressed in hematologic and solid tumors, and correlates with poor prognosis.²⁷¹ It binds to its receptor CD27 and coregulates Treg mobilization or survival, leading to immunosurveillance in the TME and promoting tumor growth.²⁷² Therefore, the safety and tolerability of e CD70-targeted ADCs such as AMG 172,²⁷³ MDX-1203,²⁷⁴ SGN-CD70A,²⁷⁵ SGN-75²⁷⁶ were tested in RCC and showed modest efficacy, manageable adverse effects, and favorable overall tolerability. Two drugs targeting ectonucleotide pyrophosphatase/phosphodiesterase 3, AGS-16M8F and AGS-16C3F, were evaluated in advanced refractory RCC. Indeed, AGS-16C3F, an improved type of the former, was tolerated and had durable antitumor activity at 1.8 mg/kg every 3 weeks.²⁷⁷ Overall, research on ADCs in RCC is still in its early stages. More studies are needed to support ADC treatment in RCC, especially in relapsed or refractory cases. Discovering more valuable and specific targets may advance this progress (Table 2).

3.12 Stereotactic body radiation therapy

SBRT, also known as stereotactic ablative radiation therapy (SABR), is a precise, high-dose radiation technique that can exert cytoreductive and immunomodulatory effects to improve survival in mRCC patients undergoing systemic therapy. It has been used for radical treatment of inoperable primary RCC and for bone and brain metastases of mRCC.^{278, 279} Although RCC is considered insensitive to radiotherapy, the high doses of radiation delivered by SBRT effectively control tumor growth.²⁸⁰ Analysis of resected RCC treated with SBRT by Ali et al.²⁸¹ identified enrichment of immune response pathways, T-cell accumulation, and dynamic remodeling of circulating T-cell pools after treatment. Studies have shown that SBRT is safe and effective for treating locally recurrent RCC, and that localized therapy can slow disease progression compared with systemic therapy alone.²⁸² Currently, attention is focused on the combination of SBRT with other therapies, especially ICI, in mRCC. A phase II randomized clinical trial (NCT04090710) is evaluating SBRT as upfront cytoreductive therapy to the primary renal mass along with combination I/N versus I/N alone therapy in patients with intermediate/poor risk mRCC who are not candidates for CN For patients with metastatic ccRCC who have failed at least one prior antiangiogenic therapy, a phase II trial (NCT02781506) is also evaluating the use of SBRT to multiple metastatic sites concurrently administered with Nivolumab. The combined therapeutic value of SBRT is expected to benefit patients with refractory RCC, such as after recurrence and metastasis (Table 2). Recent studies have further demonstrated the feasibility of using SBRT in combination with TKIs or ICIs in patients with mRCC, especially those with low metastatic tumor burden.²⁸³

3.13 Gut microbiota

The gut microbiome participates in host nutrient absorption and metabolism, maintains microcirculation, regulates immune responses, and influences tumorigenesis, progression, prognosis, and therapy through pathways like the synthesis or conversion of circulating metabolites, phagocyte activation, and production of short-chain fatty acids.^{284, 285} Increasing evidence suggests that gut microbiota a key host factor associated with the response to ICI therapy in various solid tumors, including RCC.²⁸⁶ A study analyzing baseline stool samples of patients prior to ICI therapy showed that greater microbial diversity was associated with clinical benefit from ICI therapy (p = 0.001), and multiple species were associated with clinical benefit or lack thereof.²⁸⁷ Antibiotic use resulted in lower ORR and shorter PFS with ICI treatment and adversely affected OS in patients treated with IFN or with VEGF-targeted therapies and prior cytokines.²⁸⁸ This also proves that modulation of the gut microbiota may play an important role in optimizing the prognosis of patients treated with ICIs.

Numerous studies are currently validating the potential to improve RCC treatment and prognosis through gut microbiota modulation. A phase I trial (NCT03829111) compared the effects of nivolumab and ipilimumab in treatment of RCC patients with or without daily oral CBM588, a bifidogenic live bacterial product, and found that PFS was significantly longer in patients receiving nivolumab–ipilimumab with CBM588 than without (12.7 vs. 2.5 months) and the response rate was also higher in patients receiving CBM588 (58 vs. 20%).²⁸⁹ Fecal microbiota transplantation (FMT) is another treatment option under investigation.The TACITO phase I/II trial (NCT04758507) is evaluating whether donor FMT that responds well to ICIs improves ICI efficacy in treatment-naïve patients. Another PERFORM clinical trial (NCT04163289) is investigating whether healthy-donor FMT can reduce the incidence of irAEs in mRCC patients receiving a combination of immunotherapies. Additionally, the development of engineered microbiomes has made it possible to induce antitumor activity using specific bacterial strains.²⁹⁰ Modulation of the gut microbiome through dietary adjustments has also been shown to kill tumor cells, reverse disease-related symptoms, and improve survival.²⁹¹ Overall, the gut microbiota may become future biomarkers for evaluating both the efficacy and safety of ICIs (Table 2).