Renal Cell Cancer – Insights in Drug Resistance Mechanisms

Therapeutic Strategies

Surgery is a potentially curative option for early and localized RCC. However, there remains a high risk of distant relapse, estimated at 30% (7). The 5-year survival rate for metastatic or advanced RCC is notably poor, standing at only 18% (8). Traditional chemotherapy and radiation therapy have proven ineffective in treating all subtypes of RCC (8, 9). Targeted therapies – particularly TKI – have been the mainstay of treatment for RCC patients for several years. However, the benefits of VEGF-TKI in terms of progression-free survival (PFS) have been only modestly favorable (10, 11). Moreover, approximately 70% of patients who initially respond to TKI therapy eventually develop drug resistance, while 30% of patients do not respond to TKI from the outset (12).

More recently, immune checkpoint inhibitors (ICI) have brought about a transformation in the treatment of ccRCC. Nevertheless, although some patients demonstrate robust responses, a significant portion either display inherent resistance to ICI therapy or develop resistance within a few months (13). Gaining a comprehensive understanding of the mechanisms of action of TKI and ICIs, as well as the subsequent development of resistance, holds the potential to lead to the development of effective treatment strategies for patients with advanced RCC in the future.

In ccRCC, receptor tyrosine kinases that are overexpressed in cancer cells are activated through the binding of growth factors present in the blood, such as VEGF and PDGF. This activation leads to the stimulation of signaling pathways that promote cell proliferation and survival. TKI like sunitinib and pazopanib function by inhibiting this VEGF pathway, and they have demonstrated effectiveness in improving progression free survival in patients with advanced RCC (14). Initially, these TKI were commonly used as first-line treatments for advanced ccRCC. However, the development of resistance has necessitated changes in the treatment algorithm (15).

Immunotherapy has emerged as a promising approach in the treatment of ccRCC by directly countering the tumor cells’ ability to evade and hide from the immune response mounted by the host. Clinical studies have revealed a direct correlation between the expression of programmed cell death protein 1 (PD-1) and the down-regulation of the host immune response against ccRCC. ICI, such as nivolumab and ipilimumab, act by blocking checkpoint proteins like PD-1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), thereby enabling the immune system to identify and attack cancer cells. Specifically targeting PD-1, nivolumab inhibits the interaction between PD-1 and T-cells, preventing T-cell inactivation and facilitating immune cell recognition of cancer cells (16, 17). In a randomized phase II study, nivolumab demonstrated significant anti-tumor efficacy, with an objective response rate of 20%. Notably, resistance was not detected in 40% of patients over a 24-month period, highlighting its potential long-term effectiveness (18).

In the Phase III CheckMate 025 clinical trial, nivolumab was evaluated as a second-line treatment and compared to the mammalian target of rapamycin (mTOR) inhibitor everolimus. The study showed a 25% response rate with nivolumab, compared to 5% with everolimus, indicating a superior overall long-term survival benefit with nivolumab. Furthermore, the ICI-mediated adverse effects, such as gastrointestinal toxicity and thyroiditis, were found to be less significant and less frequently reported than the side effects observed with everolimus. The favorable safety profile of nivolumab resulted in fewer patients (31 out of 406; 8%) discontinuing treatment during the trial compared to the everolimus arm (52 out of 397; 13%) (19).

The mTOR inhibitors everolimus and temsirolimus have received approval for the treatment of metastatic RCC. These medications function by inhibiting the activity of the mTOR pathway, a regulatory system involved in cell growth, proliferation, and metabolism in cancer cells. Studies have demonstrated their effectiveness in treating ccRCC, particularly when used in combination with targeted therapies like TKI and ICI. In a phase III trial involving patients with untreated advanced metastatic ccRCC and unfavorable prognostic features, temsirolimus monotherapy was correlated with significantly prolonged overall survival and progression free survival vs when used with IFN-α. The number of patients who went on to have non-progressive, stable disease for a minimum of 6 months was 32.1% in temsirolimus in comparison to 28.1% in the combination treatment group (20).

However, similarly to ICI and TKI, the treatment with mTOR inhibitors has limitations. It is susceptible to the development of resistance and has notable toxicities that can significantly impact patients’ quality of life. Reported adverse effects include hypercholesterolemia, hyperglycemia, and aphthous-like stomatitis. These toxicities can be dose-limiting, thereby reducing the potency and efficacy of the drugs (21).

Future research on mTOR inhibitors should prioritize the identification of predictive biomarkers to determine drug effectiveness and explore innovative strategies to enhance efficacy while minimizing toxicity. Notably, studies have revealed that the down-regulation of lactotransferrin, a significant protein in the innate immune system, promotes metastasis in ccRCC. Interestingly, this down-regulation also renders the ccRCC tumor cells more responsive to mTOR inhibitors, suggesting its potential as a predictor for therapeutic effectiveness (22).

The prognostic model developed by the Memorial Sloan-Kettering Cancer Center has long been regarded as the gold standard for predicting the outcomes of metastatic RCC patients undergoing targeted therapies. To further enhance the accuracy of prognostic predictions, the International Metastatic RCC Database Consortium score expanded upon the previous factors and incorporated platelet and neutrophil evaluations, resulting in a total of six risk factors. The classification of prognosis based on points assigned to each risk factor can be found in Table I (23).

VEGF-targeted therapies, including bevacizumab and the TKI sunitinib and pazopanib, have exhibited remarkable advancements in progression-free survival, primarily conducted on patients classified as favorable and intermediate risk. Extensive research findings have led to their approval as first-line treatments, as depicted in Table II (14, 24, 25).

Furthermore, TKI monotherapy continues to be a viable first-line treatment option for a significant number of patients who may not be eligible for immunotherapy. The findings from the STAR trial directly apply to this patient population (15). Finally, tivozanib – a selective TKI – has shown enhanced and durable response rates, as compared to sorafenib. As a result, it has been approved as a first-line treatment specifically for patients in the favorable risk group. Still, TKI are associated with a range of adverse events that can have a substantial impact on patients’ health-related quality of life (26).

c-Met serves as the TKI for hepatocyte growth factor and plays a pivotal role in cancer cell proliferation, VEGF-induced angiogenesis, and the metastatic progression of tumors. The expression of c-Met has been notably observed in sarcomatoid and papillary type 1 RCC, as well as in advanced-stage and low-grade differentiation tumors. Immunohistochemical staining for c-Met is also commonly observed in collecting duct carcinoma and urothelial carcinoma of the renal pelvis. Existing literature suggests that c-Met expression is unfavorably associated with various survival endpoints (27). Activating mutations and amplifications of the c-Met gene have been documented in patients with papillary RCC, and c-Met-driven RCC tumors may benefit from treatment with c-Met TKI. In a phase II study, the potent oral multikinase inhibitor foretinib was investigated in patients with metastatic papillary RCC. A total of 74 patients, who had received no more than one prior systemic therapy, were stratified based on their c-Met mutational status. Notably, patients with germline c-Met mutations exhibited a more favorable tumor response compared to those without mutations, while c-Met amplification did not show a significant correlation with tumor response. The safety profile of foretinib was characterized by manageable toxicities, and an overall response rate of 13.5% was observed among patients with c-Met mutations (28).

Mechanisms of Resistance

RCC is a heterogeneous tumor characterized by varying blood flow conditions within different tissues. The presence of hypoxia can impair cellular responses, leading to dysfunctional vascularization and promoting metastasis. This, in turn, contributes to therapy resistance by inducing cell quiescence. Hypoxia plays a crucial role in RCC and is associated with a poor prognosis. One mechanism through which RCC cells develop resistance to hypoxia involves the up-regulation of HIF, a transcription factor that becomes activated in response to low oxygen levels (27, 29). HIF controls the expression of genes involved in angiogenesis, glucose metabolism, and cell survival (30). Through the up-regulation of these genes, HIF assists RCC cells in adapting to the hypoxic environment, enabling their continued growth and proliferation. Consequently, this resistance to hypoxia contributes to the resistance of tumor cells to VEGF-TKI and ICI, further exacerbating therapy resistance (29).

ICI-mediated antiangiogenic therapy in the treatment of metastatic RCC involves suppressing the production of proangiogenic factors or inhibiting their binding to their respective receptors, thereby halting angiogenesis (31). However, the prolonged use of this therapy can lead to secondary hypoxia as a result of decreased vasculature caused by the drug treatment. In response to sustained hypoxia, tumor cells can initiate complex intracellular signaling mechanisms, leading to the development of several VEGF- and PDGF-independent proangiogenic factors such as epidermal growth factor receptor (EGFR), placental growth factor (PlGF), fibroblast growth factor 2 (FGF2), erythropoietin (EPO), transforming growth factor (TGF)-α, interleukin (IL)-6, and IL-8. These factors induce acquired resistance and therapy failure, primarily by activating the HIF pathway (32).

Another mechanism through which RCC cells develop resistance to hypoxia involves the activation of the PI3K/AKT/mTOR pathway, a signaling pathway crucial for cell growth and survival. Dysregulation of this pathway is commonly observed in RCC, and its activation can lead to the down-regulation of anti-angiogenic factors like thrombospondin-1 (TSP-1) and endostatin (33, 34). This down-regulation contributes to the induction of resistance against chemotherapeutic and antiangiogenic drugs. Some researchers posit that PTEN acts as a tumor inhibitor by restraining PI3K activity. Loss of PTEN function can enhance the tumor’s expression of immunosuppressive cytokines, which, in turn, diminishes tumor T-cell infiltration and fosters drug resistance. Additionally, it has been demonstrated that IFN-mediated inhibition of cell proliferation and apoptosis is compromised in IFNGR1 knockout cells, and the absence of IFNGR1 in tumor cells can promote tumor growth in vivo while reducing the response to anti-CTLA-4 therapy (35).

RCC is characterized by hypervascular nature and rely on increased production of growth factors, such as VEGF and PDGF-β. Inactivation of VHL leads to deregulated stimulation of HIF-1α and HIF-2α genes, resulting in the activation of various oncogenes. However, it has been observed that a subgroup with only HIF-2α expression demonstrates primary resistance to antiangiogenic drugs (36). Furthermore, additional studies have indicated that the presence or absence of a VHL mutation does not significantly impact the response of patients with metastatic ccRCC to medications targeting VEGF (37, 38). This suggests that although hypoxia and HIF may contribute to resistance against targeted therapeutics, factors beyond VHL mutation status likely play a more critical role in predicting patient response to VEGF-targeted therapies.

Lysosomes are organelles enclosed by a membrane and contain hydrolytic enzymes, including acid hydrolases. These enzymes play a crucial role in the breakdown of various biomolecules such as proteins, lipids, nucleic acids, and carbohydrates. Their optimal activity occurs within the acidic pH range of 4.6–5.0, which is maintained by proton pumps located in the lysosomal membrane (39). Lysosomal sequestration is a physiological process where hydrophobic weak base compounds accumulate within acidic lysosomes. This process is vital for the elimination of certain drugs and chemicals from the body, as well as for the regulation of cellular metabolism. Weak base compounds are lipophilic and can easily traverse cell membranes. However, when they reach the acidic lysosomal compartment, they undergo protonation due to their weak basic properties. As a result, they are unable to cross the lysosomal lipid membrane (40). Figure 1 depicts the mechanisms of action of sunitinib and highlights the mechanisms of drug resistance in RCC.

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Figure 1.

Mechanisms of action of sunitinib (VEGF-tyrosine kinase inhibitor) and mechanisms of drug resistance in renal cell carcinoma. Created in BioRender.com. Adapted from (13, 15-17, 21). Sunitinib is a small molecule TKI, inhibiting activation of VEGF-R and PDGF-R, blocking intracellular pathways mediating cellular growth and angiogenesis. Mechanisms of resistance include upregulation of HIF pathways to stimulate angiogenesis and subsequent tumor growth. Lysosome sequestration of sunitinib contributes to reduced drug efficacy and resistance. Pro-angiogenic factors are inhibited by ICI-mediated anti-angiogenic therapy, which contributes to hypoxia. VEGF, Vascular endothelial growth factor; TKI, tyrosine kinase inhibitor; ICI, immune checkpoint inhibitor; HIF, hypoxia-inducible factor; PI3K, phosphoinositide 3-kinase; VEGF-R, vascular endothelial growth factor receptor; PDGF-R, platelet-derived growth factor receptor; mTOR, mammalian target of rapamycin; AKT, protein kinase B.

Since many TKI are weak bases that can penetrate cell membranes, they become trapped in their protonated forms within the acidic lysosomal compartment, away from their intended targets. This leads to reduced drug efficacy and development of drug resistance. It is important to address this lysosomal sequestration phenomenon in order to optimize drug effectiveness and combat resistance. Numerous studies have indicated that specific TKI, including sunitinib, erlotinib, and pazopanib, can undergo lysosomal sequestration (41, 42). Interestingly, sorafenib, unlike sunitinib, does not fall into the same category of hydrophobic, membrane-permeable weak bases. This observation suggests that a different mechanism might be responsible for the lysosomal sequestration of sorafenib, potentially involving the action of drug pumps (43). In fact, both sorafenib and sunitinib have been found to be sequestered by the ABC transporter P-glycoprotein (P-gp) (44). Notably, the administration of verapamil, a P-gp inhibitor, led to enhanced antitumor activity for both sorafenib and sunitinib, providing further evidence of the involvement of P-gp in TKI resistance (45).

Pathway bypass mechanisms can contribute to drug resistance by activating alternative effector pathways that sustain oncogenic transcription and translational output. The tumor suppressor PTEN serves as a negative regulator of the PI3K/Akt/mTOR pathway (46). Mutations in PTEN function result in the constitutive activation of AKT/mTOR signaling downstream of the cellular targets of TKI. Although PTEN mutations are rare in RCC, several studies have demonstrated reduced PTEN gene expression in many cases of RCC (42). Notably, renal cells that exhibit resistance to sunitinib treatment often lack PTEN expression. However, restoring PTEN function or utilizing medications to inhibit AKT/mTOR signaling can restore sensitivity to sunitinib in PTEN-deficient ccRCC cells once again (47).

Mounting evidence indicates that the tumor microenvironment (TME) plays a pivotal role in the development of resistance to targeted therapies. This microenvironment encompasses tumor cells, the extracellular matrix (ECM), signaling molecules, and various types of stromal cells, including fibroblasts, vascular endothelial cells, pericytes, and immune cells. Notably, myeloid-derived suppressor cells (MDSCs) constitute a significant component that contributes to the intricate nature of the TME. Tumors have been shown to recruit MDSCs, which produce several pro-angiogenic factors that stimulate VEGF-independent angiogenesis, thereby promoting resistance to anti-angiogenic drugs (45-48). Studies conducted by Ko et al. have demonstrated that, despite a significant decrease in MDSC levels in the peripheral blood of RCC patients undergoing sunitinib treatment, the levels of MDSCs in tumor tissues of these patients remain unchanged (49). Furthermore, the resistance of MDSCs to sunitinib has been associated with the local presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) within renal tumors (50). These findings shed light on the complex interactions within the TME that contribute to resistance mechanisms.

Cells from both the innate and adaptive immune systems infiltrate the TME, forming an interconnected network that governs various aspects of tumor growth (51). ccRCC is particularly characterized by high infiltration of T-cells, exhibiting diverse phenotypes and functions (52). Resistance to ICI treatments has been associated with the tumor environment (53), potentially influenced by significant immunosuppression-mediated in part by MDSCs (54). Despite sufficient antigen presentation and T-cell infiltration, interactions between immune cells and tumor cells within the TME can suppress the activity of ICI. MDSCs play a role in suppressing anti-tumor T cell activity through mechanisms such as, reactive oxygen species (ROS) production, arginase and IL-10 secretion, nitrosylation of chemokines, and depletion of essential nutrients like cysteine and tryptophan, which are crucial for T-cell function (55). Additionally, MDSCs can promote tumor progression by stimulating regulatory T cells (Tregs), which inhibit T-cell activity (56, 57). Several studies have shown that depleting or impairing Tregs or MDSCs can restore the anti-cancer efficacy of ICI (56, 58). The ability of MDSCs to suppress the immune system in cancer patients may serve as an indicator for predicting the effectiveness of ICI therapy (53). The reduction of major histocompatibility complex (MHC) antigens presents another impediment to the recognition of RCC cells by T-cells. This dysregulation is linked to mutations in MHC genes within tumor cells, occurring as their malignant phenotype advances (59). Figure 2 illustrates the mechanisms of action of nivolumab, as well as the mechanisms of drug resistance in RCC within the TME.

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Figure 2.

Mechanisms of action of nivolumab and mechanisms of drug resistance in RCC in the context of the tumor microenvironment. Created in BioRender.com. Adapted from (32, 36-38). Nivolumab is an anti-PD-1 monoclonal antibody acting to activate T-lymphocyte-mediated anti-tumor activity by blocking the PD-1-PD-L1 T-cell regulation pathway. Tumor-secreted cytokines recruit myeloid-derived suppressor cells, which cause immunosuppression to counteract the effects of nivolumab. Pro-angiogenic factors secreted by myeloid-derived suppressor cells stimulate angiogenesis and subsequent tumor growth. These contribute to mechanisms of resistance. ICI, Immune checkpoint inhibitor; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; IL-10, interleukin-10.

Furthermore, mutations within the tumor can impact T-cell recruitment in the TME. Tumors characterized by PTEN loss exhibit overexpression of VEGF, leading to reduced infiltration of CD8+ T-cells (60). Mutations activating the MAPK pathway result in increased expression of VEGF and IL-8, which inhibit T-cell recruitment within the tumor (58). Mutations that elevate β-catenin levels contribute to the depletion of CD103+ dendritic cells (61). These mutations are likely to contribute to primary resistance to ICI by creating an environment where T-cells are absent within the TME.

Numerous studies have consistently highlighted the pivotal roles played by ABC family genes in maintaining cellular homeostasis, regulating cholesterol metabolism, influencing disease susceptibility, and impacting tumor resistance mechanisms (62). These ABC transporter genes are known to facilitate drug efflux, thereby bolstering the chemical resistance of cancer cells. Mutations within ABC genes can significantly alter cancer cell phenotypes, including proliferation, differentiation, migration, and invasion. Notably, ABCA1 and ABCD1 have been linked to the onset and progression of tumors. Additionally, ABCA13, ABCB1, ABCC1, and ABCC2 have shown associations with drug resistance. ABCB2 and ABCB3 have been identified as participants in evading the immune response within tumors. Furthermore, ABCG2 has exhibited correlations with tumor progression, prognosis, and drug resistance, while ABCB10 has been associated with both tumor progression and prognosis. Recently has been presented that ABCG1 holds diagnostic and prognostic value for ccRCC patients (63).

Overcoming Resistance

To address the challenges posed by resistance and treatment failure, researchers are exploring various strategies to enhance the efficacy of current drug treatments for ccRCC. Newer generations of TKI, such as tivozanib and cabozantanib, exhibit increased potency and target a broader range of tyrosine kinases. However, it is important to consider that higher potency and broader activity may be associated with an elevated risk of side-effects and toxicity (14).

Sequential treatment using different TKI has shown promise in overcoming resistance in patients with advanced RCC. TKI have varying affinities for similar and distinct targets, and sequential treatment leverages this diversity. Phase II studies have demonstrated the clinical activity of axitinib in patients with refractory metastatic RCC following prior use of sorafenib, as well as in patients previously treated with other therapies such as sunitinib, cytokines, temsirolimus, or bevacizumab with interferons (64). Extensive research supports the successful sequential use of TKI followed by everolimus, leading to its authorization for systemic treatment of metastatic ccRCC (65).

Modulating the immune system through the administration of cytokines in combination with ICI has emerged as an effective strategy to overcome resistance. Cytokines, such as interleukins, are crucial signaling molecules that regulate immune responses. In the context of ccRCC, interleukins like IL-2 and IL-15 have been investigated as potential agents to augment the anti-cancer activity of immune cells. Combining ICI with interleukins, such as IL-2 or IL-15, holds promise for enhancing immune cell activity against RCC and improving response rates in patients who have developed resistance to ICI monotherapy. However, it is important to exercise caution in the use of interleukins in RCC due to the potential for toxicity. Careful patient selection and management are necessary to mitigate adverse effects (66, 67). Further research is warranted to determine the optimal dosage, sequencing, and combination strategies for these therapies. Additionally, identifying biomarkers that can reliably predict patient response to these treatments is a crucial area of investigation.

Gene screening can be conducted using the clustered regularly interspaced palindromic repeats CRISPR/Cas9 system, which is a microbial RNA-guided adaptive immune system capable of cleaving genetic material. In the context of drug resistance in tumor cells, various mechanisms can contribute to their ability to evade treatment, such as enhancing drug efflux, improving DNA repair processes, attenuating apoptosis, and promoting stemness, thereby enabling cancer cell proliferation and survival (68). Excitingly, recent studies have demonstrated the potential of the CRISPR/Cas9 technique in identifying and targeting critical cellular factors associated with these drug resistance mechanisms. By leveraging CRISPR/Cas9, it becomes possible to enhance the effectiveness of anticancer drugs by specifically addressing these underlying factors. This innovative approach holds promise for improving treatment outcomes by better understanding the genetic basis of drug resistance and developing strategies to overcome it (69). Further research and exploration of the CRISPR/Cas9 system in the context of cancer therapy are necessary to fully unlock its potential in combating drug resistance.

A study conducted on patients with primary refractory ccRCC utilized the CRISPR/Cas9 technique to identify the role of farnesyltransferase, a critical enzyme essential for tumor cell survival. As a monotherapy, the efficacy of targeting farnesyltransferase alone did not reach a significant level for licensing. However, when combined with sunitinib, an approved treatment for ccRCC, the antitumor potency was significantly enhanced. Furthermore, through CRISPR/Cas9 screening, the protein UBE21 was identified as being highly expressed in ccRCC tissue and playing a crucial role in tumor cell proliferation. This finding not only holds therapeutic value but also suggests the potential use of UBE21 as a diagnostic tool for ccRCC (70). These discoveries highlight the potential of CRISPR/Cas9 technology in identifying key molecular targets that can enhance the efficacy of existing treatments and provide valuable insights for both therapeutic and diagnostic applications. Further research is warranted to explore the full potential of targeting farnesyltransferase and UBE21 in ccRCC management.

In the United States, sunitinib is no longer recommended as the first-line treatment for metastatic RCC due to its lower efficacy and higher rates of resistance. However, there is potential for reinstating the use of sunitinib and other TKI that are prone to resistance by combining them with drugs such as trebananib, an inhibitor of the Ang/Tie-2 pathway. That pathway plays a critical role in basal vascular development and angiogenesis. By targeting Ang/Tie-2 pathway, cell sensitivity to sunitinib can be increased, leading to a potential halt in tumor progression. The combination of sunitinib and a drug like trebananib holds promise for improving the effectiveness of TKI treatment and overcoming resistance in metastatic RCC (71). Further research and clinical trials are needed to assess the safety and efficacy of this combination therapy approach. If successful, it could offer new treatment options and improve outcomes for patients with metastatic RCC.

In the pursuit of overcoming resistance in metastatic ccRCC, various approaches are being explored. These include the development of novel variants of existing therapies that target alternative signaling pathways, combination and sequential therapy involving both new and established targeted agents or immunotherapy (72, 73). Additionally, promising research is being conducted to utilize tumor biomarkers for guiding and optimizing targeted therapy, enabling the identification of patients who will derive the most benefit from different targeted treatments (74, 75). Furthermore, efforts are underway to gain a deeper understanding of the molecular mechanisms underlying drug resistance in RCC. This knowledge is pivotal in developing highly specific targeted therapies that can effectively counteract resistance, ultimately leading to improved prognostic outcomes for patients. These multifaceted strategies, encompassing therapeutic advancements, biomarker-guided treatment selection, and enhanced comprehension of drug resistance mechanisms, hold promise in tackling resistance and elevating the efficacy of treatment options for metastatic ccRCC.