Sunitinib resistance in renal cell carcinoma

Of the many targeted therapies introduced since 2006, sunitinib has carved its way to become the most commonly used first-line therapy for the treatment of metastatic renal cell carcinoma (RCC). Despite significant improvements in progression-free survival, 30% of the patients are intrinsically resistant to sunitinib and the remaining 70% who respond initially will eventually become resistant in 6–15 months. While the molecular mechanisms of acquired resistance to sunitinib have been unravelling at a rapid rate, the mechanisms of intrinsic resistance remain elusive. Combination therapy, sunitinib rechallenge and sequential therapy have been investigated as means to overcome resistance to sunitinib. Of these, sequential therapy appears to be the most promising strategy. This mini review summarises our emerging understanding of the molecular mechanisms, and the strategies employed to overcome sunitinib resistance.


Introduction
The past decade has witnessed tremendous improvements in the understanding of the role of angiogenesis in renal cell carcinoma (RCC), leading to the development and implementation of many angiogenesisinhibitors, also known as targeted therapies, in clinical practice (1-7). These achievements largely stem from the elucidation of two inter-connected molecular pathways that regulate angiogenesis and proliferation in RCC, an inactivated von Hippel Lindau (VHL) gene and activated mammalian target of rapamycin (mTOR) (6-11). These pathways have been extensively reviewed (8-18). Of the many targeted therapies, sunitinib has carved its way to become the most frequently used first-line therapy for the treatment of metastatic RCC. However, the initial enthusiasm is hampered by the development of intrinsic and extrinsic resistance to therapy. In this mini review, a summary of the angiogenesis pathway in RCC, the emerging molecular mechanisms of sunitinib resistance, and the approaches to overcome resistance to sunitinib in RCC are discussed.

The VHL-HIF axis in RCC
The VHL gene is inactivated in 70-80% of sporadic clear cell RCC either through mutations, hyper-methylations or loss of heterozygosity (8-10). Subsequently, the production of its functional protein, pVHL, is either inhibited or decreased in these cases. The best studied function of pVHL is the degradation of the transcription factor hypoxia-inducible factor (HIF). As the microenvironment of solid tumors is often hypoxic, tumor cells undergo adaptive changes to facilitate their survival. One such survival mechanism under hypoxic conditions is the up-regulation of HIFalpha (HIF-α). Under normoxic conditions, pVHL forms complexes with elongin B, elongin C, Rbx1 and cullin 2 to form a pVHL-E3 ubiquitin ligase complex (pVHL-E3 complex) (15-23). The pVHL-E3 complex then binds to HIF-α, leading to its polyubiquitination and proteasomal degradation. In the absence of a functional pVHL, secondary to VHL mutations, the formation of the pVHL-E3 complex and its binding to HIF are inhibited and therefore, the degradation of HIF-α is prevented even in normoxic conditions (15)(16)(17)(18)(19)(20)(21)(22)(23). This leads to the stabilization and accumulation of HIF in cells ( Figure 1). Subsequently, HIF is translocated to the nucleus, where it binds to hypoxia-responsive elements of the DNA and transactivates a plethora of molecules that regulate angiogenesis ( Figure 1). The best studied of these molecules is the vascular endothelial growth factor (VEGF), a crucial regulator of vascular development during embryogenesis (vasculogenesis) and blood vessel formation from the existing endothelium in adults (angiogenesis) (24-31).

The role of VEGF in RCC
In humans, the VEGF system consists of five secreted ligands, VEGF A-D and placenta growth factor-1 (PlGF), and three receptor tyrosine kinases, VEGF R1-R3. The binding of the ligands to the receptors initiates VEGF-mediated angiogenesis which involves endothelial cell proliferation, migration, permeability and capillary formation. However, VEGF alone is not sufficient for the maintenance and stabilization of the newly formed vessels, and requires input from the surrounding microenvironment. This support comes from surrounding peri-endothelial cells such as vascular smooth muscles (VSMC) and pericytes that stabilize the newly formed vasculature and support endothelial cell survival (32-34). This is achieved by the cross talk between plateletderived growth factor-B (PDGF-B) secreted by the endothelial cells and the receptor tyrosine kinases of PDGF, especially PDGFR-B, of the VSMC and pericytes (32-35). Thus the interplay between VEGF, PDGF and their tyrosine kinase receptors plays a crucial role in angiogenesis secondary to VHL inactivation in RCC.

The role of mTOR in RCC
mTOR, which exists as mTORC1 and mTORC2 complexes, is a key component of the phosphoinositide 3-kinase (PI3K)/Akt signalling pathway that regulate cell cycle, proliferation and angiogenesis (12, 36). mTOR signalling can be activated via a number of mechanisms, including overexpression of growth factor receptors, mutations in PI3K/Akt, tuberous sclerosis tumor suppressor genes TSC1/2 or phosphatase and tensin homolog (PTEN) (37-42). mTOR activates the translation of the pro-proliferative factors such as cyclin D1 and cMyc, and the angiogenic factor HIF through phosphorylation of ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) (6, 43-45). The mTORC2, through protein kinase Cα, regulates cell morphology, motility, adhesion, invasion and metastasis (45). Furthermore, inactivation of VHL increases mTOR activity which in turn exacerbates the loss of VHL function leading to enhanced HIF activity (38) (Figure 1). VHL inactivation itself leads to deregulation of cyclin D1 (6, 44). Thus mTOR activation is involved in cell proliferation through cyclin D1 and c-Myc, and angiogenesis through HIF ( Figure  1).

Targeted therapy in RCC
Elucidation of these pathways had identified the potential of angiogenesis inhibition as a promising therapeutic option for metastatic RCC leading to the development and implementation of angiogenesis and mTOR inhibitors in clinical practice. These targeted therapies are broadly classified as VEGF inhibitors, multi-tyrosine kinase inhibitors and mTOR inhibitors. The most successful VEGF inhibitor is the humanized VEGFneutralizing antibody bevacizumab, which A non-functional VHL is the major risk factor for the development and progression of RCC. The functional protein of VHL, pVHL, complexes with E3-ligase and degrades HIF. When the VHL is non-functional, HIF is stabilized and translocated to nucleus where it binds with HIF responsive elements of the DNA and activates many pro-angiogenic factors including VEGF and PDGF. They interact with their respective tyrosine kinase receptors VEGFR (mostly at endothelial cells) and PDGFR (mostly at vascular smooth muscle cells and pericytes) and promote angiogenesis. The PI3K/AKT/mTOR pathway is activated by many factors including growth factor receptors. mTOR in turn activates cyclin D1 and cMyc and promotes cell proliferation and survival. Furthermore, VHL inactivation also activates mTOR, which in turn up-regulates HIF and subsequent angiogenesis. GFR, growth factor receptor; HIF, hypoxiainducible factor; PDGF platelet-derived growth factor; PDGFR, receptor for PDGF; VEGF, vascular endothelial growth factor; VEGFR, receptor for VEGF; VHL,von Hippel Lindau gene. exerts its anti-angiogenic activity by acting against the angiogenic endothelial cells surrounding the tumor, rather than the tumor per se, thus blocking the supply of oxygen and nutrients to the tumors (46)(47)(48). The multi-tyrosine kinase inhibitors are sunitinib, sorafenib, pazopanib and axitinib. Many more are in various phases of clinical trials. They inhibit multiple tyrosine kinase receptors and neutralize the downstream signalling pathways activated by ligand-receptor binding that leads to angiogenesis. Two of the most successful mTOR inhibitors are temsirolimus and everolimus. Both are rapamycin analogues and bind to FK506-binding protein 12 (FKBP12), which in turn binds to mTOR leading to the inhibition of the PI3K/Akt/mTOR pathway (45,49). In addition, temsirolimus has been shown to have a direct inhibitory effect on HIF and VEGF (50). Of these targeted therapeutics, sunitinib has become the most frequently used first-line targeted therapeutic for the treatment of metastatic RCC.

Intrinsic and acquired resistance to sunitinib
Despite the benefits achieved through sunitinib in terms of progression-free survival and disease stabilization, all patients develop resistance to sunitinib and eventually relapse. While the criteria for defining resistance or response to therapy based on RECIST (response evaluation criteria in solid tumors) are debatable as recently pointed out (56, 57), and investigators implement variations, based on the available data it is reasonable to conclude that approximately 70% of patients respond to therapy initially and the remaining 30% show primary resistance (intrinsic resistance) (57-59). In the 70% of patients who show initial response, durable responses are rare, and acquired resistance (extrinsic resistance) to treatment develops in almost all of them in 6-15 months (56-63).
The mechanisms of sunitinib resistance are varied and multifactorial. Table  1 summarizes our emerging understanding of the molecular mechanisms of resistance to sunitinib. It should be noted that most of these mechanisms are based on preclinical studies, and therefore, their relevance in clinical settings is yet to be verified. Taken together, the functional significance of these mechanisms can be summarized under two categories: restoration of angiogenesis through the activation of VEGF-independent pathways (64-69, 71-80) and reduced bioavailability either through increased efflux or lysosomal sequestration (70,81). Of these, activation of tyrosine kinase-independent alternate angiogenesis pathways leading to restoration of angiogenesis appears to be gaining consensus.
While the mechanisms of sunitinibmediated alternate angiogenesis are still elusive, hypoxia is emerging as the major culprit. Sunitinib inhibits angiogenesis, largely through the inhibition of VEGF and its receptors. This helps in the stabilization or regression of the tumor in the short term. However, this also results in hypoxia. Sustained hypoxia by sunitinib 'resets' the tumor microenvironment and leads to the development of a VEGF/VEGFRindependent alternate angiogenic pathway through the up-regulation of angiogenic factors other than VEGF (60, 83). For example, as shown in Table 1, upregulation of IL-8 or down-regulation of IFN-γ (65, 69), may circumvent the antiangiogenic effects of sunitinib, and functionally compensate for the VEGF/VEGFR-mediated inhibition of angiogenesis. Apart from contributing to acquired resistance through alternate angiogenesis pathways, hypoxia could also contribute to intrinsic resistance by selecting a more malignant RCC phenotype, which may accelerate metastatic development and prone cells to insensitivity for anti-angiogenic treatment (59, 84).

Overcoming resistance to sunitinib
The pre-clinical studies (Table 1)

Conclusion
Intrinsic and extrinsic resistance to sunitinib remains a challenge in the effective treatment of metastatic RCC. The mechanisms of intrinsic resistance remain elusive, and much research is warranted in this largely unexplored area. At the same time, pre-clinical studies have been unravelling the molecular mechanisms of extrinsic or acquired resistance at a rapid rate. However, there is a wide gap between the bench and the bedside. While optimism remains that this gap will narrow in the years to come, at present, the best strategy to overcome resistance to sunitinib appears to be sequential therapy. There is no universal consensus as to the optimal sequence of therapy. While establishing a universal consensus for sequential therapy appears logical, the practicality of implementing such approach would be difficult, at least in part, due the heterogeneity of RCC. Thus personalised medicine could be one way to overcome resistance. To achieve this, discovery of a sunitinib resistance 'biomarker map' would be of utmost value. A close interaction between clinicians and basic scientists aimed at designing clinically relevant experiments will enable a speedy resolution to overcome sunitinib resistance in the days to come.