SPOP, a CRL3 substrate adaptor protein, plays an important role in the development of some cancers[10–12]. Over the past decade, the potential functions of SPOP in urologic cancers have gradually attracted much attention from investigators. SPOP was found to be the most common missense mutated gene in human prostate cancers and has been shown to be associated with the pathogenesis of primary prostate tumours, but SPOP mutations in RCC tumours have not yet been reported[15, 16, 23, 24]. Recent studies have shown that SPOP is an oncoprotein that is overexpressed in RCC[17, 18, 25]. However, the results of our preliminary cell experiments showed that overexpression of SPOP inhibited RCC cell proliferation, migration and invasion and increased cellular apoptosis rates. Similar to our cell experiment results, some studies also found that high expression of SPOP suppressed the malignant biological behaviour of cancer cells in vitro via ubiquitin-dependent proteolysis of the signalling pathway[14, 26–28]. Therefore, SPOP may be associated with inhibition of the aggressiveness of RCC cells.
SPOP plays key roles in cancer development by promoting ubiquitination and degradation of the substrate protein of specific signalling pathways. For example, SPOP has a definitive tumour suppressing role in gastric cancer by promoting the degradation of the transcription factor Gli2 of the Hedgehog (Hh)/Gli2 signalling pathway. In an in vitro drug sensitivity experiment, we found that the proliferation inhibitory rates of cells were significantly increased and cellular apoptosis was induced when SPOP was overexpressed in RCC cells that were treated with sunitinib or IFN-α2b. Sunitinib, a tyrosine kinase inhibitor targeting the VEGF receptor, has been the first-line targeted therapy for patients with mRCC who have been classified as having MSKCC intermediate-risk or poor-risk disease and has shown an improvement in survival[6, 29]. Cellular migration, proliferation and survival of cancer cells as well as endothelial cell differentiation are driven mainly by VEGF/VEGFR activation, which in turn activates the PI3K/Akt/mTOR signalling pathway[30, 31]. One study showed that 4-chloro fascaplysin, a marine sponge alkaloid derivative, inhibited tumour growth and VEGF-mediated angiogenesis by disrupting the PI3K/Akt/mTOR signalling cascade. The PI3K/Akt/mTOR axis, which is involved in cancer cell proliferation, differentiation and cellular metabolism, is frequently activated in many cancers and is one of the most significant molecular pathways in mRCC[33, 34]. Activation of the PI3K/Akt/mTOR pathway is correlated with aggressive behaviour and poor prognosis of RCC tumours and is more significantly altered in ccRCC, high TNM stage tumours, and tumours with poor prognostic features[35, 36]. SPOP binding to the substrate is a crucial event for E3 ligase-mediated ubiquitination and subsequent proteasome degradation. Levels of the PI3K/Akt pathway have been found to be correlated with SPOP expression, which could inhibit colorectal cancer and osteosarcoma invasion by significantly reducing the levels of PI3K and p-Akt[37, 38]. In the present study, there was a significant difference in the sensitivity of different cell lines overexpressing SPOP to several concentrations of sunitinib. It could be a promising potential molecular mechanism that may provide an effective therapeutic strategy for patients with advanced kidney cancer by exploring the relationships among the SPOP, VEGF and PI3K/Akt/mTOR pathways.
In addition, the Hedgehog signalling pathway, which increases tumour invasion and metastatic potential, is another important molecular mechanism that is worth investigating in the future. Aberrant activation of the Hedgehog pathway is associated with tumorigenesis in some cancers, including RCC, and plays an important role in RCC development[39–41]. Limited studies suggest that SPOP suppresses tumour development by negatively regulating the Hedgehog/Gli2 signalling pathway in gastric cancer. In addition, the expression levels of the Hedgehog signalling pathway component genes Gli1 and Gli2, which are activated by the PI3K/Akt signalling pathway in RCC, are significantly elevated in ccRCC and provide a promising therapeutic strategy for RCC. Currently, the Hedgehog inhibitors (HHIs) vismodegib and sonidegib are approved for use in advanced BCC, and other potential uses for the treatment of solid tumours beyond BCC are under development or in clinical trials. Given the above, SPOP, as a tumour suppressor protein, plays an important role in inhibiting tumorigenesis by regulating different signalling pathways. However, studies on the molecular mechanism of the SPOP protein in RCC are still limited. Exploring the underlying mechanisms of signalling pathways in kidney cancer in detail is the best approach to provide a theoretical basis for the development of novel therapeutic strategies for mRCC patients in the future.
Differential expression levels or mutation profiles of SPOP in tumours play different roles in tumorigenesis and cancer progression [10, 11, 13]. Several studies have shown that SPOP expression is downregulated in some primary tumours, including gastric cancer, liver cancer, colorectal cancer, pancreatic cancer and non‑small cell lung cancer, and low expression of SPOP is associated with poor prognosis in patients[14, 26–28, 44]. In the current study, immunohistochemical staining demonstrated that the SPOP protein was mainly expressed at low levels in the cytoplasm of ccRCC tissues and was relatively highly expressed in most adjacent nontumour tissues and normal kidney tissues. The inhibitory role of SPOP was confirmed by an earlier study that showed that downregulation of SPOP expression in cancers might inhibit its functions as a tumour suppressor gene and might promote cancer development. On the basis of the results of immunohistochemical staining and cell culture experiments in the present study, SPOP may act as a potential tumour suppressor protein in the tumorigenesis of RCC. However, the findings of the survival analysis did not provide supportive evidence showing a correlation of SPOP expression and overall survival, suggesting that high expression of SPOP could not be regarded as a hallmark of RCC and could not yet predict the prognosis of patients. The exact role of the SPOP protein in RCC is controversial and still needs to be confirmed by further research based on a large cohort of samples.
Although there are some important discoveries in the present study, some limitations need to be discussed. First, the concentration gradient of drug experiments was too large to accurately reflect the significant concentration. Second, it is widely accepted that RCC is a heterogeneous tumour with distinct pathological tissue subtypes, including clear cell, papillary, and chromophobe subtypes. The TMA tissues used in this study consisted of a single pathological tissue subtype and could not be used to explore the expression of SPOP in the different subtypes of RCC. More pathological tissue subtypes should be included to analyse the expression of SPOP in RCC tissue in the future, especially fresh frozen tissue from RCC radical nephrectomy.