The PI3K/AKT/mTOR pathway plays a central role in regulating cell growth and participates in the control of other numerous cellular processes, including mRNA initiation and protein translation. PI3K, the first member of the pathway, can be directly activated by tyrosine kinase receptors, leading to phosphorylation and consequent activation of AKT; in turn, activation of AKT is negatively controlled by PTEN. Activated AKT (phos-AKT) exerts its downstream effects by phosphorylation of either proteins related to the tuberous sclerosus complex genes (TSC1/TSC2) or a proline-rich AKT substrate (PRAS40). A rapamycin-sensitive raptor complex (mTORC1) is then phosphorylated by any of these proteins, which in turns activates two downstream signaling pathways, S6 and 4EBP1. Phosphorylated 4EBP1 dissociates from eIF4E, allowing the latter to form a complex that facilitates cap-dependant translation of cell cycle-related proteins, such as c-MYC and cyclin D1, and hypoxia-induced factors, such as HIF-1α. On the other hand, phosphorylation of S6 ultimately leads to ribosomal protein translation and ribosome biogenesis. is a diagrammatic representation of the mTOR and hypoxia-induced pathways considering the members we evaluated in the present study.
Figure 4 Diagrammatic representation of the mTOR and hypoxia-induced pathways. In tumors with activation of the mTOR pathway the use of mTOR inhibitors, such as sirolimus (Rapamycin) and derived drugs, may be beneficial. Blunt-ended arrows indicate inhibitory (more ...)
In our cohort, PTEN levels were markedly reduced in both primary and metastatic ccRCC relative to benign controls, suggesting that, when present, PTEN loss may represent an early step in ccRCC carcinogenesis. Our results are in agreement with previous studies which have showed a marked reduction in PTEN levels in ccRCC.3,15,33
Recently, using data mining in a set of ccRCC TMAs Dahinden et al demonstrated an adverse prognosis in patients with loss of cytoplasmic PTEN staining and suggested a crucial role of PTEN and p27 inactivation in tumor progression.6
All tested markers were significantly associated with the pT stage at nephrectomy. Additionally, expression levels of 4EBP1, p27, and HIF-1α were associated with tumor size, supporting the notion of a collaborative interplay between mTOR, hypoxia-induced pathways and cell cycle-related proteins in regulating tumor growth and local aggressiveness. We also observed significantly higher levels of phos-AKT, phos-S6, and 4EBP1 in metastatic ccRCC when compared to primary lesions, indicating that activation of the mTOR pathway might be associated with the acquisition of a more aggressive phenotype. The overall expression levels of c-MYC were low in all the samples but the higher levels we observed in metastatic tumors suggest that activation of this gene may play a role in tumor progression. Activation of the c-MYC pathway has been described previously in the majority of ccRCC,12,40
although other studies have failed in demonstrated c-MYC upregulation in this subset of renal tumors.24,38,45
Attempts to predict survival in renal cell carcinoma have traditionally relied on standard clinicopathologic variables such as performance status and other clinical variables, tumor histologic type, pTNM stage, Fuhrman grade, and size.28,31
In agreement with previous studies,7
in our cohort of patients with primary ccRCC, pT stage and tumor size were significant predictors of outcome when only clinicopathologic variables were considered; Fuhrman grade also showed a significant association with prognosis when categorized as low (Fuhrman 1 and 2) and high (Fuhrman 3 and 4) grades. However, when biomarkers were included into the multivariate model, pT stage and Fuhrman grade lost significance as prognosticators of DSS and tumor progression. Additional studies, preferably in prospective cohorts, are needed to support our current observation on the role of expression levels of mTOR and hypoxia-induced pathway members as predictors of outcome independent of pT stage or Fuhrman grade alone. It would also be of great interest to evaluate the predictive performance of these biomarkers in nonmetastatic RCC against prognostic tools currently in use such as the UCLA Integrated Staging System (UISS) or the Mayo Clinic’s SSIGN, among others.46
Finally, the design of nomograms considering clinicopathological features as well as expression levels of these biomarkers could be of value following confirmation of our findings in prospective series of cases.
In our cohort of primary ccRCC higher levels of phos-S6 predicted better outcome. Our results are consistent with those published by Pantuck et al33
who investigated 375 RCC, including 323 ccRCC, and found higher phos-S6 levels to be predictors of DSS. They also identified loss of PTEN and phos-AKT expression as significant prognosticators but only phos-S6 remained an independent predictor in their multivariate model. In our series, HIF-1α and 4EBP1 levels were also predictors of outcome in primary ccRCC. 4EBP1 has shown association with malignant progression and adverse prognosis in breast, ovarian, endometrial and prostate carcinomas1
yet there are no reports on its expression or prognostic significance in RCC. On the other hand, previous studies have shown that HIF-1α expression is an adverse prognostic marker in ccRCC.8,10,19,23,30
This is of particular interest since inactivation of the VHL tumor suppressor gene results in stabilization of the HIF-1α subunit.16
Furthermore, HIF-1α is thought to downregulate the mTOR pathway independent of a hypoxic microenvironment.47
Accordingly, both HIF-1α and phos-S6 were found to be independent predictors of DSS and tumor progression. Additionally, HIF-1α expression levels were elevated to a similar extent in both primary and metastatic ccRCC while metastatic tumors demonstrated significantly higher levels of phos-S6 compared to primary lesions. This finding may provide additional evidence for the importance of the interaction between the hypoxia-induced and the mTOR pathways in tumor progression of ccRCC.
A possible weakness of the present study includes the use of nonmatched tissue for primary and metastatic tumors. Metastatic ccRCC might had different tumor grades and stages in their primary setting compared to the ones observed in the evaluated primary ccRCC of our series, and this could have influenced the distribution of the biomarker levels of expression. However, we still deem worthwhile to evaluate the mTOR and hypoxia-inducible pathways in nonmatched metastatic ccRCC, especially considering that mTOR inhibitors, such as temsirolimus, have been recently FDA-approved for the treatment of advanced RCC.25
The fact that the expression levels of the analyzed biomarkers were not predictors of outcome in metastatic ccRCC suggest that the prognostic value of these might be limited to the evaluation of nonmetastatic ccRCC. Nevertheless, given the retrospective design of the current series, these findings require further prospective confirmation. Another potential limitation of the present study is the use of TMA sampling. Given the staining heterogeneity of some markers, the use of TMA instead of whole sections could have some impact on the detected expression levels. However, several studies have supported the high throughput value of the TMA usage and the adequate representation of the overall expression levels using multiple TMA spots.5
In summary, we found evidence of activation of both the mTOR and the hypoxia-induced pathways in primary and metastatic ccRCC. PTEN loss seems to be an early event during tumorigenesis. In addition, tumor size, HIF-1α and phos-S6 expression were found to be independent predictors of both DSS and tumor progression in primary ccRCC. Our results suggest that immunohistochemical evaluation of mTOR and hypoxia-induced pathway members might be useful in predicting the outcome of patients with primary ccRCC, complementing the information provided by routine gross pathologic and microscopic analysis.