mTOR pathway is a key regulator of protein translation and cell proliferation that has been shown to be up-regulated in several solid malignancies including thyroid carcinoma 10
, small cell carcinoma of lung 11
, gastrointestinal tumors 12, 13
and clear cell renal cell carcinoma 14
. The main downstream effectors of mTOR pathway (phos S6 and 4E-BP1) have been shown to be independent predictors of prognosis in several types of solid tumors including renal cell 14
, ovarian 15
, liver 16-19
and mammary carcinomas 20
While the search for prognostic biomarkers in UrCa initially focused on cell cycle regulators, p53 and retinoblastoma (RB) alterations 21, 22
, more recent efforts have also included tyrosine kinase receptors, mTOR and microenvironment interaction pathways 23
as potential prognosticators. Several studies have addressed the prognostic potential of individual mTOR pathway members in bladder cancer 24-26
. Comprehensive simultaneous assessment of key members of PI3K- mTOR pathway and its downstream affected biomarkers, in relation to clinicopathologic parameters and outcome, in a well characterized cystectomy cohort is needed.
PTEN loss leading to activation of AKT/mTOR axis has been reported in other genitourinary tract tumors such as prostate adenocarcinoma27, 28
, renal cell carcinoma29
and upper urinary tract UrCa30
. These findings offered a rational for the limited but promising response to mTOR inhibitors therapy in such settings. However, Yoo et al 31
, using a mouse model that conditionally deletes PTEN in urogential epithelium, found AKT/mTOR pathway highly activated in prostate tumors, but not in bladder epithelium. Moreover, PTEN knock-out mice were shown to develop urothelial carcinomas in the upper urinary tract, but not in the bladder 30
. In contrast, synchronous deletions of both PTEN and p53 tumor suppressor genes have been shown to lead to the development of invasive bladder cancer in mouse models 32
Our findings of frequent loss of Pten (42%) and phos Akt (44%) expression in UrCa are in line with prior studies. Pten and phos Akt loss of expression has been previously reported in up to 50% and 70% of UrCa, respectively, while lower Pten expression has been linked to invasive behavior 24-26
. In our cohort, both Pten and phos Akt expression levels inversely correlated with pT stage and aggressive divergent histology and their loss was more likely to be seen in invasive compared to non-invasive tumor component within a given TMA spot. Our findings of lack of correlation of either Pten or phos Akt tumor expression with disease outcome have also been previously illustrated by Harris et al. and others 26, 33
. The generally lower levels of mTOR pathway members in UrCa with divergent histology suggests downregulation of mTOR pathway in less differentiated urothelial tumors.
The positive but weak correlation between Pten and phos Akt expression is intriguing and may suggest a non-conditional association between the loss of inhibitory effect of PTEN tumor suppressor gene and phos Akt expression (Akt activation) in UrCa. Bose et al 34
found no correlation between Pten and phos Akt expression in mammary carcinoma, despite finding both markers to be altered, suggesting that Akt activation is not always directly linked to PTEN loss.
AKT regulates its downstream target mTOR which in turns operates through two distinct complexes: mTORC1 and mTORC2. mTORC2 directly activates AKT in a feedback fashion while mTORC1 pathway regulates cell growth through its main downstream effectors: ribosomal S6 kinase and 4E-BP1. Activation of mTORC1 is thought to stimulate translation through phosphorilation of S6 and inhibition of 4E-BP1. 35
. Our findings of lower phos S6 and higher 4E-BP1 expression levels in UrCa compared to benign urothelium is consistent with the lower activation of AKT found in our UrCa cohort resulting in an overall downregulation of mTORC1 downstream events. The inverse correlation between phos S6 expression and tumor pT stage in our cohort is in keeping with our finding of a favorable prognostic effect of higher phos S6 levels in UrCa but is in contrast with prior findings of unfavorable prognostic effect of Phos S6 expression in other solid tumors 19
Our finding of downregulation of phos S6 in more aggressive UrCa tumors could theoretically be related to their hypoxic tolerant phenotype 36
given prior evidence for downregulation of mTOR pathway through HIF1α activation in hypoxic states 37-39
and given the association of HIF1α overexpression with poor outcome in UrCa 40-44
. We are in the process of evaluating HIF1α expression in the current cohort in relation to mTOR pathway expression hoping to test such hypothesis.
In addition to promoting translation, phos S6 is recognized to repress PIK3-AKT pathway through the inhibition of insulin receptor substrates 1 and 2 (IRS1/IRS2) 7
. Accordingly, we found phos S6 expression, but not 4E-BP1 to correlate with other AKT-regulated members: p27 and c-Myc. In fact, the strongest correlation among pathway members was between c-Myc and phos S6. Furthermore, high c-Myc expression was an independent predictor of disease progression in our cohort. These results are also in keeping with a recently suggested MYC-dependent mechanism for phos S6 translation 45
Lower p27 expression in our UrCa tumors and its inverse correlation with pT stage and aggressive divergent histology is in agreement with prior reports 46-48
. The significantly positive correlation between p27 and Pten expression levels in our UrCa cohort could be interpreted as additional evidence of a potential oncogenic role for downregulation of p27 in PTEN deficient bladders as it has been recently pointed by Yoo et al 31
. Unlike some of the prior studies on p27 in UrCa 46, 48
, we did not find loss of p27 expression to be an unfavorable predictor of disease progression or DSS. One possible reason for the difference could be that these studies had a larger proportion of superficial tumors. Interestingly, recent studies have pointed to a favorable effect on outcome for loss of p27 in UrCa especially in combination with other cell cycle markers 21, 49
. Additional large cohort studies are needed to better resolve the prognostic role if any of p27 in UrCa patients.
In summary, our study represent the first attempt at simultaneous assessment of key members of PI3K- mTOR pathway and its downstream affected biomarkers, in relation to clinicopathologic parameters and outcome. We found phos S6 expression to be an independent predictor of DSS (p=0.03) and c-Myc expression to be an independent predictor of disease progression (p=0.02) in addition to TNM pathologic stage grouping in our cohort. Our intriguing novel findings of a statistically significant prognostic role for phos S6 and c-Myc in a multivariate model that included established clincopathologic prognostic parameters are promising and certainly warrant further confirmation in an independent cystectomy cohort, preferably in a prospective setting and in combination with cell cycle markers evaluation. Additional studies to address potential value of mTOR pathway markers expression in predicting therapeutic response to mTOR inhibitors are also warrented.