Fig 1.
Schematic representation of membrane protein O-glycosylation and the PI3K/Akt/mTOR pathways.
A) Representation of membrane protein O-glycosylation with emphasis on the STn expression by cancer cells. This is a highly regulated process of critical importance for protein stability and function. Briefly, newly synthesized proteins are O-glycosylated in the Golgi apparatus by the ppGalNAcTs-mediated addition of GalNAc moiety to Ser/Thr residues. This originates the Tn antigen (GalNAc-O-Ser/Thr-protein backbone), which is the simplest O-glycan. In normal cells these chains are extended through the sequential addition of other sugars first by CGALT-1 and then other enzymes. This culminates in highly complex, heterogeneous and elongated glycans often terminated by ABO or Lewis blood group related antigens (left drawing). In cancer cells the Tn antigen is immediately sialylated by ST6GalNAc.I, originating the STn antigen (Neu5Ac-GalNAc-O-Ser/Thr-protein backbone), thereby inhibiting further chain elongation (right drawing). The expression of STn at the cell surface influences cell-cell adhesion and cancer cell recognition, favouring motility, invasion and immune escape. B) Schematic representation of the PI3K/Akt/mTOR pathway, which is ubiquitously activated in bladder tumours. This is a highly conserved pathway regulated mainly by a wide variety of extracellular signals, including mitogenic growth factors, hormones, nutrients, cellular energy levels, and stress conditions. These signals activate tyrosine receptor kinases that recruit PI3K, which catalyses the conversion of membrane-bound PIP2 to PIP3. Then Akt and PDK-1 are activated through binding to PIP3. PTEN preferentially dephosphorylates PIP3, inhibiting signalling progression. Full Akt activation requires double phosphorylation by PDK-1 itself and PDK-2 (not shown). Akt phosphorylates mTOR directly or may also inactivate TSC1/TSC2 complex, inhibiting mTOR inactivation. mTORC1 triggers cell growth and proliferation by phosphorylating eukaryotic translation regulators, among these p70S6 kinase (p70S6K or S6K1) that, in turn, phosphorylates the ribosomal protein S6 (pS6), and the eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1). For the protein mTOR to activate its signalling cascade, it must form the rapamycin-sensitive ternary complex mTORC1. Key PI3K/Akt/mTOR-pathway proteins pAkt, pmTOR and pS6 explored in this studied are highlighted by orange circles.
Table 1.
Clinical-pathological data of the studied sample (n = 96).
Table 2.
Antibodies used in the immunohistochemical analysis.
Fig 2.
Association between disease groups and cancer-specific survival (CSS) in the studied patients.
Kaplan-Meier analysis showing the CSS of NMIBC (Ta and T1) and s of MIBC (T2,T3 and T4). Comparison performed by log-rank test (p<0.001); + censored NMIBC patients; ⧫ censored MIBC patients.
Fig 3.
STn expression in different bladder tumors stages.
(A) Distribution of STn negative and positive tumors along the different stages of bladder cancer; (B) Representative images of STn staining in NMIBC and MIBC. Left—NMIBC showing a predominance of STn positive cells in the superficial layers, away from the fibrovascular support; note vessels without positive cells. Right—MIBC showing the invasion front with STn positively stained cells; note positive STn urothelial cells in the vessels (arrow), suggesting possible involvement in metastasis.
Table 3.
Association between the evaluated markers and the stage of disease.
Fig 4.
Expressions of pAkt, pmTOR, pS6 and PTEN in NMIBC and MIBC (40x magnification).
A and B) pAKT nuclear and cytoplasmatic expression in NMIBC (A) and MIBC (B). In NMIBC cases pAkt presented a heterogeneous pattern with areas of different intensity of expression. In MIBC, stromal cells mainly in the areas close to the tumour showed higher expression. C and D) pmTOR cytoplasmic expression in NMIBC (C) and MIBC (D). In NMIBC pmTOR was expressed across several layers, although there was a more intense staining in the superficial ones. In MIBC positive cases pmTOR expression was focal. E and F) pS6 cytoplasmatic expression in NMIBC (E) and MIBC (F). In NMIBC pS6 expression was observed in all the superficial layers both in umbrella and differentiated cells. In MIBC the immunoreactivity was diffuse, however more present in basal and mitotic cells. pS6 expression was higher in the invasion front and in tumour infiltrating lymphocytes and endothelial cells. G and H) PTEN cytoplasmic and nuclear expressions in NMIBC (G) and MIBC (H). PTEN expression was higher in NMIBC compared to MIBC.
Fig 5.
Effect of STn expression in cancer-specific survival (CSS).
Kaplan–Meier analysis showing the association between STn and CSS in: (A) all studied bladder cancer patients; (B) NMIBC patients. Comparison performed by log-rank test (A: p = 0.024; B: p = 0.020); + censored STn negative tumours; ⧫ censored STn positive tumours.
Fig 6.
Effect of PTEN expression and cancer-specific survival (CSS) in the studied patients.
Kaplan-Meier analysis showing the effect in CSS of PTEN expression in all studied bladder cancer patients. Comparison performed by log-rank test (p = 0.013); + censored PTEN negative tumours; ⧫ censored PTEN positive tumours.
Fig 7.
Effect of PI3K/Akt/mTOR pathway activation in cancer-specific survival (CSS) of patients with STn positive MIBC.
Kaplan–Meier analysis showing the association between pAKT, pmTOR and pS6 expressions in the CSS of STn positive tumors MIBC: Comparison performed by log-rank test (p = 0.027); + censored pAKT and pmTOR and pS6 negative tumours; ⧫ censored pAKT and/or pmTOR and/or pS6 positive tumours.
Fig 8.
STn and pS6 expressions in bladder tumours from BBN-exposed male ICR mice with or without the administration of mTOR-inhibitor sirolimus (rapamycin).
A) Experimental design to determine the sirolimus effect on STn and pS6 expressions in a model of urothelial carcinogenesis (male ICR mice). B) Expression of STn and pS6 in BBN-derived urothelial tumours in the presence and absence of sirolimus. BBN-induced bladder tumours (Groups 1 and 3) overexpressed STn and pS6, which was more pronounced in Group 3, after longer lifespan. Exposure to sirolimus decreased the number of invasive lesions in groups 2 and 4 (data not shown) and, concomitantly, decreased the expressions of STn and pS6. C) Histological sections showing the expressions of STn and pS6 in BBN-induced urothelial tumours before and after treatment.