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Mammalian target of rapamycin complex 2 (mTORC2) is a critical determinant of bladder cancer invasion.

Gupta S, Hau AM, Beach JR, Harwalker J, Mantuano E, Gonias SL, Egelhoff TT, Hansel DE - PLoS ONE (2013)

Bottom Line: Invasive behavior is a major determinant of prognosis.This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation.These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Bladder cancer is the fourth most common cause of cancer in males in the United States. Invasive behavior is a major determinant of prognosis. In this study, we identified mammalian target of rapamycin complex 2 (mTORC2) as a central regulator of bladder cancer cell migration and invasion. mTORC2 activity was assessed by the extent of phosphorylation of Ser473 in AKT and determined to be approximately 5-fold higher in specimens of invasive human bladder cancer as opposed to non-invasive human bladder cancer. The immortalized malignant bladder cell lines, UMUC-3, J82 and T24 demonstrated higher baseline mTORC2 activity relative to the benign bladder papilloma-derived cell line RT4 and the normal urothelial cell line HU1. The malignant bladder cancer cells also demonstrated increased migration in transwell and denudation assays, increased invasion of matrigel, and increased capacity to invade human bladder specimens. Gene silencing of rictor, a critical component of mTORC2, substantially inhibited bladder cancer cell migration and invasion. This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation. These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.

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Increased mTORC2 activity occurs in invasive bladder cancer.We compared A, non-invasive low-grade papillary UCa (non-inv LG; scale bar 80 microns); B, non-invasive high-grade papillary UCa (non-inv HG; scale bar 80 microns); and C, invasive high-grade UCa to determine mTORC2 activity (scale bar 100 microns). D, immunoblotting for p-Ser473 as a marker of mTORC2 activity was performed and shows higher mTORC2 activity in both non-invasive HG and invasive lesions. E, densitometry was used to quantify p-Ser473/ total AKT signal intensities; averages and standard error of the mean (SEM) were normalized to the average signal intensity for the non-invasive LG samples.
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pone-0081081-g001: Increased mTORC2 activity occurs in invasive bladder cancer.We compared A, non-invasive low-grade papillary UCa (non-inv LG; scale bar 80 microns); B, non-invasive high-grade papillary UCa (non-inv HG; scale bar 80 microns); and C, invasive high-grade UCa to determine mTORC2 activity (scale bar 100 microns). D, immunoblotting for p-Ser473 as a marker of mTORC2 activity was performed and shows higher mTORC2 activity in both non-invasive HG and invasive lesions. E, densitometry was used to quantify p-Ser473/ total AKT signal intensities; averages and standard error of the mean (SEM) were normalized to the average signal intensity for the non-invasive LG samples.

Mentions: Non-invasive low-grade (LG) UCa is defined by papillary fronds lined by urothelium with retained polarity and only minimal cytologic atypia (Figure 1A). This histology correlates with frequent mutations in FGFR3 and RAS [21]. In contrast, non-invasive high-grade (HG) UCa shows nuclear enlargement, hyperchromasia, and loss of polarity that is commonly associated with mutations in TP53 and RB genes (Figure 1B). In a subset of HG UCa cases, invasive behavior is observed (Figure 1C) and this is associated with worsened outcomes. We evaluated mTORC2 activity in samples of LG UCa, HG UCa, and HG UCa with invasion by determining phosphorylation of AKT at Ser473 (p-Ser473). Immunoblotting suggested an increase in p-Ser473 in non-invasive HG UCa compared with LG lesions (Figure 1D) and an additional increase in p-Ser473 in invasive HG lesions. Densitometry comparing p-Ser473/total AKT ratios was performed, with results normalized to non-invasive LG UCa (Figure 1E). Although the small number of samples precluded statistical significance, p-Ser473 appeared increased in the more advanced pathologies.


Mammalian target of rapamycin complex 2 (mTORC2) is a critical determinant of bladder cancer invasion.

Gupta S, Hau AM, Beach JR, Harwalker J, Mantuano E, Gonias SL, Egelhoff TT, Hansel DE - PLoS ONE (2013)

Increased mTORC2 activity occurs in invasive bladder cancer.We compared A, non-invasive low-grade papillary UCa (non-inv LG; scale bar 80 microns); B, non-invasive high-grade papillary UCa (non-inv HG; scale bar 80 microns); and C, invasive high-grade UCa to determine mTORC2 activity (scale bar 100 microns). D, immunoblotting for p-Ser473 as a marker of mTORC2 activity was performed and shows higher mTORC2 activity in both non-invasive HG and invasive lesions. E, densitometry was used to quantify p-Ser473/ total AKT signal intensities; averages and standard error of the mean (SEM) were normalized to the average signal intensity for the non-invasive LG samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842329&req=5

pone-0081081-g001: Increased mTORC2 activity occurs in invasive bladder cancer.We compared A, non-invasive low-grade papillary UCa (non-inv LG; scale bar 80 microns); B, non-invasive high-grade papillary UCa (non-inv HG; scale bar 80 microns); and C, invasive high-grade UCa to determine mTORC2 activity (scale bar 100 microns). D, immunoblotting for p-Ser473 as a marker of mTORC2 activity was performed and shows higher mTORC2 activity in both non-invasive HG and invasive lesions. E, densitometry was used to quantify p-Ser473/ total AKT signal intensities; averages and standard error of the mean (SEM) were normalized to the average signal intensity for the non-invasive LG samples.
Mentions: Non-invasive low-grade (LG) UCa is defined by papillary fronds lined by urothelium with retained polarity and only minimal cytologic atypia (Figure 1A). This histology correlates with frequent mutations in FGFR3 and RAS [21]. In contrast, non-invasive high-grade (HG) UCa shows nuclear enlargement, hyperchromasia, and loss of polarity that is commonly associated with mutations in TP53 and RB genes (Figure 1B). In a subset of HG UCa cases, invasive behavior is observed (Figure 1C) and this is associated with worsened outcomes. We evaluated mTORC2 activity in samples of LG UCa, HG UCa, and HG UCa with invasion by determining phosphorylation of AKT at Ser473 (p-Ser473). Immunoblotting suggested an increase in p-Ser473 in non-invasive HG UCa compared with LG lesions (Figure 1D) and an additional increase in p-Ser473 in invasive HG lesions. Densitometry comparing p-Ser473/total AKT ratios was performed, with results normalized to non-invasive LG UCa (Figure 1E). Although the small number of samples precluded statistical significance, p-Ser473 appeared increased in the more advanced pathologies.

Bottom Line: Invasive behavior is a major determinant of prognosis.This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation.These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Bladder cancer is the fourth most common cause of cancer in males in the United States. Invasive behavior is a major determinant of prognosis. In this study, we identified mammalian target of rapamycin complex 2 (mTORC2) as a central regulator of bladder cancer cell migration and invasion. mTORC2 activity was assessed by the extent of phosphorylation of Ser473 in AKT and determined to be approximately 5-fold higher in specimens of invasive human bladder cancer as opposed to non-invasive human bladder cancer. The immortalized malignant bladder cell lines, UMUC-3, J82 and T24 demonstrated higher baseline mTORC2 activity relative to the benign bladder papilloma-derived cell line RT4 and the normal urothelial cell line HU1. The malignant bladder cancer cells also demonstrated increased migration in transwell and denudation assays, increased invasion of matrigel, and increased capacity to invade human bladder specimens. Gene silencing of rictor, a critical component of mTORC2, substantially inhibited bladder cancer cell migration and invasion. This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation. These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.

Show MeSH
Related in: MedlinePlus