Platelet-derived growth factor receptor-α (PDGFRα) is a novel biomarker along with smooth myosin heavy chain for the pacemaker cells (previously termed 'atypical' smooth muscle cells) in the murine and cynomolgus monkey pelvis-kidney junction. r PDGFRα + cells present in adventitial and urothelial layers of murine renal pelvis do not express smooth muscle myosin heavy chain (smMHC) but are in close apposition to nerve fibres. r Most c-Kit + cells in the renal pelvis are mast cells. Mast cells (CD117 + /CD45 +) are more abundant in the proximal renal pelvis and pelvis-kidney junction regions whereas c-Kit + interstitial cells (CD117 + /CD45 −) are found predominantly in the distal renal pelvis and ureteropelvic junction. r PDGFRα + cells are distinct from c-Kit + interstitial cells. r A subset of PDGFRα + cells express the Ca 2+-activated Cl − channel, anoctamin-1, across the entire renal pelvis. r Spontaneous Ca 2+ transients were observed in c-Kit + interstitial cells, smMHC + PDGFRα cells and smMHC − PDGFRα cells using mice expressing genetically encoded Ca 2+ sensors.

Overexpression of Rictor has been demonstrated to result in increased mTORC2 nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However the mechanisms regulating Rictor expression in these tumors is not clearly understood. In this report, we demonstrate that Rictor is regulated at the level of mRNA translation via HSF1-induced HuR activity. HuR is shown to directly bind the 3′ UTR of the Rictor transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive Rictor expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate Rictor and inhibit GBM growth and invasive characteristics in vitro and suppresses xenograft growth in mice. Modulation of AKT or HSF1 activity via the ectopic expression of mutant alleles support the ability of AKT to activate HSF1 and demonstrate continued HSF1/HuR/Rictor signaling in the context of AKT knockdown. We further show that constitutive overexpression of HuR is able to maintain Rictor expression under conditions of AKT or HSF1 loss. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates Rictor translational efficiency via an AKT/HSF1/HuR signaling cascade resulting in enhanced mTORC2 activity in these tumors.

Running title: Feed-forward loop signaling regulates mTORC2 Word count (text) = 4483 Word count (abstract) = 227 Figure count = 7 Table count = 1 Reference count = 49 2 ABSTRACT Overexpression of Rictor has been demonstrated to result in increased mTORC2 nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However the mechanisms regulating Rictor expression in these tumors is not clearly understood. In this report, we demonstrate that Rictor is regulated at the level of mRNA translation via HSF1-induced HuR activity. HuR is shown to directly bind the 3' UTR of the Rictor transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive Rictor expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate Rictor and inhibit GBM growth and invasive characteristics in vitro and suppresses xenograft growth in mice. We further demonstrate that constitutive overexpression of HuR is able to maintain Rictor expression under conditions of AKT or HSF1 loss. In an additional level of regulation, miR-218, a known Rictor targeting miRNA is shown to be subject to mTORC2/STAT3mediated repression. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates Rictor translational efficiency and suppresses miR-218 resulting in enhanced mTORC2 activity in these tumors.

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