Dynamic Regulation of Caveolin-1 Phosphorylation and Caveolae Formation by Mammalian Target of Rapamycin Complex 2 in Bladder Cancer Cells

Dynamic Regulation of Caveolin-1 Phosphorylation and Caveolae Formation by Mammalian Target of Rapamycin Complex 2 in Bladder Cancer Cells

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Dynamic Regulation of Caveolin-1 Phosphorylation and Caveolae Formation by Mammalian Target of Rapamycin Complex 2 in Bladder Cancer Cells Andrew M. Hau,* Sounak Gupta,* Mariah Z. Leivo,* Kazufumi Nakashima,* Jesus Macias,* Weidong Zhou,y Alex Hodge,y Julie Wulfkuhle,y Brian Conkright,z Krithika Bhuvaneshwar,z Shruti Rao,z Subha Madhavan,z Emanuel F. Petricoin, III,y and Donna E. Hansel* From the Department of Pathology,* University of California, San Diego, La Jolla, California; the Center for Applied Proteomics and Personalized Medicine,y George Mason University, Manassas, Virginia; and The Innovation Center for Biomedical Informatics,z Georgetown University, Washington, District of Columbia Accepted for publication May 7, 2019. Address correspondence to Donna E. Hansel, M.D., Ph.D., Department of Pathology, University of California, San Diego, 9500 Gilman Dr., MC 0612, La Jolla, CA 92093. E-mail: [email protected] ucsd.edu.

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The mammalian target of rapamycin (mTOR) and associated phosphatidylinositol 3-kinase/AKT/mTOR signaling pathway is commonly up-regulated in cancer, including bladder cancer. mTOR complex 2 (mTORC2) is a major regulator of bladder cancer cell migration and invasion, but the mechanisms by which mTORC2 regulates these processes are unclear. A discovery mass spectrometry and reverse-phase protein arrayebased proteomics dual approach was used to identify novel mTORC2 phosphoprotein targets in actively invading cancer cells. mTORC2 targets included focal adhesion kinase, protooncogene tyrosine-protein kinase Src, and caveolin-1 (Cav-1), among others. Functional testing shows that mTORC2 regulates Cav-1 localization and dynamic phosphorylation of Cav-1 on Y14. Regulation of Cav-1 activity by mTORC2 also alters the abundance of caveolae, which are specialized lipid raft invaginations of the plasma membrane associated with cell signaling and membrane compartmentalization. Our results demonstrate a unique role for mTORC2-mediated regulation of caveolae formation in actively migrating cancer cells. (Am J Pathol 2019, -: 1e16; https://doi.org/ 10.1016/j.ajpath.2019.05.010)

Bladder cancer is the most common urinary tract malignancy, with an estimated 81,000 new bladder cancer cases and 17,000 bladder cancereassociated deaths this year alone in the United States.1 Although most patients present with noninvasive disease, progression to invasive cancer occurs in approximately 40% to 60% of patients and results in an increased risk of metastasis and reduced diseasespecific survival.2 Current treatment modalities include bladder removal with neoadjuvant chemotherapy or radiation therapy for locally advanced, invasive disease and chemotherapy or immunotherapy for metastatic disease.3 Understanding the cell signaling pathways relevant to the development of invasive bladder cancer behavior may enhance the ability to develop strategic approaches in bladder cancer therapy.

The mammalian target of rapamycin complex 2 (mTORC2) is a major protein complex that drives bladder cancer invasion and key effector of the mTOR pathway.4e6 mTOR is an evolutionary conserved serine/threonine kinase that can integrate extracellular and environmental cues, such as growth factor signaling and nutrient status, to affect a diverse array of cell processes, such as cell proliferation, Supported by the Department of Pathology, University of California, San Diego. Disclosures: E.F.P. has US governmente and George Mason Universityeassigned patents concerning the reverse-phase protein array technology and receives licensing and royalty distribution from these patents. E.F.P. is a consultant to ADVX Investors Group, LLC, which has licenses in reverse-phase protein arrayebased immunoprecipitation from George Mason University and US governmenteowned patents.

Copyright ª 2019 Published by Elsevier Inc. on behalf of the American Society for Investigative Pathology. https://doi.org/10.1016/j.ajpath.2019.05.010

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metabolism, and motility. Specifically, mTOR mediates these functions as an essential component of two multiprotein complexes, mTORC1 and mTORC2. These complexes can be distinguished by their subunit composition (raptor and PRAS40 for mTORC1 and rictor, mSin1, and protor for mTORC2) and their downstream functions.7 Whereas mTORC1 regulates cell growth and metabolism through phosphorylation of p70-S6K and 4EBP1, mTORC2 phosphorylates AKT S473, increases Rho-GTPase activity, and activates protein kinase C (PKC).7,8 We previously described a promigratory and proinvasive function of mTORC2 on in vitro bladder cancer cell migration and invasion, which was mediated through Rho-GTPase activation,4 as well as increased mTORC2 activity occurring in invasive, high-stage human bladder cancers. Given the important role of mTORC2 in regulating bladder cancer cell migration and invasion, a proteomics-based approach using mass spectrometry (MS) and reverse-phase protein array (RPPA) was used to discover and identify novel targets of mTORC2 signaling in motile and nonmotile conditions. Phosphopeptide enrichment coupled to MS and RPPA represent two powerful complementary phosphoproteomic approaches that allow for discovery-based, quantifiable detection of proteins within biological samples. Although these technologies are able to effectively uncover important signaling events and map the activated signaling architecture of input samples, each have their own limitations.9 Although an MS approach can provide a global unbiased view of phosphoprotein expression, it is also possible that important signaling-related phosphoproteins may be undetected because of low relative protein abundance within a sample, low stoichiometry of phosphorylated signaling proteins compared with phosphorylated high abundance proteins and/or rapid degradation or dephosphorylation of a protein before analysis. By contrast, RPPA has greater analytical sensitivity than MS because of its ability to measure the phosphorylation state of very low abundance signaling proteins from microscopic quantities of cells. However, the biggest limitation of RPPA is the availability and dependence of antibodies for detection of proteins. Furthermore, RPPA can be viewed as a somewhat biased approach because arrays of specific antibodies are selected to probe a biological sample(s) for changes in protein expression and/or activity. Nevertheless, both MS and RPPA serve as increasingly important and complementary technologies for protein signaling analysis in cancer and other diseases. Here, a parallel MS- and RPPA-based proteomics discovery approach was used to elucidate novel downstream targets of mTORC2 signaling in motile bladder cancer cells, followed by validation and functional testing of a subset of protein signaling networks that appeared relevant for migration and invasion. Multiple classes of proteins regulated by mTORC2, including mediators of cell morphology, cell assembly and organization, cell adhesion, cytoskeletal rearrangement, and cell motility, were identified. A subset of putative mTORC2-mediated

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targets were validated, and the ability of mTORC2 to regulate dynamic phosphorylation of caveolin-1 (Cav-1) on Y14 and Cav-1 cellular localization, both of which represent novel downstream effects of mTORC2, was functionally tested. Furthermore, mTORC2 can also induce up-regulation of cavin1, an essential component for caveolae formation and function. The results of this study have identified a novel role for mTORC2-regulation of caveolae formation during bladder cancer cell motility.

Materials and Methods Cell Culture Human J82 and T24 bladder cancer cells were obtained and authenticated from the ATCC (Manassas, VA). Cells were grown in RPMI 1640 medium (Thermo Fisher Scientific, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) and maintained at 37 C in a humidified chamber containing 5% CO2.

Gene Silencing and Serum Stimulation SMARTpool siGENOME siRNA against RICTOR (siRictor; catalog number M-016984-02) and CAV1 (catalog number M-003467-01) were purchased from GE Dharmacon (Lafayette, CO) and transfected into cells using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA). Pooled siGENOME nontargeting control siRNA (siNTC) numbers 2-5 (catalog numbers D-001210-02 to -05) was used for controls. Briefly, cells were plated onto 100-mm dishes and incubated overnight. Each plate of cells was transfected in Opti-MEM reduced serum media (Gibco) containing siRNA (160 pmol) and maintained for 72 hours. For serum stimulation, media was aspirated from plates and cells were washed three times with phosphate-buffered saline (PBS) before the addition of RPMI 1640 medium supplemented with 10% fetal bovine serum during the last hour of gene silencing.

Immunoblotting Whole cell extracts were prepared using radioimmunoprecipitation assay buffer (25 mmol/L Tris hydrochloride, pH 7.6, 150 mmol/L NaCl, 1% Triton X-100, Q8 0.5% sodium deoxycholate, 0.1% SDS) containing PhosSTOP phosphatase and cOmplete Mini EDTA-free protease inhibitor cocktails (Roche, Mannheim, Germany). Cell lysates were cleared by centrifugation at 16,000  g for 15 minutes at 4 C, and protein concentration was determined by the bicinchoninic acid method. Equal amounts (10 mg) of protein were separated by SDS-PAGE using 4% to 15% Tris-Glycine Gradient gels (Bio-Rad, Hercules, PA) and immobilized onto polyvinylidene fluoride membranes. Membranes were blocked in 5% (w/v) nonfat dry milk in 50 mmol/L Tris hydrochloride, pH 7.4, 150 mmol/L NaCl

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(TBS) plus 0.1% Tween-20 (TBS-T) and then incubated at 4 C overnight with the appropriate primary antibody diluted in 5% (w/v) nonfat dry milk or bovine serum albumin. The next day, membranes were washed with TBS-T (three times for 5 minutes each) then incubated with the appropriate horseradish peroxidaseeconjugated secondary antibody for 1 hour at room temperature. Membranes were washed again in TBS-T (three times for 5 minutes each) and proteins detected using Clarity Western ECL blotting substrate (BioRad). Primary antibodies for 4EBP1 (1:1000; catalog number 9644), pan-AKT (1:1000; catalog number 4685), autocrine motility factor receptor (1:1000; catalog number 9590), Cav-1 (1:1000; catalog number 3267), cofilin (1:1000; catalog number 5175), epidermal growth factor receptor (EGFR) (1:1000; catalog number 4267), FAK (1:1000; catalog number 13009), HER2 (1:1000; catalog number 4290), mTOR (1:1000; catalog number 2983), Ncadherin (1:1000; catalog number 13116), phosphorylated (p-) 4EBP1 T37/46 (1:2000; catalog number 2855), p-AKT S473 (1:2000; catalog number 4060), p-AKT T308 (1:1000; catalog number 4056), p-CAV-1 Y14 (1:1000; catalog number 3251), p-cofilin S3 (1:1000; catalog number 3313), p-EGFR Y1173 (1:1000; catalog number 4407), p-ezrin T567/radixin T564/moesin T558 (1:1000; catalog number 3141), p-FAK Y397 (1:1000; catalog number 8556), p-FAK Y567/577 (1:1000; catalog number 3281), p-FAK Y925 (1:1000; catalog number 3284), p-HER2 Y1248 (1:1000; catalog number 2247), p-HER2 Y877 (1:1000; catalog number 2241), p-mTOR S2448 (1:1000; catalog number 5536), p-p70-S6K T389 (1:1000; catalog number 9234), peplatelet-derived growth factor receptor (p-PDGFR)-a Y754 (1:1000; catalog number 2992), p-PDGFR-b Y751 (1:1000; catalog number 4549), p-S6 S235/236 (1:2000; catalog number 4858), p-S6 S240/244 (1:2000; catalog number 5364), p-SRC Y527 (1:1000; catalog number 2105), p-SRC Y416 (1:1000; catalog number 6943), p-Talin S425 (1:1000; catalog number 5426), pevascular endothelial growth factor receptor 2 Y1175 (1:1000; catalog number 3770), p70-S6K (1:1000; catalog number 2708), rictor (1:1000; catalog number 2140), S6 (1:2000; catalog number 2217), snail (1:1000; catalog number 3879), SRC (1:1000; catalog number 2123), ZO-1 (1:1000; catalog number 8193), and b-catenin (1:1000; catalog number 8480) were purchased from Cell Signaling (Danvers, MA). Primary antibodies for liprin a-1 (1:1000; catalog number ab26192), liprin b-1 (1:1000; catalog number ab104117), and cavin-1 (1:1000; catalog number ab48824) were purchased from AbCam (Cambridge, UK). Primary antibodies for p-Adducin S662 (1:1000; catalog number 06-820) and mSin1 (1:1000; catalog number NBP1-89569) were purchased from EMD Millipore (Burlington, MA) and Novus (Centennial, CO), respectively. Actin antibody (1:5000; Sigma-Aldrich, St. Louis, MO) was routinely used as a loading control. Densitometry analysis was performed using ImageJ software version 1.48 (NIH, Bethesda, MD; http:// imagej.nih.gov/ij).

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Immunoprecipitation of Rictor Immunoprecipitation of rictor-containing mTOR complexes was performed as previously described with minor modification.10 T24 and J82 cells were lyzed for 30 minutes on ice with 0.3% CHAPS lysis buffer (40 mmol/L HEPES, pH 7.5, 120 mmol/L NaCl, and 1 mmol/L EDTA) containing protease and phosphatase inhibitors. After centrifugation at 16,000  g for 15 minutes, fresh whole cell lysates (1 mg) were incubated with 3 mg of rictor (catalog number 5379; Cell Signaling) or 3 mg of rabbit IgG isotype control Sepharose bead conjugates (catalog number 3423; Cell Signaling) for 2 hours with rotation at 4 C. Beads were then collected by centrifugation at 2000  g for 5 minutes and washed three times with lysis buffer. The captured immunoprecipitates were eluted by heating in 35 mL 2 Laemmli sample buffer containing 50 mmol/L 2-mercaptoethanol for 5 minutes at 95 C and then analyzed by immunoblot analysis. Co-immunoprecipitation of endogenous Cav-1 was detected with a monoclonal mouse antieCav-1 antibody (catalog number MAB5736; R&D Systems, Minneapolis, MN).

Cell Migration Assay Modified scratch-wound migration assays were performed as previously described.4

Phosphoprotein Enrichment and MS Phosphoproteomic analysis of cell lysates was performed as previously described.11 Cell lysates were prepared by resuspension for 1 hour in lysis buffer consisting of Tris hydrochloride (50 mmol/L, pH 7.4), NaCl (150 mmol/L), Triton X-100 (0.5% w/v), NP-40 (0.5% w/v), 80 mmol/L dithiothreitol, 10 mL/mL of protease inhibitor cocktails (Sigma-Aldrich), 1 mmol/L phenylmethylsulfonyl fluoride, Q12 1 mmol/L Na3VO4, and PhosSTOP phosphatase inhibitor cocktail (Roche, Mannheim, Germany), sonicated for 30 seconds, and centrifuged at 16,000  g for 10 minutes. The supernatants were precipitated with 4 volumes of acetone (Sigma-Aldrich) overnight at 20 C and centrifuged at 9000  g for 5 minutes. The pellets were dried by lyophylization (Heto, Dry Winner) for 2 hours. The cell pellets were resuspended in 200 mL of 8 mol/L urea, and protein concentrations were measured by Bradford Assay (Bio-Rad). Protein samples were then reduced with 10 mmol/L dithiothreitol for 30 minutes at 37 C and then alkylated by 50 mmol/L iodoacetamide for 20 minutes at room temperature. The concentrated urea in the sample was diluted to a final concentration of 2 mol/L, and the proteins were digested by trypsin at 37 C for 6 hours in a buffer containing ammonium bicarbonate (50 mmol/L, pH 9). The digestion mixture was then acidified by adding glacial acetic acid to a final concentration of 2% and desalted by SepPak C18 column (Waters Corp., Milford, MA).

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Phosphopeptides were enriched from the desalted 1-mg tryptic peptides using TiO2 column (200 mm  2 cm) packed in-house.11 A total of 100 fmol of standard phosphopeptide angiotensin II phosphate was added to the SepPak-cleaned sample. The sample was then mixed with an equal volume of loading buffer (200 mg/mL DHB, 5% trifluoroacetic acid, 80% acetonitrile), and loaded into the TiO2 column using the Pressure Cell (Brechbühler Inc., Schlieren, Switzerland) with flow rate of 3 mL/minute. The column was washed by 200 mL of Wash Buffer 1 (40 mg/ mL dihydroxybenzoic acid, 2% trifluoroacetic acid, 80% acetonitrile) and 2  200 mL of a second wash buffer 2 (2% trifluoroacetic acid, 50% acetonitrile) to remove nonphosphopeptides. Phosphopeptides were eluted from the column with the elution buffer (5% ammonia solution). Ammonia in the eluate was removed by lyophilization (approximately 3 minutes), and the sample was acidified by adding glacial acetic acid to a final concentration of 2% and desalted by ZipTip (EMD Millipore). The purified phosphopeptides were analyzed by highsensitive reversed-phase liquid chromatography coupled nanospray tandem MS using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). LTQ-Orbitrap provides high-accuracy mass measurement that is essential for the validation of modified peptide identification and the reduction of false-positive identification. The reversed-phase LC column was slurry-packed in house with 5 mm of 200 Å pore size C18 resin (Michrom BioResources, Inc., Auburn, CA) in a 100 mm i.d.  10-cm-long piece of fused silica capillary (Polymicro Technologies, Phoenix, AZ) with a laser-pulled tip. After packing, the new column, the highperformance liquid chromatography system (Surveyor MS Pump Plus from Thermo Fisher Scientific), and the LTQOrbitrap were tested by analyzing 100 fmol of Yeast Enolase Standard & Tryptic Digestion (catalog number PTD/00001/46; Michrom Bioresources, Inc.) to ensure that stable ESI, desired mass accuracy, peak resolution, peak intensity, and retention time could be obtained. Additional iteration was performed to ensure reproducibility. A total of 100 fmol of standard peptide angiotensin I was spiked into the sample as an internal standard. After sample injection, the column was washed for 5 minutes with mobile phase A (0.1% formic acid), and peptides were eluted using a linear gradient of 0% mobile phase B (0.1% formic acid, 80% acetonitrile) to 40% B in 120 minutes at 200 nL/minute then to 100% B in an additional 10 minutes. The highperformance liquid chromatography gradient was shallower than that of general proteomic analysis because phosphopeptides are relatively hydrophilic. Before and after analyzing one sample, the column was washed with highperformance liquid chromatography mobile phase B for 30 minutes, then mobile phase A for 20 minutes at a high flow rate (1 mL/minute) to reduce potential carryover. The LTQOrbitrap mass spectrometer was operated in a datadependent mode in which each full MS scan (60,000 resolving power) was followed by eight MS/MS scans

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where the eight most abundant molecular ions were dynamically selected and fragmented in by collisioninduced dissociation using a normalized collision energy of 35%. The fragmented ions were detected by LTQ. The dynamic exclusion time was 30 seconds, and the dynamic exclusion size was 200. The FT master scan preview mode, charge state screening, monoisotopic precursor selection, and charge state rejection were enabled so that only the 1þ, 2þ, and 3þ ions were selected and fragmented by collisioninduced dissociation. Tandem mass spectra collected by Xcalibur version 2.0.2 were searched against the National Center for Biotechnology Information human protein database (released in September 2009 with 37,391 entries) using Proteome Discoverer software version 2.1 (Thermo Fisher Scientific) with full tryptic cleavage constraints, static cysteine alkylation by iodoacetamide, variable methionine oxidation, and variable phosphorylation of Ser/Thr/Tyr. Mass tolerance for precursor ions was 5 ppm, and mass tolerance for fragment ions was 0.25 Da. The Proteome Discoverer search results were filtered by criteria Xcorr versus charge 1.5, 1.8, 2.5 for 1þ, 2þ, 3þ ions; ranked top #1; probability of randomized identification of peptide <0.1. Confident peptide identification were determined using these stringent filter criteria for database match scoring followed by manual evaluation of the results. The false discovery rate was estimated by searching a combined forward-reversed database as described by Elias and Gygi.12 The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD013473.13

Reverse-Phase Protein Microarray Analysis Cellular lysates were printed in triplicate onto nitrocellulosecoated slides (Grace Bio-labs, Bend, OR) using an Aushon 2470 arrayer (Aushon BioSystems, Billerica, MA). Before proceeding with immunostaining, each array was treated with Reblot antibody stripping solution (Chemicon, Temecula, CA) for 15 minutes and blocked in I-block solution (Tropix, Bedford, MA) for 1 hour to reduce nonspecific binding. Each array was probed with one primary antibody on an automatic Autostainer (Dako Cytomation, Carpinteria, CA) using the Catalyzed Signal Amplification System kit (Dako Cytomation). Antibody specificity was tested for single-band specificity and ligand induction via Western blot analysis. Fluorescent detection was achieved using the streptavidin-conjugated IRDye680 (LI-COR Biosciences, Lincoln, NE) according to the manufacturer’s instructions. The total amount of protein contained in each sample was measured by Sypro Ruby Protein Blot Stain (Molecular Probes, Eugene, OR) as previously described.14 Images were acquired using the PowerScanner (TECAN, Mönnedorf, Switzerland), and spot intensity values were quantified using MicroVigene software version 5.1.0.0 (VigeneTech, Carlisle, MA) as previously described.14 The

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Cav-1 Regulation in Bladder Cancer Cells RPPA raw data from J82 and T24 bladder cancer cells are available in Supplemental Tables S1 and S2, respectively.

Immunofluorescence Microscopy Immunofluorescence staining of bladder cancer cells grown on poly-L-lysine coated glass coverslips was performed using standard procedures. Cells were fixed with ice-cold methanol for 10 minutes and blocked with 10% normal goat serum (Gibco) in PBS with 0.1% Triton X-100 for 30 minutes at room temperature. Cav-1 was stained using an Alexa Fluor 488econjugated mouse antieCav-1 monoclonal antibody (1:10; catalog number IC5736G; R&D Systems) for 1 hour at room temperature. Coverslips were mounted onto glass slides using ProLong Gold Antifade mountant with DAPI for nuclear counterstaining (Molecular Probes). Images were obtained on a Leica DM IRE2 inverted fluorescent microscope (Buffalo Grove, IL) with a Hamamatsu ORCA-100 digital camera (Sewickley, PA) using SimplePCI software version 6 (Hamamatsu Corp.).

Electron Microscopy Preparation of cell lysates for transmission electron microscopy was performed using standard procedures. J82 cells were collected by trypsinization and fixed with Karnovsky’s fixative (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 mol/L PBS, pH 7.4) at 4 C for a minimum of 3 hours. After rinsing with PBS, cells were postfixed in 1.5% osmium tetroxide in PBS for 45 minutes. Cells were then washed with deionized water and dehydrated in ascending concentrations (35% to 100%) of ethanol for 15 minutes each. Samples were then immersed in propylene oxide, 1:1 propylene oxide, and epoxy resin mixture (Quetol 651 embedding kit; Electron Microscopy Sciences, Hatfield, PA), 2:3 propylene oxide and epoxy resin mixture, and then overnight at 75 C with pure epoxy resin mixture under constant agitation and vacuum to optimize specimen infiltration. Thin sections (60 nm) were then cut using an RMC MTX-L ultra-microtome (Boeckeler Instruments, Inc., Tucson, AZ) and mounted on thin-bar 200-mesh copper grids. The grids were then stained with methanolic uranyl acetate saturated solution followed by bismuth subnitrate staining for contrast enhancement. Samples were examined with a Zeiss EM 10 C transmission electron microscope (Oberkochen, Germany) and images captured using a GATAN model 785 digital camera (Pleasanton, CA).

Human Samples

Clinicopathologic Features

Feature Age at diagnosis, years Mean, median Range Sex, No./Total No. (%) Male Female Signs and/or symptoms, No./Total No. (%)* Hematuria Microhematuria Gross hematuria Obstruction Irritative bladdery Other (pain, weight loss) None Pathological T stage, No./Total No. (%) pT1 pT2 pT3 pT4 Tumor size, cm3 Mean, median Range

All patients 65.9, 64 43e84 39/53 (74) 14/53 (26) 30/53 2/53 28/53 3/53 12/53 4/53 15/53

(57) (3.8) (53) (5.7) (23) (7.5) (28)

1/53 13/53 32/53 7/53

(1.9) (25) (60) (13)

3.5, 2.9 0.5e8

*In many cases, patients reported multiple signs and/or symptoms. y Irritative bladder signs and symptoms include frequency, urgency, dysuria, nocturia, polyuria, and urinary tract infection.

Immunohistochemistry Immunohistochemical staining of sectioned human tissue with Cav-1 (catalog number 3267; Cell Signaling) was performed using procedures as previously described.6 Briefly, tissue sections from paraffin-embedded blocks were cut onto precoated slides, followed by deparaffinization, rehydration, and heat-induced antigen retrieval using sodium citrate buffer. Sections were then blocked with 10% normal goat serum in TBS for 1 hour at room temperature and then incubated overnight at 4 C with primary antibody against Cav-1 diluted in blocking buffer. After four washes in TBS, blocking of endogenous peroxidase was performed by incubation with 3% H2O2 in TBS for 10 minutes. For enzymatic detection, tissue was counterstained with speciesappropriate prediluted horseradish peroxidase polymer secondary antibody and chromagen developed with diaminobenzidine using a Rabbit/Mouse specific horseradish peroxidase/diaminobenzidine (ABC) Detection IHC Kit Q14 according to the manufacturer’s instructions (AbCam).

Ingenuity Pathway Analysis Analysis

Formalin-fixed, paraffin-embedded tumor blocks from 53 patients were curatively obtained following University of California, San Diego Institutional Review Board approval. Invasive disease (pT2þ) was enriched in this patient population. Clinicopathologic information for this patient ½T1 cohort is provided in Table 1.

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Table 1

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The Ingenuity Pathway Analysis program (Qiagen, Venlo, Netherlands) was used to analyze phosphoproteins that were elevated in the control or RICTOR knockdown group. P < 0.05 was used to select differentially expressed phosphoproteins. The shortlisted phosphoprotein names were then mapped to corresponding Entrez gene identification

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Hau et al Table 2 Phosphoproteins Identified by Mass Spectrometry Analysis Putatively Regulated by mTORC2 Protein name

Gene name

Increased target protein phosphorylation by mTORC2 BCL2-associated transcription factor 1 isoform1 Bifunctional coenzyme A synthase Liprin a-1* Multiple PDZ domain protein Myotubularin-related protein 14 Probable E3 ubiquitin-protein ligase HERC1 R3H domain-containing protein 2 Staphylococcal nuclease domain-containing protein 1 Talin-1* Telomeric repeat-binding factor 2einteracting protein 1 Decreased target protein phosphorylation by mTORC2 60S acidic ribosomal protein P0 AMFR* Coiled-coil domain containing 6 cAMP-dependent transcription factor ATF-2 Liprin b-1* Mediator of DNA damage checkpoint protein 1 P44/42 mitogen-activated protein kinase 1 Protein scribble homolog Solute carrier family 35, family C2 isoform b Transcription factor AP-1 Transcription factor ELYS Transmembrane protein 43

Results BCLAF1

MS-Based Analytics to Identify mTORC2 Targets COASY PPFIA1 MPDZ MTMR14 HERC1 R3HDM2 SND1 TLN1 TERF2IP

RPLP0 AMFR CCDC6 ATF2 PPFIBP1 MDC1 MAPK1 SCRIB SLC35C2 JUN AHCTF1 TMEM43

*Protein was validated by immunoblot analysis. All phosphoproteins were identified in at least two of three biological replicates. AMFR, autocrine motility factor receptor; mTORC2, mammalian target of rapamycin complex 2.

numbers. Both pathway and gene ontology enrichment analyses were conducted to identify candidates involved in some type of molecular and cellular component function as classified by the gene ontology nomenclature.

Data Mining The cBio Portal for Cancer Genomics web tool was used to analyze the provisional RNA sequencing data from The Cancer Genome Atlas (TCGA; https://portal.gdc.cancer. gov/projects/TCGA-BLCA, last accessed January 25, 2019) for bladder urothelial carcinoma. This study includes 408 tumor samples. All searches were performed according to the cBioPortal instructions.15,16

Statistical Analysis Tests for statistical significance were determined using the t-test with P < 0.05. Comparison between Kaplan-Meier survival curves was performed using the Mantel-Cox log

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rank test. All statistical analyses were performed using GraphPad Prism software version 6 (GraphPad Software, La Jolla, CA).

Pharmacologic inhibitors of mTOR, such as PP242, KU0063795, and Torin-2, have served as valuable tools to study various biological processes associated with phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling.17,18 However, these inhibitors affect both mTOR-containing complexes, making it difficult to distinguish mTORC1versus mTORC2-mediated effects. Accordingly, targeted gene silencing of RICTOR was performed to selectively ablate mTORC2 activity, which was confirmed by reduced AKT S473 phosphorylation. This approach does not alter mTORC1-dependent phosphorylation of downstream signaling effectors.4 A migration model was implemented whereupon serum stimulation after serum starvation was used to induce cell migration, and this was compared with nonmotile, serum-starved cells transfected with siNTC.4 MS was used as a phosphoproteomic discovery approach to capture global changes of phosphorylation events in these cell populations that were increased or decreased by the presence or absence of mTORC2. After downstream workflows, stringent filter criteria from MS analysis identified 212 and 199 unique peptides in control and rictorsilenced cells, respectively. Ten unique phosphoproteins in control siRNA-transfected cells and 12 unique phosphoproteins in rictor-silenced were identified by MS analysis in at least two of the three biological replicates (Table 2). ½T2 Ingenuity Pathway Analysis was used to perform functional enrichment of these proteins to determine the biological and/or signaling pathways affected by the presence or absence of mTORC2 activity. Ingenuity Pathway Analysis analysis revealed 26 and 22 significantly altered (P < 0.05) cellular functions up-regulated or downregulated by mTORC2 activity, respectively (Figure 1, A ½F1 and B). Among these, the top five categories up-regulated by mTORC2 activity were cellular assembly and organization, cell death and survival, cellular growth and proliferation, cellular function and maintenance, and gene expression. By contrast, the top five most overrepresented categories down-regulated by mTORC2 activity included cell death and survival, cellular growth and proliferation, cellular development, cell morphology, and cellular assembly and organization. Given the role of mTORC2 in regulating cytoskeletal remodeling, cell adhesion, and cell motility, the investigation was focused on the novel targets of mTORC2 for phosphoproteins involved in cellular assembly and organization, cell morphology, and cellular movement. Among these functions, mTORC2 increased phosphorylation of eight proteins and decreased phosphorylation of 32 proteins

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(Figure 2A and Table 3). One protein, nonmuscle myosin heavy chain 10, was identified in both categories. The STRING database was used to assemble a protein interaction network of the 39 commonly represented phosphoproteins.19 Thirty-six phosphoproteins formed one large distinct network with significantly enriched interactions (Figure 2B). Two of the top five gene ontology biological processes that were significantly enriched within this network included movement of cell or subcellular components (n Z 19/35; P Z 1.26  109) and regulation of cell motility (n Z 14/35; P Z 7.76  109).

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Toward the goal of identifying novel signaling effectors of mTORC2 activity, the differential protein and phosphoprotein changes that occur within the bladder cancer cell migration model were examined using RPPA analysis. This method serves as a complementary approach to MS and the global analysis of mTORC2 signaling perturbations in the rictor-silenced cell migration model. Lysates were probed with 150 antibodies that represented a diverse set of signaling pathways, biological functions, and protein classes, including PI3K/AKT/mTOR signaling, mitogenactivated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) signaling, Janus kinase (JAK)/STAT signaling, AMP-activated protein kinase (AMPK) signaling and energy sensing, PKC signaling, cell stress and immune

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807 808 809 810 811 812 813 814 815 Figure 1 Mass spectrometryebased ana- Q23 816 817 lytics to identify mammalian target of rapa818 mycin complex 2 (mTORC2) targets. Ingenuity 819 Pathway Analysis identified 26 and 22 signif820 icantly altered cellular functions putatively increased (A) or decreased (B) by mTORC2 821 activity, respectively. The values adjacent to 822 each bar indicate the number of phospho823 proteins represented in each respective 824 cellular function. See also Table 1. 825 826 827 828 829 830 831 832 833 834 835 836 837 response, focal adhesion and cell motility, cell cycle regu838 lation, cell death and apoptosis, translation and transcrip839 tion, tumor suppressors and oncogenes, and receptor 840 841 tyrosine kinases (RTKs) (Supplemental Figure S1). Rictor 842 silencing and consequent loss of mTORC2 activity 843 decreased AKT S473 phosphorylation, whereas downstream 844 mTORC1 targets (p-AKT T308, p-PRAS40 T246, p-S6 845 S235/236 and S240/244, p-p70-S6K S371, T389 and T412, 846 and p-4EBP1 S65 and T70) were unaffected in serum847 stimulated cells (Figure 3). ½F3 848 RPPA analysis also identified that mTORC2 can regulate 849 additional phosphoprotein targets across multiple signaling 850 pathways. Loss of mTORC2 activity resulted in activation 851 of cell death and apoptosis-signaling effectors, including 852 caspases 3, 6, 7, and 9, poly (ADP-ribose) polymerase, and Q17 853 854 increased phosphorylation of several members of the Bcl-2 855 family of apoptosis proteins, which confirmed previous 856 reports linking mTORC2 with apoptosis in leukemic, 857 breast, and nonesmall cell lung carcinoma cell line mod858 20e22 els. By contrast, reduced pathway activation of 859 numerous signaling effectors associated with the MAPK/ 860 ERK, JAK/STAT, AMPK, and PKC pathways, as well as 861 proteins involved in cell motility and focal adhesion 862 (p-adducin S662, p-cofilin S3, p-CrkII Y221, and p-SRC 863 Y416), cell cycle regulation (cyclins A and D1, p-Chk-1 864 865 S345, and p-Rb S780), and transcription and translation 866 (p-eIF2a S51, p-eIF4E S209, and p-FKHR S256 and T24) 867 was observed. 868

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Figure 2 Pathway and gene ontology analysis of mammalian target of rapamycin complex 2 (mTORC2)eregulated targets. A: Venn diagrams depicting the number of phosphoproteins represented among the indicated functions in control or rictor-silenced cells, respectively. B: Common unique phosphoproteins queried through the STRING database reveals one large protein interaction network. The STRING database assembles information about known and predicted protein interactions from numerous sources. Thicker lines represent stronger associations between proteins.

Validation of mTORC2 Effects on Discovery Phosphoprotein Targets To validate the findings from parallel MS and RPPA analysis, a large subset of proteins and phosphoproteins was selected to confirm the directional change in expression or phosphorylation status between siNTC or siRictortransfected bladder cancer cells through immunoblot analysis under nonmotile (serum starved) and motile (serum stimulated) conditions in J82 and T24 bladder cancer cells.23,24 The effects of mTORC2 signaling were confirmed on known targets to verify the specificity of rictor silencing

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in selectively reducing mTORC2 activity. Rictor silencing ablated mTORC2 activity as evidenced by reduced phosphorylation of AKT S473 (Figure 3A). In addition, mTORC1 phosphorylation targets were unaffected by rictor silencing, with no phosphorylation alterations evident in AKT T308, S6 235/236 and S240/244, p70-S6K T389, and 4EBP1 T37/46, consistent with data from the RPPA analysis and prior studies.4 Given the previously described role of mTORC2 in regulating bladder cancer cell migration and invasion, immunoblot validation was performed to confirm the effects Q18 of mTORC2-driven expression and phosphorylation changes of proteins known or proposed to be associated with these cellular processes. In some cases, discrepancies between RPPA and representative immunoblot signals may be the result of using different antibodies with different epitope specificities and/or interassay variation. Loss of rictor affected the expression and/or phosphorylation of proteins involved in cell motility and focal adhesion, many of which have not been previously associated with mTORC2 activity (Figure 3B). In rictor-ablated cells, increased expression or phosphorylation of the cell adhesion factors FAK Y397, Y576/577 and Y925, total FAK, Ncadherin, b-catenin, p-ezrin T567, total talin-1, and snail was observed in both serum-starved and serum-stimulated conditions when compared with NTC control cells. By contrast, expression of liprin b-1 and TJP1/ZO-1 and phosphorylation of cofilin S3 were increased in rictorsilenced cells in the absence of serum, suggesting differential effects of mTORC2 activity under motile versus nonmotile conditions. Loss of mTORC2 activity resulted in reduction of talin-1 S425 and adducin S662 phosphorylation and protein expression of liprin a-1 and autocrine motility factor receptor in both serum-starved and stimulated conditions. These immunoblot findings are consistent with results obtained by RPPA analysis and represent validation of novel signaling effectors and downstream mediators of mTORC2. A subset of these proteins (N-cadherin, b-catenin, snail, and TJP1/ZO-1) have been implicated in epithelial-mesenchymal transition and with increased metastatic potential in a number of tumor systems, suggesting programmatic changes in cell function may also be regulated by mTORC2.25e32 These findings were also validated in human T24 bladder cancer cells (Supplemental Figures S2 and S3).

Phosphorylation of Cav-1 and Modification of Caveolae-Associated Receptor Tyrosine Kinases Are Regulated by mTORC2 A major motivation of this study was to identify novel targets of mTORC2 activity in the context of active bladder cancer cell migration. The unbiased global view of phosphoprotein expression changes due to mTORC2 silencing afforded by MS complemented by the high analytical sensitivity of RPPA to measure phosphorylation statuses of

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Figure 3 Protein pathway activation mapping of mammalian target of rapamycin complex 2 (mTORC2) ablated J82 bladder cancer cells. Validation of mass spectrometry and reverse-phase protein array (RPPA) results by immunoblot analysis for proteins and phosphoproteins involved in phosphatidylinositol 3kinase, AKT, and mTOR signaling (A) and cell motility and focal adhesion (B). Selected proteins and phosphoproteins representing their respective signaling pathway or cellular processes from RPPA analysis are also shown by each heat map for side-by-side comparison. All immunoblots are representative of at least three independent experiments. See also Supplemental Figure S1. p-, phosphorylated; siNTC, nontargeting control siRNA.

proteins relevant to cell motility and focal adhesion would allow for discovery of proteins using our bladder cancer cell migration model. To this end, MS analysis identified Cav-1 (Table 3) as a candidate downstream target of mTORC2 and is functionally associated with pathways relevant to cell migration, including cellular assembly and organization, cell morphology, and cellular movement. Cav-1 belongs to a family of integral membrane proteins and is an essential component of small (50 to 100 nm) flask-shaped invaginations of the plasma membrane called caveolae.33 Aside from roles in caveolae formation and stability, Cav1 acts as a scaffolding protein within caveolae and noncaveolar subdomains of lipid rafts to cluster and regulate numerous signaling molecules, such as G proteins, integrins, SRC family tyrosine kinases, PI3Ks, and RTKs.33 On the basis of identification of an abundance of phosphorylated Cav-1 in rictor-expressing control cells using MS, upregulation of Cav-1 activity by mTORC2 was anticipated. In particular, two well-characterized phosphorylation sites, Y14 and S80, were considered as possible residues that are potentially phosphorylated after serum stimulation of

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siNTC-transfected control cells. Residue Y14 phosphorylation is essential for Cav-1 association with SH2domainecontaining adaptor proteins, such as Grb7, at focal adhesion sites that promote integrin-mediated cell migration.34,35 Phosphorylation of this residue is also required for caveolae-mediated endocytosis of cell surface signaling proteins. To test whether Cav-1 Y14 is differentially phosphorylated in rictor-expressing versus rictor-silenced cells, immunoblot analysis of Y14 phosphorylation was performed in response to serum stimulation. A rapid and robust up-regulation of Cav-1 Y14 phosphorylation was observed after the addition of serum in rictor-expressing J82 cells as evidenced by immunoblot and corresponding densitometry analyses (Figure 4, A and B). By contrast, mTORC2silenced cells showed constitutive phosphorylation of Y14 in both the presence and absence of serum, suggesting that rictor knockdown results in loss of dynamic regulation of Cav-1. Given the effect of mTORC2 silencing on Cav-1 phosphorylation, the expression of cavin-1, a necessary component for caveolae formation,36 was subsequently

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Hau et al Table 3 Common Phosphoproteins Represented Among Cellular Assembly and Organization, Cell Morphology, and Cellular Movement as Determined by IPA Analysis Protein name

Gene name

Represented in control cells Caveolin-1* Serine/threonine-protein kinase MRCK b Catenin (cadherin-associated protein), D1 Filamin C, g Huntingtin Myosin, heavy chain 10, nonmuscle Protein kinase C, D Vesicle-associated membrane protein 7 Represented in rictor-silenced cells Tyrosine-protein kinase ABL1 Abelson tyrosineeprotein kinase 2 a-actinin-4 A-kinase anchor protein 12 Rho GTPase-activating protein 5 Rho guanine nucleotide exchange factor 12 Girdin CD2-associated protein Dystonin Epidermal growth factor receptor* Abl interactor 1 ARF GTPase-activating protein GIT1 78-kDa glucose-regulated protein Heat shock protein b-1 Transcription factor AP-1 Keratin, type II cytoskeletal 8 Prelamin-A/C Mitogen-activated protein kinase 1 MARCKS-related protein Myosin, heavy chain 10, nonmuscle Neurofibromin Presenilin-1 Tyrosine-protein phosphatase nonreceptor type 12 Receptor-type tyrosine-protein phosphatase a* Rho-associated protein kinase 1 Septin-2 Vinexin Signal transducer and activator of transcription 3 SUN domain-containing protein 2 Tight junction protein ZO-1* Utrophin Vinculin

CAV1 CDC42BPB CTNND1 FLNC HTT MYH10 PRKCD VAMP7 ABL1 ABL2 ACTN4 AKAP12 ARHGAP5 ARHGEF12 CCDC88A CD2AP DST EGFR EPS8 GIT1 HSPA5 HSPB1 JUN KRT8 LMNA MAPK1 MARCKSL1 MYH10 NF1 PSEN1 PTPN12 PTPRA ROCK1 SEPT2 SORBS3 STAT3 SUN2 TJP1 UTRN VCL

*Denotes that protein was validated by immunoblot analysis. IPA, Ingenuity Pathway Analysis.

investigated and its expression found to be markedly upregulated on rictor silencing and serum stimulation. To verify the potential interaction of Cav-1 with mTORC2, mTORC2-associated proteins were immunoprecipitated from T24 and J82 bladder cancer cell lysates using a rictor antibodyeSepharose bead conjugate. Intact mTORC2 was confirmed based on co-immunoprecipitation

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of enriched amounts of mTOR and the mTORC2-specific component, mSIN1 (Figure 4C). In both cell lines, coimmunoprecipitation of Cav-1 was observed with rictor antibody, confirming an interaction between the mTORC2 complex and Cav-1. Growth factor RTKs coordinate numerous biological processes and are subjected to multiple levels of regulation, including attenuation of receptor autophosphorylation by protein tyrosine phosphatases, receptor sequestration and degradation, and up-regulation of inhibitory proteins that counteract downstream signaling effectors.37 Both mTORC1 and mTORC2 signaling complexes coordinate negative feedback signals to some growth factor RTKs, such as insulin and insulin-like growth factor 1 receptors, PDGFR-b, and ERBB/HER kinase receptors.38e42 Consistent with these reports, reduced phosphorylation status of Y1135/36 on insulin-like growth factor 1 receptor, Y751 and Y716 on PDGFR-b, and Y1248 on ERBB2 was detected in rictor-silenced J82 cells by RPPA and immunoblot analysis (Figure 4D and Supplemental Figure S1). Because Cav-1 functions as a scaffolding protein to regulate signal transduction and rictor silencing eliminates dynamic Cav-1 phosphorylation, we hypothesized that mTORC2 feedback may regulate RTKs through modulation of Cav-1 activity. The effect of Cav-1 silencing was therefore evaluated on the phosphorylation status of EGFR and ERBB2. No apparent differences in EGFR Y1173, ERBB2 Y877, and ERBB2 Y1248 phosphorylation and EGFR and ERBB2 expression were observed in Cav-1esilenced J82 cells compared with rictor silencing, suggesting that loss of mTORC2 activity may directly affect regulation of EGFR and ERBB2 through Cav-1 (Figure 4E). Lastly, the role of Cav-1 was evaluated on bladder cancer cell migration using a 24-hour modified scratch-wound migration assay. Cav-1 silencing had a small but significant (8%; P Z 0.01, two-tailed t-test) inhibitory effect on J82 cell migration (Figure 4F) in contrast to a modest and significant decrease (35%; P Z 0.0002, two-tailed t-test) in T24 cells (Figure 4G). These results are consistent with previous reports establishing a promigratory role of Cav-1 in bladder cancer cells.43e45

mTORC2 Can Induce Cav-1 Redistribution and Modify Caveolae Abundance Whereas tyrosine-14 phosphorylation of Cav-1 is linked to the regulation of cell signaling and caveolae-mediated endocytosis, serine-80 phosphorylation localizes Cav-1 to endoplasmic reticulum membranes.46 Because commercial antibodies are not currently available for immunoblot detection of p-Cav-1 S80 that would allow directly determining whether this residue is activated by mTORC2, immunofluorescence microscopy was used to determine the localization pattern of Cav-1 in response to mTORC2 ablation and serum stimulation. In both control and

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1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 Figure 4 Phosphorylation of caveolin-1 (Cav-1) and modification of caveolae-associated receptor tyrosine kinases are regulated by mTORC2. A: One-hour 1285 time-course serum stimulation of nontargeting control siRNA (siNTC)e or siRNA against RICTOR (siRictor)etransfected J82 cells to assess Cav-1 Y14 phos1286 phorylation and cavin-1 protein expression levels. Phosphorylated (p)-AKT S473 levels were evaluated to confirm down-regulation of mammalian target of 1287 rapamycin complex 2 (mTORC2) activity. B: Corresponding densitometry analysis of p-Cav-1 Y14 expression. C: J82 and T24 cells were subjected to immu1288 noprecipitation (IP) with an IgG isotype control or anti-rictor Sepharose bead conjugate, and immunoprecipitates were immunoblotted with the indicated antibodies. D and E: Co-IP of Cav-1 was observed with endogenous intact mTORC2. Immunoblot analyses of select growth factor receptors in response to rictor 1289 (D) or Cav-1 (E) silencing in J82 cells. All immunoblots are representative of at least three independent experiments. F and G: Representative micrographs and 1290 quantification of distance migrated of control (siNTC) and Cav-1esilenced J82 (F) and T24 (G) cells serum starved for 12 to 16 hours before serum stimulation 1291 to promote cell migration during 24 hours. Dashed lines indicate the migratory cell front at t Z 0 hours. Data are expressed as means  SE from three 1292 independent experiments (B) and means  SEM of 50 random measurements per field from three independent experiments (F and G). *P < 0.05, ***P < 0.001 1293 (two-tailed t-test). Scale bar Z 200 mm. EGFR, epidermal growth factor receptor; ERBB, --; IN, input; PDGFR, platelet-derived growth factor receptor; 1294 RPPA, reverse-phase protein array; SIN, ---; VEGFR, vascular endothelial growth factor receptor. 1295 1296 rictor-silenced cells, a punctate staining pattern of Alexa Together, these results suggest that the Y14 and not the 1297 Fluor 488elabeled Cav-1 was present throughout the cell S80 residue of Cav-1 may be modified by mTORC2 1298 1299 ½F5 (Figure 5A). However, in only control cells, Cav-1 was also signaling because a reticular staining pattern consistent with 1300 endoplasmic reticulum localization was not observed in localized to the edge and rear of cells in a manner consistent 1301 either experimental condition. with polarization of Cav-1 during cell migration.47 1302

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Figure 5 Mammalian target of rapamycin complex 2 (mTORC2) can induce caveolin-1 (Cav-1) redistribution and modify caveolae abundance. A: Nontargeting control siRNA (siNTC)e or siRNA against RICTOR (siRictor)etransfected J82 bladder cancer cells were grown on glass coverslips and stimulated with serum for 1 hour. Cells were then fixed for immunofluorescence staining of Cav-1. The overlay panels depict the merged images for Cav-1 (green) and DAPI (blue). BeG: Electron micrographs of siNTC (B, D and F) and rictor silenced (C, E and G) cells. Caveolae are detected as flask-shaped invaginations of the plasma membrane (PM) and fully invaginated vesicles in the cytosol. Dotted lines outline the PM. Arrows denote invaginating caveolae on the PM and arrowheads indicate vesicular caveolae. Scale bars: 200 nm (B and C), 1 mm (D and E), and 0.5 mm (F and G). Nuc, nucleus.

Because the data showed increased cavin-1 expression, modification of Y14 Cav-1 phosphorylation, and redistribution of Cav-1 with rictor silencing, we hypothesized that loss of mTORC2 activity might also alter the abundance of caveolae. Transmission electron microscopy was used to evaluate changes in caveolae abundance in mTORC2-ablated J82 cells. In both NTC and rictor siRNAetransfected cells, membrane-dense flask-shaped invaginations were observed

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on the plasma membrane and vesicular and fully-invaginated caveolae within the cell cytosol (Figure 5, B and C). The size of these vesicles (approximately 100 nm) was consistent with caveolae, as previously described.48 However, compared with rictor-expressing control cells (Figures 5, D and F), an increased abundance of caveolae was observed in rictorsilenced cells (Figures 5, E and G), suggesting that mTORC2 activity affects caveolae formation through

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1489 regulation of cavin-1 expression and/or Cav-1 Y14 1490 phosphorylation. 1491 1492 Association of Caveolin-1 with Overall Survival in 1493 Human Bladder Cancer 1494 1495 Having identified Cav-1 as a potential target of mTORC2, 1496 which can promote bladder cancer cell invasion,4 Cav-1 1497 expression was analyzed in a cohort of patients with uro1498 thelial carcinoma to determine its association with out1499 comes. Previous studies have found that increased Cav-1 1500 expression correlates with increasing bladder tumor grade 1501 1502 and stage,49,50 although there are limited reports of Cav-1 1503 association with outcomes. Cav-1 expression was assessed 1504 using immunohistochemistry in 53 primary high-grade 1505 bladder cancers that were associated with metastasis in a 1506 subset of cases. Cav-1 was expressed in the cell cytoplasm 1507 and/or cell membrane in 49 of 53 cases (92.5%) 1508 ½F6 (Figure 6A). The remaining four patients (7.5%) lacked 1509 detectable Cav-1 expression. Representative images with 1510 matching hematoxylin and eosin stains of Cav-1enegative 1511 and epositive specimens are shown in Figure 6, B and C. 1512 It was next tested whether Cav-1 expression can predict 1513 1514 patient outcomes. High Cav-1 expression was associated 1515 with a 5-year survival rate of 18.5% compared with a 46.1% 1516 5-year survival rate associated with low Cav-1 expression. 1517 The publicly available transcriptomics bladder cancer data 1518 from the provisional TCGA RNA sequencing data set were 1519 next examined. High and low CAV1 expression (n Z 101 1520 each) was defined as the top and bottom quartiles, respec1521 tively, among the patient cohort (n Z 408). A significant 1522 reduction in overall survival (P Z 0.006) was observed in 1523 patients with high CAV1 expression compared with low 1524 CAV1 expression, with median survival rates of 2.0 and 7.2 1525 1526 years, respectively (Figure 6D). 1527 1528 Discussion 1529 1530 mTOR plays a central role in cellular growth and prolifer1531 ation, metabolism, and cell motility through two distinct 1532 multiprotein complexes, mTORC1 and mTORC2. Given its 1533 significance in maintaining cellular homeostasis, it is fitting 1534 1535 that dysregulation of mTOR activity is associated with 1536 myriad malignancies, including bladder cancer. Although 1537 much is known about the regulation and functions of 1538 mTORC1, studies that define mTORC2 activity are more 1539 limited. Our laboratory has previously described a critical 1540 role for mTORC2 in promoting bladder cancer migration 1541 and invasion, with downstream effects on RhoA and Rac1 1542 likely responsible in part for mediating this response.4 To 1543 identify additional potential targets of mTORC2 signaling 1544 during bladder cancer motility, MS- and RPPA-based pro1545 teomic analysis was used concomitantly. Diverse categories 1546 1547 of phosphoproteins pivotal in a wide range of cellular pro1548 cesses were identified, including those involved in cell 1549 motility, such as autocrine motility factor receptor; 1550

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Figure 6

Caveolin-1 (Cav-1) is expressed in high-grade invasive bladder cancer. Immunohistochemical staining was performed with an antieCav-1 monoclonal antibody. A: Cav-1 positivity is primarily observed as diffuse cytoplasmic staining though membranous staining is present in some cases. B and C: Negative Cav-1 tumor immunoreactivity with matching hematoxylin and eosin (H&E) staining (B) in contrast to positive tumor expression (C). D: Kaplan-Meier analysis showing association between CAV1 mRNA expression and overall survival based on the provisional data set from The Cancer Genome Atlas on bladder cancer. The top and bottom quartiles of CAV1 expression among all patients were defined as high and low CAV1 expressors, respectively. A significant difference in overall survival is observed between high and low CAV1 expression. P Z 0.006.

cytoskeletal arrangement, such as adducin and cofilin; and cell and focal adhesion, including FAK, SRC, N-cadherin, b-catenin, ezrin, talin-1, liprin a-1, liprin b-1, TJP1/ZO-1, and snail. Cav-1 was also identified as a novel

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Hau et al downstream target of mTORC2, with mTORC2 regulation of Cav-1 phosphorylation and localization contributing to altered expression of known cancer-associated RTKs, such as EGFR and focal adhesion complexes, which were also identified through our screen. Cav-1 has diverse functions that can positively or negatively regulate cell signaling pathways. For example, both EGFR and the insulin receptor can be negatively and positively activated by Cav-1 expression.51e53 The differential regulatory roles of Cav-1 on cell surface receptors underscore the complexity of Cav-1 involvement in disease and biological processes as additionally evidenced by the variable effects on bladder cancer cell migration observed when Cav-1 is silenced. Cav-1 was first described as a direct substrate of SRC kinase that localizes to caveolae.54 Subsequent studies also revealed the localization of Cav-1 to noncaveolar lipid rafts at or near focal adhesion sites where Y14-phosphorylated Cav-1 regulates focal adhesion complex dynamics to control directional migration.34 The identification of an enrichment of phosphorylated Cav-1 in the rictor-expressing control bladder cancer cell model by MS suggested that mTORC2 could regulate Cav-1 activity through phosphorylation of the Y14 and/or S80 residues. Co-immunoprecipitation of Cav-1 was observed with mTORC2, and immunoblot analysis showed that Cav-1 phosphorylation on Y14 was robustly up-regulated after serum-stimulation in rictor-expressing control cells and could be ablated when rictor was silenced. However, this does not exclude the possibility of an indirect interaction between mTORC2 and Cav-1. To further support the hypothesis that mTORC2 regulates Cav-1, the subcellular distribution pattern of Cav-1 was analyzed in cells with or without rictor silencing after serum stimulation to promote cell motility. In serum-stimulated control cells, Cav-1 was frequently polarized to the membrane and trailing edge of cells. By contrast, in rictorsilenced cells, Cav-1 staining appeared punctate within the cell cytosol with no apparent evidence of endoplasmic reticulum localization indicative of S80 phosphorylation.46,47 Therefore, the observed difference in Cav-1 polarization is likely the result of Y14 rather than S80 phosphorylation. However, this assessment of mTORC2 regulation of S80 phosphorylation is limited because of the lack of a commercial antibody to this site. Given the observed localization of Cav-1 to the plasma membrane under motile conditions in control cells and the mTORC2-driven cavin-1 expression change identified with rictor silencing, whether mTORC2 could regulate formation or abundance of caveolae in this model system was tested. Rictor silencing resulted in increased caveolae formation seen by transmission electron microscopy. On the basis of these data, mTORC2 appears to be an upstream mediator of Cav-1 Y14 phosphorylation and may be responsible for regulating caveolae formation and abundance. Because caveolae can cluster and enhance the signaling of numerous RTKs within the cell, mTORC2 may also have a role in mediating bladder cancer caveolar-

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associated RTK protein expression and/or stability, including EGFR and ERBB2. Cell migration is a complex process that requires spatial and temporal regulation of numerous signaling components at integrin-mediated adhesion sites. These adhesions dynamically form and turn over at the leading edge of a cell to generate traction forces while they disassemble at the rear to produce rear-end retraction and detachment for cell body translocation. This highly regulated process is understood to be primarily mediated by FAK and SRC tyrosine kinases that signal to numerous downstream effectors, including Cav-1 and the Rho family GTPases Rac1 and RhoA, the latter of which was previously found to be regulated by mTORC2 in bladder cancer cells.4,55 Under serum-free conditions in which cells do not migrate, Y14 of Cav-1 was not phosphorylated in control cells but was markedly phosphorylated on serum stimulation. Because mTORC2related expression changes in focal adhesion complex proteins were seen, whether mTORC2 could spatially regulate Cav-1 localization to cell adhesions to prevent focal adhesion turnover, which thereby maintains a resting (nonmotile) state, was examined. This theory is supported by findings reported by Beardsley et al47 that suggest that loss of Cav-1 polarity in stationary cells and targeted silencing of Cav-1 impede directional movement. The authors also found that immunofluorescence staining for Cav-1 in stationary cells appeared punctate throughout the cell, a similar result observed in rictor-silenced, serum-stimulated bladder cancer cells. Prior studies have failed to demonstrate an association between Cav-1 expression and overall survival for patients with bladder cancer,49,50 although the patient numbers were limited. Public gene expression data from the provisional TCGA bladder cancer study that includes a large patient data set were therefore examined. A highly significant association was found between high CAV1 expression and reduced median and overall survival. In summary, two parallel phosphoproteomics approaches were used to identify protein and phosphoprotein targets of mTORC2, including regulators of cell motility, cell morphology, cellular assembly and organization, and several other functions that have not been previously linked with mTORC2 activity. mTORC2 was found to dynamically regulate Cav-1 Y14 phosphorylation, alter Cav-1 localization, and mediate caveolae formation. Although known to regulate some components of focal adhesion complexes, additional cell adhesion and cell motility proteins were also identified as potential targets of mTORC2, and these proteins may be regulated in conjunction with Cav-1. Specifically, the effects on the activities of FAK and SRC on rictor silencing would suggest a functional association among these kinases and mTORC2. Indeed, a link between mTORC2 activation and Fyn, a SRC family kinase, in cooperation with FAK was previously described during focal adhesion signaling that defines marrow-derived mesenchymal stem cell fate and cytoskeletal structure

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Cav-1 Regulation in Bladder Cancer Cells emanating from mechanical stimuli, such as cell stress or strain.56,57 However, it is unclear whether the same association exists during growth factoreinduced activation of mTORC2. Future studies that dissect the role of mTORC2 in the regulation of the caveolar microenvironment are important to determine effects on RTK signaling in the context of cell motility and stasis and the regulation of additional proteins associated with focal adhesions.

Acknowledgments We thank Dr. Henry C. Powell (University of California, San Diego) for his assistance and expert advice for the electron microscopy experiments, Drs. David M. Sabatini (Massachusetts Institute of Technology) and Dudley W. Lamming (University of Wisconsin) for technical protocols for rictor co-immunoprecipitation, and Dr. Kun-Liang Guan (University of California, San Diego) for critically reviewing the manuscript. A.M.H., S.G., S.M., E.F.P., and D.E.H. designed Q20 Q21 research; A.M.H., S.G., M.Z.L., K.N., J.M., W.Z., A.H., and J.W. performed research; B.C., K.B., and S.R. analyzed data; A.M.H, K.B., S.R, S.M., E.F.P., and D.E.H. reviewed and/or wrote the article; and D.E.H. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Supplemental Data Supplemental material for this article can be found at https://doi.org/10.1016/j.ajpath.2019.05.010.

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