RICTOR Amplification Promotes NSCLC Cell Proliferation Through Formation and Activation of Mtorc2 at the Expense of Mtorc1

Published OnlineFirst August 14, 2020; DOI: 10.1158/1541-7786.MCR-20-0262

MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS

RICTOR Ampliﬁcation Promotes NSCLC Cell Proliferation through Formation and Activation of mTORC2 at the Expense of mTORC1 Laura C. Kim1, Christopher H. Rhee2, and Jin Chen1,3,4,5,6

ABSTRACT ◥ Non–small cell lung cancer (NSCLC) is characterized by genomic mTORC1. Additionally, Rictor overexpression increases the pro- alterations, yet a targetable mutation has not been discovered in liferation and growth of NSCLC 3D cultures and tumors in vivo. nearly half of all patients. Recent studies have identified amplifi- Conversely, knockout of RICTOR leads to decreased mTORC2 cation of RICTOR, an mTORC2-specific cofactor, as a novel formation and activity, but increased mTORC1 function. Because actionable target in NSCLC. mTORC2 is one of two distinct mTOR Rictor has mTOR-dependent and -independent functions, we also complexes to sense environmental cues and regulate a variety of knocked out mLST8, a shared mTOR cofactor but is specifically cellular processes, including cell growth, proliferation, and metab- required for mTORC2 function. Inducible loss of mLST8 in RIC- olism, all of which promote tumorigenesis when aberrantly regu- TOR-amplified NSCLC cells inhibited mTORC2 integrity and lated. Interestingly, other components of mTORC2 are not coam- signaling, tumor cell proliferation, and tumor growth. Collectively, plified with RICTOR in human lung cancer, raising the question as these data identify a mechanism for Rictor-driven tumor progres- to whether RICTOR amplification-induced changes are dependent sion and provide further rationale for the development of an on mTORC2 function. To model RICTOR amplification, we over- mTORC2-specific inhibitor. expressed Rictor using the Cas9 Synergistic Activation Mediator system. Overexpression of Rictor increased mTORC2 integrity and Implications: RICTOR amplification drives NSCLC proliferation signaling, but at the expense of mTORC1, suggesting that over- through formation of mTORC2, suggesting mTORC2-specific expressed Rictor recruits common components away from inhibition could be a beneficial therapeutic option.

Introduction The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that acts in two distinct complexes, rapamycin-sensitive Lung cancer is the leading cause of cancer-related deaths worldwide, mTORC1 and rapamycin-insensitive mTORC2. Both complexes share despite significant advances in therapeutic options for these patients. the mTOR kinase and scaffolding protein mLST8, whereas mTORC1 Lung cancer is divided into two subtypes, non–small cell lung cancer contains scaffolding protein Raptor and mTORC2 contains Raptor- (NSCLC) and small cell lung cancer (SCLC), which account for analogous scaffolding protein Rictor and regulatory component approximately 85% and 15% of cases, respectively. NSCLC is a disease Sin1 (6, 7). Growth factors, amino acids, and cellular energy activate typically characterized by its genomic alterations, although nearly half mTORC1, which has well-characterized functions including regula- of all patients lack a known targetable alteration (1). Recent advances tion of cell growth, protein translation, metabolism, and autop- in tumor sequencing technologies have identified amplification of hagy (6, 7). Although less well understood, mTORC2 is activated by mTORC2-specific component RICTOR as potential targetable alter- growth factors and the PI3K pathway, primarily through binding of ation in several types of cancer including NSCLC, SCLC, breast cancer, PIP3 in the plasma membrane via the PH domain of cofactor and gastric cancer (2–5). Sin1 (8). Once localized to the membrane, mTORC2 can phos- phorylate (S473) and activate its downstream effector AKT (9), also situated at the membrane. In addition to AKT, mTORC2 targets include PKCa and SGK (10, 11). mTORC2 and its downstream 1Program in Cancer Biology, Vanderbilt University, Nashville, Tennessee. effectors act together to regulate cell proliferation, metabolism, and 2Vanderbilt University, Nashville, Tennessee. 3Division of Rheumatology and cytoskeletal organization (6, 7). Immunology, Department of Medicine, Vanderbilt University Medical Center, Aberrant signaling of both mTOR complexes has been implicated 4 Nashville, Tennessee. Vanderbilt-Ingram Cancer Center, Vanderbilt University in many types of cancers, although most studies have focused on 5 Medical Center, Nashville, Tennessee. Veterans Affairs Medical Center, mTORC1. mTORC2’s role in cancer has also been reported; for Tennessee Valley Healthcare System, Nashville, Tennessee. 6Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee. example, mTORC2 is required for tumor formation in PTEN-null prostate cancer (12, 13) and drives tumor progression and resis- Note: Supplementary data for this article are available at Molecular Cancer tance in HER2-positive breast cancer (3, 14). Additionally, the Research Online (http://mcr.aacrjournals.org/). recent identification of RICTOR amplification as a potential action- Corresponding Author: Jin Chen, Vanderbilt University Medical Center, able target in several cancer types suggests an additional subtype of T-3207D, Medical Center North, Vanderbilt University School of Medicine, cancer may rely on mTORC2 signaling for tumorigenesis (2–5). 1161 21st Avenue South, Nashville, TN 37232. Phone: 615-343-3819; Fax: 615- fi 343-8648; E-mail: [email protected] However, the mechanism by which ampli cation of a single scaffolding component within mTORC2 drives its oncogenic function Mol Cancer Res 2020;XX:XX–XX remains unknown. doi: 10.1158/1541-7786.MCR-20-0262 In this report, we investigate the roles of RICTOR amplification as a 2020 American Association for Cancer Research. driver of mTORC2 formation and promoter of NSCLC tumor growth.

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Using the Cas9 Synergistic Activation Mediator (SAM) system (15) to Use Committee and followed all state and federal rules and increase expression of Rictor or the CRISPR-Cas9 system to knock out regulations. Rictor, we show that alterations in Rictor lead to corresponding changes in not only mTORC2 formation, but also mTORC1. Further, Western blot and coimmunoprecipitation overexpression of Rictor in NSCLC cells leads to increased growth of Cell lysates for Western blotting only were collected in RIPA buffer. 3D cultures and in vivo tumor xenografts. We also found that RICTOR- Tumor lysates were collected in Triton X lysis buffer (1% Triton X-100, amplified NSCLC is sensitive to loss of mLST8, an mTOR cofactor 0.5 mmol/L EDTA, 50 mmol/L Tris-Cl). All lysis buffers were sup- specifically required for mTORC2 function (13), both in vitro and plemented with protease inhibitors and phosphatase inhibitors (Com- in vivo. Collectively, these data show that Rictor promotes NSCLC plete Mini and PhosStop inhibitor cocktail, Roche). Protein concen- through mTORC2 formation and could potentially be targeted with an tration was determined by Pierce BCA Protein Assay kit, and equal mTORC2-specific inhibitor. amounts of protein extracts were mixed with 4 Laemmli sample buffer and separated by electrophoresis on an SDS-PAGE gel, and then transferred onto nitrocellulose membranes. Membranes were blocked Materials and Methods with 5% milk in TBST buffer and incubated with corresponding Cell lines and cell culture primary antibodies and IRdye-conjugated or HRP-conjugated sec- H1975, H358, H23, and H1703 cells were obtained from ATCC ondary antibodies. Immunoreactivity was detected using the Odyssey and maintained in RPMI-1640 media containing 10% fetal bovine scanner (Li-cor Biosciences) or enhanced chemiluminescence. To serum (FBS) and 1% penicillin/streptomycin. BEAS-2B and H460 perform immunoprecipitation, equal amounts of input lysates (500 cells were obtained from the Vanderbilt-Ingram Cancer Center mg) collected in CHAPS buffer (40 mmol/L Tris, pH 7.5, 120 mmol/L Core Facility and maintained in the same conditions. 293T cells NaCl, 1 mmol/L EDTA, 0.3% CHAPS) were incubated with the were obtained from ATCC and maintained in DMEM containing primary antibodies (1–2 mg) for 2 hours to overnight at 4C. Protein 10% FBS. All cells were cultured in a humidified incubator with 5% G Dynabeads (Thermo Fisher Scientific #10-003-D) were added and CO2 at 37 C. Cell lines were used between passages 1 and 50 lysates were incubated for 1 hour and washed four times with CHAPS after thaw. Cell lines from ATCC were authenticated using short lysis buffer. Immunoprecipitate and whole-cell lysates were then tandem repeat profiling. Mycoplasma testing was performed every subjected to Western blot analysis. All antibodies are listed in Sup- 6 months, most recently in November 2019, using the PlasmoTest plementary Table S2. Quantification of Western blots was performed kit (Invitrogen). using ImageJ software.

Plasmids and sgRNA sequences Proximity ligation assay CRISPR-Cas9 backbone vectors were obtained from Addgene, and Cells cultured on glass coverslips were fixed with 4% PFA, permea- guide RNA sequences were cloned into the vector according to bilized with 1% Triton X-100 in PBS, and stained with the Duolink depositor’s instructions. Plasmids and guide RNA primers are listed (Sigma-Aldrich #92102) proximity ligation assay according to the in Supplementary Table S1. manufacturer’s protocol using antibodies listed in Supplementary Table S2 and counterstained and mounted with DAPI (Thermo Lentivirus production and transduction Scientific #P36941). Proximity ligation assay (PLA) puncta and CRISPR SAM, knockout, and inducible Cas9 cell lines were estab- DAPI-stained nuclei were enumerated using ImageJ software. lished using the lentiviral delivery system. Briefly, lentiviruses were packaged in HEK293T cells by transfecting cells with CRISPR or Cell growth assays expression plasmids together with psPAX2 (lentiviral packaging) and Cell growth was measured by MTT, colony formation, and BrdUrd pCMV-VSV-G (envelope) plasmids at a 1:1:1 molar ratio using the assays. For MTT assays, 2,000 cells were plated into each well of a 96- Lipofectamine 2000 Reagent. Media were changed after 16 hours of well plate in 100 mL of complete growth medium. Cell viability was transfection, and virus was collected after 24 to 48 hours. Indicated measured by incubating cells with 20 mLof5mg/mL Tetrazolium salt 3- cells were transduced with 1:1 virus and complete growth media with (4,5-dimethylthiazol-2-yL)-2,5-diphenyltetrazolium bromide (MTT, polybrene (8 mg/mL) for 24 hours and selected with puromycin (1– Sigma-Aldrich) and quantified by reading absorbance at 590 nm after 2 mg/mL), blasticidin (10 mg/mL), or hygromycin (40–50 mg/mL) for at resuspending in MTT solvent. For colony formation assay, 500 cells least 48 hours to establish stable cell lines before being used for were plated in complete growth medium in each well of a 12-well plate. experiments. Cells were grown for 10 to 14 days, and the media were changed every 3 days. Colonies were stained with 0.5% crystal violet in methanol and Xenograft assay colony area was quantified. For the BrdUrd assay, cells were grown on 2.5 106 cells suspended in 100 mL of Matrigel and PBS (1:1) were glass coverslips for 24 hours, then incubated with BrdUrd (10 ng/mL) injected into the hind flanks of 6-week-old athymic nude (Foxn1nu; for 30 minutes. Cells were fixed in 4% paraformaldehyde for 15 Envigo) or Rag1 / C57BL/6J mice. For xenograft experiments using minutes and then permeabilized with 1% Triton X-100 in PBS for 5 an inducible Cas9, doxycycline feed (Envigo #TD.00426) was given for minutes. DNA was denatured by incubation with 2N HCl for 30 10 days. Tumor measurements were started 10 days after injection and minutes at 37C, followed by rinsing in PBS. Cells were blocked for measured for 30 to 60 days after injection. Tumors were measured 1 hour using 2.5% goat serum in PBS and then probed with Alexa 647- every 2 to 3 days with digital calipers, and tumor volume was calculated conjugated anti-BrdUrd antibody (1:50, Invitrogen) overnight at 4C. according to the formula (V ¼ 4/3p(l/2)(h/2)(w/2)). Data are Coverslips were mounted onto slides using ProLong Gold antifade presented as mean SEM. Two-way ANOVA with Bonferroni reagent with DAPI (Thermo Scientific #P36941). 40 images were correction was used for statistical analysis. Experiments with mice quantified by counting BrdUrd-positive nuclei/total nuclei using were pre-approved by the Vanderbilt Institutional Animal Care and ImageJ software.

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3D cultures correlate with copy-number values (Fig. 1F). In all lines, SAM Rictor 60 mL of Matrigel (Corning #354230) was added to each well of cells exhibited increased mRNA (Fig. 1G) and protein (Fig. 1H) levels an 8-well chamber slide (Thermo Scientific) and incubated at 37C of Rictor compared with SAM empty vector (EV) control cells. Across to solidify. Cells (5,000) were suspended in 400 mLofcomplete all cell lines analyzed, RICTOR mRNA was upregulated approximately media with 2.5% Matrigel and 5 ng/mL of EGF then added to the 2- to 4-fold with the CRISPR SAM system ( Fig. 1G). Analysis of chamber slides. Cultures were monitored for 20 days, and media mRNA expression in RICTOR-amplified samples compared with were replaced every 4 days. Area of cultures was quantified using diploid samples of TCGA lung adenocarcinoma samples (Fig. 1C) ImageJ software. showed an approximately 2.25-fold increase in Rictor expression. This correlates well with the expression changes in Rictor when using IHC staining the CRISPR SAM system in Fig. 1G, suggesting that SAM-induced Xenograft tumor sections were paraffin-embedded and sectioned. Rictor expression is comparable to endogenous Rictor levels in human Rehydrated paraffin sections were subjected to antigen retrieval lung cancer patients. (Retrievagen A, BD Pharmingen), and endogenous peroxidases were blocked by 3% H2O2 for 30 minutes. Sections were blocked in 2.5% Alterations in Rictor expression lead to corresponding changes goat serum in PBS and stained with primary antibody and biotinylated in mTORC2 and mTORC1 secondary antibody, followed by avidin–peroxidase reagent and DAB. We hypothesized that amplification of RICTOR could promote Antibodies are listed in Supplementary Table S2. Sections were then tumorigenesis by driving formation of mTORC2. To test this hypoth- counterstained with hematoxylin and mounted with Cytoseal-XYL esis, we used the PLA to quantitate the interactions between mTOR (Thermo Scientific Richard-Allan Scientific). and either Rictor (mTORC2) or Raptor (mTORC1). The same rabbit antibody against mTOR was used in all PLA experiments, whereas TCGA data analysis mouse anti-Rictor or anti-Raptor were used to differentiate between Alteration frequency and copy-number segmentation plots and the two complexes (Supplementary Table S2). Compared with SAM analysis were generated using the online platform cbioportal.org. EV cells, SAM Rictor cells exhibited increased fluorescent foci indi- Proportion of copy-number gain/loss was analyzed using the GenVisR cating an increase in mTOR–Rictor interactions (Fig. 2A), thereby package (16). suggesting an increase in the formation of mTORC2. In contrast, mTOR–Raptor interactions were decreased in Rictor-overexpressing cells, consistent with a previous report showing an increase in mTOR– Results Rictor precipitation when mTORC1 was inhibited (18). To comple- RICTOR is amplified and overexpressed in NSCLC ment the PLA studies, we also immunoprecipitated for mTOR and Amplification of RICTOR has been identified in several different found that Rictor and Sin1 binding to mTOR was increased in SAM types of cancers, including breast cancer, gastric cancer, SCLC, and Rictor cells compared with control, whereas binding of Raptor to NSCLC (2–5). Analysis of the TCGA PanCancer Atlas identified mTOR was reduced (Fig. 2B). Furthermore, this Rictor-driven RICTOR amplification in 12% of total NSCLC cases, and 15% of increase in mTORC2 formation increased downstream phosphoryla- squamous cell carcinoma and 11% of adenocarcinoma cases, with tion of AKT (S473) in Rictor-overexpressing cells compared with amplification being the most frequent alteration of RICTOR (Fig. 1A). control (Fig. 2C), suggesting the increased mTORC2 was indeed RICTOR is located on the 5p chromosome, a site of frequent copy- functional. number gain in NSCLC (17). In addition to the overall 5p gain, copy- In order to assess the effect of Rictor overexpression on mTORC1 number segment and GISTIC analysis reveals a focal amplification of activity while excluding the effects of cross-talk between the mTOR the chromosomal locus surrounding the RICTOR gene (Supplemen- complexes, we stimulated SAM Rictor or EV cells with glutamine, a tary Fig. S1A and S1B). Interestingly, no other components of the known activator of mTORC1, but not mTORC2 (19). SAM Rictor cells mTOR complexes were coaltered with RICTOR amplification showed a reduction in phosphorylation of S6K1, a direct substrate of (Fig. 1B). Further analysis of adenocarcinoma cases, which accounts mTORC1, compared with SAM EV cells in response to glutamine for the majority of NSCLC cases, showed a positive correlation stimulation (Fig. 2D), confirming a reduction in mTORC1 activity between copy-number and mRNA expression of RICTOR, suggesting caused by a loss of mTORC1 upon Rictor overexpression. These data amplification does indeed lead to an increase in Rictor expression in suggest amplification of Rictor in NSCLC promotes the formation and NSCLC cases (Fig. 1C). activity of mTORC2 at the expense of mTORC1. In order to examine how amplification of a single mTOR complex To complement overexpression experiments, we used CRISPR- component might drive tumorigenesis, we used the Cas9 Synergistic Cas9–mediated genome editing (20–22) to target RICTOR for loss of Activation Mediator system (SAM) to upregulate expression of RIC- function in H23 cells, a cell line with verified RICTOR amplification TOR (15). This system utilizes an enzymatically inactive Cas9 (dCas9) (Fig. 1E; ref. 2). Rictor-deficient H23 cells showed a reduction in fused to VP64, coexpressed with MS2-P65-HSF1 activation helper mTOR–Rictor interactions and an increase in mTOR–Raptor inter- proteins. A small guide RNA (sgRNA) with two MS2 aptamers actions compared with sgLacZ control cells when assessed by PLA targeting the promoter region of the gene of interest is also expressed, ( Fig. 2E). Additionally, immunoprecipitation of mTOR in sgRictor such that all components of the system colocalize and activate tran- cells showed a loss of Rictor and Sin1 binding to mTOR, whereas scription of the targeted gene. Multiple sgRNAs targeting the promoter Raptor binding was increased (Fig. 2F). As expected with a loss of region of RICTOR (Fig. 1D) were tested in a panel of nontumorigenic, mTORC2, phosphorylation of AKT (S473) was reduced in H23 immortalized lung epithelial (BEAS-2B) and NSCLC (H1975, H358, sgRictor compared with sgLacZ control cells in response to serum H460, and H2030) cell lines, all of which have lower copy-number stimulation (Fig. 2G). Glutamine stimulation, however, increased the values compared with the H23 cell line (Fig. 1E), an NSCLC cell with a levels of p-S6RP (S235/236) in Rictor-deficient cells (Fig. 2H), sug- verified amplification of RICTOR (2). Western blot analysis of these gesting mTORC1 activity was increased. Together, these results dem- cell lines at baseline demonstrated Rictor protein levels that largely onstrate that changes in cellular Rictor alter the amount of mTORC2,

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Figure 1. CRISPR SAM system can be used to model RICTOR amplification in NSCLC. A–C, Patient data from the TCGA Pan-Lung Cancer Atlas were analyzed using the online platform at cbioportal.org. A, Alteration frequencies of RICTOR in NSCLC, squamous cell carcinoma, and adenocarcinoma. B, Alterations in mTOR complex components in patient-matched samples. C, Correlation of RICTOR mRNA levels with copy-number values in lung adenocarcinoma. Red, amplified samples based on GISTIC scoring. D–H, CRISPR SAM (15) was used to target the promoter region of Rictor 100 bp (R5) and 121 bp (R6) upstream of the transcription start site. D, A panel of immortalized lung epithelium and NSCLC cell lines. E, RICTOR copy-number values of utilized cell lines according to the CCLE database. F, Western blot analysis of Rictor protein levels in parental cell lines. mRNA relative to Actin (G) and protein levels of Rictor (H) were measured by qRT-PCR or Western blotting, respectively. Quantification of Rictor expression normalized to tubulin is indicated in numbers below the blots. EV, empty vector. , P < .05; , P < .01.

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Figure 2. Rictor alterations promote corresponding changes in mTORC2 and mTORC1. A, PLA were used to assess mTOR–Rictor or mTOR–Raptor interactions in SAM Rictor or EV H1975 cells. Data presented as mean SD. B, Interactions among components of mTOR complexes were measured by coimmunoprecipitation (co-IP) followed by Western blot analysis in SAM Rictor-overexpressing or EV control H1975 cells with the indicated antibodies. Densitometry quantifications of the co-IP relative to mTOR pulldown are displayed below as a fold change compared with SAM EV cells (n ¼ 3). C and D, SAM Rictor or SAM EV control H1975 or H358 whole-cell lysates were assessed by Western blot analysis using the indicated antibodies. Quantification of phospho/total AKT or S6K1 normalized to tubulin is plotted below. C, Cells were serum starved overnight and then stimulated with serum for 15 minutes. D, Cells were starved of glutamine overnight followed by stimulation with 4 mmol/L glutamine for 3 hours. E, PLAs were used to assess mTOR–Rictor or mTOR–Raptor interactions in sgLacZ control or sgRictor knockout H23 cells. Data, mean SD. F, Interactions among components of mTOR complexes were measured by coimmunoprecipitation followed by Western blot analysis in H23 sgRictor knockout or sgLacZ control cells with the indicated antibodies. Densitometry quantifications of the co-IP relative to mTOR pulldown are displayed below as a fold change compared with SAM EV cells (n ¼ 2). G and H, sgLacZ control or sgRictor knockout H23 whole-cell lysates were assessed by Western blot analysis using the indicated antibodies. Quantification of phospho/total AKT or S6RP normalized to tubulin is plotted below. G, Cells were serum starved overnight followed by stimulation with serum for 15 minutes. H, Cells were starved of glutamine overnight followed by stimulation with 4 mmol/L glutamine for 3 hours. , P < 0.05; , P < 0.0001.

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consequently increasing or decreasing the amount of mTORC1 in the Cas9-mediated LOF of mLST8, although continuous treatment was opposite direction. required for sustained expression of Cas9 (Fig. 4D). Inducible knockout of mLST8 in H23 resulted in a reduction in colony formation after a Rictor promotes proliferation of NSCLC cells single dose of doxycycline in sgMLST8 cells compared with sgLacZ mTORC2 is a known regulator of cell proliferation and promoter of (Fig. 4E). tumorigenesis (6, 7). However, we found that Rictor overexpression For xenograft studies, H23 cells expressing a doxycycline-inducible did not alter cell number when grown in 2D (data not shown). To Cas9 and either sgLacZ control or sgMLST8 were injected into the hind determine if Rictor-driven mTORC2 provides a growth advantage to flanks of athymic nude mice. Doxycycline was given for 10 days to NSCLC cells, control or Rictor-overexpressing H1975 or H358 cells induce Cas9 expression and mLST8 LOF and tumor growth was were grown as Matrigel-embedded 3D cultures for 20 days. Imaging of measured every 2 to 4 days for 2 months. At the end of the study, cultures on the final day showed the area of SAM Rictor cultures was tumors were removed, weighed, and subjected to further analysis by significantly larger than SAM EV cultures (Fig. 3A), suggesting that IHC. Tumor volume and weight of sgMLST8 tumors were significantly Rictor overexpression promotes growth of NSCLC. reduced compared with sgLacZ (Fig. 4F and G). Western blot analysis Conversely, loss of Rictor in H23 RICTOR-amplified cells signif- of tumor lysates showed sustained reduction of mLST8 to varying icantly reduced the number of viable cells compared with control when degrees in tumors expressing sgMLST8 (Fig. 4H). Additionally, IHC measured by MTT assay (Fig. 3B). The reduction in the cell number of staining of Ki-67 was reduced in sgMLST8 tumors compared with Rictor-deficient cells was due to an inhibition of cell proliferation, as sgLacZ tumors (Fig. 4I), recapitulating the in vitro results of loss of cell judged by BrdUrd incorporation (Fig. 3C). These results are consistent proliferation caused by inhibition of mTORC2 activity. with the possibility that Rictor-driven mTORC2 formation promotes the cell proliferation of NSCLC tumor cells. Discussion Increased Rictor expression promotes NSCLC tumor growth Amplification of RICTOR has been identified as an actionable target in vivo in NSCLC, yet no other mTORC2 component is coamplified with Next, we investigated whether the growth advantage conferred by RICTOR in human lung cancer data sets (Fig. 1). Because Rictor was Rictor overexpression would result in faster growing tumors in vivo. shown to have mTOR-independent functions (24), the mechanism by SAM Rictor #5 or EV control H1975 cells were injected into the hind which amplification of RICTOR could drive oncogenesis has not been flanks of Rag1-null immunodeficient mice and tumor growth was clearly defined. We hypothesized that overexpression of Rictor would monitored for approximately 1 month. Rictor overexpression signif- suppress Raptor–mTOR interactions and increase Rictor–mTOR icantly increased tumor volume compared with control in the xeno- interactions, driving activity of mTORC2, a known activator of AKT graft model (Fig. 3D). Tumors were harvested and weighed upon and cell proliferation. Here we show that overexpression of Rictor in removal. The mass of SAM Rictor tumors was significantly larger than NSCLC increases the amount of mTORC2 at the expense of mTORC1, control tumors (Fig. 3E). Western blot analysis of tumor lysates thereby increasing mTORC2 downstream signaling and facilitating showed that SAM R5 tumors retained the increased expression of tumor cell proliferation. Rictor throughout the course of tumor development and exhibited Aberrant mTORC2 signaling has been identified as an important increased levels of p-AKT, a readout of mTORC2 activity (Fig. 3F). mediator of tumorigenesis in the context of other cancer-causing Furthermore, SAM Rictor tumors had an increase in Ki-67 staining mutations such as loss of PTEN or activation of PI3K (12, 25). (Fig. 3G), consistent with studies showing mTORC2 activity drives cell Amplification of RICTOR in NSCLC also co-occurs with common proliferation. oncogenic driver mutations, including KRAS and EGFR (2). In this study, we performed all experiments with both KRAS (H358 and H23) Targeting mTORC2 for loss of function inhibits tumor growth of and EGFR (H1975)-mutant cell lines. In H358 or H1975 cells har- RICTOR-amplified NSCLC boring these mutations, overexpression of Rictor increased 3D culture Increased proliferation of Rictor-overexpressing tumors suggested growth, yet 3D cultures of immortalized, nontumorigenic lung epi- that mTORC2 inhibition might be beneficial for treatment of RIC- thelial cells (BEAS2B) were unchanged in response to overexpression TOR-amplified tumors. However, an mTORC2-specific small mole- of Rictor (data not shown). Conversely, in H23 RICTOR-amplified and cule is not currently available. A previous study from our lab identified KRAS-mutant NSCLC, loss of mTORC2 inhibited tumor growth, mLST8, a cofactor associated with both complexes, to be selectively suggesting that mTORC2 activity can promote tumor cell proliferation required for mTORC2 integrity and function, but not mTORC1 (13); but may not be sufficient to cause cellular transformation on its own. In thus, targeting of mLST8 could be used to specifically inhibit addition to proliferation, mTORC2 has known roles in regulation of mTORC2. As shown in Fig. 4A, CRISPR-Cas9–mediated targeting metabolism and cytoskeletal organization. Additional studies focused of mLST8 in H23 cells inhibited coimmunoprecipitation of mTORC2- on understanding the impact of RICTOR amplification on these other specific components Rictor and Sin1 with the mTOR kinase, whereas aspects of tumor biology are warranted. Raptor coimmunoprecipitation increased (Fig. 4A). Western blot Our study shows that increased mTORC2 formation occurs at the analysis of downstream signaling also confirmed that p-AKT was expense of mTORC1 formation, a finding that seems counter to the decreased upon mLST8 knockout (Fig. 4A). Consistent with Rictor large body of work demonstrating the importance of mTORC1 in knockout in Fig. 3, mLST8 knockout also reduced the relative number promoting tumor growth. It is important to note that the increased of viable cells and inhibited cell proliferation as measured by MTT proliferative effect of Rictor overexpression was observed in 3D culture assays and BrdUrd uptake assays, respectively (Fig. 4B and C). experiments, a condition that more closely mimics the nutrient To achieve equal cell numbers between WT and mLST8 cells for gradients present in the in vivo tumor microenvironment. A recent implantation in vivo, we utilized a doxycycline-inducible Cas9 system study has suggested mTORC1 may inhibit tumor growth in nutrient- coexpressed with an sgRNA targeting mLST8 (23). In vitro experi- starved conditions, offering a potential explanation to how reduced ments showed that a single dose of doxycycline was enough to induce mTORC1 could still promote tumor growth (26). Thus, further

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Figure 3. Rictor promotes NSCLC proliferation and tumor growth. A, SAM Rictor or EV H1975 or H358 cells were grown as Matrigel-embedded 3D cultures supplemented with 5 ng/mL of EGF. Representative images of cultures after 20 days are shown. Each point indicates an individual colony, with mean and SD denoted. B, Relative number of viable sgLacZ control and sgRictor H23 knockout cells was assessed by MTT assay. C, Proliferation of sgLacZ control and sgRictor knockout H23 cells was measured by BrdUrd uptake. Representative images are shown. Data presented as mean SD. D–G, 2.5 106 SAM Rictor #5 or EV control H1975 cells were injected subcutaneously into the hind flanks of Rag1-null immunodeficientmice.Tumorvolume(D)andtumormass(E)were measured. Data, mean SEM (n ¼ 9/group). F, Tumor lysates from 3 individual tumors per group were analyzed by Western blotting using the indicated antibodies. G, Proliferation of tumors was assessed by IHC staining of Ki-67. Representative images are shown, and quantification is presented as mean SEM. , P < 0.05; , P < 0.01; , P < 0.0001.

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Figure 4. mLST8 loss of function inhibits growth of RICTOR-ampliﬁed tumors in vivo. A–C, sgControl or sgMLST8 knockout H23 cells were generated by CRISPR-mediated gene editing. A, Interactions among components of mTOR complexes were measured by coimmunoprecipitation followed by Western blot analysis with the indicated antibodies. WCL, whole-cell lysate. B, Relative number of viable cells was measured by MTT assay. C, Proliferation was measured by BrdUrd uptake assay. Representative images are shown. Data presented as mean SEM. D–I, H23 cells were generated to express a doxycycline-inducible Cas9 and either sgLacZ or sgMLST8 #2. D, Western blot analysis of cells treated with doxycycline (1 mg/mL). Number of dox treatments over 10 days is indicated. E, Colony formation assay of 2,000 cells treated with doxycycline for 72 hours and then cultured for 2 weeks. Data presented as mean SEM. F–I, 5 106 cells were implanted in the hind ﬂanks of athymic nude mice that were fed doxycycline food for 10 days. F, Tumor volume was monitored 3 times a week. Data presented as mean SEM. G, Tumor mass was measured after tumors were removed 5 weeks after implantation. Data presented as mean SEM. H, Western blot analysis of mLST8 expression in 3 pairs of tumor lysates (M1–M3). M1, mouse #1. I, Tumor sections were analyzed by IHC for proliferation by Ki-67. , P < 0.05; , P < 0.01.

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Rictor Promotes NSCLC through mTORC2

investigation of nutrient availability in 3D or in vivo tumor settings is loop on PI3K/AKT signaling (34, 35), which could potentially lead required to better understand how mTORC2 upregulation impacts to an unwanted upregulation of mTORC2 signaling. In our study, tumor cell proliferation. we target mLST8 to specifically inhibit mTORC2 and show that RICTOR is located on the p arm of chromosome 5 (5p13.1), a tumor growth of RICTOR-amplified H23 NSCLC cells is reduced, common site of copy-number gain in lung cancer and other dis- suggesting inhibition of the mTOR–mLST8 interaction (13) may be eases (17). Recent studies, including our own analysis, show that an efficacious way to specifically inhibit mTORC2 and treat RIC- in addition to this overall copy-number gain, the loci surrounding TOR-amplified NSCLC. the RICTOR gene is a site of focal amplification (Supplementary Fig. S1; ref. 2). Amplification of RICTOR often co-occurs with ampli- Disclosure of Potential Conflicts of Interest fi cation or copy-number gain of several other genes along the 5p L.C. Kim reports grants from National Cancer Institute during the conduct of the chromosome, including SKP2 and GOLPH3, both of which have been study. J. Chen reports grants from VA and grants from NCI during the conduct of the implicated in cancer progression and modulating the PI3K–mTOR– study. No potential conflicts of interest were disclosed by the other author. AKT signaling axis (27–29). Other coamplified genes with known tumorigenic properties include OSMR and LIFR, also located at the Authors’ Contributions 5p13.1 cytoband (30–32). Although we were able to show that over- L.C. Kim: Conceptualization, data curation, formal analysis, investigation, – – expression of Rictor alone was able to increase the proliferative writing original draft, writing review, and editing. C.H. Rhee: Data curation. J. Chen: Conceptualization, resources, funding acquisition, project administration, capacity of lung cancer cell lines, further studies exploring the inter- writing–review, and editing. action between RICTOR and its coaltered genes in cancer are necessary to fully understand the oncogenic effects of amplification occurring at Acknowledgments the 5p chromosome. We would like to acknowledge the Vanderbilt University Medical Center Trans- The identification of Rictor overexpression as a driver of lational Pathology Share Resource for their help with IHC experiments. This work was mTORC2 formation and tumor progression suggests that specific supported by a VA Merit Award 5101BX000134 and a VA Research Career Scientist inhibition of mTORC2 would be a logical therapeutic strategy for Award (J. Chen), NIH grants R01 CA177681 (J. Chen), R01 CA95004 (J. Chen), T32 patients with RICTOR-amplified cancers. However, only mTORC1- CA009592 (L.C. Kim), and F31 CA2220804-01 (L.C. Kim). specific inhibitors (rapamycin analogues) or mTOR kinase inhibi- The costs of publication of this article were defrayed in part by the payment of page tors that block the activity of both complexes are available (33). Our charges. This article must therefore be hereby marked advertisement in accordance data suggest that increased mTORC2 formation can reduce the with 18 U.S.C. Section 1734 solely to indicate this fact. levels of mTORC1, thus inhibition of both mTOR complexes may not be necessary for the treatment of RICTOR-amplified cancers. Received March 20, 2020; revised June 10, 2020; accepted August 7, 2020; Additionally, inhibition of mTORC1 releases a negative feedback published first August 14, 2020.

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OF10 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH

RICTOR Amplification Promotes NSCLC Cell Proliferation through Formation and Activation of mTORC2 at the Expense of mTORC1

Laura C. Kim, Christopher H. Rhee and Jin Chen

Mol Cancer Res Published OnlineFirst August 14, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-20-0262

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