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Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death

Open AccessPublished:October 08, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102580
      Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth muscle–like “LAM” cells in the lungs. LAM cells bear loss-of-function mutations in tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2, causing hyperactivation of the proliferation promoting mammalian/mechanistic target of Rapamycin complex 1 pathway. Additionally, LAM-specific active renin-angiotensin system (RAS) has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear. Here, we report that TSC2-deficient cells are sensitive to the blockade of angiotensin II receptor type 1 (Agtr1). We show that treatment of these cells with the AGTR1 inhibitor losartan or silencing of the Agtr1 gene leads to increased cell death in vitro and attenuates tumor progression in vivo. Notably, we found the effect of Agtr1 blockade is specific to TSC2-deficient cells. Mechanistically, we demonstrate that cell death induced by Agtr1 inhibition is mediated by an increased expression of Klotho. In TSC2-deficient cells, we showed overexpression of Klotho or treatment with recombinant (soluble) Klotho mirrored the cytocidal effect of angiotensin blockade. Furthermore, Klotho treatment decreased the phosphorylation of AKT, potentially leading to this cytocidal effect. Conversely, silencing of Klotho rescued TSC2-deficient cells from cell death induced by Agtr1 inhibition. Therefore, we conclude that Agtr1 and Klotho are important for TSC2-deficient cell survival. These findings further illuminate the role of the RAS in LAM and the potential of targeting Agtr1 inhibition in TSC2-deficient cells.

      Keywords

      Abbreviations:

      ACE (angiotensin-I converting enzyme), AGTR (angiotensin II receptor), AML (angiomyolipomas), cPARP (cleaved PARP), DMSO (dimethyl sulfoxide), EV (empty vector), KD (knockdown), LAM (lymphangioleiomyomatosis), LDH (lactate hydrogenase), MEF (mouse embryonic fibroblasts), mTORC1 (mammalian/mechanistic target of Rapamycin complex 1), NT-shRNA (nontarget shRNA), qPCR (quantitative PCR), RAS (renin-angiotensin system), sKlotho (soluble Klotho), TSC (tuberous sclerosis complex)
      Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome characterized by hamartoma-like tumor growths in various organs, cerebral calcifications, seizures, and mental retardation, and the development of cystic lung disease called lymphangioleiomyomatosis (LAM). TSC is caused when one of the TSC genes TSC1 or TSC2 is mutated, more often TSC2 than TSC1 (
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      Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis.
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      Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.
      ), resulting in increased activation of the mammalian/mechanistic target of Rapamycin complex 1 (mTORC1) (
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      Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM).
      ). LAM is a multisystem disorder primarily affecting women of child-bearing age, characterized by cystic lung destruction, axial lymphatic abnormalities, and abdominal angiomyolipomas (AML) (
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      ). LAM occurs sporadically in patients with no evidence of germline genetic abnormality and by the age of 40 in about 80% of women with TSC (
      • Costello L.C.
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      High frequency of pulmonary lymphangioleiomyomatosis in women with tuberous sclerosis complex.
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      Prevalence and clinical characteristics of lymphangioleiomyomatosis (LAM) in patients with tuberous sclerosis complex.
      ). Inhibitors of the mTORC1 pathway have been shown to have therapeutic benefits in LAM and other TSC manifestations; however, there is a need for continuous therapy for persistent benefit, since mTORC1 inhibitors have cytostatic, and not cytotoxic, effects on TSC2-deficient cells (
      • Dabora S.L.
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      Multicenter phase 2 trial of sirolimus for tuberous sclerosis: kidney angiomyolipomas and other tumors regress and VEGF- D levels decrease.
      ,
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      Efficacy and safety of sirolimus in lymphangioleiomyomatosis.
      ). Identifying novel therapies that alone or in combination with mTORC1 inhibitors can induce TSC2-deficient cell death is of critical importance. We recently demonstrated that targeting E26 transformation–specific (ETS) variant transcription factor 2, an ETS family transcription factor specifically in TSC2-deficient cell promotes cytocidal response via regulation of poly(ADP-ribose) polymerase (PARP)-1 binding protein and therefore can have therapeutic potential in LAM or other TSC manifestations (
      • Shrestha S.
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      ETV2 regulates PARP-1 binding protein to induce ER stress-mediated death in tuberin-deficient cells.
      ).
      Multiple TSC-related manifestations have also been linked to the renin-angiotensin system (RAS). For instance, LAM lung nodules have been shown to possess a functional RAS, including the presence of renin, angiotensin I converting enzyme (ACE), angiotensinogen, angiotensin II, and angiotensin II receptors (
      • Valencia J.C.
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      • Bruneval P.
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      Tissue-specific renin-angiotensin system in pulmonary lymphangioleiomyomatosis.
      ). A retrospective review of LAM patients receiving ACE inhibitors showed slower decline in lung function (
      • Steagall W.K.
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      • Pacheco-Rodriguez G.
      • Moss J.
      Angiotensin-converting enzyme inhibitors may affect pulmonary function in lymphangioleiomyomatosis.
      ). Furthermore, patients with TSC2-polycystic kidney disease 1 deletion syndrome and hypertension treated with inhibition of the RAS pathway via angiotensin receptor blockade had decreased renal AML development compared to those who did not receive this therapy (
      • Siroky B.J.
      • Yin H.
      • Dixon B.P.
      • Reichert R.J.
      • Hellmann A.R.
      • Ramkumar T.
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      Evidence for pericyte origin of TSC-associated renal angiomyolipomas and implications for angiotensin receptor inhibition therapy.
      ). However, the precise mechanisms underpinning these effects of angiotensin II receptor (AGTR) blockade have not been elucidated. Here, we seek to exploit the RAS present in LAM or TSC to overcome the limitations of rapalogs.
      Klotho, an aging-suppressor gene, encodes for α-Klotho protein (Klotho), which associates with fibroblast growth factor receptor family protein to regulate metabolic processes (
      • Sachdeva A.
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      Klotho and the treatment of human malignancies.
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      • et al.
      Klotho: a tumor suppressor and a modulator of the IGF-1 and FGF pathways in human breast cancer.
      ,
      • Zhou X.
      • Fang X.
      • Jiang Y.
      • Geng L.
      • Li X.
      • Li Y.
      • et al.
      Klotho, an anti-aging gene, acts as a tumor suppressor and inhibitor of IGF-1R signaling in diffuse large B cell lymphoma.
      ). Klotho is a transmembrane protein with a short cytoplasmic domain and two large homologous extracellular domains, KL1 and KL2. The proteolytic cleavage of extracellular domains by beta-secretase generates the soluble form of Klotho that is detected in circulation in various bodily fluids (
      • Kimura T.
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      Impact of renal transplantation and nephrectomy on urinary soluble klotho protein.
      ,
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      A decreased level of serum soluble Klotho is an independent biomarker associated with arterial stiffness in patients with chronic kidney disease.
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      Alpha-klotho expression in human tissues.
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      Association between serum soluble klotho levels and mortality in chronic hemodialysis patients.
      ,
      • Pedersen L.
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      Soluble serum Klotho levels in healthy subjects. Comparison of two different immunoassays.
      ,
      • Xu Y.
      • Sun Z.
      Molecular basis of klotho: from gene to function in aging.
      ). Soluble Klotho (sKlotho) has been associated with regulation of transporters, oxidative stress, and signaling factors, as well as inhibition of insulin-like growth factor-1 receptor (
      • Tai N.C.
      • Kim S.A.
      • Ahn S.G.
      Soluble klotho regulates the function of salivary glands by activating KLF4 pathways.
      ). Klotho’s function as a tumor suppressor has also been demonstrated in various malignancies, including colorectal, pancreatic, gastric, renal, breast, and ovarian cancers (
      • Sachdeva A.
      • Gouge J.
      • Kontovounisios C.
      • Nikolaou S.
      • Ashworth A.
      • Lim K.
      • et al.
      Klotho and the treatment of human malignancies.
      ,
      • Wolf I.
      • Levanon-Cohen S.
      • Bose S.
      • Ligumsky H.
      • Sredni B.
      • Kanety H.
      • et al.
      Klotho: a tumor suppressor and a modulator of the IGF-1 and FGF pathways in human breast cancer.
      ,
      • Dai D.
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      • Liu J.
      • Ma X.
      • Xu W.
      Klotho inhibits human follicular thyroid cancer cell growth and promotes apoptosis through regulation of the expression of stanniocalcin-1.
      ,
      • Li X.X.
      • Huang L.Y.
      • Peng J.J.
      • Liang L.
      • Shi D.B.
      • Zheng H.T.
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      Klotho suppresses growth and invasion of colon cancer cells through inhibition of IGF1R-mediated PI3K/AKT pathway.
      ,
      • Sun H.
      • Gao Y.
      • Lu K.
      • Zhao G.
      • Li X.
      • Li Z.
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      Overexpression of Klotho suppresses liver cancer progression and induces cell apoptosis by negatively regulating wnt/beta-catenin signaling pathway.
      ,
      • Wang Y.
      • Chen L.
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      • He D.
      • He J.
      • Xu W.
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      Klotho sensitizes human lung cancer cell line to cisplatin via PI3k/Akt pathway.
      ,
      • Xie B.
      • Chen J.
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      Klotho acts as a tumor suppressor in cancers.
      ,
      • Xie B.
      • Zhou J.
      • Shu G.
      • Liu D.C.
      • Chen J.
      • Yuan L.
      Restoration of klotho gene expression induces apoptosis and autophagy in gastric cancer cells: tumor suppressive role of klotho in gastric cancer.
      ). Furthermore, multiple lines of evidence show that Klotho is downstream of both mTORC1 and angiotensin signaling (
      • Lim S.C.
      • Liu J.J.
      • Subramaniam T.
      • Sum C.F.
      Elevated circulating alpha-klotho by angiotensin II receptor blocker losartan is associated with reduction of albuminuria in type 2 diabetic patients.
      ,
      • Lin Y.
      • Kuro-o M.
      • Sun Z.
      Genetic deficiency of anti-aging gene klotho exacerbates early nephropathy in STZ-induced diabetes in male mice.
      ,
      • Luo Y.
      • Yang C.
      • Lu W.
      • Xie R.
      • Jin C.
      • Huang P.
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      Metabolic regulator betaKlotho interacts with fibroblast growth factor receptor 4 (FGFR4) to induce apoptosis and inhibit tumor cell proliferation.
      ,
      • Mitani H.
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      • Aizawa T.
      • Ohno M.
      • Usui S.
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      In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage.
      ,
      • Sato T.
      • Seyama K.
      • Fujii H.
      • Maruyama H.
      • Setoguchi Y.
      • Iwakami S.
      • et al.
      Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis.
      ,
      • Yoon H.E.
      • Ghee J.Y.
      • Piao S.
      • Song J.H.
      • Han D.H.
      • Kim S.
      • et al.
      Angiotensin II blockade upregulates the expression of Klotho, the anti-ageing gene, in an experimental model of chronic cyclosporine nephropathy.
      ). However, the role of Klotho in TSC2-related manifestations remains to be examined.
      Herein, we explored the roles of AGTR1 and Klotho in TSC2-deficient cells. We show that blocking AGTR1 in TSC2-deficient cells results in cell death in vitro and inhibition of tumor growth in vivo. Crucially, we also show that treatment with losartan, an AGTR1-blocker, as well as shRNA-mediated Agtr1 knockdown (KD) leads to induction of Klotho expression. Further, genetic manipulation of Klotho (silencing or overexpressing) leads to protection against or induction of cell death, respectively. Additionally, treatment with sKlotho increased TSC2-deficient cell death by reducing AKT phosphorylation. Based on our data, Klotho-dependent cytotoxic effect of AGTR1 blockade may highlight a potential therapeutic target for the treatment of LAM and other manifestations of TSC.

      Results

      AGTR1 expression is TSC2 loss–independent

      Tuberin deficiency results in mTORC1 hyperactivation, leading to uncontrolled cell growth (
      • Cui Y.
      • Steagall W.K.
      • Lamattina A.M.
      • Pacheco-Rodriguez G.
      • Stylianou M.
      • Kidambi P.
      • et al.
      Aberrant SYK kinase signaling is essential for tumorigenesis induced by TSC2 inactivation.
      ). As we hypothesize that AGTR1 is involved in LAM pathogenesis, we first sought to determine whether AGTR1 expression is regulated upon Tsc2 loss. We performed quantitative PCR (qPCR) and Western blot using TSC2-deficient ELT3V and TSC2-addback ELT3T cells derived from Eker rat uterine leiomyoma (
      • Howe S.R.
      • Everitt J.L.
      • Gottardis M.M.
      • Walker C.
      Rodent model of reproductive tract leiomyomata: characterization and use in preclinical therapeutic studies.
      ). Both cell types expressed Agtr1 mRNA and AGTR1 protein at similar levels, suggesting that AGTR1 expression is Tsc2 loss–independent (Fig. S1, AD).

      AGTR1 inhibition induces TSC2-deficient cell death in vitro

      Next, to determine the effects of AGTR1 blockade on TSC2-deficient cells, we treated ELT3V cells with losartan and examined the effects on cell survival. Treatment with losartan resulted in a decrease in P38 MAPK phosphorylation, a known downstream effector of AGTR1 signaling (Fig. S2, A and B) (
      • Imani J.
      • Bodine S.P.M.
      • Lamattina A.M.
      • Ma D.D.
      • Shrestha S.
      • Maynard D.M.
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      Dysregulated myosin in Hermansky-Pudlak syndrome lung fibroblasts is associated with increased cell motility.
      ). Western blot analysis demonstrated an increase in cleaved PARP (cPARP) levels compared to dimethyl sulfoxide (DMSO) control, suggesting increased cell death (Fig. 1, A and B). We further evaluated the levels of Caspase7, an upstream regulator of PARP cleavage (
      • Boucher D.
      • Blais V.
      • Denault J.B.
      Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis.
      ,
      • Genchi G.
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      • Catalano A.
      Nickel: human health and environmental toxicology.
      ) and found that treatment with losartan increased cleaved Caspase7 levels (Fig. S2, C and D). Additionally, treatment with losartan induced a significant increase in lactate dehydrogenase (LDH) release, suggesting disruption of the cell membrane and increased cytotoxicity (Fig. 1C). The deep blue cell viability assay also confirmed that losartan treatment led to reduced TSC2-deficient cell viability (Fig. S2E). To demonstrate that these effects are specific to TSC2-deficient cells, we treated TSC2-addback ELT3T cells with losartan and showed that in contrast to the effects of AGTR1 blockade in ELT3V cells, ELT3T cells treated with losartan did not have an increase in cell death as demonstrated by the lack of increase in cPARP level (Fig. S3, A and C) as well as no difference in LDH release compared to DMSO treatment (Fig. S3D).
      Figure thumbnail gr1
      Figure 1Inhibition of AGTR1 induces TSC2-deficient cell death in vitro. A, TSC2-deficient ELT3V cells were starved overnight and treated with DMSO (vehicle control) or losartan (100 nM) for 24 h in 0.5% serum supplemented media. Equal amounts of protein from whole-cell lysates of treated cells were analyzed by Western blot. Representative blots for PARP, cleaved PARP (cPARP), and β-ACTIN (loading control) are shown. B, histogram for cPARP/PARP is presented as the fold change relative to DMSO-treated ELT3V cells. C, scatter plot for LDH release by ELT3V cells treated with DMSO or losartan is presented as percent cytotoxicity. No drug (or water control) samples were used as “low control” for LDH measurement. D, Sh-RNA–mediated Agtr1 knockdown (AT1-shRNA) in ELT3V cells. D, quantitative PCR analysis of shRNA-mediated knockdown of Agtr1. Histogram for Agtr1 mRNA expression in ELT3V cells targeted with control (NT-shRNA) and AT1-shRNA is presented. B2m was used as a housekeeping gene. E, representative blots for AGTR1, cPARP, PARP, and β-ACTIN (loading control). Equal amounts of protein from whole-cell lysates of NT-shRNA and AT1-shRNA cells were analyzed by Western blot. Histograms for (F) AGTR1/β-ACTIN and (G) cPARP/PARP are expressed as the fold change relative to NT-shRNA cells. H, scatter plot for LDH release by NT-shRNA and AT1-shRNA presented as percent cytotoxicity. NT-shRNA samples were used as “low control” for LDH measurement. All graphs represent mean ± SEM of at least three independent experiments. Each biological replicate value is presented as a full circle. Statistical significance of ∗p < 0.05 or ∗∗p < 0.01 was determined by (B, D, and F–H) one-sample t test or (C) two-tailed t test. AGTR, angiotensin II receptor; cPARP, cleaved PARP; DMSO, dimethyl sulfoxide; LDH, lactate hydrogenase; NT-shRNA, nontarget shRNA; PARP, poly(ADP-ribose) polymerase; TSC, tuberous sclerosis complex.
      Since losartan can also affect transforming growth factor-β signaling in a nonreceptor-dependent manner (
      • Diop-Frimpong B.
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      Effect of angiotensin II receptor blocker on plasma levels of TGF-beta 1 and interstitial fibrosis in hypertensive kidney transplant patients.
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      • Kim M.D.
      • Baumlin N.
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      • Salathe S.F.
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      Losartan rescues inflammation-related mucociliary dysfunction in relevant models of cystic fibrosis.
      ,
      • Podowski M.
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      • Poonyagariyagorn H.
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      Angiotensin receptor blockade attenuates cigarette smoke-induced lung injury and rescues lung architecture in mice.
      ) and to ascertain that the observed effects of losartan on TSC2-deficient cell death are driven by the AGTR1, we silenced Agtr1 using shRNA in ELT3V cells. Agtr1 KD cells, denoted by AT1-shRNA, showed significantly reduced Agtr1 mRNA and AGTR1 protein expressions compared to nontarget control cells (NT-shRNA) (Fig. 1, DF). Like ELT3V cells treated with losartan, AT1-shRNA cells demonstrated increased level of cPARP expression compared to NT-shRNA cells (Fig. 1, E and G), increased LDH release (Fig. 1H), and decreased cell viability (Fig. S2F). Together, these data strongly suggest that Agtr1 gene silencing leads to increased TSC2-deficient cell death.
      These data demonstrate that, contrary to TSC2-addback cells, TSC2-deficient cells require continuous AGTR1 signaling for cell survival.

      AGTR1 regulates Klotho expression in TSC2-deficient cells

      Angiotensin receptor blockade has been shown to induce Klotho expression in the kidney (
      • Yoon H.E.
      • Ghee J.Y.
      • Piao S.
      • Song J.H.
      • Han D.H.
      • Kim S.
      • et al.
      Angiotensin II blockade upregulates the expression of Klotho, the anti-ageing gene, in an experimental model of chronic cyclosporine nephropathy.
      ,
      • Zhou Q.
      • Lin S.
      • Tang R.
      • Veeraragoo P.
      • Peng W.
      • Wu R.
      Role of fosinopril and valsartan on klotho gene expression induced by angiotensin II in rat renal tubular epithelial cells.
      ). Like Agtr1, comparison between TSC2-deficient ELT3V and TSC2-addback ELT3T cells showed no significant differences in Klotho expression (Fig. S1, B and D). Next, to evaluate whether Klotho is downstream of AGTR1 in TSC2-deficient cells, we treated ELT3V and ELT3T cells with losartan and analyzed cell lysates for Klotho expression with Western blotting. Our data showed that AGTR1 inhibition with losartan resulted in increased Klotho levels only in TSC2-deficient cells (Fig. 2, A and B) but not in TSC2-addback cells (Fig. S3, A and B). Further, to confirm that these effects are AGTR1-dependent, we examined Klotho protein levels in Agtr1 KD cells and found a corresponding increase in Klotho levels in AT1-shRNA cells compared to NT-shRNA cells (Fig. 2, C and D). To confirm the specificity of AT1-shRNA, a second construct (AT1-shRNA-2) was used to corroborate that Agtr1 silencing leads to increased Klotho expression and cell death (Fig. S4, AE). Taken together, our data show that Klotho is downstream of the AGTR1 receptor in TSC2-deficient cells.
      Figure thumbnail gr2
      Figure 2AGTR1 blockade–mediated TSC2-deficient cell death is Klotho-dependent. A, TSC2-deficient ELT3V cells were starved overnight and treated with DMSO or losartan (100 nM) for 24 h in 0.5% serum supplemented media. Equal amounts of protein isolated from whole-cell lysates of treated cells were analyzed by Western blot. Representative blots for Klotho and β-ACTIN (same membrane as A) are shown. B, histogram for Klotho/β-ACTIN is presented as the fold change relative to DMSO-treated ELT3V cells. C, equal amounts of lysates extracted from NT-shRNA and AT1-shRNA ELT3V cells were analyzed by Western blot. Representative blots for Klotho and β-ACTIN (same membrane as E) are shown. D, histogram for Klotho/β-ACTIN is presented as the fold change relative to NT-shRNA–treated ELT3V cells. E, ELT3V cells were transfected with Scr or Klotho siRNA, starved overnight, and treated with losartan (100 nM) for 24 h. Equal amounts of lysates were analyzed by Western blot. Representative blots for Klotho, cPARP, PARP, and β-ACTIN (loading control) are shown. Histograms for (F) Klotho/β-ACTIN and (G) cPARP/PARP are presented as fold change relative to Scr siRNA-transfected cells treated with DMSO. H, ELT3V cells were transfected with an empty vector or Klotho construct for 48 h. Equal amounts of lysates extracted were subjected to Western blot analysis. Representative blots for Klotho, cPARP, PARP, and β-ACTIN (loading control) are shown. Histogram for (I) Klotho/β-ACTIN and (J) cPARP/PARP are presented as fold change relative to vector transfected cells. All graphs represent mean ± SEM of at least three independent experiments. Each biological replicate value is presented as a full circle. Statistical significance of ∗p < 0.05 for each graph was determined by one-sample t test. AGTR, angiotensin II receptor; cPARP, cleaved PARP; DMSO, dimethyl sulfoxide; NT-shRNA, nontarget shRNA; PARP, poly(ADP-ribose) polymerase; TSC, tuberous sclerosis complex.

      AGTR1-dependent cell death is driven by Klotho in TSC2-deficient cells

      To elucidate Klotho’s involvement in Agtr1 blockade–mediated cell death, we silenced Klotho using siRNA in ELT3V cells (Fig. 2, E and F), followed by treatment with losartan. We observed that cells targeted with Klotho siRNA had a marked decrease in cPARP compared to cells targeted with Scr siRNA (Fig. 2, E and G), suggesting that Klotho is necessary for AGTR1 blockade–dependent cell death in TSC2-deficient cells. These results were also confirmed with a second Klotho siRNA (Fig. S4, FH).
      Finally, to examine if increased Klotho levels are sufficient to induce TSC2-deficient cell death, ELT3V cells were transiently transfected with Klotho or empty vector (EV) construct for 48 h and subjected to Western blotting. Cells overexpressing Klotho demonstrated increased cell death as shown by the increase in cPARP levels compared to vector transfected cells (Fig. 2, HJ). Taken together, these data show that Klotho is necessary and sufficient to induce TSC2-deficient cell death and that AGTR1 signaling is necessary for mTORC1-hyperactive cell survival.
      Next, we wanted to examine if sKlotho affects TSC2-deficient cell survival. First, we performed an LDH release assay with increasing sKlotho concentrations and determined that 100 ng/ml was the optimal dose (Fig. S2G), which is consistent with previous studies (
      • Batlahally S.
      • Franklin A.
      • Damianos A.
      • Huang J.
      • Chen P.
      • Sharma M.
      • et al.
      Soluble Klotho, a biomarker and therapeutic strategy to reduce bronchopulmonary dysplasia and pulmonary hypertension in preterm infants.
      ,
      • Miao J.
      • Huang J.
      • Luo C.
      • Ye H.
      • Ling X.
      • Wu Q.
      • et al.
      Klotho retards renal fibrosis through targeting mitochondrial dysfunction and cellular senescence in renal tubular cells.
      ). Next, we exposed ELT3V cells with 100 ng/ml sKlotho for 24 h. Treatment with sKlotho significantly increased cPARP level, suggesting increased cell death (Fig. 3, A and B). Similar to AGTR1 inhibition, treatment of TSC2-addback ELT3T cells with sKlotho did not result in cell death (Fig. 3, C and D). Treatment with sKlotho also induced a significant increase in LDH release in TSC2-deficient ELT3V cells but not in TSC2-addback ELT3T cells (Fig. 3, E and F). Finally, sKlotho-driven cell death was also confirmed by demonstrating the decreased cell viability in TSC2-deficient cells (Fig. S2H).
      Figure thumbnail gr3
      Figure 3Soluble Klotho treatment induces cell death only in TSC2-deficient cells but not TSC2-addback cells. Representative blots for cPARP, PARP, TUBERIN, and β-ACTIN (loading control) in (A) TSC2-deficient ELT3V cells and (C) TSC2-addback ELT3T cells starved overnight and then treated with vehicle control (water, 0 ng/ml sKlotho) or sKlotho (100 ng/ml) for 24 h in serum starved conditions are shown. Equal amounts of lysates extracted from treated cells were subjected to Western blot analysis. Histograms for cPARP/PARP expression in (B) ELT3V and (D) ELT3T cells are presented as fold change relative to control (0 ng/ml sKlotho). Scatter plot of LDH release presented as percent cytotoxicity after exposure of (E) ELT3V and (F) ELT3T cells to water and sKlotho treatment. Water-treated samples are used as “low control” for percent cytotoxicity calculation. All graphs represent mean ± SEM of at least three independent experiments. Each biological replicate value is presented as a full circle. Statistical significance of ns p > 0.05 or ∗∗p < 0.01 were determined by one-sample t test. cPARP, cleaved PARP; LDH, lactate hydrogenase; PARP, poly(ADP-ribose) polymerase; sKlotho, soluble Klotho; TSC, tuberous sclerosis complex.

      Klotho-dependent cell death is mediated by AKT

      AKT signaling pathway is associated with proliferation/survival of various cancer cells, and Klotho overexpression has been shown to decrease activation of prosurvival phospho(p)-AKT (
      • Sachdeva A.
      • Gouge J.
      • Kontovounisios C.
      • Nikolaou S.
      • Ashworth A.
      • Lim K.
      • et al.
      Klotho and the treatment of human malignancies.
      ,
      • Li X.X.
      • Huang L.Y.
      • Peng J.J.
      • Liang L.
      • Shi D.B.
      • Zheng H.T.
      • et al.
      Klotho suppresses growth and invasion of colon cancer cells through inhibition of IGF1R-mediated PI3K/AKT pathway.
      ,
      • Wang Y.
      • Chen L.
      • Huang G.
      • He D.
      • He J.
      • Xu W.
      • et al.
      Klotho sensitizes human lung cancer cell line to cisplatin via PI3k/Akt pathway.
      ,
      • Wan X.
      • Harkavy B.
      • Shen N.
      • Grohar P.
      • Helman L.J.
      Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism.
      ). First, we found that pAKT (Ser473) levels were decreased in TSC2-deficient cells with Agtr1 silencing (Fig. 4, A and B). We then tested the effect of sKlotho treatment at different time points on pAKT at Ser473 (not shown). Reduced activation of AKT was observed in TSC2-deficient ELT3V cells (Fig. 4, C and D) with significant decrease in pAKT level (relative to AKT) within 30 min. Additionally, to test if the induction of cell death in TSC2-deficient cells was pAKT-dependent, we treated TSC2-deficient mouse embryonic fibroblasts (MEF) transfected with EV or with constitutively active (myristoylated) AKT (MEF-AKT1) cells with sKlotho for 24 h. Similar to ELT3V cells, MEF-EV cells showed increased cell death when treated with sKlotho (Fig. 4, E and F) compared to vehicle (water) control. However, constitutive activation of AKT1 in MEF-AKT1 cells prevented the Klotho-dependent cell death (Fig. 4, E and F). These data coupled with our data showing the increased cleavage of Caspase7 upon treatment with losartan, and the known downstream effect of AKT signaling on Caspase7 cleavage (
      • Lee J.E.
      • Ahn S.
      • Jeong H.
      • An S.
      • Myung C.H.
      • Lee J.A.
      • et al.
      Olig2 regulates p53-mediated apoptosis, migration and invasion of melanoma cells.
      ), strongly support our hypothesis that Klotho-mediated cell death is AKT-dependent.
      Figure thumbnail gr4
      Figure 4TSC2-deficient cell death is induced by a Klotho-mediated effect on pAKT. A, equal amounts of lysates extracted from NT-shRNA and AT1-shRNA ELT3V cells were analyzed by Western blot. Representative blots for pAKT, AKT, and β-ACTIN (loading control) are shown. B, histograms for pAKT/AKT is presented as the fold change relative to NT-shRNA–treated ELT3V cells. C, TSC2-deficient ELT3V cells were starved overnight and treated with vehicle control (water) or sKlotho (100 ng/ml) for indicated amount of time in serum-starved condition. Equal amounts of lysates extracted from treated cells were subjected to Western blot analysis. Representative blots for phosphorylated(p)AKT, AKT, and β-ACTIN (loading control) are shown. D, histogram for pAKT/AKT is presented as fold change relative to control (0 ng/ml sKlotho). E, TSC2-deficient MEF-AKT and MEF-EV cells were starved overnight and treated with vehicle control (water) or sklotho (100 ng/ml) for 24 h. Equal amounts of lysates extracted from MEF-EV– and MEF-AKT–treated cells were subjected to Western blot analysis. Representative blots for cPARP, total PARP, and β-ACTIN (loading control) are shown. (F) histogram for cPARP/PARP expressions in MEF-EV and MEF-AKT is presented as fold change relative to water control (0 ng/ml sKlotho). All graphs represent mean ± SEM of at least three independent experiments. Each biological replicate value is presented as a full circle. Statistical significance of ns p > 0.05 or ∗p < 0.05 were determined by one-sample t test. cPARP, cleaved PARP; EV, empty vector; MEF, mouse embryonic fibroblasts; NT-shRNA, nontarget shRNA; PARP, poly(ADP-ribose) polymerase; TSC, tuberous sclerosis complex.

      AGTR inhibition suppresses TSC2-deficient xenograft tumor development in vivo

      To evaluate the effects of AGTR1 inhibition on the growth of TSC2-deficient xenografts, TSC2-deficient NT-shRNA and AT1-shRNA cells were subcutaneously injected in immunodeficient mice. After 60 days of follow-up and monitoring, mice harboring Agtr1 KD cells (AT1-shRNA) showed reduced tumor development compared with mice harboring NT-shRNA cells (Fig. 5A). Moreover, when animals were sacrificed, there was a distinct difference in gross tumor appearance (Fig. 5B), as well as significantly decreased tumor size (Fig. 5C) and weight (Fig. 5D) in AT1-shRNA group compared to NT-shRNA group. As expected, qPCR and Western blot analysis of lysates from tumor homogenates showed a decreased expression of AGTR1 (Fig. 5, E, F, and H), increased expression of Klotho (Fig. 5, F and I), and increased cPARP levels (Fig. 5, G and J). These data demonstrate that TSC2-deficient xenografts are sensitive to AGTR1 inhibition and provide rationale for future clinical trials.
      Figure thumbnail gr5
      Figure 5AGTR1 blockade impairs TSC2-deficient null xenograft tumor development. Female CB17-SCID mice were inoculated with 2.5 million control (NT-shRNA) or Agtr1 shRNA (AT1-shRNA)-transfected ELT3V cells subcutaneously into suprascapular region (n = 10 per group). Tumor length and width were measured daily with a caliper by a blinded investigator, and surface area was calculated. A, tumor-free survival analyses of mice in each group are plotted. Statistical significance ∗∗p < 0.01 was determined by Log-rank test. B, representative gross appearance of excised tumors is displayed. The scale bar represents 1 cm. Comparison of (C) average volume and (D) weight of the excised tumors. E, RNA was extracted from excised tumors and qPCR was performed analyzing Agtr1 expression. B2m was used as housekeeping gene. F, representative blots for AGTR1, Klotho, and (G) cPARP, PARP, and β-ACTIN (loading control) for protein isolated from excised tumors are shown. Histograms for (H) AGTR1/β-ACTIN, (I) Klotho/β-ACTIN, and (J) cPARP/PARP expressions are presented as mean ± SEM. Each biological replicate value is presented as a full circle. Statistical significance of ∗p < 0.05 or ∗∗p < 0.01 was determined by a two-tailed t test. AGTR, angiotensin II receptor; cPARP, cleaved PARP; NT-shRNA, nontarget shRNA; PARP, poly(ADP-ribose) polymerase; TSC, tuberous sclerosis complex.
      Finally, we also evaluated these findings in another TSC2-deficient 105K cell line. In vitro, we showed that TSC2-deficient 105K cells, when treated with losartan, demonstrated increased Klotho expression (Fig. 6, A and B). We also observed increased cell death as indicated by increased cPARP levels (Fig. 6, A and C), increased LDH release (Fig. 6D), and decreased cell viability (Fig. 6E) with losartan treatment. Moreover, treatment with sKlotho resulted in decreased AKT phosphorylation (Fig. 6, F and G). Further, and like TSC2-addback ELT3T cells, inhibition of AGTR1 in TSC2-addback 105K cells with losartan treatment showed no difference in Klotho and cPARP levels (Fig. S5, AC) as well as in percent LDH release compared to DMSO treatment (Fig. S5D). We also evaluated the activity of losartan in an immunodeficient mouse model bearing subcutaneous TSC2-deficient 105K xenograft tumors (
      • Chu S.C.
      • Horiba K.
      • Usuki J.
      • Avila N.A.
      • Chen C.C.
      • Travis W.D.
      • et al.
      Comprehensive evaluation of 35 patients with lymphangioleiomyomatosis.
      ,
      • Johnson S.
      Rare diseases. 1. Lymphangioleiomyomatosis: clinical features, management and basic mechanisms.
      ). Mice harboring TSC2-deficient 105K xenograft tumors treated with vehicle control (DMSO) showed progressive tumor growth. In contrast, mice treated with losartan showed a persistent stabilization in tumor size (Fig. 6I).
      Figure thumbnail gr6
      Figure 6AGTR1 blockade induces cell death in TSC2-deficient 105K cells in vitro and impairs xenograft tumor development in vivo. A, TSC2-deficient 105K cells were starved overnight and treated with DMSO or losartan (100 nM) for 24 h in 0.5% serum supplemented media. Equal amounts of lysates from treated cells were analyzed by Western blot. Representative blots for Klotho, cleaved PARP, total PARP, and β-ACTIN are shown. Histograms for (B) Klotho/β-ACTIN and (C) cPARP/PARP are presented as fold change relative to DMSO. D, scatter plot of 105K cells LDH release treated with DMSO or losartan presented as percent cytotoxicity. No drug (or water control) samples were used as “low control” for LDH measurement. E, cell viability was measured by the deep blue cell viability assay and values for losartan treatment are presented as percent of DMSO-treated cells. F, equal amounts of lysates extracted from 105K cells treated with sKlotho were subjected to Western blot analysis. Representative blots for pAKT, AKT, and β-ACTIN (loading control) are shown. G, histogram for pAKT/AKT is presented as fold change relative to control (0 ng/ml sKlotho). H, cell viability was measured using a deep blue cell viability assay and values for sKlotho treatment are presented as percent of water (vehicle)-treated cells. All graphs represent mean ± SEM of at least three independent experiments. Each biological replicate value is presented as a full circle. Pairwise comparisons are presented for significant differences: statistical significances of ∗p < 0.05 or ∗∗p < 0.01 were determined by (B, C, E, and G–H) one-sample t test or (D) two-tailed t test. I, growth comparison of xenograft tumors TSC2-deficient 105K tumors in vivo treated with vehicle (DMSO) or losartan (30 mg/kg) by oral gavage. Arrow indicates the start of losartan treatment. Statistical significance of ∗∗∗p <0.001 for each day was determined by two-tailed t test. AGTR, angiotensin II receptor; cPARP, cleaved PARP; DMSO, dimethyl sulfoxide; LDH, lactate hydrogenase; PARP, poly(ADP-ribose) polymerase; TSC, tuberous sclerosis complex.

      Discussion

      In this study, we demonstrate that there is an important role for the AGTR1–Klotho axis in TSC2-deficient cells. Our data provide evidence that blocking AGTR1 using losartan, a commercially available angiotensin receptor blocker, induces cell death in TSC2-deficient cells from two different origins, ELT3 cells derived from an TSC2-deficient Eker rat uterine leiomyoma (
      • Howe S.R.
      • Everitt J.L.
      • Gottardis M.M.
      • Walker C.
      Rodent model of reproductive tract leiomyomata: characterization and use in preclinical therapeutic studies.
      ), and 105K cells derived from a renal tumor in a TSC2 heterozygous mouse (
      • Filippakis H.
      • Alesi N.
      • Ogorek B.
      • Nijmeh J.
      • Khabibullin D.
      • Gutierrez C.
      • et al.
      Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R.
      ). It is important to note that losartan has additional off-target receptor-independent cellular effects especially on transforming growth factor-β signaling (
      • Diop-Frimpong B.
      • Chauhan V.P.
      • Krane S.
      • Boucher Y.
      • Jain R.K.
      Losartan inhibits collagen I synthesis and improves the distribution and efficacy of nanotherapeutics in tumors.
      ,
      • el-Agroudy A.E.
      • Hassan N.A.
      • Foda M.A.
      • Ismail A.M.
      • el-Sawy E.A.
      • Mousa O.
      • et al.
      Effect of angiotensin II receptor blocker on plasma levels of TGF-beta 1 and interstitial fibrosis in hypertensive kidney transplant patients.
      ,
      • Kim M.D.
      • Baumlin N.
      • Yoshida M.
      • Polineni D.
      • Salathe S.F.
      • David J.K.
      • et al.
      Losartan rescues inflammation-related mucociliary dysfunction in relevant models of cystic fibrosis.
      ,
      • Podowski M.
      • Calvi C.
      • Metzger S.
      • Misono K.
      • Poonyagariyagorn H.
      • Lopez-Mercado A.
      • et al.
      Angiotensin receptor blockade attenuates cigarette smoke-induced lung injury and rescues lung architecture in mice.
      ). However, shRNA-mediated knockdown of AGTR1 resulted in similar effects to losartan on TSC2-deficient cell death, indicating that effects of losartan are less likely due to an off-target effect and more likely due to inhibition of the AGTR1. Consistently, losartan treatment or shRNA-mediated receptor silencing significantly reduced tumor burden in an immunodeficient xenograft model, suggesting a potential role for targeting AGTR1 in LAM and other manifestations of TSC. Interestingly, our data show that there are no differences in AGTR1 expression in TSC2-deficient and TSC2-addback cells. However, inhibiting AGTR1 resulted in cell death only in TSC2-deficient cells but not TSC2-addback cells, suggesting that only TSC2-deficient cells require a continued AGTR1 signaling for survival. Our data pave the way to potentially use this vulnerability to target tumors, resulting from tuberin deficiency and hyperactivation of the mTORC1 pathway.
      The potential therapeutic effects of targeting renin-angiotensin in LAM and TSC have been demonstrated in retrospective studies of lung function (
      • Steagall W.K.
      • Stylianou M.
      • Pacheco-Rodriguez G.
      • Moss J.
      Angiotensin-converting enzyme inhibitors may affect pulmonary function in lymphangioleiomyomatosis.
      ). Patients with TSC2-polycystic kidney disease 1 deletion syndrome and hypertension treated with ACE inhibitors or angiotensin receptor blockers had decreased renal AML development compared to control (
      • Siroky B.J.
      • Yin H.
      • Dixon B.P.
      • Reichert R.J.
      • Hellmann A.R.
      • Ramkumar T.
      • et al.
      Evidence for pericyte origin of TSC-associated renal angiomyolipomas and implications for angiotensin receptor inhibition therapy.
      ). In vitro evidence suggested that stimulation of AGTR1 by angiotensin II drives VEGF-A secretion in mTORC1-activated, TSC2-deficient angiomyolipoma cells, leading to increased cell proliferation, which was shown to be blocked by valsartan, an AGTR1 inhibitor (
      • Siroky B.J.
      • Yin H.
      • Dixon B.P.
      • Reichert R.J.
      • Hellmann A.R.
      • Ramkumar T.
      • et al.
      Evidence for pericyte origin of TSC-associated renal angiomyolipomas and implications for angiotensin receptor inhibition therapy.
      ). The losartan concentration that we used in vitro (0.0479 μg/ml) is well within the 1 μg/ml serum concentration achieved with the current therapeutic dosage of 100 mg orally daily (
      • Ohtawa M.
      • Takayama F.
      • Saitoh K.
      • Yoshinaga T.
      • Nakashima M.
      Pharmacokinetics and biochemical efficacy after single and multiple oral administration of losartan, an orally active nonpeptide angiotensin II receptor antagonist, in humans.
      ). Losartan has an excellent lung bioavailability (
      • Tronde A.
      • Norden B.
      • Jeppsson A.B.
      • Brunmark P.
      • Nilsson E.
      • Lennernas H.
      • et al.
      Drug absorption from the isolated perfused rat lung--correlations with drug physicochemical properties and epithelial permeability.
      ); however, our in vivo proof of concept experiments using a 30 mg/kg dose would be the equivalent to ∼2.4 mg/kg human equivalent dose (
      • Nair A.B.
      • Jacob S.
      A simple practice guide for dose conversion between animals and human.
      ), a higher losartan dose than the commonly used 100 mg orally daily (
      • Tronde A.
      • Norden B.
      • Jeppsson A.B.
      • Brunmark P.
      • Nilsson E.
      • Lennernas H.
      • et al.
      Drug absorption from the isolated perfused rat lung--correlations with drug physicochemical properties and epithelial permeability.
      ). In clinical practice and based on their use for hypertension, the safety profile of angiotensin receptor blockers is well-documented. In addition, losartan has been used in clinical trials at 200 mg orally daily with no difference in adverse side effects between the high dose (200 mg daily) compared to the standard dose of 100 mg orally daily (
      • Hou F.F.
      • Xie D.
      • Zhang X.
      • Chen P.Y.
      • Zhang W.R.
      • Liang M.
      • et al.
      Renoprotection of optimal antiproteinuric doses (ROAD) study: a randomized controlled study of benazepril and losartan in chronic renal insufficiency.
      ). Collectively, these previous data and our data provide compelling evidence of the viability of a therapeutic strategy using angiotensin receptor blockade, perhaps in early diseases affecting patients with LAM or TSC.
      Our data also indicates that the cytocidal effects triggered by blocking the AGTR1 are mediated by an increase in Klotho protein expression. Klotho is a tumor suppressor that has previously been shown to interact with the mTOR pathway (
      • Lin Y.
      • Kuro-o M.
      • Sun Z.
      Genetic deficiency of anti-aging gene klotho exacerbates early nephropathy in STZ-induced diabetes in male mice.
      ). Klotho has previously been implicated in lung disease with effects on muco-ciliary clearance (
      • Garth J.
      • Easter M.
      • Skylar Harris E.
      • Sailland J.
      • Kuenzi L.
      • Chung S.
      • et al.
      The effects of the anti-aging protein klotho on mucociliary clearance.
      ), airway inflammation (
      • Krick S.
      • Grabner A.
      • Baumlin N.
      • Yanucil C.
      • Helton S.
      • Grosche A.
      • et al.
      Fibroblast growth factor 23 and Klotho contribute to airway inflammation.
      ), recovery from acute lung injury (
      • Gagan J.M.
      • Cao K.
      • Zhang Y.A.
      • Zhang J.
      • Davidson T.L.
      • Pastor J.V.
      • et al.
      Constitutive transgenic alpha-Klotho overexpression enhances resilience to and recovery from murine acute lung injury.
      ) and interstitial lung disease (
      • Barnes J.W.
      • Duncan D.
      • Helton S.
      • Hutcheson S.
      • Kurundkar D.
      • Logsdon N.J.
      • et al.
      Role of fibroblast growth factor 23 and klotho cross talk in idiopathic pulmonary fibrosis.
      ,
      • Buendia-Roldan I.
      • Machuca N.
      • Mejia M.
      • Maldonado M.
      • Pardo A.
      • Selman M.
      Lower levels of alpha-Klotho in serum are associated with decreased lung function in individuals with interstitial lung abnormalities.
      ) amongst others. Our data show the importance of Klotho upregulation in TSC2-deficient cells. In vitro, both membrane and sKlotho induces TSC2-deficient cell death. We also showed that Klotho silencing rescues the cell death phenotype induced by losartan treatment, suggesting Klotho is necessary and sufficient for regulating TSC2-deficient cell survival. Additionally, blockade of the AGTR1 also results in an increase in Klotho mRNA levels and the effects are specific to TSC2-deficient cells. Both membrane and sKlotho influence several signaling pathways, including insulin-like growth factor 1–mediated PI3K/AKT signaling pathway; notably, an increase in Klotho has been shown to decrease phosphorylation of AKT, a known prosurvival pathway (
      • Li X.X.
      • Huang L.Y.
      • Peng J.J.
      • Liang L.
      • Shi D.B.
      • Zheng H.T.
      • et al.
      Klotho suppresses growth and invasion of colon cancer cells through inhibition of IGF1R-mediated PI3K/AKT pathway.
      ,
      • Wang Y.
      • Chen L.
      • Huang G.
      • He D.
      • He J.
      • Xu W.
      • et al.
      Klotho sensitizes human lung cancer cell line to cisplatin via PI3k/Akt pathway.
      ,
      • Wang Y.
      • Sun Z.
      Current understanding of klotho.
      ,
      • Zhu Y.
      • Xu L.
      • Zhang J.
      • Xu W.
      • Liu Y.
      • Yin H.
      • et al.
      Klotho suppresses tumor progression via inhibiting PI3K/Akt/GSK3beta/Snail signaling in renal cell carcinoma.
      ). The role of AKT in TSC2-deficient cells remains understudied. In TSC2-deficient cells, mTORC1 hyperactivation negatively impacts AKT phosphorylation via inhibition of the mTORC2 complex and disruption of PI3K signaling. However, minimal activation of mTORC2 is sufficient to phosphorylate AKT, and AKT rebound phosphorylation when these cells are treated with rapamycin represents a key prosurvival pathway in TSC2-deficient cells (
      • Wan X.
      • Harkavy B.
      • Shen N.
      • Grohar P.
      • Helman L.J.
      Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism.
      ). While much remains to be discovered regarding the regulation of AKT signaling by the TSC1/2 complex (
      • Huang J.
      • Manning B.D.
      A complex interplay between Akt, TSC2 and the two mTOR complexes.
      ), our data suggest a prosurvival role for AKT in TSC2-deficient cells, as a decrease in pAKT induced by Klotho treatment was associated with increased cell death. Consistent with this result, Klotho-mediated suppression of AKT phosphorylation was associated with increased cell death, and expression of constitutively phosphorylated AKT protected TSC2-deficient cells from Klotho-mediated cell death. Taken together, these data suggest that AGTR1 inhibition drives Klotho expression, which decreases phosphorylation of AKT, a known prosurvival pathway, resulting in TSC2-deficient cell death.
      In conclusion, consistent with previous data showing the importance of AGTR1 (
      • Valencia J.C.
      • Pacheco-Rodriguez G.
      • Carmona A.K.
      • Xavier J.
      • Bruneval P.
      • Riemenschneider W.K.
      • et al.
      Tissue-specific renin-angiotensin system in pulmonary lymphangioleiomyomatosis.
      ,
      • Steagall W.K.
      • Stylianou M.
      • Pacheco-Rodriguez G.
      • Moss J.
      Angiotensin-converting enzyme inhibitors may affect pulmonary function in lymphangioleiomyomatosis.
      ) in LAM and AML (
      • Siroky B.J.
      • Yin H.
      • Dixon B.P.
      • Reichert R.J.
      • Hellmann A.R.
      • Ramkumar T.
      • et al.
      Evidence for pericyte origin of TSC-associated renal angiomyolipomas and implications for angiotensin receptor inhibition therapy.
      ), this study demonstrates both in vitro and in vivo and offers the scientific rationale to target AGTR1 in LAM and in TSC. We delineated the importance of AGTR1 signaling in TSC2-deficient cells and demonstrated that AGTR1 blockade could be a potential therapy in LAM and TSC, especially early in the disease when there could be a potential hesitation to commit patients to lifelong therapy with inhibitors of the mTORC1 pathway. Because AGTR1 inhibitors have a remarkable safety profile, evaluating their efficacy in clinical trials in LAM, AML, and other manifestations of TSC is warranted.

      Experimental procedures

      Cell lines, cell culture, and reagents

      Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 atmosphere. TSC2-deficient ELT3V and TSC2-addback ELT3T cells are derived from Eker rat uterine leiomyoma, as described previously (
      • Howe S.R.
      • Everitt J.L.
      • Gottardis M.M.
      • Walker C.
      Rodent model of reproductive tract leiomyomata: characterization and use in preclinical therapeutic studies.
      ). TSC2-deficient and TSC2-addback cystadenoma 105K cell lines were obtained from Dr Elizabeth Henske’s laboratory (
      • Filippakis H.
      • Alesi N.
      • Ogorek B.
      • Nijmeh J.
      • Khabibullin D.
      • Gutierrez C.
      • et al.
      Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R.
      ). TSC2-deficient MEF infected with pBabe-Puro-Myr-Flag-AKT1, to generate TSC2-deficient MEF with myristoylated AKT1 (MEF-AKT1), and control TSC2-deficient MEF (MEF-EV) infected with EV were obtained from Dr Carmen Priolo’s laboratory (
      • Priolo C.
      • Pyne S.
      • Rose J.
      • Regan E.R.
      • Zadra G.
      • Photopoulos C.
      • et al.
      AKT1 and MYC induce distinctive metabolic fingerprints in human prostate cancer.
      ). Myristoylation directs AKT to the membrane, keeping it constitutively phosphorylated at both Ser473 and Thr308 sites (
      • Priolo C.
      • Pyne S.
      • Rose J.
      • Regan E.R.
      • Zadra G.
      • Photopoulos C.
      • et al.
      AKT1 and MYC induce distinctive metabolic fingerprints in human prostate cancer.
      ,
      • Feng Y.
      • Mischler W.J.
      • Gurung A.C.
      • Kavanagh T.R.
      • Androsov G.
      • Sadow P.M.
      • et al.
      Therapeutic targeting of the secreted lysophospholipase D autotaxin suppresses tuberous sclerosis complex-associated tumorigenesis.
      ). Cells were serum starved overnight before treatment with indicated drugs/compounds, including losartan (100 nM (
      • Imani J.
      • Bodine S.P.M.
      • Lamattina A.M.
      • Ma D.D.
      • Shrestha S.
      • Maynard D.M.
      • et al.
      Dysregulated myosin in Hermansky-Pudlak syndrome lung fibroblasts is associated with increased cell motility.
      ,
      • Liu Y.
      • Leri A.
      • Li B.
      • Wang X.
      • Cheng W.
      • Kajstura J.
      • et al.
      Angiotensin II stimulation in vitro induces hypertrophy of normal and postinfarcted ventricular myocytes.
      ); Tocris Bioscience), DMSO vehicle (Sigma-Aldrich), water (vehicle), or Klotho (100 ng/ml (
      • Batlahally S.
      • Franklin A.
      • Damianos A.
      • Huang J.
      • Chen P.
      • Sharma M.
      • et al.
      Soluble Klotho, a biomarker and therapeutic strategy to reduce bronchopulmonary dysplasia and pulmonary hypertension in preterm infants.
      • Miao J.
      • Huang J.
      • Luo C.
      • Ye H.
      • Ling X.
      • Wu Q.
      • et al.
      Klotho retards renal fibrosis through targeting mitochondrial dysfunction and cellular senescence in renal tubular cells.
      ); R&D Systems) for indicated durations.

      Lentiviral expression system

      To establish stable Agtr1 KD TSC2-deficient ELT3V cells, we obtained a psi-LVRU6GH vector harboring nontargeting shRNA oligonucleotide sequence (NT-shRNA) and a psi-LVRU6GH vector harboring Agtr1-targeting shRNA oligonucleotides sequences (AT1-shRNA or AT1-shRNA-2). The shRNA oligonucleotide sequences are listed in Table S1. Each vector consisted of EGFP and hygromycin selection markers. Lentiviral particles were produced by cotransfecting each shRNA vector together with third generation lentiviral packaging plasmids (Addgene), including pMDLg/pRRE (Addgene plasmid #12251), pRSV-Rev (Addgene plasmid #12253), and pCMV-VSV-G (Addgene plasmid #8454) into 293T cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific). Lentiviral packaging plasmids were gifts from Didier Trono (
      • Dull T.
      • Zufferey R.
      • Kelly M.
      • Mandel R.J.
      • Nguyen M.
      • Trono D.
      • et al.
      A third-generation lentivirus vector with a conditional packaging system.
      ). Agtr1 shRNA oligonucleotide constructs cloned into psi-LVRU6GH vectors were designed and generated by GeneCopoeia, Inc. The media were replaced 16 h posttransfection and supernatants were collected 48 h posttransfection, which were used to infect cultured cells for 48 h. The culture media was replaced with 100 μg/ml hygromycin B (Sigma-Aldrich) containing media every 2 days for 10 days. Complete cell death of no virus control was ensured before the end of the selection. After selection, knockdown efficiency was assessed using qPCR and Western blot analyses of endogenous Agtr1 mRNA and AGTR1 protein expression.

      RNA interference

      Predesigned MISSION siRNA for Klotho gene (Klotho siRNA) and MISSION siRNA Universal Negative control (Scr siRNA) were purchased from Sigma-Aldrich. RNA interference was performed in ELT3V cells with the indicated concentration of each siRNA using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific). Media containing transfection mix was replaced with no serum media 8 h posttransfection. Cells were starved overnight and treated with DMSO or losartan in 0.5% serum media for additional 24 h. Total siRNA transfection duration before harvest was 48 h. Sequences and concentrations for siRNA used are listed in Table S1.

      Plasmid

      For transient expression of Klotho (membrane), sequence cloned into pcDNA3.1/V5/His-TOPO expression vector was a gift from Dietz (Addgene plasmid # 17712; http://n2t.net/addgene:17712; RRID:Addgene_17712) (
      • Feng Y.
      • Mischler W.J.
      • Gurung A.C.
      • Kavanagh T.R.
      • Androsov G.
      • Sadow P.M.
      • et al.
      Therapeutic targeting of the secreted lysophospholipase D autotaxin suppresses tuberous sclerosis complex-associated tumorigenesis.
      ). EV, pcDNA3.1 was used as control. Two micrograms of each plasmid was transfected into ELT3V cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific). Transfection media was replaced at 8 h posttransfection and cells were harvested for analysis at 48 h after transfection.

      Quantitative RT-PCR

      Total RNA from cultured cells for each experiment were isolated using RNeasy Plus Mini kit (Qiagen), and cDNA was synthesized using amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT), according to the manufacturers’ protocol. Real-time qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories). Primers used for RT-qPCR for target genes and housekeeping genes are listed in Table S2.

      Protein extraction and Western blotting

      Cells in culture dishes were placed on ice, washed twice with cold PBS, scraped, and centrifuged into pellets. Then, the cell pellets were lysed in RIPA lysis buffer (Invitrogen) supplemented with protease and phosphatase inhibitors (Invitrogen) for 30 min with constant agitation and centrifuged at 13,000g for 15 min at 4 °C. The supernatants were collected for Western blot analysis. Equal amounts of total protein were loaded onto NuPage 4% to 12% Bis-Tris Protein Gels (Invitrogen) and then subsequently immunoblotted with the primary antibodies listed in Table S3.

      LDH colorimetric and deep blue cell viability assays

      To investigate if treatment with losartan or sKlotho induced cytotoxicity, LDH-Cytox Assay Kit (BioLegend) was used following the “homogeneous assay using viable cells” protocol for LDH measurement. Additionally, the Deep Blue Cell Viability kit (BioLegend) was used as a secondary measure of cytotoxicity induced by losartan or sKlotho treatment. For both assays, cells were cultured in a 96-well plate for 24 h, serum starved overnight, and treated with control (Water), DMSO, losartan, or sKlotho in 0.5% serum media for 24 h. For shRNA-transduced cells, an equal number of NT-shRNA or AT1-shRNA or AT1-shRNA-2 cells were cultured in a 96-well plate for 24 h, serum starved overnight, and media replaced with 0.5% serum media for additional 24 h. The measurements (luminescence or fluorescence readings) were taken and analyzed according to the manufacturers’ protocols. For LDH assay, water-treated samples were used as “low control” for percent cytotoxicity calculation. Comparisons were made between DMSO and losartan or water and sKlotho. For shRNA-transduced cells LDH assay, NT-shRNA samples were used as “low control” for percent cytotoxicity calculation. For viability assay, fluorescence levels for losartan, sKlotho, or AT1-shRNA/AT1-shRNA-2 were determined relative to DMSO, water, or NT-shRNA respectively.

      Animal studies

      All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee at Brigham and Women’s Hospital. LAM is a disease that affects women; therefore, all animal studies were performed on female mice. For the in vivo xenograft study, female immunodeficient C.B17 SCID mice (6–7 weeks old) were obtained from Taconic Biosciences and randomly divided into two groups for injection (NT-shRNA or AT1-shRNA). A total of 2.5 × 106 NT-shRNA or AT1-shRNA cells in 200 μl cell culture media supplemented with 25% Matrigel (Corning Inc) were subcutaneously injected into the suprascapular area (10 mice/group). Mice were then closely monitored for body weight and general health status every 3 days until the tumor development and every other day after. Tumors’ sizes/volumes were measured using calipers and calculated using the standard equation (1/2)(L × W2), where W is the smaller side of the tumor. All mice were euthanized when one of the mice met the institutional euthanasia criteria for xenograft tumor size (i.e., ≥2 cm in diameter). The tumors were removed, photographed, weighed, and cut into sections for qPCR and Western blot. Total RNA was isolated using a RNeasy Plus Mini kit (Qiagen), according to the manufacturer’s protocol and total protein was isolated using RIPA lysis buffer supplemented with protease and phosphatase inhibitors as previously described in section “Protein extraction and Western blotting”.
      For in vivo losartan treatment, the experiment was conducted in collaboration with the TSC Alliance Preclinical Consortium. A total of 2.5 × 106 TSC2-deficient cystadenoma 105K cells in a 1:1 ratio of DMEM and Matrigel were injected into female nude mice via subcutaneous injection. Fourteen mice were used per treatment group. Once the average xenograft tumor volume reached about 100 mm3 (day 15), mice were administered with losartan (30 mg/kg) or vehicle control daily by p.o. route for 28 consecutive days. After the treatment phase, 10 mice selected at random were monitored for an additional 28-day period for tumor regrowth analysis.

      Statistical analyses

      All data are presented as mean ± SEM of at least three independent experiments or biological replicates. Details of statistical tests and significance for each experiment are presented in the corresponding figure legends. One-sample t test was used to compare control and treatment groups when data were normalized to the control group (i.e., mean of control group expressed as 1). t test (two-tailed) was utilized to compare means between any two groups. For in vivo data, t test (two-tailed) was used to compare data between two groups. Log-rank (Mantel-Cox) test was utilized to compare the tumor-free survival. All analyses were done using GraphPad Prism 9.2.0 (GraphPad Prism Software, San Diego, CA, www.graphpad.com). p-value of less than 0.05 was considered significant.

      Data availability

      All the relevant data are contained within the article and the supporting information.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Author contributions

      S. S., M. A. P., and S. E.-C. conceptualization; S. S., E. P. H., M. A. P., C. P., and S. E.-C. methodology; E. P. H. and C. P. resources; S. S., E. A., D. J. A., D. D. T., and S. E.-C. data curation; D. J. A. formal analysis; S. E.-C. supervision; S. E.-C. funding acquisition; S. S., J. I., D. J. A., and A. M. L., investigation; S. S. writing – original draft; E. A., J. I., D. J. A., A. M. L., D. D. T., E. P. H., M. A. P., C. P., and S. E.-C. writing – review and editing.

      Funding and additional information

      This work was funded by CDMRP grant W81XWH-19-1-0250 and the Anne Levine LAM Research Fund (both to S. E.-C.).

      Supporting information

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