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

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.

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.
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 (1, 2), resulting in increased activation of the mammalian/mechanistic target of Rapamycin complex 1 (mTORC1) (3)(4)(5)(6). LAM is a multisystem disorder primarily affecting women of child-bearing age, characterized by cystic lung destruction, axial lymphatic abnormalities, and abdominal angiomyolipomas (AML) (7)(8)(9)(10)(11)(12)(13). 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 (14)(15)(16). 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 (17,18). 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 TSC2deficient 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 (19).
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 (20). A retrospective review of LAM patients receiving ACE inhibitors showed slower decline in lung function (21). 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 (22). 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.
Herein, we explored the roles of AGTR1 and Klotho in TSC2-deficient cells. We show that blocking AGTR1 in TSC2deficient 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.

AGTR1 expression is TSC2 loss-independent
Tuberin deficiency results in mTORC1 hyperactivation, leading to uncontrolled cell growth (45). 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 (46). Both cell types expressed Agtr1 mRNA and AGTR1 protein at similar levels, suggesting that AGTR1 expression is Tsc2 lossindependent (Fig. S1, A-D).

AGTR1 inhibition induces TSC2-deficient cell death in vitro
Next, to determine the effects of AGTR1 blockade on TSC2deficient 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) (47). 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 (48,49) 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).
Since losartan can also affect transforming growth factor-β signaling in a nonreceptor-dependent manner (50)(51)(52)(53) and to ascertain that the observed effects of losartan on TSC2deficient 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, D-F). 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 (44,54). 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, A-E). Taken together, our data show that Klotho is downstream of the AGTR1 receptor in TSC2-deficient cells.

AGTR1-dependent cell death is driven by Klotho in TSC2-deficient cells
To elucidate Klotho's involvement in Agtr1 blockademediated 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 AGTR1 blockade induces klotho-mediated TSC2 -/cell death with Scr siRNA (Fig. 2, E and G), suggesting that Klotho is necessary for AGTR1 blockade-dependent cell death in TSC2deficient cells. These results were also confirmed with a second Klotho siRNA (Fig. S4, F-H).
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, H-J). Taken together, these data show that Klotho is necessary and sufficient to induce TSC2-deficient cell death and that AGTR1 signaling is necessary for mTORC1hyperactive cell survival.
Next, we wanted to examine if sKlotho affects TSC2deficient 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 (55, 56). 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).

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 (23,34,36,57). 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 Figure 1. Inhibition 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. AGTR1 blockade induces klotho-mediated TSC2 -/cell death treatment at different time points on pAKT at Ser473 (not shown). Reduced activation of AKT was observed in TSC2deficient 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 TSC2deficient cells was pAKT-dependent, we treated TSC2deficient 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 Cas-pase7 cleavage (58), strongly support our hypothesis that Klotho-mediated cell death is AKT-dependent.

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.
Finally, we also evaluated these findings in another TSC2deficient 105K cell line. In vitro, we showed that TSC2deficient 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 TSC2addback ELT3T cells, inhibition of AGTR1 in TSC2-addback 105K cells with losartan treatment showed no difference in Klotho and cPARP levels (Fig. S5, A-C) 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 AGTR1 blockade induces klotho-mediated TSC2 -/cell death tumors (7,9). 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).

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 (46), and 105K cells derived from a renal tumor in a TSC2 heterozygous mouse (59). It is important to note that losartan has additional off-target receptor-independent cellular effects especially on transforming growth factor-β signaling (50)(51)(52)(53). However, shRNA-mediated knockdown of AGTR1 resulted in similar effects to losartan on TSC2deficient 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 reninangiotensin in LAM and TSC have been demonstrated in retrospective studies of lung function (21). 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 (22). 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 (22). 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 (60). Losartan has an excellent lung bioavailability (61); 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 (62), a higher losartan dose than the commonly used 100 mg orally daily (61). 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 (63). 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 (40). Klotho has previously been implicated in lung disease 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.
with effects on muco-ciliary clearance (64), airway inflammation (65), recovery from acute lung injury (66) and interstitial lung disease (67,68) 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 TSC2deficient 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 (34,36,69,70). 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 (57). While much remains to be discovered regarding the regulation of AKT signaling by the TSC1/2 complex (71), 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, Klothomediated 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 (20,21) in LAM and AML (22), 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- Figure 4. TSC2-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. AGTR1 blockade induces klotho-mediated TSC2 -/cell death 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.

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 (75). 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.

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 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. AGTR1 blockade induces klotho-mediated TSC2 -/cell death 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 shRNAtransduced 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 × 10 6 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 × W 2 ), 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 × 10 6 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 mm 3 (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 Graph-Pad 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.