Stress-Induced Catecholamines Attenuate Sorafenib Efficacy by Inhibiting Ferroptosis in β2-adrenergic Receptor Positive Renal Cell Carcinoma

Stress-Induced Catecholamines Attenuate Sorafenib Efficacy by Inhibiting Ferroptosis in β2-adrenergic Receptor Positive Renal Cell Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Stress-Induced Catecholamines Attenuate Sorafenib Efficacy by Inhibiting Ferroptosis in β2-adrenergic Receptor Positive Renal Cell Carcinoma Sei Naito, Masaki Ushijima, Hiromi Ito, Takafumi Narisawa, Osamu Ichiyanagi, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7131878/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ferroptosis, an iron-dependent form of non-apoptotic cell death, has emerged as a promising target for novel cancer therapy. While sorafenib, a molecular targeted agent for renal cell carcinoma (RCC), has been implicated in ferroptosis induction, conflicting reports persist. Chronic stress, mediated by catecholamines via β2-adrenergic receptors (ADRB2), is known to promote cancer progression, yet its influence on drug resistance and ferroptosis remains elusive. In this study, we first confirmed ADRB2 expression in RCC through immunohistochemistry of surgical specimens. Using the ADRB2-expressing RCC cell line ACHN, we found that stimulation with the β-adrenergic agonist isoproterenol (ISO) conferred resistance to sorafenib both in vitro and in a mouse stress model—an effect abrogated by ADRB2 knockdown. To assess the involvement of ferroptosis in this resistance mechanism, we employed the ferroptosis inhibitor ferrostatin-1 (Fer-1). Similar to ISO, Fer-1 diminished sorafenib sensitivity in ACHN cells. Ferroptosis inducers erastin and RSL3 markedly reduced cell viability; however, ISO attenuated ferroptosis in ACHN cells. Mechanistically, ISO upregulated DUSP1 expression and inhibited the phosphorylation of ERK1/2, p38, and JNK, all of which contribute to ferroptosis suppression. These findings provide compelling evidence that chronic stress-induced ADRB2 activation promotes sorafenib resistance in certain RCC subtypes by mitigating ferroptotic cell death. Biological sciences/Cancer/Urological cancer/Renal cancer/Renal cell carcinoma Biological sciences/Cell biology/Cell death Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In recent years, remarkable advances have been seen in the pharmacotherapy of renal cell carcinoma (RCC), including immune checkpoint inhibitors and molecular targeted drugs (MTDs). Despite these development, long-term efficacy is observed in only approximately 30% of metastatic RCC patients 1 , and many eventually develop resistant to treatment. Ferroptosis is a non-apoptotic cell death process in which phospholipids on the cell membrane are converted to lipid peroxide by the oxidant iron, and the accumulation of lipid peroxide leads to cell membrane rupture 2 , 3 . Glutathione peroxidase (GPX4) detoxifies lipid peroxides, relying on glutathione (GSH) for its function. The Cystine/Glutamic acid transporter (xCT) plays a crucial role in maintaining GSH levels. Inhibition of xCT reduces cystine availability, depleting GSH and leading to GPX4 inactivation, ultimately resulting in ferroptosis 4 . While sorafenib inhibits xCT and some studies have reported that sorafenib induces ferroptosis 5 , 6 , other reports disputed this claim 7 , leaving the discussion unresolved. Cancer patients frequently experience chronic psychological stress 8 , which triggers the secretion of catecholamine and cortisol through the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis 9 . These stress-induced hormones have been implicated in tumor progression and the development of aggressive malignancies with poor prognosis 10 , 11 , 12 , 13 . Chronic stress responses, particularly β-adrenergic receptor (ADRB) activation, are associated with cancer progression via activation of signaling pathways such as cAMP-protein kinase, extracellular signal-regulated kinase (ERK), and Akt 12 , 14 . In breast cancer, β-blockers usage has been linked to improved prognosis 15 , 16 , 17 , 18 , while tumors with abundant sympathetic innervation often exhibit worse outcomes 19 . In hepatocellular carcinoma, stimulation of β2-adrenergic receptors (ADRB2) has been reported to contribute to sorafenib treatment 20 . Despite growing evidence from other cancers, the role of ADRB2 in RCC remains poorly understood. Xie et al. demonstrated that ADRB2 undergoes lysosomal degradation mediated by the von Hipple-Lindau (VHL) protein 21 , which is commonly inactivated in clear cell RCC 22 . Secondary analysis using The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have shown that lower ADRB2 mRNA expression correlates with higher tumor stage and grade, whereas higher ADRB2 expression is associated with better overall survival (OS) 23 . In another report, ADRB2 inhibitors have tumor suppressive effects in VHL −/− RCC cells by upregulating BAX and caspase-3/7, inducing apoptosis, inhibiting angiogenesis via HIF-2α suppression, and reducing inflammation thorough p65/NF-capper B inhibition 24 . In this study, we first investigated ADRB expression through immunohistochemistry of surgical specimens. Then ADRB2-mediated stress catecholamine responses and drug resistance in RCC. We also explored the role of ferroptosis in sorafenib-induced cytotoxicity against RCC and examined how ADRB2 stimulation affects ferroptosis. Materials/Subjects and Methods Drugs and reagents Isoproterenol (ISO) was purchased from Sigma-Aldrich (MO, USA). Sorafenib, cabozantinib, lenvatinib and rapamycine were purchased from LC Laboratories (MA, USA). Ferroptosis inhibitor Ferrostatin-1 (Fer-1), a selective ferroptosis inducer RSL3, and p38 inhibitor SB203580 were purchased from Cayman Chemical (MI, USA). Apoptosis inhibitor Z-VAD-FMK (z-VAD), autophagy inhibitor 3-Methyladenin (3-MA), necroptosis inhibitor Necrostatin-1 (Nec-1), ERK1/2 pathway inhibitor PD0325901, and c-Jun N-terminal kinase (JNK) inhibitor SP600125 were purchased from Adipogen Life Sciences (CA, USA). Ferroptosis inducer erastin was purchased from Chemscene (NJ, USA). Propranolol (PRO) hydrochloride was kindly gifted from Sumitomo Seisaku (Osaka Japan). Cell culture Human RCC cell lines A-498 (VHL mutant clear cell RCC [ccRCC]), 769-P (VHL mutant ccRCC), Caki-1 (VHL null ccRCC), Caki-2 (VHL null papillary RCC [pRCC]), and ACHN (VHL null pRCC) were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 50 µg/mL kanamycin and 10% fetal bovine serum in a 5% CO2, 37°C incubator. All cell lines were verified to be free of mycoplasma contamination. Immunohistochemical staining We investigated the expression of ADRB1 and ADRB2 in primary tumors of 35 patients diagnosed with mRCC who underwent nephrectomy at our institute between 2010 and 2016 using immunohistochemical (IHC) staining. This study was approved by the Ethics Committee of Yamagata University based on the Declaration of Helsinki (approval number: 2020 − 163) and written informed consent for the use of clinical samples was obtained from all patients. Detailed procedures are shown in Supplementary Text 1. The IHC specimens were scored on a 3-point scale for the highest expression in the specimens as follows: no expression, expression, and high expression. Additionally, we examined the relationship between expression levels and clinical factors, including grade, stage, disease-free survival, and cause-specific survival. Quantitative reverse transcriptional-polymerase chain reaction (qRT-PCR) The detailed procedures of qRT-PCR are shown in Supplementary Text 2 and the primer sequences are shown in Supplementary Figure S2 . Western blot analysis Western blot analysis was performed according to the our institution's protocol 25 and the materials are shown in Supplementary Text 3. Small interfering RNA (siRNA) We performed RNA interference using siRNA to silence ADRB1 and ADRB2. The detailed procedures are shown in Supplementary Text 4. Cell viability assay (MTS assay) Cell viability was measured using MTS assay. The detailed procedures are shown in Supplementary Text 5. cyclic adenosine monophosphate (cAMP) assay GloSensor cAMP 20F™ (Promega) consists of the cAMP-binding domain of PKA and a variant of firefly luciferase. When these components bind to cAMP, a structural change occurs that leads to an increase in light output. The detailed procedures are shown in Supplementary Text 6. Stressed mouse model and sorafenib administration The detailed procedures are shown in Supplementary Text 7. All procedures were performed using female BALB/c nude mice (6–7 week old, 20–25 g) according to the animal welfare regulations of Yamagata University Faculty of Medicine based on the “Guidelines for the implementation of animal experiments” established by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. All animal studies were approved by the institutional review board of Yamagata University. The mice were randomly divided into the following four groups: control + vehicle (n = 5), control + sorafenib (n = 5), stress + vehicle (n = 5), stress + sorafenib (n = 5). Blinding was not performed in this study. The stress group of mice were subjected to stress by placed in a well ventilated 50 ml conical tube for 6 hours a day (from 9 am to 3 pm), following the previous study reported by Thaker et al. 14 . The mice were scheduled to be euthanized if their tumor size exceeded 1500 mm 3 . However, no mice reached this threshold, and none were excluded from the experiment for any reason. The sample size was calculated on a tumor volume difference of 110 mm 3 between sorafenib-treated stressed mice and sorafenib-treated non-stressed mice, with a standard deviation of 60 mm 3 , a significance level of 0.05, and a statistical power of 0.8. ADRB2-knockdown using shRNA Lentivirus vectors containing the interfering sequence of ADRB2 (shADRB2: SHCLNV MISSION shRNA TRCN0000008085) and negative control (mock:SHC001 MISSION pLKO.1-puro Empty vector control plasmid DNA) were purchased from Sigma-Aldrich. The detailed procedures are shown in Supplementary Text 8. Assessment of Malondialdehyde (MDA) Levels We detected MDA levels to assess the level of ferroptosis. The detailed procedures are shown in Supplementary Text 9. Assessment of reactive oxygen species (ROS) Measurement We detected ROS levels to assess the level of ferroptosis. The detailed procedures are shown in Supplementary Text 10. Statistical Analysis Comparisons between immunostaining levels and clinical data were performed using cross tabulation and Chisquare or Fisher’s exact tests. A Kaplan–Meier survival curve analysis and a log-rank test were used to estimate cause-specific survival (CSS). Continuous variables were expressed as median, mean ± standard deviation (SD). Normality was assessed using the Kolmogorov-smirnov test, and all data sets showed P-values greater than 0.05. Homogeneity of variance was evaluated using the F-test, and all data sets also showed P-values greater than 0.05. Group comparisons of means were statistically analyzed using either t-tests or analysis of variance (ANOVA), with post-hoc Dunnett and Bonferroni tests conducted as needed for multiple comparisons. P-value < 0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism 9.1.1 software and EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics 26 . Results Immunohistochemical staining in RCC surgical tissues In primary RCC tissues from patients with metastases, both ADRB1 and ADRB2 expressions were confirmed in all cases. Higher ADRB1 expression relative to normal tissue was observed in 29 of 35 cases (82.9%), while higher ADRB2 expression was detected in 28 of 35 cases (80.0%). No significant associations were found between ADRB expression levels and clinical factors, including grade, stage, and cause-specific survival (Fig S1 A, Table S1 ). In clear cell RCC, even within individual tissues, there was a mixture of areas that expressed ADRB and areas that did not. No clear relationship was observed between high-expression regions and specific anatomical locations, such as near the pseudo-capsule, near tumor vessels, or near necrotic areas. In papillary RCC tissue (N = 1), both ADRB1 and ADRB2 were highly expressed throughout the lesion (Fig S1 B). Expression of ADRB1 and ADRB2 mRNA and protein in RCC cell lines The mRNA expression levels of ADRB1 and ADRB2 in RCC cell lines were quantified relative to ADRB2 expression in 769-P. ADRB1 mRNA levels were comparable among A-498, Caki-1, and Caki-2, but were notably lower in 769-P. In contrast, ACHN cells exhibited an 11.7-fold higher ADRB1 expression compared to ADRB2 expression in 769-P. ADRB2 mRNA levels were elevated by 6.2-fold higher in A-498, 100-fold in Caki-1, and 640-fold in ACHN. (Fig. 1 B). Western blot analysis confirmed high protein expression of both ADRB1 and ADRB2 in ACHN (Fig. 1 C and Fig. S2 ). While ADRB1 was detected across all examined cell lines, ADRB2 expression was minimal except in ACHN. Notably, mRNA and protein expression levels were not directly correlated (Fig. 1 B, C, and Fig. S2 ). Although a previous study reported that pVHL facilitates ADRB2 degradation 24 , no correlation was observed between ADRB2 levels and VHL gene status in the present study (Fig. 1 B, C, and Fig. S2 ). Based on these findings, ACHN, a pRCC cell line with high ADRB1/2 expression, and A-498, a typical ccRCC cell line with VHL gene mutation, were selected for subsequent experiments. The effect of ADRB stimulation on RCC cell activity and therapeutic drug efficacy To assess the response of RCC cells to ADRB stimulation, cAMP levels were measured in ACHN and A-498 cells following ISO treatment using the GloSensor cAMP 20F™. In both cell lines, ISO treatment led to an increase in cAMP levels (Fig. 2 A). Cell viability was evaluated in ACHN and A-498 cells treated with ISO using the MTS assay; however, no significant changes were observed (Fig. 2 B). To examine the potential relationship between ADRB stimulation and drug resistance, an MTS assay was conducted in ACHN and A-498 with clinically relevant MTDs, including sorafenib (0–10 µM), cabozantinib (0–20 µM), lenvatinib (0–20 µM), and rapamycin (0-100 nM). The assay was performed under ISO-free conditions and under conditions mimicking physiological ISO concentrations—10 nM (blood levels) and 10 µM (neuronal terminal levels) 27 . Cell viability ratios were calculated relative to MTD-free conditions. No significant difference in cell viability was observed with 10 nM ISO compared to ISO-free conditions for sorafenib treatment. However, at the neuronal terminal level (10 µM ISO), the efficacy of sorafenib was attenuated in ACHN cells. In contrast, A-498 cells, even 10 µM ISO did not affect the effect of sorafenib. Additionally, ISO did not influence the efficacy of the other tested drugs in either ACHN or A-498 cells (Fig. 2 C). To determine whether ADRB1/2 knockdown could reverse the attenuation of sorafenib efficacy by ISO (Fig. 3 A, B, and Fig. S3 ), cell viability was assessed in ADRB1/2 knockdown ACHN cells treated with 5 µM sorafenib, with or without the addition of 10 µM ISO. While sorafenib treatment led to decrease in cell viability, the addition of ISO attenuated its effect. Among the knockdown conditions, ADRB2 knockdown—but not ADRB1 knockdown—reduced the attenuating effect of ISO on sorafenib efficacy (Fig. 3 C). Furthermore, to explore the potential therapeutic application of ADRB antagonism, cell viability was examined following co-administration of a non-specific ADRB antagonist (PRO). Although 10 µM PRO, which is not clinically achievable, slightly decreased cell viability, low concentrations of PRO failed to reverse ISO-induced attenuation of sorafenib efficacy (Fig. 3 D). Effect of stress-induced catecholamine on sorafenib resistance in rodent xenograft model To investigate the impact of stress-induced catecholamine stimulation on sorafenib resistance and the role of ADRB2 in an in vivo model, we first generated ADRB2 knockdown ACHN cells using the shRNA method. ADRB2 expression was low at both mRNA and protein levels in ADRB2 knockdown ACHN cells (Fig. S4 A). Next, mock ACHN cells were inoculated into the right flank of the mice, while ADRB2 knockdown ACHN cells were implanted into the left flank. The mice were then administered either sorafenib or vehicle under conditions of stress exposure or standard rearing. Under stress conditions, mice treated with sorafenib exhibited a significant increase in urinary adrenaline, noradrenaline, and dopamine levels (Fig. 4 A), whereas cortisol levels remained unchanged (Fig. 4 B). In control-reared mice with mock ACHN cells, sorafenib treatment effectively inhibited tumor growth. In the vehicle-treated group, stress exposure did not significantly affect tumor volume. However, in sorafenib-treated mice, the stress-exposed group demonstrated a significantly greater increase in tumor volume compared to the control-reared group. In contrast, in mice baring shADRB2 ACHN tumors, no significant differences in tumor volume changes were observed between the control-reared and stress-exposed groups under sorafenib treatment, with both groups showing suppressed tumor growth (Fig. 4 C and D). These findings suggest that stress-induced ADRB2 activation attenuates the anti-tumor effect of sorafenib. ISO mitigates ferroptosis induced by sorafenib, erastin, or RSL3 To investigate the mechanisms underlying sorafenib-induced suppression of cell viability, we assessed its effects by introducing various inhibitors, including the ferroptosis inhibitor Fer-1, the apoptosis inhibitor z-VAD, the autophagy inhibitor 3-MA, and the necroptosis inhibitor Nec-1. In ACHN, the ferroptosis inhibitor counteracted the reduction in cell viability caused by sorafenib, whereas the apoptosis, autophagy, and necroptosis inhibitors had no impact on sorafenib-induced suppression of cell viability (Fig. 5 A). These findings suggest that the sorafenib decreases cell viability in ACHN cells via ferroptosis induction. However, in A498 cells, Fer-1 did not reverse sorafenib-induced suppression of cell viability, indicating that sorafenib-induced ferroptosis exhibits cell line-dependent variability. Next, we examined the effects of ISO on established ferroptosis inducers, erastin and RSL3. Although these inducers decreased cell viability, ISO mitigated their effects (Fig. 5 B). Additionally, erastin increased levels of the lipid peroxidation marker malondialdehyde (MDA), but this increase was inhibited by ISO (Fig. 5 C). Similarly, erastin treatment led to increased reactive oxygen species (ROS) levels, which were suppressed by ISO (Fig. 5 D). These results indicate that ISO suppresses ferroptosis induced by erastin and RSL3. Mechanism of ISO-induced ferroptosis resistance A previous study demonstrated that adrenaline increases dual-specificity phosphatase 1 (DUSP1), which inhibits the phosphorylation of mitogen-activated protein kinases (MAPKs) 28 . Additionally, multiple studies have shown that MAPK pathways—ERK1/2, p38, and JNK pathways—enhance ferroptosis induction 6 , 29 , 30 . To investigate the mechanisms through which ISO modulates ferroptosis, we first treated cells with ISO and examined its effect on DUSP1 expression and MAPK phosphorylation. As expected, ISO up-regulated DUSP1. Phosphorylation of JNK and ERK was inhibited at both 10 nM and 10 µM ISO concentrations, whereas inhibition of p38 phosphorylation was observed only at the higher concentrations, not at the lower concentrations (Fig. 6 A). Next, we assessed cell viability following treatment with ferroptosis inducers and MAPK inhibitors. The JNK inhibitor SP600125 and the ERK1/2 inhibitor PD0325901 attenuated the effects of sorafenib and erastin, and inhibition of two or more MAPK pathways further suppressed ferroptosis induction (Fig. 6 B and C). Discussion In this study, we demonstrated that ADRB2 is expressed in RCC cells and that its activation mitigates sorafenib-induced ferroptosis both in vitro and in vivo. ADRB1/2 proteins were detected in all examined RCC specimens. While transcriptomic data from TCGA and ICGC indicated that higher ADRB2 mRNA expression correlated with lower stage and improved OS 23 , our findings did not establish an association between ADRB2 protein expression and clinical factors. This discrepancy may stem from widely observed lack of correlation between protein and mRNA expression levels in G protein coupled receptors 31 , 32 , a trend consistent with our findings that ADRB1/2 mRNA and protein levels were not aligned. A previous study reported that VHL protein promoted ADRB2 degradation, leading to ADRB2 accumulation in clear cell RCC, which harbors VHL gene inactivation 21 . However, in our cell line study, ADRB2 protein expression did not correlate with VHL gene status. In most clinical samples, there were regions within the same specimen where ADRB2 was expressed and others where it was not, suggesting the presence of an unidentified mechanism regulating ADRB2 expression that warrants further investigation. Chronic stress is well-documented to negatively influence cancer progression 8 , 14 , largely via catecholamine-mediated mechanisms 10 , 11 , 12 . In our study, while ISO alone did not affect cell viability, it suppressed the anti-tumor effect of sorafenib specifically in ACHN cells, an ADRB2-expressing cell line. This ISO-induced suppression was inhibited by high doses of PRO and ADRB2 knock-down. These findings suggest that ADRB2 activation underlines this phenomenon; however, clinically relevant doses of PRO failed to restore the sorafenib effect. Additionally, ISO-induced suppression required high-dose ISO, consistent with concentrations expected in nerve terminals 27 , but not lower ISO levels corresponding to blood concentrations. A prior study reported that PRO alone suppressed RCC cell viability 24 , yet our findings did not corroborate this effect. Albiñana et al. demonstrated that adding PRO or an ADRB2 inhibitor to 786O cells reduced ROS levels by enhancing the activity of enzymes involved in ROS metabolism, particularly glutathione peroxidase 33 . While this result may seem contradictory to our observation that ADRB2 activation attenuates ferroptosis, it does not necessarily negate their findings within the context of their experimental system, in which GSH was present. This distinction is critical, as xCT inhibition by sorafenib, erastin, or RSL3 suppresses GSH synthesis, a vital factor in mitigating ROS-induced damage. Furthermore, ROS plays a complex role in cellular processes, with high ROS levels triggering ferroptosis-induced cell death in tumor cells, as observed in our study, whereas moderate ROS levels have been reported to promote tumor proliferation and activate pro-survival signaling pathways 34 . Our investigation into the inhibitory effect of ISO on ferroptosis revealed the involvement of MAPKs in ferroptotic regulation, consistent with prior research 35 . Previous studies have shown that adrenalin increases DUSP1, thereby inhibiting MAPK phosphorylation 28 . Additionally, noradrenaline-induced DUSP1 expression is mediated through the activation of the cAMP-PLC-PKC-CREB signaling pathway via ADRB2, which subsequently inhibits JNK-mediated c-Jun phosphorylation and protects ovarian cancer cells from apoptosis 36 . In our study, high concentration of ISO suppressed MAPK phosphorylation, thereby inhibiting ferroptosis. Currently, immune checkpoint inhibitors and MTDs targeting for vascular endothelial growth factor receptor (VEGFR) constitute the primary therapeutic approaches for RCC, while agents with direct cytotoxic effects remain limited and often exhibit insufficient efficacy. Sorafenib, an MTD that targets multiple kinases—including VEGFR family, platelet-derived growth factor receptor family, c-kit, raf, p38, FMS-like tyrosine kinase 3, and xCT—was historically widely used for RCC treatment 5 but is no longer prioritized in contemporary clinical practice, as per current clinical guidelines 1 . Although sorafenib has been reported to induce ferroptosis via xCT inhibition 37 , 38 , findings regarding its cytotoxic effects remain inconclusive 7 . Our findings further revealed that sorafenib induced ferroptosis in ACHN cells, but not in A498 cells, highlighting cell line-dependent variability in ferroptosis sensitivity. This variability may contribute to the mixed clinical responses to sorafenib, with certain RCC tumors undergoing sorafenib-induced ferroptosis, while others remain resistant. The mechanism by which sorafenib induces ferroptosis appears to vary among cell lines and remains unresolved, posing a key challenge for future research. Another limitation of this study is the use of a restraint model for stress induction in mice, which may introduce confounding factors beyond catecholamine-mediated effects. In conclusion, Catecholamine stimulation attenuates ferroptosis. Consequently, ADRB2 activation under chronic stress compromises sorafenib efficacy by suppressing ferroptosis in RCC. Declarations Conflict of Interest This work was supported by JSPS KAKENHI Grant Number JP20K18085, which Hidenori Kanno received, and JSPS KAKENHI Grant Number 23K15777, which Atsushi Yamagishi received. Sei Naito received honoraria from Pfizer Japan Inc., Merk biopharma Japan Inc., Bristol Meier’s squib Japan Inc., Ono Pharma, MSD Japan, and Eisai Co. as their sponsored speaker. Norihiko Tsuchiya received honoraria from Pfizer Japan Inc. Janssen, Novartis, Ono, Bayer, Sanofi, Takeda Pharm, Bristol-Myers Squibb Japan, and Astelas Pharma., as their sponsored speaker and research funds from Pfizer Japan Inc. and Eisai outside the submitted work. The other authors have declared that no conflict of interest exists. Availability of Data and Materials The data generated in this study are publicly available in Sei Naito at [email protected] Acknowledgements The authors would like to thank the patients for consenting to the use of their surgical specimens. 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Additional Declarations (Not answered) Supplementary Files SupplementaryText8.docx Supplementary Text 8 SupplementaryText2.docx Supplementary Text 2 SupplementaryText3.docx Supplementary Text 3 SupplementaryText6.docx Supplementary Text 6 SupplementaryText5.docx Supplementary Text 5 SupplementaryText4.docx Supplementary Text 4 SupplementaryText1.docx Supplementary Text 1 SupplementaryText9.docx Supplementary Text 9 SupplementaryText7.docx Supplementary Text 7 SupplementTableS1.tif Supplement Table S1 SupplementaryText10.docx Supplementary Text 10 SupplementFig.S4.tif Supplement Fig. S4 SupplementFig.S5.tif Supplement Fig. S5 SupplementFig.S2.tif Supplement Fig. S2 SupplementFig.S1.tif Supplement Fig. S1 SupplementFig.S3.tif Supplement Fig. S3 TableS1.png Supplement Table S1 SupplementaryTableS3.xlsx Supplementary Table S3 FigS1.png Supplement Fig. S1 FigS3.png Supplement Fig. S3 FigS5.png Supplement Fig. S5 SupplementaryTableS2.xlsx Supplementary Table S2 FigS2.png Supplement Fig. S2 FigS4.png Supplement Fig. S4 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7131878","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":501211917,"identity":"006c457c-7493-4c67-9c45-9f4d2e904153","order_by":0,"name":"Sei 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14:55:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7131878/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7131878/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89975900,"identity":"95a1b8a2-85f2-482e-8a23-47606ccbc765","added_by":"auto","created_at":"2025-08-27 05:59:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127079,"visible":true,"origin":"","legend":"\u003cp\u003eADRB1/2 expression in RCC. A: Immunohistochemical staining of ADBR1/2 in surgical specimens. The pictures show representative staining in normal and tumor tissues. All specimens expressed both ADRB1/2, around 80% of which showed highly expression. B: ADRB1/2 mRNA expression in RCC cell lines measured by qRT-PCR method. C: ADRB1/2 protein expression in RCC cell lines measured by Western blot analysis. Abbreviations: ADRB, β2-adrenergic receptors; RCC, renal cell carcinoma; qRT-PCR, quantitative reverse transcriptional-polymerase chain reaction\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/95c7f19e96243509ec10e0c7.jpg"},{"id":89975898,"identity":"39be06b4-814e-4935-9871-d589123d7ef8","added_by":"auto","created_at":"2025-08-27 05:59:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97249,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ADRB stimulation in RCC cell lines. A: cAMP levels after ISO stimulation. B: Cell viability before and after ISO stimulation measured by MTS assay. C: Cell viability after treatment of sorafenib, cabozantinib, lenvatinib, or rapamycin under ISO stimulation in ACHN or A498 cells. Abbreviations: ADRB, β2-adrenergic receptors; RCC, renal cell carcinoma; cAMP. cyclic adenosine monophosphate; ISO, isoproterenol.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/22f1e97059dddc116faa3e6b.png"},{"id":89976850,"identity":"90b89c9e-4163-46f3-a392-303b5c0c0dbf","added_by":"auto","created_at":"2025-08-27 06:07:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92406,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of ADRB1/2. A: ADRB1/2 mRNA expression. B: ADRB1/2 protein expression. C. Cell viability of ACHN cells treated by sorafenib or ISO + sorafenib under ADRB1/2 knockdown. D: Cell viability of ACHN cells treated by PRO and/or sorafenib and/or ISO. .Abbreviations: ADRB, β2-adrenergic receptors; RCC, renal cell carcinoma; ISO, isoproterenol; PRO, propranolol.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/3b84562ff544c34beec640b6.png"},{"id":89975944,"identity":"292fbc75-ae85-4048-92fc-61c1cf648961","added_by":"auto","created_at":"2025-08-27 05:59:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":145345,"visible":true,"origin":"","legend":"\u003cp\u003eStress-induced catecholamine on sorafenib resistance in rodent model. A: Urine catecholamine levels under conditions of sorafenib and/or stress. B: Serum cortisol levels under conditions of sorafenib and/or stress. C: Tumor volume in mice treated stress and/or sorafenib in mock and shADRB2 tumor. D: Pictures of tumor treated stress and/or sorafenib in mock and shADRB2 tumor. Abbreviations: ADRB, β2-adrenergic receptors\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/ec2a9b1df5c96376e09e3c73.jpg"},{"id":89975947,"identity":"fca4a55b-60ad-48b7-b243-682db9483693","added_by":"auto","created_at":"2025-08-27 05:59:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":97132,"visible":true,"origin":"","legend":"\u003cp\u003eISO mitigates ferroptosis induced by sorafenib, erastin, or RSL3. A: Cell viability of ACHN or A498 treated by sorafenib under treatment of the ferroptosis inhibitor Fer-1, the apoptosis inhibitor z-VAD, the autophagy inhibitor 3-MA, and the necroptosis inhibitor Nec-1. B: Cell viability of ACHN treated by ISO and erastin or RSL3. C: MDA treated by erastin ± ISO. D: ROS level treated by erastin ± ISO. Abbreviations: ISO, isoproterenol; MDA, malondialdehyde; ROS, reactive oxygen species.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/5ed46e43555a229f105bddb0.png"},{"id":89975908,"identity":"92c07d3d-9a57-47fb-b783-28483d069a98","added_by":"auto","created_at":"2025-08-27 05:59:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":82613,"visible":true,"origin":"","legend":"\u003cp\u003eISO upregulates DUSP1 and suppresses MAPK phosphorylation, which mitigates the effect of sorafenib and erastin. A: The levels of DUSP1 and MAPK phosphorylation treated by ISO. B: Cell viability of ACHN treated by sorafenib and/or MAPK inhibitors, including p38 inhibitor SB203580, JNK inhibitor SP600125, and ERK1/2 inhibitor PD0325901. C: Cell viability of ACHN treated by erastin and/or MAPK inhibitors, including p38 inhibitor SB203580, JNK inhibitor SP600125, and ERK1/2 inhibitor PD0325901. Abbreviations: ISO, isoproterenol; DUSP1, dual-specific phosphatase 1; JNK, c-JUN N-terminus kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/fa23b9ae998d86e3ce693df6.jpg"},{"id":91098733,"identity":"54aace5e-caa6-4955-8ec4-c538db5bbd8d","added_by":"auto","created_at":"2025-09-11 14:28:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1573022,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/38ad1f7c-8a1f-48e2-b057-135ed6b51bd6.pdf"},{"id":89975913,"identity":"f70b83e0-bdf7-427e-9673-b5ee2641a2c9","added_by":"auto","created_at":"2025-08-27 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05:59:42","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":872395,"visible":true,"origin":"","legend":"Supplement Table S1","description":"","filename":"TableS1.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/8d7831cf757e2532fe82f7d0.png"},{"id":89975914,"identity":"f0a33a1f-5591-49c1-b541-f0dfa1fdc9d3","added_by":"auto","created_at":"2025-08-27 05:59:41","extension":"xlsx","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":18525,"visible":true,"origin":"","legend":"Supplementary Table S3","description":"","filename":"SupplementaryTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/1f7f7b2f8f49cf64442f0033.xlsx"},{"id":89975937,"identity":"678990ce-66be-42dc-9458-92ce3f9e3891","added_by":"auto","created_at":"2025-08-27 05:59:42","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":18812221,"visible":true,"origin":"","legend":"Supplement Fig. S1","description":"","filename":"FigS1.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/195dd5b92af1cb03be3b2520.png"},{"id":89976867,"identity":"84912f2f-3a2b-46bd-8b94-a7c54936efec","added_by":"auto","created_at":"2025-08-27 06:07:42","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":8993087,"visible":true,"origin":"","legend":"Supplement Fig. S3","description":"","filename":"FigS3.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/3e9322d5438a269f3669eac4.png"},{"id":89975954,"identity":"1796e100-0925-4662-953f-cfbb0490194c","added_by":"auto","created_at":"2025-08-27 05:59:43","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"supplement","size":11719126,"visible":true,"origin":"","legend":"Supplement Fig. S5","description":"","filename":"FigS5.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/37aff53f71a900919d765150.png"},{"id":89975957,"identity":"5fae2d06-afe5-458d-b3d2-8173db7ff983","added_by":"auto","created_at":"2025-08-27 05:59:43","extension":"xlsx","order_by":22,"title":"","display":"","copyAsset":false,"role":"supplement","size":13921,"visible":true,"origin":"","legend":"Supplementary Table S2","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/ef847a4790dd3e6008844358.xlsx"},{"id":89975960,"identity":"ee6c8d2d-af27-492d-911d-ab770044a5cc","added_by":"auto","created_at":"2025-08-27 05:59:43","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"supplement","size":12150047,"visible":true,"origin":"","legend":"Supplement Fig. S2","description":"","filename":"FigS2.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/35a2ac6ecb43493ed1211436.png"},{"id":89978620,"identity":"0d11a20b-184c-4aa5-b990-746636f28457","added_by":"auto","created_at":"2025-08-27 06:15:43","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"supplement","size":1230875,"visible":true,"origin":"","legend":"Supplement Fig. S4","description":"","filename":"FigS4.png","url":"https://assets-eu.researchsquare.com/files/rs-7131878/v1/b88b6903a80d700415ff92e5.png"}],"financialInterests":"(Not answered)","formattedTitle":"Stress-Induced Catecholamines Attenuate Sorafenib Efficacy by Inhibiting Ferroptosis in β2-adrenergic Receptor Positive Renal Cell Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, remarkable advances have been seen in the pharmacotherapy of renal cell carcinoma (RCC), including immune checkpoint inhibitors and molecular targeted drugs (MTDs). Despite these development, long-term efficacy is observed in only approximately 30% of metastatic RCC patients\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and many eventually develop resistant to treatment.\u003c/p\u003e\u003cp\u003eFerroptosis is a non-apoptotic cell death process in which phospholipids on the cell membrane are converted to lipid peroxide by the oxidant iron, and the accumulation of lipid peroxide leads to cell membrane rupture\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Glutathione peroxidase (GPX4) detoxifies lipid peroxides, relying on glutathione (GSH) for its function. The Cystine/Glutamic acid transporter (xCT) plays a crucial role in maintaining GSH levels. Inhibition of xCT reduces cystine availability, depleting GSH and leading to GPX4 inactivation, ultimately resulting in ferroptosis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. While sorafenib inhibits xCT and some studies have reported that sorafenib induces ferroptosis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, other reports disputed this claim\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, leaving the discussion unresolved.\u003c/p\u003e\u003cp\u003eCancer patients frequently experience chronic psychological stress\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, which triggers the secretion of catecholamine and cortisol through the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These stress-induced hormones have been implicated in tumor progression and the development of aggressive malignancies with poor prognosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Chronic stress responses, particularly β-adrenergic receptor (ADRB) activation, are associated with cancer progression via activation of signaling pathways such as cAMP-protein kinase, extracellular signal-regulated kinase (ERK), and Akt\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In breast cancer, β-blockers usage has been linked to improved prognosis \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, while tumors with abundant sympathetic innervation often exhibit worse outcomes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In hepatocellular carcinoma, stimulation of β2-adrenergic receptors (ADRB2) has been reported to contribute to sorafenib treatment\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite growing evidence from other cancers, the role of ADRB2 in RCC remains poorly understood. Xie et al. demonstrated that ADRB2 undergoes lysosomal degradation mediated by the von Hipple-Lindau (VHL) protein\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, which is commonly inactivated in clear cell RCC\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Secondary analysis using The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have shown that lower ADRB2 mRNA expression correlates with higher tumor stage and grade, whereas higher ADRB2 expression is associated with better overall survival (OS)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In another report, ADRB2 inhibitors have tumor suppressive effects in \u003cem\u003eVHL\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e RCC cells by upregulating BAX and caspase-3/7, inducing apoptosis, inhibiting angiogenesis via HIF-2α suppression, and reducing inflammation thorough p65/NF-capper B inhibition\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In this study, we first investigated ADRB expression through immunohistochemistry of surgical specimens. Then ADRB2-mediated stress catecholamine responses and drug resistance in RCC. We also explored the role of ferroptosis in sorafenib-induced cytotoxicity against RCC and examined how ADRB2 stimulation affects ferroptosis.\u003c/p\u003e"},{"header":"Materials/Subjects and Methods","content":"\u003cp\u003e\u003cb\u003eDrugs and reagents\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIsoproterenol (ISO) was purchased from Sigma-Aldrich (MO, USA). Sorafenib, cabozantinib, lenvatinib and rapamycine were purchased from LC Laboratories (MA, USA). Ferroptosis inhibitor Ferrostatin-1 (Fer-1), a selective ferroptosis inducer RSL3, and p38 inhibitor SB203580 were purchased from Cayman Chemical (MI, USA). Apoptosis inhibitor Z-VAD-FMK (z-VAD), autophagy inhibitor 3-Methyladenin (3-MA), necroptosis inhibitor Necrostatin-1 (Nec-1), ERK1/2 pathway inhibitor PD0325901, and c-Jun N-terminal kinase (JNK) inhibitor SP600125 were purchased from Adipogen Life Sciences (CA, USA). Ferroptosis inducer erastin was purchased from Chemscene (NJ, USA). Propranolol (PRO) hydrochloride was kindly gifted from Sumitomo Seisaku (Osaka Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHuman RCC cell lines A-498 (VHL mutant clear cell RCC [ccRCC]), 769-P (VHL mutant ccRCC), Caki-1 (VHL null ccRCC), Caki-2 (VHL null papillary RCC [pRCC]), and ACHN (VHL null pRCC) were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 50 \u0026micro;g/mL kanamycin and 10% fetal bovine serum in a 5% CO2, 37\u0026deg;C incubator. All cell lines were verified to be free of mycoplasma contamination.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemical staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe investigated the expression of ADRB1 and ADRB2 in primary tumors of 35 patients diagnosed with mRCC who underwent nephrectomy at our institute between 2010 and 2016 using immunohistochemical (IHC) staining. This study was approved by the Ethics Committee of Yamagata University based on the Declaration of Helsinki (approval number: 2020\u0026thinsp;\u0026minus;\u0026thinsp;163) and written informed consent for the use of clinical samples was obtained from all patients. Detailed procedures are shown in Supplementary Text 1. The IHC specimens were scored on a 3-point scale for the highest expression in the specimens as follows: no expression, expression, and high expression. Additionally, we examined the relationship between expression levels and clinical factors, including grade, stage, disease-free survival, and cause-specific survival.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantitative reverse transcriptional-polymerase chain reaction (qRT-PCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe detailed procedures of qRT-PCR are shown in Supplementary Text 2 and the primer sequences are shown in Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWestern blot analysis was performed according to the our institution's protocol \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and the materials are shown in Supplementary Text 3.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSmall interfering RNA (siRNA)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe performed RNA interference using siRNA to silence ADRB1 and ADRB2. The detailed procedures are shown in Supplementary Text 4.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell viability assay (MTS assay)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell viability was measured using MTS assay. The detailed procedures are shown in Supplementary Text 5.\u003c/p\u003e\u003cp\u003e\u003cb\u003ecyclic adenosine monophosphate (cAMP) assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGloSensor cAMP 20F\u0026trade; (Promega) consists of the cAMP-binding domain of PKA and a variant of firefly luciferase. When these components bind to cAMP, a structural change occurs that leads to an increase in light output. The detailed procedures are shown in Supplementary Text 6.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStressed mouse model and sorafenib administration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe detailed procedures are shown in Supplementary Text 7. All procedures were performed using female BALB/c nude mice (6\u0026ndash;7 week old, 20\u0026ndash;25 g) according to the animal welfare regulations of Yamagata University Faculty of Medicine based on the \u0026ldquo;Guidelines for the implementation of animal experiments\u0026rdquo; established by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. All animal studies were approved by the institutional review board of Yamagata University. The mice were randomly divided into the following four groups: control\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;5), control\u0026thinsp;+\u0026thinsp;sorafenib (n\u0026thinsp;=\u0026thinsp;5), stress\u0026thinsp;+\u0026thinsp;vehicle (n\u0026thinsp;=\u0026thinsp;5), stress\u0026thinsp;+\u0026thinsp;sorafenib (n\u0026thinsp;=\u0026thinsp;5). Blinding was not performed in this study. The stress group of mice were subjected to stress by placed in a well ventilated 50 ml conical tube for 6 hours a day (from 9 am to 3 pm), following the previous study reported by Thaker et al.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The mice were scheduled to be euthanized if their tumor size exceeded 1500 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, no mice reached this threshold, and none were excluded from the experiment for any reason. The sample size was calculated on a tumor volume difference of 110 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e between sorafenib-treated stressed mice and sorafenib-treated non-stressed mice, with a standard deviation of 60 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, a significance level of 0.05, and a statistical power of 0.8.\u003c/p\u003e\u003cp\u003e\u003cb\u003eADRB2-knockdown using shRNA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLentivirus vectors containing the interfering sequence of ADRB2 (shADRB2: SHCLNV MISSION shRNA TRCN0000008085) and negative control (mock:SHC001 MISSION pLKO.1-puro Empty vector control plasmid DNA) were purchased from Sigma-Aldrich. The detailed procedures are shown in Supplementary Text 8.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of Malondialdehyde (MDA) Levels\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe detected MDA levels to assess the level of ferroptosis. The detailed procedures are shown in Supplementary Text 9.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of reactive oxygen species (ROS) Measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe detected ROS levels to assess the level of ferroptosis. The detailed procedures are shown in Supplementary Text 10.\u003c/p\u003e\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eComparisons between immunostaining levels and clinical data were performed using cross tabulation and Chisquare or Fisher\u0026rsquo;s exact tests. A Kaplan\u0026ndash;Meier survival curve analysis and a log-rank test were used to estimate cause-specific survival (CSS). Continuous variables were expressed as median, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Normality was assessed using the Kolmogorov-smirnov test, and all data sets showed P-values greater than 0.05. Homogeneity of variance was evaluated using the F-test, and all data sets also showed P-values greater than 0.05. Group comparisons of means were statistically analyzed using either t-tests or analysis of variance (ANOVA), with post-hoc Dunnett and Bonferroni tests conducted as needed for multiple comparisons. P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism 9.1.1 software and EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eImmunohistochemical staining in RCC surgical tissues\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn primary RCC tissues from patients with metastases, both ADRB1 and ADRB2 expressions were confirmed in all cases. Higher ADRB1 expression relative to normal tissue was observed in 29 of 35 cases (82.9%), while higher ADRB2 expression was detected in 28 of 35 cases (80.0%). No significant associations were found between ADRB expression levels and clinical factors, including grade, stage, and cause-specific survival (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn clear cell RCC, even within individual tissues, there was a mixture of areas that expressed ADRB and areas that did not. No clear relationship was observed between high-expression regions and specific anatomical locations, such as near the pseudo-capsule, near tumor vessels, or near necrotic areas. In papillary RCC tissue (N\u0026thinsp;=\u0026thinsp;1), both ADRB1 and ADRB2 were highly expressed throughout the lesion (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of ADRB1 and ADRB2 mRNA and protein in RCC cell lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mRNA expression levels of ADRB1 and ADRB2 in RCC cell lines were quantified relative to ADRB2 expression in 769-P. ADRB1 mRNA levels were comparable among A-498, Caki-1, and Caki-2, but were notably lower in 769-P. In contrast, ACHN cells exhibited an 11.7-fold higher ADRB1 expression compared to ADRB2 expression in 769-P. ADRB2 mRNA levels were elevated by 6.2-fold higher in A-498, 100-fold in Caki-1, and 640-fold in ACHN. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWestern blot analysis confirmed high protein expression of both ADRB1 and ADRB2 in ACHN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). While ADRB1 was detected across all examined cell lines, ADRB2 expression was minimal except in ACHN. Notably, mRNA and protein expression levels were not directly correlated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough a previous study reported that pVHL facilitates ADRB2 degradation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, no correlation was observed between ADRB2 levels and \u003cem\u003eVHL\u003c/em\u003e gene status in the present study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Based on these findings, ACHN, a pRCC cell line with high ADRB1/2 expression, and A-498, a typical ccRCC cell line with \u003cem\u003eVHL\u003c/em\u003e gene mutation, were selected for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe effect of ADRB stimulation on RCC cell activity and therapeutic drug efficacy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the response of RCC cells to ADRB stimulation, cAMP levels were measured in ACHN and A-498 cells following ISO treatment using the GloSensor cAMP 20F\u0026trade;. In both cell lines, ISO treatment led to an increase in cAMP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cell viability was evaluated in ACHN and A-498 cells treated with ISO using the MTS assay; however, no significant changes were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To examine the potential relationship between ADRB stimulation and drug resistance, an MTS assay was conducted in ACHN and A-498 with clinically relevant MTDs, including sorafenib (0\u0026ndash;10 \u0026micro;M), cabozantinib (0\u0026ndash;20 \u0026micro;M), lenvatinib (0\u0026ndash;20 \u0026micro;M), and rapamycin (0-100 nM). The assay was performed under ISO-free conditions and under conditions mimicking physiological ISO concentrations\u0026mdash;10 nM (blood levels) and 10 \u0026micro;M (neuronal terminal levels)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Cell viability ratios were calculated relative to MTD-free conditions. No significant difference in cell viability was observed with 10 nM ISO compared to ISO-free conditions for sorafenib treatment. However, at the neuronal terminal level (10 \u0026micro;M ISO), the efficacy of sorafenib was attenuated in ACHN cells. In contrast, A-498 cells, even 10 \u0026micro;M ISO did not affect the effect of sorafenib. Additionally, ISO did not influence the efficacy of the other tested drugs in either ACHN or A-498 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether ADRB1/2 knockdown could reverse the attenuation of sorafenib efficacy by ISO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, and Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), cell viability was assessed in ADRB1/2 knockdown ACHN cells treated with 5 \u0026micro;M sorafenib, with or without the addition of 10 \u0026micro;M ISO. While sorafenib treatment led to decrease in cell viability, the addition of ISO attenuated its effect. Among the knockdown conditions, ADRB2 knockdown\u0026mdash;but not ADRB1 knockdown\u0026mdash;reduced the attenuating effect of ISO on sorafenib efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, to explore the potential therapeutic application of ADRB antagonism, cell viability was examined following co-administration of a non-specific ADRB antagonist (PRO). Although 10 \u0026micro;M PRO, which is not clinically achievable, slightly decreased cell viability, low concentrations of PRO failed to reverse ISO-induced attenuation of sorafenib efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of stress-induced catecholamine on sorafenib resistance in rodent xenograft model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the impact of stress-induced catecholamine stimulation on sorafenib resistance and the role of ADRB2 in an \u003cem\u003ein vivo\u003c/em\u003e model, we first generated ADRB2 knockdown ACHN cells using the shRNA method. ADRB2 expression was low at both mRNA and protein levels in ADRB2 knockdown ACHN cells (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eNext, mock ACHN cells were inoculated into the right flank of the mice, while ADRB2 knockdown ACHN cells were implanted into the left flank. The mice were then administered either sorafenib or vehicle under conditions of stress exposure or standard rearing. Under stress conditions, mice treated with sorafenib exhibited a significant increase in urinary adrenaline, noradrenaline, and dopamine levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), whereas cortisol levels remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn control-reared mice with mock ACHN cells, sorafenib treatment effectively inhibited tumor growth. In the vehicle-treated group, stress exposure did not significantly affect tumor volume. However, in sorafenib-treated mice, the stress-exposed group demonstrated a significantly greater increase in tumor volume compared to the control-reared group. In contrast, in mice baring shADRB2 ACHN tumors, no significant differences in tumor volume changes were observed between the control-reared and stress-exposed groups under sorafenib treatment, with both groups showing suppressed tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). These findings suggest that stress-induced ADRB2 activation attenuates the anti-tumor effect of sorafenib.\u003c/p\u003e\u003cp\u003e\u003cb\u003eISO mitigates ferroptosis induced by sorafenib, erastin, or RSL3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the mechanisms underlying sorafenib-induced suppression of cell viability, we assessed its effects by introducing various inhibitors, including the ferroptosis inhibitor Fer-1, the apoptosis inhibitor z-VAD, the autophagy inhibitor 3-MA, and the necroptosis inhibitor Nec-1. In ACHN, the ferroptosis inhibitor counteracted the reduction in cell viability caused by sorafenib, whereas the apoptosis, autophagy, and necroptosis inhibitors had no impact on sorafenib-induced suppression of cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These findings suggest that the sorafenib decreases cell viability in ACHN cells via ferroptosis induction. However, in A498 cells, Fer-1 did not reverse sorafenib-induced suppression of cell viability, indicating that sorafenib-induced ferroptosis exhibits cell line-dependent variability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we examined the effects of ISO on established ferroptosis inducers, erastin and RSL3. Although these inducers decreased cell viability, ISO mitigated their effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Additionally, erastin increased levels of the lipid peroxidation marker malondialdehyde (MDA), but this increase was inhibited by ISO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Similarly, erastin treatment led to increased reactive oxygen species (ROS) levels, which were suppressed by ISO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results indicate that ISO suppresses ferroptosis induced by erastin and RSL3.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanism of ISO-induced ferroptosis resistance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA previous study demonstrated that adrenaline increases dual-specificity phosphatase 1 (DUSP1), which inhibits the phosphorylation of mitogen-activated protein kinases (MAPKs)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Additionally, multiple studies have shown that MAPK pathways\u0026mdash;ERK1/2, p38, and JNK pathways\u0026mdash;enhance ferroptosis induction\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To investigate the mechanisms through which ISO modulates ferroptosis, we first treated cells with ISO and examined its effect on DUSP1 expression and MAPK phosphorylation. As expected, ISO up-regulated DUSP1. Phosphorylation of JNK and ERK was inhibited at both 10 nM and 10 \u0026micro;M ISO concentrations, whereas inhibition of p38 phosphorylation was observed only at the higher concentrations, not at the lower concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Next, we assessed cell viability following treatment with ferroptosis inducers and MAPK inhibitors. The JNK inhibitor SP600125 and the ERK1/2 inhibitor PD0325901 attenuated the effects of sorafenib and erastin, and inhibition of two or more MAPK pathways further suppressed ferroptosis induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that ADRB2 is expressed in RCC cells and that its activation mitigates sorafenib-induced ferroptosis both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo.\u003c/em\u003e ADRB1/2 proteins were detected in all examined RCC specimens. While transcriptomic data from TCGA and ICGC indicated that higher ADRB2 mRNA expression correlated with lower stage and improved OS\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, our findings did not establish an association between ADRB2 protein expression and clinical factors. This discrepancy may stem from widely observed lack of correlation between protein and mRNA expression levels in G protein coupled receptors\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, a trend consistent with our findings that ADRB1/2 mRNA and protein levels were not aligned. A previous study reported that VHL protein promoted ADRB2 degradation, leading to ADRB2 accumulation in clear cell RCC, which harbors \u003cem\u003eVHL\u003c/em\u003e gene inactivation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, in our cell line study, ADRB2 protein expression did not correlate with \u003cem\u003eVHL\u003c/em\u003e gene status. In most clinical samples, there were regions within the same specimen where ADRB2 was expressed and others where it was not, suggesting the presence of an unidentified mechanism regulating ADRB2 expression that warrants further investigation.\u003c/p\u003e\u003cp\u003eChronic stress is well-documented to negatively influence cancer progression\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, largely via catecholamine-mediated mechanisms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In our study, while ISO alone did not affect cell viability, it suppressed the anti-tumor effect of sorafenib specifically in ACHN cells, an ADRB2-expressing cell line. This ISO-induced suppression was inhibited by high doses of PRO and ADRB2 knock-down. These findings suggest that ADRB2 activation underlines this phenomenon; however, clinically relevant doses of PRO failed to restore the sorafenib effect. Additionally, ISO-induced suppression required high-dose ISO, consistent with concentrations expected in nerve terminals\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, but not lower ISO levels corresponding to blood concentrations. A prior study reported that PRO alone suppressed RCC cell viability\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, yet our findings did not corroborate this effect.\u003c/p\u003e\u003cp\u003eAlbi\u0026ntilde;ana et al. demonstrated that adding PRO or an ADRB2 inhibitor to 786O cells reduced ROS levels by enhancing the activity of enzymes involved in ROS metabolism, particularly glutathione peroxidase\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. While this result may seem contradictory to our observation that ADRB2 activation attenuates ferroptosis, it does not necessarily negate their findings within the context of their experimental system, in which GSH was present. This distinction is critical, as xCT inhibition by sorafenib, erastin, or RSL3 suppresses GSH synthesis, a vital factor in mitigating ROS-induced damage. Furthermore, ROS plays a complex role in cellular processes, with high ROS levels triggering ferroptosis-induced cell death in tumor cells, as observed in our study, whereas moderate ROS levels have been reported to promote tumor proliferation and activate pro-survival signaling pathways\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur investigation into the inhibitory effect of ISO on ferroptosis revealed the involvement of MAPKs in ferroptotic regulation, consistent with prior research\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that adrenalin increases DUSP1, thereby inhibiting MAPK phosphorylation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Additionally, noradrenaline-induced DUSP1 expression is mediated through the activation of the cAMP-PLC-PKC-CREB signaling pathway via ADRB2, which subsequently inhibits JNK-mediated c-Jun phosphorylation and protects ovarian cancer cells from apoptosis\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In our study, high concentration of ISO suppressed MAPK phosphorylation, thereby inhibiting ferroptosis.\u003c/p\u003e\u003cp\u003eCurrently, immune checkpoint inhibitors and MTDs targeting for vascular endothelial growth factor receptor (VEGFR) constitute the primary therapeutic approaches for RCC, while agents with direct cytotoxic effects remain limited and often exhibit insufficient efficacy. Sorafenib, an MTD that targets multiple kinases\u0026mdash;including VEGFR family, platelet-derived growth factor receptor family, c-kit, raf, p38, FMS-like tyrosine kinase 3, and xCT\u0026mdash;was historically widely used for RCC treatment\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e but is no longer prioritized in contemporary clinical practice, as per current clinical guidelines\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Although sorafenib has been reported to induce ferroptosis via xCT inhibition\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, findings regarding its cytotoxic effects remain inconclusive\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Our findings further revealed that sorafenib induced ferroptosis in ACHN cells, but not in A498 cells, highlighting cell line-dependent variability in ferroptosis sensitivity. This variability may contribute to the mixed clinical responses to sorafenib, with certain RCC tumors undergoing sorafenib-induced ferroptosis, while others remain resistant. The mechanism by which sorafenib induces ferroptosis appears to vary among cell lines and remains unresolved, posing a key challenge for future research.\u003c/p\u003e\u003cp\u003eAnother limitation of this study is the use of a restraint model for stress induction in mice, which may introduce confounding factors beyond catecholamine-mediated effects.\u003c/p\u003e\u003cp\u003eIn conclusion, Catecholamine stimulation attenuates ferroptosis. Consequently, ADRB2 activation under chronic stress compromises sorafenib efficacy by suppressing ferroptosis in RCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Number JP20K18085, which Hidenori Kanno received, and JSPS KAKENHI Grant Number 23K15777, which Atsushi Yamagishi received.\u003c/p\u003e\n\u003cp\u003eSei Naito received honoraria from Pfizer Japan Inc., Merk biopharma Japan Inc., Bristol Meier\u0026rsquo;s squib Japan Inc., Ono Pharma, MSD Japan, and Eisai Co. as their sponsored speaker.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNorihiko Tsuchiya received honoraria from Pfizer Japan Inc. Janssen, Novartis, Ono, Bayer, Sanofi, Takeda Pharm, Bristol-Myers Squibb Japan, and Astelas Pharma., as their sponsored speaker and research funds from Pfizer Japan Inc. and Eisai outside the submitted work. The other authors have declared that no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are publicly available in Sei Naito at [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the patients for consenting to the use of their surgical specimens.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMonteiro FSM, Soares A, Debiasi M, Schutz FA, Maluf FC, Bastos DA, \u003cem\u003eet al.\u003c/em\u003e First-line Treatment of Metastatic Renal Cell Carcinoma in the Immuno-oncology Era: Systematic Review and Network Meta-analysis. \u003cem\u003eClinical Genitourinary Cancer\u003c/em\u003e 2020, 18(4): 244\u0026ndash;251.e244.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDixon J, Scott, Lemberg M, Kathryn, Lamprecht R, Michael, Skouta R, Zaitsev M, Eleina, Gleason E, Caroline, \u003cem\u003eet al.\u003c/em\u003e Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death. \u003cem\u003eCell\u003c/em\u003e 2012, 149(5): 1060\u0026ndash;1072.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang S, Wan, Sriramaratnam R, Welsch E, Matthew, Shimada K, Skouta R, Viswanathan S, Vasanthi, \u003cem\u003eet al.\u003c/em\u003e Regulation of Ferroptotic Cancer Cell Death by GPX4. \u003cem\u003eCell\u003c/em\u003e 2014, 156(1\u0026ndash;2): 317\u0026ndash;331.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, \u003cem\u003eet al.\u003c/em\u003e Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. \u003cem\u003eCell\u003c/em\u003e 2017, 171(2): 273\u0026ndash;285.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYousef EH, El Gayar AM, El-Magd NFA. 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While sorafenib, a molecular targeted agent for renal cell carcinoma (RCC), has been implicated in ferroptosis induction, conflicting reports persist. Chronic stress, mediated by catecholamines via β2-adrenergic receptors (ADRB2), is known to promote cancer progression, yet its influence on drug resistance and ferroptosis remains elusive. In this study, we first confirmed ADRB2 expression in RCC through immunohistochemistry of surgical specimens. Using the ADRB2-expressing RCC cell line ACHN, we found that stimulation with the β-adrenergic agonist isoproterenol (ISO) conferred resistance to sorafenib both \u003cem\u003ein vitro\u003c/em\u003e and in a mouse stress model\u0026mdash;an effect abrogated by ADRB2 knockdown. To assess the involvement of ferroptosis in this resistance mechanism, we employed the ferroptosis inhibitor ferrostatin-1 (Fer-1). Similar to ISO, Fer-1 diminished sorafenib sensitivity in ACHN cells. Ferroptosis inducers erastin and RSL3 markedly reduced cell viability; however, ISO attenuated ferroptosis in ACHN cells. Mechanistically, ISO upregulated DUSP1 expression and inhibited the phosphorylation of ERK1/2, p38, and JNK, all of which contribute to ferroptosis suppression. These findings provide compelling evidence that chronic stress-induced ADRB2 activation promotes sorafenib resistance in certain RCC subtypes by mitigating ferroptotic cell death.\u003c/p\u003e","manuscriptTitle":"Stress-Induced Catecholamines Attenuate Sorafenib Efficacy by Inhibiting Ferroptosis in β2-adrenergic Receptor Positive Renal Cell Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 05:59:34","doi":"10.21203/rs.3.rs-7131878/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8539b2ff-c0c1-498f-933e-944a68529181","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53241871,"name":"Biological sciences/Cancer/Urological cancer/Renal cancer/Renal cell carcinoma"},{"id":53241872,"name":"Biological sciences/Cell biology/Cell death"}],"tags":[],"updatedAt":"2025-09-11T14:20:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 05:59:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7131878","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7131878","identity":"rs-7131878","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}