Bcl-xL Inhibition Disrupts p27 Stability and Promotes Senescence Escape: Insights for Cancer Senotherapy

Bcl-xL Inhibition Disrupts p27 Stability and Promotes Senescence Escape: Insights for Cancer Senotherapy | 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 Research Article Bcl-xL Inhibition Disrupts p27 Stability and Promotes Senescence Escape: Insights for Cancer Senotherapy Xiaobai He, Xiaopan Chen, XinYi Qian, Di Cui, SongLin Zhang, Xiuli Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7250116/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 Purpose: Cellular senescence serves as a double-edged sword in oncology, acting as a tumor suppressor and a promoter of a pro-tumorigenic environment post-chemotherapy, necessitating targeted removal strategies. To investigate whether inhibition of Bcl-xL (a key anti-apoptotic protein and senescent cell survival factor) leads to therapeutic failure by enabling senescent tumor cells to resist apoptosis, re-enter the cell cycle, and potentially drive tumor progression. Methods: We utilized pharmacological Bcl-xL inhibitors and shRNA-mediated Bcl-xL knockdown to disrupt Bcl-xL function in senescent tumor cells. The effects on cell fate, cell cycle regulators, and associated molecular pathways were analyzed. Results: Contrary to the expected induction of apoptosis, Bcl-xL inhibition facilitated the cell cycle re-entry of senescent tumor cells. This escape from senescence was driven by a marked reduction in the stability of the cell cycle inhibitor p27. The reduction occurred through increased cytoplasmic localization of p27 and reduced phosphorylation, leading to its proteasome-dependent degradation. Thus, Bcl-xL disruption initiates a pathway enabling senescence escape. Conclusion: Inhibiting Bcl-xL in senescent tumor cells promotes senescence escape rather than apoptosis, representing a significant paradigm shift in understanding senolytic drug mechanisms. These findings reveal the complex dual nature of senescence in cancer and underscore the critical need for careful design when combining senolytic agents with chemotherapeutics to prevent inadvertent tumor cell proliferation. Bcl-xL Senescence Escape p27 Stability Senotherapy Chemotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Cellular senescence act as both tumor suppressor and promoter post-chemotherapy. Inhibition of Bcl-xL facilitates the cell cycle re-entry of senescent tumor cells. Inhibition of Bcl-xL resulted in degradation of p27. Accelerated p27 degradation mediates senescence escape. Consider tumor types and timing is vital for application of Bcl-2 family inhibitors. Introduction Cellular senescence, characterized by an irreversible arrest in cell growth, plays a pivotal role in tumor suppression, wound healing, and the aging process [ 1 , 2 ]. Triggered by various stressors, this cellular state was first described by Hayflick and Moorhead (1961) as a limit to the division potential of normal diploid cells [ 3 ]. While senescence acts as a defense by halting the proliferation of compromised cells, it paradoxically can promote tumor growth through the senescence-associated secretory phenotype (SASP) that remodels the tumor microenvironment by producing various secreted proteins including inflammatory cytokines, chemokines, etc., and regulating the properties of adjacent cells [ 4 – 6 ], and through intricate reprogramming processes that induce stemness in cancer cells [ 7 – 11 ]. Moreover, the accumulation of data revealed that severe genotoxic stress-induced senescence is unstable, including oncogene-induced senescence (OIS), it can reverse spontaneously or after secondary stimulation. Numerous studies have shown the phenomenon of escape-from-OIS [ 12 – 15 ]. Our pioneering research has previously revealed that exposure of senescent tumor cells to specific clinical anticancer agents can inadvertently facilitate their escape from senescence [ 12 ]. This discovery underlines a critical gap in our understanding of the mechanisms driving this escape, highlighting the need for further investigation. The Bcl2 family, traditionally recognized for its anti-apoptotic functions, has emerged as a key regulator of senescence [ 13 , 14 ]. This nuanced understanding of Bcl2 family's role underlines the therapeutic potential of senolytic drugs, like navitoclax, which target senescent cells expressing anti-apoptotic Bcl-2 family proteins to mitigate aging-related conditions and enhance cancer treatment outcomes [ 13 , 15 ]. Among the family, Bcl-xL stands out for its unique influence on both senescence and apoptosis, setting it apart from its counterparts [ 16 – 18 ]. Importantly, Bcl-xL, along with Bcl-2, contributes to cell cycle regulation, particularly through modulating p27, a key factor in cell cycle control and senescence [ 19 – 21 ]. These functions of Bcl2/Bcl-xl underline a comprehensive role beyond apoptosis inhibition, situating p27 not only as a senescence marker but also as a critical determinant in the maintenance of the senescent cell cycle arrest. Understanding the intricate relationship between Bcl2/Bcl-xL and p27 is crucial for unraveling the complex mechanisms of senescence and its implications for cancer development and aging. Our previous work has highlighted the potential unintended consequences of ABT737, a prototype BH3-mimetic that inhibits anti-apoptotic BCL-2 family proteins, pointing to the complex effects of senolytic drugs [ 12 ]. Furthermore, recent findings have demonstrated that sub-lethal doses of ABT737 can induce mitochondrial DNA (mtDNA) release, stimulating the SASP and potentially contributing to a pro-tumorigenic environment [ 22 ]. This raises the concern that senescent cells, when treated with Bcl-2 family inhibitors, may develop resistance and re-enter the cell cycle, leading to tumor progression [ 23 ]. Our current study addresses these concerns by revealing that inhibition of Bcl-xL, a key member of the Bcl-2 family, paradoxically facilitates the cell cycle re-entry of senescent tumor cells, bypassing apoptosis. Focusing on the critical roles of Bcl-xL and p27 in the orchestration of senescence and cell cycle dynamics, our study investigates their interplay, particularly under conditions of Bcl-xL inhibition. We aim to elucidate how Bcl-xL inhibition affects p27^Kip1 stability and the potential consequences for senescent tumor cells regarding cell cycle re-entry. This investigation seeks to illuminate the underlying mechanisms through which Bcl-xL influences senescence and cell cycle progression, contributing to the development of targeted approaches for cancer therapy and the management of aging-related pathologies. Materials and Methods Cell lines, compounds, plasmids, and antibodies Human lung adenocarcinoma A549 cells was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco Modified Eagle Medium (Hyclone) supplemented with 10% FBS (Hyclone) and Penicillin (100 units/ml)-Streptomycin (100 µg/ml) (Hyclone). Cells were cultured in 37°C incubator with 5% CO2. A549 cells with stable knockdown of Bcl2 and Bcl-xL were generated by infection with pLKO.1-Bcl2 shRNAs and pLKO.1-Bcl-xL shRNAs virus followed by puromycin (2 µg/ml) selection. Compounds, including doxorubicin (Dox, Cat#: D107159), ABT-199 (Cat#: A124869), and CX5461 (Cat#: C127663) were obtained from Aladdin (Shang Hai, P. R. China). Cisplatin (DDP) and ABT-737 (Cat#: S1002) were purchased from Selleck Chemicals (Houston, TX, USA). WEHI-539 (Cat#: HY-15607), cycloheximide (Cat#: HY-12320), and MG-132 (Cat#: HY-13259) were purchased from MCE (MedChemExpress, Monmouth Junction, NJ, USA). p27 siRNAs and siRNA control were synthesized by GenePharma (Suzhou, P. R. China). Bcl2 and Bcl-xL shRNAs were synthesized by Tsingke Biological technology (Beijing, P. R. China). All primer sequences were listed in Table S1 . Plasmids of pCDNA3.1-Bcl2 and pCDNA3.1-Bcl-xL were purchased from Heyin (Hangzhou, P. R. China). Rabbit polyclonal antibodies for Bcl2 (Cat#: 2876S), Bcl-xL (Cat#: 2762S), p27 (Cat#: 3688S), Histone H3 (Cat#: 4620S), and β-tublin (Cat#: 2128S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibody of p27 (Cat#: 25614-1-AP) used for immunofluorescence staining (IF) and p-p27 (Ser10, Cat# AF3326)) were obtained from Proteintech (Rosemont, IL, USA). Goat-anti-rabbit IgG FITC (Cat#: HA1004) was from Hangzhou HuaAn Biotechnology (Hangzhou, P. R. China). Mouse monoclonal antibodies for p21 (Cat#: sc-397), p130 (Cat#: sc-374521), and β-actin (Cat#: sc-47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Goat-anti-mouse IgG (HRP conjugate, Cat#: IH-0031) and goat-anti-Rabbit IgG (HRP conjugate, Cat#: IH-0011) were purchased from Beijing Dingguo Changsheng Biotechnology (Beijing, P. R. China). JC-1 (5,6 -Dichloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) was bought from Beyotime (C2006,, Shanghai, P. R. China). Western Blot Chemotherapy-induced senescent cells and non-senescent cells were lyzed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate). Samples were centrifuged at 14,000 × g for 10 min at 4°C to remove insoluble debris. About 20 µg of protein quantified by BCA kit was boiled in Laemmli loading buffer for 5 min and loaded to the SDS-PAGE gel for further investigation. The expression of indicated proteins were detected using their specific antibodies. RNA isolation and RT-qPCR Total RNA was extracted from cells using Trizol (Cat#: 10606ES60, Yeasen Biotechnology, Shanghai, P. R. China). Briefly, cells were collected after the indicated treatment and lysed in proper volume of Trizol reagent according to the cell numbers. Then cells were centrifuged at 12,000 × g for 10 min at 4°C to remove insoluble debris. Proper volume of chloroform (about 1/5 of Trizol volume) was added to the supernatant to separate RNA from the mixture of nucleoprotein complexes and DNA. Phase separation was achieved by centrifuged at 12,000 g for 15 min at 4°C. The RNA containing phase was transferred to a fresh tube and similar volume of 2-propanol was added to each sample to precipitate the RNA. The precipitated RNA pellet was then washed once with 75% ethanol and dried by air-drying. RNA was dissolved in RNase-free water and quantified using NanoDrop microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was transcribed to cDNA using PrimeScript RT Master Mix and analyzed by TB Green Premix Ex Taq II (Takara Bio USA, Inc. CA, USA). A panel of PCR primers listed in Table S1 were designed using the Primer3 (v. 0.4.0) software and synthesized by TsingKe Biological Technology (Beijing, P. R. China). Colony formation assay Cells (8 × 10 4 cells/well) were seeded to 6 well plates overnight before the treatment of Dox (100 ng/ml) or DDP (5 µM) for 7 days. Then cells were washed twice with 1 × PBS to remove the residual drugs and cultured in regular DMEM medium in the presence or absence of indicated additional treatment, including Bcl2 family inhibitors and transient knockdown of p27. After the second treatment, the senescent cells were cultured in normal medium for 2–6 weeks with refeeding every 3 days until colonies were visible. Colonies were detected using crystal violet staining. β-galactosidase staining Cells (8 × 10 4 cells/well) were seeded to 6 well plates overnight before the treatment of Dox (100 ng/ml) or DDP (5 µM) for 7 days. The β-galactosidase activity in senescent cells was measured by senescence β-galactosidase staining kit (Cat#: C0602, Beyotime, Shanghai, P. R. China) according to manufacturer protocol. Briefly, after being washed with 1 × PBS, cells were fixed in the fixation buffer for 10 min at room temperature. Then, cells were washed three times with 1 × PBS and incubated with staining buffer containing X-gal at 37°C overnight before being photographed. RNA interference Dox or DDP-induced senescent cells were transfected with 20 pmol control siRNA or p27 siRNAs using lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA) following manufacturer instruction. The cells were cultured in regular DMEM medium after 48 h of transfection and the formed colonies were observed by crystal violet staining. The p27 knockdown efficiency in senescent cells was confirmed by western blot. Cycloheximide chase assay The protein stability of p27 in both non-senescent or senescent cells with or without Bcl2 family inhibitors treatment were analyzed by cycloheximide (CHX), a protein synthesis inhibitor. Cells pre-treated with or without indicated inhibitors for 2 h were incubated with CHX (2 µM) for 0–7 h. Then, cells at each time point were immediately collected and lysed, and protein level was analyzed by western blot. Subcellular localization analysis The localization of p27 was analyzed by western blot and IF staining. Nuclear and cytoplasmic protein extraction kit combined with western blot was employed to detect p27 subcellular localization in non-senescent and senescent cells with or without Bcl-xL inhibitor treatment. The protein preparation was performed following manufacturer’s instruction and the western blot was as measured above. Histone H3 was served as the nuclear reference and β-tublin was used as the cytoplasm reference. For IF staining, about 2 × 10 4 cells were seeded to each chamber of 35 mm confocal dishes (Cellvis, Sunnyvale, California, USA) for 24 h. Then cells were rinsed twice with 1 × PBS containing 0.1% Triton X-100 (PBST) and fixed in 4% paraformaldehyde (PFA) solution for 10 min at room temperature (RT). The fixed cells were rinsed twice with PBST and incubated in PBST for another 5 min. After blocking with 1% BSA dissolved in PBS for 30 min at RT, cells were incubated with indicated primary antibodies diluted in 1% BSA (1 : 200, v/v) for 4 h at RT or overnight at 4°C. Then, the cells were washed three times with PBST to remove unbound primary antibodies and incubated with FITC-conjugated secondary antibody for another 2 h at RT. Anti-fade solution containing DAPI (Cat#: P0131, Beyotime, Shanghai, P. R. China) were added to each chamber after the cells were washed three times with PBST. A minimum of 100 cells were imaged using confocal microscopy (Leica, Wetzlar, Germany). JC-1 stainning Mitochondrial membrane potential (MtMP) in senescent cells was measured by staining with JC-1. In brief, A549 cells treated with DDP (5 µM), or Dox (100 ng/ml) for 7 days were incubated in pre-heated MOPS containing 1 µM JC-1 for 15 min at 37°C in the dark, washed with PBS, then observed at either 510 nm (green mitochondria/J-monomer) or 590 nm (red-to-orange mitochondria/J-aggregate) using a confocal microscope (Leica, Wetzlar, Germany). A minimum of 100 cells were imaged. Statistical Analysis All results were reproducible in at least three independent experiments. Values were shown as mean ± SD. Student’s t-test was used for analyzing p value. p value < 0.05 was considered as statistically significant (marked as *). Results Inhibition of Bcl-xL induces senescence escape In our investigation into the effects of Bcl-xL inhibition on senescent tumor cells, we employed both pharmacological and genetic approaches to modulate Bcl-xL activity. To induce senescence, cells were treated with one of three agents: the RNA polymerase I inhibitor CX5461 (Fig. 1 a, reported in our previous paper [ 12 ], supplementary Fig. S1 a-b), cisplatin (DDP), or doxorubicin (Dox) (Fig. 1 b, supplementary Fig. S1 b-d). These agents were selected for their diverse mechanisms of action, providing a broad spectrum of senescence induction to ensure the observed effects on cell cycle re-entry were not specific to a single pathway of senescence stimuli. Following the establishment of a senescent phenotype, confirmed by increased SA-β-galactosidase staining and halted proliferation, we proceeded with the inhibition of Bcl-xL. This was achieved through the administration of WEHI-539, a specific inhibitor of Bcl-xL (Fig. 1 c, Fig. S2 a-b), and ABT-737, which targets multiple members of the Bcl-2 family, including Bcl-xL (Fig. 1 a, 1 d and supplementary Fig. S2 c). Remarkably, both inhibitors facilitated the escape of senescent cells from their growth arrest, evident from a significant decrease in senescent cells and an increase in cloning formation, indicative of cell cycle re-entry. Besides, Bcl2-specific inhibitor ABT199 didn’t shows the effect, underline the Bcl-xl involved the senescence escape (supplementary Fig. S2 d). To validate these findings, we further silenced Bcl-xL using shRNA in cells rendered senescent by Dox treatment (Fig. 1 e-f). Mirroring the effects observed with pharmacological inhibition, genetic knockdown of Bcl-xL led to a marked upsurge in cloning formation. Similar results were also found in PC-9 cells (supplementary Fig. S3a-b). These results underline Bcl-xL’s critical role in maintaining the senescent state and suggest its inhibition as a potential mechanism to promote cell cycle re-entry in senescent tumor cells. A significant insight from our experiments, particularly highlighted in Fig. 1 g was the notable increase in Bcl-xL levels within the cytoplasm of cells treated with DDP and Dox. This shift in Bcl-xL localization from a potentially nuclear to a more cytoplasmic presence upon senescence induction adds a layer of complexity to our understanding of Bcl-xL's role in cellular senescence. It implies a regulatory mechanism at play, where Bcl-xL's cellular distribution may influence the senescence outcome and, consequently, the cell's fate upon Bcl-xL inhibition. p27 involved in Bcl-xL inhibition induced senescence escape Given the pronounced role of Bcl-xL in the regulation of senescence, our investigation next sought to uncover the mechanisms through which Bcl-xL inhibition facilitates the escape from senescence. To this end, senescent cells, both untreated and treated with Bcl-xL inhibitors, underwent proteome analysis to pinpoint changes in protein expression critical for cell cycle control. Among the most significantly down-regulated proteins, p27 stood out as a key factor in cell cycle regulation, drawing our attention to its potential involvement in senescence dynamics. The down-regulation of p27 was found to be specific to senescent cells. Western blot analyses, as depicted in Fig. 2 a-c and Supplemental Fig. S4a, confirmed a noticeable decrease in p27 protein levels in senescent cells exposed to various Bcl-xL inhibitors. This effect was not observed in non-senescent cells, underscoring the senescence-specific impact of Bcl-xL inhibition. In contrast, the levels of another cell cycle regulator, p130 protein, remained unchanged across both senescent and non-senescent cells, as shown in Supplemental Fig. S4c-e, highlighting the selective down-regulation of p27 upon Bcl-xL inhibition. Further substantiating the specificity of this effect, the reduction in p27 protein expression in senescent cells was notably induced by the Bcl-xL inhibitor WEHI-539, consistent with observations from the use of the pan-specific inhibitor ABT-737. This contrasted with the slight reduction seen after 48 hours of treatment with the Bcl-2-specific inhibitor ABT-199, as documented in Supplemental Fig. S4b, indicating a unique sensitivity of p27 to Bcl-xL-targeted inhibition. To further confirm the role of Bcl-xL in regulating p27, we performed shRNA-mediated inhibition of Bcl-xL in PC-9 cells (Fig. S3c). The results demonstrated that inhibition of Bcl-xL by shRNA led to a significant reduction in p27 levels, while p21 remained unchanged, reinforcing the specificity of Bcl-xL's effect on p27 stability. To ascertain the role of p27 in maintaining senescence, we employed siRNA to transiently knock down p27. Among several targeted siRNAs, only siRNA1 reduce half p27 protein and siRNA3 markedly reduced p27 expression in senescent cells. Notably, only efficient knockdown of p27 led to significant changes in cell behavior, including enhanced colony formation, as evidenced in Fig. 2 d-e. This outcome reinforces the critical role of p27 in senescence maintenance and suggests that its down-regulation, triggered by Bcl-xL inhibition, is a key event facilitating senescence escape. Bcl-xL regulate p27 protein stability in senescent and senescent escape cells In our detailed examination of Bcl-xL's role in cellular senescence, we found both mRNA and protein levels of Bcl-xL to be significantly elevated in cells rendered senescent by DDP and Dox treatment, aligning with findings from other studies [ 16 ]. Notably, this increase in Bcl-xL was observed regardless of the p53 status, as evidenced by the elevated levels of Bcl-xL in both H358 (p53 null) and PC-9 (p53 mutant) cell lines following treatment with senescence-inducing drugs (Fig. 3 a-b, supplementary Fig. S5a-b). This underlines the independence of Bcl-xL upregulation from p53-mediated pathways in the context of drug-induced senescence. Contrastingly, while p27 protein levels saw a significant increase post-treatment, p27 mRNA levels did not exhibit a similar upsurge (Fig. 3 c-g). This discrepancy, particularly in the absence of down-regulation in p27 mRNA within Bcl-xL inhibitor-treated senescent cells, points to a post-transcriptional mechanism of regulation, suggesting that p27's stability is modulated as a downstream effect of Bcl-xL activity. To further elucidate this mechanism, we assessed p27's stability across different cellular contexts. In non-senescent cells, p27 exhibited a half-life of approximately 2 hours, which markedly extended to 7 hours in DDP-induced senescent cells, and surpassed 7 hours in Dox-induced counterparts (Fig. 4 a-c). This enhancement in p27 stability underlines its role in the senescent phenotype. However, the introduction of Bcl-xL inhibitors notably disrupted this stability, reducing p27's half-life back to around 2 hours, as demonstrated in cells treated with either ABT-737 or WEHI-539 (Fig. 4 d-f). Intriguingly, in non-senescent cells, WEHI-539 appeared to marginally increase p27 stability (Fig. S6a), while ABT-199 showed no significant impact on p27's half-life in senescent cells, highlights the unique regulatory pathway mediated by Bcl-xL (supplementary Fig. S6b). These observations collectively highlight Bcl-xL's critical involvement in regulating p27 stability, a key factor in maintaining the senescent state. By modulating p27 levels through a post-transcriptional mechanism, Bcl-xL emerges as a critical player in the dynamics of senescence and its potential reversal, offering insightful perspectives into the molecular intricacies of senescence and the escape from this cellular fate. p27 Ser10 phosphorylation and re-localization in senescent and senescent escape cells In this study, we examined the role of Bcl-xL in modulating the function of p27, a key regulator of the cell cycle, focusing on changes in its stability and subcellular localization. Initial fractionation studies in non-senescent A549 cells revealed that p27 is predominantly cytoplasmic. However, upon inducing senescence with chemotherapy drugs, there was a significant increase in p27 levels in both the cytoplasm and nucleus, indicating a shift in localization in response to senescence induction (Fig. 5 a). In contrast, p130, another cell cycle regulator, remained nuclear across all conditions. Treatment with Bcl-xL inhibitors, ABT-737 and WEHI-539, resulted in a significant decrease in nuclear p27 within 24 hours, suggesting Bcl-xL's involvement in controlling p27's nuclear localization (Fig. 5 b). This effect was corroborated in cells with Bcl-xL knocked down, where nuclear p27 levels also significantly decreased, unlike in cells lacking Bcl-2, where the nuclear/cytoplasmic ratio of p27 remained unchanged (Fig. 5 c, supplementary Fig. S7d). Immunofluorescence staining further confirmed these observations, showing an increase in nuclear p27 in DDP and Dox-induced senescent cells. The inactivation of Bcl-xL led to a shift in p27 localization to the cytoplasm, as observed in Bcl-xL knockdown cells, particularly in the presence of MG-132 (Fig. 5 d-f, Fig. S7a-b). This re-localization effect of Bcl-xL inhibition on p27 was not observed in non-senescent cells (Fig. S7c). Moreover, the study investigated the role of Ser10 phosphorylation in p27 stability. Phosphorylation of p27 on Ser10, which influences its stability, was found to be higher in senescent cells compared to non-senescent cells. This phosphorylation was notably reduced by treatment with Bcl-xL inhibitors, indicating a mechanism through which Bcl-xL may regulate p27 stability and thereby affect its function (Fig. 6 a-d). These findings highlight Bcl-xL's critical role in influencing p27's subcellular localization and stability, including the modulation of Ser10 phosphorylation. By affecting the distribution and availability of p27 for cell cycle control, Bcl-xL contributes to the cellular decision-making process regarding senescence induction and escape, providing important insights into the molecular dynamics of cellular senescence. Discussion Cellular senescence acts as a double-edged sword in oncology, both suppressing tumors and promoting a pro-tumorigenic environment following chemotherapy, thus necessitating targeted removal strategies. Our research contributes to this complex landscape by revealing an unanticipated consequence of Bcl-xL inhibition. Instead of inducing apoptosis, inhibiting Bcl-xL facilitates the cell cycle re-entry of senescent tumor cells. This discovery suggests that while Bcl-xL is typically understood as a survival factor, its role in senescence is crucially linked to p27 stability. The persistent presence of senescent cells may foster a pro-tumorigenic environment through the secretion of inflammatory factors and initiate reprogramming processes that induce stemness in cancer cells, complicating tumor therapy [ 8 , 24 ]. These findings highlight the therapeutic potential of senolytic drugs, particularly Bcl-2 family inhibitors like ABT-737 and ABT-263, which have shown promising efficacy in preclinical studies for eliminating senescent cells [ 16 , 25 ]. However, our study underlines the complexity of targeting senescent cells, as Bcl-xL deficiency, while enhancing senescent cell death, also allows a subset of these cells to survive and proliferate rapidly. The complexities of cancer senolytic therapy are highlighted by the phenomenon of cell cycle reactivation in senescent tumor cells, influenced by cytotoxic drugs, which might contribute to the observed discrepancy in long-term survival benefits despite favorable initial responses. This underlines the importance of understanding the mechanisms behind senescence escape triggered by senolytic drugs to refine cancer therapeutic strategies. Since our previous study that observed subjecting senescent tumor cells to cytotoxic treatments stimulates the clonogenic proliferation of remaining survivors [ 12 ], subsequent research by other research groups have suggest WNT /β-catenin pathways induction, as a promoter, initiate the senescence-escape reprogram [ 8 , 9 ]. Our present findings delineate a specific reduction in p27 expression in senescent cells upon Bcl-xL inhibition, without affecting p130, identifying p27 as a crucial blockage element in the process of senescence escape associated with Bcl2 family inhibition. The observed regulatory complexity of p27, influenced by Bcl-xL inhibition, suggests a nuanced interaction between Bcl-xL and p27 stability. Our investigation into p27 stability demonstrated its post-translational accumulation in chemotherapy-induced senescent cells, unaffected at the mRNA level by Bcl-xL inhibitors, as confirmed by cycloheximide chase assays. This significant reduction in p27 stability upon Bcl-xL inhibition unveils a mechanism that potentially promotes senescence escape. The differential activity of E3 ubiquitin ligases responsible for p27 degradation across the cell cycle suggests that Bcl-xL's regulation of p27 stability and localization could significantly impact senescence dynamics and cell cycle re-entry. Moreover, we explored the role of Ser10 phosphorylation in p27 stability, observing an increase in senescent cells. Bcl-xL inhibition notably reduced this phosphorylation, indicating a possible mechanism through which Bcl-xL modulates p27 stability, thus affecting its cellular function. In conclusion, our study reveals that Bcl-xL inhibition mediates senescence escape predominantly through accelerated p27 degradation, either by promoting its nuclear export for cytoplasmic degradation or by inhibiting its phosphorylation and subsequent nuclear degradation. These insights into the mechanism of senescence escape facilitated by Bcl-xL inhibition are critical for the development of more effective cancer therapies. Given the complex role of Bcl-xL in tumor biology, careful consideration of tumor types and timing of drug application is essential to harness the full therapeutic potential of Bcl-2 family inhibitors. Declarations Authors Contributions Yang designed and performed the experiments, analyzed the data, supervised the project, and wrote the manuscript; X. He, X. Chen designed and performed the experiments, analyzed the data; D. Cui analyzed the data; X. Qian, S. Zhang. X. Yang, W. Fan, N. Huang performed the experiments; M.Du contributed to sample preparation; L. Chen analyzed the data, discussed the results and wrote the manuscript. All the authors contributed to revision the manuscript. Funding This work was supported in part by grants from the Natural Science Foundation of Zhejiang Province (LY20H160046 to L. Yang); Research Program for Medicine and Health Science and Technology of Zhejiang Province (2021KY080 to L. Yang, 2022KY585 to X. Yang); and Zhejiang People’s Hospital Research Startup Fund (ZRY2019A003 to L. Yang). Acknowledgements The authors would like to thank Clinical Research Institute, Zhejiang Provincial People’s Hospital for providing the research platform and experimental equipment. The authors also thank Xue Yang for technical assistance in confocal microscopy. Data Availability The data generated in this study are available within the article and its supplementary data files. Conflict of Interest The authors declare no conflicts of interest. Ethics declarations No Human/Animal Subjects: The research does not involve experiments on human participants or animals, nor does it use their tissues. Ethics Committee Confirmation: The relevant institutional ethics committee (Zhejiang Provincial People's Hospital Research Ethics Committee) has explicitly reviewed the study and confirmed that formal ethical approval is not required based on the nature of the work. Clinical trial number: not applicable. References Hernandez-Segura A, Nehme J, Demaria M: Hallmarks of Cellular Senescence . 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Yosef R, Pilpel N, Tokarsky-Amiel R, Biran A, Ovadya Y, Cohen S, Vadai E, Dassa L, Shahar E, Condiotti R et al : Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL . Nature communications 2016, 7 :11190. Rahman M, Olson I, Mansour M, Carlstrom LP, Sutiwisesak R, Saber R, Rajani K, Warrington AE, Howard A, Schroeder M et al : Selective Vulnerability of Senescent Glioblastoma Cells to BCL-XL Inhibition . Mol Cancer Res 2022, 20 (6):938-948. Greider C, Chattopadhyay A, Parkhurst C, Yang E: BCL-x(L) and BCL2 delay Myc-induced cell cycle entry through elevation of p27 and inhibition of G1 cyclin-dependent kinases. Oncogene 2002, 21(51):7765-7775. Vairo G, Soos TJ, Upton TM, Zalvide J, DeCaprio JA, Ewen ME, Koff A, Adams JM: Bcl-2 retards cell cycle entry through p27(Kip1), pRB relative p130, and altered E2F regulation . Mol Cell Biol 2000, 20 (13):4745-4753. Du X, Fu X, Yao K, Lan Z, Xu H, Cui Q, Yang E: Bcl-2 delays cell cycle through mitochondrial ATP and ROS . Cell cycle 2017, 16 (7):707-713. Victorelli S, Salmonowicz H, Chapman J, Martini H, Vizioli MG, Riley JS, Cloix C, Hall-Younger E, Machado Espindola-Netto J, Jurk D et al : Apoptotic stress causes mtDNA release during senescence and drives the SASP . Nature 2023, 622 (7983):627-636. Martin N, Popgeorgiev N, Ichim G, Bernard D: BCL-2 proteins in senescence: beyond a simple target for senolysis? Nature reviews Molecular cell biology 2023, 24 (8):517-518. Triana-Martinez F, Loza MI, Dominguez E: Beyond Tumor Suppression: Senescence in Cancer Stemness and Tumor Dormancy. Cells 2020, 9(2). van Deursen JM: Senolytic therapies for healthy longevity . Science 2019, 364 (6441):636-637. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials1.docx OriginalimagesforWesternblot.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7250116","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":500085012,"identity":"743b5bd2-b95f-4b1f-9ad6-68c352d57d40","order_by":0,"name":"Xiaobai He","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiaobai","middleName":"","lastName":"He","suffix":""},{"id":500085013,"identity":"21cf8176-e570-47ed-9539-954f5d829de8","order_by":1,"name":"Xiaopan Chen","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiaopan","middleName":"","lastName":"Chen","suffix":""},{"id":500085014,"identity":"07ef2857-8e06-4c41-8618-794f33f27581","order_by":2,"name":"XinYi Qian","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"XinYi","middleName":"","lastName":"Qian","suffix":""},{"id":500085015,"identity":"7e602212-f80b-47b7-aedb-19085d927fd0","order_by":3,"name":"Di Cui","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Cui","suffix":""},{"id":500085016,"identity":"c5a8e937-66bf-4e4b-b432-eab8728ca346","order_by":4,"name":"SongLin Zhang","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"SongLin","middleName":"","lastName":"Zhang","suffix":""},{"id":500085017,"identity":"01f90fad-b5c6-49fb-a35b-d21456e22bc8","order_by":5,"name":"Xiuli Yang","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiuli","middleName":"","lastName":"Yang","suffix":""},{"id":500085018,"identity":"f16274bb-4138-4193-89c3-10d650ac4366","order_by":6,"name":"Weijiao Fan","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Weijiao","middleName":"","lastName":"Fan","suffix":""},{"id":500085019,"identity":"f513d045-21d3-45be-9489-588566a98b59","order_by":7,"name":"Nan Huang","email":"","orcid":"","institution":"Li’an People's Hospital (Affiliated People's Hospital, Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Huang","suffix":""},{"id":500085020,"identity":"cf36260a-b7ec-45c9-a38d-cfd7b938ca16","order_by":8,"name":"Miaomiao Du","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Miaomiao","middleName":"","lastName":"Du","suffix":""},{"id":500085021,"identity":"1d8fbe93-7050-4b29-be40-ea776d5c14ca","order_by":9,"name":"Linjie Chen","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Linjie","middleName":"","lastName":"Chen","suffix":""},{"id":500085022,"identity":"1004ef9b-9e09-4aea-b075-69ffb8ecea39","order_by":10,"name":"Leixiang Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYLCCBwwSDGwgxscGMN+AsJYEqBbGmSRogQBmXmK0GBw/e/hFQoVFYh/72cOvbXdsk2dgb94mwVBzB7eWM3lpFglnJIzZePLSrHPP3DZs4DlWJsFw7BluLQdyzAwS2yTk2BhyzIxz224D/ZVjJsHYcBi3lvNvgFr+SfCw8b8xM7YEaZF/Q0DLjRzjB4kNQFskcowfM4Jt4cGvRfLGGzOGhGNAv0i8MWPsbbtt2MaTVmyRcAy3Fr7zOcYfPtTUJc7vBzJ+tt2W52c/vPHGhxrcWhQOMLBJQNkQBjgZJODUwMAg38DA/AHKhjNGwSgYBaNgFKAAADZWU+HOeUYSAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College","correspondingAuthor":true,"prefix":"","firstName":"Leixiang","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-07-30 07:58:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7250116/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7250116/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89318949,"identity":"222b8271-18b4-4a37-992f-60aac73fb5a8","added_by":"auto","created_at":"2025-08-18 17:51:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":536007,"visible":true,"origin":"","legend":"\u003cp\u003eBcl-xL inhibition promotes senescence escape. (\u003cstrong\u003ea\u003c/strong\u003e) A549 cells were treated with 100 nM CX5461 for 7 days to induce senescence, followed by treatment with different concentrations of ABT-737 for 3 days. The cells were then cultured in drug-free medium for 5 days after the drug was removed and stained for SA-β-galactosidase activity. Colonies were marked with red circles. (\u003cstrong\u003eb\u003c/strong\u003e) A549 cells were treated with DDP (5 μM) or Dox (50 ng/ml) for 7 days to induce senescence. The β-galactosidase activity was detected by SA-β-galactosidase staining kit and photographed under phase-contrast microscope. (\u003cstrong\u003ec\u003c/strong\u003e) DDP-induced senescent cells were treated with different concentrations of WEHI-539 for 3 days. Then cells were cultured in drug-free medium until visible colonies were observed. Cell colonies were stained with crystal violet, and cell morphology was imaged under phase-contrast microscope. (\u003cstrong\u003ed\u003c/strong\u003e) Non-senescent and DDP-induced senescent A549 cells were treated with different concentrations of ABT-737 for 3 days. The cells were cultured in regular medium until visible colonies formed. Cell morphology was recorded after 3 days of ABT-737 treatment and 7 days after ABT-737 was removed using microscopy and the visible colonies were marked with red circles. Cell colonies were stained with crystal violet. (\u003cstrong\u003ee\u003c/strong\u003e) Knockdown efficiency of Bcl-xL by shRNAs was detected by western blot. Original blots are presented in Supplementary Figure S8. (\u003cstrong\u003ef\u003c/strong\u003e) A549 cells with stable Bcl-xL knockdown were treated with Dox (100 ng/ml) for 7 days to induce senescence. Then cells were cultured in drug-free medium until colonies were visible. Cell colonies were stained with crystal violet. (\u003cstrong\u003eg\u003c/strong\u003e) Representative IF images of Bcl-xL expression in non-senescent and drug-induced senescent A549 cells. Nuclear Bcl-xL was marked with arrows.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/bed649b0f20bcd6f5898c582.png"},{"id":89319394,"identity":"d666b550-71d1-48eb-beba-6e581424091f","added_by":"auto","created_at":"2025-08-18 17:59:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1460657,"visible":true,"origin":"","legend":"\u003cp\u003eInactivation of Bcl-xL reduces p27 expression in senescent cells. (\u003cstrong\u003ea-c\u003c/strong\u003e) The expression of p27 in DDP-induced senescent (\u003cstrong\u003ea\u003c/strong\u003e), Dox-induced senescent (\u003cstrong\u003eb\u003c/strong\u003e), and non-senescent (\u003cstrong\u003ec\u003c/strong\u003e) A549 cells treated with or without different concentrations of Bcl2 or Bcl-xL inhibitors for 24 h was analyzed by western blot. Representative images were quantified using the Image Lab 3.0 software. (\u003cstrong\u003ed\u003c/strong\u003e) Drug induced senescent A549 cells were transfected with control siRNA or p27 siRNAs. The knockdown efficiency of p27 siRNAs was confirmed by western blot. Original blots are presented in Supplementary Figure S9. (\u003cstrong\u003ee\u003c/strong\u003e) Colony formation in senescent cells with p27 transient knockdown was detected by crystal violet staining.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/4b0f6fdda223ad2e8aabc253.png"},{"id":89318953,"identity":"fea47f08-e37c-4994-a1fc-44193fd5c07a","added_by":"auto","created_at":"2025-08-18 17:51:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":95051,"visible":true,"origin":"","legend":"\u003cp\u003eBcl-xL regulates p27 expression in senescent cells. (\u003cstrong\u003ea-b\u003c/strong\u003e) The expression of Bcl-xL in DDP and Dox induced senescent A549 cells was analyzed by western blot (\u003cstrong\u003ea\u003c/strong\u003e) and RT-qPCR (\u003cstrong\u003eb\u003c/strong\u003e). Values are mean ± SD of triplicates. *, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs control. (\u003cstrong\u003ec\u003c/strong\u003e) The expression of p27 and p130 in A549 cells treated with DDP or Dox at different time points were detected by western blot. Original blots are presented in Supplementary Figure S10. (\u003cstrong\u003ed\u003c/strong\u003e) The mRNA expression of p27 in non-senescent and drug-induced senescent A549 cells was detected by RT-qPCR. Values are mean ± SD of triplicates. (\u003cstrong\u003ee\u003c/strong\u003e) Drug-induced senescent A549 cells were treated with ABT-737 (2 μM) for 24 h. The mRNA expression of p27 was analyzed by RT-qPCR. Values are mean ± SD of triplicates. (\u003cstrong\u003ef-g\u003c/strong\u003e) Dox (\u003cstrong\u003ef\u003c/strong\u003e) or DDP (\u003cstrong\u003eg\u003c/strong\u003e) induced senescent A549 cells were treated with different concentrations of WEHI-539 for 24 h. The mRNA level of p27 was detected by RT-qPCR. Values are mean ± SD of triplicates.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/3e26febfb0a1c9c0ffb05453.png"},{"id":89318951,"identity":"1140391a-07fa-40e9-877a-12b8e84ea85b","added_by":"auto","created_at":"2025-08-18 17:51:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71778,"visible":true,"origin":"","legend":"\u003cp\u003eBcl-xL regulates p27 stability in senescent cells. (\u003cstrong\u003ea\u003c/strong\u003e) A549 cells with normal proliferation were incubated with CHX (2 μM) for 0 - 6 h. Cells were collected at each time point and the protein level was analyzed by western blot. (\u003cstrong\u003eb-c\u003c/strong\u003e) DDP (\u003cstrong\u003eb\u003c/strong\u003e) or Dox (\u003cstrong\u003ec\u003c/strong\u003e)-induced A549 senescent cells were incubated with CHX (2 μM) for 0 - 7 h. The expression of p27 at each time point was detected by western blot. (\u003cstrong\u003ed-e\u003c/strong\u003e) DDP-induced A549 senescent cells were pre-treated with 5 μM of ABT-737 (\u003cstrong\u003ed\u003c/strong\u003e) or 5 μM of WEHI-539 (\u003cstrong\u003ee\u003c/strong\u003e) for 2 h before incubation with CHX (2 μM). The expression of p27 at each time point was analyzed by western blot. (\u003cstrong\u003ef\u003c/strong\u003e) Summary of the half-life of p27 in non-senescent cells, senescent cells, and senescent cells treated with Bcl-xL inhibitors. Original blots are presented in Supplementary Figure S11. All representative western blot images of p27 half-life were qualified by Image Lab 3.0 software.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/81a0765aa28d57e3eda4f92a.png"},{"id":89320345,"identity":"7bf54650-2a58-49b2-9e02-395c089fdf4f","added_by":"auto","created_at":"2025-08-18 18:23:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":175908,"visible":true,"origin":"","legend":"\u003cp\u003eBcl-xL affects p27 subcellular localization in senescent cells. (\u003cstrong\u003ea\u003c/strong\u003e) Proteins from non-senescent and senescent cells were separated into nuclear and cytoplasmic fractions to analyze the subcellular localization of p27. H3 served as the nuclear protein marker and b-tublin the cytoplasmic protein marker. (\u003cstrong\u003eb\u003c/strong\u003e) DDP-induced senescent cells were treated with ABT-737 or WEHI539 for 24 h, then cells were collected to extract nuclear proteins. The indicated proteins were analyzed by western blot. (\u003cstrong\u003ec\u003c/strong\u003e) A549 cells with or without Bcl-xL stable knockdown were collected to analyze the subcellular localization of p27 after nuclear/cytoplasmic fractionation, followed by western blot. Original blots are presented in Supplementary Figure S12. (\u003cstrong\u003ed\u003c/strong\u003e) Non-senescent and drug-induced senescent A549 cells were incubated with 10 μM of MG-132 for 4 h, followed by IF staining to observe the localization of p27 in cells using confocal microscopy. (\u003cstrong\u003ee\u003c/strong\u003e) DDP-induced senescent cells were treated with 5 μM of Bcl-xL inhibitors for 24 h, cells were fixed and stained with p27 antibody. The p27 localization was observed using confocal microscopy. (\u003cstrong\u003ef\u003c/strong\u003e) A549 cells with stable Bcl-xL knockdown were treated with 10 μM of MG-132 for 4 h, followed by staining with p27 antibody. The expression of p27 was detected using confocal microscopy. S stands for short exposure; L stands for long exposure.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/3789414c41e3c69d7f3d2448.png"},{"id":89318960,"identity":"d4225806-674b-40a9-a7c3-56ca9c7c90f5","added_by":"auto","created_at":"2025-08-18 17:51:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61292,"visible":true,"origin":"","legend":"\u003cp\u003eInactivation of Bcl-xL decreases the phosphorylation on Ser10 of p27. (\u003cstrong\u003ea\u003c/strong\u003e) A549 cells were treated with Dox (100 ng/ml) or DDP (5 μM) for 7 days to induce senescence. The cells were collected to analyze the Ser10 phosphorylation of p27 using western blot. (\u003cstrong\u003eb-c\u003c/strong\u003e) Non-senescent (\u003cstrong\u003eb\u003c/strong\u003e) and drug-induced senescent (\u003cstrong\u003ec\u003c/strong\u003e) A549 cells were treated with ABT-737 (5 μM) or WEHI-539 (5 μM) for 24 h. The expression of Ser10 phosphorylation of p27 was detected by western blot. (\u003cstrong\u003ed\u003c/strong\u003e) The expression of phospho-Ser10 p27 in A549 cells with stable Bcl-xL knockdown were detected by western blot. Original blots are presented in Supplementary Figure S13. Representative western blot images of p-p27 (Ser10) were qualified by Image Lab 3.0 software.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/9682fe9e21baf748b965d4dc.png"},{"id":94165100,"identity":"924e2eff-b610-4a3b-8c68-160f7d243c91","added_by":"auto","created_at":"2025-10-23 06:08:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2689118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/3d9d8316-06ee-404e-89c1-e76db0b10963.pdf"},{"id":89319396,"identity":"2ceb245b-6f4b-4619-b1f1-75b480c16bf5","added_by":"auto","created_at":"2025-08-18 17:59:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3487528,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/178a3ea41904c30ae9733b83.docx"},{"id":89318974,"identity":"1d07a04a-a747-44cf-92a6-fd6f83df8787","added_by":"auto","created_at":"2025-08-18 17:51:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11961203,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalimagesforWesternblot.docx","url":"https://assets-eu.researchsquare.com/files/rs-7250116/v1/17e499fe95571c88119b1a95.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bcl-xL Inhibition Disrupts p27 Stability and Promotes Senescence Escape: Insights for Cancer Senotherapy","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eCellular senescence act as both tumor suppressor and promoter post-chemotherapy.\u003c/li\u003e\n \u003cli\u003eInhibition of Bcl-xL facilitates the cell cycle re-entry of senescent tumor cells.\u003c/li\u003e\n \u003cli\u003eInhibition of Bcl-xL resulted in degradation of p27.\u003c/li\u003e\n \u003cli\u003eAccelerated p27 degradation mediates senescence escape.\u003c/li\u003e\n \u003cli\u003eConsider tumor types and timing is vital for application of Bcl-2 family inhibitors.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eCellular senescence, characterized by an irreversible arrest in cell growth, plays a pivotal role in tumor suppression, wound healing, and the aging process [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Triggered by various stressors, this cellular state was first described by Hayflick and Moorhead (1961) as a limit to the division potential of normal diploid cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. While senescence acts as a defense by halting the proliferation of compromised cells, it paradoxically can promote tumor growth through the senescence-associated secretory phenotype (SASP) that remodels the tumor microenvironment by producing various secreted proteins including inflammatory cytokines, chemokines, etc., and regulating the properties of adjacent cells [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and through intricate reprogramming processes that induce stemness in cancer cells [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, the accumulation of data revealed that severe genotoxic stress-induced senescence is unstable, including oncogene-induced senescence (OIS), it can reverse spontaneously or after secondary stimulation. Numerous studies have shown the phenomenon of escape-from-OIS [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Our pioneering research has previously revealed that exposure of senescent tumor cells to specific clinical anticancer agents can inadvertently facilitate their escape from senescence [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This discovery underlines a critical gap in our understanding of the mechanisms driving this escape, highlighting the need for further investigation.\u003c/p\u003e\u003cp\u003eThe Bcl2 family, traditionally recognized for its anti-apoptotic functions, has emerged as a key regulator of senescence [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This nuanced understanding of Bcl2 family's role underlines the therapeutic potential of senolytic drugs, like navitoclax, which target senescent cells expressing anti-apoptotic Bcl-2 family proteins to mitigate aging-related conditions and enhance cancer treatment outcomes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among the family, Bcl-xL stands out for its unique influence on both senescence and apoptosis, setting it apart from its counterparts [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Importantly, Bcl-xL, along with Bcl-2, contributes to cell cycle regulation, particularly through modulating p27, a key factor in cell cycle control and senescence [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These functions of Bcl2/Bcl-xl underline a comprehensive role beyond apoptosis inhibition, situating p27 not only as a senescence marker but also as a critical determinant in the maintenance of the senescent cell cycle arrest. Understanding the intricate relationship between Bcl2/Bcl-xL and p27 is crucial for unraveling the complex mechanisms of senescence and its implications for cancer development and aging.\u003c/p\u003e\u003cp\u003eOur previous work has highlighted the potential unintended consequences of ABT737, a prototype BH3-mimetic that inhibits anti-apoptotic BCL-2 family proteins, pointing to the complex effects of senolytic drugs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, recent findings have demonstrated that sub-lethal doses of ABT737 can induce mitochondrial DNA (mtDNA) release, stimulating the SASP and potentially contributing to a pro-tumorigenic environment [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This raises the concern that senescent cells, when treated with Bcl-2 family inhibitors, may develop resistance and re-enter the cell cycle, leading to tumor progression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our current study addresses these concerns by revealing that inhibition of Bcl-xL, a key member of the Bcl-2 family, paradoxically facilitates the cell cycle re-entry of senescent tumor cells, bypassing apoptosis. Focusing on the critical roles of Bcl-xL and p27 in the orchestration of senescence and cell cycle dynamics, our study investigates their interplay, particularly under conditions of Bcl-xL inhibition. We aim to elucidate how Bcl-xL inhibition affects p27^Kip1 stability and the potential consequences for senescent tumor cells regarding cell cycle re-entry. This investigation seeks to illuminate the underlying mechanisms through which Bcl-xL influences senescence and cell cycle progression, contributing to the development of targeted approaches for cancer therapy and the management of aging-related pathologies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eCell lines, compounds, plasmids, and antibodies\u003c/p\u003e\u003cp\u003eHuman lung adenocarcinoma A549 cells was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco Modified Eagle Medium (Hyclone) supplemented with 10% FBS (Hyclone) and Penicillin (100 units/ml)-Streptomycin (100 \u0026micro;g/ml) (Hyclone). Cells were cultured in 37\u0026deg;C incubator with 5% CO2. A549 cells with stable knockdown of Bcl2 and Bcl-xL were generated by infection with pLKO.1-Bcl2 shRNAs and pLKO.1-Bcl-xL shRNAs virus followed by puromycin (2 \u0026micro;g/ml) selection. Compounds, including doxorubicin (Dox, Cat#: D107159), ABT-199 (Cat#: A124869), and CX5461 (Cat#: C127663) were obtained from Aladdin (Shang Hai, P. R. China). Cisplatin (DDP) and ABT-737 (Cat#: S1002) were purchased from Selleck Chemicals (Houston, TX, USA). WEHI-539 (Cat#: HY-15607), cycloheximide (Cat#: HY-12320), and MG-132 (Cat#: HY-13259) were purchased from MCE (MedChemExpress, Monmouth Junction, NJ, USA). p27 siRNAs and siRNA control were synthesized by GenePharma (Suzhou, P. R. China). Bcl2 and Bcl-xL shRNAs were synthesized by Tsingke Biological technology (Beijing, P. R. China). All primer sequences were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Plasmids of pCDNA3.1-Bcl2 and pCDNA3.1-Bcl-xL were purchased from Heyin (Hangzhou, P. R. China). Rabbit polyclonal antibodies for Bcl2 (Cat#: 2876S), Bcl-xL (Cat#: 2762S), p27 (Cat#: 3688S), Histone H3 (Cat#: 4620S), and β-tublin (Cat#: 2128S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibody of p27 (Cat#: 25614-1-AP) used for immunofluorescence staining (IF) and p-p27 (Ser10, Cat# AF3326)) were obtained from Proteintech (Rosemont, IL, USA). Goat-anti-rabbit IgG FITC (Cat#: HA1004) was from Hangzhou HuaAn Biotechnology (Hangzhou, P. R. China). Mouse monoclonal antibodies for p21 (Cat#: sc-397), p130 (Cat#: sc-374521), and β-actin (Cat#: sc-47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Goat-anti-mouse IgG (HRP conjugate, Cat#: IH-0031) and goat-anti-Rabbit IgG (HRP conjugate, Cat#: IH-0011) were purchased from Beijing Dingguo Changsheng Biotechnology (Beijing, P. R. China). JC-1 (5,6 -Dichloro-1,1\u0026prime;,3,3\u0026prime;-tetraethyl-imidacarbocyanine iodide) was bought from Beyotime (C2006,, Shanghai, P. R. China).\u003c/p\u003e\u003cp\u003eWestern Blot\u003c/p\u003e\u003cp\u003eChemotherapy-induced senescent cells and non-senescent cells were lyzed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate). Samples were centrifuged at 14,000 \u0026times; g for 10 min at 4\u0026deg;C to remove insoluble debris. About 20 \u0026micro;g of protein quantified by BCA kit was boiled in Laemmli loading buffer for 5 min and loaded to the SDS-PAGE gel for further investigation. The expression of indicated proteins were detected using their specific antibodies.\u003c/p\u003e\u003cp\u003eRNA isolation and RT-qPCR\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from cells using Trizol (Cat#: 10606ES60, Yeasen Biotechnology, Shanghai, P. R. China). Briefly, cells were collected after the indicated treatment and lysed in proper volume of Trizol reagent according to the cell numbers. Then cells were centrifuged at 12,000 \u0026times; g for 10 min at 4\u0026deg;C to remove insoluble debris. Proper volume of chloroform (about 1/5 of Trizol volume) was added to the supernatant to separate RNA from the mixture of nucleoprotein complexes and DNA. Phase separation was achieved by centrifuged at 12,000 g for 15 min at 4\u0026deg;C. The RNA containing phase was transferred to a fresh tube and similar volume of 2-propanol was added to each sample to precipitate the RNA. The precipitated RNA pellet was then washed once with 75% ethanol and dried by air-drying. RNA was dissolved in RNase-free water and quantified using NanoDrop microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was transcribed to cDNA using PrimeScript RT Master Mix and analyzed by TB Green Premix Ex Taq II (Takara Bio USA, Inc. CA, USA). A panel of PCR primers listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e were designed using the Primer3 (v. 0.4.0) software and synthesized by TsingKe Biological Technology (Beijing, P. R. China).\u003c/p\u003e\u003cp\u003eColony formation assay\u003c/p\u003e\u003cp\u003eCells (8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded to 6 well plates overnight before the treatment of Dox (100 ng/ml) or DDP (5 \u0026micro;M) for 7 days. Then cells were washed twice with 1 \u0026times; PBS to remove the residual drugs and cultured in regular DMEM medium in the presence or absence of indicated additional treatment, including Bcl2 family inhibitors and transient knockdown of p27. After the second treatment, the senescent cells were cultured in normal medium for 2\u0026ndash;6 weeks with refeeding every 3 days until colonies were visible. Colonies were detected using crystal violet staining.\u003c/p\u003e\u003cp\u003eβ-galactosidase staining\u003c/p\u003e\u003cp\u003eCells (8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded to 6 well plates overnight before the treatment of Dox (100 ng/ml) or DDP (5 \u0026micro;M) for 7 days. The β-galactosidase activity in senescent cells was measured by senescence β-galactosidase staining kit (Cat#: C0602, Beyotime, Shanghai, P. R. China) according to manufacturer protocol. Briefly, after being washed with 1 \u0026times; PBS, cells were fixed in the fixation buffer for 10 min at room temperature. Then, cells were washed three times with 1 \u0026times; PBS and incubated with staining buffer containing X-gal at 37\u0026deg;C overnight before being photographed.\u003c/p\u003e\u003cp\u003eRNA interference\u003c/p\u003e\u003cp\u003eDox or DDP-induced senescent cells were transfected with 20 pmol control siRNA or p27 siRNAs using lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA) following manufacturer instruction. The cells were cultured in regular DMEM medium after 48 h of transfection and the formed colonies were observed by crystal violet staining. The p27 knockdown efficiency in senescent cells was confirmed by western blot.\u003c/p\u003e\u003cp\u003eCycloheximide chase assay\u003c/p\u003e\u003cp\u003eThe protein stability of p27 in both non-senescent or senescent cells with or without Bcl2 family inhibitors treatment were analyzed by cycloheximide (CHX), a protein synthesis inhibitor. Cells pre-treated with or without indicated inhibitors for 2 h were incubated with CHX (2 \u0026micro;M) for 0\u0026ndash;7 h. Then, cells at each time point were immediately collected and lysed, and protein level was analyzed by western blot.\u003c/p\u003e\u003cp\u003eSubcellular localization analysis\u003c/p\u003e\u003cp\u003eThe localization of p27 was analyzed by western blot and IF staining. Nuclear and cytoplasmic protein extraction kit combined with western blot was employed to detect p27 subcellular localization in non-senescent and senescent cells with or without Bcl-xL inhibitor treatment. The protein preparation was performed following manufacturer\u0026rsquo;s instruction and the western blot was as measured above. Histone H3 was served as the nuclear reference and β-tublin was used as the cytoplasm reference. For IF staining, about 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells were seeded to each chamber of 35 mm confocal dishes (Cellvis, Sunnyvale, California, USA) for 24 h. Then cells were rinsed twice with 1 \u0026times; PBS containing 0.1% Triton X-100 (PBST) and fixed in 4% paraformaldehyde (PFA) solution for 10 min at room temperature (RT). The fixed cells were rinsed twice with PBST and incubated in PBST for another 5 min. After blocking with 1% BSA dissolved in PBS for 30 min at RT, cells were incubated with indicated primary antibodies diluted in 1% BSA (1 : 200, v/v) for 4 h at RT or overnight at 4\u0026deg;C. Then, the cells were washed three times with PBST to remove unbound primary antibodies and incubated with FITC-conjugated secondary antibody for another 2 h at RT. Anti-fade solution containing DAPI (Cat#: P0131, Beyotime, Shanghai, P. R. China) were added to each chamber after the cells were washed three times with PBST. A minimum of 100 cells were imaged using confocal microscopy (Leica, Wetzlar, Germany).\u003c/p\u003e\u003cp\u003eJC-1 stainning\u003c/p\u003e\u003cp\u003eMitochondrial membrane potential (MtMP) in senescent cells was measured by staining with JC-1. In brief, A549 cells treated with DDP (5 \u0026micro;M), or Dox (100 ng/ml) for 7 days were incubated in pre-heated MOPS containing 1 \u0026micro;M JC-1 for 15 min at 37\u0026deg;C in the dark, washed with PBS, then observed at either 510 nm (green mitochondria/J-monomer) or 590 nm (red-to-orange mitochondria/J-aggregate) using a confocal microscope (Leica, Wetzlar, Germany). A minimum of 100 cells were imaged.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll results were reproducible in at least three independent experiments. Values were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Student\u0026rsquo;s t-test was used for analyzing \u003cem\u003ep\u003c/em\u003e value. \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant (marked as *).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eInhibition of Bcl-xL induces senescence escape\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn our investigation into the effects of Bcl-xL inhibition on senescent tumor cells, we employed both pharmacological and genetic approaches to modulate Bcl-xL activity. To induce senescence, cells were treated with one of three agents: the RNA polymerase I inhibitor CX5461 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, reported in our previous paper [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-b), cisplatin (DDP), or doxorubicin (Dox) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb-d). These agents were selected for their diverse mechanisms of action, providing a broad spectrum of senescence induction to ensure the observed effects on cell cycle re-entry were not specific to a single pathway of senescence stimuli.\u003c/p\u003e\u003cp\u003eFollowing the establishment of a senescent phenotype, confirmed by increased SA-β-galactosidase staining and halted proliferation, we proceeded with the inhibition of Bcl-xL. This was achieved through the administration of WEHI-539, a specific inhibitor of Bcl-xL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea-b), and ABT-737, which targets multiple members of the Bcl-2 family, including Bcl-xL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ec). Remarkably, both inhibitors facilitated the escape of senescent cells from their growth arrest, evident from a significant decrease in senescent cells and an increase in cloning formation, indicative of cell cycle re-entry. Besides, Bcl2-specific inhibitor ABT199 didn\u0026rsquo;t shows the effect, underline the Bcl-xl involved the senescence escape (supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eTo validate these findings, we further silenced Bcl-xL using shRNA in cells rendered senescent by Dox treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-f). Mirroring the effects observed with pharmacological inhibition, genetic knockdown of Bcl-xL led to a marked upsurge in cloning formation. Similar results were also found in PC-9 cells (supplementary Fig. S3a-b). These results underline Bcl-xL\u0026rsquo;s critical role in maintaining the senescent state and suggest its inhibition as a potential mechanism to promote cell cycle re-entry in senescent tumor cells.\u003c/p\u003e\u003cp\u003eA significant insight from our experiments, particularly highlighted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg was the notable increase in Bcl-xL levels within the cytoplasm of cells treated with DDP and Dox. This shift in Bcl-xL localization from a potentially nuclear to a more cytoplasmic presence upon senescence induction adds a layer of complexity to our understanding of Bcl-xL's role in cellular senescence. It implies a regulatory mechanism at play, where Bcl-xL's cellular distribution may influence the senescence outcome and, consequently, the cell's fate upon Bcl-xL inhibition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ep27 involved in Bcl-xL inhibition induced senescence escape\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the pronounced role of Bcl-xL in the regulation of senescence, our investigation next sought to uncover the mechanisms through which Bcl-xL inhibition facilitates the escape from senescence. To this end, senescent cells, both untreated and treated with Bcl-xL inhibitors, underwent proteome analysis to pinpoint changes in protein expression critical for cell cycle control. Among the most significantly down-regulated proteins, p27 stood out as a key factor in cell cycle regulation, drawing our attention to its potential involvement in senescence dynamics.\u003c/p\u003e\u003cp\u003eThe down-regulation of p27 was found to be specific to senescent cells. Western blot analyses, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c and Supplemental Fig. S4a, confirmed a noticeable decrease in p27 protein levels in senescent cells exposed to various Bcl-xL inhibitors. This effect was not observed in non-senescent cells, underscoring the senescence-specific impact of Bcl-xL inhibition. In contrast, the levels of another cell cycle regulator, p130 protein, remained unchanged across both senescent and non-senescent cells, as shown in Supplemental Fig. S4c-e, highlighting the selective down-regulation of p27 upon Bcl-xL inhibition.\u003c/p\u003e\u003cp\u003eFurther substantiating the specificity of this effect, the reduction in p27 protein expression in senescent cells was notably induced by the Bcl-xL inhibitor WEHI-539, consistent with observations from the use of the pan-specific inhibitor ABT-737. This contrasted with the slight reduction seen after 48 hours of treatment with the Bcl-2-specific inhibitor ABT-199, as documented in Supplemental Fig. S4b, indicating a unique sensitivity of p27 to Bcl-xL-targeted inhibition. To further confirm the role of Bcl-xL in regulating p27, we performed shRNA-mediated inhibition of Bcl-xL in PC-9 cells (Fig. S3c). The results demonstrated that inhibition of Bcl-xL by shRNA led to a significant reduction in p27 levels, while p21 remained unchanged, reinforcing the specificity of Bcl-xL's effect on p27 stability.\u003c/p\u003e\u003cp\u003eTo ascertain the role of p27 in maintaining senescence, we employed siRNA to transiently knock down p27. Among several targeted siRNAs, only siRNA1 reduce half p27 protein and siRNA3 markedly reduced p27 expression in senescent cells. Notably, only efficient knockdown of p27 led to significant changes in cell behavior, including enhanced colony formation, as evidenced in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-e. This outcome reinforces the critical role of p27 in senescence maintenance and suggests that its down-regulation, triggered by Bcl-xL inhibition, is a key event facilitating senescence escape.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBcl-xL regulate p27 protein stability in senescent and senescent escape cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn our detailed examination of Bcl-xL's role in cellular senescence, we found both mRNA and protein levels of Bcl-xL to be significantly elevated in cells rendered senescent by DDP and Dox treatment, aligning with findings from other studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, this increase in Bcl-xL was observed regardless of the p53 status, as evidenced by the elevated levels of Bcl-xL in both H358 (p53 null) and PC-9 (p53 mutant) cell lines following treatment with senescence-inducing drugs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b, supplementary Fig. S5a-b). This underlines the independence of Bcl-xL upregulation from p53-mediated pathways in the context of drug-induced senescence.\u003c/p\u003e\u003cp\u003eContrastingly, while p27 protein levels saw a significant increase post-treatment, p27 mRNA levels did not exhibit a similar upsurge (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-g). This discrepancy, particularly in the absence of down-regulation in p27 mRNA within Bcl-xL inhibitor-treated senescent cells, points to a post-transcriptional mechanism of regulation, suggesting that p27's stability is modulated as a downstream effect of Bcl-xL activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further elucidate this mechanism, we assessed p27's stability across different cellular contexts. In non-senescent cells, p27 exhibited a half-life of approximately 2 hours, which markedly extended to 7 hours in DDP-induced senescent cells, and surpassed 7 hours in Dox-induced counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). This enhancement in p27 stability underlines its role in the senescent phenotype. However, the introduction of Bcl-xL inhibitors notably disrupted this stability, reducing p27's half-life back to around 2 hours, as demonstrated in cells treated with either ABT-737 or WEHI-539 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f). Intriguingly, in non-senescent cells, WEHI-539 appeared to marginally increase p27 stability (Fig. S6a), while ABT-199 showed no significant impact on p27's half-life in senescent cells, highlights the unique regulatory pathway mediated by Bcl-xL (supplementary Fig. S6b).\u003c/p\u003e\u003cp\u003eThese observations collectively highlight Bcl-xL's critical involvement in regulating p27 stability, a key factor in maintaining the senescent state. By modulating p27 levels through a post-transcriptional mechanism, Bcl-xL emerges as a critical player in the dynamics of senescence and its potential reversal, offering insightful perspectives into the molecular intricacies of senescence and the escape from this cellular fate.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ep27 Ser10 phosphorylation and re-localization in senescent and senescent escape cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, we examined the role of Bcl-xL in modulating the function of p27, a key regulator of the cell cycle, focusing on changes in its stability and subcellular localization. Initial fractionation studies in non-senescent A549 cells revealed that p27 is predominantly cytoplasmic. However, upon inducing senescence with chemotherapy drugs, there was a significant increase in p27 levels in both the cytoplasm and nucleus, indicating a shift in localization in response to senescence induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In contrast, p130, another cell cycle regulator, remained nuclear across all conditions.\u003c/p\u003e\u003cp\u003eTreatment with Bcl-xL inhibitors, ABT-737 and WEHI-539, resulted in a significant decrease in nuclear p27 within 24 hours, suggesting Bcl-xL's involvement in controlling p27's nuclear localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This effect was corroborated in cells with Bcl-xL knocked down, where nuclear p27 levels also significantly decreased, unlike in cells lacking Bcl-2, where the nuclear/cytoplasmic ratio of p27 remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, supplementary Fig. S7d).\u003c/p\u003e\u003cp\u003eImmunofluorescence staining further confirmed these observations, showing an increase in nuclear p27 in DDP and Dox-induced senescent cells. The inactivation of Bcl-xL led to a shift in p27 localization to the cytoplasm, as observed in Bcl-xL knockdown cells, particularly in the presence of MG-132 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-f, Fig. S7a-b). This re-localization effect of Bcl-xL inhibition on p27 was not observed in non-senescent cells (Fig. S7c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, the study investigated the role of Ser10 phosphorylation in p27 stability. Phosphorylation of p27 on Ser10, which influences its stability, was found to be higher in senescent cells compared to non-senescent cells. This phosphorylation was notably reduced by treatment with Bcl-xL inhibitors, indicating a mechanism through which Bcl-xL may regulate p27 stability and thereby affect its function (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-d).\u003c/p\u003e\u003cp\u003eThese findings highlight Bcl-xL's critical role in influencing p27's subcellular localization and stability, including the modulation of Ser10 phosphorylation. By affecting the distribution and availability of p27 for cell cycle control, Bcl-xL contributes to the cellular decision-making process regarding senescence induction and escape, providing important insights into the molecular dynamics of cellular senescence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCellular senescence acts as a double-edged sword in oncology, both suppressing tumors and promoting a pro-tumorigenic environment following chemotherapy, thus necessitating targeted removal strategies. Our research contributes to this complex landscape by revealing an unanticipated consequence of Bcl-xL inhibition. Instead of inducing apoptosis, inhibiting Bcl-xL facilitates the cell cycle re-entry of senescent tumor cells. This discovery suggests that while Bcl-xL is typically understood as a survival factor, its role in senescence is crucially linked to p27 stability.\u003c/p\u003e\u003cp\u003eThe persistent presence of senescent cells may foster a pro-tumorigenic environment through the secretion of inflammatory factors and initiate reprogramming processes that induce stemness in cancer cells, complicating tumor therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These findings highlight the therapeutic potential of senolytic drugs, particularly Bcl-2 family inhibitors like ABT-737 and ABT-263, which have shown promising efficacy in preclinical studies for eliminating senescent cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, our study underlines the complexity of targeting senescent cells, as Bcl-xL deficiency, while enhancing senescent cell death, also allows a subset of these cells to survive and proliferate rapidly.\u003c/p\u003e\u003cp\u003eThe complexities of cancer senolytic therapy are highlighted by the phenomenon of cell cycle reactivation in senescent tumor cells, influenced by cytotoxic drugs, which might contribute to the observed discrepancy in long-term survival benefits despite favorable initial responses. This underlines the importance of understanding the mechanisms behind senescence escape triggered by senolytic drugs to refine cancer therapeutic strategies. Since our previous study that observed subjecting senescent tumor cells to cytotoxic treatments stimulates the clonogenic proliferation of remaining survivors [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], subsequent research by other research groups have suggest WNT /β-catenin pathways induction, as a promoter, initiate the senescence-escape reprogram [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our present findings delineate a specific reduction in p27 expression in senescent cells upon Bcl-xL inhibition, without affecting p130, identifying p27 as a crucial blockage element in the process of senescence escape associated with Bcl2 family inhibition.\u003c/p\u003e\u003cp\u003eThe observed regulatory complexity of p27, influenced by Bcl-xL inhibition, suggests a nuanced interaction between Bcl-xL and p27 stability. Our investigation into p27 stability demonstrated its post-translational accumulation in chemotherapy-induced senescent cells, unaffected at the mRNA level by Bcl-xL inhibitors, as confirmed by cycloheximide chase assays. This significant reduction in p27 stability upon Bcl-xL inhibition unveils a mechanism that potentially promotes senescence escape. The differential activity of E3 ubiquitin ligases responsible for p27 degradation across the cell cycle suggests that Bcl-xL's regulation of p27 stability and localization could significantly impact senescence dynamics and cell cycle re-entry. Moreover, we explored the role of Ser10 phosphorylation in p27 stability, observing an increase in senescent cells. Bcl-xL inhibition notably reduced this phosphorylation, indicating a possible mechanism through which Bcl-xL modulates p27 stability, thus affecting its cellular function.\u003c/p\u003e\u003cp\u003eIn conclusion, our study reveals that Bcl-xL inhibition mediates senescence escape predominantly through accelerated p27 degradation, either by promoting its nuclear export for cytoplasmic degradation or by inhibiting its phosphorylation and subsequent nuclear degradation. These insights into the mechanism of senescence escape facilitated by Bcl-xL inhibition are critical for the development of more effective cancer therapies. Given the complex role of Bcl-xL in tumor biology, careful consideration of tumor types and timing of drug application is essential to harness the full therapeutic potential of Bcl-2 family inhibitors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYang\u0026nbsp;\u003c/strong\u003edesigned and performed the experiments, analyzed the data, supervised the project, and wrote the manuscript; \u003cstrong\u003eX. He,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eX. Chen\u0026nbsp;\u003c/strong\u003edesigned and performed the experiments, analyzed the data; \u003cstrong\u003eD. Cui\u0026nbsp;\u003c/strong\u003eanalyzed the data; \u003cstrong\u003eX. Qian, S. Zhang. X. Yang,\u003c/strong\u003e \u003cstrong\u003eW. Fan, N. Huang\u0026nbsp;\u003c/strong\u003eperformed the experiments; \u003cstrong\u003eM.Du\u003c/strong\u003e contributed to sample preparation; \u003cstrong\u003eL. Chen\u003c/strong\u003e analyzed the data, discussed the results and wrote the manuscript. All the authors contributed to revision the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by grants from the Natural Science Foundation of Zhejiang Province (LY20H160046 to L. Yang); Research Program for Medicine and Health Science and Technology of Zhejiang Province (2021KY080 to L. Yang, 2022KY585 to X. Yang); and Zhejiang People\u0026rsquo;s Hospital Research Startup Fund (ZRY2019A003 to L. Yang).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Clinical Research Institute, Zhejiang Provincial People\u0026rsquo;s Hospital for providing the research platform and experimental equipment. The authors also thank Xue Yang for technical assistance in confocal microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are available within the article and its supplementary data files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo Human/Animal Subjects: The research does not involve experiments on human participants or animals, nor does it use their tissues.\u003c/p\u003e\n\u003cp\u003eEthics Committee Confirmation: The relevant institutional ethics committee (Zhejiang Provincial People\u0026apos;s Hospital Research Ethics Committee) has explicitly reviewed the study and confirmed that formal ethical approval is not required based on the nature of the work.\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHernandez-Segura A, Nehme J, Demaria M: \u003cstrong\u003eHallmarks of Cellular Senescence\u003c/strong\u003e. \u003cem\u003eTrends in cell biology \u003c/em\u003e2018, \u003cstrong\u003e28\u003c/strong\u003e(6):436-453.\u003c/li\u003e\n\u003cli\u003eChaib S, Tchkonia T, Kirkland JL: \u003cstrong\u003eCellular senescence and senolytics: the path to the clinic\u003c/strong\u003e. \u003cem\u003eNature medicine \u003c/em\u003e2022, \u003cstrong\u003e28\u003c/strong\u003e(8):1556-1568.\u003c/li\u003e\n\u003cli\u003eHayflick L: The Limited in Vitro Lifetime of Human Diploid Cell Strains. \u003cem\u003eExp Cell Res \u003c/em\u003e1965, 37:614-636.\u003c/li\u003e\n\u003cli\u003eCoppe JP, Desprez PY, Krtolica A, Campisi J: \u003cstrong\u003eThe senescence-associated secretory phenotype: the dark side of tumor suppression\u003c/strong\u003e. 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al\u003c/em\u003e: \u003cstrong\u003eApoptotic stress causes mtDNA release during senescence and drives the SASP\u003c/strong\u003e. \u003cem\u003eNature \u003c/em\u003e2023, \u003cstrong\u003e622\u003c/strong\u003e(7983):627-636.\u003c/li\u003e\n\u003cli\u003eMartin N, Popgeorgiev N, Ichim G, Bernard D: \u003cstrong\u003eBCL-2 proteins in senescence: beyond a simple target for senolysis?\u003c/strong\u003e \u003cem\u003eNature reviews Molecular cell biology \u003c/em\u003e2023, \u003cstrong\u003e24\u003c/strong\u003e(8):517-518.\u003c/li\u003e\n\u003cli\u003eTriana-Martinez F, Loza MI, Dominguez E: Beyond Tumor Suppression: Senescence in Cancer Stemness and Tumor Dormancy. \u003cem\u003eCells \u003c/em\u003e2020, 9(2).\u003c/li\u003e\n\u003cli\u003evan Deursen JM: \u003cstrong\u003eSenolytic therapies for healthy longevity\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e2019, \u003cstrong\u003e364\u003c/strong\u003e(6441):636-637.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Bcl-xL, Senescence Escape, p27 Stability, Senotherapy, Chemotherapy","lastPublishedDoi":"10.21203/rs.3.rs-7250116/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7250116/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e Cellular senescence serves as a double-edged sword in oncology, acting as a tumor suppressor and a promoter of a pro-tumorigenic environment post-chemotherapy, necessitating targeted removal strategies. To investigate whether inhibition of Bcl-xL (a key anti-apoptotic protein and senescent cell survival factor) leads to therapeutic failure by enabling senescent tumor cells to resist apoptosis, re-enter the cell cycle, and potentially drive tumor progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe utilized pharmacological Bcl-xL inhibitors and shRNA-mediated Bcl-xL knockdown to disrupt Bcl-xL function in senescent tumor cells. The effects on cell fate, cell cycle regulators, and associated molecular pathways were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eContrary to the expected induction of apoptosis, Bcl-xL inhibition facilitated the cell cycle re-entry of senescent tumor cells. This escape from senescence was driven by a marked reduction in the stability of the cell cycle inhibitor p27. The reduction occurred through increased cytoplasmic localization of p27 and reduced phosphorylation, leading to its proteasome-dependent degradation. Thus, Bcl-xL disruption initiates a pathway enabling senescence escape.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eInhibiting Bcl-xL in senescent tumor cells promotes senescence escape rather than apoptosis, representing a significant paradigm shift in understanding senolytic drug mechanisms. These findings reveal the complex dual nature of senescence in cancer and underscore the critical need for careful design when combining senolytic agents with chemotherapeutics to prevent inadvertent tumor cell proliferation.\u003c/p\u003e","manuscriptTitle":"Bcl-xL Inhibition Disrupts p27 Stability and Promotes Senescence Escape: Insights for Cancer Senotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-18 17:50:59","doi":"10.21203/rs.3.rs-7250116/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":"586fae9d-b8bd-4f66-88d6-73dd6f4bf86a","owner":[],"postedDate":"August 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-23T06:08:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-18 17:50:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7250116","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7250116","identity":"rs-7250116","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}