CBX2 Enhances Paclitaxel Resistance via PI3K/AKT Signaling Pathway in Breast Cancer

CBX2 Enhances Paclitaxel Resistance via PI3K/AKT Signaling Pathway in Breast Cancer | 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 CBX2 Enhances Paclitaxel Resistance via PI3K/AKT Signaling Pathway in Breast Cancer Yishan He, Mengquan Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6902662/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 Objective Paclitaxel resistance is a significant contributor to poor prognosis in breast cancer treatment. The expression of CBX2 is upregulated in breast cancer with paclitaxel resistance. But the role and mechanism of CBX2 in promoting drug resistance in breast cancer is still unclear. Methods The CBX2 gene expression was inhibited by gene knockdown technology. Cell activity was assessed through CCK8 assay. The apoptosis levels evaluated via flow cytometry. The gene expression levels were detected using Q-PCR. The protein expression level was detected with WB. Transcriptome sequencing was employed to identify differences in gene expression and pathways regulated by CBX2. Mitochondrial membrane potential was analyzed by JC-1 assay. Results With CBX2 gene knockdown, the p-gp protein expression level was decreased, cell activity was declined, apoptosis level was reduced, PI3K and AKT protein levels were decreased, Bcl-2 protein level was decreased, mitochondrial membrane potential loss, and the pro-apoptotic proteins BAX, Caspase9 and Caspase3 levels were increased. Conclusion CBX2 activates the PI3K-AKT signaling pathway, thereby enhancing the anti-apoptotic capacity of cells and promoting resistance to paclitaxel in breast cancer. Therefore, targeting CBX2 may represent a promising strategy for overcoming drug resistance in breast cancer therapy. breast cancer Paclitaxel resistance CBX2 PI3K Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Breast cancer has emerged as a significant threat to women's health. According to the national cancer statistics published by the National Cancer Center in 2023, the incidence of female breast cancer is 29.05/100,000, making it the most prevalent tumor among women. Furthermore, both incidence and mortality rates have shown a continuous increase from 2000 to 2016[ 1 ]. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by extensive intratumor heterogeneity, high metastasis, and chemoresistance, leading to the highest mortality rate[ 2 , 3 ]. Drug resistance remains one of the foremost clinical challenges in breast cancer treatment and is a critical factor contributing to poor clinical outcomes[ 4 ]. Despite advancements in research and therapy, our understanding of the underlying mechanisms driving drug resistance is still limited. Therefore, how to reverse the drug resistance in breast cancer and enhance the therapeutic efficacy has become an important clinical issue that requires immediate attention. CBX2 protein is an important component of the Polycomb Repressive Complex 1 (PRC1), which can regulate the transcriptional states of genes through chromatin remodeling and histone modification[ 5 ]. Notably, CBX2 is overexpressed in breast tumors, with the highest levels observed in triple-negative breast cancer[ 6 ]. Knockdown of CBX2 leads to a reduction in mTORC1 activity, thereby inhibiting E2F signal and promoting the aging of breast cancer cells[ 7 ]. Furthermore, knockout of the CBX2 gene results in compromised genomic stability, increased spontaneous chromosome breakage, and a tendency towards polyploidy. This disruption is particularly associated with cell cycle abnormalities, notably blocking G2/M transition, ultimately leading to apoptosis in ovarian cancer cells[ 8 , 9 ]. The study revealed that CBX2 is overexpressed and enhances the activity of leukemia cells. The stability of CBX2 is regulated by SAHA [ 10 ]. Notably, downregulation of CBX2 significantly reduced leukemia cell activities and increased the cell apoptosis rates [ 11 ]. Furthermore, CBX2 could promote the demethylation of H3K27me3 by recruiting Jmjd3, and enhance the transcription level of IFN-β[ 12 ]. The CBX2 could interact with SMARCE1 to inhibit EGFR transcription [ 13 ]. CBX2 was highly expressed in chemotherapy-resistant ovarian tissues and promoted the cell proliferation[ 14 ]. The related study indicated that inhibition of CBX2 could enhance the sensitivity of hepatocellular carcinoma (HCC) cells to oxaliplatin both in vitro and in vivo [ 15 ]. This research has confirmed that CBX2 plays a crucial role as a regulator of paclitaxel resistance in triple-negative breast cancer cells. Downregulating CBX2 expression could significantly decrease the expression level of P-gp protein and reverse paclitaxel resistance in breast cancer cells, but the underlying mechanism is still unclear. 2. Materials and Methods 2.1 The Cell culture and treatment The MDA-MB-231/PTX cells were cultured in DMEM supplemented with 10%FBS and 1%PS, under a humidified atmosphere containing 5% (v/v) CO 2 at 37℃. The plasmid was transfected into cells using Lipofectamine™ 3000 (Invitrogen, L3000001). 2.2 CCK8 assay The MDA-MB-231/PTX cells were seeded into 96 well plates, and subjected to various treatment conditions for 24h, 48h and 72h. Following the treatments, the cells were incubated with 10% CCK8 for a period of 1 to 2 hours. The optical density (OD) value was subsequently measured at a wavelength of 450nm by microplate reader. 2.3 Quantitative real-time PCR assay MDA-MB-231/PTX cells were lysed with TRIzol reagent (Life technogies, 15596018). the cDNA was synthesized with PrimeScript™RT reagent Kit (TaKaRa, RR047A). The PCR was conducted with Novostart SYBR qPCR SuperMix Plus (Novoprotein, E096-01B). The amplification conditions included an initial denaturation at 95°C for 1 minute, followed by 40 cycles of denaturation at 95°C for 20 seconds and annealing/extension at 60°C for 1 minute. The sequences of the sense and antisense primers are as follows: 5ʹ-AGAAGGAACATGAGAAGGAGGTG-3ʹ and 5ʹ-GACTTGGATTTGGAGGGAG C-3ʹ (CBX2); 5ʹ-TGACTTCAACAGCGACACCCA-3ʹ and 5ʹ- CACCCTGTTGC TGTAGCCAAA-3ʹ (GAPDH). Relative expression was assessed utilizing the comparative threshold cycle method and determined by the value of 2 –ΔΔCt . 2.4 Western blot assay The protein lysates from MDA-MB-231/PTX cells were obtained using RIPA lysis buffer (Beyotime Institute of Biotechnology, P0013B), and subsequently separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). The membranes were blocked with 5% nonfat milk, and probed with a primary antibody and probed with a primary antibody followed by HRP-conjugated goat anti-rabbit/mouse secondary antibodies (ZB-2301/ZB-2305, Zsbio). The membranes were visualized with an ECL Kit (34095, Thermo). The proteins densities were analyzed by image J software. The primary antibody: anti-CBX2 antibody (15579-1-AP, proteintech), anti-P-gp antibody (22336-1-AP, proteintech), anti-PI3K antibody (ab154598, abcam), anti-AKT antibody (ab8805, abcam), anti-Bcl-2 antibody (ab182858, abcam), anti-Caspae9 antibody (ab32539, abcam), anti-Caspae3 antibody (ab32351, abcam), and anti-GAPDH antibody (10494-1-AP, proteintech). 2.5 Mitochondrial membrane potential assay The MDA-MB-231/PTX cells were cultured in a confocal dish and subjected to various treatment conditions for 48 hours. Subsequently, the cells were incubated with JC-1(C2006, Beyotime) for 20 minutes. Images were captured using a fluorescent microscope. 2.6 Cell apoptosis assay The MDA-MB-231/PTX treated under various conditions for 48h. Following treatment, the cells were digested using pancreatic enzymes, collected by centrifugation, and subsequently incubated with Annexin V/PI (C1062L, Beyotime). The analysis was performed using flow cytometry. 2.7 Statistical analysis All results are represented as the means ± standard deviation (SD) derived from a minimum of three independent experiments. The Statistical significance was assessed using Student's t-test for comparison between two groups. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001 were deemed statistically significant. All statistical analyses were conducted utilizing GraphPad Prism version 8.0. 3. Results 3.1 The expression difference of CBX2 in breast cancer The expression difference of CBX2 in different tumors was analyzed using an online database ( https://www.proteinatlas.org ), revealing that, in comparison to normal tissues, CBX2 expression was significantly upregulated in tumors to varying extents (Fig. 1 A). The expression of CBX2 in different subtypes of breast cancer was analyzed with Timer2.0. The findings indicated that CBX2 expression was elevated in all breast cancer subtypes compared to normal tissue, with particularly high levels observed in basal subtype, which belongs to triple-negative breast cancer (Fig. 1 B). The expression level of CBX2 in breast cancer tissues was further analyzed alongside pathological data, revealing that CBX2 is significantly overexpressed in these tissues (Fig. 1 C). Survival prognosis analysis indicated that patients exhibiting high levels of CBX2 expression had a poorer survival outcome (Fig. 1 D). Finally, an analysis utilizing the NGDC single-cell database demonstrated that the expression of CBX2 was markedly upregulated in cancer cells (Fig. 1 E). 3.2 The changes of CBX2 expression before and after PTX resistance in breast cancer were assessed MDA-MB-231 cells and MDA-MB-231/PTX cells were observed (Fig. 2 A). The cell activity was checked using CCK8 assay. The results showed that the half maximal inhibitory concentration (IC50) for MDA-MB-231 and MDA-MB-231/PTX cells were 9.399 and 23.40nM (Fig. 2 A). The expression levels of CBX2 protein were examined via WB. The result showed that the expression level of CBX2 was up-regulated in MDA-MB-231/PTX cell lines (Fig. 2 B). The transcription level of CBX2 was detected by Q-PCR, and the results showed that the expression of CBX2 was up-regulated in MDA-MB-231/PTX cell lines (Fig. 2 C). To further investigate the role of CBX2 in drug resistance, the three gene knockdown vectors were designed and constructed. Both Q-PCR and WB results showed that the three knockdown vectors significantly inhibited the expression of CBX2, with the shCBX2-2 vector exhibiting the highest efficiency in knockdown (Fig. 2 D-E). 3.3 The effect of CBX2 on the activity of drug-resistant cells was analyzed The results from the CCK8 assay indicated that knockdown of the CBX2 gene significantly reduced the activity of MDA-MB-231/PTX cells (Fig. 3 A). Flow cytometry analysis revealed an increase in apoptosis levels in CBX2-knockdown cells (Fig. 3 B). These results suggest that CBX2 plays an important role in the process of drug resistance and anti-apoptosis in breast cancer. Subsequently, CBX2 was further overexpressed in MDA-MB-231/PTX cell, resulting in an increase in cellular activity. In contrast, the knockdown of CBX2 led to a decrease in cell activity (Fig. 3 D). Combined with the analysis of DRESIS database, it was observed that the resistance gene ABCB1 (p-gp) is upregulated in breast cancer exhibitin resistance to paclitaxel (Fig. 3 C). Furthermore, Western blot analysis revealed an increased expression level of P-glycoprotein (Fig. 3 E). With CBX2 overexpression, the expression level of P-gp was increased. Conversely, following the knockdown of CBX2, a decrease in the expression level of P-gp was observed. 3.4 Transcriptome sequencing analysis The difference genes were analyzed by transcriptome sequencing following the knockdown of the CBX2 gene. The genes expression levels were presented in heat maps (Fig. 4 A). Furthermore, the signaling pathway enrichment analysis using KEGG revealed that the PI3K-AKT pathway underwent significant changes (Fig. 4 B). Meanwhile, an analysis of the DRESIS database revealed that the PI3K-AKT pathway is implicated in breast cancer's resistance to paclitaxel (Fig. 4 C). Furthermore, Western blot results indicated that the PI3K and AKT proteins were high levels, but the difference is not significant following the overexpression of CBX2. In contrast, upon knockdown of CBX2, there was a marked decrease in the protein expression levels of both PI3K and AKT. (Fig. 4 D). 3.5 The effects of CBX2 on anti-apoptosis of breast cancer resistant cells were analyzed The DRESIS database analysis result showed that the apoptosis pathway was activated in breast cancer cells treated with paclitaxel (Fig. 5 A). A decreased mitochondrial membrane potential is a significant characteristic of apoptosis. So that, mitochondrial membrane potential was detected with JC-1 staining. The results showed that there are no significant alterations in membrane potential associated with CBX2 overexpression. however, a marked reduction in membrane potential was observed following CBX2 knockdown (Fig. 5 B). To further investigate whether the knockdown of CBX2 could activate the apoptosis pathway by inhibiting the PI3K-AKT signaling pathway, we assessed key proteins involved in apoptosis through Western blot analysis in this study. The result showed that following CBX2 knockdown, the expression level of anti-apoptosis protein BCL-2 was significantly reduced, while the levels of pro-apoptotic proteins BAX, Caspase-9, and Caspase-3 were markedly increased. In contrast, with the overexpression of CBX2, the expression of anti-apoptosis protein BCL-2 was a high level. However, the expressions of pro-apoptotic proteins BAX, Caspase 9, and Caspase 3 were low levels (Fig. 5 C). These results showed that CBX2 is related with anti-apoptosis abilities in BC. 4. Discussion Breast cancer has emerged as a significant health threat to women[ 16 ]. Drug resistance in breast cancer represents a significant factor contributing to poor prognosis and high mortality rates among patients in clinical settings[ 17 ]. Paclitaxel is a first-line therapeutic agent for breast cancer. Paclitaxel resistance has long posed a significant challenge in clinical treatment. This study found that the expression of gene CBX2 was upregulated in breast cancer cells exhibiting paclitaxel resistance. Relevant studies have confirmed that the drugs sensitivity enhance with CBX2 knocking down[ 14 , 15 ]. In this project, CBX2 gene knockdown vectors were constructed, and subsequently transfected into MDA-MB-231/PTX cells. With CBX2 gene knockout, the cell activity decreased, apoptosis level increased, and the expression of P-gp, a key drug-resistant protein, was down-regulated. These findings indicate that cellular drug sensitivity has increased and that drug toxicity has heightened, ultimately leading to enhanced apoptosis rates. DRESIS database also showed that breast cancer cells activate the pro-survival pathway by PI3K during the process of paclitaxel resistance. Survival signals induced by various receptors are primarily mediated mainly by PI3K/AKT pathway [ 18 ]. Consequently, the PI3K/AKT pathway contributes to the multidrug resistance observed in cancers [ 19 ]. The PI3K/Akt signaling pathway represents a targeted therapeutic approach for cancer treatment [ 20 , 21 ]. In this study, transcriptome sequencing results were analyzed, revealing that knockdown of CBX2 led to inhibition of the PI3K-AKT pathway. The WB experiment confirmed that the expression levels of PI3K and AKT were down-regulated in the CBX2 knockdown group. These findings suggest that CBX2 inhibits cell viability by intervening in the PI3K-AKT pathway, thereby enhancing cellular sensitivity to paclitaxel. Furthermore, activation of the apoptosis pathway was observed through down-regulation of BCL-2 expression. Apoptosis is a form of programmed cell death that is intricately regulated by the balance between pro-survival and pro-apoptotic members of the BCL-2 protein family [ 22 ]. The apoptosis programmed process highly regulated by the BCL-2 family of proteins [ 23 ]. Cancer cells commonly evade apoptosis through upregulation of the BCL-2 anti-apoptotic proteins [ 24 ]. Therefore, targeting the inactivation of BCL-2 has demonstrated significant therapeutic advantages by enhancing apoptotic sensitivity and reversing drug resistance [ 25 ]. BCL-2 is an anti-apoptotic protein, while the pro-apoptotic protein BAX serves as a key effector in the apoptosis process. BCL-2-associated X protein (BAX) serves as a pivotal executor of mitochondrial-regulated cell death, exerting its lethal function by permeabilizing the outer membrane of mitochondria (MOM) [ 26 ]. The physiological role of BAX is crucial for maintaining tissue homeostasis; however, dysregulation of BAX can result in abnormal cell death [ 27 ]. Combined with WB results, it was confirmed that the expression level of anti-apoptotic protein BCL-2 was down-regulated, while the expression levels of pro-apoptotic proteins Bax, Caspase9 and Caspase3 were up-regulated. Additionally, a significant decrease in mitochondrial membrane potential was observed following CBX2 knockdown. These findings indicate that the apoptosis pathway is activated. 5. Conclusion CBX2 enhances the resistance of breast cancer to paclitaxel (PTX) through the PI3K/AKT signaling pathway and increases the anti-apoptotic capacity of breast cancer cells by upregulating Bcl-2 levels. Consequently, CBX2 represents a critical target for drug resistance therapy in breast cancer (Fig. 6 ). Declarations Author contribution Y.S.H-Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Writing-Original draft; M.Q.L- Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing-review & editing. All authors reviewed the manuscript, and agreed to the published version of the manuscript. Funding This study was supported by the Key scientific research project of higher educational institutions in Henan Province (NO.21A320043) and the 2020 Henan Province Medical Science and Technology Research Plan Key Projects of Jointly Constructed by the Province and Ministry (NO.SBGJ202002038). Ethics approval This cell line-based study was exempt from ethics approval. Conflict of interest The authors declare no conflict of interest. Data availability statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgments We acknowledge the proteinatlas, TIMER2.0, NGDC and DRESIS databases for free use. References Zheng, R., Zhang, S., Zeng, H., Wang, S., Sun, K., Chen, R., Li, L., Wei, W., and He, J. (2022). Cancer incidence and mortality in China, 2016. Journal of the National Cancer Center 2 , 1-9. Mohammed, I., Arun, M., Arran K, T., J Michael, D., Rachael, N., and Vijay K, T. (2024). Basal-epithelial subpopulations underlie and predict chemotherapy resistance in triple-negative breast cancer. EMBO Mol Med 16 . Yang, F., Xiao, Y., Ding, J., Jin, X., Ma, D., Li, D., Shi, J., Huang, W., Wang, Y., Jiang, Y., et al. (2023). Ferroptosis heterogeneity in triple-negative breast cancer reveals an innovative immunotherapy combination strategy. Cell metabolism 35 , 84-100.e108. 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Targeting Bcl-2 for cancer therapy. Biochim Biophys Acta Rev Cancer 1876 . Katia, C., Vanessa, H., Andreas, J., Timo, D., Milos, G., Aida, P.-B., Shashank, D., Noel, W., John S H, D., Jervis Vermal, T., et al. (2022). The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial-DNA-mediated inflammation. Mol Cell 82 . Adam Z, S., and Evripidis, G. (2021). Physiological and pharmacological modulation of BAX. Trends Pharmacol Sci 43 . Additional Declarations No competing interests reported. Supplementary Files uncroppedGelsandBlotsimages.pdf 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6902662","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487474006,"identity":"ae933151-f9ac-49f0-bce2-ba8cf44c7ed7","order_by":0,"name":"Yishan He","email":"","orcid":"","institution":"The First Afffliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yishan","middleName":"","lastName":"He","suffix":""},{"id":487474007,"identity":"ded0dbcd-9301-4fa0-8aed-52cc6c921f58","order_by":1,"name":"Mengquan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACNv7+hw8+VNjU88sfPvggoaKGsBY+iTPMhjPOpCVIzmBLNnhw5hhhLXIMOWzSvG2HEwxm8JhJPmxhJsJhDGePSc44czjPQLrBrCKxgY2Bv707Ab8W5r5kiw8V6cXmMgfSbiTukGGQOHN2AwFbDhjenHHGmnFnQ8KxG4ln2BgMJHIJaUkwAPqFmXHDgcS2gsQ2ZmK05BgBtTgnbriRzMZAnBaJY8mgQDaW7DnGLJFw5hgPQb/I9zcfBEWlHD97/8ePPypq5Pjbe/FrwQA8pCkfBaNgFIyCUYAVAADWS1Ab/5+gcAAAAABJRU5ErkJggg==","orcid":"","institution":"The First Afffliated Hospital of Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Mengquan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-06-16 07:23:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6902662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6902662/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87324770,"identity":"33cc00f0-59c0-4108-9afa-7afdea8f9fa3","added_by":"auto","created_at":"2025-07-22 17:22:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":505198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ethe expression levels of CBX2 in breast cancer. \u003c/strong\u003e(A) The expression difference of CBX2 in different tumors; (B) The expression of CBX2 in different subtypes of breast cancer was analyzed with Timer2.0; (C) The expression level of CBX2 in breast cancer tissues; (D) Survival prognosis analysis; (E) The expression of CBX2 in malignant cells of breast cancer.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6902662/v1/b8909fcaa29e46bab9dbcc4a.png"},{"id":87324328,"identity":"2fc51dce-9dcd-4390-8d86-8b22e4af6c98","added_by":"auto","created_at":"2025-07-22 17:14:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":236867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CBX2 expression difference in breast cancer cells with PTX resistance. \u003c/strong\u003e(A) The cell activities were checked by CCK8; (B-C) The CBX2 expression difference was checked by WB and Q-PCR; (D-E) The CBX2 gene knockdown efficiency was checked by WB and Q-PCR.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6902662/v1/95d7566bdb8312cfd08bec04.png"},{"id":87324334,"identity":"a1e7f496-ebb7-4756-b639-bed4a1b2d5d8","added_by":"auto","created_at":"2025-07-22 17:14:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":387276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of CBX2 on cell activity. \u003c/strong\u003e(A) The cell activities were checked by CCK8; (B) The cell apoptosis was checked by flow cytometer; (C) The resistance genes were analyzed with DRESIS database; (D) The cell activities were checked by CCK8; (E) The p-gp was checked by WB.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6902662/v1/c0be607966763708d93ae602.png"},{"id":87324330,"identity":"33f28d59-5852-41c7-8a92-87fb9c88e743","added_by":"auto","created_at":"2025-07-22 17:14:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":422203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe PI3K-AKT pathway was regulated by CBX2. \u003c/strong\u003e(A) The genes difference was checked with transcriptome sequencing; (B) The pathway was analyzed by KEGG; (C) The relationship between PI3K-AKT pathway and PTX resistance was analyzed with DRESIS database; (D) The PI3K and AKT proteins were checked by WB.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6902662/v1/7a02dbc0380c55d900b67756.png"},{"id":87324335,"identity":"0d1ad47a-8886-4d6f-977c-6f3e831902e3","added_by":"auto","created_at":"2025-07-22 17:14:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":349650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe apoptosis pathway was regulated by CBX2. \u003c/strong\u003e(A) The relationship between apoptosis pathway and PTX resistance was analyzed with DRESIS database; (B) The mitochondrial membrane potential was detected with JC-1; (C) The Bcl-2, BAX, Caspase9 and Caspse3 proteins were checked by WB.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6902662/v1/db8b73192f34cb9517b3af87.png"},{"id":87324332,"identity":"937a01b9-285e-4436-bb89-b0fb0663dc3d","added_by":"auto","created_at":"2025-07-22 17:14:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":298153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe regulatory mechanism diagram of CBX2\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6902662/v1/c8fb9f79741545a6459264c9.png"},{"id":107697735,"identity":"cd797419-760c-4882-adaf-f6dd988a6cce","added_by":"auto","created_at":"2026-04-24 07:26:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2407513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6902662/v1/4bbc467c-8f54-47c3-b591-2e17129d171f.pdf"},{"id":87324329,"identity":"66e2a82e-ddbc-4463-b0d1-3f517bc4b70b","added_by":"auto","created_at":"2025-07-22 17:14:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":206968,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6902662/v1/89e09517f68386e322a237d9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CBX2 Enhances Paclitaxel Resistance via PI3K/AKT Signaling Pathway in Breast Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer has emerged as a significant threat to women's health. According to the national cancer statistics published by the National Cancer Center in 2023, the incidence of female breast cancer is 29.05/100,000, making it the most prevalent tumor among women. Furthermore, both incidence and mortality rates have shown a continuous increase from 2000 to 2016[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by extensive intratumor heterogeneity, high metastasis, and chemoresistance, leading to the highest mortality rate[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Drug resistance remains one of the foremost clinical challenges in breast cancer treatment and is a critical factor contributing to poor clinical outcomes[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite advancements in research and therapy, our understanding of the underlying mechanisms driving drug resistance is still limited. Therefore, how to reverse the drug resistance in breast cancer and enhance the therapeutic efficacy has become an important clinical issue that requires immediate attention.\u003c/p\u003e\u003cp\u003eCBX2 protein is an important component of the Polycomb Repressive Complex 1 (PRC1), which can regulate the transcriptional states of genes through chromatin remodeling and histone modification[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, CBX2 is overexpressed in breast tumors, with the highest levels observed in triple-negative breast cancer[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Knockdown of CBX2 leads to a reduction in mTORC1 activity, thereby inhibiting E2F signal and promoting the aging of breast cancer cells[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, knockout of the CBX2 gene results in compromised genomic stability, increased spontaneous chromosome breakage, and a tendency towards polyploidy. This disruption is particularly associated with cell cycle abnormalities, notably blocking G2/M transition, ultimately leading to apoptosis in ovarian cancer cells[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The study revealed that CBX2 is overexpressed and enhances the activity of leukemia cells. The stability of CBX2 is regulated by SAHA [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, downregulation of CBX2 significantly reduced leukemia cell activities and increased the cell apoptosis rates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, CBX2 could promote the demethylation of H3K27me3 by recruiting Jmjd3, and enhance the transcription level of IFN-β[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The CBX2 could interact with SMARCE1 to inhibit EGFR transcription [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. CBX2 was highly expressed in chemotherapy-resistant ovarian tissues and promoted the cell proliferation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The related study indicated that inhibition of CBX2 could enhance the sensitivity of hepatocellular carcinoma (HCC) cells to oxaliplatin both in vitro and in vivo [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis research has confirmed that CBX2 plays a crucial role as a regulator of paclitaxel resistance in triple-negative breast cancer cells. Downregulating CBX2 expression could significantly decrease the expression level of P-gp protein and reverse paclitaxel resistance in breast cancer cells, but the underlying mechanism is still unclear.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 The Cell culture and treatment\u003c/h2\u003e\u003cp\u003eThe MDA-MB-231/PTX cells were cultured in DMEM supplemented with 10%FBS and 1%PS, under a humidified atmosphere containing 5% (v/v) CO\u003csub\u003e2\u003c/sub\u003e at 37℃. The plasmid was transfected into cells using Lipofectamine\u0026trade; 3000 (Invitrogen, L3000001).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 CCK8 assay\u003c/h2\u003e\u003cp\u003eThe MDA-MB-231/PTX cells were seeded into 96 well plates, and subjected to various treatment conditions for 24h, 48h and 72h. Following the treatments, the cells were incubated with 10% CCK8 for a period of 1 to 2 hours. The optical density (OD) value was subsequently measured at a wavelength of 450nm by microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Quantitative real-time PCR assay\u003c/h2\u003e\u003cp\u003eMDA-MB-231/PTX cells were lysed with TRIzol reagent (Life technogies, 15596018). the cDNA was synthesized with PrimeScript\u0026trade;RT reagent Kit (TaKaRa, RR047A). The PCR was conducted with Novostart SYBR qPCR SuperMix Plus (Novoprotein, E096-01B). The amplification conditions included an initial denaturation at 95\u0026deg;C for 1 minute, followed by 40 cycles of denaturation at 95\u0026deg;C for 20 seconds and annealing/extension at 60\u0026deg;C for 1 minute. The sequences of the sense and antisense primers are as follows: 5ʹ-AGAAGGAACATGAGAAGGAGGTG-3ʹ and 5ʹ-GACTTGGATTTGGAGGGAG C-3ʹ (CBX2); 5ʹ-TGACTTCAACAGCGACACCCA-3ʹ and 5ʹ- CACCCTGTTGC TGTAGCCAAA-3ʹ (GAPDH). Relative expression was assessed utilizing the comparative threshold cycle method and determined by the value of 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Western blot assay\u003c/h2\u003e\u003cp\u003eThe protein lysates from MDA-MB-231/PTX cells were obtained using RIPA lysis buffer (Beyotime Institute of Biotechnology, P0013B), and subsequently separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). The membranes were blocked with 5% nonfat milk, and probed with a primary antibody and probed with a primary antibody followed by HRP-conjugated goat anti-rabbit/mouse secondary antibodies (ZB-2301/ZB-2305, Zsbio). The membranes were visualized with an ECL Kit (34095, Thermo). The proteins densities were analyzed by image J software. The primary antibody: anti-CBX2 antibody (15579-1-AP, proteintech), anti-P-gp antibody (22336-1-AP, proteintech), anti-PI3K antibody (ab154598, abcam), anti-AKT antibody (ab8805, abcam), anti-Bcl-2 antibody (ab182858, abcam), anti-Caspae9 antibody (ab32539, abcam), anti-Caspae3 antibody (ab32351, abcam), and anti-GAPDH antibody (10494-1-AP, proteintech).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Mitochondrial membrane potential assay\u003c/h2\u003e\u003cp\u003eThe MDA-MB-231/PTX cells were cultured in a confocal dish and subjected to various treatment conditions for 48 hours. Subsequently, the cells were incubated with JC-1(C2006, Beyotime) for 20 minutes. Images were captured using a fluorescent microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Cell apoptosis assay\u003c/h2\u003e\u003cp\u003eThe MDA-MB-231/PTX treated under various conditions for 48h. Following treatment, the cells were digested using pancreatic enzymes, collected by centrifugation, and subsequently incubated with Annexin V/PI (C1062L, Beyotime). The analysis was performed using flow cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll results are represented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) derived from a minimum of three independent experiments. The Statistical significance was assessed using Student's t-test for comparison between two groups. * \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001 were deemed statistically significant. All statistical analyses were conducted utilizing GraphPad Prism version 8.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 The expression difference of CBX2 in breast cancer\u003c/h2\u003e\u003cp\u003eThe expression difference of CBX2 in different tumors was analyzed using an online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), revealing that, in comparison to normal tissues, CBX2 expression was significantly upregulated in tumors to varying extents (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The expression of CBX2 in different subtypes of breast cancer was analyzed with Timer2.0. The findings indicated that CBX2 expression was elevated in all breast cancer subtypes compared to normal tissue, with particularly high levels observed in basal subtype, which belongs to triple-negative breast cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The expression level of CBX2 in breast cancer tissues was further analyzed alongside pathological data, revealing that CBX2 is significantly overexpressed in these tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Survival prognosis analysis indicated that patients exhibiting high levels of CBX2 expression had a poorer survival outcome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Finally, an analysis utilizing the NGDC single-cell database demonstrated that the expression of CBX2 was markedly upregulated in cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 The changes of CBX2 expression before and after PTX resistance in breast cancer were assessed\u003c/h2\u003e\u003cp\u003eMDA-MB-231 cells and MDA-MB-231/PTX cells were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The cell activity was checked using CCK8 assay. The results showed that the half maximal inhibitory concentration (IC50) for MDA-MB-231 and MDA-MB-231/PTX cells were 9.399 and 23.40nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The expression levels of CBX2 protein were examined via WB. The result showed that the expression level of CBX2 was up-regulated in MDA-MB-231/PTX cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The transcription level of CBX2 was detected by Q-PCR, and the results showed that the expression of CBX2 was up-regulated in MDA-MB-231/PTX cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To further investigate the role of CBX2 in drug resistance, the three gene knockdown vectors were designed and constructed. Both Q-PCR and WB results showed that the three knockdown vectors significantly inhibited the expression of CBX2, with the shCBX2-2 vector exhibiting the highest efficiency in knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 The effect of CBX2 on the activity of drug-resistant cells was analyzed\u003c/h2\u003e\u003cp\u003eThe results from the CCK8 assay indicated that knockdown of the CBX2 gene significantly reduced the activity of MDA-MB-231/PTX cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Flow cytometry analysis revealed an increase in apoptosis levels in CBX2-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results suggest that CBX2 plays an important role in the process of drug resistance and anti-apoptosis in breast cancer. Subsequently, CBX2 was further overexpressed in MDA-MB-231/PTX cell, resulting in an increase in cellular activity. In contrast, the knockdown of CBX2 led to a decrease in cell activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Combined with the analysis of DRESIS database, it was observed that the resistance gene ABCB1 (p-gp) is upregulated in breast cancer exhibitin resistance to paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, Western blot analysis revealed an increased expression level of P-glycoprotein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). With CBX2 overexpression, the expression level of P-gp was increased. Conversely, following the knockdown of CBX2, a decrease in the expression level of P-gp was observed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Transcriptome sequencing analysis\u003c/h2\u003e\u003cp\u003eThe difference genes were analyzed by transcriptome sequencing following the knockdown of the CBX2 gene. The genes expression levels were presented in heat maps (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, the signaling pathway enrichment analysis using KEGG revealed that the PI3K-AKT pathway underwent significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Meanwhile, an analysis of the DRESIS database revealed that the PI3K-AKT pathway is implicated in breast cancer's resistance to paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, Western blot results indicated that the PI3K and AKT proteins were high levels, but the difference is not significant following the overexpression of CBX2. In contrast, upon knockdown of CBX2, there was a marked decrease in the protein expression levels of both PI3K and AKT. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5 The effects of CBX2 on anti-apoptosis of breast cancer resistant cells were analyzed\u003c/h2\u003e\u003cp\u003eThe DRESIS database analysis result showed that the apoptosis pathway was activated in breast cancer cells treated with paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). A decreased mitochondrial membrane potential is a significant characteristic of apoptosis. So that, mitochondrial membrane potential was detected with JC-1 staining. The results showed that there are no significant alterations in membrane potential associated with CBX2 overexpression. however, a marked reduction in membrane potential was observed following CBX2 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To further investigate whether the knockdown of CBX2 could activate the apoptosis pathway by inhibiting the PI3K-AKT signaling pathway, we assessed key proteins involved in apoptosis through Western blot analysis in this study. The result showed that following CBX2 knockdown, the expression level of anti-apoptosis protein BCL-2 was significantly reduced, while the levels of pro-apoptotic proteins BAX, Caspase-9, and Caspase-3 were markedly increased. In contrast, with the overexpression of CBX2, the expression of anti-apoptosis protein BCL-2 was a high level. However, the expressions of pro-apoptotic proteins BAX, Caspase 9, and Caspase 3 were low levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results showed that CBX2 is related with anti-apoptosis abilities in BC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eBreast cancer has emerged as a significant health threat to women[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Drug resistance in breast cancer represents a significant factor contributing to poor prognosis and high mortality rates among patients in clinical settings[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Paclitaxel is a first-line therapeutic agent for breast cancer. Paclitaxel resistance has long posed a significant challenge in clinical treatment. This study found that the expression of gene CBX2 was upregulated in breast cancer cells exhibiting paclitaxel resistance. Relevant studies have confirmed that the drugs sensitivity enhance with CBX2 knocking down[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In this project, CBX2 gene knockdown vectors were constructed, and subsequently transfected into MDA-MB-231/PTX cells. With CBX2 gene knockout, the cell activity decreased, apoptosis level increased, and the expression of P-gp, a key drug-resistant protein, was down-regulated. These findings indicate that cellular drug sensitivity has increased and that drug toxicity has heightened, ultimately leading to enhanced apoptosis rates.\u003c/p\u003e\u003cp\u003eDRESIS database also showed that breast cancer cells activate the pro-survival pathway by PI3K during the process of paclitaxel resistance. Survival signals induced by various receptors are primarily mediated mainly by PI3K/AKT pathway [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Consequently, the PI3K/AKT pathway contributes to the multidrug resistance observed in cancers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The PI3K/Akt signaling pathway represents a targeted therapeutic approach for cancer treatment [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this study, transcriptome sequencing results were analyzed, revealing that knockdown of CBX2 led to inhibition of the PI3K-AKT pathway. The WB experiment confirmed that the expression levels of PI3K and AKT were down-regulated in the CBX2 knockdown group. These findings suggest that CBX2 inhibits cell viability by intervening in the PI3K-AKT pathway, thereby enhancing cellular sensitivity to paclitaxel. Furthermore, activation of the apoptosis pathway was observed through down-regulation of BCL-2 expression.\u003c/p\u003e\u003cp\u003eApoptosis is a form of programmed cell death that is intricately regulated by the balance between pro-survival and pro-apoptotic members of the BCL-2 protein family [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The apoptosis programmed process highly regulated by the BCL-2 family of proteins [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cancer cells commonly evade apoptosis through upregulation of the BCL-2 anti-apoptotic proteins [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, targeting the inactivation of BCL-2 has demonstrated significant therapeutic advantages by enhancing apoptotic sensitivity and reversing drug resistance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. BCL-2 is an anti-apoptotic protein, while the pro-apoptotic protein BAX serves as a key effector in the apoptosis process. BCL-2-associated X protein (BAX) serves as a pivotal executor of mitochondrial-regulated cell death, exerting its lethal function by permeabilizing the outer membrane of mitochondria (MOM) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The physiological role of BAX is crucial for maintaining tissue homeostasis; however, dysregulation of BAX can result in abnormal cell death [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Combined with WB results, it was confirmed that the expression level of anti-apoptotic protein BCL-2 was down-regulated, while the expression levels of pro-apoptotic proteins Bax, Caspase9 and Caspase3 were up-regulated. Additionally, a significant decrease in mitochondrial membrane potential was observed following CBX2 knockdown. These findings indicate that the apoptosis pathway is activated.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eCBX2 enhances the resistance of breast cancer to paclitaxel (PTX) through the PI3K/AKT signaling pathway and increases the anti-apoptotic capacity of breast cancer cells by upregulating Bcl-2 levels. Consequently, CBX2 represents a critical target for drug resistance therapy in breast cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.S.H-Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Writing-Original draft; M.Q.L- Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing-review \u0026amp; editing. \u0026nbsp;All authors reviewed the manuscript, and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Key scientific research project of higher educational institutions in Henan Province (NO.21A320043) and the 2020 Henan Province Medical Science and Technology Research Plan Key Projects of Jointly Constructed by the Province and Ministry (NO.SBGJ202002038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis cell line-based study was exempt from ethics approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the proteinatlas, TIMER2.0, NGDC and DRESIS databases for free use.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZheng, R., Zhang, S., Zeng, H., Wang, S., Sun, K., Chen, R., Li, L., Wei, W., and He, J. 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Cell research \u003cem\u003e25\u003c/em\u003e, 445-458.\u003c/li\u003e\n \u003cli\u003eHu, K., Yao, L., Xu, Z., Yan, Y., and Li, J. (2022). Prognostic Value and Therapeutic Potential of CBX Family Members in Ovarian Cancer. Frontiers in cell and developmental biology \u003cem\u003e10\u003c/em\u003e, 832354.\u003c/li\u003e\n \u003cli\u003eFu, Y., Yang, K., Wu, K., Wang, H., Li, Q., Zhang, F., Yang, K., Yao, Q., Ma, X., Deng, Y., et al. (2022). Identification of hepatocellular carcinoma subtypes based on PcG-related genes and biological relevance with cancer cells. Clinical epigenetics \u003cem\u003e14\u003c/em\u003e, 184.\u003c/li\u003e\n \u003cli\u003eJavier David, B.F., Eileen, M., Alicia, d.L.A., Allini, M., Richa, S., Francesco, G., Jérôme, V., Ariana, Z., Carina, M., Cheng-Har, Y., et al. (2023). Global Stage Distribution of Breast Cancer at Diagnosis: A Systematic Review and Meta-Analysis. JAMA Oncol \u003cem\u003e10\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eMuhammad Muzamil, K., Satya Siva Kishan, Y., Bharat Ashok, R., Nina, F., and Vladimir P, T. (2024). Recent strategies to overcome breast cancer resistance. Crit Rev Oncol Hematol \u003cem\u003e197\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eFresno Vara, J., Casado, E., de Castro, J., Cejas, P., Belda-Iniesta, C., and González-Barón, M. (2004). PI3K/Akt signalling pathway and cancer. Cancer treatment reviews \u003cem\u003e30\u003c/em\u003e, 193-204.\u003c/li\u003e\n \u003cli\u003eLiu, R., Chen, Y., Liu, G., Li, C., Song, Y., Cao, Z., Li, W., Hu, J., Lu, C., and Liu, Y. (2020). PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers. Cell death \u0026amp; disease \u003cem\u003e11\u003c/em\u003e, 797.\u003c/li\u003e\n \u003cli\u003eHe, Y., Sun, M., Zhang, G., Yang, J., Chen, K., Xu, W., and Li, B. (2021). Targeting PI3K/Akt signal transduction for cancer therapy. Signal transduction and targeted therapy \u003cem\u003e6\u003c/em\u003e, 425.\u003c/li\u003e\n \u003cli\u003eYu, L., Wei, J., and Liu, P. (2022). Attacking the PI3K/Akt/mTOR signaling pathway for targeted therapeutic treatment in human cancer. Seminars in cancer biology \u003cem\u003e85\u003c/em\u003e, 69-94.\u003c/li\u003e\n \u003cli\u003eDiepstraten, S., Anderson, M., Czabotar, P., Lessene, G., Strasser, A., and Kelly, G. (2022). The manipulation of apoptosis for cancer therapy using BH3-mimetic drugs. Nature reviews. Cancer \u003cem\u003e22\u003c/em\u003e, 45-64.\u003c/li\u003e\n \u003cli\u003eShirin, H., and Mohamed, R. (2021). Targeting BCL-2 in Cancer: Advances, Challenges, and Perspectives. Cancers (Basel) \u003cem\u003e13\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eAndrea, L., Denis E, R., Nadege, G., Felix, K., Hua, Z., Miguel A, M.-R., Lars Ulrik, N., Swathi-Rao, N., Ping, C., Eduardo, V., et al. (2022). Co-targeting of BAX and BCL-XL proteins broadly overcomes resistance to apoptosis in cancer. Nat Commun \u003cem\u003e13\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eLinlin, Z., Zaiming, L., and Xiangxuan, Z. (2021). Targeting Bcl-2 for cancer therapy. Biochim Biophys Acta Rev Cancer \u003cem\u003e1876\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eKatia, C., Vanessa, H., Andreas, J., Timo, D., Milos, G., Aida, P.-B., Shashank, D., Noel, W., John S H, D., Jervis Vermal, T., et al. (2022). The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial-DNA-mediated inflammation. Mol Cell \u003cem\u003e82\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eAdam Z, S., and Evripidis, G. (2021). Physiological and pharmacological modulation of BAX. Trends Pharmacol Sci \u003cem\u003e43\u003c/em\u003e.\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":"breast cancer, Paclitaxel resistance, CBX2, PI3K, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-6902662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6902662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003ePaclitaxel resistance is a significant contributor to poor prognosis in breast cancer treatment. The expression of CBX2 is upregulated in breast cancer with paclitaxel resistance. But the role and mechanism of CBX2 in promoting drug resistance in breast cancer is still unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe CBX2 gene expression was inhibited by gene knockdown technology. Cell activity was assessed through CCK8 assay. The apoptosis levels evaluated via flow cytometry. The gene expression levels were detected using Q-PCR. The protein expression level was detected with WB. Transcriptome sequencing was employed to identify differences in gene expression and pathways regulated by CBX2. Mitochondrial membrane potential was analyzed by JC-1 assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWith CBX2 gene knockdown, the p-gp protein expression level was decreased, cell activity was declined, apoptosis level was reduced, PI3K and AKT protein levels were decreased, Bcl-2 protein level was decreased, mitochondrial membrane potential loss, and the pro-apoptotic proteins BAX, Caspase9 and Caspase3 levels were increased.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eCBX2 activates the PI3K-AKT signaling pathway, thereby enhancing the anti-apoptotic capacity of cells and promoting resistance to paclitaxel in breast cancer. Therefore, targeting CBX2 may represent a promising strategy for overcoming drug resistance in breast cancer therapy.\u003c/p\u003e","manuscriptTitle":"CBX2 Enhances Paclitaxel Resistance via PI3K/AKT Signaling Pathway in Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 17:14:27","doi":"10.21203/rs.3.rs-6902662/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":"7b91bc2c-d1ef-417a-8ce2-fe17ccc6fd97","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T07:24:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 17:14:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6902662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6902662","identity":"rs-6902662","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}