Knockdown of PPP2R2B inhibits pancreatic cancer progression via the ERK/MAPK pathway by modulating EMT and apoptosis

Knockdown of PPP2R2B inhibits pancreatic cancer progression via the ERK/MAPK pathway by modulating EMT and apoptosis | 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 Knockdown of PPP2R2B inhibits pancreatic cancer progression via the ERK/MAPK pathway by modulating EMT and apoptosis Fangfang Han, Zhou Chen, Cheng Ye, Chunlu Dong, Yan Du, Huaqing Shi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5324510/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Pancreatic cancer (PC) is one of the most lethal types of cancer, as current treatments are largely ineffective. Our research uncovers that PPP2R2B is overexpressed in a majority of PC cases, playing a significant role in the growth and spread of PC tumors. Knockdown of PPP2R2B inhibits PC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), while promoting cell apoptosis. Conversely, overexpression of PPP2R2B enhances these processes, leading to increased proliferation, migration, invasion, and EMT, and reduced apoptosis. Further analysis showed that reducing PPP2R2B levels in PC inactivates the MAPK pathways—ERK, JNK, and p38, ultimately promoting PC growth. The addition of an ERK inhibitor reverses the effects of PPP2R2B knockdown, restoring cell proliferation, migration, and invasion. Our experiments in live subjects demonstrate that removing PPP2R2B inhibits tumor growth in PC mouse models and alters the levels of proteins involved in EMT and cell death. These findings demonstrate that PPP2R2B contributes to PC progression by modulating EMT and apoptosis through the ERK/MAPK pathway. Targeting PPP2R2B or its downstream signaling pathways may offer a promising therapeutic strategy for pancreatic cancer. Pancreatic cancer PPP2R2B MAPK pathway EMT Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pancreatic cancer (PC) ranks as the third primary cause of cancer-related fatalities across the globe, known for its aggressive nature and tendency for late detection. This results in a high annual mortality rate of around 400,000 globally. Patients with advanced or inoperable PC have a five-year survival rate of approximately 13%, dropping to 3% for those with distant metastasis 1,2 . Despite improvements in treatment approaches in recent years, the delayed diagnosis often limits the potential for surgical resection as a curative option 3,4 . Therefore, a comprehensive comprehension of the biological processes behind PC is fundamental for the creation of more potent biomarkers and treatment strategies. PPP2R2B, part of the B family of regulatory subunit 2 phosphatases, encodes the beta version of the B55 subfamily. Its expression peaks during the fetal stage and decreases as development advances. Predominantly found in the brain and testes, lower levels are present in the lungs and spleen 5,6 . The upregulation of PPP2R2B during neural maturation in embryos highlights its crucial role in embryonic growth. In the realm of cancer, PPP2R2B might carry out functions akin to those seen in development or differentiation 7-11 . While PPP2R2B has been implicated in the initiation and progression of multiple cancers via mutations or elevated expression, its specific effects on prostate cancer growth and metastasis remain to be fully elucidated. The transition from epithelial to mesenchymal cells (EMT) is a crucial event in the advancement and dissemination of cancer, encompassing a sequence of complex and dynamic stages. It involves a complex series of steps that are dynamic in nature. Among the different pathways involved in EMT 12-14 , the Mitogen-Activated Protein Kinase (MAPK) pathway is particularly significant in regulating important cellular processes that contribute to the growth and dissemination of tumors [15,16]. Consequently, targeting the MAPK/EMT axis has emerged as a central focus in contemporary cancer research 17-20 . Our study uncovers a new finding of PPP2R2B being overexpressed in the majority of pancreatic cancer cases, highlighting its essential function in promoting tumor growth and dissemination. In addition, our findings illustrate the capability of PPP2R2B to stimulate the proliferation and metastasis of PC cells through the MAPK/EMT pathway. The purpose of this research is to decipher the role of PPP2R2B, a recently identified factor promoting cancer growth, in regulating EMT and apoptosis through the MAPK pathway in driving the advancement of pancreatic cancer. With the limited options for treating PC, our findings may pave the way for innovative treatment approaches focused on inhibiting PPP2R2B. Results PPP2R2B's increased expression is observed in human PC cell lines and tissues Our previous RNA sequencing (RNA-seq) study revealed a significant down-regulation of PPP2R2B in PANC-1 cells upon treatment with a tumor suppressor drug. Furthermore, PPP2R2B is found to be highly expressed in PC samples from both the Cancer Genome Atlas (TCGA) and GEPIA datasets. In this research, we used qRT-PCR to assess the PPP2R2B expression in human PC tissues. The findings indicated a notable rise in PPP2R2B levels in PC tissues in contrast to nearby normal samples. (Fig. 1E). Additionally, the upregulation of PPP2R2B was observed in four PC cell lines (PANC-1, BxPC-3, SW1990, and AsPC-1) in comparison to HPDE6-C7 cells (Fig.1D). The Kaplan-Meier analysis revealed that patients with PC who exhibited high levels of PPP2R2B had reduced disease-free survival rates in comparison to individuals with low PPP2R2B expression levels ( Fig. 1C). These results indicate that the elevated presence of PPP2R2B could potentially contribute significantly to the progression of PC. Correlation of PPP2R2B expression with clinical and pathological characteristics To assess the potential therapeutic relevance of PPP2R2B expression in prostate cancer (PC) patients, we investigated its levels in a cohort of 60 subjects, with a median age of 61. The accompanying table outlines the important clinical, pathological, and radiographic findings in this study population. It is noteworthy that a majority of the tumors (76.7%) examined in our investigation were categorized as stage I-II, with sizes ranging from 2 to 4 cm. As PPP2R2B demonstrates variable expression levels in this PC group, we proceeded to investigate its connection with established tumor traits and indicators of prognosis. Our findings found a correlation between PPP2R2B expression and nerve invasion as well as tumor dimensions. Notably, there was no significant relationship observed between the immunohistochemical score of PPP2R2B and tumor stage or nodal status (Table 1). The inhibition of PPP2R2B inhibit the proliferation, migration, and invasion of PC cells Previous studies utilizing qRT-PCR revealed elevated PPP2R2B expression notable in both PANC-1 and BxPC-3 cells (Fig. 1). Based on these results, we selected these two cell lines for further investigations. To study the impact of PPP2R2B on PC development, we established stable knockdown cell lines named sh-PPP2R2B in BxPC-3 and PANC-1 cells (Fig. 2 A, B). From the qRT-PCR data, sh-PPP2R2B-2 and sh-PPP2R2B-3 were chosen for subsequent experiments. Following this, we evaluated the growth of PC cells using CCK8 and EdU assays, which indicated that inhibiting PPP2R2B resulted in decreased proliferation of PANC-1 and BxPC-3 cells (Fig. 2 B, C). We also examined the impact of PPP2R2B on cell migration and invasion. Our wound healing assays revealed a notable decrease in PC cell motility upon PPP2R2B silencing (Fig.2D). Migration and invasion capabilities were further assessed using Transwell assays, showing reduced activity in both processes following PPP2R2B knockdown (Fig.2E). Knockdown of PPP2R2B suppresses EMT and promotes apoptosis in PC cells To further investigate the effects of PPP2R2B knockdown on the EMT process in pancreatic cancer cells, this study utilized Western blot analysis to examine the expression of EMT markers E-cadherin, N-cadherin, and Vimentin. The experimental results demonstrated that compared to the control group (sh-NC), the experimental group with reduced PPP2R2B expression significantly increased the protein level of E-cadherin in pancreatic cancer cell lines PANC-1 and BxPC-3, while the levels of N-cadherin and Vimentin were decreased (Fig. 3A). Furthermore, apoptosis-related proteins Bcl-2 and Bax were analyzed using WB, and flow cytometry was employed to assess the apoptotic rates of cells. The results of flow cytometry indicated that compared to the control group (sh-NC), reduced PPP2R2B expression significantly increased the apoptosis rate in pancreatic cancer cell lines PANC-1 and BxPC-3 (Fig. 3C). Western blot analysis showed that PPP2R2B knockdown notably upregulated the expression of Bax protein and downregulated the expression of Bcl-2 protein in PANC-1 and BxPC-3 cells (Fig. 3B). Overexpression of PPP2R2B promotes proliferation, migration, and invasion, suppresses EMT, and inhibits apoptosis in PC cells To further investigate the biological role of PPP2R2B in pancreatic cancer cells, we constructed a lentiviral vector for PPP2R2B overexpression (oe-PPP2R2B) and a negative control lentiviral vector (oe-NC). The results showed that the expression level of PPP2R2B was significantly increased in the oe-PPP2R2B group compared to the control group (Fig. 4A). Moreover, upregulating PPP2R2B significantly promoted the proliferation of PC cells compared to the oe-NC group (Fig. 4B-C). Wound-healing and transwell assay demonstrated that oe-PPP2R2B markedly enhanced the migration and invasion ability of PC cells (Fig. 4D-E). The results of WB indicated that oe-PPP2R2B significantly decreased the protein levels of E-cadherin while increasing the expression of N-cadherin and Vimentin in PC cells (Fig. 5A). Flow cytometry showed that oe-PPP2R2B significantly inhibited apoptosis in PC cells compared to the oe-NC group (Fig. 5C). WB results revealed that PPP2R2B overexpression decreased the levels of Bax protein while increasing the levels of Bcl-2 protein in PANC-1 and BxPC-3 cells (Fig. 5B). PPP2R2B knockdown inhibited the MAPK signaling pathway activity in PC Further investigation into the role of PPP2R2B in pancreatic cancer was conducted. Analysis of GO and KEGG pathways indicated enrichment of PPP2R2B in the MAPK signaling pathway (Fig. 6A-B). Results from GSEA showed a positive correlation between elevated PPP2R2B levels and MAPK pathway activation (Fig. 6C). PPP2R2B was discovered as a potential activator of the MAPK signaling pathway, which is recognized to impact both EMT and apoptosis, ultimately having a crucial impact on the growth, movement, and infiltration of pancreatic cancer cells. Furthermore, the impact of PPP2R2B on the ERK, JNK, and p38 MAPK pathways was evaluated. WB analysis demonstrated significantly higher levels of ERK, JNK, and p38 in the PPP2R2B knockdown group compared to the sh-NC group (Fig. 6D). These results highlight the role of PPP2R2B in modulating MAPK signaling pathways in pancreatic cancer. Overexpression of PPP2R2B had the opposite effect on MAPK pathway (Fig. 6E). The effect of PPP2R2B on PC cells after adding an ERK inhibitor We found that PPP2R2B most significantly affects ERK protein expression among the three MAPK pathways. To confirm whether PPP2R2B regulates pancreatic cancer cell proliferation, invasion, and migration through the ERK/MAPK pathway, we treated PANC-1 and BxPC-3 cells with an ERK inhibitor after overexpressing PPP2R2B. The CCK8 assay showed that PPP2R2B overexpression significantly promoted cell proliferation, but this effect was reduced with ERK inhibitor treatment (Fig. 7A). The wound-healing and transwell assay showed that migration and invasion were also weakened by the ERK inhibitor (Fig. 7B-C). These results suggest that PPP2R2B enhances pancreatic cancer cell proliferation, migration, and invasion, likely through the ERK/MAPK pathway. The ERK inhibitor's ability to reduce these effects further supports this pathway's role in regulating cancer cell behavior. PPP2R2B stimulated PC tumor growth in vivo To explore the effects of PPP2R2B on the progression of PC, BALB/c mice were injected with PANC-1 cells in which PPP2R2B was either silenced or expressed as a negative control. After 28 days, the tumors were removed and observed. Our results indicated a notable decrease in the size, volume, and weight of tumors in the mice injected with sh-PPP2R2B compared to those in the control group (Fig. 8A). Immunohistochemical studies revealed reduced levels of markers associated with EMT and apoptosis in the sh-PPP2R2B group as opposed to the control, consistent with previous western blot findings. The inhibition of tumor growth in vivo following PPP2R2B knockdown can be seen in Figure 8. Discussion In the present research, we uncover a new role that promotes tumor growth for PPP2R2B in the advancement of PC. Our results show that PPP2R2B is upregulated in PC cell lines and tumor samples in comparison to normal pancreatic cells and neighboring non-tumor tissues, respectively. Increased PPP2R2B levels are associated with reduced disease-free survival in PC patients, highlighting its potential as a predictive marker. Significantly, we demonstrate that PPP2R2B enhances PC cell growth, movement, and infiltration by modulating the MAPK/EMT signaling pathway, representing a notable advancement in comprehending the molecular characteristics of PC. The functional implications of PPP2R2B in normal and abnormal states pose an interesting contradiction. Although its involvement in fetal development emphasizes its crucial function in early human growth, particularly in the brain and reproductive system 21 , PPP2R2B's reappearance in cancer brings attention to a more sinister aspect of its role 8, 22, 23 . This dual functionality is prominently displayed in the setting of PC, where PPP2R2B appears to revert to its developmental functions, leading to abnormal cell differentiation and proliferation. Our research suggests that knocking down PPP2R2B in PC cells results in decreased cell proliferation, migration, and invasion capabilities, as demonstrated by CCK-8, EdU, wound healing, and transwell assays. Conversely, upregulating PPP2R2B enhances these malignant characteristics, further supporting its role in promoting tumorigenesis. In animal studies, the tumor-suppressive effects of PPP2R2B depletion were confirmed. Mouse xenografts injected with PC cells lacking PPP2R2B showed significantly inhibited tumor growth compared to control cells. In line with our lab findings, tumors originating from PPP2R2B-deficient cells displayed decreased expression of EMT-triggering and anti-apoptotic markers, further highlighting PPP2R2B's involvement in regulating these processes. Extensive research has focused on the ERK/MAPK signaling pathway in various cancer types 24,25. The ERK/MAPK system plays a key role in numerous biological processes, serving as a vital cell signaling pathway 26-28 . These functions encompass embryonic development, tissue regeneration, cell division, and motility. EMT is indispensable for the invasion and metastasis of malignant tumors 29-31 . The activation of the MAPK pathway can trigger EMT, prompting cancer cells to relocate and acquire invasive capabilities 32,33 . By inducing changes in cellular structure and function, EMT facilitates distant organ metastasis and enhances the ability of tumor cells to traverse vascular barriers and access the circulatory or lymphatic systems. Our research outlines the detailed mechanisms by which PPP2R2B contributes to its oncogenic effects, specifically by influencing the ERK, JNK, and p38 branches of the MAPK signaling pathway to regulate processes related to EMT and apoptosis. In particular, The inhibition of PPP2R2B leads to a decrease in the expression of the epithelial marker E-cad and an increase in the expression of the mesenchymal markers N-cad and Vimentin, suggesting a promotion of the EMT transition process. Furthermore, knockdown of PPP2R2B leads to the promotion of apoptosis, characterized by a increase in pro-apoptotic markers such as Bax and Beclin, and an decrease in the levels of the anti-apoptotic protein Bcl-2. By analyzing clinical data, a strong correlation was discovered between elevated PPP2R2B levels and nerve infiltration. Prior research has indicated that abnormal PPP2R2B methylation levels play a crucial role in neurological conditions like Spinocerebellar Ataxia Type 12 22,34 . PPP2R2B has the ability to modify mitochondrial division/fusion patterns by modulating kinase/phosphatase equilibrium, apoptotic and anti-apoptotic protein levels, ultimately leading to neurotoxicity and prompting neuronal demise 35 . Suppression of gene activity exhibits a neuroprotective impact, aligning with our observation that heightened PPP2R2B expression in tumors could hasten nerve cell differentiation and spread. Through our research into the role of PPP2R2B in the progression of PC, we have uncovered the intricate interplay between genetic factors and the pathways that promote cancer growth. Our findings emphasize PPP2R2B's pivotal function in promoting the spread and growth of tumors. This study demonstrates how PPP2R2B influences the signaling pathway MAPK/EMT, providing new perspectives on the molecular processes driving the advancement of PC. Furthermore, due to the intricate nature of cancer development, it is crucial to thoroughly explore the interplay between PPP2R2B and various signaling molecules and pathways within the tumor microenvironment. These investigations have the potential to unveil further levels of control and reveal innovative treatment strategies that could potentially counteract resistance mechanisms and improve treatment efficacy. In summary, our research makes a substantial contribution to the increasing evidence highlighting the crucial importance of PPP2R2B in the development of pancreatic cancer. By elucidating how PPP2R2B impacts the MAPK/EMT signaling pathway and the progression of tumors, we set the stage for additional studies on therapies targeting PPP2R2B that may bring renewed optimism to individuals with pancreatic cancer. As we delve deeper into the complex role of PPP2R2B in cancer, the potential for developing improved and specialized treatments for pancreatic cancer, as well as other types of cancer, appears to be increasingly optimistic. Materials and Methods Data and resources The Cancer Genome Atlas (TCGA) data repository (https://gdc-portal.nci.nih.gov/) was utilized to access level-three transcriptome RNA sequencing data of patients with PC. In addition, RNA-sequence information from both normal and PC samples was obtained from Datasets GSE62165 and GSE15471 retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). The potential prognostic significance of PPP2R2B in PC was investigated using the Gene Expression Profiling Interactive Analysis (GEPIA2) tool (http://gepia.cancer-pku.cn/), a web-based server for customizable and interactive large-scale expression analysis. Functional enrichment analysis The deeper exploration of potential functional implications of PPP2R2B associated with PC involved the application of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Moreover, Gene Set Enrichment Analysis (GSEA) was employed to assess the intrinsic molecular mechanisms distinguishing low- and high-risk cohorts. Patients and s ample s Between 2021 and 2022, a total of 60 pairs of tissue samples from PCs and nearby tissues were gathered from individuals who underwent pancreaticoduodenectomy at the First Hospital of Lanzhou University. Prior to participating in the research, all subjects had given their consent after being informed about the study's objectives. Approval for the research was granted by the Institutional Review Board (IRB) of the First Hospital of Lanzhou University (LDYYLL-2024-244). Cell lines and cell culture Human four PC cell lines including PANC-1, BxPC-3, SW1990, AsPC-1, and the HPDE6-C7 normal human pancreatic duct epithelial cell line were acquired from the BNCC and the Shanghai Branch of the Chinese Academy of Sciences. In order to ensure cell survival, all cell lines were grown in DMEM at 37°C with 95% humidity and 5% CO 2 . Q uantitative r ea l- ti me PCR (qRT-PCR) After total RNA extraction with the TRIzol Reagent (TaKaRa), cDNA synthesis was carried out using 5 µg of the mentioned total RNA and the PrimeScript RT Reagent Kit (TaKaRa). A Bio-Rad RT-PCR amplification system along with a SYBR Green PCR Kit (Takara) was utilized for qRT-PCR. The quantification of mRNA expression levels was conducted using the 2ΔΔCt method, with normalization to the endogenous reference gene GAPDH. The PCR primer sequences for the human PPP2R2B were as follows: (forward) 5′-TGCAGCTTACTTTCTTCTGTCT-3′ and (reverse) 5′-GTAGCCTTCTGGCCTCTTATC-3′. For the GAPDH primers, the sequences were (forward) 5′-AAGGTGAAGGTCGGAGTCAAC-3′ and (reverse) 5′-GGGGTCATTGATGGCAACAATA-3′. Western blot ting (WB) Antibodies against PPP2R2B, N-cadherin (N-cad), E-cadherin (E-cad), Vimentin, Bax, and Bcl-2 were bought from Wuhan Sanying (Wuhan, China). ERK, JNK and p38 antibodies, were sourced from Boser Bio (Wuhan, China). Proteins were extracted from cells and tissues using SDS-PAGE gels for separation. Following separation, the proteins were transferred onto a PVDF membrane and then incubated with primary antibodies targeting PPP2R2B, accompanying biomarkers, and GAPDH at 4 degrees Celsius for 48 hours. After blocking the membrane with BSA at room temperature for 2 hours, secondary antibodies were added and incubated for another 2 hours. The membrane underwent three TBST washes before exposure to a luminescent solution, using GAPDH as an internal control. Immunohistochemistry (IHC) staining A senior pathologist reviewed tissue slides to detect and label tumor regions. Cylinder replicas (1.0 mm wide) were collected from the designated tumor sites and adjacent non-malignant pancreatic tissue in the donor's tissue samples. These samples were then embedded in a fresh paraffin block. Standard protocols were followed for immunohistochemical staining, employing specific Vimentin, Beclin, and PPP2R2B antibodies. Cell counting Kit‑8 (CCK-8) assay Cells were first plated onto 96-well culture dishes with a concentration of 5 × 10 3 cells per well. Following plating, the dishes were placed in the incubator for durations spanning from 24 to 72 hours. A CCK-8 solution obtained from Boster Bio (China) was utilized for the experiment. A volume of 10 µL of the CCK-8 solution was added to each separate well and permitted to incubate within the incubator for a duration of one hour. In order to guarantee precise evaluation of cellular growth, the absorbance at a wavelength of 450 nm was measured for each well utilizing a plate reader. 5-Ethynyl-2’-deoxyuridine ( EdU ) assay The Seville Biotech (Wuhan, China) provided the necessary reagents for the EdU assay. Pancreatic cancer cells were seeded in confocal dishes at a density of 3×10 5 cells per well. Subsequently, the cells were treated with 4% paraformaldehyde (Beyotime, China) for 10 minutes to fix them. After fixing, the cells underwent three washes with PBS and were permeabilized for 5 minutes using 1% Triton (Beyotime, China). Subsequently, the cells were exposed to the dyeing agent in the dark for 30 minutes, then subjected to DAPI staining (Olympus, Tokyo, Japan) at 37 °C for 5 minutes. Imaging was carried out with a microscope at 400× magnification. Wound healing assay An exact count of cells were uniformly spread in a 6-well dish and cultured for a duration of 24 hours. A gap was created between the cells by drawing a single line using the tip of a crystal pipette. After washing with PBS, images were captured; then, drug-containing and standard media were added. Gap growth was monitored and captured at 0 and 24 hours. The area was determined by dividing the width by the length. Mobility of cells (%) was calculated as 100 times the difference in width between 0 h and 24 h, divided by the width at 0 h. The Image J program was utilized to gauge the gap between the opposing ends of the wound. Analysis of the data from three distinct trials involving two sets of samples was conducted for statistical purposes. Migration and invasion assay The Transwell system filter (8.0 µm pore size; BD Biosciences, USA) was used for the migration assay. In a 24-well culture plate (Corning, USA), 200 µl of either treated or untreated 1×10 5 cells were added to the upper chamber of Transwell inserts for 24 hours, followed by the addition of 700 µl of medium containing 30% FBS to the lower chamber. The invasion assay utilized chambers with inserts coated uniformly with Matrigel (BD Biosciences). The chambers were then fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 30 minutes. Following this, the upper surface of the inserts was carefully cleaned using absorbent paper, and the stained cells were imaged randomly using a microscope after rinsing with PBS. Cell migration and invasion capabilities were assessed by quantifying the average number of labeled cells across five different fields. Statistical analysis was performed utilizing data from two groups across three independent experiments. Animal experiment The nude mice used in this study were purchased from Spefo (Beijing) Biotechnology Co., Ltd. Four to six-week-old BALB/c mice, weighing between eighteen and twenty grams, were kept in a sterile setting with unrestricted access to food and water. Through unbiased randomization, ten mice were evenly split into two groups, with each group consisting of five mice. Then, both groups were injected subcutaneously in the side with 5×10 6 PANC-1 cells that expressed various PPP2R2B shRNAs and control NC shRNAs. Weekly measurements were taken of the tumor's dimensions, which were determined using the formula of multiplying the length by the square of the width, and then multiplying by 0.5. Afterward, the mice were put to sleep with isoflurane overdose, followed by measuring the tumor’s size and weight in a lab setting. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals, and were granted approval by the Ethics Committee at the First Hospital of Lanzhou University (LDYYLL-2024-244). Statistical analysis Statistical analyses were conducted using the SPSS 22.0 software from Chicago, USA, and GraphPad Prism software version 9.5 from the USA. The results were presented as mean ± SEM (standard error of the mean). A comparison of means between two groups was made using an unpaired t-test. To assess differences between two or more groups, one-way ANOVA and the Bonferroni test for multiple comparisons were applied. Statistical significance was defined as p < 0.05. Declarations Acknowledgements Not applicable. Author contributions Study concepts: FFH, WCZ; Study design: FFH, ZC; Experimental studies: CY, HQS; Data analysis: HQS, ZC; Statistical analysis: FFH, YD; Manuscript preparation: FFH, CLD; Manuscript editing: FFH, PFX; Manuscript review: FFH, WCZ. All authors contributed to the article and approved the submitted version. Data availability statement Data will be made available upon request by contacting the corresponding author. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Statement of Ethics All animal experiments were performed following the ethical standards in the 1964 Declaration of Helsinki and its later amendments. The study including human sample and animal experimental protocols has been approved by the Institutional Review Board (IRB) of the First Hospital of Lanzhou University (LDYYLL-2024-244), in compliance with the First Hospital of Lanzhou University guidelines for the care and use of animals. The informed consent was exempted by the IRB. Funding The work was supported by National Natural Science Foundation of China (82260555), The First Hospital of Lanzhou University Intra-Hospital Fund (ldyyyn2020-93). References Bray, F., Ferlay, J., Soerjomataram, I., et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 68, 394–424 (2018). Siegel, R. L., Giaquinto, A. N., Jemal, A. Cancer statistics, 2024. CA: A Cancer Journal for Clinicians 74, 12–49 (2024). Cass, S. H., Tzeng, C. D., Prakash, L. R., et al. Trends Over Time in Recurrence Patterns and Survival Outcomes after Neoadjuvant Therapy and Surgery for Pancreatic Cancer. Annals of Surgery (2024) [Epub ahead of print]. Van Bodegraven, E. A., Balduzzi, A., van Ramshorst, T. M. E., et al. Prophylactic abdominal drainage after distal pancreatectomy (PANDORINA): an international, multicentre, open-label, randomised controlled, non-inferiority trial. The Lancet Gastroenterology & Hepatology 9, 438–447 (2024). Johnson, H., Narayan, S., Sharma, A. K. Altering phosphorylation in cancer through PP2A modifiers. Cancer Cell International 24, 11 (2024). Zheng, H., Qi, Y., Hu, S., et al. Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase. Science 370, e4122 (2020). Hao, Y., Wang, C., Xu, D. Identification and validation of a novel prognostic model based on platinum resistance-related genes in bladder cancer. Int. Braz. J. Urol. 49, 61–88 (2023). Ishibashi, K., Ishii, K., Sugiyama, G., et al. Regulation of β-Catenin Phosphorylation by PR55β in Adenoid Cystic Carcinoma. Cancer Genomics & Proteomics 15, 53–60 (2018). Li, Z., Li, Y., Wang, X., et al. PPP2R2B downregulation is associated with immune evasion and predicts poor clinical outcomes in triple-negative breast cancer. Cancer Cell International 21, 13 (2021). Tan, J., Lee, P. L., Li, Z., et al. B55β-associated PP2A complex controls PDK1-directed MYC signaling and modulates rapamycin sensitivity in colorectal cancer. Cancer Cell 18, 459–471 (2010). Vazquez, A., Kulkarni, D., Grochola, L. F., et al. A genetic variant in a PP2A regulatory subunit encoded by the PPP2R2B gene associates with altered breast cancer risk and recurrence. Int. J. Cancer 128, 2335–2343 (2011). Celià-Terrassa, T., Kang, Y. How important is EMT for cancer metastasis? PLoS Biol. 22, e3002487 (2024). Guo, Z., Ashrafizadeh, M., Zhang, W., et al. Molecular profile of metastasis, cell plasticity, and EMT in pancreatic cancer: a pre-clinical connection to aggressiveness and drug resistance. Cancer Metastasis Rev. 43, 29–53 (2023). Khan, A. Q., Hasan, A., Mir, S. S., et al. Exploiting Transcription Factors to Target EMT and Cancer Stem Cells for Tumor Modulation and Therapy. Semin. Cancer Biol. 100, 1–16 (2024). Brown, B. A., Myers, P. J., Adair, S. J., et al. A histone methylation-MAPK signaling axis drives durable epithelial-mesenchymal transition in hypoxic pancreatic cancer. Cancer Res. 84, 1764–1780 (2024). Sugimoto, T., Iwagami, Y., Kobayashi, S., et al. Skeletal Muscle-Derived Irisin Enhances Gemcitabine Sensitivity and Suppresses Migration Ability in Pancreatic Ductal Adenocarcinoma. Ann. Surg. Oncol. 31, 3718–3736 (2024). Du, Q., Lin, Y., Ding, C., et al. Pharmacological activity of matrine in inhibiting colon cancer cells VM formation, proliferation, and invasion by downregulating claudin-9 mediated EMT process and MAPK signaling pathway. Drug Des. Devel. Ther. 17, 2787–2804 (2023). Lee, Y. S., Kim, H. S., Kim, H. J., et al. The role of LOXL2 induced by glucose metabolism-activated NF-κB in maintaining drug resistance through EMT and cancer stemness in gemcitabine-resistant PDAC. J. Mol. Med. 101, 1449–1464 (2023). Mi, K., Zeng, L., Chen, Y., et al. DHX38 enhances proliferation, metastasis, and EMT progression in NSCLC through the G3BP1-mediated MAPK pathway. Cell Signal. 113, 110962 (2024). Zhao, C., Li, X., Zhang, R., et al. Sense and anti-sense: Role of FAM83A and FAM83A-AS1 in Wnt, EGFR, PI3K, EMT pathways and tumor progression. Biomed. Pharmacother. 173, 116372 (2024). Xu, F., Chen, A., Pan, S., et al. Systems genetics analysis reveals the common genetic basis for pain sensitivity and cognitive function. CNS Neurosci. Ther. 30, e14557 (2024). Shen, D., Kang, S. Comprehensive analysis of mitochondria-related genes indicates that PPP2R2B is a novel biomarker and promotes the progression of bladder cancer via Wnt signaling pathway. Biol. Direct 19, 17 (2024). Malvi, P., Chava, S., Cai, G., et al. HOXC6 drives a therapeutically targetable pancreatic cancer growth and metastasis pathway by regulating MSK1 and PPP2R2B. Cell Rep. Med. 4, 101285 (2023). Sheng, W., Shi, X., Lin, Y., et al. Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling. J. Exp. Clin. Cancer Res. 39, 16 (2020). Sheng, W., Chen, C., Dong, M., et al. Calreticulin promotes EGF-induced EMT in pancreatic cancer cells via Integrin/EGFR-ERK/MAPK signaling pathway. Cell Death Dis. 8, e3147 (2017). Xu, J., Wang, Y., Jiang, J., et al. ADAM12 promotes clear cell renal cell carcinoma progression and triggers EMT via EGFR/ERK signaling pathway. J. Transl. Med. 21, 56 (2023). Ungefroren, H., Konukiewitz, B., Braun, R., et al. TAp73 inhibits EMT and cell migration in pancreatic cancer cells through promoting SMAD4 expression and SMAD4-dependent inhibition of ERK activation. Cancers 15, 3791 (2023). Sun, X. Y., Li, H. Z., Xie, D. F., et al. LPAR5 confers radioresistance to cancer cells associated with EMT activation via the ERK/Snail pathway. J. Transl. Med. 20, 456 (2022). Janta, S., Pranweerapaiboon, K., Vivithanaporn, P., et al. Holothurin A inhibits RUNX1-enhanced EMT in metastasis prostate cancer via the Akt/JNK and P38 MAPK signaling pathway. Mar. Drugs 21, 345 (2023). Wang, J. F., Zhao, K., Chen, Y. Y., et al. NKCC1 promotes proliferation, invasion and migration in human gastric cancer cells via activation of the MAPK-JNK/EMT signaling pathway. J. Cancer 12, 253–263 (2021). Xin, Y., Li, K., Yang, M., et al. Fluid shear stress induces EMT of circulating tumor cells via JNK signaling in favor of their survival during hematogenous dissemination. Int. J. Mol. Sci. 21, 8115 (2020). Bao, S., Ji, Z., Shi, M., et al. EPB41L5 promotes EMT through the ERK/p38 MAPK signaling pathway in esophageal squamous cell carcinoma. Pathol. Res. Pract. 228, 153682 (2021). Song, H., Tian, D., Sun, J., et al. circFAM120B functions as a tumor suppressor in esophageal squamous cell carcinoma via the miR-661/PPM1L axis and the PKR/p38 MAPK/EMT pathway. Cell Death Dis. 13, 361 (2022). Zhou, C., Liu, H. B., Jahanbakhsh, F., et al. Bidirectional transcription at the PPP2R2B gene locus in spinocerebellar ataxia type 12. Mov. Disord. 38, 2230–2240 (2023). Madera-Salcedo, I. K., Sánchez-Hernández, B. E., Svyryd, Y., et al. PPP2R2B hypermethylation causes acquired apoptosis deficiency in systemic autoimmune diseases. JCI Insight 5, e126457 (2019). Tables Table 1 Association between the expression of PPP2R2B in pancreatic cancer and adjacent tissues and clinical characteristics in 60 PC patients Variables n(%) GENE low GENE high p Value expression n(%) expression n(%) Age 60 13 47 <65 39(65.0) 10(76.9) 29 (61.7) 0.49 ≥65 21(35.0) 3(23.1) 18(38.3) Gender Female 40(66.7) 7(53.8) 33(70.2) 0.438 Male 20(33.3) 6(46.2) 14(29.8) Primarysite Pancreatic head 46(76.7) 12(92.3) 34(72.3) 0.256 Pancreatic body tail 14 (23.3) 1(7.7) 13(27.7) AJCC stage I,II 46(76.7) 10(76.9) 36(76.6) l II,IV 14(23.3) 3(23.1) 11(23.4) Vascular invasion No 42(70.0) 9(69.2) 33(70.2) 1 Yes 18(30.0) 4(30.8) 14(29.8) Nerve invasion No 18(30.0) 8(61.5) 10(21.3) 0.014 Yes 42(70.0) 5(38.5) 37(78.7) Lymph node metastasis No 30 (50.0) 7(53.8) 23(48.9) 1 Yes 30(50.0) 6(46.2) 24(51.1) Tumor size(cm) ≤2 9(15.0) 6(46.2) 3(6.4) 0.002 2-4 36(60.0) 5(38.5) 31(66.0) ≥4 15(25.0) 2(15.4) 13(27.7) Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-5324510","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371075984,"identity":"30a5b230-d4bb-4edb-a882-254ad8231b26","order_by":0,"name":"Fangfang Han","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Fangfang","middleName":"","lastName":"Han","suffix":""},{"id":371075985,"identity":"f8a3dd11-45c3-4354-9028-8f9a29651448","order_by":1,"name":"Zhou Chen","email":"","orcid":"","institution":"First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Chen","suffix":""},{"id":371075986,"identity":"5dde13da-e8c9-462d-8a2a-d6d97ab8c8e8","order_by":2,"name":"Cheng Ye","email":"","orcid":"","institution":"First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Ye","suffix":""},{"id":371075987,"identity":"92b9d44e-e3d9-4848-9ec8-daf6f96dc8e2","order_by":3,"name":"Chunlu Dong","email":"","orcid":"","institution":"First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chunlu","middleName":"","lastName":"Dong","suffix":""},{"id":371075988,"identity":"8d6808ae-67ab-47c5-951f-e3f1bce8c985","order_by":4,"name":"Yan Du","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Du","suffix":""},{"id":371075989,"identity":"bec3c936-bf11-40fb-930f-709a4ccd607c","order_by":5,"name":"Huaqing Shi","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Huaqing","middleName":"","lastName":"Shi","suffix":""},{"id":371075990,"identity":"b2989f30-a015-4ffa-bab8-9d5dff6a4ce6","order_by":6,"name":"Pengfei Xin","email":"","orcid":"","institution":"First Hospital of Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Pengfei","middleName":"","lastName":"Xin","suffix":""},{"id":371075991,"identity":"87f697b6-e712-4768-87ad-1b30f808d315","order_by":7,"name":"Wence Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACCQgpx8/e2PjwAwlaLIwlew43G0uQoKUiccON9DYBHmJ0yM9uPibNUyPBuOHmwzagfjs53QYCWhjnHEuT5jkmwSx5O7HtQQFDsrHZAQJamCVyzKRzGyTY+G4nthtIMBxI3EZICxtUCw/DzYNtQJIILTxQLRICNxiJ1CIhkZZs/eeYhIFkTyIwkA2I8Iv8jOSDN2fU1NX3sx9/+PBDhZ0cQS1owIA05aNgFIyCUTAKcAAAo8I86dkdjDUAAAAASUVORK5CYII=","orcid":"","institution":"Lanzhou University","correspondingAuthor":true,"prefix":"","firstName":"Wence","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-10-24 09:23:23","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5324510/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-5324510/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78542190,"identity":"fceca33d-c66e-4947-acd3-036549f1f0f8","added_by":"auto","created_at":"2025-03-14 16:13:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":949322,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of PPP2R2B is upregulated in pancreatic cancer (PC) tissues and cell lines. (A-B) By comparing the mRNA levels of PPP2R2B in PC samples to normal pancreas tissues from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), as well as PC samples from TCGA, through the use of Gene Expression Profiling Interactive Analysis (GEPIA). (C) The analysis of survival time utilizing Kaplan-Meier curves to establish the relationship between PPP2R2B expression and disease-free survival in PC patients. (D) Investigating PPP2R2B mRNA levels in the human pancreatic ductal epithelial cell line (HPDE6-C7) and various pancreatic adenocarcinoma cell lines (AsPC-1, BxPC-3, SW1990, and PANC-1). (E) Presentation of typical images showing immunohistochemical staining for PPP2R2B protein expression in PC and neighboring normal pancreatic tissues. The scale bar is set at 50 µm. All quantitative data shown as mean ± SEM. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. PC, pancreatic cancer.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/26b131d0fb5aa17cd8ac45b9.jpg"},{"id":78542192,"identity":"b8f84b26-b2b6-4a7f-9f71-c9449f07ede0","added_by":"auto","created_at":"2025-03-14 16:13:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":756676,"visible":true,"origin":"","legend":"\u003cp\u003eThe reduction in PPP2R2B inhibits cell growth, movement, and infiltration, leading to cell death. Pancreatic cancer cells PANC-1 and BxPC-3 were transfected with sh-PPP2R2B or sh-NC, respectively. (A) The levels of PPP2R2B expression in pancreatic cancer cells were quantified using qRT-PCR. (B) CCK8 assay was utilized at various time intervals (0 h, 24 h, 48 h, and 72 h) to evaluate the viability of pancreatic cancer cells. (C) An EdU assay was conducted to measure the proliferation capability of pancreatic cancer cells. (D) The migration capacity of pancreatic cancer cells transfected with sh-PPP2R2B or sh-NC was assessed using a wound healing assay. (E) Transwell assays were employed to determine the migration and invasion potentials of pancreatic cancer cells transfected with sh-PPP2R2B or sh-NC. All numerical data are noted as the mean ± SEM. * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/a7a60b848d76d17fbf852a87.jpg"},{"id":78542361,"identity":"322fc6a3-5566-486c-96ff-0d4852f7f463","added_by":"auto","created_at":"2025-03-14 16:21:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":307177,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of PPP2R2B inhibits the EMT process and promotes apoptosis in pancreatic cancer cells.\u003cstrong\u003e \u003c/strong\u003e(A) Western blot was used to detect the protein levels of E-cadherin, N-cadherin, and Vimentin in pancreatic cancer cells PANC-1 and BxPC-3. (B) Flow cytometry was performed to assess apoptosis in PANC-1 and BxPC-3 cells. E-cad, E-cadherin, N-cad, N-cadherin. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/a150217d1869670fd229014b.jpg"},{"id":78541174,"identity":"cd8d109d-e9c7-4f5b-bc6d-9deef0de0aea","added_by":"auto","created_at":"2025-03-14 16:05:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":531432,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of PPP2R2B enhances the proliferation, migration, and invasion abilities of PANC-1 and BxPC-3 cells post-individual transfections with oe-PPP2R2B or oe-NC. (A) qRT-PCR was applied to analyze the relative levels of PPP2R2B expression in pancreatic tumor cells. (B) The cell survival rate in pancreatic cancer cells was gauged via a CCK8 assay at time intervals of 0, 24, 48, and 72 hours. (C) The replicative capability of pancreatic carcinoma cells was assessed using an EdU assay. (D) The migratory potential of pancreatic tumor cells transfected with oe-PPP2R2B or oe-NC was determined utilizing a wound closure experiment. (E) Transwell analyses were utilized to evaluate the movement and penetration abilities of pancreatic cancer cells transfected with oe-PPP2R2B or oe-NC. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/58f21b0443f7d202d77f65c9.jpg"},{"id":78541171,"identity":"3def9148-1c97-445d-b2f7-31fab83358a0","added_by":"auto","created_at":"2025-03-14 16:05:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":230054,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of PPP2R2B inhibits the EMT process and promotes apoptosis in pancreatic cancer cells. (A) Protein expressions of apoptosis regulators (Bax, Bcl-2) were assessed in PANC-1 and BxPC-3 cells for the oe-PPP2R2B and oe-NC group. (B) Flow cytometry was performed to assess apoptosis in PANC-1 and BxPC-3 cells. \u0026nbsp;E-cad, E-cadherin, N-cad, N-cadherin. * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/ab75d76b0b4ce473760e3913.jpg"},{"id":78542188,"identity":"b2887a29-b885-46e5-84d7-f23778fe23c6","added_by":"auto","created_at":"2025-03-14 16:13:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":373306,"visible":true,"origin":"","legend":"\u003cp\u003ePPP2R2B serves as a positive enhancer of MAPK signaling pathway in PC cells. (A-C) The results from GO, KEGG, and GSEA analyses indicated a connection between PPP2R2B and MAPK signaling pathways. (D) Protein expressions of PPP2R2B, MAPK signaling pathway proteins (ERK, p38, JNK) were assessed in PANC-1 and BxPC-3 cells for the PPP2R2B-knockdown groups (sh-PPP2R2B-2, sh-PPP2R2B-3) and the negative control group (sh-NC). (E) Protein expressions of PPP2R2B, MAPK signaling pathway proteins (ERK, p38, JNK) were assessed in PANC-1 and BxPC-3 cells for the oe-PPP2R2B and oe-NC group. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/dea4474e83f50d5bc858889d.jpg"},{"id":78542360,"identity":"3e0e6db5-8edc-41c5-bd8a-1b6059bd5802","added_by":"auto","created_at":"2025-03-14 16:21:13","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":848691,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of PPP2R2B on pancreatic cancer cells after ERK inhibitor treatment. (A) CCK8 assay was utilized to evaluate the viability of pancreatic cancer cells. (B) The migration capacity of pancreatic cancer cells was assessed using a wound healing assay. (C) Transwell assays were employed to determine the migration and invasion potentials of pancreatic cancer cells. ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/f3b3da018d47a82f97ae3542.jpg"},{"id":78541177,"identity":"4cf7b887-9c21-47f2-8be2-b7016b6fc528","added_by":"auto","created_at":"2025-03-14 16:05:13","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":805826,"visible":true,"origin":"","legend":"\u003cp\u003eSuppression of tumour growth in vivo by silencing PPP2R2B. (A-C) Tumor volumes and masses were assessed in both sh-NC (control) and sh-PPP2R2B treated groups. (D) Immunohistochemistry (IHC) staining was conducted to detect the levels of PPP2R2B expression, as well as epithelial-mesenchymal transition and apoptosis-related markers.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/5715b4efb24fc9a59e4989c4.jpg"},{"id":78543022,"identity":"f2d4aff1-bd5c-4b0b-9bc5-559003b291e2","added_by":"auto","created_at":"2025-03-14 16:29:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5733866,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5324510/v2/0339d5af-ffe7-44da-9708-40f63d632dec.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eKnockdown of PPP2R2B inhibits pancreatic cancer progression via the ERK/MAPK pathway by modulating EMT and apoptosis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer (PC) ranks as the third primary cause of cancer-related fatalities across the globe, known for its aggressive nature and tendency for late detection. This results in a high annual mortality rate of around 400,000 globally. Patients with advanced or inoperable PC have a five-year survival rate of approximately 13%, dropping to 3% for those with distant metastasis\u003csup\u003e1,2\u003c/sup\u003e. Despite improvements in treatment approaches in recent years, the delayed diagnosis often limits the potential for surgical resection as a curative option\u003csup\u003e3,4\u003c/sup\u003e. Therefore,\u0026nbsp;a comprehensive comprehension of the biological processes behind PC is fundamental for the creation of more potent biomarkers and treatment strategies.\u003c/p\u003e\n\u003cp\u003ePPP2R2B, part of the B family of regulatory subunit 2 phosphatases, encodes the beta version of the B55 subfamily. Its expression peaks during the fetal stage and decreases as development advances. Predominantly found in the brain and testes, lower levels are present in the lungs and spleen\u003csup\u003e5,6\u003c/sup\u003e. The upregulation of PPP2R2B during neural maturation in embryos highlights its crucial role in embryonic growth. In the realm of cancer, PPP2R2B might carry out functions akin to those seen in development or differentiation\u003csup\u003e7-11\u003c/sup\u003e. While PPP2R2B has been implicated in the initiation and progression of multiple cancers via mutations or elevated expression, its specific effects on prostate cancer growth and metastasis remain to be fully elucidated.\u003c/p\u003e\n\u003cp\u003eThe transition from epithelial to mesenchymal cells (EMT) is a crucial event in the advancement and dissemination of cancer, encompassing a sequence of complex and dynamic stages.\u0026nbsp;It involves a complex series of steps that are dynamic in nature. Among the different pathways involved in EMT\u003csup\u003e12-14\u003c/sup\u003e, the Mitogen-Activated Protein Kinase (MAPK) pathway is particularly significant in regulating important cellular processes that contribute to the growth and dissemination of tumors [15,16]. Consequently, targeting the MAPK/EMT axis has emerged as a central focus in contemporary cancer research\u003csup\u003e17-20\u003c/sup\u003e. Our study uncovers a new finding of PPP2R2B being overexpressed in the majority of pancreatic cancer cases, highlighting its essential function in promoting tumor growth and dissemination. In addition, our findings illustrate the capability of PPP2R2B to stimulate the proliferation and metastasis of PC cells through the MAPK/EMT pathway.\u003c/p\u003e\n\u003cp\u003eThe purpose of this research is to decipher the role of PPP2R2B, a recently identified factor promoting cancer growth, in regulating EMT and apoptosis through the MAPK pathway in driving the advancement of pancreatic cancer. With the limited options for treating PC, our findings may pave the way for innovative treatment approaches focused on inhibiting PPP2R2B.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePPP2R2B\u0026apos;s increased expression is observed in human PC cell lines and tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous RNA sequencing (RNA-seq) study revealed a significant down-regulation of PPP2R2B in PANC-1 cells upon treatment with a tumor suppressor drug. Furthermore, PPP2R2B is found to be highly expressed in PC samples from both the Cancer Genome Atlas (TCGA) and GEPIA datasets. In this research, we used qRT-PCR to assess the PPP2R2B expression in human PC tissues. The findings indicated a notable rise in PPP2R2B levels in PC tissues in contrast to nearby normal samples. (Fig. 1E). Additionally, the upregulation of PPP2R2B was observed in four PC cell lines (PANC-1, BxPC-3, SW1990, and AsPC-1) in comparison to HPDE6-C7 cells (Fig.1D). The Kaplan-Meier analysis revealed that patients with PC who exhibited high levels of PPP2R2B had reduced disease-free survival rates in comparison to individuals with low PPP2R2B expression levels ( Fig. 1C). These results indicate that the elevated presence of PPP2R2B could potentially contribute significantly to the progression of PC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation of PPP2R2B expression with clinical and pathological characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the potential therapeutic relevance of PPP2R2B expression in prostate cancer (PC) patients, we investigated its levels in a cohort of 60 subjects, with a median age of 61. The accompanying table outlines the important clinical, pathological, and radiographic findings in this study population. It is noteworthy that a majority of the tumors (76.7%) examined in our investigation were categorized as stage I-II, with sizes ranging from 2 to 4 cm. As PPP2R2B demonstrates variable expression levels in this PC group, we proceeded to investigate its connection with established tumor traits and indicators of prognosis. Our findings found a correlation between PPP2R2B expression and nerve invasion as well as tumor dimensions. Notably, there was no significant relationship observed between the immunohistochemical score of PPP2R2B and tumor stage or nodal status (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe inhibition of PPP2R2B inhibit the proliferation, migration, and invasion of PC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies utilizing qRT-PCR revealed elevated PPP2R2B expression notable in both PANC-1 and BxPC-3 cells (Fig. 1). Based on these results, we selected these two cell lines for further investigations. To study the impact of PPP2R2B on PC development, we established stable knockdown cell lines named sh-PPP2R2B in BxPC-3 and PANC-1 cells (Fig. 2 A, B). From the qRT-PCR data, sh-PPP2R2B-2 and sh-PPP2R2B-3 were chosen for subsequent experiments. Following this, we evaluated the growth of PC cells using CCK8 and EdU assays, which indicated that inhibiting PPP2R2B resulted in decreased proliferation of PANC-1 and BxPC-3 cells (Fig. 2 B, C). We also examined the impact of PPP2R2B on cell migration and invasion. Our wound healing assays revealed a notable decrease in PC cell motility upon PPP2R2B silencing (Fig.2D). Migration and invasion capabilities were further assessed using Transwell assays, showing reduced activity in both processes following PPP2R2B knockdown (Fig.2E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of PPP2R2B suppresses EMT and promotes apoptosis in PC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the effects of PPP2R2B knockdown on the EMT process in pancreatic cancer cells, this study utilized Western blot analysis to examine the expression of EMT markers E-cadherin, N-cadherin, and Vimentin. The experimental results demonstrated that compared to the control group (sh-NC), the experimental group with reduced PPP2R2B expression significantly increased the protein level of E-cadherin in pancreatic cancer cell lines PANC-1 and BxPC-3, while the levels of N-cadherin and Vimentin were decreased (Fig. 3A). Furthermore, apoptosis-related proteins Bcl-2 and Bax were analyzed using WB, and flow cytometry was employed to assess the apoptotic rates of cells. The results of flow cytometry indicated that compared to the control group (sh-NC), reduced PPP2R2B expression significantly increased the apoptosis rate in pancreatic cancer cell lines PANC-1 and BxPC-3 (Fig. 3C). Western blot analysis showed that PPP2R2B knockdown notably upregulated the expression of Bax protein and downregulated the expression of Bcl-2 protein in PANC-1 and BxPC-3 cells (Fig. 3B). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of PPP2R2B promotes proliferation, migration, and invasion, suppresses EMT, and inhibits apoptosis in PC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the biological role of PPP2R2B in pancreatic cancer cells, we constructed a lentiviral vector for PPP2R2B overexpression (oe-PPP2R2B) and a negative control lentiviral vector (oe-NC). The results showed that the expression level of PPP2R2B was significantly increased in the oe-PPP2R2B group compared to the control group (Fig. 4A). Moreover, upregulating PPP2R2B significantly promoted the proliferation of PC cells compared to the oe-NC group (Fig. 4B-C). Wound-healing and transwell assay demonstrated that oe-PPP2R2B markedly enhanced the migration and invasion ability of PC cells (Fig. 4D-E). The results of WB indicated that oe-PPP2R2B significantly decreased the protein levels of E-cadherin while increasing the expression of N-cadherin and Vimentin in PC cells (Fig. 5A). Flow cytometry showed that oe-PPP2R2B significantly inhibited apoptosis in PC cells compared to the oe-NC group (Fig. 5C). WB results revealed that PPP2R2B overexpression decreased the levels of Bax protein while increasing the levels of Bcl-2 protein in PANC-1 and BxPC-3 cells (Fig. 5B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePPP2R2B knockdown inhibited the MAPK signaling pathway activity in PC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther investigation into the role of PPP2R2B in pancreatic cancer was conducted. Analysis of GO and KEGG pathways indicated enrichment of PPP2R2B in the MAPK signaling pathway (Fig. 6A-B). Results from GSEA showed a positive correlation between elevated PPP2R2B levels and MAPK pathway activation (Fig. 6C). PPP2R2B was discovered as a potential activator of the MAPK signaling pathway, which is recognized to impact both EMT and apoptosis, ultimately having a crucial impact on the growth, movement, and infiltration of pancreatic cancer cells. Furthermore, the impact of PPP2R2B on the ERK, JNK, and p38 MAPK pathways was evaluated. WB analysis demonstrated significantly higher levels of ERK, JNK, and p38 in the PPP2R2B knockdown group compared to the sh-NC group (Fig. 6D). These results highlight the role of PPP2R2B in modulating MAPK signaling pathways in pancreatic cancer. Overexpression of PPP2R2B had the opposite effect on MAPK pathway (Fig. 6E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of PPP2R2B on PC cells after adding an ERK inhibitor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe found that PPP2R2B most significantly affects ERK protein expression among the three MAPK pathways. To confirm whether PPP2R2B regulates pancreatic cancer cell proliferation, invasion, and migration through the ERK/MAPK pathway, we treated PANC-1 and BxPC-3 cells with an ERK inhibitor after overexpressing PPP2R2B. The CCK8 assay showed that PPP2R2B overexpression significantly promoted cell proliferation, but this effect was reduced with ERK inhibitor treatment (Fig. 7A). The wound-healing and transwell assay showed that migration and invasion were also weakened by the ERK inhibitor (Fig. 7B-C). These results suggest that PPP2R2B enhances pancreatic cancer cell proliferation, migration, and invasion, likely through the ERK/MAPK pathway. The ERK inhibitor\u0026apos;s ability to reduce these effects further supports this pathway\u0026apos;s role in regulating cancer cell behavior.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePPP2R2B stimulated PC tumor growth in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the effects of PPP2R2B on the progression of PC, BALB/c mice were injected with PANC-1 cells in which PPP2R2B was either silenced or expressed as a negative control. After 28 days, the tumors were removed and observed. Our results indicated a notable decrease in the size, volume, and weight of tumors in the mice injected with sh-PPP2R2B compared to those in the control group (Fig. 8A). Immunohistochemical studies revealed reduced levels of markers associated with EMT and apoptosis in the sh-PPP2R2B group as opposed to the control, consistent with previous western blot findings. The inhibition of tumor growth in vivo following PPP2R2B knockdown can be seen in Figure 8.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present research, we uncover a new role that promotes tumor growth for PPP2R2B in the advancement of PC. Our results show that PPP2R2B is upregulated in PC cell lines and tumor samples in comparison to normal pancreatic cells and neighboring non-tumor tissues, respectively. Increased PPP2R2B levels are associated with reduced disease-free survival in PC patients, highlighting its potential as a predictive marker. Significantly, we demonstrate that PPP2R2B enhances PC cell growth, movement, and infiltration by modulating the MAPK/EMT signaling pathway, representing a notable advancement in comprehending the molecular characteristics of PC.\u003c/p\u003e\n\u003cp\u003eThe functional implications of PPP2R2B in normal and abnormal states pose an interesting contradiction. Although its involvement in fetal development emphasizes its crucial function in early human growth, particularly in the brain and reproductive system\u003csup\u003e21\u003c/sup\u003e, PPP2R2B\u0026apos;s reappearance in cancer brings attention to a more sinister aspect of its role\u003csup\u003e8, 22, 23\u003c/sup\u003e. This dual functionality is prominently displayed in the setting of PC, where PPP2R2B appears to revert to its developmental functions, leading to abnormal cell differentiation and proliferation.\u003c/p\u003e\n\u003cp\u003eOur research suggests that knocking down PPP2R2B in PC cells results in decreased cell proliferation, migration, and invasion capabilities, as demonstrated by CCK-8, EdU, wound healing, and transwell assays. Conversely, upregulating PPP2R2B enhances these malignant characteristics, further supporting its role in promoting tumorigenesis. In animal studies, the tumor-suppressive effects of PPP2R2B depletion were confirmed. Mouse xenografts injected with PC cells lacking PPP2R2B showed significantly inhibited tumor growth compared to control cells. In line with our lab findings, tumors originating from PPP2R2B-deficient cells displayed decreased expression of EMT-triggering and anti-apoptotic markers, further highlighting PPP2R2B\u0026apos;s involvement in regulating these processes.\u003c/p\u003e\n\u003cp\u003eExtensive research has focused on the ERK/MAPK signaling pathway in various cancer types\u003csup\u003e24,25.\u003c/sup\u003e The ERK/MAPK system plays a key role in numerous biological processes, serving as a vital cell signaling pathway\u003csup\u003e26-28\u003c/sup\u003e. These functions encompass embryonic development, tissue regeneration, cell division, and motility. EMT is indispensable for the invasion and metastasis of malignant tumors\u003csup\u003e29-31\u003c/sup\u003e. The activation of the MAPK pathway can trigger EMT, prompting cancer cells to relocate and acquire invasive capabilities\u003csup\u003e32,33\u003c/sup\u003e. By inducing changes in cellular structure and function, EMT facilitates distant organ metastasis and enhances the ability of tumor cells to traverse vascular barriers and access the circulatory or lymphatic systems.\u003c/p\u003e\n\u003cp\u003eOur research outlines the detailed mechanisms by which PPP2R2B contributes to its oncogenic effects, specifically by influencing the ERK, JNK, and p38 branches of the MAPK signaling pathway to regulate processes related to EMT and apoptosis. In particular, The inhibition of PPP2R2B leads to a decrease in the expression of the epithelial marker E-cad and an increase in the expression of the mesenchymal markers N-cad and Vimentin, suggesting a promotion of the EMT transition process. Furthermore, knockdown of PPP2R2B leads to the promotion of apoptosis, characterized by a increase in pro-apoptotic markers such as Bax and Beclin, and an decrease in the levels of the anti-apoptotic protein Bcl-2.\u003c/p\u003e\n\u003cp\u003eBy analyzing clinical data, a strong correlation was discovered between elevated PPP2R2B levels and nerve infiltration. Prior research has indicated that abnormal PPP2R2B methylation levels play a crucial role in neurological conditions like Spinocerebellar Ataxia Type 12\u003csup\u003e22,34\u003c/sup\u003e. PPP2R2B has the ability to modify mitochondrial division/fusion patterns by modulating kinase/phosphatase equilibrium, apoptotic and anti-apoptotic protein levels, ultimately leading to neurotoxicity and prompting neuronal demise\u003csup\u003e35\u003c/sup\u003e. Suppression of gene activity exhibits a neuroprotective impact, aligning with our observation that heightened PPP2R2B expression in tumors could hasten nerve cell differentiation and spread.\u003c/p\u003e\n\u003cp\u003eThrough our research into the role of PPP2R2B in the progression of PC, we have uncovered the intricate interplay between genetic factors and the pathways that promote cancer growth. Our findings emphasize PPP2R2B\u0026apos;s pivotal function in promoting the spread and growth of tumors. This study demonstrates how PPP2R2B influences the signaling pathway MAPK/EMT, providing new perspectives on the molecular processes driving the advancement of PC.\u003c/p\u003e\n\u003cp\u003eFurthermore, due to the intricate nature of cancer development, it is crucial to thoroughly explore the interplay between PPP2R2B and various signaling molecules and pathways within the tumor microenvironment. These investigations have the potential to unveil further levels of control and reveal innovative treatment strategies that could potentially counteract resistance mechanisms and improve treatment efficacy.\u003c/p\u003e\n\u003cp\u003eIn summary, our research makes a substantial contribution to the increasing evidence highlighting the crucial importance of PPP2R2B in the development of pancreatic cancer. By elucidating how PPP2R2B impacts the MAPK/EMT signaling pathway and the progression of tumors, we set the stage for additional studies on therapies targeting PPP2R2B that may bring renewed optimism to individuals with pancreatic cancer. As we delve deeper into the complex role of PPP2R2B in cancer, the potential for developing improved and specialized treatments for pancreatic cancer, as well as other types of cancer, appears to be increasingly optimistic.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eData and resources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cancer Genome Atlas (TCGA) data repository (https://gdc-portal.nci.nih.gov/) was utilized to access level-three transcriptome RNA sequencing data of patients with PC. In addition, RNA-sequence information from both normal and PC samples was obtained from Datasets GSE62165 and GSE15471 retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). The potential prognostic significance of PPP2R2B in PC was investigated using the Gene Expression Profiling Interactive Analysis (GEPIA2) tool (http://gepia.cancer-pku.cn/), a web-based server for customizable and interactive large-scale expression analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe deeper exploration of potential functional implications of PPP2R2B associated with PC involved the application of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Moreover, Gene Set Enrichment Analysis (GSEA) was employed to assess the intrinsic molecular mechanisms distinguishing low- and high-risk cohorts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients and s\u003c/strong\u003e\u003cstrong\u003eample\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBetween 2021 and 2022, a total of 60 pairs of tissue samples from PCs and nearby tissues were gathered from individuals who underwent pancreaticoduodenectomy at the First Hospital of Lanzhou University. Prior to participating in the research, all subjects had given their consent after being informed about the study\u0026apos;s objectives. Approval for the research was granted by the Institutional Review Board (IRB) of the First Hospital of Lanzhou University (LDYYLL-2024-244).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;lines and cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman four PC cell lines including PANC-1, BxPC-3, SW1990, AsPC-1, and the HPDE6-C7 normal human pancreatic duct epithelial cell line were acquired from the BNCC and the Shanghai Branch of the Chinese Academy of Sciences. In order to ensure cell survival, all cell lines were grown in DMEM at 37\u0026deg;C with 95% humidity and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQ\u003c/strong\u003e\u003cstrong\u003euantitative\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003er\u003c/strong\u003e\u003cstrong\u003eea\u003c/strong\u003e\u003cstrong\u003el-\u003c/strong\u003e\u003cstrong\u003eti\u003c/strong\u003e\u003cstrong\u003eme\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePCR\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter total RNA extraction with the TRIzol Reagent (TaKaRa), cDNA synthesis was carried out using 5 \u0026micro;g of the mentioned total RNA and the PrimeScript RT Reagent Kit (TaKaRa). A Bio-Rad RT-PCR amplification system along with a SYBR Green PCR Kit (Takara) was utilized for qRT-PCR. The quantification of mRNA expression levels was conducted using the 2\u0026Delta;\u0026Delta;Ct method, with normalization to the endogenous reference gene GAPDH. The PCR primer sequences for the human PPP2R2B were as follows: (forward) 5\u0026prime;-TGCAGCTTACTTTCTTCTGTCT-3\u0026prime; and (reverse) 5\u0026prime;-GTAGCCTTCTGGCCTCTTATC-3\u0026prime;. For the GAPDH primers, the sequences were (forward) 5\u0026prime;-AAGGTGAAGGTCGGAGTCAAC-3\u0026prime; and (reverse) 5\u0026prime;-GGGGTCATTGATGGCAACAATA-3\u0026prime;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003cstrong\u003eting (WB)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibodies against PPP2R2B, N-cadherin (N-cad), E-cadherin (E-cad), Vimentin, Bax, and Bcl-2 were bought from Wuhan Sanying (Wuhan, China). ERK, JNK and p38 antibodies, were sourced from Boser Bio (Wuhan, China).\u003c/p\u003e\n\u003cp\u003eProteins were extracted from cells and tissues using SDS-PAGE gels for separation. Following separation, the proteins were transferred onto a PVDF membrane and then incubated with primary antibodies targeting PPP2R2B, accompanying biomarkers, and GAPDH at 4 degrees Celsius for 48 hours. After blocking the membrane with BSA at room temperature for 2 hours, secondary antibodies were added and incubated for another 2 hours. The membrane underwent three TBST washes before exposure to a luminescent solution, using GAPDH as an internal control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003estaining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA senior pathologist reviewed tissue slides to detect and label tumor regions. Cylinder replicas (1.0 mm wide) were collected from the designated tumor sites and adjacent non-malignant pancreatic tissue in the donor\u0026apos;s tissue samples. These samples were then embedded in a fresh paraffin block. Standard protocols were followed for immunohistochemical staining, employing specific Vimentin, Beclin, and PPP2R2B antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell counting Kit‑8\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(CCK-8)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were first plated onto 96-well culture dishes with a concentration of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well. Following plating, the dishes were placed in the incubator for durations spanning from 24 to 72 hours. A CCK-8 solution obtained from Boster Bio (China) was utilized for the experiment. A volume of 10 \u0026micro;L of the CCK-8 solution was added to each separate well and permitted to incubate within the incubator for a duration of one hour. In order to guarantee precise evaluation of cellular growth, the absorbance at a wavelength of 450 nm was measured for each well utilizing a plate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5-Ethynyl-2\u0026rsquo;-deoxyuridine (\u003c/strong\u003e\u003cstrong\u003eEdU\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Seville Biotech (Wuhan, China) provided the necessary reagents for the EdU assay. Pancreatic cancer cells were seeded in confocal dishes at a density of 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well. Subsequently, the cells were treated with 4% paraformaldehyde (Beyotime, China) for 10 minutes to fix them. After fixing, the cells underwent three washes with PBS and were permeabilized for 5 minutes using 1% Triton (Beyotime, China). Subsequently, the cells were exposed to the dyeing agent in the dark for 30 minutes, then subjected to DAPI staining (Olympus, Tokyo, Japan) at 37 \u0026deg;C for 5 minutes. Imaging was carried out with a microscope at 400\u0026times; magnification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn exact count of cells were uniformly spread in a 6-well dish and cultured for a duration of 24 hours. A gap was created between the cells by drawing a single line using the tip of a crystal pipette. After washing with PBS, images were captured; then, drug-containing and standard media were added. Gap growth was monitored and captured at 0 and 24 hours. The area was determined by dividing the width by the length. Mobility of cells (%) was calculated as 100 times the difference in width between 0 h and 24 h, divided by the width at 0 h. The Image J program was utilized to gauge the gap between the opposing ends of the wound. Analysis of the data from three distinct trials involving two sets of samples was conducted for statistical purposes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMigration and invasion assay\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Transwell system filter (8.0 \u0026micro;m pore size; BD Biosciences, USA) was used for the migration assay. In a 24-well culture plate (Corning, USA), 200 \u0026micro;l of either treated or untreated 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were added to the upper chamber of Transwell inserts for 24 hours, followed by the addition of 700 \u0026micro;l of medium containing 30% FBS to the lower chamber. The invasion assay utilized chambers with inserts coated uniformly with Matrigel (BD Biosciences). The chambers were then fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 30 minutes. Following this, the upper surface of the inserts was carefully cleaned using absorbent paper, and the stained cells were imaged randomly using a microscope after rinsing with PBS. Cell migration and invasion capabilities were assessed by quantifying the average number of labeled cells across five different fields. Statistical analysis was performed utilizing data from two groups across three independent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nude mice used in this study were purchased from Spefo (Beijing) Biotechnology Co., Ltd. Four to six-week-old BALB/c mice, weighing between eighteen and twenty grams, were kept in a sterile setting with unrestricted access to food and water. Through unbiased randomization, ten mice were evenly split into two groups, with each group consisting of five mice. Then, both groups were injected subcutaneously in the side with 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e PANC-1 cells that expressed various PPP2R2B shRNAs and control NC shRNAs. Weekly measurements were taken of the tumor\u0026apos;s dimensions, which were determined using the formula of multiplying the length by the square of the width, and then multiplying by 0.5. Afterward, the mice were put to sleep with isoflurane overdose, followed by measuring the tumor\u0026rsquo;s size and weight in a lab setting. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals, and were granted approval by the Ethics Committee at the First Hospital of Lanzhou University (LDYYLL-2024-244).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using the SPSS 22.0 software from Chicago, USA, and GraphPad Prism software version 9.5 from the USA. The results were presented as mean\u0026nbsp;\u0026plusmn;\u0026nbsp;SEM (standard error of the mean). A comparison of means between two groups was made using an unpaired t-test. To assess differences between two or more groups, one-way ANOVA and the Bonferroni test for multiple comparisons were applied. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy concepts: FFH, WCZ; Study design: FFH, ZC; Experimental studies: CY, HQS; Data analysis: HQS, ZC; Statistical analysis: FFH, YD; Manuscript preparation: FFH, CLD; Manuscript editing: FFH, PFX; Manuscript review: FFH, WCZ. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request by contacting the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed following the ethical standards in the 1964 Declaration of Helsinki and its later amendments. The study including human sample and animal experimental protocols has been approved by the Institutional Review Board (IRB) of the First Hospital of Lanzhou University (LDYYLL-2024-244), in compliance with the First Hospital of Lanzhou University guidelines for the care and use of animals. The informed consent was exempted by the IRB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by National Natural Science Foundation of China (82260555), The First Hospital of Lanzhou University Intra-Hospital Fund (ldyyyn2020-93).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBray, F., Ferlay, J., Soerjomataram, I., et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 68, 394\u0026ndash;424 (2018).\u003c/li\u003e\n \u003cli\u003eSiegel, R. L., Giaquinto, A. N., Jemal, A. Cancer statistics, 2024. CA: A Cancer Journal for Clinicians 74, 12\u0026ndash;49 (2024).\u003c/li\u003e\n \u003cli\u003eCass, S. H., Tzeng, C. D., Prakash, L. R., et al. Trends Over Time in Recurrence Patterns and Survival Outcomes after Neoadjuvant Therapy and Surgery for Pancreatic Cancer. Annals of Surgery (2024) [Epub ahead of print].\u003c/li\u003e\n \u003cli\u003eVan Bodegraven, E. A., Balduzzi, A., van Ramshorst, T. M. E., et al. Prophylactic abdominal drainage after distal pancreatectomy (PANDORINA): an international, multicentre, open-label, randomised controlled, non-inferiority trial. The Lancet Gastroenterology \u0026amp; Hepatology 9, 438\u0026ndash;447 (2024).\u003c/li\u003e\n \u003cli\u003eJohnson, H., Narayan, S., Sharma, A. K. Altering phosphorylation in cancer through PP2A modifiers. Cancer Cell International 24, 11 (2024).\u003c/li\u003e\n \u003cli\u003eZheng, H., Qi, Y., Hu, S., et al. Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase. Science 370, e4122 (2020).\u003c/li\u003e\n \u003cli\u003eHao, Y., Wang, C., Xu, D. Identification and validation of a novel prognostic model based on platinum resistance-related genes in bladder cancer. Int. Braz. J. Urol. 49, 61\u0026ndash;88 (2023).\u003c/li\u003e\n \u003cli\u003eIshibashi, K., Ishii, K., Sugiyama, G., et al. Regulation of\u0026nbsp;\u0026beta;-Catenin Phosphorylation by PR55\u0026beta;\u0026nbsp;in Adenoid Cystic Carcinoma. Cancer Genomics \u0026amp; Proteomics 15, 53\u0026ndash;60 (2018).\u003c/li\u003e\n \u003cli\u003eLi, Z., Li, Y., Wang, X., et al. PPP2R2B downregulation is associated with immune evasion and predicts poor clinical outcomes in triple-negative breast cancer. Cancer Cell International 21, 13 (2021).\u003c/li\u003e\n \u003cli\u003eTan, J., Lee, P. L., Li, Z., et al. B55\u0026beta;-associated PP2A complex controls PDK1-directed MYC signaling and modulates rapamycin sensitivity in colorectal cancer. Cancer Cell 18, 459\u0026ndash;471 (2010).\u003c/li\u003e\n \u003cli\u003eVazquez, A., Kulkarni, D., Grochola, L. F., et al. A genetic variant in a PP2A regulatory subunit encoded by the PPP2R2B gene associates with altered breast cancer risk and recurrence. Int. J. Cancer 128, 2335\u0026ndash;2343 (2011).\u003c/li\u003e\n \u003cli\u003eCeli\u0026agrave;-Terrassa, T., Kang, Y. How important is EMT for cancer metastasis? PLoS Biol. 22, e3002487 (2024).\u003c/li\u003e\n \u003cli\u003eGuo, Z., Ashrafizadeh, M., Zhang, W., et al. Molecular profile of metastasis, cell plasticity, and EMT in pancreatic cancer: a pre-clinical connection to aggressiveness and drug resistance. Cancer Metastasis Rev. 43, 29\u0026ndash;53 (2023).\u003c/li\u003e\n \u003cli\u003eKhan, A. Q., Hasan, A., Mir, S. S., et al. Exploiting Transcription Factors to Target EMT and Cancer Stem Cells for Tumor Modulation and Therapy. Semin. Cancer Biol. 100, 1\u0026ndash;16 (2024).\u003c/li\u003e\n \u003cli\u003eBrown, B. A., Myers, P. J., Adair, S. J., et al. A histone methylation-MAPK signaling axis drives durable epithelial-mesenchymal transition in hypoxic pancreatic cancer. Cancer Res. 84, 1764\u0026ndash;1780 (2024).\u003c/li\u003e\n \u003cli\u003eSugimoto, T., Iwagami, Y., Kobayashi, S., et al. Skeletal Muscle-Derived Irisin Enhances Gemcitabine Sensitivity and Suppresses Migration Ability in Pancreatic Ductal Adenocarcinoma. Ann. Surg. Oncol. 31, 3718\u0026ndash;3736 (2024).\u003c/li\u003e\n \u003cli\u003eDu, Q., Lin, Y., Ding, C., et al. Pharmacological activity of matrine in inhibiting colon cancer cells VM formation, proliferation, and invasion by downregulating claudin-9 mediated EMT process and MAPK signaling pathway. Drug Des. Devel. Ther. 17, 2787\u0026ndash;2804 (2023).\u003c/li\u003e\n \u003cli\u003eLee, Y. S., Kim, H. S., Kim, H. J., et al. The role of LOXL2 induced by glucose metabolism-activated NF-\u0026kappa;B in maintaining drug resistance through EMT and cancer stemness in gemcitabine-resistant PDAC. J. Mol. Med. 101, 1449\u0026ndash;1464 (2023).\u003c/li\u003e\n \u003cli\u003eMi, K., Zeng, L., Chen, Y., et al. DHX38 enhances proliferation, metastasis, and EMT progression in NSCLC through the G3BP1-mediated MAPK pathway. Cell Signal. 113, 110962 (2024).\u003c/li\u003e\n \u003cli\u003eZhao, C., Li, X., Zhang, R., et al. Sense and anti-sense: Role of FAM83A and FAM83A-AS1 in Wnt, EGFR, PI3K, EMT pathways and tumor progression. Biomed. Pharmacother. 173, 116372 (2024).\u003c/li\u003e\n \u003cli\u003eXu, F., Chen, A., Pan, S., et al. Systems genetics analysis reveals the common genetic basis for pain sensitivity and cognitive function. CNS Neurosci. Ther. 30, e14557 (2024).\u003c/li\u003e\n \u003cli\u003eShen, D., Kang, S. Comprehensive analysis of mitochondria-related genes indicates that PPP2R2B is a novel biomarker and promotes the progression of bladder cancer via Wnt signaling pathway. Biol. Direct 19, 17 (2024).\u003c/li\u003e\n \u003cli\u003eMalvi, P., Chava, S., Cai, G., et al. HOXC6 drives a therapeutically targetable pancreatic cancer growth and metastasis pathway by regulating MSK1 and PPP2R2B. Cell Rep. Med. 4, 101285 (2023).\u003c/li\u003e\n \u003cli\u003eSheng, W., Shi, X., Lin, Y., et al. Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling. J. Exp. Clin. Cancer Res. 39, 16 (2020).\u003c/li\u003e\n \u003cli\u003eSheng, W., Chen, C., Dong, M., et al. Calreticulin promotes EGF-induced EMT in pancreatic cancer cells via Integrin/EGFR-ERK/MAPK signaling pathway. Cell Death Dis. 8, e3147 (2017).\u003c/li\u003e\n \u003cli\u003eXu, J., Wang, Y., Jiang, J., et al. ADAM12 promotes clear cell renal cell carcinoma progression and triggers EMT via EGFR/ERK signaling pathway. J. Transl. Med. 21, 56 (2023).\u003c/li\u003e\n \u003cli\u003eUngefroren, H., Konukiewitz, B., Braun, R., et al. TAp73 inhibits EMT and cell migration in pancreatic cancer cells through promoting SMAD4 expression and SMAD4-dependent inhibition of ERK activation. Cancers 15, 3791 (2023).\u003c/li\u003e\n \u003cli\u003eSun, X. Y., Li, H. Z., Xie, D. F., et al. LPAR5 confers radioresistance to cancer cells associated with EMT activation via the ERK/Snail pathway. J. Transl. Med. 20, 456 (2022).\u003c/li\u003e\n \u003cli\u003eJanta, S., Pranweerapaiboon, K., Vivithanaporn, P., et al. Holothurin A inhibits RUNX1-enhanced EMT in metastasis prostate cancer via the Akt/JNK and P38 MAPK signaling pathway. Mar. Drugs 21, 345 (2023).\u003c/li\u003e\n \u003cli\u003eWang, J. F., Zhao, K., Chen, Y. Y., et al. NKCC1 promotes proliferation, invasion and migration in human gastric cancer cells via activation of the MAPK-JNK/EMT signaling pathway. J. Cancer 12, 253\u0026ndash;263 (2021).\u003c/li\u003e\n \u003cli\u003eXin, Y., Li, K., Yang, M., et al. Fluid shear stress induces EMT of circulating tumor cells via JNK signaling in favor of their survival during hematogenous dissemination. Int. J. Mol. Sci. 21, 8115 (2020).\u003c/li\u003e\n \u003cli\u003eBao, S., Ji, Z., Shi, M., et al. EPB41L5 promotes EMT through the ERK/p38 MAPK signaling pathway in esophageal squamous cell carcinoma. Pathol. Res. Pract. 228, 153682 (2021).\u003c/li\u003e\n \u003cli\u003eSong, H., Tian, D., Sun, J., et al. circFAM120B functions as a tumor suppressor in esophageal squamous cell carcinoma via the miR-661/PPM1L axis and the PKR/p38 MAPK/EMT pathway. Cell Death Dis. 13, 361 (2022).\u003c/li\u003e\n \u003cli\u003eZhou, C., Liu, H. B., Jahanbakhsh, F., et al. Bidirectional transcription at the PPP2R2B gene locus in spinocerebellar ataxia type 12. Mov. Disord. 38, 2230\u0026ndash;2240 (2023).\u003c/li\u003e\n \u003cli\u003eMadera-Salcedo, I. K., S\u0026aacute;nchez-Hern\u0026aacute;ndez, B. E., Svyryd, Y., et al. PPP2R2B hypermethylation causes acquired apoptosis deficiency in systemic autoimmune diseases. JCI Insight 5, e126457 (2019).\u003cbr\u003e\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eAssociation between the expression of PPP2R2B in pancreatic cancer and adjacent tissues and clinical characteristics in 60 PC patients\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"472\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003en(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGENE\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u003cstrong\u003elow\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGENE\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cstrong\u003ehigh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eexpression\u003c/strong\u003e \u003cstrong\u003en(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eexpression\u003c/strong\u003e \u003cstrong\u003en(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026lt;65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e39(65.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e10(76.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e29 (61.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026ge;65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e21(35.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e3(23.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e18(38.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e40(66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e7(53.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e33(70.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.438\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e20(33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e6(46.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e14(29.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 472px;\"\u003e\n \u003cp\u003ePrimarysite\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003ePancreatic head\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e46(76.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e12(92.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e34(72.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e0.256\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003ePancreatic body tail\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e14 \u0026nbsp; \u0026nbsp; (23.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e1(7.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e13(27.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 472px;\"\u003e\n \u003cp\u003eAJCC stage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eI,II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e46(76.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e10(76.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e36(76.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003el\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eII,IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e14(23.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e3(23.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e11(23.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003eVascular invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e42(70.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e9(69.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e33(70.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e18(30.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e4(30.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e14(29.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 472px;\"\u003e\n \u003cp\u003eNerve invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e18(30.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e8(61.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e10(21.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e42(70.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e5(38.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e37(78.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 472px;\"\u003e\n \u003cp\u003eLymph node metastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e30 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e7(53.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e23(48.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e30(50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e6(46.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e24(51.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 472px;\"\u003e\n \u003cp\u003eTumor size(cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026le;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e9(15.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e6(46.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e3(6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e2-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e36(60.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e5(38.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e31(66.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026ge;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e15(25.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e2(15.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e13(27.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 64px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\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":"Pancreatic cancer; PPP2R2B; MAPK pathway; EMT; Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-5324510/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5324510/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePancreatic cancer (PC) is one of the most lethal types of cancer, as current treatments are largely ineffective. Our research uncovers that PPP2R2B is overexpressed in a majority of PC cases, playing a significant role in the growth and spread of PC tumors. Knockdown of PPP2R2B inhibits PC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), while promoting cell apoptosis. Conversely, overexpression of PPP2R2B enhances these processes, leading to increased proliferation, migration, invasion, and EMT, and reduced apoptosis. Further analysis showed that reducing PPP2R2B levels in PC inactivates the MAPK pathways—ERK, JNK, and p38, ultimately promoting PC growth. The addition of an ERK inhibitor reverses the effects of PPP2R2B knockdown, restoring cell proliferation, migration, and invasion. Our experiments in live subjects demonstrate that removing PPP2R2B inhibits tumor growth in PC mouse models and alters the levels of proteins involved in EMT and cell death. These findings demonstrate that PPP2R2B contributes to PC progression by modulating EMT and apoptosis through the ERK/MAPK pathway. Targeting PPP2R2B or its downstream signaling pathways may offer a promising therapeutic strategy for pancreatic cancer.\u003c/p\u003e","manuscriptTitle":"Knockdown of PPP2R2B inhibits pancreatic cancer progression via the ERK/MAPK pathway by modulating EMT and apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-03-14 16:05:08","doi":"10.21203/rs.3.rs-5324510/v2","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}},{"code":1,"date":"2024-10-28 07:11:08","doi":"10.21203/rs.3.rs-5324510/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":"d93848c9-e75c-472b-899c-d685cb3409eb","owner":[],"postedDate":"March 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-30T06:53:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-14 16:05:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-5324510","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5324510","identity":"rs-5324510","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}