Study On Perioperative Comprehensive Treatment Strategy And Mechanism Exploration Of Prognosis-Associated Gene CPSF3 In Non-Small Cell Lung Cancer

Lung cancer is one of the most common malignant tumors and the largest cause of cancerrelated death worldwide.There are over one million new cases diagnosed every year,of which about 80%-90%are non-small cell lung cancer(NSCLC).Currently,the main treatments for NSCLC include surgery,chemotherapy,radiation therapy,targeted therapy,and immunotherapy.Developing the best comprehensive treatment plan,exploring effective prognostic molecular markers,and developing new targeted and immunotherapeutic drugs have always been important research directions of NSCLC.This article focuses on the treatment strategies for NSCLC.We compared the perioperative effects of minimally invasive surgery and conducts a long-term survival study of perioperative adjuvant therapy.We explored the potential of perioperative platelet count to predict lymph node metastasis and further investigates the mechanism of the prognosis-related gene CPSF3,finding that it may become a new target for molecular targeted therapy.The standard treatment for early-stage resectable NSCLC is lung cancer radical surgery,which aims to safely and completely remove the primary tumor and lymph nodes at risk.Open surgery used to be the main method of NSCLC surgery.With the development of technology,minimally invasive surgery represented by video-assisted thoracoscopic surgery(VATS)has been widely applied in the treatment of NSCLC and has shown significant advantages.Compared to open surgery,VATS can reduce intraoperative bleeding,alleviate postoperative pain,significantly improve patients’ perioperative quality of life,and shorten hospital stay.At present,minimally invasive surgery mainly includes traditional VATS and robot-assisted thoracic surgery(RATS).Since its first application in lung cancer treatment in 2002,RATS has become more and more common in NSCLC surgery.RATS has better visibility than traditional VATS and reduces natural tremors in limbs,making the operation more accurate.Patients have to pay a high cost for RATS.We conducted a clinical retrospective study of perioperative minimally invasive surgery treatment for NSCLC and hoped to help patients choose the most suitable minimally invasive surgery method and to help patients maximize their benefits during surgery.Surgical treatment of NSCLC has made significant progress over the past decades.However,even with complete resection of the tumor,about 50%-80%of patients still face the risk of recurrence.Therefore,systemic adjuvant therapy for NSCLC is of great importance.Chemotherapy is currently the most important perioperative treatment for NSCLC.A large number of clinical studies have shown that adjuvant chemotherapy can produce long-term survival benefits.Radiotherapy also plays an important role in perioperative treatment.In palliative treatments,more than half of the patients need to undergo radiotherapy.Platinumbased chemotherapy represents standard adjuvant therapy for operable stage Ⅱ and Ⅲ A NSCLC.Among them,patients with stage ⅢA-N2 NSCLC constitute a special group,and adjuvant therapy occupies an important position in the comprehensive treatment of this group.However,the recurrence rate of N2 patients is still high,and the overall survival rate is poor.Several studies have reported that for patients with ⅢA-N2,neoadjuvant combined radiotherapy and chemotherapy can improve long-term outcome.It is generally believed that pneumonectomy will limits the implementation of adjuvant therapy regimens and may lead to more complications and perioperative death events.For NSCLC patients requiring pneumonectomy,the determination of the perioperative comprehensive treatment plan should still be a major focus of discussion and should be based on multidisciplinary discussions.At present,there is little research on comprehensive treatment plan for pneumonectomy.We conducted this retrospective study on the efficacy of perioperative adjuvant therapy regimens for NSCLC patients,hoping to help establish the best adjuvant therapy plan for pneumonectomy patients and improve their long-term survival benefitsResearches have found that platelets can be new target for tumor intervention.Regular use of antiplatelet drugs such as aspirin can reduce the risk of distant metastasis in various cancers,and this conclusion has also been confirmed in lung adenocarcinoma.Additionally,several studies have observed an increase in platelet count in advanced stage patients with breast cancer,colon cancer,stomach cancer,lung cancer,ovarian cancer,and esophageal cancer.Meanwhile,there have been reported that elevated platelet count may be associated with poor prognosis in patients.There have been many studies on hematological indicators related to the perioperative prognosis of NSCLC patients,but the role of platelets has not been fully investigated.For decades,numerous studies have shown the correlation between platelets and cancer.In addition to being a recognized predictive marker for venous thrombosis risk in cancer patients,platelets are considered to be a significant participant in tumor progression.Recent studies suggest that platelets can affect tumor growth and metastasis,and increased platelet count is closely associated with poor prognosis of malignant tumors.Literatures have reported that circulating tumor cells in cancer patients can educate platelets.Educated platelets can assist tumor cell proliferation and metastasis through various ways,including the release of growth factors,helping tumor cell adhesion and aggregation,protecting circulating tumor cells from immune surveillance,and reducing the body’s immune response.Previous studies have reported that elevated preoperative platelet counts can promote hematogenous metastasis of lung cancer,but the potential role of platelets in predicting lymph node metastasis has not been fully investigated.We conducted this study on the correlation between preoperative platelet and lymph node metastasis in NSCLC patients,hoping to further explore the biological role of platelets in lung adenocarcinoma.As one of the essential adjuvant treatment methods,molecular targeted therapy has always been a focused field in lung adenocarcinoma research.At present,various types of protein tyrosine kinases are used as targets for molecular targeted therapy,playing a significant role in clinical treatment and providing substantial help to patients.However,current molecular targeted therapy still faces many challenges.Some lung adenocarcinoma patients do not have gene targets corresponding to existing drugs.Drug resistance emergence in many patients during treatment.Therefore,exploring new targets with better prognosis and treatment potential,and developing molecular targeted drug are of great importance for the clinical treatment of lung adenocarcinoma.CPSF3 belongs to the metal β-lactamase family and is the core component of specific factors required for cleavage and polyadenylation of pre-mRNA.Studies have found that knocking down CPSF3 in prostate cancer cells and colon cancer cells can induce apoptosis.But there is no research exploring the biological regulatory role of CPSF3 in lung adenocarcinoma.In this study,we analyzed transcriptional information in the TCGA database and found that p53 is likely to be the downstream target of CPSF3 regulation in lung adenocarcinoma.p53,also known as the guardian of the genome,is a transcription factor capable of activating a large number of genes.p53 can function as a tumor suppressor to activate or inhibit various cellular biological functions such as cell cycle arrest,DNA repair,senescence,apoptosis,metabolism,autophagy,or ferroptosis.We conducted this study to determine the biological functions of CPSF3 in lung adenocarcinoma and further explore the molecular mechanisms.The four parts of this study are relatively independent but interconnected.This research focus on the minimally invasive surgical treatment strategy for NSCLC,radiotherapy and chemotherapy regimen,feasibility of antiplatelet therapy under current research,and the development of new molecular targets for targeted therapy.The main content of the study is as follows.Part 1.Comparison of perioperative efficacy of minimally invasive surgery for NSCLC Objective:To compare the perioperative efficacy and safety of minimally invasive surgery for NSCLC,aiming to help patients choose the best surgical approach and improve perioperative benefits.Methods:The study retrospectively collected and analyzed the clinical information of 849 NSCLC patients who underwent minimally invasive lung cancer radical surgery at Shandong University Qilu Hospital from August 2020 to April 2021.Propensity score matching was used to balance the baseline data of VATS and RATS groups.Logistic regression analysis and subgroup analysis were used to compare the perioperative outcomes of the two groups of patients,determine their influencing factors,and then analyze the potential impact of patients’BMI on minimally invasive surgery.Results:Compared with the VATS group,RATS increased the number of lymph node dissections(P<0.001),reduced intraoperative blood loss(P<0.001),shortened the duration of chest tube(P<0.001),and reduced postoperative hospital stay(P<0.001)and postoperative complication rate(P=0.027),and facilitated the patient’s recovery(P=0.003).But RATS group had significantly higher hospitalization costs(P<0.001).Multivariate logistic regression analysis showed that RATS(P=0.027)was an independent risk factor for reducing the risk of postoperative complications.Subgroup analysis showed that in patients with a BMI of 24kg/m228kg/m2,RATS had significant advantages in perioperative outcomes,with a greater number of lymph node dissections(P<0.001)and a lower risk of postoperative complications(P=0.030)compared to the VATS group.Conclusion:Both RATS and VATS can be safely applied to NSCLC patients.Statistical results show that compared to VATS,patients receiving RATS have better perioperative outcomes but higher hospitalization costs,so patients need to choose based on their individual circumstances.In overweight patients with a BMI of 24kg/m2-28kg/m2,RATS has a significant perioperative benefit and a better cost-effectiveness ratio,but this advantage is not evident in obese patients with a BMI≥ 28kg/m2.Part 2.Long-term survival study of perioperative adjuvant therapy regimen for NSCLC Objective:To compare the long-term survival of patients with NSCLC who received different adjuvant therapy during the perioperative period of pneumonectomy,hoping to develop the best adjuvant therapy for patients and improve their survival benefits.Methods:This study retrospectively collected clinical information of NSCLC patients undergoing pneumonectomy in Surveillance,Epidemiology,and End Results database.A total of 4,308 patients were included in this study.Propensity score matching was used to balance the patients’ data.Kaplan-Meier survival analysis and Cox hazard ratio regression model were used to identify risk factors associated with overall survival.Restricted cubic splines were used to detect possible nonlinear correlations between survival risk with age,and to perform subgroup analysis based on the optimal cutoff point.Results:For N0-N1 stage NSCLC patients undergoing pneumonectomy,chemotherapy brought significant long-term survival benefits(P<0.001),but radiotherapy did not bring significant benefits(P=0.614).In N2 patients,the chemotherapy+postoperative radiotherapy group had the worst long-term survival outcomes(P=0.014).Restricted cubic splines and subgroup analysis showed that the effect of age on long-term survival outcomes was in patients receiving chemotherapy+neoadjuvant radiotherapy(P=0.004).For patients aged≤65 years who were preparing to undergo pneumonectomy,this treatment regimen had potential clinical value,but for patients aged>65 years,this treatment regimen resulted in poorer prognosis(P=0.005).Conclusion:In summary,for NSCLC patients undergoing pneumonectomy,chemotherapy according to guideline recommendation can bring better benefits,but radiotherapy should be very cautiously performed on top of chemotherapy.Chemotherapy combined with neoadjuvant radiotherapy may have potential clinical value for patients aged ≤65 years,but the decision to receive chemotherapy combined with neoadjuvant radiotherapy for better preoperative preparation may not be suitable for elderly patients aged>65 years.From current relevant clinical research,the combination of immunotherapy and chemotherapy seems to be a promising adjuvant therapy strategy for pneumonectomy.Part 3.Platelets may be a new target for lung adenocarcinoma,and their preoperative count is associated with lymph node metastasisObjective:To research the correlation between preoperative platelet level and lymph node metastasis in patients with NSCLC,and explore the biological role of platelets in the progression of lung adenocarcinoma,hoping to provide clinical evidence for the effectiveness of antiplatelet therapy.Methods:This retrospective study collected data of 852 lung adenocarcinoma patients who underwent lobectomy and systematic lymph node dissection,and analyzed the association between preoperative platelet count within 7 days and lymph node metastasis.Multiple logistic regression analysis was used to identify risk factors for lymph node metastasis,and the correlation between platelet count and subtypes of lung adenocarcinoma was analyzed.Results:Multiple logistic regression analysis revealed that platelet count within 7 days before surgery were independent risk factors for lymph node metastasis in lung adenocarcinoma patients(P<0.05).The lymph node metastasis rate in patients with preoperative platelet count<300 × 109/L was significantly lower than patients with platelet count≥ 300 × 109/L(P<0.001).Moreover,even elevated preoperative platelet count within the normal reference range was significantly associated with an increased lymph node metastasis rate(P<0.001).Subgroup analysis found that solid subtype patients had significantly higher platelet count levels than other subtypes(P<0.001).Meanwhile,the platelet count was significantly higher in solidpredominant subtype than in solid-minor subtype(P<0.001).Conclusions:Higher preoperative platelet within 7 days before surgery may be a potential hematological indicator for predicting lymph node metastasis in lung adenocarcinoma patients.The preoperative platelet count in the solid subtype of lung adenocarcinoma is significantly higher than in other subtypes,which suggests that solid subtype may have unique mechanisms of promoting platelet production.The increased platelets may also contribute to tumor lymph node metastasis,leading to poor prognosis.Lung adenocarcinoma patients may get benefits from antiplatelet treatments.Part 4.CPSF3 is a potential target and a prognostic biomarker in lung adenocarcinoma,targeting p53 signaling pathway to regulate cell cycle,proliferation and apoptosisObjective:To research the effects of CPSF3 expression level in lung adenocarcinoma on patients’ prognosis and tumor biological function,hoping to discover new prognostic indicators for the treatment of lung adenocarcinoma and identify new gene targets for molecular targeted therapy.Methods:We analyzed the expression of CPSF3 in 516 lung adenocarcinoma tissues and 637 normal tissues from the TCGA and GTEx datasets.Then we used immunohistochemical staining on lung adenocarcinoma tissues and matched normal tissues to verify the protein expression of CPSF3.Chi-square test was used to analyze the association between CPSF3 expression and pathological stage and prognosis information of patients.Kaplan-Meier survival analysis was performed to analyze overall survival.GO analysis and KEGG enrichment analysis were used to study the biological functions and downstream signaling pathways influenced by CPSF3.We knocked down CPSF3 in lung adenocarcinoma cell lines,then used flow cytometry,EDU,colony-forming assay and CCK8 assay to detect cell cycle,proliferation,and apoptosis.Subsequently,transcriptome sequencing,KEGG enrichment analysis,and Western Blot were performed on lung adenocarcinoma cells to analyze the molecular mechanism of CPSF3-induced apoptosis,cell cycle arrest,and inhibition of cell proliferation.Results:1.CPSF3 was significantly overexpressed in lung adenocarcinoma tissues and was significantly associated with increased pathological stage and poor prognosis.2.Knocking down CPSF3 induced apoptosis,cell cycle arrest,and inhibited cell proliferation in lung adenocarcinoma cells.The activation of the p53 signaling pathway might play a crucial role in this regulatory process.3.Knocking down CPSF3 activated the expression level of p53 protein in lung adenocarcinoma cells,then regulated the expression of downstream target proteins,including upregulation of cyclin-dependent kinase inhibitor p21 and apoptosis-inducing proteins BAX and APAF1,as well as downregulation of apoptosis inhibitory protein Bcl-2 and various cell cycle-related proteins.Conclusion:CPSF3 overexpression in lung adenocarcinoma tissues was significantly associated with increased pathological stage and poor prognosis in patients.CPSF3 may not only serve as a novel molecular biomarker for evaluating the prognosis but also become an important target for molecular targeted therapy in lung adenocarcinoma.Knocking down CPSF3 activated the p53 signaling pathway in lung adenocarcinoma cells,relieved the transcriptional repression and excessive degradation of p53.So that p53 can targeted the expression of various downstream genes and exert its tumor-suppressive effects.The small molecule inhibitor JTE-607 targeting CPSF3 may be a potential anti-tumor drug,but more researches are needed to explore its role in lung adenocarcinoma.