研究背景

胰腺癌是生存率极低的消化系统恶性肿瘤。虽然手术作为胰腺癌患者的根治性治疗手段，但是大部分患者往往被诊断时已发生远处转移而丧失手术机会。而无论是早期还是晚期胰腺癌患者，化疗均是有效的治疗方式，但常常因患者出现化疗耐药，导致其治疗效果不佳。因此，如何克服胰腺癌化疗耐药是亟待解决的科学难题，也是广大胰腺癌研究者所聚焦的热点问题。本课题组前期利用胰腺癌人源异种移植瘤模型（Patient-derived tumor xenograft, PDX）进行吉西他滨药效学实验，筛选出对吉西他滨化疗敏感和耐药的PDX模型。通过对其进行高通量测序，构建耐药相关分子调控网络，从中我们发现布鲁顿酪氨酸激酶抑制剂（Inhibitor of Bruton’s tyrosine kinase, IBTK）在吉西他滨耐药组中明显高表达。既往研究显示，IBTK具有促癌作用且与肿瘤细胞增殖、凋亡、化疗敏感性等生物学作用息息相关。因此，我们选择IBTK作为研究靶点，拟对其在胰腺癌中介导吉西他滨化疗耐药的分子机制进行深入探索，希冀为今后胰腺癌的治疗提供新希望。

研究目的

       检测IBTK在胰腺癌组织中的表达及临床意义；明确IBTK对胰腺癌细胞增殖、凋亡、化疗敏感性等生物学行为的影响；探索吉西他滨诱导IBTK表达上调的具体机制；探索IBTK是否通过调控自噬及泛素化蛋白酶体降解途径影响胰腺癌化疗耐药的发生。

研究方法

       利用生物信息学分析，绘制吉西他滨化疗敏感和耐药PDX模型间差异基因热图，筛选出吉西他滨耐药PDX中高表达的IBTK作为研究靶点。利用qRT-PCR及Western Blot对IBTK在耐药PDX模型的组织及耐药胰腺癌细胞株中的mRNA及蛋白表达进行检测。然后，对胰腺癌组织芯片进行免疫组织化学染色检测胰腺癌组织中IBTK的表达水平，使用Kaplan–Meier（K–M）生存分析及Log-rank检验，检验高/低表达IBTK患者间生存时间是否具有统计学差异。采用单因素及多因素Cox回归分析筛选影响胰腺癌患者预后的独立风险因素，评估IBTK作为胰腺癌患者预后评估的临床应用价值。在IBTK的上游调控机制中，利用qRT-PCR检测随吉西他滨药物浓度及作用时间梯度增高后，胰腺癌中IBTK的mRNA表达情况。分析吉西他滨化疗用药前及用药后PDX模型间KEGG信号通路富集情况，使用Western Blot验证吉西他滨诱导内质网应激调控IBTK表达的具体机制。

另外，IBTK在胰腺癌中具体功能的探索中，我们通过在胰腺癌细胞株中上调或下调IBTK的表达水平，采用CCK8法、流式细胞仪检测、克隆形成实验检测IBTK对胰腺癌细胞增殖、凋亡、吉西他滨化疗敏感性的影响；然后，通过构建裸鼠皮下移植瘤模型在体内明确IBTK对胰腺癌吉西他滨化疗敏感性的影响。在IBTK的下游调控机制中，通过对敲除IBTK的胰腺癌细胞株进行转录组测序及蛋白组质谱分析，推测IBTK可能调控的具体分子机制；然后，我们使用Western Blot、GFP/mRFP-LC3B双荧光染色蛋白、电子显微镜、Lyso-Tracker探针等方法，明确IBTK在胰腺癌中对自噬的调控作用；最后，我们利用CO-IP、CHX及MG132药物作用等手段探索IBTK通过调控ARID2影响自噬发生，进而介导胰腺癌化疗耐药的分子机制。

研究结果

IBTK在吉西他滨耐药PDX模型及耐药细胞株中表达量上调，且在胰腺癌组织中，其表达量较正常胰腺组织明显升高；IBTK的表达水平的高低是胰腺癌患者预后评估的独立危险因素。在功能学实验结果中，IBTK在胰腺癌中表达量升高可以促进胰腺癌细胞增殖、提高对吉西他滨化疗的耐受程度并抑制凋亡的发生；另外，敲低IBTK的表达能够抑制胰腺癌细胞增殖、增强对吉西他滨化疗的敏感性并促进凋亡的发生。同时，在体内实验中，利用小鼠胰腺癌皮下移植瘤模型证实过表达IBTK可以提高胰腺癌对吉西他滨的化疗耐受性。接着，在IBTK的上游调控机制中，我们发现随着吉西他滨用药浓度的提高及作用时间的延长，IBTK的mRNA表达量越来越高，同时在胰腺癌细胞中吉西他滨能够触发内质网应激的发生，通过PERK/CHOP通路激活IBTK的表达；其次，在IBTK的下游调控机制中，我们发现在胰腺癌细胞中过表达IBTK后，可导致溶酶体功能增强，促使自噬体降解程度加强，自噬体数量减少进而促进自噬流的发生。在对IBTK下游机制的更为深入的研究中，我们发现IBTK能够与抑癌蛋白ARID2相互结合，通过影响ARID2的泛素化水平致使其被蛋白酶体水解，进而调控溶酶体功能，使胰腺癌细胞的的自噬水平发生改变，最终介导胰腺癌吉西他滨化疗耐药的发生。

研究结论

IBTK在胰腺癌患者中表达上调，其表达量的高低可作为评估胰腺癌患者预后的独立风险因素。在机制上，吉西他滨的刺激可以通过触发内质网应激中PERK/CHOP通路介导IBTK表达升高，进而IBTK可通过影响ARID2泛素化水平，促进抑癌蛋白ARID2被蛋白酶体水解，从而影响胰腺癌细胞的自噬状态改变，导致胰腺癌化疗耐药的发生。IBTK可能作为胰腺癌患者的预后评估标志物及潜在治疗靶点，为今后相关研究提供新思路。

Background

Pancreatic cancer is a malignant tumor in the gastrointestinal system with a very low survival rate. Although surgery is used as a radical treatment for pancreatic cancer patients, most patients often have distant metastases at the time of diagnosis and lose the opportunity for surgery. Chemotherapy is an effective treatment method for both early and advanced pancreatic cancer patients, but the patients are often resistant to chemotherapy, resulting in poor treatment effects. Therefore, how to overcome the chemoresistance of pancreatic cancer is a scientific problem that needs to be solved urgently, and it is also a hot issue focused on the majority of pancreatic cancer researchers. In the previous study, we used the patient-derived tumor xenograft (PDX) model of pancreatic cancer to conduct a gemcitabine pharmacodynamic experiment and screened out the PDX models that were sensitive and resistant to gemcitabine chemotherapy. Through the high-throughput RNA-sequencing, a gemcitabine-resistance-related molecular regulatory network was constructed, from which we found that the Inhibitor of Bruton's tyrosine kinase (IBTK) was significantly overexpressed in the gemcitabine-resistant group. Previous studies have shown that IBTK was a carcinogenic gene and is closely related to biological effects such as tumor cell proliferation, apoptosis, and chemosensitivity. Therefore, we chose IBTK as a research target to further explore the molecular mechanism of its mediating gemcitabine chemoresistance in pancreatic cancer, expecting to provide new hope for the treatment of pancreatic cancer in the future.

Objective

Detect the expression and clinical significance of IBTK in pancreatic cancer tissues; clarify the effects of IBTK on the biological behaviors of pancreatic cancer cells, such as proliferation, apoptosis, and chemosensitivity; explore the specific mechanism of gemcitabine induce over-expression of IBTK; exploration of whether autophagy and ubiquitinated proteasomal degradation pathways affect the chemoresistance in pancreatic cancer.

Methods

Using the bioinformatics analysis, the heat map of differential genes between gemcitabine chemotherapy-sensitive and chemotherapy-resistant PDX models was drawn, and IBTK was overexpression in gemcitabine-resistant PDX was screened out as the research target. The mRNA and protein expressions of IBTK in chemoresistant PDX model tissues and gemcitabine-resistant pancreatic cancer cell lines were detected by qRT-PCR and Western Blot. Then, immunohistochemical staining was performed on pancreatic cancer tissue to detect the expression level of IBTK. Kaplan–Meier (K–M) survival analysis and Log-rank test were used to test differences in the survival time between patients with high or low expression of IBTK. Univariate and multivariate Cox regression analysis was used to screen independent risk factors affecting the prognosis of pancreatic cancer patients, and to evaluate the clinical application value of IBTK predicting prognosis in pancreatic cancer patients. In the upstream regulatory mechanism of IBTK, qRT-PCR was used to detect the mRNA expression of IBTK in pancreatic cancer with the increase of gemcitabine drug concentration and medication time gradient. The KEGG enriched signaling pathway between the previous and later gemcitabine treatment was analyzed, and Western Blot was used to verify the specific mechanism of gemcitabine-induced endoplasmic reticulum stress regulating IBTK expression. In addition, in the exploration of the specific function of IBTK in pancreatic cancer, we up-regulated or down-regulated the expression level of IBTK in pancreatic cancer cell lines, and used the CCK8 method, flow cytometry detection, and clone formation assay to detect the effect of IBTK on pancreatic cancer cell proliferation, apoptosis and the chemosensitivity of gemcitabine; then, the effect of IBTK on gemcitabine chemoresistance was clarified in vivo by subcutaneous xenografts of nude mice. In the downstream regulation mechanism of IBTK, the specific molecular mechanism of IBTK regulation was speculated by transcriptome sequencing and proteome mass spectrometry analysis of IBTK knockout pancreatic cancer cell lines. Then, we used Western Blot, GFP/mRFP-LC3B double fluorescence staining protein, electron microscopy, Lyso-Tracker probe, and other methods to clarify the regulation of IBTK on autophagy in pancreatic cancer. Finally, we used CO-IP, CHX, and MG132 drugs to explore the effect of IBTK on autophagy by regulating ubiquitylation degradation of ARID2.

Results

The expression level of IBTK is up-regulated in gemcitabine-resistant PDX model and gemcitabine-resistant cell lines, and its expression level is significantly higher in pancreatic cancer tissue than in normal pancreatic tissue; the level of IBTK expression is an independent risk for prognosis evaluation of pancreatic cancer patients. In the results of functional experiments, the increased expression of IBTK in pancreatic cancer can promote the proliferation, improve the resistance to gemcitabine chemotherapy and inhibit the apoptosis in pancreatic cancer cells; in addition, inhibiting the expression of IBTK can inhibit proliferation, enhance sensitivity to gemcitabine chemotherapy and promote the apoptosis in pancreatic cancer cells. In vivo, we used the subcutaneous xenograft model to confirm that overexpression of IBTK can induce the gemcitabine chemoresistance of pancreatic cancer. Next, in the upstream regulation mechanism of IBTK, we found that with the increase of gemcitabine drug concentration and the medication time, the mRNA expression of IBTK became higher and higher, and gemcitabine could trigger endoplasmic reticulum stress in pancreatic cancer cells. In addition, in the downstream regulatory mechanism of IBTK, we found that overexpression of IBTK in pancreatic cancer cells can enhance lysosomal function and enhance autophagosome degradation leading to the decreased number of autophagosomes to promote the autophagic flux. In a more in-depth study of the downstream mechanism of IBTK, we found that IBTK can bind to the tumor suppressor protein ARID2, and affected the ubiquitination level of ARID2 which is hydrolyzed by the proteasome. Finally, IBTK could regulate the function of lysosomes and change the autophagy inducing the gemcitabine chemoresistance in pancreatic cancer.

Conclusions

The expression of IBTK is up-regulated in pancreatic cancer patients, and its expression level can be used as an independent risk factor for evaluating the prognosis of pancreatic cancer patients. Mechanistically, the stimulation of gemcitabine can mediate the increase of IBTK expression by the PERK/CHOP pathway in endoplasmic reticulum stress, and then IBTK can promote the proteasome hydrolysis of the tumor suppressor protein ARID2 by affecting its ubiquitylation level, thereby affecting the autophagy status leads to chemoresistance in pancreatic cancer. IBTK may serve as a prognostic marker and potential therapeutic target for pancreatic cancer patients, providing new thought for related research in the future.