Profiling of a novel circadian clock-related prognostic signature and its role in immune function and response to molecular targeted therapy in pancreatic cancer.

www.aging-us.com AGING 2023, Vol. 15, No. 1 Research Paper Profiling of a novel circadian clock-related prognostic signature and its role in immune function and response to molecular targeted therapy in pancreatic cancer Yu Jin1, Shuang Gong2, Guochen Shang1, Lilin Hu1, Gangping Li1 1 Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2 First School of Clinic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China Correspondence to: Gangping Li; email: Keywords: circadian clock, neutrophil, pancreatic cancer, prognosis, targeted therapy Received: October 11, 2022 Accepted: December 20, 2022 Published: January 9, 2023 Copyright: © 2023 Jin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Background: Pancreatic ductal adenocarcinoma (PADA) represents a devastating type of pancreatic cancer with high mortality. Defining a prognostic gene signature that can stratify patients with different risk will benefit cancer treatment strategies. Methods: Gene expression profiles of PADA patients were acquired from the Cancer Genome Atlas and Gene Expression Omnibus, including GSE62452 and GSE28735. Differential expression analysis was carried out using the package edgeR in R. Intro-tumor immune infiltrates were quantified by six different computational algorithms XCELL, TIMER, QUANTISEQ, MCPCOUNTER, EPIC, and CIBERSORT. Biological processes were investigated based on R package “clusterProfiler”. Results: 13 genes (ARNTL2, BHLHE40, FBXL17, FBXL8, PPP1CB, RBM4B, ADRB1, CCAR2, CDK1, CSNK1D, KLF10, PSPC1, SIAH2) were eligible for the development of a prognostic gene signature. Performance of the prognostic gene signature was assessed in the discovery set (n = 210), validation set (n = 52), and two external data set (GSE62452, n = 65, and GSE28735, n = 84). Area under the curve (AUC) for predicting 3-year overall survival was 0.727, 0.732, 0.700, and 0.658 in the training set, the validation set, and the two test sets, respectively. KM curve revealed that the low-risk group had an improved prognosis than the high-risk group in all four datasets. PCA analysis demonstrated that the low-risk group was apparently separated from the high-risk group. CD8 T cell and B cell were significantly reduced in the high-risk group than in the low-risk group, while neutrophils were significantly augmented in the high-risk group than in the low-risk group. BMS-536924, Foretinib, Linsitinib, and Sabutoclax were more sensitive in the low-risk group, whereas Erlotinib was more effective in the high-risk group. Conclusions: We successfully established and verified a novel circadian clock-related gene signature, which could stratify patients with different risk and be reflective of the therapeutic effect of molecular targeted therapy. Our findings could incorporate the pharmacological modulation of circadian clock into future therapeutic strategies. mortality annually, and a less than 5% five-year survival rate [1, 2]. Current standard treatment for PADA is a combination therapy using chemotherapy, molecular targeted therapy, immunotherapy and surgical operation. Unfortunately, despite advancement in therapeutic strategies, a median survival time for INTRODUCTION Pancreatic ductal adenocarcinoma (PADA) accounts for approximately ninety percent of pancreatic cancers, and represents a lethal cancer, with an estimated 232000 newly diagnosed cases, 22700 cancer-associated www.aging-us.com 119 AGING MATERIALS AND METHODS PADA patients is only 23 months. On the other hand, we should pay attention to this phenomenon that approximate 27% of patients with resected PDAD can survive for five years. One underlying rationale for this phenomenon is heterogeneity of tumor phenotypes of pancreatic cancer itself. Another potential explanation might be that some patients’ response to some treatment regiment, while the others fail to response to the therapy strategies. Therefore, it seems essential to distinguish between patients who would most benefit from the current therapy and those who would be not fit for the treatment [3, 4]. To this end, the present study aimed to define an indicator of distinct survival outcomes. Acquisition of data Gene expression information of PADA patients was acquired from the Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO), including GSE62452 [17] and GSE28735 [18]. TCGA contains 172 PADA patients with a follow-up time of more than one month, among including of 171 primary tumors and one metastatic tumor. These samples were divided into the discovery set (n = 120) and validation set (n = 52). GSE62452 contains 69 PADA and adjacent non-tumor samples, among which 65 PADA samples had a follow-up time of more than one month. These samples were snap-frozen and sequenced using Affymetrix Human Gene 1.0 ST Array (GPL6244). GSE28735 contains 45 PAD patients, among which 42 PADA patients had a follow-up time of more than one month. These samples were also snap-frozen and sequenced using Affymetrix Human Gene 1.0 ST Array (GPL6244). Gene expression information was got through Cancer Cell Line Encyclopedia (CCLE) database. IC50 values of pancreatic cancer cell lines was got from Genomics of Drug Sensitivity in Cancer (GDSC). In the present study, gene expression profiling from TCGA was subjected to TPM normalization, and all arrays were subjected to RMA normalized in GSE62452 and GSE28735. A total of 104 circadian clock-associated genes that were retrieved in MSigDB database [19]. Basic information for gene expression data was shown in the Table 1. The circadian clock endows humans the ability to perceive temporal changes of the external environment through a series of physiological activities [5]. The circadian rhythm is a sophisticated biological process, and supervises a series of cyclic biological functions via regulating a diversity of molecular activity and signal transduction processes [6]. In the case that circadian clock is disrupted, the hazard of conditions such as metabolic disorders, cardiovascular disease and cancer will increase [1]. Literatures have revealed a link between circadian rhythm disturbance and human cancer [7]. Although the negative effect of circadian clock disruption has gradually begun to be known [8– 10], it remains underappreciated to exploit it for making more effective chronotherapy strategies. Besides, the role of circadian clock in PADA is elusive; therefore, we sought to interrogate the specific association between circadian clock and the pathogenesis of PADA. Establishment and evaluation of the circadian clockrelated indicator In addition, PADA is an exceptionally heterogeneous carcinoma [11] (...truncated)