The Effects of WISP1 Polymorphisms on the Prognosis of Lung Cancer Patients with Platinum-Based Chemotherapy

Introduction

Lung cancer is the most common incidence and the leading cause of cancer related deaths worldwide.¹ Two major types of lung cancer are classified as small cell lung cancer (SCLC) and non–small cell lung cancer and (NSCLC).² NSCLC patients, approximately 85% of the total lung cancer cases, are often diagnosed in advanced stage and metastatic.^3,4 At present, the main therapies of lung cancer are surgery, radiation therapy, immunotherapy and chemotherapy.⁵ Immunomodulatory therapies have been approved as second-line agents for patients with advanced lung cancer as well as first-line therapy for patients with high level (>50%) of PD-L1 expression and absence of sensitizing EGFR mutations or ALK rearrangements.⁶ However, lung cancer patients with advanced NSCLC who do not fit an approved molecular targeted therapy, the standard first-line treatment remains platinum-based doublet therapy with or without bevacizumab.⁵ The five-year overall survival rate for lung cancer is still approximately 18%, the prognosis for the advanced-stage disease is even poorer, with a median overall survival of approximately one year.^7,8

Wnt1 inducible signaling pathway protein 1 (WISP1), as a member of the CCN family of proteins, is known as CCN4.⁹ The human WISP1 gene is located on chromosome 8q24.1–q24.3.¹⁰ The CCN family of proteins possess six secreted extracellular matrix associated proteins. Each family member contains four structural domains: an insulin-like growth factor binding protein-like module (IGFBP), a von Willebrand factor type C repeat (VWC), a thrombospondin domain (TSP1), and a C-terminal cysteine-knot-containing (CT) domain.¹¹ Wnt1 signaling pathways can modulate multiple processes that involve neuronal development, angiogenesis, immune cell modulation, tumorigenesis, and stem cell proliferation.^12–15 Activation of WISP1 transcript and protein were detected in carcinomas in vivo, such as hepatocellular carcinoma, colon adenocarcinomas, lung carcinoma, and breast cancer.^10,16–18 However, the role of WISP1 in clinical lung cancer with platinum-based chemotherapy still needs to be clarified.

Single Nucleotide Polymorphisms (SNPs) represent the most common type of variation in the human genome, a single base in the DNA differs from the usual base at that position. SNPs are the marker of choice in genetic analysis and also useful in locating genes association with diseases.¹⁹ While the reason of SNPs effects on gene expression, protein binding is not completely understood.²⁰ Previous study reported that WISP1 has been detected in scirrhous gastric carcinoma and invasive cholangiocarcinoma.²¹ Overexpression of WISP1 variants increases the growth rate of mouse embryonic fibroblasts rat kidney fibroblasts, hBMSCs and human esophageal cancer cells.^21,22 In lung cancer cells overexpression WISP1 leads to an inhibition in vitro cell invasion and motility, as well as lung metastasis.¹⁷ The roles of WISP1 were marked varieties in difference type of cancers. For example, high expression levels of WISP1 were correlated with poor prognosis in breast,¹⁸ rectal,²³ esophageal cancer²⁴ and colon cancer,²⁵ but WISP1 expressed lower levels in melanoma with poor prognosis.²⁶ Despite the role of WISP1 affects lung cancer progression is not clear.

In the present study, we evaluated whether genotypes of WISP1 in lung cancer patients could be involved in prognosis of lung cancer patients with platinum-based chemotherapy.

Methods and Materials

Study Populations

The study subjects consist of 363 patients that are diagnosed lung cancer in the Affiliated Cancer Hospital or Xiangya Hospital of Central South University (Changsha, Hunan, China) during 2011 to 2013. Patients clinical and laboratory findings were collected from medical records. The recruitment criteria included the following: Eligible patients were histologically or cytologically proven lung cancer who had experienced disease progression. Patients with a history of previous malignant tumor or concomitant malignancies, metastases were excluded from this cohort. All patients were treated with platinum-based chemotherapy at least 2 cycles and were not given radiotherapy and/or biological therapy before and during chemotherapy. All patients were signed informed consents were obtained from all study participants before chemotherapy. The clinical and laboratory data including age, sex, smoking history, TNM stage, histological type and stage, chemotherapy regimens were collected from medical records.

The study was in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration, and the study protocol was approved by the Ethics Committee of Xiangya School of Medicine, Central South University with a registration number of CTXY-110008-2.

SNP Selection, DNA Extraction and Genotyping

According to the data from the HapMap, or previous studies, we selected 39 SNPs (Table S1) of the WISP1 gene which described previously.^27,28 In brief, the following criteria: (1) A total of them with minor allele frequency (MAF) ≥0.05 in the Chinese population were selected. (2) The tag SNP selection was performed using the Tagger program implemented in Haploview version 4.2. (3) SNPs located in the promoter region, exon region, and the 3ʹ untranslated region (UTR). Genomic DNA was extracted from the whole blood by using the FlexiGene DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA sample was stored at −20 °C before usage. WISP1 polymorphism was genotyped using the Sequenom Mass ARRAY System (Sequenom, San Diego, CA, USA).

Statistical Analysis

The statistical analysis was conducted by the SPSS version 25.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism (version 8, GraphPad Software Inc., San Diego, CA). We used Cox proportional hazards regression model to analysis each genetic variant and OS/PFS, measured as HRs with their corresponding 95% CIs. The covariates used for adjusted HR for PFS included age, gender, smoking status, histology, and clinical stage between the OS and PFS. Kaplan-Meier test was used to assess each genetic variant on the cumulative probability of PFS and OS. All association analyses were evaluated by three models including additive, dominant, and recessive. All P-values presented were two-sided, and a level of P < 0.05 was considered statistically significant.

Results

Characteristics and Survival Status of the Lung Cancer Patients

In order to investigate the association of WISP1 Polymorphisms and clinical results of platinum-based chemotherapy, the present study consisted of 363 patients diagnosed with lung cancer, who had DNA samples, complete data on demographic, clinical characteristics, progression free survival (PFS) and overall survival (OS). All patients who received at least 2 cycles of platinum-based chemotherapy were enrolled to investigate the association of WISP1 polymorphisms with chemotherapy. As shown in Table 1, 289 were males and 74 were females, with the median age of cohort was 56 (a range of 21 to 77 years old). The patients who ever or current smokers accounted for 63.6%, and the ones who were never smokers accounted for 36.4%. For the patient group, 244 (67.2%) patients with presented advanced NSCLC and 119 (32.8%) patients with SCLC, and most of which were with stage III/IV (65.8%).

Table 1 Association Between Clinical Characteristics and PFS and OS of Patients with Lung Cancer

Association of WISP1 Polymorphisms with Platinum‑based Chemotherapy Prognosis in the Lung Cancer Patients

To investigate the association between WISP1 Polymorphisms and the prognosis of lung cancer patients received platinum-based chemotherapy, we selected 39 SNPs and those SNPs under investigation are shown in Table 2. After adjustments for age, sex, smoking status, histological type and TNM stage 7 SNPs (rs16904853, rs10956697, rs2929965, rs2929973, rs7828685, rs2977551, rs2977549) consistently showed a significant association with PFS and ∕ or OS in lung cancer patients using Log rank test and multivariate Cox regression analysis (Table 2). Unconditional logistic regression analysis with adjustments revealed that WISP1 rs16904853 (P for log-rank=0.010), rs10956697 (P for log-rank=0.014), rs2929965 (P for log-rank=0.036), rs7828685 (P for log-rank=0.025) were significantly associated with lung cancer PFS in the additive model. WISP1 rs10956697 (P for log-rank=0.004), rs2929973 (P for log-rank=0.015), rs7828685 (P for log-rank=0.027), rs2977551 (P for log-rank=0.032), rs2977549 (P for log-rank=0.017) were significantly related to lung cancer PFS in the dominant model. rs16904853 (P for log-rank=0.003), rs2929965 (P for log-rank=0.013), rs7828685 (P for log-rank=0.023) were significant association with PFS in the recessive model (Figure 1). For OS analysis, our results demonstrated that lung cancer patients with rs10956697 were significantly related to better OS (HR=0.728, 95% CI=0.557–0.953, P =0.021) in the dominant model. Additionally, patients carrying TT genotype in rs2977551 were significantly related to better OS (HR=0.725, 95% CI=0.552–0.913, P =0.020) (Table 2) (Figure 2).

Table 2 Association Between Single Nucleotide Polymorphisms (SNPs) and Clinical Outcomes in Lung Cancer Patients

Figure 1 Kaplan–Meier plots of lung cancer patients with different genotypes of in WISP1 gene. (A) PFS of lung cancer patients by rs16904853, rs10956697, rs7828685, rs2929965 genotypes using additive model. (B) PFS of lung cancer patients by rs10956697, rs2929973, rs7828685, rs2977551, rs2977549 genotypes using dominant model. (C) PFS of lung cancer patients by rs16904853, rs2929965, rs7828685 genotypes using recessive model.

Figure 2 Kaplan–Meier plots of lung cancer patients with different genotypes of in WISP1 gene. (A) OS of lung cancer patients by rs10956697 using dominant model. (B) OS of lung cancer patients by rs2977551 using dominant model.

Association Between SNPs and Lung Cancer Progression

Next, we evaluated the relationship between the single nucleotide polymorphisms were related to tumor progression with patients receiving platinum-based chemotherapy. As described in Table 3, the rs2929973, rs2977551 and rs2977549 in WISP1 gene still significantly contributed to T stage of lung cancer patients. Both in the univariate Cox regression analysis and after adjusted by age, gender, smoking status, histological type and stage, we found that patients carrying GT or GG genotype in rs2929973 were a lower hazard to early T stage (T1 or T2) when compared with TT genotype (OR=0.486, 95% CI=0.271–0.871, P =0.015). Patients who carried the rs2977551 CT or CC genotype had a more significantly association with early T stage than patients with TT genotype (OR=0.428, 95% CI=0.238–0.771, P =0.005). For rs2977549 compared with CT or CC genotype carries, individuals with TT genotype tended to be association with early T stage (OR=0.438, 95% CI=0.246–0.781, P =0.005).

Table 3 Association Between Single Nucleotide Polymorphisms (SNPs) and Disease Progression in Lung Cancer Patients

Subgroup Analysis of Association Between WISP1 Polymorphisms and Prognosis

To better explore the WISP1 polymorphisms with OS and PFS of patients received chemotherapy, stratified analysis was performed based on the ages, gender, smoking status, histological type and TNM stage.

As shown in Table 4, the risk genotype group carries with rs2929973 GT or GG genotypes tended to have a significantly increased risk of disease progression and PFS in subgroups of patients with ≤60 years old. For patients who had never smoked, carrying TT genotype had a significantly decreased risk of death (HR=0.617, 95% CI=0.383–0.994, P =0.047). In the presence of GT or GG genotype, patients with NSCLC were markedly increased than those with TT genotype (HR=0.693, 95% CI=0.501–0.959, P=0.027). For patients who had TNM stage III/IV, carrying GT or GG genotype had a significantly increased risk of death (HR=0.649, 95% CI=0.470–0.896, P =0.009) and PFS (HR=1.377, 95% CI=1.024–1.852, P =0.034). For rs2977551, we found that carriers of CT or CC genotype tended to have a significantly increased risk of PFS in subgroups of younger (≤60 years old) (HR=1.413, 95% CI=1.022–1.954, P =0.037), never smokers (HR=1.572, 95% CI=1.007–2.455, P =0.047). For rs2977549, the risk genotype group carriers with CT or CC genotypes tended to have a significantly increased risk of disease progression in subgroups of patients with ≤60 years old. Patients carrying TT genotype tend to a lower risk of PFS (HR=1.480, 95% CI=1.071–2.047, P =0.018). Compared with CT or CC genotype tend to be sensitive to platinum-based chemotherapy, patients with NSCLC were markedly increased risk of death than those with TT genotype (HR=1.502, 95% CI=1.087–2.075, P=0.014) and PFS (HR=1.358, 95% CI=1.005–1.835, P=0.046). For patients who had TNM stage III/IV, rs2977549 carrying TT genotype had a significantly decreased risk of death (HR=1.601, 95% CI=1.157–2.216, P =0.005) and PFS (HR=1.419, 95% CI=1.053–1.913, P =0.022).

Table 4 Stratified Analysis of the Association Between rs2929973, rs2977551, rs2977549 and Prognosis in Lung Cancer Patients

Discussion

Our present study systematically investigated the clinical significances of the WISP1 polymorphisms on the prognosis of lung cancer patients that underwent platinum-based chemotherapy. In the current study, we found that WISP1 rs2929973, rs2977551 and rs2977549 contribute to a significantly correlated with platinum-based prognosis. In conclusion, our investigation provided the useful basics to make more precise evaluation of the chemotherapy efficacy and then further design personalized therapy.

The differential expression status of WISP1 between the tumor tissue and normal healthy tissue.⁹ High levels of WISP1 correlated with survival in prostate adenocarcinoma and overall survival in primary melanoma, low-grade glioma, and kidney papillary cell carcinoma.²⁹ WISP1 could regulate complex biological process during disease by effecting proliferation, cell survival, and cell differentiation, development and disease.^10,16,30,31 It had been pointed out that WISP1 protects lung carcinoma cells from intrinsic p53-dependent, but not extrinsic Fas ligand-activated.³² Increased epithelial cell proliferation and EMT induction highlight WISP1 as a possible linker of organ fibrosis and cancer.³³ The correlation of its expression with clinical outcome makes WISP1 a promising target for the evaluation of clinical diagnosis and prognosis of cancers. WISP1 signaling is critically involved in cell survival and growth, through regulating PI3K and Akt.¹² WISP1 led to Akt phosphorylation in epithelial and mesenchymal cells.^15,34 In our study, we found that WISP1 was related to platinum-based chemotherapy response of lung cancer.

Previous association studies that polymorphism is one of the factors that may affect prognosis, mainly focused on the mis-sense variants or coding regions of individual genes. In this study, we focused on the genetic alteration of WISP1, a total of 39 SNPs to study the prognosis effect platinum-based chemotherapy. Our study found WISP1 rs2929973 carrying G variant allele are more likely to have an increased susceptibility of lung cancer. Patients with younger age (≤60), or patients who had never smoked, NSCLC, or advanced tumor (TNM stage III/IV), it had better OS when rs2929973 carrying TT genotype. Patients who carry the rs2977551 CT or CC genotype tended to have a significantly increased risk of PFS in subgroups of younger (≤60 years old), never smokers. Patients carrying TT genotype in rs2977549 better OS in lung cancer patient with NSCLC, had TNM stage III/IV. In conclusion, the WISP1 rs2929973 T allele, rs2977551 T allele and rs2977549 T allele maybe a potential protective allele in the prognosis of lung cancer patients treated with platinum-based chemotherapy.

WISP1 is a downstream gene of the canonical Wnt-β-catenin pathway, and its mutations were reported to be associated with multiple diseases, including asthma, hypertension and spinal osteoarthritis.^13,34–36 Chen et al revealed that WISP1 was overexpressed in non-small cell lung carcinoma (NSCLC) samples compared with their normal lung tissue counterparts. Our previous studies revealed that WISP1 genetic polymorphisms were related to susceptibility and the platinum-based chemotherapy response of lung cancer, and we hypothesized that WISP1 polymorphisms may also be associated with the chemotherapy toxicity of lung cancer.²⁸ Most previous studies about the relationships of WISP1 polymorphisms and diseases were focused on the polymorphisms of WISP1 rs2929973. However, results from different studies often provide contradicting results or statistically non-significant associations. Previous study indicated that WISP1 rs2929973 was associated with asthma and individuals carrying the G allele of rs2929973 conferring lower forced expiratory volume in the first second.³⁷ Patients carrying the WISP1 rs2929973 GG and TT variant were almost twice as likely as those carrying the GT genotype to have estrogen receptor (ER)- and progesterone receptor (PR)-positive tumors.³⁸ In addition, the genetic polymorphisms of WISP1 (rs2977549) variants were associated with lung and smoking-related cancer prognosis.²⁷ Previous research found that patient carrying the rs10956697 AC genotype had a significantly decreased risk of gastric cancer (OR = 0.58, 95% CI 0.35–0.98). Smokers with the rs10956697 AC and AC + AA genotypes exhibited a 0.28-fold lower and 0.32-fold lower risk of gastric cancer.³⁹ Rs16904853, rs2977549, and rs2977551 polymorphisms were significantly associated with hematologic toxicity.²⁷

However, our study has several limitations. Independent validation of correlations between association of SNPs with lung cancer prognosis are needed. No SNPs remained significant by considering multiple-testing correction that calculated the p value of the SNPs by FDR-BH (Benjamini & Hochberg (1995) step-up FDR control) correction, it perhaps the sample size for the study is not large enough. Moreover, the function represent interconnections between polymorphisms and prognosis of lung cancer patients with platinum-based chemotherapy was not determined in our study.

Conclusion

In conclusion, lung cancer patients with WISP1 rs2929973 GT or GG, rs2977551 CT or CC and rs2977549 CT or CC genotypes had increased risk of disease progression, and patients with rs2977551 CC or CT had a significantly longer OS. Our results showed that those polymorphisms were significantly associated with the chemotherapy prognosis of lung cancer patients. Thus, we thought that the genotypes of WISP1 may be contributed to a potential candidate biomarker for the prediction of prognosis in lung cancer patients.