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Protein Kinase D1 Is Increased in Tumor Tissue, Correlates With Advanced Tumor Features and Worse Prognosis of Non-Small Cell Lung Cancer

Objective: This study aimed to assess protein kinase D1 expression and its association with tumor characteristics as well as prognosis in patients with non-small cell lung cancer. Methods: Protein kinase D1 expression in tumor tissues and adjacent tissues from 172 patients with non-small cell lung cancer who underwent surgical resection were analyzed by immunohistochemical staining. Based on the total immunohistochemical score, protein kinase D1 expression was classified as protein kinase D1 high expression (further divided into protein kinase D1 high+++, protein kinase D1 high++, and protein kinase D1 high+ expressions) and protein kinase D1 low expression. Clinical characteristics of patients with non-small cell lung cancer were acquired from the database. Accumulating disease-free survival and overall survival were calculated based on patients’ relapse/survival status. Results: Protein kinase D1 expression was increased in tumor tissues compared to adjacent tissues (P < .001). Tumor protein kinase D1 high expression correlated with poorer pathological differentiation (P = .041), increased tumor size (P = .003), the presence of lymph node metastasis (P = .001), and elevated tumor, nodes and metastases stage (P < .001). Besides, both accumulating disease-free survival and overall survival were decreased in patients with tumor protein kinase D1 high expression compared to patients with tumor protein kinase D1 low expression (P = .010 for disease-free survival and P = 0.005 for overall survival). Moreover, they were lowest in patients with tumor protein kinase D1 high+++ expression, followed by patients with tumor protein kinase D1 high++ expression, then patients with tumor protein kinase D1 high+ expression, and highest in patients with tumor protein kinase D1 low expression (P < .001 for disease-free survival and P = .001 for overall survival). Notably, higher tumor protein kinase D1 expression was an independent predictive factor for decreased disease-free survival (P = .001) and overall survival (P = .004). Conclusions: Protein kinase D1 might be a potential marker to identify patients with non-small cell lung cancer with worse tumor features and prognosis.


Introduction
Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, is listed as both one of the most prevalent cancers and one of the primary causes of cancer-related deaths worldwide. 1 Meanwhile, it is estimated that the prevalence of NSCLC will continue to rise, partially due to the rising tobacco use in the past decades and elevated air pollution. 2 Thus, NSCLC is presently, and still will be one of the leading concerns of public health in the future. Despite the advancement in therapies for NSCLC during the past decades, the prognosis of patients with NSCLC remains unsatisfactory with a 5-year survival rate of 15% to 20% in total patients and about 4% in advanced patients. 3 Therefore, searching for novel prognostic biomarkers might be beneficial for patients with NSCLC by improving the management and overall prognosis.
Protein kinase D1 (PKD1) is a serine/threonine kinase that belongs to the PKD family. 4 As a kinase, PKD1 is able to phosphorylate its downstream targets and thus regulates several signaling pathways (such as the phosphatidylinositol-3-kinase/ protein kinase B, the mitogen-activated protein kinase/ extracellular-regulated protein kinases 1/2, and the nuclear factor-kB). 5 By adjusting these pathways, PKD1 is able to critically regulate cancer cell proliferation, migration, invasion, and angiogenesis in several solid tumors including breast cancer, pancreatic cancer, and NSCLC. [6][7][8] Moreover, the previous study suggests that elevated tumor PKD1 expression independently predicts worse prognosis in patients with cancer. 9 Considering the regulation of PKD1 in the malignant progression of NSCLC, and its role as a biomarker in patients with cancer, we hypothesized that PKD1 might act as a potential biomarker in NSCLC. However, the clinical application of PKD1 in NSCLC largely remains to be discovered. In the present study, we aimed to investigate PKD1 expression, its association with clinical characteristics and prognosis in patients with NSCLC, thus further identifying a potential prognostic marker for NSCLC.

Patients
A total of 172 patients with NSCLC who received surgical resection in our hospital from January 2015 to December 2019 were retrospectively analyzed in this study. The screening criteria included: (1) confirmed primary NSCLC, (2) age within 18 to 80 years old, (3) TNM stage I-IIIA and treated by surgical resection, (4) complete data of tumor features and follow-up, and (5) paraffin-embedded tumor and adjacent specimens were available. Patients were excluded from this study if they were secondary or relapsed NSCLC, complicated with or history of other cancers, underwent neoadjuvant therapy, or presented with severe abnormalities in hemogram, liver, or kidney before surgery. This study was approved by the institutional review board of our hospital, and written informed consent was collected from patients or their family members.

Data Collection
By reviewing the database of our hospital, following characteristics of patients were collected: demographics (age and gender), history of smoke and drink, chronic comorbidities (hypertension, hyperlipidemia, and diabetes), tumor features (differentiation, tumor size, lymph node [LYN] metastasis, TNM stage, and carcinoembryonic antigen [CEA] level).

Immunohistochemical Staining
Formalin-fixed paraffin-embedded (FFPE) tumor and adjacent specimens were collected from the sample storeroom of our hospital. The PKD1 expression in the tumor and adjacent tissue was detected by immunohistochemical (IHC) staining. In brief, the FFPE specimens were cut into 4-mm slices and then were baked overnight in an air oven. After deparaffinized by xylene and hydrated by graded ethanol, the slices were treated with fresh hydrogen peroxide to inhibit endogenous tissue peroxidase activity. Then heat-induced epitope retrieval was performed in antigen retrieval citrate buffer by bringing up to boil. After that, the slices were blocked using normal goat serum. Subsequently, the slices were incubated with PKD1 polyclonal antibody in 1:200 dilution (Invitrogen) overnight at 4 C, followed by incubation with horseradish peroxidaseconjugated goat-anti-rabbit immunoglobulin G antibody (Abcam) at 37 C for 60 minutes. Thereafter, diaminobenzidine (Invitrogen) was used for chromogenic reaction, and the hematoxylin (Thermo Scientific) was used for counterstaining. Finally, the slices were viewed and photographed on a microscope (Leica).

Immunohistochemical Staining Analysis
Immunohistochemical staining results were assessed with the use of a semi-quantitative scoring method according to a previous study, 10 which was scored on the basis of staining intensity and positively stained cell density. Briefly, the staining density was scored as 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. And the positively stained cell density was scored as: 0, 0%; 1, <25%; 2, 26% to 50%; 3, 51% to 75%; and 4, >75%. Multiplying the density score by the intensity score, a total IHC score was obtained, which was ranging from 0 to 12. Based on the total IHC score, PKD1 low and high expressions were defined as follows: IHC score 3, PKD1 low expression and IHC score >3, PKD1 high expression. Additionally, PKD1 high expression was further classified as: IHC score 4 to 6, high þ expression; IHC score 7 to 9, high þþ expression; and IHC score 10 to 12, high þþþ expression. 10

Follow-Up and Survival Assessment
Patients were followed up to December 31, 2019, by clinic visit or telephone contact, and history and physical (H&P) examination as well as a chest-enhanced computed tomography (CT) scan were performed every 6 to 12 months for 2 years, then H&P and a non-contrast-enhanced chest CT scan were conducted annually. Patients' survival status was documented in the follow-up records. Disease-free survival (DFS) and overall survival (OS) were evaluated. The DFS was defined as the time interval from the surgery to the disease relapse, disease progression, or patients' death; OS was defined as the time interval from the surgery to the patients' death.

Statistical Analysis
SPSS 24.0 software (IBM) was used for statistical analysis, and GraphPad Prism 7.01 software (GraphPad Inc) was used for figure construction. Data were described as mean and SD, median and interquartile range, or number (percentage) as appropriate. The comparison of PKD1 expression between tumor tissue and adjacent tissue was determined by McNemar test. Comparison of clinical features between PKD1 high expression patients and PKD1 low expression patients was determined by w 2 test or Wilcoxon rank-sum test. Survival curves were plotted using the Kaplan-Meier method, and the survival comparison was determined by the log-rank test. Factors predicting DFS or OS were assessed by univariate and forward stepwise multivariate Cox's proportional hazards regression model analysis. P value < .05 was considered as statistically significant.

PKD1 Expression
The example images of PKD1 IHC staining in tumor and adjacent tissues of patients with NSCLC were shown in Figure 1A. In tumor tissues, PKD1 high expression was detected in 105 (61.0%) cases and PKD1 low expression was detected in 67 (39.0%) cases. Meanwhile in adjacent tissues, PKD1 high expression was detected in 54 (31.4%) cases and PKD1 low expression was detected in 118 (68.6%) cases. The following comparison analysis revealed that PKD1 expression was increased in tumor tissues compared to adjacent tissues of patients with NSCLC (P < .001; Figure 1B). Further classification showed that in tumor tissues, PKD1 high þþþ expression was detected in 17 (9.9%) cases, PKD1 high þþ expression was detected in 38 (22.1%) cases, PKD1 high þ expression was detected in 50 (29.0%) cases, and PKD1 low expression was detected in 67 (39.0%) cases. Meanwhile in adjacent tissues, PKD1 high þþþ expression was detected in 1 (0.6%) case, PKD1 high þþ expression was detected in 12 (7.0%) cases, PKD1 high þ expression was detected in 41 (23.8%) cases, and PKD1 low expression was detected in 118 (68.6%) cases. Subsequent comparison analysis also displayed that PKD1 expression was elevated in tumor tissues compared to adjacent tissues of patients with NSCLC (P < .001; Figure 1C).

Association of Tumor PKD1 With Accumulating DFS
Accumulating DFS was decreased in patients with NSCLC with tumor PKD1 high expression compared to patients with tumor PKD1 low expression (P ¼ .010; Figure 3A). Moreover, accumulating DFS was lowest in patients with tumor PKD1 high þþþ expression, then in patients with tumor PKD1 high þþ expression, followed by patients with tumor PKD1 high þ expression, and highest in patients with tumor PKD1 low expression (P < .001; Figure 3B).

Association of Tumor PKD1 With Accumulating OS
Accumulating OS was reduced in patients with NSCLC with tumor PKD1 high expression compared to patients with tumor PKD1 low expression (P ¼ .005; Figure 4A). Moreover, accumulating OS was shortest in patients with tumor PKD1 high þþþ expression, then in patients with tumor PKD1 high þþ expression, followed by patients with tumor PKD1 high þ expression, and longest in patients with tumor PKD1 low expression (P ¼ .001; Figure 4B).   (Table 4).

Discussion
Protein kinase D1, a serine/threonine kinase that belongs to the PKD family, is involved in the progression of cancers by regulating angiogenesis, cell proliferation, apoptosis, and so on. 11 In breast cancer, PKD1 promotes the proliferation and anchorage-independent growth of MCF-7 cells through activating the extracellular-regulated protein kinases (ERK)1/2 pathway, thus supporting the tumorigenesis of breast cancer. 6 In pancreatic cancer, the overexpression of PKD1 attenuates the apoptosis induced by the cluster of differentiation 95 in Colo357 cells. 7 In pancreatic ductal adenocarcinoma, the overexpression of PKD1 is induced by vascular endothelial growth factor secretion in PDAC cells, which further promotes human umbilical vein endothelial cell tube formation when cocultured. 12 In NSCLC, previous study suggests that PKD1 regulates the malignant potential of NSCLC cell line A549. 8 Therefore, PKD1 is a critical regulator of several solid tumors including NSCLC.  Although the abovementioned studies have displayed the effect of PKD1 on the progression of several solid tumors, the clinical role of PKD1 in patients with cancer is quite ambiguous. For its expression in tumor tissues, one previous study suggests that PKD1 is downregulated in the tumor tissues of patients with head and neck squamous cell carcinoma compared to the paired normal counterparts. 13 Meanwhile, another study reveals that the PKD1 messenger RNA level is increased in tumor tissues of patients with esophageal squamous cell cancer (ESCC) compared with paired adjacent tissues. 14 Therefore, PKD1 is dysregulated in several solid tumor tissues, and its expression might be organ-specific. However, the potential clinical application of PKD1 in NSCLC is still unclear. In this study, we observed that PKD1 was upregulated in tumor tissues compared to adjacent tissues in patients with NSCLC. Possible explanations for our data might be that the high expression of PKD1 might activate the ERK1/2 pathway or interact with its effector proteins that contributed to tumorigenesis (such as metadherin 15 to promote the malignant proliferation and anchorage-independent growth of lung fibroblasts (as in breast cancer), which enhances the tumorigenesis of NSCLC. 6 As to the association of PKD1 with tumor characteristics, it is unclear in almost all sorts of cancer (including NSCLC), only one previous study shows that tumor PKD1 is positively correlated with pathological T category in patients with ESCC. 14 Therefore, we performed this study and found that tumor PKD1 was correlated with worse pathological differentiation, increased tumor size, the presence of LYN metastasis, and higher TNM stage in patients with NSCLC. Our data could be explained by that (1) high tumor PKD1 expression might enhance malignancy of NSCLC cells, 8 and worse pathological differentiation is also characterized by increased malignancy. Therefore, high tumor PKD1 was correlated worse pathological differentiation in patients with NSCLC; (2) upregulation of tumor PKD1 might enhance the activity ERK1/2 pathway (as in breast cancer cells 6 ) to increase the proliferation of NSCLC cells, which accelerated tumor growth in patients with NSCLC. Therefore, it was correlated with increased tumor size in patients with NSCLC; (3) high tumor PKD1 might induce angiogenesis (as in pancreatic ductal adenocarcinoma 12 ) and facilitate the formation of the pro-tumor microenvironment by enhancing fibroblast proliferation, 16 which are 2 key factors for the metastasis of NSCLC. 9 Therefore, tumor PKD1 was  Regarding the prognostic value of PKD1 in patients with tumor, one previous study suggests that high tumor PKD1 mRNA expression is correlated with decreased metastasisfree survival in patients with breast cancer and low OS in patients with triple-negative breast cancer. 9 Another study proposes that patients with ESCC with reduced tumor PKD1 mRNA expression have longer cancer-specific survival, and lower tumor PKD1 mRNA expression is an independent prognostic factor. 14 However, no previous study has been conducted to investigate the prognostic value of PKD1 in patients with NSCLC. In the present study, we found that both accumulating DFS and OS were decreased in patients with tumor PKD1 high expression and the higher tumor PKD1 expression they present, the worse accumulating DFS and OS they had. It was worth noting that higher tumor PKD1 was an independent predictive factor for both reduced DFS and OS in patients with NSCLC. Possible explanations might be that: (1) Tumor PKD1 was correlated with deteriorated tumor characteristics (mentioned above), which might lead to a worse prognosis in patients with NSCLC. (2) It is proposed that the upregulation of PKD1 could induce chemoresistance in pancreatic cancer cells and prostate cancer cells. 17,18 Hence, we thought high tumor PKD1 expression might also induce chemoresistance, which may cause the worse prognosis of patients with NSCLC. Therefore, it was correlated with an inferior prognosis in patients with NSCLC.
There were several limitations in this study. Firstly, the sample size of this study was relatively small, which might cause low statistic power, especially in the comparison of accumulating DFS and OS among patients with NSCLC with tumor PKD1 high þþþ , high þþ , high þ , and low expressions. Secondly, in order to minimize bias, advanced patients with NSCLC were excluded from this study. However, most of the patients with NSCLC present advanced disease when diagnosed according to previous study, 19 and excluding advanced patients with NSCLC might limit the possible application of this study. Thirdly, the embedded molecular mechanisms of PKD1 in NSCLC progression were not investigated in this  Abbreviations: CEA, carcinoembryonic antigen HR, hazard ratio; LYN, lymph node; OS, overall survival; PKD1, protein kinase D1. a Factors affecting OS were analyzed by univariate and forward stepwise multivariate Cox proportional hazard regression model. b PKD1 expression was categorized as low ¼ 0, high þ ¼ 1, high þþ ¼ 2, high þþþ ¼ 3. c Abnormal CEA level: >5.0 ng/mL; normal CEA level: 5.0 ng/mL. study, which could be studied further. Finally, the follow-up duration in this study was relatively short, and the association of tumor PKD1 with long-term prognosis in patients with NSCLC was unclear, which could be investigated further.
Collectively, PKD1 is increased in tumor tissue compared to adjacent tissue, and its high tumor expression is correlated with worse tumor characteristics and prognosis in patients with NSCLC. Tumor PKD1 might be a potential indicator for tumor feature and prognosis to improve the management of patients with NSCLC, thus enhancing their overall prognosis.

Authors' Note
Jing Yao and Yan Jiang contributed equally to this work. This study was approved by the Institutional Review Board of The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology with approval number of whzxh-20200101, and written informed consent was collected from patients or their family members.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.