SENP1 Aberrance and Its Linkage to Clinical Features, Adjuvant Regimen, and Prognosis in Patients With Surgical Non-small Cell Lung Cancer Receiving Adjuvant Chemotherapy

Background Small ubiquitin-like modifier-specific protease 1 (SENP1) plays vital roles in cancer progression and chemoresistance, but its prognostic value in non-small cell lung cancer (NSCLC) is vague. This study aimed to explore the correlation of SENP1 with clinical features, adjuvant chemotherapy regimen, and prognosis in patients with surgical NSCLC receiving adjuvant chemotherapy. Methods Tumor and adjacent tissues were collected from 157 patients with surgical NSCLC receiving adjuvant chemotherapy. Meanwhile, tumor tissue and paired adjacent tissue specimens were obtained to evaluate SENP1 protein expression by immunohistochemistry (IHC) assay; among which, 102 pairs were used to detect SENP1 messenger RNA (mRNA) by reverse transcription quantitative PCR. Results SENP1 IHC score and SENP1 mRNA expression were increased in tumor tissue than adjacent tissue (p < 0.001). Besides, elevated SENP1 IHC score was correlated with > 5 cm tumor size (p = 0.045), lymph node metastasis occurrence (p = 0.003), and advanced tumor-node-metastasis (TNM) stage (p = 0.012); meanwhile, increased SENP1 mRNA expression was associated with histopathological subtype (p = 0.011), lymph node metastasis occurrence (p = 0.008), and higher TNM stage (p = 0.015). Besides, no correlation was found in SENP1 IHC score (p = 0.424) or mRNA expression (p = 0.927) with specific adjuvant chemotherapy regimen. Additionally, both the SENP1 protein (high) (p = 0.003) and mRNA high (p = 0.028) were correlated with poor disease-free survival (DFS), while SENP1 protein high was also associated with shorter overall survival (OS) (p = 0.029). Furthermore, SENP1 protein (high vs. low) was independently associated with unsatisfying DFS [p = 0.009, hazard ratio (HR) = 1.798] and OS (p = 0.049, HR = 1.735). Conclusion SENP1 may serve as a potential biomarker to improve the management of patients with surgical NSCLC receiving adjuvant chemotherapy.


INTRODUCTION
Lung cancer is a common human cancer with an incidence of nearly two million cases per year and a death number of 1,796,144 in 2020 worldwide (1,2). Accounting for the majority (80% to 85%) of all the cases of lung cancer, non-small cell lung cancer (NSCLC) is often caused by environmental and genetic factors with cigarette smoking being the major one (3,4). Over the past two decades, advancements in treating NSCLC have been achieved and adjuvant chemotherapy is recommended for patients with NSCLC at stage II and stage IIIA following surgery (5)(6)(7). Although adjuvant chemotherapy improves their survival outcome to some extent, their prognosis remains unsatisfactory due to the high incidence of recurrence (8)(9)(10). Therefore, to improve the survival outcome and the management of patients with NSCLC, it is necessary to find out new biomarkers.
Small ubiquitin-like modifier-specific protease 1 (SENP1) is a nuclear protease, which deconjugates small ubiquitin-like modifier (SUMO) ylated proteins (11). It is reported that SENP1 participates in the progression of various cancers. For instance, by mediating deSUMOylation of ubiquitin-conjugating enzyme E2T (UBE2T) and the subsequent protein kinase B (Akt) pathway, SENP1 promotes tumor progression in hepatocellular carcinoma (HCC) (12); meanwhile, by modulating epithelial-mesenchymal transition (EMT), SENP1 plays a vital role in invasion and migration of HCC cells (13); by regulating phosphatase and tensin homolog (PTEN) stability, SENP1 facilitates prostate cancer progression (14); SENP1 also involves in irinotecan resistance in colon cancer (15). Besides, in terms of NSCLC, it is suggested that overexpression of SENP1 in NSCLC relates to chemotherapy resistance. For example, one study suggests that SENP1 expression in tumor is negatively correlated with treatment response in patients with NSCLC (16); another study reports that SENP1 is a potential predictive factor for chemosensitivity in patients with NSCLC (17). Based on the above information, we hypothesized that SENP1 might be a potential biomarker for patients with surgical NSCLC receiving adjuvant chemotherapy. However, no previous study investigated this issue.
Therefore, this study measured the expression of SENP1 by immunohistochemistry (IHC) assay and reverse transcription quantitative PCR (RT-qPCR) detection, with the objective to explore the correlation of SENP1 expression with clinical features, chemotherapy regimen, and prognosis in patients with surgical NSCLC receiving adjuvant chemotherapy.

Patients
This retrospective study reviewed 157 patients with NSCLC who received surgical resection and adjuvant chemotherapy in our hospital between January 2016 and December 2019. The screening criteria were: (i) diagnosed as primary NSCLC according to the European Society for Medical Oncology (ESMO) clinical recommendation (18); (ii) aged over 18 years; (iii) tumor-node-metastasis (TNM) stages II-III; (iv) the Eastern Cooperative Oncology Group Performance Status (ECOG PS) scores 0-1; (v) underwent NSCLC surgical resection and adjuvant chemotherapy; and (vi) had available specimens to perform IHC assay. The exclusion criteria were: (i) had history of other cancers or malignancies at diagnosis; (ii) underwent chemotherapy or radiotherapy before surgical resection; and (iii) without complete clinical characteristics and survival data for analysis. This study was approved by the Institutional Review Board.

Collection of Clinical Data
The following clinical characteristics of all the patients were recorded in this study: age, gender, smoking, drinking, hypertension, hyperlipidemia, diabetes, histopathological subtype, differentiation, tumor size, lymph node metastasis, TNM stage, the ECOG PS score, carcinoembryonic antigen (CEA), and cancer antigen 125 (CA125). Besides, the adjuvant chemotherapy regimen was also recorded, which included vinorelbine + cisplatin (NP), taxol + cisplatin or carboplatin (TP), gemcitabine + cisplatin or carboplatin (GP), and docetaxel + cisplatin or carboplatin (DP). The follow-up was performed by clinic visit or telephone and the final date of follow-up was June 30, 2021. The median follow-up duration was 4.2 years with a 95% CI of 3.8 to 4.6 years, which was estimated using the reverse Kaplan-Meier (KM) method (19). Survival data were collected to assess disease-free survival (DFS) and overall survival (OS).

Reverse Transcription Quantitative PCR Assay
Among 157 patients with NSCLC, a total of 102 tumor tissue and paired adjacent tissue specimens, which were frozen at −80 • C, were accessible for RT-qPCR assay to detect the expression of SENP1 messenger RNA (mRNA). The sample was treated by TRIzol TM Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) to extract total RNA, which was then submitted to perform reverse transcription using the iScript TM cDNA Synthesis Kit (with random primer) (Bio-Rad, Hercules, California, USA). After that, qPCR was carried out with the QuantiNova SYBR Green PCR Kit (Qiagen, Duesseldorf, Nordrhein-Westfalen, Germany). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as reference gene. The quantitative analysis of SENP1 mRNA expression was conducted with the use of 2 − Ct method. Primers were designed referring to a previous study (21). According to the median value (2.593) of SENP1 mRNA in NSCLC tumor, the expression of SENP1 mRNA was classified as high (>2.593) and low (≤2.593).

Statistical Analysis
The SPSS software version 24.0 (IBM Corporation, Armonk, New York, USA) and the GraphPad Prism version 7.01 (GraphPad Software Incorporation, San Diego, California, USA) were used for data analysis and graph plotting, respectively. SENP1 expression between tumor tissue specimens and paired adjacent tissue specimens was compared using the paired samples t-test and the Wilcoxon signed-rank test. Variance equality was assessed by F-test. Comparison of SENP1 expression between/among patients with different clinical characteristics was analyzed using the Student's t-test, the one-way ANOVA test, the Mann-Whitney U test, or the Kruskal-Wallis H ranksum test. The KM curve was plotted to display survival profile and log-rank test was used to determine accumulating DFS and OS differences between patients. The Cox proportional hazards regression analysis was used for prognostic analysis. Statistical significance was defined as p < 0.05.

Clinical Characteristics
A total of 157 patients with NSCLC who received surgical resection and adjuvant chemotherapy were included for analyses; the mean age of these patients was 61.

Small Ubiquitin-Like Modifier-Specific Protease 1 Expression in the Tumor Tissue and Adjacent Tissue
The expression of SENP1 protein was assessed in the tumor tissue and paired adjacent tissue by IHC assay (Figure 1A). Meanwhile, SENP1 IHC score was higher in the tumor tissue than in the adjacent tissue (n = 157) (p < 0.001) ( Figure 1B); SENP1 mRNA expression in the tumor tissue was also elevated than that in the adjacent tissue (n = 102) (p < 0.001) ( Figure 1C).

Correlation Between SENP1 Expression and Clinical Features
Elevated SENP1 IHC score was correlated with > 5 cm tumor size (p = 0.045), the occurrence of lymph node metastasis (p = 0.003), and more advanced TNM stage (p = 0.012). However, no correlation was found in the SENP1 IHC score with other clinical features (p > 0.05). In addition, increased SENP1 mRNA expression was associated with histopathological subtype (p = 0.011), the occurrence of lymph node metastasis (p = 0.008), and higher TNM stage (p = 0.015). However, no association of SENP1 mRNA expression with other clinical features was observed (p > 0.05) ( Table 2).

Association of SENP1 Expression With Adjuvant Chemotherapy Regimen
Adjuvant chemotherapy regimen was recorded in this study and analysis of the correlation of SENP1 expression with adjuvant chemotherapy regimen was conducted, which discovered that   both the SENP1 IHC score (p = 0.424) and SENP1 mRNA expression (p = 0.927) showed no correlation with adjuvant chemotherapy regimen (Figures 2A,B).

Association of SENP1 Expression With Accumulating OS
Small ubiquitin-like modifier-specific protease 1 protein high was associated with shorter accumulating OS (p = 0.029).

DISCUSSION
With respect to the SENP1 expression in cancer tissues and adjacent tissues, it is suggested that SENP1 upregulates in pancreatic ductal adenocarcinoma tissues than in adjacent tissues (22). Additionally, the expression of SENP1 is higher in tumor tissues than paracarcinoma tissues in patients with HCC (12). This study found that SENP1 expression was increased in NSCLC tumor tissues than adjacent tissues in patients with surgical NSCLC receiving adjuvant chemotherapy. A possible reason could be that: SENP1 reflected the higher proliferation rate of cells; meanwhile, the proliferation rate in NSCLC cells in the tumor tissue was increased than that in the adjacent tissue cells. Thus, SENP1 expression was higher in NSCLC tumor tissues compared with adjacent tissues in patients with surgical NSCLC receiving adjuvant chemotherapy.
In terms of the correlation of SENP1 with clinical features, a previous study shows that SENP1 expression positively correlates with lymph node metastasis and TNM stage in patients with pancreatic cancer (22). Another study suggests that plasma exosome-derived SENP1 associates with higher tumor diameter and tumor stage in patients with osteosarcoma (23). Besides, SENP1 overexpression is correlated with moderate and low differentiation of NSCLC tumors (11). In this study, we observed that in patients with surgical NSCLC receiving adjuvant chemotherapy, SENP1 expression was correlated with larger tumor size, histopathological subtype, the occurrence of lymph node metastasis, and higher TNM stage. Possible explanations could be that: (1) through the hypoxia-induced factor-1α (HIF-1α) signaling pathway, SENP1 could promote the proliferation of NSCLC cancer cells, resulting in larger tumor size; (2) SENP1 could regulate matrix metalloproteinase-9 (MMP-9) to  promote NSCLC cancer metastasis; meanwhile, SENP1 might enhance NSCLC cell invasive ability via modulating epithelialmesenchymal transition marked genes, which contributed to the occurrence of lymph node metastasis (22,24). Thus, SENP1 expression was associated with larger tumor size and occurrence of lymph node metastasis (17); and (3) SENP1 expression was correlated with larger tumor size and lymph node metastasis, which were features of more advanced TNM stage. Therefore, SENP1 expression was associated with higher TNM stage (25). Moreover, no correlation was found in SENP1 expression with adjuvant chemotherapy regimen in this study, which could be explained by that: adjuvant chemotherapy regimen was applied after surgical resection and the sample for analyzing was collected before but not during or after adjuvant chemotherapy, thus no correlation in SENP1 expression with adjuvant chemotherapy regimen was found.
Concerning the association of SENP1 expression with prognosis in patients with cancer, higher plasma exosome-derived SENP1 correlates with worse DFS and OS in patients with osteosarcoma (23); additionally, SENP1 overexpression independently correlates with poor prognosis in patients with NSCLC (11). This study discovered that both the SENP1 protein high and SENP1 mRNA high were correlated with poor accumulating DFS, while SENP1 protein high was also associated with shorter accumulating OS in patients with surgical NSCLC receiving adjuvant chemotherapy. Meanwhile, SENP1 protein (high vs. low) was an independent risk factor for unsatisfying accumulating DFS and OS. The explanation could be that: (1) as mentioned earlier, SENP1 was related to chemotherapy resistance in patients with surgical NSCLC receiving adjuvant chemotherapy (11), further causing unsatisfying DFS and OS and (2) SENP1 expression was correlated with the occurrence of lymph node metastasis and higher TNM stage (as mentioned above), which could indirectly cause poor prognosis.