CENPF driven lung adenocarcinoma and lung squamous cell carcinoma with immune inltrates

Background: CENPF (centromere protein F) is a critical gene that associates with the centromere-kinetochore complex and plays an important role in the tumor development. However, the associations of CENPF expression and tumor inltrating lymphocytes in lung cancer remain unknown. Methods : CENPF expression and prognostic factor was analyzed via the Gene Expression Proling Interactive Analysis (GEPIA) site. The correlation between CENPF and cancer immune inltrates was investigated via and Tumor Immune Estimation Resource (TIMER) site. Further, correlations between CENPF expression and gene marker sets of immune inltrates were analyzed by TIMER. Results: The TCGA database of Lung adenocarcinoma(LUAD) and Lung squamous cell carcinoma(LUSC) patients showed that high CENPF expression was associated with poorer overall survival (OS HR=1.5,P=0.01) and disease-free survival (DFS HR=1.4,P=0.027) in LUAD. Specically, high CENPF expression have no correlated with worse OS(OS HR=0.78,P=0.071) and DFS(DFS HR=1,P=0.87) in LUSC. CENPF expression was positively correlated with inltrating levels of B cells, macrophage in LUAD, B cells, and CD8+ T cells, macrophages, neutrophils, and dendritic cells (DCs) in LUSC. CENPF expression showed strong correlations with diverse immune marker sets in LUAD, and LUSC. After down-regulating the expression of CENPF, the proliferative capacity of Lung adenocarcinoma and Lung squamous cell carcinoma cells was inhibited. Conclusions: This report suggest that CENPF is high expression, correlated with poor prognosis and immune inltrating levels of, including those of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and DCs in in LUAD and LUSC. In addition, CENPF expression is potentially closely related to the proliferation and metastasis of lung cancer cells. These studies suggest that CENPF can be used as a new prognostic target for determining prognosis and immune inltration in Lung adenocarcinoma and Lung squamous cell carcinoma.


Background
Lung cancer is a malignant tumor with a high incidence in the world, Chemotherapy resistance, metastasis and rapid proliferation and are important biological characteristics leading to poor prognosis [1]. Except for prostate cancer in males and breast cancer in females, lung cancer is the second most commonly diagnosed cancer in men and women [2]. Due to the biological role of immune-related mechanisms in tumor disease, more immunotherapy strategies are considered to be a promising direction for the cancer treatment [3]. Immunotherapy displayed effective antitumor effects in non-smallcell lung carcinoma (NSCLC),mainly including programmed death ligand-1 (PD-L1) inhibitors, programmed death-1 (PD-1) and cytotoxic T lymphocyte associated antigen 4 (CTLA4) [4,5]. In addition, Immunotherapy that has been approved for use, such as anti-PD-L1 showed a partial response in advanced lung cancer [6]. Therefore, further researches are needed to develop treatments for lung cancer, including the identi cation of prognostic factors for lung cancer Immunotherapy.
Centromere protein F (CENPF) located on chromosome 1q41, is a protein encoding gene acts as part of the centromere-kinetochore complex and a component of the nuclear matrix during G2 of interphase [7].
The express of CENPF accumulates during the cell cycle, reaches peak levels in the G2/M phase, and then declined upon completion of mitosis in a cell cycle-dependent manner via chromosome segregation regulating [8]. Previous studies indicate that CENPF is one gene that is strongly associated with aggressive prostate cancer, and that its expression is positively correlated with metastasis tumor cells as well as plays a critical role in metabolic of prostate cancer cells [8,9]. Moreover, CENPF also signi cantly plays a role in cancer chemotherapeutic sensitivity. The previous ndings demonstrate that the CENPF plays a critical role in cancer progression.
In the current study, we aim to analysis CENPF expression and correlation with prognosis of lung cancer patients in databases including GEPIA and TIMER. Moreover, we evaluated the correlation of CENPF with tumor-in ltrating immune cells in the Lung adenocarcinoma and Lung squamous cell carcinoma microenvironments via TIMER. And CENPF could promote the proliferation of Lung adenocarcinoma and Lung squamous cell carcinoma. The ndings of this report reveal the important role of CENPF in lung adenocarcinoma and lung squamous cell carcinoma and provide a potential relationship and potential mechanism between CENPF and tumor immune interactions.

Cell culture
The A549 and H1299 cell line were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). The two cells were cultured in DMEM (Gibco, NY, USA) supplemented with 10% FBS in a 37 °C, 5% CO 2 incubator.

Wound healing analysis
The migration ability of NSCLC cells was evaluated using the scratch assay. After sh-Control and sh-CENPF transfection, cells were seeded in six-well plastic plates at a density of 5×10 5 cells/well. After 24 hours, the cells reached 90%-100% monolayer con uence. A straight scratch was arti cially created in the cell monolayers with a sterile 200μL pipette tip. Cell debris was removed with PBS three times. The cell growth medium was FBS free and cultured for 48 hours at 37°C. Scratch wound widths were measured under the microscope, and the relative wide of wound closure was determined by comparing to sh-Control cells.

Transwell invasion assay
The effect of CENPF on NSCLC cell invasion was evaluated by Transwell invasion assay. 2×10 4 cells were transfected with sh-Control and sh-CENPF for 48 hours and then added into the Transwell upper chamber with Matrigel and DMEM(1:8) (BD, Matrigel™) polycarbonate membrane (8.0 μm; Corning Incorporated, Corning, NY, USA). A total of 700μL medium containing 20% FBS was added to the lower Transwell chamber. After incubation for 24 hours, cells on the lower surface of the polycarbonate membrane were xed with 4% paraformaldehyde and stained CCK-8 assay cells were seeded at a density of 3,000cells/well in 96-well plates. After treatment with 10μL CCK-8 in 100μL complete culture medium, absorbance was measured at 450 nm using a enzyme-labeled instrument. Each experi ment was performed in triplicate.

Statistical Analysis
Statistical signi cance was tested using two-tailed Student's t-test and chi-squared test. Survival analyses were performed using Kaplan-Meier plots. Statistical analysis was performed using the SPSS22.0, with *P<0.05, ** P<0.05.

CENPF was downregulated in LUAD and LUSC
To evaluate the CENPF expression in TCGA, we analyzed CENPF expression base on the RNA-seq data of kinds of malignancies cancers. The differential expression between the tumor and normal tissues for CENPF in all TCGA tumors is shown in Figure. Prognostic role of CENPF in LUAD and LUSC Further, In order to elucidate the Prognostic role of CENPF in LUAD and LUSC. When CENPF expression level was divided into top 50% versus lower 50%, we found that higher CENPF (top 50% vs bottom 50%) expression was signi cantly associated with worsened OS and DFS in LUAD (Figure 2A, B). We then explored whether CENPF expression was associated with survival in LUSC, we found that higher CENPF expression was signi cantly associated with improved OS and DFS (Figure 2C D).

Prognostic role of immune In ltration Level in LUAD and LUSC
To explore the prognostic role of immune in ltration level in LUAD and LUSC. The B cells, CD8 + T cells, CD4 + T cells, macrophage, neutrophils, and dendritic cells (DCs) was analyzed in the prognostic role. Accordingly, increased in ltrating levels of B cells, macrophage was signi cantly associated with improved prognosis in LUAD ( Figure 3A). We then explored whether immune in ltration level was associated with survival in LUSC cohort and found that higher B cells, and CD8+ T cells, macrophages, neutrophils, and dendritic cells (DCs) in ltrating levels was signi cantly associated with improved survival ( Figure 3B).

Association of CENPF with cancer immunity in LUAD and LUSC
We therefore hypothesized that immunity in LUAD and LUSC could be linked to CENPF expression. Cases with higher CENPF expression showed signi cantly increased in ltration of B cells, CD8+T cells in LUAD ( Figure 4A) and increased in ltration of B cells, and CD8+ T cells, macrophages, neutrophils, and dendritic cells in LUSC ( Figure 4B).We analyzed correlation between CENPF expression and tumor immune in ltration level abundance indicated by decreased tumor purity. We found that higher CENPF expression was signi cantly associated with increased purity only in LUSC ( Figure 4C). We then illustrated whether CENPF copy number between tumor immune in ltration level abundance. We found that CENPF copy number was signi cantly associated with increased tumor immune in ltration level (B cells, and CD4 + T cells, macrophages, neutrophils, and dendritic cells) in LUAD ( Figure 4D). In the LUSC, CENPF copy number was signi cantly associated with increased tumor immune in ltration level (B cells, CD8 + T and CD4 + T cells, macrophages, neutrophils, and dendritic cells) in LUAD ( Figure 4E).

CENPF knockdown suppresses the proliferation, migration and invasion of NSCLC cells
We next determined the effects of CENPF knockdown on the proliferation potential of NSCLC cells in vitro. Our CCK8 assay results showed that CENPF knockdown group inhibited the growth abilities of H1299 and A549 cells than that of control group ( Figure 6A). In addition, the results of colony formation assays showed that CENPF knockdown also inhibited the clone formation abilities of H1299 and A549 cells ( Figure 5B). In order to nd whether CENPF is related to the cancer metastasis in NSCLC cells, H1299 and A549 cells were seeded in the culture palte for cell scratch assay to detect cell migration ability. The results indicated that CENPF knockdown group inhibited the migration of H1299 and A549 cells ( Figure   6C). Further, the transwell migration and invasion assay was used to analyze the function of CENPF in NSCLC cells migration and invasion. It was also demonstrated that CENPF knockdown inhibited the migration and invasion of H1299 and A549 cell ( Figure 6D).

Discussion
CENPF is a component of the nuclear matrix during the G2 phase of interphase. CENPF localization suggests that it is important in chromosome segregation during mitosis [10]. It localizes to the intracellular bridge and the spindle in the cell cycle, respectively. Although the biological function and molecular mechanism of CENPF have not been studied in depth, it is known that CENPF is up-regulated and is associated with poor prognosis of prostate cancer and hepatocellular carcinoma [11,12]. In addition, CENPF might act as mitochondria and other cellular cargoes by attaching them during both polymerization and depolymerization of microtubule [13]. Previous study demonstrated that CENPF express highly, may be as an key regulator in lung cancer progress [14][15][16]. Here, we conclude that changes in CENPF expression levels are associated with prognosis of lung cancer from the perspective of immune in ltration. High expression level of CENPF correlates to the poorer prognosis in LUSC and LUAD. Further analysis showed that in LUAD and LUSC, tumor immune in ltration levels and different immunological markers (PDL1.PD1, CTLA4) were signi cantly associated with CENPF expression levels. Therefore, our research provides insights into the potential role of CENPF in tumor immunology and its use as a biomarker for cancer.
In present study, we measured CENPF expression and survival rate based on TCGA data from GEPIA. The level of CENPF expression in LUAD and LUSC based in TCGA data underlying unclear mechanisms to different biological properties. Furthermore, the data from TCGA showed high level of CENPF expression was correlated with poorer prognosis in LUAD, LUSC. Another intriguing nding of this study is that CENPF expression is correlated with diverse immune in ltration levels in lung cancer. Moreover, the correlation between CENPF expression and the marker genes of immune cells implicate the role of CENPF in tumor immunotherapy in LUAD and LUSC. The tumor microenvironment(TME) plays a key role in the occurrence and development of tumors, and immune in ltration is one of the most important features [17,18]. In addition, the latest research found that tumor immune cell in ltration signi cantly affects the prognosis and e cacy of immunotherapy, mainly including tumor macrophages and neutrophils in ltrating [19,20]. Therefore, it is particularly important to identify new immunotherapeutic targets in lung cancer by studying gene networks and tumor immunity [21,22].

Conclusion:
Taken together, high CENPF expression correlates with poorer prognosis and increased immune in ltration levels of LUAD and LUSC, especially in in non-small-cell lung carcinoma (NSCLC). In addition, in NSCLC cell lines A549 and H1299, CENPF knockdown potentially contributes to the inhibition of tumor proliferation. Therefore, CENPF may play an important role in tumor immune cell in ltration and may serve as a prognostic biomarker for lung cancer patients.

Notes
Lei Li and Pengchao Zheng contributed equally to this work.