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The Relationship Between UBE2C and AGGF1 Overexpression and Tumor Angiogenesis in Non-Small Cell Lung Cancer

Background Tumor infiltration and metastasis are the leading causes of death for patients with tumors. Angiogenesis is a prerequisite for tumor growth and metastasis. Angiogenic factor with G patch and FHA domains 1 (AGGF1) is an angiogenic factor, whereas ubiquitin-conjugating enzyme E2C (UBE2C) functions in protein ubiquitination. Microvessel density (MVD) is the most common indicator of tumor microvessels, and vasculogenic mimicry (VM) facilitates blood supply to tumors. This study explored UBE2C and AGGF1 expression in non-small cell lung cancer (NSCLC) and their relationship with angiogenesis and prognosis to identify biological factors that might predict NSCLC infiltration, metastasis, and prognosis. Methods The specimens and clinical pathological data of patients with NSCLC confirmed by pathology after surgical resection between January 2013 and December 2015 were collected. UBE2C and AGGF1 expression, as well as microvessel formation and VM in NSCLC, was observed using immunohistochemistry. The relationships between UBE2C, AGGF1, MVD, VM, and clinical pathological parameters and their relationships with overall survival (OS) and disease-free survival (DFS) were analyzed. Results UBE2C and AGGF1 levels in NSCLC tissues were significantly higher than those in corresponding normal tissues (57.1% vs 15.6 and 59.7% vs 25.3%, respectively; P < 0.05). UBE2C, AGGF1, MVD, and VM were positively correlated with each other (P < 0.05) and were all related to tumor size, lymph node metastasis, and tumor-node-metastasis stage (P < 0.05). Kaplan–Meier analysis showed that patient OS and DFS in the UBE2C, AGGF1, VM-positive, and high-MVD groups were reduced (all P < 0.001). Univariate and multivariate analyses showed that UBE2C, AGGF1, VM, and MVD were independent risk factors for NSCLC prognosis. Conclusion UBE2C and AGGF1 overexpression is associated with angiogenesis and poor prognosis and may be important for predicting NSCLC invasion, metastasis, and prognosis.


Introduction
The latest research results show that there were approximately 10 million cancer deaths worldwide in 2020, of which approximately 1.8 million were due to lung cancer. 1 Lung cancer remains the most common cause of cancer-related deaths, and its incidence continues to increase. 1 In most countries, the 5-year survival rate of lung cancer is only 10-19%. 2 More than 80% of lung cancers are further classified as non-small cell lung cancer (NSCLC) and are already in an advanced stage when the diagnosis is confirmed. 3 Chemotherapy is no longer the most sought-after important treatment modality for patients with advanced and metastatic NSCLC because targeted therapy has less adverse reactions and can improve the prognosis. 4 Tumor infiltration and metastasis are hallmarks of malignant tumors and are the main causes of death in patients with these diseases. 5 Therefore, it is essential to identify the biological factors that might predict the infiltration, metastasis, and prognosis of NSCLC.
The study of tumor angiogenesis has always been an important topic in the field of oncology. When the oxygen provided by the vascular system in the tumor tissue cannot meet the needs for rapid tumor cell growth, new blood vessels are formed based on the original vascular system, and this process is called tumor angiogenesis. 6 Angiogenesis is closely related to tumor progression and it makes tumors more prone to metastasis and recurrence; thus, highly vascularized NSCLC tends to be associated with a poor prognosis. 5,7 Currently, microvessel density (MVD) is the most commonly used indicator to evaluate angiogenesis. 8 In vasculogenic mimicry (VM), a tube surrounded by tumor cells lined with a periodic acid-Schiff (PAS)-positive basement membrane is formed. The blood in this vessel is transferred via the tumor microvessels. 9 Because the tumor cells constituting VM are in direct contact with the blood, they can easily enter circulation and contribute to distant metastasis. 10 Ubiquitin-conjugating enzyme E2C (UBE2C) is a key component of the ubiquitin-proteasome system. It binds ubiquitin molecules to target proteins that are then degraded by the 26-S proteasome. Overexpression of UBE2C induces tumorigenesis by inducing chromosomal hysteresis and the production of aneuploid cells. 11 UBE2C level is almost undetectable in normal tissues; however, it is overexpressed in many cancers. [12][13][14][15][16] Studies have shown that UBE2C is related to multiple biological behaviors, such as tumor occurrence, proliferation, invasion, and apoptosis. 11,14,15 Previous studies have shown that UBE2C may have a regulatory effect on tumor angiogenesis; 17,18 however, the relationship between UBE2C and MVD and VM in NSCLC remains unclear.
The angiogenic factor with G patch and FHA domains 1 (AGGF1) gene was discovered by Tian et al in 2004; they reported that the gene was highly expressed in vascular endothelial cells in patients with congenital venous malformation osteotome syndrome. 19 Studies have confirmed that AGGF1 plays a vital role in maintaining vascular functions, including vascular integrity and stability, and it is necessary for tumor angiogenesis; furthermore, the AGGF1-PI3K-AKT signaling pathway also plays a key role in tumor angiogenesis. 20 It has been reported that AGGF1 level is upregulated in various types of human cancers, such as glioblastoma, colorectal cancer, liver cancer, gastric cancer, and esophageal cancer. [21][22][23][24][25] However, the expression of AGGF1 in NSCLC and its relationship with angiogenesis have not yet been reported.
As the role of UBE2C and AGGF1 in promoting angiogenesis in NSCLC has yet to be elucidated, we used immunohistochemistry to simultaneously detect the levels of UBE2C, AGGF1, MVD, and VM in NSCLC to explore the effects of UBE2C and AGGF1 on angiogenesis, their relationship, and prognosis.

Patients and Tissue Specimens
We randomly selected 154 patients who underwent radical surgery for lung cancer between January 2013 and December 2015, who did not receive any anti-cancer treatment before surgery, and were confirmed to have lung squamous cell carcinoma (SCC) or lung adenocarcinoma (Ade) by immunohistochemistry after surgery. Paraffin specimens with cancerous and corresponding normal tissues were selected from these patients. To evaluate the survival status of patients after surgery in a timely manner, the patients were followed-up by telephone every 6 months. The overall survival (OS) was recorded as ending in the patient's death by December 2020, and the disease-free survival (DFS) was recorded as ending in the patient's relapse or death by December 2020. This study was conducted with the written informed consent of all patients and approval from the Ethics Committee of Bengbu Medical College (NO. 2020KY035) in accordance with the ethical guidelines of the Declaration of Helsinki. The tumor-nodemetastasis (TNM) stage was determined according to the 8th edition of the American Joint Commission on Cancer lung cancer staging system. The clinical pathological parameters of the patients are presented in Table 1.

Immunohistochemistry
The selected paraffin specimens were successively cut into 4-μm-thick tissue sections. Slices were soaked for 2 h, dewaxed with xylene, dehydrated with gradient alcohol, and then washed with distilled water and phosphatebuffered saline (PBS) thrice, each for 3 min. To retrieve the antigens, the sections were subjected to boiling in citric https://doi.org/10.2147/CMAR.S320393

DovePress
Cancer Management and Research 2021:13 5920 acid buffer (pH 6.0), cooled to 20°C, and rinsed again with PBS, as described above. To inhibit endogenous peroxidase, 3% H 2 O 2 was added dropwise to the sections and rinsed with PBS for 9 min after 10-20 min. Rabbit polyclonal anti-AGGF1 (1:100; AB_2844914, Affinity Biosciences), rabbit monoclonal anti-UBE2C (1:1000; ab252940, Abcam), and anti-CD34 (1:200; ab762, Abcam) antibodies were added dropwise onto the different sections and incubated overnight at 4°C. Subsequently, the sections were incubated with secondary antibodies for 30 min. Finally, the 3,3′-diaminobenzidine (DAB) solution and the hematoxylin solution were sequentially added dropwise to the slices, which were washed with distilled water and then placed in a gradient of alcohol for dehydration.

Evaluation of Staining
The immunohistochemical results of the sections were evaluated by two experienced pathologists. The immunohistochemical scores for UBE2C and AGGF1 proteins were determined by the proportion of positive cells in tumor tissue (0, < 10%; 1, 11-50%; 2, 51-75%; 3, > 75%) and staining intensity (0, no staining; 1, pale yellow staining; 2, tan staining; 3, brown staining); the final score was the product of these two values. If the total score was ≥ 3, it was determined positive; otherwise, it was considered negative. MVD was determined according to the method described by Weidner et al. 26 The vascular endothelium was labeled with CD34, and the most densely populated area of microvessels was observed under 100fold magnification. The microvessels in three different areas were then counted under 400-fold magnification, and the average number was calculated as the MVD of the tumor. We demonstrated the structure of VM by double staining with PAS and CD34. When CD34 was negative and PAS was positive, VM was considered to exist in the tumor.

Statistical Methods
Statistical analyses were performed using SPSS 25.0. The relationships among UBE2C, AGGF1, MVD, VM, and clinical pathological parameters were analyzed using the Chi-square test. The correlations among UBE2C, AGGF1, MVD, and VM were determined using Spearman correlation analysis. Kaplan-Meier analysis was used to compare the factors with OS and DFS. Univariate and multivariate Cox regression models were used to identify risk factors affecting prognosis. Statistical significance was set at P < 0.05.

Univariate and Multivariate Analyses
We then analyzed the factors affecting the prognosis of NSCLC using the Cox risk regression model. Univariate analysis revealed that UBE2C expression, AGGF1 expression, MVD value, positive VM, tumor size, LNM, and TNM stage were significantly correlated with OS and DFS, and were important factors affecting the prognosis of patients with NSCLC (Tables 4 and 5). Multivariate analysis showed that UBE2C expression, AGGF1 expression, MVD value, positive VM, tumor size, LNM, and TNM stage were closely related to OS and DFS and could be used as independent prognostic indicators of NSCLC (Tables 4  and 5).

Discussion
Research on invasion and metastasis of NSCLC can facilitate more effective measures and improve its prognosis. Huang et al found that exosomes could promote the invasion and metastasis of NSCLC. 27 In this study, for the first time, in order to identify novel biological factors that might predict the infiltration and metastasis of NSCLC, the expressions of UBE2C and AGGF1 proteins were detected simultaneously and their relationships with VM, MVD, and prognosis were explored. UBE2C is essential for ubiquitination. Under the control of the UBE2C protein and after a series of enzymatic reactions, the substrate protein is ubiquitinated and degraded. The normal progress of this process is related to successful completion of the cell cycle and signal transduction. 28 Our results showed that UBE2C is overexpressed in NSCLC tissues (57.1%) and is rarely expressed in normal tissues (15.6%). We found that UBE2C overexpression was associated with a larger tumor volume, LNM, and later clinical stage, suggesting that it might be involved in the growth and metastasis of NSCLC. Through Kaplan-Meier survival analysis and univariate and multivariate analyses, we observed that the overexpression of UBE2C leads to a shorter OS and     29 Wang et al and Guo et al confirmed through cytological experiments that the overexpression of UBE2C in pancreatic cancer and NSCLC was related to the proliferation and invasion of tumor cells and was negatively correlated with OS. 13,30 Therefore, our findings are consistent with those of existing studies. AGGF1 is an important angiogenic factor that promotes angiogenesis, and has been verified in many studies. Yao et al confirmed that AGGF1 plays an important role in regulating mouse endothelial progenitor cells, and knocking out this gene was found to lead to angiogenesis disorders. 31 Zhang et al indicated that AGGF1 is crucial for both physiological and pathological angiogenesis, and defects in this gene were shown to inhibit tumor growth. 20 Studies have shown that AGGF1 is overexpressed in liver, colorectal, and gastric cancer and is negatively correlated with OS and DFS. However, the expression of AGGF1 in NSCLC and its relationship with prognosis remain unclear. In this study, we used immunohistochemistry to observe the expression of AGGF1 in NSCLC for the first time. The positive rates of AGGF1 in NSCLC tissues and normal tissues were 59.7% and 25.3%, respectively, and the overexpression of AGGF1 was related to tumor size, LNM, and later TNM stage, indicating that AGGF1 is involved in the progression of NSCLC. Kaplan-Meier survival analysis, as well as univariate and multivariate analyses, showed that the overexpression of AGGF1 resulted in a shorter OS and DFS. Accordingly, the expression of AGGF1 and its prognostic implications are similar between NSCLC and other tumors.
We aimed to explore the effects of UBE2C and AGGF1 on angiogenesis by analyzing the relationships among UBE2C, AGGF1, MVD, and VM in NSCLC and further evaluating the value of UBE2C and AGGF1 in predicting the infiltration, metastasis, and prognosis of this disease. This study is the first to jointly detect the expression of UBE2C and AGGF1, as well as MVD and VM, in NSCLC and it is therefore innovative. Our results, consistent with those of previous studies, demonstrate that high MVD and VM positivity are associated with a larger tumor volume, LNM, later clinical stage, and a shorter OS and DFS, suggesting that tumor angiogenesis promotes the growth and metastasis of NSCLC and leads to poor prognosis. 18,32,33 In addition, we found that both UBE2C and AGGF1 were positively correlated with MVD and VM in NSCLC, indicating that both UBE2C and AGGF1 could possibly promote angiogenesis in this disease. Zhang et al found that after silencing UBE2C, the expression levels of VEGF and MMP-9, which are recognized vascular factors, were also decreased, suggesting that UBE2C might have a regulatory effect on angiogenesis. 17 Shen et al found that UBE2C was positively correlated with MVD and VM in breast cancer. 18 Furthermore, Tu et al demonstrated a positive correlation among AGGF1, VEGF, and MVD in hepatocellular carcinoma. 23 From these results, it could be concluded that UBE2C and AGGF1 might be involved in the promotion of angiogenesis in various tumors, including NSCLC. In addition, relevant literature has shown that the ubiquitination of UBE2C can regulate angiogenesis by regulating VEGFR, NOTCH signaling, and WNT signaling, among others. 34 Zhang et al found that AGGF1 promotes angiogenesis by regulating the PI3K-AKT pathway in mice. 20 However, further experiments are required to identify the pathways through which UBE2C and AGGF1 promote angiogenesis in NSCLC. Furthermore, the aforementioned effects of UBE2C and AGGF1 on angiogenesis are mainly mediated by vascular endothelial cells. To date, there has been no study on the mechanisms by which UBE2C and AGGF1 promote the formation of endothelial cellindependent VM. Studies have shown that high expression of both UBE2C and AGGF1 promotes epithelialmesenchymal transition (EMT). 13,15,35 The formation of VM is closely related to EMT, and the upregulation of EMT-related factors can promote the formation of VM. 36,37 Therefore, we concluded that UBE2C and AGGF1 might promote VM formation by promoting EMT. In addition, from our results, we can see that there is a positive correlation between UBE2C and AGGF1, further illustrating the importance of UBE2C in angiogenesis. However, it is still unclear how these two interact. Studies have shown that UBE2C, like AGGF1, can regulate the PI3K-AKT-mTOR pathway, which plays a key role in angiogenesis, [38][39][40] suggesting that UBE2C and AGGF1 may have similar effects on angiogenesis. In this study, owing to the limited conditions, the research sample size was not large enough and only one experimental method was used. Thus, the specific mechanisms by which UBE2C and AGGF1 promote angiogenesis need to be studied further.
In conclusion, we have demonstrated that the overexpression of UBE2C and AGGF1 in NSCLC is associated with angiogenesis and leads to a poor prognosis, suggesting that UBE2C-and AGGF1overexpressing NSCLC may be more prone to invasion and metastasis. Therefore, UBE2C and AGGF1 can be considered as possible biological markers for predicting the infiltration, metastasis, and prognosis of NSCLC.

Funding
There is no funding to report.