The prognostic value of the lysyl oxidase family in ovarian cancer

Abstract Background Our study intended to evaluate the prognostic value of lysyl oxidase (LOX) and its four relevant members, the lysyl oxidase–like genes (LOXL1‐4), in ovarian cancer (OC) patients. Material and Methods The Kaplan‐Meier plotter (KM plotter) database was used to investigate the prognostic power of the LOX family for OC patients. Overall survival (OS) and progression‐free survival (PFS) were the clinical endpoints. The prognostic roles of the LOX family in OC patients were also analyzed according to various clinicopathological characteristics, including histological subtypes, clinical stages, pathological grades, and chemotherapeutic treatments. Results Overexpression of LOX, LOXL1, LOXL2, and LOXL3 mRNA indicated poor OS and PFS in OC patients, particularly in serous and grade II + III OC patients. Overexpression of LOXL4 mRNA resulted in worse PFS in OC patients. Overexpression of LOX and LOXL1 mRNA showed worse OS and PFS in stage III + IV OC patients, and overexpression of LOXL3 mRNA indicated worse OS and PFS in stage I + II OC patients. Overexpression of LOX, LOXL3, and LOXL4 mRNA indicated worse OS and PFS among OC patients who received platinum, taxol, and taxol + platinum chemotherapy. Overexpression of LOXL1 and LOXL2 mRNA was related to lower OS and PFS in OC patients who received platinum chemotherapy. Conclusion LOX, LOXL1, LOXL2, and LOXL3 may become potential predictive markers for negative outcomes in OC patients. Moreover, the LOX family can serve as new molecular predictors for the efficiency of platinum‐based chemotherapy in OC patients.

of paclitaxel-and platinum-based agents. 4 However, the majority of advanced high-grade serous ovarian cancer (HGSOC) patients experience disease relapse within 3 years and die within 5 years with the gold standard treatment strategy. 5 Currently, interval debulking surgery following neoadjuvant chemotherapy is an alternative therapeutic regimen to the gold standard clinical therapy in advanced-stage OC patients. 6,7 Therefore, exploring potential prognostic markers for OC patients and probing molecular predictors for the efficiency of chemotherapy regimens in OC patients are necessary.
The lysyl oxidase (LOX) family consists of five members: LOX, the first described member of this family, and its four related members called lysyl oxidase-like genes (LOXL1-4). The LOX family proteins have two highly conserved sequences in the C-terminus: a unique copper-binding (Cu) region and a cytokine receptor-like (CRL) region. 8 The LOX family proteins are characterized by a variable N-terminal domain, which was determined to exert distinct functions. 9 The LOX family is important for extracellular matrix (ECM) cross-linking and remodeling 10 and is involved in the process of angiogenesis 11 and tubulogenesis. 12 Furthermore, the LOX family has different impacts on the migration, 13 invasion, 14 and metastasis 15 of cancer cells.
Previously published studies have focused on the effects of the LOX family in human tumors and implicated that the LOX family exhibited divergent expression patterns and prognostic functions in diverse cancers. [16][17][18][19] Low oxygen tension stimulates the activation of the hypoxia-inducible factor(HIF)pathway in the metastatic microenvironment of ovarian carcinoma and subsequently elevates LOX expression in a HIF-dependent pattern to facilitate collagen remodeling and tumor invasion. 20 Wu et al 21 illustrated that the rs1800449 G473A polymorphism of LOX might increase the susceptibility and recurrence of ovarian carcinoma.
Currently, the prognostic functions of the LOX family in patients suffering from OC have not been systematically and comprehensively determined. Therefore, our study intended to evaluate the prognostic power of the LOX family in OC patients, specifically regarding their mRNA expression patterns.

| MATERIAL S AND ME THODS
This study protocol obtained ethical approval from the ethical committee of the Second Affiliated Hospital of Wenzhou Medical University (No. L-2020-08).

| Establishment of the ovarian cancer microarray database
The Kaplan-Meier plotter (KM plotter) database is a freely available online tool (www.kmplot.com) that is capable of assessing the potential impact of cancer-associated genes on survival for breast cancer, 22 gastric cancer, 23 lung cancer, 24 and ovarian cancer patients. 25 The available gene mRNA expression data and clinical

| Kaplan-Meier survival analysis
In our study, the prognostic power of the LOX family in patients suffering from OC was assessed by utilizing the KM plotter database. According to higher or lower mRNA expression levels than the automatically selected best cutoff values of the chosen gene, the OC patients were classified into high (upregulation) or low (downregulation) mRNA expression cohorts. Overall survival (OS) and progression-free survival (PFS) were the studied clinical endpoints, and Kaplan-Meier survival plots were subsequently generated for the two patient cohorts. 27 In addition, LOX, LOXL1, LOXL2, LOXL3, and LOXL4 were analyzed for their associations with various clinicopathological features of OC patients, including histological subtypes, clinical stages, pathological grades, and chemotherapeutic treatments.

| Statistical analysis
The KM plotter database was developed by using the PostgreSQL server, which can simultaneously process gene expression and clinical data. The data were imported into R software for calculations and analysis. Kaplan-Meier analysis was performed to draw survival curves, and the log-rank test was employed to analyze the differences, with P < .05 considered statistically significant. The hazard ratios (HRs) with 95% confidence intervals (CIs) were computed.
HR > 1 suggested a worse clinical prognosis, and HR < 1 suggested a better clinical prognosis for OC patients. When the 95% CI of the HR contains 1, the results were not considered to be significantly different. Moreover, the KM plotter webpage explained that the generated P-value does not include correction for multiple hypothesis testing by default. 28

| Different prognostic values of the LOX family in OC patients
The study results showed that the elevated mRNA expression of LOX was associated with lower OS ( Figure 1A) and PFS ( Figure 1B Table 2.

| Prognostic functions of the LOX family in OC patients with different histological subtypes
The study explored the prognostic functions of the LOX family in serous and endometrioid ovarian carcinoma patients ( Table 3). The

| Prognostic power of the LOX family in OC patients with different clinical stages
The study results demonstrated the prognostic correlation of the LOX family with clinical stage I + II (early stages) and clinical stage III + IV (advanced stages) OC patients ( Table 4). The overexpression of LOX and LOXL1 mRNA was associated with worse OS and PFS in stage III + IV OC patients. The high expression of LOXL2 mRNA was related to unfavorable OS in stage III + IV OC patients but had no effect on PFS. In addition, the elevated mRNA expression of LOXL3 was associated with lower OS and PFS in stage I + II OC patients and showed worse PFS in stage III + IV OC patients. The upregulated mRNA expression of LOXL4 was illustrated to be associated with worse PFS in stage III + IV OC patients.
The above findings suggested that the elevated mRNA expression of LOX and LOXL1 was associated with worse OS and PFS in stage III + IV OC patients, and the increased mRNA expression of LOXL3 was related to poor OS and PFS in stage I + II OC patients.

| Prognostic roles of the LOX family in OC patients with different pathological grades
The study results revealed the prognostic power of the LOX family for pathological grade I (well differentiation) and pathological grade Parameters LOX, LOXL1, LOXL2 LOXL3, LOXL4 Cases for OS (n)

Cases for PFS (n)
Histological subtypes

| Prognostic significance of the LOX family in OC patients treated with different chemotherapeutic strategies
The study results implicated the prognostic significance of the LOX family in OC patients undergoing platinum-based chemotherapy, taxol-based chemotherapy, and taxol + platinum chemotherapy (

| D ISCUSS I ON
Our research proposed to explore the prognostic functions of the LOX family in OC patients, focusing on the mRNA expression levels.
The results showed that the elevated expression of LOXL4 mRNA was related to worse PFS in OC patients, and the elevated expression of LOX, LOXL1, LOXL2, and LOXL3 mRNA was associated with unfavorable OS and PFS in OC patients.
Among this five-protein family, LOX is the best-studied isoform.
LOX is situated and exerts functions in the nuclei of fibrogenic cells, 29 catalyzing the covalent cross-linking of collagens and elastin and subsequently increasing the extracellular matrix tension. 30 The role of LOX as a potential predictive factor in cancer metastasis and progression was evidenced in several human cancers, such as pancreatic carcinoma, 31  In addition, some limitations of our present study also need to be discussed. First, our study documented the prognostic value of the LOX family in OC patients only at the mRNA expression level. We will further study the LOX family at the protein level to certify the prognostic functions of this family in OC patients. Second, the data we collected were merely from a freely available online database, and the mechanisms of how the LOX family exerts its functions on the prognosis of OC patients are unknown. More efforts are needed to further illustrate the mechanisms and pathways of the LOX family that are related to the biological behaviors (metastasis, proliferation, migration, invasion, etc) of ovarian cancer based on biochemical (cellular function), physiological (animal models), and pathological (human cancer specimens) researches.

| CON CLUS ION
In summary, LOX, LOXL1, LOXL2, and LOXL3 may be negative prognostic indicators for OC patients, particularly for serous, grade II + III and platinum-based chemoresistant OC patients. This finding may help to accurately predict the prognosis of OC patients, and the discovery of LOX family gene-target inhibitors may be an efficient way to adjuvantly treat OC patients.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.