Pancreatic adenocarcinoma (PDAC), a highly fatal disease with a poor prognosis, ranks as the seventh leading cause of cancer mortality globally (Sung et al., 2021), is projected to become the second leading cancer-related death in America and Europe in 2030 (Rahib et al., 2014; Quante et al., 2016). However, over the past decades, albeit considerable advances have been made in the diagnosis, surgery, chemotherapy, radiotherapy and systemic therapy, however, due to non-specific symptoms, lacking reliable markers, low surgical resection rate and therapy resistance, only months of overall survival (OS) achieved (Conroy et al., 2011; Winter et al., 2012), with the 5-years survival remaining a grim 10% (Siegel et al., 2021). Therefore, further investigation of pathological features and the pathogenesis of PDAC is imperative for exceptional therapeutic strategies.

Such poor outcomes of PDAC have fueled ongoing efforts to exploit the tumor microenvironment (TME). The TME is a highly complex ecosystem composed of cancer cells, stromal cells, immune cells, and the extracellular matrix (ECM) interaction. Growing evidence has illustrated the pivotal roles of TME components in immune suppression and tumor progression. It is worth noting that the surrounding ECM has long been implicated in accelerating PDAC progression by directly promoting cellular transformation and metastasis. Furthermore, the ECM and its reorganization are of paramount importance in the evolution of both tumor and immune microenvironment and their interaction (Setargew et al., 2021). These alterations may greatly affect the biological behavior of tumor cells and stromal cells, and ultimately influence the prognosis of PDAC patients.

Lysyl oxidase (LOX) family includes five copper-dependent amino enzymes: LOX and LOX-like (LOXL) 1-4 (Molnar et al., 2003; Barker et al., 2012), of which the canonical function is catalyzing collagen and elastin cross-linking (Harris, 1976; Kagan and Li, 2003; Zhao et al., 2018). Lysyl Oxidases (LOXs) contribute to ECM stiffness and homeostasis, and their dysregulation is involved in several diseases, including tissue fibrosis (Al-U’datt et al., 2019; Li et al., 2020) and cancer (Wang et al., 2016; Tan et al., 2020). In reality, the activity of LOXs integrates a complex network within the TME linking the ECM and the immunological components. Intriguingly, LOXs play different roles in various tumor progression, exhibiting either enhancing (Wilgus et al., 2011; Kasashima et al., 2016)or suppressing (Johnston and Lopez, 2018; Zhao et al., 2020) function in different types of human malignancies.

It has been demonstrated that overexpressed LOXs are critically involved in the PDAC progression. Le Calve et al. (Le Calve et al., 2016) found higher mRNA expression of LOX, LOXL1 and LOXL2 in PDAC. Ma et al. (Ma et al., 2019) found that LOX expression might be an independent prognostic factor of progression-free survival (PFI) in PDAC patients. Higher LOXL2 expression elevated the invasiveness of PDAC cells and correlated with lower survival of PDAC patients (Kim et al., 2019). However, the potential roles of LOXs in the PDAC immune response have not been elucidated. Herein, various large-scale open-accessed databases were applied to explore the expression pattern, cancer-related functional aspects, prognostic value, and relationship with the immune infiltrating of LOXs in PDAC.

As a kind of secreted protein family, LOXs play a pivotal role in PDAC pathogenesis by regulating the TME in two aspects, ECM remodeling and immune-related responses. It has been well known that the TME could significantly affect the behavior of tumor cells and ultimately influence the outcomes of patients (Setargew et al., 2021). The TME of PDAC is characterized by dense desmoplasia and extensive immunosuppression (Neesse et al., 2015; Ren et al., 2018). LOXs are major drivers in the biogenesis of the desmoplastic matrix in cancer. LOXs prevent collagen degradation and improve tissue stiffness through catalyzing the cross-linking process of collagen and elastin, and finally contribute to dense desmoplasia in PDAC. Dense desmoplasia plays a critical role in metastasis and limiting chemotherapy efficacy (Incio et al., 2016; Ren et al., 2018; Schizas et al., 2020). With the increasing interest in anti-stromal therapies, the LOX family has emerged as a potential target. Although the role of the LOX family in tumorigenesis and prognosis of several cancers has been partially confirmed, further bioinformatics analysis of PADC has yet to be performed. In this study, we used multitalented public databases to reveal the dysregulated expression of the LOXs and their relationship with tumor stage and prognosis. As for extensive immunosuppression in PDAC, we found that LOXs, particularly LOX and LOXL3 are closely related to TILs, various immune signatures, and immune checkpoints, however, the function in the immune microenvironment remains unclear which calls for further studies.

Elevated LOX expression has been noted in PDAC tissues compared to adjacent tissues and correlated with poor OS and DSS (Miller et al., 2015; Ma et al., 2019). A high expression of LOX was detected in metastatic pancreatic cancer in the mice model. Conversely, LOX inhibition increased immune cell recruitment, vascularization, and decreased fibrillar collagen (Miller et al., 2015). In line with this finding, Le Calve et al. (2016) found that LOX protein was upregulated in PDAC tissues. Compared to adjacent pancreas tissues, we found higher mRNA expression of LOX in PDAC tissues, which was correlated with tumor stage and grade of PDAC. The IHC showed a higher LOX protein level in PDAC tissues than normal pancreatic tissues. The functional enrichment analysis showed that LOX and its ligands were mainly involved in the ECM regulatory. The transcriptional level of LOX is strongly related to the infiltration of CD8⁺ T cell, macrophage, NK cell, neutrophil, dendritic cell infiltration, as well as various immune biomarkers and immune checkpoints. Furthermore, a high LOX expression was significantly associated with poor OS, PFI, and DSS in PDAC patients. These results together indicate the oncogenic role and prognostic value of LOX in PDAC, as well as the role in the immune response.

LOXL1 transcript was also detected upregulated in PDAC patients and associated with chemotherapy resistance (Le Calve et al., 2016). In our study, the mRNA and protein expression of LOXL1 in PDAC was higher than that in normal tissues. However, the mRNA expression of LOXL1 showed no relationship with tumor stage, grade, and outcomes of PDAC patients. We also revealed the role of LOXL1 in ECM shaping and a close relationship with infiltrating levels of CD8⁺ T cell, macrophage and NK cell, neutrophil, and dendritic cell. However, more studies should be carried out to identify the potential function of LOXL1 in PDAC progression.

Higher LOXL2 expression correlated with clinicopathological features of advanced disease and predicted a worse OS of PDAC patients (Park et al., 2016; Kim et al., 2019). Previous studies have revealed that LOXL2 induced epithelial-mesenchymal transition (EMT) through the PI3K-AKT signaling pathway (Wu and Zhu, 2015) to enhance the invasive and migratory capacity of PDAC cells (Park et al., 2016). In the current study, the expression of LOXL2 was higher in human pancreatic cancer tissues than in normal tissues, which highly correlated to the tumor stage. The functional enrichment analysis showed LOXL2 also had a close relationship with ECM regulation. In addition, LOXL2 expression showed a significant relationship with macrophage and NK cell, neutrophil, and dendritic cell infiltration. And Minici’s study (Minici et al., 2020) has demonstrated LOXL2 is expressed by infiltrating plasmablasts in PDAC. It is worth noting the high expressed LOXL2 predicted an inferior OS, PFI, and DSS in PDAC patients. Consequently, LOXL2 might be a representative biomarker for PDAC. However, phase II clinical trials failed to demonstrate the utility of LOXL2 inhibition in PDAC (Benson et al., 2017), which may be due to cancer-associated fibroblasts (CAFs). The discovery of the tumor-restraining functions of CAFs provides a potential explanation for the unsuccessful clinical trials of therapeutic agents targeting CAFs or stromal components (Van Cutsem et al., 2020). These observations suggest that future therapeutic strategies should avoid generic targeting of tumor-restraining CAF subpopulations in favor of precise reprogramming and normalization of tumor-promoting CAF subsets (Chen et al., 2021).

In this study, overexpressed mRNA of LOXL3 was identified in PDAC tissues and correlated with tumor grade, but showed no relationship with the outcomes of PDAC patients according to the GEPIA database and CPTAC database. Intriguingly, the LOXL3 protein level was found to be downregulated according to our IHC results and HPA database as well, which was controversial. As is known, the expression of LOXL3 was detected mainly in the nucleus correlated with tumor invasion, lymph node metastasis, and poorer prognosis of patients (Kasashima et al., 2018) through interacting with SNAIL and contributing to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells (Eiseler et al., 2012). Additionally, TGF-induced LOXL3 upregulation in gastric cancer cells suggested that LOXL3 was downstream from the TGF-signaling pathway (Kasashima et al., 2018). However, studies on LOXL3 were mainly based on evidence from cell lines, limited evidence from in vivo studies was found. Meanwhile, LOXL3 expression in some tumors was contrary as in some cases LOXL3 was downregulated. Li Ma (Ma et al., 2017) found that LOXL3 was a dual-specificity enzyme involved in STAT3 deacetylation and deacetylimination modulation which reduced the activity of signal transducer and activator of transcription 3 (STAT3). STAT3 activation helps to promote tumor progression (Sun et al., 2021) and is associated with PDAC through PDAC patients’ biopsy study(Laklai et al., 2016). De-activation of STAT3 by LOXL3 would be a protective role in PDAC pathogenesis. Furthermore, unlike other LOX family members, the functional analysis of LOXL3 and its co-expressed genes showed enrichment in the immune response. LOXL3 expression showed a high correlation with CD8⁺ T cell, macrophage, NK cell, neutrophil, and dendritic cell infiltrating levels. In addition, LOXL3 was highly related to biomarkers of CD8⁺ T cell, T cell, B cell, monocyte, TAM, M1macrophage, M2 macrophage, neutrophil, DC cell, Th1, Th2, and Th17. In addition, the LOXL3 expression was positively associated with immune checkpoints. LOXL3 was also found to induce downregulation of Th17 and Treg activation as well (Ma et al., 2017) which play a role in cancer pathogenesis (Marques et al., 2021). Thus, we speculate LOXL3 plays a crucial role in the modulation and recruitment of immune cells and affects the expression of immune signatures in PDAC, and more studies should be carried out to validate.

Accumulating evidence suggest LOXL4 an oncoprotein in several tumors, such as hepatocellular carcinoma (Li et al., 2019), lung cancer (Zhang et al., 2019) and breast cancer (Choi et al., 2017). However, the expression and prognostic role of LOXL4 in PDAC have yet to be investigated. This report demonstrated a higher mRNA expression of LOXL4 in PDAC tissues with no correlation with tumor stage, grade, or outcomes in PDAC patients. The protein expression of LOLX4 showed no difference in PDAC and normal pancreas. The functional analysis of LOXL4 identified similar results to the canonical function of LOXs. In addition, the expression of LOXL4 showed a weak correlation with immune biomarkers. These results together suggest that LOXL4 function in pancreatic tumorigenesis needs to be further determined.

The involvement of one or more LOX members was verified in different cancers. Thus, the LOX family members are likely to present distinct functions specific for each member and partially overlapping activity. We further analyzed the synergy function of LOXs with overlapping co-expressed genes by GO enrichment analysis and KEGG pathway enrichment. GO analysis demonstrated the role of LOXs in ECM regulation. The cancer network showed that changes in LOXs expression mainly affect pathways in cancer (hsa05200), PI3K-AKT signaling pathway (has04151), focal adhesion (has04510), ECM-receptor interaction (hsa04512). These pathways are significantly associated with PDAC progression. Considering the major ECM component in PDAC, the predominant role of LOXs in the ECM remodeling, and involvement in the cancer-related pathways, we speculate that differentially expressed LOXs in PDAC are potential targets for drug therapy.

Current therapeutic strategies aim to deconstruct the surrounding desmoplastic stroma and target immunosuppressive pathways that have largely failed in PDAC. Even immunotherapeutic strategies, such as immune-checkpoint blockade, gained remarkable success in many malignancies that have not yet translated to PDAC (Johnson et al., 2017; Stromnes et al., 2017; Morrison et al., 2018). In addition, ablating the stromal barriers that restrict drug delivery has also demonstrated disappointing and contradictory responses. The key contributor is the multi-faceted functions of the complex TME components (Chronopoulos et al., 2016; Stromnes et al., 2017). Thus, combination strategies that target multiple features of the TME simultaneously might succeed. LOXs inhibitors reduce the fibrotic ECM and desmoplasia and overcome chemoresistance in triple-negative breast cancer by increasing the diffusion of chemotherapeutics into the tumor (Saatci et al., 2020). As demonstrated LOXs played a crucial role in modulating immune cells and affecting the expression of immune signatures in PDAC. Considering the close relationship of LOXs and the immune checkpoints, LOXs inhibitors might also play a role in promoting the anti-tumor effect of immune checkpoint inhibitors. A combination of LOXs inhibitor treatment may improve patient outcomes by priming the TME. However, further preclinical in vivo investigations and clinical trials need to be carried out to confirm our speculation.

In conclusion, our research identifies upregulated LOX, LOXL1 and LOXL2 mRNA and protein expression in PDAC tissue. LOX and LOXL2 could serve as novel biomarkers with prognostic significance in PDAC patients. The function of LOXs and their ligands is mainly involved in ECM regulation and pathways in cancer. Intriguing, LOXL3 and its co-expressed genes hold remarked relationship with immune response. Furthermore, a close association between the expression of LOX and LOXL3 with tumor immune infiltration was verified, uncovering the potential molecular mechanism underlying the carcinogenesis in PDAC. Our findings inspire new insight into prognostic biomarkers and new immunotherapeutic targets for PDAC. However, more experimental studies are needed to reveal the link between LOXs and PDAC, thereby promoting the clinical application of LOXs as prognostic indicators and combine therapy targets in PDAC.