Neutrophil Elastase and Neutrophil Extracellular Traps in the Tumor Microenvironment

Abstract

Tumor-associated neutrophils (TANs) play a major role during cancer development and progression in the tumor microenvironment. Neutrophil elastase (NE) is a serine protease normally expressed in neutrophil primary granules. Formation of neutrophil extracellular traps (NETs), a mechanism used by neutrophils, has been traditionally associated with the capture and killing of bacteria. However, there are recent discoveries suggesting that NE secretion and NETs formation are also involved in the tumor microenvironment. Here, we focus on how NE and NETs play a key regulatory function in the tumor microenvironment, such as tumor proliferation, distant metastasis, tumor-associated thrombosis, and antitumor activity. Additionally, the potential use of NETs, NE, or associated molecules as potential disease activity biomarkers or therapeutic targets will be introduced.

2.1. Introduction of Tumor-Associated Neutrophils (TANs)

Neutrophils, also known as polymorphonuclear cells (PMNs), are the most abundant white blood cells in human circulation accounting for approximately 60% of all leukocytes [1]. They are the first immune cells to respond to inflammatory or infectious etiologies as they are crucial participants in the proper functioning of both innate and adaptive immune responses [2]. As part of their key role, neutrophils are the first line of immune defense against invading pathogens and also participate in the development of inflammatory cascades [3, 4]. Neutrophils have a unique polymorphic nucleus that is segregated into 3–5 lobules in humans, with each lobule having a diameter of approximately 2 μm [5]. In healthy individuals, most neutrophils are destined to be cleared even without ever performing their function. During infection, they have crucial roles in the clearance of microorganisms [4, 6].

Currently, there is increasing evidence demonstrating that not only do neutrophils play key roles in inflammatory diseases but also in cancer as neutrophils have both pro- and antitumor properties [7–9]. Neutrophils accumulate in many types of human and murine tumors and regulate nearly all steps of tumor progression. Neutrophils are involved in cancer through multiple mechanisms and have been implicated in almost all stages of the oncogenic process including tumor initiation, growth, proliferation, and metastases [10–13]. Human and mice studies have shown that there are two neutrophil phenotypes: antitumor N1 neutrophils and protumorigenic N2 neutrophils [14, 15]. Tumor-associated neutrophils (TAN) can be activated under various conditions resulting in antitumor and protumor functions [14]. Although the ability of TANs to promote or prevent cancer progression is not fully understood, we know that TANs consist of various polarization states, and the mechanism behind this polarization is ill defined. Here we aim to review the existing evidence of neutrophil recruitment, functions as well as the different regulators of neutrophil in the tumor microenvironment.

Neutrophils found within the tumor play a central role in inflammation and are attracted by CXCR2 ligands such as CXCL1, CXCL2, and CXCL5 [13, 16]. The initiation of tumor growth can be promoted by neutrophils through the release of reactive oxygen species (ROS), reactive nitrogen species (RNS), or proteases [17]. Neutrophils have been implicated in various cancers. Human colorectal cancer liver metastases and murine gastrointestinal liver metastases exhibit infiltration by neutrophils. The depletion of neutrophils in a murine liver metastases model has been shown to diminish metastatic growth through fibroblast growth factor 2 [18]. In human hepatocellular carcinoma (HCC) samples, over-expression of CXCL5 was well correlated with intratumoral neutrophil infiltration, shorter overall survival, and tumor recurrence [19]. The depletion of hepatic neutrophils via antibody has been shown to protect the liver from diethylnitrosamine (DEN)-induced HCC in murine models, since neutrophils stimulate hepatocellular ROS and telomere DNA damage [20]. In a mice flank tumor model, neutrophils contribute to the antitumor activity of TGF-b blockade [14]. Neutrophil-derived ROS are important regulators of protumorigenic γδ17 T cells that have been identified as immunosuppressive and antitumoral in a mice melanoma model [21].

Alternatively, neutrophil recruitment has been shown to be a key component of the antitumor efficacy of bovis Bacillus Calmette-Guerin treatment in bladder cancer, chemotherapy in mouse lung cancer, radiotherapy in several syngeneic mouse tumor models, rituximab and trastuzumab treatments in human non-Hodgkin’s lymphoma, and breast cancer, respectively [22–26]. Activated TANs can also elicit antitumor functions either directly through lysis of tumor cells or by antibody-dependent cell-mediated cytotoxicity [23, 27, 28].

2.2. Introduction of Neutrophil Elastase in Health and Disease

Neutrophil elastase (NE) belongs to the family of serine protease normally expressed in polymorphonuclear neutrophils (PMNs) [29–31]. It plays a key role in numerous physiological and pathological processes, including antimicrobial defenses, inflammation, and cancer progression [2, 32]. During neutrophil degranulation or neutrophil extracellular traps (NETs) formation, NE is released into the extracellular space in a process known as NETosis. Not only is NE a necessary component of NETs, but activated NE is also required for NET formation [33]. Although NE can also be released from neutrophils independent of NET formation, and rapidly inactivated by plasma antiproteases, DNA-associated NE appears to retain its proteolytic activity for extended periods [34, 35]. NE has been implicated as a biomarker for the diagnosis and prognosis of inflammatory bowel disease as it has been detected in the colonic mucosa of patients with ulcerative colitis [36–40].

It has been shown that activated NE protects against infection by destroying pathogenic bacteria [4]. Mutations in the ELA2 gene encoding neutrophil elastase (NE) occur in most cases of severe congenital neutropenia as well as sporadic and autosomal-dominant cyclic neutropenia [41, 42]. The release of NE is responsible for the activation of epithelial protease-activated receptors, which leads to cell shrinking and reduction of barrier function [43]. NE and ROS are required for TNFα-primed neutrophils and antineutrophil cytoplasmic antibodies to cause increased pulmonary endothelial permeability and lung edema in a model of acute Wegener’s granulomatosis [44]. In addition, NE is also important in the reciprocal coupling between innate immunity and coagulation necessary for thrombus formation [45]. NE has been shown to decrease the allostimulatory ability of human monocyte-derived dendritic cells [2]. Finally, how neutrophil elastase is integrated in the activation, regulation, and effector mechanisms of cancers continues to be explored [46].

2.2.1. Neutrophil Elastase in Primary Tumor Initiation and Growth

NE has been shown to have a protumorigenic role in breast, lung, prostate, and colon cancers [47–52]. Blocking the activity of NE in mouse models of numerous cancer types can significantly reduce the effect of neutrophils on tumor progression, and metastasis [47, 49, 50, 53]. NE promotes tumor growth in different ways either by increasing cancer cell proliferation, migration, invasion, or by inducing angiogenesis within the tumor microenvironment. NE may also contribute to tumorigenesis by inactivating tumor suppressors [47, 52, 54–57]. Also, there is increased expression of NE within the tumor and in circulation during tumorigenesis, which promotes tumor growth [58–60]. NE may directly induce tumor cell proliferation in both human and mouse lung adenocarcinomas by gaining access to an endosomal compartment within tumor cells where it degrades insulin receptor substrate-1 [47]. It has been shown that the production of NE from TANs may be involved in tumor invasion, which is associated with a poor prognosis in patients with non-small cell lung cancer. Lastly, NE may facilitate the invasion of cancer cell either by directly dissolving the tumor matrix or indirectly by activating a protease cascade [61].

NE released from activated neutrophils may also mediate PI3K-associated signaling pathway for tumor cell proliferation and promote the growth and progression of cancer cells. The treatment of esophageal cancer cell lines with NE induces the release of growth factors, including protransforming growth factor-alpha, platelet-derived growth factor-AA, platelet-derived growth factor-BB, and vascular endothelial growth factor. Furthermore, use of Sivelestat, a specific NE inhibitor, significantly inhibits the release of these growth factors [47, 57]. EMILIN1, a multidomain glycoprotein expressed in several tissues, exerts a crucial regulatory tumor suppression function through the engagement of α4/α9 integrins. This tumor suppressor function of EMILIN1was impaired through its cleavage by NE, a process that has been implicated in the digestion of sarcomas and ovarian cancers [54].

2.2.2. Neutrophil Elastase in Cancer Metastases

In human studies, NE has been associated with breast cancer metastasis, and the detection of tumor NE level might be helpful in selecting the appropriate individualized treatment for patients with breast cancer [51, 62]. The release of NE can also facilitate non-small cell lung cancer metastasis by degrading basement membranes and allowing egress of tumor cells into the circulation [63, 64]. ONO-5046.Na is a specific NE inhibitor that has been reported to reduce hepatic ischemia-reperfusion injury by inhibiting accumulation of neutrophils [65, 66]. Ischemia-reperfusion injury has been implicated in liver metastasis and ONO-5046.Na may reduce the burden of metastasis through reduction of ischemia-reperfusion injury [67].

2.2.3. Neutrophil Elastase as a Therapeutic Target in Cancer

It has been reported that NE released by TANs can be taken up by breast cancer cells, which could enhance their susceptibility to cytotoxic T lymphocyte lysis [60, 68]. In a colorectal cancer mouse model, a higher expression of active NE was detected in the tumor and giving the NE inhibitor Sivelestat inhibited tumor growth. Within human colorectal cancer tissue, increased amounts of NE expression were detected compared to the adjacent nontumor tissues. In addition, the serum NE concentration in colorectal cancer patients was significantly higher than that in the healthy controls, indicating that the NE levels in serum may also potentially be a diagnostic marker of colorectal cancer in patients [49]. Investigation of Sivelestat use for cancer therapy should be further explored as its safety and efficacy have been tested in treatment of patients with acute lung injury and acute respiratory distress [69, 70]. The NE inhibitors, AZD9668 and BAY-85–8501, were developed and studied in phase II clinical trials for pulmonary diseases. AZD9668 has the potential to reduce lung inflammation and the associated structural and functional changes in some other human diseases. BAY 85–8501 was shown to be safe and efficacious in a mouse acute lung injury model and recently is being tested in clinical studies for the treatment of pulmonary diseases [71, 72].

Taken together, NE could be of great value as a potential diagnostic marker or therapeutic target for cancer patients. Given that NE plays a key role in normal immune response to bacterial pathogens, there is a potential detrimental risk for infections if the drug is given chronically. Currently, there has been no clinical trial on the use of NE in cancer but in designing such trial, every step must be carefully considered in order to gain the greatest benefit [46].

2.3. Introduction of Neutrophil Extracellular Traps in Health and Disease

Neutrophils form neutrophil extracellular traps (NETs), which are large, extracellular, web-like structures composed of cytosolic and granule proteins that are assembled on a scaffold of decondensed chromatin [73]. The granular components are 25 nm in diameter and are normally stored in distinctive neutrophil granules that reside on the DNA backbone structure of NETs and provide antimicrobial activity [73, 74]. NETs can capture and kill bacteria, fungi, viruses, parasites and are thought to prevent bacterial and fungal dissemination [75–79].

NET formation, also known as “NETosis,” describes the process by which neutrophils produce and release active NETs. It is important to note that NETosis is distinct from necrosis and apoptosis. This same process, which leads to neutrophil death, is also a mechanism of bacterial sequestration [74, 80–82]. During NETosis, the nucleus first delobulates, while the granules disappear, followed by membrane vesiculation (Fig. 2.1). After nuclear disintegration, the chromatin expands, allowing contact between granular and cellular components. Finally, the cytoplasmic membrane ruptures, releasing NETs into the extracellular space [83]. In summary, NETosis is a form of neutrophil-specific cell death where neutrophils extrude extracellular fibers composed of chromatin and granule proteins that are characterized by the release of large web-like structures [84, 85].

Fig. 2.1.

Formation of neutrophil extracellular traps

NETs have been shown to contribute to the pathogenesis of immune-related diseases such as lupus nephritis when dysregulated [86–88]. Increasing evidence suggests that the process by which neutrophils produce and release NETs not only happens in infections such as sepsis, but also has key role in noninfectious diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), diabetes, atherosclerosis, acute lung injury, hepatic ischemia reperfusion injury, thrombosis, and chronic liver disease [87, 89–100]. In the past few years, there has been increasing evidence for a role of NETs in cancer [86].

2.3.1. Neutrophil Extracellular Traps in Primary Tumor Initiation and Growth

The first association of NETs in cancer was described in 2012 when neutrophils from tumor-bearing mice showed an increased propensity for NETosis upon stimulation with LPS [101]. NET-induced coagulation is a sequela of some malignancies and in an intestinal cancer model, coagulation and tumorigenesis was reduced by DNase [102, 103]. In a Lewis lung carcinoma model with hemorrhagic tumors, a large area of neutrophils and NET-like structures were found within the hemorrhagic tumors [104]. These hemorrhagic areas contained intact and primed hypercitrullinated neutrophils ready for NET formation [104]. In mouse insulinoma and breast cancer models, NET accumulation extends to the peripheral circulation, causing systemic inflammation and impaired vessel function in organs not infiltrated by tumor cells. DNase I treatment abolished these remote effects, suggesting that NETs mediate the collateral effects of tumors in distal organs [105]. Our group also recently found that blocking NETs can reduce the risk of HCC in the setting of non-alcoholic steatohepatitis (NASH) [100].

2.3.2. Neutrophil Extracellular Traps in Metastases

NETs have been implicated in cancer metastasis in the context of systemic infection. In a cecal ligation and puncture (CLP) model, microvascular NET deposition and consequent trapping of circulating lung carcinoma cells within DNA webs were associated with increased formation of hepatic micrometastases. This effect was abolished by NET inhibition with DNase or neutrophil elastase inhibitor [106]. Our laboratory has reported that NET formation is accelerated immediately after major liver resection in patients with metastatic colorectal cancer. Circulating MPO–DNA levels, a NET biomarker, was associated with a significant increase in early metastatic recurrence. Mechanistic investigations in vitro indicated that mouse neutrophil–derived NET triggered HMGB1 release and activated TLR9-dependent pathways in cancer cells to promote their adhesion, proliferation, migration, and invasion [107]. These effects can be abrogated by inhibiting NET formation in mice via DNase I treatment or inhibition of peptidyl arginine deiminase type IV (PAD4). PAD4 is an essential enzyme in NET formation that catalyzes the citrullination of histones-H3, a critical step for chromatin decondensation and expulsion [97].

2.3.3. Antitumor Properties of Neutrophil Extracellular Traps

Increasing evidence suggests that NETs play a key role in cancer; however, the antitumorigenic and the protumorigenic roles of NET formation appear to depend on various conditions. For example, NETs induced within the vasculature by systemic bacterial infection or surgical stress facilitates cancer metastasis in the liver and the lung. In contrast, neutrophils that express high CD16 and low CD62 have shown increased migratory and NET-producing capacity and this unique feature correlates with better survival in patients with head and neck squamous cell carcinoma [108]. These neutrophils and NETs are presumed to have destroyed the tumor cells. Based on these reports, the functions of NETs and neutrophils seem to differ based on the type of cancer or the degree of progression [109]. Further studies are needed to characterize factors that influence NET production and the difference between various forms of NETS and neutrophils.

2.3.4. Neutrophil Extracellular Traps as a Therapeutic Target in Cancer

NETs are involved in tumor proliferation, distant metastasis, and tumor-associated thrombosis, strongly suggesting that NETs are a potential therapeutic target in cancer [109, 110]. Multiple studies have shown that DNase can digest NET formation, thereby reducing tumor proliferation, distant metastasis, and tumor-associated thrombosis [100, 102, 105–107]. In human studies, PAD4 has been shown to be overexpressed in some tumors [111, 112]. Inhibiting PAD4 by using PAD4 inhibitors or PAD4 KO mice significantly reduces tumor progression [100, 107, 113–115]. However, PAD4 inhibition or the use of DNase for cancer treatment in patients is still in the rudimentary stage. Additionally, it is unknown if inhibiting NET formation has a synergistic effect with current cancer treatments, such as chemotherapy, radiotherapy, molecule-targeted therapy, and immunotherapy [110]. More work is needed to investigate the relationship between NET and already established cancer treatments.

2.4. Conclusions

NE secretion and NET formation in the tumor microenvironment could be the initiators of disease or a side effect of the general overwhelming response of the immune system. There is potential for NE or NET-related molecules to be used as biomarkers and as targets for therapeutic intervention in cancer-related diseases. Although additional studies on the pathogenicity of NE and NETs in tumor microenvironment are still needed, important progress has been made to show that both NE and NETs may induce tumor proliferation, distant metastasis, and tumor-associated thrombosis (Fig. 2.2). The capacity of NE and NETs to potentiate or suppress inflammation may have a beneficial function in cancer and other pathologies. A better understanding of the function and impact of NE and NETs on health will enable the suppression of detrimental attributes without interfering with beneficial ones and ultimately, and allow us to exploit NE and NETs to treat diseases. Continued investigations into the relevance of NE and NETs in disease will reveal new functions and shed further light on the role(s) of extracellular chromatin.

Fig. 2.2.

Neutrophil elastase and neutrophil extracellular traps can induce tumor proliferation, distant metastasis, and tumor-associated thrombosis

Abbreviations

CXCL

C-X-C motif chemokine

CXCR

C-X-C chemokine receptor

DNase

Deoxyribonuclease

LPS

Lipopolysaccharide

MPO

Myeloperoxidase

NE

Neutrophil elastase

NET

Neutrophil extracellular trap

PAD4

Protein arginine deiminase 4

PMA

Phorbol-12-myristate-13-acetate

PMN

Polymorphonuclear neutrophil

RA

Rheumatoid arthritis

ROS

Reactive oxygen species

SLE

Systemic lupus erythematosus

TAN

Tumor-associated neutrophil

TGF-β

Transforming Growth factor-β

TLR

Toll-like receptor