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# Revisiting Neutrophil Gelatinase-Associated Lipocalin (NGAL) in Cancer: Saint or Sinner?

Brigitte Bauvois, Santos A. Susin

► To cite this version:

Brigitte Bauvois, Santos A. Susin. Revisiting Neutrophil Gelatinase-Associated Lipocalin (NGAL) in Cancer: Saint or Sinner?. Cancers, 2018, 10 (9), pp.336. 10.3390/cancers10090336. hal-02365894

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Submitted on 15 Nov 2019

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# Revisiting Neutrophil Gelatinase-Associated Lipocalin (NGAL) in Cancer: Saint or Sinner?

Brigitte Bauvois<sup>a-c</sup> and Santos A. Susin<sup>a-c</sup>

Corresponding author: Dr Brigitte Bauvois, Centre de Recherche des Cordeliers INSERM UMRS1138, 15 rue de l'Ecole de Médecine, F-75270 Paris cedex 06, France. E-mail address: brigitte.bauvois@crc.jussieu.fr. Phone: +33-144-273-138.

## ABSTRACT

Neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein present in a wide variety of tissues and cell types. Dysregulated expression of NGAL in human malignancies suggests its value as clinical marker. A growing body of evidence is highlighting NGAL's paradoxical (i.e. both beneficial and detrimental) effects on cellular processes associated with tumor development (proliferation, survival, invasion, and multidrug resistance). This review (i) summarizes our current knowledge of NGAL's expression profiles in solid tumors and leukemias, and (ii) critically evaluates the beneficial and detrimental activities of NGAL having been documented in a large diverse range of cancer-derived cell lines. A better understanding of the causal relationships between NGAL dysregulation and tumor development will require a fine analysis of the molecular aspects and biological role(s) of NGAL both in primary tumors and at different stages of disease. Having an accurate picture of NGAL's contribution to tumor progression is a prerequisite for attempting to modulate this protein as putative therapeutic target.

**Keywords:** neutrophil gelatinase-associated lipocalin (NGAL); MPP-9; cancer; survival; invasion; signaling; drug resistance.

## Highlights

NGAL can be employed as diagnostic and/or prognostic biomarker in human cancers

NGAL's roles in cancer raise more questions than answers

<footnote><sup>a</sup> INSERM UMRS 1138, Cell Death and Drug Resistance in Lymphoproliferative Disorders Team, Centre de Recherche des Cordeliers, Paris, France.</footnote>

<footnote><sup>b</sup> Sorbonne Universités Paris Cité, F-75006 Paris, France</footnote>

<footnote><sup>c</sup> Université Paris Descartes Paris 05, F-75006 Paris, France</footnote>
# 1. Introduction

Human neutrophil gelatinase-associated lipocalin (NGAL) is a 25 kDa glycosylated protein from the lipocalin family.¹ The lipocalins' common secondary and tertiary structure corresponds to a single, eight-stranded antiparallel β-barrel around a central pocket that is capable of binding low-molecular-weight ligands.¹ NGAL was initially characterized as an antibacterial immune factor via the pocket's ability to capture siderophores (such as bacterial enterochelin and mammalian endogenous catechols) that bind iron with high affinity, causing iron depletion and thus the inhibition of bacterial cell growth.¹,² A 30 kDa isoform of NGAL has been described, and probably results from differential glycosylation.³ NGAL also exists as a 46 kDa disulfide-linked homodimer and a 130 kDa heterodimer bound to the inactive zymogen form of the matrix metalloproteinase MMP-9 (proMMP-9).¹,⁴ Indeed, NGAL was first purified from human neutrophils because of its ability to bind proMMP-9 into a disulfide-linked complex;⁵ the Cys-87 in NGAL forms a disulfide bond with an as yet unidentified cysteine residue in MMP-9's hemopexin domain.¹ Excellent reviews have described the genomic organization of the NGAL gene and the protein's three-dimensional structure, as well as its expression profiles in adult and fetal tissues, and biological fluids.¹, ⁴, ⁶

There is now evidence to suggest that NGAL may be a marker of disease status in chronic and acute pathological conditions in general and in inflammatory, metabolic, neurologic and cancer diseases in particular.², ⁴, ⁷⁻⁹ For example, urine and blood levels of NGAL monomer increase following acute kidney injury (nephron epithelia damage).⁷, ¹⁰, ¹¹

The initial functional studies investigated the role of lipocalin-2 (Lcn-2, the murine homolog of human NGAL) in a mouse model.¹, ¹², ¹³ However, Lcn-2 exhibits little homology with human NGAL (62%), and notably does not contain the unpaired cysteine that can form the NGAL homodimer and the NGAL/proMMP-9 heterodimer in humans.¹ These facts are crucial when analyzing the specific roles attributed to NGAL in humans which might be distinct from that of Lcn-2 in mice.¹, ², ⁴, ¹², ¹³ For example, human NGAL is not involved in myeloid cell apoptosis or acute response in contrast to what was previously reported for Lcn-2 in mice.¹⁴ A growing number of studies have explored the NGAL's possible roles in various models of cancer, and suggest that the protein has both beneficial and detrimental functions.¹, ², ⁴, ¹³ Although ongoing studies are investigating the value of the NGAL-proMMP-9 complex as a marker of disease status in inflammation and cancer, there are still no detailed data on its full functional significance in this disease.¹, ¹⁵ Here, we briefly review the current literature on NGAL's expression profiles (both free and complexed to proMMP-9) in solid tumors and leukemias, evaluate the positive or negative effects of free NGAL observed in various models of cancer, consider the remaining challenges, and discuss the prospects for determining NGAL's functional value in cancer.

# 2. NGAL as a Biomarker in Cancer

NGAL is regularly expressed in a large variety of cell types (including adipocytes, hepatocytes, pneumocytes, splenocytes, mesangial and microglial cells, renal epithelial cells and vascular smooth muscle cells).¹,⁴ Concerning the immune system, during hematopoiesis, immature (CD34⁺) bone marrow progenitor cells express NGAL.¹⁶ During the maturation of granulocyte precursors in the bone marrow, NGAL is synthesized almost exclusively by myelocytes and metamyelocytes.¹⁷ Expression of NGAL and its 130 kDa complex is also observed in activated monocytes and neutrophils,⁵,¹⁶ and the dimer is the major molecular form of free NGAL secreted by neutrophils.¹⁸ To date, NGAL protein expression has never been reported in resting B and T lymphocytes. Circulating low levels of the protein (mainly as the dimer) are detected in the urine and blood of healthy subjects.¹⁸, ¹⁹ The main sources of circulating NGAL are thought to be neutrophils and renal epithelial cells.⁷, ¹⁸

## 2.1 Solid Tumors

A number of recent reviews have reported NGAL dysregulation in cancer.¹, ⁴, ¹², ¹³, ²⁰ Quantitative measurements of NGAL protein and mRNA levels performed in blood, urine and tissues, show that NGAL is overexpressed in non-microbe-associated cancers (including breast, brain, ovarian, endometrial, pancreatic, colorectal, bladder, prostate, liver, lung and skin cancers).¹, ⁴, ¹², ¹³, ²⁰⁻²³ In striking contrast, one study shows that NGAL is downregulated in primary malignant and metastatic tissues of oral cancer compared to normal tissues.²⁴

The abnormally elevated levels of NGAL in most cancers appear significantly correlated with disease severity and poor survival.¹, ⁴, ⁷⁻⁹, ¹³, ²⁰, ²⁵ For example, studies on breast cancer suggest the usefulness of serum NGAL in monitoring disease progression²⁶ and the association of serum NGAL with reduced survival.²⁷ NGAL appears to be a diagnostic biomarker of advanced or recurrent ovarian cancer²⁸ and pancreatic cancer.²² Although serum NGAL levels increase in patients with colorectal cancer,²⁹, ³⁰ NGAL does not seem to be
suitable as a diagnostic biomarker,²⁹ but can have a prognostic utility in metastatic patients.³⁰ ³¹ NGAL levels in gastric tumor tissues are associated with worse survival.³² NGAL is not useful for diagnosing renal cell carcinoma,³³ but it may be helpful to select a proper therapy in cases of metastatic disease without the need for pretreatment biopsy.³⁴ Interestingly, Roli et al recently reevaluated the potential value of NGAL as a prognostic and diagnostic marker in cancer.³⁵ Their meta-analysis shows that high NGAL levels in biological fluids (serum, urine) could be useful to predict disease-free survival for patients with colorectal and breast cancer, but its prognostic and diagnostic accuracy remains uncertain for other human tumors (including pancreatic, thyroid, liver, lung, esophageal, oral and kidney).³⁵ Note that in the cancers evaluated, the inclusion of single studies enrolling a limited number of patients influence the results by overestimating the effect size.³⁵

Few studies investigated the value of the 130 kDa NGAL complex as a marker of disease status in several solid tumors.²⁶, ³³, ³⁶⁻³⁸ The expression of the NGAL complex often correlates with the aggressive behavior of cancer cells (gastric, anaplastic thyroid, breast, kidney, oral).²⁶, ³², ³³, ³⁶, ³⁸ In endometrial cancer, the NGAL complex may be useful in the assessment of tumor stage before surgical treatment.³⁹

## 2.2 Leukemias

Acute and chronic lymphoid/myeloid leukemias are clonal disorders that result from the neoplastic transformation of hematopoietic progenitor cells. These diseases are characterized by the survival and expansion of clonal progenitors, cell dissemination from the bone marrow into the blood and peripheral tissues, and often resistance to chemotherapy.⁴⁰⁻⁴³ More precisely, acute lymphoblastic leukemia (ALL) is a heterogeneous disease that includes B and T-ALL cancers.⁴⁰ Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B lymphocytes (CD19⁺, CD5⁺, and CD23⁺) in the peripheral blood, bone marrow, and secondary lymphoid organs.⁴¹ Acute myeloid leukemia (AML) is a highly heterogeneous disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development.⁴² Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder that originates from a pluripotent stem cell expressing the Ph chromosome (t(9;22) chromosomal translocation) with a constitutively active BCR-ABL fusion gene, leading to the production of the p210 BCR-ABL protein.⁴³ The NGAL complex is found in blood tumor cells from patients with all these types of leukemia.¹⁵, ⁴⁴⁻⁴⁷ While overexpression of free NGAL is observed in blood cells from AML, CLL and CML patients,¹⁵, ⁴⁴⁻⁴⁷ there are still no data on NGAL's expression in ALL. Paradoxically, the levels of bone marrow NGAL transcript are found to be lower in AML patients than in healthy individuals, and these levels recover to normal values following complete remission and then decline again at relapse.⁴⁸ In patients with AML, higher bone marrow NGAL mRNA expression is observed in individuals with a good prognosis than in individuals with a poor prognosis, and independently of the French-American-British (FAB) classification.⁴⁹ Moreover, patients with higher expression levels of NGAL mRNA in the bone marrow in combination with the wild type FLT3-ITD sequence have better prognoses.⁴⁹ The prognostic or predictive value of NGAL (dimer and/or monomer, free and complexed) in the serum of patients with AML, ALL or CLL remains to be determined. With regard to CML, inhibition of the constitutive tyrosine kinase activity of p210 BCR-ABL⁴³ with imatinib alleviates the hyperproliferation-induced symptoms.⁵⁰ Furthermore, serum levels of NGAL are significantly higher in CML patients than in healthy individuals.⁴⁶, ⁴⁷, ⁵¹, ⁵² If CML patients achieve complete molecular remission after imatinib therapy, NGAL serum levels fall and are significantly lower than during the full-blown disease.⁴⁶, ⁵¹ These findings indicate that NGAL is a useful marker of CML's response to treatment, and strongly suggest the existence of a functional link between NGAL and BCR-ABL.

## 3. Detrimental and Beneficial Effects of NGAL in Cancer

In normal tissues, NGAL serves to provide protection against bacterial infection and modulate oxidative stress.¹ Few studies have investigated NGAL's physiological function(s) in the hematopoietic system by treating cells with recombinant human NGAL (rhNGAL).¹⁴, ⁵³⁻⁵⁵ NGAL does not appear to influence the balance between survival and death of bone marrow stem and progenitor CD34⁺ cells, mature granulocytes, and T and B lymphocytes,¹⁴, ⁵³ whereas it blocks the maturation of lineage-committed myeloid cells into mature erythrocytes and monocytes.⁵³ In contrast, a study performed by Lu et al.⁵⁴ suggests that NGAL induces the death of bone marrow CD34⁺ cells through the production of reactive oxygen species. Moreover, it seems that NGAL may favor the differentiation of bone marrow and mesenchymal stem cells into osteoblasts and fibroblasts, respectively.⁵⁴ Another study demonstrated that NGAL increases the number of T-regulatory cells (Tregs) in the peripheral blood mononuclear cell population by up-regulating the human leukocyte antigen G (HLA-G) complex, which is a mediator of tolerance.⁵⁵ This observation may have important implications for cancer, since a number of preclinical and clinical studies have linked the presence of Tregs to an increased risk of carcinogenesis and cancer development.⁵⁶
Below, we summarize current knowledge of NGAL's divergent functions in cancer (Table 1). Several investigative techniques have been used to study NGAL's role in tumor models: (i) treatment of cells with rhNGAL or neutralizing NGAL antibodies (NGAL Abs), and (ii) stable overexpression or knockdown (using siRNA) of NGAL expression with sense or antisense NGAL cDNA.

In different models of human cancer (lung, thyroid, gastric, and breast cancer), NGAL facilitates the survival and proliferation of malignant cells.<sup>37, 57-61</sup> In lung carcinoma, NGAL might protect against oxidative stress by activating the nuclear factor E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway<sup>58</sup> and inducing the expression of heme oxygenase-1 and superoxide dismutase 1,2.<sup>62</sup> Paradoxically, NGAL inhibits the proliferation and invasion of liver carcinoma cells, and this inhibition is associated with the blockade of the JNK and PI3/Akt signaling pathways.<sup>63</sup> In a model of advanced pancreatic cancer, NGAL reduced invasion (by suppressing FAK activation) and inhibits angiogenesis (by blocking VEGF production).<sup>64</sup> In contrast, NGAL increases the motility and invasion of colon carcinoma cells by modifying the subcellular localization of E-cadherin and Rac1 (one of the Rho small GTPases) through an iron-dependent mechanism.<sup>65</sup> These data are consistent with reports in which NGAL favors the migration and invasion of endometrial cancer and cholangiocarcinoma cells.<sup>61, 66</sup> Several research groups have already analyzed NGAL's role in multidrug resistance.<sup>24, 49, 61, 67-71</sup> While NGAL does not interfere with doxorubicin resistance in breast cancer cells,<sup>71</sup> it might favor the intracellular accumulation of other chemotherapeutic drugs in breast cancer,<sup>23</sup> renal cancer,<sup>69</sup> glioblastoma,<sup>68</sup>, oral squamous cancer<sup>24</sup> and leukemic AML<sup>49</sup> cell lines. In contrast, elevated NGAL levels might contribute to drug resistance in endometrial cancer,<sup>61</sup> and non-small cell lung cancer cells.<sup>67</sup> Thus, NGAL appears to exhibit negative or positive effects on tumor progression, depending on the type of cancer in question (Figure 1).

Whether NGAL bound to proMMP-9 retains a function has not yet been established. By binding cell surface receptors, NGAL and proMMP-9 can initiate signal transducing events that control tumour cell processes.<sup>15</sup> It is therefore legitimate to suggest that the NGAL complex could interfere with the binding of NGAL and/or proMMP-9 to their respective receptors, thus modulating signalling events induced by free NGAL and/or proMMP-9.

## 4. Conclusion and Future Questions

In molecular terms, there is increasing evidence of crosstalk between NGAL and cancer. There is a growing body of evidence suggesting that NGAL overexpression in tumors results from stimuli (including hypoxia and inflammatory cytokines) present in the tumor microenvironment.<sup>72</sup> The NF-κB signaling pathway, activated in most cancers including leukemias, regulates the transcription of NGAL<sup>1</sup> and the MAPK pathway may cooperate with NF-κB to up-regulate the expression of NGAL.<sup>1</sup> Moreover, epigenetic modifications might be important in initiating NGAL expression in the tumor cells. This may explain the increased levels of NGAL in most cancers. It remains to identify the specific molecular forms of NGAL (in serums and in cells) associated with a specific type of cancer (solid or liquid).

In functional terms, NGAL appears to exhibit either beneficial or detrimental effects through the modulation of proliferation, survival, migration, invasion, angiogenesis and drug resistance (Figure 1) - all cellular events considered to be hallmarks of cancer.<sup>73</sup> How, then, can NGAL's opposing effects in cells be explained? One may consider that the one or more functional roles of NGAL expressed by tumor cells are intrinsically linked to a specific type of cancer. However, several parameters have to be taken into account when assessing NGAL's roles in malignant cells. Firstly, all of the above-mentioned functional studies used cancer-derived cell lines that might not reflect the progressively malignant stages of a given cancer. Secondly, it is possible that our understanding of NGAL's pro-/anti-tumor activities has been conditioned by the different technical approaches used in the studies. Indeed, NGAL overexpression and silencing probably target intracellular NGAL localization, whereas rhNGAL and NGAL antibodies mimic and target extracellular NGAL. As seen in leukocytes, inside-out and outside-in signaling mechanisms often overlap.<sup>74</sup> Alternatively, NGAL's actions inside and outside cells may be exerted through distinct mechanisms. For example, the study by Tong et al.<sup>57</sup> indicated that inhibiting intracellular NGAL expression with siRNA in A549 lung cancer cells increases cell death, whereas cell treatment with rhNGAL (even at high doses) had no effect on cell viability. Finally, in the functional studies using rhNGAL, the NGAL receptor involved in the cellular events has not been characterized; this might also explain NGAL's divergent effects.

Indeed, two distinct cell surface receptors were initially identified for NGAL: low-density lipoprotein receptor-related protein-2 (LRP-2, also known as megalin)<sup>75</sup> and the solute carrier family 22 member 17 (also known as SLC22A17 and NGAL-receptor 2).<sup>76</sup> LRP-2 internalizes a variety of unrelated ligands, including nutrients, hormones and their carrier proteins, signaling molecules, and extracellular proteins.<sup>77</sup> Both LRP-2 and NGAL-R2 bind both free and iron-bound NGAL, and promote the latter's endocytosis.<sup>1, 75, 76</sup> Two splice variants of SLC22A17 (designated as NGAL-R1 and NGAL-R3) have also been identified, and appear to be involved in NGAL-mediated transport inside cells.<sup>78</sup> It remains to be determined whether all these receptors are able to bind all the different forms of NGAL (monomer, dimer and complex). Accordingly, the paradoxical effect of
rhNGAL on the survival of bone marrow CD34⁺ cells might possibly be related to molecular differences between the distinct sources of purified rhNGAL (Table 1).¹⁴,⁵⁴ Moreover, the existence of distinct NGAL receptors suggests that the signaling pathways through which cellular responses are induced might be distinct or similar. These key signaling pathways might be involved singly or concurrently in tumor progression. Importantly, the NGAL receptors have not been characterized in most cancers - including leukemias -. For each type of cancer studied, characterization of NGAL's molecular forms, receptors and related signaling mechanisms will be essential for gaining a better understanding of the role of tumor NGAL in cancer progression. Furthermore, greater knowledge of the intracellular signaling pathways induced by NGAL might provide a molecular basis for targeted therapeutic approaches.

Novel therapies are needed to overcome resistance to chemotherapeutic drugs, and the identification of novel, eligible cancer targets is always of general interest. Given the abnormally high levels of NGAL secreted in disease settings, it has been suggested that NGAL is a therapeutic target. Few studies have investigated the role of NGAL in cancer drug resistance (Table 1). Unexpectedly, NGAL contributes to both drug resistance and sensitivity, depending on the chemotherapeutic drug and the type of cancer in question. These findings therefore provide the rationale for further investigations of NGAL putative utility as a drug target.

In conclusion, dysregulation of NGAL expression is observed in tumor cells. NGAL now appears to be a pleiotropic cytokine, but an essential question for clinicians and scientists is whether NGAL participates to the disease process as a "saint" or a "sinner". The challenge is now to solve all the above-mentioned questions and thus establish an integrated model of NGAL's actions in cancer.

**5. Search Strategy and Selection Criteria**

Data for this Review were identified by searches of PubMed, and references from relevant articles using the search terms "neutrophil gelatinase-associated lipocalin", "NGAL", "lipocalin", "cancer", "solid tumor", "neoplasma", "hematological disease" and "leukemia". Only articles published in English between 1993 and 2018 were included.

**Funding**

Not applicable in the preparation of this review. The author's published work cited in the review was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM).

**Author's contributions**

BB and SAS contributed equally to the writing the manuscript. BB performed the table and the figure.

**Conflict of interest**

The authors declare no conflict of interest.

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<table><caption>Table 1. Beneficial and Detrimental Effects of NGAL in Cancer.</caption><thead><tr><th>Cell type</th><th>Investigative technique</th><th>Impact of NGAL on cell processes</th><th>Ref.</th></tr></thead><tbody><tr><td><strong>Solid Cancer</strong></td><td></td><td></td><td></td></tr><tr><td>Lung (A549 cells)</td><td>siRNA anti-NGAL</td><td>Survival ↑ Antioxidant (ROS ↓)</td><td>57</td></tr><tr><td>Lung (A549 and PC9 cells)</td><td>siRNA</td><td>Proliferation ↑ Antioxidant (Nrf2/HO-1 ↑)</td><td>58</td></tr><tr><td>Lung and liver (A549 and HepG2 cells)</td><td>siRNA NGAL overexpression</td><td>Antioxidant (HO-1 & SOD1/2 ↑)</td><td>62</td></tr><tr><td>Anaplastic thyroid (FRO cells)</td><td>rhNGAL (*) siRNA anti-NGAL</td><td>Survival ↑</td><td>59</td></tr><tr><td>Gastric (MGC-803 and SGC-7901 cells)</td><td>siRNA</td><td>Survival & Proliferation ↑</td><td>60</td></tr><tr><td>Liver (SK-Hep-1 cells)</td><td>rhNGAL (R&D) NGAL overexpression</td><td>Proliferation & Migration ↓</td><td>63</td></tr><tr><td>Pancreatic (Paca cells)</td><td>siRNA NGAL overexpression</td><td>Invasion & Angiogenesis ↓</td><td>64</td></tr><tr><td>Colon (KM12C, HCT116, and DLD1 cells)</td><td>siRNA NGAL overexpression</td><td>Cell-cell adhesion ↓ Migration ↑</td><td>65</td></tr><tr><td>Cholangiocarcinoma (RMCCA-1 cells)</td><td>siRNA</td><td>Migration & Invasion ↑</td><td>66</td></tr><tr><td>Endometrial (HHUA and RL95-2 cells)</td><td>siRNA overexpression</td><td>Survival & Migration ↑ Cisplatin resistance ↑</td><td>61</td></tr><tr><td>Squamous cell carcinoma (SAS cells)</td><td>siRNA</td><td>Survival & Migration ↓ Cisplatin Resistance ↓</td><td>24</td></tr><tr><td>Non-small-cell lung (H441, and H3255 cells)</td><td>siRNA NGAL overexpression</td><td>Erlotinib Resistance ↑</td><td>67</td></tr><tr><td>Glioblastoma (U87MG, U373MG, and T98G cells)</td><td>NGAL overexpression</td><td>Carmustine Resistance ↓ Cell Death ↑</td><td>68</td></tr><tr><td>Renal cancer (Caki-1, A498, and ACHN cells)</td><td>rhNGAL(Pfizer, Sigma) NGAL overexpression</td><td>Sunitinib Resistance ↓</td><td>69</td></tr><tr><td>Breast (MDA-MB-23 cells)</td><td>rhNGAL (R&D, Sino Biological Inc.) siRNA</td><td>Rhodamine-123 Resistance ↓</td><td>70</td></tr><tr><td>Breast (MCF-7 cells)</td><td>NGAL overexpression</td><td>Proliferation & Angiogenesis ↑ No effect on Doxorubicin Resistance</td><td>37<br>71</td></tr><tr><td>Colorectal (HT-29 cells)</td><td>NGAL overexpression</td><td>No effect on Doxorubicin Resistance</td><td>71</td></tr><tr><td><strong>Leukemia</strong></td><td></td><td></td><td></td></tr><tr><td>AML (THP1 and OCI-AML3 cells)</td><td>NGAL overexpression</td><td>Cytarabine Resistance ↓ Cell death ↑</td><td>49</td></tr></tbody></table>

rhNGAL: recombinant human NGAL; ROS, reactive oxygen species; *, not commercially available rhNGAL; Nrf2: nuclear factor E2-related factor-2. Cisplatin and cytarabine inhibit DNA replication; carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea) and doxorubicin prevent DNA replication and transcription; erlotinib is an EGFR tyrosine kinase inhibitor; sunitinib is a multi-targeted receptor tyrosine kinase inhibitor. ↑ stimulation; ↓ inhibition.
**Figure 1. Schematic Diagram Illustrating the Putative Roles of NGAL in Modulating Major Cellular Processes.**

The synthesis and release of tumor NGAL (monomer, dimer or complex) increase in response to various stimuli (inflammatory cytokines, hypoxia etc). Extracellular NGAL binds to specific cell surface receptors (megalin, SLC22A7 isoforms) on tumor cells, and thus may activate or inactivate signaling pathways. In turn, this modulates proliferation, survival, migration, invasion, angiogenesis, immunotolerance and multidrug resistance - all events involved in tumor biology. A given cell type may be involved in the NGAL-mediated actions reported here to a variable extent. Intracellular NGAL might be directly involved in the modulation of cell responses. The NGAL complex's possible effects on tumor cells remain to be identified.