Role of Pirin, an Oxidative Stress Sensor Protein, in Epithelial Carcinogenesis

Simple Summary Pirin is a protein which is detected at low levels in normal tissues. However, it is detected at high levels in multiple cancers, particularly in melanomas, cervical cancer or squamous cell lung carcinomas. Essentially, its role in cancer is related to the host response against factors causing oxidative stress, favoring cell migration and metastasis. Here we summarize the biological functions of Pirin in relation to its role in cancer, suggesting that Pirin is a potential therapeutic target. Abstract Pirin is an oxidative stress (OS) sensor belonging to the functionally diverse cupin superfamily of proteins. Pirin is a suggested quercetinase and transcriptional activator of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Its biological role in cancer development remains a novel area of study. This review presents accumulating evidence on the contribution of Pirin in epithelial cancers, involved signaling pathways, and as a suggested therapeutic target. Finally, we propose a model in which Pirin is upregulated by physical, chemical or biological factors involved in OS and cancer development.


Introduction
The cupin superfamily of proteins is considered to be one of the most functionally diverse protein superfamilies. These proteins are characterized by their conserved β-barrel structure and two distinctive short signature sequence motifs: PGX5HXHX3,4-EX6G and GX5PXGX2HX3N, which contain residues responsible for binding to metal ions. There is also a 15-50 amino acid sequence located between both motifs [1][2][3][4]. Interestingly, a range of metal ions bind to the cupin active site, including nickel, iron, manganese, copper, zinc, and cadmium, accounting for biological functions of cupin proteins [5]. They display diverse enzymatic functions such as dioxygenases, isomerases, oxalate oxidases and non-enzymatic activities such as auxin binding, sucrose binding, seed storage, and transcriptional factor, in Archaea, Eubacteria, and Eukaryota [1,6,7]. Although the focus was initially given to the physiological functions of cupin proteins, new insights reveal this superfamily may The second function attributed to Pirin is acting as a transcriptional co-regulator. Initially, it was reported that Pirin interacts with the nuclear factor I/CCAAAT box transcription factor (NF-I) and binds to the hematologic oncogene B-cell lymphoma 3-encoded protein (BCL-3), forming complexes along with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p50 [11,17,18]. NF-κB is a ubiquitous transcriptional regulator considered central in immune response, apoptosis, inflammation, and oxidative stress (OS) [19][20][21]. More recently, it was described that under oxidizing conditions, Pirin metal structurally changes from Fe2+ (inactive form) to Fe3+ (active form) and functionally enhances the binding affinity to NF-κB p65, which suggests a previously unknown role of Pirin as an iron-dependent OS modulator [22]. However, the Pirin Fe center does not represent its enzymatic active site, but functions as an allosteric control site. Meanwhile, the R-shaped surface loop area of Pirin is structurally modified depending on the metal oxidation state, and thus, the two resulting conformational states, active and inactive, are crucial for Pirin's ability to regulate NF-κB [22]. In addition, the Fe3+ form of Pirin shows restricted conformational space and electrostatic complementarity which are crucial for NF-kB binding [23,24]. Notably, these redox mediator functions are also observed in plants and prokaryotes in which Pirin-like proteins regulate oxidative pathways and are closely related to cell death [25,26]. Additionally, Pirin is expressed at high levels in the kidney and spleen of transgenic cytosolic superoxide dismutase (Sod1)-deficient mice [27]. All the above findings are summarized in Table 1.
A bioinformatic analysis demonstrated that Pirin is functionally associated with Wiskott-Aldrich syndrome protein family member 2 (WASF2), Proteasome subunit alpha type-7 (PSMA7), Ras-related C3 botulinum toxin substrate 1 (RAC1), Nck-associated protein 1 (NCKAP1), Cytoplasmic protein (NCK1) and Abelson tyrosine-protein kinase 2 (ABL2) (Figure 2). WASF2 is involved in signal transmission from tyrosine kinase receptors to actin filaments leading to lamellipodia formation [28,29]. PSMA7 participates in the ATP-dependent degradation of ubiquitinated proteins [30]. RAC1 is a GTPase which is involved in a plethora of cellular processes such as cell growth, cytoskeletal reorganization, and protein kinase activation, among others [31,32]. NCKAP1 is part of the WAVE complex that regulates lamellipodia formation through the reorganization of actin filaments, is involved in cell invasion, and promotes cancer cell malignancy [33,34]. NCK1 is an adapter protein which interacts with tyrosine-phosphorylated growth factor receptors, playing a role in DNA damage response by activating downstream effectors. In addition, NCK1 is involved in actin cytoskeletal remodeling, podosome formation and cancer invasion [35,36]. ABL2 is a cytoplasmic tyrosine kinase which coordinates actin remodeling through tyrosine phosphorylation of proteins controlling cytoskeleton dynamics. Taken together, the functional association of Pirin with proteins involved in cytoskeleton reorganization partially explains the link between Pirin overexpression and increased cell migration in some cellular models [37,38]. Further experimental settings are warranted to elucidate the molecular mechanisms involved in these functional associations. Pirin functional association network. Filled nodes represent proteins with predicted 3D structures. Edges color, Purple: experimentally determined; light blue: association in curated databases; green: co-mentioned in PubMed abstracts (STRING V11.0, full STRING network, confidence 0.900) [39]. Abbreviations: WASF2: Wiskott-Aldrich syndrome protein family member 2; PSMA7: proteasome subunit alpha type-7; RAC1: Ras-related C3 botulinum toxin substrate 1; NCK1: cytoplasmic protein; NCKAP1: Nck-associated protein 1; ABL2: Abelson tyrosine-protein kinase 2.

Role of Pirin in Cancer Development
Recent investigations have been carried out on the contribution of Pirin in various types of cancer, including epithelial tumors [40] as well as those from the hematopoietic and neurological systems [41,42]. Additionally, Pirin has been reported as a potential molecular target of metastatic suppressors [43]. The following sections summarize the interaction of Pirin with carcinogenic agents and its potential role in epithelial carcinogenesis.

Lung Cancer
Lung cancer is the most commonly diagnosed cancer in women and men worldwide, with tobacco smoke (TS) being the predominant cause [44,45]. It has been reported that Pirin levels are remarkably raised in the airway epithelium of chronic smokers [46]. Likewise, cigarette smoke extracts upregulate Pirin levels in human bronchial epithelial cells in a dose-dependent manner [47]. Such Pirin upregulation induces apoptosis of epithelial cells which may be explained by an interaction between Pirin and NF-κB [47]. Another study showed increased Pirin levels in human small airway epithelial cells exposed to TS, suggesting a potential role of Pirin in TS-associated injury [48]. Accordingly, other studies demonstrated that Pirin is upregulated in bronchial epithelial cells exposed to cigarette smoke extracts and is accompanied by ferritin gene upregulation, a well-known marker of ferroptosis [49]. Furthermore, PIR has been identified as a novel nuclear factor erythroid 2-related factor 2 (NRF2)-modulated gene in the small airway epithelium of healthy smokers [50]. NRF2, an oxidant responding transcription factor highly active in lung cancer cell lines [51,52], can bind to the PIR promoter which contains four functional antioxidant response elements (ARE) [50,53]. This association between NRF2 and PIR may explain a possible OS-regulation in the tobacco smoke-exposed airway epithelial cells [50]. However, NRF2 activation/addiction may be involved in other Pirin-independent activities such as cisplatin resistance in lung cancer cells [54,55]. Additionally, increased Pirin expression is found in alveolar macrophages of mice exposed to TS for 1 to 3 months, suggesting that Pirin may also be involved in macrophage activation [56]. Of note, Pirin levels show a strong correlation with smoking, as well as chronic obstructive pulmonary disease (COPD) [57], which frequently progresses to lung cancer. Moreover, particulate matter in outdoor air pollution, considered to be a potent oxidative agent and a significant risk factor for respiratory diseases and lung cancer [58], is associated with Pirin overexpression in human respiratory fibroblasts [57]. Additionally, the telomerase RNA component (TERC) encoding gene, upregulated in non-small cell lung carcinoma (NSCLCs) [59], was found to be regulated by Pirin [60]. Taken together, the above findings lead us to suggest a potential role of Pirin in lung cancer, although additional studies are necessary to confirm these observations.

Cervical Cancer
Cervical cancer is the fourth most prevalent cancer among women, causing a total of 311,000 deaths in 2018 [61]. The persistent infection of high-risk human papillomavirus (HPV) is considered a necessary condition for cervical cancer development [62]. We demonstrated for the first time that Pirin is expressed in a HPV load-dependent manner in cervical cancer cells [63]. Additionally, it was demonstrated that PIR knockdown increases Ecadherin levels and reduces Slug, zinc finger E-box-binding homeobox protein (ZEB) and Snail in cervical cancer cells, suggesting its contribution to epithelial-mesenchymal transition (EMT) and cell migration [63]. However, there is an interaction between Pirin, B-cell lymphoma 3-encoded protein (BCL-3), and Slug in melanoma cells, whereas in cervical cancer cells Pirin induces EMT by decreasing E-cadherin expression independent of BCL-3-Slug signaling [38,64]. Interestingly, we found that Pirin levels are increased by HPV16 E6 and E7 oncoproteins in infected cervical cancer cells, unlike HPV-negative cells [63]. Moreover, curcumin, a common food additive and well-known antioxidant agent [65,66], has been reported to interfere with EMT in HeLa cells (HPV-18) and breast cancer cell lines [67][68][69]. Notably, we demonstrated that curcumin decreases Pirin levels and reduces EMT and cell migration, suggesting a novel Pirin-dependent mechanism wherein curcumin rescues cervical cancer cells from EMT [70]. It has been shown that basal Pirin expression is strongly dependent on NRF2, due to its crucial interaction with an ARE site located in a short region downstream of the transcription start site (TSS) [71]. Considering that Pirin is a NF-κB activator, we hypothesize that Pirin may act as a mediator between NRF2 and NF-κB in cervical cancer cells [71].

Skin Cancer
Skin cancer is the most common cancer in the United States when non-melanoma carcinomas are included in the registries [72], and approximately 100,000 new melanoma cases are diagnosed every year [73]. In this type of skin cancer, a relevant role in cell migration and progression was identified for Pirin [38,74]. Miyasaki et al. identified high expression of Pirin in melanoma cell lines. In addition to this discovery, they found that a small molecule named Triphenyl compound A (TPhA) blocks the interaction between Pirin and BCL-3, inhibiting cell migration [38]. It was also established that Pirin localizes in the nucleus or cytoplasm, depending on the stage of melanoma progression. In fact, a significant proportion of cytoplasmatic Pirin was found in metastatic melanoma cells compared to primary melanoma cells, suggesting that this pattern of Pirin localization may represent a cancer progression biomarker [74]. Moreover, the same group identified that Pirin is barely expressed in mature nevus samples compared to the high levels found in primary and metastatic melanomas. By inducing PIR knockdown, it was also found that metastatic melanoma cells change both morphology and size, which was compatible with a senescent phenotype. Altogether, these results suggest that Pirin contributes to the metastatic properties of melanoma cells [75].
It has been extensively reported that ultraviolet (UV) radiation is a major risk factor for melanomas [76]. It is important to note that UV radiation, specifically UV-A, induces NRF2 nuclear translocation and accumulation and may activate NRF2-controlled proteins in human skin cells [77]. Moreover, UV-A radiation promotes reactive oxygen species (ROS) production and subsequently triggers the activation of NRF2 in melanocytes [78]. Even though the mentioned findings consider Pirin regulator-NRF2 signaling pathways, there are still no studies that specifically assess the effect of UV radiation in Pirin levels. In miR-155-overexpressing melanocytic cancer cells (CL16-miR-155), Pirin shows less intensity stain than controls by immunohistochemistry (IHC) assays [79]. Since miR-155 overexpression may inhibit tumor dissemination, extravasation, and colonization, Pirin may mediate, whether directly or indirectly, metastasis development [79,80]. In addition to these findings, after treatment with Hsc025, a novel molecule that improves hepatic fibrosis-degree and stimulates wound healing, Pirin levels modestly increase in fibroblasts [81,82]. However, PIR knockdown significantly counteracts the effect of Hsc025 on fibroblast migration, suggesting that Pirin may be a key intermediary in this process [82]. More recently, another novel compound known as bisamide (CCT251236) was identified as a potent ligand of Pirin [83]. It is important to note that these molecules do not directly interact; in contrast, bisamide amide groups and Pirin Fe bind through a water-mediated interaction. Additionally, there is consistent evidence that bisamide modulates the expression of the transcription factor HSF1 and subsequently inhibits the migration of human melanoma cells WM266.4; however, Pirin participation remains unclear [83]. In light of these findings, it was recently demonstrated that both CCG-222740 and CCG-257081, which are compounds structurally similar to CCT251236 and possible inhibitors of metastatic and fibrotic signals, are also able to bind to Pirin [43]. Moreover, they show that such CCG compounds may disrupt Pirin expression, and subsequently interfere with MRTF/SRF/DNA signaling, which has been associated with melanoma metastasis [43].

Breast Cancer
In 2018, breast cancer was the second most prevalent cancer and the second most common cause of death among women in the United States [44,84]. Several genes have been shown to alter their expression pattern in human breast tumors, and some of these may have a role in predicting clinical prognosis [85,86]. One of these candidates includes the potential participation of the PIR gene [87]. Based on a previous report in which Pirin expression significantly varied between metastasis patients and non-metastasis patients [87], Shubbar et al. showed that Pirin levels in normal breast cells do not differ from invasive breast cancer samples. Nevertheless, Pirin levels are highly correlated with positive axillary lymph nodes status, suggesting a connection between high Pirin levels and local metastasis [88]. Furthermore, PIR knockdown resulted in a noticeably reduced cell proliferation rate in breast cancer cells and decreased xenograft tumor growth in mice [89]. In addition, Pirin mediates breast tumorigenesis by promoting E2F1 expression, a key cell cycle regulator that is abnormally active in malignant tumors [89][90][91]. In fact, Pirin binds to the 3 -terminal region of the E2F1 promoter and subsequently facilitates G1 to S phase transition in breast cancer cells [89].

Head and Neck and Gastrointestinal Cancers
Other epithelial cancers such as oral, pancreas, biliary tract, and colorectal carcinomas have also been linked with changes in Pirin expression. First, we demonstrated that Pirin is overexpressed in oral cells expressing HPV16 E6 and E7 oncoproteins [63]. To confirm these results in clinical specimens, we evaluated the relationship between PIR gene expression and HPV status in head and neck carcinomas from The Cancer Genome Atlas (TCGA) consortium [92]. This analysis showed a statistically significant increase in PIR gene expression in HPV positive cases (evaluated by in situ hybridization) when compared with HPV negative ones (p = 0.02028, Welch's t-test) (Figure 3). Furthermore, both epidermal growth factor receptor/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (EGFR/MEK/ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathways may be involved in the activation of Pirin by HPV-E7 oncoprotein in oral cells [63], which is in line with a previous report on transformed rat fibroblasts [93]. More recently, we demonstrated that EGFR/PI3K/AKT1/NRF2 signaling promotes Pirindependent canonical NF-κB activation, which, in turn, promotes oral cell migration [37]. Altogether, this strongly suggests a novel carcinogenic mechanism by which Pirin is involved in oral cancer, although the clinical consequences remain to be determined. Similar to the aforementioned findings in lung cells [50], Pirin is strictly regulated by NRF2 in pancreas cancer cells [94]. Moreover, high Pirin levels are associated with a reduced survival probability in cholangiocarcinoma patients, suggesting that Pirin is a plausible prognostic biomarker [95].

Non-Epithelial Cancers
While Pirin has been widely studied in epithelial cancers, other studies have proposed it as a promising prognostic marker as well as an anti-proliferative therapeutic target of curcumin in hematopoietic and neurological tumors [42,97]. Pirin is downregulated in acute myeloid leukemias, and indeed, its ablation may impair myeloid differentiation and maturation in both humans and mice [41]. Furthermore, Pirin is associated with contrasting high levels of peripheral white blood cells in B-precursor acute lymphoblastic leukemia, which entails a distinctive prognosis in leukemia patients [98,99]. In Figure 4, PIR gene expression in 10,071 patients with epithelial and non-epithelial tumors extracted from TCGA consortium is shown [92]. As observed, PIR transcripts are differentially detected in tumors from a different origin (p < 0.0001, Welch's ANOVA test). Additionally, PIR is highly expressed in tumors including melanomas, lung, cervical or head and neck cancers among others when compared with leukemia, thymoma or diffuse large B-cell lymphoma (DOBC).

Conclusions
Pirin is an established OS sensor and part of the functionally diverse cupin superfamily. Similar to other cupin proteins, Pirin shows enzymatic properties and acts as a nuclear transcriptional regulator. However, its potential oncogenic activity has been a growing topic of discussion in the past years. Recent findings have shown that Pirin plays a role in the development of cancer in epithelial lung, skin, cervical, and oral tumors. Diverse factors (such as environmental and viral) promote ROS increase, consequently raising Pirin levels for activating NF-κB, cell proliferation, cell migration and EMT. In addition, HPV E7 oncoprotein promotes EGFR/PI3K/AKT/NRF2 pathway activation, leading to NRF2 recruitment in the PIR promoter, in turn increasing Pirin expression (Table 2 and Figure 5). This suggests a key participation of Pirin in cancer promotion and progression, although additional experimental approaches, including animal models, for testing Pirin-mediated tumorigenesis are warranted. Thus, this accumulating evidence provides auspicious signs of Pirin as a significant potential biomarker or therapeutic target in the years to come. Lung TS Activation Pirin levels are increased in airway epithelium of chronic smokers [46] TS Activation Pirin overexpression occurs in a dose-dependent manner [47] TS Activation Interaction with NF-κB resulting in a pro-apoptotic response [47] TS Activation Pirin overexpression is accompanied by ferroptosis markers upregulation [49] TS Activation Interaction with NRF2 in smoke-exposed airway epithelial cells [50] Cervical