The Autotaxin—Lysophosphatidic Acid Axis Promotes Lung Carcinogenesis

Lung cancer is the most prevalent form of malignancy and the leading cause of global cancer-related mortality; the prognosis for patients with lung cancer remains dismal, with a 5-year survival rate below 20%, for all disease stages combined. The major (85%) histological subtype of lung cancer is non–small cell lung cancer (NSCLC), which is further subdivided to adenocarcinoma (ADC; 40%), squamous cell carcinoma (SCC; 25%–30%), and large cell carcinoma (LCC; 10%–15%; ref. 1).

The molecular origins of lung cancer lie in complex interactions between the environment and host genetic susceptibility, further modulated by genetic and epigenetic changes, leading to changes in the activation status of oncogenes and tumor suppressor genes (1). However, lung cancer, and especially NSCLC, is considered as a group of distinct diseases with vast genetic and cellular heterogeneity and although some genomic alterations are shared among various histologic types, most alterations remain distinct (2). The identified genomic alterations are not always associated with the activation of the relevant cellular pathways and the corresponding phenotypic aberrations (3), thus further increasing the complexity of correlating genotypic and phenotypic data.

Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), more commonly known as autotaxin (ATX), is a secreted glycoprotein widely present in biological fluids. ATX catalyzes the extracellular hydrolysis of circulating and/or de novo produced lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA; ref. 4). LPA, whose levels are catabolized by phospholipid phosphatases (PLPP; ref. 5), is a pleiotropic phospholipid mediator that evokes growth factor–like responses such as cell growth, survival, differentiation and motility, in most cell types (6). The large variety of LPA effector functions is attributed to at least six G-protein coupled LPA receptors (LPAR), exhibiting overlapping specificities and widespread distribution (7). The orphan GPR87 and P2Y10 receptors, as well as the receptor for advanced glycation end products (RAGE) and the intracellular peroxisome proliferator-activated receptor γ (PPARγ), have also been suggested to mediate LPA signaling (4, 7).

ATX was originally isolated as an autocrine motility stimulation factor from the supernatant of metastatic melanoma cells; because increased ATX expression has been detected in several chronic inflammatory and/or malignant diseases (4, 8). ATX expression and LPA production have been shown to be essential for embryonic development (9), but largely dispensable for adult life (10), suggesting ATX/LPA as a developmental pathway aberrantly re-activated in pathophysiological situations. Transgenic overexpression of ATX or LPARs specifically in the mammary gland resulted in spontaneous breast cancer in aged mice (11), suggesting that ATX/LPA may amplify oncogenic age-related signals. Moreover, conditional genetic deletion of ATX from hepatocytes was recently shown to attenuate the development of both liver fibrosis and hepatocellular carcinoma (HCC; ref. 12).

Increased ATX and LPA levels have been identified in the lung tissue and bronchoalveolar lavage fluid (BALF), respectively, of patients with idiopathic pulmonary fibrosis (IPF), whereas genetic deletion of Enpp2 or Lpar1 have been shown to attenuate the development of the modeled disease (13, 14). Therefore, given that pulmonary fibrosis is associated with an increased risk for lung cancer (15), we herein aimed to further investigate the potential involvement of the ATX/LPA axis in lung cancer pathogenesis. ENPP2 was in silico discovered to be genetically altered in some patients with lung cancer, whereas increased ATX staining and activity were detected in patients and animal models, accompanied by a downregulation of PLPP3 expression. Most importantly, genetic deletion of Enpp2 or Lpar1 in mice resulted in disease attenuation, thus confirming a procarcinogenic role of ATX/LPA in the lung.

Demographics and clinicopathological characteristics of patients and controls are presented at Supplementary Tables S1 and S2. Clinical investigations were conducted according to the principles of the Declaration of Helsinki, after approval by an Institutional Review Board. All patients and controls consented in writing for anonymous research usage of their biological samples.

All mice were bred under specific pathogen-free conditions in their respective genetic backgrounds for over 10 generations at the animal facilities of the Alexander Fleming Biomedical Sciences Research Center. Mice were housed at 20–22°C, 55% ± 5% humidity, and a 12-h light-dark cycle; water and food were given ad libitum. All reported experimentation in mice for this project, in line with the ARRIVE guidelines, was approved by the Institutional Animal Ethical Committee of BSRC Alexander Fleming and the Veterinary service and Fishery Department of the local governmental prefecture. All randomly-assigned experimental controls were littermate sex- and age-matched mice.

The generation and genotyping protocols for Enpp2^n/n (9), Lpar1^−/− (16), Lpar2^−/− (17), K-ras^G12D/+ (18), TgCC10-Cre and TgLysM-Cre (14) have been described previously. All mice, except for Lpar1^−/−, were bred in a C57BL/6J genetic background. The oncogenic allele of K-ras (G12D; ref. 18) was activated upon the intratracheal delivery of 2.5 × 10⁷ pfu/mouse of an adenovirus expressing Cre recombinase, as previously reported (19) and as described in detail in an Online Data Supplement. Chemically induced lung cancer was provoked upon weekly intraperitoneal (i.p.) injections of 1 g/Kg urethane (URE; ethyl carbamate) for ten consecutive weeks, as previously reported (20) and as described in an Online Data Supplement.

Histology, immunohistochemistry, ATX activity assay (TOOS), HPLC-ESI/MS/MS lipid analysis, RNA Extraction and Real-time RT-PCR were performed essentially as previously reported (12, 14, 20, 21) and as detailed in an Online Data Supplement.

Microarray hybridization and expression data analysis are described in an Online Data Supplement. The MIAME-compliant dataset is publicly available (#E-MTAB-6706) at ArrayExpress.

Unless otherwise indicated, statistical significance was estimated through pair-wise comparisons with control values using an unpaired, two-sided Student t test or with a Mann-Whitney Rank Sum Test in cases of not normal distribution.

To explore a possible role of the ATX/LPA axis in lung cancer, we first examined the genomic status of ENPP2, as well as of LPARs and PLPPs, in patients with lung cancer. Genomic Identification of Significant Targets in Cancer (GISTIC) analysis was performed online in cBioPortal (22) on publicly available whole-exome sequencing data from the recent landmark genomic study of lung cancer (23), as deposited to The Cancer Genome Atlas (TCGA). ENPP2 was found genetically altered in 8% of patients with lung cancer (n = 1,144; Supplementary Fig. S1A); almost 50% of the identified alterations were gene amplifications that were accompanied by MYC, as well as other genes in the 8q24 genomic locus, amplification in the same patients (Supplementary Fig. S1B). Alterations in other genes of the ATX/LPA axis were much fewer (Supplementary Fig. S1A).

To assess steady-state mRNA expression of ENPP2, as well as LPARs and PLPPs, in human lung cancer patients' lung tissue, we performed data mining in 41 lung cancer patient microarray datasets publicly available at NCBI/GEO (Supplementary Table S1) with the embedded tool GEO2R. In sharp contrast with a similar in silico query in HCC (12), ENPP2 mRNA expression was found to be statistically significantly downregulated in lung cancer samples, as compared with controls, in most of the patient cohorts' datasets (Supplementary Table S1 and Supplementary Fig. S2A). PLPP3 expression in patients with lung cancer was consistently found downregulated, as previously shown with the same methodology in patients with HCC (12). Identical results were obtained from qRT-PCR analysis of 14 patients with NSCLC paired lung samples, where ENPP2 and PLPP3 mRNA expression was found lower in cancer biopsies as compared with adjacent healthy lung tissue isolated from the same patients (Supplementary Fig. S2B), thus suggesting deregulated LPA metabolism in patients with lung cancer.

To examine pulmonary ATX protein levels, we performed ATX immunohistochemistry in lung tissue biopsies of 281 patients with lung cancer (Table 1), in comparison with 10 healthy controls, spotted in a tissue microarray (TMA) to ensure identical procedures and facilitate direct comparisons. As previously shown (14), ATX immunostaining in the healthy lung tissue was localized mainly to the bronchial epithelium and to a lesser extent in alveolar macrophages (Fig. 1A). Moderate to intense ATX staining was detected in lung cancer as compared to healthy lung tissue (Fig. 1A–C), found localized in cancer cells and negligible in the surrounding stroma (Fig. 1A). However, no statistically significant associations of the intensity of ATX staining with any demographic or clinicopathological characteristics were observed (Table 1). Moreover, increased serum ATX activity levels, as detected with the TOOS assay on natural LPC substrates, were found in a different patient with lung cancer cohort (n = 68) in comparison with control samples (n = 19; Supplementary Table S2). Therefore, lung cancer in humans is associated with increased levels of ATX protein and activity, corroborating a possible involvement of the ATX/LPA axis in lung cancer pathogenesis.

Remarkably, a similarly paradoxical ATX expression profile was detected as well in two animal models of lung cancer, the genetically engineered model of the conditionally activated K-ras^G12D oncogene (18) and the URE-induced chemical carcinogenesis model (20). In both these models ATX staining was detected in lung cancer cells (Fig. 2A and B; Supplementary Fig. S3A and S3B), whereas ATX activity levels were found increased in the BALFs of the same mice (Fig. 2C and D), correlating with total protein levels (Fig. 2E and F; r² = 0,79/0,78, P = 0.001/0.002, respectively). In the K-ras^G12D–driven model, unlike the URE-induced one, higher ATX activity levels were also observed in the plasma (Fig. 2G and H). In sharp contrast, pulmonary Enpp2, as well as Plpp3, mRNA expression was found downregulated in both animal models (Supplementary Fig. S4A–S4D), as shown for human patients. No major changes in the expression profile of LPARs was noted (Supplementary Fig. S4E and S4F).

To examine whether the observed increased pulmonary ATX levels in lung cancer play a functional role in lung carcinogenesis, Enpp2 was conditionally deleted from bronchial epithelial cells and macrophages, the main pulmonary cells expressing ATX (14). To this end, Enpp2^n/n mice (9) were mated with transgenic mouse strains expressing the Cre recombinase under the control of the CC10 (Scgb1a1) or the LysM promoters (14). As previously shown, CC10/LysM-directed genetic deletion of ATX, as verified with recombination assays, did not have any appreciable effects on lung development and architecture under healthy conditions (14). URE (1 g/Kg) was then injected intraperitoneally (IP) for 10 consecutive weeks to C57BL6/J CC10Enpp2^−/−or LysMEnpp2^−/−mice, as well as to control littermates (Enpp2^n/n), and disease severity was examined 15 weeks after the last injection.

As expected (20), all control mice that received URE developed neoplastic lesions (100% incidence), ranging from atypical adenomatous hyperplasias (AAH) to adenomas (AD; Fig. 3A). Inflammation was not a prominent histopathologic feature, although many macrophages (giant cells) were noted near AAH lesions (Fig. 3A). Genetic deletion of Enpp2 in both CC10Enpp2^−/−or LysMEnpp2^−/−mice resulted in a statistically significant decrease in the number of URE-induced AAH (Fig. 3B and C), indicating a role for pulmonary ATX expression at the initial phases of URE-induced carcinogenesis. However, no statistically significant decreases in the number of AD were noted (Fig. 3D and E); moreover, the genetic deletions diminished but did not attenuate ATX BALF levels (Fig. 3F and G), suggesting extra-pulmonary sources of ATX.

Genetic deletion of Lpar1 has been shown to attenuate the development of bleomycin-induced pulmonary fibrosis (13), as was also shown for ATX (14), whereas pulmonary fibrosis is associated with an increased risk for lung cancer (15). Therefore, we next examined a possible contribution of LPAR1 in lung cancer development, through the administration of URE to Lpar1^−/− mice and their WT littermates. It should be noted that since C57BL/6J Lpar1^−/− mice are embryonically lethal (data not shown and ref. 16), Lpar1^−/− mice were bred and maintained on a mixed C57BL/6J/129 genetic background, which is known to be more susceptible to URE-induced carcinogenesis (24). In accordance, WT C57BL/6J/129 mice developed an increased number of ADs, as compared to WT C57BL/6J mice, upon URE administration (Fig. 4A–C). Deletion of Lpar1 resulted in a reduction of the numbers of URE-induced ADs (Fig. 4C), confirming the participation of the ATX/LPA axis in lung cancer development. On the contrary, no such effect was observed in C57BL/6J Lpar2^−/− mice (Fig. 4D–F), perhaps in line with a suggested anti-inflammatory role for LPAR2 in allergic airway inflammation (25).

To confirm the procarcinogenic role of the ATX/LPA axis in another animal model of lung cancer, we next used the genetically engineered model of K-ras^G12D/+(18). A replication-deficient adenovirus expressing the cre recombinase (Ad-Cre) was administered intratracheally to K-ras^LSL-G12D/+Enpp2^n/nmice, as well as to control K-ras^LSL-G12D/+littermates, thus removing the synthetic stop element (LSL; lox-stop-lox) and allowing the expression of the oncogene and the overtime development of lung cancer, ranging from AAH to AD and ADC (18). At the same time, the adenoviral-delivered cre also deleted Enpp2 in the infected cells. Mice were then sacrificed 8 or 16 weeks post Ad-Cre administration and examined for neoplastic lesions (Fig. 5A and B). The stochastic genetic deletion of Enpp2 from pulmonary cells resulted in a significant reduction of AAHs at both time points (Fig. 5C and D), confirming the results from the URE model. A statistically non-significant reduction of ADs was observed at 8w post Ad-Cre administration, but not at 16w (Fig. 5E and F).

Noteworthy, the effect of the Enpp2 genetic deletion on ATX BALF levels in K-ras^G12D/+Enpp2^−/−mice was prominent at 8w post Ad-Cre administration but negligible at 16w (Fig. G and H), suggesting that pulmonary cells have a significant contribution to ATX BALF levels at the initial phases of lung cancer development, likely complemented by extra-pulmonary sources at the late stages. In accordance, LPC levels, as measured with LC/MS/MS, were almost doubled in BALFs at 8w post lung cancer induction (Fig. 5I), and diminished in the corresponding plasmas (Fig. 5J); noteworthy, LPC has been shown to be a potent inducer of ATX expression in hepatocytes (12). BALF LPA levels were found increased at 16w in BALFs but not plasmas (Fig. 5K and L), indicating a role for LPA in K-ras^G12D–driven lung cancer pathogenesis and stressing the importance of the downregulated PLPP3 levels, observed at this time point (Supplementary Fig. S4D), in LPA metabolism and lung cancer development. LPA levels were found diminished upon Enpp2 genetic deletion (Fig. 5K), reconfirming the key role of ATX in extracellular LPA production.

Consistent with the reported LPA effects in various cell types in vitro (4, 26), genetic deletion of Enpp2 and the attenuation of LPA levels resulted in a decrease in the proliferation of cancer cells, as assessed with PCNA staining, as well as a similar decrease in VEGFR2 staining, a marker of angiogenesis (Fig. 6A–D, respectively). However, no major changes were observed in p53 and PTEN staining (data not shown), that their levels have been suggested to be modulated from LPA in lung cancer cell lines in vitro.

To obtain additional mechanistic insights into the mode of action of ATX/LPA in K-ras^G12D–driven lung cancer pathogenesis, we performed mRNA expression profiling in whole lungs of K-ras^G12D/+Enpp2^+/+and K-ras^G12D/+Enpp2^−/−mice 16w post induction. Limited by the heterogeneity of the cancerous lungs and the relatively small spread of cancer cells, 303 bona fide deregulated genes were identified (Supplementary Table S3; Fold change > 1.5; P < 0.05); only 60 out of them were found upregulated. Interestingly, gene set enrichment analysis indicated that, in the relative absence of ATX and upon lower LPA levels, the 243 downregulated genes were enriched in metabolic genes, including lipid metabolism (Supplementary Table S4). Therefore, these results suggest that the procarcinogenic effects of the ATX/LPA axis in lung cancer pathogenesis include metabolic rewiring, as was recently suggested for HCC (12). However, further studies are required to validate and dissect this intriguing hypothesis, that has multiple implications in LPA physiology and cancer biology.

The ENPP2 gene resides in the human chromosomal region 8q24, one of the most frequent regions of somatic copy number alterations (SCNA; ref. 27) and a region that contains potential susceptibility loci for various cancers, including lung cancer (28). The 8q24 locus has been suggested to regulate the expression of the proto-oncogene MYC (29), whereas LPA has been reported to stimulate Myc expression and was suggested to cooperate with it in cell transformation (30). Here, ENPP2 was in silico discovered to be genetically altered in 8% of a large cohort of patients with lung cancer (Supplementary Fig. S1), co-amplified, in 50% of the cases, with MYC that resides in the same region, one of the most frequently focally amplified genes in lung ADC (23). However, ENPP2 mRNA expression was found lower in cancerous biopsies than in normal lung tissue, in most patients with lung cancer cohorts' datasets and all samples examined (Supplementary Table S1 and Supplementary Fig. S2). Noteworthy, ENPP2 was also reported as underexpressed in breast carcinomas despite a detected gene amplification, most likely due to the observed hypermethylation in promoter regions of the gene (8, 31), suggesting that epigenetic modifications could also downregulate ATX mRNA levels at the terminal stages of lung cancer.

Striking immunostaining for ATX, which has been scrutinized elsewhere (12, 14, 21), was detected in both ADCs and SCCs (Fig. 1). However, and given the reported downregulated ENPP2 mRNA levels in lung cancer, it is unlikely that the observed increased ATX levels are derived from the cancer cells themselves, as previously reported for breast cancer (32). ATX has been shown to engage integrins β1 and β3 at the surface of platelets and cancer cells (33–35), both of which correlate with short survival in patients with early NSCLC (36) and participate in heterodimers associated with disease progression in various cancers (37). Therefore, it cannot be excluded that locally secreted or extravasated ATX can bind to the surface of lung cancer cells via integrins, thus avoiding clearance while exerting locally produced LPA effects and establishing a permissive microenvironment for cancer growth. In addition, PLPP3 expression was discovered to be lower in all lung cancer datasets and samples examined (Supplementary Table S1 and Supplementary Fig. S2), suggesting an additional mechanism to maintain high levels of LPA on the surface of tumor cells, because PLPP3 is a transmembrane protein shown to degrade extracellular LPA (5).

Mouse models for lung cancer are valuable tools for deciphering tumor biology, as well as for the development and validation of new tumor intervention strategies. In this study, we employed the two most widely used, the carcinogen (URE)–induced and the genetically engineered K-Ras^G12D-driven, that represent the two major types of human lung cancer from a genomic perspective, one principally with somatic mutations and the other with SCNAs respectively (27, 38). Identical results regarding Enpp2 expression have been obtained in both models, remarkably phenocopying the human data: increased ATX activity was measured in BALFs and ATX staining was detected in cancer cells (Fig. 2); however, Enpp2, as well as Plpp3, mRNA levels were found downregulated in whole lung samples (Supplementary Fig. S4). Importantly, the mouse studies revealed that ATX levels correlated with total protein in BALFs, indicative of endothelial permeability and pulmonary edema, suggesting that a large part of pulmonary ATX derives from the circulation during carcinogenesis. Moreover, ATX has been reported to be released from human platelets upon tumor cell–induced aggregation (35), suggesting an additional possible source of ATX. Noteworthy, 40% of plasma ATX is thought to derive from the adipose tissue (39), known to secrete various hormones and cytokines (adipokines) that exhibit significant systemic effects on various pathophysiological processes, including lipid metabolism, inflammation, and cancer (40).

Genetic deletion of Enpp2 from its major pulmonary source—the bronchial epithelium (CC10⁺), as well as from monocytic (LysM⁺) cells, resulted in a marked reduction of AAHs (Fig. 3), the first neoplastic lesions that develop upon carcinogen-induced carcinogenesis. Comparable results were obtained upon the stochastic genetic deletion of Enpp2 in pulmonary (most likely epithelial) cells upon K-ras^G12D-driven lung carcinogenesis (Fig. 5), suggesting that local pulmonary ATX expression plays a role in tumor initiation. ATX deletion was accompanied by lower LPA BALF levels (Fig. 5), reaffirming that ATX exerts its effects through the local production of LPA. It has been proposed that LPA can downregulate ATX expression levels in some cells and conditions (41), providing an additional explanation for the low Enpp2 mRNA levels at the terminal stages of both human (Supplementary Table S1 and Supplementary Fig. S2) and mouse (Supplementary Fig. S4) lung cancer. LPA levels remained high most likely due to the enzymatic action of extravasated ATX and/or due to a marked reduction in Plpp3 expression upon carcinogenesis (Supplementary Fig. S4).

The genetic deletion of ATX from pulmonary cells in both models did not result in a significant reduction in AD development, most likely due to the recruitment of ATX from the circulation that complements or even outweighs local pulmonary expression. However, the participation of the ATX/LPA axis in lung cancer development was ultimately proven in Lpar1 knock out mice, where a significant decrease of ADs upon URE-induced carcinogenesis was observed (Fig. 4). LPAR1 expression and signaling has been also shown as a pathogenic determinant of pulmonary fibrosis (13); therefore, these results further extend the similarities between lung fibrosis and cancer (15). LPAR2, which is hardly expressed in pulmonary cells (Supplementary Fig. S4; ref. 7), was shown to have minor effects in URE-induced lung cancer (Fig. 4). LPAR6 is highly expressed in the liver and HCC (12, 42) and has been shown to support liver tumorigenesis (42). LPAR6 is also the highest expressing LPAR in the lung (Supplementary Fig. S4), but its role in pulmonary pathophysiology has not been explored yet.

LPAR1 couples to at least three distinct G proteins (G_q, G_i and G_12/13), which in turn feed into multiple effector pathways, including activation of the mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades, leading to cytoskeletal rearrangements and to increased proliferation (Fig. 6) and motility (4, 6, 7, 26). Noteworthy, these LPA-stimulated signaling pathways are shared with many of the established players and therapeutic targets in lung carcinogenesis, such as K-RAS and EGFR. Therefore, costimulation by LPAR1 and its associated G-proteins could lower their activation thresholds, thus promoting disease initiation and progression. In addition to these direct effects to cancer cells, LPA has been also suggested to regulate angiogenesis (Fig. 6; ref. 43), as well as pulmonary vascular leak (13), facilitating the influx of inflammatory and growth factors. An involvement of the ATX/LPA axis in cancer immunoregulation is also likely, since ATX has been reported to adhere to lymphocytes facilitating their homing (44), whereas increased ATX staining has been reported at lymphoid aggregates in patients tissue, including NSCLC (21, 45). Moreover, all immune cells were shown to possess some LPARs, and LPA has been recently proposed to convert monocytes into macrophages (46).

Beyond these established or suggested ATX/LPA effects in various hallmarks of cancer, a novel metabolic function of the axis in carcinogenesis is emerging. ATX/LPA were recently shown, both in vitro and in vivo, to play a role in liver lipid metabolism during HCC development, regulating triglyceride (TGA) levels (12). Expression profiling of modeled HCC in the absence of hepatocyte ATX expression, indicated genes that are involved in lipid metabolism (12). Intriguingly, similar deregulated metabolic pathways were also discovered in lung cancer (Supplementary Table S4), and several related liver specific genes were found activated by ATX/LPA in the lung (Supplementary Table S3), suggesting that LPA-induced metabolic rewiring is not confined to the liver. However, further studies are needed to establish a direct causative relationship and to dissect a possible involvement of these complex and redundant pathways. Finally, many genes involved in drug metabolism and/or resistance were discovered to be affected by ATX/LPA during lung (Supplementary Tables S3 and S4) and liver (12) carcinogenesis. Accordingly, ATX has been suggested to protect cancer cells from taxol-induced apoptosis (47) and to mediate the acquired resistance against sunitinib, cisplatin and adriamycin (48, 49). Moreover, LPA signaling was shown to induce the Nrf2-mediated expression of multi-drug resistance transporters and antioxidant genes (50), suggesting that therapeutic targeting of the ATX/LPA axis could be useful as an adjuvant therapy.

In summary, strong indications on a possible involvement of the ATX/PLPP3-LPA/LPAR1 axis in lung carcinogenesis, in both human patients and mouse animal models were provided. Furthermore, genetic deletion of Enpp2 and Lpar1 was shown to attenuate cancer development in animal models, thus establishing a procarcinogenic role of the axis in lung cancer pathogenesis, expanding the mechanistic links of pulmonary fibrosis and cancer, while further revealing possible LPA effects in tumor metabolism, an emerging hallmark.

No potential conflicts of interest were disclosed.

Conception and design: D. Bouros, K. Syrigos, V. Aidinis

Development of methodology: C. Magkrioti, N. Oikonomou, G.T. Stathopoulos, E. Bouros, D. Bouros

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Magkrioti, N. Oikonomou, E. Kaffe, M.-A. Mouratis, I. Barbayianni, V. Harokopos, C. Valavanis, J. Chun, A. Kosma, E. Bouros, D. Bouros, K. Syrigos

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Magkrioti, N. Oikonomou, E. Kaffe, M.-A. Mouratis, V. Harokopos, C. Valavanis, G.T. Stathopoulos, K. Syrigos

Writing, review, and/or revision of the manuscript: C. Magkrioti, M.-A. Mouratis, J. Chun, E. Bouros, D. Bouros, K. Syrigos, V. Aidinis

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Magkrioti, N. Xylourgidis, I. Barbayianni, P. Megadoukas

Study supervision: D. Bouros, K. Syrigos, V. Aidinis

Other (performing immunohistochemistry evaluation of human and mouse pathology and immunohistochemical data): C. Valavanis

This research has been co-financed in part by the European Union (European Regional Development Fund) and Greek national funds through the National Strategic Reference Framework (NSRF) and the Operational Programs "Competitiveness and Entrepreneurship" (#09SYN-11-679 and 09SYN-12-680 to V. Aidinis; MIS 5002562 to BSRC Fleming), as well as the National co-funding of European programs (#2006ΣE01330024—ΣAE 013/3). C. Magkrioti was supported by a scholarship from the State Scholarships Foundation (#4688; IKY/Siemens-excellence).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.