This article has Open Peer Review reports available.
Phosphorylation of pyruvate kinase M2 and lactate dehydrogenase A by fibroblast growth factor receptor 1 in benign and malignant thyroid tissue
© Kachel et al.; licensee BioMed Central. 2015
Received: 19 June 2014
Accepted: 24 February 2015
Published: 18 March 2015
Lactate dehydrogenase A (LDHA) and Pyruvate Kinase M2 (PKM2) are important enzymes of glycolysis. Both of them can be phosphorylated and therefore regulated by Fibroblast growth factor receptor 1 (FGFR1). While phosphorylation of LDHA at tyrosine10 leads to tetramerization and activation, phosphorylation of PKM2 at tyrosine105 promotes dimerization and inactivation. Dimeric PKM2 is found in the nucleus and regulates gene transcription. Up-regulation and phosphorylation of LDHA and PKM2 contribute to faster proliferation under hypoxic conditions and promote the Warburg effect.
Using western blot and SYBR Green Real time PCR we investigated 77 thyroid tissues including 19 goiter tissues, 11 follicular adenomas, 16 follicular carcinomas, 15 papillary thyroid carcinomas, and 16 undifferentiated thyroid carcinomas for total expression of PKM2, LDHA and FGFR1. Additionally, phosphorylation status of PKM2 and LDHA was analysed. Inhibition of FGFR was performed on FTC133 cells with SU-5402 and Dovitinib.
All examined thyroid cancer subtypes overexpressed PKM2 as compared to goiter. LDHA was overexpressed in follicular and papillary thyroid cancer as compared to goiter. Elevated phosphorylation of LDHA and PKM2 was detectable in all analysed cancer subtypes. The highest relative phosphorylation levels of PKM2 and LDHA compared to overall expression were found in undifferentiated thyroid cancer. Inhibition of FGFR led to significantly decreased phosphorylation levels of PKM2 and LDHA.
Our data shows that overexpression and increased phosphorylation of PKM2 and LHDA is a common finding in thyroid malignancies. Phospho-PKM2 and Phospho-LDHA could be valuable tumour markers for thyroglobulin negative thyroid cancer.
The Warburg effect describes a general feature of cancer cells to show elevated glucose uptake and lactate production even in the presence of oxygen . Warburg proposed an impaired glucose oxidation, which leads to extensive excretion of lactate under normoxia . However, recent data showed that the Warburg effect is common not only in cancer cells but also in induced pluripotent stem cells  and in proliferating T cells . These findings raise many questions related to cancer specific alterations in glycolysis and their possible use as prognostic or therapeutic targets.
Pyruvate Kinase (PK), catalysing the step from phosphoenolpyruvate to pyruvate, is a key enzyme of glycolysis. Furthermore it is an important regulator of the Warburg effect. In thyroid tissue there are two isoenzymes: pyruvate kinase M1 (PKM1) and pyruvate kinase M2 (PKM2), which result from alternative splicing of the PKM gene . Bluemlein et al. showed that PKM2 is the dominant isoenzyme in all examined benign and malignant thyroid tissues  (Additional file 1). Higher levels of PKM2 in tumour tissues contribute to growth advantage and faster progression in xenograft models as compared to cancer cells expressing PKM1 . However, elevated levels of inactivated dimeric PKM2 are found in cancer cells . This inactivation may be promoted by different mechanisms [9,10] and suggests that PKM2 may possess other, non-glycolytic functions such as regulation of transcription. In addition to these effects involvement of other proteins, which may dramatically affect the function of PKM2, has been reported [10,11]. It has been demonstrated that phosphorylation of tyrosine 105 of PMK2 by fibroblast growth factor receptor 1 (FGFR1) prevents tetramerization and inactivates PKM2. As a consequence this leads to faster proliferation under hypoxic conditions and increased tumour growth in xenograft models . The enzymatically inactivated dimeric form of PKM2 can be translocated to the nucleus and may act as a protein kinase regulating gene transcription implicated in tumour growth [13-16]. Inactivation of PKM2 leads to accumulation of upstream glycolytic metabolites and activation of the pentose-phosphate pathway, hexosamine-pathway and serine biosynthesis. This results in increased availability of metabolites for redox control and nucleotide biosynthesis [11,17]. With regard to clinical employment as a tumourmarker, a great diagnostic and prognostic potential of PKM2 has been demonstrated for several malignancies including oesophagus, pancreas or colorectal cancer [18,19]. However, data concerning PKM2 in thyroid cancer is still lacking.
Lactate dehydrogenase (LDH) catalyses the conversion from pyruvate to lactate. Active LDH consists of four monomers. The two different monomers lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB) are expressed in an organ depending manner. While LDHA preferentially turns pyruvate into lactate, LDHB works the opposite way .
LDHA is up-regulated in a wide range of tumour tissues including lung, breast, endometrium, urinary bladder, testicular germ cell and large intestine cancers [20,21]. Down-regulation or inhibition of LDHA resulted in decreased ATP levels, reduced mitochondrial membrane potential and an increase in oxidative stress that is linked to cell death. Furthermore, decreased levels of LDHA were related to inhibition of tumour xenograft maintenance and progression [22,23]. Investigations on thyroid tissues showed a decreased LDHA/LDHB ratio in thyroid oncocytoma and follicular tumours compared to normal thyroid tissue which was altered by estrogen related receptor alpha. Those tumours were more dependent on oxidative phosphorylation . In contrast to PKM2, FGFR1-mediated phosphorylation of LDHA at tyrosine 10 (y10) promotes tetramerization and turns LDHA into the active enzyme .
Fibroblast growth factor receptors (FGFR) play an important role in many human malignancies, such as bladder or breast cancer [26,27]. They are involved in the regulation of cellular proliferation, differentiation, migration and cell survival . With regard to thyroid tissue, the most studied receptor of this family, FGFR1, is overexpressed in differentiated thyroid cancer and in thyroid hyperplasia [29,30].
So far, FGFR1-mediated phosphorylation effects on LDHA and PKM2 have only been demonstrated in cell culture experiments without any relevance to human cancer tissue, especially thyroid carcinoma.
In this study we investigated the expression of PKM2, LDHA and FGFR1 in thyroid benign and malignant tissues by employment of qPCR and western blot. Additionally, a possible impact of FGFR1-mediated phosphorylation of PKM2 and LDHA on thyroid malignancy was evaluated.
A total of 77 thyroid tissue samples, including 19 goiter tissues, 11 follicular adenomas (FA), 16 follicular carcinomas (FTC), 15 papillary thyroid carcinomas (PTC) and 16 undifferentiated thyroid carcinomas (UTC) were collected from patients at the surgical institute of the Martin Luther University Halle Wittenberg. The study was approved by the ethical committee of the medical department of the Martin Luther University Halle Wittenberg. All patients gave written consent.
Protein isolation and western blot analysis
Cell Signaling Technology, Cambridge, England
Phospho-PKM2 (Tyr105) Antibody
Cell Signaling Technology, Cambridge, England
LDHA (C4B5) Rabbit mAb
Cell Signaling Technology, Cambridge, England
Phospho-LDHA (Tyr10) Antibody #8176
Cell Signaling Technology, Cambridge, England
FGF Receptor 1 (D8E4)
Cell Signaling Technology, Cambridge, England
Monoclonal Anti-ß-Aktin Clone AC15
Sigma Aldrich, Saint Louis, USA
Goat anti Rabbit IgG HRP
Santa Cruz Biotechnology, Santa Cruz, USA
Goat anti Mouse IgG HRP
Santa Cruz Biotechnology, Santa Cruz, USA
PKM1 Rabbit Polyclonal Ab
Signalway Antibody, Maryland, USA
5′CTG GGA AGC CTG TCA TCT GT-3′
5′- AGT CCC CTT TGG CTG TTT CT-3′
5′-GGC CTC TGC CAT CAG TAT CT-3′
5′-GCC GTG ATA ATG ACC AGC TT-3′
5′-ACA CTG CGC TGG TTG AAA A- 3′
5′-TGG TAT GTG TGG TTG ATG CTC- 3′
5′-AGC AGG CTC AGC GAT ATG AT-3′
5′-TCT CAG CAC CTT CCG TCT TT-3′
5′-ACC CAG AAG ACT GTG GAT GG-3′
5′-TTC TAG ACG GCA GGT CAG GT-3′
Cell culture experiments
8505C and FTC133 cells were grown in DMEM/F12 suppplemented with 10% FCS and 1% PenStrep and incubated at 37°C, 5% CO2. For B-CPAP RPMI 1640 medium was used. FGFR1 inhibition experiments were performed on FTC133 cells by employment of Receptor Tyrosine Kinase Inhibitors TKI-258 (Dovitinib, Biomol) and SU-5402 (Sigma-Aldrich). Inhibition was conducted over 4 h with the indicated inhibitor concentrations. Control cells received corresponding concentrations of DMSO.
Statistical analyses were performed with IBM SPSS (version 20) software by employment of a priori test. In case of p < 0.05 a Kruskal Wallice and Mann Whitney U test for subgroup analysis was performed. For correlation analysis the Pearson coefficient was used. Boxplots were performed according to Tukey’s definition.
Expression and phosphorylation of y105 of pyruvate kinase M2 (PKM2)
Expression and phosphorylation of lactate dehydrogenase A (y 10)
Analysis of phosphorylated LDHA revealed higher levels of LDHA in each cancer subgroup (PTC, FTC and UTC) in contrast to goiter. The median expression of total LDHA was 54.9% in goiter, 75% in FA, 81.5% in FTC, 97.4% in PTC and 72.5% in UTC. Median Phospho-LDHA levels were 25.1% in goiter, 43% in FA, 60.8% in FTC, 69.7% in PTC, 63.6% in UTC. PTC showed the highest median expression of total LDHA (97,4%) and the highest median level of Phospho-LDHA (69.7%). UTC revealed a noticeable, however not significant up-regulation of LDHA as compared to goiter. Phospho-LDHA / total LDHA ratio showed significantly increased relative phosphorylation in all cancer subgroups in comparison to goiter (Figure 8). UTC and FTC showed significantly increased Phospho-LDHA/total-LDHA ratio in comparison to follicular adenoma (FA) (Figure 8).
The correlation between the FGFR1 expression and Phospho-LDHA levels was r 0.311 (p < 0.05) (data not shown) and was even lower using a Phospho-LDHA/LDHA ratio; r 0.226 (p < 0.05) (Figure 5). In histological subgroups only FTC showed a significant correlation between FGFR1 and Phospho-LDHA/LDHA ratio at r 0.648 (p < 0.05) (data not shown). All other groups did not show a significant correlation. However, in the group of goiter tissue a correlation of r 0.444 (p = 0.057) (data not shown) was found, being just outside of agreed statistical significance.
Expression of fibroblast growth factor receptor 1 (FGFR1)
Analysis of FGFR1 mRNA expression revealed a noticeable increase in FA and all cancer subgroups compared to goiter tissue. LDHA was found markedly higher in FTC compared to FA or goiter without a statistical difference observed. Only the differences between UTC and goiter were statistically significant (Figure 9).
Expression of phosphorylated PKM2 and LDHA under FGFR1 inhibition
In this study we demonstrated that total and phosphorylated PKM2 and LDHA proteins are significantly up-regulated in thyroid cancer tissues as compared to goiter. To our knowledge, in addition to previous PKM2 results in thyroid cancer , this is the first report demonstrating an increased phosphorylation status of both proteins in thyroid cancer.
It is well known that in addition to the Warburg effect, expression of PKM2 and LDHA in tumour tissues may be regulated by Hypoxia inducible factor 1-alpha (HIF1a) and c-Myc. Both oncogenes were also reported to be elevated in thyroid malignancies [31-36]. With regards to our data, we found that in thyroid carcinoma tissues PKM2 is expressed more abundantly than LDHA. Based on these observations we suggest that the Warburg effect is not the only factor affecting PKM2 regulation in thyroid cancer, especially in UTC. Furthermore, as reported in our study, different expressional changes observed between PKM2 and LDHA may result from other than the enzymatic role of PKM2 in gene transcription and proliferation [16,37,38].
Various other studies had previously demonstrated increased levels of LDHA and PKM2 in tumour tissues and their important role in proliferation and survival of malignant cells [7,23,39]. In accordance with these findings we were able to show that thyroid carcinoma samples revealed not only significantly elevated levels of total PKM2 and LDHA compared to goiter or FA, but this expressional increase correlated directly with phosphorylated forms of both proteins. Furthermore, our data suggests that in patients with thyreoglobulin-negative thyroid cancer, phosphorylated PKM2 and LDHA may represent a more valuable diagnostic potential than total expressions of these proteins. Indeed, we found that the FGFR1 expression correlated well with phosphorylation status of both proteins. These results suggest a possible, but not only FGFR1-mediated phosphorylation mechanism of PKM2 and LDHA in thyroid carcinoma. However, increased phosphorylation of various proteins and up-regulation of FGFR are known to occur in cancer, being a possible confounder of our conclusion.
We therefore conducted additional cell culture experiments and were able to demonstrate that phosphorylation of PKM2 and LDHA occurs in an FGFR1-specific manner. Inhibition of FGFR1 in the thyroid cancer cell line FTC133 resulted in significantly decreased phosphorylation status of both investigated enzymes. It is worth noting that tyrosine kinase inhibitors like Dovitinib or SU-5402 could be a therapeutic option to target the Warburg effect in thyroid cancer cells.
Discrimination between follicular adenoma (FA) and FTC is often a challenge. Based on our data we noticed a significantly increased relative phosphorylation of PKM2 and LDHA (Figures 3 and 8) in FTC in comparison to FA. However, studies with early stage follicular thyroid cancer are necessary to determine whether relative phosphorylation could be a tool to discriminate FTC from FA.
In summary, we demonstrated that increased levels of total and phosphorylated forms of PKM2 and LDHA in malignant tissues represent a novel expressional signature with diagnostic potential for thyroid cancer.
We would like to express our gratitude for Kathrin Hammje for her excellent technical assistance and Dr. Gabriel Magnucki for his stimulating intellectual input. We would like to thank Thea Hoeschel for her great help with the manuscript and Juli Geber for her outstanding language skills.
- Warburg O. über den Stoffwechsel der Carcinomzelle. Naturwissenschaften. 1924;12:1131–7.View ArticleGoogle Scholar
- Warburg O. On the origin of cancer cells. Science. 1956;123:309–14.View ArticlePubMedGoogle Scholar
- Prigione A, Fauler B, Lurz R, Lehrach H, Adjaye J. The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells. 2010;28:721–33.View ArticlePubMedGoogle Scholar
- Colombo SL, Palacios-Callender M, Frakich N, De Leon J, Schmitt CA, Boorn L, et al. Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes. Proc Natl Acad Sci U S A. 2010;107:18868–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Noguchi T, Inoue H, Tanaka T. The M1-and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem. 1986;261:13807–12.PubMedGoogle Scholar
- Bluemlein KK, Grüning N-MN, Feichtinger RGR, Lehrach HH, Kofler BB, Ralser MM. No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget. 2011;2:393–400.View ArticlePubMedPubMed CentralGoogle Scholar
- Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R, et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature. 2008;452:230–3.View ArticlePubMedGoogle Scholar
- Mazurek S. Pyruvate kinase type M2, a key regulator within the tumour metabolome and a tool for metabolic profiling of tumours. Ernst Schering Found Symp Proc. 2007;99–124.Google Scholar
- Christofk HRH, Vander Heiden MGM, Wu NN, Asara JMJ, Cantley LCL. Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature. 2008;452:181–6.View ArticlePubMedGoogle Scholar
- Mazurek S, Zwerschke W, Jansen-Dürr P, Eigenbrodt E. Effects of the human papilloma virus HPV-16 E7 oncoprotein on glycolysis and glutaminolysis: role of pyruvate kinase type M2 and the glycolytic-enzyme complex. Biochem J. 2001;356:247–56.View ArticlePubMedPubMed CentralGoogle Scholar
- Grüning N-M, Rinnerthaler M, Bluemlein K, Mülleder M, Wamelink MMC, Lehrach H, et al. Pyruvate kinase triggers a metabolic feedback loop that controls redox metabolism in respiring cells. Cell Metab. 2011;14:415–27.View ArticlePubMedPubMed CentralGoogle Scholar
- Hitosugi T, Kang S, Vander Heiden MG, Chung TW, Elf S, Lythgoe K, et al. Tyrosine phosphorylation inhibits PKM2 to promote the warburg effect and tumor growth. Sci Signal. 2009;2:ra73.View ArticlePubMedPubMed CentralGoogle Scholar
- Gao X, Wang H, Yang JJ, Liu X, Liu Z-R. Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell. 2012;45:598–609.View ArticlePubMedPubMed CentralGoogle Scholar
- Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R, et al. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell. 2011;145:732–44.View ArticlePubMedPubMed CentralGoogle Scholar
- Luo W, Semenza GL. Pyruvate kinase M2 regulates glucose metabolism by functioning as a coactivator for hypoxia-inducible factor 1 in cancer cells. Oncotarget. 2011;2:551–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Lv L, Xu Y-P, Zhao D, Li F-L, Wang W, Sasaki N, et al. Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol Cell. 2013;52:340–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Luo W, Semenza GL. Emerging roles of PKM2 in cell metabolism and cancer progression. Trends Endocrinol Metab. 2012;23:560–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Hathurusinghe HR, Goonetilleke KS, Siriwardena AK. Current Status of Tumor M2 Pyruvate Kinase (Tumor M2-PK) as a Biomarker of Gastrointestinal Malignancy. Ann Surg Oncol. 2007;14:2714–20.View ArticlePubMedGoogle Scholar
- Dhar DK, Olde Damink SWM, Brindley JH, Godfrey A, Chapman MH, Sandanayake NS, et al. Pyruvate kinase M2 is a novel diagnostic marker and predicts tumor progression in human biliary tract cancer. Cancer. 2012;119:575–85.View ArticlePubMedPubMed CentralGoogle Scholar
- Koukourakis MI, Giatromanolaki A, Sivridis E. Lactate dehydrogenase isoenzymes 1 and 5: differential expression by neoplastic and stromal cells in non-small cell lung cancer and other epithelial malignant tumors. Tumour Biol. 2003;24:199–202.View ArticlePubMedGoogle Scholar
- Hilf R, Rector WD, Orlando RA. Multiple molecular forms of lactate dehydrogenase and glucose 6-phosphate dehydrogenase in normal and abnormal human breast tissues. Cancer. 1976;37:1825–30.View ArticlePubMedGoogle Scholar
- Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell. 2006;9:425–34.View ArticlePubMedGoogle Scholar
- Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, et al. Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci. 2010;107:2037–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Mirebeau-Prunier D, Le Pennec S, Jacques C, Fontaine J-F, Gueguen N, Boutet-Bouzamondo N, et al. Estrogen-related receptor alpha modulates lactate dehydrogenase activity in thyroid tumors. PLoS One. 2013;8:e58683.View ArticlePubMedPubMed CentralGoogle Scholar
- Fan J, Hitosugi T, Chung TW, Xie J, Ge Q, Gu TL, et al. Tyrosine phosphorylation of lactate dehydrogenase a is important for NADH/NAD+ redox homeostasis in cancer cells. Mol Cell Biol. 2011;31:4938–50.View ArticlePubMedPubMed CentralGoogle Scholar
- di Martino E, Tomlinson DC, Knowles MA. A decade of FGF receptor research in bladder cancer: past, present, and future challenges. Ther Adv Urol. 2012;2012:1–10.View ArticleGoogle Scholar
- Reis-Filho JS, Simpson PT, Turner NC, Lambros MB, Jones C, Mackay A, et al. FGFR1 emerges as a potential therapeutic target for lobular breast carcinomas. Clin Cancer Res. 2006;12:6652–62.View ArticlePubMedGoogle Scholar
- Eswarakumar VP, Lax I, Schlessinger J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 2005;16:139–49.View ArticlePubMedGoogle Scholar
- St Bernard R. Fibroblast growth factor receptors as molecular targets in thyroid carcinoma. Endocrinology. 2004;146:1145–53.View ArticlePubMedGoogle Scholar
- Thompson SD, Franklyn JA, Watkinson JC, Verhaeg JM, Sheppard MC, Eggo MC. Fibroblast growth factors 1 and 2 and fibroblast growth factor receptor 1 are elevated in thyroid hyperplasia. J Clin Endocrinol Metab. 1998;83:1336–41.View ArticlePubMedGoogle Scholar
- Shim H, Dolde C, Lewis BC, Wu CS, Dang G, Jungmann RA, et al. c-Myc transactivation of LDH-A: implications for tumor metabolism and growth. Proc Natl Acad Sci U S A. 1997;94:6658–63.View ArticlePubMedPubMed CentralGoogle Scholar
- David CJ, Chen M, Assanah M, Canoll P, Manley JL. HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature. 2010;463:364–8.View ArticlePubMedGoogle Scholar
- Semenza GL. Hypoxia-inducible factors: mediators of cancer progression and targets for cancer therapy. Trends Pharmacol Sci. 2012;33:207–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen M, Zhang J, Manley JL. Turning on a fuel switch of cancer: hnRNP proteins regulate alternative splicing of pyruvate kinase mRNA. Cancer Res. 2010;70:8977–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Burrows N, Resch J, Cowen RL, Wasielewski von R, Hoang-Vu C, West CM, et al. Expression of hypoxia-inducible factor 1 in thyroid carcinomas. Endocr Relat Cancer. 2010;17:61–72.View ArticlePubMedPubMed CentralGoogle Scholar
- Terrier P, Sheng ZM, Schlumberger M, Tubiana M, Caillou B, Travagli JP, et al. Structure and expression of c-myc and c-fos proto-oncogenes in thyroid carcinomas. Br J Cancer. 1988;57:43.View ArticlePubMedPubMed CentralGoogle Scholar
- Israelsen WJ, Dayton TL, Davidson SM, Fiske BP, Hosios AM, Bellinger G, et al. PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells. Cell. 2013;155:397–409.View ArticlePubMedGoogle Scholar
- Yang W, Xia Y, Hawke D, Li X, Liang J, Xing D, et al. PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis. Cell. 2012;150:685–96.View ArticlePubMedPubMed CentralGoogle Scholar
- Parra-Bonilla G, Alvarez DF, Alexeyev M, Vasauskas A, Stevens T. Lactate dehydrogenase a expression is necessary to sustain rapid angiogenesis of pulmonary microvascular endothelium. PLoS One. 2013;8:e75984.View ArticlePubMedPubMed CentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.