BACKGROUND: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. New drug targets and proteins that would assist sensitive PPGL imagining could improve therapy and quality of life of patients with PPGL, namely those with recurrent or metastatic disease. Using a combined proteomic strategy, we looked for such clinically relevant targets among integral membrane proteins (IMPs) upregulated on the surface of tumor cells and non-membrane druggable enzymes in PPGL. METHODS: We conducted a detailed proteomic analysis of 22 well-characterized human PPGL samples and normal chromaffin tissue from adrenal medulla. A standard quantitative proteomic analysis of tumor lysate, which provides information largely on non-membrane proteins, was accompanied by specific membrane proteome-aimed methods, namely glycopeptide enrichment using lectin-affinity, glycopeptide capture by hydrazide chemistry, and enrichment of membrane-embedded hydrophobic transmembrane segments. RESULTS: The study identified 67 cell surface integral membrane proteins strongly upregulated in PPGL compared to control chromaffin tissue. We prioritized the proteins based on their already documented direct role in cancer cell growth or progression. Increased expression of the seven most promising drug targets (CD146, CD171, ANO1, CD39, ATP8A1, ACE and SLC7A1) were confirmed using specific antibodies. Our experimental strategy also provided expression data for soluble proteins. Among the druggable non-membrane enzymes upregulated in PPGL, we identified three potential drug targets (SHMT2, ARG2 and autotaxin) and verified their upregulated expression. CONCLUSIONS: Application of a combined proteomic strategy recently presented as "Pitchfork" enabled quantitative analysis of both, membrane and non-membrane proteome, and resulted in identification of 10 potential drug targets in human PPGL. Seven membrane proteins localized on the cell surface and three non-membrane druggable enzymes proteins were identified and verified as significantly upregulated in PPGL. All the proteins have been previously shown to be upregulated in several human cancers, and play direct role in cancer progression. Marked upregulation of these proteins along with their localization and established direct roles in tumor progression make these molecules promising candidates as drug targets or proteins for sensitive PPGL imaging.

BIOCEV 1st Faculty of Medicine Charles University Vestec 25250 Czech Republic

Department of Urology University Hospital Olomouc and Faculty of Medicine and Dentistry Palacky University Olomouc Olomouc 77900 Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital Prague 12800 Czech Republic

Proteomics Core Facility Faculty of Science BIOCEV Charles University Vestec 25250 Czech Republic

Section on Medical Neuroendocrinology Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH Bethesda MD 20892 USA

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Nölting S, Bechmann N, Taieb D, Beuschlein F, Fassnacht M, Kroiss M, et al. Personalized Management of Pheochromocytoma and Paraganglioma. Endocr Rev. 2022;43(2):199–239. doi: 10.1210/endrev/bnab019. PubMed DOI PMC

Fishbein L. Pheochromocytoma and paraganglioma: Genetics, diagnosis, and treatment. Hematol Oncol Clin North Am. 2016;30(1):135–50. doi: 10.1016/j.hoc.2015.09.006. PubMed DOI

Hamidi O, Raman R, Lazik N, Iniguez-Ariza N, McKenzie TJ, Lyden ML, et al. Clinical course of adrenal myelolipoma: a long-term longitudinal follow-up study. Clin Endocrinol (Oxf) 2020;93(1):11–8. doi: 10.1111/cen.14188. PubMed DOI PMC

Kumar S, Lila AR, Memon SS, Sarathi V, Patil VA, Menon S, et al. Metastatic cluster 2-related pheochromocytoma/paraganglioma: a single-center experience and systematic review. Endocr Connect. 2021;10(11):1463–76. doi: 10.1530/EC-21-0455. PubMed DOI PMC

Wachtel H, Hutchens T, Baraban E, Schwartz LE, Montone K, Baloch Z, et al. Predicting Metastatic potential in pheochromocytoma and paraganglioma: a comparison of PASS and GAPP Scoring Systems. J Clin Endocrinol Metab. 2020;105(12):e4661–70. doi: 10.1210/clinem/dgaa608. PubMed DOI PMC

Zielinska DF, Gnad F, Wiśniewski JR, Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell. 2010;141(5):897–907. doi: 10.1016/j.cell.2010.04.012. PubMed DOI

Tian Y, Zhou Y, Elliott S, Aebersold R, Zhang H. Solid-phase extraction of N-linked glycopeptides. Nat Protoc. 2007;2(2):334–9. doi: 10.1038/nprot.2007.42. PubMed DOI PMC

Vit O, Man P, Kadek A, Hausner J, Sklenar J, Harant K, et al. Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments. J Proteom. 2016;149:15–22. doi: 10.1016/j.jprot.2016.03.016. PubMed DOI

Vit O, Harant K, Klener P, Man P, Petrak J. A three-pronged pitchfork strategy enables an extensive description of the human membrane proteome and the identification of missing proteins. J Proteom. 2019;204:103411. doi: 10.1016/j.jprot.2019.103411. PubMed DOI

Masuda T, Tomita M, Ishihama Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 2008;7(2):731–40. doi: 10.1021/pr700658q. PubMed DOI

Hebert AS, Richards AL, Bailey DJ, Ulbrich A, Coughlin EE, Westphall MS, et al. The one hour yeast proteome. Mol Cell Proteomics. 2014;13(1):339–47. doi: 10.1074/mcp.M113.034769. PubMed DOI PMC

Hallgren J, Tsirigos KD, Pedersen MD, Almagro Armenteros JJ, Marcatili P, Nielsen H et al. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. bioRxiv 2022.04.08.487609.

Fagerberg L, Jonasson K, von Heijne G, Uhlén M, Berglund L. Prediction of the human membrane proteome. Proteomics. 2010;10(6):1141–9. doi: 10.1002/pmic.200900258. PubMed DOI

Nagaraj N, Wisniewski JR, Geiger T, Cox J, Kircher M, Kelso J, et al. Deep proteome and transcriptome mapping of a human cancer cell line. Mol Syst Biol. 2011;7:548. doi: 10.1038/msb.2011.81. PubMed DOI PMC

Vit O, Petrak J. Integral membrane proteins in proteomics. How to break open the black box? J Proteom. 2017;153:8–20. doi: 10.1016/j.jprot.2016.08.006. PubMed DOI

Blackler AR, Speers AE, Ladinsky MS, Wu CC. A shotgun proteomic method for the identification of membrane-embedded proteins and peptides. J Proteome Res. 2008;7(7):3028–34. doi: 10.1021/pr700795f. PubMed DOI PMC

Vit O, Patel M, Musil Z, Hartmann I, Frysak Z, Miettinen M, et al. Deep membrane proteome profiling reveals overexpression of prostate-specific membrane Antigen (PSMA) in high-risk human paraganglioma and pheochromocytoma, suggesting New Theranostic Opportunity. Molecules. 2021;26(21):6567. doi: 10.3390/molecules26216567. PubMed DOI PMC

Joshkon A, Heim X, Dubrou C, Bachelier R, Traboulsi W, Stalin J, et al. Role of CD146 (MCAM) in physiological and pathological angiogenesis-contribution of New antibodies for Therapy. Biomedicines. 2020;8(12):633. doi: 10.3390/biomedicines8120633. PubMed DOI PMC

Wang Z, Xu Q, Zhang N, Du X, Xu G, Yan X. CD146, from a melanoma cell adhesion molecule to a signaling receptor. Signal Transduct Target Ther. 2020;5(1):148. doi: 10.1038/s41392-020-00259-8. PubMed DOI PMC

Sharma A, Joshkon A, Ladjimi A, Traboulsi W, Bachelier R, Robert S, et al. Soluble CD146 as a potential target for preventing Triple negative breast Cancer MDA-MB-231 cell growth and dissemination. Int J Mol Sci. 2022;23(2):974. doi: 10.3390/ijms23020974. PubMed DOI PMC

Bardin N, Francès V, Combes V, Sampol J, Dignat-George F. CD146: biosynthesis and production of a soluble form in human cultured endothelial cells. FEBS Lett. 1998;421(1):12–4. doi: 10.1016/S0014-5793(97)01455-5. PubMed DOI

Stalin J, Nollet M, Garigue P, Fernandez S, Vivancos L, Essaadi A, et al. Targeting soluble CD146 with a neutralizing antibody inhibits vascularization, growth and survival of CD146-positive tumors. Oncogene. 2016;35(42):5489–500. doi: 10.1038/onc.2016.83. PubMed DOI

Stalin J, Traboulsi W, Vivancos-Stalin L, Nollet M, Joshkon A, Bachelier R, et al. Therapeutic targeting of soluble CD146/MCAM with the M2J-1 monoclonal antibody prevents metastasis development and procoagulant activity in CD146-positive invasive tumors. Int J Cancer. 2020;147(6):1666–79. doi: 10.1002/ijc.32909. PubMed DOI

Obu S, Umeda K, Ueno H, Sonoda M, Tasaka K, Ogata H, et al. CD146 is a potential immunotarget for neuroblastoma. Cancer Sci. 2021;112(11):4617–26. doi: 10.1111/cas.15124. PubMed DOI PMC

Nollet M, Stalin J, Moyon A, Traboulsi W, Essaadi A, Robert S, et al. A novel anti-CD146 antibody specifically targets cancer cells by internalizing the molecule. Oncotarget. 2017;8(68):112283–96. doi: 10.18632/oncotarget.22736. PubMed DOI PMC

Wang H, Zou L, Ma K, Yu J, Wu H, Wei M, et al. Cell-specific mechanisms of TMEM16A Ca2+-activated chloride channel in cancer. Mol Cancer. 2017;16(1):152. doi: 10.1186/s12943-017-0720-x. PubMed DOI PMC

Li H, Yu Z, Wang H, Wang N, Sun X, Yang S, et al. Role of ANO1 in tumors and tumor immunity. J Cancer Res Clin Oncol. 2022;148(8):2045–68. doi: 10.1007/s00432-022-04004-2. PubMed DOI

Duvvuri U, Shiwarski DJ, Xiao D, Bertrand C, Huang X, Edinger RS, et al. TMEM16A induces MAPK and contributes directly to tumorigenesis and cancer progression. Cancer Res. 2012;72(13):3270–81. doi: 10.1158/0008-5472.CAN-12-0475-T. PubMed DOI PMC

Britschgi A, Bill A, Brinkhaus H, Rothwell C, Clay I, Duss S, et al. Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling. Proc Natl Acad Sci U S A. 2013;110(11):E1026–34. doi: 10.1073/pnas.1217072110. PubMed DOI PMC

Shi S, Ma B, Sun F, Qu C, Li G, Shi D, et al. Zafirlukast inhibits the growth of lung adenocarcinoma via inhibiting TMEM16A channel activity. J Biol Chem. 2022;298(3):101731. doi: 10.1016/j.jbc.2022.101731. PubMed DOI PMC

Bastid J, Regairaz A, Bonnefoy N, Déjou C, Giustiniani J, Laheurte C, et al. Inhibition of CD39 enzymatic function at the surface of tumor cells alleviates their immunosuppressive activity. Cancer Immunol Res. 2015;3(3):254–65. doi: 10.1158/2326-6066.CIR-14-0018. PubMed DOI

Häusler SF, Montalbán del Barrio I, Strohschein J, Chandran PA, Engel JB, Hönig A, et al. Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity. Cancer Immunol Immunother. 2011;60(10):1405–18. doi: 10.1007/s00262-011-1040-4. PubMed DOI PMC

Michaud M, Martins I, Sukkurwala AQ, Adjemian S, Ma Y, Pellegatti P, et al. Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice. Science. 2011;334(6062):1573–7. doi: 10.1126/science.1208347. PubMed DOI

Li XY, Moesta AK, Xiao C, Nakamura K, Casey M, Zhang H, et al. Targeting CD39 in Cancer reveals an extracellular ATP- and inflammasome-driven tumor immunity. Cancer Discov. 2019;9(12):1754–73. doi: 10.1158/2159-8290.CD-19-0541. PubMed DOI PMC

Perrot I, Michaud HA, Giraudon-Paoli M, Augier S, Docquier A, Gros L, et al. Blocking antibodies targeting the CD39/CD73 immunosuppressive pathway unleash Immune responses in Combination Cancer Therapies. Cell Rep. 2019;27(8):2411–25e9. doi: 10.1016/j.celrep.2019.04.091. PubMed DOI

Allard D, Allard B, Stagg J. On the mechanism of anti-CD39 immune checkpoint therapy. J Immunother Cancer. 2020;8(1):e000186. doi: 10.1136/jitc-2019-000186. PubMed DOI PMC

Jayaprakash P, Vignali PDA, Delgoffe GM, Curran MA. Hypoxia reduction sensitizes refractory cancers to Immunotherapy. Annu Rev Med. 2022;73:251–65. doi: 10.1146/annurev-med-060619-022830. PubMed DOI

Giordano M, Cavallaro U. Different shades of L1CAM in the pathophysiology of Cancer Stem cells. J Clin Med. 2020;9(5):1502. doi: 10.3390/jcm9051502. PubMed DOI PMC

Kiefel H, Bondong S, Hazin J, Ridinger J, Schirmer U, Riedle S, et al. L1CAM: a major driver for tumor cell invasion and motility. Cell Adh Migr. 2012;6(4):374–84. doi: 10.4161/cam.20832. PubMed DOI PMC

Altevogt P, Doberstein K, Fogel M. L1CAM in human cancer. Int J Cancer. 2016;138(7):1565–76. doi: 10.1002/ijc.29658. PubMed DOI

Zander H, Rawnaq T, von Wedemeyer M, Tachezy M, Kunkel M, Wolters G, et al. Circulating levels of cell adhesion molecule L1 as a prognostic marker in gastrointestinal stromal tumor patients. BMC Cancer. 2011;11:1–7. doi: 10.1186/1471-2407-11-189. PubMed DOI PMC

Wu JD, Hong CQ, Huang WH, Wei XL, Zhang F, Zhuang YX, et al. L1 cell adhesion molecule and its Soluble Form sL1 exhibit poor prognosis in primary breast Cancer patients. Clin Breast Cancer. 2018;18(5):e851–61. doi: 10.1016/j.clbc.2017.12.011. PubMed DOI

Wachowiak R, Krause M, Mayer S, Peukert N, Suttkus A, Müller WC, et al. Increased L1CAM (CD171) levels are associated with glioblastoma and metastatic brain tumors. Med (Baltim) 2018;97(38):e12396. doi: 10.1097/MD.0000000000012396. PubMed DOI PMC

Yasumatsu R, Nakashima T, Masuda M, Ito A, Kuratomi Y, Nakagawa T, et al. Effects of the angiotensin-I converting enzyme inhibitor perindopril on tumor growth and angiogenesis in head and neck squamous cell carcinoma cells. J Cancer Res Clin Oncol. 2004;130(10):567–73. doi: 10.1007/s00432-004-0582-7. PubMed DOI

Yoshiji H, Noguchi R, Ikenaka Y, Kaji K, Aihara Y, Yamazaki M, et al. Combination of branched-chain amino acids and angiotensin-converting enzyme inhibitor suppresses the cumulative recurrence of hepatocellular carcinoma: a randomized control trial. Oncol Rep. 2011;26(6):1547–53. PubMed

Fendrich V, Chen NM, Neef M, Waldmann J, Buchholz M, Feldmann G, et al. The angiotensin-I-converting enzyme inhibitor enalapril and aspirin delay progression of pancreatic intraepithelial neoplasia and cancer formation in a genetically engineered mouse model of pancreatic cancer. Gut. 2010;59(5):630–7. doi: 10.1136/gut.2009.188961. PubMed DOI

Araújo WF, Naves MA, Ravanini JN, Schor N, Teixeira VP. Renin-angiotensin system (RAS) blockade attenuates growth and metastatic potential of renal cell carcinoma in mice. Urol Oncol. 2015;33(9):389e1–7. doi: 10.1016/j.urolonc.2014.11.022. PubMed DOI

Pinter M, Jain RK. Targeting the renin-angiotensin system to improve cancer treatment: implications for immunotherapy. Sci Transl Med. 2017;9(410):eaan5616. doi: 10.1126/scitranslmed.aan5616. PubMed DOI PMC

Almutlaq M, Alamro AA, Alamri HS, Alghamdi AA, Barhoumi T. The Effect of Local Renin Angiotensin System in the common types of Cancer. Front Endocrinol (Lausanne) 2021;12:736361. doi: 10.3389/fendo.2021.736361. PubMed DOI PMC

Kato U, Inadome H, Yamamoto M, Emoto K, Kobayashi T, Umeda M. Role for phospholipid flippase complex of ATP8A1 and CDC50A proteins in cell migration. J Biol Chem. 2013;288(7):4922–34. doi: 10.1074/jbc.M112.402701. PubMed DOI PMC

Li D, Xu T, Wang X, Ma X, Liu T, Wang Y, et al. The role of ATP8A1 in non-small cell lung cancer. Int J Clin Exp Pathol. 2017;10(7):7760–6. PubMed PMC

van Blitterswijk WJ, Verheij M. Anticancer mechanisms and clinical application of alkylphospholipids. Biochim Biophys Acta. 2013;1831(3):663–74. doi: 10.1016/j.bbalip.2012.10.008. PubMed DOI

Lu Y, Wang W, Wang J, Yang C, Mao H, Fu X, et al. Overexpression of arginine transporter CAT-1 is associated with accumulation of L-arginine and cell growth in human colorectal cancer tissue. PLoS ONE. 2013;8(9):e73866. doi: 10.1371/journal.pone.0073866. PubMed DOI PMC

Dai R, Peng F, Xiao X, Gong X, Jiang Y, Zhang M, et al. Hepatitis B virus X protein-induced upregulation of CAT-1 stimulates proliferation and inhibits apoptosis in hepatocellular carcinoma cells. Oncotarget. 2017;8(37):60962–74. doi: 10.18632/oncotarget.17631. PubMed DOI PMC

Abdelmagid SA, Rickard JA, McDonald WJ, Thomas LN, Too CK. CAT-1-mediated arginine uptake and regulation of nitric oxide synthases for the survival of human breast cancer cell lines. J Cell Biochem. 2011;112(4):1084–92. doi: 10.1002/jcb.23022. PubMed DOI

Okita K, Hara Y, Okura H, Hayashi H, Sasaki Y, Masuko S, et al. Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene. Cancer Sci. 2021;112(2):563–74. doi: 10.1111/cas.14741. PubMed DOI PMC

Locasale JW. Serine, glycine and one-carbon units: cancer metabolism in full circle. Nat Rev Cancer. 2013;13(8):572–83. doi: 10.1038/nrc3557. PubMed DOI PMC

Labuschagne CF, van den Broek NJ, Mackay GM, Vousden KH, Maddocks OD. Serine, but not glycine, supports one-carbon metabolism and proliferation of cancer cells. Cell Rep. 2014;7(4):1248–58. doi: 10.1016/j.celrep.2014.04.045. PubMed DOI

Lucas S, Chen G, Aras S, Wang J. Serine catabolism is essential to maintain mitochondrial respiration in mammalian cells. Life Sci Alliance. 2018;1(2):e201800036. doi: 10.26508/lsa.201800036. PubMed DOI PMC

Ye J, Fan J, Venneti S, Wan YW, Pawel BR, Zhang J, et al. Serine catabolism regulates mitochondrial redox control during hypoxia. Cancer Discov. 2014;4(12):1406–17. doi: 10.1158/2159-8290.CD-14-0250. PubMed DOI PMC

Minton DR, Nam M, McLaughlin DJ, Shin J, Bayraktar EC, Alvarez SW, et al. Serine catabolism by SHMT2 is required for proper mitochondrial translation initiation and maintenance of Formylmethionyl-tRNAs. Mol Cell. 2018;69(4):610–21e5. doi: 10.1016/j.molcel.2018.01.024. PubMed DOI PMC

Xie M, Pei DS. Serine hydroxymethyltransferase 2: a novel target for human cancer therapy. Invest New Drugs. 2021;39(6):1671–81. doi: 10.1007/s10637-021-01144-z. PubMed DOI

Wu ZZ, Wang S, Yang QC, Wang XL, Yang LL, Liu B, et al. Increased expression of SHMT2 is Associated with Poor Prognosis and Advanced Pathological Grade in oral squamous cell carcinoma. Front Oncol. 2020;10:588530. doi: 10.3389/fonc.2020.588530. PubMed DOI PMC

Zhang P, Yang Q. Overexpression of SHMT2 predicts a poor prognosis and promotes Tumor Cell growth in bladder Cancer. Front Genet. 2021;12:682856. doi: 10.3389/fgene.2021.682856. PubMed DOI PMC

Liu Y, Yin C, Deng MM, Wang Q, He XQ, Li MT, et al. High expression of SHMT2 is correlated with tumor progression and predicts poor prognosis in gastrointestinal tumors. Eur Rev Med Pharmacol Sci. 2019;23(21):9379–92. PubMed

Liao Y, Wang F, Zhang Y, Cai H, Song F, Hou J. Silencing SHMT2 inhibits the progression of tongue squamous cell carcinoma through cell cycle regulation. Cancer Cell Int. 2021;21(1):220. doi: 10.1186/s12935-021-01880-5. PubMed DOI PMC

Woo CC, Chen WC, Teo XQ, Radda GK, Lee PT. Downregulating serine hydroxymethyltransferase 2 (SHMT2) suppresses tumorigenesis in human hepatocellular carcinoma. Oncotarget. 2016;7(33):53005–17. doi: 10.18632/oncotarget.10415. PubMed DOI PMC

Ducker GS, Ghergurovich JM, Mainolfi N, Suri V, Jeong SK, Hsin-Jung Li S, et al. Human SHMT inhibitors reveal defective glycine import as a targetable metabolic vulnerability of diffuse large B-cell lymphoma. Proc Natl Acad Sci U S A. 2017;114(43):11404–9. doi: 10.1073/pnas.1706617114. PubMed DOI PMC

Niu F, Yu Y, Li Z, Ren Y, Li Z, Ye Q, et al. Arginase: an emerging and promising therapeutic target for cancer treatment. Biomed Pharmacother. 2022;149:112840. doi: 10.1016/j.biopha.2022.112840. PubMed DOI

Dowling JK, Afzal R, Gearing LJ, Cervantes-Silva MP, Annett S, Davis GM, et al. Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages. Nat Commun. 2021;12(1):1460. doi: 10.1038/s41467-021-21617-2. PubMed DOI PMC

Colleluori DM, Ash DE. Classical and slow-binding inhibitors of human type II arginase. Biochemistry. 2001;40(31):9356–62. doi: 10.1021/bi010783g. PubMed DOI

Zhang X, Li M, Yin N, Zhang J. The expression regulation and biological function of Autotaxin. Cells. 2021;10(4):939. doi: 10.3390/cells10040939. PubMed DOI PMC

Aiello S, Casiraghi F. Lysophosphatidic acid: promoter of Cancer Progression and of Tumor Microenvironment Development. A Promising Target for Anticancer Therapies? Cells. 2021;10(6):1390. doi: 10.3390/cells10061390. PubMed DOI PMC

Tang X, Wuest M, Benesch MGK, Dufour J, Zhao Y, Curtis JM, et al. Inhibition of Autotaxin with GLPG1690 increases the efficacy of Radiotherapy and Chemotherapy in a mouse model of breast Cancer. Mol Cancer Ther. 2020;19(1):63–74. doi: 10.1158/1535-7163.MCT-19-0386. PubMed DOI

Benesch MG, Ko YM, Tang X, Dewald J, Lopez-Campistrous A, Zhao YY, et al. Autotaxin is an inflammatory mediator and therapeutic target in thyroid cancer. Endocr Relat Cancer. 2015;22(4):593–607. doi: 10.1530/ERC-15-0045. PubMed DOI