Carbonic anhydrase 9 explained

Carbonic anhydrase IX (CA9/CA IX) is an enzyme that in humans is encoded by the CA9 gene.[1] [2] [3] It is one of the 14 carbonic anhydrase isoforms found in humans and is a transmembrane dimeric metalloenzyme with an extracellular active site that facilitates acid secretion in the gastrointestinal tract.[4] CA IX is overexpressed in many types of cancer including clear cell renal cell carcinoma (RCC) as well as carcinomas of the cervix, breast and lung where it promotes tumor growth by enhancing tumor acidosis.[5] [6]

Function

Carbonic anhydrases (CAs) are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide. They participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. They show extensive diversity in tissue distribution and in their subcellular localization.[3]

CA IX is mainly expressed in the gastrointestinal tract where it facilitates acid secretion.[7] The CA IX enzyme, along with the CA II enzyme, binds to Anion Exchanger 2 (AE2) which increases bicarbonate transport and maximizes the rate of acid secretion by gastric parietal cells.

Structure

CA IX is a transmembrane glycoprotein with an extracellular active site. The cytoplasmic tail of the enzyme contains three residues that may be phosphorylated (Thr-443, Ser-448, and Tyr-449) and participate in signal transduction.[8] Phosphorylated tyrosine 449 can interact with PI3K which activates protein kinase B to affect cellular glucose metabolism.[9]

Under physiological conditions, the enzyme exists as two nearly identical dimers.[10] Both dimers are stabilized by two hydrogen bonds between Arg-137 and the Ala-127 carbonyl oxygen as well as many Van der Waals interactions. One dimer, however, has additional stabilization due to a disulfide bridge formed by two cysteine residues. One face of the dimer contains proteoglycan (PG) domains-a feature that is unique from other CA enzymes- and the opposite face contains the C-termini which help the enzyme attach to the cell membrane.[11] CA IX contains an N-linked glycosylation site bearing mannose-type glycan structures on Asn-309 as well as an O-linked glycosylation site on Thr-78.[12]

Regulation

Expression of CA IX is primarily regulated at the transcriptional level.[13] The promoter region of the CA9 gene contains an HRE (hypoxia responsive element) where HIF-1 can bind, which allows hypoxic conditions to increase CA IX expression. Expression can also be regulated post-translationally by metalloproteinases which cause shedding of the enzyme's ectodomain.[14] Unlike other CA isozymes, CA IX is not inhibited by high lactate concentrations.[15] However, it is inhibited by bicarbonate.

Clinical significance

CA IX is a transmembrane protein and is a tumor-associated carbonic anhydrase isoenzyme. It is over-expressed in VHL mutated clear cell renal cell carcinoma (ccRCC) and hypoxic solid tumors, but is low-expressed in normal kidney and most other normal tissues. It may be involved in cell proliferation and transformation. This gene is mapped to 9p13-p12.[3]

CA IX is a cellular biomarker of hypoxia. Furthermore, recent studies examining the association between CA IX levels and various clinicopathological outcomes suggest that CA IX expression may also be a valuable prognostic indicator for overall survival[16] although this association has been questioned.[17]

CA IX shows high expression in carcinomas of the uterine cervix, kidney, oesophagus, lung, breast, colon, brain, and vulva compared to expression in few noncancerous tissues.[18] Its overexpression in cancerous tissues compared to normal ones is due to hypoxic conditions in the tumor microenvironment caused by abnormal vasculature and subsequent transcriptional activation by HIF-1 binding. In clear cell renal carcinomas, CA IX shows high expression under normoxia due to a mutation in the VHL gene that normally negatively regulates HIF-1. Because of its overexpression in many types of cancer and low expression in normal tissues, CAIX has become a useful target for clear cell RCC and breast cancer tumor imaging in mice.[19] [20]

CA IX plays a very significant role in tumor acidification as it has very high catalytic activity with the highest rate of proton transfer of the known CAs.[12] The enzyme converts carbon dioxide outside of the tumor into bicarbonate and protons, contributing to extracellular acidosis and promoting tumor growth by regulating the pH of the cytosol.

As a drug target

Because of its low expression in normal tissues and overexpression in many cancer tissues, CA IX has also become a desirable drug target. Girentuximab, an antibody that binds to CA IX, failed to improve disease-free as well as overall survival of patients with clear cell RCC in Phase III clinical trials.[21]

However, a number of small molecules have been used to inhibit CA IX. The main classes of these inhibitors are inorganic anions, sulfonamides, phenols, and coumarins. Anions and sulfonamides inhibit CA IX by coordinating the zinc ion within CA IX while phenols bind to the zinc-coordinated water molecule. Coumarins serve as mechanism-based inhibitors that are hydrolyzed by the enzyme to form a cis-2-hydroxy-cinnamic acid derivative that then binds to the active site.[22]

References

Further reading

Notes and References

  1. Opavský R, Pastoreková S, Zelník V, Gibadulinová A, Stanbridge EJ, Závada J, Kettmann R, Pastorek J . Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships . Genomics . 33 . 3 . 480–7 . May 1996 . 8661007 . 10.1006/geno.1996.0223 .
  2. Nakagawa Y, Uemura H, Hirao Y, Yoshida K, Saga S, Yoshikawa K . Radiation hybrid mapping of the human MN/CA9 locus to chromosome band 9p12-p13 . Genomics . 53 . 1 . 118–9 . October 1998 . 9787087 . 10.1006/geno.1998.5483 .
  3. Web site: Entrez Gene: CA9 carbonic anhydrase IX.
  4. Morgan PE, Pastoreková S, Stuart-Tilley AK, Alper SL, Casey JR . Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters . American Journal of Physiology. Cell Physiology . 293 . 2 . C738-48 . August 2007 . 17652430 . 10.1152/ajpcell.00157.2007 .
  5. Book: Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications. Frost. Susan C.. McKenna. Robert. Oct 2013. Springer Science & Business Media. 9789400773592. en.
  6. Chiche J, Brahimi-Horn MC, Pouysségur J . Tumour hypoxia induces a metabolic shift causing acidosis: a common feature in cancer . Journal of Cellular and Molecular Medicine . 14 . 4 . 771–94 . April 2010 . 20015196 . 3823111 . 10.1111/j.1582-4934.2009.00994.x .
  7. Web site: Tissue expression of CA9 - Summary - The Human Protein Atlas. www.proteinatlas.org. 2019-03-14.
  8. Ditte P, Dequiedt F, Svastova E, Hulikova A, Ohradanova-Repic A, Zatovicova M, Csaderova L, Kopacek J, Supuran CT, Pastorekova S, Pastorek J . Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors . Cancer Research . 71 . 24 . 7558–67 . December 2011 . 22037869 . 10.1158/0008-5472.CAN-11-2520 . free .
  9. Dorai T, Sawczuk IS, Pastorek J, Wiernik PH, Dutcher JP . The role of carbonic anhydrase IX overexpression in kidney cancer . en . European Journal of Cancer . 41 . 18 . 2935–47 . December 2005 . 16310354 . 10.1016/j.ejca.2005.09.011 .
  10. Alterio V, Hilvo M, Di Fiore A, Supuran CT, Pan P, Parkkila S, Scaloni A, Pastorek J, Pastorekova S, Pedone C, Scozzafava A, Monti SM, De Simone G . Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX . Proceedings of the National Academy of Sciences of the United States of America . 106 . 38 . 16233–8 . September 2009 . 19805286 . 2752527 . 10.1073/pnas.0908301106 . 2009PNAS..10616233A . free .
  11. De Simone G, Supuran CT . Carbonic anhydrase IX: Biochemical and crystallographic characterization of a novel antitumor target . Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics . 1804 . 2 . 404–9 . February 2010 . 19679200 . 10.1016/j.bbapap.2009.07.027 .
  12. Hilvo M, Baranauskiene L, Salzano AM, Scaloni A, Matulis D, Innocenti A, Scozzafava A, Monti SM, Di Fiore A, De Simone G, Lindfors M, Jänis J, Valjakka J, Pastoreková S, Pastorek J, Kulomaa MS, Nordlund HR, Supuran CT, Parkkila S . Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes . The Journal of Biological Chemistry . 283 . 41 . 27799–809 . October 2008 . 18703501 . 10.1074/jbc.M800938200 . free .
  13. Book: Tafreshi NK, Lloyd MC, Bui MM, Gillies RJ, Morse DL . Subcellular Biochemistry . Carbonic Anhydrase IX as an Imaging and Therapeutic Target for Tumors and Metastases . Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications . 75 . 221–54 . 2014 . 24146382 . 4282494 . 10.1007/978-94-007-7359-2_12 . Springer Netherlands . 9789400773585 .
  14. Zatovicova M, Sedlakova O, Svastova E, Ohradanova A, Ciampor F, Arribas J, Pastorek J, Pastorekova S . Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17 . British Journal of Cancer . 93 . 11 . 1267–76 . November 2005 . 16278664 . 2361518 . 10.1038/sj.bjc.6602861 .
  15. Innocenti A, Vullo D, Scozzafava A, Supuran CT . Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate . Bioorganic & Medicinal Chemistry Letters . 15 . 3 . 567–71 . February 2005 . 15664814 . 10.1016/j.bmcl.2004.11.056 .
  16. Kirkpatrick JP, Rabbani ZN, Bentley RC, Hardee ME, Karol S, Meyer J, Oosterwijk E, Havrilesky L, Secord AA, Vujaskovic Z, Dewhirst MW, Jones EL. Mark Dewhirst . Elevated CAIX Expression is Associated with an Increased Risk of Distant Failure in Early-Stage Cervical Cancer . Biomarker Insights . 3 . 45–55 . February 2008 . 19578493 . 2688355 . 10.4137/bmi.s570.
  17. Li J, Zhang G, Wang X, Li XF . Is carbonic anhydrase IX a validated target for molecular imaging of cancer and hypoxia? . Future Oncology . 11 . 10 . 1531–41 . 2015 . 25963430 . 4976829 . 10.2217/fon.15.11 .
  18. Pastorekova S, Ratcliffe PJ, Pastorek J . Molecular mechanisms of carbonic anhydrase IX-mediated pH regulation under hypoxia . BJU International . 101 Suppl 4 . s4 . 8–15 . June 2008 . 18430116 . 10.1111/j.1464-410X.2008.07642.x . 8780292 .
  19. Stillebroer AB, Franssen GM, Mulders PF, Oyen WJ, van Dongen GA, Laverman P, Oosterwijk E, Boerman OC . ImmunoPET imaging of renal cell carcinoma with (124)I- and (89)Zr-labeled anti-CAIX monoclonal antibody cG250 in mice . Cancer Biotherapy & Radiopharmaceuticals . 28 . 7 . 510–5 . September 2013 . 23697926 . 3741422 . 10.1089/cbr.2013.1487 .
  20. Kijanka MM, van Brussel AS, van der Wall E, Mali WP, van Diest PJ, van Bergen En Henegouwen PM, Oliveira S . Optical imaging of pre-invasive breast cancer with a combination of VHHs targeting CAIX and HER2 increases contrast and facilitates tumour characterization . EJNMMI Research . 6 . 1 . 14 . December 2016 . 26860296 . 10.1186/s13550-016-0166-y . 4747965 . free .
  21. Chamie K, Donin NM, Klöpfer P, Bevan P, Fall B, Wilhelm O, Störkel S, Said J, Gambla M, Hawkins RE, Jankilevich G, Kapoor A, Kopyltsov E, Staehler M, Taari K, Wainstein AJ, Pantuck AJ, Belldegrun AS . Adjuvant Weekly Girentuximab Following Nephrectomy for High-Risk Renal Cell Carcinoma: The ARISER Randomized Clinical Trial . JAMA Oncology . 3 . 7 . 913–920 . July 2017 . 27787547 . 10.1001/jamaoncol.2016.4419 . 5824229 .
  22. Maresca A, Temperini C, Vu H, Pham NB, Poulsen SA, Scozzafava A, Quinn RJ, Supuran CT . Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide inhibitors . Journal of the American Chemical Society . 131 . 8 . 3057–62 . March 2009 . 19206230 . 10.1021/ja809683v . 2158/594272 . 207136680 . free .

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