Aldehyde Dehydrogenase, more than a marker for cancer stem cells Marta Ferrer Fernández – Biochemistry Degree – Ba helor s Thesis – June 2016 INTRODUCTION • Can arise from mutations in normal stem cells, progenitor or mature differentiated cells • Are responsible for therapy failure, recurrence and metastasis • Give another perspective in the treatment against cancer Aldehyde dehydrogenase as a candidate target CANCER STEM CELLS (CSCs)... • Are a small tumour cell subpopulation that can initiate and drive tumourigenic growth • Carry out an asymmetric division: they are able to renew themselves and they can also differentiate 1. CANCER STEM CELLS AND THEIR ORIGIN 2. ALDEHYDE DEHYDROGENASE Normal stem cell Cancer stem cell ASYMMETRIC DIVISION Normal progenitor cell • Aldehyde dehydrogenases (ALDH) are enzymes that catalyse the oxidation of aldehydes to their consequent acids by a NAD(P)+-dependent irreversible reaction • This enzyme oxidises and detoxifies aldehydes that can be harmful to the organism • ALDH is considered a marker for CSCs, identifiable by the Aldefluor assay Mutated progenitor cell Normal differentiated cell ASYMMETRIC DIVISION Therapy failure (resistance) • Chemotherapy • Cooperation between ALDH and multi-drug resistance mechanism • Oxidation and inactivation of chemotherapeutic agents (cyclophosphamide) • Radiotherapy Metastasis • ALDH+ cells have demonstrated metastasic potential • This role may be due to the ALDH1A3 isoform Mutated differentiated cell Mutation De-differentiation ALDH is a CSC marker correlated with HETEROGENOUS TUMOUR FORMATION Conventional therapies CSC directed therapies TUMOUR RELAPSE TUMOUR REGRESSION Figure 1 | Origin of cancer stem cells. CSCs can renew themselves to preserve the stem cell population and they can also differentiate to form various specialised cells in an asymmetric division. In fact, they can arise from mutations in normal stem cells, progenitor cells or differentiated cells, and they are responsible for tumour formation and growth. For this reason, CSC directed therapies would lead to tumour regression, whereas conventional therapies cause tumour relapse. Tumour initiation and growth Existence of CSCs Figure was produced using Servier Medical Art (www.servier.com) Conventional therapies failure Tumour recurrence 3. RETINOIC ACID SIGNALLING PATHWAY Figure 2 | Aldefluor Assay. BODIPYaminoacetaldehyde can diffuse into the cell to be converted to a negativelycharged fluorescent product named BODIPY-aminoacetate. Need for targeted therapies against CSCs Metastasis 4. THERAPIES THAT TARGET CSCs RA ADH Retinol (vitamin A) ALDH CYP26 Retinaldehyde Retinoic acid (RA) v CRABP2 RA The interest of finding a CSC target is focused on the retinoic acid signalling pathway, in which ALDH plays an essential role. Oxidized products v FABP5 RA Nucleus Co-activator Figure 3 | Synthesis, degradation and mode of action of retinoic acid. Retinol is oxidised to retinaldehyde by a reversible reaction given by alcohol dehydrogenase (ADH). Then, retinaldehyde is oxidised to RA by ALDH in the cytosol, and free RA goes into the nucleus attached to a chaperone protein(CRABP2 or FABP5) in order to bind to RAR or RXR. When RA binds to the heterodimer, histone acetylation and activation of transcription is given. This signalling pathway is stopped when the degradation of RA is given by CYP26 enzymes. M E T H O D Targeting CSCdependent of signalling pathways Targeting the tumour microenvironment Immunotherapy Differentiation therapy S T R A T E G Y Some specific signalling pathways (TGF-β, Wnt, Notch and Hedgehog) are involved in the maintenance of the cancer stem cell state Affecting the microenvironment of the tumour, the maintenance of the stem cell state is lost and the EMT programme is altered Activation of immune response against specific antigens in CSCs This therapy induces the exit from the CSC state into a more differentiated state Elimination of signals that permit the stemness : inhibition of cytokines (such as IL-6) and growth factors Cytotoxic T lymphocytes, natural killers and antibodies All-trans retinoic acid (ATRA) treatment inhibits proliferation and invasion by activating retinoic acid pathway complex RAR RXR RARE Differentiation RAR PPAR PPRE Proliferation Cellular retinoic acid binding protein 2 (CRABP2); fatty acid binding protein 5 (FABP5); peroxisome proliferator-activated receptor (PPAR); response element (RE); retinoic acid receptor (RAR); retinoid X receptor (RXR) 5. CONCLUSIONS 1. CSCs open new frontiers in the treatment against cancer 2. ALDH is a good marker for CSCs because its identification and isolation is easier thanks to the Aldefluor Assay 3. Resistance to conventional therapies and metastasis can be explained by CSCs, where ALDH plays a valuable role on this matter 4. ATRA could be a novel cancer therapy by producing retinoic acid and causing differentiation, although nonCSCs also have to be eliminated to eradicate all the bulk of the tumour by conventional therapies ALDH is not only a marker for cancer stem cells, and further studies are needed to resolve more questions about cancer E X A M P L E S PRI-724 is an inhibitor of Wnt signalling that lo kades the βcatenin-CREB binding protein interaction proliferation RELEVANT REFERENCES 1. Allahverdiyev, A., Bagirova, M. & Oztel, O. in Dehydrogenases 3–28 (Intech, 2012). 2. Croker, A. K. The Role of ALDHhiCD44+ Cells in Breast Cancer Metastasis and Therapy Resistance. Doctoral Thesis, The University of Western Ontario, Canada, 2012. 3. Dr. Robert Weinberg - "Cancer Stem Cells: A New Target in the Fight Against Cancer". [Online]. Whitehead Pulse, 14/11/2011. Available in: https://www.youtube.com/watch?v=Nou8VWpWba4 4. Januchowski, R., Wojtowicz, K. & Zabel, M. The role of aldehyde dehydrogenase (ALDH) in cancer drug resistance. Biomed. Pharmacother. 67, 669–680 (2013). 5. Pattabiraman, D. R. & Weinberg, R. A. Tackling the cancer stem cells - what challenges do they pose? Nat. Rev. Drug Discov. 13, 497–512 (2014). Molecules in Figure 2 are from: Stem Cell Technologies. Technical Bulletin - Identification of viable stem and progenitor cells with Aldefluor [Online]. France; 2009. [Consultation 7/4/2016]. Available in: https://www.stemcell.com/~/media/Technical%20Resources/0/0/28728_aldefluor_July%202009.pdf?la=en Acknowledgements | I would like to thank my supervisor, Jaume Farrés; Carlota Colomer for the interview; R. Weinberg, D. Pattabiraman and I. Skvortsova for answering my emails and being so helpful.