1. Chemotherapy : Drug resistance Gabriela M. Almeida Cancer Biology Group IPATIMUP Encontro Ciência 2010 Lisboa, 4 Junho 2010 ChemotherapyRecurrence Drug Resistance

2. Principles of chemotherapy Chemotherapy given with a curative intent, to prolong life or to palliate symptoms Chemotherapy generally acts by killing cells that divide rapidly, characteristic of cancer cells. However it will also affect normal cells that divide rapidly (e.g. bone marrow, digestive tract, hair follicles). Conventional chemotherapy regimens usually involve several cycles of therapy with cytotoxic agents, the purpose being to kill tumour cells but allowing time for normal cells to recover from the damage. Most agents used in chemotherapy affect cell division or DNA synthesis. More recently targeted therapies (towards specific molecules involved in cancer progression) became available.

3. Pros and cons of chemotherapy Pros: cure patients, prolong life or to improve quality of life (palliation of symptoms); able to remove micro-metastasis far from the site of origin Cons: Lacks selective toxicity - Toxicity towards normal cells/tissues (sometimes life- threatening side effects); Long-term toxicity/carcinogenicity (particularly relevant when treating young patients) Selection of drug resistant clones that may lead to tumour recurrence General toxic side effects of anticancer drugs Most suppress the bone marrow and immune system Many cause nausea & vomiting Oral and GI ulceration, and diarrhea Hair may fall out (alopecia) Sterility Teratogenicity Carcinogenic

4. Mechanisms of chemoresistance, platinum-based agents as an example Platinum based agents are widely used in chemotherapy; Cisplatin was first administered to patients in the early 70s and is still widely used in chemotherapy (e.g. testicular, lung and ovarian cancer, etc); Carboplatin and Oxaliplatin, less toxic platinum based compounds, are used in many chemotherapy protocols; Mechanism of action of cisplatin Administered intravenously, inactive whilst in the bloodstream; Becomes active upon entering the cell; Forms protein, RNA, and DNA adducts; DNA adducts are the key toxic lesions formed by Cisplatin; Adducts cause inhibition of DNA replication, RNA transcription, arrest at the G2 phase of the cell cycle, and/or apoptosis. Kartalou and Essigmann, 2001

5. Detecting platinum-induced crosslink formation and repair in vitro and in the lymphocytes of cancer patients receiving platinum-based chemotherapy

6. Cisplatin Resistance by inadequate levels of cisplatin reaching target DNA Kelland, Nat Rev Cancer, 2007 Reduced intracellular accumulation of cisplatin; Increased inactivation by intracellular proteins (e.g. glutathione);

7. Cisplatin Resistance mediated after DNA binding Kelland, Nat Rev Cancer, 2007 Increased repair of cisplatin adducts; Increased ability to replicate past cisplatin adducts; Defects in the apoptotic response pathway.

8. Cancer stem cells (CSCs) The cancer stem cell (CSC) hypothesis is an attractive model to explain the functional heterogeneity that is commonly observed in solid tumours. It proposes a hierarchical organization of cells within the tumour, in which a subpopulation of stem-like cells is responsible for sustaining tumour growth. First evidence for CSCs came from acute myeloid leukaemia. There is now increasing evidence for CSCs in a variety of solid tumours (both mouse and human). The frequency of CSCs in solid tumours is highly variable, reflecting biological variation as well as technical issues. “All roads lead to Rome”? Fábián et al. 2009, Cytometry Part A 75A:67-74 The CSC phenotype could be acquired by normal tissue stem, progenitor or differentiated cells through transforming mutations, which activate/deregulate certain signalling pathways;

9. CSCs and Therapy Resistance CD133+ cells were significantly resistant to chemotherapeutic agents (e.g. carboplatin, Taxol and etoposide) compared to autologous CD133- cells. CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumours.

10. Cancer stem cells: Key players in chemoresistance? CSCs are believed to be, in part, responsible for therapy resistance as they are generally more resistant than the cells that constitute the bulk of the tumour. The chemoresistant phenotype of CSCs is believed to be due to: 1. Overexpression of drug efflux pumps 2. Alterations in apoptosis proteins: Overexpression of anti-apoptotic genes and members of the inhibitor of apoptosis protein 3. Increased telomerase expression 4. Increased antioxidant capacity/enhanced resistance to oxidative stress

11. Schatton et al. 2009, BioEssays 31:1038-1049 Currently believed that chemotherapeutic regimens are not able to effectively eradicate CSCs (but only the cancer cells that constitute the bulk of the tumour) and that this will ultimately be responsible for recurrence. Crucial to effectively target and eradicate these cells in order to improve the outcome of cancer patients Cancer stem cells: Key players in chemoresistance? FCT Financed Project: PTDC/EBB-BIO/099672/2008 (Biotecnologia) “Desenvolvimento de nanopartículas encapsuladas com siRNAs para modular a resistência a agentes quimioterapêuticos em células estaminais cancerígenas” IPATIMUP,CEQUIMED-UP, IBMC

12. Circumvention of chemoresistance in CSCs CSCs Non-CSCs Specific cell surface markers % Cell Survival Chemotherapeutic Drug Mechanisms/Proteins responsible for chemoresistance? Targeting by siRNAs Chemotherapy Coupling with CSC targeted nanoparticles Recurrence

13. Circumvention of chemoresistance in CSCs No Recurrence Chemotherapy + siRNA encapsulated CSC targeted nanoparticles ChemotherapyRecurrence

14. The Economist, 2008 Research Team: IPATIMUP: GM Almeida, MH Vasconcelos, LF Santos-Silva, RT Lima, H Seca CEQUIMED-UP: CM Barbosa, M Teixeira, R Pereira, E Sousa, E Tiritan IBMC: TL Duarte, M Pinto