Biological responses to tumor hypoxia & their potential as therapeutic targets

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Lecture by Dr. Brad Wouters in the context of the Course: "Tumour Hypoxia: From Biology to Therapy III".
For the complete e-Course see http://www.myhaikuclass.com/MaastroClinic/metoxia

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  • A key challenge for treating cancer effectively arises from phenotypic diversity within individual cancers. This is especially true when we are considering potentially curative therapies.
  • The genetic and epigenetic changes in tumor cells can tell us a lot about the nature of tumors, but is not enough to fully understand them. This is because the tumor microenvironment plays a critical role on the behavior of tumors by regulating many phenotypes that we associate with cancer.
  • Understanding the role and function of the genes and pathways altered in cancer requires us to consider them in context. There is evidence to suggest that genes/pathways are altered in cancer not only to facilitate cell autonomous functions (proliferation/survival) but also to influence their microenvironment. Simialrly, the genes that do provide cell autonmous advantages can have dramatic consequences for the micorenvironment. Eg apopotosis. Consequently the phenotype of both normal tissues, and tumors is a complex interaction between genes and their environment.
  • Importance goes beyond therapy resistance
  • Several key pathways have been recognized as important determinants of hypoxia tolerance, and thus the proportion of viable hypoxic cells in human tumors. These pathways are distinct, show unique dependencies on oxygen concentration, and influence many different downstream signalling pathways. However, a common theme that emerges from our current understanding is the ability of these pathways to transiently reprogram metabolism in a way that promotes energy homeostasis and cell survival.
  • Ubiquitin like molecule that is turned over
  • We are addressing this question – what is the importance of mTOR/4F regulation. We hypothesize that tumor cells maintain control over mTOR mediated translation to faciliate survival
  • Biological responses to tumor hypoxia & their potential as therapeutic targets

    1. 1. Biological responses to tumorhypoxia and their potential as therapeutic targets Brad Wouters This course is funded with the support of the METOXIA project under the FP7 Programme.
    2. 2. Learning ObjectivesUnderstand the importance of hypoxia to phenotypicdiversity in tumoursIdentify mechanisms that promote hypoxia tolerance intumoursUnderstand why mTOR regulation by hypoxia isimportantUnderstand why UPR signaling during hypoxia isimportant This course is funded with the support of the METOXIA project under the FP7 Programme.
    3. 3. Phenotypic diversity exists within tumorsGenetic, epigenetic,microenvironmentalCellular ‘individuality’Therapy ‘escape’Therapies are notequally effectiveagainst all tumorcells
    4. 4. Tumor hypoxia influences cancer biologyGene expressionCell phenotype• Cell signaling• Proliferation• Metabolism• Metastasis• Angiogenesis• DNA Repair,• Stem cells• Treatment resistance
    5. 5. Heterogeneity in OxygenationAmount SeverityDistribution Photos courtesy of Bert van der Kogel Time
    6. 6. The microenvironment is patient specific Cancer genetics influence the microenvironment
    7. 7. • Metabolism Genes • Angiogenesis • Genetic Instability Phenotype • Growth/proliferation • Metastasis • Stemness?Environment
    8. 8. Hypoxia is a stable phenotype of pancreaticcancers associated with aggressive disease Chang and Hedley, 2011,Cancer Research
    9. 9. Hypoxia varies widely amongst patients Hypoxic fraction in head and neck cancer Hypoxic fraction ranges from 0-100%
    10. 10. Hypoxia predicts for poor outcome 397 head and neck patients – 7 centers Nordsmark M et al, Radiother Oncol. 2005
    11. 11. Hypoxia and Treatment OutcomeCervix cancer – hypoxia predicts for overall survival Disease-free survival Metastasis Hypoxic Hypoxic
    12. 12. PSA Failure-Free Rate by Tumor Hypoxia All Patients (n=247) Bulk Patients (n=144) 1.0Biochemical failure free rate 0.9 0.8 p=0.03 p=0.009 0.7 Oxic (HP10=20%) Oxic (HP10=20%) Hypoxic (HP10=80%) Hypoxic (HP10=80%) 0.6 0 20 40 60 0 20 40 60 Time (month) Time (month) Milosevic et al, in press
    13. 13. What determines tumour hypoxia?
    14. 14. 1) Selection for hypoxia tolerant cells Graeber TG et al. Nature 379(6560): 88-91, 1996
    15. 15. Hypoxia and Selection – The case of p53 Hypoxia
    16. 16. Hypoxia-mediated mutation selection Graeber TG et al. Nature 379(6560): 88-91, 1996
    17. 17. 2) Hypoxia Initiates Adaptive Responses Wouters and Koritzinsky, Nature Reviews Cancer 2008
    18. 18. mTOR is a central metabolic regulator that responds to environmental signals Hypoxia Metabolism Protein synthesis Cell growth/proliferation
    19. 19. mTOR inhibition in hypoxic tumor areas Hedley et al, unpublished
    20. 20. Hypoxia suppresses mTOR and metabolism PTEN Metabolism Growth Proliferation Survival Koritzinsky et al, EMBO, 2006
    21. 21. mTOR inhibition promotes hypoxia tolerance in vivo Measure hypoxia Measure hypoxia Inject cells +/- Dox 4E 4EOE Rouschop et al, 2011
    22. 22. mTOR inhibition during hypoxia promotes therapy resistance IR response Inject cells +/- Dox 4E 4EOE 4 4 3 3 Relative tumour size Relative tumour size 15Gy 15Gy 2 2 1 PCDNA5 1 eIF4E Dox Dox 0 0 -7 0 7 14 21 28 35 42 49 56 63 70 -7 0 7 14 21 28 35 42 49 56 63 70 Days Days Rouschop et al, 2011
    23. 23. Hypoxia activates all 3 arms of the UPR Anoxia [hrs] 0 1 2 4 8eIF2α-P AnoxiaeIF2α [hrs] 0 1 2 4 6 * ATF-6 Anoxia [hrs] 0 .5 2 6 XBP-1u XBP-1s Wouters and Koritzinsky, Nature Reviews Cancer 2008
    24. 24. Targeting the UPR sensitizes cells to hypoxiaInject cells +/- Dox DN OE
    25. 25. Targeting the UPR improves treatment IR Inject cells +/- Dox 4DN 3 15Gy Relative tumor size 2 1 0 Dox -7 0 7 14 21 28 35 42 49 56 63 70 Days
    26. 26. Why is UPR signaling important ?Phenotype Tolerance Metastasis Autophagy LAMP3 LC3B, ATG5 pH control ROS, redox CA9 Glutathione biosynthesis Amino acid biosynthesis
    27. 27. Hypoxia inhibits translation through PERK HeLa C 1 2 4 8 C DTT eIF2 - P actin Koumenis et al., MCB, 2002
    28. 28. Hypoxia inhibits mRNA translation transcription translation DNA RNA Protein Hypoxia Hypoxia Hypoxia
    29. 29. Hypoxia inhibits overall protein synthesis Hypoxia Preserve energy Change which genes (ATP) are made into proteins
    30. 30. PERK/UPR signaling protects against ROS induced by fluctuating hypoxia
    31. 31. The UPR regulates CA9van den Beucken and Wouters, unpublished data
    32. 32. The UPR Enables Autophagy1 – Provide energy and nutrients during starvation2 – Remove damaged organelles and protein aggregates Both processes are cytoprotective
    33. 33. Autophagy is activated in hypoxic tumors NSCCNij172 Lc3b/pimo/vessels
    34. 34. The UPR induces LC3B via ATF4
    35. 35. LC3 is turned over during autophagy Geng and Klionsky, EMBO reports, 2008
    36. 36. LC3B protein is lost in UPR deficient cells HCT116 U373 Wildtype WildtypeLC3B protein LC3B protein PERK defective PERK defective
    37. 37. Hypoxia and the UPR stimulate autophagy Energy Removal of toxins Rouschop, JCI, 2011
    38. 38. PERK regulates energy metabolism
    39. 39. Inhibition of proteinsynthesis Bioenergetic homeostasis and protection against ROS Metabolism
    40. 40. Summary• Hypoxia causes phenotypic diversity in tumours• Hypoxia in patients is variable and determined by mechanisms that influence hypoxia tolerance• Hypoxia tolerance is influenced by mTOR and UPR regulation• UPR influences hypoxia tolerance through multiple mechanisms – ROS, pH, autophagy, metabolism

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