Endocrinology & Cancer

1. Endocrinology & Cancer Endocrinology 317/319 Department of Biology University of Massachusetts Boston Kenneth L. Campbell & Victoria Del Gaizo Moore

2. Overview of main points to be covered: Cell Cycle Control What can go wrong Stages of Cancer Development Specific Examples of Cancers

3. Oncogenesis - Chemical Signaling and Cancer • Basic Concepts • Tumor • Benign (self contained) • Malignant (migratory, prone to seeding tumors at other sites) • Hypertrophy - hypertrophic cells; hyperplastic tissue • Neoplasia - new, often irregular, growth or tissue • Proto-oncogenes = Normal gene precursors of oncogenes I V Mutational AgentsI V • Oncogenes = Gene associated with abnormal cell growth • Oncogene Product = Expressed protein coded by an oncogene • Mutagens • Radiation, Chemical - tend to be small changes, insertions, deletions, or base changes • Chromosome Rearrangements (in meiosis) - can be large changes, deletions, inversions • Viral Rearrangement - viruses can become lysogenic & excise & carry genes or foreign promoter DNA to subsequent cellular hosts where these insert into non-homologous sites & are expressed in a non-regulated or inappropriately regulated fashion, often leading to oncogenesis via disruption of the normal events of the cell cycle or cell cycle regulatory points.

4. Mitotic Cell Cycle & Its Controls

5. Normal Cell Cycle & Controls Rb p53, p21 route to apoptosis role of DNA repair

7. Normal Stages of Mitosis shown by time. Cells receive cues from their environment (surrounding cells and tissues) that help direct it to undergo division Each cell must monitor DNA replication to make sure that synthesis is correct, fixing any mistakes along the way.

8. During DNA replication the two strands are copied in opposite directions using different methods. Topoisomerase is also involved and makes cuts in one or both strands that must be ligated to maintain sequence integrity. Thus, there are opportunities for sequence mismatches or replication mistakes even during mitosis.

9. The anti-parallel, helical, frequently coiled and supercoiled nature of DNA within cells presents topological problems during replication that can result in mistakes that must be monitored by the cell cycle machinery.

10. Rb protein: the first major checkpoint

11. pRB- retinoblastoma protein named so because in retinoblastoma cancer both alleles of the gene are mutated so no protein is produced pRb prevents the cell from dividing or progressing through the cell cycle when DNA is damaged. Control occurs at S (DNA synthesis phase) because pRb binds and inhibits transcription factors of the E2F family When pRb is ineffective at this role, mutated cells can continue to divide and may become cancerous.

12. pRB regulation depends on phosphorylation pRb can actively inhibit cell cycle progression when it is dephosphorylated Therefore phosphorylation inactivates its function. At the end of mitosis (M phase) pRB depends on a phosphatase to dephosphyorylate it, allowing it to bind to E2F

13. pRb the Master Controller: The First Checkpoint Robert A. Weinberg, How Cancer Arises, Scientific American 275(3):62-70, September 1996.

14. Phosphorylation of pRb by cyclin/cyclin-kinase complexes allows release of pRb-bound transcription factors such as E2F. Now free, the transcription factors can alter expression of genes necessary for cell growth and DNA synthesis. Rachel A. Freiberg, Susannah L. Green, Amato J. Giaccia Hypoxia and Cell Cycle In: Cell Cycle Checkpoints and Cancer Mikhail V. Blagosklonny, Ed.ISBN: 1-58706-067-1

15. Human Papilloma Virus, HPV, perturbs the pRb checkpoint allowing cells to enter S Phase under conditions that may not be optimal or safe for DNA synthesis or cell replication. Hoenil Jo, Jae Weon Kim, Implications of HPV infection in uterine cervical cancer, Cancer Therapy 3: 419-434, 2005 Sequential phosporylation of Rb by cyclin/cdk complex inhibits the repressor activity of pRb. The HPV E7 binds to the hypophosphorylated form of the pRb proteins. This binding disrupts the complex between pRB & the cellular transcription factor E2F, resulting in the liberation of E2F, which allows the cell to enter the S phase of the cell cycle.

16. p53: the second major checkpoint

17. p53, p21 & The Second Checkpoint Robert A. Weinberg, How Cancer Arises, Scientific American 275(3):62-70, September 1996.

18. Hypoxia, cellular exposure to reactive oxygen species, mitochondrial damage, or direct damage of DNA by chemicals or radiation can stimulate p53 actions that halt cell division, activate DNA repair mechanisms, &/or stimulate the cell to undergo apoptosis (programmed cell death). This protects the organism from clonal expansion of mutated cells. Olivier Pluquet, Pierre HainautGenotoxic and non-genotoxic pathways of p53 induction Oncoserve Online, 2004 Oncoserve Online p53.mht

19. Hoenil Jo, Jae Weon Kim, Implications of HPV infection in uterine cervical cancer Cancer Therapy 3: 419-434, 2005 HPV infection also perturbs the second, p53, checkpoint preventing p53 from diverting cells with damaged DNA toward cell cycle arrest or cell death. Damaged cells can then proliferate unchecked. DNA damage induces p53 activation, leading to either cell cycle arrest or apoptosis. The HPV E6 binds to E6-AP & redirects it to p53, which results in the E6-AP-mediated ubiquitination & rapid proteasomal degradation of p53.

20. RB/p53 Interactions To Regulate Cell Cycle & Apoptosis Cell cycle transition from G1-S phase is mediated by Rb interactions with the E2F transcription factor family, an important regulator of the cell cycle. Growth factors lead to phosphorylation of Rb in late G1 phase by cdk/cyclin. This is followed by release of E2F, allowing transcriptional activation of E2F target genes; this promotes S-phase entry & cell proliferation. HPV E7 & Simian Virus 40 (SV40) promote release of E2F from Rb. In contrast HPV E6 & the dominant negative, DN-p53 inhibit p53 activity leading to cell proliferation. Shehata, Cancer Cell International 2005 5:10   doi:10.1186/1475-2867-5-10

21. p53 Rb www.physiomics-plc.com/cell%20cycle%20model.htm Cell Cycle Checkpoints

22. http://www.wellesley.edu/Chemistry/chem227/nucleicfunction/cancer/cancer.htmlhttp://www.wellesley.edu/Chemistry/chem227/nucleicfunction/cancer/cancer.html http://www.eurogene.org/etext/cancgen/lectures/Bernards.htm http://www.fhcrc.org/science/education/courses/cancer_course/basic/approaches/fundamentals/cellcycle.html www-medchem.ch.cam.ac.uk/picture.html

23. Summary of Cancer and Cell Cycle: Carcinogenesis seems to be a multistage process where normal cells progress to cancer through a gradual accumulation of genetic errors. This has led to the hypothesis that cancer may be caused by mutations that cause genetic instability. Cell cycle controls, checkpoints, maintain the genetic stability of dividing cells. There are at least two important known checkpoints, located in G1/S and G2/M involving tumor suppressors Rb and p53, respectively. p53 is activated by DNA damage. During carcinogenesis, these checkpoints fail to prevent abnormal cells from going through the cell cycle.

24. Extracellular factors may alter the status of the pRb checkpoint & thereby change transcriptional activities. http://www.benbest.com/health/cancer.html

25. Disruption of the cell cycle can result in replication of cells with: damaged DNA increased susceptibility to further DNA damage ability to avoid apoptosis ability to avoid immune surveillance activated telomerase

26. Proto-oncogene Gene mutation, gene movement, or change in regulatory sequences or position Oncogene transcription Oncogene-Product Mechanisms of DNA damage/mutation chemical – reactive agents, reactive oxygen species, environmental agents radiation replication damage – translocations, deletions, insertions viral insertion/excision

27. Oncogene: modified gene that is believed to cause cancer • The proto-oncogene can become an oncogene by a relatively small • modification of its original function. There are three basic activation types: • A mutation within a proto-oncogene can cause a change in the protein structure, causing • an increase in protein (enzyme) activity • a loss of regulation(pRB) • An increase in protein concentration, caused by • an increase of protein expression (through misregulation)(BCL-2 with miRNA’s) • an increase of protein stability, prolonging its existence and thus its activity in the cell • a gene duplication (one type of chromosome abnormality), resulting in an • increased amount of protein in the cell • A chromosomal translocation (another type of chromosome abnormality), causing • an increased gene expression in the wrong cell type or at wrong times • the expression of a constitutively active hybrid protein. This type of aberration in a • dividing stem cell in the bone marrow leads to adult leukemia(BCL-2 with t(14;18))

28. Apoptotic Pathways: All roads lead to mitochondria • Extrinsic • Extracellular signals cause intracellular changes • Caspase activation (8), eventually leads to mitochondria • Instrinsic • Starts with signals within the cell • Caspase activation (9 and 3), Cytochrome c release

29. ABCDEFH2FL-1FL-2H2 • comparable to levels found in follicular lymphoma and H2 cells, which have a t(14;18) BCL-2 actin BCL-2, a different kind of oncogene BCL-2 was originally discovered in Follicular Lymphoma patients a chromosomal translocation between chromosomes 14 and 18 placed BCL-2 under constitutive control of Ig heavy chain results: much BCL-2 protein is made all of the time in these cells This was the first time where a mutation that caused a protein to be overexpressed caused cancer (in the past only down-regulation models were known, like pRB) In Chronic Leukemia BCL-2 is also overexpressed, despite the absence of t(14;18) microRNAs are responsible for BCL-2 overexpression in these cancers loss of mir15 and 16 cause uncontrolled BCL-2 protein expression Del Gaizo Moore, et al. JCI 2007

30. How does BCL-2 overexpression lead to cancer? There are pro and anti-apoptotic proteins those that promote and those that oppose cell death BCL-2 is an anti-apoptotic protein DNA damage, environmental stress, or other cellular stresses cause death signaling molecules to be expressed. These death signals are able to be bound up by BCL-2. In a normal cell with regular amounts of BCL-2, the BCL-2 gets “full” of death signals Easily and there are excess death molecules to trigger apoptosis BUT, if a cell has extra BCL-2 then it can absorb ALL of the death molecules thereby preventing cell death

31. Normal cell BCL-2 overexpressing cell Death Cancer - death signals - other proteins bound to BCL-2 - cytochrome c - BCL-2 protein - BAX/BAK protein

32. Genes often involved in cancer formation, are hot-spots for primary or secondary mutations Tumor Suppressors Receptors Transducers Hormones Transcription Factors

33. Sigma-Aldrich

34. Sigma-Aldrich

36. The Process of Carcinogenesis

37. Cancer: inappropriately controlled cell replication leading to disruption of normal physiology, metabolism or structure ultimately disrupting homeostasis irreversibly Neoplasm: new cell growth; may be benign (not unlimited or invasive) or malignant (cancerous leading toward metastasis) Hypertrophy – elevated size, for cell or tissue Hyperplasia – elevated cell number

38. Path to Cancer: Completion is rare due to endogenous controls & mechanisms! Induction/Initiation/primary mutation – fixation into the genome -- avoidance of apoptosis -- avoidance of immune rejection Promotion – stimulation of cell expansion of mutated clone -- continued avoidance of apoptosis -- continued avoidance of immune surveillance Conversion/Transformation -- epigenetic &/or secondary mutations -- immortalization, activation of telomerase -- loss of cell contact inhibition -- angiogenesis of primary tumor Progression -- tertiary mutations -- outgrowth of tumor Metastases -- breach of vascular endothelium -- lodging & binding to capillary beds -- invasion of secondary sites

39. Changes in Cancer These range from the molecular through cellular & organ levels to the entire organism. What begins in a single cell, possibly even a single alteration of one chemical bond, ultimately manifests as a terminal physiological imbalance for the host organism. This might be termed the “Butterfly Effect” in cancer. Fabio Grizzi, Maurizio Chiriva-Internati, Cancer: looking for simplicity and finding complexity, Cancer Cell International 2006, 6:4, doi:10.1186/1475-2867-6-4.

40. James E. Trosko, Randall J. Ruch, Cell-cell communication in carcinogenesis, Frontiers in Bioscience, 3, d208-236, 2/15/1998. Schematic of the route from a tissue stem cell through the process of mutation & clonal expansion to malignancy

41. James E. Trosko, Randall J. Ruch, Cell-cell communication in carcinogenesis, Frontiers in Bioscience, 3, d208-236, 2/15/1998. Mathematical model of the route from a normal cell through the various steps & processes leading to overt cancer

42. Ball diagram of Nowell's hypothesisGreen balls represent cells that have developed a genetic abnormality & are expanding or growing into a clone of cells. One of these cells develops a 2nd genetic abnormality, blue ball. This then expands into its own clone of cells, subclones of the green population. A third genetic mistake, dark red ball, leads to clonal expansion of a sub-subclone. Eventually, a genetic mistake is made in one of the cells of the subclones or sub-subclones that allows that cell to spin off a cancerous subclone. http://www.barrettsinfo.com/content/5_how_does_cancer_develop_in_barretts.htm The site was funded by AstraZeneca LP through an unrestricted educational grant to theRyan Hill Research Foundation, Seattle, WA

43. Another schematic of carcinogenesis emphasizes the early steps in tumor formation including the role of cellular repair & suppression of apoptosis but fails to note the importance of secondary or tertiary mutations in allowing clonal expansion, evasion of immune suppression, & establishment of unlimited growth potential. Where might those occur? http://www.belleonline.com/n2v91.html James E. Klaunig, Lisa M. Kamendulis, Yong Xu, Epigenetic Mechanisms of Chemical CarcinogenesisDivision of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN

44. Another schematic emphasizing the cellular stages in carcinogenesis, cellular mutation, & agents that may be involved in the various steps. http://www2.scitech.sussex.ac.uk/undergrad/coursenotes/ehh/lec4/4.html

45. This depiction of the steps in the in vivo process compares these changes with what occurs during the in vitro transformation of cells grown in tissue culture. Cell Transformation Assays as Predictors of Human Carcinogenicity The Report and Recommendations of ECVAM Workshop 391-3 ATLA27: 745-767

46. The upper row represents disturbances in growth, differentiation, & tissue integrity that lead to the phenotypes that characterize the different stages of cancer, shown in the lower row. Multiple genetic alterations underlie cancer development, including oncogene activation & tumor suppressor loss of function. Sigma-Aldrich

47. Note that even in well developed experimental models we still lack clear markers or unique triggers for the Promotion & Progression stages of carcinogenesis. Zbigniew Walaszek, Margaret Hanausek, Thomas J. Slaga, Combined natural source inhibitors in skin cancer prevention, Cellscience Reviews 1(3), ISSN 1742-8130.

48. This description comes a bit closer to what has been observed; there still is obvious discussion of which molecular or cellular events contribute specifically to the Promotion, Progression, Conversion/ Transformation, Invasion/ Metastasis steps.

49. The interaction of carcinogens with cells that can lead cells toward formation of malignant tumors. Note the multiple points at which a carcinogen or cells damaged by a carcinogen may be eliminated or repaired. Tobacco smoking and cancer: The promise of molecular epidemiology Sophia S. Wang, B.S., Jonathan M. Samet, M.D., M.S. Salud Publica Mex 1997;39:331-345.

50. Carcinogen exposure is not simply the amount of compound applied to an organism but the amount to which the reactive molecules in target cells are exposed, moreover, multiple steps of response are involved in activation of cancer. http://www.iem.cas.cz/Data/Img/big/genetic_exotox_fig1.jpg Department Of Genetic Ecotoxicology, Institute of Experimental Medicine,Academy of Sciences of the Czech Republic