1. BioSci 145A lecture 18 page 1 © copyright Bruce Blumberg 2000. All rights reserved BioSci 145A Lecture 18 - Oncogenes and Cancer Topics we will cover today –Oncogenes and cell growth –tumor suppressor genes Final exam keys are posted evaluations will be done on 3/13 so please come and give us your candid assessment of the course

2. BioSci 145A lecture 18 page 2 © copyright Bruce Blumberg 2000. All rights reserved Cells in culture (contd) How does one judge the “normalcy” of cultured cells? –much can be surmised from morphology –what is the chromosomal constitution of the cells? chromosomal duplications, deletions and translocations are common in culture cells that have changed from normal, diploid state are aneuploid –are the cells anchorage dependent? most normal cells (except blood cells) are anchorage dependent many transformed cells can grow in soft agar –are the cells serum dependent? many abnormal cells are serum independent but many “normal” cell lines can be adapted to low or serum-free conditions –do the cells express normal protein complement? –do the cells form tumors if injected into animals? if not, they are not “transformed” cells originating from tumors are typically transformed –reduced serum dependence –reduced anchorage independence –reduced contact inhibition - cells grow in foci –will cause tumors if injected into animals typically use nude mice (lack significant part of immune system). somewhat cheating

3. BioSci 145A lecture 18 page 3 © copyright Bruce Blumberg 2000. All rights reserved Cancerous changes in cells benign vs malignant tumors –Tumor = swelling caused by new tissue growth –benign tumors contain cells that look and function like normal cells express normal complement of proteins typically remain localized to appropriate tissues –often surrounded by a fibrous capsule of connective tissues can become problematic if: –their size interferes with normal function of the tissue (e.g. brain tumor) –they secrete excessive amounts of biologically active substances such as hormones (e.g. pituitary tumor) –malignant tumors look qualitatively different from normal tissues of origin close enough to determine tissue of origin but not identical to normal tissue express only a subset of normal proteins many grow and divide more rapidly than normal can remain encapsulated in situ for a time (e.g. carcinoma in situ) later become invasive and metastatic (definition of malignant) –many tumors produce growth factors that increase the local blood supply Inhibiting angiogenesis is promising treatment

4. BioSci 145A lecture 18 page 4 © copyright Bruce Blumberg 2000. All rights reserved Cancerous changes in cells (contd) Induction of tumors –discovery of oncogenes led to the model that genetic changes could cause cancer –tumor incidence increases with age -> a series of events is required to cause a tumor believed that 6-7 discrete genetic events are required to get a cancer –agents that increase frequency of cell transformation are called carcinogens can be classified according to properties tumor initiators cause tumors –typically cause DNA damage (e.g. benzapyrene-diol-epoxide) tumor promoters aid in the growth of transformed cells, typically by inhibiting growth control (e.g. phorbol esters)

5. BioSci 145A lecture 18 page 5 © copyright Bruce Blumberg 2000. All rights reserved Cancerous changes in cells (contd) –two classes of genes are targets of mutations that cause transformation oncogenes encode proteins that can transform cells or cause cancer in animals –most are dominant gain of function mutations - three basic types –point mutations that cause constitutively active protein products –gene amplification that leads to overexpression –translocations that result in inappropriate expression (Dr. La Morte) tumor suppressor genes are recessive, loss-of- function mutations that inactivate cellular genes that regulate growth or cell cycle –five classes of tumor suppressor genes –intracellular proteins that regulate or inhibit progression through the cell cycle –receptors for secreted hormones that should inhibit cell proliferation (e.g. TGF-beta) –checkpoint control proteins that arrest the cell cycle if DNA is damaged or chromosomes are abnormal –proteins that promote apoptosis (programmed cell death) –enzymes that participate in DNA repair

6. BioSci 145A lecture 18 page 6 © copyright Bruce Blumberg 2000. All rights reserved Viruses can harbor transforming genes Peyton Rous (1911) –took chicken fibrosarcomas, ground them up, filtered out all cells, cellular debris and bacteria –injected this filtrate into other chickens -> fibrosarcomas Rous sarcoma virus remains one of the most virulent tumor viruses ever discovered –received the Nobel Prize in 1966 (55 years later, at age 86) when it was finally discovered that a virus was the cause of the cancer –http://www.nobel.se/medicine/laureates/1966/rous- bio.html –RSV contains an oncogene v-src that was demonstrated to be required for cancer induction RSV is a retrovirus with only 4 genes so this was relatively easy to demonstrate Bishop and Varmus (1977) –showed that normal cells from chickens and other species contained a cellular homolog of v-src. –This c-src (cellular src) was the first proto-oncogene –fundamental discovery that revolutionized the field (and got them a Nobel prize in 1989) was that cancer may be induced by the action of normal, or nearly normal cellular genes that were incorporated into transducing viruses –turns out that c-src is a protein tyrosine kinase that is constitutively active when mutated

7. BioSci 145A lecture 18 page 7 © copyright Bruce Blumberg 2000. All rights reserved Viral oncogenes (contd) many acutely transforming retroviruses exist –affect a variety of species –impact many cellular signaling pathways fundamental mechanism is transduction of cellular gene and later mutation due to inaccurate viral reverse transcriptases

8. BioSci 145A lecture 18 page 8 © copyright Bruce Blumberg 2000. All rights reserved Viral oncogenes (contd) oncogenes may be involved in many types of cancers –same c-onc (cellular oncogene) may be represented as v-onc (viral oncogenes) in a variety of cancers sis in both simian and feline sarcoma viruses –viruses may contain related v-onc genes Harvey and Kirsten sarcoma viruses contain v- ras genes derived from two different members of the c-ras family evidence exists directly linking oncogenes from acutely transforming retroviruses with cancer –first obtained from RSV using temperature sensitive mutations in v-src that allowed the phenotype to be reverted and regained identification of dominant oncogenes from acutely transforming retroviruses led to the model that single gene changes could cause cancers –major opponent to this idea was Peter Duesberg who later became somewhat infamous for his criticism of the involvement of HIV in AIDS in this case, Duesberg was correct –although the data linking acutely transforming retroviruses with cancer are strong, this mechanism is considered to be a relatively minor cause of cancer in humans

9. BioSci 145A lecture 18 page 9 © copyright Bruce Blumberg 2000. All rights reserved Viral oncogenes (contd) most acutely transforming retroviruses require normal retroviruses to get packaged into infective particles growth-promoting genes transduced by retroviruses confer a selective advantage because they increase the proliferation of infected tissues –retroviruses cannot replicate unless cell is proliferating viruses can be transferred laterally from one organism to another, carrying the cancer potential along viruses can also be transferred to offspring

10. BioSci 145A lecture 18 page 10 © copyright Bruce Blumberg 2000. All rights reserved Viral oncogenes (contd) Not all viruses are acutely carcinogenic –slow-acting retroviruses cause cancers by integrating near cellular protooncogenes and activating them inappropriately act slowly because integration into cellular protooncogenes is a rare event and other mutations may be required –various DNA viruses oncogenic potential resides in a single function or group of related functions that are activated early in viral lytic cycle many oncogenes act by inactivating tumor suppressor genes –polyoma T antigens –papilloma virus E6,7 antigens and cervical cancer –adenovirus E1A,B

11. BioSci 145A lecture 18 page 11 © copyright Bruce Blumberg 2000. All rights reserved Viral oncogenes (contd) Models for differences in properties between c-onc and v-onc –quantitative model viral genes are functionally indistinguishable from normal cellular genes oncogenesis comes from –overexpression –expression in inappropriate cell types –failure to turn expression off –qualitative model c-onc genes are not intrinsically oncogenic mutations can convert proto-oncogenes into oncogenes –that acquire new properties –or lose old properties –as usual, both models are correct mos, sis and myc genes can confer oncogenesis without significant mutation ras and src are changed by point mutations into dominant transforming oncogenes

12. BioSci 145A lecture 18 page 12 © copyright Bruce Blumberg 2000. All rights reserved Tumor cell DNA can transform cultured cells DNA from any of a variety of tumors can be transfected into cultured cells (typically NIH 3T3 cells) –a small number take up DNA and form foci of transformed cells –DNA is extracted from these foci and re-transfected into fresh cells to enrich for the specific human sequence –genomic library is prepared and human clones selected by hybridizing with repetitive DNA (Alu) –oncogene responsible is isolated and characterized

13. BioSci 145A lecture 18 page 13 © copyright Bruce Blumberg 2000. All rights reserved Tumor cell DNA can transform cultured cells (contd) Using such methods, a variety of human oncongenes were identified –two important properties were identified in oncogenes isolated in this way many have closely related sequences in the DNA of normal cells –this argues that the transformation was caused by mutation of a normal cellular gene (proto-oncogene) –could be a point mutation or reorganization of genomic DNA many have counterparts in the oncogenes carried by acutely transforming retroviruses –e.g.mutations were found in human bladder cancer DNA that corresponded those in the Ha-ras gene from harvey sarcoma virus. –oncogenes found in this manner frequently do not cause tumors when introduced into normal cells NIH-3T3 cells already have a mutation in a tumor suppressor gene that, in combination with the introduced oncogene, could lead to transformation It is important to note that DNA with transforming activity can only be isolated from tumorigenic cells –not present in normal DNA –in general, this is not such a great way to identify oncogenes

14. BioSci 145A lecture 18 page 14 © copyright Bruce Blumberg 2000. All rights reserved Oncogenes and cell growth Seven classes of proteins control cell growth –Collectively, these genes comprise the known set of genes involved in tumor formation

15. BioSci 145A lecture 18 page 15 © copyright Bruce Blumberg 2000. All rights reserved Oncogenes and cell growth (contd) Dominant transforming oncogenes are frequently created from proteins involved in regulating cell growth –Growth factors –Growth factor receptors –Intracellular transducers of above –Transcription factors that mediate the terminal effects of extracellular signaling

16. BioSci 145A lecture 18 page 16 © copyright Bruce Blumberg 2000. All rights reserved Oncogenes and cell growth (contd) Growth factors - proteins secreted by one cell that act on another cell (eg sis, wnt, int) –oncoprotein growth factors can only transform cells that harbor the specific receptor Growth factor receptors - transmembrane proteins that are activated by binding to extracellular ligand (protein) –very frequently protein tyrosine kinases –oncogenicity usually results from constitutive (ligand-independent) activation Intracellular transducers - several classes –protein tyrosine kinases, e.g. src –G-protein signal transduction pathways - primary effectors of activated growth factors (e.g. ras) –protein serine/threonine kinases (e.g. mos, raf) Transcription factors - these regulate gene expression directly –myc - HLH protein –fos, jun - b-ZIP proteins –erbA - nuclear receptor common feature among these is that each type of protein can trigger general changes in cell phenotypes by: –initiating changes that lead to cell growth –respond to signals that cause cell growth –altering gene expression directly

17. BioSci 145A lecture 18 page 17 © copyright Bruce Blumberg 2000. All rights reserved Oncogenes and cell growth (contd) One example signaling pathway - MAPK (Bardwell lab) growth factor receptor tyrosine kinase ras kinase cascase (serine/threonine) transcription factors since the signal passes from one component to the next, inappropriate activation of one element in the cascade canl lead to widespread changes in gene expression –these pathways are not strictly linear but branch and interact with many other signaling pathways can cause wider effects may require mutations in parallel pathways to get oncogenesis central importance of this pathway is illustrated by the number of components that can be mutated into oncogenes –aberrant activation of mitogenic pathways can contribute to oncogenicity

18. BioSci 145A lecture 18 page 18 © copyright Bruce Blumberg 2000. All rights reserved Oncogenes and cell growth (contd) Growth factor receptors are ligand modulated dimers –EGF receptor (v-erbB) is the prototype member EGF binding stimulates dimerization and activates tyrosine kinase cascade one oncogenic variant can dimerize in the absence of ligand and signals constitutively another lacks an internal regulatory domain resulting in constitutive signaling –activated kinase domain autophosphorylates and can then interact with src family proteins

19. BioSci 145A lecture 18 page 19 © copyright Bruce Blumberg 2000. All rights reserved Oncogenes and cell growth (contd) transforming activity of src-family kinases is related to kinase activity –autophosphorylation controls activity Y 416 -> active Y 527 -> weak, normally suppresses phosphorylation of Y 416 –some oncoproteins activate src by interfering with phosphorylation of Y 527

20. BioSci 145A lecture 18 page 20 © copyright Bruce Blumberg 2000. All rights reserved Oncogenes and cell growth (contd) modulation of transcription factor activity is important for oncogenesis –can’t cause cancer without altering gene expression!

21. BioSci 145A lecture 18 page 21 © copyright Bruce Blumberg 2000. All rights reserved Oncogenes and cell growth (contd) transcription factors and cancer –several prominent families of oncogenes are transcription factors - rel, jun, fos, erbA, myc, myb –actions may be quantitative or qualitative effects may be to increase activity of the oncoprotein –increased expression could upregulate target genes and influence growth, e.g. AP-1 alternatively, the mutations could make the oncoprotein a dominant negative inhibitor of other cellular transcription factors (e.g. v-erbA) –many members are “immediate early” genes transcription is immediately upregulated without the requirement for new protein synthesis when cells are treated with mitogens –likely to be involved with initiating or promoting growth increased activity would be expected to increase oncogenesis and it does with some but not others

22. BioSci 145A lecture 18 page 22 © copyright Bruce Blumberg 2000. All rights reserved Tumor suppressor genes oncogenesis is not typically dominant. A growing number of “tumor suppressor” genes have been identified that confer a genetic predisposition to cancers –several types of genes are involved apoptosis proteins (eg p53) cell-cycle control proteins (RB) DNA-repair proteins (p53) –classic examples are RB (retinoblastoma) and p53 –loss of tumor suppressor genes is implicated in several infrequent cancers of childhood retinoblastoma Wilm’s tumor

23. BioSci 145A lecture 18 page 23 © copyright Bruce Blumberg 2000. All rights reserved Tumor suppressor genes (contd) RB is a nuclear phosphoprotein that influences the cell cycle –unphosphorylated RB prevents cell proliferation by binding to E2F and blocking G1/S transition –phosphorylation of RB inhibits binding to E2F and releases block –some oncogenes (e.g. SV40 T-antigen, E1A) function by sequestering RB and removing block to cell growth –similar effects by loss of both alleles in human disease

24. BioSci 145A lecture 18 page 24 © copyright Bruce Blumberg 2000. All rights reserved Tumor suppressor genes (contd) A variety of other cell-cycle control proteins are tumor suppressor genes –p16, p21 and D cyclins –shown by identification of inactivating mutations in a variety of human tumors in quiescent cells –RB is not phosphorylated –D cyclin levels are low or absent –p16, p21 and p27 prevent activity of cdk-cyclin complexes cdc2, cdk2 and cdk4,6 interact with cyclins and promote cell cycle this is blocked by tumor suppressor genes

25. BioSci 145A lecture 18 page 25 © copyright Bruce Blumberg 2000. All rights reserved Tumor suppressor genes (contd) P53 suppresses cell growth or triggers apoptosis –more than 50% of human tumors have lost p53 protein or harbor mutations in the gene –a variety of mutations are possible recessive mutations cause loss of p53 function allowing unrestrained growth (eg. ko mice) others are dominant negative p53 mutants that interfere with normal p53 subunits in cells and allow unrestrained growth (eg rare cancers)

26. BioSci 145A lecture 18 page 26 © copyright Bruce Blumberg 2000. All rights reserved Tumor suppressor genes (contd) p53 has dual functions –cells normally have low levels of p53 –DNA damage induces large increase in p53 levels –increased p53 leads to growth arrest until DNA is repaired if cells are in G1 –cells in S-phase or later are triggered to become apoptotic p53 is a transcription factor that typically activates –one target is p21 -> cell cycle arrest –another is GADD45 - a DNA repair protein –role in inducing apoptosis is unknown at present apoptosis is an important pathway in preventing tumor formation - blocking it is a common strategy

27. BioSci 145A lecture 18 page 27 © copyright Bruce Blumberg 2000. All rights reserved Cancer - putting it all together