BioSci 145A Lecture 15 - Oncogenes and Cancerblumberg-lab.bio.uci.edu/past teaching/bio145a... ·...

BioSci 145A lecture 15 page 1 ©copyright Bruce Blumberg 2000. All rights reserved

BioSci 145A Lecture 15 - Oncogenes and Cancer

• Topics we will cover today– Introduction to normal and cancer cells– Characteristics of cells in culture– Cancerous changes in cells– Viruses can harbor transforming genes– DNA from tumor cells can transform normal

cultured cells– Oncogenes and cell growth– tumor suppressor genes

• Last year’s final exam is posted

• Don’t forget that the next two lectures will be held in theBeckman Laser Institute Library

• No office hours on the following dates (I will be out oftown)– 3/1– 3/6

• Office hours for 3/8 will be held on 3/9 from 2-3


Introduction to normal and cancer cells

• Most cells in the organism have a finite lifetime– majority of differentiated cells are postmitotic

• stem cells can divide nearly endlessly• other cell types that typically divide

– skin– lining of gut– hematopoeitic stem cells– hair follicles

• liver cells can dedifferentiate, re-enter the cellcycle

– cell growth and division are tightly controlled• most cells that can divide are only capable of a

finite number of cell divisions– so-called Hayflick limit

• cancer cells are a notable exception• Cancer cells have lost their ability to regulate their own

growth or to respond to normal growth regulatory cues orto sense their proper location in the organism– each of these characteristics of cancer cells

contributes to disease progression– a variety of genetic events are responsible– generally speaking, different genetic events can be

associated with characteristics of the developingtumor.


Introduction to normal and cancer cells (contd)• Three types of changes occur

as a cell becomestumorigenic

– immortalization - cellsretain the ability todivide endlessly

• not necessarilydetrimental toorganism

• telomerase– transformation - cells

stop responding tonormal growth controls

• do not need growthfactors and/or

• do not respond togrowth inhibitors

• transformed cellstypically formtumors in situ

– metastasis - cells gainthe ability to move fromtheir normal location andinvade other tissues

• very dangerousfeature of cancercells

• aberrant regulationof extracellularmatrix proteases


Cells in culture

• growth characteristics of normal and tumor cells differ– normal cells do not grow well, in vitro, typical

cancer cells grow very well– primary cells are the immediate descendents of cells

taken directly from a tissue.• such cells divide a small number of times and

then stop growing - senescence• subsequently, most cells will die.

– Lewin calls this the crisis stage– if the cells are kept and fed for a long time,

a small number may begin to grow– cell lines are cells that successfully pass through

crisis and gain the ability to divide indefinitely• many, if not most, overexpress telomerase

• Fundamental rule - Cells (even primary cells) changetheir phenotype almost immediately when they are placedin culture– degree of difference depends on the similarity of

their microenvironment to their usual environment• extracellular matrix• type and density of surrounding cells

– change usually comes after several cell divisions.• primary cells that stop dividing will maintain

more of their phenotype than those that divide


Cells in culture (contd)

• Characteristics of cells in culture - most cells grow as amonolayer for the following reasons:– anchorage dependence - cells require a substrate to

grow on• solid or semi solid medium

– serum dependence - cells require substances in serumto grow

• commonly called growth factors but in realitythere are two different types

– mitotic factors - required for cells to growand divide

» typically peptide growth factors, e.g.FGF, EGF, PDGF, etc

– survival factors - not strictly required forcell division, but required for cells tosurvive in culture

» typically lipids or other smallmolecules, e.g. retinol, 14-hydroxyretroretinol

– density-dependent inhibition (contact inhibition) -cells only grow until confluence

• surface is completely covered• at this time cells go into G0 and exit the cell

cycle– cytoskeletal organization - cells are flat and extended

on the surface



• illustrates– morphological differences

• flat vs rounded up– contact inhibition

• transformed cells pile up on plates and cluster in3D

– nuclear morphology• note strong staining in transformed cells, a

higher resolution picture would showmultinucleate cells and mitotic figures



• 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 andtranslocations are common in culture

• cells that have changed from normal, diploidstate 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 ofimmune system). somewhat cheating


Cancerous changes in cells

• benign vs malignant tumors– 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 ofconnective 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 ashormones (e.g. pituitary tumor)

– malignant tumors look qualitatively different fromnormal tissues of origin

• close enough to determine tissue of origin butnot 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


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 oftransformed cells, typically by inhibiting growthcontrol (e.g. phorbol esters)


Cancerous changes in cells (contd)

– two classes of genes are targets of mutations thatcause transformation

• oncogenes encode proteins that can transformcells or cause cancer in animals

– most are dominant gain of functionmutations - three basic types

– point mutations that cause constitutivelyactive protein products

– gene amplification that leads tooverexpression

– translocations that result in inappropriateexpression (Dr. La Morte)

• tumor suppressor genes are recessive, loss-of-function mutations that inactivate cellular genesthat 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 orchromosomes are abnormal

– proteins that promote apoptosis(programmed cell death)

– enzymes that participate in DNA repair


Viruses can harbor transforming genes

• Peyton Rous (1911)– took chicken fibrosarcomas, ground them up, filtered

out all cells, cellular debris and things as small asbacteria

– injected this filtrate into other chickens ->fibrosarcomas

• Rous sarcoma virus remains one of the mostvirulent tumor viruses ever discovered

– received the Nobel Prize in 1966 (55 years later)when it was finally discovered that a virus was thecause of the cancer

• RSV contains an oncogene v-src that was demonstratedto 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 otherspecies 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) was that cancer may beinduced by the action of normal, or nearly normalcellular genes that were incorporated intotransducing viruses

– turns out that c-src is a protein tyrosine kinase that isconstitutively active when mutated


Viral oncogenes (contd)

• many acutelytransformingretroviruses exist– affect a variety of

species– impact many

cellular signalingpathways

• fundamentalmechanism istransduction ofcellular gene and latermutation due toinaccurate viralreverse transcriptases



• 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 ofthe c-ras family

• evidence exists directly linking oncogenes from acutelytransforming retroviruses with cancer– first obtained from RSV using temperature sensitive

mutations in v-src that allowed the phenotype to bereverted and regained

• identification of dominant oncogenes from acutelytransforming retroviruses led to the model that singlegene changes could cause cancers– major opponent to this idea was Peter Duesberg who

later became somewhat infamous for his criticism ofthe involvement of HIV in AIDS

• in this case, Duesberg was correct– although the data linking acutely transforming

retroviruses with cancer are strong, this mechanismis considered to be a relatively minor cause of cancerin humans



• most acutely transforming retroviruses require normalretroviruses to get packaged into infective particles

• growth-promoting genes transduced by retrovirusesconfer a selective advantage because they increase theproliferation of infected tissues– retroviruses cannot replicate unless cell is

proliferating

• viruses can be transferredlaterally from one organismto another, carrying thecancer potential along

• viruses can also betransferred to offspring



• Not all viruses are acutely carcinogenic– slow-acting retroviruses

• cause cancers by integrating near cellularprotooncogenes and activating theminappropriately

• act slowly because integration into cellularprotooncogenes is a rare event and othermutations may be required

– various DNA viruses• oncogenic potential resides in a single function

or group of related functions that are activatedearly in viral lytic cycle

• many oncogenes act by inactivating tumorsuppressor genes

– polyoma T antigens– papilloma virus E6,7 antigens and cervical

cancer– adenovirus E1A,B



• Models for differences in properties between c-onc andv-onc– quantitative model

• viral genes are functionally indistinguishablefrom 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 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


Tumor cell DNA can transform cultured cells

• DNA from any of avariety of tumors can betransfected into culturedcells (typically NIH 3T3cells)– a small number take

up DNA and formfoci of transformedcells

– DNA is extractedfrom these foci andre-transfected intofresh cells to enrichfor the specifichuman sequence

– genomic library isprepared and humanclones selected byhybridizing withrepetitive DNA(Alu)

– oncogeneresponsible isisolated andcharacterized


Tumor cell DNA can transform cultured cells (contd)

• Using such methods, a variety of human oncongenes wereidentified– 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 reorganizationof genomic DNA

• many have counterparts in the oncogenes carriedby acutely transforming retroviruses

– e.g.mutations were found in human bladdercancer DNA that corresponded those in theHa-ras gene from harvey sarcoma virus.

– oncogenes found in this manner frequently do notcause tumors when introduced into normal cells

• NIH-3T3 cells already have a mutation in a tumorsuppressor gene that, in combination with theintroduced oncogene, could lead to transformation

• It is important to note that DNA with transforming activitycan only be isolated from tumorigenic cells– not present in normal DNA– in general, this is not such a great way to identify

oncogenes


Oncogenes and cell growth

• Seven classes of proteins control cell growth– Collectively, these genes comprise the known set of

genes involved in tumor formation


Oncogenes and cell growth (contd)• Dominant

transformingoncogenes arefrequently createdfrom proteinsinvolved inregulating cellgrowth– Growth factors– Growth factor

receptors– Intracellular

transducers ofabove

– Transcriptionfactors thatmediate theterminal effectsof extracellularsignaling


Oncogenes and cell growth (contd)

• Growth factors - proteins secreted by one cell that act onanother 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 expressiondirectly– myc - HLH protein– fos, jun - b-ZIP proteins– erbA - nuclear receptor

• common feature among these is that each type of proteincan 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



• 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 cascadecanl 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 intooncogenes– aberrant activation of mitogenic pathways can

contribute to oncogenicity



• Growth factor receptors are ligand modulated dimers– EGF receptor (v-erbB) is the prototype member

• EGF binding stimulates dimerization andactivates tyrosine kinase cascade

• one oncogenic variant can dimerize in theabsence of ligand and signals constitutively

• another lacks an internal regulatory domainresulting in constitutive signaling

– activated kinase domain autophosphorylates and canthen interact with src family proteins


Oncogenes and cell growth (contd)• transforming activity of

src-family kinases isrelated to kinase activity– autophosphorylation

controls activity• Y416 -> active• Y527 -> weak,

normallysuppressesphosphorylationof Y416

– some oncoproteinsactivate src byinterfering withphosphorylation ofY527



• modulation of transcription factor activity is importantfor oncogenesis– can’t cause cancer without altering gene expression!



• 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 theoncoprotein

– increased expression could upregulatetarget genes and influence growth, e.g. AP-1

• alternatively, the mutations could make theoncoprotein a dominant negative inhibitor ofother cellular transcription factors (e.g. v-erbA)

– many members are “immediate early” genes• transcription is immediately upregulated without

the requirement for new protein synthesis whencells are treated with mitogens

– likely to be involved with initiating orpromoting growth

• increased activity would be expected to increaseoncogenesis and it does with some but not others


Tumor suppressor genes

• oncogenesis is nottypically dominant.

• A growing number of“tumor suppressor” geneshave been identified thatconfer a geneticpredisposition to cancers– several types of

genes are involved• apoptosis

proteins (eg p53)• cell-cycle

control proteins(RB)

• DNA-repairproteins (p53)

– classic examples areRB (retinoblastoma)and p53

– loss of tumorsuppressor genes isimplicated in severalinfrequent cancers ofchildhood

• retinoblastoma• Wilm’s tumor


Tumor suppressor genes (contd)• RB is a nuclear

phosphoprotein thatinfluences the cell cycle– unphosphorylated

RB prevents cellproliferation bybinding to E2F andblocking G1/Stransition

– phosphorylation ofRB inhibits bindingto E2F and releasesblock

– some oncogenes (e.g.SV40 T-antigen,E1A) function bysequestering RB andremoving block tocell growth

– similar effects byloss of both alleles inhuman disease


Tumor suppressor genes (contd)

• A variety of other cell-cycle control proteins are tumorsuppressor 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



• 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 functionallowing unrestrained growth (eg. ko mice)

• others are dominant negative p53 mutants thatinterfere with normal p53 subunits in cells andallow unrestrained growth (eg rare cancers)



• p53 has dual functions– cells normally have

low levels of p53– DNA damage induces

large increase in p53levels

– increased p53 leads togrowth arrest untilDNA is repaired ifcells are in G1

– cells in S-phase or laterare triggered tobecome apoptotic

• p53 is a transcription factorthat typically activates– one target is p21 ->

cell cycle arrest– another is GADD45 - a

DNA repair protein– role in inducing

apoptosis is unknownat present

• apoptosis is an importantpathway in preventingtumor formation - blockingit is a common strategy


Cancer - putting it all together

BioSci 145A Lecture 15 - Oncogenes and Cancerblumberg-lab.bio.uci.edu/past teaching/bio145a... ·...

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