What Viruses Taught Us About Cancer The story of Src What Viruses Taught Us About Cancer

What Causes Cancer? - Random mutations (mistakes at the assembly line) - Inherited mutations (pre-disposition) - Viral infections - Environmental factors (chemical; physical) Viral infection as a cause for cancer is not common in humans, but it does exist (human papillomavirus can cause carcinoma of the uterine cervix)

Viruses cause cancer in animals The key to our understanding of the molecular and cellular mechanisms of cancer in humans

Bacteria and viruses are infectious agents Infection ? If infected by a filtrate = a virus

Viruses use the cellular machinery for replication and protein synthesis

The Rous Sarcoma Virus (RSV) A virus can transform a normal cell into a tumor Nobel prize in Physiology or Medicine 1966 Peyton Rous

Chemicals can directly induce cancer Carcinogens Chemicals can directly induce cancer 1920s Viral Infection Out Chemical Induction In Yamagiwa

RSV can transform cells in culture 30 Years Later: Rebirth of RSV research RSV can transform cells in culture RSV stock Howard Temin Immortality Harry Rubin

Studying cancer at the cellular level No contact inhibition on cell division

Changes in cell property and morphology Normal Cancer Normal Cancer

RSV can transform cells in culture - RSV can induce cancer - Cancer is a disease of malfunctioning cells rather than abnormally developing tissues - Cancer can be studied relatively easily at the cellular level

The central dogma DNA Transcription mRNA Translation Protein

The problem: RSV contains RNA, not DNA Howard Temin Nobel prize in physiology and medicine 1975 Temin: 1964 David Baltimore

One organism missed the lecture on the central dogma The retrovirus

Mutant RSVs, mutated in the src sequence, fail to induce cancer The viral genome of the normal chicken virus (ALV) contains 3 viral genes RSV, a relative of ALV, contains an additional sequence: Src Peter Duesberg Mutant RSVs, mutated in the src sequence, fail to induce cancer

Conclusions RSV transforms normal cells into cancerous cells by infecting them with the src gene The src gene (and its product, the Src protein) is probably responsible for the other viral-associated cancers

Let’s label the src gene and follow its dynamics inside the host cell, after infection Bishop and Varmus Nobel prize in physiology and medicine 1989

The cellular src gene is highly conserved between different species There is a cellular src that probably has healthy, normal roles in the cellular machinery There is a viral src that promotes cancer We distinguish between c-src v-src (cellular) (viral)

src was only the first one Jennifer Harvey found a murine leukemia Virus, inducing sarcoma in rats: v-ras (later named H-ras) Other viral transforming genes (14) showed similar patterns c-src v-src (cellular) (viral) Proto-oncogene Oncogene v-src is similar, but NOT identical to c-src

How was v-src generated? RSV exploited a kidnapped cellular gene v-src is similar, but NOT identical to c-src

It is not all about mutations Retroviruses can transform cells by altering gene expression (e.g. amplification)

So, what is src ? Cells transformed by the v-src oncogene exhibit various cellular alterations v-src affects a wide variety of cellular targets The first BIG step: using serum to immunoprecipitate the v-Src protein

Protein kinases and protein phosphatases add and remove phosphate groups from target proteins Lodish et al. Fig. 20-5

Src is a Tyrosine Kinase As a kinase, it can affect many cellular events Figure 15-18a Molecular Biology of the Cell (© Garland Science 2008)

Normally, Src kinase intrinsic activity is low What do we have here? A _______ blot These are proteins identified by antibodies that recognize phosphorylated tyrosine

Compare c-Src with conserved Src proteins Src contains three domains that are shared with other proteins Binds polyproline motifs Phosphorylates other proteins Binds peptides phosphorylated on Tyr

Src is phosphorylated on its Tyr residue

Phosphorylated Tyr 527 inactivates Src RCSB Protein databank

How is Src activated normally? Martin, Nature Reviews MCB (2001) - Dephosphorylation of Y527 - Configuration changes (e.g. SH2 binds to other proteins)

Recent work has provided a more detailed model of Src activation Closed = OFF Open = ON Cowan-Jacob et al. Structure 13, 861-871 (2005)

Cowan-Jacob et al. Structure 13, 861-871 (2005) - Myristylation of Src to the cell membrane is the first step in its activation

And what about v-Src ?

The kidnapped cellular gene was only a partial gene

Summary - RSV and other retroviruses can transform cells into tumors - Transformed cells exhibit changes in cell shape and behavior - A viral oncogene is responsible for transformation - Viral oncogenes are mutated versions of cellular proto-oncogenes - The v-Src protein is a constitutively active tyrosin kinase

c- Src is a tyrosine kinase What does it do in the cell? What are its targets?

Identifying the targets of Src Myristylation of Src is essential for transformation

p120 catenin: modulates cell-cell adhesion Identifying The Targets of Src Western blotting with anti-phosphotyrosine antibodies V = v-Src transfected cells 2A/V = non-myristylated v-Src transfected cells p120 catenin: modulates cell-cell adhesion Reynolds et al. MCB (1989)

Identifying the targets of Src Few Examples - p120 catenin: modulates cell-cell adhesion - Cortactin A: regulates actin polymerization - Focal Adhesion Kinase: involved in cell-matrix interactions Mike Schaller, ex-UNC

Src modulates both cell-cell and cell matrix adhesion junctions Cell-matrix junctions Basal lamina

Epithelial cells secrete a special ECM called the basal lamina Connective tissue

Integrins cluster into sites of contact with the ECM called focal adhesions

Focal adhesions are sites of intense protein tyrosine phosphorylation Actin: Green Phosphotyrosine: Red

Olga Krylyshkina

The ECM serves as a substrate for migrating cells Revathi Ananthakrishnan 07

Albert Harris, Biology UNC Matrix affects cell Cells affect matrix Albert Harris, Biology UNC Pioneering the studies of forces exerted by cells on the matrix (e.g. cells can remodel the matrix)

focal adhesion proteins Zoom into the focal adhesions actin filaments focal adhesion proteins integrins Plasma membrane

FAK is recruited by integrins to the membrane and is autophosphorylated - Src binds to phophorylated FAK - Src changes conformation and becomes active - Src further phosphorylates FAK - Src-FAK phosphorylate target proteins

Src and FAK act together to regulate other focal adhesion proteins Src-FAK signals to regulate adhesion turnover Src-FAK active = less adhesion, more migration

Src- FAK also promote cell survival and growth

Summary - Viruses can act as infectious “cancer agents” via viral oncogenes - Viral oncogenes are mutated versions of cellular proto-oncogenes - The v-Src protein is a constitutively active tyrosin kinase, as a result of lack of Tyr at the C-terminus - c-Src phosphorylates target proteins, including FAK - FAK and c-Src act in the focal adhesions - Focal adhesions are sites of cell-matrix interactions (both ways)

Rules of evidence How do we know this? Src is localized at the right place and time (correlative evidence) src is sufficient (gain-of-function evidence) src is necessary (loss-of-function evidence)

v-Src promotes adhesion dynamics and cell migration focal adhesions actin normal cells v-Src transformed cells lose actin-membrane attachments lose adhesion Frame et al. Nature Review MCB (2002)

Rules of evidence How do we know this? Src is localized at the right place and time (correlative evidence) src is sufficient (gain-of-function evidence) src is necessary (loss-of-function evidence)

If Src is a critical regulator of cell adhesion, what happens to an animal without any Src? Knockout mice Oliver Smithies UNC Nobel prize in physiology and medicine 2007

If Src is a critical regulator of cell adhesion, what happens to an animal without any Src? Knockout mice - Defects in bone remodeling - No lethality Soriano et al. Cell (1991)

Ubiquitous expression: Src Yes Fyn 9 different murine Src family protein kinases compensate for each other Ubiquitous expression: Src Yes Fyn How can we test their functions?

Klinghoffer et al. EMBO (1999) src; fyn; yes triple mutant mice die at embryonic day 9.5 with multiple defects Triple mutant Wild-type Klinghoffer et al. EMBO (1999)

Wound healing/Scratch assay

src; fyn; yes (SYF) triple mutant cells fail to migrate Scratch assay

Rules of evidence How do we know this? Src is localized at the right place and time (correlative evidence) src is sufficient (gain-of-function evidence) src is necessary (loss-of-function evidence)

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Constitutively active Src Src as a target for anti-cancer drugs Dasatinib Regulated Src Constitutively active Src

Src as a target for anti-cancer drugs 50 clinical trials, 14 types of tumors Usually, in combination with other drugs dasatinib bosutinib saracatinib Inhibit Src active site

Before we leave, what do we know? Viruses kidnapped a normal proto-oncogene During the “kidnapping”, the proto-oncogene became an oncogene A new viral infection inserted an oncogene into the recipient, leading to cancer