Mark Peifer and Bob Duronio

Mark Peifer and Bob Duronio
BIOL 445 Cancer Biology Spring 2015 Mark Peifer and Bob Duronio

BIOL 445 In Bio 445 we combine the approaches of Bio 202 + Bio 205
with the current scientific literature to study the family of diseases known as cancer Studying cancer not from the clinical point of view. We will combine the fields of cell biology, dev biol and genetics and learn how they all affect cancer. In many ways, we will go back to 202 and 205. You will meet a lot of stuff that you are familiar with. Sometimes, we will just mention processes to make sure we are on the same page and sometimes we will go much more in depth than you did previously. What I am saying basically is that there would be times when you will feel you are studying cell biology and not cancer biology (we will dedicate 3-4 lectures on the normal cell cycle w/o mentioning the word cancer). The issue here is that in order to really understand the cellular and molecular mechanisms of cancer we need to understand those mechanisms in normal cells. In addition to learn some of the material in more depth, we will take a different approach, not talking about the facts, but about the research. CDKs and cyclins. Thus, you must know what you are entering. This is a very challenging course. The first two lectures: introducing cancer: properties causes and an overview of the entire course. For those who took dev bio with me or Peifer, you will recognize many of the slides. After the introductory lectures: First part: we will focus on the inputs coming into the cells, normal and cancer cells, including the signaling pathways in the cytoplasm. We will especially focus on the role of genes called oncogenes in those pathways. Those inputs affect cell behavior. They affect changes in the cell shape, changes in adhesion between cells. More than anything, they change gene expression. In the second part, we will look on the reaction of the cells to these inputs, focusing on gene expressing and especially on the cell cycle and its control. We will then talk about the other major group of genes involved in cancer, the TSGs. In the third part, we will tie them together and go out of the cell to understand how the cells behave at the level of the tissue, the organ and the body. We will learn about cell adhesion, migration and metastasis. Finally, we will talk about cancer therapy, traditional and modern ways by which we try to fight this dreadful disease.

You may not believe it but by the end of the semester
This will make sense! Hanahan and Weinberg, Cell 100:57-70 (2000)

Virtually everything you’ll need is found at:
It’s a REALLY good idea to look through this carefully THIS WEEK AND Check back frequently for updates or changes

BIOL 445 Textbooks What are you responsible for?
The Biology of Cancer by Robert Weinberg 2nd Edition What are you responsible for? - Classes (attendance AND participation) Papers and assigned textbook reading(on website) -Your project- literature search and presentation Grading - Exams (20% X 2 midterms + 25% final) = 75 Presentations = 15 Classroom discussion & in-class evaluations = 10

Your Project - Choose a topic - Read the posted review paper
- Do a thorough literature research, including primary data on the gene and the disease - Make a poster and present it to your peers - The final exam will cover ALL posters

As you learned in the Online Introduction
and the assigned readings for today, Cancer is a family of diseases caused by our own cells gone wrong

and rising relative to heart disease!
Cancer is number 2 and rising relative to heart disease! Cancer accounts for nearly one-quarter of deaths in the United States, exceeded only by heart diseases. In 2004, there were 553,888 cancer deaths in the US. CDC 8

But as we’ll see, Cancer is not one disease, its many
Lung cancer is, by far, the most common fatal cancer in men (31%), followed by prostate (9%), and colon & rectum (9%). In women, lung (26%), breast (15%), and colon & rectum (10%) are the leading sites of cancer death. The most abundant cancer in male is prostate, the most abundant cancer in women is breast (both= ~33%) Source: American Cancer Society

In the Online Introduction
You learned about - Properties of cancer cells - Tumor progression - What causes cancer? - Accumulation of mutations It is a very challenging task to squeeze a whole course into lectures, so today we will try to learn key concepts in cancer biology - Molecular genetics of cancer

Cancer often starts with a single mutation
11

One mutation is not enough !!
However One mutation is not enough !! Heard of natural selection? Accumulation of mutations

What types of genes are mutated in cancers?

Two broad categories Oncogenes Mutational activation of proteins that normally Promote cell proliferation 14

Two broad categories Oncogenes Mutational activation of proteins that normally Promote cell proliferation Tumor suppressor genes Mutational inactivation of proteins that normally inhibit cell proliferation 15

A proto-oncogene: a normal cellular gene that can become an oncogene,
Oncogenes proto-oncogene oncogene A proto-oncogene: a normal cellular gene that can become an oncogene, upon mutational change Following this discovery, many genes from infectious viruses that can transform animal (not human) cells , were found in animal (and human) normal cells. We distinguish between them as oncogenes (e.g. v-src) and proto-oncogenes (c-src).

cells instruct one another
As you know from Bio 205, cells instruct one another via cell-cell signals

& other cellular targets
Signal transduction moves information from the cell surface to the nucleus & other cellular targets

Signal transduction occurs in a series of steps

Cell-cell signals can regulate cell proliferation

Cancer at the Cellular Level
Signal transduction drives information from the cell surface to the nucleus & other cellular targets

It’s time for a Bio 202 review

How many copies of each gene do we have?

Are most mutations dominant or recessive?

Oncogenes result from rare dominant mutations
That lock signaling machinery in the ON state The molecular basis of cancer. When v-src was introduced to chicken, the cells became cancerous, although there was still a normal, endogenous copy of c-src. This is also true for proto=oncogenes that become oncgones due to mutations and not because of viruses. Thus, a cellular Oncogene is dominant. One mutated copy is enough. If a proto-oncogene has a normal, cellular role and when it is mutated into an oncogene, it can promote cancer, what would you expect its cellular role is? Affecting functions that are critical for cell proliferation and survival, but functions that are normally under control. In a cell that carries an oncogene, the same “normal” function lost its control.

Src in its normal context
Genes did not evolve to cause cancer! Src in its normal context Normal skin cell tightly adherent to ECM Signaling is OFF Alberts et al.

Wounding->platelet recruitment->
cell migration and proliferation Alberts et al. Signaling is ON

Cellular signaling machinery is stuck ON
Oncogenes Cellular signaling machinery is stuck ON One mutated copy = over active protein = over proliferation, over-survival, etc.

Two broad categories Oncogenes Tumor suppressor genes Mutational inactivation of proteins that normally inhibit cell proliferation 31

The good guys become bad guys
Oncogenes The good guys become bad guys Tumor suppressor genes We lose the good guys

cell proliferation by acting as A transcriptional co-repressor
Rb puts the brakes on cell proliferation by acting as A transcriptional co-repressor DO NOT transcribe genes required for DNA replication Rb E2F DP

frees E2F/DP to turn on genes required for proliferation
Rb phosphorylation frees E2F/DP to turn on genes required for proliferation P P Transcribe gene required for DNA replication DP E2F

Retinal tumor in patient that inherited a mutant copy of Rb
Lodish et al. Fig

Retinoblastoma is inherited in
a dominant fashion Lodish et al. Fig

However, at a cellular level Retinoblastoma is Recessive!

still puts the brakes on
The remaining Rb still puts the brakes on cell proliferation DO NOT transcribe genes required for DNA replication Rb DP E2F

It’s only when both copies of Rb are knocked out
in a single cell that a tumor develops

frees E2F/DP to turn on genes required for proliferation continuously
Loss of functional Rb frees E2F/DP to turn on genes required for proliferation continuously Transcribe gene required for DNA replication DP E2F

Thus oncogenes and tumor suppressors
differ in both cellular function and genetics dominant In the case of tumor suppressor genes (TSG), the function is recessive. In this case we need mutations in both chromosomes to promote formation of tumors. The reason, while in oncogenes, we activated genes that are not supposed to be active all the time (we made the “ “good guys” to become “bad guys”), in the case of the TSGs, we are talking about guardians, regulators that make sure everything will work properly. Here, mutations lead to loss o function of the “good guys”. recessive Figure Molecular Biology of the Cell (© Garland Science 2008)

What Viruses and Nobel Laureates Taught Us About Cancer
The story of Src What Viruses and Nobel Laureates Taught Us About Cancer 42

Peyton Rous discovered a virus that causes cancer in chickens
No thank you! Peyton Rous discovered a virus that causes cancer in chickens

The Rous Sarcoma Virus (RSV)
A virus can transform a normal cell into a tumor 44

The Rous Sarcoma Virus (RSV)
A virus can transform a normal cell into a tumor Nobel Prize in Physiology or Medicine 1966 45

But what’s a virus??? The Rous Sarcoma Virus (RSV)
A virus can transform a normal cell into a tumor But what’s a virus??? 46

However the field then deviated-->Carcinogens
Chemicals can directly induce cancer 1920s Viral Infection Out Chemical Induction In Yamagiwa 47

RSV can transform cells in culture
30 Years Later There was a Rebirth of RSV research RSV can transform cells in culture RSV stock Howard Temin Immortality Harry Rubin 48

This allowed us to study cancer at the cellular level
RSV infection Changed cells No contact inhibition on cell division 49

No contact inhibition of cell division and significant alterations in cell adhesion and behavior
Normal RSV infected= Cancer RSV infected= Cancer Normal 50

But how??? Normal RSV infected= Cancer RSV infected= Cancer Normal 51

I HOPE you remember the central dogma
OK, product = protein and this is during the 60s, the classic dogma has already been established. DNA-RNA-PROTEIN

This is one of those biology facts
That you need to have permanently stored OK, product = protein and this is during the 60s, the classic dogma has already been established. DNA-RNA-PROTEIN

The central dogma DNA Transcription mRNA Translation Protein
OK, product = protein and this is during the 60s, the classic dogma has already been established. DNA-RNA-PROTEIN Protein

These viruses reverse the central dogma,
RSV is a retrovirus These viruses reverse the central dogma, making a DNA copy of their RNA genome and inserting it into your DNA Alberts et al. Fig

This earned the Nobel Prize in Physiology and Medicine in 1975!!!
Howard Temin and David Baltimore Alberts et al. Fig

Your genome is a retrovirus graveyard:
Living and dead retroviruses make up 8% of your genome, with ~100,000 whole or partial copies! Alberts et al. Fig

NEXT Breakthrough discovery
Retroviruses can cause cancer by picking up mutated versions of normal cellular genes Alberts et al. Fig

The paper that created two more Nobel laureates and founded the modern field of Cancer biology

Let’s take a very short detour

Retroviruses can also cause cancer by inserting next to and thus
activating the expression of proto-oncogenes Retroviral insertion sites in different tumors Transcribe to mRNA 5 kilobases exons wnt-1 gene Alberts et al. Fig

very valuable discovery (for science and for Roel  )
This turned out to be a very valuable discovery (for science and for Roel  )

There are two mechanisms of gene activation by retroviral insertion
Lodish et al. Fig

OK—Back to src You know mis-expressing this gene Can initiate cancer What do you want to know now??

the protein encoded by src do within the cell?
So, what job does the protein encoded by src do within the cell? The first BIG step: using antibodies to immunoprecipitate the v-Src protein 65

Who can tell us what “immunoprecipitation” means?
The first BIG step: using antibodies to immunoprecipitate the v-Src protein 66

This led to the discovery that Src
is post-translationally modified

What’s translation?? This led to the discovery that Src
is post-translationally modified What’s translation??

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

Adding labeled ATP to immunoprecipitated Src showed that Src can phophorylate a substrate
Src is a kinase! in the presence of P32-ATP A substrate is phosphorylated 70

Which amino acids can be phosphorylated?
And Why those amino acids??

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) 72

Normally, Src kinase intrinsic activity is low
What makes Src so active in transformed cells? Western Blot with antibody that recognizes Tyr phosphorylated proteins 73

What is a Western blot? Western Blot with antibody that recognizes
Tyr phosphorylated proteins 74

The structures of c-src and v-src provided an important clue!
Lodish et al. Fig

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

Scientists have determined the precise 3-dimensional structure of Src
Xu et al. Nature :

This is the bit missing in v-src

We now know that tyrosine phosphorylation of the C-terminus
creates an intramolecular and inhibitory interaction Lodish et al. Fig

Thus Src is normally inactive due to intramolecular inhibition
Lodish et al. Fig

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

v-src lacks the C-terminal Tyr and thus cannot be inactivated!
Lodish et al. Fig

has multiple consequences
Activation of Src has multiple consequences From Schwartzenberg, Oncogene 17, (1998)

“Where is Src within cells?”
Scientists next asked “Where is Src within cells?”

This is a covalently attached lipid
what might that mean?

Experiments revealed that
Myristylation of Src is essential for transformation 86

This was built into an even more detailed model of Src activation
Cowen-Jacob et al. Structure 13, (2005)

OK-- c- Src is a tyrosine kinase
What does it do in the cell? What are its targets? 88

Remember, we are still in the late 70s
Bishop and Varmus 89

Western blotting with anti-phosphotyrosine antibodies
To Identify The KEY Targets of Src, let’s look for Proteins ONLY modified by biologically active Src Western blotting with anti-phosphotyrosine antibodies V = v-Src transfected cells 2A/V = non-myristylated v-Src transfected cells Reynolds et al. MCB (1989) 90

Identifying The Targets of Src-look for
Proteins ONLY modified by biologically active 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) 91

Here are a sampling of the targets of Src
- p120 catenin: modulates cell-cell adhesion - Cortactin A: regulates actin polymerization - Focal Adhesion Kinase: involved in cell-matrix interactions Mike Schaller, ex-UNC Professor 92

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

Src modulates both cell-cell and cell matrix adhesion: The basics
Lodish et al. Fig. 22-2

Epithelial cells secrete a special ECM called the basal lamina
Alberts et al. Fig

Cells interact with the ECM via Focal adhesions, which also
Anchor the actin cytoskeleton Focal Adhesions (orange) Actin: Green Alberts et al. Fig

Focal adhesions are linked to the actin cytoskeleton Alberts et al.
Fig

A complex network of proteins links the focal adhesion to actin
and regulates actin polymerization Alberts et al. Fig

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

An oversimplified model of Src function
Normal skin cell tightly adherent to ECM Wounding->platelet recruitment-> cell migration and proliferation Alberts et al.

A less oversimplified model
Migratory growth factors e.g., EGF, PDGF Extracellular matrix Src RTKs Integrins FAK PI-3- kinase Adaptors Actin Remodel cell-matrix junctions -> cell motility From Jones et al. Eur J. Cancer 36, (2000)

FAK is recruited by integrins to the membrane and is autophosphorylated
- Src binds to phosphorylated FAK - Src changes conformation and becomes active - Src further phosphorylates FAK - Src and FAK phosphorylate target proteins 102

Src and FAK act together to regulate other focal adhesion proteins
They regulate focal adhesion turnover Src-FAK active = less adhesion, more migration 103

If Src is a critical regulator of cell adhesion, what happens to
an animal without any Src?

If Src is a critical regulator of cell adhesion, what happens to
an animal without any Src? Cell : Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Soriano P, Montgomery C, Geske R, Bradley A.

Why is this phenotype so modest?
Redundancy!!

Redundancy!! Src has two very close relatives: Fyn and Yes
Why is this phenotype so modest? Redundancy!! Src has two very close relatives: Fyn and Yes

Different Src family kinases work downstream of different receptors
Alberts et al. Fig

Fyn mutant mice are viable but have defects in
myelination of brain neurons Yes mutant mice are viable but have subtle changes in B-cell function

Src; Fyn; Yes triple mutant mice die
at embryonic day 9.5 with multiple defects Triple mutant Wild-type

That’s more like it! Triple mutant Wild-type

However, triple mutant cells
still make focal adhesions

But src; fyn; yes (SYF) triple mutant cells
fail to migrate! Scratch Wound assay 113

Now let’s take all this basic science and put it to work to discover
new drugs to treat cancer

Active site

This aided identification of kinase inhibitors
that block Src action Active site SU6656

dasatinib In leukemia, adding Src inhibition
to inhibition of the related kinase Abl improved prognosis in phase II trials and was FDA-approved to help get around drug resistance in CML dasatinib Ottmann et al. Blood 110, 2309 (2007)

dasatinib This same Src inhibitor is in
Phase II trials for advanced breast and Lung cancer dasatinib Ottmann et al. Blood 110, 2309 (2007)

Another Src inhibitor failed in Phase I/II trials for
metastatic pancreatic, breast, ovarian, and prostate cancers Active site AZD0530

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