Cellular Oncogenes

We made it to the 70s

Viral oncogenes paved the way c-src v-src proto-oncogene oncogene

Viral oncogenes paved the way c-src v-src proto-oncogene oncogene The concept: Viruses kidnap a normal proto-oncogene During the “kidnapping”, the mutated proto-oncogene became an oncogene A new viral infection inserted an oncogene into the recipient, leading to cancer 4

no known viral tumors in humans However…. At the time, there were no known viral tumors in humans

We ultimately want to know the cause of human diseases like colon cancer, rather than curing chicken sarcomas http://www.clevelandclinic.org/registries/inherited/fap.htm

We now know one oncogene mutated in the progression to malignancy is K-ras Lodish et al. Fig. 24-6

How did we get there? 8

Bob Weinberg long before he wrote our textbook Onto the stage stepped a bright and ambitious young assistant professor with a crazy idea Bob Weinberg long before he wrote our textbook 9

But how can we identify oncogenes that are not viral oncogenes?

Add DNA from carcinogen-treated cell? But how can we find oncogenes that are not viral oncogenes? Add DNA from carcinogen-treated cell? 11

First you need a way to get DNA into cells How would you do that?

DNA can be “transfected” into cells using Calcium Phosphate

As you know, oncogenes relieve contact inhibition. This can be visualized in culture by “focus formation” Transfect with oncogene J Biol Chem 2002 277:10813-23 Fiordalisi et al. J Virol 2000 74:1008-13 Yoshioka et al.

One can then test if the cells induce tumors

But it was a crazy idea 1. you need to find the right needle in a very large haystack What if you need to find two or more needles??

Chiaho Shih Bob Weinberg

Maybe it wasn’t So crazy after all! Soon everybody was trying it! Chiaho Shih Bob Weinberg 18

a focus after transfection They got transformation using DNA from chemically mutagenized mouse cells a focus after transfection cells from the focus cells near the focus Without a virus: cells can be transformed

It’s time to move on- the 80s

Could this work with DNA from human tumors? Transfect DNA from human cancer cell lines Human Cancer Cell Line Chiaho Shih Bob Weinberg

Somewhere here, among the normal mouse genes, we have a human oncogene Human Cancer Cell Line Somewhere here, among the normal mouse genes, we have a human oncogene Low transformation rates suggest that we may be dealing with a single oncogene

How to find a needle in a haystack Figure 4.8 The Biology of Cancer (© Garland Science 2007)

How to find a needle in a haystack? Generate a bacteriophage genomic library Search for the one human gene among many mouse genes using a human- specific mobile element as a probe Lodish et al. Fig. 24-4

The Race is ON Wigler lab Barbacid lab Weinberg lab December 1981 Barbacid lab Weinberg lab An Oncogene is Cloned From a Human Tumor !! But what does it encode?

In 1981 there was no way to sequence 25 kilobases The cloned gene is ~25,000 bp In 1981 there was no way to sequence 25 kilobases

In 1981 there was no way to sequence 25 kilobases The cloned gene is ~25,000 bp In 1981 there was no way to sequence 25 kilobases Let’s try a long-shot shortcut--what if the cellular oncogene is one of the known viral oncogenes? Its very unlikely - 14 v-oncogenes, ~30,000 human genes Bob Weinberg Luis Parada

What probe should we use ? Use Southern blot analysis to look for one human gene in the otherwise mouse genome What probe should we use ?

Just go through the viral oncogenes one by one mouse cells human cells human RAS mouse RAS Der et al. PNAS 82 Channing Der, now at UNC

transformed by a human oncogene The transforming oncogene is Ras Cellular oncogenes = Viral oncogenes mouse cells human cells Mouse cells transformed by a human oncogene human RAS mouse RAS Der et al. PNAS 82 Channing Der, UNC 30

c-ras v-ras (cellular ) (viral) Proto-oncogene Oncogene Carcinogens Random mutations

But what does Ras do in the cell??

But what does Ras do in the cell?? What do we want to know?

Where does it do it’s work?

Ras is postranslationally modified --where will that put it? by addition of a lipid --where will that put it?

targets Ras to the plasma membrane Lipid modification targets Ras to the plasma membrane

Farnesyltransferase inhibitors thus offered a way of reducing Ras activity

Clinicians then tried them in a variety of tumors with activated Ras involvement Tipifarnib Or Lonafarnib Examples of Phase II trials: Leukemias (esp. AML/CML): Metastatic breast cancer(with capecitabine): Pancreatic cancer (with gemcitabine): Ovarian cancer (with current 2 drug combo): Neuroblastoma and Small cell lung cancer (with Taxol):

Sadly, it was largely an epic fail Tipifarnib Or Lonafarnib Examples of Phase II trials: Leukemias (esp. AML/CML)May have some efficacy Metastatic breast cancer(with capecitabine): no significant improvement Pancreatic cancer (with gemcitabine): no improvement Ovarian cancer (with current 2 drug combo): no effect Neuroblastoma and Small cell lung cancer (with Taxol): Discontinued.

We now know parallel pathways can add related lipids to Ras, Restoring membrane targeting Tipifarnib Or Lonafarnib

What else could we ask about Ras?

Scientists found Ras binds GTP Is it a new type of kinase? Lodish et al. Fig. 20-5

Ras is an enzyme and hydolyzes GTP Lodish et al. Fig. 20-5 43

of another well studied signaling pathway Does this remind you of another well studied signaling pathway that was one of the first identified? Lodish et al. Fig. 20-5 44

2012 Nobel Prize in Chemistry! Nobel Prize.org 45

This allows Ras to act as a molecular switch Lodish et al. Fig. 20-5 46

The activity of Ras is regulated by GEFs and GAPs

How is oncogenic ras different from the normal proto-oncogene? A new race starts

They made chimeras to identify the region of Ras that confers activity

They made chimeras to identify the region of Ras that confers activity

One amino acid!!!!

Ras is a key player in >50% of human tumors! Table 4.2 The Biology of Cancer (© Garland Science 2007)

A note for the future—Ras is mutated in <5% of human breast cancers—we’ll see why later Table 4.2 The Biology of Cancer (© Garland Science 2007)

The G12V mutation prevents endogenous and GAP-stimulated GTPase activity

Thus the Ras oncogene is constitutively active i.e. stuck in the ON state! G12V Lodish et al. Fig. 20-5

Why would constitutively active Ras lead to cancer ? How does Ras act in our body, in vivo ? From cell culture to model organisms