Cellular Oncogenes

We made it to the 70s

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

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

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

We ultimately want to know the cause of human diseases like colon cancer, rather than curing chicken sarcomas

One oncogene mutated in the progression to malignancy is K-ras Lodish et al. Fig. 24-6

How did we get there?

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

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

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

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 Virol : Yoshioka et al. J Biol Chem : Fiordalisi et al.

One can then test if the cells induce tumors

But it’s a crazy idea 1. you need to find the right needle in a very large haystack 2.What if you need to find two or more needles??

Bob Weinberg Chiaho Shih

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

DNA from chemically treated mouse cells can transform normal mouse cells Without a virus: cells can be transformed a focus after transfection cells from the focus cells near the focus

It’s time to move on- the 80s

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

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

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

Lodish et al. Fig How to find a needle in a haystack? Generate a bacteriophage genomic library Searching for the one human gene among many mouse genes

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

The cloned gene is ~25,000 bp 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 Luis Parada Bob Weinberg 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

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

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

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

v-rasc-ras (cellular ) (viral) Proto-oncogeneOncogene Viruses 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?

But what does ras do in the cell?? Where does it do its work?

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

Lipid modification targets Ras to the plasma membrane

Farnesyltransferase inhibitors offer a way of reducing Ras activity

Clinicians then tried them in a variety of tumors With activated Ras involvement 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): Tipifarnib Or Lonafarnib

Sadly, it was largely an epic fail 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. Tipifarnib Or Lonafarnib

What else could we ask about Ras?

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

Lodish et al. Fig Ras is an enzyme and hydolyzes GTP

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

Nobel Prize.org 2012 Nobel Prize in Chemistry!

Lodish et al. Fig This allows Ras to act as a molecular switch

The activity of Ras is regulated by GEFs and GAPs

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

How is oncogenic ras different from the normal proto-oncogene?

Table 4.2 The Biology of Cancer (© Garland Science 2007) 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

The G12V mutation prevents endogenous and GAP-stimulated GTPase activity

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

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