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

Model organisms teach us about ourselves Drosophila melanogaster Caenorhabditis elegans Xenopus laevis Mus musculus

5 Key Signal transduction pathways Are critical for development and homeostasis All are involved in Cancer

5 Key Signal transduction pathways RTK- Ras (me) Critical for development and homeostasis All are involved in Cancer Wnt (me) TGF-ß (you) Hedgehog (you) Notch (you)

Signal transduction pathways RTK= Receptor tyrosine kinases Src=non-receptor tyrosine kinase RTKs

We’ll start by walking through the RTK pathway and then talk about how we got to this knowledge RTKs

In the absence of Ligand RTKs are monomers with an inactive kinase

Ligand binding activates RTKs by dimerization Lodish et al. Fig

RTKs are their own substrates-- i.e., they autophosphorylate

Remember SH2 domains? What did they bind??

SH2 domains allow “ effector ” proteins to bind activated receptors

One adaptor with an SH2 domains is Grb2 It also has SH3 domains--what do they bind?

One adaptor with an SH2 domains is Grb2 It also has SH3 domains--what do they bind? Ras

Step by step--think dominos Ras

Remember-Ras is anchored to the membrane through a lipid

And SOS is a GEF--remember them??

Figure Molecular Biology of the Cell (© Garland Science 2008) Each activated protein activates the next

Figure Molecular Biology of the Cell (© Garland Science 2008) MAPK/ERK enters nucleus Each activated protein activates the next

Figure Molecular Biology of the Cell (© Garland Science 2008) MAPK/ERK enters nucleus Each activated protein activates the next

Figure Molecular Biology of the Cell (© Garland Science 2008) Each activated protein activates the next MAPK/ERK enters nucleus

The RTK pathway

We made it to the 90s 10 years-old Britney Spears Kurt Cobain A new graduate from Harvard Travolta is still dancing

Model organisms teach us about ourselves Drosophila melanogaster Caenorhabditis elegans Xenopus laevis Mus musculus Californias governorus

Model organisms teach us about ourselves Drosophila melanogaster Caenorhabditis elegans Xenopus laevis Mus musculus

The eye of a fly: One Key to learning how Ras and RTKs work Lodish et al. Fig

Did you say flies?

Did you say flies?

This is all I need to know

But wait--flies Helped us Understand The single Most important Human oncogene Lodish et al. Fig

I told you the RTK pathway is key in MANY developmental decisions

Each ommatidium contains all the cells needed to see the world Including the eight photoreceptors

These cells choose fate one by one, each telling the next what fate to adopt

Wild-typesevenless mutant sevenless mutants lack an R7 photoreceptor

Sevenless encodes an RTK Lodish et al. Fig

To get a cellular response, There is a threshold level of pathway activity Gian Garriga sev pathway activity wild type sev - sev ts sev ts sev ts ; enh*/ o C 24.3 o C 22.7 o C sev threshhold R7 present R7 absent

Scientists figured out how to tune RTK activity using a temperature sensitive mutant Gian Garriga sev pathway activity wild type sev - sev ts sev ts sev ts ; enh*/ o C 24.3 o C 22.7 o C sev threshhold R7 present R7 absent

They then looked for mutants In other genes that would drop Pathway activity below the threshold Gian Garriga sev pathway activity wild type sev - sev ts sev ts sev ts ; enh*/ o C 24.3 o C 22.7 o C sev threshhold R7 present R7 absent

The mutations identified were candidates to encode things in the RTK pathway

Wow--Ras, the adapter Grb2 and a GEF are all in the RTK pathway! Sos Ras Grb2

Further, Ras acts downstream of the RTK Sevenless Lodish et al. Fig

The signal transduction pathway should look familiar Grb2 Alberts et al. Fig

Because that ’ s how we figured it out! Grb2 Alberts et al. Fig

Caenorhabditis elegans Flies were bad enough, but worms!?

Caenorhabditis elegans Luckily the Nobel Committee thinks they are cool

lineage and programmed cell death Bob Horvitz John Sulston Sydney Brenner Physiology and Medicine 2002 RNAi Physiology and Medicine 2006 Andy Fire Craig Mello GFP Chemistry 2008 Marty Chalfie Nobel Prize

A simple model for organogenesis Formation of the vulva in C. elegans (“ask Gidi Shemer what he did in graduate school?”) vulva Only 22 cells! sperm oocytes embryos early oocytes

The Key Players One gonadal anchor cell (AC) 6 vulval precursor cells (VPCs) The anchor cell induces vulval fates Sherwood and Sternberg (2003) Dev Cell

Cell Induction The AC signals the VPCs to adopt vulval fates Only 3 VPCs will actually form the vulva

How can we figure out how to build a vulva?

How can we figure out how to build a vulva? GENETICS!

No vulva induction wild type multivulvae (Muv) vulvaless (Vul) Mutants with no vulval signaling Mutants with too much vulval signaling

multivulvae (Muv) vulvaless (Vul) Loss of function mutants in the signaling pathway Gain of function mutants in the signaling pathway or loss-of-function mutations in pathway negative regulators

some examples A Screen for Vul and Muv mutants Vul Muv let-23 lin-3 lin-15 let-60 Next step: cloning and sequencing the genes Bob Horvitz physiology and medicine 2002 Nobel Prize

The first two vul mutations identify the anchor cell signal and its receptor let-23 is an EGFR homologue = RTK lin-3 is an EGF homologue

Alberts et al Where do they function? Lin-3 is expressed in the AC Let-23 is expressed in all the VPCs

lin-15 was found to be an negative regulator of vulval induction (loss of function mutation) Muv lin-15 let-60 lin-15

Next step: clone the let-60 gene let-60 was found to be a gain of function mutation that promoted vulval induction

First evidence: Ras has an in vivo role as part of the RTK pathway

The ras gain-of-function mutation = Glycine 13 Glutamine Sound familiar?

The ras gain-of-function mutation = Glycine 13 Glutamine Sound familiar?

The gain-of-function mutation = Glycine 13 Glutamine All the VPCs make vulvae G13Q Constitutively active Ras

Suppressor and enhancer screens [suppressors (or enhancers) of the mutated phenotype] Other mutations of ras lin-15 How can we find the rest of the proteins in the pathway? ? ?

Vul = “ bag of worms ” Muv Normal vulva ” For example, second mutations that turn Muv mutants into normal or Vul worms

lin-15 This worked GREAT!

The fly and worm work allowed cell biologists and biochemists to return to mammalian cells to identify the ways these new proteins worked as machines

The RTK-Ras pathway also offers drug targets for cancer treatment Alberts et al. Fig

The RTK-Ras pathway offers drug targets for cancer treatment e.g., the Raf kinase inhibitor sorafenib (also inhibits the RTKs VEGFR, PDGFR, and Kit)

The RTK-Ras pathway offers drug targets for cancer treatment e.g., the Raf kinase inhibitor sorafenib (also inhibits the RTKs VEGFR, PDGFR, and Kit) Approved for treatment of advanced renal cell carcinoma (Jan. 2006) and approved for inoperable hepatocellular carcinoma (Nov. 2007) And radioactive iodine resistant thyroid cancer (Nov. 2013)

The RTK-Ras pathway offers drug targets for cancer treatment e.g., or the Raf kinase inhibitor Vemurafenib Approved for treatment of Late stage melanoma (August 2011)

Summary - Cellular oncogenes = viral oncogenes

Summary - Cellular oncogenes = viral oncogenes - Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch

Summary - Cellular oncogenes = viral oncogenes - Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch - Ras is a major component of the RTK pathway

Summary - Cellular oncogenes = viral oncogenes - Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch - Ras is a major component of the RTK pathway -Basic and Clinical Science provide a VERY powerful partnership