Signal Transduction and Cancer

SURP ProgramJuly 8, 2008

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Outline of Today’s Lecture

✫ Signal transduction overview✫ Why important in cancer?✫ Examples of important pathways✫ How signaling is studied in the lab✫ Signal Transduction pathways as

therapeutic targets

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What is signal transduction?

✫ The process by which extracellular signals are transmitted across the cell membrane and converted into a cellular response (e.g. gene expression, apoptosis…)

✫ or even more simply: “the process by which a cell converts a signal or stimulus into another”. (www.wikipedia.com)

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Typical Components of Signaling Pathways

Ligand (Stimulus)

Receptor

Biological Response

Second Messengers

Effector/Adaptor proteins

Amplification of signal

Target

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Types of Signals

✫ Proteins:– Cytokines (e.g. IL-1)– Growth factors (e.g. EGF, TGFβ)

✫ Nutritional factors:– Sugars– Amino Acids– Lipids– Vitamins

✫ Hormones (e.g. adrenaline, estrogen)✫ Other:

– Stress, heat, pH, radiation, etc

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Signals can act in a cell-type specific manner

✫ Important to note specific context when evaluating signaling literature!

TGFβ

Mesenchymal cells (e.g. fibroblasts)

Epithelial cellsEndothelial cellsHemopoietic cellsNeural cells

Growth promoting

Growth inhibiting

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Receptors✫ Growth factors signal via receptor tyrosine kinases

(RTKs/RPTKs).✫ Transmembrane proteins, containing an extracellular

domain of several hundred AAs, short alpha helix structured hydrophobic domain spanning the membrane, and an intracellular catalytic domain that is highly conserved among family members.

✫ Ligand binding induces a change in conformation of receptors, allowing dimerization and transphosphorylation of Tyr residues.

✫ Signaling intermediates are recruited to the phosphorylated (“active”) receptor to propagate the signal.

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Receptor Activation Schematic

Molecular Biology of the Cell, Alberts et al. Ch 3

Ligand

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Phosphorylation and dephosphorylation regulate the activity of many proteins

✫ Kinases: Enzymes that catalyze the removal of a phosphate group from ATP which is transferred to serine, threonine or tyrosine residues in proteins.– 2 classes: Ser/Thr and

Tyr kinases✫ Phosphatases: Enzymes

which catalyze the removal of phosphate groups from proteins

Molecular Biology of the Cell, Alberts et al. Ch 3

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Effectors

✫ Anchor proteins localize protein kinases and phosphatases in particular places in the cell– Increases efficiency and specificity of

responses✫ Protein-protein complexes form via

interactions between specific protein domains (e.g. SH2 domains which recognize phospho-tyrosines)

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Biological outcomes✫ Changes in gene

expression✫ Regulate

translation✫ Regulate the cell

cycle✫ Migrate✫ Reorganize the

cytoskeleton✫ Initiate

angiogenesis etc

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Cancer and growth control

✫ Normal cells respond to their environment to maintain a balance between proliferation, differentiation and cell death.

✫ Cancer cells have defects in growth control, leading to…

Hahn and Weinberg Cell 2000

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Why study signal transduction in cancer?

Nearly all proto-oncogenes are members of signal transduction pathways involved in regulating

cell proliferation, differentiation or apoptosis

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What goes wrong in cancer?

✫ Tumors cells inappropriately secrete growth factors that activate cognate receptors on their cell surface - allows signaling independent of “true cell-cell communication”

Example: Renal cell carcinoma cells have an autocrine TGFa - EGFR autocrine loop

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What goes wrong in cancer (2)✫ Upregulation of growth factor

receptors✫ Deletions in receptor domains

causing defects in ligand binding, or causing constitutive dimerization.

✫ Normal cells contain 20,000-200,000 copies of EGFR; cancers cells can contain >1,000,000!– Greatly increased upstream

mitogenic signaling.

Classic example: Breast cancer cells can overexpress HER2 (an EGFR family member)

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What goes wrong in cancer? (3)✫ Increase in signal transduction

– Loss of negative regulators (e.g. mutation or epigenetic silencing of PTEN in endometrial carcinoma)

– Defects in protein turnover– Defects in protein localization (e.g. sequestration of

p27 in to the cytoplasm in RCC)– Novel oncogenes from chromosomal translocations

that result in constitutive activity of a kinase (e.g. Bcr-Abl)

✫ Increase in activation of transcription– Constitutive activation of transcription factors (e.g.

STAT3)

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Signal transduction pathways are targets of carcinogens

✫ Carcinogen (e.g. DMBA) causes an activating mutation in codon 61 of H-ras.

✫ Tumor promoters like TPA promote clonal expansion of initiated cells. Multiple signaling pathways targeted by TPA including PI3K-Akt, and protein kinase C.

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Overview of most important signal transduction pathways in cancer

✫ PI3K-Akt-mTOR SURVIVAL (regulate translational, cell growth, autophagy)

✫ Ras-MAPK PROLIFERATION✫ JAK-STAT PROLIFERATION

(anti-apoptosis, immune response)

✫ NFκB INFLAMMATION✫ WNT-βCatenin DIFFERENTIATION

CROSSTALK

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How signal transduction is studied✫ Cell culture studies:

– Use of inhibitors (e.g. rapamycin, LY294002)

– Phospho-specific antibodies– If no antibodies available,

can immunoprecipitate and western blot with broad phospho-Tyr or phospho-ser/thr antibodies

– In vitro kinase reactions: determine whether a specific kinase can phosphorylate a given substrate in a test tube.

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How signal transduction is studied (2)

✫ In vivo studies:– Phospho-specific

antibodies for immunohistochemistry

– Commercially available/custom tissue microarrays to look at many samples at once

– Transgenic/knockout mouse models

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Signal tranduction as a therapeutic target

✫ Traditional cytotoxic chemotherapy has problems:– Non-specific, unpleasant side effects --> kills

proliferating cells, even normal ones such as hair follicles, GI epithalial cells

– Cells develop resistance mechanisms (e.g. upregulate proteins that pump drugs out of the cells so they can no longer accumulate)

✫ Rationally designed targeted therapies that interfere with signaling the current rage in cancer therapeutics research.– Ideal target = a protein not involved in normal cellular

function (Bcr-Abl example in next few slides)

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Determining a good drug target

✫ Determine what pathways are active in your tumor type

✫ Determine whether any of these pathways are critical for tumor survival– “Oncogene addiction” phenomenon: Despite the fact

that tumors contain multiple genetic and epigenetic defects, their growth/survival can often be impaired by inactivation of a single oncogene (the “Achilles Heel”)!

✫ Financially worthwhile - enough of a target market (large enough prevalence)

✫ 2 examples: Bcr-Abl and HER2/neu

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Bcr-Abl in CML

✫ Chromosomal translocation results in a constitutively active tyrosine kinase.

✫ Found in 95% of CML patients

✫ Signals via a multitude of growth-promoting pathways (PI3K, JAK-STAT, Myc, NFκB)

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Gleevec

✫ Gleevec is a small molecule designed to bind to the ATP cleft and inhibit the tyrosine kinase activity of Bcr-Abl at low conc’s (40nM).– Later found to also inhibit

PDGFR and c-Kit✫ Successfully used so far:

– 98% complete response rate in chronic phase CML (NEJM, 2001)

– Reduce total tumor cells from 1010/1012 to 106 (minimal residual disease)

– Now is the standard of care!

QuickTimeª and a decompressor

are needed to see this picture.

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HER2 as a target in breast cancer✫ Some breast cancers

(~25%) overexpress the HER2 gene, a growth factor receptor in the EGFR family.

✫ These tumors have a worse prognosis than non HER2-overexpressing tumors.

✫ Antibodies such as Herceptin (trastuzumab) have been developed to block the aberrant mitogenic signaling.

✫ Good results are seen in synergy with standard therapies (chemo and radiation)

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Sample of what pharma has done

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Take home points✫ Signal transduction is the mechanism by which cells

communicate.✫ Signal transduction networks are often bizarre in

cancer and result in an imbalance between cell death and cell survival.

✫ Multiple mechanisms are employed by cancer cells to subvert normal regulation of proteins involved in signaling, including gene amplification, mutations, chromosomal translocations and epigenetic silencing of tumor suppressors.

✫ Rationally designed signaling inhibition is a relevant therapeutic approach.

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Additional Resources

✫ Cell Signaling Technologies website - www.cellsignal.com (lots of signaling diagrams) and www.phosphosite.org (information about kinases and their substrates)

✫ Science Signal Transduction Knowledge Environment - http://stke.sciencemag.org

✫ Calbiochem interactive pathways - http://www.emdbiosciences.com/html/EMD/interactivepathways.htm

Questions? Aalexand@mdanderson.org