Growth Factors, Receptors, and Signal Transduction I Tumor Biology: Cellular and Molecular Aspects of the Transformed Cell Growth Factors, Receptors, and Signal Transduction I Rebecca Riggins 202.687.7451 rbr7@georgetown.edu

Overview Peptide growth factors Growth factor receptors - structure and function Receptor activation and its consequences Growth factor and receptor defects in cancer Targeted therapies to growth factors, receptors Intracellular signaling “downstream” of receptors - key pathways: Ras/MAPK, Src, PI3K Intracellular signaling defects in cancer Targeted therapies to intracellular signaling molecules TNF and TRAIL

Polypeptide Growth Factors small peptides, or proteins interact specifically with a receptor on the cell surface secreted by multiple cell types most are secreted as inactive precursors that need to be cleaved or fragmented before they can act some are plasma membrane-bound or found in the extracellular matrix transported by blood and lymphatic systems by several different routes…3 major ones we will discuss are Endocrine Paracrine Autocrine

Endocrine: Synthesis by specific cells, target (receptor) is far away Autocrine Paracrine Endocrine Juxtacrine BLOOD VESSEL Intracrine Different modes of action for growth factors (Bafico and Aaronson, Cancer Medicine, 2002)

Paracrine: Synthesis by many cell types, receptor is nearby Autocrine Paracrine Endocrine Juxtacrine BLOOD VESSEL Intracrine Different modes of action for growth factors (Bafico and Aaronson, Cancer Medicine, 2002)

Autocrine: Synthesis by many cell types, receptor is on same cell that synthesized the growth factor Autocrine Paracrine Endocrine Juxtacrine BLOOD VESSEL Intracrine CANCER! Different modes of action for growth factors (Bafico and Aaronson, Cancer Medicine, 2002)

GROWTH FACTOR FUNCTIONS IN VIVO 1. Early development 2. Tissue differentiation 3. Wound healing and tissue repair 4. Immune responses 5. Stromal mediators of sex and other hormones . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

GROWTH FACTOR FUNCTIONS IN VITRO 1. Proliferation 2. Differentiation 3. Chemo-attraction 4. Cell death 5. Cell migration . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

REGULATION OF GROWTH FACTOR/RECEPTOR INTERACTIONS 1. Determined by growth factor availability and receptor expression levels 2. Different modes of growth factor action - autocrine, paracrine, other 3. Secretory properties - secretory signal; proteoglycan or serum protein binding 4. More than one member of same growth factor gene family may act on the same receptor 5. Same growth factor may cluster more than one receptor member of the same receptor family 6. Interactions regulated by alternative growth factor/receptor products . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

ErbB/HER Family Epidermal Growth Factor Receptor (EGFR) Seminars in Cancer Biology, Volume 14, Issue 4, August 2004, Pages 262-270

Other Growth Factors platelet-derived growth factor (PDGF) nerve growth factor (NGF) hepatocyte growth factor (HGF) insulin-like growth factor (IGF) fibroblast growth factor (FGF) vascular endothelial growth factor (VEGF) named according to what they do or where they act most stimulate growth, or are mitogenic

Transforming Growth Factor (TGF)-b subgroup of a large family…25 members TGF-b1 is the most studied in cancer in some cells, TGF-b1 is mitogenic, while in others it actually inhibits growth TGF-b can cooperate with PDGF… Smooth muscle (mesenchymal) TGF-b increases PDGF, cells proliferate Keratinocyte (epithelial) TGF-b inhibits cell proliferation No PDGFR

Tumor Necrosis Factor (TNF) again, in some cells, TNF is mitogenic, but in most it promotes cell death, or apoptosis this growth factor is not soluble, but is inserted into the plasma membrane Juxtacrine Intracrine

Growth Factor Receptors all are transmembrane proteins 3 major features: extracellular domain (ectodomain) transmembrane region intracellular domain where, when, and how they are expressed determines their biological function

Figure 5.10 The Biology of Cancer (© Garland Science 2007)

Growth Factor Receptors catalytic domain has kinase activity kinases add phosphate groups to (phosphorylate) specific amino acids Receptor Tyrosine kinases phosphorylate Tyrosine Receptor Serine/Threonine kinases phosphorylate Serine and/or Threonine ATP-binding Phospho-transferase ATP P Amino Acid P ADP Autophosphorylation: receptor phosphorylates itself

Growth Factor Receptors Transphosphorylation: receptor phosphorylates its binding partner Figure 5.15 The Biology of Cancer (© Garland Science 2007)

“Different” Growth Factor Receptors Some receptors have no catalytic domain… TNF receptors rely instead on other intracellular proteins to transmit their signal Adhesion receptors (integrins) also do this

Integrins bind Extracellular Matrix (ECM)

G protein-coupled receptors Another Example: G protein-coupled receptors Have 7 transmembrane domains Ligand may bind to extracellular OR transmembrane region (depending on receptor) Also do not have a catalytic domain

GPCR - Inactive State Figure 5.25a The Biology of Cancer (© Garland Science 2007)

GPCR – Activation Process Figure 5.25bc The Biology of Cancer (© Garland Science 2007)

However, “classical” growth factor receptors have catalytic domains that must be activated

Receptor Tyrosine Kinase (RTK) Activation Homodimer: 2 identical receptors cluster together Heterodimer: 2 different receptors from the same family cluster together Nature Reviews Cancer, Vol. 5, May 2005, pg. 341-354

RECEPTOR ACTIVATION BY GROWTH FACTORS 1. Growth factors induce receptor clustering a. High affinity binding b. Ligand mediated receptor cross-linking c. Ligand/receptor crystal structures 2. Receptor activation by homodimer or heterodimer formation a. Activation requires the ability to cross-link . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

Tyrosine Kinase Receptor Activation by Dimerization EGFR Figure 1. Ligand Binding Stabilizes the Formation of Activated Dimers(A) Inactive receptor monomers (green) are in equilibrium with inactive (green) or active (blue) receptor dimers. The active receptor dimers exist in a conformation compatible with trans-autophosphorylation and stimulation of PTK activity (blue). Ligand binding stabilizes active dimer formation and hence PTK activation.(B) Inactive disulfide bridged insulin-receptor (IR) dimers (green) are in equilibrium with active dimers (blue). Insulin binding stabilizes the active dimeric state leading to PTK activation. Insulin receptor Schlessinger J., Cell. 2000 Oct 13;103(2):211-25.

Consequences of RTK activation GROWTH FACTOR PIP3 PIP3 RTK SOS Grb2 RAS RAS P P P PI3K p85 p110 Akt PDK1 Raf P MEK P P GSK3 P P P P BAD NF-ĸB MDM2 p70S6K FKHR P ERK PROLIFERATION CELL SURVIVAL PROTEIN SYNTHESIS . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

RTK Signaling in Cancer Autocrine Transforming Loops Receptor Gene Amplification Receptor Gene Mutation Paracrine Acting Growth Factors in Tumor Progression . Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

Figure 5.12b The Biology of Cancer (© Garland Science 2007)

Figure 5.12a The Biology of Cancer (© Garland Science 2007)

Historical Perspective

In the beginning… Oncogenic retroviruses caused tumors in animal models (monkeys, chickens, etc.) These viruses contained transforming genes that had nothing to do with the viral life cycle… http://www.stanford.edu/group/nolan/index.html

Growth Factor Oncogenes eg. PDGF Cancer cells, and cells in culture that were transformed by simian sarcoma virus (SSV), were less dependent on PDGF for growth PDGF-B was cloned…its sequence was very close to that of the v-sis oncogene from SSV Thus, cellular PDGF-b (or c-sis-B) is a protooncogene Other oncogenic growth factors include the FGFs: FGF3 – activated by mouse mammary tumor virus insertion FGF4 – cloned from Kaposi’s sarcoma virus (skin tumors assoc. with HIV/AIDS) FGF5 – originally isolated from DNA of bladder tumors

Receptor Oncogenes Mechanisms v-erbB = mutated EGFR (avian erythroblastosis virus) v-fms = viral form of CSF-1 receptor v-kit = viral form of c-kit receptor Mechanisms deletions, and/or insertions (v-erbB, v-kit) chromosomal translocation, fusions with other proteins “Neu” mutation of erbB2 = change of Val → Glu in the transmembrane domain…constitutive dimerization, activation

Table 5.1 The Biology of Cancer (© Garland Science 2007)

Table 5.2 The Biology of Cancer (© Garland Science 2007)

Table 5.3 The Biology of Cancer (© Garland Science 2007)

Receptors can be good targets: Easy Access! Seminars in Cancer Biology, Volume 14, Issue 4, August 2004, Pages 262-270

Targeted Therapy in Clinical Use Nature Reviews Cancer, Vol. 5, May 2005, pg. 341-354

Understanding Targeted Therapy: ErbB receptor family Herceptin: anti-ErbB2 Erbitux: anti-EGFR Iressa, Tarceva : EGFR kinase inhibitor

Consequences of Inhibitor Action in vitro, these drugs can individually inhibit cell growth and induce tumor shrinkage in mouse models However, cooperation between ErbB family members in vivo means one inhibitor is usually not enough metastatic breast cancer, positive for ErbB2 expression…Herceptin used alone = 34% response rate Herceptin + cytotoxic chemotherapy drugs = better than either drug alone…BUT there are many side effects in cell culture studies, Herceptin + Tarceva yield much better response rates

UCLA Clinical Trial open to women with metastatic breast cancer must demonstrate amplified ErbB2 (excess gene copies) will be treated with Herceptin and Tarceva Phase I study: 14 patients, all received H+T…T at various doses to determine the best 3 patients of 14 showed partial response or stable disease…21% Phase II study: standard Herceptin + 150mg/day Tarceva

Eastern Cooperative Oncology Group Trial open to women with metastatic breast cancer must demonstrate amplified ErbB2 (excess gene copies) will be treated with Herceptin and Iressa Phase I study: standard Herceptin + 250mg/day Iressa is both active and well-tolerated by patients Phase II study: still ongoing

New Developments 2nd-generation tyrosine kinase inhibitors that target BOTH EGFR and ErbB2…pan-ErbB inhibitors ATP-binding Phospho-transferase Highly conserved between EGFR and ErbB2 Canertinib, Lapatinib, others…target ATP binding site Lapatinib is active in breast cancer cells that have acquired resistance to Herceptin In phase I/II trials, Lapatinib has also shown some effect in patients resistant to Herceptin and Iressa

More New Developments blocking the interaction of receptor family members Pertuzumab = antibody against ErbB2, prevents dimerization with other receptors, ie. EGFR This may be active in tumors without over-expressed or amplified ErB2…currently used in trials for ovarian, prostate, islet cell cancer

A few words about VEGF… VEGF interactions with its receptor are critical for angiogenesis (formation of new blood vessels by tumors) anti-VEGF antibodies block the growth factor, not the receptor Could be effective in treating both the primary tumor and the metastases that develop elsewhere in the body Currently, Bevacizumab is approved for treatment of metastatic colon cancer, and is undergoing phase I/II clinical trials in breast and other cancers

On Wednesday Intracellular signaling “downstream” of receptors - key pathways: Ras/MAPK, Src, PI3K Intracellular signaling defects in cancer Targeted therapies to intracellular signaling molecules TNF