The Biology of Cancer Chapter 5,6 : Growth Factors, Receptors, Robert A. Weinberg The Biology of Cancer First Edition Chapter 5,6 : Growth Factors, Receptors, and Cancer, signaling pathways Copyright © Garland Science 2007

A signaling pathway reaches from the cell surface into nucleus Signaling circuitry shows how the cells sense the signals and amplify and convey to nucleus ( ligand-Surface receptor-amplifier and effectors) Simple Linear pathway ? In fact, more integrated pathway (Crosstalk) Negative feedback and positive feedback pathway Different signaling pathway share the common downstream effectors. In a Tissue specific manners( different cell types use different combinations of pathways Cancer cells creates their ability to signaling unbalance and modified them to do uncontrolled cell division Figure 6.1 The Biology of Cancer (© Garland Science 2007)

How do cells communicate with cellular environments How do cells communicate with cellular environments ? Normal cells receive growth-stimulatory signal and these signals are integrated by complex networks to decide whether cells are divided or not ? Figure 5.1 The Biology of Cancer (© Garland Science 2007)

Normal cells proliferation is governed for maintenance of tissue structure: proliferation and differentiation A number of distinct cell types should be tightly controlled by signaling molecules to create normal tissue. If dysregulated in this pathway? Figure 5.2 The Biology of Cancer (© Garland Science 2007)

Effects of growth factors on cells Ligands and receptors Platelets secrets PDGF, which is a potent stimulator of fibroblast, thereby increasing cell proliferation during wound-healing Figure 5.4a The Biology of Cancer (© Garland Science 2007)

Expression of Immediate early genes is induced within a hours after serum starvation and delayed early genes are induced later by early genes(TFs) DNA chip analysis after adding serum onto serum starved cells (G0) Table 6.1 The Biology of Cancer (© Garland Science 2007)

Immediate vs. delayed early genes delayed early genes can be blocked by cyclohexamide treatment Figure 6.3 The Biology of Cancer (© Garland Science 2007)

V-Src is the first oncoprotein as a protein kinase (Tyrosine) How v-Src kinase induce the many transformed phenotypes Loss of contact inhibition Anchorage-independent fashion ? Figure 5.5a The Biology of Cancer (© Garland Science 2007)

Many substrates ? Many functional roles ? What is the difference in the regulation of cell proliferation between c-Src and v-Src ? Binds peptides phosphorylated on Tyr Binds polyproline motifs The structures of c-src and v-src and Deletion of the C-terminal region leads to activation of v-Src Figure 5.7a The Biology of Cancer (© Garland Science 2007)

Domain structure of Src SH2 domain: recognize and binds to p-Ty residue and specific oligopeptides. Many signaling proteins contains SH2 domain with a specificity to their ligands SH3 domain; binds to certain proline-rich sequence domains in partner protein Kinase domain (SH1) domain; catalytic activity Figure 6.7 The Biology of Cancer (© Garland Science 2007)

Structure and function of SH2 domain: Figure 6.8a The Biology of Cancer (© Garland Science 2007)

Activation mechanism of Src (SH2, SH3 domain, kinase domain) Inactive Partial active Full active Figure 6.11 The Biology of Cancer (© Garland Science 2007)

EGF receptor functions as a Tyr kinase Oncogenes related to Growth factor receptors Oncogenes related to growth factor receptors: bypass the growth factor requirement. The majority of oncogenes derived from growth factor receptors Tyrosine kinase activity. Tyrosine protein kinases. sequence homology of catalytic kinase domain. : Regulate key events in signal transduction pathways that control cell shape and growth. Growth factor receptor tyrosine kinases : Extracellular ligand binding domain : a single transmembrane domain : an intracellular catalytic domain : Binding of growth factor to receptor tyrosine kinases receptor dimerization, activation of tyrosine kinase activity intermolecular phosphorylation of each receptor on specific tyrosine residues recruitment of signaling molecule containing Src homology 2 (SH2) domain like PLC, PI3K, p120GAP, Grb2 ; signal transduction. Structure of EGF and Tyr kinases Figure 5.9a The Biology of Cancer (© Garland Science 2007)

An altered growth factor receptor functions as an oncoprotein Oncogenes : The mutant forms of receptor tyrosine kinases Epidermal growth factor receptor v-erbB Colony stimulating factor-1 receptor v-fms Hepatocyte growth / scatter factor receptor met Nerve growth factor receptor kit Neuroregulin receptor HER2/Neu GDNF receptor ret Receptor-like proteins with unknown ligands los Table 5.1 The Biology of Cancer (© Garland Science 2007)

Deregulation of Receptor firing constitutively sends signals to cells to proliferate or survive Figure 5.11 The Biology of Cancer (© Garland Science 2007)

Invasive human breast carcinoma Red: EFG receptor Green: TGF-a Figure 5.12c The Biology of Cancer (© Garland Science 2007)

sis as an concoprotein V-sis protein from simian sarcoma virus is closely related to PDGF PDGF stimulates mesenchymal cells such as fibroblasts,adipocytes, muscle cells, endothelial cells. V-sis activate the PDGF receptor in fibroblast to transform it, but not in epithelial cells

Oncogenic activation of receptor tyrosine kinases Receptors isolated as retrovirally transduced oncogenes : v-erbB, v-fms, v-kit Deletion of the extracellular ligand binding domain C- terminal deletions or mutations that remove negative regulatory domains Mutation that promote ligand independent dimerization A single point mutation in the transmembrane domain of the Neu/HER2 oncogene product receptor dimerization and kinase activation in the absence of ligand The loss of a single cystein residue in the EC domain of Ret receptor intrareceptor disulfide bond formation continually activated receptor A single point mutation in the kinase domain in Ret receptor increase the basal kinase activity, alters the substrate specificity : trk, met, ret, PDGFR : Genomic rearrangement of the kinase domain of the receptor with novel sequences like a domain capable of protein: protein interaction mediate dimerization Amplification and overexpression in human tumors : EGFR, HER2/neu, met, bek

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

Receptor dimerization and transphosphorylation upon ligand binding Figure 5.14 The Biology of Cancer (© Garland Science 2007)

Gene fusion may cause active dimerized receptor trk, met, ret, PDGFR : Genomic rearrangement of the kinase domain of the receptor with novel sequences like a domain capable of protein: protein interaction mediate dimerization Figure 5.17 The Biology of Cancer (© Garland Science 2007)

Structural alterations affect receptor firing Structural alterations affect receptor firing. Kits from feline sarcoma virus encodes SCF receptor mutated in Gatsrointestinal stromal tumor (GIST) of human Figure 5.18 The Biology of Cancer (© Garland Science 2007)

Hereditary cancer by mutation Ret genes encoding Tyr receptor that is found in mutant form in papillary thyroid carcinoma: Ret-fusion protein Met gene: hereditary papillary renal carcinoma Kit receptor mutant: Familial gastrointestinal stromal tumors Figure 5.19 The Biology of Cancer (© Garland Science 2007)

Other type of receptor signaling and cancer development TGF-beta; suppress the proliferation in normal Epithelial cells, but promotes the invasive activity after transformation Figure 5.20 The Biology of Cancer (© Garland Science 2007)

The Ras stands in the middle of a complex signaling cascade Adding some clues of signaling protein to make a network. sevenless (EGF homolog) and son of sevenless (sos) were discovered in Drosophila. They have functions in R7 development Shc and Grb2 function as a adaptor in this pathway TKR-Shc-Grb-Sos-Ras, but little insight into biochemical interactions for signaling. So, biochemical or cell biological experiments revealed their biochemical functions Figure 6.5 The Biology of Cancer (© Garland Science 2007)

Ras-signaling Three ras gene family members : c-H-ras, c-K-ras, N-ras; Ras proteins are targeted to the plasma membrane through a highly conserved sequence. Model for regulation of the ras p21 product Activation of receptor tyrosine kinase during signal transduction binding of the Grb2 adaptor (SH-2 vs phosphotyrosine) localization of Sos protein to the plasma membrane Sos stimulate the exchange of GDP for GTP on ras activation of ras : interact with effecter/substrate molecules (Raf, Rho, Rac). Inactivation by the intrinsic GTPase activity of ras *SH-2 containing GTPase-activating protein recruited by activated receptor tyrosine kinase. * GTPase-activating protein (GAP) stimulate low GTPase activity of ras conversion of ras from the active GTP-bound form to the inactive GDP-bound form Oncogenic forms of ras mutation that result in amino acid substitution at position 12, 13, 59, 61 in phosphate binding domain an increased exchange of GDP for GTP or an inability to interact with GAPs, locked in the active GTP-bound state no longer require activation by the Sos GDP/GTP exchange factor Figure 5.30 The Biology of Cancer (© Garland Science 2007)

Structure of Ras Oncoprotein

Alternative transformation mechanism by Ras Figure 5.32a The Biology of Cancer (© Garland Science 2007)

Figure 6.12 The Biology of Cancer (© Garland Science 2007)

Many proteins binds to p-Ty peptides of TKR Figure 6.9 The Biology of Cancer (© Garland Science 2007)

Many proteins contain protein interaction domain (SH2, SH3,PTB etc) Figure 6.10a The Biology of Cancer (© Garland Science 2007)

Table 6.2 The Biology of Cancer (© Garland Science 2007)

Three downstream signaling of Ras Ras-Raf-MEK-MAPK(Erk1/2) Figure 6.13 The Biology of Cancer (© Garland Science 2007)

Ras contols inositol lipids and the Akt/PTB for cell survival Figure 6.16b The Biology of Cancer (© Garland Science 2007)

The pleiotropic actions of a protein kinases derives from their ability to phosphorylates many targets Figure 5.7b The Biology of Cancer (© Garland Science 2007)

Pten +/- epithelial cells p-Akt Figure 6.19bc The Biology of Cancer (© Garland Science 2007)

Table 6.3 The Biology of Cancer (© Garland Science 2007)

Endocrine beta cells in the pancreatic islets; secrete Insulin Wt Constitutive Akt TG mice Endocrine beta cells in the pancreatic islets; secrete Insulin Figure 6.20 The Biology of Cancer (© Garland Science 2007)

The Jak-Stat pathway: IF, EPO, TPO signals, STAT1/2 dimer’ SH2 domain bind p-Typ that is phosphorylated by JAK and translocate into nucleus STAT3 active form: Introduction of the pair of Cys form a spontaneous dimer, which is a active form STAT3 is constitutively active in many melanoma, breast cancers activated by other Ty kinases (Src) rather than JAK Figure 6.22 The Biology of Cancer (© Garland Science 2007)

Notch signaling, Patched-smoothened signaling Mutant in T-cell leukemia Mutant in basal cell carinoma Figure 5.22 The Biology of Cancer (© Garland Science 2007)

Notch signal is important for self-renewal and differentiation Long time activation rather than short time activation like TKR Irreversible activation because of cleavage Constitutive active forms are found in tumor Elevated Jagged in prostate metastatic cancer Figure 6.29b The Biology of Cancer (© Garland Science 2007)

Gli overexpression was found in glioma Mutation was found in basal cell carcinoma Germline mutation in Gorlin syndrome Figure 6.29c The Biology of Cancer (© Garland Science 2007)

Wnt: int-1genes discovered by insertional mutagenesis by MMTV Figure 5.24 The Biology of Cancer (© Garland Science 2007)

Wnt-b-catenin pathway Two unrelated role of b-cantenin Cell adhesion form Cytoplasmic form/TFs Apc mutation in colon cancer results in accumulation of b-cantenin to activate the targets Why are there two independent function of b-cantenin ? Figure 6.26a The Biology of Cancer (© Garland Science 2007)

Signaling by G-coupled receptor and serpentine receptors Figure 5.25 The Biology of Cancer (© Garland Science 2007)

G-Protein coupled receptors can drive cell proliferations Mitogenic or anti-mitogenic, depdending on cell types Figure 6.28 The Biology of Cancer (© Garland Science 2007)

Signaling by Integrin receptors, which is involved in sensing ECM : Cell morphological change, cell migration and Metastasis Figure 5.26 The Biology of Cancer (© Garland Science 2007)

Table 5.4 The Biology of Cancer (© Garland Science 2007)

Figure 5.28b The Biology of Cancer (© Garland Science 2007)

Integrin signaling FAK(focal adhesion kinase) provides a docking site for Src and this Src phosphorylate the FAK and this p-Ty is another docking site for Grb2,Shc, PI3K ECM-Integrin-Sos-Ras-ERK pathway Figure 6.24a The Biology of Cancer (© Garland Science 2007)

Anoikis and morphogenesis Epithelial cells should be dead if they lose their contact. Ex) Epithelial cells will die in suspension culture: Anoikis These cells survive if they attach the basement membrane through their integrin, but without their attachment to the basement membrane undergo anoikis due to a cell death activation Typical breast cancer may derive from a defect of anoikis Figure 6.25a The Biology of Cancer (© Garland Science 2007)

Nuclear factor- kB Signals; TNF, LPS,IL,ROS,ant-cancer drug Targets genes: Bcl-2, IAP1/2, Myc, Cyclin D Amplified in B cell lymphoma Figure 6.29a The Biology of Cancer (© Garland Science 2007)

Arrest growth in many cell types TGF-b; dual functions Arrest growth in many cell types But contributes the phenotypes of tumor invasion (EMT pathway) Sma4 was lost in many cancer cells Smad 4 expression Figure 6.29d The Biology of Cancer (© Garland Science 2007)

Figure 6.32 The Biology of Cancer (© Garland Science 2007)

Figure 6.33 The Biology of Cancer (© Garland Science 2007)

Oncogenes of proteins with GTPase activity Three ras gene family members : c-H-ras, c-K-ras, N-ras; Ras proteins are targeted to the plasma membrane through a highly conserved sequence. Model for regulation of the ras p21 product Activation of receptor tyrosine kinase during signal transduction binding of the Grb2 adaptor (SH-2 vs phosphotyrosine) localization of Sos protein to the plasma membrane Sos stimulate the exchange of GDP for GTP on ras activation of ras : interact with effecter/substrate molecules (Raf, Rho, Rac). Inactivation by the intrinsic GTPase activity of ras *SH-2 containing GTPase-activating protein recruited by activated receptor tyrosine kinase. * GTPase-activating protein (GAP) stimulate low GTPase activity of ras conversion of ras from the active GTP-bound form to the inactive GDP-bound form Oncogenic forms of ras mutation that result in amino acid substitution at position 12, 13, 59, 61 in phosphate binding domain an increased exchange of GDP for GTP or an inability to interact with GAPs, locked in the active GTP-bound state no longer require activation by the Sos GDP/GTP exchange factor

Oncogenes of GTPase exhange factors Oncogenes act as exchange factors for Rho-like GTPase : Dbl, Vav, Ect-2, Ost, Tiam, Lbc, Lfc, Dbs. Discovered by gene tranfer methods by their ability to transform fibroblasts in culture Tiam-2 :directly influence the invasive capacity of T lymphoma cells : adjacent to a proviral insertion site in retrovirally induced invasive T lymphomas variants. Dbl : shared homology with a yeast cell division cycle protein Cdc24 --> dbl homology domain cf) Cdc24 : a exchange protein for a yeast Rho-like snall GTP binding protein Dbl homology domain : found in all of the oncogenic exchange proteins for small GTP-binding proteins, essential for the transforming activity

Oncogenes related to nonreceptor protein tyrosine kinase v-src, v-fes, v-fps, v-fgr, v-yes, lck : associated with the plasma membrane, but not transmembrane proteins Myristilated N-terminal glycine residues : Mutation of the myristoylation signal abolish transforming activity Cytoplasmic tyrosine protein kinase domain in the carboxy terminus The src subfamily : two additional domains SH-2 and SH-3 SH-2 domain - highly conserved in proteins involved in signal transduction, - recognize phosphotyrosine residues SH-3 domain - recognize prolin-rich motifs in signaling and cytoskeletal proteins Oncogenic activation of src-like kinases by point mutation and/or deletion of negative regulatory protein domains located at the carboxy terminus The abl subfamily : localized to both the nucleus and cytoplasm In Bcr-abl fusion oncoproteins, Bcr mediates olimerization of Bcr-abl consitutive activation of Abl kinase

Oncogenes related to adaptor proteins Only SH-2 and Sh-3 domains, lack any catalytic activity v-crk : cause an increase in tyrosine phosphorylated proteins in the cell Crk and other SH-2/SH-3-containing adaptor proteins SH-2 domain - bind to activated receptor tyrosine kinases or other kinases SH-3 domain : bind to other proteins Heteromeric protein complex subsequent phosphorylation of proteins relay the signal from the cell surface to the nucleus

Oncogenes of cytoplasmic serine-threonine protein kinases. Mos, cot, pim-1, raf, protein kinase C oncogenes : soluble cytoplasmic protein. V-raf serine kinase oncogenes : lost amino terminal regulatory sequences constitute activation of the kinase activity and mitogenic MAP kinase pathway. Stimulation of the PKC family by several tumor promoters. activation of the Raf signaling pathway mutant PKC enzymes are rare in human cancer Mos serine kinase oncogene : function as a cytostatic factor (CSF) in Xenopus eggs : Requires for meiotic maturation of Xenopus oocytes : The expression of c-mos normally is restricted to germ cells : Expression of mos in fibroblast by retroviral transduction prematurely enter mitosis ,chromosome fragmentation, aneuploid nature of many tumor cells

Nuclear protein family oncogenes Oncogenes in control of gene expression involved in cellular proliferation and differentiation. Many nuclear family oncogens : constitutively activated form of transcription factors. The mammalian transcription factor AP-1, a complex between c-jun and c-fos : oncogenic jun and fos transcription factors , mutation that lead to loss of negative regulatory elements constitutively active transcription factor. * V-rel : related to the NF-kB DNA binding subunit : transform B cells by binding to NF-kB recognition sites in DNA : blocking transcription of various genes required for the differentiation of B cells. * Proto-oncogenes like c-myc, c-fos, c-jun, c-myb are required for cells to transit from G0 to G1; The retroviral transductions with myc, myb, fos or c-myc amplification constitutive expression of transcription factors unregulated and/or ectopic expression in a differentiated cell constant proliferate signal in the absence of growth factors

Oncogenes and the cell cycle 1. The checkpoint of late G1 (the restriction point) : From the balance between growth inhibitory signals (TGF, cell-to-cell contacts, adhesion to matrix) and growth stimulatory signals (growth factors) : A cell decide between continued proliferation and escape from cell cycle : The deregulation of transition through the restriction point in late G1 is critical to malignant growth 2. The escape mechanisms from growth inhibition of cancer cells 1) The activation of growth promoting genes like growth factors, receptors, ras, nuclear oncoproteins 2) The loss of receptors for growth inhibitory genes such as TGF 3. The mechanism by that regulation of the G1-S checkpoint is lost 1) The overexpression of cyclin D 2) The loss of function of the tumor suppressor gene Rb

Oncogenes and cell death Induced suicide (Apoptosis) : a major mechanism for the elimination of cells with DNA damage or an aberrant cell cycle : The deregulated c-myc expression : promote apoptosis as well as proliferation : Cytokines : stimulate proliferation, inhibit apoptosis Mechanism coupling proliferation with apoptosis: the balance between proliferation and cell death. p53 protein : transcription factor that regulates the expression of proteins that negatively regulate the cyclin-dependent kinases required for cell cycle progression. : shut down the cell cycle clock until the problem (such as DNA damage) is corrected or apoptosis occurs : Loss or mutation of p53 : cells with DNA damage or oncogene activation no longer trigger apoptosis Bcl-2 proteins : inhibit the action of ICE-like proteases that trigger apoptosis (Figure 158-11) : The deregulated expression of the bcl-2 oncogene by Bcl-2 translocation cell immortalization inhibit p53-mediated apoptosis induced by growth factor deprivation, deregulated myc or genotoxic agents

Transgenic mouse models for cancers Transgenic mouse strains carrying a single oncogene generally show a strongly enhanced level of neoplasia or hyperplasia but only in specific tissues : Oncogene expression proceeds tumor formation by many months : little evidence that in vivo expression of a single oncogene can induce polyclnoal tumors Tumor-prone transgenic mice provide insight into oncogene collaboration - Crossing of two strains of oncogene-bearing mices - greatly increased tumor incidence -In mice bearing myc oncogenes (Eu-myc), insertional mutagenesis with a retrovirus - only myc, apoptosis of lymphoid cells : not sufficient for full neoplastic transformation - retrovirus promote tumorigenesis by chance integration - Several genes cooperate with myc to transform lymphoid cells : ras, raf, pim-1, abl, bcl-2, bmi-1, p53 Synergistic expression of v-H-ras and c-myc construct Induces breast cancer, but by clonal outgrowth : but nonmalignant cells expressing both oncogenes predominate : Ras mutation is rare in human breat cancer At least three proto-oncogenes implicated in human breast carcinogenesis : myc, HER2/Neu, cyclin D1 - develop mammary tumor, but need events in addition to the transgenes