1. Signal Transduction and Cancer Ali Sultan August 2009

2. Regulation of Protein Function Regulation of protein function plays a key role in governing cell behavior. Many cellular proteins are regulated by GTP or GDP binding. e.g; the Ras oncogene proteins, which involves in the control of cell proliferation. This small difference in protein conformation determines whether Ras (in the active GTP-form) can interact with its target molecule, which signals the cell to divide. The importance of such subtle differences in protein conformation is illustrated by the fact that mutations in ras genes contribute to the development of about 15-20% of human cancers. Such mutations alter the structure of the Ras proteins so that they are locked in the active GTP-bound conformation and continually signal cell division, thereby driving the uncontrolled growth of cancer cells. In contrast, normal Ras proteins alternate between the GTP- and GDP-bound conformations, such that they are active only following stimulation by the hormones and growth factors that normally control cell proliferation in multicellular organisms.

3. Phosphorylation regulates protein function Protein phosphorylation is catalyzed by protein kinases, which transfer phosphate groups from ATP to the hydroxyl groups of the side chains of serine, threonine, or tyrosine residues. Protein phosphorylation is reversed by protein phosphatases, which catalyze the hydrolysis of phosphorylated amino acid residues.

4. The prototype of the action of protein kinases came from studies of glycogen metabolism by Ed Fischer and Ed Krebs in 1955. In muscle cells the hormone epinephrine (adrenaline) signals the breakdown of glycogen to glucose-1- phosphate, providing an available source of energy for increased muscular activity. Regulation of glycogen breakdown by protein phosphorylation

5. Protein phosphorylation and signal transduction Protein kinases function as components of signal transduction pathways in which one kinase activates a second kinase, which may act on yet another kinase. The sequential action of a series of protein kinases can transmit a signal received at the cell surface to target proteins within the cell, resulting in changes in cell behavior in response to environmental stimuli. Mutational changes of protein kinases, are responsible for many diseases associated with improper regulation of cell growth and differentiation, particularly the development of cancer.

6. Oncogenes Cancer results from alterations in critical regulatory genes that control cell proliferation, differentiation, and survival. Studies of tumor viruses revealed that specific genes (called oncogenes) are capable of inducing cell transformation, thereby providing the first insights into the molecular basis of cancer. However, the majority of human cancers are not induced by viruses and apparently arise from other causes, such as radiation and chemical carcinogens. Studies of viral oncogenes led to the identification of cellular oncogenes, which are involved in the development of non-virus-induced cancers. The key link between viral and cellular oncogenes was provided by studies of the highly oncogenic retroviruses.

7. Retroviral Oncogenes Viral oncogenes were first defined in Rous Sarcoma Virus (RSV), which transforms chicken embryo fibroblasts in culture and induces large sarcomas within 1-2 weeks after inoculation into chickens. In contrast, the closely related avian leukosis virus (ALV) replicates in the same cells as RSV without inducing transformation. Both RSV and ALV infect and replicate in chicken embryo fibroblasts, but only RSV induces cell transformation.

8. RSV Gene The genomic RNA of RSV is about 10 kb, whereas that of ALV is smaller, about 8.5 kb. In addition to the genes necessary for viral replication, RSV carries additional gene called v-src. V-src is an oncogene which led to transformation of the susceptible cell type. The src gene is an addition to the genome of RSV; it is not present in ALV (see next slide). The v-Src protein is similar in amino acid sequence to a protein normally found in chicken muscle cells referred to as c-Src (for cellular Src). The c-src gene does not induce cell transformation and is termed a proto-oncogene. src gene encodes a 60-kd protein that was the first protein-tyrosine kinase to be identified. More than 100 different highly oncogenic retroviruses have been isolated from a variety of animals. Like src, many of these genes (such as ras and raf) encode proteins that are now recognized as key components of signaling pathways that stimulate cell proliferation.

9. The RSV genome contains an additional gene, src, that is not present in ALV and encodes the Src protein-tyrosine kinase.

10. How did the RSV acquire the mutated version of src It has been demonstrated (see next slide) that the retroviral oncogenes are derived from genes of the host cell, and that occasionally such a host cell gene becomes incorporated into a viral genome, yielding a new, highly oncogenic virus as the product of a virus-host recombination event. The normal-cell genes from which the retroviral oncogenes originated are called proto-oncogenes. They are important cell regulatory genes, in many cases encoding proteins that function in the signal transduction pathways controlling normal cell proliferation (e.g., src, ras, and raf).

11. Isolation of Abelson leukemia virus The highly oncogenic virus Ab-MuLV was isolated from a rare tumor that developed in a mouse that had been inoculated with a non-transforming virus (Moloney murine leukemia virus, or MuLV). MuLV contains only the gag, pol, and env genes required for virus replication. In contrast, Ab-MuLV has acquired a new oncogene (abl), which is responsible for its transforming activity. The abl oncogene replaced some of the viral replicative genes and is fused with a partially deleted gag gene, designated Δgag, in the Ab-MuLV genome.

12. Oncogenes in Human Cancer Direct evidence for the involvement of cellular oncogenes in human tumors was first obtained by gene transfer experiments. DNA of a human bladder carcinoma was found to efficiently induce transformation of recipient mouse cells in culture, indicating that the human tumor contained a biologically active cellular oncogene.

13. The 2 main types of genes that are now recognized as playing a role in cancer are oncogenes and tumor suppressor genes. Oncogenes are mutated forms of genes that cause normal cells to grow out of control and become cancer cells. They are mutations of certain normal genes of the cell called proto-oncogenes. Proto-oncogenes are the genes that normally control how often a cell divides and the degree to which it differentiates (or specializes). When a proto-oncogene mutates into an oncogene, it becomes permanently "turned on" or activated when it is not supposed to be. When this occurs, the cell divides too quickly, which can lead to cancer. Oncogenes (i)

14. The seven types of proteins that participate in controlling cell growth. Cancer can result from expression of mutant forms of these proteins: - growth factors (I), - growth factor receptors (II), - signal-transduction proteins (III), - transcription factors (IV), - pro- or anti-apoptotic proteins (V), - cell-cycle control proteins (VI), - DNA repair proteins (VII).

15. The pathway for normal cell growth starts with growth factor, which locks onto a growth factor receptor. The signal from the receptor is sent through a signal transducer. A transcription factor is produced, which causes the cell to begin dividing. If any abnormality is detected, the cell is made to commit suicide by a programmed cell death (apoptosis) regulator. More than 100 oncogenes are now recognized, and undoubtedly more will be discovered in the future. Oncogenes are divided into 5 different classes: 1)Growth factors: e.g. sis. It leads to the overproduction of a protein called platelet-derived growth factor (PDGF), which stimulates cells to grow. 2)Growth factor receptors: - These are normally turned "on" or "off" by growth factors. The best known examples of growth factor receptor gene are: erb B and erb B-2. (These are sometimes known as epidermal growth factor receptor), and HER2/neu. HER2/neu gene amplification is an important abnormality seen in about one third of breast cancers. - Both of these oncogenes are targets of newly developed anti-cancer treatments. Oncogenes (ii)

16. Oncogenes (iii) 3)Signal transducers: These are the intermediate pathways between the growth factor receptor and the cell nucleus where the signal is received. Like growth factor receptors, these can be turned on or off. When they are abnormal in cancer cells, they are turned on. Two well known signal transducers are abl and ras. Abl is activated in chronic myelocytic leukemia and is the target of the most successful drug for this disease, imatinib or Gleevec. Abnormalities of ras are found in many cancers. 4) Transcription factors: These are the final molecules in the chain that tell the cell to divide. These molecules act on the DNA and control which genes are active in producing RNA and protein. The best known of these is called myc. In lung cancer, leukemia, lymphoma, and a number of other cancer types, myc is often overly activated and stimulates cell division. 5)Apoptotic regulators: These molecules prevent a cell from committing suicide when it becomes abnormal. When these genes are overactive they prevent the cell from going through the suicide process. This leads to an overgrowth of abnormal cells, which can then become cancerous. The most well described one is called bcl-2. It is often activated in lymphoma cells.

17. Proto-oncogenes that Encode for Signalling Proteins GTP receptor associated proteins: Ras family - H-ras (Harvey rat sarcoma virus, H-RaSV) Cytoplasmic (Non-receptor) Tyrosine Kinases - src (Avian or Rous sarcoma virus) - abl (Abelson murine leukemia virus) Serine/Threonine Kinases raf family akt

18. Ras Oncogenes (i) The first human oncogene identified in gene transfer assays was subsequently identified as the human homolog of the rasH oncogene of Harvey sarcoma virus. Three closely related members of the ras gene family (rasH, rasK, and rasN) are the oncogenes most frequently encountered in human tumors. These genes are involved in approximately 20% of all human malignancies, including about 90% of pancreas, 50% of colon and 25% of lung carcinomas. the ras genes encode guanine-nucleotide binding proteins that function in transduction of mitogenic signals from a variety of growth factor receptors. The activity of the Ras proteins is controlled by GTP or GDP binding, such that they alternate between active (GTP-bound) and inactive (GDP-bound) states. The ras oncogenes are not present in normal cells; rather, they are generated in tumor cells as a consequence of mutations that occur during tumor development.

20. The ras oncogenes differ from their proto-oncogenes by point mutations resulting in single amino acid substitutions at critical positions. The first such mutation discovered was the substitution of valine for glycine at position 12 (detected in bladder carcinoma). In animal models, it has been shown that mutations that convert ras proto-oncogenes to oncogenes are caused by chemical carcinogens, providing a direct link between the mutagenic action of carcinogens and cell transformation. The mutations characteristic of ras oncogenes have the effect of maintaining the Ras proteins constitutively in the active GTP-bound conformation, which drives unregulated cell proliferation. Ras Oncogenes (ii)

21. Point mutations are only one of the ways in which proto-oncogenes are converted to oncogenes in human tumors. - Chromosomal abnormalities - Gene Amplification

22. The gene rearrangements resulting from chromosome translocations frequently lead to the generation of oncogenes. The first characterized example of oncogene activation by chromosome translocation was the involvement of the c-myc oncogene in human Burkitt's lymphomas and mouse plasmacytomas, which are malignancies of antibody- producing B lymphocytes (immunoglobulins). For example, virtually all Burkitt's lymphomas have translocations of a fragment of chromosome 8 to one of the immunoglobulin gene loci, which reside on chromosomes 2, (  light chain), 14 (heavy chain), and 22 (  light chain). Chromosomal abnormalities and cancer The c-myc proto-oncogene is translocated from chromosome 8 to the immunoglobulin heavy-chain locus (IgH) on chromosome 14 in Burkitt's lymphomas, resulting in abnormal c-myc expression.

23. Translocations of other proto-oncogenes frequently result in rearrangements of coding sequences, leading to the formation of abnormal gene products. The prototype is translocation of the abl proto-oncogene from chromosome 9 to chromosome 22 in chronic myelogenous leukemia. This translocation leads to fusion of abl with its translocation partner, a gene called bcr (breakpoint cluster gene), on chromosome 22. The result is production of a Bcr/Abl fusion protein in which the normal amino terminus of the Abl proto-oncogene protein has been replaced by Bcr amino acid sequences. The fusion of Bcr sequences results in unregulated activity of the Abl protein-tyrosine kinase, leading to cell transformation.

24. Gene amplification and Cancer A distinct mechanism by which oncogenes are activated in human tumors is gene amplification, which results in elevated gene expression. Gene amplification is common in tumor cells, occurring more than a thousand times more frequently than in normal cells, and amplification of oncogenes may play a role in the progression of many tumors to more rapid growth and increasing malignancy. A prominent example of oncogene amplification is the involvement of the N-myc gene, which is related to c-myc, in neuroblastoma (a childhood tumor of embryonal neuronal cells). Amplification of another oncogene, erbB-2 (erythroblastosis A, B, C), which encodes a receptor protein-tyrosine kinase, is similarly related to progression of breast and ovarian carcinomas.

25. Growth factors in normal cells serve as environmental signals. Growth factors regulate growth, proliferation, and survival. These are all deregulated in cancer. which bind enzyme-linked receptors. Signal diverse cellular responses including: proliferation, differentiation, growth, survival, and angiogenesis. Can signal to multiple cell types or be specific to one cell. Growth Factor: ST and Cancer Hanahan and Weinberg, (2000) Hallmarks of Cancer, Cell (100) 57

26. FactorPrincipal Source Primary ActivityComments PDGFplatelets, endothelial cells, placenta promotes proliferation of connective tissue, glial and smooth muscle cells two different protein chains form 3 distinct dimer forms; AA, AB and BB EGFsubmaxillary gland, Brunners gland promotes proliferation of mesenchymal, glial and epithelial cells TGF-  common in transformed cells may be important for normal wound healing related to EGF FGFwide range of cells; protein is associated with the ECM promotes proliferation of many cells; inhibits some stem cells; induces mesoderm to form in early embryos at least 19 family members, 4 distinct receptors NGF promotes neurite outgrowth and neural cell survival several related proteins first identified as proto-oncogenes; trkA (trackA), trkB, trkC Erythropoietinkidneypromotes proliferation and differentiation of erythrocytes TGF-  activated TH 1 cells (T-helper) and natural killer (NK) cells anti-inflammatory (suppresses cytokine production and class II MHC expression), promotes wound healing, inhibits macrophage and lymphocyte proliferation at least 100 different family members IGF-Iprimarily liverpromotes proliferation of many cell types related to IGF-II and proinsulin, also called Somatomedin C IGF-IIvariety of cellspromotes proliferation of many cell types primarily of fetal origin related to IGF-I and proinsulin Growth Factors

27. Most growth factors bind Receptor Tyrosine Kinases

29. Growth factors, signal transduction and Cancer The majority of oncogene proteins function as elements of the signaling pathways that regulate cell proliferation and survival in response to growth factor stimulation. Oncogene proteins act as growth factors (e.g., EGF), growth factor receptors (e.g., ErbB), and intracellular signaling molecules (Ras and Raf). Ras and Raf activate the ERK MAP kinase pathway (see next 2 slides), leading to the induction of additional genes (e.g., fos) that encode potentially oncogenic transcriptional regulatory proteins. Proteins with known oncogenic potential are highlighted with a yellow glow

30. Activation of the ERK MAP kinases: Stimulation of growth factor receptors leads to activation of the small GTP-binding protein Ras, which interacts with the Raf protein kinase. Raf phosphorylates and activates MEK, a dual- specificity protein kinase that activates ERK by phosphorylation on both threonine and tyrosine residues (Thr-183 and Tyr-185). ERK then phosphorylates a variety of nuclear and cytoplasmic target proteins.

31. Induction of immediate-early genes by ERK Activated ERK translocates to the nucleus, where it phosphorylates the transcription factor Elk-1. Elk-1 binds to the serum response element (SRE) in a complex with serum response factor (SRF). Phosphorylation stimulates the activity of Elk-1 as a transcriptional activator, leading to immediate-early gene induction.

32. Growth factors with Oncogenic Potential PDGF: originally shown to regulate proliferation, was also shown to have homology to v-sis, the simian sarcoma virus. Neurotrophins: a family of neurotrophic factors in the brain that encourage neuron differentiation and survival. Implicated in medulloblastomas, and also in lung, prostate and pancreatic cancers. Fibroblast Growth Factor Family: (FGF2) amplified in 5-10% of breast cancer patients Epidermal Growth Factor (EGF) Family: (EGF, TGF  )

33. Growth Factors Recptors, ST and Cancer Oncogenes encode growth factor receptors, (most of which are protein-tyrosine kinases) are converted to oncogene proteins by alterations of their amino-terminal domains, which would normally bind extracellular growth factors. For example, the receptor for platelet-derived growth factor (PDGF) is converted to an oncogene in some human leukemias by a chromosome translocation in which the normal amino terminus of the PDGF receptor is replaced by the amino terminal sequences of a transcription factor called Tel. The Tel sequences of the resulting Tel/PDGFR fusion protein dimerize in the absence of growth factor binding, resulting in constitutive activity of the intracellular kinase domain and unregulated production of a proliferative signal from the oncogene protein.

34. Growth Factor Receptors

35. GF Receptors with Oncogenic Potential EGFR: kinase activity stimulated by EGF-1 and TGF-  involved in cell growth and differentiation, was linked via sequence homology to a known avian erythroblastosis virus onocgene, v-erbB. Since then, many oncogenes have been shown to encode for GFRs. EGFR family erbB1 (c-erbB) erbB2 (neu) FGF Family FGFR-1(fig) NT3 R (trk-C) PDGFR Family CSF-1R (c-fms) SLF R (c-kit) Insulin Receptor family IGF-1 (c-ros) Neurotrophins NGFR (trk) BDNFR (trk-B) NT3 R (trk-C)

36. Transcription regulatory proteins and cancer Many oncogenes encode transcriptional regulatory proteins that are normally induced in response to growth factor stimulation. For example, 1)Transcription of the fos proto-oncogene is induced as a result of phosphorylation of Elk-1 by the ERK MAP kinase. Fos and the product of another proto-oncogene, Jun, are components of the activator protein-1 (AP-1), a transcription factor, which activates transcription of a number of target genes in growth factor-stimulated cells. Constitutive activity of AP-1, resulting from unregulated expression of either the Fos or Jun oncogene proteins, is sufficient to drive abnormal cell proliferation, leading to cell transformation. 2) The Myc proteins similarly function as transcription factors regulated by mitogenic stimuli, and abnormal expression of myc oncogenes contributes to the development of a variety of human tumors. 3) Another example, mutations of the gene encoding the thyrotropin receptor convert it to an oncogene in thyroid tumors. Thyrotropin is a pituitary hormone that stimulates proliferation of thyroid cells through a G protein-coupled receptor that activates adenylyl cyclase. Mutations of the thyrotropin receptor in thyroid tumors result in constitutive activity of the receptor, which then drives cell proliferation via activation of the cAMP signaling pathway.

37. ST intermediates can be targets for anti-cancer drugs Gleevec (Novartis): A new drug used in the treatment of Chronic Myeloid Leukemia (CML) and other cancers. Imatinib mesylate is a protein- tyrosine kinase inhibitor that inhibits the Bcr- Abl tyrosine kinase, the constitutive abnormal tyrosine kinase created by the Philadelphia chromosome abnormality in CML. It inhibits proliferation and induces apoptosis in Bcr-Abl positive cell lines as well as fresh leukemic cells from Philadelphia chromosome positive CML. It has been shown to be effective in creating lasting remissions.

38. ST intermediates can be targets for anti-cancer drugs BAY 43-9006 (Sorafenib): is a "targeted drug" specifically engineered to inhibit RAF kinase within the cancer cells. It turns out that in addition to targeting RAF Kinase, BAY 43-9006 also inhibits the VEGF and PDGF receptors on blood vessel cells. Both of these receptors are also kinases. Clear cell kidney cancer is well known for vastly over-producing VEGF, which in turn stimulates blood vessel growth which supplies the tumor with oxygen and nutrients. Most clear cell RCC has a defect in what is known as the VHL (Von Hippel-Lindau) gene which causes the cell to think it is short on oxygen and to pump out huge amounts of VEGF. BAY 43-9006 interrupts this signal on the blood vessel cells.

39. Growth Factor Receptors can be drug targets HERCEPTIN ( Trastuzumab ): is a monoclonal antibody that acts on the HER2/neu (erbB2) receptor. Trastuzumab's principal use is as an anti-cancer therapy in breast cancer in patients whose tumors over express this receptor. The drug binds to the extracellular segment of the erbB2 receptor. Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle so there is reducedcell cycle proliferation. It has been suggested that trastuzumab induces some of its effect by downregulation of erbB2downregulation leading to disruption of receptor dimerization and signaling through the downstream PI3K cascade causing cell cycle arrest. Also, trastuzumab suppresses angiogenesis by both induction ofangiogenesis antiangiogenic factors & repression of proangiogenic factors.