From anomalous cell cycle regulation to malignant tumors

From anomalous cell cycle regulationto malignant tumors • Regulation of cell proliferation • Regulation of cell death • Oncogenes and tumor suppressors • viral oncogenes • Telomerase activity • Angiogenesis • Immune modulation • Metastasis • Multi drug resistance • Genetic instability

Anomalies of cell cycle regulation- continuously proliferating cells • Somatic cells divide only „by permission”: they require growth factors (GF). • Cell types, which divide permanently exhibit very limited lifespan and have a tendency to undergo apoptosis. • Mutations are continuously repaired. Irreparable mutations elicit cell cycle arrest, cells with mutant genomes undego apoptosis.

There is a dynamic balance between lost/destroyed cells and cell divisions in our tissues. Stem cells and regenerating cells replace cells lost through injuries or wear. High degree of differentiation usually blocks cell division, while proliferation inhibits differentiation. Cell surface receptors bind tissue specific growth factors and elicit cascades of signaling events, eventually leading to DNA synthesis and cell division. Unambiguous growth signals or withdrowal of growth factors during the cell cycle force cells into apoptosis. Continuously dividing cells are killed in the absence of growth factors. Regulation of the cell cycle

Anomalies of cell cycle regulation: autocrine growth factors • Production of autocrin growth factors can lead to uncontroled proliferation of a certain cell type • Usually the mutation affects the enhancer-promoter region of the growth factor gene • Chromosome aberrations andrecombinational events mightreplace the regulatory region of the gene orleading to gene multiplication • EGF – hepatocellular ccgastrin – colorectal ccPDGF – embryonic ccIGF-1 – esophageal cc

Anomalies of cell cycle regulation: receptor mutations • Mutations affecting the structure of the receptor („active” conformation is stable in the absence of ligand) • Mutation affecting the regulatory region of the gene, or gene amplification: overproduction of the receptor molecule • Chromosome aberrations andrecombinational events mightreplace the regulatory region of the gene • erb B2/HER-2 over-expressed in mammary carcinoma

Anomalies of cell cycle regulation: G-protein mutations • Mutations affecting the structure or GTP-ase activity of G-proteins (if GTP can not be split, G-protein remains active) • Ras is mutated in many human tumors. In Ras gene most mutations affect aa 12 (61 or 13), the GTP-ase site • Effects of Ras mutations: Raf (MAPK) pathway,PI-3K (Akt/PKB pathway)RasGEF (in humas very important)

Anomalies of cell cycle regulation: mutations affecting signal molecules • Most mutations found in the coding region of the genes of signal molecules • Mutations affecting the balance of kinases/phosphatases orkinases/ubiquitin ligases • Mutations altering the length of the signal • Mutations affecting the compartmentalization of activated signal molecules • 33 human lung tumors (141 PK genes tested) - 41 PK mutations found typically CG/AT mutations: caused by smoking

Anomalies of cell cycle regulation: mutations affecting transcription factors • Mutations both in the control and coding region of the genes of transcription factors are found in human tumors • TFs are master regulatory genes: affecting activity of many different genes • TFs have direct effect on cell cycle • Myc in AML, pancreatic ccabnormal STAT3 activity found in the majority of human malignancies

Anomalies of cell cycle regulation Mutations affecting regulatory molecules: cyclins, cdk’s and inhibitors of cell cycle progression (p21, p27KIP, etc) Activity of these proteins is normally regulated by MAPKs, transcription factors

Anomalies of cell cycle regulation A large number ofgenes participate incell cycle control Their mutation might cause deregulated cell proliferation

Anomalies of cell cycle regulation Two key control points: G1 / S phase transition governed by Rb DNA synthesis is not allowed in the absence of mitogenic signals Two key Non-phosphorylated Rb binds E2F, S phase genes are inactive

Anomalies of cell cycle regulation G2 / M phase transition, controlled by p53 Mitosis can start only after repair of all mutation If reparation is impossible, p53 induces apoptosis

Loss of the apoptotic tendency in continuously dividing cells: a way to „immortalization” • Terminal differentiation of permanently proliferating cell types includes apoptosis. Extended survival of these cell types tips the balance. • Abnormal level of survival factors (eg. autocrine survival factors) or absence of death signals (mutations causing loss of response to death signals) • Mutations abrogating dependence on survival factors • In the absence of pro-apoptotic mediators the number of these cell types increases to abnormal ranges causing severe symptoms (lpr/gld mutations in mice). • Increased production of anti-apoptotic factors leads to similar effects (bcl2 system).

The balance of pro- and anti-apoptotic proteins in the mitochondria The bcl2/bax family of proteins regulate death (release of cytochrome C, activation of the caspase cascade) or survival In cells of human B-cell lymphoma bcl2 production is upregulated, B cell accumulate

Pro-apoptotic ligands and their receptors belong to the TNF family of proteins Killing of (mutant) cells is triggered by TNF-family ligands acting on death receptors. Immune cells use amembrane destructing enzyme, Granzyme Bto kill tumor cells (and parasite-infected cells)

Loss of the apoptotic tendency Apoptosis is an active process,dependent on ATP and co-ordinated action of many proteins Mutations leading to inactivationof any of pro-apoptotic proteins might cause immortalization of the cells

Telomers and telomerase Unlimited proliferation of somatic cells is prevented, because chromosome replication is accompanied by the shortening of telomers Only cells with active telomerase (germinal cells, stem cells and progenitor cells) display telomerase activity

Telomers and telomerase Telomerase can be activated because of mutations affecting the regulatory region of the gene Telomerase „positivity” is a general property of malignant cells Telomerase has an RNA template and produces telomeris sequences of DNA by reverse transcription

Oncogenes, protooncogenes, viral oncogenes P. Rous isolated a „filtratable” agent causing sarcoma of chickens almost acentury ago Mouse virus transmitter by mothers milk Leukemia and sarcoma viruses of birds, rodents and primates One single gene of Rous sarcoma virus is capable of inducing oncogenic transformation of chicken cells. Src gene encodes a protein tyrosine kinase Sarcoma viruses carry oncogenes. Retrovirus genomes encode processed oncogenes The „normal”, cellular gene is a protooncogene Retroviruses use reverse transcriptase to convert RNA into dsDNA

Retroviral oncogenes Retroviral oncogenes are of host origin Each type of these viruses cause a special form of malignant disease (a few carry two genes, causing two kind of tumors) Retroviruses without oncogenes can cause tumor growth by enhancer-insertion (into regultory elements of cellular protooncogenes) B cell lymphoma

Viral oncogenes of DNA viruses Papillomaviruses DNA viruses carry viral oncogenes: these genes increase the accident of malignant transformationEach tumor is different – one virus strain can caause many types of malignancies Papillomavirus proteins interfere with the action of p53 and Rb Tumor suppressors are host genes preventing oncogenic transformation: both genes must be inactivated in order to allow uncontroled proliferation! Cervical cancer Oral papilloma

Viral oncogenes of DNA viruses Hepatitis B and C viruses Hepatitis B and C viruses tend to cause persistant infection which might convert into hepatocellular carcinoma Both viruses are blood-borne viruses do not (infrequently) infectious orally A good HBV vaccine is available

Viral oncogenes of DNA viruses Herpesviruses Herpesvirus-8 – in collaboration with retroviruses (leukemia viruses or HIV) – might cause Kaposi sarcoma, a pigmented skin tumor. It is it is not rare among AIDS patients

Viral oncogenes of DNA viruses Herpesviruses Another herpesvirus, Epstein-Barr virus (EBV) is the causative agent of Burkitt lymphoma. Though EBV is infecting more than half of the population everywhere, BL is much more frequented among starving children in third world countries and people simultaneously infected by parasites of malaria, sleeping sickness or other devastating diseases.

Anti-tumor mechanisms Cells proliferation is stricktly controlled. Mitogenic signals are needed. Checkpoints prevent unscheduled cell division and mitosis of mutant cells Continuously proliferating cells have a limited life span, dfferentiation is linked to apoptosis Senescence of telomers prevent long term proliferation of somatic cells Tumor suppressors inhibit false mitotic signals or uncontrolled cell division The proofreading of new DNA and repair of mutations is continuous. Absence of blood vessels in neplastic tissue inhibit growth of cell mass Immune cells produce death signals to induce apoptosis of mutant cells. Complement activation lyses membranes of cell labelled by antibodies against tumor antigens Cytotoxic lymphocytes eliminate cells presenting tumor antigens Natural killer cells destroy mutant cells non-presenting cells, resistant to apoptosis TNF induces death in cells of new endothelium of tumor tissues

Cellular mechanisms leading to immortalization and tumor cell formation Mutations affecting mitotic factor production, receptor function, normal duration and direction of signaling, activity of enzyme activities controlling signal events and acivity of transcription factors might disturb control of proliferation Dominant mutations of protooncogenes (converting them into oncogenes) and concommittant loss of tumor suppressor functions (recessive mutations!) are also needed. Telomerase must be activated. Further mutation are necessary which cause loss of death receptors, induce resistance to apoptosis, CTL, NK and complement-induced killing, capacity to induce growth of new blood vessels, to block action of death signals on tumor cells and capillary endothel. Local immunosuppression should be achieved by producing cytokines. Still further mutations are necessary to upregulate many genes essential for mobilization and metastatic growth, multi-drug resistance, radiation resistance, etc. How is it possible to collect so many mutations in relatively short time?

Genetic instability is cause, not result of abnormal proliferation! Increased mutation rate is essential for the emergence of malignant cells Mutations of DNA polymerases, proofreading and repair enzymes (or long term exposure to mutagenic agents, especially if combined with repair-inhibitors) might only result in an excessive number of mutation Retroviruses are able to immortalize specific cell types causing well defined malignancies (erythroleukemia, bone tumors, sarcoma, myeloblastosis, etc.) in animal models and human patients. Polyoma viruses induce many different kinds of tumors in immune- suppressed or –incompetent animals or individuals. Several human viruses (HPV, EBV, CMV, HHV-8, HBV és HCV, HLTV) and bacteria (Helicobacter, Chlamydia, stb.) are established etiological agents of malignancies. Virulence factors of pathogenic organisms can help in the development of tumor cells and in their immune-escape

Mutation rate and tumor formation Loss of repair enzyme activity leads to early death of experimental mice Heterozygotes show very little effect in the absence of mutagenic agents, but became vulnerable if exposed to DNA- damaging compounds or effects (X ray)

DNA repair is vital The genome-protecting gene network A large number of gene products are involved in keeping the genome free of mutations, genedefects and chromosome aberrations.

Genetic and environmental factors in oncogenesis • Human genetic background: Gene defects affecting • DNA synthesis • or repair enzymes • or proto-oncogenes, anti-oncogenes, • Environmental factors • mutagenic compounds, UV, X-ray, etc., • repair-inhibitors • immunosuppressive compounds, effects, • Living environment • viruses, pathogenes • immunosuppressive agents and treatments, starving, ageing • social stress

From anomalous cell cycle regulation to malignant tumors