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Cell cycle and tumors. Oncogenes Oncogene is a gene that encodes a protein which hyperactivity is one step to cancer A proto-oncogene is a normal gene.

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Presentation on theme: "Cell cycle and tumors. Oncogenes Oncogene is a gene that encodes a protein which hyperactivity is one step to cancer A proto-oncogene is a normal gene."— Presentation transcript:

1 Cell cycle and tumors

2 Oncogenes Oncogene is a gene that encodes a protein which hyperactivity is one step to cancer A proto-oncogene is a normal gene that can become an oncogene due to mutations changing structure- function abilities of its product or increasing its expression Oncogene is a dominant allele of a proto-oncogene = loose of a (normal) function is dominant Structural that cause an increase in protein (enzyme ) activity a loss of regulation (constant activity) Regulatory due to an increase of protein expression an increase of protein (mRNA) Chromosomal translocation Mutations that result in proto- oncogene – oncogene transformation

3 Proto-oncogene activation

4 TRKs as proto-oncogenes EGFR signaling causes increased proliferation, decreased apoptosis, and enhanced tumor cell motility and neo-angiogenesis. The EGFR is expressed or highly expressed in a variety of human tumors of epithelial origin. Carlos L. Arteaga The Oncologist 2002;7:31-39 Increased expression. Heterodimerization Mutations in EGFR Ligand autocrine loop. Mutations in the inhibition system (phosphatase)

5 GPCRs can exert positive effects on EGFR signaling in several ways, such as - activation of matrix metalloproteinases, which cleave membrane-tethered EGFR ligands - GPCRs indirectly activate Src, which can phosphorylate the EGFR at tyrosines. Steroid hormones can also influence EGFR signaling by activating the transcription of genes encoding EGFR ligands Estrogen can transactivate EGFR via the GPCR GPR30, potentially explaining the EGF-like effect of estrogen Carlos L. Arteaga The Oncologist 2002;7:31-39 Heterologic control

6 EGFR Mutations The EGFR mutation, EGFRvIII, involves deletion of exons 2 to 7 and loss of residues 6 to 276 in the receptor’s ectodomain. This mutant yields a constitutively active receptor that is not downregulated by endocytosis and is potently transforming

7 Ras-protooncogene Mutations of the Ras proto- oncogene occurs in 20% of human cancers Most of mutations results in loss of the response of oncogenic Ras to GTPase-activating proteins (GAPs). The constitutively activated, GTP-bound Ras activates downstream effectors including the MAP kinase. In addition, Ras activity in tumor cells can be increased by other mechanisms, including mutation of GAP proteins and in downstream proteins.

8 Proto-oncogene Raf Raf serine/threonine kinase=MAPKKK Activation of the Raf serine/threonine kinase is induced by binding to Ras and leads to activation of the MAP kinase pathway 66% of melanomas and 10% of colon cancers harbor activating mutations in the BRAF proto-oncogene The mutations lead to constitutive activation of the downstream MEK ( MAPKK) and extracellular signal- regulated kinases (ERK1/2=MAPK). This results in the phosphorylation of MAPK's cytoplasmic and nuclear targets and alters the pattern of normal cellular gene expression.

9 Protooncogenes of the PI3K/Akt signaling pathway Amplification or activating point mutation of the gene encoding the catalytic subunit of PI3K (p110) is observed in 20–30% of cancers, and amplification of the Akt2 (p85) gene occurs in others Akt promotes cell survival by activation of the transcription factor NF- kB; preventing inactivation of Myc, -catenin, cyclin D1, and cyclin E, and blocking upregulation of p27 Kip1 and Bim. Furthermore, the growth of cancer cells requires the activation of mTOR The PI3K/Akt pathway is activated in 30–40% of human cancers and is thought to play a critical role in tumor cell survival, proliferation, growth, and glucose utilization.

10 Oncogene silencing by anticancer drugs Inhibitors of TRKs – monoclonal antibodies; Imatinib, Erlotinib – block ATP binding to tyrosine kinase active site. Ras requires posttranslational modifications, including addition of a farnesyl lipid moiety. - Inhibition of RAS farnesylation. Inhibitors of Raf kinases (e.g., sorafinib) Inhibitors of MEK (MAPKK) Inhibitors of TOR - repamycin Harrison's Internal Medicine > Chapter 80. Cancer Cell Biology and Angiogenesis > Harrison’s Internal Medicine

11 Tumor suppressor genes Tumor suppressor gene is a gene encoding a protein which function is required to prevent cancer Loose of function by both tumor suppressor gene alleles is one step to cancer Unlike oncogenes, cancer- associated mutations in tumor suppressor genes are recessive = loose of a (normal) function is recessive Tumor-suppressor geneseither have a repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both including Genes that are active at checkpoints Genes that products are involved in DNA reparartion Some genes that product are involved in cell adhesion

12 p53 p53 signaling pathways Gasco et al. Breast Cancer Res 2002 4:70-76 p53 is a key tumor suppressor p53 is a transcription factor p53 activity is regulated by the inhibitor MDM2 and by phosphorylation Under the non-stressed condition, the p53 protein is maintained at a low level in cells by the proteasome degradation pathway due to activity of MDM2, an E3 ubiquitin ligase. Once activated, p53 binds to a specific DNA sequence, termed the p53-responsive element, in its target genes to regulate their expression.

13 5 The ataxia–telangiectasia- mutated protein (ATM) abd Rad3-related protein kinase (ATR) are kinases that get active in response to DSB and phosphorylate histone H2AX, p53 and some other targets ATM is active at G1/G0 phase ATR is active at G2 phase Lucy C. Riches et al. Mutagenesis 2008;23:331-339

14 Activation of the G2/M checkpoint after DNA damage. In response to DNA damage, the ATM/ATR phosphorylate and activate Chk1 and Chk2 Chk1 and Chk2 phosphorylate Cdc25. Phosphorylated Cdc25 is sequestered in the cytoplasm by 14- 3-3 proteins, which prevents activation of cyclinB/Cdk1 by Cdc25 and results in G2 arrest. ATM/ATR also activates p53- dependent signaling. This contributes to the maintenance of G2 arrest by upregulating 14-3-3. In addition, p53 induces p21, a Cdk inhibitor that binds to and inhibits cyclinB/Cdk1 complexes. P: phosphorylation. Wang et al. Molecular Cancer 2009 8:8 doi:10.1186/1476-4598-8-8

15 Genetic lesions that render the retinoblastoma pathway nonfunctional are thought to occur in all human cancers. Loss of function of pRB as guardian of the G 1 restriction point enables cancer cells to enter a mitotic cycle without the normal input from external signals. pRB –retinoblastoma protein

16 The Hallmarks of Cancer Figure 1 Acquired Capabilities of Cancer. We suggest that most if not all cancers have acquired the same set of functional capabilities during their development, albeit through various mechanistic strategies. Douglas Hanahan, Robert A Weinberg. The Hallmarks of Cancer. Cell, Volume 100, Issue 1, 2000, 57 - 70

17 Hallmark 1. Self-sufficiency in growth signals Mechanisms developed by tumor cells Examples Selfproduction of corresponding mitogens PDGF (platelet-derived growth factor) and TGF α (tumor growth factor α ) by glioblastomas and sarcomas, respectively Overexpression or structural alternations in mitogen receptors EGFR is upregulated or mutated in stomach, brain, and breast tumors Changes in the mitogen- activated cascades Up to 25% human tumors activate MAPK w/o upstream signaling via mutations in Ras, Raf, MEK Mutations in inhibitory systemsMutations in EGDR controlling phosphatase Cooperation with normal neighbors

18 Hallmark 2. Insensitivity to Antigrowth Signals MechanismExample Disruption of the retinoblastoma protein (PRb) pathway Loose of functional PRb mutations in CDK4 PRb sequestration by viral oncoproteins Insensibility to contact inhibition Fibroblasts Antigrowth signals act in two different ways, either to force cells to enter a quiescent phase or to differentiate.

19 Transforming growth factor- β (TGF- β ) TGF- β regulates differentiation and proliferation of different cell types. The TGF- β family play a key role in control of maintenance of metabolic homeostasis in the bone tissue and in the fracture healing process. Poniatowski et al., 2015

20 Antigrowth signaling Osteoarthritis and Cartilage 2009 17, 1539-1545DOI: (10.1016/j.joca.2009.06.008) TGFBRs (I and II types) are Ser/Thr kinase TGFBRs phosphorylate Smads on C-end Phosphorylated SMADs interact with the common signaling transducer Smad4, and translocate to the nucleus. SMAD complex activate genes required for chondrocyte differentiation

21 TGFRB control TGF- β ligand-bound receptor complexes are also subject to constitutive control related to their internalization on the clathrin- dependent or lipid- raft-dependent endocytic pathway, and this ensures their correct physiological response, activity, and distribution on a cell surface Zhao and Chen, 2014

22 Contact inhibition of locomotion (CIL) In 1954 Abercrombie & Heasyman found that when moving fibroblasts met, they stopped moving in the direction that led to contact, and little overlap between opposing populations was observed. Besides fibroblasts, neural crest cells and haemocytes exhibit CIL When a collision occurs between the leading edges of two fibroblasts, both cells stop moving and retract their leading edges from the point of contact When the leading edge of one fibroblast contacts the rear side of another, the movement of the second fibroblast is not affected As for cancer cells, while many, but not all, cancer cells fail to undergo CIL towards normal cells, the same cancer cells do display CIL during collisions with one another

23 Quantification of CIL. CIL is measured by comparing the contact acceleration indices (Cx) for free moving (F) and contacting (C) cells. Cells were tracked for 50 ′ before collision (A) and 50 ′ after collision (B). Free moving cells were tracked for the same time periods. The component Cx of vector B–A represents the difference between how far the cell has progressed in the direction of A ′ and how far it would have gone had there been no collision. CIL is indicated by a negative Cx value because cells change direction and move backwards following the collision. A more negative Cx value indicates a more significant CIL response. Journal of Microscopy Volume 251, Issue 3, pages 232-241, 15 MAR 2013 DOI: 10.1111/jmi.12024 Volume 251, Issue 3,

24 Molecular mechanisms of CIL Upon contact, cells stop migrating, retract their actin-driven protrusions, repolarize and form a new protrusion to reinitiate migration in a new direction. CIL is mediated by Eph-ephrin signaling Eph receptor–ephrin ligand interactions depend upon direct cell– cell interactions and are unique in that they trigger bidirectional signalling within the receptor and ligand-expressing cell. Activation of EphA receptors leads to repulsive migratory behaviour via the GTPase RhoA whereas EphBs can trigger Cdc42 activation and this can facilitate attractive migration Many cancer cells hyperproduced EphB receptors Contact-unimpeded migration could facilitate tumour invasion through the stroma

25 Hallmark 3. Evading Apoptosis MechanismExample Inactivation of p53More than50% of all cancers Titration of the FAS ligand via hyperexpression of the nonsignaling FAS receptor homolog (oncogene) Lung and colon carcinoma Upregulation of Bcl-2 (oncogene)B cell lymphomas

26 p53 and cancer Fifty percent of cancer harbours p53 mutations, while in the remaining 50%, the wild type p53 is deemed to lose its function via various mechanisms that affect the expression and activity of p53. Xu-Monette et al., 2012

27 p53 and cancer The main inhibition mechanism of p53 is amplification or overexpression of its negative regulator MDM2. MDM2 is an E3 ubiquitin ligase that induces polyubiquitination of p53 protein and subsequently promotes its proteolytic degradation in the 26S proteasome complex Overexpression of MDM2 will lead to a high production of polyubiquitinated p53 ready proteasomal degradation. Bortezomib inhibits the proteasome Nutlin competitively displaces p53 from the binding on MDM2 and effectively causes the stabilization of p53. RITA binds to the amino terminal on p53 domain instead of on MDM2 protein.

28 Reactivation of p53 mutants PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis) is a small molecule drug that reactivates mutant p53 by restoring its wild type conformation and transcriptional functions, by addition thiols in mutant p53 MIRA-1 structurally distinct from PRIMA-1, is a maleimide compound that shifts the equilibrium between the native and unfolded conformation of p53 towards the native conformation

29 Bcl-2 family and intrinsic pathway in cancer Mitochondrial outer membrane permeabilization (MOMP) Cancer cells can modulate apoptotic pathways transcriptionally, translationally, and post-translationally.

30 Hallmark 4. Limitless Replicative Potential MechanismExample Telomere maintenanceAll kind of cancers Mammalian telomeres end has single-stranded (TTAGGG)-rich 3'-overhangs that are tucked back into the preceding double stranded region to form a T- loop. The T-loop is stabilized by telomere- specific proteins TRF1 and TRF2

31 Telomere shortening The hexanucleotide repeat TTAGGG tandomly repeated from less than one hundred to several thousand times and telomere-binding proteins. T Every division, the cell looses 50-200 repeats

32 Telomere shortening T-loop

33 Telomerase Cell 2004 116, 273-279DOI: (10.1016/S0092-8674(04)00038-8) Telomerase is not expressed in most somatic cells Telomerase is expressed in most (>90 %) cancer cells The catalytic TERT subunit includes RNA and possesses reverse transcriptase activity

34 Hallmark 5. Induced angiogenesis Virtually all cells in a tissue have to reside within 100 μ m of a capillary blood vessel In order to progress to a larger size, malignant tumors develop angiogenic ability Angiogenesis is regulated by positive and negative signaling circuits Angiogenesis-regulating growth factors Positive - vascular endothelial growth factor (VEGF) and acidic and basic fibroblast growth factors (FGF1/2). Negative - thrombospondin-1, β -interferon Angiogenesis-regulating receptors - Integrins MechanismExample Increased expression of VEGF and/or FGFs Many tumors Decreased expression of thrombospondin-1, β -interferon

35 Spannuth WA et al. (2008) Angiogenesis as a strategic target for ovarian cancer therapy Nat Clin Pract Oncol doi:10.1038/ncponc1051 Angiogenesis (A) Endothelial sprouting is the dominant process of vessel growth. Luminal endothelial cells migrate through the vessel basement membrane into the underlying extracellular matrix, developing an elongated 'sprouting' morphology. (B) Vasculogenic mimicry is the development of microvascular channels by aggressive tumor cells. (C) Vessel co-option involves the use of the pre-existing vasculature in the host tissue. (D) The process of tumor neovascularization, involving the release of proangiogenic factors (e.g. VEGF) by tumor cells to cause endothelial activation, blood vessel growth, and subsequent tumor expansion.


37 Regulation of neovascularization Vascular endothelial growth factor (VEGF) activates the eNOS, SRC, RAS-ERK and PI3K-AKT signaling cascades through VEGFR2 receptor on endothelial cells, which induce vascular permeability, endothelial migration, proliferation and survival, respectively JAG1-Notch signaling promotes angiogenic sprouting. TGF- β signaling through TGFBR1/ALK5 receptor to the Smad2/3 cascade inhibits endothelial cell activation, maintaining endothelial quiescence, whereas TGF- β signaling through the ACVRL1/ALK1 receptor to the Smad1/5 cascade promotes the migration and proliferation of endothelial cells

38 To inhibit VEGF/VEGFR human/humanized monoclonal antibodies are used: Bevacizumab (Avastin) - anti-VEGF ; Sunitinib (Sutent) and sorafenib (Nexavar) are multi-kinase inhibitors, targeting VEGFR2

39 TGF β paradox Daniel R. Principe et al. JNCI J Natl Cancer Inst 2014;106:djt369

40 Hallmark 6. Tissue Invasion and Metastasis Most types of human cancer are able to release malignant cells that move out, invade adjacent tissues, and spread to remote body parts to found new colonies (metastases). Metastases arise as admixture of cancer cells and normal supporting cells mobilized from the host tissue. Metastases are the cause of 90% of human cancer deaths

41 Epithelial–mesenchymal transition (EMT) Tumor cells achieve the potential to invade neighboring tissue and to disseminate throughout the body by activating an epithelial– mesenchymal transition (EMT) program The tissue remodeling processes occurring during EMT are shared by embryonic development, wound healing, and metastasis formation. Molecular hallmarks of EMT are the loss of cell–cell adhesion, the gain of cell migration capabilities to evade from the primary tumor Among many growth factors, TGF- β is one of the most potent inducers of EMT. Singh and Settleman, 2010

42 The loss of cell polarity, the loss of epithelial markers, such as E-cadherin and ZO-1, the gain of the expression of mesenchymal markers, such as N- cadherin, vimentin, and fibronectin, a dramatic cytoskeletal reorganization accompanied by the change from an epithelial, differentiated morphology to a fibroblast-like, motile, and invasive cell behavior. Jonathan M. Lee et al. J Cell Biol 2006;172:973-981 EMT features

43 Jonathan M. Lee et al. J Cell Biol 2006;172:973-981 Signaling events during EMT Cleavage of E-cadherin activation of Snail1 Snail1 localization to the nucleus Repression of E-cadherin by Snail1, Twist, or other repressors expression of vimentin and other mesenchymal gene products, partly because of β -catenin/Tcf–Lef1 activation. Translocation of β -catenin to the nucleus TGF- β activate Tcf–Lef1 transcription complex The c-Met receptor tyrosine kinase, through the Crk adaptor, also stimulates EMT.

44 Hallmark 7. Genomic instability Genomic instability is defined as the tendency of the genome to acquire mutations and epimutations as well as alterations in gene or chromosome dosage Age-related telomere shortening increases genome instability: with short telomeres dividing cells undergo end-to end telomeric fusions which can lead to chromosomal breakage at mitosis More generally - Age-related alterations in nuclear architecture and chromatin structure input in acquisition of genomic instability Defects in the integrity of mitochondria profoundly impact on the stability of our genome, by generating ROS that damage DNA and oxidize proteins.

45 Genomic instability associated with lamins Mutations or alterations in the processing of lamins affect ability to maintain the integrity of the genome. Lamins associate with telomeres, lamins- deficient cells exhibit accumulation of telomeres towards the nuclear periphery during interphase. Gonzalo, 2014

46 Cancer stem cells are pluri-potent progenitor cells that can self-renew and divide by asymmetric cell division to give rise to differentiated or committed progenitors. CSCs can be isolated or identified by distinct marker expression profiles CSCs arise from existing stem/progenitor cells or from acquisition of oncogenic lesions in terminally differentiated cells resulting in dedifferentiation to a primitive stem cell-like state Cancer stem cells

47 Strategies to combat a cancer

48 miRNAs and cancer MicroRNAs regulate gene expression by forming duplexes with the 3’ UTRs of target mRNAs, facilitating their degradation by the RISC complex of proteins. miRNAs are transcribed as precursors, which are subsequently processed by the DROSHA complex Georgia Sotiropoulou et al. RNA 2009;15:1443-1461

49 miRNAs regulates oncogene activity miRNAs might play oncogenic or tumor suppressor roles Each miRNA recognizes hundreds of different transcript targets. Most miRNAs in animals function to inhibit effective mRNA translation through imperfect base pairing at their 3 ′ -untranslated region miRNAs are up- or down-regulated in various forms of cancer, including prostate, breast, ovarian,colorectal, kidney, and bladder cancer let-7 microRNA negatively regulates Ras expression Smad proteins can directly regulate microRNA processing by binding to the DROSHA complex Sotiropoulou et al., 2009

50 The Ames test The Ames test is a biological assay to assess the mutagenic potential of chemical compounds Technically, it uses bacteria (auxotrotrophs on histidine) to test whether a given chemical can cause mutations in the DNA.

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