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					Lecture 7




            Cancer Genetics and
                Genomics
 Cancer is not a single disease, it is used to describe
  the more virulent forms of neoplasia, a disease
  process characterized by uncontrolled cellular
  proliferation leading to a mass or tumor (neoplasm).
 For a neoplasm to be a cancer, however, it must also
  be malignant, which means its growth is no longer
  controlled and the tumor is capable of progression by
  (1) invading neighboring tissues, (2) spreading
  (metastasizing) to more distant sites, or both.
 Tumors that do not invade or metastasize are not
  cancerous but are referred to as benign tumors.
   There are three main forms of cancer:
    – sarcomas, in which the tumor has arisen in
      mesenchymal tissue, such as bone, muscle, or
      connective tissue, or in nervous system tissue;
    – carcinomas, which originate in epithelial
      tissue, such as the cells lining the intestine,
      bronchi, or mammary ducts; and
    – hematopoietic and lymphoid malignant
      neoplasms, such as leukemia and lymphoma,
      which spread throughout the bone marrow,
      lymphatic system, and peripheral blood.
   Within each of the major groups, tumors are
    classified by site, tissue type, histological
    appearance, and degree of malignancy.
GENETIC BASIS OF CANCER
 Neoplasia is an abnormal accumulation of
  cells that occurs because of an imbalance
  between cellular proliferation and cellular
  attrition.
 Cells proliferate as they pass through the
  cell cycle and undergo mitosis. Attrition,
  due to programmed cell death, removes
  cells from a tissue.
The Genetic Basis of Cancer
Regardless of whether a cancer occurs
 sporadically in an individual, as a result of
 somatic mutation, or repeatedly in many
 individuals in a family as a hereditary trait,
 cancer is a genetic disease.
Genes in which mutations cause cancer fall
 into two distinct categories: oncogenes and
 tumor-suppressor genes (TSGs). TSGs in
 turn are either "gatekeepers" or
 "caretakers".
 An oncogene is a mutant allele of a proto-
  oncogene, a class of normal cellular protein-
  coding genes that promote growth and survival of
  cells.
 Oncogenes facilitate malignant transformation by
  stimulating proliferation or inhibiting apoptosis.
 Oncogenes encode proteins such as:
   – proteins in signaling pathways for cell proliferation
   – transcription factors that control the expression of
     growth-promoting genes
   – inhibitors of programmed cell death machinery
 Gatekeeper TSGs control cell growth.
  Gatekeeper genes block tumor development by
  regulating the transition of cells through
  checkpoints ("gates") in the cell cycle or by
  promoting programmed cell death and, thereby,
  controlling cell division and survival.
 Loss-of-function mutations of gatekeeper genes
  lead to uncontrolled cell accumulation. Gatekeeper
  TSGs encode:
   – regulators of various cell-cycle checkpoints
   – mediators of programmed cell death
Caretaker TSGs protect the integrity of the
 genome. Loss of function of caretaker genes
 permits mutations to accumulate in
 oncogenes and gatekeeper genes, which, in
 concert, go on to initiate and promote
 cancer. Caretaker TSGs encode:
  – proteins responsible for detecting and repairing
    mutations
  – proteins involved in normal chromosome
    disjunction during mitosis
  – components of programmed cell death
    machinery
 Tumor initiation. Different types of genetic
  alterations are responsible for initiating cancer.
  These include mutations such as:
   – activating or gain-of-function mutations, including gene
     amplification, point mutations, and promoter mutations,
     that turn one allele of a proto-oncogene into an
     oncogene
   – ectopic and heterochronic mutations of proto-
     oncogenes
   – chromosome translocations that cause misexpression
     of genes or create chimeric genes encoding proteins
     with novel functional properties
   – loss of function of both alleles, or a dominant negative
     mutation of one allele, of TSGs.
 Tumor progression. Once initiated, a cancer
  progresses by accumulating additional genetic
  damage, through mutations or epigenetic
  silencing, of caretaker genes that encode the
  machinery that repairs damaged DNA and
  maintains cytogenetic normality.
 A further consequence of genetic damage is
  altered expression of genes that promote
  vascularization and the spread of the tumor
  through local invasion and distant metastasis.
   Some tumor-suppressor genes directly regulate proto-oncogene
    function (gatekeepers); others act more indirectly by maintaining
    genome integrity and correcting mutations during DNA replication
    and cell division (caretakers). Activation of an antiapoptotic gene
    allows excessive accumulation of cells, whereas loss of function of
    apoptotic genes has the same effect. Activation of oncogenes or
    antiapoptotic genes is dominant. Mutations in tumor-suppressor genes
    are recessive; when both alleles are mutated or inactivated, cell
    growth is unregulated or genomic integrity is compromised. Loss of
    pro-apoptotic genes may occur through loss of both alleles or through
    a dominant negative mutation in one allele.
 The development of cancer (oncogenesis) results
  from mutations in one or more of the vast array of
  genes that regulate cell growth and programmed cell
  death.
 When cancer occurs as part of a hereditary cancer
  syndrome, the initial cancer-causing mutation is
  inherited through the germline and is therefore
  already present in every cell of the body.
 Most cancers, however, are sporadic because the
  mutations occur in a single somatic cell, which then
  divides and proceeds to develop into the cancer.
 It is not surprising that somatic mutations can cause
  cancer. Large numbers of cell divisions are required
  to produce an adult organism of an estimated 1014
  cells from a single-cell zygote.
 Given a frequency of 10-10 replication errors per base
  of DNA per cell division, and an estimated 1015 cell
  divisions during the lifetime of an adult, replication
  errors alone result in thousands of new DNA
  mutations in the genome in every cell of the organism.
 Genome and chromosome mutations add to the
  mutational burden.
 The genes mutated in cancer are not inherently more
  mutable than other genes. Many mutations doubtlessly
  occur in somatic cells and cause one cell among many
  to lose function or die, but they have no phenotypic
  effects because the loss of one cell is masked by the
  vast majority of healthy cells in an organ or tissue.
 What distinguishes oncogenic mutations is that, by
  their very nature, they allow even one mutant cell to
  develop into a life-threatening disease.
Micro-RNA genes

 The catalogue of genes involved in cancer also
  includes genes that are transcribed into noncoding
  RNAs from which regulatory microRNAs
  (miRNAs) are generated.
 There are at least 250 miRNAs in the human
  genome that carry out RNA-mediated inhibition of
  the expression of their target protein-coding genes,
  either by inducing the degradation of their targets'
  mRNAs or by blocking their translation.
 Approximately 10% of miRNAs have been found to
  be either greatly overexpressed or down-regulated in
  various tumors, and are referred to as oncomirs.
 One example is the 100-fold overexpression of the
  miRNA miR-21 in glioblastoma multiforme, a
  highly malignant form of brain cancer.
 Overexpression of some miRNAs can suppress the
  expression of tumor-suppressor gene targets,
  whereas loss of function of other miRNAs may
  allow overexpression of the oncogenes they
  regulate.
 Since each miRNA may regulate as many as 200
  different gene targets, overexpression or loss of
  function of miRNAs may have widespread
  oncogenic effects because many genes will be
  dysregulated.
Initiation and Progression of Cancer

 Once it is initiated, a cancer progresses by
  accumulating additional genetic damage
  through mutations in caretaker genes
  encoding the cellular machinery that repairs
  damaged DNA and maintains cytogenetic
  normality.
 Damage to these genes produces an ever-
  widening cascade of mutations in an
  increasing assortment of the genes that
  control cellular proliferation and repair
  DNA damage.
 In this way, the original clone of neoplastic
  cells serves as a reservoir of genetically
  unstable cells, referred to as cancer stem
  cells. These give rise to multiple
  sublineages of varying degrees of
  malignancy, each carrying a set of
  mutations that are different from but overlap
  with mutations carried in other sublineages.
 In this sense, cancer is fundamentally a
  "genetic" disease, and mutations are central
  to its etiology and progression.
 A paradigm for the development of cancer, as
  illustrated in Figure 16-2, provides a useful
  conceptual framework for considering the role
  of genetic changes in cancer.
 It is a general model that probably applies to
  many if not most cancers, although it is best
  elucidated in the case of colon cancer.
   Figure 16-2. Stages in the evolution of cancer. Increasing degrees
    of abnormality are associated with sequential loss of tumor-
    suppressor genes from several chromosomes and activation of
    proto-oncogenes, with or without a concomitant defect in DNA
    repair. Multiple lineages carrying somewhat different mutational
    spectra and epigenetic changes are likely, particularly once
    metastatic disease appears.
Cancer in Families
 Many forms of cancer have a higher
  incidence in relatives of patients than in the
  general population.
 Most prominent among these familial forms
  of cancer are the nearly 50 mendelian
  hereditary cancer syndromes in which the
  risk of cancer is very high and the
  approximately 100 additional mendelian
  disorders listed in Online Inheritance in
  Man that predispose to cancer.
 Extensive epidemiological studies have shown,
  however, that some families have an above-average
  risk of cancer even in the absence of an obvious
  mendelian inheritance pattern.
 For example, an increased incidence of cancer, in
  the range of 2- to 3-fold, has been observed in first-
  degree relatives of probands with most forms of
  cancer, which suggests that many cancers are
  complex traits resulting from both genetic and
  environmental factors.
 Thus, a family history of cancer in multiple first-
  degree or second-degree relatives of a patient
  should arouse the physician's suspicion of increased
  cancer risk in the patient.
 Although individuals with a hereditary cancer
  syndrome represent probably less than 5% of all
  patients with cancer, identification of a genetic basis
  for their disease has great importance both for clinical
  management of these families and for understanding
  cancer in general.
 First, the relatives of individuals with strong
  hereditary predispositions, which are most often due
  to mutations in a single gene, can be offered testing
  and counseling to provide appropriate reassurance or
  more intensive monitoring and therapy, depending on
  the results of testing.
 Second, as is the case with many common diseases,
  understanding the hereditary forms of the disease
  provides crucial insights into disease mechanisms that
  go far beyond the rare hereditary forms themselves.
ONCOGENES
 An oncogene is a mutant gene whose altered
  function or expression results in abnormal
  stimulation of cell division and proliferation.
 The mutation can be an activating gain-of-function
  mutation in the coding sequence of the oncogene
  itself, a mutation in its regulatory elements, or an
  increase in its genomic copy number, leading to
  unregulated heterochronic or ectopic function of
  the oncogene product.
 Oncogenes have a dominant effect at the cellular
  level; that is, when it is activated or
  overexpressed, a single mutant allele is sufficient
  to initiate the change in phenotype of a cell from
  normal to malignant.
   Activated oncogenes encode proteins that act in
    many steps in the pathway that controls cell
    growth, including growth factors that stimulate
    cell division, the receptors and cytoplasmic
    proteins that transduce these signals, the
    transcription factors that respond to the
    transduced signals, and the proteins that
    counteract programmed cell death (apoptosis).
   Fig 16-3. Mechanisms of
    tumorigenesis by
    oncogenes of various
    classes. Unregulated
    growth factor signaling
    may be due to mutations
    in genes encoding
    growth factors
    themselves (1), their
    receptors (2), or
    intracellular signaling
    pathways (3).
    Downstream targets of
    growth factors include
    transcription factors (4),
    whose expression may
    become unregulated.
    Both telomerase (5) and
    antiapoptotic proteins
    that act at the
    mitochondria (6) may
    interfere with cell death
    and lead to
    tumorigenesis.
Table 16-1. Mechanisms of Activation of Proto-oncogenes

Mechanism                         Type of Gene Activated        Result

Regulatory mutation               Growth factor genes           Increased expression


Structural mutation               Growth factor receptors,      Allows autonomy of
                                  signal-transducing proteins   expression


Translocation, retroviral         Transcription factors         Overexpression
insertion, gene amplification

Regulatory mutation,                Oncomirs                    Overexpression, down-
translocation, retroviral insertion                             regulates tumor-suppressor
                                                                genes
Deletion, inactivating mutation   Oncomirs                      Loss of expression, up-
                                                                regulates oncogenes
    TUMOR-SUPPRESSOR GENES

 Whereas the proteins encoded by oncogenes
  promote cancer, mutations in tumor-suppressor
  genes (TSGs) contribute to malignancy by a
  different mechanism, that is, through loss of
  function of both alleles of the gene.
 TSGs are highly heterogeneous. Some truly
  suppress tumors by regulating the cell cycle or
  causing growth inhibition by cell-cell contact;
  TSGs of this type are gatekeepers because they
  regulate cell growth directly.
   Other TSGs, the caretakers, are involved in
    repairing DNA damage and maintaining
    genomic integrity. Loss of both alleles of
    genes that are involved in repairing DNA
    damage or chromosome breakage leads to
    cancer indirectly by allowing additional
    secondary mutations to accumulate either in
    proto-oncogenes or in other TSGs.
Table 16-3. Selected Tumor-Suppressor Genes
Gene    Gene product and possible             DISORDERS IN WHICH THE GENE IS
        function sporadic                     AFFECTED

Gatekeepers                                    Familial             Sporadic
RB1     p110                                  Retinoblastoma    Retinoblastoma, small
        Cell cycle regulation                                   cell lung carcinomas,
                                                                breast cancer
TP53    p53                                   Li-Fraumeni       Lung cancer, breast
        Cell cycle regulation                 syndrome          cancer, many others

DCC     Dcc-receptor                          None known        Colorectal cancer
        Decreases cell survival in the
        absence of survival signal from its
        netrin ligands
VHL     Vhl                                   von Hippel-       Clear cell renal
        Forms part of a cytoplasmic           Lindau syndrome   carcinoma
        destruction complex with APC that
        normally inhibits induction of
        blood vessel growth when oxygen
        is present
Gene         Gene product and       DISORDERS IN WHICH THE GENE IS
             possible function      AFFECTED
             sporadic

Caretakers                           Familial                 Sporadic


 BRCA1,      Brca1, Brca2            Familial breast   Breast cancer, ovarian
BRCA2        Chromosome repair in    and ovarian       cancer
             response to double-     cancer
             stranded DNA breaks


 MLH1,       Mlh1, Msh2              Hereditary        Colorectal cancer
MSH2         Repair nucleotide       nonpolyposis
             mismatches between      colon cancer
             strands of DNA
 The Two-Hit Origin of Cancer
 The existence of TSG mutations leading to cancer
  was originally proposed in the 1960s to explain
  why certain tumors can occur in both hereditary
  and sporadic forms.
 For example, it was suggested that the hereditary
  form of the childhood cancer retinoblastoma
  might be initiated when a cell in a person
  heterozygous for a germline mutation in a tumor-
  suppressor retinoblastoma gene, required to
  prevent the development of the cancer, undergoes
  a second, somatic event that inactivates the other
  allele.
 As a consequence of this second somatic event,
  the cell loses function of both alleles, giving rise
  to a tumor. The second hit is most often a somatic
  mutation, although loss of function without
  mutation, such as occurs with transcriptional
  silencing, has also been observed in some cancer
  cells.
 In the sporadic form of retinoblastoma, both
  alleles are also inactivated, but in this case, the
  inactivation results from two somatic events
  occurring in the same cell.
 The "two-hit" model is now widely accepted as
  the explanation for many familial cancers besides
  retinoblastoma, including familial polyposis coli,
  familial breast cancer, neurofibromatosis type 1
  (NF1), hereditary nonpolyposis colon carcinoma,
  and a rare form of familial cancer known as Li-
  Fraumeni syndrome.
 In all of these syndromes, the second hit is often
  but not always a mutation.
 Silencing due to epigenetic changes such as DNA
  methylation, associated with a closed chromatin
  configuration and loss of accessibility of the DNA
  to transcription factors, is another important,
  alternative molecular mechanism for loss of
  function of a TSG.
 Because an alteration in gene function due to
  methylation is stably transmitted through mitosis,
  it behaves like a mutation; because there is no
  change in the DNA itself, however, the alteration
  is referred to as an epigenetic rather than a genetic
  change.
 Epigenetic silencing of gene expression is a
  normal phenomenon that explains such widely
  diverse phenomena as X inactivation, genomic
  imprinting, and regulation of a specialized
  repertoire of gene expression in the development
  and maintenance of differentiation of specific
  tissues.
Gatekeeper Tumor-Suppressor Genes in
Autosomal Dominant Cancer Syndromes

Retinoblastoma
 Retinoblastoma, the prototype of diseases caused
  by mutation in a TSG, is a rare malignant tumor of
  the retina in infants, with an incidence of about 1
  in 20,000 births.
 Diagnosis of a retinoblastoma must usually be
  followed by removal of the affected eye, although
  smaller tumors, diagnosed at an early stage, can be
  treated by local therapy so that vision can be
  preserved.
 About 40% of cases of retinoblastoma are of the
  heritable form, in which the child inherits one mutant
  allele at the retinoblastoma locus (RB1) through the
  germline.
 A somatic mutation or other alteration in a single
  retinal cell leads to loss of function of the remaining
  normal allele, thus initiating development of a tumor.
 The disorder is inherited as a dominant trait because
  the large number of primordial retinoblasts and their
  rapid rate of proliferation make it very likely that a
  somatic mutation will occur in one or more of the
  more than 106 retinoblasts.
 Since the chance of the second hit in the heritable
  form is so great, the hit occurs frequently in more
  than one cell, and thus heterozygotes for the
  disorder are often affected with multiple tumors,
  often affecting both eyes.
 On the other hand, the occurrence of the second
  hit is a matter of chance and does not occur 100%
  of the time; the penetrance of retinoblastoma,
  therefore, although high, is not complete.
 The other 60% of cases of retinoblastoma are
  nonheritable (sporadic); in these cases, both RB1
  alleles in a single retinal cell have been inactivated
  independently.
 Because two hits in the same cell is a rare event,
  there is usually only a single clonal tumor and the
  retinoblastoma is found in one eye only.
 Although sporadic retinoblastoma usually occurs
  in one place in one eye only, 15% of patients with
  unilateral retinoblastoma have the heritable type
  but by chance develop a tumor in only one eye.
 Another difference between hereditary and
  sporadic tumors is that the average age at onset of
  the sporadic form is in early childhood, later than
  in infants with the heritable form.
   Figure 16-5
    Retinoblastoma
    in a young girl,
    showing as a
    white reflex in
    the affected eye
    when light
    reflects directly
    off the tumor
    surface.
        Loss of Heterozygosity
 Geneticists studying DNA polymorphisms in the
  region close to the RB1 locus made an unusual but
  highly significant genetic discovery when they
  analyzed the alleles seen in tumor tissue from
  retinoblastoma patients.
 Individuals with retinoblastoma who were
  heterozygous at polymorphic loci near RB1 in
  normal tissues, such as in their white blood cells,
  had tumors that contained alleles from only one of
  their two chromosome 13 homologues, revealing a
  loss of heterozygosity (LOH) in the region of the
  gene.
 In familial cases, the retained chromosome
  13 markers were the ones inherited from the
  affected parent, that is, the one with the
  abnormal RB1 allele. Thus, LOH
  represented the second hit of the remaining
  allele.
 LOH may occur by interstitial deletion, but
  there are other mechanisms, such as mitotic
  recombination or nondisjunction.
 LOH is the most common mutational
  mechanism by which the function of the
  remaining normal RB1 allele is disrupted in
  heterozygotes.
 When LOH is not seen, the second hit is
  usually a second somatic gene mutation or,
  occasionally, transcriptional inactivation of
  a nonmutated allele through methylation.
 LOH is a feature of a number of other
  tumors, both heritable and sporadic, and is
  often considered evidence for the existence
  of a TSG, even when that gene is unknown.
   Figure 16-6 Comparison of mendelian and sporadic
    forms of cancers such as retinoblastoma and familial
    polyposis of the colon.
   Figure 16-7 Chromosomal mechanisms that could lead to identical
    DNA markers at or near a tumor-suppressor gene in an individual
    heterozygous for an inherited germline mutation. The figure depicts
    the events that constitute the "second hit" that leads to retinoblastoma.
    Local events such as mutation, gene conversion, or transcriptional
    silencing, however, could cause loss of function of both RB1 genes
    without producing LOH. + is the normal allele, rb the mutant allele.
 The RB1 gene maps to chromosome 13, in
  band 13q14. In a small percentage of
  patients with retinoblastoma, the first
  mutation is a cytogenetically detectable
  deletion or translocation of this portion of
  chromosome 13.
 Such chromosomal changes, if they also
  disrupt genes adjacent to RB1, may lead to
  dysmorphic features in addition to
  retinoblastoma.
         Li-Fraumeni syndrome
 There are rare "cancer families" in which there is a
  striking history of many different forms of cancer
  (including several kinds of bone and soft tissue
  sarcoma, breast cancer, brain tumors, leukemia,
  and adrenocortical carcinoma), affecting a number
  of family members at an unusually early age,
  inherited in an autosomal dominant pattern.
 This highly variable phenotype is known as the
  Li-Fraumeni syndrome (LFS). Because the TSG
  TP53, encoding the protein p53, is inactivated in
  the sporadic forms of many of the cancers found
  in LFS, TP53 was considered a candidate for the
  gene defective in LFS.
 DNA analysis of several families with LFS has
  now confirmed this hypothesis; affected members
  in more than 70% of families with LFS carry a
  mutant form of the TP53 gene as a germline
  mutation.
 As seen also in retinoblastoma, one of the two
  mutations necessary to inactivate the TP53 gene is
  present in the germline in familial LFS, whereas in
  many sporadic cancers, both mutations are
  somatic events.
   Figure 16-8 A pedigree of the Li-Fraumeni
    syndrome, in which breast cancer, sarcomas,
    and other malignant tumors have occurred.
    Ages at diagnosis are shown.
 The p53 protein is a DNA-binding protein that
  appears to be an important component of the
  cellular response to DNA damage.
 In addition to being a transcription factor that
  activates the transcription of genes that stop cell
  division and allow repair of DNA damage, p53
  also appears to be involved in inducing apoptosis
  in cells that have experienced irreparable DNA
  damage.
 Loss of p53 function, therefore, allows cells with
  damaged DNA to survive and divide, thereby
  propagating potentially oncogenic mutations. The
  TP53 gene can therefore be considered to also be a
  gatekeeper TSG.