Download Molecular basis of cancer (adapted from Robbins and Cotran, 2009

Molecular basis of cancer (adapted from Robbins and Cotran, 2009)
Objectives:
1. Know the principles and examples of molecular events that are involved in malignant
transformation
2. Understand the role of stroma in the development and progression of neoplasia
3. Understand the cellular metabolic changes that accompany malignant transformation
4. Know the steps involved in metastasis and the general cellular genotype/phenotype that enables
metastasis
A. Principle concepts of the molecular events in cancer:
1. Non lethal genetic damage
a. Acquired from environmental agents (exogenous or endogenous), viruses or can be inherited in
the germ line
2. Tumor that is formed by clonal expansion had been genetically damaged
a. Implications are tumors start off (but may not end up as) monoclonal—ie possess the same
allele(s)
3. Normal regulatory genes are the principle targets of genetic damage
a. Proto-oncogenes: growth promoting genes, mutations considered dominant
b. Tumor suppressor genes: mutations considered recessive b/c both allelles need to be lost for
transformation generally speaking but there are exceptions….
c. Genes involved in apoptosis: can behave as tumor suppressor or proto-oncogenes
d. Genes involved in DNA repair: indirectly involved in transformation. Defects in these genes
allow for DNA replication in the face of non-lethal damage to genome and thus pre dispose
cell to perpetuating mutations and transformation. These cells are deemed to have a
“mutator” phenotype
e. miRNAs: regulatory molecules, can act as oncogenes or tumor suppressor genes via regulation
of translation of genes
4. Carcinogenesis is a multistep process both phenotypically and genetically with the acquisition of
multiple mutations
a. Over time, many tumors can have malignant “progression”
b. Accumulation of independently acquired mutations that general subclones of cells that have
varying abilities to divide and invade.
c. By the time tumors are clinically evident, they are very heterogenous genetically and
phenotypically
B. Essential alterations for malignant transformation: 8 changes in cellular physiology that determine a
malignant phenotype
1. Self sufficiency in growth signals  consequence of oncogene activation
a. Cell proliferation review in brief: growth factor binding  limited activation of GF receptor 
signal transduction  nuclear signals that initiate DNA transcription  entry into the cell
cycle
b. Oncogenes: genes that promote autonomous cell growth in cancer cells  oncoproteins
c. Proto-ongenes: unmutated cellular counter parts. When mutated, proto-oncogenes can
become oncogenes
d. Oncoproteins lack internal regulatory elements, production does not depend on external
signals.
e. Classes of oncogenes include
i. Growth factors: PDGF, fibroblast growth factor, HGF
ii. Growth factor receptors: EGF-r, PGDF-r, stem cell factor receptor (proto-oncogene =
KIT)
iii. Signal transduction proteins: GTP binding (protooncogenes: KRAS, HRAS), WNT signal
transduction (b-catenin), nonreceptor tyrosine kinase (ABL). In human cancers, point
mutations are RAS commonly found.
1. C-ABL non receptor tyrosine kinase gene translocation fuses with the BCR
gene  constitutively active TK activity; key mutation in some leukemias.
Imatinab mesylate (Gleevec) blocks the kinase activity with low toxicity and
high therapeutic effects.
iv. Transcriptional activators: C-MYC
v. Cell cycle regulators: Cyclin D, E, CDK4 ( formation of G1 restriction point with cyclin
D)
vi. In addition to individual gene mutations, cancers are also associated with
chromosomal changes: chromosomes can be lost or gained, portions deleted, or
portions can be translocated. Translocations can lead to activation of genes through
the rearrangement of regulatory elements or through the production of fusion genes
that encode chimeric proteins that promote tumorgenesis.
vii. Other genetic changes in tumors include: gene amplification
viii. Epigenetic changes also occur: chromatin pattern contributions to tumorgenesis is
not well understood.
ix. Role of miRNAs: small non coding aa RNAs that are a part of the RNA induced
silencing complex (RISC). Mediate post transcriptional gene silencing: especially genes
that involve cell growth and proliferation. Many cancers have deletions in miRNA
sequences
f. Mechanisms of activation of above genes include: overexpression, point mutations and
translocations.
2. Loss of response to growth inhibitory signals: the brakes on cell proliferation.
a. Non-nuclear: TGF-b receptor (growth inhibitor), NF-1 (cytoskeletal stability), PTEN (signal
transduction), SMAD2 and 4 (signal transduction)
b. Nuclear: RB1 (cell cycle), p53 (cell cycle and apoptosis), WT1 (transcription), BRCA1 and 2
(DNA repair)
c. Prototypic tumor suppressor gene is RB: point is that a heterozygote for a deactivation
mutation in RB is at an increased risk for developing retinoblastoma. Normal function of RB is
a gatekeeper that regulates the G1 to S transition.
d. P53 reduces risk of transformation by these related mechanisms: activation of cell cycle
pause, induction of permanent cell cycle arrest, or triggering apoptosis. P53 has a short ½ life
b/c it is complexed with a protein that targets it for destruction. Under conditions of cellular
stress s and DNA damage mediated through other proteins, p53 is released from this protein
that increases its ½ life and activates it as a transcriptional factor.
i. Activates transcription of proteins involved in cell cycle arrest, gene repair and
apoptosis
ii. Activates transcription of miRNA, mir34 family  block translation of proproliferative genes such as cyclins and anti-apoptosis gene such as BCL-2
iii. Not clear what how the cell decides when to stop and repair versus launch into
apoptosis: in general it is thought that the DNA repair pathway activated first then if
signals that allow p53 levels to rise continue, transcription of pro-apoptotic genes
occur.
iv. Cancers with normal p53 respond better to treatments that induce apoptosis than
those with p53 mutations
v. P53 is related to 2 other proteins p63 and p73: there is significant cross talk between
the proteins—p63 and p73 have more tissue specific expression and can act in
concert or antagonize each other’s activities.
3. Evasion of apoptosis  consequence of p53 inactivation or activation of anti-apoptosis genes
a. Review of apoptotic pathways: extrinsic and intrinsic
b. BCL-2 mutations in some types of human lymphomas  accumulation of BCL-2 allowing cells
to escape apoptosis. Not cause massive growth of B cells but accumulation  slow indolent
course of disease
c. P53’s role in apoptosis and cell repair: discussed in the section above
4. Limitless replicative potential  avoid cellular senescence through elongation of telomeres
a. Senescence from telomere shortening: see by cell as double DNA breaks and triggers p53 and
RB to stop cell cycle. Cell can attempt to repair ends but chromosomes break apart during
mitosis and triggers cell death.
b. Virtually all cancer cells have upregulation of telomerase that maintains the length of the
telomeres
5. Angiogenesis  induction of blood supply for nutrients and removal of metabolic wastes
a. Solid tumors cannot be larger than 1-2mm without own vascular supply.
b. Stimulation of vascular growth: from pre-existing capillaries or recruitment of bone marrow
progenitors
c. Provides nutrients and source growth factors: IGF-1, PDGF etc as well as a route for
metastasis
d. Factors that spur blood vessel growth or loss of inhibition of blood vessel growth can be
produced by the neoplastic cells, inflammatory cells, or other stromal cells
i. Proteases can release pro angiogenic factors stored in the stroma along with releasing
angiogenesis inhibitors
ii. Hypoxia can induce angiogenesis through induction of cytokines such as VEGF
iii. Anti VEGF antibody: bevacizumab: anti angiogenesis drug used in cancer treatment
6. Invasion and metastatic potential:
a. Theories of how metastatic populations arise in a tumor
i. Clonal evolution: development of heterogenous population and subclones with
metastatic potential
ii. Primary tumor has already has a heterogenous population some of which are capable
of metastasis
iii. Influence of genetic background on the development of metastasis: mouse models of
cancer differ with the same cancer type in different strains of mice
iv. Metastasis occurring through cancer stem cells
v. No single or cluster of “metastastic” genes discovered to date although there are a
number of cell signaling molecules that are lost in cancer.
b. Metastatic cascade: invasion of ECM, vascular dissemination, homing of tumor cells and
colonization
i. Invasion: changes in cell-cell interactions from loss of intercellular adhesion molecules
(E cadherins) , degradation of ECM via direct or indirect elaboration of proteases (eg
capthesins, metallomatrix proteinases), attachment to ECM and migration of cells
(actin cytoskeletal rearrangements that allow for movement). Stromal interactions
key to allowing or preventing invasion!!
1. Neoplastic cells are only one of the cells that compose the tumor: other
players include ECM, immune cells, blood vessels. All these elements can
contain or elaborate factors that can promote the growth and metastasis of
the neoplastic cells. Stroma represents a current target of therapeutic
intervention.
ii. Vascular dissemination and homing: neoplastic cell circulate in clumps, can activate
coagulation cascade. Cells must adhere to site of extravasation (eg using molecules
analogous to CD44 on hyaluronate substrates) and escape the basement membrane.
Homing does not always correlate with the vascular draining of the primary site. Mets
of differing cancers may have tropisms for particular organs: possibly as a result of
differentially expressed vascular ligands, chemokine signaling, and non-permissive
environments (avascular tissues such as cartilage)
7. Defects in DNA repair  leads to genomic instability and further mutation accumulation Individuals
born with DNA repair pathway (mismatch repair, nucleotide excision repair and recombination repair)
mutations are at increased risk of developing cancer.
8. Alterations in metabolism to support rapid growth and suboptimal environmental conditions.
a. Warburg effect: tumor cells uptake more glucose and put it through aerobic glycolysis
lactate and pyruvate production with lower use of oxidative phosphorylation (TCA cycle).
Tumor cells rely on glycolysis for ATP generation even in the face of adequate oxygenation:
glycolysis is energically less efficient than oxidative phos. Why?
b. Glucose  carbon, oxygen and hydrogen for anabolic processes and energy. Glutamine 
nitrogens for purines, pyrimidines and non-essential amino acids as well as NADPH for fatty
acid synthesis and control of redox potential. Glucose can also generate NADPH.
c.
Normal cells usually metabolize glucose to water and CO2 through glycolysis and the TCA
cycle. When there is high need for cell division (eg development, regeneration) cell needs
more substrates to synthesize membranes, and nucleic acids  using glucose as source for
building blocks of cell growth through and not energy therefore slow rate of pyruvate into the
TCA cycle  secreted as lactate. Lactate lowers pH of environment  influences stroma,
enables motility of cells?
d. In majority of cancers, the tumor mitochrondria is not defective: the shunting of glucose away
from the mitochondria is not a result of mt dysfunction.
e. Oncogenes and tumor suppressor gene products mediate this metabolic switch: can stimulate
the transcription of genes that mediate the glycolysis and glutaminolysis pathway as well as
increase # of glucose transporters.
i. Hypoxia inducible factor: regulate many genes that function in glycolysis, triggered by
hypoxia. Loss of PTEN (tumor suppressor gene) can also lead to HIF transcription.
ii. P53 also is involved in the cellular adaptation to stress through the transcription of a
number of genes: normal p53 suppresses the glucose transporter transcription and
decreases glycolysis in a number of ways  decrease in cell growth.
C. Causes of cancer:
a. Chemical carcinogenesis:
i. Initiators: cause permanent DNA damage in a group of cells. For the change to be
heritable, the DNA template needs to be replicated.
ii. Promoters: induce reversible cellular changes, do not affect cellular DNA directly. Usually
enhances proliferation of damaged cells.
iii. Examples of such agents include:
1. Direct acting: no metabolic conversion needed to be carcinogenic
2. Indirect acting: Most known carcinogens are metabolized by the p450
enzyme system—which contain many genetic polymorphisms-- so the
susceptibility of an individual to carcinogenesis depend in part on the
individual’s genetic composition.
3. Most chemical carcinogens are mutagenic: eg aflatoxin B1 produces
characteristic mutations in p53 gene  associated with high rates of liver
cancer in some countries where there is aflatoxin contamination of food.
b. Radiation carcinogens: UV rays and ionizing radiation
c. Microbial carcinogens: Oncogenic viruses (covered by Dr. Westmoreland)
d. Communicable tumors: Transmissible venereal tumor in the dog and Devil Facial Tumor in
Tasmanian Devils. Both are clonal tumors (TVT thought to be of histiocytic origin and DFT of
neuroendocrine origin) that are communicated from animal to animal via coitus, biting or other
physical contact. TVTs can undergo spontaneous regression through the immune mediated
mechanisms where as the Devil Facial tumor rarely resolves.
e. Other: Feline injection site sarcomas and the issue of vaccine adjuvants
D. Tumoral immunity:
a. Tumors are not entirely “self”: can elicit an immune reaction possible to completely eliminate
tumors via the immune system?
b. Case of TVTs and Devil Facial Tumors:
c. Tumor antigens
i. Specific ones: only on tumor cells
ii. Related: on tumor and normal cells
iii. Mutated gene products: don’t seem to major targets in of cytotoxic T cells experimentally
iv. Aberrant or overexpressed proteins—eg tyrosinase in melanomas, targeted in vaccine
trials in melanoma
v. Antigens expressed by oncogenic viruses
vi. Oncofetal antigens—normally expressed by fetal tissues and aberrant expression by
cancer cells and in some inflammatory conditions. Aid in tumor diagnosis—tumor marker
vii. Altered cell surface glycoproteins/glycolipids—tumors expressed higher than normal
levels, may be used as diagnostic markers or therapy targets
viii. Cell specific differentiation antigens—used for diagnosis, possible immunotherapeutic
target.
d. Anti tumor mechanisms: effector cells include cytotoxic T cells (CTLs), NK cells, macrophages as
well as antibodies
e. Immune surveillance and escape :
i. Increased frequency of cancer in immunosupression
ii. Cancer cells develop able to evade immune response
1. Antigen negative variants
2. Loss or reduction of MHC molecules: escape detection by CTLs
3. Lack of co stimulation: prevents sensitization of T cells and may result in T cell
anergy or apoptosis
4. Immunsupression: production of products such as TGF-B can be an
immunosuppressant
5. Antigen masking: Antigens may be hidden by polysaccharide molecules.