Cancer in a Nutshell Anish Patel

Anish Presents Cancer in a Nutshell

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Everything you ever need to know about cancermaybe

The foundation for all development in understanding of cancer over the past few
decades is the discovery of two sets of genes that influence cancer development.

Oncogenes: An abnormal version of a proto-oncogene, that accelerates and
promotes the development of cancer. They show dominant gain of function
that is, they only require one allele to be mutated to have their effect.
Example: Ras
RET (in Type 2 MEN)

Tumour suppressor genes: A gene that inhibits the development of cancer, by
regulating and controlling the normal progression through the cell cycle and
encouraging the death of abnormal cells. They display recessive loss of
function. That is, they require the loss of both alleles to lose their effect
however, new examples have emerged where this is not the case (e.g. PTEN
these are said to show haploinsufficiency)
Example: Retinoblastoma protein (pRb)
P53

There are 6 characteristics that a normal cell needs to acquire to become
cancerous. These have become known as the hallmarks of cancer.

1) Autonomy of growth signals
2) Insensitivity to anti-growth signals
3) Resistance to apoptosis
4) Sustained angiogenesis
5)Limitless replicative potential
6) Tissue invasion and metastasis

1) Normal cells require exogenous mitogenic growth signals before they can
move from a quiescent state (G0) to a proliferative one. However, tumour cells
create their own signals. To do this they could:
Alter the receptors for growth signals (e.g. increased
expression of HER-2 receptor in gastric cancer -- makes the
cells more sensitive)
Alter the signal transduction pathway (e.g. mutant Ras in
about 25% of cancers)
Make their own growth factors (e.g. TGF- by sarcoma)

n.b. many cancerous cells will induce their neighbouring normal cells to
produce growth factors/mitogens!
Cancer in a Nutshell Anish Patel

2) Within a normal tissue, many antiproliferative signals act to keep cells in the
quiescent (G0) state and also to enter them into the post-mitotic state, where
they lose the ability to divide.

The most important STOP signal is retinoblastoma protein
(pRb).

In the quiescent state, pRb binds a transcription factor E2F.
This means that E2F cannot bind to DNA and so cannot
promote the transcription of proteins necessary for mitosis.



However, pRb can be inactivated by
phosphorylation. This phosphorylation is carried
out by cyclin-CDK complexes. E2F is then free to
act as a transcription factor on genes.





However, in the normal cell, there are ways of
disabling cyclin-CDKs for example, the TGF-
can activate factors such as p21 and p15 that
disable cyclin-CDK and so pRb remains
unphosphorylated and so ACTIVE.


For a cell to acquire resistance to anti-proliferative signals, it can use several
strategies:
o Some lose responsiveness to TGF-
o Some produce dysfunctional TGF- receptors
o Some produce mutant, ineffective pRb


Note: the Human Papilloma Virus (HPV) induces cervical cancer by
producing E7 protein, that disables pRb.







Cancer in a Nutshell Anish Patel

















3) In normal cell populations, programmed cell death, apoptosis, represents a
major wall of controlling cell number. In apoptosis, cell membranes are disrupted,
the cytoskeletons are broken down, the cytosol extruded and the chromosome
and nucleus are destroyed. The remains are phagocytosed. The apoptotic
machinery consists of sensors that detect damaged cells and the effectors, that
mediate the apoptosis. Death signals are conveyed by death molecules, such as
FASL and TNF-..The signal is then transferred to the mitochondria, which
release cytochrome C, which mediates the apoptosis.

The key tumour suppressor gene that upregulates
pro-apoptotic signals is p53.

Therefore, most cancer cells have a mutated form of p53 and so
they evade apoptosis. Other methods of evading death include
alteration in the cell-death signalling pathway and upregulation
of anti-apoptotic signals (e.g. bcl-2 upregulation in follicular cell
lymphoma).

4) Normal cells can only multiply a certain number of times that is, they have
limited replicative potential. When they exceed this potential, they become
senescent. However, if you force cells to multiple when senescent, they enter a
state called crisis. These cells multiply very badly, with a huge number of
genetic errors. Problem: by chance, one of these errors can lead to a mutation
that means the cell has infinite replicative ability it is said to be immortalised.
Oh gay.
This occurs, because the cancer cells gain a mutation that allows them to
produce telomerase enzyme in huge amounts. This allows them to keep
rebuilding the telomere of chromosome (usually this would get degraded with
every division and so eventually, the cell cant replicate anymore)
Retinoblastoma:

Retinoblastoma is a rare childhood cancer of the retina, caused by the mutation of BOTH
pRb alleles one mutation is usually inherited, the other is by chance (except some
unlucky fuckers who have two random mutations sporadic RB). This demonstrates the
recessive loss of function of tumour suppressor genes. A child with one allele lost is said to
have loss of heterogeneity. Note the leukocoria in the childs right eye:


Cancer in a Nutshell Anish Patel

5) Every cell need a blood supply to stay alive. Tumour cells are no different. If
they are to grow above a tiny tumour, they need to
create blood vessels the process of angiogenesis.
There are both pro-angiogenic signals (e.g. VEGF and
FGF) and anti-angiogenic signals (angiostatin and
thrombospondin). Mutations in a tumour tip the
balance, so that pro-angiogenic factors outweigh anti-
angiogenic factors.
There also needs to be cell-cell interactions, via
integrins, for successful angiogenesis. Quiescent
capillaries express a different integrin to sprouting capillaries this is therefore a
potential treatment for cancer!
Metalloproteinase enzyme are also essential. They must be present to break
down the endothelial cell membranes and ECM, so that new capillaries can join
up with existing systemic circulation again, MP inhibitors are a potential therapy
for cancer.

What are the stages of angiogenesis?

Tumour derived growth factors stimulate local endothelial cell
proliferation these sprouts grow towards the tumours
Proteolytic breakdown and remodelling of the ECM by serine
proteases and metaloproteinases.
Maturation and differentiation of endothelial cells and formation of
the basement membrane.

There are some important differences between normal capillaries and tumour
vessels. Because they are proliferating and growing so fast, tumour vessels are
disorganised, contorted, leaky, contain many shunts and dead ends. This leads
to inefficient, sluggish blood flow within a tumour this means most cells have
hypoxic and necrotic areas in their centre.

Why are blood vessels a good target for anti-cancer therapy?

Destruction of one vessel destroys many dependent tumour cells
Easy access (shove it in the systemic circulation!)
No development of drug resistance
Applicable to all solid tumours

How can we target tumour blood vessels?

Block cell-ECM adhesion molecules e.g. integrins
Inhibit pro-angiogenic factors e.g. antibodies directed against VEGF
Matrix metalloproteinase inhibitors also prevent metastasis!
Administer endogenous inhibitors e.g. angiostatin
Cancer in a Nutshell Anish Patel
Antivascular drugs to destroy established tumour vessels

6) The final acquired ability of cancer cells is the ability to invade and
metastasise. To metastasise, a tumour must transform itself from a carcinoma
in situ to an invasive carcinoma. The 5 stages of metastasis are as follows:

1 Hyperproliferation and breakdown of cell-cell interactions (normally,
cells are held together by molecules such as cadherins). Once the
interactions are broken, the tumour cells break through the basement
membrane using matrix metalloproteinases. The mutation that leads to
this is the switch from an adhesive molecule to a non-adhesive one (e.g.
N-CAM switch in Wilms tumour and small cell carcinoma) There is also
increased expression of MMP not only by the cancer cells, but by their
neighbouring cells also!

2 Movement through the stroma and invasion of the capillary/lymph
vessel intravastation.

3 Movement to a distant site and then adherence to the endothelium,
again by mutated integrins e.g. V3 expression.

4 Extravastation the tumour cells leave the blood vessel and enter
their new tissue

5 tumour cells need to seed in their new tissue and then continue to
proliferate




Where do cancer cells metastasise to?

Depending on the primary tumour types, cancer cells metastasize to different
tissues. E.g. Carcinomas spread to the lymph nodes, whereas sarcomas spread
to the lung. The breast, liver, colon and kidney are all common sites of
metastases usually the first organ that the cells encounter after leaving the
primary source is where the metastases spread!


OK, so that was the 6 hallmarks of cancer but there are other enabling
factors that influence cancer formation. The most importance of these is genetic
instability. You may have noticed that there need to be at least 6 mutations in
the same cell for a cancer to be successful. This is remarkably difficult to do,
because the body has excellent mechanism to prevent mutations!

Cancer in a Nutshell Anish Patel
Mistakes in DNA are usually picked up by caretaker proteins and then corrected
(or if the damage is too big, the cell is apoptosed) Again, the most important of
these is p53 which when DNA damage is detected, halts the progression of the
cycle to give time for repair or induces apoptosis.

How can DNA be repaired?

Mismatch repair a type of repair for when an erroneous base is
inserted/deleted or paired with the wrong partner e.g. G-T pairing. When there is
an error in mismatch repair, hereditary nonpolyposis colorectal cancer results.

Nucleotide excision repair under the influence of UV light, pyrimidines form
dimers. These need to be excised this is usually performed by the nucleotide
excision machinery.












It can therefore be seen that wither a mutation in the caretaker genes (such as
p53) or in the repair genes can lead to genetic instability that is, it is easier for
the cell to acquire the required mutations.

It should also be noted that there are many pathways to the same result the
pathways that cells take o their way to become malignant are highly variable.
Within a given cancer type, mutations of particular target genes such as ras or
p53 may be found in only a subset of histologically identical tumours. Remember
there are often different ways of achieving the same hallmark so a
histologically similar tumour could have an entirely different mutation.
Furthermore, some mutations can occur early in some tumours, but late in others
it is, after all, a random process.


Staging of cancers TNM staging:
T = Primary tumour T1-2 localized
T 3-4 locally advanced
N = lymph nodes N0 no invasion
N1 invasion
M = metastases M0 absent
Clinical highlight:
Xeroderma Pigmentosum
If there is a mutation in the genes that code for
the nucleotide excision enzymes, the syndrome
of xeroderma pigmentosum results, The person
is unable to undo damage to the DNA in
response to sunlight and the result is multiple
melanomas and basiliomas.
Cancer in a Nutshell Anish Patel
M1 present


Leukaemias

All blood cells come from a common pluripotent stem cell. This then differentiates
into a common myeloid progenitor and a common lymphoid progenitor. The CMP
leads to RBCs, platelets, neutrophils and monocytes. The CLP produces B-cells
and T-cells.
Haematological malignancies arise from a single tumour-initiating cells that have
undergone malignant transformation. In acute leukaemias there is a block in
differentiation, so a large number of immature precursor cells appear in the
blood. In chronic leukaemias, cell differentiation is not affected, but there is a
huge increase in the number of mature cells present in the blood.

Chronic myeloid leukaemia

Symptoms: CML is often asymptomatic, but it may present with malaise, low-
grade fever, increased susceptibility to infections, anemia, and thrombocytopenia
with easy bruising (although an increased platelet count (thrombocytosis) may
also occur in CML). Splenomegaly may also be seen.
Cell of origin: common myeloid progenitor
Translocation: t(9;22), Bcr-Abl gene produced increased proliferation and
decreased apoptosis. The so-called philadelphia chromosome is produced.

Acute myeloid leukaemia

Symptoms: similar to CML, but on microscopic examination of a blood flm, there
are immature cells in the blood.
Cell of origin: ??common myeloid precursor
Translocation: t(15;17) produces PML-RARA protein, which leads to a
differentiation block

Acute lymphoid leukaemia

Symptoms: similar to CML, but is far more common in children.
Cell of origin: lymphoid progenitor

Other leukaemia translocations:
Burkitts NH lymphoma t(8;12) IgH-Myc
Follicular NH Lymphoma t(14;18) IgH-Bcl2
Cervical Cancer

This is the 2
nd
most common cancer in women. It is diagnosed by cervical biopsy.
Cervical cancer is given cervical intraepithelial neoplasia grades (CIN Grade)

Cancer in a Nutshell Anish Patel
CIN1 (Grade I), the least risky type, represents only mild dysplasia, or
abnormal cell growth and is considered a low grade squamous
intraepithelial lesion (LGSIL). It is confined to the basal 1/3 of the
epithelium.
CIN2 (Grade II) as well as the two higher grades are considered high
grade squamous intraepithelial lesions (HSIL). CIN2 represents
moderate dysplasia, and is confined to the basal 2/3 of the epithelium
CIN3 (Grade III): Severe dysplasia spans greater than 2/3 of the entire
epithelium, including the full thickness. It was previously known as
carcinoma in situ.
CIN4 (Grade IV) : Invasive carcinoma
Only a very small proportion (1%) of cervical neoplasia progress from CIN 3 to 4.
The biggest risk factor for cervical cancer is infection with Human Papilloma
Virus, but multiple sexual partners, early onset of sexual activity, oral
contraceptive use and smoking are also thought to increase the risk.
HPV is a non-enveloped double stranded DNA virus that infects mucous
membranes (mucosantropic) Other papilloma viruses are cutaneous (i.e. they
infect the skin). The virus enters the cell in endosomes and then escapes into the
cell nucleus. Here it expresses viral proteins importantly the proteins E6 and E7
which are viral oncogenes. The types of HPV most associated with cancer are
HPV 16 and 18. They cause dyskaryosis, which can lead to malignancy. Note,
however, that HPV is necessary but not sufficient. There are now several
vaccines available against HPV such as Gardasil and Cervarix, which protect
against the most potent strains, 16 and 18, as well as 6 and 11 which cause
genital warts.
Other bacteria/virus associated cancers:
Gastric cancer H. pylori
Liver cancer -- Hepatitis B/C
Kaposis Sarcoma Herpes (full body KS definitive of HIV/AIDS)
B-cell lymphoma Epstein Barr Virus

Tumour markers:

Some tumours produce unique proteins, which are not produced by normal cells.
These are known as tumour specific antigens (TSAs) e.g. CEA by
adenocarcinomas.

Cancer in a Nutshell Anish Patel
Some tumours produce normal proteins in a much larger amounts than normal
tissues these are known as tumour associated antigens (TAAs) e.g. hCG in
choriocarcinoma and germ cell tumours.

These markers can be used in the diagnosis and monitoring of cancer. They can
also be used in rational drug design and targeted drug therapy. The SEREX
database is a log of known TSAs and TAAs.

Cancer therapy:

Chemotherapy the goal of anti-cancer drugs is to selectively kill malignant
cells and spare normal host cells. Selective toxicity is not possible to the degree
seen in antibacterial therapy, because essentially, cancer cells are self.
Therefore, anti-cancer drugs tend to suppress all proliferating cells, including
bone marrow and GIT epithelia etc

Problems with chemotherapy:

1) Side-effects of treatment: bone marrow toxicity, impaired wound healing,
alopecia (hair loss), malabsorption, depression of growth, sterility, teratogenicity,
severe nausea and vomiting.

2) Most anti-cancer drugs are only effective against cells that are rapidly dividing
(that is, tumours with a high growth fraction) this is a big problem as the larger
the tumour, the smaller the growth fraction!

3) Cells within a tumour are heterogeneous they all differ from each other in
subtle ways which can make treatment difficult, as different cells will be
sensitive to different.

4) Cancer cells can development drug resistance they gain mutations that
render the drugs ineffective, much like the development of bacterial resistance.

Classes of anti-cancer drugs

1) Alkylating agents
2) Anti-metabolites
3) Cytotoxic antibiotics
4) Plant derivatives
5) Others

1) Alkylating agents
This group of drugs cross-links nucleotides within the DNA strand, by covalent
bond formation. This inhibits DNA replication.

e.g. Chlorambucil, cisplatin
Cancer in a Nutshell Anish Patel
Alkylating agents are useful in the treatment of Non-Hodgkin,s and Burkitts
lymphoma. Cisplatin has the extra use of treatment of prostate and ovarian
cancer.

2) Anti-metabolites
These agents inhibit one or more of the pathways involved with DNA synthesis.

e.g. Methotrexate an inhibitor of dihydrofolate reductase (the enzyme that
converts folate to tetrahydrofolate). Tetrahydrofolate is essential in the
conversion of dUMP to dTMP. Useful in AML, osteosarcoma, lymphoma.

5-Fluorouracil inhibits the enzyme thymidylate synthase and so stops DNA
synthesis. Used for breast, prostate, ovarian, pancreatic and hepatic carcinoma.

Cyatarabine -- gets incorporated into the extending DNA chain and so
terminates elongation. Treatment for AML.

3) Cytotoxic Antibiotics
These are microbial derived agents that inhibit mammalian cell division.

e.g. Doxorubicin interacts with the topoisomerase enzyme and freezes DNA
replication in the unwound step. Try it in rhabdomyosarcoma and Wilms tumour

Bleomycin breaks the phosphate backbone of the DNA and generates free
radicals, which fragment the DNA. We love it for testicular and ovarian cancer!

4) Plant derived agents
Inhibition of microtubule function.

e.g. Vinca alkaloids act to inhibit the assembly of microtubules and therefore
blocks spindle formation. Used in LOADS of stuff inc leukaemias, NHL,
testicular/ovarian carcinoma.

Etoposide acts in a similar way to doxorubicin

5) Others

e.g. Imatinib mesylate a tyrosine kinase inhibitor that is selective for Bcr-Abl
positive tumours e.g. CML.

Generally, anti-cancer drugs are used in combination with each other, as this
increases the toxicity towards tumour cells (but hopefully not to normal cells!)
Combine drugs that act at different targets and at different stages of the cell
cycle.


Cancer in a Nutshell Anish Patel
Antibody-targeted cancer therapy

Recall the TSAs and TAAs these can be targeted by antibodies. There are
several types of antibodies that you need to know the definitions of:

Monoclonal antibody: An antibody with a particular specificity that is produced
from a single B-cell/plasma cell
Murine monoclonal antibody: A monoclonal antibody produced by
a mouse. The problem with these is that they are immunogenic (they
generate an immune reaction)

Chimeric antibody: When a murine V (variable) region and the
complemenatarity determining region (CDR) region are grafted onto a human
constant region.

Humanized antibody: When just the murine CDR region is grafted onto a
human variable region framework and constant region.

How do you use antibodies in cancer therapy?

Use the antibodies to invoke the normal immune response e.g.
sytotoxicity, antibody dependent cellular cytotoxicity, complement
activation.
Neutralise growth factors e.g the anti-VEGF antibody, bevacizumab
(Avastin)
Attach cytotoxic agents to the antibody targets the drug to the tumour
cells.
Radioimmunotherapy attach a radioisotope to the antibody. Tis
allows a much more accurate deliverance of radiotherapy. With both
cytotoxic drug attachment and RIT, there is the important bystander
effect. This is the effect by which surrounded cells can also be killed,
even if they do not display the antigen.
ADEPT Antibody-Directed Enzyme Prodrug Therapy the antibody is
joined to an enzyme. A prodrug is given, which is only converted to the
active form by the antibody-attached enzyme. This allows targeting of
dangerous cytotoxic drugs to tumour cells and so reduces the toxicity to
normal host cells.


DNA repair and cancer
Because of inherent limitations in the DNA repair mechanisms, if humans lived long
enough, they would all eventually develop cancer.
[48][49]
There are at least 34 Inherited
human DNA repair gene mutations that increase cancer risk. Many of these mutations
cause DNA repair to be less effective than normal. In particular, Hereditary nonpolyposis
colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA
Cancer in a Nutshell Anish Patel
mismatch repair pathway. BRCA1 and BRCA2, two famous genes whose mutations
confer a hugely increased risk of breast cancer on carriers, are both associated with a
large number of DNA repair pathways, especially NHEJ and homologous recombination.
Cancer therapy procedures such as chemotherapy and radiotherapy work by
overwhelming the capacity of the cell to repair DNA damage, resulting in cell death.
Cells that are most rapidly dividing most typically cancer cells are preferentially
affected. The side-effect is that other non-cancerous but rapidly dividing cells such
as stem cells in the bone marrow are also affected. Modern cancer treatments attempt to
localize the DNA damage to cells and tissues only associated with cancer, either by
physical means (concentrating the therapeutic agent in the region of the tumor) or by
biochemical means (exploiting a feature unique to cancer cells in the body).