DRTBALU

Tumor biology of head

and neck cancers
The future of cancer management
Dr. T. Balasubramanian

2009

WWW.DRTBALU.CO.IN
 Tumor biology of head and neck cancers

Introduction:

Most head and neck cancers result from multistep accumulation of genetic
alterations resulting in clonal outgrowth of transformed cells. These
alterations take place at the level of DNA. The DNA molecule is actually a self
replicating chemical information system based on a quaternary code. The
genetic information is contained in a sequence of four nitrogen based
molecules (adenine, guanine, thiamine and cytosine). Any alteration in the
code of DNA can cause far reaching effect on the cellular biology. These
nitrogen based molecules are bonded with each other by hydrogen. It should
be borne in mind that it is the very same hydrogen atom that binds the water
molecules together. Water when boiled beyond its boiling point these
hydrogen molecules breaks thereby separating the water molecules. This very
same effect occurs also in a double stranded DNA molecule also. Heating or
exposure to alkaline environment melts these bonds causing the double
stranded DNA molecule to separate. The reverse effect occurs on cooling and
is known as annealing or hybridization.

Gene:
The term gene denotes a stretch of DNA that codes a protein. Human genome
project has managed to identify 35,000 genes, Out of these genes only 5% code
for protein synthesis and regulatory proteins. The function of the rest of the
genes is not known. Residing inside the genes are sequences that code for
amino acids and are known as exons and some sequences don’t code for any
protein and are known as introns. These introns should be considered to be a
full stop in the gene sequence. During gene transcriptions both exons and
introns are transcribed into the messenger RNA. The introns are excised later.
Malignancy may be considered to be due to deregulation of growth control
aspect of the genome.

Operon model of gene functioning:

This gene regulation system has been extensively studied in lactose

metabolizing bacteria. This model was first studied in bacteria E coli. Lactose
Operon coded production of enzymes that allowed the bacteria to metabolize
lactose in high concentrations.
An Operon is defined as a cluster of related genes that coded for enzymes
necessary for metabolism of a substance. This model helps in increasing the
output of enzymes when there is need and to reduce its output when the need
is not there.

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Structure of an Operon:

1. The region at the beginning of an Operon is a promoter zone. This is

precisely the area where the enzyme RNA polymerase attaches to the
DNA. This attachment stimulates the transcription of the gene bearing
area into the messenger RNA.
2. The next segment of an Operon is the operator region. This area is
considered to be a control switch that can switch on / off the
transcription process. If a repressor protein binds to this area it
effectively stops the enzyme RNA polymerase from moving on to the
gene bearing area, thus effectively stopping the transcription process.
3. Structurally speaking the repressor has two sites. I.e. signal receptor
binding site and an operator binding site. If the signal receptor site is
occupied by the correct chemical the operator binding site is distorted
so that it cannot bind to the operator. This causes the transcription
process to begin. On the contrary if the signal receptor binding site is
vacant the operator binding site can bind to the operator portion of the
Operon thereby blocking the whole transcription process.

Figure showing an Operon

Unregulated Operon may occur if the repressor fails to bind to the operator.
This can be due to:

a. Mutation in the repressor gene code, so that the repressor protein

doesn’t bind to the operator region
b. Mutation of the operator zone of the promoter gene so that the repressor
protein cannot bind to the operator region
c. Mutation of the Operon genes. This causes a change in the gene
products affecting the regulatory control of the Operon.

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Proto-oncogenes:

These are normal cellular genes that are involved in normal growth regulation
and cellular differentiation. Mitogenic signals affect these genes. The
Mitogenic signals could be growth factors, growth factor receptors,
cytoplasmic signal transduction proteins, and nuclear proteins.
Proto-oncogenes that function along the pathway of normal growth and
cellular differentiation have been identified and they are known to play a
regulatory network that extends from the cell surface up to the cell nucleus.
When these genes are mutated or undergo deregulation they can destabilize
normal cell growth promoting tumerogenesis. These mutated / deregulated
proto-oncogenes are known as oncogenes.

Activation of oncogenes:

The activation of oncogenes is vital in the Pathophysiology of tumerogenesis.

It is known to occur in the following 4 ways:

1. Gene acquisition of a novel transcriptional promoter. This leads to over

expression of the gene concerned with a resultant increase in its
byproduct.
2. Chromosomal translocation with deregulation of proto-oncogenes. This
produces unregulated stimulation to cell growth.
3. Gene amplification due to increase in the gene number

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Diagram showing various stages of cell regulation by proto-oncogenes.

Examples of Proto-oncogenes:

1. Growth factor – Platelet derived growth factor

2. Growth factor receptor – erb-B epidermal growth receptor
3. Membrane protein – Used for signal transduction (ras)
4. Cytoplasmic protein – e.g. MOS
5. Nuclear protein – MYC

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Diagram showing conversion of proto-oncogenes to oncogenes

As illustrated in the diagram above proto-oncogene can be converted to

oncogenes by:

1. Gene amplification
2. Point mutation
3. Acquisition of promoter / enhancer sequences
4. Chromosome translocation

Gene amplification: Also known as gene duplication is a process by which

multiple copies of the same gene are produced. This causes an amplification
of the enzymes / reactions coded by the gene.

Point mutation: In this type of mutation a single genetic nucleotide is replaced

by another one. It also indicates insertion / deletion of a single base pair of
nucleotide. Point mutation can be categorized as transitions and transversions.
Transitions – In this type of point mutation a purine base is replaced by
another purine base while a pyramidine base is replaced by another
pyramidine base.
Transversion – In this type of point mutation a purine base is replaced by a
pyramidine one and vice versa.

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Functionally point mutations can be classified as:
a. Nonsense mutation: It codes for a stop and truncates the protein
molecule
b. Missence mutation: This sequence codes for a different amino acid
c. Silent mutation: This sequence codes for same / different protein
without any functional change in the protein
Point mutations can be spontaneously caused during DNA replication. The
rate of point mutations can be increased by mutagens. These mutagens can be
physical / chemical. Physical mutagens include UV radiation X-ray radiation,
extreme heat etc.

Acquisition of Promoter / Enhancer sequences: By transfer of genetic material

a gene can acquire abnormal promoter and enhancer sequences. This would
lead to abnormal coding and expression of proteins.

Chromosomal translocation: This is caused by rearrangement of parts between

two non homologous chromosomes. This is one process which enables
exchange of genetic material between two chromosomes.
Translocation can be of two types:
1. Reciprocal (Non Robertsonian)
2. Robertsonian

In Reciprocal translocation the exchange of genetic material is between two

Non homologous chromosomes. They are usually harmless, and usually have
increased risks of creating abnormal gametes leading on to miscarriages.

In Robertsonian translocation reallocation takes place between two acrocentric

chromosomes. This causes fusion of two chromosomes at the centromere zone
with loss of their short arms. This translocation causes a reduction in the
human karyotype chromosomal number i.e. 45 chromosomes instead of
normal 46.

Tumor suppressor genes:

Genes belonging to this group are known to cause suppression of malignant

transformation of cells. Expression of these genes plays a protective role in
preventing malignant cells from developing. Mutations involving these genes
are known to cause Retinoblastoma.
Classic example of this group of genes is the p53 tumor suppressor gene which
has been implicated in various malignant tumors of head and neck. P53 is
known as the guardian angel of the genome protecting it from abnormal
changes.

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Stages of tumerogenesis:

Tumerogenesis is a multistage procedure. Classically described stages

include: Stage of initiation, stage of promotion and stage of progression.

Diagram showing the stages of tumerogenesis

Tumor initiation: This is a rapid and irreversible process due to genetic

changes within the cells. This is commonly caused by the cell’s interaction
with the carcinogen. These cells should be considered as primed to undergo
malignant transformation. These initiated cells themselves donot express
Neoplastic potential. For this to occur they must undergo promotion.

Tumor promotion: This process is reversible and has a prolonged latency

period. The initiated cells usually develop into neoplasm on being exposed to
the promoting agent.

Tumor progression: This is a feature of already established malignant tumors.

Tumors down the line manage to acquire propensity to distant metastasis,
radio resistance, resistance to chemotherapeutic agents.

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Genetic alterations:

Genetic susceptibility to head and neck cancers:

Genes that code for activation of Glutathione – S – transferase protects an
individual from head and neck malignancy. It is only this enzyme that
neutralizes tobacco carcinogens. Absence / inactivation of this gene may
predispose an individual to tobacco induced malignancies.

Genetic alterations include a variety of changes in the structure and sequence

of cellular DNA within the offending clonal population. These changes result
in activation of proto-oncogenes and inactivation of tumor suppressor genes.
These DNA changes are caused by a variety of mechanisms like endogenous
mutation and exogenous mutation. Exogenous mutations are caused by potent
environmental carcinogens.
These genetic mutations cause changes in the biologic characteristics of any
neoplasm like cell growth, death, motility and invasion. These mutations also
influence the host’s defense mechanism and immunological status. Another
important aspect of tumor biology in head and neck cancers is the role played
by Circadian rhythm. Circadian rhythm enables humans to adapt to daily
environmental changes and also manages to synchronize various biochemical
and physiologic processes with each other. These circadian clocks have
known to interfere with cell cycle. An intact circadian rhythm is necessary for
normal cell growth and cell death. It is hence necessary to study the role
played by circadian rhythm in the tumerogenesis.

Ras proto-oncogene:

There are three oncogenes in this family. They are N-ras, H-ras and K-ras.
These oncogenes encode membrane associated G-proteins and guanosine
triphosphatases. These two proteins signals cell surface growth receptors. It
has been proved that nearly 20% of oropharyngeal malignancies are due to
point mutations involving N-ras genes.

Chromosome region 9p21 loss is the most common aberration detected in

patients with head and neck cancers. It has been demonstrated that 9p21 loss is
one of the earliest detectable events in head and neck cancer patients. This
chromosomal region loss is also commonly seen in patients with squamous
hyperplastic lesions (benign) of head and neck.

Inhibition of gene p16: This gene is a critical inhibitor of cyclin CDK

complexes. Inactivation of this gene permits inappropriate progression
through critical G1/S cell cycle check points allowing cell division to occur

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unimpeded and relentlessly. This gene can be inhibited by complete deletion
or by methylation of the promoter region.

CCND1 /PRAD1:

In cell division transition from G1 to S phase (phase of DNA synthesis) and

from G2 to M phase (mitosis phase) are critical control points in a growing
cell. A group of proteins called cyclic AMP dependent kinases are responsible
in the regulation of these stages of cell division. Cyclin D1 is one such kinase.
The CCND1 gene encodes cyclin D1 protein and is located in chromosome
11q13. This particular focus gets amplified in 50% of patients with squamous
cell carcinoma of head and neck region.
CCND1 amplification and over expression are seen commonly in patients with
head and neck malignancies.

EMS1 oncogene:

This is located in the same area as CCND1. This gene encodes cytoskeletal
protein (cortactin). Amplification of this oncogene leads to over expression of
cortactin causing it to migrate from cytoplasm into the cellular matrix. This
affects the functioning of cytoskeleton thus contributing to the invasive nature
of the tumor cells. Activation of this gene predicts higher recurrence rate and
poor prognosis.

MPP11:

Amplification of this gene is an important even in the progression of head and

neck malignant tumors.

Deletion of several discrete regions in chromosome 3p: This has been

identified in 60% of head and neck cancer patients. The precise nature of
function of this gene is still unknown.

Loss of chromosome 17p: This has been shown to occur in more than 50% of
head and neck malignancies. It also correlates with p53 inactivation.

Mutation / inactivation of p53 genes: This is a tumor suppressor gene. It is of

course the most extensively studied of all genes. This gene is known to
suppress cell division. It induces G1 arrest till genetic repair is effected. If
genetic repair is not possible it directs the cell into apoptotic pathway. It has
been shown that Human papilloma virus E6 gene interacts with p53 protein
causing it to degrade thus essentially inactivating it. Carcinogenesis of HPV is
due to this action.

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Loss of chromosome 13q: Is seen in about 60% of patients with tumors of head
and neck. This portion of the chromosome is supposed to contain the RB gene.
Human papilloma virus E7 another oncogenic protein is known to deactivate
this RB gene. This gene has been known to negatively modulate transcription
factor E2F.

Amplification of 11q13: This has commonly been implicated in 40% of head

and neck cancer patients. This chromosomal area is responsible for the
production of cyclin D1. Cyclin D1 is considered to be an important oncogene
in the tumerogenesis of head and neck malignant tumors. Cyclin D1 is known
to activate cell cycle progression.

Squamous cell carcinoma related onco-gene: This gene is activated by

amplification of 3q26.3 gene. This onco-gene has been identified as one of the
important initiator of squamous cell carcinoma of head and neck.

Role of growth factors in tumerogenesis of head and neck malignancies:

It is the presence of Growth factors and their receptors signals stimulus to
cell division and growth in normal cells under physiologic conditions. Over
expression of growth factors and their receptors can cause pathologic
proliferation of cells and hence considered as products of proto-oncogenes.
More than 90% of head and neck cancers over express epidermal growth factor
receptor. This growth factor is encoded by c-erb
The HER-2/neu gene encodes transmembrane receptor called tyrosine
kinase. Tyrosine kinase belongs to epidermal growth factor receptor group.
Hence HER -2/neu gene amplification plays a role in tumor genesis.

It has been shown that nearly 50% of tumors arising from oropharynx contain
oncogenic human papilloma virus DNA. As described above the
tumorogenecity of HPV is due to the action of this protein on p53
chromosome. It has also been shown that patients with antibodies to HPV
showed overall better survival rates. Recently the antiviral agent cidofovir
when used in combination with tumor irradiation resulted in increased radio
sensitivity of tumor cells.
The concept of molecular staging has been introduced. This procedure
makes use of these commonly present tumor markers described above. These
elements can also be used in early diagnosis of malignancies / potential
malignancies. Recently therapeutic strategies have been evolved to target thee
p53 mutant tumors. This is done by developing adenovirus with E1b 55 kd
gene deleted. This virus is known to selectively replicate and lyse p53
deficient cells.

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Cyclidin D1 has been found to be commonly over expressed in patients with
head and neck cancers. Flavopiridol a CDK inhibitor has been used to repress
transcription of cyclin D. This causes a cellular arrest at G2 and G1 phases of
cell division. This chemical is also found to promote p53 independent cellular
apoptosis. It is also known to increase the chemo / radio sensitivity of tumor
cells.

Over expression of epidermal growth factor in head and neck tumors is

associated with poorer prognosis. Various EGFR blockers have been devised
in order to improve the prognosis. These blockers include antibodies, tyrosine
kinase etc.

The knowledge regarding tumor biology will be of immense help in

formulating diagnostic and prognostic testing tools.

Investigational tools in molecular biology:

Southern Blot technique: Named after the British biologist Edwin Southern
who invented this process.
This analysis compares the electrophoretic patterns of DNA fragments. In this
test the DNA extracted from the tumor sample is enzymatically digested into
small fragments. These fragments can be compared by their travel rate in the
gel plate. Larger fragments of DNA remain close to the well of origin while
the smaller segments of DNA travel the farthest in the gel plate. The enzyme
used for this process is usually restriction endonuclease. If the DNA
fragments are larger than 15kb, prior to blotting the gel should be treated with
dilute HCL. This acid environment breaks the DNA molecule into smaller
pieces making their movement in the gel plate more efficient.
The DNA fragments can be blotted into nylon or other similar synthetic
membranes. For this purpose the sheet of nylon / nitrocellulose is placed on
top of the gel. Even pressure is applied over the sheet to ensure good even
contact between the gel and the membrane. DNA moves from the gel to the
membrane due to capillary action. The membrane is baked in vacuum inside a
regular oven at a temperature of 80⁰ C for 2 hours. This permanently attaches
the transferred DNA to the membrane.
The membrane is exposed to hybridization probe. This is usually a single
DNA fragment with a specific sequence and is tagged by incorporating
radioactivity / dyes. Hybridization probe is usually prepared from RNA.
After hybridization the excess probe is washed away and the membrane is
studied by taking an X-ray film. If dyes are used then color of the dye can be
used to study the probe.
Hybridization probe to a specific DNA fragment indicates that this fragment
contains a DNA sequence that is complementary to the probe.

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Diagram showing methodology of Southern Blot technique

Northern blot technique: This method is used to analyze and compare the
fragments of RNA molecules. This is helpful in the study of gene expression.
It is possible to observe cellular control over structure and function by
observing a particular gene expression levels during differentiation. This
procedure involves the use of electrophoresis to separate RNA samples by size
and use of hybridization probe to identify the target RNA sequence.

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Diagram showing Northern blot technique

Western blot technique:

This is also known as protein immunoblast. This technique detects specific
proteins in a tissue sample. It uses gel electrophoresis to separate these
proteins by the length of their polypeptide chain. These detected proteins are
transferred on to a membrane where they can be probed by using antibodies
specific to the target protein.

Diagram showing western blot technique

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Polymerase chain reaction:

This procedure creates multiple copies of DNA segment which is amplified

from a very small quantity of DNA. It was first developed by Kary Mullis in
1984. This involves a series of biochemical reactions like denaturation,
annealing and extension. This method relies on thermal cycling. Each of these
thermal cycles consists of heating and cooling of the reaction. These thermal
cycles cause melting of DNA and its replication. The enzyme used for the
reaction is heat stable DNA polymerase. This heat stable DNA polymerase
assembles new DNA strands from DNA building blocks.

Role of tumor molecular biology:

a. Early diagnosis of head and neck malignancies. Head and neck tumors
happen to be the most antigenic of all malignant lesions. Several
markers have been suggested to be of value in the early diagnosis of
head and neck malignancies. They include squamous cell carcinoma
antigen, cytokeratin fragment 19. High levels of Tumor growth factor α
have been identified in urine of patients with advanced head and neck
cancers.
b. Molecular biology can offer new tools to effectively identify caner
locations. The following criteria should be fulfilled.
1. The protein in question should be easily accessed by the injected
antibody. In other words it should be expressed over the cell
surface.
2. The protein should be specific to the cancer cell
3. The target protein should be over expressed relative to the
background levels
Currently available tools belonging to this category is indium 111
labeled antibodies which can be directed against epidermal cell growth factor.
This test could be considered to be specific for squamous cell carcinomas of
head and neck areas.

c. These molecular tools may successfully be used to stage malignant

lesions. Over expression of epidermal growth factor is commonly seen
in stage III and stage IV head and neck malignancies.
d. Molecular biology can help to predict with reasonable degree of
accuracy tumor behavior also. It has been demonstrated that activation
of certain proto oncogenes may predict radio / chemo resistance of the
lesion. Altered raf proto oncogenes have been demonstrated in patients
with radio resistant head and neck tumors.
e. Therapeutic applications: Current knowledge of molecular biology can
help us to specifically direct treatment. Theoretically speaking by
preventing the flow of information from oncogene DNA to RNA

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 malignant transformation of a cell can be arrested. It is also possible to
replace / compensate for the defective tumor suppressor gene. This will
go a long way in preventing malignant lesions from forming. Another
possibility is suicide gene therapy wherein a gene can be introduced
into the tumor cell to cause destruction of that particular cell line.
f. Gene therapy can be used as immune moderators. This will help the
normal immune mechanism of the body in destroying these potentially
distorted tumerogenic cells.

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