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 tranocl"iption pl"~ess.

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.

,G"."" :3

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 playa 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 I 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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••

1

a--

Growth factor

Growth factor receptor ~ ~Membrane

d PlF-9tein

. ~--...... ~ cytciplasmic /1 "protein\

b--

e - ~uclear protein

P -. ;{St!"IJc.l\! r'ad

rllteln .

produce p«lI:em

- -.. i!nzym~

----

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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On,oglt'ne

Tumeur development

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

N on 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.

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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. Tn.ese 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 GljS 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.

CCNDl jPRADl:

In cell division transition from Gl 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 cyelic AMP dependent kinases are responsible in the regulation of these stages of cell division. Cyelin Dl is one such kinase. The CCNDl gene encodes cyelin Dl protein and is located in chromosome llql3. This particular focus gets am.plified in 50Dh. of patients with squamous cell carcinoma of head and neck region.

CCNDI amplification and over expression are seen commonly in patients with head and neck malignancies.

EMSl oncogene:

This is located in the same area as CCNDl. 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.

MPPll:

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 l7p: 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 Gl 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 I3q: 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 Ilql3: This has commonly been implicated in 40% of head and neck cancer patients. This chromosomal area is responsible for the production of cyelin DI. Cyelin DI is considered to be an important oncogene in the tumerogenesis of head and neck malignant tumors. Cyelin DI 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,lneu gene encodes transmembrane receptor called tyrosine kinase. Tyrosine kinase belongs to epidermal growth factor receptor group. Hence HER -2,lneu 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 Elb 55 kd gene deleted. This virus is known to selectively replicate and lyse p53 deficient cells.

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Cyclidin Dl 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 Gl 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 80° 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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Membrane with separated nucleic acids hybridized with labeled probe

StaiCllo:: or pa. per tow@lls

NIUlcleic acids separat,edl accorci~lng to size by el~ectro'ptiu:W~siis in an agaroSfl gel

Niitroce IIII ulose membrane

U rn I a b ell e ci -----,,...=-~=----

. IDINIA

Labeled ~ NA standards as----..--.~ size markers

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$e,;Mlrated nucleic acid$. b.lottedl onto membrane by suction of buffer throuqh both gel and pape,

sealled plastic --=~ ba,g

After removal of unbound probe, bands complementary to labeled probe are revealed by specific detection of I.abell

® Detected ~bandlS

,.,

Positions /' _ oflabeled~ markers

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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o

.J,. RNA Extraction

~ Electrophoresis

RNA separated by size

~--~~ ,,--~

I Northern blotting ~ (transfer of RUA to

membrane)

labeled probes

~uaHzation of labeled RNA on X-rw film

RNA filled to membrane with UV or heat

Membrane hybridized with labeled probes

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 rumors 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 a 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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