Genes and the Environment

Viruses and Cancer.

Environmental Exposure and Cancer.

Carcinogenesis

What Causes Cancer?

Carcinogens  Age  genetic make up  immune system  diet  day-to-day environment  Viruses

Age

Diet

Factors in Carcinogenesis

Chromosomes/DNA

In addition to chemicals and radiation, a few viruses also can trigger the development of cancer. In general, viruses are small infectious agents that cannot reproduce on their own, but instead enter into living cells and cause the infected cell to produce more copies of the virus. Like cells, viruses store their genetic instructions in large molecules called nucleic acids. In the case of cancer viruses, some of the viral genetic information carried in these nucleic acids is inserted into the chromosomes of the infected cell, and this causes the cell to become malignant.

Viruses

Viruses contribute to development of some cancers.  Typically, the virus can cause genetic changes in cells that make them more likely to become transformed.

These cancers and viruses are linked

Cervical cancer and the genital wart virus, HPV Primary liver cancer and the Hepatitis B virus  T cell leukaemia in adults and the Human T cell

leukaemia virus

HTLV-1 naturally infects CD4+ T lymphocytes and can be

transmitted between close contacts through blood transfer or from mother to infant through cells in breast milk. In most cases the infection is harmless. However, as many as

1 in 20 infected individuals eventually develop a type of adult T cell leukaemia in which every tumour cell carries a clonally integrated HTLV1 provirus.

HTLV1 differs from the standard 'chronically oncogenic' and 'acutely oncogenic' retroviruses in its mechanism of action; it appears to drive cell growth through expression of a

particular viral protein, Tax, in latently-infected cells

HTLV retrovirus and adult T-cell leukaemia

Mode of action Tax can transactivate expression of a

number of key cellular genes that enhance cell growth.The best examples are the genes encoding

interleukin 2 (a T cell growth factor) and the interleukin 2 receptor (a molecule that allows

cells to respond to the growth factor).As a consequence, the infected cells not only

make their own growth signals, but also respond to them

Mode of action

HTLV1 induces a weak growth transformation of T cells in the laboratory but, in the body, is probably never sufficiently strong to induce T cell leukaemia on its own.

BUT, a virally infected cell in which growth controls have even partly broken down, is more susceptible to further genetic accidents.

During persistent infection a gradual build-up of HTLV1-positive T cells which have accumulated additional genetic changes may occur.

Eventually this can lead to selection and outgrowth of a fully malignant, HTLV1-positive clone

At this stage malignant cell growth can occur in the absence of tax gene expression.

Epstein Barr Virus

Epstein-Barr virus (EBV), also called Human herpesvirus 4 (HHV-4), is a virus of the herpes family (which includes Herpes simplex virus and Cytomegalovirus),

one of the most common viruses in humans. Most people become infected with EBV,

often asymptomatic but commonly causes infectious mononucleosis.

It is named after Michael Epstein and Yvonne Barr, who together with Bert Achong discovered the virus in 1964.

On infecting the B-lymphocyte, the linear virus genome circularises and the virus subsequently persists within the cell as an episome.

The virus can execute several distinct programmes of virally-encoded gene expression broadly categorised as being lytic cycle or latent cycle. The lytic cycle or productive infection results in staged expression

of a host of viral proteins with the ultimate objective of producing infectious virions. Formally, this phase of infection does not inevitably lead to lysis of the host cell as EBV virions are produced by budding from the infected cell.

The latent cycle programmes are those that do not result in production of virions.

EBV-associated malignancies

The strongest evidence linking EBV and cancer formation is found in Burkitt's lymphoma and Nasopharyngeal carcinoma

Burkitts Lymphoma

a type of Non-Hodgkin's lymphoma most common in equatorial Africa co-existent with the presence of malaria. Malaria infection causes reduced immune surveillance of EBV

immortalised B cells, so allowing their proliferation. This proliferation increases the chance of a mutation to occur. Repeated mutations can lead to the B cells escaping the body's cell-cycle control, allowing the cells to proliferate unchecked, resulting in the formation of Burkitt's lymphoma. Burkitt's lymphoma commonly affects the jaw bone, forming a huge tumour mass. It responds quickly to chemotherapy treatment, namely cyclophosphamide, but recurrence is common.

Other B cell lymphomas arise in immunocompromised patients such as those with AIDS or who have undergone organ transplantation with associated immunosuppression.

Smooth muscle tumours are also associated with the virus.

Nasopharyngeal carcinoma

found in the upper respiratory tract, most commonly in the nasopharynx, and is linked to the EBV virus.

It is found predominantly in Southern China and Africa, due to both genetic and environmental factors. It is much more common in people of Chinese ancestry (genetic), but is also linked to the Chinese diet of a high amount of smoked fish, which contain nitrosamines, well known carcinogens (environmental).

Kaposi's sarcoma

form of skin cancer that can involve internal organs. It most often is found in patients with acquired immunodeficiency syndrome (AIDS), and can be fatal.

K.S.

Kaposi's sarcoma (KS) was once a very rare form of cancer, primarily affecting elderly men of Mediterranean and eastern European background (tumours on lower legs), until the 1980s, when it began to appear among AIDS patients.

AIDS-related KS, emerged as one of the first illnesses observed among those with AIDS. Unlike classic KS, AIDS-related KS tumours generally appear on the upper body, including the head, neck, and back. The tumours also can appear on the soft palate and gum areas of the mouth, and in more advanced cases, they can be found in the stomach and intestines, the lymph nodes, and the lungs.

Kaposi Sarcoma and HHV8

Studies in 2000 showed that HHV-8 was the culprit behind KS.

It does not work alone. In combination with a patient's altered response to

cytokines (regulatory proteins produced by the immune system) and the HIV-1 transactivating protein Tat which promotes the growth of endothelial cells, HHV-8 can then encode interleukin 6 viral proteins, specific cytokines that stimulate cell growth in the skin.

This becomes KS.

HHV-8

HHV-8 destroys the immune system further by directing a cell to remove the major histocompatibility complex (MHC-1) proteins that protect it from invasion.

These proteins are then transferred to the interior of the cell and are destroyed.

This leaves the cell unguarded and vulnerable to invaders which would normally be targeted for attack by the immune system.

The natural history of HPV infection and cervical cancer

Cervical Screening

(von Knebel Doberitz/European Journal of Cancer 2002; 38: 2229-2242).

Genomic Map of HPV

The interacting worlds of HPV and p16

The Hepatitis B Virus (HBV)

HBV is a mostly double-stranded DNA virus in the Hepadnaviridae family.

HBV causes hepatitis in human. The HBV genome has four genes: pol, env, pre-core and

X that respectively encode the viral DNA-polymerase, envelope protein, pre-core protein (which is processed to viral capsid) and protein X.

The function of protein X is not clear but it may be involved in the activation of host cell genes and the development of cancer.

Consequences of HBV Infection

HBV causes acute and chronic hepatitis. The chances of becoming chronically infected

depends upon age. About 90% of infected neonates and 50% of

infected young children will become chronically infected.

In contrast, only about 5% to 10% of immunocompetent adults infected with HBV develop chronic hepatitis B.

Hepatocellular carcinoma

cancer that arises from hepatocytes, the major cell type of the liver.

Hepatocellular carcinoma is one of the major cancer killers.

It affects patients with chronic liver disease who have established cirrhosis, and currently is the most frequent cause of death in these patients.

The main risk factors for its development are hepatitis B and C virus infection, alcoholism and aflatoxin intake.

integrated HBV can generate chromosomal instability

it has been suggested that viral DNA sequences encompassing the encapsidation signal may exhibit intrinsic recombinogenic activity via binding to a putative ‘recombinogenic’ cellular protein

Genes and the Environment –Thyroid Disease

The thyroid gland is a small gland located at the front and sides of the neck.

It is made up of right and left lobes connected by a bridge called the isthmus and weighs between 20 and 30 grams.

It is slightly heavier in females and becomes enlarged during menstruation and pregnancy.

The thyroid gland produces three hormones called thyroxine (T4), tri-iodothronine (T3) and calcitonin. These hormones help to regulate the body's metabolism.

Iodine is essential for the production of the thyroid hormones.

Good sources of iodine include fish such as cod, sea bass and haddock.

Dairy products and plants grown in soil that is rich in iodine are also good sources.

The thyroid participates in these processes by producing thyroid hormones, principally thyroxine (T4) and triiodothyronine (T3).

These hormones regulate the rate of metabolism and affect the growth and rate of function of many other systems in the body.

Iodine is an essential component of both T3 and T4. The thyroid also produces the hormone calcitonin, which

plays a role in calcium homeostasis.

Physiology

The primary function of the thyroid is production of the hormones thyroxine (T4), triiodothyronine (T3), and calcitonin.

Up to 80% of the T4 is converted to T3 by peripheral organs such as the liver, kidney and spleen.

T3 is about ten times more active than T4.

The function of the thyroid gland is to take iodine, found in many foods, and convert it into thyroid hormones: thyroxine (T4) and triiodothyronine (T3).

Thyroid cells are the only cells in the body which can absorb iodine.

These cells combine iodine and the amino acid tyrosine to make T3 and T4.

T3 and T4 are then released into the blood stream and are transported throughout the body where they control metabolism (conversion of oxygen and calories to energy).

Every cell in the body depends upon thyroid hormones for regulation of their metabolism.

The normal thyroid gland produces about 80% T4 and about 20% T3, however, T3 possesses about four times the hormone "strength" as T4.

The thyroid gland is under the control of the pituitary gland, a small gland at the base of the brain.

When the level of thyroid hormones (T3 & T4) drops too low, the pituitary gland produces Thyroid Stimulating Hormone (TSH) which stimulates the thyroid gland to produce more hormones.

Under the influence of TSH, the thyroid will manufacture and secrete T3 and T4 thereby raising their blood levels.

The thyroid gland is invested by a thin capsule of connective tissue, which projects into its substance and imperfectly divides it into masses of irregular form and size.

Cut surface reveals a number of closed vesicles, containing a yellow fluid, and separated from each other by intermediate connective tissue

Significance of iodine

In areas of the world where iodine (essential for the production of thyroxine, which contains four iodine atoms) is lacking in the diet, the thyroid gland can be considerably enlarged, resulting in the swollen necks of endemic goitre.

Significance of iodine

Because of the thyroid's selective uptake and concentration of what is a fairly rare element, it is sensitive to the effects of various radioactive isotopes of iodine produced by nuclear fission.

In the event of large accidental releases of such material into the environment, the uptake of radioactive iodine isotopes by the thyroid can, in theory, be blocked by saturating the uptake mechanism with a large surplus of non-radioactive iodine, taken in the form of potassium iodide tablets.

Significance of iodine

While biological researchers making compounds labelled with iodine isotopes do this, in the wider world such preventive measures are usually not stockpiled before an accident, nor are they distributed adequately afterward.

One consequence of the Chernobyl disaster was an increase in thyroid cancers in children in the years following the accident.

Chernobyl

On April 26, 1986, the fourth reactor of the Chernobyl Nuclear Power Plant, exploded at 01:23 AM local time. All permanent residents of Chernobyl and Zone of alienation were evacuated because radiation levels in the area had become unsafe.

Chernobyl

Chernobyl

Chernobyl

Identifying molecular mechanisms that reveal underlying pathobiology in Thyroid Neoplasia

Background

Thyroid Cancer most frequently occurring endocrine malignancy, sub-divided into a number of diagnostic /morphological

categories. Papillary thyroid carcinoma (PTC)

Most common thyroid malignancy Ireland<100 cases/yr, U.S.~24,000 cases/yr Incidence on the rise - global estimate 0.5 million new

cases this year Fastest growing cancer in women worldwide

Pathological Pathways

Follicular Epithelial Cell

Papillary Thyroid Carcinoma

Follicular Carcinoma

Molecular markers in PTC ret/PTC

To date 15 chimeric mRNAs involving 10 different genes have been described

Ret/PTC-1 and ret/PTC-3 are the most common types, accounting for 90%.

Morphological variants are likely to reflect variations in tumour biology which have yet to be fully defined.

ret/PTC-1 expression and associated thryoiditis.

0

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Hashimotothyroiditis ret

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Hashimotothyroiditis ret

neg

Lymphocyticthyroiditis ret

pos

Lymphocyticthyroiditis ret

neg

Graves diseaseret pos

Graves diseaseret neg

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E-cadherin expression and radiation dose

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Radiation Dose (Gy)

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Ret/PTC-1 activation

ret/PTC-3 activation

commonly seen in children exposed to ionizing radiation. ? Radiation signature

Literature demonstrates correlation with solid/follicular variant morphology, poorer prognosis and aggressive tumor behavior.

Correlation between tumor morphology and specific ret rearrangements

Ret/PTC-3 activation

commonly seen in children exposed to ionising radiation. Literature demonstrates correlation with solid/follicular variant

morphology, poorer prognosis and aggressive tumour behavior.

? prevalence of the common ret chimeric transcripts (ret/PTC-1 and ret/PTC-3) in a group of “sporadic” PTC. Correlation between tumour morphology and specific ret

rearrangements

Ret/PTC-3 analysis in sporadic cases

Ret/PTC-1 and ret/PTC-3 transcripts were detected in 43% and 18 % of PTCs respectively.

Ret/PTC-3 correlated strongly with the solid/follicular variant [55%] and was not detected in conventional PTC.

Ret/PTC chimeric transcripts in an Irish Cohort of Sporadic Papillary Thyroid Carcinoma.

SP Finn, P Smyth, O’ Leary JJ, Sweeney EC, Sheils O.

J Clin Endocrinol Metab 2003 Feb; 88(2):938-41.PMID: 12574236 [PubMed - in process]

Real time analysis of beta and gamma catenin mRNA expression in ret/PTC-1 activated and non-activated thyroid tissues. Smyth P, Finn S, O’Leary J, Sheils O.

Diagn Mol Pathol 2003 Mar;12(1):44-9 PMID: 12605035 [PubMed - in process]

Conclusion of ret/PTC 3 analysis in sporadic cases

Significant prevalence of ret/PTC transcripts in this sporadic group of PTC.

Higher incidence than previously reported in areas not affected by ionising radiation ? More sensitive technique ? Effect of exposure to ionising radiation…..

Ret activation and morphology

In the setting of radiation induced PTC it is apparent that specific ret/PTC rearrangements are associated with specific tumour morphology ret/PTC-1 associated with classic morphology

?low dose/long latency

ret/PTC-3 associated with solid/follicular morphology and adverse prognosis.

?higher dose/short latency

BRAF

Raf kinases

Serine/threonine kinases Function in

Ras/Raf/MEK/ERK pathway

3 isoforms: A-Braf, B-Raf, C-Raf/Raf-1

BRAF implicated in many cancers

Temporal trends?

1982

-84

1985

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1988

-90

1991

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1994

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1997

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2000

-02

0123456789

Year

ret/PTC

T1799A mutation

Microarray analysis

Effect of BRAF mutation on transcriptome

‘Normal’ thyroid cell line (left) PTC Cell Lines with BRAF

mutation (right)

BRAF (mut) transfection experiment

• Left cluster = wt braf- –normal cell line

•Right Cluster = (mut) braf-–Normal cell line transfected with braf (mut).

Effect of ret/PTC-1 activation on transcriptome

ret/PTC-1 transfection experiment

Left cluster = wt ret normal cell line

Right Cluster = ret/PTC-1- Normal cell line

transfected with ret/PTC-1

Name Panther Function

Galectin 3 Signalling molecule, cell adhesion molecule

Calcium Calmodulin-dependant protein kinase 1

Non-receptor serine/threonine protein kinase

TGFB1 Angiogenesis

Syndecan 3 Cell adhesion, proliferation, differentiation

TGFb1 Receptor protein serine/threonine kinase signalling. Cell cycle control

Calgizzarin (S100A11) Tumour suppressor, calmodulin related protein

CD44 Cell adhesion, inhibition of apoptosis

TIMP1 Metalloprotease and its inhibitor

MMP14 Metalloprotease and its inhibitor

Genes differentially expressed (upregulated) in PTC vs benign

Data derived from array analysis of surgically resected lesions

Genes differentially expressed (downregulated) in PTC vs benign

Name Panther Function

TIMP4 Metalloprotease Inhibitor

RAB23 Small GTPase – Member RAS oncogene family

FGFR2 Tyrosine Protein Kinase receptor, MAPKKK cascade, JAK-STAT cascade

Metallothionein Homeostasis activities

Syndecan 2 Cytoskeletal protein, defence and immunity protein, extracellular matrix glycoprotein

MAPK4

TSHr G-protein receptor

Lectin, mannose-binding1 (LMAN1)

Immunity and Defence

Data derived from array analysis of surgically resected lesions

Archival ffpe Tissues

TSHr Expression

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1015202530

0 1 2 3 4 5 6 7

TSH

r in

dex 1=FTC/well diff

2=FTC/poorly diff3=PTC/well diff4=PTC/poorly diff5=FA/norm6=ATC7=MTC

Archival FFPE Tissues

Normalised E-cadherin expression vs Tissue type

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PTC PTC PTC HT FTC ATC FA/NORM

ret-ve ret+ve