Additional Cancer Lecture Slides

CANCER

Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. If the spread is not controlled, it can result in death. (American Cancer Society)

INCIDENCE of CANCER

- is a common disease - 1 person in 3 can expect to contract cancer at some stage in their life -1 person in 5 can expect to die from it

-worldwide, 100-350 of every 100,000 people will die of cancer each year - given a global population of about 6.4x109, this implies 6.4 t0 22.4 million people will die this year

TUMOUR CLASSIFICATION

BENIGN TUMOURS Develop in any tissue • grow locally • May cause problems by pressure (brain) or obstruction ( colon) • Histologically resemble the tissue of origin • Covering or lining tissues of skin, intestine, bladder etc may produce wart-like outgrowths containing all cell types • In other situations only one cell type may be present- may produce an excess of particular hormone

• Benign does not mean ‘completely harmless’ • Do not spread to distant sites

TUMOUR CLASSIFICATION IN SITU TUMOURS

Usually develop in the epithelium Usually small • Have altered histological appearance • Loss of normal arrangement of cells • Variations in cell size and shape, increase in nucleus size and staining ( increased DNA ), presence of abnormal chromosomes • Do not invade basement membrane and supporting mesenchyme

TUMOUR CLASSIFICATION CANCERS

Fully developed malignant tumours with the specific capacity to invade and destroy the underlying mesenchyme. •

Metastasise

•

Stimulate angiogenesis and development of blood supply

•

difficult to treat.

Two Broad Classes of Genes are Involved in the Onset of Cancer

1).

Proto-oncogenes activated by mutation to become oncogenes,- excessively active in growth promotion.

2).

Tumour Suppressor genes normally restrain cell growth- damage to these genes allows inappropriate growth

Many of the genes in both classes code for proteins involved in •

entry into, and passage through, the cell cycle

•

cell death by apoptosis

•

repair of damaged DNA

Chemical Carcinogens

-Earliest example 1775 coal tar and skin cancer -Later, 2 – naphthylamine as a bladder carcinogen

-Wide chemical diversity and many ( eg polycyclic aromatic hydrocarbons) show great chemical stability.

- Now known to be converted to highly reactive compounds by detoxification enzymes in the liver.

-Guanine is often converted to methyl guanine, acts like adenine and pairs with thymidine in the copied strandhence G-C pair is converted to A-T pair as point mutation.

3 ways in which cancer cells become growth signal autonomous.

- modulation of growth factor provision

- modulation of growth factor receptor activity

- modulation of intracellular signalling pathways

Self Sufficiency in Growth Signals ii). Modulation of growth factor receptor activity Many growth factor receptors are protein tyrosine kinases Overexpression allows tumours to respond to low levels of growth factor that would not normally produce a growth response.

EGF-R ( the receptor for EGF) and Erb-B ( the receptor for hereglulin ) are upregulated in stomach, brain and breast tumours.

HER2/neu is overexpressed in stomach and breast tumours

Overexpression of GF receptors may result in ligand –independent signalling.

Self Sufficiency in Growth Signals ii). Modulation of growth factor receptor activity (cont) Receptors may become structurally altered- ligand independent as a result. Truncated versions of the EGF receptor lacking the cytoplasmic domain are constitutively active

Alteration of integrins expression (ECM receptors) to those favouring growth.

Ligand activated GF receptors and pro-growth integrins attached to ECM often activate the SOS-Ras-Raf-MAP Kinase pathway.

Self Sufficiency in Growth Signals iii). Modulation of intracellular signalling pathways

Frequently involves the SOS-Ras –Raf- MAPK cascade About 25% of human tumours have a mutated Ras protein. (90% pancreas, 50% colon, 30% lung -- the first oncogene discovered in human tumours)) --- mitogenic signals are transmitted without any upstream activation of the pathway

Ras also interacts with PI3 kinase This enables growth signals to simultaneously generate survival signals ie. Signals which protect against apoptosis

Insensitivity to Antigrowth Signals

Tumours have developed several ways to block TGFß action

•Expression of TGFß receptor is down regulated •The receptor is mutated to a less active form •Intra-cellular signalling is disrupted by•Mutation of Smad •Loss of p15 •Mutation of CDK4 to be less p15 sensitive •Mutation of Rb

Insensitivity to Antigrowth Signals

Tumours have developed several ways to block TGFß acting through Rb

In some DNA – virus induced tumours ( cervical carcinomas) Rb is inactivated by being complexed with a viral protein .

In human cervical tumours this is the E7 protein of human papilloma virus

Insensitivity to Antigrowth Signals

Cancer cells can also •Turn off the expression of cell adhesion molecules that transmit antigrowth signals •These probably act through Rb also

Insensitivity to Antigrowth Signals

Some tumours have developed mechanisms for differentiation

One such mechanism involves the c-myc oncogene

Evasion of Apoptosis

Hormone dependent tumours undergo massive apoptosis if the hormones were removed.

Suggested that increased cell growth and apoptosis occurred at the same time

Apoptosis may be switched on by oncogene overexpression Elimination of cells with activated oncogenes by apoptosis may be the primary means by which mutant cells are continually removed from the body’s tissues.

For a tumour to progress it has to inactivate the apoptopic machinery

Evasion of Apoptosis

In 50% of lymphomas, there is a mutation in c-myc and a mutation in bcl-2

Further evidence for a myc-bcl-2 interaction

Fibroblasts overexpressing myc grown in culture In low serum the c-myc expressing cells show high apoptosis Increased apoptosis could be abolished by •Addition of survival factors such as IGF-1 to the medium •Overexpression of Bcl-2 or Bcl-XL •Disruption of the FAS pathway

Evasion of Apoptosis

In transgenic mice, •Inactivation of Rb ( expected to increase cell proliferation) produced slow growing microscopic tumours with a high rate of apoptosis •Additional inactivation of p53( a key mediator of apoptosis) in the same cells produced rapidly growing tumours

•Mutation of p53 and the presence of a mutated p53 protein is extremely common in human tumours ( greater than 50%)

Some lung and colon cancers produce a decoy nonsignalling receptor for the FAS ligand

Limitless Replicative Potential Why should tumour cells need to become ‘immortalised’? Normal human cell types have the capacity for 60–70 doublings. This should enable clones of tumor cells to expand to numbers that vastly exceed the number of cells in the human body. There seems to be no sense in the idea that the tumour cells have to become immortal in order for malignant tumour growth to occur But…… During tumour development there is widespread apoptosis along side the increased cell division. The number of cells in a tumour greatly under represents the cell divisions required to produce it. Thus the generational limit of normal somatic cells may be a barrier to cancer development.

Limitless Replicative Potential Telomeres

Telomeres are simple-sequence DNA repeats found at the end of chromosomes Human telomeres contain 250-1500 copies (6-12 kb) of the sequence TTAGGG

At each cell division, 50–100 bp of telomeric DNA are lost from the ends of every chromosome DNA polymerases are unable to completely replicate the 3′ ends. Progressive shortening of the telomeres occurs with each division. Eventually the telomeres lose the ability to protect the ends of the chromosomes This results in end to end chromosomal fusion and the death of the affected cell.

Limitless Replicative Potential Telomeres

…………..and Telomerase

Telomere maintenance occurs in just about all malignant cells The majority (85%–90%) upregulated expression of an enzyme called telomerase This adds hexanucleotide repeats onto the ends of telomeric DNA.

The telomeres are thus kept at a length above a critical threshold which allows for unlimited multiplication of descendant cells.

Sustained Angiogenesis Normal Cells and Tissues

Cells in a tissue need to be within 100μ of a capillary blood vessel This closeness is achieved during organ development and once a tissue is formed, the growth of new blood vessels—the process of angiogenesis—is transitory and carefully regulated. Cancer Cells Cancer cells initially lack angiogenic capacity this limits initial expansion of the tumour. To develop to a clinically detectable size the tumours usually develop angiogenic ability.

Sustained Angiogenesis Cancer Cells

A cancer cell mass of about 2 millimeters emits signals that recruit surrounding connective tissue and vascular cells to the tumor and induce them to grow into blood vessels.

The blood supply to the tumour provides nutrients and oxygen, and provides a route to the rest of the body, ie. A route to metastasis.

Sustained Angiogenesis Angiogenesis is controlled by both positive and negative signals. Important ones are • vascular endothelial growth factor (VEGF) • acidic and basic fibroblast growth factors (FGF1 and FGF2)

• thrombospondin-1 •binds to a transmembrane receptor( CD36) on endothelial cells.

Sustained Angiogenesis Tumors activate angiogenesis by changing the balance of inducers and inhibitors. Many tumors increase expression of VEGF and/or FGFs. - activation of ras oncogene may upregulate VEGF expression. Some down regulate expression of inhibitors such as thrombospondin-1 -thrombospondin-1 is to positively regulated by the p53. -Loss of p53 function, which occurs in most human tumors, causes thrombospondin-1 levels to fall, releasing the endothelial cells from inhibition.

Some do both.

The mechanisms remain largely incompletely understood.

Metastasis

An exception to the immobilised format of tissue structure is the white blood cell.

This can migrate out of the blood stream and enter into other tissues.

For tumour cells to metastasise, they must acquire the migratory properties usually restricted to white-blood cells.

Metastasis

Spread may occur via the blood or the lymphatics. Localised spread to lymph nodes is a sign of poor prognosis.

Certain tumors spread to particular organs.

- prostate cancer to the bones. - colon cancer to the liver. -stomach cancer to the ovary. -occular melanoma to the liver.

Metastasis Metastatic cells must become mobile the cytoskeleton has to be re-configured for motility. Normal epithelial cells have several shapes—cylindrical, cuboid or flattened. Cancer cells are usually star-shaped and elongated. Similar fibroblastic morphology is also observed in epithelial cells during development of the embryo. Many tumour cells resemble cells seen only during the very early stages of embryonic development.

Metastasis Metastatic cells must loose connections with the other cells and ECM of the primary tumour

Proteases used to degrade the basal membrane Plasminogen activators ( serine proteases) - leads to conversion of plasminogen to plasmin

Cathepsin B ( cysteine protease)

Matrix metalloproteinases ( MMPs) - often converted from a pro- (inactive ) form by plasmin

Metastasis Cancer cells in the blood stream

2 mechanisms for surviving in the bloodstream.

Travel in clusters, increases the possibility that at least one will survive. Surround themselves with blood cells such as platelets, -- masks the cancer cells from immune surveillance.

Metastasis The secondary site The secondary site is often specific. Specific interactions between the cancer-cell surface and the endothelial cells that line the blood vessels in the new host tissue. Carbohydrates on the cancer-cell surface bind to a specific receptor on the endothelial cells called a selectin. Different selectins recognise different carbohydrates on the cancer cell surface. Normally, the carbohydrate-selectin interactions are used by white blood cells to identify particular tissues to combat local infection. Each cancer-cell type expresses a different set of carbohydrates on its surface, attracted to different selectin molecules. The specificity of these interactions helps account for the differential homing specificities of different types of cancer cells.

Metastasis The secondary site

The cancer cell contacts a surface where the cells express the appropriate selectins. More bonds, mediated by integrins, form between the cells. The cancer cell migrates through the blood-vessel wall, degrading the connective-tissue matrix with proteases. The cancer cell is now ready to proliferate and form a new tumor in its new host tissue

Cancer Therapy Current Therapy is fairly crude Surgery if possible some cancer cells remain at the original site others may have already started to metastasise to distant organs So the patient is given Radiation eradicates cells by inducing apoptosis can be directed very specifically to where the primary tumor was located in order to destroy any remaining cancer cells. However, undetected metastases elsewhere go untreated Radiation is often given in conjunction with Chemotherapy designed to curtail division and proliferation many normal cells with high turnover rates, such as skin, hair and blood cells, are affected along with the cancer cells Sometimes cancer cells develop resistance to chemotherapy Current therapeutic approaches are thus fairly 'blunderbus-like'

Cancer Therapy Potential Improved Therapeutic Approaches Stimulate the patient's immune system

Boost the patient's immune system, and direct it against molecules expressed on the cancer cells but not on healthy cells. Tumor-specific antigens have been hard to identify Many of the immune agents now in use target healthy cells as well In the test tube, immune cells are effective in killing the cancer cells. Unfortunately, in vivo once the immune cells meet the cancer cells, nothing further seems to happen. The immune cells fail to mediate any attack.

Cancer Therapy Potential Improved Therapeutic Approaches Stimulate the patient's immune system

Cancer cells ward off possible attack by secreting large amounts of immunosuppressive messenger molecules, such as interleukin-10 transforming growth factor b and prostaglandin E2. They also secrete molecules such as α2-macroglobulin, which inhibit cancer-celldestroying proteases.

May travel through the circulation in clusters, (increasing the possibility of survival) or may surround themselves with platelets to escape immune surveillance.

Cancer Therapy Potential Improved Therapeutic Approaches Stimulate the patient's immune system

The cancer cells may also attack the immune cells using the Fas/ Fas-L system.

Cancer Therapy Potential Improved Therapeutic Approaches Doxorubicin ( Adriamycin)

Doxorubicin induces the expression of both Fas and FasL on cancer cells –

- causes cancer cells to kill themselves by inducing apoptosis.

Cancer Therapy Potential Improved Therapeutic Approaches

Some modest successes with immunostimulants. Injecting the bacterial preparation BCG seems to be effective in early stage bladder tumours.

Interleukin 2 (IL-2) or with alpha-interferon (IFN-α) has been beneficial in some instances.

Generally attempts to stimulate the host immune system sufficiently to beat off the cancer have been disappointing.

Cancer Therapy Potential Improved Therapeutic Approaches Monoclonal Antibodies as therapeutic agents

The receptor Her2 is overexpressed in a subset of breast tumours. Anti-Her2 monoclonal antibody (Herceptin) has proved highly successful. The antibody combines with the receptor and blocks it so that growth factors ( EGF) no longer bind. May also cause the receptor it to be internalised This leads to the selective death of the cancer cell

Cancer Therapy Potential Improved Therapeutic Approaches Immunotoxins Monoclonal antibodies coupled to a cytotoxic drug. Mylotarg. MAb against a cell-surface molecule (called CD33), found on acute myelogenous leukemia cells coupled to a complex oligosaccharide called calicheamicin Makes double stranded breaks in DNA.

BL22, a MAb against CD22 (found on some leukemias and lymphomas) joined to a bacterial endotoxin that blocks protein synthesis.

Cancer Therapy Potential Improved Therapeutic Approaches Radioimmunotherapy

Monoclonal antibodies against tumor antigens can also be coupled to radioactive atoms such as indium -111 , yttrium -90 or iodine-131.

Cancer Therapy Potential Improved Therapeutic Approaches Allografts of T cells

Treat leukemia with massive doses of chemotherapy and radiation - the leukemic cells are killed. - also kills the patient's own bone marrow. The patient must be then given a transplant of donor bone marrow containing the stem cells from which all blood cells are derived.

Cancer Therapy Potential Improved Therapeutic Approaches Autografts of T cells: Tumor-Infiltrating Lymphocytes (TIL)

The idea here is that T cells isolated from within solid tumours are specific for tumour antigens. T cells are isolated from the solid tumour, - grown to large numbers outside the body ( in culture) -activated - and reintroduced back into the patient. Has been successful with malignant melanomas of the skin. Successful in about 77% of patients, and worked against metastases as well as the primary tumours.

Cancer Therapy Potential Improved Therapeutic Approaches Cancer vaccines

1).

Dendritic-cell Vaccines

Dendritic cells are the most potent of the antigen-presenting cells. -harvest dendritic cells from the patient -expose these in vitro to antigens associated with the type of tumour in the patient -inject the "pulsed" dendritic cells back into the patient -hope they kick the immune system into action. So far have shown promise against melanomas, prostate cancer and lymphoma.

Cancer Therapy Potential Improved Therapeutic Approaches Cancer vaccines

2).

Tumour-specific Antigen Vaccines

Tumour cells are removed from the patient, - treated with heat or chemicals so they are not viable, - mixed with an adjuvant (such as BCG) - injected back into the patient, -hopefully to now stimulate the patient's immune system. Such vaccines are currently in clinical trials for use against chronic myelogenous leukemia CML).

Cancer Therapy Potential Improved Therapeutic Approaches Inhibit Tumour Angiogenesis

Two angiogenesis inhibitors with potential clinical applications

1)

angiostatin (an amino-terminal fragment of plasminogen)

2)

endostatin (a 20 kD protein derived from the carboxyl-terminal domain of collagen XVIII)

Both inhibit angiogenesis in laboratory animals

Cancer Therapy Potential Improved Therapeutic Approaches Inhibit Tumour Angiogenesis

Much of the research has been centered on VEGF. Antisense constructs against VEGF inhibit experimental angiogenesis. Monoclonal antibodies against VEGF receptors have also been successful in stopping angiogenesis. Genetically engineered cells secreting a soluble form of the VEGF receptor have been shown capable of inhibiting angiogenesis at distant sites.

Cancer Therapy Potential Improved Therapeutic Approaches Inhibit Tumour Angiogenesis

Small molecule inhibitor of receptor tyrosine phosphorylation - inhibits the tyrosine phosphorylation of VEGF receptors - and PDGF receptor and thus inhibits signalling . Has potent anti-angiogenic effects in pre-clinical models and is undergoing clinical trial.

Cancer Therapy Potential Improved Therapeutic Approaches Inhibit Tumour Angiogenesis

Various interferons also are reported to inhibit angiogenesis Interferon alfa-2a given to a 5-year-old girl with a large rapidly growing tumor of the mandible. Resulted in dramatic decreases in urinary excretion of FGF, and complete tumor regression over a three year treatment-free interval

Cancer Therapy Potential Improved Therapeutic Approaches Correct Mutated Signalling Pathways Recent experimental strategies attempt to compensate for or correct defective genes. Eg with ras The ras protein has to be post-translationally modified. This requires that ras has a lipid molecule attached to it. This is catalyzed by an enzyme called farnesyl transferase. Inhibitors of farneslyl transferase are being tested in clinical trials as anti -cancer agents.

Cancer Therapy Potential Improved Therapeutic Approaches Restore Growth Inhibitory Pathways Another (theoretical) approach is to restore the function of growth-inhibiting genes inactivated through mutation.

Gene therapy, -- replace the mutated gene with a functioning one. To date the clinical results have been disappointing.

Cancer Therapy Potential Improved Therapeutic Approaches Halt Metastasis

2 general targets. Adhesion molecules Proteases

Cancer Therapy Potential Improved Therapeutic Approaches Halt Metastasis

Restore adhesion-molecule synthesis

Gene-replacement therapy has worked in the laboratory, but it is still a long way from being useful clinically.

Cancer Therapy Potential Improved Therapeutic Approaches Halt Metastasis Other potential targets are the proteases that metastatic cells use. Marimastat, an inhibitor of matrix metalloproteinases (MMPs) caused great excitement. Marimastat is water-soluble. Marimastat inhibited tumour development and metastasis in animals. Treated animals showed increased amounts of connective tissue around the tumors, consistent with the drug inhibiting MMPs. Early clinical trials were promising. Unfortunately the early results were not borne out by more extensive studies. Despite the initial optimism, the usefulness of marimastat may be limited to a small subset of patients.

Cancer Therapy Potential Improved Therapeutic Approaches Halt Metastasis

Another approach involves a specific small, water-soluble inhibitor of cathepsin-B. - claimed to inhibit only the cathepsin-B on the cell membrane - not the internal lysosomal enzyme - and to have minimal effect on normal cells. Inhibition of extracellular, but not intracellular, activity suggests therapeutic promise, because it blocks only the pathologically expressed cathepsin-B and not the physiologically required stores.

Cancer Therapy Potential Improved Therapeutic Approaches Halt Metastasis Given orally to rats with colon cancer that had metastasized to the liver the number of tumors was reduced by one-third and the size of the tumors was reduced by two-thirds. Strongly suggests that cathepsin-B is involved in colon-cancer metastasis to the liver. It has yet to be shown that this drug has any use therapeutically,

but it seems possible.

Cancer Therapy Potential Improved Therapeutic Approaches Induce Apoptosis in the Tumour Attempts to stimulate apoptosis specifically in tumour cells seem to work in the laboratory, but have not yet been useful clinically SoOverexpression of BAX (apoptosis stimulator) in tumor cells increases the sensitivity to conventional chemotherapy and to decrease the growth rate when implanted into mice. Another approach has been to stimulate apoptosis by transfection of wild type p53 with the addition of a construct expressing FAS-L. This approach led to dramatically enhanced apoptosis in transfected glioma cells in culture Yet another approach has been to inhibit the activity of bcl-2 by the use of drugs, which seems to work in cells overexpressing Bcl-2

Cancer Therapy Potential Improved Therapeutic Approaches Telomerase as a Potential Target A second approach assumes that tumor cells which strongly express telomerase should drive a telomerase promoter.

Transfection with a cytotoxic transgene in which the telomerase promoter is attached to the gene for Bax (to promote apoptosis) should produce tumour-specific killing.

This was found to be so. Induction of Bax elicited tumor-specific apoptosis in vitro and suppressed tumor growth in nude mice. Normal, telomerase negative, cells were not affected .