1. Cancer cell, horrifying but intriguing Magdalena CHECHLIŃSKA Department of Immunology Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology Warszawa

2. Cells Your body is made up of approx. 10 13 -10 14 cells that can only be seen under a microscope. These cells are grouped together to make up the tissues and organs of our bodies. Cells are basically similar. They all have a centre called a nucleus. Inside the nucleus are the genes. Genes are really bits of code. The information they carry can be switched on or off. The genes control the cell. They decide when it will reproduce, what it does and even when it will die.

3. Body tissues Body tissues grow by increasing the number of cells that make them up. The cells reproduce themselves exactly. But once we are grown up, most cells mature, become specialised for their particular job in the body and lose the ability to reproduce. There are always immature cells around (called stem cells) to replace cells that are damaged or killed. Some cells carry on reproducing. These include sperm cells, hair cells, cells in the gut and cells that make blood in the bone marrow.

4. How do the cells know when to stop growing? Normal growth and healing is very orderly and precise. The cells send chemical messages to each other. The messages come from the genes inside the cells.

5. How do new cells end up in the right place? The cells have a natural ability to stick together in the right place. Scientists call this cell adhesion. If the cell does find itself in a place where its surface molecules are different from its neighbours, it will die.

6. How cells reproduce Cells double up very precisely so that the new cells are exactly the same as the old ones. Each cell makes copies of all its genes. Then it splits into two with one set of genes in each new cell. This is called mitosis

7. Can cells carry on doubling for ever? No they can't! It seems human cells are pre-programmed to reproduce up to 50 or 60 times maximum. Then they will become senescent and eventually die. 40 population doublings = 1 cell gives rise to 10 12 cells

8. What cancer is Cancer is a disease caused by normal cells changing so that they grow in an uncontrolled way. The uncontrolled growth causes a lump called a tumour to form. There are over 200 different types of cancer because there are over 200 different types of body cells. For example, cells that make up the lungs can cause a lung cancer. There are different cells in the lungs, so these may cause different types of lung cancer.

9. How cancer starts - MUTATIONS One cell (?) that has lost a number of vital control systems due to mutations. Mutation means a gene has been damaged or lost. One gene 'codes' for one protein. Mutation  too much protein is made  a protein is not made at all.  the properties of a protein are changed 3 types of genes that are important in making a cell cancerous: »Genes that encourage the cell to multiply »Genes that stop the cell multiplying »Genes that repair the other damaged genes

10. How cancer starts Oncogenes Some genes encourage cells to multiply (proliferate). If these genes become abnormal, they tell the cell to multiply all the time. Tumour suppressor genes (anti-oncogenes) Some genes specifically to stop the cell multiplying - they act as the brake to the oncogene's accelerator. If one of these 'tumour suppressor genes' becomes damaged and stops working, then the cell may carry on and on multiplying and it becomes immortal, which is one of the properties of a cancer cell. p53 normally stops cells with other damaged genes from reproducing and encourages them to commit suicide (apoptosis). p53 is damaged or missing in most human cancers. Genes that repair other damaged genes These genes normally repair any damage to the DNA that the cell's genes are made of. If these genes are damaged, then other mutations are not repaired and the cell can reproduce the mutations in its daughter cells. These genes have been found to be damaged in some human cancers, including bowel cancer.

11. How cancer starts How do mutations happen? By chance when a cell is reproducing. It is not easy for a normal cell to turn into a cancer cell. There have to be about half a dozen different mutations before this happens. Cells often self destruct if they carry a mutation. Or they might be recognised by the immune system as abnormal and killed. This means most pre-cancerous cells die before they can cause disease. Only a few can turn into a cancer. It can take a long time before enough mutations happen for a cell to become cancerous. This is why many cancers are more common in older people. There has been more time to be exposed to carcinogens. And more time for accidents when cells reproduce.

12. How cancer starts Genetic make up There have to be a number of genetic mutations within a cell before it becomes cancerous. Sometimes we are born with one of these mutations already. This does not mean we will get cancer. But with one mutation from the outset, it makes it more likely statistically that we will ( 'genetic predisposition‘). Examples: the BRCA1 and BRCA2 breast cancer genes. Women who carry one of these faulty genes have a higher chance of developing breast cancer than women who do not. BUT: Most women with breast cancer do not have a mutated BRCA1 or BRCA 2 gene. Less than 5% of all breast cancer is due to these genes. So most breast cancer is not caused by a high risk inherited gene fault.

14. Progression of epithelial cancer (carcinoma) Thiery, 2002

15. Normal cells Reproduce themselves exactly (FIDELITY) Stop reproducing at the right time (CONTACT INHIBITION) Stick together in the right place Self destruct if they are damaged (APOPTOSIS) Become specialised or 'mature‘ (DIFFERENTIATION) Cancer cells Carry on reproducing Don't obey signals from other neighbouring cells Don't stick together Don't become specialised, but stay immature Don't die if they move to another part of the body

16. Cancer cell – acquired capabilities Hanahan, 2000

17. Self-sufficiency in growth signals Synthesis of growth factors Expression of growth factor receptors Receptor structure changes resulting in signal transmission in spite of the lack of a growth factor Intracellular signals Membrane

18. Kassis, 2001 Normal cells: Tightly packed Autocrine interactions - blocked Dysplasia Cells do not stick together Autocrine activation and further detachment of cells EGFR

19. Tumour Kunz-Schugart, 2002 ECM

20. „successful neoplastic cells are those able to engage other cells to promote their growth ”. Skobe i Fusenig

21. Tumour microenvironment influence cancer cell growth dośw.Mueller i wsp. 2001 Wounding stimulates the growth of primary and secondary tumours. (Experimental models of human cancers) Hofer, 1999

24. TGFRII - mice, selectively on fibroblasts, excessive fibroblasts in prostate, followed by prostate cancer. Conclusion: defective stromal fibroblasts stimulate epithelial tumour growth due to the lack of TGF  inhibitory activity Bhowmick i wsp., Science, 2004

25. Inhibits: ● nomal melanocytes, ● melanoma cells – early stages czerniaka ● lung and liver epithelial cells Insensitivity to anti-growth signals stimulates: ● advanced melanoma cells Does not inhibit cell lines in vitro: ● lung cancer ● liver cancer IL-6

26. Apoptosis = programmed cell death = = gene-directed cellular self-destruction Cells are programmed to die at a particular point. This cell suicide mechanism enables metazoans (multicellular organisms) to control cell number. „Apoptosis” is of greek origin, meaning "falling off or dropping off", in analogy to leaves falling off trees. This analogy emphasizes that the death of living matter is an integral and necessary part of the life cycle of organisms.

27. Apoptosis Functions of apoptosis –Development –Homeostasis: Immune cell regulation Cell damage or infection Response to stress or DNA damage In the human body 100.000 cells are produced every second by mitosis and a similar number die by apoptosis, i.e. 50 to 70 billion cells die each day due to apoptosis in the average human adult. In a year, this amounts to the proliferation and subsequent destruction of a mass of cells equal to an individual's body weight.

28. Evading apoptosis : example: TNF – pro- and anti-apoptotic

29. Apoptosis Cell damage or infection Response to stress or DNA damage DNA damage Damage response p53 Repair Arrest Apoptosis

30. Evading apoptosis – ineffective effectors IL-10 TGF-  mTNF sTNF TNFR FasL Fas FasL Cytotoxic/effector cell Tumour cell IL-6 No apoptosis Apoptosis

31. Limitless proliferative potential – cancer cells are immortal Avoiding the progressive erosion of chromosomes (all cancer cells) by telomerase expression (85-90%) and other mechanism.

32. What telomeres are Telomeres are extensions of the linear, double- stranded DNA molecules of which chromosomes are composed, and are found at each end of both of the chromosomal strands. Telomeres are essential for chromosome stability Telomeres shorten with each cell division each round of DNA replication leaves 50-200 bp DNA unreplicated

33. Telomeres, normal metaphase

34. Telomere shortening as a mitotic counter Telomere lenght Sperm, eggs 20 kilobases Foetal cells~15 kilobases Adult cells~10 kilobases Senescent cells ~5 kilobases Each round of DNA replication leaves 50-200 base pairs DNA unreplicated Cells with telomeres that are 10-12 kb in length (average) divide 50-60 times

35. Telomere maintenance is evident in all types of malignant cells. The main mechanism: upregulation of telomerase (85-90% of malignant cells). Telomerase is an enzyme, which is able to extend telomeres.

36. Progression of epithelial cancer (carcinoma) Thiery, 2002

37. Cancer cells stimulate angiogenesis, i.e. new vessels’ development

38. Angiogenesis – the growth of new blood vessels < 100  m from capillaries

39. Pro-and anti-angiogenic factors There are at least: 20 angiogenic growth factors 30 known natural angiogenesis inhibitors found in the body. Tumours present „proangiogenic” profile, i.e the balance between angiogenic inducers and inhibitors is disturbed in favour of the inducers.

40. Proangiogenic factors Hypoxia VEGF increased expression: breast, ovarian, pancreatic,prostate cancer High serum levels correlate with adverse prognosis.

41. Progression of epithelial cancer (carcinoma) Thiery, 2002

42. Cancer cells invade and colonise other sites Adhesion to extracellular matrix components and basement membrane Basement membrane degradation Extracellular matrix modification and degradation Active migration in extracellular matrix Penetration thorough:  blood vessel walls,  lymphatic vessel walls,  body cavities walls Implantation, survival and growth at distant sites

43. Carcinoma cells in primary mammary tumour move along ECM fibres Scale bar 25  m

44. Intravasation in primary mammary tumour Condeelis and Segal, 2003

45. Adhesion molecules Growth factors released in tumour microenvironment induce changes of adhesion molecules on: Cancer cells Fibroblasts Endothelial cells

46. Proteases - enzymes Released not only by cancer cells but also by other cells in the microenvoronment Role: extracellular matrix degradation Activation and release of matrix-associated growth factors Protease activity correlates with the ability to form distant metstases. Protease inhibitors inhibit cell migration Active migration

47. Distant metastases

48. Organ-specific pattern of metastasis Site10%10-30%30-50%50-70%70% BreastKidney, skin, brain AdrenalLiver, bone, lung Lymph nodes BladderBrain, skinKidney, boneAdrenal, lung CervixBrain, skinKidney, boneAdrenal, lung ColorectumSkinBrain, kidney, lung Bone, adrenal, liver Lymph nodes KidneySkin, boneBrain, kidneyLiverLung Kidney, distant nodes Adrenal, brainBoneLiver, local lymph nodes MelanomaKidneyAdrenal, brain, bone, skin Lung. Liver nodes OvaryBrain, skin, kidney Bone, adrenalLung, liver nodes ProstateBrain, skinKidney, adrenal, liver, lung Bone, nodes

49. „What is that decides which organs shall suffer in a case of disseminated cancer?” (S. Paget, The Lancet, 1889) „When a plant goes to seed, its seeds are carried in all directions, but they can only live and grow if they fall in congenial soil.” Cancer cell dissemination – an ineffective process New microenvironment may be favourable to cancer cells, e.g.  in the bone marrow breast cancer cells are stimulated by IGF 1, prostate cancer cells - by TGF  ;  in the liver, colorectal cancer cells have an increased ability to grow due to TGF  presence.

50. Molecules determining cancer dissemination Constitutively expressed in the metastasis development site Promote cancer cell adhesion to endothelium and migration in and out the vessels Induce invasion into the new, favourable environment Cells colonising new sites must express the receptors relevant to the growth factors present in the secondary sites.

51. Cancer cells express patterns of chemokine receptors that match chemokines specifically expressed in organs to which they commonly metastasise. Muller, 2001

52. Immunosuppression Impairment of immune reponse, local and systemic

53. FasL Immunosupresja

54. Mutation rate: 2.2 x 10 -9 per base pair per year X 3 billion (3x10 9 ) chemical base pairs that make up human DNA X 5% of genome encoding X 10 14 cells in the average human = 3.3 x 10 13 mutations per person per year. Still, cancer arises in only 1 in 3 lifetimes. The rarity of cancer highlights the efficacy of potent anti-tumorigenic mechanisms presiding over somatic cells. Cancers prevail only when these mechanisms have failed. We perceive only the rare surviving clones that beat all the odds and appear as clinical disease. We see only successes of cancer cells, not the failures. The relative rarity of the cancer cell

55. Cancer progression and clinical appearance Liotta, 2001

56. „Biology and Cancer Research have developed together. Invariably, at each stage, the characteristics of the cancer cell have been ascribed to some defect in whatever branch of biology happens at the time to be fashionable and exciting ” John Cairns