Environmental Cancers Melanoma and Lung Cancer - PowerPoint Presentation

1. Environmental CancersMelanoma and Lung CancerLara K. Goudsouzian, Ph.D.Associate Professor of BiologyDeSales UniversityLara.goudsouzian@desales.edu

2. Melanoma

3. Melanocytes Produce MelaninMelanin is a dark pigment. The melanin travels in vesicles called melanosomes into the keratinocytes (skin cells). Dark skinned people have more melanin than light skinned people.Question: Where else in the body do we see melanin?

4. The Sun vs. The SkinUVA and UBV rays both do damage to DNA, but UVB does the most.

5. DNA Repair is a Pretty Good SolutionThe pyrimidine dimers formed by UVB irradiation are “bulky lesions” (remember those?). Nucleotide excision repair repairs them.

6. Melanin Protects from the SunThe more sun exposure an individual has, the greater the production of melanin. This eases the burden on the nucleotide excision repair system by reducing DNA damage.Question: What happens when you have LOTS of sun exposure in a short amount of time?

7. Sunburn is Not a BurnBecause the sun is hot and because the skin turns red and blisters, it is a common misconception that sunburn is caused by excessive heat. In fact, sunburn results when excessive DNA damage activates p53, which in turn activates apoptosis.Question: Why would so much UV lead to apoptosis?

8. ProblemIn this study, mice homozygous for a p53 mutation (-/-), heterozygotes (-/+) or wild-type (+/+) were exposed to UVB radiation 3 times per week for 30 weeks. Interpret the results on this experiment.Jiang, W., Ananthaswamy, H. N., Muller, H. K., & Kripke, M. L. (1999).  Oncogene,18(29), 4247-4253.

9. Melanoma is Cancer of MelanotcytesThe first historical descriptions of melanoma came from the great Greek physician Hippocrates (460 – 375 BCE). The word melanoma comes from the Greek melas (dark), and oma (tumor).

10. As Long as We’ve Had Skin, We’ve Had MelanomaThe preserved skin of 2400 year old mummies found in Peru have melanomas!

11. It Took Some Time to Figure Out the Cause of Melanoma“As to the remote and exciting causes of melanosis, we are quite in the dark, nor can more be said of the methodus medendi. We are hence forced to confess the incompetency of our knowledge of the disease under consideration, and to leave to future investigators the merit of revealing the laws which govern its origin and progress….and pointing out the means by which its ravages may be prevented or repressed” - Thomas Fawdington, The Manchester Royal Infirmary, 1826.

12. This Type of Map Helped Determine the CauseWhat do you notice about the distribution pattern of melanoma?

13. ProblemThis table lists the results of a large scale study where researchers correlated melanoma incidence with sun exposure. What can you conclude from this data?Newton-Bishop, J. A., Chang, Y., Elliott, F., Chan, M., Leake, S., Karpavicius, B., Barrett, J. H. (2011). European Journal of Cancer,47(5), 732-741. Control = no melanomaCase = melanomaOR = Odds Ratio, which determines if exposure to a risk factor actually causes the outcomeOR=1 Exposure does not affect odds of outcomeOR>1 Exposure associated with higher odds of outcomeOR<1 Exposure associated with lower odds of outcome95% CI = 95% Confidence Interval, which means that you can be 95% confident that the range contains the mean of your data

14. ProblemIn this same study, researchers tried to correlate melanoma development with other factors. What can you conclude from this data?

15. ProblemThis figure is from a meta-analysis of risk for melanoma associated with a high use of sunbeds (aka tanning beds). What can you conclude?This is a forest plot, which is a good way to represent a meta-analysis of many studies. The y-axis lists each study.The x-axis shows the OR (Odds Ratio). The square represents the OR of each study. The area of the square relates to the weight of the study in the meta-analysis. The horizontal lines represent the 95% CI for each study. The vertical line is OR=1. The diamond shows the overall OR and 95% CI for the meta-analysisBoniol, M., Autier, P., Boyle, P., & Gandini, S. (2012). Cutaneous melanoma attributable to sunbed use: Systematic review and meta-analysis. BMJ,345(Dec13 6).

16. ProblemNodular melanoma is the most lethal subtype of melanoma, causing about half of all deaths due to skin cancer.Interpret this Kaplan-Meier survival curveCan you postulate at least 3 reasons for the racial disparity in survival?Mahendraraj, Krishnaraj & Sidhu, Komal & Lau, Christine & McRoy, Georgia & Chamberlain, Ronald & Smith, Franz. (2017). Malignant Melanoma in African–Americans: A Population-Based Clinical Outcomes Study Involving 1106 African–American Patients from the Surveillance, Epidemiology, and End Result (SEER) Database (1988–2011). Medicine 96(15) April 2017.

17. Most Melanomas Involve a Mutation in the BRAF GeneWhen a melanocyte surface Receptor Tyrosine Kinase (RTK) is activated by growth factors, it activates Ras by converting it to RAS-GTP. RAS-GTP activates BRAF, which in turn activates the pathway that leads to cell proliferation. In melanoma, a mutation in BRAF means that it is constitutively active. No growth factor stimulation is necessary.Question: How is it that BRAF causes melanoma and p53 also causes melanoma?

18. Xeroderma Pigmentosum is a Hereditary Disorder Which Increases Melanoma IncidenceThere are many other mutations which can result in melanoma. Humans with a mutation in the gene which encodes XPA are very sensitive to the sun. XPA is a protein necessary for nucleotide excision repair. Median age of skin cancer onset in XP population is 10 (compared to 60 in general population).

19. ProblemResearchers discovered that XPA abundance cycles in mice who are kept in natural light. The protein’s levels are lowest before dawn and highest in late afternoon. In this study, hairless mice were exposed to a large dose of UV irradiation at either 4AM or at 4PM, then their rates of tumor formation tracked.Interpret this figure.How would adaptive evolution have led to this finding?Question: Do you think XP is inherited in a dominant or a recessive fashion?

20. ProblemIf ultraviolet radiation is so deleterious, why haven’t all human populations evolved to produce as much melanin as those closest to the Equator?

21. ProblemWhat were the results of this large-scale study of Swedish women?Lindqvist, P. G., Epstein, E., Landin-Olsson, M., Ingvar, C., Nielsen, K., Stenbeck, M., & Olsson, H. (2014).  Journal of Internal Medicine,276(1), 77-86.

22. The Sun Makes Vitamin DVitamin D can also be obtained through diet, but most unenriched foods just don’t have enough. We evolved to get Vitamin D from sunlight, not food.

23. ProblemIn this study, mice were injected with human colon cancer cells. Half the mice were fed a vitamin D-deficient chow (black line) and half were fed a chow supplemented with vitamin D (dashed line). Tumor volumes were measured from 7 – 20 days post-injection.What information can you gain from this figure?Can you make a connection between this figure and adaptive evolution in humans?Tangpricha, V., Spina, C., Yao, M., Chen, T. C., Wolfe, M. M., & Holick, M. F. (2005). The Journal of Nutrition,135(10), 2350-2354.

24. ProblemIn this study, 1179 women of age 66+7 years were split into three groups. One group was given a placebo, one group was given a calcium supplement only, and one group was given a calcium supplement and vitamin D. Their health was tracked over five years. What was the result of this study?What was the purpose of the placebo?Lappe, J. M., Travers-Gustafson, D., Davies, K. M., Recker, R. R., & Heaney, R. P. (2007). The American Journal of Clinical Nutrition,85(6), 1586-1591.

25. Vemurafenib is a Therapy for MelanomaVemurafenib binds to the most common mutant of BRAF (BRAFV600E) and prevents it from activating its downstream target.

26. ProblemThis waterfall plot shows132 melanoma patients with the BRAFV600E mutation who were treated with Vemurafenib. Their tumor size was measured and plotted on this graph. Color relates to the metastatic stage of the melanoma. What is the relationship between severity of the cancer and efficacy of the drug?What is going on with the 11 patients at the far right of the graph, the ones whose lines go all the way down to -100?What is going on with the first 9 patients? What might explain their results?Would you take this drug if you had melanoma?M1a: Subcutaneous metastasisM2b: Lung metastasisM1c: Other visceral organ metastasis (brain, liver, kidney, etc.)Sosman, J. A., Kim, K. B., Schuchter, L., Gonzalez, R., Pavlick, A. C., Weber, J. S., Ribas, A. (2012). New England Journal of Medicine,366(8), 707-714.

27. Lung Cancer

28. LungsLungs are spongy because they are filled with alveoli. Each alveolus is a small sac that can inflate with air, like a tiny balloon. Blood vessels wrap around the alveoli to allow gas exchange via simple diffusion.

29. Lung CancerLung cancer is unusual in that it is often diagnosed very late. The two most common symptoms, cough and fatigue, are easily dismissed as unimportant health concerns.

30. There is Almost No Lung Cancer in HistoryBefore 1900, only 1/1000 autopsies showed lung cancer. That’s 0.1% of deaths due to lung cancer.

31. World War I Before World War I, pipes and cigars were the most common means of tobacco consumption. However, these were not convenient to use in the trenches. Soldiers began to switch to cigarettes, as the small packages were easy to carry and keep dry. An explosion of cigarette advertising around this time helped cement the transition.

32. Anything Boys Can Do…After World War I, men were smoking a lot of cigarettes, but smoking was still considered an unladylike activity. Tobacco companies saw an opportunity with the women’s suffrage movement. They began marketing directly to women, associating cigarette smoking with female independence and power. Women took the bait.

33. Nazis Got Exactly One Thing RightFritz Lickint (1898-1960) was a German physician who first reported a link between cigarette smoking and lung cancer. As a direct result of his work, the Nazi government launched the first ever state-sponsored anti-tobacco campaign in 1933.Question: Why did this knowledge and related anti-tobacco campaign not spread?

34. The Good Guys Figure it OutIn 1950, British scientists Austin Hill and Richard Doll were given the job of surveying lung cancer patients in London to try to figure out the cause of their disease. It was assumed that atmospheric pollution from cars and factories was the reason why lung cancer cases had spiked. Instead, they correlated lung cancer with cigarette consumption. Rumor has it that they only added the question about smoking history because the last page of the survey they were about to give out to lung cancer patients was half-empty!Question: If cigarette smoking began to increase in 1920, why did the British Medical Research Council wait until 1950 to sponsor this report?

35. ProblemInterpret these this figure. Explain the difference in the graphs between men and women.Doll, R., & Hill, A. B. (1950). Smoking and Carcinoma of the Lung. BMJ,2(4682), 739-748.

36. Tobacco Smoke Contains >60 Known CarcinogensThe most mutagenic is likely benzopyrene, which forms adducts (covalently linked groups) with DNA.Question: How will this adduct affect key DNA processes?

37. Most Smoking-Derived Cancers Involve EGFR MutationEGFR stands for epidermal growth factor receptor. Normally, EGFR becomes activated by auto-phosphorylation when bound by its ligand. In lung cancer, EGFR become constitutively activated.Question: How would the addition of a phosphate group activate a protein?

38. ProblemA population of never-smokers with lung carcinoma was surveyed for their exposure to Environmental Tobacco Smoke (aka Secondhand Smoke). These individuals were then tested for the presence of activating EGFR mutations in their lung tumors: “EGFR (+)”.What does this data show you about the relationship between ETS and EGFR activating mutations?How do you explain the data in the 0-27 year exposure category?Kawaguchi, T., Ando, M., Kubo, A., Takada, M., Atagi, S., Okishio, K., Sasaki, H. (2010). Clinical Cancer Research,17(1), 39-45.

39. Tarceva (Erlotinib) is a Therapy for Lung CancerErlotinib binds directly to the EGFR protein, preventing its activation.

40. ProblemIn Figure A of this study, mice injected with HCC827 lung carcinoma cells formed tumors. These mice were then treated with erlotinib (black) or control (white).In Figure C, protein extracts from HCC827 lung carcinoma cells were run out on a protein gel and the amount of protein determined via Western Blot. The p-EGF lane shows the amount of phosphorylated EGFR protein. The EGFR lane shows total EGFR protein. GAPDH is the loading control.Is Tarceva an effective therapy?By what mechanism do you think Tarceva acts?Tarceva therapy costs $100,000 per year. Do you think insurance companies should be required to pay?Iwai, T., Moriya, Y., Shirane, M., Fujimoto-Ouchi, K., & Mori, K. (2011). Oncology Reports,27(4), 923-928.

41. It’s Working!Aggressive public health campaigns, high cigarette taxes, bans on advertising, and social pressure have worked to drive down cigarette consumption. Smoking rates are way down in the USA as well as most developed countries, and still falling.

42. So…Smoking is Over, Right?Wrong. The cigarette companies just went elsewhere. More cigarettes are smoked now than ever before, just not here.Question: Why would people in countries such as China and India make good customers for cigarette manufacturers?

43. There are Other Ways to Get Lung Cancers~90% of lung cancers are associated with smoking. The other 10% come from other environmental factors.

44. Mining Has Long Been Associated with Lung DiseaseGeorgius Agricola, a pioneering German metallurgist and physician, wrote the book De re metallica in 1550. It is considered a pioneering work of mining and geology. In it, he makes the first mention of high incidence of respiratory disease among miners.

45. Uranium Miners Died in DrovesIn 1879, scientists Harting and Heese published a report of disease among the uranium miners of the Schneeberg Mines, in Germany. They determined that between 1869-1877, 150 out of 650 miners had died of a mysterious disease they named “Miner’s Disease”.

46. Radon is the CulpritRadon is a colorless, odorless gas. It is the natural breakdown product of uranium.

47. Radon Produces Ionizing RadiationIonizing radiation damages DNA in two ways:It can induce breaks directly.It increases production of free radicals, which also induce breaks.

48. ProblemIn this study, Czechoslovakian uranium miners were tracked from 1948-1975. At the end of this period, they were separated into 2 groups for analysis:Group A: High rate of exposure early on in their careers, then low rateGroup B: Constant rate of radon exposure throughout their mining careerWhat is the relationship between radon exposure and lung cancer?Is there a difference in lung cancer risk if overall radon exposure is the same but the time distribution is different?Additional Lung Cancer: The number of cancers that is more than statistically expected given the demographics of the groupWLM: A measure of radon exposure (1 WLM = 3.54 mJ h m-3)Kunz, E., Sevc, J., Placek, V., & Horacek, J. (1979). Health Physics,36(6), 699-706.

49. Bulldogs…We Have RadonRadon is a threat to everyone, not just miners. Radon is high in many parts of the country, especially right here. Radon gas from soil and bedrock collects in homes. Radon pumps are almost ubiquitous in our area (I have one!).Question: If radon predates cigarettes, why was the death rate from lung cancer only 0.1% until the 1900’s?

50. Radon-Derived Lung Cancer Often Show an EML4-ALK FusionThese are two unrelated genes who are located close to one another on Chromosome 2. Their proteins don’t seem to interact under normal circumstances, but radon’s ionizing radiation induces a breakage and religation event that places the two genes next to each other. This results in the transcription and translation of a single, fusion protein.

51. A Closer LookALK is a receptor tyrosine kinase (ring a bell?). It localizes to the cell membrane. When bound by its ligand, the ALK protein dimerizes and auto-phosphorylates, then activates growth pathways.EML4 is a microtubule-associated protein.

52. ProblemIn this study, column A shows the protein that was tagged with Yellow Fluorescent Protein (YFP). In B, the first column shows the cellular distribution of YFP, both magnified (rectangle) and zoomed out (square). The second column shows the cellular distribution of tubulin (tagged with a red fluorescent protein). The third column shows the merge of the two colored images. The overlap appears yellow, due to the magic of microscopy.In C, the localization of the YFP and RFP signals was plotted by imaging software. An R=1 indicates 100% overlap of the two signals.In your own words, interpret the four horizontal lines of this figure.This is wild-type EML4 (non-fusion) attached to YFPThis is wild-type ALK (non-fusion) attached to YFPThese are 2 different cancerous EML4-ALK fusions tagged with YFP (see how that gray tail is now attached?)Richards, M., Oregan, L., Roth, D., Montgomery, J., Straube, A., Fry, A., & Bayliss, R. (2015). Biochemical Journal,467(3), 529-536.

53. A Double-WhammyThe EML4-ALK fusion results in mis-localization and trimerization of ALKMislocalizationTrimerization

54. Result?The EML4-ALK fusion protein goes crazy, and starts activating growth through RAS and other downstream signaling molecules.

55. ProblemIn this study, the genomes of a number of lung tumors were sequenced, and their cancer-causing mutations identified. The mutations are represented as Venn diagrams. Overlapping regions indicate >1 common cancer mutation identified in a single tumor.Why don’t EGFR and ALK overlap?Sequist, L. V., Heist, R. S., Shaw, A. T., Fidias, P., Rosovsky, R., Temel, J. S., Dias-Santagata, D. (2011). Annals of Oncology,22(12), 2616-2624.

56. ProblemThis Kaplan-Meier curve shows the results of regular cytotoxic chemotherapy on lung cancer patients with the EGFR mutation, cancer patients with the EML4-ALK mutation, or lung cancer patients with neither mutation. What can you conclude about the use of chemotherapy in these patients?Morodomi Y., Takenoyama M., Inamasu E., Toyozawa R., Kojo M., Toyokawa G., Shiraishi Y., Takenaka T., Hirai F., Yamaguchi M., Taguchi K., Seto T., Sugio K.,and  Ichinose Y (2014). Anticancer Res. 34(7):3825-30

57. Alectinib is a Targeted Therapy for EML4-ALK Derived Lung CancerAlectinib prevents the phosphorylation of the EML4-ALK fusion, which prevents activation.