Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene

1. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene The journal of Clinical Investigation 112:1809-1820 (2003) Lien Hsu

2. Outlines • Introduction---- AutophagyBeclin 1Hypothesis • Methods and Results • Discussion • Critics

3. Introduction----what is autophagy?

4. Autophagy (autonomous phagocytosis) Functions: I. allows cells to survive during starvationII.enables cells to undergo structural remodeling during differentiation and developmentIII.prevents aging • Defects of autophagy--?--Development of cancer Malignant cells----lower basal autophagic activity ; no increased protein degradation rates

5. Beclin 1 I. promotes starvation-induced autophagy in human breast carcinoma cells II. 17q21, a tumor-susceptibility locus III. Monoallelically deleted----in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer

6. Hypothesis Inference----tumor suppressor? *biallelic mutations of beclin 1 have not been demonstrated in human cancer~~ haplo-insufficient tumor suppressor gene?

7. Methods and Results • Knock-out mice beclin 1 +/- • +/- x +/- => F1----embryonic lethality of homozygous-deficient mice

8. Beclin 1 heterozygous disruption in mice results in increased spontaneous tumorigenesis Prevalence of macroscopic malignancies Macroscopic malignancy any malignancy All malignancies lung carcinoma hepatocellular carcinoma +/ - +/+ Lymphomas (gray) andlymphoproliferative disease (black or white)

9. Lung carcinoma anti-TTF-1(lung carcinoma):specific transcription factor in bronchial and type II alveolar epithelial cells well-differentiated papillary lung carcinoma in beclin 1(+/-) anti-Beclin 1(lung) Hepatocellular carcinoma Gross pathology of liver tumor anti-Beclin 1(hepatocellular carcinoma)

10. Lymphomas inset shows lymphoma adjacent to normal kidney Lymphoproliferative disease in the thymus anti-Pax5 (dark purple anti-CD3 (brown): DLCL anti-BCL-6: transcriptional repressor controls germinal center formation: human B cell lymphoma

11. Southern blot to detect wt and disrupted beclin 1 allele in tumor and normal tissuse *no deletion or rearrangement of remaining wt beclin 1 allele

12. Results suggest: • functional inactivation of one beclin 1 is sufficient to promote tumorigenesis • beclin 1 is a haplo-insufficient tumor-suppressor gene

13. Beclin 1 heterozygous disruption in mice “accelerates” the development of HBV (hepatisis B virus)-induced premalignant lesions The model---- I. Cross beclin +/- X beclin +/+ with HBV transgenesis (13m)II.liver is a major site of nutrient starvation-induced autophagy Extent of small-celldysplasia in liver HBV transgenic mice (13m) preneoplastic small-cell dysplasia in the liver (beclin 1+/- express HBV) +/+ HBV trangenic mice(white)+/- HBV transgenic mice(black)

14. Results suggest: • Beclin 1 heterozygous disruption in mice accelerates the development of HBV-induced premalignant lesions

15. Beclin 1 heterozygous disruption results in increased cellular proliferation in vivo intraepithelial Epithelial ductneoplasia adenomyoepithelioma acinar neoplasia beclin 1 heterozygous deficiency results in abnormal cellular proliferation in the TEBs and mammary ducts. Terminal end budTEB Mammary ducts Number Size Studies for pro-proliferation affects in germinal center formation: B lymphocyte

16. Result suggest: • beclin 1 heterozygous disruption increases cellular proliferation in vivo, beginning at an early age. Inference:the increased cellular proliferation in beclin 1+/– mice may increase the number of genetic mutations that occur over the lifetime of the animals, thereby contributing to the increased spontaneous tumorigenesis that occurs in older beclin 1+/– mice

17. Q: whether beclin 1 +/- affects its known function in autophagy? Beclin 1 heterozygous disruption decrases autophagy in vivo GFP-LC3 marker----Upon stimulation of autophagy, LC3 localizes to pre-autophagosomal membranes* The muscle has been shown to be an important site of starvation-induced autophagy 2m old 24hr starvation Q: whether beclin 1 heterozygous deletion affects autophagy in any of the tissues associated with increased spontaneous tumorigenesis?

18. Lymphocyte---no; liver---variably expressed; lung----typeII aveolar and bronchial epithelial cells • Well-differentiated papillary lung carcinoma----show in bronchial cell origin

19. Results suggest---- • beclin 1 heterozygous deletion reduces autophagic activity in a tissue that undergoes starvation-induced increases in autophagy (i.e. muscle)

20. Discussion • Autophagy genes may represent a novel class of tumor-suppressor genes. • The precise mechanisms by which the autophagy fuction of Beclin 1 contributes to tumor suppression is not known. • Autophagy may also contribute to tumor suppression by degrading specific cellular organelles and long-lived proteins that are essential for regulating cell growth, thereby functioning as a brake on cell growth in response to mitogenic signals.

21. Critics • No normal histologic slides to compare. • Why didn’t the authors mention if expression of Beclin 1 decreases in all neoplastic lesions or not? • Is there any other possible autophagy-related gene involved in tumorigenesis? • Is tumorigenesis really through any funtion of autophagy? or just because of beclin 1?