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---- Autophagy Beclin 1 Hypothesis ► Methods and Results ► Discussion ► Critics

3. Introduction----what is autophagy?

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

5. Beclin 1 I. promotes starvation-induced autophagy in human breast carcinoma cells I. promotes starvation-induced autophagy in human breast carcinoma cells II. 17q21, a tumor-susceptibility locus II. 17q21, a tumor-susceptibility locus III. Monoallelically deleted----in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer 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? * 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. any malignancy +/ - +/+ Prevalence of macroscopic malignancies All malignancies lung carcinoma hepatocellular carcinoma Lymphomas (gray) and lymphoproliferative disease (black or white) Beclin 1 heterozygous disruption in mice results in increased spontaneous tumorigenesis Macroscopic malignancy

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

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

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. preneoplastic small-cell dysplasia in the liver (beclin 1+/- express HBV) Extent of small-cell dysplasia in liver HBV transgenic mice (13m) +/+ HBV trangenic mice(white) +/- HBV transgenic mice(black) 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

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 duct neoplasia adenomyoepithelioma acinar neoplasia Terminal end bud TEB Mammary ducts Number Size Studies for pro- proliferation affects in germinal center formation: B lymphocyte beclin 1 heterozygous deficiency results in abnormal cellular proliferation in the TEBs and mammary ducts.

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. 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? Q: whether beclin 1 +/- affects its known function in autophagy ?

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?