Promotion of tumorigenesis by Promotion of tumorigenesis by heterozygous disruption of the heterozygous disruption of the

beclin 1beclin 1 autophagy gene autophagy gene

The journal of Clinical InvestigationThe journal of Clinical Investigation 112:1809-1820 (2003)112:1809-1820 (2003)

Lien HsuLien Hsu

OutlinesOutlines

► Introduction----Introduction---- Autophagy Autophagy Beclin 1Beclin 1 HypothesisHypothesis

►Methods and ResultsMethods and Results►DiscussionDiscussion►CriticsCritics

Introduction----what is autophagy?

Autophagy (Autophagy (autonomous phagocytosis)

Functions: Functions:

I. I. allows cells to survive during starvationallows cells to survive during starvation II.II.enables cells to undergo structural remodeling enables cells to undergo structural remodeling during differentiation and development during differentiation and developmentIII.III.prevents agingprevents aging

► Defects of autophagy--?--Development of Defects of autophagy--?--Development of cancer cancer

Malignant cells-Malignant cells-------lower basal autophagic lower basal autophagic activity ; no increased protein degradation ratesactivity ; no increased protein degradation rates

Beclin 1Beclin 1 I.I. promotes starvation-induced autophagy in promotes starvation-induced autophagy in

human breast carcinoma cells human breast carcinoma cells

II.II. 17q21, a tumor-susceptibility locus 17q21, a tumor-susceptibility locus

III.III. Monoallelically deleted----in 40-75% of cases Monoallelically deleted----in 40-75% of cases of human sporadic breast, ovarian, and prostate of human sporadic breast, ovarian, and prostate cancercancer

HypothesisHypothesis

Inference----tumor suppressor?Inference----tumor suppressor?

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

Methods and ResultsMethods and Results

► Knock-out mice Knock-out mice beclin 1beclin 1 +/- +/-► +/- x +/- => F1----embryonic lethality of homozygous-deficient mice+/- x +/- => F1----embryonic lethality of homozygous-deficient mice

any malignancy

+/ -

+/+

Prevalence of macroscopic malignancies

All malignancies lung carcinoma hepatocellular carcinoma

Lymphomas (gray) andlymphoproliferative disease (black or white)

Beclin 1 heterozygous disruption in mice results in increased spontaneous tumorigenesis

Macroscopic malignancy

well-differentiated papillary lung well-differentiated papillary lung carcinoma in carcinoma in beclin 1beclin 1(+/-)(+/-) anti-Beclin 1(lung)anti-Beclin 1(lung)

anti-TTF-1(lung carcinoma):anti-TTF-1(lung carcinoma):specific transcription factor in specific transcription factor in bronchial and type II alveolar bronchial and type II alveolar epithelial cellsepithelial cells

Gross pathology of liver tumorGross pathology of liver tumor anti-Beclin 1(hepatocellular carcinomaanti-Beclin 1(hepatocellular carcinoma))

Lung carcinoma

Hepatocellular carcinoma

anti-Pax5 (dark purpleanti-Pax5 (dark purple

anti-CD3 (brown): DLCLanti-CD3 (brown): DLCL

anti-BCL-6: transcriptional repressor anti-BCL-6: transcriptional repressor controls germinal center formation: human B controls germinal center formation: human B cell lymphomacell lymphoma

Lymphoproliferative Lymphoproliferative disease in the disease in the thymusthymus

inset shows lymphoma adjacent to inset shows lymphoma adjacent to normal kidneynormal kidney

Lymphomas

Southern blot to detect wt and disrupted Southern blot to detect wt and disrupted beclin 1beclin 1 allele in tumor and normal tissuse allele in tumor and normal tissuse

*no deletion or rearrangement of remaining wt beclin 1 allele

Results suggest:Results suggest:►functional inactivation of one functional inactivation of one beclin 1beclin 1 is is

sufficient to promote tumorigenesis sufficient to promote tumorigenesis

►beclin 1beclin 1 is a haplo-insufficient tumor-suppressor is a haplo-insufficient tumor-suppressor genegene

preneoplastic small-cell dysplasia in the liver preneoplastic small-cell dysplasia in the liver (beclin 1+/- express HBV)(beclin 1+/- express HBV)

Extent of small-celldysplasia 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 lesionsThe model----I. Cross beclin +/- X beclin +/+ with HBV transgenesis (13m)II.liver is a major site of nutrient starvation-induced autophagy

Results suggest:►Beclin 1 heterozygous disruption in

mice accelerates the development of HBV-induced premalignant lesions

Beclin 1 heterozygous disruption results in increased cellular proliferation in vivo intraepithelial

Epithelial duct neoplasia adenomyoepithelioma acinar neoplasia

Terminal end budTEB

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.

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

Beclin 1 heterozygous disruption decrases autophagy in vivoGFP-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?

► Lymphocyte---no; liver---variably expressed; lung----typeII aveolar and bronchial epithelial cells

► Well-differentiated papillary lung carcinoma----show in bronchial cell origin

Results suggest----

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

DiscussionDiscussion► Autophagy genes may represent a novel class of tumor-Autophagy genes may represent a novel class of tumor-

suppressor genes.suppressor genes.

► The precise mechanisms by which the autophagy fuction The precise mechanisms by which the autophagy fuction of Beclin 1 contributes to tumor suppression is not of Beclin 1 contributes to tumor suppression is not known.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.

CriticsCritics►No normal histologic slides to compare.No normal histologic slides to compare.

►Why didn’t the authors mention if expression Why didn’t the authors mention if expression of Beclin 1 decreases in all neoplastic lesions of Beclin 1 decreases in all neoplastic lesions or not?or not?

►Is there any other possible autophagy-related Is there any other possible autophagy-related gene involved in tumorigenesis?gene involved in tumorigenesis?

►Is tumorigenesis really through any funtion of Is tumorigenesis really through any funtion of autophagy? or just because of autophagy? or just because of beclin 1?beclin 1?