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Beclin-1 expression is retained in high-grade serous ovarian cancer yet is not essential for autophagy induction in vitro.

Correa RJ, Valdes YR, Shepherd TG, DiMattia GE - J Ovarian Res (2015)

Bottom Line: Surprisingly, efficient siRNA-mediated Beclin-1 knockdown did not attenuate autophagy induction, whereas knockdown of other autophagy-related genes blocked the process.Beclin-1 knockdown instead decreased cell viability without inducing apoptosis.Taken together, these data demonstrate that despite its sustained expression, Beclin-1 is dispensable for autophagy induction in ovarian tumor cells in vitro, yet may be retained to promote cell viability by a mechanism independent of autophagy or apoptosis regulation.

View Article: PubMed Central - PubMed

Affiliation: Translational Ovarian Cancer Research Program, London Regional Cancer Program, London, Ontario, Canada. rcorrea2016@meds.uwo.ca.

ABSTRACT

Background: Autophagy is a conserved cellular self-digestion mechanism that can either suppress or promote cancer in a context-dependent manner. In ovarian cancer, prevalent mono-allelic deletion of BECN1 (a canonical autophagy-inducer) suggests that autophagy is impaired to promote carcinogenesis and that Beclin-1 is a haploinsufficient tumor suppressor. Nonetheless, autophagy is known to be readily inducible in ovarian cancer cells. We sought to clarify whether Beclin-1 expression is in fact disrupted in ovarian cancer and whether this impacts autophagy regulation.

Methods: BECN1 expression levels were assessed using The Cancer Genome Atlas (TCGA) datasets from 398 ovarian high-grade serous cystadenocarcinomas (HGSC) and protein immunoblot data from HGSC samples obtained at our institution. Knockdown of BECN1 and other autophagy-related gene expression was achieved using siRNA in established human ovarian cancer cell lines (CaOV3, OVCAR8, SKOV3, and HeyA8) and a novel early-passage, ascites-derived cell line (iOvCa147-E2). LC3 immunoblot, autophagic flux assays, transmission electron microscopy and fluorescence microscopy were used to assess autophagy.

Results: We observed prevalent mono-allelic BECN1 gene deletion (76%) in TCGA tumors, yet demonstrate for the first time that Beclin-1 protein expression remains relatively unaltered in these and additional samples generated at our institution. Surprisingly, efficient siRNA-mediated Beclin-1 knockdown did not attenuate autophagy induction, whereas knockdown of other autophagy-related genes blocked the process. Beclin-1 knockdown instead decreased cell viability without inducing apoptosis.

Conclusions: Taken together, these data demonstrate that despite its sustained expression, Beclin-1 is dispensable for autophagy induction in ovarian tumor cells in vitro, yet may be retained to promote cell viability by a mechanism independent of autophagy or apoptosis regulation. Overall, this work makes novel observations about tumor expression of Beclin-1 and challenges the accepted understanding of its role in regulating autophagy in ovarian cancer.

No MeSH data available.


Related in: MedlinePlus

Beclin-1 knockdown does not block autophagy induction in ovarian cancer cell lines with low, intermediate, and high levels of Beclin-1 expression. CaOV3, OVCAR8 eGFP-LC3, and HeyA8 cells were each transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. a Adherent cells were allowed to attach overnight, treated with DMSO or Akti-1/2 (5 μM) the next day, and harvested 24 h later to generate protein lysates. Immunoblots were performed for indicated proteins. b Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Protein lysates were generated and immunoblot performed for indicated proteins. Immunoblots depicted are representative of duplicate experiments
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Fig4: Beclin-1 knockdown does not block autophagy induction in ovarian cancer cell lines with low, intermediate, and high levels of Beclin-1 expression. CaOV3, OVCAR8 eGFP-LC3, and HeyA8 cells were each transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. a Adherent cells were allowed to attach overnight, treated with DMSO or Akti-1/2 (5 μM) the next day, and harvested 24 h later to generate protein lysates. Immunoblots were performed for indicated proteins. b Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Protein lysates were generated and immunoblot performed for indicated proteins. Immunoblots depicted are representative of duplicate experiments

Mentions: Rather than a single siRNA targeting BECN1, we chose to use a ‘SMARTpool’ of siRNAs in an attempt to ensure efficient knockdown. Transfection with siRNAs against BECN1 efficiently reduced its protein level in CaOV3, OVCAR8, and HeyA8 cells. Expression of the canonical autophagy regulatory proteins ATG5 and ATG7 was also efficiently reduced using RNA interference (Fig. 4). In adherent cells, knockdown significantly reduced levels of Beclin-1 protein, but this had no effect on Akti-1/2-mediated autophagy upregulation as LC3-II accumulation was not correspondingly blocked. This phenomenon was observed in all lines tested: CaOV3, OVCAR8, HeyA8 (Fig. 4a) as well as SKOV3 (Additional file 4: Figure S2A). Replicate immunoblots were quantified and statistically analyzed, demonstrating significant Beclin-1 knockdown (p < 0.001) but unchanged LC3-II (Additional file 5: Figure S3A). In contrast, knockdown of ATG5, ATG7, or their combination, effectively blocked LC3-II accumulation as expected (Fig. 4a). In SKOV3 cells, only knockdown of ATG7 effectively blocked LC3-II accumulation (Additional file 4: Figure S2A).Fig. 4


Beclin-1 expression is retained in high-grade serous ovarian cancer yet is not essential for autophagy induction in vitro.

Correa RJ, Valdes YR, Shepherd TG, DiMattia GE - J Ovarian Res (2015)

Beclin-1 knockdown does not block autophagy induction in ovarian cancer cell lines with low, intermediate, and high levels of Beclin-1 expression. CaOV3, OVCAR8 eGFP-LC3, and HeyA8 cells were each transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. a Adherent cells were allowed to attach overnight, treated with DMSO or Akti-1/2 (5 μM) the next day, and harvested 24 h later to generate protein lysates. Immunoblots were performed for indicated proteins. b Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Protein lysates were generated and immunoblot performed for indicated proteins. Immunoblots depicted are representative of duplicate experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4524172&req=5

Fig4: Beclin-1 knockdown does not block autophagy induction in ovarian cancer cell lines with low, intermediate, and high levels of Beclin-1 expression. CaOV3, OVCAR8 eGFP-LC3, and HeyA8 cells were each transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. a Adherent cells were allowed to attach overnight, treated with DMSO or Akti-1/2 (5 μM) the next day, and harvested 24 h later to generate protein lysates. Immunoblots were performed for indicated proteins. b Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Protein lysates were generated and immunoblot performed for indicated proteins. Immunoblots depicted are representative of duplicate experiments
Mentions: Rather than a single siRNA targeting BECN1, we chose to use a ‘SMARTpool’ of siRNAs in an attempt to ensure efficient knockdown. Transfection with siRNAs against BECN1 efficiently reduced its protein level in CaOV3, OVCAR8, and HeyA8 cells. Expression of the canonical autophagy regulatory proteins ATG5 and ATG7 was also efficiently reduced using RNA interference (Fig. 4). In adherent cells, knockdown significantly reduced levels of Beclin-1 protein, but this had no effect on Akti-1/2-mediated autophagy upregulation as LC3-II accumulation was not correspondingly blocked. This phenomenon was observed in all lines tested: CaOV3, OVCAR8, HeyA8 (Fig. 4a) as well as SKOV3 (Additional file 4: Figure S2A). Replicate immunoblots were quantified and statistically analyzed, demonstrating significant Beclin-1 knockdown (p < 0.001) but unchanged LC3-II (Additional file 5: Figure S3A). In contrast, knockdown of ATG5, ATG7, or their combination, effectively blocked LC3-II accumulation as expected (Fig. 4a). In SKOV3 cells, only knockdown of ATG7 effectively blocked LC3-II accumulation (Additional file 4: Figure S2A).Fig. 4

Bottom Line: Surprisingly, efficient siRNA-mediated Beclin-1 knockdown did not attenuate autophagy induction, whereas knockdown of other autophagy-related genes blocked the process.Beclin-1 knockdown instead decreased cell viability without inducing apoptosis.Taken together, these data demonstrate that despite its sustained expression, Beclin-1 is dispensable for autophagy induction in ovarian tumor cells in vitro, yet may be retained to promote cell viability by a mechanism independent of autophagy or apoptosis regulation.

View Article: PubMed Central - PubMed

Affiliation: Translational Ovarian Cancer Research Program, London Regional Cancer Program, London, Ontario, Canada. rcorrea2016@meds.uwo.ca.

ABSTRACT

Background: Autophagy is a conserved cellular self-digestion mechanism that can either suppress or promote cancer in a context-dependent manner. In ovarian cancer, prevalent mono-allelic deletion of BECN1 (a canonical autophagy-inducer) suggests that autophagy is impaired to promote carcinogenesis and that Beclin-1 is a haploinsufficient tumor suppressor. Nonetheless, autophagy is known to be readily inducible in ovarian cancer cells. We sought to clarify whether Beclin-1 expression is in fact disrupted in ovarian cancer and whether this impacts autophagy regulation.

Methods: BECN1 expression levels were assessed using The Cancer Genome Atlas (TCGA) datasets from 398 ovarian high-grade serous cystadenocarcinomas (HGSC) and protein immunoblot data from HGSC samples obtained at our institution. Knockdown of BECN1 and other autophagy-related gene expression was achieved using siRNA in established human ovarian cancer cell lines (CaOV3, OVCAR8, SKOV3, and HeyA8) and a novel early-passage, ascites-derived cell line (iOvCa147-E2). LC3 immunoblot, autophagic flux assays, transmission electron microscopy and fluorescence microscopy were used to assess autophagy.

Results: We observed prevalent mono-allelic BECN1 gene deletion (76%) in TCGA tumors, yet demonstrate for the first time that Beclin-1 protein expression remains relatively unaltered in these and additional samples generated at our institution. Surprisingly, efficient siRNA-mediated Beclin-1 knockdown did not attenuate autophagy induction, whereas knockdown of other autophagy-related genes blocked the process. Beclin-1 knockdown instead decreased cell viability without inducing apoptosis.

Conclusions: Taken together, these data demonstrate that despite its sustained expression, Beclin-1 is dispensable for autophagy induction in ovarian tumor cells in vitro, yet may be retained to promote cell viability by a mechanism independent of autophagy or apoptosis regulation. Overall, this work makes novel observations about tumor expression of Beclin-1 and challenges the accepted understanding of its role in regulating autophagy in ovarian cancer.

No MeSH data available.


Related in: MedlinePlus