Limits...
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 reduces cell viability without altering autophagy or apoptosis. a iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. 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. Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Immunoblots were performed (n = 3 repeated experiments) with a representative blot depicted (unrelated, intervening lanes cropped). b iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1 and immunoblot performed to verify Beclin-1 knockdown in adherent cells (96 h post-transfection). c Viable cells were also counted at this time using Trypan Blue exclusion and normalized to control. Bars: Mean ± SEM (n = 3, *p < 0.05). d In spheroid cultures, viability was assessed using the CellTiter-Glo assay (7 days post-transfection and 72 h post-seeding to non-adherent culture). Cell viability data are normalized to controls. Bars: Mean ± SEM (n = 3, *p < 0.05). e Apoptosis was quantified using a Caspase 3/7 activity assay in adherent cultures at 96 h post-transfection. f Transfected cells were then seeded to non-adherent culture and apoptosis quantified in spheroids at indicated time points. Caspase 3/7 activity data were normalized to controls. Bars: Mean ± SEM (n = 3)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4524172&req=5

Fig6: Beclin-1 knockdown reduces cell viability without altering autophagy or apoptosis. a iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. 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. Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Immunoblots were performed (n = 3 repeated experiments) with a representative blot depicted (unrelated, intervening lanes cropped). b iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1 and immunoblot performed to verify Beclin-1 knockdown in adherent cells (96 h post-transfection). c Viable cells were also counted at this time using Trypan Blue exclusion and normalized to control. Bars: Mean ± SEM (n = 3, *p < 0.05). d In spheroid cultures, viability was assessed using the CellTiter-Glo assay (7 days post-transfection and 72 h post-seeding to non-adherent culture). Cell viability data are normalized to controls. Bars: Mean ± SEM (n = 3, *p < 0.05). e Apoptosis was quantified using a Caspase 3/7 activity assay in adherent cultures at 96 h post-transfection. f Transfected cells were then seeded to non-adherent culture and apoptosis quantified in spheroids at indicated time points. Caspase 3/7 activity data were normalized to controls. Bars: Mean ± SEM (n = 3)

Mentions: Our findings were confirmed in the ascites-derived, early-passage cell line iOvCa147-E2, which was also transfected with siRNAs against BECN1, ATG5, and ATG7. As before, significant Beclin-1 knockdown (p < 0.05) could not disrupt autophagy induced by Akti-1/2 (Fig. 6a, Additional file 5: Figure S3C) or spheroid formation (Fig. 6a, Additional file 5: Figure S3D). However, autophagy could be blocked by knockdown of ATG5 and ATG7, or their combination (Fig. 6a).Fig. 6


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 reduces cell viability without altering autophagy or apoptosis. a iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. 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. Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Immunoblots were performed (n = 3 repeated experiments) with a representative blot depicted (unrelated, intervening lanes cropped). b iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1 and immunoblot performed to verify Beclin-1 knockdown in adherent cells (96 h post-transfection). c Viable cells were also counted at this time using Trypan Blue exclusion and normalized to control. Bars: Mean ± SEM (n = 3, *p < 0.05). d In spheroid cultures, viability was assessed using the CellTiter-Glo assay (7 days post-transfection and 72 h post-seeding to non-adherent culture). Cell viability data are normalized to controls. Bars: Mean ± SEM (n = 3, *p < 0.05). e Apoptosis was quantified using a Caspase 3/7 activity assay in adherent cultures at 96 h post-transfection. f Transfected cells were then seeded to non-adherent culture and apoptosis quantified in spheroids at indicated time points. Caspase 3/7 activity data were normalized to controls. Bars: Mean ± SEM (n = 3)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4524172&req=5

Fig6: Beclin-1 knockdown reduces cell viability without altering autophagy or apoptosis. a iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. 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. Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Immunoblots were performed (n = 3 repeated experiments) with a representative blot depicted (unrelated, intervening lanes cropped). b iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1 and immunoblot performed to verify Beclin-1 knockdown in adherent cells (96 h post-transfection). c Viable cells were also counted at this time using Trypan Blue exclusion and normalized to control. Bars: Mean ± SEM (n = 3, *p < 0.05). d In spheroid cultures, viability was assessed using the CellTiter-Glo assay (7 days post-transfection and 72 h post-seeding to non-adherent culture). Cell viability data are normalized to controls. Bars: Mean ± SEM (n = 3, *p < 0.05). e Apoptosis was quantified using a Caspase 3/7 activity assay in adherent cultures at 96 h post-transfection. f Transfected cells were then seeded to non-adherent culture and apoptosis quantified in spheroids at indicated time points. Caspase 3/7 activity data were normalized to controls. Bars: Mean ± SEM (n = 3)
Mentions: Our findings were confirmed in the ascites-derived, early-passage cell line iOvCa147-E2, which was also transfected with siRNAs against BECN1, ATG5, and ATG7. As before, significant Beclin-1 knockdown (p < 0.05) could not disrupt autophagy induced by Akti-1/2 (Fig. 6a, Additional file 5: Figure S3C) or spheroid formation (Fig. 6a, Additional file 5: Figure S3D). However, autophagy could be blocked by knockdown of ATG5 and ATG7, or their combination (Fig. 6a).Fig. 6

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