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Breast cancer genes PSMC3IP and EPSTI1 play a role in apoptosis regulation.

Capdevila-Busquets E, Badiola N, Arroyo R, Alcalde V, Soler-López M, Aloy P - PLoS ONE (2015)

Bottom Line: A key element to delineate the biology of individual tumors is the regulation of apoptosis.We first explore the existence of direct physical interactions with annotated BC-apoptotic genes.Our results show that PSMC3IP and EPSTI1 are able to modulate the extrinsic apoptotic pathway in estrogen receptor positive and triple negative breast cancer cell lines, highlighting them as potential therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Joint IRB-BSC-CRG Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Catalonia, Spain.

ABSTRACT
A key element to delineate the biology of individual tumors is the regulation of apoptosis. In this work, we functionally characterize two breast cancer associated genes, the proteasome 26S subunit ATPase 3 interacting protein (PSMC3IP) and the epithelial-stromal interaction 1 (EPSTI1), to explore their potential apoptotic role in breast cancer. We first explore the existence of direct physical interactions with annotated BC-apoptotic genes. Based on the generated interaction network, we examine several apoptotic markers to determine the effect of PSMC3IP and EPSTI1 gene expression modulation in two different human breast cancer cell lines to suggest potential molecular mechanisms to unveil their role in the disease. Our results show that PSMC3IP and EPSTI1 are able to modulate the extrinsic apoptotic pathway in estrogen receptor positive and triple negative breast cancer cell lines, highlighting them as potential therapeutic targets.

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Caspase-3 activity modulation and analysis of cleaved PARP protein levels.(A) Caspase-3 activity was measured by colorimetric quantification of fluorescent products released from caspase-3 cleaved substrates.in TRAIL-treated MDA-MB-231 cells overexpressing XIAP, PSMC3IP or EPSTI1. None of the overexpressed genes was able to significantly decrease the activity relative to MYC-tag empty transfection vector (Vector) as control. (B) Caspase-3 activity was also measured in MDA-MB-231 cells under basal or TRAIL-treated conditions after gene silencing using specific siRNA targeting XIAP, PSMC3IP or EPSTI1. (C) Immunoblot analysis of cleaved PARP protein levels in gene-overexpressing MDA-MB-231 cells under TRAIL conditions, using MYC-tag transfection vector (Vector) as a negative control, reveals an attenuation of downstream apoptotic cascades. (D) This effect is much more pronounced in TRAIL-treated MCF-7 cells. (E) Analysis of cleaved PARP protein levels after gene silencing in MDA-MB-231 cells. siLUC was used as a negative control. (F) The same analysis in MCF-7 cells shows a more pronounced effect, even under basal conditions. EPSTI1-depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (*P <0.05, **P <0.01, ***P <0.001 vs MYC-tag vector in overexpression assays and vs siLUCIFERASE in silencing).
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pone.0115352.g005: Caspase-3 activity modulation and analysis of cleaved PARP protein levels.(A) Caspase-3 activity was measured by colorimetric quantification of fluorescent products released from caspase-3 cleaved substrates.in TRAIL-treated MDA-MB-231 cells overexpressing XIAP, PSMC3IP or EPSTI1. None of the overexpressed genes was able to significantly decrease the activity relative to MYC-tag empty transfection vector (Vector) as control. (B) Caspase-3 activity was also measured in MDA-MB-231 cells under basal or TRAIL-treated conditions after gene silencing using specific siRNA targeting XIAP, PSMC3IP or EPSTI1. (C) Immunoblot analysis of cleaved PARP protein levels in gene-overexpressing MDA-MB-231 cells under TRAIL conditions, using MYC-tag transfection vector (Vector) as a negative control, reveals an attenuation of downstream apoptotic cascades. (D) This effect is much more pronounced in TRAIL-treated MCF-7 cells. (E) Analysis of cleaved PARP protein levels after gene silencing in MDA-MB-231 cells. siLUC was used as a negative control. (F) The same analysis in MCF-7 cells shows a more pronounced effect, even under basal conditions. EPSTI1-depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (*P <0.05, **P <0.01, ***P <0.001 vs MYC-tag vector in overexpression assays and vs siLUCIFERASE in silencing).

Mentions: It is well known that the activation of initiator caspases, like caspase-8, leads to the activation of the executioner caspases, such as caspase-3 in MDA-MB-231 cells [42]. Therefore, we investigated whether PSMC3IP or EPSTI1 expression affects caspase-3 activity under basal or apoptotic conditions in MDA-MB-231 cells. We observed that overexpression of either gene does not alter caspase-3 activity levels (Fig. 5A). Yet, EPSTI1 silencing results in an increased caspase-3 activity in both basal conditions (2.6-fold, P<0.001) and upon TRAIL treatment (2.4-fold, P<0.001), giving similar results as the silencing of the anti-apoptotic gene XIAP. On other hand, PSMC3IP silencing is only able to increase caspase-3 activity under TRAIL treatment (1.5-fold, P<0.05) (Fig. 5B). These results indicate that indeed EPSTI1 and PSMC3IP modulate caspase-3 activity in MDA-MB.231 cells, albeit at varying degrees of apoptotic stimulation.


Breast cancer genes PSMC3IP and EPSTI1 play a role in apoptosis regulation.

Capdevila-Busquets E, Badiola N, Arroyo R, Alcalde V, Soler-López M, Aloy P - PLoS ONE (2015)

Caspase-3 activity modulation and analysis of cleaved PARP protein levels.(A) Caspase-3 activity was measured by colorimetric quantification of fluorescent products released from caspase-3 cleaved substrates.in TRAIL-treated MDA-MB-231 cells overexpressing XIAP, PSMC3IP or EPSTI1. None of the overexpressed genes was able to significantly decrease the activity relative to MYC-tag empty transfection vector (Vector) as control. (B) Caspase-3 activity was also measured in MDA-MB-231 cells under basal or TRAIL-treated conditions after gene silencing using specific siRNA targeting XIAP, PSMC3IP or EPSTI1. (C) Immunoblot analysis of cleaved PARP protein levels in gene-overexpressing MDA-MB-231 cells under TRAIL conditions, using MYC-tag transfection vector (Vector) as a negative control, reveals an attenuation of downstream apoptotic cascades. (D) This effect is much more pronounced in TRAIL-treated MCF-7 cells. (E) Analysis of cleaved PARP protein levels after gene silencing in MDA-MB-231 cells. siLUC was used as a negative control. (F) The same analysis in MCF-7 cells shows a more pronounced effect, even under basal conditions. EPSTI1-depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (*P <0.05, **P <0.01, ***P <0.001 vs MYC-tag vector in overexpression assays and vs siLUCIFERASE in silencing).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4295872&req=5

pone.0115352.g005: Caspase-3 activity modulation and analysis of cleaved PARP protein levels.(A) Caspase-3 activity was measured by colorimetric quantification of fluorescent products released from caspase-3 cleaved substrates.in TRAIL-treated MDA-MB-231 cells overexpressing XIAP, PSMC3IP or EPSTI1. None of the overexpressed genes was able to significantly decrease the activity relative to MYC-tag empty transfection vector (Vector) as control. (B) Caspase-3 activity was also measured in MDA-MB-231 cells under basal or TRAIL-treated conditions after gene silencing using specific siRNA targeting XIAP, PSMC3IP or EPSTI1. (C) Immunoblot analysis of cleaved PARP protein levels in gene-overexpressing MDA-MB-231 cells under TRAIL conditions, using MYC-tag transfection vector (Vector) as a negative control, reveals an attenuation of downstream apoptotic cascades. (D) This effect is much more pronounced in TRAIL-treated MCF-7 cells. (E) Analysis of cleaved PARP protein levels after gene silencing in MDA-MB-231 cells. siLUC was used as a negative control. (F) The same analysis in MCF-7 cells shows a more pronounced effect, even under basal conditions. EPSTI1-depleted cells were previously treated with IFN-α at 1000 U/ml for 8h. In apoptosis-induced conditions, MDA-MB-231 and MCF-7 cells were treated with TRAIL for 24h at 80 or 100ng/mL, respectively. XIAP was used as an anti-apoptotic reference in all experiments. Each bar represents the mean ±SD of three experiments performed in duplicate (*P <0.05, **P <0.01, ***P <0.001 vs MYC-tag vector in overexpression assays and vs siLUCIFERASE in silencing).
Mentions: It is well known that the activation of initiator caspases, like caspase-8, leads to the activation of the executioner caspases, such as caspase-3 in MDA-MB-231 cells [42]. Therefore, we investigated whether PSMC3IP or EPSTI1 expression affects caspase-3 activity under basal or apoptotic conditions in MDA-MB-231 cells. We observed that overexpression of either gene does not alter caspase-3 activity levels (Fig. 5A). Yet, EPSTI1 silencing results in an increased caspase-3 activity in both basal conditions (2.6-fold, P<0.001) and upon TRAIL treatment (2.4-fold, P<0.001), giving similar results as the silencing of the anti-apoptotic gene XIAP. On other hand, PSMC3IP silencing is only able to increase caspase-3 activity under TRAIL treatment (1.5-fold, P<0.05) (Fig. 5B). These results indicate that indeed EPSTI1 and PSMC3IP modulate caspase-3 activity in MDA-MB.231 cells, albeit at varying degrees of apoptotic stimulation.

Bottom Line: A key element to delineate the biology of individual tumors is the regulation of apoptosis.We first explore the existence of direct physical interactions with annotated BC-apoptotic genes.Our results show that PSMC3IP and EPSTI1 are able to modulate the extrinsic apoptotic pathway in estrogen receptor positive and triple negative breast cancer cell lines, highlighting them as potential therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Joint IRB-BSC-CRG Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Catalonia, Spain.

ABSTRACT
A key element to delineate the biology of individual tumors is the regulation of apoptosis. In this work, we functionally characterize two breast cancer associated genes, the proteasome 26S subunit ATPase 3 interacting protein (PSMC3IP) and the epithelial-stromal interaction 1 (EPSTI1), to explore their potential apoptotic role in breast cancer. We first explore the existence of direct physical interactions with annotated BC-apoptotic genes. Based on the generated interaction network, we examine several apoptotic markers to determine the effect of PSMC3IP and EPSTI1 gene expression modulation in two different human breast cancer cell lines to suggest potential molecular mechanisms to unveil their role in the disease. Our results show that PSMC3IP and EPSTI1 are able to modulate the extrinsic apoptotic pathway in estrogen receptor positive and triple negative breast cancer cell lines, highlighting them as potential therapeutic targets.

Show MeSH
Related in: MedlinePlus