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The BIRC6 gene as a novel target for therapy of prostate cancer: dual targeting of inhibitors of apoptosis.

Luk SU, Xue H, Cheng H, Lin D, Gout PW, Fazli L, Collins CC, Gleave ME, Wang Y - Oncotarget (2014)

Bottom Line: Two dASOs, targeting BIRC6+cIAP1 and BIRC6+survivin, showed substantial inhibition of CRPC cells proliferation, exceeding that obtained with single BIRC6 targeting.The growth inhibition was associated with increased apoptosis, cell cycle arrest and suppression of NFkB activation.Moreover, treatment with both dASOs led to significantly lower viable tumor volume in vivo, without major host toxicity.

View Article: PubMed Central - PubMed

Affiliation: The Vancouver Prostate Centre, Vancouver General Hospital and Department of Urologic Sciences, the University of British Columbia, Vancouver, BC, Canada; Department of Experimental Therapeutics, BC Cancer Agency, Vancouver, BC, Canada.

ABSTRACT
Treatment resistance, the major challenge in the management of advanced prostate cancer, is in part based on resistance to apoptosis. The Inhibitor of Apoptosis (IAP) family is thought to play key roles in survival and drug resistance of cancer via inhibition of apoptosis. Of the IAP family members, cIAP1, cIAP2, XIAP and survivin are known to be up-regulated in prostate cancer. BIRC6, a much less studied IAP member, was recently shown to be elevated in castration-resistant prostate cancer (CRPC). In the present study, we showed a correlation between elevated BIRC6 expression in clinical prostate cancer specimens and poor patient prognostic factors, as well as co-upregulation of certain IAP members. In view of this, we designed antisense oligonucleotides that simultaneously target BIRC6 and another co-upregulated IAP member (dASOs). Two dASOs, targeting BIRC6+cIAP1 and BIRC6+survivin, showed substantial inhibition of CRPC cells proliferation, exceeding that obtained with single BIRC6 targeting. The growth inhibition was associated with increased apoptosis, cell cycle arrest and suppression of NFkB activation. Moreover, treatment with both dASOs led to significantly lower viable tumor volume in vivo, without major host toxicity. This study shows that BIRC6-based dual IAP-targeting ASOs represent potential novel therapeutic agents against advanced prostate cancer.

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Dual IAP-targeting ASOs knockdown BIRC6, cIAP1 or survivin proteins and lead to marked suppression of CRPC cell proliferation(A-B) Western blotting showing protein levels of BIRC6, cIAP1 and survivin in two CRPC cell lines (A) PC-3 cells and (B) C4-2 cells transfected with Mock or increasing dosages of scrambled ASO (Scrb), dASOs 6w2 and 6w5 for 72 hr. (C) Comparison of dual IAP targeting and single IAP-targeting. Cell viability of PC-3 cells transfected with dASOs 6w2, 6w5 and siRNA-targeting BIRC6, cIAP1 or survivin, was determined by MTS assay at 72 hr after transfection. Cell viabilities of ASO- and siRNA-treated cells were normalized with corresponding Scrb ASO and siRNA controls. Error bars represent mean percentage cell viability ± S.D. Western blotting of 3 IAPs showing comparable amounts of reduced protein expression obtained with dASO and siRNA single IAP-targeting. (D) Proliferation of PC-3 cells transfected with mock, Scrb ASO, dASOs 6w2 and 6w5. Error bars represent mean cell number ± S.D. (E) MTS viability assay of C4-2 cells treated with dASOs.
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Figure 2: Dual IAP-targeting ASOs knockdown BIRC6, cIAP1 or survivin proteins and lead to marked suppression of CRPC cell proliferation(A-B) Western blotting showing protein levels of BIRC6, cIAP1 and survivin in two CRPC cell lines (A) PC-3 cells and (B) C4-2 cells transfected with Mock or increasing dosages of scrambled ASO (Scrb), dASOs 6w2 and 6w5 for 72 hr. (C) Comparison of dual IAP targeting and single IAP-targeting. Cell viability of PC-3 cells transfected with dASOs 6w2, 6w5 and siRNA-targeting BIRC6, cIAP1 or survivin, was determined by MTS assay at 72 hr after transfection. Cell viabilities of ASO- and siRNA-treated cells were normalized with corresponding Scrb ASO and siRNA controls. Error bars represent mean percentage cell viability ± S.D. Western blotting of 3 IAPs showing comparable amounts of reduced protein expression obtained with dASO and siRNA single IAP-targeting. (D) Proliferation of PC-3 cells transfected with mock, Scrb ASO, dASOs 6w2 and 6w5. Error bars represent mean cell number ± S.D. (E) MTS viability assay of C4-2 cells treated with dASOs.

Mentions: As BIRC2 (cIAP1), BIRC4 (XIAP) and BIRC5 (survivin) tended to be co-upregulated in prostate cancer in addition to BIRC6, simultaneous targeting of BIRC6 plus one of these IAP members was more likely to give superior anti-cancer effects. Accordingly, dual-targeting antisense oligonucleotides (dASOs), specifically targeting combinations of BIRC6 with each of the other three IAPs, i.e. 6w2, 6w4, 6w5, were tested for reduction of BIRC6 protein expression. As shown in Supplementary Fig. S3, only 6w2 and 6w5-1 markedly reduced BIRC6 protein levels in prostate cancer cells. The effects of these two dASOs on BIRC6, BIRC2 and BIRC5 protein levels were then tested by treating PC-3 and C4-2 cells with increasing doses of the dASOs. As shown in Figures 2A and 2B, treatment with dASO 6w2 (100 and 200 nM) resulted in marked, dose-dependent reductions in both BIRC6 and cIAP1 protein expression; similarly dASO 6w5-1 (100 and 200 nM) (in the following text referred to as 6w5), led to marked reductions in both BIRC6 and survivin protein expressions. A time course experiment showed that treatment of PC-3 cells with dASOs 6w2 and 6w5 resulted in a marked reduction in BIRC6 protein expression after 48 hours of transfection, whereas reduction in cIAP1 and survivin protein expressions by these dASOs started at 72 hours after transfection (Fig. S4).


The BIRC6 gene as a novel target for therapy of prostate cancer: dual targeting of inhibitors of apoptosis.

Luk SU, Xue H, Cheng H, Lin D, Gout PW, Fazli L, Collins CC, Gleave ME, Wang Y - Oncotarget (2014)

Dual IAP-targeting ASOs knockdown BIRC6, cIAP1 or survivin proteins and lead to marked suppression of CRPC cell proliferation(A-B) Western blotting showing protein levels of BIRC6, cIAP1 and survivin in two CRPC cell lines (A) PC-3 cells and (B) C4-2 cells transfected with Mock or increasing dosages of scrambled ASO (Scrb), dASOs 6w2 and 6w5 for 72 hr. (C) Comparison of dual IAP targeting and single IAP-targeting. Cell viability of PC-3 cells transfected with dASOs 6w2, 6w5 and siRNA-targeting BIRC6, cIAP1 or survivin, was determined by MTS assay at 72 hr after transfection. Cell viabilities of ASO- and siRNA-treated cells were normalized with corresponding Scrb ASO and siRNA controls. Error bars represent mean percentage cell viability ± S.D. Western blotting of 3 IAPs showing comparable amounts of reduced protein expression obtained with dASO and siRNA single IAP-targeting. (D) Proliferation of PC-3 cells transfected with mock, Scrb ASO, dASOs 6w2 and 6w5. Error bars represent mean cell number ± S.D. (E) MTS viability assay of C4-2 cells treated with dASOs.
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Show All Figures
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Figure 2: Dual IAP-targeting ASOs knockdown BIRC6, cIAP1 or survivin proteins and lead to marked suppression of CRPC cell proliferation(A-B) Western blotting showing protein levels of BIRC6, cIAP1 and survivin in two CRPC cell lines (A) PC-3 cells and (B) C4-2 cells transfected with Mock or increasing dosages of scrambled ASO (Scrb), dASOs 6w2 and 6w5 for 72 hr. (C) Comparison of dual IAP targeting and single IAP-targeting. Cell viability of PC-3 cells transfected with dASOs 6w2, 6w5 and siRNA-targeting BIRC6, cIAP1 or survivin, was determined by MTS assay at 72 hr after transfection. Cell viabilities of ASO- and siRNA-treated cells were normalized with corresponding Scrb ASO and siRNA controls. Error bars represent mean percentage cell viability ± S.D. Western blotting of 3 IAPs showing comparable amounts of reduced protein expression obtained with dASO and siRNA single IAP-targeting. (D) Proliferation of PC-3 cells transfected with mock, Scrb ASO, dASOs 6w2 and 6w5. Error bars represent mean cell number ± S.D. (E) MTS viability assay of C4-2 cells treated with dASOs.
Mentions: As BIRC2 (cIAP1), BIRC4 (XIAP) and BIRC5 (survivin) tended to be co-upregulated in prostate cancer in addition to BIRC6, simultaneous targeting of BIRC6 plus one of these IAP members was more likely to give superior anti-cancer effects. Accordingly, dual-targeting antisense oligonucleotides (dASOs), specifically targeting combinations of BIRC6 with each of the other three IAPs, i.e. 6w2, 6w4, 6w5, were tested for reduction of BIRC6 protein expression. As shown in Supplementary Fig. S3, only 6w2 and 6w5-1 markedly reduced BIRC6 protein levels in prostate cancer cells. The effects of these two dASOs on BIRC6, BIRC2 and BIRC5 protein levels were then tested by treating PC-3 and C4-2 cells with increasing doses of the dASOs. As shown in Figures 2A and 2B, treatment with dASO 6w2 (100 and 200 nM) resulted in marked, dose-dependent reductions in both BIRC6 and cIAP1 protein expression; similarly dASO 6w5-1 (100 and 200 nM) (in the following text referred to as 6w5), led to marked reductions in both BIRC6 and survivin protein expressions. A time course experiment showed that treatment of PC-3 cells with dASOs 6w2 and 6w5 resulted in a marked reduction in BIRC6 protein expression after 48 hours of transfection, whereas reduction in cIAP1 and survivin protein expressions by these dASOs started at 72 hours after transfection (Fig. S4).

Bottom Line: Two dASOs, targeting BIRC6+cIAP1 and BIRC6+survivin, showed substantial inhibition of CRPC cells proliferation, exceeding that obtained with single BIRC6 targeting.The growth inhibition was associated with increased apoptosis, cell cycle arrest and suppression of NFkB activation.Moreover, treatment with both dASOs led to significantly lower viable tumor volume in vivo, without major host toxicity.

View Article: PubMed Central - PubMed

Affiliation: The Vancouver Prostate Centre, Vancouver General Hospital and Department of Urologic Sciences, the University of British Columbia, Vancouver, BC, Canada; Department of Experimental Therapeutics, BC Cancer Agency, Vancouver, BC, Canada.

ABSTRACT
Treatment resistance, the major challenge in the management of advanced prostate cancer, is in part based on resistance to apoptosis. The Inhibitor of Apoptosis (IAP) family is thought to play key roles in survival and drug resistance of cancer via inhibition of apoptosis. Of the IAP family members, cIAP1, cIAP2, XIAP and survivin are known to be up-regulated in prostate cancer. BIRC6, a much less studied IAP member, was recently shown to be elevated in castration-resistant prostate cancer (CRPC). In the present study, we showed a correlation between elevated BIRC6 expression in clinical prostate cancer specimens and poor patient prognostic factors, as well as co-upregulation of certain IAP members. In view of this, we designed antisense oligonucleotides that simultaneously target BIRC6 and another co-upregulated IAP member (dASOs). Two dASOs, targeting BIRC6+cIAP1 and BIRC6+survivin, showed substantial inhibition of CRPC cells proliferation, exceeding that obtained with single BIRC6 targeting. The growth inhibition was associated with increased apoptosis, cell cycle arrest and suppression of NFkB activation. Moreover, treatment with both dASOs led to significantly lower viable tumor volume in vivo, without major host toxicity. This study shows that BIRC6-based dual IAP-targeting ASOs represent potential novel therapeutic agents against advanced prostate cancer.

Show MeSH
Related in: MedlinePlus