KLF6-SV1 inhibition induces apoptosis and synergizes with cisplatin in cancer cells. A, top, percentage of apoptosis in A2780 or A2780CP20 cells following treatment with increasing doses of cisplatin (1–50 μmol/L); bottom, Western blot analysis of A2780 or A2780CP20 cells after treatment with cisplatin (1–50 μmol/L). B, fluorescence-activated cell sorting of cells after treatment with siNTC, siSV1, cisplatin (50 μmol/L), or siSV1 plus cisplatin (50 μmol/L). C, SKOV3 cells were treated with the indicated agents and the percentage of apoptosis was determined. D, KLF6-SV1 inhibition does not induce apoptosis in noncancerous 293T fibroblasts.

KLF6-SV1 inhibition induces apoptosis through a NOXA-dependent mechanism. A, top, Western blot analysis of SKOV3 cell lysates following treatment with siNTC, siNTC plus cisplatin, siSV1, or siSV1 plus cisplatin; bottom, caspase-9 activity assay. B, suppression of siSV1-dependent apoptosis by caspase inhibitors. Following treatment of siSV1 with either a pan-caspase inhibitor, a caspase-8–specific (extrinsic marker) inhibitor, or caspase-9–specific (intrinsic marker) inhibitor, the percentage of apoptotic cells was measured. C, NOXA RNA and protein levels in SKOV3 cells following siNTC, siNTC plus cisplatin, siSV1, or siSV1 plus cisplatin treatment. D, quantitative reverse transcription-PCR (qRT-PCR) and Western blot analysis of NOXA and KLF6-SV1 following RNA interference (RNAi) transfection. Bottom, percentage of apoptosis followed by inhibition of NOXA, KLF6-SV1, or KLF6-SV1 in combination with NOXA. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

In vivo antitumor effects of KLF6-SV1 inhibition. A, top, treatment regimen; middle, quantitative real-time PCR for KLF6-SV1 and KLF6 expression levels in i.p. tumors; bottom, Western blot analysis of KLF6-SV1 protein levels in i.p. tumors after treatment with siNTC or siSV1. B, rate of growth of tumors treated with siNTC or siSV1, measured by in vivo molecular imaging. C, total bioluminescent signal from the abdominal region of treated mice at day 19 after the final dose of siSV1 or siNTC. D, total tumor mass. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

KLF6-SV1 inhibition increases survival in mice bearing i.p. tumors. A, top, treatment regimen; bottom, in vivo whole-body bioluminescence imaging of a subset of mice before (day 7) and after treatment (day 28) with either siNTC (3 mg/kg) plus cisplatin (5 mg/kg), siSV1 (3 mg/kg) plus cisplatin (5 mg/kg), or cisplatin (5 mg/kg) alone. B, rate of tumor growth of all three groups (cisplatin, n = 10; siNTC + cisplatin, n = 5; siSV1 + cisplatin, n = 9) using whole-body bioluminescence imaging values compared with the initial bioluminescent signal before treatment (day 7). C, Kaplan-Meier survival curves comparing PBS-treated (n = 8), siNTC plus cisplatin–treated (n = 5), siSV1 plus cisplatin–treated (n = 9), or cisplatin alone–treated (n = 5) mice. D, Kaplan-Meier survival curves comparing siNTC and siSV1 at escalating doses. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

KLF6-SV1 binds and regulates NOXA. A, top, Western blot analysis of KLF6, KLF6-SV1, NOXA, Mcl-1, PUMA, BAX, and actin in cell lysates from cells stably expressing pBABE (empty vector control) or pSV1 (KLF6-SV1) and in the presence or absence of cisplatin (50 μmol/L); bottom, qRT-PCR analysis of KLF6-SV1 and NOXA in pBABE or pSV1 cells treated with cisplatin (50 μmol/L). B, percentage of apoptosis in cells stably expressing pBABE or pSV1 in the presence or absence of cisplatin (50 μmol/L). C, top, coimmunoprecipitation assay of NH₂-terminal flag-tagged KLF6-SV1 (SV1-flag) and COOH-terminal V5-tagged NOXA (NOXA-V5); bottom, TAP assay of COOH-terminal streptavidin-tagged (SBP) and calmodulin-tagged (CBP) KLF6-SV1 (pCTAP-SV1) or pCTAP-empty. D, half-life studies examining the effect of KLF6-SV1 overexpression on NOXA stability. Cells overexpressing NOXA alone or in combination with KLF6-SV1 were treated with cycloheximide (CHX) and harvested at various time points (0, 30, 60, and 120 min). ***, P < 0.0005.

HDM2 mediates the degradation of KLF6-SV1 and NOXA following cisplatin. A, top, KLF6-SV1 and NOXA accumulation following MG132 treatment (proteasome inhibitor). KLF6-SV1 is ubiquitinated. 293T cells were transiently transfected with pV5-empty, pSV1-V5, and HA-tagged ubiquitin (HA-Ub) expression vectors and then treated with MG132 for 6 h. Bottom, equal protein amounts were immunoprecipitated with anti-HA antibody and Western blotted with anti-KLF6 antibody. B, MDM2 inhibition leads to KLF6-SV1 and NOXA accumulation. Cells were treated with either increasing amounts of a synthetic inhibitor of MDM2 (EMD) for 6 h or RNAi targeting nontargeting control or HDM2 and cell lysates were immunoblotted with KLF6-SV1, NOXA, and actin. C, suppression of MDM2 abolished cisplatin-induced degradation of KLF6-SV1 and NOXA. Cells were simultaneously treated with cisplatin (50 μmol/L) and the MDM2 inhibitor (40 μmol/L) for 12 h and for protein analysis. D, HDM2 coimmunoprecipitates with KLF6-SV1. Reciprocal coimmunoprecipitations were performed using 293T cell extracts overexpressing pSV1-V5 and pMDM2-flag or pSV1-V5 alone. Top, soluble protein extracts were immunoprecipitated (IP) with anti-V5 or anti-flag antibodies and analyzed by immunoblotting. Endogenous immunoprecipitation using SKOV3 cell extracts. Bottom, soluble protein extracts were immunoprecipitated using anti-MDM2 antibody and immunoblotted with an anti-KLF6 antibody or an anti-KLF6 antibody and immunoblotted with anti-MDM2 antibody. IgG immunoprecipitation was used as control.