Systemic administration of antisense oligonucleotides simultaneously targeting CK2α and α' subunits reduces orthotopic xenograft prostate tumors in mice

Abstract

CK2 is a highly conserved, ubiquitous, signal responsive protein serine/threonine kinase. CK2 promotes cell proliferation and suppresses apoptosis, and increased CK2 expression is observed in all cancers examined. We previously reported that direct injection of antisense (AS) CK2α phosphorothioate oligonucleotides (PTO) into xenograft prostate tumors in mice significantly reduced tumor size. Downregulation of CK2α in tumor cells in vivo appeared to result in overexpression of CK2α' protein. This suggested that in cancer cells downregulation of CK2α might be compensated by CK2α' in vivo, prompting us to design a bispecific (bs) AS PTO (bs-AS-CK2) targeting both catalytic subunits. bs-AS-CK2 reduced CK2α and α' protein expression, decreased cell proliferation, and induced apoptosis in cultured cells. Biodistribution studies of administered bs-AS-CK2 oligonucleotide demonstrated its presence in orthotopic prostate xenograft tumors. High dose injections of bs-AS-CK2 resulted in no damage to normal liver or prostate, but induced extensive cell death in tumor tissue. Intraperitoneal treatment with bs-AS-CK2 PTO decreased orthotopic tumor size and downregulated both CK2 mRNA and protein expression. Tumor reduction was accomplished using remarkably low doses and was improved by dividing the dose using a multi-day schedule. Decreased expression of the key signaling pathway proteins NF-κB p65 and AKT was also observed. We propose that the molecular downregulation of CK2 through bispecific targeting of the two catalytic subunits may be uniquely useful for therapeutic elimination of tumors.

Fig. 1 a

Downregulation of CK2α in prostate tumors causes increased expression of CK2α′. Whole cell lysates from orthotopic xenograft prostate tumors treated via tail vein injection with 200 μl of 2 mg/ml AS-CK2α phosphodiester OGN in PBS or 200 μl of PBS were subjected to immunoblot analysis using antibodies directed against CK2α/α′ and actin. Signal density was quantitated using Image J and normalized to actin. Average expression in AS-CK2α treated tumors for CK2α and α′ was 0.38 ± 0.10 and 0.88 ± 0.02 compared to 0.58 and 0.24 for PBS treated tumor. b A bispecific antisense sequence reduces cell viability better than α or α′-specific sequences alone. PTO (7.5 μM) were directly administered to PC3-LN4 cells grown on tenascin and fibronectin. Cell proliferation was assessed by means of ³H-Thy incorporation over a 24 h period. Both sense-CK2α and AS-GAPDH were included as controls. bs-AS-CK2 #1 and #2 represent different bispecific OGN sequences designed to simultaneously target CK2α and α′. Data are presented as the mean ± SEM

Fig. 2 a

bs-AS-CK2 downregulates both CK2α and α′ proteins in cultured PC3-LN4 prostate cancer cells. PC3-LN4 cells were transfected with 80 or 160 nM bs-AS-CK2 or 160 nM AS-RFP (red fluorescent protein) PTO using DOTAP reagent. Cells were collected at 48 and 72 h post-transfection and analyzed by immunoblot for CK2α and α′ and actin expression. b bs-AS-CK2 decreases DNA synthesis in cultured prostate cells. Cultured PC3-LN4 cells grown on tenascin and fibronectin and BPH-1 cells grown on laminin cells were transfected using Dharmafect reagent with 50 or 100 nM bs-AS-CK2 or AS-RFP PTO. After 48 h, ³H-Thy was added, and cells were harvested for counting at 72 h. *Significant difference from Dharmafect (P < 0.005). ^ Significant difference from AS-RFP (P < 0.05). c bs-AS-CK2 induces apoptosis in cultured PC3-LN4 prostate cancer cells. PC3-LN4 cells were transfected with 160 or 320 nM bs-AS-CK2 or 160 nM AS-GAPDH PTO using Dharmafect reagent. After 48 h, the cells were plated onto cover slips. Cells were fixed at 72 h post-transfection, and analyzed using a TUNEL staining kit

Fig. 3 a

Biodistribution of FITC-bs-AS-CK2 in prostate tumor-bearing mice. Mice (n = 3) bearing orthotopic xenograft PC3-LN4 prostate tumors were injected i.p. with 2 doses of FITC-labeled bs-AS-CK2 (66 μg/kg) 24 h apart. Tissues were collected 24 h after the second dose, weighed, and homogenized in lysis buffer. FITC fluorescence was quantified against a standard curve. Background was subtracted from mice processed equivalently but treated with PBS and dose ratios were calculated as % injected dose per gram wet tissue weight. Data is presented as the mean ± SEM. b Localization of FITC-bs-AS-CK2 in tumor tissue. Representative mouse tissue from the biodistribution study illustrates that FITC-labeled OGN was present in tumor at 24 h following the second dose. Tumors were paraffin embedded and processed for confocal immunofluorescence microscopy using a double-label strategy of anti-FITC (green) and anti-CK8 (red) antibodies to mark OGN and tumor, respectively. A–C represents a section processed with primary antibodies and D–F represents a section processed without primary antibodies at ×600

Fig. 4

Acute dosing regimens with bs-AS-CK2 indicate no damage to normal tissues. Tissue from normal, non-tumor-bearing animals (a, c, e) or tumor-bearing (b, d, f) were processed for H&E staining 10 days following acute cumulative dosing of bs-ASCK2, AS-GAPDH or saline given as two injections with a 24 h interval as labeled in the figure. Each treatment group included three mice. Images were captured at ×400

Fig. 5

Dose response of orthotopic prostate tumors following bs-ASCK2 PTO treatment. Mice bearing orthotopic PC3-LN4 prostate tumors were treated i.p. with decreasing cumulative doses starting from 132 μg/kg of PTO, either AS-GAPDH or bs-AS-CK2. Mice were dosed twice with a 24 h interval. Mice were killed 10–13 days after starting treatment. a Tumor weight comparisons between different treatment groups. Results are reported as means ± SEM for group sizes of 3–8 animals. b Immunoblot analyses of fractionated tumor lysates. Representative signals from nuclear matrix of two tumors in each treatment groups are shown. Lamin B was used as a loading control

Fig. 6

Tumor response to bs-AS-CK2 PTO treatment is schedule-dependent. Mice bearing orthotopic PC3-LN4 prostate tumors were treated i.p. with a single 66 μg/kg dose of AS-GAPDH PTO or with bs-AS-CK2 PTO using 1 of 4 different schedules: single dose of 66 μg/kg, 2 × 33 μg/kg doses spaced 1 d apart (q1d), 2 × 33 μg/kg doses spaced 4 d apart (q4d), or 4 × 16.5 μg/kg doses spaced 1 d apart (q1d). Mice were killed 13 days after starting treatment. a Tumor weight comparisons between different treatment groups. Results are reported as means ± SEM for group sizes of five animals. *Significant difference from control (P < 0.05). b Immunoblot analyses of fractionated tumor lysates. Representative signals from nuclear matrix or cytosol of two tumors in each treatment group are shown. The upper four blots represent nuclear matrix fraction, and the lower two blots represent cytosolic fraction. Lamin B and actin were used as loading controls for nuclear matrix and cytosol, respectively