Addiction to protein kinase Cɩ due to PRKCI gene amplification can be exploited for an aptamer-based targeted therapy in ovarian cancer

PRKCI, the gene for protein kinase Cι (PKCι), is frequently amplified in ovarian cancer and recent studies have shown that PKCι participates in ovary tumorigenesis. However, it is unknown whether PKCι is differentially involved in the growth/survival between PRKCI-amplified and non-amplified ovarian cancer cells. In this study, we analyzed ovarian cancer patient dataset and revealed that PRKCI is the only PKC family member significantly amplified in ovarian cancer and PRKCI amplification is associated with higher PKCι expression. Using a panel of ovarian cancer cell lines, we found that abundance of PKCι is generally associated with PRKCI amplification. Interestingly, silencing PKCι led to apoptosis in PRKCI-amplified ovarian cancer cells but not in those without PRKCI amplification, thus indicating an oncogenic addiction to PKCɩ in PRKCI-amplified cells. Since small-molecule inhibitors characterized to selectively block atypical PKCs did not offer selectivity nor sensitivity in PRKCI-amplified ovarian cancer cells and were even cytotoxic to non-cancerous ovary surface or fallopian tube epithelial cells, we designed an EpCAM aptamer-PKCι siRNA chimera (EpCAM-siPKCι aptamer). EpCAM-siPKCι aptamer not only effectively induced apoptosis of PRKCI-amplified ovarian cancer cells but also greatly deterred intraperitoneal tumor development in xenograft mouse model. This study has demonstrated a precision medicine-based strategy to target a subset of ovarian cancer that contains PRKCI amplification and shown that the EpCAM aptamer-delivered PKCι siRNA may be used to suppress such tumors.

amounts to a final concentration of ~10-50µM of each oligonucleotide. The mixture was brought to 95°C in a heat block for a few minutes and then allowed to cool to room temperature.
Afterwards, these annealed, partially single-stranded templates generated were then used in the in vitro transcription kits (Promega). Aptamers were generated using RNA generated from 2 sequences, hence Oligo 1 and Oligo 2 listed above.

Visualization for Aptamers
The Forna package is an RNA secondary structure visualization tool that was used to edit and display the RNA-based structures of the aptamers generated in this study (http://rna.tbi.univie.ac.at/forna/).

Aptamer Synthesis
Control and PKCiota aptamers were individually synthesized by in vitro transcription (Promega) with phage promoter-containing synthetic oligonucleotides as templates (IDT). The oligonucleotides were PAGE or HPLC purified to remove partial sequences. The T7 RNA polymerase promoter is underlined and the EpCAM aptamer is bolded.

5' TAATACGACTCACTATAGCGACTGGTTACCCGGTCGT 3'
Each aptamer is composed of two RNA structures that were generated in separate in vitro transcription reactions but then were annealed together in a 1:1 molar ratio. Normal purine and pyrimidines were added to the mixture but 2'fluoro (F)-pyrimidines (TriLink Biotechnologies) were also added in a (1:4) ratio and the reaction mixture was adjusted to compensate for the additional volumes. The two RNAs were annealed to form one entity by heating at 94°C for 3 minutes followed by slowly cooling to room temperature within 1 hour. Annealed aptamers were stored in -80C. a b c d Figure S1. The status of PRKCI amplification is not associated with ovarian cancer patient survival or tumorigenic behaviors in established ovarian cancer cells. a. Overnight-cultured cells were trypsinized and added into 96-well plates and MTT assay was performed at 12 and 84 h after plating. The percent growth was expressed as OD at 84 h relative to value at 12 h. Data are mean ± SD. Mann-Whitney U-test was used to compare the difference between PRKCI-amplified and non-amplified groups. NS indicate no statistical significance. b. Overnight-cultured cells were subjected to Transwell assay. Cells on undersurface of upper chambers were stained and counted using the Imaris 7.0 imaging software. Data are means ± SD. The Mann-Whitney U-test was performed to compare the difference between PRKCI-amplified and non-amplified groups.  Cell growth analysis was performed on OSE and FTECs. The percent growth was expressed as 84-h values relative to 12-h values. The value of PKCι siRNAtreated cells were compared relative to Control siRNA-treated ones, which were normalized to 100%. Data are means ± SD. n = 4. Two-way ANOVA was used to analyze and NS indicates no statistical significance.          Once tumor was detected in the mice, they were divided into two groups (5 per group) and received either EpCAM-control or EpCAM-siPKCι aptamer thrice a week intraperitoneally (200nmole/mouse). Tumor outgrowth was monitored weekly using the Xenogen IVIS-200 In Vivo bioluminescence imaging system. Error bars represent standard errors. * indicates P < 0.005 vs EpCAM control.

Weeks of Treatment
Supplementary Data Figure S10