Lethality of PAK3 and SGK2 shRNAs to Human Papillomavirus Positive Cervical Cancer Cells Is Independent of PAK3 and SGK2 Knockdown

<div><p>The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects.</p></div

Inhibition of HPV+ cervical cancer cells by lentiviral shRNAs that do not target human genes.

<p>Cell viability was assessed using a CellTiter Blue assay 5 days after infection. Each of the viruses was titrated for infection to determine its potency of inducing HeLa cell proliferation/viability inhibition. Percent cell viability (fluorescence) in the presence of lentiviral shRNAs was calculated by comparing with a control lentivirus without shRNA expression. Data represent the average ± standard deviation of 4 replicates from one of three independent experiments with similar results; (A) Inhibition of HeLa cell proliferation/viability; (B) Inhibition of proliferation/viability of CaSki cells.</p

Knockdown of PAK3 and SGK2 by shRNAs.

<p>mRNA levels were determined by quantitative reverse transcription PCR analysis at 72 hours after infection with the respective shRNA vectors; control denotes infection with a vector encoding non-target shRNA. Each bar on the bar graph represents the average ± standard deviation of 4 replicates from one of three independent experiments with similar results. mRNA levels were normalized with GAPDH expression; PAK3 and SGK2 protein expression were determined 72 hours after infection using a Western immunoblot. GAPDH protein acted as a protein loading control for each sample. (A) PAK3 shRNA decreased PAK3 mRNA levels in HeLa cells; (B) PAK3 shRNAs reduced PAK3 mRNA expression in DMS-79 cells; (C) PAK3 shRNAs reduced PAK3 protein expression in DMS-79 cells. DMS-79 cell lysates were analyzed with PAK3 monoclonal antibody N-19; (D) SGK2 shRNAs reduced SGK2 mRNA levels in HeLa cells; (E) SGK2 shRNAs reduced SGK2 mRNA levels in GTL16 cells; (F) SGK2 shRNAs reduced SGK2 protein levels in GTL16 cells. GTL16 cell lysates were analyzed with SGK2 monoclonal antibody 3Q-2.</p

Loss of cell viability induced by PAK3 or SGK2 shRNAs.

<p>(A) HeLa cells were infected with PAK3 shRNA at a 1:10 dilution. Cell apoptosis was determined using a caspase 3/7 glo luciferase assay 72 hours after infection. The bar graph presents fold changes of caspase 3/7 activity (luminescence) induced by various shRNAs compared with a no-shRNA control. Data represent the average ± standard deviation of 4 replicates from one of two separate experiments with similar results; (B) Inhibition of proliferation/viability of HeLa cells by PAK3 shRNAs was assessed using a CellTiter blue assay 5 days after infection. The bar graph presents percent viability (fluorescence) of lentiviral shRNA-infected cells compared with a control lentivirus without shRNA expression. Data represent the mean ± standard deviation of three independent experiments; (C) SGK2 lentiviral shRNAs induced HeLa cell apoptosis. Data represent 4 replicates from one of two separate experiments with similar results; (D) SGK2 shRNAs inhibited proliferation/viability of HeLa cells. Data represent the average ± standard deviation of two independent experiments; (E) SGK2 lentiviral shRNAs induced autophagy of HeLa cells. Cells were infected with SGK2 lentiviral shRNAs at 1:16 and 1:32 dilution, respectively. 1 μM Rapamycin was included as an autophagy control on each plate. Cell plates were fixed 72 hour after infection and immunostained for induction of autophagy with a LC3B primary antibody, followed with an Alexa 488-conjugated secondary antibody. Images were captured using the confocal Opera High Content Imager. Images of SGK2 lentiviral shRNA-infected HeLa cells (~1200 cells/well) were captured and cell numbers counted. The average intensity of LC3B staining per cell was measured and calculated using a modified Capella (Perkin Elmer) algorithm. The bar graph presents the average ± standard deviation of LC3B staining intensity derived from two wells.</p

Rescue of SGK2 shRNA-induced phenotypes.

<p>(A) HeLa cells were transfected with serially diluted SGK2 expressing plasmids harboring silent mutations at the shRNA 2111 annealing site. Six hours after transfection, the cells were infected with SGK2 lentiviral shRNA 2111 (1:15 dilution). SGK2 mRNA expression levels were determined 72 hours after infection. The bar graph presents quantities of SGK2 mRNA normalized with GAPDH mRNA. Data represent the average ± standard deviation of 4 replicates from one of two experiments with similar results; (B) HeLa cell apoptosis induced by SGK2 shRNA 2111 was quantified with a caspase 3/7 glo assay 72 hours after infection. Two variants of SGK2 (alpha and beta) were used for the phenotype rescue analysis. Data represent the average ± standard deviation of two independent experiments; (C) HeLa cell proliferation/ viability inhibition induced by SGK2 shRNA 2111 was determined using the CellTiter Blue assay 5 days after infection. Data represents the average ± standard deviation of two independent experiments.</p

Study shRNA lentivirus vectors.

<p>^a NA—not available</p><p>Study shRNA lentivirus vectors.</p

A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses

<div><p>A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC₅₀) values of 2 to 8 μM relative to IC₅₀ values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.</p></div