PP4 inhibition sensitizes ovarian cancer to NK cell-mediated cytotoxicity via STAT1 activation and inflammatory signaling

Abstract

Background: Increased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in ovarian cancer (OC), the frequency of responses to immune checkpoint blockade (ICB) therapy in OC is much lower than other cancer types. Recent studies have highlighted that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity, which leads to enhanced responses to ICB. Protein phosphatase 4 (PP4) is a critical regulator of the DDR; however, its potential role in antitumor immunity is currently unknown.

Results: Our results show that the PP4 inhibitor, fostriecin, combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using multiple OC cell lines, we show that PP4 inhibition or PPP4C knockdown combined with carboplatin triggers inflammatory signaling via Nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 1 (STAT1) activation. This resulted in increased expression of the pro-inflammatory cytokines and chemokines: CCL5, CXCL10, and IL-6. In addition, IFNB1 expression was increased suggesting activation of the type I interferon response. Conditioned media from OC cells treated with the combination of PP4 inhibitor and carboplatin significantly increased migration of both CD8 T cell and natural killer (NK) cells over carboplatin treatment alone. Knockdown of stimulator of interferon genes (STING) in OC cells significantly abrogated the increase in CD8 T-cell migration induced by PP4 inhibition. Co-culture of NK-92 cells and OC cells with PPP4C or PPP4R3B knockdown resulted in strong induction of NK cell interferon-γ, increased degranulation, and increased NK cell-mediated cytotoxicity against OC cells. Stable knockdown of PP4C in a syngeneic, immunocompetent mouse model of OC resulted in significantly reduced tumor growth in vivo. Tumors with PP4C knockdown had increased infiltration of NK cells, NK T cells, and CD4⁺ T cells. Addition of low dose carboplatin treatment led to increased CD8⁺ T-cell infiltration in PP4C knockdown tumors as compared with the untreated PP4C knockdown tumors.

Conclusions: Our work has identified a role for PP4 inhibition in promoting inflammatory signaling and enhanced immune cell effector function. These findings support the further investigation of PP4 inhibitors to enhance chemo-immunotherapy for OC treatment.

Keywords: Genital Neoplasms, Female; Genome Instability; Immunity, Innate; Inflammation Mediators.

Figure 1. PP4 subunits are amplified in ovarian cancer. (A) Genomic profiling of PP4 across human cancers using TCGA data from cBioPortal. (B) PPP4C mRNA expression versus copy number from pan-cancer TCGA data is plotted as a bar graph, ****p<0.0001. (C) Comparison between transcript level expression of PP4 subunits (transcripts per million) compared between TCGA ovarian tumor cohort (n=418) and normal ovary (n=88) and fallopian tube (n=5) (GTEx); ns, not significant, ****p<0.0001. (D) i and ii, western blot showing expression of PP4 subunits across a panel of ovarian cancer cell lines. mRNA, messenger RNA; PP4, protein phosphatase 4; TCGA, The Cancer Genome Atlas.

Figure 2. PP4 inhibition or knockdown sensitizes ovarian cancer cell lines to carboplatin. (A) The mouse ovarian cancer cell line, ID8 (ID8-p53WT) and the isogenic p53 knockout cell line (ID8-p53KO) were transfected with control or PPP4C siRNA. Cell viability was measured by MTS assay at 96 hours. Results represent three replicates per experiment group, mean±SEM, *p<0.05, **p<0.01. PP4C knockdown following siRNA transfection was verified by western blot (inset). (B) ID8-p53KO cells were stably transduced with two shRNA constructs targeting PPP4C or control plasmid using lentivirus. The stable cell lines were treated with increasing doses of carboplatin and cell viability was determined at 96 hours by MTS assay. Western blot confirming the reduction in PP4 expression in shRNA transfected cell lines compared with vector control is shown (inset) (C) ID8-p53WT cells were pretreated±fostriecin (Fos) (1 nM) for 24 hours, followed by carboplatin treatment at the indicated doses. Cell proliferation was monitored using Incucyte S3 and the difference in mean cell confluence across treatment groups (n=6) is plotted, *p<0.0001. (D) Clonogenic survival assay of ID8-p53KO cells pretreated±Fos (1 nM), followed by carboplatin (10 µM) treatment, mean±SEM, ns, not significant, **p<0.01. Representative images are shown adjacent to the graph. (E) Human ovarian cancer cell lines OVCAR8 and OVCAR3 cells were pretreated±Fos (1 nM), followed by carboplatin (1 µM) and colony formation was determined. Mean values are shown from three independent experiments. Error bars show SEM for each group, *p<0.05, **p<0.01. (F) Cellular thermal shift assay assay with OVCAR4 and OVCAR3 cells±Fos (10 nM). The increase in protein stability was determined by western blot. Relative band intensities were calculated from images and represented as percentage relative to control. Data shown is an average of three biological replicates, *p<0.0001. MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PP4, protein phosphatase 4.

Figure 3. Pharmacological inhibition of PP4 augments carboplatin-induced DNA damage. (A) Ovarian cancer cell lines were pretreated with Fos (1 nM) for 24 hours followed by carboplatin at indicated doses, ID8-p53KO (10 µM), OVCAR4 (1 µM), OVCAR3 (1 µM), and OVCAR8 (2.5 µM). At 96 hours, micronuclei formation was quantified. Representative micrographs from ID8-p53KO cells depicting micronuclei are shown. The micronuclei count per 45–50 sites was averaged across conditions and represented as violin plots,*p<0.05, **p<0.01, **p<0.001, ****p<0.0001. (B) OVCAR3 cells were pretreated±Fos (1 nM), followed by carboplatin treatment at 2.5 µM for 96 hours and co-immunostained with FANCD2 and γ-H2AX. Representative images of γ-H2AX and FANCD2 foci across treatment conditions are shown. Image-based quantitation of foci counts was done across 70–90 sites and are shown in violin plots. ns, not significant, ****p<0.0001. (C) Left, schematic of DR-GFP assay. Right, representative flow plots showing GFP expression in OVCAR8-DR-GFP cells following transfection with pCβASceI plasmid and indicated treatment conditions. pCDNA3.1 was used as a transfection control. The percentage of cells expressing GFP fluorescence were quantified and efficiencies in homologous recombination were normalized to either untreated controls or control siRNA transfected cells. Values are mean+SEM. ***p<0.001, ****p<0.0001. Fos, fostriecin; PP4, protein phosphatase 4.

Figure 4. Loss of PP4 activity potentiates DNA damage-induced inflammatory signaling. (A) Representative western blot showing STAT1 phosphorylation at Y701 and S727 in OC cells pretreated±Fos (1 nM) for 24 hours followed by carboplatin treatment at the indicated doses on day 5. (B) STAT1 signaling shown following PPP4C siRNA transfection and carboplatin treatment on day 5. PP4C expression is also shown. (C) Representative western blot showing p65 phosphorylation at S536 in OC cells pretreated±Fos (1 nM) for 24 hours followed by carboplatin treatment at indicated doses on day 5. (D) Immunoblots of p-p65 following PPP4C siRNA transfection and carboplatin treatment on day 5. Actin is used as loading control. (E) RNA transcript levels of CCL5, CXCL10, IL-6 and IFNB1 were measured in OVCAR3 (left) and OVCAR8 (right) cells±PPP4C siRNA transfection and carboplatin treatment by quantitative, real-time PCR and relative fold change was calculated from three replicates. Values are mean±SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. (F) Cytokine array analysis with conditioned media collected from OVCAR3 cells transfected with either control or PPP4C siRNA±carboplatin. Spot densities of select cytokines are shown. Fos, fostriecin; OC, ovarian cancer; PP4, protein phosphatase 4; STAT, signal transducer and activator of transcription.

Figure 5. Protein phosphatase 4 knockdown in ovarian cancer cells enhances effector immune cell migration. (A) Left, schematic diagram of in vitro immune cell migration assay. Conditioned media was collected on day 5 from either ID8-p53KO (mouse) or OVCAR3 (human) cells pretreated±Fos for 24 hours followed by carboplatin treatment at the indicated doses: 10 µM (ID8-p53KO) and 1 µM (OVCAR3). The conditioned media from each treatment group was loaded in the bottom chamber. Right, fold change in migration was calculated relative to control, mean±SEM (n=3) is shown. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. (B) Conditioned media was collected on day 5 from either ID8-p53KO or OVCAR3 cells that were treated as described in (A). In parallel, CD8 T or NK-92 cells were pretreated with either MEKi (7.5 µM) or Raci (10 µM) or STAT3i (2 µM) for 15 min prior to adding in the transwell chamber. Conditioned media from each treatment group was loaded in the bottom chamber. Fold change in migration was calculated relative to control, mean±SEM (n=3) is shown. *p<0.05, ***p<0.001 and ****p<0.0001. (C) Left, schematic diagram of in vitro CD8 T-cell migration assay following STING1 siRNA transfection in ovarian cancer cells. OVCAR8 cells were transfected with control or STING1 siRNA±Fos and carboplatin. On day 5, conditioned media was collected and used in the lower chamber as chemoattractant. Human CD8 T cells were isolated from the ascites of a patient with ovarian cancer and used in the upper chamber. Right, fold change in migration (n=3) is shown, ns, not significant, *p<0.05 and ****p<0.0001. Fos, fostriecin; NK, natural killer; STAT, signal transducer and activator of transcription; STING, stimulator of interferon genes.

Figure 6. Loss of PP4 promotes NK cell activation and NK cell-directed ovarian cancer killing. OVCAR3 and OVCAR8 cells were transfected with either control or PPP4C or PPP4R3B siRNA, followed by carboplatin treatment at indicated doses: OVCAR3 (0.5 µM), OVCAR8 (2.5 µM). On day 5, cells were co-cultured with NK-92 (1:1) and immunostained for IFN-γ and TNF-α following PMA restimulation. (A) representative scatter plots of the cytokine production in OVCAR3 are shown. (B) relative fold changes in IFN-γ+ cells were calculated for OVCAR3 (n=3) and OVCAR8 (n=3), mean±SD is shown, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. (C) Left, representative flow scatter plots and, right, the percentage of CD45⁺ CD107a⁺ cells following NK-92 and OVCAR8 co-culture are shown as bar graph, ***p<0.001 and ****p<0.0001 (D) NK-92 mediated cytotoxicity assays. OVCAR8 cells (n=3) were transfected with control or PPP4C or PPP4R3B siRNA±carboplatin treatment (2.5 µM). NK-92 cells were added in a 1:1 ratio. After 3 hours, the cells were stained with annexin V and PI and the CD45-negative cells were analyzed. Representative flow plots and percentage of early and total apoptotic cells are shown for all treatment groups, mean±SD, ****p<0.0001. IFN, interferon; NK, natural killer; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; PP4, protein phosphatase 4; SSC-A, side scatter-area; TNF, tumor necrosis factor.

Figure 7. PP4C knockdown augments immune cell infiltration. (A) Left, schematic. Immune proficient C57BL/6 mice were injected with ID8-p53KO control and ID8-p53KO-ppp4shRNA clone 2 cells (n=6–7 mice). (i) Representative images for mice from respective groups showing relative tumor burden on day 26. (ii) Tumor growth was monitored by weekly measurement of luminescence (total flux) using IVIS, mean±SEM, *p<0.5, **p<0.01, ***p<0.001. (iii) Luminescence curves of individual mice measured over time. (B) i–ii, Primary omental tumor along with metastatic nodules were collected at time of necropsy, weighed, and shown as a bar graph, mean±SEM, *ns, not significant, p<0.05, **p<0.01. (C) Volume of ascites collected at necropsy is shown, ns, not significant, *p<0.05, **p<0.01. Tumors were disassociated and immune cell populations were identified by flow cytometry. (D, E) CD3^–CD161⁺ and CD3⁺CD161⁺ per 10³ live cells, mean±SEM, ns, not significant, *p<0.05. (F) Quantitation of IFN-γ in CD3−CD161+natural killer cells following ex vivo restimulation with PMA/ionomycin for 4 hours, mean±SEM, ns, not significant, *p<0.05. (G, H) CD4⁺ and CD8⁺ T cells per 10³ live cells, mean±SEM, **p<0.01, ****p<0.0001. (I) CD4⁺CD25⁺ Treg population per 10³ live cells, mean±SEM, IFN, interferon; IVIS, in vivo imaging system; ns, not significant; PMA, phorbol 12-myristate 13-acetate; PP4, protein phosphatase; Treg, regulatory T cell; .

Figure 8. Schematic diagram showing mechanism by which PP4C is involved in antitumor immunity. Loss of PP4 augments chemotherapy-induced DNA damage triggering a type I interferon response mediated by STING and STAT1 signaling. Increased chemokine production on inhibition or knockdown of PP4C led to improved immune cell migration both in vitro and in vivo and augmented natural killer cell-directed ovarian cancer cell killing in vitro. Created with Biorender.com. cGAS, cyclic GMP-AMP synthase; DSB, double strand break; Gzm, granzyme; IFN, interferon; IRF, interferon regulatory factor; NF-κB, nuclear factor kappa B; PFN, perforin; PP4, protein phosphatase 4; STAT, signal transducer and activator of transcription; STING, stimulator of interferon genes; TNF, tumor necrosis factor.