Cell surface CD55 traffics to the nucleus leading to cisplatin resistance and stemness by inducing PRC2 and H3K27 trimethylation on chromatin in ovarian cancer

Abstract

Background: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers.

Methods: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing.

Results: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2.

Conclusions: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.

Fig. 1

CD55 protein localizes in the nucleus of human ovarian cancer cells. (A) Generation of A2780 cancer stem cells from A2780 ovarian cancer cells using an established NANOG-GFP reporter system. Cancer stem cells were injected into NSG mice and tumors harvested at necropsy and fixed for FFPE analysis. (B) Tumor specimen from A2780 CSCs non-targeted control (NT), shRNA#1 and shRNA#2 were processed for hematoxylin & eosin (H&E) and immunohistochemical analysis for CD55. Yellow arrowheads denote CD55 nuclear staining. (C) Human ovarian tumor specimen (Clear cell carcinomas and Endometrioid ovarian carcinoma) collected and fixed. (D) Hematoxylin and eosin staining, and CD55 immunohistochemistry were performed. Yellow arrowheads denote CD55 nuclear staining. (E, F) CD55 protein expression in ascites cells from chemoresistant OC patients. CCF OC45, CCF OC61 and CCF OC88 ascites cells cultured, harvested, and lysed for immunoblot analysis of CD55. (G) CCF OC45, CCF OC61 and CCF OC88 cells were harvested, fractionated for nuclear and cytoplasmic pools, and immunobloted for CD55.  Lamin A/C and tubulin used as nuclear and cytoplasmic loading controls, respectively

Fig. 2

Cell surface CD55 is enriched in the nucleus of chemoresistant ovarian cancer cells. (A) A2780, CP70, and SKOV3 ovarian cancer (OC) were analyzed by immunofluorescence (IF) for CD55 protein localization. After fixation, cells were treated either with or without a permeabilizing agent (Triton X-100), followed by IF processing using a CD55 antibody. The cells were then counterstained with DAPI to visualize the nucleus. Cell surface CD55 expression is indicated with arrowhead and nuclear CD55 with an arrow. (B, C) Platinum sensitive (Designated as S) and resistant (Designated as R) ovarian cancer cells were lysed and whole cell, cytoplasmic, and nuclear fractions isolated followed by SDS-PAGE and immunoblotting for CD55 protein. Lamin A/C and Tubulin were used as nuclear and cytoplasmic marker respectively. (D) HEK293 and Jurkat cells were fractionated to enrich cytoplasmic and nuclear compartments, resolved by SDS-PAGE, and blotted for CD55. Lamin A/C was used as nuclear marker and Tubulin was used as cytoplasmic marker. (E) HEK293 cells were grown on coverslips, fixed with paraformaldehyde, treated with/without permeabilizing agent (Triton X-100) followed by IF for CD55. DAPI counterstaining was used to detect the nuclei. (F) Cytoplasmic and nuclear expression of CD59, a GPI-anchored protein in CP70 cells. (G) CP70 cells were treated with cycloheximide (50 µg/ml) for 0, 1, 3, and 6 h, followed by cell fractionation for cytoplasmic and nuclear isolation. Samples separated on SDS-PAGE followed by western blotting for CD55 protein expression corrected to 0 time point. Percentage of CD55 expression relative to initial CD55 protein are presented (Blots shown in supplementary Fig. 1I). Tubulin and lamin A/C immunoblots show relative enrichment of cytoplasmic and nuclear fractions respectively

Fig. 3

Nuclear CD55 is glycosylated and originates from the cell surface. (A) Cytoplasmic and nuclear proteins from ovarian cancer cells were fractionated. Fractionated proteins were then treated with protein deglycosylation mix II enzyme to remove glycosylation from CD55 proteins. Protein samples were processed for immunoblot analysis. (B) Ovarian cancer cells (OV81 and CP70) were subjected to 24-hour tunicamycin treatment. Subsequently, cytoplasmic, and nuclear proteins were fractionated, and CD55 protein expression was assessed through immunoblot analysis. (C) PIPLC treatment strategy to shed surface CD55 protein. (D, E, and F) CP70 cells were treated with increasing dose of PIPLC (0, 10, 25, 35 Unit/ml) at 37 °C for 2, 8, and 12 h. At indicated times, cells were harvested and fractionated, followed by separation by SDS-PAGE and CD55 immunoblotting. The resulting blots were quantified using Image J software. Data are representative of an experiment that was repeated 3 times

Fig. 4

CD55 S/T domain binds chromatin and drives cell proliferation, CSC frequency, and cisplatin resistance. (A) Wild type CD55 and domain deletion mutants were engineered and cloned into lentiviral vector, pLenti CMV Puro DEST. Lentiviruses were transduced into CD55 CRISPR KO CP70 cells and stable cell lines generated. Cells were cultured and fractionated to obtain cytoplasmic and nuclear pools. Cytoplasmic and nuclear fractions were separated on SDS-PAGE and immunoblotted for CD55 (Blots shown in supplementary Fig. 5A). Summary of findings displayed as either positive (+) or negative (-) for nuclear localization. (B) Immunofluorescence analysis of CD55 OE, ΔST, and Δ1234 (S/T only) transduced CP70 cells. CD55 protein localization was shown in red color. Nucleus was counter stained with DAPI (Blue). (C) CD55 immunoblot of CD55 OE, ΔST, and Δ1234 transduced CP70 KO cells. LaminA/C was used as loading control for nuclear fraction and Tubulin was used as a control for cytoplasmic fraction. (D) Cell proliferation analysis using Incucyte. KO, OE, and mutants ΔST, Δ1234 were analyzed over a 4-day period. (E) Stem cell frequency in tumorspheres was analyzed by limiting dilution assay. One way ANOVA was performed, and Tukey’s multiple comparison test was performed to determine p values (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (F) Cisplatin sensitivity assay of OE, KO, and mutants ΔST, Δ1234 transduced CP70 cells. Data was analyzed with GraphPad Prism and IC₅₀ values are indicated in parentheses. (G) CP70 parental and ∆1234 cells were harvested and cytosolic, membrane, soluble nuclear, and chromatin bound protein fractions were analyzed by immunoblot for CD55. Tubulin (Cytosolic), Na⁺/K⁺ ATPase (Membrane), Histone 3 (Chromatin) and Lamin AC (Nuclear) were used as loading markers for each fraction

Fig. 5

CD55 nuclear localization accelerates tumor growth and induce chemoresistance. (A) CP70 CD55 KO, OE, ∆1234, ∆S/T cells were intraperitoneally injected into NSG mice. Once tumors were detected, mice were randomized into either saline (Veh) or cisplatin (2 mg/kg) twice weekly. (B, C, D, E) Representative bioluminescence images of 3 tumor bearing mice from each cohort at 11, 25, and 39 days of the treatment in (B) OE cohort (C), KO cohort (D), ∆1234 cohort and (E) ∆S/T cohort. (F) Tumor growth kinetics of KO vs. OE mice treated with cisplatin or veh. (G) Tumor growth kinetics of ∆1234 vs. ∆ST mice treated with cisplatin or veh. (N = 9 mice/treatment). (H) Tumor FFPE sections from Veh treated KO, OE, ∆S/T and ∆1234 mice were analyzed for apoptosis (TUNEL assay), cell proliferation (Ki-67 immunohistochemistry, and H&E staining. (I) Tumor FFPE sections from Cisplatin treated KO, OE, ∆S/T and ∆1234 mice were analyzed for apoptosis (TUNEL assay), cell proliferation (Ki-67 immunohistochemistry, and H&E staining. (J) Analysis of Ki-67 positive cells in FFPE sections from KO, OE, ∆S/T and ∆1234 mice. Representative data from 5 different fields of Ki-67 FFPE section of each treatment group. (K) Analysis of TUNEL positive cells in FFPE sections from KO, OE, ∆S/T and ∆1234 mice. Representative data from 10 different fields of Ki-67 FFPE section of each treatment group. One way ANOVA was performed, and Tukey’s multiple comparison test was performed to determine p values (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Fig. 6

ZMYND8 is a binding partner of nuclear CD55. (A, B) CP70 cells were cultured and cytoplasmic and nuclear proteins were extracted. CD55 protein was immunoprecipitated (IP) from cytoplasmic and nuclear fractions. The immunoprecipitated protein samples were used to run SDS PAGE. The gels were sent for LCMS analysis to detect binding partners of CD55 protein. Spectral counts obtained from LCMS analysis indicated relative abundance of CD55 binding partners. ZMYND8 was identified as the most abundant binding partner of CD55 protein in the nucleus. (C) CP70 cells were transduced with empty vector (EV) or CD55, harvested and lysed followed by immunoprecipitation with CD55 and western blot for CD55 and ZMYND8. (D) CP70 cells, transduced with empty vector (EV) or CD55 were harvested and lysed followed by immunoprecipitation with ZMYND8 and western blot for ZMYND8 and CD55. (E) Expression of ZMYND8 and CD55 in KO cells. (F) ZMYND8 was knocked out by CRISPR, and cell proliferation analyzed by Incucyte. (G) Cisplatin sensitivity was assayed in CP70 cells. 95% Confidence Interval (CI) is 10.77–12.97. in ZMYND8 KO, 3.97–5.41 in parental cells. (H) Cisplatin sensitivity assay. (I) CSC frequency assay. Unpaired t test * p < 0.05. (J) CP70 cells (CD55 OE, CD55 KO, CD55∆1234, and CD55∆ST) were grown and immunoblot was performed. Quantification of ZMYND8 expression was also performed using image J software. One way ANOVA was performed, and Tukey’s multiple comparison test was performed to determine p values (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001). (K) Real time PCR was performed to check ZMYND8 gene expression in CP70 cells. (L) Progression free survival in relation to ZMYND8 expression in 1435 HGSOC and endometrioid patients determined using KMPlot. Number at risk, low = 1017 and high = 418. Hazards ratio and Logrank P indicated in the graph. (M) Overall survival in relation to ZMYND8 expression in 516 HGSOC and endometrioid patients determined using KMPlot. Number at risk, low = 1140 and high = 692. Hazards ratio and Logrank P indicated in the graph. (N) Reciprocal regulation of CD55 and ZMYND8

Fig. 7

Nuclear CD55 associates with PRC2 members and regulates H3K27me3 mark. (A) Expression of H3K27Me3 in cytoplasmic and nuclear fractions of CP70 CD55 KO and CP70 CD55 OE cells. Lamin A/C was used as nuclear marker and tubulin was used as cytoplasmic loading control. (B) Expression of PRC2 complex members, EZH2, SUZ12, EED, JRID2, AEBP2, and EZH1 in cytoplasmic and nuclear fractions of CP70 CD55 KO and CP70 CD55 OE cells. (C) GSEA plot after bulk RNA sequencing in CP70 CD55 KO and OE cells. PRC2 target genes were positively correlated with CD55 overexpression. (D) Differential gene expression of PRC2 target genes in OE and KO cells. (E) CD55 OE cells were cultured, and nuclear fractions were prepared. ZMYND8 protein was immunoprecipitated followed by immunoblot analysis for EZH2, SUZ12, and ZMYND8. (F) Nuclear lysates of OE cells were prepared followed by SUZ12 protein immunoprecipitation and immunoblot analysis of SUZ12, CD55, and EZH2. (G) Nuclear fractions were prepared from OE cells followed by immunoprecipitation with EZH2 protein and immunoblot analysis of EZH2, CD55, and SUZ12. Each experiment was performed three times. (H) CD55 interacts with ZMYND8 and PRC2 complex to modulate PRC2 target genes in ovarian cancer

Fig. 8

CD55 induces CSC activity and chemoresistance in Ovarian Cancers. Nuclear CD55 traffics to the nucleus from the cell surface. The S/T domain of CD55 is necessary for nuclear trafficking. In the nucleus, CD55 binds chromatin and interacts with ZMYND8 and PRC2 members and regulates PRC2 target gene expression. Nuclear CD55 drives epigenetic modifications, self-renewal, and platinum resistance in ovarian cancer cells