Ecdysoneless Protein Regulates Viral and Cellular mRNA Splicing to Promote Cervical Oncogenesis

Abstract

High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, we identified Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, as a novel HPV16 E6-interacting protein. Here, we show that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. We demonstrate that ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, we show that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. We demonstrate that ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis-linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, our results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. IMPLICATIONS: This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.

Figure 1.

Interaction between HPV E6 Proteins and ECD. A,³⁵S-labeled E6 proteins were incubated with GST, GST-ECD, GST-E6AP, or GST-E6APmut and binding assay was done. B, 293T cells were transfected with FLAG-ECD or myc-HPV16 E6 alone or in combination and cell lysates were subjected to IP with anti-FLAG antibody and resolved by SDS-PAGE (right). 40 μg aliquots of lysates (left) were subjected to IB with anti-Myc (top) and anti-FLAG (bottom left). C, Schematic representation of deletion mutants. D,³⁵S-labeled ECD FL or mutant proteins were incubated with GST or GST-E6 and binding experiments were performed. E, 293T cells were transfected with FL-ECD or indicated mutants with or without Myc-16E6 or Myc-16E6 alone. Cell lysates were subjected to IP with anti-FLAG antibody and WB with anti-MYC or anti-FLAG antibodies.

Figure 2.

ECD expression in cervical cancers and HNSCC lines (A and B) and cervical tissues (C). A and B, WB of cell lysates from primary HFKs (lanes 1–3), immortalized HFKs by E6+E7 (lanes 4–6) or by TERT (lane 7) and cervical cancer cell lines (lanes 8 and 9), immortal keratinocytes HaCaT (B, lane 4), and HNSCC lines (B, lanes 5–11). C, Representative IHC staining of cervical tissue array for ECD expression in normal (i, ii); adenocarcinoma (iii, iv); adenosquamous carcinoma (v, vi); squamous cell carcinoma (vii and viii). Images, 100× magnification (inset 400×). D, IHC was scored using semi-quantitative H-score and histogram were plotted on the basis of the expression of ECD in different histopathologic types of cervical cancer. The number in the histogram represents number of patients included in the study. E, Kaplan–Meier survival analysis from TCGA data reveal ECD mRNA overexpression correlates with short patient OS (F) as well as disease-specific survival.

Figure 3.

ECD KD decreases oncogenic traits of HeLa cell line. Scrambled or two different ECD shRNAs (#1 and #2′ A–C,) or control or siRNA#1 or #2 expressing cells (D–F) were WB (A and D), and then analyzed for cell proliferation (B). C, Anchorage dependence assay followed by staining of colonies with crystal violet, counted, and plotted as histograms cells. Cells were plated on Boyden chambers for assessing their ability to migrate (E) or invade (F). Twenty-four hours later, migrated and invaded cells were stained with propidium iodide, counted, and plotted as histograms. ANOVA analysis indicated that mean number of colonies of control versus ECD KD cells were statistically different (all Tukey-adjusted P < 0.001). *, P ≤ 0.05; **, P ≤ 0.01, ***, P ≤ 0.001; and ns, nonsignificant. Mean ± SE was derived from three independent experiments, each done in triplicates. G and H, ECD cooperates with E7 to immortalize HFKs. G, WB of cell lysates with indicated antibodies; β-actin was used as a loading control. H, Cumulative population doublings (PD) is plotted over times from two independent experiments (Ex-I and Ex-II). I and J, qRT-PCR using specific primers of indicated genes from RNA samples of indicated cells over increasing passages (P). β-Actin was used as an internal control. Fold change with respect to control after normalizing with β-actin was calculated and plotted. Each experiment was repeated three times.

Figure 4.

ECD regulates E6 intron splicing. A, IGV visualization of HPV E6/E7 reads-distribution along with the HPV reference genome (left) and number of the averaged splice junction reads of E6*I and E6*II (right) in SiHa cells upon control or KD of ECD or PRPF8 (source data Supplementary Table S5). B, Number of the averaged E6 reads from the E6 intron region upon control, ECD KD or PRPF8 KD (source data Supplementary Table S5). SD of triplicate samples. C and E, SiHa cells were treated with control or siRNA against ECD or PRPF8, followed by RT-PCR analyses, and endpoint PCR products were run on 2% agarose gel. Representative images are shown. GAPDH was used as an internal control. Quantification of gel bands were determined by using ImageJ software (D and F) and WB of SiHa cell lysates (G–I) from control or KD of ECD, PRPF8, or DDX39A. GAPDH was used as a loading control.

Figure 5.

ECD regulates RNA splicing. A, Differential splicing events as determined by rMATS after KD of ECD or PRPF8 as compared with control. B, Overlap of DSGs in cells with KD of ECD or PRPF8. C, Percentage of exon/intron splicing defects in KD of ECD or PRPF8. D, Heat map shows concordance of splicing defects between KD of ECD and PRPF8. Colors represent fraction of genes in each ECD KD category and the overlap with each splicing category in PRPF8 KD. E–G, RT-PCR followed by endpoint PCR products run on 2% agarose gel electrophoresis in SiHa cells upon control or ECD KD. Quantification using ImageJ software. The values presented are normalized with actin (used as control). Sashimi plots were generated using RNA-seq datasets.

Figure 6.

ECD KD alters differential gene expression in SiHa cells. A, Venn diagram shows overlap of DEGs upon ECD or PRPF8 KD as compared with scrambled KD. B, GO results for genes that are increased (blue) or decreased (yellow) in ECD KD cells compared with control KD. C, GSEA results for the gene sets: response to type I IFN, and response to virus. Rank order is increased to decreased expression (left to right). D, IFN-related genes that are turned on upon ECD KD. Signal represents BPM normalized reads. E–H, qRT-PCR validation of indicated genes in SiHa cells upon control or ECD KD, data is represented as relative fold change normalized with β-actin. Significance of the ratios were calculated using Student two-tailed t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ns, nonsignificant. Mean ± SE was derived from three independent experiments, each done in triplicates.