Article Figures & Data
Figures
- Figure 1.
Decreased 5-hmC levels are an epigenetic hallmark for malignancy and tumor progression in HGSOC. A–C, Comparative evaluation of 5-hmC staining in normal ovarian surface epithelium (OSE), normal fallopian tubal epithelium (FTE), and HGSOC patient tumors, respectively, as assessed by IHC studies. The bold arrows indicate normal OSE and ovarian stroma; positive stromal staining was used as an internal staining control (20 μm scale bar, 100× objective lens magnification). D, Quantification of 5-hmC staining scores for normal OSE (n = 5), normal FTE (n = 5), and HGSOC cases (n = 203). Interestingly, HGSOC tumors show significantly lower 5-hmC levels when compared with normal OSE and FTE, respectively (***, P < 0.001; **, P < 0.01). E, Representative 5-hmC levels in primary tumors and corresponding metastatic disease. Shown are examples of a primary tumor with low 5-hmC staining in both the primary tumor and metastatic lesion as well as a primary tumor with higher 5-hmC levels, which decrease upon metastasis (20 μm scale bar, 100× objective lens magnification). F, Quantification of 5-hmC staining scores in primary tumors and corresponding metastatic lesions when patient cohorts are separated by a primary tumor 5-hmC score of 6. Interestingly, patients with low primary tumor 5-hmC scores (n = 90) maintain low scores in metastatic disease, whereas patients with higher 5-hmC (n = 22) scores have significantly reduced 5-hmC levels in metastatic disease (**, P < 0.01; ***, P < 0.001). G, Same conclusions are reached if patient cohorts are separated by a primary tumor 5-hmC staining score of 2. Patients with low primary tumor 5-hmC scores (n = 71) tend to maintain low scores in metastatic disease, whereas patients with higher 5-hmC (n = 41) scores have significantly reduced 5-hmC levels in metastatic disease (**, P < 0.01; ***, P < 0.001).
- Figure 2.
Loss of 5-hmC is correlated with a shorter time to relapse following platinum-based chemotherapy and poor prognosis in patients newly diagnosed with HGSOC. A, Progression-free survival of patients with platinum response data (n = 59) shows a significant decrease in average time to disease relapse for patients with low 5-hmC (<2, n = 30) when compared to patients with high 5-hmC (≥2, n = 29). Patients with high 5-hmC levels experience a significantly longer time to initial disease relapse following platinum-based chemotherapy relative to low 5-hmC patients (**, P < 0.01). B, Kaplan–Meier survival curve of all patients with HGSOC included in the BWH TMA (n = 107). Patients with higher 5-hmC levels (primary tumor score ≥2, n = 54) have a significantly increased overall survival when compared with patients with low 5-hmC levels (primary tumor score <2, n = 53; ***, P < 0.0001).
- Figure 3.
Reversal of low 5-hmC profiles via TET2 overexpression restores platinum sensitivity. A, Representative immunoblot assay results showing global 5-hmC levels in A2780 platinum (CDDP) resistant (A2780Res), sensitive (A2780Sen), and resistant tumor cells with TET2 overexpression (A2780Res+pTET2). Platinum-resistant tumor cells exhibit lower 5-hmC levels when compared with their sensitive counterpart (**, P < 0.01). However, TET2 overexpression in A2780Res cells increases global 5-hmC to statistically higher levels than those seen in the parental A2780Res line (**, P < 0.01) and comparable with those seen in the platinum-sensitive A2780Sen line. B, Immunofluorescence analysis of 5-hmC levels in A2780Res, A2780Sen, and A2780Res+pTET2 tumor lines. Blue color indicates DAPI counterstain of DNA and green color shows 5-hmc levels. C, Relative TET2 mRNA expression in platinum A2780Res, A2780Sen, and resistant tumor cells with TET2 overexpression (A2780Res+pTET2) as assessed by qRT-PCR. Platinum resistant A2780Res tumor cells have significantly lower TET2 levels when compared with their sensitive A2780Sen counterpart (***, P < 0.001) and A2780Res+pTET2 line (***, P < 0.001). D, Comparative chemosensitivity profiles of A2780Res, A2780Sen, and A2780Res+pTET2 tumor lines as assessed by MTT. The CDDP IC50 value of the A2780Res+pTET2 line is similar to the CDDP sensitive counterpart (n.s. P > 0.05) and is significantly lower than the resistant parental line (**, P < 0.01). Thus, TET2 overexpression in the chemoresistant A2780Res line restores the 5-hmC levels and platinum sensitivity of the A2780Sen counterpart. E, A2780Res, A2780Sen, and A2780Res+pTET2 tumor lines were labeled with Hoechst 33342 dye and analyzed by flow cytometry. Control cells were treated with verapamil prior to Hoechst 333422 dye incubation. The A2780Res line has significantly more cancer stem-like cells (SP) when compared with its sensitive counterpart or resistant+pTET2 line (***, P < 0.001). F, A2780Res, A2780Sen, and A2780Res+pTET2 tumor lines were labeled with Aldefluor and analyzed by flow cytometry. Similarly, the A2780Res line has significantly more ALDH+ tumor cells when compared with its sensitive A2780Sen counterpart (*, P < 0.05) or resistant+pTET2 line (**, P < 0.01), respectively. G, A2780Res, A2780Sen, and A2780Res+pTET2 tumor lines were labeled with the LGR5 antibody and analyzed by flow cytometry. The A2780Res line has significantly more LGR5+ tumor cells as compared with its sensitive A2780Sen counterpart or A2780Res+pTET2 tumor lines (*, P < 0.05).
- Figure 4.
Pretreatment with 5-azacytidine increases global 5-hmC levels and platinum sensitivity. A, 5-hmC immunoblot and quantification of 5-hmC levels in A2780Res, CaOV3, OVCAR4, OVSAHO tumor lines treated for 72 hours with 5-azacytidine (5-aza). Methylene blue staining was used as a DNA loading control. Pretreatment with 5-aza significantly increases 5-hmC levels at all concentrations when compared with untreated (NT) controls in the tested cancer cell lines (*, P < 0.05; **, P < 0.01; ***, P < 0.001). B, The platinum sensitivity of ovarian cancer cell lines increases following pretreatment with 5-aza as shown by MTT analysis. Tumor lines, which were treated for 72 hours with 5, 10, or 20 μmol/L 5-aza followed by CDDP therapy, show statistically significant increases in CDDP IC50 values (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
- Figure 5.
Pretreatment with 5-azacytidine increases TET2 and TET3 expression in ovarian cancer cell lines. A, Comparative mRNA expression levels of TET1, TET2, and TET3 in the A2780Res tumor line following 5-aza treatment (concentrations of 5, 10, and 20 μmol/L, respectively) at various timepoints (treatment time of 12, 24, and 48 hours, respectively) as assessed by qRT-PCR. TET2 and TET3 are significantly upregulated at all concentrations and timepoints relative to untreated cells (NT) (**, P < 0.01; ***, P < 0.001). B, TET2 protein levels in A2780Res cells treated with 5-aza at concentrations of 5, 10, and 20 μmol/L for 12, 24, and 48 hours, respectively, as assessed by Western blot analysis. There is an increase in TET2 expression when compared with untreated (NT) cells. C–E, Comparative mRNA expression levels of TET1, TET2, and TET3 in OVCAR4, CaOV3, and OVSAHO ovarian cancer cell lines, respectively, following 12 hours of 5-aza (20 μmol/L) treatment as assessed by qRT-PCR. TET2 and TET3 are significantly upregulated in all cell lines following 5-aza treatment compared with untreated (NT) cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001). F, TET2 protein levels in ovarian cancer cell lines following treatment with 5-aza as assessed by Western blot analysis.
- Figure 6.
Pretreatment with 5-azacytidine followed by CDDP therapy increases overall survival in vivo in an aggressive platinum A2780Resistant tumor xenograft model. A, Comparative analysis of the tumor development timeline in animal models. A2780Res controls (n = 5) and animals treated with either 5-aza (n = 5) or CDDP monotherapy (n = 8) developed visible tumors in a significantly shorter time than mice treated with combination therapy (n = 8; **, P < 0.01), which received pretreatment with 5-aza followed by CDDP therapy. B, Kaplan–Meier curve showing survival for A2780Res controls (n = 5), 5-aza monotherapy (n = 5), CDDP monotherapy (n = 8), and 5-aza pretreatment + CDDP combination therapy (n = 8) cohorts. The combination group showed a statistically significant survival benefit compared with all the other groups. Of note, the overall survival of mice treated with combination therapy was significantly increased compared with either CDDP alone (*, P < 0.05) or 5-aza monotherapy (***, P < 0.001). C, Comparative analysis in a separate experiment of the average tumor weight in mice treated for 3 treatment cycles with either 5-aza (n = 4) or CDDP monotherapy (n = 6) when compared with combination therapy (n = 6). Most importantly, the analysis shows a significantly reduced tumor burden in A2780Res mice pretreated with 5-aza followed by CDDP therapy when compared with either CDDP alone (*, P < 0.05) or 5-aza alone (**, P < 0.01). D, Comparative IHC analysis of tumor markers in the various treatment cohorts. Tumors in the combination therapy group show increased 5-hmC levels, a lower Ki-67 cellular proliferation index, and higher tumor apoptosis as assessed by cleaved caspase-3 IHC (20× objective lens magnification).
Additional Files
Supplementary Data
- Additional Materials and Methods - Additional Materials and Methods
- Supplementary Figure 1 - IHC composite scoring system, cohort 5-hmC scoring system compared to normal OSE and FTE
- Supplementary Table 1 - Characteristics of all HGSOC cases included in the BWH TMA
- Supplementary Table 2 - Characteristics of HGSOC cases with platinum relapse data included in the BWH TMA
- Supplementary Figure 2 - Immunofluorescence studies on patient tumor ascites, quantification of 5-hmC from patient ascites by dot blot, 5-hmC measured with mass spectrometry
- Supplementary Figure 3 - Western blot analysis of A2780, IF quantification, immunoblot quantification in human and murine cell lines
- Supplementary Figure 4 - A2780 immunoblot and IC50 one week after 5-aza treatments, additional cell line IC50's
- Supplementary Figure 5 - In vivo IHC scores quantified
Volume 24, Issue 6
