KDM4C, a H3K9me3 Histone Demethylase, is Involved in the Maintenance of Human ESCC-Initiating Cells by Epigenetically Enhancing SOX2 Expression

Abstract

Our studies investigating the existence of tumor-initiating cell (TIC) populations in human esophageal squamous cell carcinoma (ESCC) had identified a subpopulation of cells isolated from ESCC patient-derived tumor specimens marked by an ALDHbri+ phenotype bear stem cell-like features. Importantly, KDM4C, a histone demethylase was enhanced in ALDHbri+ subpopulation, suggesting that strategies interfering with KDM4C may be able to target these putative TICs. In the present study, by genetic and chemical means, we demonstrated that, KDM4C blockade selectively decreased the ESCC ALDHbri+ TICs population in vitro and specifically targeted the TICs in ALDHbri+-derived xenograft, retarding engraftment. Subsequent studies of the KDM4C functional network identified a subset of pluripotency-associated genes (PAGs) and aldehyde dehydrogenase family members to be preferentially down-regulated in KDM4C inhibited ALDHbri+ TICs. We further supported a model in which KDM4C maintains permissive histone modifications with a low level of H3K9 methylation at the promoters of several PAGs. Moreover, ectopic expression of SOX2 restored KDM4C inhibition-dependent ALDHbri+ TIC properties. We further confirmed these findings by showing that the cytoplasmic and nuclear KDM4C staining increased with adverse pathologic phenotypes and poor patient survival. Such staining pattern of intracellular KDM4C appeared to overlap with the expression of SOX2 and ALDH1. Collectively, our findings provided the insights into the development of novel therapeutic strategies based on the inhibition of KDM4C pathway for the eliminating of ESCC TIC compartment.

Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

A subset of ESCC cells contained ALDHbri+ components which endowed with stem cell properties as evidenced in clonogenicity and self-renewal in vitro. (A) Representative Aldefluor analysis in primary patient-derived ESCC cells and KYSE-150 cell line. Control samples incubated with the inhibitor, DEAB, were used to ensure identification of ALDHbri+ and ALDHlow- cells. (B) Sphere formation efficiency of fluorescence-activated cell sorting (FACS)-sorted ALDHbri+ and ALDHlow- cells isolated from fresh samples and cell lines. (C) Representative images of ALDHbri+ and ALDHlow- derived spheres of EC-2 (scale bar = 50 μm). (D) Representative Aldefluor analysis in dissociated esospheres of EC-2 in serial propagations. Control samples incubated with the inhibitor, DEAB, were used to ensure identification of ALDHbri+ and ALDHlow- cells. (E) Representative images of ALDHbri+ and ALDHlow- spheres of EC-2 in three passages (scale bar = 50 μm). (F). ALDHbri+ and unseparated subpopulations of EC-2 were capable of self-renewal in vitro, as shown by similar esosphere-initiating capacity in three passages.

Figure 2

A subset of ESCC cells contained ALDHbri+ components which were tumorigenic in vivo. (A) Tumorigenesis of ALDHbri+ and ALDHlow- in NOD/SCID mice. Tumor growth curves were plotted for the numbers of engrafted cells (10,000, 1000, and 100 cells) and for each subpopulation (ALDHbri+ and ALDHlow-). Tumor growth kinetics correlated with the latency and size of tumor formation and the number of ALDHbri+ cells. No tumor was detected at the ALDHlow- cells' injection site (100 cells injected). Data represent mean ± SEM (n = 3). P < .05 compared with ALDHlow-. (B) Representative bioluminescent images of mice harboring tumors are shown. To evaluate the tumorigenesis of ALDHbri+ and ALDHlow-in vivo, we infected both cells with a lentivirus expressing luciferase, and inoculated 10,000, 1000 and 100 luciferase-tagged cells into NOD/SCID mice. Tumor formation was measured at weekly intervals following inoculation, revealed a statistically significant increase (P < .01) in tumoral formation in ALDHbri+ compared with ALDHlow- cells. (C) Differentiation of ALDHbri+ and ALDHlow- cells in vivo. Sorted ALDHbri+ and ALDHlow- cells from EC-2 and EC-3 were implanted into NOD/SCID mice and the xenografts derived from ALDHbri+ and those from ALDHlow- cells were analyzed by Aldefluor assay. (D) Histology and KDM4C IHC analysis of primary tumor and tumor xenograft generated from ALDHbri+ cells isolated from EC-2 and EC-3. The ALDHbri+ population was capable of regenerating the phenotypic heterogeneity of the initial tumor after a passage in NOD/SCID mice.

Figure 3

KDM4C inhibition decreased the percentage, clonogenicity and self-renewal of ESCC ALDHbri+ TICs in vitro. (A) Quantification of the fraction of ALDHbri+ cells, determined by Aldefluor assay in patient-derived ESCC cells transduced with LV-c, LV-shKDM4C-5 or LV-shKDM4C-7. Significant reduction in the percentage of ALDHbri+ cells after silencing of KDM4C is shown. (B) Representative images of spheres formed by patient 2-derived ALDHbri+ TICs transduced with LV-c, LV-shKDM4C-5 or LV-shKDM4C-7. (Scale bar = 50 μm). Decreased Number and size of spheres are shown in ALDHbri+ cells after silencing of KDM4C. (C) ALDHbri+ cells were capable of self-renewal in vitro, as shown by similar esosphere-initiating capacity in three passages of EC-2. Significant reduction in the self-renewal of ALDHbri+ cells after silencing of KDM4C is shown. (D) Quantification of the fraction of ALDHbri+ cells, determined by Aldefluor assay in patient-derived ESCC cells treated with vehicle, 5 μM, 10 μM or 20 μM KDM4C inhibitor CaA. Significant reduction in the percentage of ALDHbri+ cells after CaA treatment is shown. (E) Representative images of spheres formed by patient 2-derived ALDHbri+ cells treated with vehicle, 5 μM, 10 μM or 20 μM CaA. (Scale bar = 50 μm). Decreased Number and size of spheres are shown in ALDHbri+ cells upon CaA treatment in a dose-dependent manner. (F) ALDHbri+ cells of EC-2 were capable of self-renewal in vitro, as shown by similar esosphere-initiating capacity in serial passages. Significant reduction in the self-renewal of ALDHbri+ cells after CaA treatment is shown. Data shown are the mean ± S.E.M. of at least three independent experiments. *P⩽0.05; **P⩽0.01.

Figure 4

The KDM4C inhibitor CaA preferentially abolished TIC in ALDHbri+-derived xenograft model in vivo. (A) Representative images of subcutaneous xenografts in nude mice taken at the same time for each group. For each xenograft, 10,000 cells were injected into the mammary fat pad of nude mice. There is a statistically significant size reduction of the tumors treated with 5 mg/kg or 10 mg/kg CaA compared with the control tumors (P < .01, respectively). (B) Tumor growth curves were plotted for the numbers of engrafted cells (10,000 FACS-sorted ALDHbri+ cells). There is a statistically significant size reduction of the tumor treated with 5 mg/kg or 10 mg/kg CaA compared with the controls during the course of each indicated time points (P < .01, respectively). (C) Mice bearing injection were treated with either vehicle or CaA (5 mg/kg or 10 mg/kg) three times a week for a total of 8 weeks. Cells were dissociated from tumors and subject to Aldefluor assay. Shown are cytograms of percent ALDHbri+ cells. (D) Xenotransplants from each group were collected, and ALDH1 IHC staining was done. Cytoplasmic ALDH1 expression was detected in the controls (arrowheads denote positive staining), whereas low expression was detected in the tumors treated with CaA. (E) Representative bioluminescent images of mice harboring tumors are shown (Upper panel). To evaluate the effect of CaA treatment on tumor initiation, we infected ALDHbri+ cells with a lentivirus expressing luciferase, and inoculated 10,000 luciferase-tagged cells into NOD/SCID mice. One week after the initial tumor inoculation, tumor-bearing animals were begun administered intraperitoneally with CaA (5 mg/kg or 10 mg/kg) or saline controls three times a week for a total of 8 weeks. Tumor formation was monitored using bioluminescence imaging. Quantification of the normalized photon flux (Lower panel), measured at weekly intervals following inoculation, revealed a statistically significant decrease (P < .01, respectively) in tumoral formation in CaA-treated compared with controls for mice inoculated with luciferase-tagged ALDHbri+ cells.

Figure 5

Profiling of KDM4C knockdown and control ALDHbri+ ESCC TICs. (A) Venn diagrams for genes that are down-regulated by KDM4Csh knockdown or CaA treatment. ALDHbri+ cells were treated with shKDM4C or CaA. The mRNA levels in KDM4C-depleted cells were measured by Agilent Human Whole Genome Microarrays and compared with those in the controls. (B) Heatmap shows unsupervised clustering of down-expressed transcripts obtained for the ALDHbri+ ESCC cells after KDM4C knockdown (right) or mock transfection (left) using Agilent Human Whole Genome Microarrays. Colored spots indicate up-regulated (red) or down-regulated (blue) genes from microarray analysis. (C) Heatmap shows Gene ontology analysis of genes that are co-down-regulated by KDM4C knockdown and CaA treatment. Gene ontology analysis was performed using the program DAVID. (D) qPCR validation of differentially expressed genes. Expression levels were normalized to GAPDH. White and black and bars represent expression in controls and KDM4C knockdown ALDHbri+ cells, respectively (P < .01, respectively). (E) Immunostaining of control and KDM4C-shRNA ALDHbri+ cells. KDM4C-shRNA cells have decreased KDM4C, SOX2 and ALDH1 expression relative to control cells. Nuclei were stained with DAPI. (Scale bar = 50 μm).

Figure 6

Increased H3K9 methylation levels at the pluripotency-associated gene promoters in response to KDM4C inhibition in ALDHbri+ cells. ChIP assays were performed on ALDHbri+ cells following KDM4C knockdown (via transfection with control shRNA, KDM4CshRNA-5, KDM4CshRNA-7) or exposure or not exposure to CaA for various concentrations, as indicated. Cells were then collected for ChIP analyses using antibodies to the indicated H3K9 methylation forms to determine H3K9 methylation levels at the SOX2, c-Myc, and Pou5f1 promoters. (A) Representative agarose gels showing PCR amplification products corresponding to the SOX2 promoter region is shown. Specific antibodies that individually recognize either the di- (H3K9me2) or the trimethylated form of H3K9 (H3K9me3) were used. GST antibody was used as a control. (B) ChIP analysis of H3K9 methylation levels at the SOX2 promoter after KDM4C inhibition as quantified by real-time PCR. Relative promoter occupancies (% input) are shown with error bars based on standard errors (SEs) calculated from at least three replicates. The input signal is set as 100% (not depicted in graphs) for each set of assays. (C) Representative agarose gels showing PCR amplification products corresponding to the c-Myc promoter region is shown. Specific antibodies that individually recognize either the di- or the trimethylated form of H3K9 were used. (D) ChIP analysis of H3K9 methylation levels at the c-Myc promoter after KDM4C inhibition as quantified by real-time PCR. (E) ChIP analysis of H3K9 methylation levels at the SOX2 promoter after 48 h of exposure or not exposure to CaA for various concentrations, as indicated, as quantified by real-time PCR. Relative promoter occupancies (%input) by the indicated H3K9 methylated forms are shown with error bars based on SEs calculated from at least three replicates. (F) ChIP analysis of H3K9 methylation levels at the c-Myc promoter after 48 h of exposure or not exposure to CaA for various concentrations, as indicated, as quantified by real-time PCR. (G) Xenotransplants from each group were collected, Western blot analysis and IHC staining(H) were done. Representative immunoblots of H3K9me3 and H3K9me2 protein expression in whole tissue extracts from ALDHbri+-derived xenograft treated with or without CaA. Note the increase in H3K9me3 and H3K9me2 immunoreactivity in CaA-treated xenografts compared with controls. β-Actin was used as loading control. (H) Expression of SOX2 and H3K9me3 protein in paraffin sections of CaA-treated xenografts analyzed by IHC. Sections were counterstained with hematoxylin (blue nuclei). Note the increase in nuclear H3K9me3 methylation and decrease in SOX2 immunostaining in CaA-treated xenografts compared with controls (arrowheads denote positive staining).

Figure 7

SOX2 is the key downstream effector of KDM4C responsible for maintaining ALDHbri+ ESCC cells. (A) Representative images of spheres formed by ALDHbri+ cells transduced with LV-c or LV-shSOX2. (Scale bar = 50 μm, Upper panel). Esosphere formation analysis of ALDHbri+ cells reveal SOX2 knockdown lead to a reduction in clonogenic efficiency (Lower panel). (B) ALDHbri+ cells were capable of self-renewal in vitro, as shown by similar esosphere-initiating capacity in serial passages. Significant reduction in the self-renewal of ALDHbri+ cells upon SOX2 knockdown is shown. (C-E) Enforced SOX2 over-expression could rescue the differentiation phenotype induced by KDM4C depletion. ALDHbri+ cells were transfected with vehicle or a SOX2-overexpressing vector and challenged with shRNA directing against KDM4C. The cells were subjected to Aldefluor analysis, spheroid formation test and expand in subsequent serial propagations to examine self-renewal. Note the rescue and the maintenance of ALDHbri+ populations (C), spheroid formation (D) and self-renewal capability (E) in SOX2-overexpressing-KDM4C-shRNA treated cells. Little or no rescue was observed when the cells were challenged with vehicle. (F-G) Enforced SOX2 over-expression could rescue the differentiation phenotype induced by CaA treatment. ALDHbri+ cells were transfected with vehicle or a SOX2-overexpressing vector and treated with CaA. The cells were subjected to Aldefluor analysis and spheroid formation test. Note the rescue and the maintenance of ALDHbri+ populations (F) and spheroid formation capability (G) in SOX2-overexpressing-CaA treated cells. Little or no rescue was observed when the cells were challenged with vehicle. (H) Enforced SOX2 over-expression could rescue the engraftment capability upon CaA treatment. ALDHbri+ cells were transfected with vehicle or SOX2-overexpressing vector and subjected to engraftment capability analyses. Note the maintenance of engraftment capability in SOX2-overexpressing cells upon CaA treatment. Little or no rescue was observed when the cells were challenged with vehicle. Data shown are the mean ± S.E.M. of at least three independent experiments. *P⩽0.05; **P⩽0.01.