HDAC1 controls CIP2A transcription in human colorectal cancer cells

Abstract

This work describes the effectiveness of HDAC-inhibitor (S)-2 towards colorectal cancer (CRC) HCT116 cells in vitro by inducing cell cycle arrest and apoptosis, and in vivo by contrasting tumour growth in mice xenografts. Among the multifaceted drug-induced events described herein, an interesting link has emerged between the oncoprotein histone deacetylase HDAC1 and the oncogenic Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) which is overexpressed in several cancers including CRCs. HDAC1 inhibition by (S)-2 or specific siRNAs downregulates CIP2A transcription in three different CRC cell lines, thus restoring the oncosuppressor phosphatase PP2A activity that is reduced in most cancers. Once re-activated, PP2A dephosphorylates pGSK-3β(ser9) which phosphorylates β-catenin that remains within the cytosol where it undergoes degradation. The decreased amount/activity of β-catenin transcription factor prompts cell growth arrest by diminishing c-Myc and cyclin D1 expression and abrogating the prosurvival Wnt/β-catenin signaling pathway. These results are the first evidence that the inhibition of HDAC1 by (S)-2 downregulates CIP2A transcription and unleashes PP2A activity, thus inducing growth arrest and apoptosis in CRC cells.

Keywords: CIP2A transcription; CRC cells; HDAC-inhibitor; HDAC1; PP2A.

Figure 1. (S)-2 induced growth arrest and apoptosis in HCT116 cells

(A) HCT116 cells were incubated for 6, 15, 24 and 48 h without/with 5 μM (S)-2 and then processed by Western blot and immunostained for the acetylated histone H3/H4 and nonhistone protein acetyl-α-tubulin, while α-tubulin as such was taken as the loading control. (B) Cells (10⁵/well) were seeded in 6-well plates and allowed to attach overnight. The day after, increasing amounts of (S)-2 (0, 2.5 and 5 μM) were added to the wells and viable cells (trypan blu-negative) were counted with the aid of a Bürker chamber at the indicated time points (results were the mean ± SD of experiments done in quadruplicate). (C) Phase contrast pictures of companion cultures showed that (S)-2 induced morphological changes and a marked decrease in cell density (a typical experiment out of three). (D) For cell cycle distribution HCT116 cultures were treated without/with 5 μM (S)-2 for 48 h and then incubated with a PI/RNase solution for 30 min at 4°C prior to the flow cytometric analysis. The percentage of cells in the different phases of the cell cycle was calculated by the ModFit program and shown in each panel were reproduced in three separate experiments. (E) top – Apoptosis in cell cultures treated without/with the drug was also assessed cytofluorimetrically by using the Annexin-V-Fluos/PI assay and the calculated percentages of four different assays yielded comparable values. (E) bottom – Cell extracts of HCT116 incubated for the indicated time points with 5 μM (S)-2 were subjected to Western immunoblot analysis of the PARP cleaved fragment; GAPDH was the loading control. (F) Cell cultures were pre-incubated for 2 h with the pan-caspase inhibitor Z-VAD-fmk (30 μM) and then were treated without/with 5 μM (S)-2 for 24 h. Cell lysates were analyzed by Western immunoblot to detect the cleavage of PARP and of caspase 9; GAPDH was the loading control.

Figure 2. The effects of (S)-2 on GSK-3β/β-catenin pathway

(A) Cell extracts were analyzed by Western blot to detect phospho-GSK-3β(ser9), GSK-3β and active-β-Catenin levels; GAPDH was used as the loading control. (B) The total, cytosolic and nuclear extracts of HCT116 cells treated for 48 h without/with 5 μM (S)-2 were obtained (see Materials and Methods) and probed for the active-β-Catenin levels. Purity of the two subcellular fractions was assessed by the presence of GAPDH and fibrillarin as the markers of the cytosolic and nuclear compartment, respectively. (C) mRNA and protein levels of c-Myc and cyclin D1 in HCT116 cells treated without/with 5 μM (S)-2 for 24 h and 48 h were determined by quantitative real-time PCR (***P ≤ 0.01) and Western blot, respectively.

Figure 3. PP2A is responsible for drug-mediated pGSK-3β dephosphorylation

(A) HCT116 cells transfected with pool-siRNAs towards either PP1 or PP2A for 24 h were incubated for additional 24 h without/with 5 μM (S)-2; then cell extracts were analyzed by Western immunoblot to detect levels of pGSK-3β(ser9), PP1 and PP2A; α-tubulin was used as the loading control. (B) HCT116 cell cultures were pre-incubated for 2 h with 50 μM Cantharidin and then treated without/with 5 μM (S)-2 for 24 h. Cell lysates were analyzed by Western blot an probed with specific antibodies against pGSK-3β(ser9) and cleaved PARP fragment; GAPDH was the reference protein. (C and D) mRNA and protein levels of either I2PP2A and CIP2A from HCT116 cells treated without/with 5 μM (S)-2 were determined at the indicated time points by quantitative real-time PCR (**P ≤ 0.05; ***P ≤ 0.01) and Western blot, respectively.

Figure 4. Inhibition of HDAC1 induces CIP2A downregulation

(A) HCT116, HT-29 and HCT8 cell cultures were treated without/with 5 μM (S)-2 for 24 h or transfected with either HDAC1-specific and scrambled siRNAs for 48 h. Cell lysates were submitted to Western immunoblot to detect HDAC1 and CIP2A levels; GAPDH was used as the control protein. (B) Cells were first treated with HDAC1-specific siRNA and scrambled siRNA for 48 h, and then co-transfected with either 1082CIP2ALuc-pGL4.10 or pGL4.10 plasmids and with pGL4.70 up to 16 h. Luc2 and Renilla mRNA levels were determined by quantitative real-time PCR (**P ≤ 0.05; ***P ≤ 0.01).

Figure 5. Tumour xenograft

(A) top – Aliquots of HCT116 cell suspension (2.5 × 10⁶ cell/100 μl RPMI) were injected subcutaneously in both flanks of male nude mice (see also as Materials and Methods). Positively-xenografted mice were randomized into two groups and then injected ip with either the drug or DMSO as the vehicle. Treatments were administered three times a week for the first two weeks and twice in a row on the last week when mice were sacrificed by cervical dislocation. Variations in the tumour volumes along with the experiment and after the sacrifice were measured by a caliper. (A) bottom – Excised tumours were weighed and volumes were calculated according to the formula (length (mm)) × (width (mm)) × (depth (mm)) × p/6. Statistical analyses of changes in tumour volumes (mean ± SD) after 0, 3, 6 and 8 treatments were as follows: [for untreated mice: #0 (25.7 ± 10.0); #3 (103.3 ± 20.6); #6 (201.7 ± 64.2); #8 (188.6 ± 62.1)] and [for treated mice: #0 (28.2 ± 11.7); #3 (76.1 ± 25.5); #6 (128.6 ± 26.5); #8 (142.2 ± 24.7)]. According to these values the drug was capable of reducing tumour volumes of about 26.4% (1st week), 36.2% (2nd week) and 24.6% (3rd week), as compared to control. Photographs are representative of tumour masses from mice treated with either the vehicle or (S)-2, respectively. (B) top – Immunohistochemistry was performed on specimens of human colon cancer xenografts by using primary antibodies against acetyl-H3 and γ-H2AX, and also the monoclonal antibody MB-1 that recognizes the nuclear marker Ki-67 associated to cell proliferation (see Materials and Methods) followed by a peroxidase-conjugated IgG preparation; 3,3′-diaminobenzidine was employed as the chromogen for development. Slides were counterstained with aqueous Meyer hematoxylin and mounted with glycerol for visual inspection and photography. (B) bottom – Statistical analyses of data on both top panels of the figure were carried out by Student's t-test and significant differences between the two groups were indicated by the asterisks (**P < 0.05; ***P < 0.01).

Figure 6. Schematic diagram of (S)-2-induced growth arrest and apoptosis in colon cancer cells

The oncogenic protein HDAC1 acts as a transcription factor for CIP2A that afterward complexes and inactivates PP2A. The latter becomes unable to dephosphorylate pGSK-3β(ser9) thus allowing the translocation of β-catenin in the nucleus where it induces the transcription of c-Myc and cyclin D1 and, consequently, cell proliferation. The inhibition of HDAC1 by (S)-2 leads to the downregulation of CIP2A expression and, thus, restores the activity of PP2A that dephosphorylates pGSK-3β(ser9). The activated kinase GSK-3β phosphorylates β-catenin that undergoes degradation by the ubiquitin-proteasome system. The decrease in β-catenin levels downregulates c-Myc and cyclin D1 expression and, consistent with this, leads to cell growth arrest.