A role for CITED2, a CBP/p300 interacting protein, in colon cancer cell invasion

Abstract

A thorough understanding of histone acetyltransferase CBP/p300-mediated regulation of gene expression and cell growth is essential to identify mechanisms relevant to the development of histone deacetylase (HDAC) inhibitor-based preventive and therapeutic strategies. We found that knockdown of CBP/p300 interacting coactivator with glutamic acid/aspartic acid-rich tail 2 (CITED2) increased colon cancer cell invasiveness in vitro. Gene expression profiling revealed that CITED2 knockdown induced matrix metalloproteinase-13 (MMP-13) gene expression in colon cancer cells. Butyrate, a naturally occurring HDAC inhibitor, induced CITED2 expression and downregulated MMP-13 expression in RKO cells. Additionally, ectopic expression of CITED2 arrested RKO cell growth. Thus, CITED2 regulates colon cancer invasion and might be a target for HDAC inhibitor-based intervention of colon cancer.

Fig 1. CITED2 knockdown induced morphological changes in RKO cells

(A) RKO cells were transduced with plko1 empty lentiviral vector or plko1-shCITED2, and selected with puromycin for 3 weeks. The pools of puromycin-resistent cells were used for total RNA isolation and RT-PCR analyses. (B) Whole cell extracts were prepared and mouse anti-CITED2 (Novus Biologicals) and anti-GAPDH (Chemicon) antibodies were used for immunoblotting (IB). (C) Phase contrast microscopy of live cells (magnification: 200×). (D) Cells were stained with Alexa fluor 488 phalloidin (Molecular Probes) and examined with confocal microscope (magnification: 400×).

Fig 2. CITED2 knockdown increased cell invasive capacity

(A) RKO cells were first cultured in serum free medium for 20–24 h, then collected and resuspended in medium with 0.1% BSA. Culture medium with 10% FBS was added to the lower chamber of BD Matrigel Invasion Chamber and the resuspended cells were added onto the top of the Matrigel. Forty hours later, cells on the lower surface of the membrane were fixed with methanol, stained with H&E and viewed with microscope (magnification: 20×). Representative images from three independent experiments are shown. (C) Cells on the lower surface of the membrane were counted under a microscope. Data shown are means ± SEM for numbers of cells from three independent experiments, and ten fields were counted from each experiment.

Fig 3. CITED2 knockdown does not affect the β-catenin pathway

(A) Whole cell extract (WCE), cytoplasmic (Cyto) and nuclear (Nucl) fractions of RKO cells were prepared for immunoblotting. Rabbit anti-HDAC1 (Santa Cruz Biotechnology) and mouse anti-β-catenin (BD Biosciences) were used for immunoblotting. HDAC1 was used as a marker for the nuclear fraction, and GAPDH was used as a marker for the cytoplasmic fraction. (B) Total RNA was isolated from cells for RT-PCR as described above.

Fig 4. CITED2 knockdown increased MMP-13 expression and enzyme activity in RKO cells

(A) RKO cells were transfected with a control siRNA (Ambion) or CITED2-specific siRNA. Twenty hours after transfection, total RNA was isolated for RT-PCR. (B) Cells were seeded in 12-well plates. Twelve hours later, cells were washed and incubated in serum-free medium for another 24 h. The medium was collected, centrifuged; and the supernatant was used to assay MMP-13 activity.

Fig 5. CITED2 knockdown increased MMP-13 expression and cell invasiveness in SW480 cells

(A) SW480 cells were transfected with a control siRNA (Ambion) or CITED2-specific siRNA. Twenty hours after transfection, total RNA was isolated for RT-PCR. (B) SW480 cells were transduced with plko1 empty lentiviral vector or plko1-shCITED2, and selected with puromycin for 3 weeks. The pools of puromycin-resistent cells were used for RT-PCR (upper panels) and Western blot (lower panels) analyses. (C) Matrigel invasion assays of cells in (B) were performed as described in Fig 2B. Twenty-four hours after invasion, cells on the lower surface of the membrane were stained with H&E and counted. Data shown are means ± SEM of cells from three independent experiments, and ten fields were counted from each experiment.

Fig 6. Butyrate upregulated CITED2 expression and downregulated MMP-13 expression in RKO cells

(A) RKO cells were treated with 5 mM sodium butyrate for 16 h. Total RNA was isolated for RT-PCR. (B) RKO cells transfected with pMK17 reporter, then treated with 2.5 mM sodium butyrate for 20 h prior to luciferase reporter assays that were normalized to protein. Results are presented as means ± SEM of three experiments. (C) RKO and SW480 cells were transfected with pCMV10 vector (Vec) or pCMV10/HA-CITED2 (HA-C2). Two days later, cells were collected and total RNA was isolated for RT-PCR analyses.

Fig 7. Ectopic expression of CITED2 arrested RKO cells

(A) RKO cells were transfected with pCMV10 vector or pCMV10/HA-CITED2 and selected with G418 at 0.5 mg/ml for 3 weeks. Cells were then seeded in 12-well plates at a confluency of ~25%. Forty hours later, cells were collected for flow cytometry. Data shown are mean±SEM of three repeats. * p<0.05. (B) Cells were incubated with 2.5 mM sodium butyrate for up to 8 h. Western blot was performed.