The positive feedback between Snail and DAB2IP regulates EMT, invasion and metastasis in colorectal cancer

DAB2IP has been identified as a tumor suppressor in several cancers but its oncogenic role and transcriptionally regulatory mechanisms in the progression of colorectal carcinoma (CRC) remain unknown. In this study, DAB2IP was down-regulated in CRC tissues and a valuable prognostic marker for survival of CRC patients, especially in the late stage. Moreover, DAB2IP was sufficient to suppress proliferation, epithelial-mesenchymal transition (EMT), invasion and metastasis in CRC. Mechanically, the linear complex of EZH2/HDAC1/Snail contributed to DAB2IP silencing in CRC cells. The study further proved that the positive feedback loop between Snail and DAB2IP existed in CRC cells and DAB2IP was required for Snail-induced aggressive cell behaviors. Finally, DAB2IP correlated negatively with Snail and EZH2 expressions in CRC tissues. Our findings reveal the suppressive role and a novel regulatory mechanism of DAB2IP expression in the progression of CRC. DAB2IP may be a potential, novel therapeutic and prognostic target for clinical CRC patients.


Patients
This study was conducted on a total of 200 paraffinembedded CRC samples from 2004 to 2005 year, which were histologically and clinically diagnosed from the Department of Pathology, Nanfang Hospital affiliated with Southern Medical University. They were not pretreated with radiotherapy or chemotherapy prior to surgery. They were followed up for 5 years and their complete clinical data were collected. For the use of these clinical materials for research purposes, prior patient's consent and approval from the Institute Research Ethics Committee were obtained. Clinical information of the samples was described in detail in Table 1. Patients included 111 males and 89 females, of ages ranging from 31 to 88 years (mean, 62 years old). All cases were with no metastasis at its presence at original presentation with CRC. The tables on metastasis pertain to its presence at any time in followup. A total of 75 (37.5%) patients died during follow up and 46 (23%) patients experienced distant metastasis. Fresh CRC tissues and the corresponding normal tissues were collected from 20 patients who underwent CRC resection without prior radiotherapy and chemotherapy at the Department of General Surgery in Nanfang Hospital in 2008 year. These samples were collected immediately after resection, snap-frozen in liquid nitrogen, and then stored at −80°C until needed.

Real-Time RT-PCR
Total RNA was extracted using Trizol reagent (Invitrogen) and cDNA was synthesized using an access RT system (Promega). Real-time PCR was performed using Mx3000P Real-time PCR System (Stratagene) and SYBR PremixExTaqTM (TaKaRa). The primers were selected as the following: For DAB2IP, forward 5′-TGG ACG ATG TGC TCT ATG CC-3′, reverse 5′-GGA TGG TGA TGG TTT GGT AG-3′. The PCR condition was 95°C for 30s, followed by 40 cycles of amplification (95°C for 5s, 60°C for 34s, 72°C for 34s). The comparative quantification was determined using the 2 −ΔΔCt method. Each sample was tested three times.

Immunohistochemical Analysis (IHC)
The sections were deparaffinized and rehydrated, and endogenous peroxidase was inhibited with 0.3% H 2 O 2 methanol. For antigen retrieval, slides were boiled in 0.01 M, pH 6.0 sodium citrate buffer for 15 min in a microwave oven. After blocked with the 5% normal goat serum, primary anti-DAB2IP polyclonal antibody (1:400, Abcam, Cambridge, UK), anti-Ezh2 monoclonal antibody (1:200, Cell Signal Technology, USA) and anti-Snail monoclonal antibody (Abcam, Cambridge, UK) was applied and the slides were incubated at 4°C overnight. Following incubation with biotinylated secondary antisera, the streptavidinbiotin complex/horseradish peroxidase was applied. Finally, the visualization signal was developed with DAB staining, and the slides were counterstained in hematoxylin. The stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters. The staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The extent of staining was scored as 0 (0%),

SuPPlemenTARy mATeRIAlS AnD meTHODS
www.impactjournals.com/oncotarget/ 1 (1-25%), 2 (26-50%), 3 (51-75%) and 4 (76-100%), according to the percentages of the positive staining areas in relation to the entire carcinoma-involved area or the entire section for the normal samples. The sum of the intensity and extent scores was used as the final staining score (0-7) for DAB2IP or Ezh2. The staining of DAB2IP or Ezh2 was assessed as follows: (−) means a final staining score of <3; (+) a final staining score of 3; (+ +) a final staining score of 4; and (+ + +) a final staining score of ≥5. Tumors having a final staining score of 3 or higher were considered to be positive. This relatively simple, reproducible scoring method gives highly concordant results between independent evaluators and has been used in previous studies [1,2]. Cutoff values for DAB2IP were chosen based on a measure of heterogeneity by using log-rank statistical analysis with respect to overall survival. An optimal cutoff value was identified: Tumors with a final staining score 0~+ were classified as tumors with low expression of DAB2IP, and tumors with a final staining score ++~+++ were classified as tumors with high expression of DAB2IP.

Chromatin immunoprecipitation assay (ChIP)
Cells were lysed using SDS lysis buffer and DNA was sheared by sonication to lengths between 200 and 1000 base pairs. Protein-DNA complexes were precipitated by antisnail antibody (Abcam, Cambridge, UK), and then recovered using protein G agarose beads, washed, and eluted. Crosslinks in protein-DNA complexes were then reversed by NaCL. The immunoprecipitated DNA was amplified by PCR for specific sequences containing snail-binding sites.