Loss of the 14-3-3σ is essential for LASP1-mediated colorectal cancer progression via activating PI3K/AKT signaling pathway

LIM and SH3 protein 1 (LASP1) can promote colorectal cancer (CRC) progression and metastasis, but the direct evidence that elucidates the molecular mechanism remains unclear. Here, our proteomic data showed that LASP1 interacted with 14-3-3σ and decreased the expression of 14-3-3σ in CRC. Deletion of 14-3-3σ was required for LASP1-mediated CRC cell aggressiveness. In vitro gain- and loss-of-function assays showed that 14-3-3σ suppressed the ability of cell migration and decreased the phosphorylation of AKT in CRC cells. We further observed clearly co-localization between AKT and 14-3-3σ in CRC cells. Treatment of PI3K inhibitor LY294002 markedly prevented phosphorylation of AKT and subsequently counteract aggressive phenotype mediated by siRNA of 14-3-3σ. Clinically, 14-3-3σ is frequently down-regulated in CRC tissues. Down-regulation of 14-3-3σ is associated with tumor progression and poor prognosis of patients with CRC. Multivariate analysis confirmed low expression of 14-3-3σ as an independent prognostic factor for CRC. A combination of low 14-3-3σ and high LASP1 expression shows a worse trend with overall survival of CRC patients. Our research paves the path to future investigation of the LASP1-14-3-3σ axis as a target for novel anticancer therapies of advanced CRC.

as previously described. 1 For 2-D difference gel electrophoresis (2-D DIGE), the proteins were labelled with fluorescent cyanine dyes (GE Healthcare, Milwaukee, Wisconsin, USA) following the manufacturer's instructions. In brief, 50 mg of extracted protein to be compared was labelled with 400 pmol Cy3 or Cy5, while 400 pmol Cy2 was employed to label 50 mg of internal standard protein of each sample at an equal amount. The labelling was performed on ice in the dark for 30 min, and then quenched with 1 ml of 10 mM lysine (Sigma) for 10 min. A 50 mg aliquot of Cy3and Cy5-labelled samples was combined before mixing with 50 mg of Cy2-labelled internal standard. The 2× sample buffer with an equal volume (8 M urea, 2 M thiourea, 4% CHAPS, 2% Bio-lyte, pH 4-7, 130 mM dithiothreitol (DTT)) was added to the sample, and the final volume was adjusted to 450 ml with rehydration buffer (8 M urea, 4% CHAPS, 1% Biolyte, pH 4e7, 40 mM DTT). The proteins were applied to IPG (immobilised pH gradient) strips (pH 4-7, 24 cm) and focused on an IPGphor (GE Healthcare). Focused IPG strips were equilibrated, and then loaded onto 12% SDS-polyacrylamide gels (SDS-PAGE) using low-fluorescence glass plates on an Ettan DALT II system (GE Healthcare). All electrophoresis procedures were performed in the dark. The biological triplicates were run on three gels as analytical gels. In addition, another strip was performed in parallel as a preparative gel for picking spots as described in 2-D DIGE, except that the IPG strip was loaded with 1000 mg of proteins, and the gel was stained with Coomassie brilliant blue. After SDS-PAGE, the three analytical gels were scanned with a Typhoon 9410 scanner (GE Healthcare) with appropriate excitation/emission wavelengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm) and Cy5 (633/670 nm) to generate nine protein spot maps.
DeCyder 5.0 software (GE Healthcare) was used for 2-D DIGE analysis according to the manufacturer's recommendation. The DeCyder differential in-gel analysis (DIA) module was used for pairwise comparisons of each sample with the internal standard in each gel. The DeCyder biological variation analysis (BVA) module was then used to simultaneously match all nine protein spot maps, using the Cy3/Cy2 and Cy5/Cy2 DIA ratios, to calculate average abundance changes and paired Student t test p values for the variance of these ratios for each protein pair across all samples. The differential protein spots (|ratio|>2, P<0.05) which were altered consistently in all three protein spot maps were selected for further identification.

Immunohistochemistry (IHC)
Immunohistochemistry was performed, as previously described 2 , to investigate the localization and expression of 14-3-3σ and LASP1 in 116 human CRC tissues.

Plasmid constructs
All eukaryotic expression vectors were constructed in pEX-3 (pGCMV/MCS/Neo). Generation of 14-3-3σ was achieved by using oligonucleotides in-frame to amino-terminus of pEX-3 template by standard PCR techniques (Shanghai GenePharma Co., Shanghai, China). The coding sequence for the constructs was verified by sequencing analysis.

siRNA-mediated gene silencing
Expression of human 14-3-3σ and LASP1 was knocked down with siRNA duplexes targeting the sequence. The siRNAs were designed and chemically synthesized (Shanghai GenePharma Co., Shanghai, China) for targeting different coding regions of the genes. The negative control siRNA targeting unknown mRNA sequence was used as a control. The sequences used are shown in Table S2

Co-immunoprecipitation (Co-IP)
The cells were transiently or stably transfected with the indicated constructs.
Cells were harvested and lysed in 1 ml of lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). Resulting lysates were subjected to immunoprecipitation with antibodies directed to the epitope tag. Immunoprecipitates were washed in lysis buffer, resolved by SDS-polyacrylamide gel electrophoresis, and subsequently analyzed by mass spectrum or protein immunoblotting.

Desorption/ionization Time of Flight Mass Spectrometry
The identified protein bands in the gel were excised from the gel and were in-gel digested. Briefly, the gel bands were destained in 30 mmol/L K 3 Fe(CN) 6