Experimental study on differences in clivus chordoma bone invasion: an iTRAQ-based quantitative proteomic analysis.

Although a bone tumor, significant differences in the extent of bone invasion exist in skull base chordoma, which directly affect the extent of surgical resection, and have an impact on its prognosis. However, the underlying mechanism of the phenomenon is not clearly understood. Therefore, we used an iTRAQ-based quantitative proteomics strategy to identify potential molecular signatures, and to find predictive markers of discrepancy in bone invasion of clivus chordoma. According to bone invasive classification criteria, 35 specimens of clivus chordoma were calssified to be either endophytic type (Type I) or exophytic type (Type II). An initial screening of six specimens of endophytic type and six of exophytic was performed, and 250 differentially expressed proteins were identified. Through the GO and IPA analysis, we found evidence that the expression of inflammatory activity-associated proteins up-regulated in endophytic type, whereas the expression of cell motility-associated proteins up-regulated in exophytic ones. Moreover, TGFβ1 and mTOR signal pathway seemed to be related with bone invasion. Thus, TGFβ1, PI3K, Akt, mTOR, and PTEN were validated in the following 23 samples by immune histochemistry and Western blot. The expression levels of TGFβ1 and PTEN were significantly lower in the endophytic type than in the exophytic ones. It was found that TGFβ1 may play an important role in its bone invasion. The mechanisms may be related with conducting an increased inflammatory cell response and a decline in cytoskeletal protein expression. PTEN is confirmed to be associated with the degree of bone invasion. The PI3K/AKT/mTOR signaling pathway might be associated with the bone invasion, but still needs a larger sample size to be verified These results, for the first time, not only demonstrate the biological changes that occur in different growth patterns from the perspective of proteomics, but also provide novel markers that may help to reveal the mechanisms behind clivus chordomas

Workflow of the iTRAQ proteomic strategy.

<p>In this work, six pathologically verified tissue samples of endophyticclivus chordoma (Type I) and six samples of exophyticclivus chordoma (Type II)were digested with trypsin. The peptides were then sequencedusing 2D-LC/MS/MS and iTRAQ proteomic analysis. Subsequently, the peptide sequences were compared with the existing human database to acquire the protein list. The proteins were quantitatively analyzed usingPanther, and IPA was used to analyze biological functions. Several candidate proteins with interestingbiological functions were selected and further validated using IHC ofadditional 23 samples.Abbreviation: GO&IPA Analysis, Gene Ontology &Ingenuity Pathway Analysis; IHC,immunohistochemistry.</p

The Bone invasive classification criteria of clivus chordoma.

<p>Graph a shows the standard region in the baseline level of an anatomy image. Graph b shows a representative lesion of endophytic type (Type I); and Graph c shows a representative lesion of exophytic type (Type II).</p

The list of differentially expressed proteins.

<p>The list of differentially expressed proteins.</p

Protein synthesis and cellular function networks from IPA analysis.

<p>It shows the cellular function network and includes the functions “Cellular Movement, Cell Morphology, Connective Tissue, and Development and Function”, and “Cellular Movement, Cellular Development, Skeletal& Muscular System Development and Functio”. Proteins in red were up-regulated in endophytic clivus chordomas compared with exophytic ones, and proteins in green were down-regulated in endophytic clivus chordomas compared with exophytic ones.</p

Panther analysis of endophyticclivus chordomas vs exophytic ones.

<p>Graph a shows cellular compartment analysis; Graph b shows molecular function analysis; and Graph c shows biological process analysis.</p

The basic information of included patients.

<p>Note: Group: 1, experimental group; 2, confirmation group. Type: I, endophytic type (type I); II, exophytic type (type II). Gender: 1, male; 2, female.</p><p>The basic information of included patients.</p

The list of molecules that are related to cellular movement.

<p>The list of molecules that are related to cellular movement.</p

Results of immunohistochemical analysis ofTGFβ1, PI3K, Akt,mTOR and PTEN in tissue samples.

<p>Magnification: 200X. Representative images of clivus chordomas tissue that were immunostained for TGFβ1, PI3K, Akt, mTOR and PTEN. Graph a shows the representative positive image of TGFβ1 in exophytic type. Graph b shows the representative positive image of TGFβ1 in endophytic type. Graph c is a negative control.</p