Src nuclear localization and its prognostic relevance in human osteosarcoma

Abstract

Osteosarcoma is the most common malignant bone tumor in children and young adults. The identification of proteins which exhibit different subcellular localization in low- versus high-risk osteosarcoma can be instrumental to obtain prognostic information and to develop innovative therapeutic strategies. Beside the well-characterized membrane and cytoplasmic localization of Src protein, this study evaluated the prognostic relevance of its so-far unknown nuclear compartmentalization. We analyzed the subcellular distribution of total and activated (pY418) Src in a tissue microarray including 60 osteosarcoma samples. Immunohistochemical analyses revealed a variable pattern of Src expression and localization, ranging from negative to high-stained nuclei combined with a substantial cytoplasmic staining for total and activated forms. The analysis of Kaplan-Meier survival curves in relationship to the diverse permutations of cytoplasmic and nuclear staining suggested a correlation between Src subcellular localization and the overall survival (OS) of osteosarcoma patients. In order to explain this different subcellular localization, normal osteoblasts and three osteosarcoma cell lines were used to investigate the molecular mechanism. Once confirmed a variable Src localization also in these cell lines, we demonstrated a correlation between the N-myristoyltransferase enzymes expression and activity and the Src nuclear content. In conclusion, these results described a so-far unknown Src nuclear localization in osteosarcoma cells, suggesting that the combined detection of nuclear and cytoplasmic Src levels can be used as a prognostic marker for osteosarcoma patient survival. A correlation between the N-myristoyltransferase enzymes and the Src subcellular localization was described as well.

Nuclear Functions of the Tyrosine Kinase Src

“…Taken together, this evidence suggested that the SFKs could be useful to create an "open" chromatin more accessible to transcriptional factors [52]. In this context, we recently demonstrated the Src nuclear localization in osteoblasts and low aggressive osteosarcoma cells [46], and in particular we observed nuclear Src accumulation in hypocondensated chromatin, as demonstrated by the low DRAQ5 staining (Figure 2). This finding, together with the work by Takahashi, strongly suggests a function for nuclear Src in the regulation of transcription.…”

“…Indeed, in osteosarcoma and in hormone-positive breast cancer the Src nuclear compartmentalization is associated with improved patients' overall survival. This evidence suggests a sort of physiological relevance for Src nuclear localization, confirmed by the high Src nuclear content observed in normal osteoblasts [46].…”

“…These results suggest that immunohistochemical analysis of Src subcellular localization, together with its expression, can provide more accurate information in the assessment of osteosarcoma prognosis [46].…”

“…In our recent work, we described Src nuclear compartmentalization as a good prognosis factor for osteosarcoma patients' overall survival as assessed by tissue microarray analysis [46]. Indeed, a high nuclear Src accumulation is detected in normal osteoblasts as well as in low-aggressive osteosarcoma cell line SaOS2 cells, while its nuclear localization decreases in relationship to tumor aggressiveness, being very low in high metastatic 143B cells.…”

“…In support of David-Pfeuty hypothesis, we recently demonstrated that Src nuclear localization in osteoblasts and osteosarcoma cell lines is related to the myristoylation status of the cells. Indeed, low aggressive osteosarcoma SaOS-2 cells show high content of nuclear Src with a low myristoylation and low expression of N-myristoyltransferase (NMT) enzymes, in comparison to high metastatic 143B osteosarcoma cells, in which nuclear Src is lower while myristoylation and NMT expression is very high [46].…”