In Situ Analysis of mTORC1/C2 and Metabolism-Related Proteins in Pediatric Osteosarcoma

Abstract

Activation of the mTOR pathway has been observed in osteosarcoma, however the inhibition of mammalian target of rapamycin (mTOR) complex 1 has had limited results in osteosarcoma treatment. Certain metabolic pathways can be altered by mTOR activation, which can affect survival. Our aim was to characterize the mTOR profile and certain metabolic alterations in pediatric osteosarcoma to determine the interactions between the mTOR pathway and metabolic pathways. We performed immunohistochemistry on 28 samples to analyze the expression of mTOR complexes such as phospho-mTOR (pmTOR), phosphorylated ribosomal S6 (pS6), and rapamycin-insensitive companion of mTOR (rictor). To characterize metabolic pathway markers, we investigated the expression of phosphofructokinase (PFK), lactate dehydrogenase-A (LDHA), β-F1-ATPase (ATPB), glucose-6-phosphate dehydrogenase (G6PDH), glutaminase (GLS), fatty acid synthetase (FASN), and carnitin-O-palmitoyltransferase-1 (CPT1A). In total, 61% of the cases showed low mTOR activity, but higher pmTOR expression was associated with poor histological response to chemotherapy and osteoblastic subtype. Rictor expression was higher in metastatic disease and older age at the time of diagnosis. Our findings suggest the importance of the Warburg-effect, pentose-phosphate pathway, glutamine demand, and fatty-acid beta oxidation in osteosarcoma cells. mTOR activation is linked to several metabolic pathways. We suggest performing a detailed investigation of the mTOR profile before considering mTORC1 inhibitor therapy. Our findings highlight that targeting certain metabolic pathways could be an alternative therapeutic approach.

Keywords: mTOR; metabolic; metabolic adaptation; osteosarcoma; pathways; pediatric.

FIGURE 1

The distribution of the pmTOR, pS6 and rictor immunostaining results and mTOR activity characterization of the samples. Most of the cases (61%) showed low mTOR activity, although 21% of the samples showed potential mTORC2 and 14% showed potential mTOR activity. We found mTORC2 activity in only one sample (4%).

FIGURE 2

Example for mTORC2, potential mTOR, potential mTORC2 and low mTOR activity. Representative pmTOR, pS6 and rictor immunohistochemical stainings are presented. The numbers in the right lower corner are the given H-scores for each sample. Pictures were taken with CaseViewer 2.3. Software (3D Histech Ltd. Budapest, Hungary). The scale bar shows 20 μm.

FIGURE 3

Metabolic marker expression in osteosarcoma cases. The distribution of H-scores are visible on the boxplot graph. “X” signs the mean, horizontal lines sign the median values of H-scores. The numbers in the right lower corner are the given H-scores for each sample. Evaluation of metabolic markers showed that LDHA, G6PDH, GLS and CPT1A expression was high in most of the cases. ATPB and GLS shows granular pattern due to mitochondrial localization. Pictures were taken with CaseViewer 2.3. Software (3D Histech Ltd. Budapest, Hungary). The scale bar shows 20 μm.

FIGURE 4

Panel (A): H-score values for pmTOR were found significantly higher in those patients who showed poor response to chemotherapy. H-score levels were significantly lower in non-osteoblastic subtype. Panel (B): H-score values for rictor were significantly higher in those patients who had metastasis at the time of the diagnosis and who were older than 14 years.

FIGURE 5

On the left side of Panel (A) Kaplan-Meier curve shows significant difference between the survival of patients with high and low GLS expression. (Log Rank p = 0.019.) On the right side we present the H-score values for GLS. The scores were significantly higher in patients, who died due to relapse or progression. On Panel (B) we present the H-score values for FASN. The expression was significantly higher in metastatic disease, and in patients who died due to relapse or progression.

FIGURE 6

The results of the Spearman’s correlation analysis. The color intensity changes according to the Spearman’s rho value. Correlation coefficient above 0.4 is considered positive, under −0.4 is considered negative correlation. p values for significance are visible in the upper triangle.