Characterizing the metabolic role of STAT3 in canine osteosarcoma

Abstract

Signal transducer and activator of transcription 3 (STAT3) dysregulation has been characterized in canine OS, with previous data suggesting that constitutive STAT3 activation contributes to survival and proliferation in OS cell lines in vitro. Recently, the contribution of STAT3 to tumour metabolism has been described across several tumour histologies, and understanding the metabolic implications of STAT3 loss may elucidate novel therapeutic approaches with synergistic activity. The objective of this work was to characterize metabolic benchmarks associated with STAT3 loss in canine OS. STAT3 expression and activation was evaluated using western blotting in canine OS cell lines OSCA8 and Abrams. STAT3 was deleted from these OS cell lines using CRISPR-Cas9, and the effects on proliferation, invasion and metabolism (respirometry, intracellular lactate) were determined. Loss of STAT3 was associated with decreased basal and compensatory glycolysis in canine OS cell lines, without modulation of cellular proliferation. Loss of STAT3 also resulted in diminished invasive capacity in vitro. Interestingly, the absence of STAT3 did not impact sensitivity to doxorubicin in vitro. Our data demonstrate that loss of STAT3 modulates features of aerobic glycolysis in canine OS impacting capacities for cellular invasions, suggesting a role for this transcription factor in metastasis.

Keywords: STAT3; canine; comparative oncology; metabolism; osteosarcoma.

FIGURE 1

STAT3 is activated in canine OS. Western blot demonstrating expression of pSTAT3 and STAT3 in canine OS cell lines. Protein lysates are representative of one biologic replicate, and were not pooled across multiple passages.

FIGURE 2

Effect of STAT3 on cellular proliferation and glycolytic rate. (A) Representative western blot confirming STAT3 deletion in canine OS cell lines. (B) Proliferation over 72 h was determined in STAT3 knockout lines using the SRB assay. Results are presented as the mean + SD across three biologic replicates. p < .05 was considered significant. (C) Basal and compensatory glycolysis were determined in STAT3 knockout cell lines using the Seahorse XFe Glycolytic Rate Assay. Results of one representative biologic replicate are shown as mean ± SD. p < .01 was considered significant. M(#) = monoclonal cell line. ⁺ p < .01; ^# p ≤ .001

FIGURE 3

Longitudinal Trends in Glycolytic Rate. Percent proton export rate (%PER) in (A) OSCA‐8 and (B) Abrams cell lines across biologic replicates using the Seahorse XFe Glycolytic Rate Assay. Error bars represent the mean ± SD.

FIGURE 4

Loss of STAT3 decreases invasive capacity of canine OS cell lines. The invasive capacity of OSCA8 (A) and Abrams (B–C) cells with and without STAT3 expression is shown as the mean of three biologic replicates. p < .05 was considered significant. M(#) = monoclonal knockout cell line. *p < .05; ⁺ p < .01; ^# p ≤ .001. Error bars represent SE. Statistical comparisons are made using the wild‐type cell line as the reference.

FIGURE 5

STAT3 deletion does not sensitize OS cells to doxorubicin. Proliferation in (A) OSCA8 and (B) Abrams cell lines exposed to increasing concentrations of doxorubicin are displayed as a fraction of an untreated control after exposure doxorubicin for 24 h. Error bars indicate the SD across three biologic replicates.