1. Glutamine mediates oncogenic transformations in high‐invasive cells by regulating STAT3 activity Comparison of OVCAR3 invasive capacity in Gln depleted and complete media conditions.
Glutamine mediates oncogenic transformations in high‐invasive cells by regulating STAT3 activity Comparison of OVCAR3 invasive capacity in Gln depleted and complete media conditions. Invasive capacity of SKOV3 is measured under complete media, Gln depleted media, and drugs inhibiting Gln's entry into tricarboxylic acid (TCA) cycle. L‐DON, BPTES, EGCG, AOA, and Rotenone decrease SKOV3's Matrigel invasive capacity. α‐Ketoglutarate (α‐KG) addition under glutamine deprivation or EGCG conditions rescues SKOV3's invasive capacity. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, n ≥ 4. Activation of Stat3 through tyrosine‐705 phosphorylation (Stat3.pY705) is elevated in high‐invasive ovarian cancer (OVCA) cells. The phosphorylation level of EGFR and Erk 1/2 increases with increasing degree of invasiveness. Tyrosine kinase signaling pathway activities can be affected by metabolic stress and in high‐invasive cells that are glutamine and glucose deprived. Stat3.pY705 is reduced along with total Jak levels. Stat3.pY705 levels are only reduced in the OVCA420 cell lines upon glucose deprivation, while the regulation of Stat3 phosphorylation by metabolic stress is absent in the low‐invasive cell line, OVCAR3. Stat3 serine‐727 phosphorylation (Stat3.pS727) is also reduced along with phosphorylation level of Erk 1/2 in the high‐invasive cell lines and only in response to glutamine deprivation. β‐actin as loading control. Cell proliferation in high‐invasive OVCA cell line is reduced when glutamine starved. Proliferation can be partially rescued by overexpression of transgenic Stat3 and a constitutively active mutant of Stat3, Stat3c, mean ± SD, n = 3, *P < 0.05. β‐actin as loading control. α‐KG addition rescues STAT3 tyrosine phosphorylation and Jak1, but cannot rescue Stat3 serine‐727 phosphorylation. β‐actin as loading control. Western blot figures are cut from same gel, but lanes are rearranged to show complete media conditions in first column, glutamine deprivation in second and α‐KG addition in the third column. The full Western blot images of the gel and the detailed description are included in the source file. OAA addition rescues STAT3 tyrosine phosphorylation. β‐actin as loading control. Western blot figures are cut from same gel, but lanes are rearranged to include only the relevant conditions. The full Western blot images of the gel and the detailed description are included in the source file. BPTES and rotenone inhibit STAT3 phosphorylation at tyrosine 705 (Y705). β‐actin as loading control. Western blot figures are cut from same gel, but lanes are arranged to show only the relevant conditions. The full Western blot images of the gel and the detailed description are included in the source file. Inhibition of Stat3 decreases OVCA metastasis. Treatment of SKOV3 cells with AG490 results in reduced invasion. Gene expression levels of targets of STAT3 involved in invasion were determined using KyotoOv38 in five OVCA cells. High‐invasive OVCA cells had higher gene expression of invasive genes. The addition of AG490, a Stat3 inhibitor enhances the effect of glucose and glutamine deprivation on proliferation in OVCA cell lines. The addition of stattic, inhibitor of STAT3, has a combinational cytotoxic effect on both cell lines. Data information: Data in (I) and (J) are expressed as mean ± SEM, n ≥ 8, *P < 0.05, **P < 0.01, ***P < 0.001.Source data are available online for this figure.
Lifeng Yang et al. Mol Syst Biol 2014;10:728
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