Figure 2: PIK3CA mutation upregulates GPT2 which renders CRC dependent on glutamine.

(a) GPT2 mRNA levels are higher in PIK3CA mutant clones. RT–PCR analyses of the indicated genes in the HCT116 and DLD1 PIK3CA mutant and WT clones. (b) GPT2 protein levels are higher in cells with PIK3CA mutations. Cell lysates of WT and mutant clones were blotted with the indicated antibodies. SLC1A5: glutamine transporter. (c,d) GPT2 expression is upregulated in PIK3CA mutant CRC cell lines. qRT–PCR (c) and western blot of GPT2 protein in the indicated cell lines (d). (e) GPT2 mRNA levels are higher in PIK3CA mutant tumours. qRT–PCR analyses of GPT2 in tumours with no mutations in PIK3CA or in PTEN, PDK1, AKTs and IRS (n=10) versus tumours with PIK3CA mutations (n=10). Data are plotted as whiskers (min to max). (f–h) GPT2 is needed for growth and for glutamine dependency in PIK3CA mutant cells. GPT2 was knocked down with two independent shRNAs in a HCT116 PIK3CA mutant clone (Mut 1). Stable pools were selected for further analysis. Western blot of GPT2 in control and GPT2 knockdown clones (f). Two thousand cells were seeded in 96-well plates and grown under normal culture conditions, and cell numbers were counted 5 consecutive days (g). Control and GPT2 knockdown clones were grown with or without glutamine for 72 h. Cell apoptosis was quantified (h). (i–k) GPT2 is sufficient for growth and for creating glutamine sensitivity in PIK3CA WT cells. A HCT116 PIK3CA WT clone (WT 1) was transfected with empty vector (control), or a FLAG-tagged WT GPT2 (GPT2) or a FLAG-tagged inactive GPT2 mutant (GPT2 K341H). Stable pools were selected for further analysis. Western blot analyses of transfected FLAG-GPT2 protein levels (i); cell proliferation under normal culture conditions (j); control and GPT2 overexpression (OE) cell pools were grown with or without glutamine for 72 h. Cell apoptosis was quantified (k). Data are presented as mean±s.e.m. of three independent cultures. *P<0.05; **P<0.01; ***P<0.001; NS, not significant; t-test.