Figure 6

mTOR-dependent PCK1 expression is associated with cancer cell survival. (a) mTOR-PCK1 subnetwork constructed from the KEGG pathways. Network consists of node as protein/gene (or complex) and edges are inhibition or activation connection. Differential expression information for (1) HCC, (2) RCC and (3) CC is provided above each node. Red represents upregulation, green represents downregulation and white represents no significant change or not found in this analysis. (b) Systematic representation gluconeogenesis pathway. Enzymes are marked in blue color and differential expression information for (1) HCC, (2) RCC and (3) CC is provided above each gene. Red represents upregulation, green represents downregulation and white represents no significant change or not found in this analysis. Reaction colored in red are rate-limiting steps in the pathway. (c) Different cancer cell lines, HCC (HepG2, HuH7), RCC (SK-RC-45, SK-RC-26B), CC (HCT116, SW480), lung carcinoma (H1299), breast cancer (MCF7), gastric adenocarcinoma (AGS) and leukemia (K562), were treated with torin1 for 24 h and the cell lysates were analyzed by immunoblotting for PEPCK. β-Actin serves as a loading control. (d) Cell lines were treated with torin1 for 24 h and total RNA was extracted for determining the expression levels of PCK1. (e) All cell lines were treated with torin1 for 24 h and apoptosis induction was measured using Annexin V/PI by flow cytometry as described in Materials and Methods. (f) Correlation plot for fold increase in PCK1 RNA expression and fold increase in cell death upon torin1 treatment. (g) Schematic representation of hypothesis. Cancer cells downregulate gluconeogenic program to use glucose via the shunt pathways. mTOR blockade induces nuclear localization of FoxO leading to the upregulation of PCK1 and inhibition in cancer cell proliferation and survival. All panels: n≥3, *P<0.05, **P<0.01 compared with Con cells.