Tumour maintenance is mediated by eNOS

Abstract

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.

Figure 1

AKT promotes tumour maintenance by phosphorylation of eNOS. a, PI3K-TtH^LacZ or c, TtH^LacZ cells expressing indicated constructs were mixed with Ras^G12V-TtH cells, injected into mice, and tumours or recultured tumour cells were stained with X-gal. n=5, mean±s.e.m. b, Protein levels of phosphorylated AKT (p-AKT), phopshorylated eNOS (p-eNOS, ect: ectopic, end: endogenous), HA-eNOS (HA), and AKT in PI3K-TtH^LacZ cells expressing wildtype or S₁₁₇₇A HA-eNOS treated with DMSO or LY294002.

Figure 2

eNOS activates wildtype HRas to promote tumour maintenance. Protein levels of S-nitrosylated (nitroso), GTP-bound, total, or input HRas or NRas, phosphorylated AKT (p-AKT), or as a loading control tubulin in a, PI3K-TtH^LacZ cells treated with DMSO or wortmannin, b, PI3K-TtH^LacZ cells transfected with wildtype or C₁₁₈S HRas or c, TtH^LacZ cells expressing the indicated combinations of p110-CAAX, vector or eNOS shRNA. d, Ras^G12V-TtH cells were mixed with PI3K-TtH^LacZ cells expressing the indicated constructs, injected into mice, and tumours or recultured tumour cells were stained with X-gal to visualize PI3K-TtH^LacZ cells. n=5, mean±s.e.m.

Figure 3

eNOS activation is required for tumour growth. Representative mice and/or tumours a, following injection with Ras^G12V-TtH cells expressing indicated constructs or b, after treatment with DMBA/TPA to induce skin tumours on mice of indicated genotype (week 20).

Figure 4

eNOS activation fosters cancer cell growth through activation of endogenous Ras. Protein levels of phosphorylated eNOS (p-eNOS) or as a loading control, tubulin, in pancreatic a, cancer lines, b, tumour and normal tissue. Protein levels of GTP-bound or total HRas, NRas, or KRas, p-eNOS or tubulin, and excised tumours of c, CFPac-1 cells expressing eNOS or scramble shRNA, d, CFPac-1 and MIAPaCa-2 cells expressing dox-inducible shRNA +/− dox, and CFPac-1 cells expressing e, HRas or f, NRas shRNA plus a vector or an RNAi-resistant wildtype or C₁₁₈S NRas or HRas, or a scramble sequence. g, Proposed signalling.