Oncogenic and drug-sensitive RET mutations in human epithelial ovarian cancer

Abstract

Background: Epithelial ovarian cancer (EOC) is a highly lethal malignancy. Improvement in genetic characterization of EOC patients is required to propose new potential targets, since surgical resection coupled to chemotherapy, presents several limits such as cancer recurrence and drug resistance. Targeted therapies have more efficacy and less toxicity than standard treatments. One of the most relevant cancer-specific actionable targets are protein tyrosine kinases (PTKs) whose role in EOC need to be better investigated.

Methods: EOC genomic datasets are retrieved and analyzed. The biological and clinical significance of RET genomic aberrations in ovarian cancer context are investigated by a series of in vitro and in vivo experiments.

Results: Epithelial ovarian cancer sequencing projects identify recurrent genomic RET missense mutations in 1.98% of patients, ranking as the top-five hit among the 100 receptor tyrosine kinases-encoding genes. RET mutants R693H and A750T show oncogenic transformation properties in NIH3T3 cells. Introduction of the RET mutants into human EOC cells increases RET signaling, cell viability, anchorage-independent cell growth and tumor xenograft growth in nude mice, demonstrating that they are activating mutations. RET mutants significantly enhance the activation of RET and its downstream MAPK and AKT signaling pathway in ovarian cancer cells. Vandetanib, a clinical approved RET inhibitor, inhibits the cell viability and decreases the activation of RET-MAPK signaling pathways in EOC cells expressing oncogenic RET mutants.

Conclusions: The discovery of RET pathogenic variants in the EOC patients, suggests a previously underestimated role for RET in EOC tumorigenesis. The identification of the gain-of-function RET mutations in EOC highlights the potential use of RET in targeted therapy to treat ovarian cancer patients.

Keywords: Ovarian cancer; RET; Targeted therapy; Vandetanib.

Fig. 1

RET alterations in ovarian cancer. a Missense mutations in the CDS (coding sequence) of RET from the TCGA, COSMIC, ICGC and CCLE databases are marked over the affected protein domains. CLD, cadherin-like domain; CRD, cysteine-rich domain; TM, transmembrane domain; TKD, tyrosine kinase domain. b Genes with a mutation frequency ranking in the top 5 of 100 PTKs in ovarian cancer are listed, and the mutation frequency, FDA approved inhibitors and representative reference are also shown. c Kaplan-Meier plot of the progression-free survival of ovarian cancer patients based on RET alterations from the TCGA database

Fig. 2

Oncogenic transformation properties of RET mutations. a HEK293T cells were transfected transiently with empty vector (EV), RET wild-type (WT) or RET mutants (14 potential active mutations and C634R mutant as the positive control). The lysates were analyzed by western blotting with anti-phospho RET (Y905) and anti-RET antibodies. b Bar graphs demonstrated the quantification of western blotting bands in (a), normalized to WT control. c NIH3T3 cells were transfected stably with empty vector (EV), RET wild-type (WT) or RET mutants (R693H and A750T) viruses. The lysates were analyzed by western blotting with anti-phospho RET (Y905) and anti-RET antibodies. d Bar graphs showed the quantification of western blotting bands in (c), normalized to WT control. e and f RET mutants promote the anchorage-independent growth of NIH3T3 cells. NIH3T3 cells stably transfected with EV, WT, or RET mutants were seeded in soft agar in triplicate for 5 weeks and stained with methyl thiazol tetrazolium (MTT), representative plates (f) and the number of colonies (f) are shown. All of the above results represent three repeated experiments *, P < 0.05 compared to WT

Fig. 3

Oncogenic potential of RET mutants in vitro and in vivo. a Lysates from epithelial ovarian cancer cell (EOC) A2780 transduced with EV or WT or mutants were analyzed by immunoblot with anti-phospho RET (Y905), anti-RET antibody, anti-phospho ERK (T202/204), anti-ERK antibody, anti-phospho AKT (S473), and anti-AKT antibody (b-d) Bar graphs showing quantification of western blot bands in (a), normalized to WT control. e R693H and A750T mutants increase the cell viability of A2780 cells. A2780 cells stably expressing EV, RET WT or RET mutants grew on 96-well plates in triplicate for 72 h, and the viability was measured by CTG assay. f and g A2780 cells transduced with EV, RET WT or mutants were seeded in 6-well plates (500 cells per well) in triplicate for 2 weeks and stained with MTT. Representative plates (f) and number of colonies are presented (g). h–j RET mutants promote the growth of ovarian cancer xenografts in nude mice. Representative pictures (h), tumor volumes (i) and tumor weights (j) are shown. *, P < 0.05 compared to WT. **, P < 0.01 compared to WT

Fig. 4

Vandetanib inhibits the viability and cell signaling of RET in EOC cells expressing RET mutants. a Drug sensitivity of RET mutants in the CTG assay. A2780 cells transduced with RET R693H or A750T were seeded in 96-well plates in triplicate and treated with 0, 500, 750, 1000, 2500, or 5000 nM vandetanib at 24 h and 72 h after plating. The inhibition effects of vandetanib are shown in the bar graph and normalized to the WT control. b Vandetanib strongly inhibits the phosphorylation of RET and ERK in A2780 cells with RET mutations. A2780 cells expressing either RET R693H or A750T mutant were treated with 500 nM vandetanib for 4 h before harvesting, and the western blotting results are shown in (b). c and d Bar graphs showing quantification of western blotting bands in (b), normalized to WT control. The results represent three repeated experiments *, P < 0.05 compared to WT. **, P < 0.01 compared to WT