NOTCH2 in the bladder promotes tumor development and leads to more malignant phenotype.
Our recently published results revealed that NOTCH2 is an oncogene that drives bladder cancer (BCa) progression. To test whether NOTCH2 can promote BCa development, we have established a constitutively active NOTCH2 intracellular domain (N2ICD) mouse model. Previous TCGA data showed that NOTCH2 expression and copy number gain are enriched in basal tumors.
We have created lentiviral constructs based on the FUGW vector containing the N2ICD transgene, driven by either uroplakin-2 (Upk2, to induce luminal tumors) or cytokeratin-5 (Krt5, to induce basal tumors). These constructs also include intraribosomal entry sequence (IRES) followed by the firefly luciferase gene. Lentiviral particles were inocluted by ultra-sound guided injections into the subepithelial space of the bladder wall. Mice were treated with the bladder specific carcinogen BBN (0.05%, in drinking water) for 12 weeks. We have also tested the metastatic potential of NOTCH2 mutants (A2025E and Q2223X) over-expressed cell lines in vivo using the zebrafish embryo model. Each wild-type, NOTCH2 mutants (A2025E and Q2223X), N2ICD over-expressed RT4 or RT112 cell lines, and NOTCH2 knock-down UC13 cell lines were injected to zebrafish embryo. After 3 days, the number of metastases were counted.
Luciferase activity was observed in the bladder of some mice 20 weeks into the experiment: 2 cases in the empty vector control group (2/15, 13.3%), 6 cases in the Upk2 group (6/15, 40%), and 5 cases in Krt5 group (5/15, 33.3%). The tumors in both Upk2 and Krt5 group showed not only urothelial but also squamous cell carcinoma features. Moreover, in both Upk2 and Krt5 group, tumorigenesis was observed in 20 weeks after BBN while tumorigenesis in control group was observed in 26 weeks. In the zebrafish model, the number of metastasis were increased in the N2ICD over-expressed groups compared to the wild-type and mutant lines, while the metastasis were decreased in NOTCH2 knock-down UC13 line.
Our results suggest that the over-expression of N2ICD in the bladder wall could accelerate tumor development and possibly lead to more malignant phenotypes. Moreover, over-expression of N2ICD promotes metastatic potential.