To support the clinical findings on the genetic duet of BRAF V600E and TERT promoter mutations and demonstrate its biological relevance, we used in vitro and in vivo models to examine the roles of BRAF V600E and TERT in oncogenic cellular activities and xenograft tumor development of PTC cells BCPAP and K1 and melanoma cells A375, which all harbored both BRAF V600E and TERT promoter mutations. As shown in Supplementary Fig. 1, BRAF shRNA effectively knocked down BRAF protein and suppressed ERK phosphorylation of the MAPK pathway; similarly, TERT siRNA knocked down more than 80% of TERT protein in the three cells. Knockdown of either BRAF or TERT significantly inhibited cell proliferation and dual knockdown of BRAF and TERT induced a further inhibition (Fig. 1a). Either BRAF or TERT knockdown decreased cell migration and invasion and dual knockdown of BRAF and TERT had more robust effects (Fig. 1b, c). Similarly, either BRAF or TERT knockdown inhibited anchorage-independent growth of K1 and A375 cells in soft agar and dual knockdown of BRAF and TERT nearly completely abolished colony growth (Fig. 1d). BCPAP cells naturally formed only a few colonies in soft agar and knockdown of either BRAF or TERT completely abolished the colony formation (Fig. 1d). We also tested the role of BRAF V600E and TERT in thyroid tumor growth using the BRAF V600E inhibitor PLX4032 and stable TERT knockdown to suppress the MAPK pathway and the TERT, respectively, in K1 cell (Fig. 1e), from which xenograft tumors were derived (Fig. 1f, g). Either administration of PLX4032 or TERT knockdown inhibited tumor growth and combination of the two nearly completely abolished tumor growth (Fig. 1f, g), particularly evident in tumor weight (Fig. 1g). These data demonstrated that, like BRAF V600E, TERT also played a robust role in cancer-hallmark oncogenic cellular activities and tumorigenesis of cancer cells; the oncogenic effect of TERT was in fact even more robust than BRAF V600E in these cells harboring both BRAF V600E and TERT promoter mutations. TERT was thus demonstrated here to be a powerful oncoprotein. These results also showed that BRAF V600E and TERT displayed a cooperative manner in promoting the oncogenic behaviors of these cells, recapitulating the clinical findings on the genetic duet of BRAF V600E and TERT promoter mutations.Fig.1Cooperative role of BRAF V600E and TERT in the oncogenic behavior and tumor growth of cancer cells. Specific shRNA against BRAF and siRNA against TERT were used to knock down BRAF and TERT in the indicated cancer cells, respectively, followed by performance of assays of MTT of cell proliferation (a), transwell cell migration (b), cell invasion (c), and colony formation in soft agar (d) (with representative images shown in the left panel and the average colony numbers in the right panel). Western blotting analysis of TERT and phosphorylation of ERK (p-ERK) was performed for K1 cells (e), from which xenograft tumors were derived to test the role of TERT and BRAF V600E in tumor development and growth (f, g). Panel f shows the time course of tumor growth and panel g shows the weights of tumors surgically excised. The “Control” in a−d represented the combination of scramble shRNA and Control siRNA. The “Control” in e−g represented the combination of scramble shRNA and DMSO. The horizontal bar in panels b and c represents 100 μm. The red horizontal bar on the left of panel d represents 100 μm; colonies larger than this size were counted and colony numbers are shown on the right of the panel. Little vertical bars in a, d, f, and g represent standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, by two-tailed Student’s t test. In panel a, the P values are for the comparison of the indicated condition with “BRAF/TERT knockdown (KD)” (red line). In panel f, the P values are for the comparison of the indicated condition with “PLX4032/shTERT” (red line)