AZD1480 Inhibits Thyroid Tumorigenesis Figure 6. JAK1/2 knockdown by siRNA does not affect proliferation nor RET and PI3K pathway activation in thyroid cancer cell lines. (A) The expression of JAK1 or/and JAK2 was downregulated by siRNA in TPC-1, MZ-CRC1 and TT cell lines. The levels of the indicated proteins and their phosphorylation status were determined after 48 hours, by western-blot analysis. (B) Cell proliferation was measured by BrdU incorporation, 48 hours after transfection with a combination of siRNAs targeting both JAK1 and JAK2. Results represent mean 6 SE of three independent experiments. *p,0.05. doi:10.1371/journal.pone.0046869.g006 tumor regression, with tumors displaying extensive areas of necrotic tissue and a significant decrease in the number of blood vessels. The latter might have caused a drop in nutrient and oxygen supply to the tumors, likely explaining the accentuated necrosis and, consequently, tumor reduction. Such effects on tumor growth have been previously docu- mented in other cancer models (lung, breast, prostate, brain) [23,36,37]. In these models, JAK inhibition was particularly effective in phospho-STAT3-positive tumors/cell lines. How- ever, AZD1480 has also been shown to inhibit the growth of cancer cell lines independently of STAT3 activation, partic- ularly at higher doses [23], possibly due to off-target effects of the drug. In a recent report, AZD1480 blocked both JAK/STAT3 and FGFR3 signaling in myeloma cells [30]. To test whether the growth inhibitory effects of AZD1480 were dependent on STAT3 in our models, we knocked-down STAT3 in TPC-1 cells. STAT3 deficiency in these cells did not affect their sensitivity to JAK inhibition as compared to control cells. Moreover, AZD1480 was similarly effective in blocking the growth of STAT3- deficient TPC-1 xenografts, which displayed extensive necrosis, similarly to AZD1480- treated parental TPC-1 tumors. Additionally, we knocked- down JAK1 and JAK2 expression in TPC-1, MZ-CRC1 and TT-cell lines by siRNA, which had no effect on their proliferation. These data demonstrate that AZD1480 inhibits the growth of RET-activated thyroid cancer cell lines in vitro PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e46869