Late intervention with anti-BRAF(V600E) therapy induces tumor regression in an orthotopic mouse model of human anaplastic thyroid cancer

Abstract

Human anaplastic thyroid cancer (ATC) is a lethal disease with an advanced clinical presentation and median survival of 3 months. The BRAF(V600E) oncoprotein is a potent transforming factor that causes human thyroid cancer cell progression in vitro and in vivo; therefore, we sought to target this oncoprotein in a late intervention model of ATC in vivo. We used the human ATC cell line 8505c, which harbors the BRAF(V600E) and TP53(R248G) mutations. Immunocompromised mice were randomized to receive the selective anti-BRAF(V600E) inhibitor, PLX4720, or vehicle by oral gavage 28 d after tumor implantation, 1 wk before all animals typically die due to widespread metastatic lung disease and neck compressive symptoms in this model. Mice were euthanized weekly to evaluate tumor volume and metastases. Control mice showed progressive tumor growth and lung metastases by 35 d after tumor implantation. At that time, all control mice had large tumors, were cachectic, and were euthanized due to their tumor-related weight loss. PLX4720-treated mice, however, showed a significant decrease in tumor volume and lung metastases in addition to a reversal of tumor-related weight loss. Mouse survival was extended to 49 d in PLX4720-treated animals. PLX4720 treatment inhibited cell cycle progression from 28 d to 49 d in vivo. PLX4720 induces striking tumor regression and reversal of cachexia in an in vivo model of advanced thyroid cancer that harbors the BRAF(V600E) mutation.

Fig. 1.

Experimental design. Orthotopic anaplastic tumors were implanted in 48 SCID mice. Each week, six mice were killed. Their tumors and lungs were evaluated by histology and GFP biomaging. Either vehicle or PLX4720 was begun at 28 d, and the response to therapy was evaluated weekly. “No surviving mice” reflects the fact that all mice at this time point showed physical criteria to be killed (e.g. cachexia and/or advanced thyroid tumors causing compression of the trachea and esophagus).

Fig. 2.

PLX4720 extends survival and reverses cachexia in orthotopic mice with human ATC. A, Mouse weight over time. B, Untreated mice developed weight loss, piloerection, and cachexia by 28 d. Vehicle-treated mice progressed to severe cachexia, whereas PLX4720-treated mice had reversal of weight loss, cachexia, and piloerection over the 3-wk course of therapy. *, P < 0.05.

Fig. 3.

PLX4720 induces tumor regression. A, Vehicle-treated mice had an exponential tumor growth up to 115 mm³ ± 14 mm³ by 35 d. No mouse in this group survived beyond 35 d. B, PLX4720-treated mice had tumor regression down to 14 mm³ ± 1 mm³ by 49 d. C, Tumor progression and regression (gross photos and GFP images). *, P < 0.05.

Fig. 4.

PLX4720 induces regression of lung metastases. Number of metastases/cm² as detected by GFP imaging. A, Vehicle-treated mice had exponential growth in the number of metastases, whereas PLX4720-treated mice had a significant reduction in the number of metastases by 49 d (B); *, P < 0.05. C, Anterior projections of the lungs with GFP imaging and gross photos.

Fig. 5.

Histology of orthotopic human ATC. Control tumors at 35 d demonstrated extrathyroidal extension (e.g. tracheal and esophageal invasion) (panels A and B), and lung metastases (asterisk, panel C). Mice treated with 3 wk of PLX4720, however, showed smaller and circumferential tumors (arrow, panels D and E), and decreases in lung metastases (asterisk, panel F). Ki67 immunostaining was significantly decreased with PLX4720 treatment (71.6% ± 6% in controls compared with 15% ± 2.9% in PLX4720-treated mice, P < 0.01) (panel G). Phospho-ERK1/2 immunostaining was significantly decreased with PLX4720 treatment (∼80% in controls compared with 10–20% in PLX4720-treated mice, P < 0.01) (panel H).

Fig. 6.

Immunoexpression of Cyclin E2 and p27/kip1. A, Control tumors show strong nuclear Cyclin E2 (CCNE2) protein levels (2+) at 28 d and 35 d. Orthotopic tumors treated with 3 wk of PLX4720 show a significant decrease of nuclear CCNE2 protein levels (moderate, 2+) at 35 d and 42 d, which progressively decrease up to 49 d (week, 1+). All magnifications, ×400. B, Control tumors show low nuclear p27/kip1 protein levels (2+) at 28 d and 35 d. Orthotopic tumors treated with 3 wk of PLX4720 show a significant increase of p27/kip1 protein levels (strong, 3+) at 35 d and 42 d, which progressively increase up to 49 d (strong, 3+). All magnifications, ×400.

Fig. 7.

Immunoexpression of total caspase-3 and cleaved caspase-3. A, Control tumors and PLX4720-treated tumors show strong (3+) cytoplasmic total caspase 3 protein levels (2+) at 28, 35, 42, and 49 d. All magnifications, ×400. B, Control tumors show moderate cytoplasmic cleaved caspase 3 protein levels (2+) at 28 d and 35 d. Orthotopic tumors treated with 3 wk of PLX4720 show a slight but not significant increase of cytoplasmic cleaved caspase 3 protein levels at 35 d (2+) and 42 d (3+), and moderate at 49 d (2+). C, TUNEL assay using peroxidase system. Slight increase of nuclear staining (apoptotic cells) in the PLX4720-treated orthotopic tumors at 42 d compared with vehicle (control)-treated tumors. All magnifications, ×400.