B-Raf(V600E) and thrombospondin-1 promote thyroid cancer progression

Abstract

Although B-Raf(V600E) is the most common somatic mutation in papillary thyroid carcinoma (PTC), how it induces tumor aggressiveness is not fully understood. Using gene set enrichment analysis and in vitro and in vivo functional studies, we identified and validated a B-Raf(V600E) gene set signature associated with tumor progression in PTCs. An independent cohort of B-Raf(V600E)-positive PTCs showed significantly higher expression levels of many extracellular matrix genes compared with controls. We performed extensive in vitro and in vivo validations on thrombospondin-1 (TSP-1), because it has been previously shown to be important in the regulation of tumor angiogenesis and metastasis and is present in abundance in tumor stroma. Knockdown of B-Raf(V600E) resulted in TSP-1 down-regulation and a reduction of adhesion and migration/invasion of human thyroid cancer cells. Knockdown of TSP-1 resulted in a similar phenotype. B-Raf(V600E) cells in which either B-Raf(V600E) or TSP-1 were knocked down were implanted orthotopically into the thyroids of immunocompromised mice, resulting in significant reduction in tumor size and fewer pulmonary metastases from the primary carcinoma as compared with the control cells. Treatment of orthotopic thyroid tumors, initiated 1 week after tumor cell implantation with PLX4720, an orally available selective inhibitor of B-Raf(V600E), caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity. In conclusion, B-Raf(V600E) plays an important role in PTC progression through genes (i.e., TSP-1) important in tumor invasion and metastasis. Testing of a patient's thyroid cancer for B-Raf(V600E) will yield important information about potential tumor aggressiveness and also allow for future use of targeted therapies with selective B-Raf(V600E) inhibitors, such as PLX4720.

Fig. 1.

TSP-1 expression in B-Raf^V600E-positive 8505c cells. (A) Real-time RT-PCR analysis in 8505c cells demonstrates a significant decrease of B-Raf^V600E relative mRNA levels by sh#1 [fold change (FC) =0.1 ± 0.01] and sh#10 (FC = 0.14 ± 0.007) vs. control (fold change = 1 ± 0.06). (B) Western blot analysis demonstrating a decrease greater than 90% of B-Raf^V600E protein levels by sh#1 and sh#10. B-Raf^V600E knockdown reduces endogenous levels of phosphorylated p-MEK1/2 (Ser217/221) and p-ERK1/2 (Thr202/Tyr204). Upon B-Raf^V600E silencing, TSP-1 protein levels are approximately 65% down-regulated by sh#1 and 50% by sh#10 upon B-Raf^V600E silencing vs. sh control 8505c cells. (C) Real-time RT-PCR analysis shows that TSP-1 relative mRNA levels are down-regulated approximately 2-fold by sh#1 (FC = 0.45 ± 0.03) and by sh#10 (FC = 0.51 ± 0.01). (D) Indirect immunofluorescence confirms that TSP-1 protein is down-regulated by sh#1 and sh#10 vs. sh control 8505c cells. These data represent the mean ± SEM of three independent experiments. *P < 0.05; ***P < 0.001.

Fig. 2.

Silencing of B-Raf^V600E and functional effects in 8505c cells. (A) Flow cytometry analysis shows a significant reduction of BrdU incorporation of 8505c cells B-Raf^V600E sh#1 and sh#10 in S-phase vs. sh control cells. (B) Cell adhesion and (C) migration and invasion assays show that B-Raf^V600E knockdown by both sh#1 and sh#10 significantly reduces the number of cells that adhered to type I collagen and the number of migrating and invading 8505c cells. These data represent the average ± SD of three independent experiments. *P < 0.05; ***P < 0.001.

Fig. 3.

TSP-1 silencing and functional effects in 8505c cells. (A) Western blot analysis shows a decrease in TSP-1 protein levels by shRNAs (sh) targeting TSP-1 in 8505c cells. TSP-1 knockdown reduces endogenous levels of phosphorylated p-ERK1/2 (Thr202/Tyr204). Real-time RT-PCR analysis shows a significant decrease of TSP-1 relative mRNA levels, 33.3-fold less by sh#7 [fold change (FC) = 0.03 ± 0.006] and 6.25-fold less by sh#8 (FC = 0.2 ± 0.013) vs. sh control cells (FC = 1.04 ± 0.1). (B) Flow cytometric analysis shows a significant reduction of BrdU incorporation in S-phase in 8505c cells sh#7 TSP-1 vs. sh control cells. (C) Cell adhesion and (D) migration and invasion assays show that knockdown of TSP-1 by both sh#7 and sh#8 significantly reduces the number of 8505c cells adherent to type I collagen substrate and the number of migrating and invading 8505c cells. These data represent the average ± SD of three independent experiments. **P < 0.01; ***P < 0.001.

Fig. 4.

Proinvasive role of B-Raf^V600E in 8505c cells grown in 3D cultures. (A) Structural and functional domains of full-length human TSP-1 protein. A single subunit is depicted as a series of structural domains based on the amino acid sequence. The vertical lines that are adjacent to the amino-terminal domain are the interchain disulfide bonds. (B) 3D cell culture–based ECM assay shows that treatment with 0.6 μM full-length human TSP-1 protein or 2 μM of recombinant amino-terminal domain of TSP-1 protein rescues the invasive phenotype (dendritic-like structures with filopodia; arrows) of 8505c cells that underwent knockdown of B-Raf^V600E (sh#1) (day 9). (C) 3D assay shows that treatment with 2 μM of recombinant amino-terminal or 3TSR domains of TSP-1 rescues the invasive phenotype (dendritic-like structures with filopodia; arrows) of 8505c cells with that underwent TSP-1 knockdown (sh#7) (day 9).

Fig. 5.

Knockdown of either B-Raf^V600E or TSP-1 in an in vivo model of thyroid carcinoma. (Ai) Volume of orthotopic thyroid carcinomas (OTC) formed by sh control 8505c cells, sh#1 B-Raf^V600E cells, or sh#7 TSP-1 8505c cells. (Aii) Number of metastatic 8505c cells foci found in each mouse that was averaged per group (n = 6). These data represent the average ± SEM. (Bi-iii) Gross images of a large OTC (arrow) of sh control 8505c cells. (Magnification, ×10.) Bii (phase contrast) and Biii (GFP excitation): tracheal impingement. (Biv–vi) Histological sections (H&E). (Biv) OTC within the right thyroid, showing histopathologic features of a high-grade neoplasm: mitoses (arrows), necrosis (asterisk), and marked cellular pleomorphism. (Magnification: ×40.) (Bv) OTC invading the neck muscles (arrows) and fat (arrowhead). (Magnification: ×40.) (Bvi) adjacent tracheal wall (asterisk) and blood vessels (arrow). (Magnification: ×60.) (Bvii–ix) Gross image of an sh#1 8505c cells small OTC (arrow). (Magnification: ×10.) Bviii: phase contrast; Bix: GFP excitation. (Bx and Bxi) Small OTC (asterisk) within the right thyroid (Bxii) with no tracheal wall invasion (asterisk). (Magnification: Bx, ×20; Bxi, ×60; Bxii, ×60.) (Bxiii) Gross picture of a sh#7 8505c cells small OTC (arrow) (Magnification: ×10.) (Bxiv and Bxv) Small OTC within the right thyroid with no tracheal wall invasion (arrow and asterisk). (Magnification: Bxiv, ×40; Bxv, ×40). (Ci) Gross lung image from a mouse with metastatic sh control 8505c cells OTC. (Magnification: ×10.) (Cii) GFP excitation reveals miliary multifocal lung micrometastases. (Magnification: ×10.) (Ciii) H&E stain of numerous lung micrometastasis of sh control 8505c cells (arrows). (Magnification: ×60.) (Civ) Gross lung image from a mouse injected with sh#1 B-Raf^V600E 8505c cells. (Magnification: ×10.) (Cv) GFP excitation and (Cvi) H&E staing do not reveal lung micrometastasis of sh#1 8505c cells small OTC. (Magnification: Cv, ×10; Cvi, ×40.) (Cvii) Gross lung image (Magnification: ×10.) and (Cviii) H&E stain from a mouse injected with sh#7 TSP-1 8505c. (Magnification: ×20.) (Di) Gross image of a large OTC of 8505c cells (arrow) in mice treated with the vehicle (control group). (Dii) GFP excitation shows a large right OTC (arrow) with extension to the left side (control group). (Diii) GFP excitation reveals miliary multifocal lung micrometastases (arrows) in mice treated with vehicle (control group). (Div) Small OTC of 8505c cells (arrow) in mice treated with PLX4720 30 mg/kg per day for 21 days. (Dv) GFP excitation shows a small right mass (arrow) upon PLX4720 treatment. (Dvi) Significant decrease of the number of lung micrometastases in mice treated with PLX4720 (arrow). (Magnification: Di–vi, ×10.) *P < 0.05; ***P < 0.001.