Pharmacologic inhibition of MEK signaling prevents growth of canine hemangiosarcoma

Abstract

Angiosarcoma is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human angiosarcoma, we studied primary cells and tumorgrafts derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive extracellular signal-regulated kinase (ERK) activation. The mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multireceptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human angiosarcoma, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was upregulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human angiosarcoma. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human angiosarcoma, and it highlights the use of spontaneous canine cancers as a model of human disease.

Figure 1

HSA primary tumors are pERK positive. pERK-immunostained sections of formalin-fixed HSA visceral (A–K), cutaneous (L–N), and cardiac tumors (O). (A–F, L, M, O) Positive for pERK. (G–K, N) Tissue is negative for pERK. Bar = 100 μm.

Figure 2

ERK is constitutively active in HSA-derived cells. Primary cells isolated from visceral, cutaneous, or cardiac HSA were incubated overnight in the presence or absence of serum. Total lysates were collected and immunoblotted against phospho-ERK1/2 and total ERK1/2. DNSTECs were serum-starved and immunoblotted as a negative control. UV-treated MDCK is a positive control for canine ERK1/2 activation.

Figure 3

Morphological and IHC analysis of HSA cutaneous-derived xenografts, cardiac-derived tumorgrafts, and primary HSA. (A) Similarity between primary cutaneous HSA and canine cutaneous-derived xenografts for the F0 and F5 generations. (B) Similarity between primary cardiac HSA with canine cardiac-derived tumorgrafts for the both F0 and F7 generations. Bar = 100 μm.

Figure 4

MEK inhibition reduces tumor growth in both cutaneous xenograft and cardiac-derived tumorgraft. (A) Growth curves of individual cutaneous xenografts. Arrowhead represents 20% decrease in drug concentration on treatment day 28. Arrow signifies a tumor estimated to be 10 mm³. (B) Box-and-whisker plot of tumor volume at treatment day 42; the dot represents the fast-growing vehicle outlier. (C) Growth curves of individual cardiac-derived tumorgrafts. (D) Box-and-whisker plot of tumor volume at treatment day 42; the dot represents a CI-1040 refractory tumor. (E) Growth curves of average cardiac-derived tumografts treated with PD0325901, vehicle, or non-treated (n=10). (F) Box-and-whisker plot of tumor volume at treatment day 22. Error bars on growth curves represent standard deviations.

Figure 5

Sensitivity of cutaneous-derived xenograft and cardiac-derived tumors to sorafenib treatment. (A) Growth curves of individual cutaneous xenografts; 6 of 10 mice developed toxicity and failed to complete the study. (B) Box-and-whisker plot of tumor volume at treatment day 43. (C) Growth curves of individual cardiac tumorgrafts. (D) Box-and-whisker plot of tumor volume at treatment day 22. (D) Box-and-whisker plot representing the treatment day at which tumorgrafts grew to 3 times their treatment-day-1 volume. Error bars on growth curves represent standard deviations.

Figure 6

Morphological and immunohistological similarities of visceral, cutaneous, and cardiac HSA and human AS tumors. (A-F) H&E- and CD31-stained sections of visceral, cutaneous, and cardiac HSA. (G-L) H&E- and CD31-stained sections of visceral, cutaneous, and cardiac AS. Bar = 100 μm.

Figure 7

MEK activity is present in human AS. (A and B) IHC assay shows that phosphorylated ERK1/2 is present in two independent cutaneous AS. (C) The 20 most up-regulated and down-regulated MAPK-responsive transcription factors in AS. RNA isolated from OCT-frozen AS were compared to RNA from normal human tissue (blood, kidney, and skeletal muscle) and pooled normal tissue (Pooled controls 1 and 2). Bar = 100 μm.