Roles for endothelin receptor B and BCL2A1 in spontaneous CNS metastasis of melanoma

Abstract

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases.

Figure 1

Confirmation of EDNRB expression in melanoma clinical tissue samples. A, RT-PCR examination of expression of EDNRB in clinical samples of melanoma CNS metastases. B, examination of EDNRB immunostaining in independent clinical samples showed lower levels of expression in primary and lymph node metastases than lung and brain metastases.

Figure 2

Confirmation of functional relevance of EDNRB. A, orthotopic implantation of cells overexpressing ENDRB (6-4EDNRB) leads to more aggressive spontaneous metastatic disease and shorter median survival (P < 0.05) when compared with controls. B, a higher incidence of brain metastases in mice implanted with cell lines overexpressing EDNRB was also noted (6-4EDNRB; 5 out of 8 mice) when compared with control 6-4vector (1 out of 10 mice; *, Fisher's exact test P < 0.05). Similar alterations in the frequency of metastatic disease were not noted n other organs such as the kidney and liver.

Figure 3

Role of EDNRB in melanoma cell proliferation and intracranial melanoma growth. A, both brain metastatic cell lines 131/4-5B1 and B2 show increased proliferation in the presence of EDNRB ligand ET3, which is reversed by the addition of EDNRB-specific inhibitor A192621 (one-way ANOVA, P < 0.05). Values are expressed as proliferation relative to the respective cell line cultured in the absence of ET3. B, brain-metastatic cell lines show enhanced proliferation in the presence of brain-CM compared with the visceral metastatic parental 113/6-4L subline. This enhanced proliferation was inhibited by A192621 (one-way ANOVA, P < 0.05). Values are expressed as proliferation relative to the respective cell line cultured in the absence of ET3. C and D, intracranially implanted 6-4 EDNRB cells formed larger tumors (indicated by arrows) when compared with empty vector control. Tumor area in brain cross-sections was significantly greater in 6-4 EDNRB group (t test P < 0.05; E) and showed higher number of Ki67-positive cells (t test, P < 0.05; F). All values are expressed as mean ±SEM. G and H, knockdown of EDNRB in 131/4-5B2cell line (knockdown lines hp2136 and hp3119, respectively) resulted in a significant decrease in intracranial tumor size (t test, P < 0.05).

Figure 4

Therapeutic targeting of EDNRB in metastatic disease. A, using the spontaneous brain metastatic 131/4-5B2 model, EDNRB-specific inhibitor A192621 mediated suppression of metastatic disease leading to significant prolongation of median survival (log rank test, P < 0.05) and improved HR when compared with mice treated with vehicle control. B, daily treatment with A192621 failed to inhibit intracranial melanoma tumors. C to E, a combination of A192621 and cyclosporin A resulted in a significant decrease in intracranial tumor size when compared with cyclosporin A + vehicle (Student's t test, P < 0.05).

Figure 5

Examination of the role of BCL2A1 in intracranial tumor growth. A, RT-PCR examination of BCL2A1 in clinical samples of melanoma CNS metastases. B, mice implanted orthotopically with 6-4BCL2A1 cells showed more aggressive spontaneous metastatic disease leading to a shorter median survival. C, the overexpression of BCL2A1 did not lead to a significant increase in incidence of metastatic disease in various organs. D to F, intracranially implanted 6-4BCL2A1 cells gave rise to larger intracranial melanomas when compared with 6-4vector cells.