RESUMO
Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy.
Assuntos
Anticorpos/uso terapêutico , DNA Recombinante/genética , Neuralgia/metabolismo , Neuralgia/terapia , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular , Animais , Anticorpos/farmacologia , Benzofuranos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Gânglios Espinais/citologia , Hiperalgesia/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Condução Nervosa/genética , Medição da Dor , Limiar da Dor/fisiologia , Quinolinas , RNA Mensageiro/genética , Ratos , Ratos Wistar , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Current therapies for metastatic melanoma are targeted either at cancer mutations driving growth (e.g., vemurafenib) or immune-based therapies (e.g., ipilimumab). Tumour progression also requires angiogenesis, which is regulated by VEGF-A, itself alternatively spliced to form two families of isoforms, pro- and anti-angiogenic. Metastatic melanoma is associated with a splicing switch to pro-angiogenic VEGF-A, previously shown to be regulated by SRSF1 phosphorylation by SRPK1. Here, we show a novel approach to preventing angiogenesis-targeting splicing factor kinases that are highly expressed in melanomas. METHODS: We used RT-PCR, western blotting and immunohistochemistry to investigate SRPK1, SRSF1 and VEGF expression in tumour cells, and in vivo xenograft assays to investigate SRPK1 knockdown and inhibition in vivo. RESULTS: In both uveal and cutaneous melanoma cell lines, SRPK1 was highly expressed, and inhibition of SRPK1 by knockdown or with pharmacological inhibitors reduced pro-angiogenic VEGF expression maintaining the production of anti-angiogenic VEGF isoforms. Both pharmacological SRPK1 inhibitors and SRPK1 knockdown reduced growth of human melanomas in vivo, but neither affected cell proliferation in vitro. CONCLUSIONS: These results suggest that selective blocking of pro-angiogenic isoforms by inhibiting splice-site selection with SRPK1 inhibitors reduces melanoma growth. SRPK1 inhibitors may be used as therapeutic agents.