Tumor-specifically hypoxia-induced therapy of SPRY1/2 displayed differential therapeutic efficacy for melanoma

Abstract

Activation of receptor tyrosine kinase (RTK) signalling pathways is frequently correlated to cancer cell proliferation, angiogenesis and cell survival. Sprouty (SPRY) proteins function as a physiological endogenous inhibitor of RTK signalling pathways, have been shown to be deregulated in most cancer cells. Here, we demonstrated that over-expression of SPRY1 and SPRY2 inhibited B16F10 cell proliferation through G1 phase arrest in vitro, and SPRY2 showed more potent inhibitory effects than SPRY1. In order to tumor-specific delivery of SPRY1/2 in vivo, two strains of attenuated Salmonella typhimurium VNP20009 (VNP-PQE-SPRY1 and VNP-PQE-SPRY2) were constructed to specifically express SPRY1 or SPRY2 under the control of a hypoxia-induced nirB promoter. The efficiency and specificity of the recombinant strains were validated in both bacteria and animal tumor models. SPRY1 and SPRY2 gene could be specifically driven by the nirB promoter under hypoxia, but not normoxia conditions. In addition, the tumor-targeting ability of VNP-PQE-SPRY1 or VNP-PQE-SPRY2 was similar with VNP. VNP-PQE-SPRY2 significantly suppressed melanoma growth in vivo, suggesting that SPRY2 is a more efficient agent for melanoma therapy. Moreover, the antitumor effect of VNP-SPRY2 is mainly mediated through the inhibition of ERK1/2 phosphorylation, which leads to the inhibition of proliferation in melanoma. Taken together, our results indicated that SPRY2 displayed more potent melanoma suppression than SPRY1 both in vitro and in vivo, and the hypoxia-induced tumor-specific gene therapy of SPRY2 delivered by VNP20009 is a promising strategy for melanoma therapy.

Keywords: SPRY1; SPRY2; attenuated Salmonella typhimurium VNP20009; melanoma gene therapy.

Figure 1

Effects of SPRY1 or SPRY2 overexpression on B16F10 melanoma proliferation in vitro. A. MTT assay of cell proliferation after transfection of SPRY1 or SPRY2 expressing or empty vectors. B. Western blot analysis shows the expression of genes of interest in each group. C. Cell cycle analysis of B16F10 cells that transfected with SPRY1 or SPRY2 expressing or empty vectors. D. Western blot analysis of expression of phospho-ERK1/2, total-ERK1/2 and cyclinD1 after transfection of SPRY1 or SPRY2 expressing or empty plasmids in B16F10 cells. α-tubulin was served as loading control. One representative of three independent experiments is displayed. The extent of ERK activation (p-ERK intensity/total ERK intensity) and relative expression of cyclinD1 were quantified by densitometric analysis.*p < 0.05; **p < 0.01; ***p < 0.001 versus empty plasmids transfection cells. Data are expressed as mean ± SD of three independent experiments.

Figure 2

Secretory expression of SPRY1 and SPRY2 by S. typhimurium strain VNP20009. A. Schematic diagram of the construction of vectors for expression of SPRY1 and SPRY2 in VNP20009. B. The indicated stable strains were grew in anaerobic or aerobic jars and their bacteria lysates and culture supernatant were examined for the expression of genes of interest through western blotting as described in the methods. C. The relative tissue distribution of indicated stable strains were detected by colony formation assay. *p < 0.001, tumor compared with the other tissues. Data are expressed as mean ± SD of three animals. D. Detection of the expression of genes of interest in the liver, spleen, lung, kidney, and tumor tissues of the mice bearing melanoma by western blotting. α-tubulin was served as loading control. One representative of three independent experiments is displayed.

Figure 3

In vivo evaluation of therapeutic efficacy of the recombinant Salmonella. A. Tumor growth curves of groups as indicated. B. Tumor doubling time (time for a tumor to double in volume) of different groups. Data are presented as mean ± SD, *p < 0.05, **p < 0.01 (n = 8 mice), N.S: NS: not significant. C. Detection of the expression of recombinant proteins in the sections of tumor tissues through immune-histochemical studies. N represents necrotic tumor area, and V represents vital tumor cells, black arrows, positive cells.

Figure 4

S. typhimurium strain VNP20009 carrying SPRY2 expressing plasmid induced down-regulation of ERK phosphorylation and inhibition of proliferation in tumor tissues of mice bearing melanoma. A. Western blotting analysis of the expression of phospho-ERK and total-ERK in tumor tissues of mice treated with indicated strains. α-tubulin was served as loading control. B. Statistical result of the extent of ERK activation for groups as indicated. C. Immunohistochemical staining of tumor sections of mice treated as indicated with Ki-67 antibody. Black arrows, Ki-67 positive cells. D. Statistical result of Ki-67 index (ratio of the number of positive staining cells to the number of all cells) for groups as indicated. Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3 mice), N.S: not significant.