Attenuated Salmonella Typhimurium Strain (VNP20009) Mediated Melanoma Gene Therapy With Sprouty1/2 And The Therapeutic Mechanisms

Melanoma is a kind of highly invasive malignant skin cancer, whose incidence worldwide has been increasing in the past thirty years. The most widely used strategy for melanoma therapy is the combination between surgical operation and chemotherapy. However, melanoma is easy to develop resistance to chemotherapy, which constrains its efficacy in melanoma therapy. Thus, it’s necessary to develop new strategies for melanoma therapy from the new point.Melanoma is highly malignant because of its characteristics of uncontrolled proliferation, invasion and metastasis. Accumulating knowledge indicated that over-activation of Receptor Tyrosine Kinase (RTK) signalling plays a pivotal role in the process of tumorigenesis and progression. One of the factors that lead to the constitutive activation of this signalling is the absence of endogenous inhibitors that would otherwise inhibit the activity of this pathway. Sprouty is a kind of endogenous RTK signalling inhibitors, which was first found in Drosophila, and four homologous genes (Sprouty 1-4) have been identified in mammals up to now. Recent studies have shown that some members of Sprouty proteins are down-regulated or dysfunction in melanomas. Thus, the exogenous expression of Sprouty proteins in melanoma cells might lead to the re-inhibition of the aberrant activation of RTK signalling and tumor growth, which might provide a new strategy for the melanoma therapy.In the first part of this thesis, we overexpressed Sproutyl and Sprouty2, two members of Sprouty family, separately in mouse B16F10 melanoma cell through liposome transfection. We found that both Sproutyl and Sprouty2 (Sproutyl 12) could lead to a significant grow inhibition of B16F10 cells. Further analysis indicated that overexpression of Sprouty 1/2 leaded to the inhibition of ERK.1/2 phosphorylation and thus arrest cell cycle in G1 phase. Besides, we found overexpression of Sproutyl 12 have on significant impacts on apoptosis in B16F10 cells. In summary, our work demonstrated that overexpression of Sproutyl 12 leaded to inhibition of B16F10 cell proliferation through G1 arrest, which lays the foundation for the further application of Sprouty proteins in melanoma therapy.Salmonella typhimurium (S. typhimurium) is a kind of facultative anaerobic gram-negative strain. Attenuated S. typhimurium strain VNP20009 could preferentially accumulate, carry exogenous plasmid and selectively expressed exogenous protein in solid tumors. Thus, it has been widely used as a tumor-targeting vector in tumor gene therapy.In the second part of this thesis, we engineered VNP20009 to express Sproutyl and Sprouty2 in the melanoma tumors under the control of the anaerobic promoter NirB in a secretory way. We first analyzed the in vivo distribution of VNP20009 harboring Sproutyl and Sprouty2 expressing plasmids (VNP-Sproutyl and VNP-Sprouty2). Both VNP-Sproutyl and VNP-Sprouty2 preferentially accumulated and replicated in the tumor tissues, which indicated a good tumor-targeting ability. Then, we demonstrated that the selective expression of genes of interest could only been detected under anaerobic conditions both in vitro and in vivo. Further immunochemical studies showed that the therapeutic genes were only expressed in the hypnotic areas of tumor tissues. Next, we evaluated the antitumor efficacy of VNP-Sproutyl and VNP-Sprouty2 in vivo, and we found VNP-Sprouty2 could significantly inhibit tumor grow and promote animal survival. The tumor doubling time and tumor delay time were extended as well. However, VNP-Sproutyl has no significant therapeutic effects in comparison with VNP-Vector.To further analysis the mechanisms involving in the anti-tumor effects of VNP-Sprouty2 and leading to the different therapeutic effects of VNP-Sproutyl and VNP-Sprouty2, we analyzed the extent of ERK1/2 phosphorylation in the tumor tissues of mice bearing melanoma. Western Blot results showed that VNP-Sprouty2 led to a significant inhibition on ERK1/2 phosphorylation, while VNP-Sproutyl could only slightly inhibit its phosphorylation. Further immunochemical studies with Ki-67 indicated that VNP-Sprouty2 induced a significant cell proliferation inhibition, while VNP-Sproutyl and VNP-Vector could not. These results were consistent with their different anti-tumor efficacy, which indicated that their different abilities on the inhibition of ERK phosphorylation might be a reason for their different anti-tumor abilities in vivo. Additionally, our results showed that Sprouty2 is a more suitable agent for melanoma gene therapy.Overall, our studies demonstrated that Sprouty2 expressing VNP20009 could significantly delay tumor growth and extend mice survival, which provided a potential effective strategy in the melanoma therapy.