RESUMO
Hepatocellular carcinoma (HCC) is one of the most common malignancies over the world. Alpha-fetoprotein (AFP) is an oncofetal protein during HCC development, which could generate weaker and less reproducible antitumor protection, and may serve as a target for immunotherapy. Therefore, it is imperative to enhance its immunogenicity and develop therapeutic vaccines to eliminate AFP-expressing tumors. In this study, by using glutaraldehyde cross-linking, we constructed a potential therapeutic peptide vaccine, heat shock protein 72 (HSP72) and AFP epitope peptide (HSP72/AFP-P). ELISPOT was applied to evaluate the quantity of AFP-specific CD8+ T cell that secreted IFN-γ in immunized BALB/C mice. Granzyme B released from natural killer cells and AFP-specific antibody responses in immunized mice were detected by ELISA. The anti-tumor effects were investigated by in vitro cytotoxic T-lymphocyte assays and in vivo tumor therapeutic experiments. The results showed that reconstructed HSP72 and AFP epitope peptide vaccine synergistically exhibited significant increases in AFP-specific CD8+ T cells, natural killer cells responses and impressive antitumor effects against AFP-expressing tumors. Immunization of BALB/C mice with HSP72/AFP-P vaccine elicited stronger T-cells responses. The numbers of IFN-γ-producing CD8+ T cells from mice immunized with HSP72/AFP-P were 30 times more than those from mice immunized with AFP-P, HSP72 or PBS (P < 0.01). The concentration of granzyme B in natural killer cells from mice immunized with HSP72/AFP-P were 15 times higher than that from other groups (P < 0.01). In vitro effector cells from mice immunized with HSP72/AFP-P showed much stronger cytolytic effect on H22 target cells than those from mice vaccinated with AFP-P, HSP72 or PBS (P < 0.01). Priming mice with the reconstructed vaccine exhibited robust strong protective immunity. Mice immunized with HSP72 or AFP-P alone demonstrated higher average tumor volumes than mice immunized with HSP72/AFP-P (P < 0.05). Our study suggests that constructing a tumor vaccine by cross-linking AFP antigen epitope peptide and HSP72 is a promising approach for cancer therapy.
RESUMO
This study identified tumorigenic processes most dependent on murine heat shock protein 72 (HSP72) in the mouse mammary tumor virus-PyMT mammary tumor model, which give rise to spontaneous mammary tumors that exhibit HSP72-dependent metastasis to the lung. RNA-seq expression profiling of Hspa1a/Hspa1b (Hsp72) WT and Hsp72-/- primary mammary tumors discovered significantly lower expression of genes encoding components of the extracellular matrix (ECM) in Hsp72 knockout mammary tumors compared to WT controls. In vitro studies found that genetic or chemical inhibition of HSP72 activity in cultured collagen-expressing human or murine cells also reduces mRNA and protein levels of COL1A1 and several other ECM-encoding genes. In search of a possible mechanistic basis for this relationship, we found HSP72 to support the activation of the tumor growth factor-ß-suppressor of mothers against decapentaplegic-3 signaling pathway and evidence of suppressor of mothers against decapentaplegic-3 and HSP72 coprecipitation, suggesting potential complex formation. Human COL1A1 mRNA expression was found to have prognostic value for HER2+ breast tumors over other breast cancer subtypes, suggesting a possible human disease context where targeting HSP72 may have a therapeutic rationale. Analysis of human HER2+ breast tumor gene expression data using a gene set comprising ECM-related gene and protein folding-related gene as an input to the statistical learning algorithm, Galgo, found a subset of these genes that can collectively stratify patients by relapse-free survival, further suggesting a potential interplay between the ECM and protein-folding genes may contribute to tumor progression.