Development of PEA-15 using a potent non-viral vector for therapeutic application in breast cancer

Abstract

Advanced breast cancer requires systemic treatment, therefore developing an efficient and safe strategy is urgently needed. To ensure the success of target therapy, we have developed a breast cancer-specific construct (T-VISA) composed of the human telomerase reverse transcriptase (hTERT; T) promoter and a versatile transgene amplification vector VISA (VP16-GAL4-WPRE integrated systemic amplifier) to target PEA-15 (phosphoprotein enriched in astrocytes) in advanced breast tumors. PEA-15 contains a death effector domain that sequesters extracellular signal-regulated kinase (ERK) in the cytoplasm, thereby inhibiting cell proliferation and inducing apoptosis. T-VISA-PEA-15 was found to be highly specific, selectively express PEA-15 in breast cancer cells, and induce cancer-cell killing in vitro and in vivo without affecting normal cells. Moreover, intravenous treatment with T-VISA-PEA-15 coupled with liposome nanoparticles attenuated tumor growth and prolonged survival in mice bearing advanced breast tumors. Importantly, there was virtually no severe toxicity when PEA-15 is expressed by our T-VISA system compared with cytomegalovirus (CMV) promoter. Thus, our findings demonstrate an effective cancer-targeted therapy that is worthy of development in clinical trials eradicating advanced breast cancer.

Keywords: Breast cancer; PEA-15; T-VISA system; Target therapy.

Fig. 1

Expression of PEA-15 is downregulated in breast tumor tissues compared with adjacent morphologically normal breast epithelial tissues from the same patient. IHC confirmed that PEA-15 protein was downregulated in the breast tumor tissue (T) compared with the paired adjacent normal breast epithelium (N) from the same patient.

Fig. 2

T-VISA-PEA-15 selectively exerts an inhibitory effect in multiple breast cancer cell lines. (A) Schematic diagram of CMV-PEA-15 and T-VISA-PEA-15 constructs in the pUK21 backbone. (B) Different advanced breast cancer cells were respectively cotransfected increasing amount (0, 0.25, 0.5, 1.0, or 2 μg/well) of T-VISA-PEA-15 and 100 ng of CMV-Luc. (C) T-VISA-PEA-15 effectively inhibits breast tumor cells proliferation. The results are expressed as a percentage of T-VISA control transfected cells (100%) and represent the mean ± SD of three independent experiments. ***indicates P < 0.001. (D) PEA-15 expression driven by T-VISA also kills primary human breast tumor cells effectively. ***indicates P < 0.001. (E) The killing effects of T-VISA-PEA-15 on normal breast cells are slower than CMV-PEA-15. ***indicates P < 0.001.

Fig. 3

T-VISA-PEA-15 erk1/2 in cyto and induce the apoptosis. (A) Western blot analysis of ERK, pERK and the downstream protein expression in MDA-MB-231 cell. GAPDH was used as an internal control. (B) Western blotting verified cleaved caspase 3 and cleaved caspase 7 protein expression. (C) Annexin V-FITC/propidium iodide staining and FACS quantification of the number of apoptotic cells in MDA-MB-231 and MCF-7 cells transfected with DNA(Ctrl, CMV-PEA-15 or T-VISA-PEA-15)-liposome complexes. FACS, fluorescence-activated cell sorting; FITC, fuorescein isothiocyanate.

Fig. 4

T-VISA-PEA-15:liposome complexes dramatically suppresses breast tumor growth and Significantly Improves Survival in the breast tumor model. BALB/c nude mice bearing MDA-MB-231-Luc tumors were treated with 20 μg of liposomal plasmid DNA (CMV-PEA-15, T-VISA-PEA-15 or Ctrl), twice weekly for three consecutive weeks. (A) The tumor cell growth photon signals were quantified (left panel) with Xenogen’s software and representative images are presented (right panel). Error bars indicate SEM. Arrows indicate the treatment time points. (B) Kaplan-Meier survival analysis from (A) was determined. Expression of PEA-15 driven by T-VISA vector prolonged mouse survival more effectively than PEA-15 driven by CMV promoter. (C) Representative images of in vivo apoptosis of tumor by TUNEL assay.

Fig. 5

T-VISA-PEA-15 nanoparticles have no systemically acute toxicity compared with CMV-PEA-15 in immunocompetent mice. (A and B) Kaplan-Meier survival analysis of female BALB/c mice who were treated with single doses of 100 μg or 50 μg plasmid in a liposomal complex via the tail vein. (C, D, and E) Kinetics serum level of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood creatinine (CRE) in mice after a single dose of 50 μg plasmid injection. Asterisk, P < 0.05.

Fig.6

Diagrammatic sketch of the T-VISA-PEA-15 system targeting to breast cancer. The T-VISA-PEA-15 system involves a series of steps in human breast cancer: Firstly, the hTERT promoter drives the GAL4-VP2 fusion protein specifically expression in breast tumor and then open target gene PEA-15 mRNA transcripts. Afterwards, the PEA-15 mRNA transcripts enhance and prolong expression duration of PEA-15 protein, which can sequesters extracellular signal-regulated kinase (ERK) in cytoplasm, thereby leading to apoptosis of breast cancer cells. However, there’s little non-specificity gene expression in normal cells for the activity of hTERT promoter is very lower, thereafter, normal cells survival.