Abstract
BackgroundPlacental like alkaline phosphate (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting.MethodsPLAP promoter alone or in combination with NFκB DNA response elements was used for expressing shRNA targeting the long control region (LCR) of human papillomavirus (HPV)-16 oncogenes E6 and E7 via transcriptional gene silencing in PLAP expressing cervical cancer cell lines, SiHa and CaSki. This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting. Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties.ResultsReduction of HPV-16 E6 and E7 expression by TGS led to the activation of the previously suppressed target genes of p53 (PUMA and NOXA) and Rb (cyclins A2 and E). Cell death was seen only in PLAP expressing HPV-16 infected SiHa and CaSki cells but not in the HPV-18 integrated HeLa and non-PLAP CHO cells. There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes. Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region.ConclusionA combination of novel PLAP promoter and antibody based specificities has the potential for being developed as a possible therapeutic strategy for PLAP positive neoplasia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0602-1) contains supplementary material, which is available to authorized users.
Highlights
Placental like alkaline phosphate (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues
Our results show that engineered immuno-virosomes and transcriptional gene silencing (TGS) inducing constructs provide dual specificity to tumour targeting in terms of delivery and cellular expression, could be foreseen as a potential gene therapy tool
HPV-18 integrated cervical cancer cell line (HeLa), HepG2 and Chinese hamster ovary (CHO) were obtained from American Type Culture Collection (ATCC) while SiHa and CaSki were obtained from National Centre for Cell Science (NCCS), Pune
Summary
Placental like alkaline phosphate (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting. Tumour specific antigens like HER2/neu and CD20 have been shown to be useful for combating tumours [1–3]. In some cases such antigens have been targeted for specific delivery by antibody and particulate delivery approaches [4–6]. We reasoned that combining an antibody based targeting modality with a construct based on antigen’s own promoter would provide a novel way for increasing tumour specificity and efficacy.
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