Eradication of Human Ovarian Cancer Cells by Transgenic Expression of Recombinant DNASE1, DNASE1L3, DNASE2, and DFFB Controlled by EGFR Promoter: Novel Strategy for Targeted Therapy of Cancer

Abstract

Introduction: Ovarian cancer is the most deadly among all gynecological cancers. Patients undergoing systemic therapies of advanced ovarian cancers suffer from horrendous side effects. Cancer survivors and their offspring suffer from iatrogenic consequences of systemic therapies: genetic mutations. The ultimate goal of our work is development of therapies, which selectively and completely eliminate cancer cells, but do not harm healthy cells. An important consideration for attaining this goal is the fact that ovarian cancer cells over-express EGFR or its mutants, what becomes the factor discriminating them from healthy cells - a potential facilitator of personalized therapy.

Specific aim: The specific aim of this project was threefold: (1) to bioengineer suicide genes' carrying vectors guided by synthetic antibodies for EGFRvIII and EGFR; (2) to genetically engineer DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, and DFFB controlled by the EGFR promoter; (3) to selectively eradicate ovarian cancer cells by intranuclear targeting of the transgenically expressed recombinant DNases.

Methods: Synthetic antibodies for EGFR and EGFRvIII were selected from the human library and used to bioengineer biotag-guided transgenes' vectors. Coding sequences for the human DNASE1, DNASE1L3, DNASE2, DFFB controlled by the EGFR promoter were amplified from the human cDNA and genetically engineered into the plasmid constructs also coding for the fusions with NLS and GFP. The vectors carrying transgenes for the DNases were delivered in vitro into human ovarian cancer cells from ascites and cultures.

Results: Synthetic antibody guided vectors delivered the transgenes for the recombinant DNases efficiently into the ovarian cancer cells. Transgenic expression and nuclear targeting of the DNases in those cells resulted in destruction of their genomes and led to their death, as validated by labeling with the molecular death tags. In healthy cells, which did not over-express EGFR, no changes were recorded.

Conclusion: Targeted expression of the recombinant DNASE1, DNASE1L3, DNASE2, DFFB in the ovarian cancers in vitro resulted in their complete eradication, but had no effects upon the healthy cells. This novel therapeutic strategy has a potential for streamlining it into in vivo trials, as personalized, targeted therapy of ovarian and other cancers.

Keywords: Apoptosis; Cancer of the ovary; DNase; Epidermal growth factor receptor; Nuclear localization signal; Personalized therapy of cancer; Suicide gene therapy of cancer.

Figure 1

The DNA constructs with the coding sequences for human recombinant DNase1, DNase1L3, DNase2, DFFB (hrDNases) were delivered by anti-EGFRvIII (DEF) and anti-EGFR (GHI) antibody guided vectors into the nuclei of EGFRvIII + or EGFR+ over-expressing ovarian cancer cells from the patients’ ascites. They were labeled with the anti-dsDNA (D,G) or anti-phosphatidylserine (F,I) and imaged with phase contrast (E,H) or Ploem’s epifluorescence (D,F,G,I). Non-transduced EGFRvIII+ ovarian cancer cells from ascites stained with bisbenzimide (A) and labeled with anti-EGFRvIII antibody (C), were imaged with phase contrast (B) or Ploem’s fluorescence (A,C) as the controls. Transgenic expression and intranuclear targeting of the hrDNases in ovarian cancer cells resulted in collapse of the chromatin architecture, as highlighted after labeling with anti-dsDNA fluorescent antibodies (E,G), as well as externalization of phospatidylserine as highlighted after labeling with anti-PS fluorescent antibodies (F,I).

Figure 2

The DNA constructs with the coding sequences for human recombinant DNase1, DNase1L3, DNase2, DFFB (hrDNases) were delivered by anti-EGFRvIII (B,E) and anti-EGFR (C,F) antibody guided vectors into the nuclei of EGFRvIII + or EGFR+ over-expressing ovarian cancer cells from the patients’ ascites. Genomic DNA from these cells was isolated, electrophoresed, and stained. The reference ladders were 100bp (A) and 200bp (D). In the targeted and transduced cells, genomic DNA was completely degraded (B,C). In the cells, which were transduced with the reversed orientation vectors (E) or non-transfected (F), genomic DNA was retained in the loading wells.

Figure 3

Deadly effects of transgenic expression of DNases in the ovarian cancer cells over-expressing EGFRvIII from ascites and culture were quantified by labeling of the cells with superparamagnetic antibodies against dsDNA followed by measuring relaxivities with NMRS. That followed by separation of the labeled cells with MACS. Lethal effects of transgenic DNases onto the ovarian cancer cells were quantified by labeling with the elemental tagged antibodies for EDXS to yield identical results. Viability of the cells remained unaffected, when the cells were transfected with the vectors coding reversed orientation sequences as compared to the cells not exposed to any vectors at all.

Figure 4

Transduction of the EGFRvIII+ over-expressing ovarian cancer cells with the DNA constructs for the human recombinant DNase1, DNase1L3, DNase2, DFFB (hrDNases) resulted in their surfaces’ topographies disfigured by multiple blebs (C,D). These could be compared for the surface blebs, which occurred as the results of ROS-induced apoptosis in the cultured OVCAR cells (A,B). Pores in the membranes’ blebs, which are only seen at seen at high magnifications, are the routes of entry for labels targeting their content. Presence of these pores in the blebs’ membranes explains observations that the blebs’ contents are labeled long before the cells’ interiors. HFW A,C: 20 μm; B,D: 7.2 μm.

Figure 5

Transduction of the EGFRvIII+ over-expressing ovarian cancer cells with the DNA constructs for the human recombinant DNase1, DNase1L3, DNase2, DFFB (hrDNases) resulted in complete destruction of their chromatin architecture (C,D). This could be compared with the state of collapse of chromatin architecture, which occurred as the results of ROS-induced apoptosis (A,B). In the rapidly cryo-immobilized EGFRvIII+ overexpressing cultured ovarian cancer cells, which were labeled with the anti-dsDNA superparamagnetic antibodies, chromatin architecture was revealed by EFTEM with the filter at the zero loss energy and contrast tuning (A) and distribution of the genomic DNA by ESI with the filter set at the Gd edge (B) [Malecki et al. 2013. WO2012048161 http://patentscope.wipo.int/search/en/WO2012048161]. HFW: 11.25 μm.