[ASCRIT] Antisense chemoradioimmunotherapy consisting of anti-POEM (Arg-Gly-Asp) scFv linked onto high-energy radioisotopes, vinorelbine-tartrate and 21-nucleotide double-stranded siRNA targeted to DNMT1 induce apoptosis in metastatic breast cancer (MBC) characterized by hypermethylated p53, p16, RASSF1A, RAR-b2, BRCA2, H1C1, ESRI1, CDH1, Trbeta1, GSTP1, CCND2 and overexpression of bcl-2, cdc25c, Raf-1 and α₈β1 integrin

Introduction

Metastatic breast cancer (MBC) is resistant to almost all cytotoxic drugs and radiation, making it one of the most aggressive malignancies in humans, with the worst mortality. The failure of tumour cells to undergo apoptosis causes resistance to chemoradiological therapies due to overexpression of oncogenes and transcriptionally repressed apoptotic tumour suppressor genes due to aberrant methylation (CIMP⁺). Also, upregulation of the ECM gene POEM is associated with adhesion, migration and invasion of highly aggressive and MBC.

Materials and method

We obtained surgically a total of 168 MBC specimens from lymph nodes and lungs of patients. Genomic DNA of tumours was analyzed for CpG island hypermethylation by using methylation specific PCR. All of the tumours showed hypermethylation of tumour suppressor genes with the following frequencies: p16 89%, RASSF1A 84%, RARb2 76%, BRCA2 54%, p53 52%, HIC1 47%, ESR1 43%, CDH1 37% and below 35% for TRbeta1, GSTP1 and CCND2. Quantitative IHC, WB, SB and RT-PCR revealed overexpression of DNMT1, α₈β₁ integrin, bcl-2, Raf-1 and cdc25c. We treated the MBC with anti-POEM (Arg-Gly-Asp) scFv attached onto high-energy radioisotopes, vinorelbine-tartrate and 21-nucleotide double-stranded siRNA segment generated against DNMT1.

Results

Post-treatment, we detected re-expression of tumour suppressor genes p53, p16, RASSF1A, RARb2, BRCA2, HIC1, ESR1, CDH1, TRbeta1, GSTP1 and CCND2 after inhibition of DNMT1mRNA. There was downregulation of metastatic ECM gene POEM (Arg-Gly-Asp) due to targeted scFv blocking binding to α₈β₁ integrin receptor with subsequent inhibition of adhesion, spreading and survival of metastatic tumour cells. There was inactivation of bcl-2,Raf-1 and cdc25c due to phosphorylation by vinorelbine. Furthermore, we detected upregulation of p21waf1, p27kip, Bid and Bak. The high energy radioisotopes induced DNA double-strand breaks in MBC cells and with MT depolymerizer agent vinorelbine they arrested their growth at the G2/M transition according to flow cytometry analysis. We detected externalization of PS, depolarization of mitochondrial transmembrane potential (ΔψM), activation of caspase-3, -7, -8 and -9 and bax, cleavage of poly(ADP-ribose)polymerase and DNA fragmentation. TEM exhibited irreversible D2 apoptotic signs, forming apoptotic bodies that were phagocytosed by adjacent tumor cells, leading to a bystander killing effect (BKE). BrdU and MTT exhibited inhibition of DNA synthesis and metabolic activity of treated MBC cells compared with untreated controls.

Conclusion

We were able to eradicate MBC cells with combined chemoradioimmunotherapy after circumvention of chemoresistance and radioresistance mechanisms such as hypermethylation of p53, p16, RASSF1A, RARb2, BRCA2, HIC1, ESR1, CDH1, TRbeta1, GSTP1 and CCND2, and overexpression of bcl-2, POEM, α₈β₁, Raf-1 and cdc25c.