Use of the Rad51 promoter for targeted anti-cancer therapy

Abstract

Rad51 protein, involved in homologous recombination, is overexpressed in a variety of tumors, and its expression is correlated with a poor prognosis. Here we propose to exploit the overexpression of Rad51 in cancer cells to design a Rad51 promoter-based anticancer therapy. On average, Rad51 mRNA and protein levels are increased in cancer cells four-and sixfold, respectively. Serendipitously, we discovered that when the Rad51 ORF is replaced with another ORF, the difference in promoter activity between normal and cancer cells increases to an average of 840-fold with a maximum difference of 12,500-fold. This dramatic difference in activity has high therapeutic potential. We demonstrate that the fusion of Rad51 promoter to diphtheria toxin A (DTA) gene kills a variety of cancer cell types, including breast cancer, fibrosarcoma, and cervical cancer cells, with minimal effect on normal breast epithelial cells and normal fibroblasts. Our results suggest that therapies based on the Rad51 promoter will be highly tumor specific and open new avenues for targeting a broad range of cancers.cancer ͉ transcriptionally targeted therapy

Regulation of Rad51 promoter

“…8,9 Expression of Rad51 is tightly controlled in normal human cells to decrease aberrant chromosomal recombination. 10,11 The opposite is seen in the majority of human tumors and cancer cell lines, where Rad51 mRNA and protein levels as well as the promoter activity are very high. 10,[12][13][14][15] Rad51 overexpression not only allows the cancer cells to survive treatment, 13,16 but is linked to metastasis 17 and aggressiveness 18 of the disease and poorer patient prognosis.…”

“…10,11 The opposite is seen in the majority of human tumors and cancer cell lines, where Rad51 mRNA and protein levels as well as the promoter activity are very high. 10,[12][13][14][15] Rad51 overexpression not only allows the cancer cells to survive treatment, 13,16 but is linked to metastasis 17 and aggressiveness 18 of the disease and poorer patient prognosis. [19][20][21] Although the role of Rad51 in cancer development, progression and pathogenesis has been well established, very few studies have examined the mechanisms behind the differential regulation of Rad51 between cancerous and non-cancerous cells.…”

“…Promoter activity was measured by placing the open reading frame (ORF) of firefly luciferase under the transcriptional control of this cloned Rad51 promoter. 10 Cancerous cells displayed an average 850-fold increase in promoter activity compared with non-cancerous cell lines, with a maximal difference of 12 500-fold. Due to such a large differential in promoter activity/ transgene expression, we further extended the use of our cloned Rad51 promoter for use in targeted anti-cancer gene therapy.…”

“…[19][20][21] Although the role of Rad51 in cancer development, progression and pathogenesis has been well established, very few studies have examined the mechanisms behind the differential regulation of Rad51 between cancerous and non-cancerous cells. 10,[22][23][24][25] We previously cloned a segment of the Rad51 promoter region spanning −2931 bp upstream to +3601 bp downstream relative to the start of transcription and analyzed promoter activity in a variety of non-cancerous and cancerous human cell lines. Promoter activity was measured by placing the open reading frame (ORF) of firefly luciferase under the transcriptional control of this cloned Rad51 promoter.…”

Development of Small Molecules that Specifically Inhibit the D-loop Activity of RAD51

“…[2][3][4] RAD51, the central protein of HR, is overexpressed in various types of human cancer cells. 5,6 These high expression levels of RAD51 can elevate HR efficiency in cancer cells, thereby inducing cellular resistance to DNA-damaging chemotherapy and radiotherapy. [7][8][9][10] Inhibition of RAD51 by antisense RNAs or RAD51 inhibitors are reported to enhance the sensitivity of cancer cells to chemotherapy and radiotherapy.…”