Small Interfering RNA Screens Reveal Enhanced Cisplatin Cytotoxicity in Tumor Cells Having both BRCA Network and TP53 Disruptions

Abstract

RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53؉/؊ matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity ϳ4-to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.

Functional Genomics Reveals Diverse Cellular Processes That Modulate Tumor Cell Response to Oxaliplatin

“…À HeLa cell line, also previously showed that the loss of SHFM1 or BRIP1 expression enhanced cisplatin cytotoxicity (26). Our study showed that the loss of either SHFM1 or BRIP1 expression increased oxaliplatin cytotoxicity in an siRNA screen by using the HCT 116 TP53 þ cell line.…”

“…S2). Likely explanations for these differences include both the underlying mutational status of TP53 in the cell lines used and the more fundamental genomic variation between HeLa and HCT 116 tumor cell lines (derived from a human cervical adenocarcinoma and CRC, respectively), resulting in a differential sensitization upon the loss of additional genes in the presence of DNA-damaging agents such as cisplatin and oxaliplatin (26). Indeed, 11 genes that were previously identified as enhancers of cisplatin cyto-…”

Hierarchical involvement of Bak, VDAC1 and Bax in cisplatin-induced cell death

“…Among 56 distinct siRNA transfections (54 single and 2 cotransfections), one siRNA species that depleted VDAC1 was consistently (in five out of five experiments) the most cytoprotective one. In this experimental setting, VDAC1 depletion was more efficient in maintaining viability and/or in inhibiting cell death than the knockdown of any other confirmed pro-apoptotic protein known to participate in CDDPelicited lethal signaling, including breast cancer 1 (BRCA1) (Bartz et al, 2006), p53 (Siddik, 2003), apoptotic peptidase-activating factor-1 , Bak and Bax (alone and in combination) (Wang et al, 2001;Kepp et al, 2007) as well as excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) (Olaussen et al, 2006). VDAC1 knockdown exhibited greater cytoprotective effects against CDDP-induced cell death than the depletion of VDAC2 and VDAC3 (which were only marginally effective or totally ineffective, respectively) or of any other constituent of the PTPC such as the three adenine nucleotide translocase isoforms, the two hexokinase isoforms or cyclophilin D (all of which are implicated in CDDP-induced cell death) (Figure 1).…”

Suppression of Rev3, the catalytic subunit of Polζ, sensitizes drug-resistant lung tumors to chemotherapy

“…Notably, human cells expressing reduced levels of REV3L are more sensitive to killing by cisplatin (14,15). Additionally, in an siRNA-based screen, a reduction in REV3L sensitized human cells to killing by cisplatin to an extent equal or greater to a reduction of BRCA1 (16). Finally, chicken DT40 cells deficient in Rev3 showed the highest sensitivity to cisplatin of any of the DNA repair or checkpoint mutants tested (17).…”