Abstract
Genomic studies have revealed that breast cancer consists of a complex biological process with patient-specific genetic variations, revealing the need for individualized cancer diagnostic testing and selection of patient-specific optimal therapies. One of the bottlenecks in translation of genomic breakthroughs to the clinic is the lack of functional genomic assays that have high clinical translatability. Anchorage-independent three-dimensional (3D) growth assays are considered to be the gold-standard for chemosensitivity testing, and leads identified with these assays have high probability of clinical success. The Cancer BioChip System (CBCS) allows for the simultaneous, quantitative, and real time evaluation of multitudes of anchorage-independent breast cancer cell growth inhibitors. We employed a Test Cancer BioChip that contains silencing RNAs (siRNAs) targeting cancer-related genes to identify 3D-specific effectors of breast cancer cell growth. We compared the effect of these siRNAs on colony growth of the hormone receptor positive (MCF7) and Human Epidermal Growth Factor Receptor 2/c- Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2/c-erb-b2) positive (SK-BR-3) cells on the Test Cancer BioChip. Our results confirmed cell-specific inhibition of MCF7 and SK-BR-3 colony formation by estrogen receptor α (ESR1) and (ERBB2) siRNA, respectively. Both cell lines were also suppressed by Phosphoinositide-3-kinase Catalytic, alpha Polypeptide (PIK3CA) siRNA. Interestingly, we have observed responses to siRNA that are unique to this 3D setting. For example, ß-actin (ACTB) siRNA suppressed colony growth in both cell types while Cathepsin L2 (CTSL2) siRNA caused opposite effects. These results further validate the importance of the CBCS as a tool for the identification of clinically relevant breast cancer targets.
Highlights
High throughput RNA interference (RNAi) screens have revealed genes essential for the growth of breast cancer cells [1,2] and sensitivity to current therapies [3,4,5]
We tested growth of two breast cancer cell lines: MCF7 and SKBR-3. Both cell lines express most genes targeted on the Test Cancer BioChip at comparable levels, with the exception of ESR1, PGR, SCUBE2, CCNB1, and Insulin-like Growth Factor 1 Receptor (IGF1R) being higher in MCF7 cells and GRB7 and ERBB2 being elevated in SK-BR-3
We identified PIK3CA silencing RNAs (siRNAs) to be a significant suppressor of anchorage-independent growth of both MCF7 and
Summary
High throughput RNA interference (RNAi) screens have revealed genes essential for the growth of breast cancer cells [1,2] and sensitivity to current therapies [3,4,5]. Anchorage-independent growth assays have been considered to be the gold-standard for chemosensitivity testing for breast cancer [6] These assays utilize different types of matrices, including soft agar, to inhibit cellular attachment and allow for 3D growth of cells. Normal epithelial cells depend on cell-cell contact and attachment to a physical support for survival and growth These unique properties of anchorage-independent growth assays allow for selective chemotoxicity testing of tumor cells in a setting that is 3D, and more relevant to the in vivo milieu [6]. Targets identified with these assays have a higher likelihood of clinical success
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