P3-16-02: Targeting Tumor Initiating Cells with siRNA/Nanotherapy in Triple Negative Breast Cancer.
Authors: Granados-Principal S , Deng X , Liu D , Chang JC , Shen H , Dave B 
Published: June 2012

Residual human breast tumor cells after conventional therapies are enriched in tumor-initiating cells (TIC) characterized by CD44+/ CD24-/low/lineage- with self-renewal capacities. Our gene expression analyses in those cells and in breast cancer cells propagated as mammospheres (MSs) reveal an epithelial-mesenchymal-transition (EMT) signature (400 genes) mainly found in claudin-low molecular subtype human breast tumors. We performed lentiviral shRNA knockdown of that signature in MS. Critical shRNAs were found to decrease the TIC subpopulation. Among those genes, we found STAT3 (signal transducer and activator of transcription 3), NTN4 (netrin 4), and RPL39L (ribosomal protein L39-like). Here, we used a multiscale siRNA delivery system targeting those three genes in order to clarify the effect of silencing those genes over the self-renewal capacity on MSs with claudin-low features. To this end, duplex small interfering RNA (siRNA) against STAT3, NTN4, RPL39L, and scrambled siRNA as control, were introduced into neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC), and using mesoporous microscale biodegradable silicon particles as carriers. This multistage siRNA delivery system has been reported as a good approach for sustained gene silencing. Claudin-low-like human breast cancer cell lines (SUM159 and BT549) were plated in 24-well ultralow attachment plates with mammary epithelial growth medium (MEGM) (5,000 cells/well). Both cell lines were then treated with 1μg/well/6 wells of silicon particles loaded with DOPC nanoliposomes/siRNA. The primary MSs were allowed to grow for 3 days. MFs were counted by day 3 with a GelCount colony counter (Oxford Optronix, Oxford, UK). Mammosphere-Forming Efficiency (MSFE) was calculated by dividing the number of MSs by the number of seeded cells. In addition, established MSs were serially passaged by dissociation, and single cells were replated on fresh 24-well ultra-low attachment plates to form secondary mammospheres, which were counted after 3 days. One-way ANOVA and Tukey test were performed. A p value less than 0.05 was considered as significant. Our results show that silencing STAT3, NTN4, and RPL39L significantly reduces the MSFE in both primary (1%, 0.9%, and 1.7% respectively) (Figure 1) and secondary MSs (0.49%, 0.51%, and 0.45% respectively) (Figure 2) when compared to the scrambled control (2.4% and 1 % respectively) in BT549 cells. For SUM159 cells, we did not find any change in primary MSs for STAT3 (2.2%) and NTN4 (2.1%), even we found a higher percentage of MSFE in those cells treated with RPL39L siRNA (3.9%) when compared to the scrambled control (2.1%) (Figure 3). Nevertheless, a lesser MSFE were observed in those cells treated with STAT3 (0.9%, not significant), NTN4 (0.8%) and RPL39L (0.78%) in comparison to control (1.1%) (Figure 4). In conclusion, knocking-down of EMT-related genes (STAT3, NTN4, and RPL39L) decreases significantly the self-renewal capacity in mammospheres derived from claudin-low-like human breast tumor cells, being BT549 cells more sensitive than SUM159 cells to that silencing with siRNA loaded in DOPC nanoliposomes into silicon particles as carriers.

Acknowledgments: We thank Fundacion Alfonso Martin Escudero for the personal support of Dr. Sergio Granados-Principal.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-02.