Overexpression and involvement of special AT-rich sequence binding protein 1 in multidrug resistance in human breast carcinoma cells

Qing-Quan Li ¹ , Zhong-Qing Chen ² , Jing-Da Xu ¹ , Xi-Xi Cao ¹ , Qi Chen ¹ , Xiu-Ping Liu ¹ and Zu-De Xu ²

¹ Department of Pathology, Shanghai Medical College, Shanghai ; ² Department of Pathology, Huashan Hospital, Fudan University, Shanghai, China

Cancer Sci, 2009. (DOI: 10.1111/j.1349-7006.2009.01372.x)

Summary

Special AT-rich sequence binding protein 1 (SATB1) is a transcription factor whose expression promotes metastasis in various cancers, including colorectal, lymphoma, and breast cancer. Multidrug-resistant cancer cells often exhibit increased metastasis and aggression, making it worthwhile to examine SATB1 expression in such cells.

Li et al. examined and characterized the relationship between SATB1 expression and multidrug resistance (MDR) in several human breast carcinoma cells lines: MCF7 vs. MCF7/ADR (adriamycin) and MCF7/MX (mitoxantrone); Hs578T vs. Hs578T/Dox (doxorubicin); MX-1 vs. MX-1/T (Taxol). Although the drug-resistant sublines were cultured and selected with the single anticancer drug indicated, they all demonstrated MDR.

Through quantitative RT-PCR and Western blot analysis, the authors showed that SATB1 had higher mRNA (Fig. 1a) and protein expression (Fig. 1b) in MCF7/ADR than in MCF7 in vitro, and its level of expression was even correlated with the degree of drug resistance (Fig. 1e). Similarly, SATB1 had higher protein levels in each of the other MDR cell lines, when compared to their respective parental controls (Fig. 1d).

To show that SATB1’s role in MDR is not only correlative but also causative in vivo, the authors selectively knocked down SATB1 in MCF7/ADR cells using shRNA (MCF7/ADR-shRNASATB1) (Fig. 2a). Using MTT assays to evaluate cell metabolism and growth, Li et al. found that adriamycin (ADM) inhibited the growth of this knocked-down cell line much more than that of the MCF7/ADR line expressing control shRNA (i.e. shRNA not specific to SATB1) (Fig. 2b). This suggests that SATB1 expression promotes ADM resistance, and that suppression of SATB1 can reverse ADM resistance.

Moreover, after in vivo ADM treatments, tumors from both MCF7 transfected with SATB1 and MCF7/ADR with control-shRNA grew faster than their SATB1-suppressed counterparts (Fig. 2c). This trend further implicates SATB1 overexpression as a cause for ADM resistance.

To determine the molecular basis for this SATB1-MDR relationship, the authors compared protein expression levels of three classical MDR molecules: breast cancer resistant protein (BCRP); P-glycoprotein (transport protein responsible for efflux of drugs from cell); and multidrug resistant protein 1 (MRP1)(Fig. 3a). The only factor which changed significantly with SATB1 expression (whether in MCF7 or MCF7/ADR) was the level of P-gp expression. Additionally, there is increased activity of multidrug resistance 1 (MDR1, codes for P-gp) mRNA in cells overexpressing SATB1 (namely, MCF7-SATB1 and MCF7/ADR-con). As such, SATB1 promotes P-gp expression at both the mRNA and protein level, consequently leading to increased multidrug resistance.

The authors further verified this finding using laser cytometry to quantify intracellular ADM. Cells overexpressing SATB1 had lower ADM concentrations, thus confirming that SATB1 increases P-gp functional expression, leading to efflux of ADM and increased MDR.

In this study, Li et al. directly link SATB1 overexpression with P-gp overexpression and consequently, with the MDR phenotype. That is, SATB1 increases cancer malignancy not only by accelerating metastasis, but also by protecting against cytotoxic anticancer drugs. Moreover, they successfully reverse this MDR trait in human breast carcinoma cell lines by disrupting SATB1 expression using RNAi, rendering the cells more susceptible to drug treatments and associated apoptosis. This study identifies new potential inhibitive points for reversing MDR in carcinomas and increasing chemotherapy efficacy.

Critique

Li et al. offered a fairly comprehensive exploration of SATB1’s role in MDR, using different permutations of SATB1 under- and overexpression to characterize its effects on both a cellular and tissue level, in vitro and in vivo. The consistent use of controls (e.g. β-actin as a positive control in all Western blots; MCF7 and MCF7/ADR-shRNASATB1 as negative controls for MCF7-SATB1 and MCF7/ADR, respectively) makes their experimental results more reliable and consistent.

However, the focus on MCF7 over the other human breast carcinoma lines warrants some concern, as the authors did not justify this decision at all. It would be worthwhile to look more closely at SATB1 expression and P-gp activity in some of the other cell lines mentioned, just to confirm that their findings are consistent across other MDR cell lines, and not just specific to MCF7/ADR.

Also, there was little discussion as to what specifically characterizes MDR, and which drugs are affected by MDR. For instance, the authors did not mention how they determined the cell sublines they cultured were indeed resistant to multiple drugs, since the lines were selected using a single drug each. Without some substantiation to this effect, their data is still useful for MCF7/ADR, but no longer generally applicable to MDR carcinoma cell lines.