Silencing the hsp25 gene eliminates migration capability of the highly metastatic murine 4T1 breast adenocarcinoma cell

Abstract

The 25-kDa heat shock protein (Hsp25) is associated with various malignancies and is expressed at high levels in biopsies as well as circulating in the serum of breast cancer patients. In this study, we used RNA interference technology to silence the hsp25 gene in 4T1 breast adenocarcinoma cells, known as a poorly immunogenic, highly metastatic cell line. We demonstrate that transfection of 4T1 cells with short interference RNA-Hsp25 dramatically inhibits proliferation as compared with control transfected cells. In addition, we show that 4T1 cells transfected with short interference RNA-Hsp25 abrogates tumor migration potential by a mechanism that is in part due to the repression of matrix metalloproteinase 9 expression and a concomitant upregulation of its antagonist, tissue inhibitor metalloproteinase 1. Taken together, these findings provide a model system for the study of metastatic potential of tumors and are suggestive of an earlier unrecognized role for Hsp25 in tumor migration.

Fig. 1

Schematic representation of cloning of siRNA-Hsp25. pSuper vector was used to introduce a 64-base oligomer to form the hairpin with a Hsp25-specific sequence. This 21-base was synthesized with defined sequences to provide two restriction sites (Bgl II and Hin d III) and, on transcription, form a hairpin, in order to initiate gene silencing. Eight clones were screened and three positive clones were sequenced. All experiments were performed with one of the matched sequences.

Fig. 2

Effect of transfection of siRNA-Hsp25 on 4T1 breast adenocarcinoma cells. 4T1 cells were transfected with GFP^plasmid plus siRNA-Hsp25 plasmid as described in ‘Materials and Methods'. After 48 h, cells were sorted using flow cytometry for either GFP-negative or GFP-positive cells. Sorted cells were stained with anti-Hsp25 antibody and a secondary isotype-matched antibody biotinylated with Texas red. Cells were counterstained for nuclei integrity with DAPI (blue fluorescence). Immunofluorescent pictograms show GFP-negative cells express Hsp25 (red fluorescence) and GFP-positive cells are negative for Hsp25. Data are representative of at least three independently performed experiments with similar results.

Fig. 3

Suppression of Hsp25 expression in 4T1 breast adenocarcinoma cells. Freshly sorted GFP^plasmid or siRNA-Hsp25 transfectants were lyzed, and extracted proteins were analyzed by 12% SDS-PAGE and immunoblotted with antibodies specific to Hsp25 or Hsc70 and visualized with a coupled secondary antibody chemiluminescence method. Anti-β-actin antibody was used as loading control on stripped Hsp25 membranes. The intensities of the bands were analyzed by densitometry with a video densitometer (Chemilmager ™ 5500; Alpha Innotech, San Leandro, Calif., USA) using the American Applied Biology software. Figures represent relative density. Results are representative of three independently performed experiments with similar results.

Fig. 4

Effect of siRNA-Hsp25 in response to heat stress in 4T1 breast adenocarcinoma cells. Exponentially growing GFP^plasmid or siRNA-Hsp25 transfectants were either exposed to non-lethal heat shock (HS) treatment (43 °C, 30 min) and allowed to recover for 18 h or maintained at 37 °C. Cells were then lyzed, and extracted proteins were analyzed by 12% SDS-PAGE and immunoblot with antibodies specific to Hsp25 or Hsc70 and visualized with a coupled secondary antibody chemiluminescence method. Anti-β-actin antibody was used as loading control on stripped Hsp25 membranes. Results are representative of two independently performed experiments with similar results.

Fig. 5

Effect of silencing the hsp25 gene on proliferation of 4T1 breast adenocarcinoma cells. Exponentially growing wild-type 4T1 cells (wt) or 4T1 cells transfected with GFP^plasmid, siRNA-Hsp25, pLXSN or Hsp25^Neo transfectants were seeded and treated with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt (MTS) to determine the proliferation as described in ‘Materials and Methods’. Data are the mean percent proliferation ± SD and represent the sum of three independently performed experiments. ^* p < 0.05 versus control (Student’s t test).

Fig. 6

Effect of siRNA-Hsp25 on cell cycle distribution. Freshly sorted 4T1 cells (10⁵) transfected with GFP^plasmid (a), siRNA-Hsp25 (b), pLXSN (c) or Hsp25^Neo (d) were fixed in ethanol and incubated with propidium iodide and RNAse A for 1 h at 37 °C. DNA content and cell cycle were performed using flow cytometric analysis as described in detail in ‘Materials and Methods'. Results are a representative of four independently performed experiments with similar results. Abscissa, fluorescence intensity; ordinate, relative cell number.

Fig. 7

Suppression of migration of 4T1 cells upon transfection with siRNA-Hsp25. a 4T1 (10⁵) cells transfected with siRNA-Hsp25, GFP^plasmid or Hsp25^Neo were placed on Matrigel invasion chambers (BD Pharmingen). Twenty-two hours later, cells were harvested and stained with 0.5% crystal violet solution and mounted in a microscope slide at 10 × magnification. Data are representative of three independently performed experiments with similar results. b 4T1 (10⁵) cells transfected with siRNA-Hsp25, GFP^plasmid or Hsp25^Neo were placed on Matrigel invasion chambers (BD Pharmingen). Twenty-two hours later, cells were harvested and stained with 0.5% crystal violet solution and mounted in a microscope slide under 10 × magnification and 15 fields were counted. Bars are the mean percent migration ± SD and represent the sum of three independently performed experiments. ^* p < 0.05 versus control (Student’s t test).

Fig. 8

Silencing of Hsp25 inhibits the expression of MMP-9 and upregulates TIMP-1 expression. Freshly sorted GFP^plasmid or siRNA-Hsp25 transfectants were lyzed. Protein concentration was determined by the Bradford method and wells were equally loaded (5μg) with the extracted proteins as shown by performed β-actin staining on stripped membranes. Proteins were analyzed by 12% SDS-PAGE and immunoblots performed. Anti-Hsp25-, anti-MMP-9- or anti-TIMP-1-specific polyclonal antibodies were used. Visualization of bands was done using enhanced chemiluminescence reagent. Results are representative of two independently performed experiments with similar results.