Downregulation of Hsp27 (HSPB1) in MCF-7 human breast cancer cells induces upregulation of PTEN

Abstract

Hsp27 (HSPB1) is usually overexpressed in breast cancers affecting the disease outcome and the sensitivity of tumors to chemotherapy and radiotherapy. Hsp27 interacts with other proteins such as β-catenin, histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3. Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene that is deleted in many human tumors. The PI3K/Akt signaling pathway is negatively regulated by PTEN. Hsp27 is described as a key component of the Akt signaling cascade: Akt, BAD, Forkhead transcription factors, Hsp27, mitogen-activated protein kinase kinase-3 and -6. Here, we have examined whether the downregulation of Hsp27 by siHsp27 affects the PTEN levels in the MCF-7 human breast cancer cell line. PTEN was detected with two different antibodies using western blots and immunocytochemistry. p-Akt was also evaluated by western blot. In addition, Hsp27 and PTEN were immunoprecipitated to know whether these proteins interact. Intracellular colocalization studies were carried out by confocal microscopy. A significant reduction in the Hsp27 levels was noted in the siHsp27 transfected cells. These Hsp27 downregulated cells showed a significant increased expression of PTEN. The MW 76 and 55 kDa PTEN forms were upregulated as revealed by two different antibodies. The phosphatase activity of PTEN seems to be active because p-Akt levels were reduced. Hsp27 immunoprecipitation was bringing PTEN and vice versa, these two proteins seem to interact at cytoplasmic level by FRET. Downregulation of Hsp27 stabilized PTEN protein levels. Chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) levels were not significantly influenced by Hsp27 downregulation. In conclusion, we report a novel function of Hsp27 modulating the PTEN levels in human breast cancer cells suggesting an interaction between these two molecules.

Fig. 1

Downregulation of Hsp27 and its effect on β-catenin expression in MCF-7 cells. a Hsp27 levels as revealed in the western blot analysis (performed as described previously; Fanelli et al. 2008). The antibodies used were: a mouse monoclonal antibody against Hsp27 (1:1,000; cat. # SPA-800, Stressgen Biotech. Corp., Victoria, Canada), b mouse monoclonal antibody against α-tubulin (1:16,000; Sigma Chem. Co., St. Louis, MO, USA), and c mouse monoclonal antibody anti-β-catenin (1:500; cat. # 18-0226; Zymed, Carlsbad, CA, USA). 1 Control untreated cells, 2 control cells treated with Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), 3 control cells transfected with the empty vector for 72 h, 4 note the decreased expression of Hsp27 in the siHsp27 transfected cells (72 h after transfection). The immunoblots images were capture using LAS-4000 imaging system (Fujifilm Life Sc., USA). b Graph showing a significant depletion of Hsp27 at 48 h (immunoblot not shown) and 72 h after siHsp27 transfection. Mock 48 and 72 h were MCF-7 cells transfected with empty vector and analyzed at 48 and 72 h after transfection, respectively. The evaluation of the immunoblots was performed using NIH image V1,62 program (NIH, Bethesda, MD, USA). The data were analyzed with the Prism computer program (Graph Pad Software, San Diego, CA, USA); data shown are means ± standard errors of the mean of three independent experiments. Statistical significance was assessed by column analyses with one-way ANOVA, the level of significance was set at p < 0.05. c Immunoblot showing the significant decrease of β-catenin. 1 Control untreated MCF-7 cells, 2 and 3 cells transfected with siHSP27 (48 and 72 h after transfection; p < 0.05). d Immunofluorescence of MCF-7 cells showing: a the basal Hsp27 levels (left panel, control cells treated with Lipofectamine™ 2000), and b the decreased Hsp27 levels after 72 h of siHsp27 transfection (right panel). Bar 10 μm. The MCF-7 human breast cancer cell line was kindly provided by Dr. MC Abba [Centro de Investigaciones Inmunológicas Básicas y Aplicadas (CINIBA), Universidad Nacional de La Plata, Argentina]. The cells were routinely cultured in Dulbecco’s Modified Eagle Medium (GIBCO, Invitrogen Corp, Argentina) supplemented with 10 % fetal calf serum (GIBCO) and 100 IU/ml penicillin and 100 μg/ml streptomycin (GIBCO) at 37 °C in an incubator with 5 % CO₂ and 100 % humidity. Subconfluent cells were split twice a week at a ratio of 1:20. For knockdown of Hsp27 expression, transient transfections were done with 2 μg/ml pSIREN-RetroQ empty vector (Mock-transfection control) and shHsp27-pSIREN-RetroQ vector for 5h using Lipofectamine^TM 2000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s recommendations. The shHsp27-pSIREN-RetroQ vector was gently provided by Dr. MY Sherman (Boston University Medical School, Boston, MA, USA; O’Callaghan-Sunol et al. 2007). The vector contained the sequence of human Hsp27 (accession number NM 001540) as target for RNA interference: shHsp27 (start 701): ATCCGATGAGACTGCCGCCAA. The transfection efficiency was evaluated in each experiment using pSIREN-DNR-DsRed-Express (a gift from Dr. MY Sherman). After the start of transfection at 48 and 72 h, cells were washed twice in ice-cold PBS, lysed with cell lysis buffer (triton-x buffer with protease inhibitors) and stored at −80 °C for immunoblotting analysis. Immunofluorescence staining: MCF-7 cells were fixed with 2 % paraformaldehyde in PBS for 10 min at 37 °C, washed with PBS and blocked with 50 mM NH₄Cl in PBS. Then the cells were permeabilized with 0.05 % saponin in PBS containing 0.5 % BSA, and incubated with primary antibody against Hsp27 (1:100). After washing, cells were incubated with secondary antibody conjugated with FITC (1:500; Jackson ImmunoResearch Laboratories Incorporated, West Grove, PA, USA). MCF-7 cells were mounted with Mowiol (Sigma-Aldrich, Argentina) and examined by confocal microscopy using an FV1000 Olympus Confocal Microscope and FV 10-ASW 1.7 software (Olympus, Japan). Images were processed using ImageJ software

Fig. 2

Depletion of Hsp27 increases PTEN content in MCF-7 cells. a Immunoblot showing a significant increase in PTEN at 24 h (2) and 48 h (3) after siHsp27 transfection. 1 PTEN in MCF-7 control cells (Lipofectamine). PTEN was detected using a rabbit polyclonal antibody (1:1,000 dilution) generated against a PTEN synthetic peptide sequence between amino acids 33 to 47 (Perandones et al. 2004). The graph shows the statistical analysis of this blot, data shown are means ± standard errors of the mean of three independent experiments (Graph Pad Prism Software, San Diego, CA, USA); p < 0.05. b Immunoblot showing again the increase in PTEN levels after different times of siHsp27 transfection. 1 Control MCF-7 cells (treated with Lipofectamine), 2 cells after 48 h of continuous transfection with siHsp27, 3 empty lane, 4 control cells (Lipofectamine), 5 cells after 24 h of continuous transfection with siHsp27

Fig. 3

After downregulation of Hsp27, PTEN is increased and p-Akt is decreased. a In this case, PTEN was revealed using an affinity purified rabbit polyclonal antibody raised against a peptide mapping at the C-terminus of PTEN of human origin (1:400; PTEN C-20 sc-6817-R; Santa Cruz Biotech., Santa Cruz, CA, USA). 1 Untreated control MCF-7 cells; 2 control cells treated with Lipofectamine; 3 mock transfection control cells (72 h); 4 siHsp27-treated cells, 72 h after transfection. b Basal p-Akt levels (MW 70 kDa) in control MCF-7 cells (1) and in siHsp27-treated MCF-7 cells at 72 h after transfection (2). p-Akt was detected using a rabbit polyclonal antibody (1:1,000 dilution) generated against an epitope corresponding to amino acids 345–480 of Akt1 of human origin. This antibody detects Akt1, Akt2 and Akt3 (H-136, sc-8312, Santa Cruz Biotech., Santa Cruz, CA, USA). p-Akt was analyzed by Mann-Whitney test (graph)

Fig. 4

Immunocytochemistry reveals upregulation of PTEN by siHsp27 and PTEN-Hsp27 interactions in MCF-7 cells. a Low-power microphotographs (upper panel; bar 10 μm) and high-power microphotographs (lower panel; bar 4 μm) to show the increased PTEN expression in the MCF-7 cells transfected with siHsp27. PTEN can be seen mainly in the nuclei but also in the cytoplasm of the tumor cells. b The confocal microscopy shows that the two proteins colocalize in the cytoplasm. FRET was done according to Kenworthy (2001). PTEN was detected using an aptamer (z7) (Moncalero et al. 2011)

Fig. 5

Immunoprecipitation studies reveal an interaction between Hsp27 and PTEN. a Hsp27 immunoprecipitation (using the mouse monoclonal antibody from Stressgen Biotech described before) is presented in two independent experiments (IP-I, IP-II). Note the presence of PTEN in IP-I and IP-II. M molecular weight markers (Full Range Rainbow from Amersham, GE Healthcare, UK). C control immunoprecipitation using a mouse monoclonal antibody generated against membranes from MCF-7 cells (own antibody). 1 MCF-7 cell supernatant (total lysate) before immunoprecipitation showing both Hsp27 and PTEN proteins. 2 Same as 1 but after immunoprecipitation. b After PTEN immunoprecipitation note the presence of Hsp27. The antibody used for PTEN IP was a rabbit polyclonal antibody (Perandones et al. 2004). This antibody recognizes PTEN and the ubiquitinated forms of PTEN. 1 MCF-7 cell supernatant (total lysate) before immunoprecipitation showing Hsp27 and a weak PTEN band. 2 Same as 1 but after immunoprecipitation. c Immunoprecipitation was performed with a p-PTEN rabbit polyclonal antibody (ser 370, sc-101787, Santa Cruz Biotech., Santa Cruz, CA, USA), note the presence of Hsp27. 1 MCF-7 cell supernatant (total lysate) before immunoprecipitation showing Hsp27 and a weak p-PTEN band. 2 Same as 1 but after immunoprecipitation. d After Hsp27 immunoprecipitation (IP-II) note a weak CHIP band (using a rabbit polyclonal antibody, N-terminal, C9118, Sigma-Aldrich, MO, USA). C control immunoprecipitation using a mouse monoclonal antibody generated against membranes from MCF-7 cells (own antibody). 1 MCF-7 cell supernatant (total lysate) before immunoprecipitation showing both Hsp27 and CHIP proteins. 2 Same as 1 but after immunoprecipitation. Immunoprecipitation was carried out with the described antibodies (12 μg) attached to the Dynabeads M-280 Tosylactivated (cat. # 142.03, from Invitrogen, Argentina), using 500 μg of total proteins in the lysate. We used the protocol provided by the company

Fig. 6

PTEN stability is maintained after Hsp27 depletion. MCF-7 cells were continuously transfected with siHsp27 for 48 h and then treated with cycloheximide (200 μg/ml) for the times indicated. Cells were lysed and cell lysates were resolved by SDS-PAGE (10 %), followed by western blot with anti-PTEN (PTEN C-20 sc-6817-R Santa Cruz Biotech., Santa Cruz, CA, USA). N Nonspecific band used as loading control. The bands were quantified with ImageJ software. Data are shown as the mean of two different experiments. Bars standard deviation. Note that the amount of PTEN remained at high levels during the time course of the experiment