SUPPLEMENTARY DATA. A 90kDa fragment of Filamin A promotes Casodex-induced growth inhibition in

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1 SUPPLEMENTARY DATA A 90kDa fragment of Filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen-receptor positive C4-2 prostate cancer cells. DETAILED MATERIALS AND METHODS Cell Culture and Pharmacological Treatments LNCaP, PC-3, DU-145 and 22Rv1 cells were purchased from American Type Culture Collection, Manassas, VA, while C4-2 cells were from UroCor, (Oklahoma City, OK). Androgen-independent clones of LNCaP cells (LNAI cells) were obtained by prolonged culture of LNCaP cells in androgen-free medium : phenol-red free RPMI 1640 with 10% charcoal stripped FBS (CSS, Hyclone, Logan, UT). All other cells were cultured in complete-medium : RPMI 1640 medium with 10% fetal bovine serum (FBS, Invitrogen) unless otherwise specified. Casodex was kindly provided by Dr. Barry Furr, AstraZeneca, UK. 4,5 -Dihydrotestosterone (DHT) was obtained from Sigma-Aldrich, St. Louis, MO. PD98059, LY and rapamycin were obtained from Calbiochem, EMD Biosciences, San Diego, CA. Transfections and plasmids: Cells were transiently transfected using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer s specifications based on established protocols using 2 g plasmid DNA as described earlier (Ghosh et al., 1999; Ghosh et al., 2005). pcmv-flna(16-24) and pcmv-flna(1-15) plasmids were kindly provided by Dr. E.W. Yong, National University of Singapore, Singapore. Constitutively active Akt (myr-akt, pcmv-6-myr-akt-ha) and dominant negative Akt (dn-akt, pcmv-6-akt-k179m) were kindly provided by Dr. Thomas Franke, Columbia University, New York, NY and has been used by us previously (Ghosh et al., 2005). A 1

2 human PSA reporter plasmid consisting of the human PSA 5 -flanking region (-631/-1) containing androgen response elements I and II (ARE I and ARE II) tagged to a luciferase construct (hpsa-luc) was kindly provided by Dr. Bandana Chatterjee, University of Texas Health Science Center at San Antonio, San Antonio, TX. RNA inhibition: LNCaP cells were plated in 60 mm dishes and transfected with 50 pmoles of one of the following: a pool of 3 duplexes sold as Filamin A sirna (sirna1p, Santa Cruz Biotechnology, Santa Cruz, CA) with the following sequences: Strand #1: 5 -CCAUCACUGACAACAAAGA-3, Strand #2: 5 -CUGCAGAGUUUAU- CAUUGA-3, Strand #3: 5 -GCUACCUCAUCUCCAUCAA-3. A second anti-flna sirna duplex (sirna2) with the following target sequence was purchased from Dharmacon Research Inc., Lafayette, CO: 5 -CAACGTTGGTAGTCATTGT-3. Control was a pool of 4 scrambled non-specific sirna duplex (sicontrol Non-Targeting sirna Pool, Dharmacon Research, Inc.). According to the manufacturer, BLAST analysis confirmed at least 4 mismatches with all known human, mouse and rat genes, and each individual sirna within this pool was extensively characterized by genomewide microarray analysis and found to have minimal off-target signatures. Determination of Cell Proliferation Rates: Flow cytometry: The fraction of cells in S- phase was determined by flow cytometry in propidium iodide stained ethanol-fixed cells as described previously (Ghosh et al., 2002). Experiments were repeated three times, unless otherwise specified. Proliferation rates are normalized to the percentage of cells in the S-phase for the control population, which was considered 100. MTT assay: Cell growth rates were estimated by MTT assay. Cells were plated in 24-well plates and treated as indicated. Following treatment, each well was incubated with 15 l of 5 mg/ml 2

3 3-[4,5-Dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT) for 1 hour in a CO 2 incubator at 37 o C. The medium was aspirated and 0.5 ml DMSO added per well. Proliferation rates were estimated by colorimetric assay reading formazan intensity in a plate reader at 560 nm. Subcellular fractionation: Cells were plated in a 100 mm dish and grown to 70% confluence. They were then treated as described and separated into cytoplasmic and nuclear fractions as follows: the plates were washed with PBS twice and the cells collected in 0.5 ml of Buffer A (10 mm HEPES, ph 7.9, 10 mm KCl, 0.1 mm EDTA, 0.5 mm DTT) containing 200 μl of 10% IGEPAL and protease inhibitors (0.1 mm benzamidine, 1 mm PMSF, 10 μg/ml each phenathroline, leupeptin, aprotinin, and pepstatin A). Following 10 min incubation at room temperature, the lysate was transferred to ice and centrifuged at 4 C at 1500 rpm in a benchtop refrigerated centrifuge (Eppendorf 5417R) for 5 min, and the supernatant collected as the cytosolic fraction. The pellet containing the nuclei was resuspended in 150 ml of Buffer B (20 mm HEPES, ph 7.9, 0.4M NaCl, 1 mm EDTA, 10% Glycerol) containing the same protease inhibitors and solubilized by vigorous shaking at 4 C for 2h. The suspension was then centrifuged at 4 C as before for 5 min and the supernatant collected as the nuclear fraction. Western blotting and immunoprecipitation: These techniques were performed as described elsewhere (Ghosh et al., 2002; Ghosh et al., 2001). Mouse monoclonal anti-ar and anti-n-terminal FlnA antibodies, rabbit polyclonal anti-total Akt, Anti-total ERK (recognizing both ERK1 and ERK2), anti-total p70s6k (recognizing phosphorylated and unphosphorylated forms of p70 and p85 S6 kinase), anti-cyclin D1, p27 Kip1, total Rb and -actin were from Santa Cruz Biotechnology, Santa Cruz, CA. Rabbit polyclonal anti- 3

4 phospho-rb (Ser 795), anti-phospho-erk(t303/y204), anti-phospho-akt (Ser 473), anti-lamin A/C, anti-phospho-p70s6k(t389), anti-phospho 4E-BP1 (Thr 37/46), ribosomal protein S6 and anti-phospho-flna (Ser 2152) were from Cell Signaling Technology, Beverly, MA. Mouse monoclonal anti-c-terminal FlnA (MAB1680) was from Chemicon, Temecula, CA). Mouse monoclonal anti-psa antibody was from Neomarkers, Lab Vision Corporation, Fremont, CA. Unless otherwise stated, all data shown is representative of three different experiments showing similar results. The bands were quantitated using Scion Image, Scion Corporation, Frederick, MD. Immunocytochemistry: Cells were trypsinized and centrifuged to form a cell pellet. The pellet was fixed in 10% buffered formalin (Medical Industries, Richmond IL) for 30 mins at RT, after which the pellet was immersed in 600 l liquefied agar at o C. The agar containing the cell pellet was paraffin-embedded and processed based on established protocols. The paraffin-embedded cell block was then sectioned and immunostained by standard immunohistochemistry procedures as described (Kreisberg et al., 2004; Malik et al., 2002), using C-terminal FlnA. Universal Negative Control Mouse (DAKO/Cytomation) was used as negative control. AR transcriptional activity: Reporter gene activity was determined by luciferase assay (Hartig et al., 2002). LNCaP and/or C4-2 cells were transfected with 2 μg of hpsa-luc using Lipofectamine PLUS (Invitrogen, Grand Island, NY) according to the manufacturer s recommendations. After 24 h, cells were trypsinized, and 100,000 cells plated in 6-well tissue culture plate. Cells grown in phenol-free medium containing charcoal-stripped serum for 24 h were treated as required for an additional 24 h. Cells were harvested, and cell lysates prepared for performing luciferase assays using a 4

5 luciferase enzyme assay system (Promega Corp., Madison, WI). Each transfection experiment was performed in duplicate or triplicate on at least three separate occasions. Results represent an average of independent experiments with data presented as relative luciferase activity using means of untreated controls as standards. SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. Immunoperoxidase staining with anti-flna (C-terminal) antibody (upper panel) and anti-flna (N-terminal) antibody (lower panel) in LNCaP, C4-2 and PC-3 cells. The C-terminal antibody detects both the 280 kda and the 90 kda proteins whereas the N-terminal antibody detects the 280 kda alone. Note that LNCaP cells display staining in both nuclei and in cell membranes (brown reaction product) with the C-terminal antibody and only the membrane with the N-terminal antibody. C4-2 and PC-3 cells show FlnA staining on cell membranes but not nuclei with both antibodies. Counter stain was with hematoxylin (blue). 40X original magnification. Supplementary Figure 2. Expression of FlnA in mouse models of androgenindependent and dependent prostate cancer. (left panel) LNAI cells were orthotopically implanted in the prostates of castrated BALB/athymic mice and developed tumors that did not express nuclear FlnA. (middle panel) In contrast, androgen-dependent CWR22 tumors expressed strong FlnA staining in the nuclei (right panel) whereas 22Rv1, a recurrent AI tumor obtained by implanting CWR22 prostate tumors in castrated nude mice, lacked nuclear staining for FlnA. All three tumors were stained with the mouse monoclonal antibody to C-terminal FlnA described earlier (20X). 5

6 Supplementary Figure 3. C4-2 cells were stably transfected with FlnA (C cells) or vector alone (C4-2V), pelleted, paraffin embedded and immunostained using the anti-flna (C-terminal) antibody (upper panel) or anti-flna (N-terminal) antibody (lower panel). Note that in C4-2V cells, the FlnA staining was mainly confined to the membrane (280 kda only) whereas in the C cells, the staining was also nuclear (280 kda + 90 kda). (40X). Supplementary Figure 4. Growth curves comparing proliferation rates of C4-2V (vector transfected) and C (FlnA transfected) cells in DMSO (control) or 1 M hydroxyflutamide (flutamide). 25,000 cells were plated in 24-well plates and cultured for 1-5 days as indicated. Proliferation rates were determined by MTT assay and show mean ± S.D. of three independent readings. Supplementary Figure 5. Inhibition of Filamin A expression in LNCaP cells with FlnAspecific sirna duplex (a pool of three individual sirna duplexes (FlnA sirna1p) or a single sirna (FlnA sirna2)) vs a non-specific control sirna duplex (control sirna). Cells were subjected to subcellular fractionation and localization of FlnA (upper panel) and the AR (middle panel) determined by Western blotting (C: cytoplasmic; N: nuclear). The presence of Lamin A/C was used as a determinant of the nuclear fraction (lower panel). 6

7 Supplementary Figure 6. (left panel) Expression of AR in PC-3 cells repressed the levels of the 90 kda band. AR null PC-3 cells were transfected with vector only, FlnA or wild-type AR, and subjected to subcellular fractionation into cytoplasmic (C) and nuclear (N) fragments. localization of the AR (upper panel) and FlnA (middle panel) was determined by Western blotting. The presence of Lamin A/C was used as a determinant of the nuclear fraction (lower panel). (right panel) Expression of 90 kda FlnA, AR and lamin A in RWPE-1 cells isolated from a normal prostate. 7

8 Supplementary Figure 1. Immunoperoxidase staining with anti-flna (C-terminal) antibody (upper panel) and anti-flna (N-terminal) antibody (lower panel) in LNCaP, C4-2 and PC-3 cells. The C-terminal antibody detects both the 280 kda and the 90 kda proteins whereas the N-terminal antibody detects the 280 kda alone. Note that LNCaP cells display staining in both nuclei and in cell membranes (brown reaction product) with the C-terminal antibody and only the membrane with the N-terminal antibody. C4-2 and PC-3 cells show FlnA staining on cell membranes but not nuclei with both antibodies. Counter stain was with hematoxylin (blue). 40X original magnification. LNCaP C4-2 PC-3 FlnA N-terminal FlnA C-terminal

9 Supplementary Figure 2 Expression of FlnA in mouse models of androgen-independent and dependent prostate cancer. (left panel) LNAI cells were orthotopically implanted in the prostates of castrated BALB/athymic mice and developed tumors that did not express nuclear FlnA. (middle panel) In contrast, androgen-dependent CWR22 tumors expressed strong FlnA staining in the nuclei (right panel) whereas 22Rv1, a recurrent AI tumor obtained by implanting CWR22 prostate tumors in castrated nude mice, lacked nuclear staining for FlnA. All three tumors were stained with the mouse monoclonal antibody to C-terminal FlnA described earlier (20X). LNAI CWR22 CWR22Rv1

10 Supplementary Figure 3. C4-2 cells were stably transfected with FlnA (C cells) or vector alone (C4-2V), pelleted, paraffin embedded and immunostained using the anti-flna (C-terminal) antibody (upper panel) or anti-flna (N-terminal) antibody (lower panel). Note that in C4-2V cells, the FlnA staining was mainly confined to the membrane (280 kda only) whereas in the C cells, the staining was also nuclear (280 kda + 90 kda). (40X). C4-2V FlnA 90 C FlnA 90 C4-2V FlnA 280 C FlnA 280

11 Supplementary Figure 4 Growth curves comparing proliferation rates of C4-2V (vector transfected) and C (FlnA transfected) cells in DMSO (control) or 1 mm hydroxyflutamide (flutamide). 25,000 cells were plated in 24-well plates and cultured for 1-5 days as indicated. Proliferation rates were determined by MTT assay and show mean ± S.D. of three independent readings. ABSORBANCE Control Flutamide C4-2V DAYS ABSORBANCE Control Flutamide C4-2 FlnA DAYS

12 Supplementary Figure 5 Inhibition of Filamin A expression in LNCaP cells with FlnA-specific sirna duplex (a pool of three individual sirna duplexes (FlnA sirna1p) or a single sirna (FlnA sirna2)) vs a non-specific control sirna duplex (control sirna). Cells were subjected to subcellular fractionation and localization of FlnA (upper panel) and the AR (middle panel) determined by Western blotting (C: cytoplasmic; N: nuclear). The presence of Lamin A/C was used as a determinant of the nuclear fraction (lower panel). LNCaP Control sirna FlnA sirna1p FlnA sirna2 C N C N C N FlnA AR Lamin A

13 Supplementary Figure 6 (left panel) Expression of AR in PC-3 cells repressed the levels of the 90 kda band. AR null PC-3 cells were transfected with vector only, FlnA or wild-type AR, and subjected to subcellular fractionation into cytoplasmic (C) and nuclear (N) fragments. localization of the AR (upper panel) and FlnA (middle panel) was determined by Western blotting. The presence of Lamin A/C was used as a determinant of the nuclear fraction (lower panel). (right panel) Expression of 90 kda FlnA, AR and lamin A in RWPE-1 cells isolated from a normal prostate. PC-3 PC-3+ FlnA16-24 PC-3/AR CN CN CN C RWPE-1 N AR FlnA 90 kda Lamin A