Activin receptor signaling regulates prostatic epithelial cell adhesion and viability

Abstract

Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum factor(s) in cell proliferation. Using prostatic cancer cell (PCC) lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII), as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS). Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic fibroblast growth factor was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-15 expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer growth and metastasis.

Figure 1

Serum-dependent activin A-induced proliferation and signaling in PCCs. (A) PC-3 cells plated at 50% confluence for 24 hours in F12 medium without serum were then treated with medium containing increasing concentrations (0, 1%, 5%, and 10%) of FBS (S) or heat-inactivated FBS (HI). Proliferation was assessed using the MTS assay after 24 hours of treatment. Results are representative of three repetitions and presented as mean ± SEM; n = 8 (S) or 4 (HI) (*significantly different from 0% serum, P < .0001; ^#significantly different from serum treated cells, P < .05). (B) PC-3 cells were plated at 40% confluence in serum free or 1% FBS medium for 24 hours before being treated with and without activin A. Cells were treated with physiologically relevant concentrations of serum hormone as indicated on the figure and in [98]. Cell proliferation was assessed using the MTS assay after 3 days. Results are presented as mean ± SEM and are representative of five separate experiments (*significantly different from 0 and 1% serum alone, n = 4, P < .05). (C) PC-3 cells were plated at 50% confluence in medium containing 1% serum for 24 hours before being treated with activin A (0.5 or 2.5 ng/ml) in 1% FBS. Whole-cell lysates collected at 1 hour and 3 days after treatment were subjected to immunoblot analysis using a rabbit antihuman P-Smad2 antibody and goat antihuman GAPDH antibody was used as the loading control.

Figure 2

Blocking antibody suppression of ActRII signaling inhibits PCC adhesion and viability. PC-3 or LNCaP cells were plated in 10% serum at 40% confluence for 24 hours, after which cells were either collected (day 0 control) or treated every day for 3 days with 1) medium containing 5% serum or with 2) medium containing 5% serum plus ActRII monoclonal antibody (25 µg/ml), before collection. (A) PC-3 (upper panel) and LNCaP (lower panel) cell viability was measured using the trypan blue staining assay. Results are presented as a percentage change from the untreated day 3 control (mean ± SEM; n = 4, P < .01; different letters indicate significant differences between treatments; experiments were repeated five times). (B) Immunoblot analysis of PC-3 cells using a mouse monoclonal antibody against human ActRII (70 kDa) and normalization against GAPDH (37 kDa). (C) Immunoblot analysis of PC-3 cells using an affinity purified rabbit polyclonal antibody against human phosphorylated Smad2 (45 kDa) and normalization against GAPDH (37 kDa). (D) PC-3 cells were cultured in F12 medium with 5% serum for 1 day before treatment ± antihuman ActRII antibody (20 µg/ml) for 3 days. Morphology was then assessed using a Zeiss Axiophot inverted microscope. Magnification is given on the figure. Scale, 50 µm. (E) PC-3 cells (left panels) were cultured in F12 medium with 5% serum, and LNCaP cells (right panels) were cultured in RPMI-1640 medium with 5% serum for 1 day before treatment ± antihuman ActRII antibody (20 µg/ml) for 3 days. Original magnification, x 100. Scale, 400 µm. (F) Immunoblot analysis of PC-3 cells described in Figure 3 for caspase 3 (32-kDa inactive and 17- to 22-kDa active forms) using a mouse monoclonal antibody. (G) Immunoblot analysis of PC-3 cells described in (A) for ADAM-15 (70 kDa) using a rabbit antihuman ADAM-15 antibody and normalization against β-actin (47 kDa) using a goat antihuman β-actin antibody. Experiments were repeated two times.

Figure 3

Antisense oligonucleotide suppression of ActRII signaling inhibits PCC adhesion and viability. PC-3 cells were plated in 10% serum at 40% confluence for 24 hours, after which cells were either collected (day 0 control) or treated every day for 3 days with 1) medium containing 10% serum plus lipofectamine (control), plus 2) sense-P oligonucleotide to ActRIIA (sense A), 3) antisense-P oligonucleotide to ActRIIA (antisense A), 4) sense-P oligonucleotide to ActRIIB (sense B), or 5) antisense-P oligonucleotides to ActRIIB (antisense B; final concentration of 0.4 µM each). (A) PC-3 cell viability was measured using the trypan blue staining assay. Results are presented as number of cells (mean ± SEM; n = 3–6; (*significantly different from day 3 control, P < .005). (B) Immunoblot analysis of ActRII (70 kDa) was performed using a mouse monoclonal antibody against human ActRII and normalization against β-actin (47 kDa) using a goat antihuman β-actin antibody. Quantitation of blot is shown on the right. (C) Immunoblot analysis of PC-3 cells for ADAM-15 (70 kDa) using a rabbit antihuman ADAM-15 antibody and normalization against β-actin (47 kDa) using a goat antihuman β-actin antibody. Quantitation of blot is shown on the right.

Figure 4

ActRII signaling regulates cell proliferation, viability, morphology, and attachment of M17 neuroblastoma cells. (A) Human M17 neuroblastoma cells cultured in OPTI-MEM medium were plated in 0.5% FBS at 40% confluence. After 24 hours, wells were treated with the antihuman ActRII mouse monoclonal antibody (0, 10, or 20 µg/ml). Cell proliferation was assessed using the MTS assay after 3 days. Results are presented as percentage change from the control (mean ± SEM; n = 3; *significantly different from control, P < .001). Ab = antibody. (B) M17 cells were plated in 10% serum at 40% confluence for 24 hours, after which cells were either collected (day 0 control) or treated every day for 3 days with 1) medium containing 10% serum plus lipofectamine (control), plus 2) sense-P oligonucleotide to ActRIIA (sense A), 3) antisense-P oligonucleotide to ActRIIA (antisense A), 4) sense-P oligonucleotide to ActRIIB (sense B), or 5) antisense-P oligonucleotides to ActRIIB (antisense B; final concentration of 0.4 µM each). M17 cell viability was measured using the trypan blue staining assay. Results are presented as number of cells (mean ± SEM; n = 4; *significantly different from day 3 control, P < .005). (C) M17 neuroblastoma cells cultured in OPTI-MEM medium were plated in 0.5% FBS for 1 day before treatment ± antihuman ActRII antibody (20 µg/ml) for 3 days. Original magnification, x 100. Scale, 400 µm. (D) M17 cells were plated exactly as in Figure 4B, and immunoblot analysis was performed for ADAM-15 as in Figure 3C. Quantitation of blot is shown on the right.

Figure 5

Basic fibroblast growth factor synergizes with activin A to induce serum-dependent PCC proliferation. PC-3 cells were plated at 40% confluence in medium ± 1% serum for 24 hours before being treated with EGF, IGF-1, and bFGF (10 or 25 ng/ml) ± activin (2.5 ng/ml). Cell proliferation was assessed using the MTS assay after 3 days. Results are presented as a mean percentage of untreated control ± SEM; n = 4(^#significantly different from 1% serum control without activin A, P < .01; *significantly different from 1% serum control without activin A, P < .01; ^@significantly different from 1% serum control with activin A, P < .001).