Activated α2-macroglobulin binding to human prostate cancer cells triggers insulin-like responses

Abstract

Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2-3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2-3-fold increase in lipogenesis as determined by 6-[(14)C]glucose or 1-[(14)C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [(14)CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy.

Keywords: Aerobic Glycolysis; Antibody Directed Against the Carboxyl-terminal Domain of GRP78; Cell Surface GRP78; Glycolysis; Lipogenesis; Prostate Cancer; Signal Transduction; Warburg Effect; α-2-Macroglobulin and Metabolic Regulation.

FIGURE 1.

Inhibition of α₂M* (black bars) and insulin (white bars)-induced protein synthesis in 1-LN (A) and DU-145 (B) prostate cancer cells is shown. DNA synthesis for 1-LN (C) and DU-145 (D) cells, respectively, is shown. Cell growth studies are shown in E and F, respectively. The bars in A–D are as follows: 1, buffer; 2, α₂M* or insulin; 3, MKK-2206 then α₂M* or insulin; 4, KU0063794 then α₂M* or insulin; 5, C-75 then α₂M* or insulin; and 6, orlistat then α₂M* or insulin. Protein synthesis data are the mean ± S.E. from three experiments. The effects of inhibitors on cell growth of 1-LN (E) and DU-145 (F) cells stimulated with α₂M* (black bars) or insulin (white bars) are shown. The bars at 16 h in E and F are as above. The effects of inhibitors alone on protein synthesis (G) and DNA synthesis (H) in 1-LN (black bars) and DU-145 (white bars) prostate cancer cells are shown. The bars in both G and H are as follows: 1, buffer; 2, MKK-2206; 3, KU0063794; 4, C-75; and 5, orlistat. The values in G and H are the mean ± S.E. from three experiments. Values significantly different at 5% level from buffer or inhibitor-treated cells are marked with an asterisk.

FIGURE 2.

Effect of time of incubation of 1-LN (A) and DU-145 cells (B) with α₂M* (100 pm) on the expression of SREBP1 (○), ATP citrate lyase (black bars), and fatty-acid synthase (▩) is shown. The values are expressed in arbitrary units ( × 10²) and are the mean ± S.E. from three to four independent experiments. The abbreviations used in the figure are as follows: ATP-CYL, ATP citrate lyase; FAS, fatty-acid synthase; ACC, CoA carboxylase; and SREBP, sterol regulatory binding protein 1. Real time mRNA PCR of SREBP1, SREBP2, ATP citrate lyase, acetyl-CoA carboxylase, and fatty-acid synthase in 1-LN and DU-145 cells stimulated with α₂M or insulin (C). The mRNA levels from three to four experiments are expressed as fold change over the mRNA levels of buffer-treated cells. The mRNA levels are expressed relative to β-actin. Values significantly different at 5% level from buffer-treated cells are marked with an asterisk.

FIGURE 3.

1-[¹⁴C]Deoxyglucose uptake by 1-LN (A) and DU-145 (B) cells is shown. 1-[¹⁴C]Deoxyglucose uptake in cells treated with buffer (black bars), α₂M* (light gray bars); insulin (dark gray bars), or anti-CTD antibody then α₂M* for 60 min (white bars) is shown. 1-[¹⁴C]Deoxyglucose uptake is expressed as cpm/mg/supernatant and is the mean ± S.E. from four experiments. Values significantly different at the 5% level at the 60-min period from buffer and anti-CTD are indicated by an asterisk. C, Glut-mRNA levels determined by real time PCR in 1-LN (black bars) and DU-145 (white bars) cells treated with α₂M* or insulin. Glut-1 mRNA levels are expressed as fold change over mRNA levels in buffer-treated cells and are significantly different (asterisk) at 5% levels from buffer-treated cells and are denoted by an asterisk. D, lactate secretion in1-LN (black bars) and DU-145 (white bars) cells treated with the following: 1, buffer; 2, α₂M*; 3, insulin; or 4, anti-CTD then α₂M*. Lactate secretion is expressed as micromolars/60 min and is the mean ± S.E. from three experiments. Values significantly different at the 5% level from buffer and anti-CTD-treated cells are indicated by an asterisk.

FIGURE 4.

Induction of lipogenesis by α₂M* or insulin in prostate cancer cells. A, 1-LN cells, inhibition of α₂M* (black bars) or insulin (white bars)-induced lipogenesis from 1-[¹⁴C]acetate in 1-LN prostate cancer cells by fatty-acid synthase inhibitors. The bars in the figure are as follows: 1, buffer; 2, α₂M* or insulin; 3, LY294002 (20 μm/25 min) and then α₂M* or insulin; 4, rapamycin (100 nm/20 min) and then α₂M or insulin; 5, torin and then α₂M* or insulin; 6, C-75 and then α₂M or insulin; 7, KU0063794 and then α₂M* or insulin; and 8, anti-CTD and then α₂M or insulin. B, inhibition of α₂M* (black bars) or insulin (white bars)-induced lipogenesis from 1-[¹⁴C]acetate in DU-145 cells by the fatty-acid synthase inhibitors in DU-145 cells. The bars in the figure are same as in A. The values are the mean ± S.E. from three experiments in both cases. Values significantly different at the 5% level in A and B from buffer and inhibitor-treated cells are denoted by an asterisk. C, inhibition of α₂M* (black bars) or insulin (white bars)-induced lipogenesis from 6-[¹⁴C]glucose in 1-LN prostate cancer cells pretreated with fatty-acid synthase inhibitors. The bars in the figure are as follows: 1, buffer; 2, α₂M* or insulin; 3, LY294002 and then α₂M* or insulin; 4, rapamycin and then α₂M* or insulin; 5, torin and then α₂M* or insulin; 6, C-75 and then α₂M or insulin; 7, KU0063794 and then α₂M* or insulin; 8, MKK2206 and then α₂M* or insulin; 9, anti-CTD and then α₂M*. D, suppression of α₂M* (black bars) or insulin (white bars)-induced lipogenesis from 6-[¹⁴C]glucose in DU-145 cells pretreated with fatty-acid synthase inhibitors as in C. The results are the mean ± S.E. from three experiments. Values significantly different at the 5% levels in from buffer-treated and inhibitor-treated cells are denoted by an asterisk. Treatment of 1-LN or DU-145 cells with inhibitors alone did not effect 1-[¹⁴C]acetate or 6-[¹⁴C]glucose measurements (data not shown).

FIGURE 5.

α₂M (black bars) and insulin (white bars) up-regulate the incorporation of 1-[¹⁴C]acetate into a nonsaponifiable lipid fraction (cholesterogenesis) in 1-LN cells (A) and in DU-145 cells (B). The bars in A and B are as follows: 1, buffer; 2, α₂M* and then α₂M* or insulin; 3, LY294002 and then α₂M* or insulin; 4, rapamycin and then α₂M or insulin; 5, torin and then α₂M* or insulin; 6, C-75 and then α₂M* or insulin; 7, KU0063794 and then α₂M* or insulin; 8, anti-CTD and then α₂M*. The incorporation of 1-[¹⁴C]acetate into nonsaponifiable fractions (cholesterogenesis) in both A and B is expressed as cpm/mg cell protein and is the mean ± S.E. from three experiments. Values significantly different at the 5% level from buffer and inhibitor-treated cells in both A and B are denoted by an asterisk. Up-regulation of 1-[¹⁴C]acetate incorporation into saponifiable fractions (fatty acids) by α₂M* (black bars) or insulin (white bars) in 1-LN cells (C) and DU-145 (D) cells. The bars in C and D are as follows: 1, buffer; 2, α₂M* or insulin; 3, LY294002 and then α₂M* or insulin; 4, rapamycin and then α₂M* or insulin; 5, torin and then α₂M* or insulin; 6, C-75 and then α₂M* or insulin; 7, KU0063794 and then α₂M* or insulin; 8, anti-CTD and then α₂M*. The incorporation of 1-[¹⁴C]acetate into saponifiable fraction (fatty acids) in C and D is expressed as cpm/mg cell protein and is the mean ± S.E. from three experiments. Values significantly different from buffer and inhibitor-treated cells in both C and D are denoted by an asterisk.

FIGURE 6.

Thin layer chromatography of 1-[lsqb]¹⁴C]acetate-labeled lipid extracts of 1-LN and DU-145 cells treated with α₂M* or insulin. A, representative thin layer chromatogram of cell lipids of 1-LN cells. A similar pattern was obtained with DU-145 cells (data not shown). B, fatty-acid synthase inhibitors inhibit α₂M-induced increased incorporation of 1-[¹⁴C]acetate into esterified cholesterol (black) triglycerides (red), fatty acids (green), cholesterol (blue), and phosphatidylcholine (white) fractions of 1-LN cells. The bars are in the figure are as follows: 1, buffer; 2, α₂M*; 3, LY294002 and then α₂M*; 4, rapamycin and then α₂M*; 5, torin and then α₂M*; 6, C-75 and then α₂M*; 7, KU0063794 and then α₂M*; 8, anti-CTD and then α₂M*. C, inhibition of insulin-induced increase 1-[¹⁴C]acetate incorporation into cellular lipids fraction of 1-LN cells by prior treatment of cells with fatty-acid synthase inhibitors. Bars in the figure are as in B except treatment with anti-CTD or KU0063794, which was not performed. Fatty-acid synthase inhibitors suppress α₂M* (D) and insulin-induced increase (E) of [¹⁴C]acetate incorporation into cellular lipids of DU-145 cells. The bars in figure in D are the same as in B, and the bars in figure in E are the same as in C. The incorporation of 1-[¹⁴C]acetate into various lipid fractions in B–E is expressed as cpm/mg protein and is mean ± S.E. from three experiments. Values significantly different at 5% levels from buffer and inhibitor-treated cells in B–E are denoted by an asterisk.

FIGURE 7.

Inhibition of proteolytic cleavage of SREBP1 by fatostatin A on α₂M*-dependent metabolic regulation. Fatostatin abrogates α₂M* or insulin-induced protein and DNA synthesis in1-LN (black bars) and DU-145 (white bars) cells (A and B). The bars are as follows: 1, buffer; 2, α₂M*; 3, insulin; 4, fatostatin A (20 μm/2 h) and then α₂M*; 5, fatostatin A and then insulin; and 6, fatostatin. Similar results were obtained with LnCAP cells (C and D). The bars are as above and the mean ± S.E. from three experiments. Values significantly different at the 5% levels from buffer and inhibitor-treated cells are denoted by an asterisk. A representative immunoblot of three experiments showing cleaved SREBP1 (kDa ∼151) into mature cleaved SREBP1 (kDa ∼74) in α₂M* or insulin-treated cells and its inhibition by fatostatin A (E and F). The lanes in immunoblots are as follows: lane 1, α₂M*; lane 2, insulin; lane 3, fatostatin A then α₂M*; lane 4, fatostatin A then insulin; or lane 5, buffer.

FIGURE 8.

Fatostatin A suppresses α₂M* or insulin-induced increased lipogenesis from 1-[¹⁴C]acetate in 1-LN (A) and DU-145 (B) and LnCap (C) prostate cancer cells. The bars in figure in A and B are as follows: 1, buffer; 2, α₂M*; 3, insulin; 4, fatostatin A and then α₂M*; 5, fatostatin A and then insulin; and 6, anti-CTD and then α₂M*. The bars in figure in C are as follows: 1, buffer; 2, α₂M*; 3, insulin; 4, torin and then α₂M* or insulin; 5, C-75 and then α₂M* or insulin; 6, fatostatin A and then α₂M* or insulin; 7, fatostatin A. 1-[¹⁴C]Acetate incorporation in A–C is expressed as cpm/mg cell protein and is mean ± S.E. from three experiments. Values significantly different at 5% levels from buffer and inhibitor-treated cells in A–C are denoted by an asterisk. Inhibition of SREBP1 cleavage by fatostatin A suppresses α₂M* or insulin-induced increased de novo phosphatidylcholine synthesis from [¹⁴CH₃]choline in 1-LN (D) and DU-145 (E). The bars in both D and E are as follows: 1, buffer; 2, α₂M*; 3, insulin; 4, fatostatin A and then α₂M*; 5, fatostatin A and then insulin; 6, anti-CTD and then α₂M*. The synthesis of phosphatidylcholine in both panels is expressed as cpm/mg cell protein and is the mean ± S.E. from three experiments. Values significantly different at 5% levels from buffer and fatostatin A-treated cells in both panels are denoted by an asterisk.

FIGURE 9.

Down-regulation of cell surface GRP78 by RNAi suppresses α₂M*-induced expression of ATP citrate lyase (black bars) and fatty-acid synthase (white bars) in 1-LN (A) and DU-145 (B) prostate cancer cells. The panels in both immunoblots are as follows: 1, Lipofectamine + α₂M*; 2, GRP78 dsRNA (100 nm/48 h)/Lipofectamine and then α₂M*; and 3, scrambled dsRNA (100 nm/48 h)/Lipofectamine and then α₂M*. The changes in expression of target proteins are in arbitrary units and are the mean ± S.E. from four experiments. Values significantly different at the 5% level for GRP78 dsRNA-transfected cells compared with the controls are denoted by an asterisk. A representative immunoblot of fatty-acid synthase and ATP citrate lyase in 1-LN (A) and DU-145 (B) cells from four experiments is shown below the respective bar diagrams.

FIGURE 10.

Schematic representation of cellular events involved in α₂M*-induced aerobic glycolysis in prostate cancer cells. The abbreviations are as in the cleaved or active form, except for mSREBPS1, which indicates the mature form.