CLU blocks HDACI-mediated killing of neuroblastoma

Abstract

Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation.

Fig. 1

CLU is differentially expressed in S-type NB cells. a Expression of CLU and selected RNAs previously shown to be specific for neuroblastic (top panel) or Schwannian stromal cells (bottom panel) in a panel of N-type [IMR-32, LA1-55n, SH-SY5Y, SK-N-BE(2)-M17, and SH-IN (FSK-treated)] and S-type (SH-EP and SMS-KCNs) NB cell lines as well as in three human NB tumor specimens (NB1, NB2, and NB3). Color indicates fold difference of each sample from the midpoint of the two means for S- and N-type cells, with red indicating higher and green indicating lower expression. P values are from two-tailed t tests, and the fold difference indicates the ratio of average N-type to S-type expression values. b SH-SY5Y cells are transfected with a full-length CLU (f-Clu) construct expressing precursor form (p-CLU) and mature form (m-CLU) of CLU. Both forms are localized in the cytoplasm. Nuclear form of CLU (n-CLU) is expressed from a CLU expression vector starting at the second methionine of the CLU sequence. Note the differences of the positions of each CLU on a SDS–polyacrylamide gel. c Immunoblot analyses of three N-type (IMR32, SH-SY5Y, and GOTO) and three S-type (SH-EP1, SK-N-AS, and LA1-5S) cell lines show endogenous expression of secreted form of CLU and mature form in S-type and not in N-type NB cell lines

Fig. 2

Clusterin is highly expressed in the neuroblastic, but not stromal, components of neuroblastic tumors. a The NB tissue is obtained from our tissue core at the University of Michigan, with one stage I, four stage II, one stage III, and three stage IV tumors. b Immunohistochemistry was performed with antibodies specific for CLU as described in the text. Strong cytoplasmic staining is seen in the small neuroblasts of a poorly differentiated NB (left) and the larger, more mature-appearing ganglion cells of an intermixed ganglioneuroblastoma (right). Staining prevalence and intensity were scored semiquantitatively from 0 to 3+, with separate scores assigned to the histologically distinct neuroblastic and stromal cells. Categorization of expression revealed that whereas the N-type components of all but one tumor had at least 2+ expression (mean 2.95), most S-type components had CLU present at less than 1+ levels (mean 0.95)

Fig. 3

Clusterin expression is increased with HDACI treatment. a NB N-type (IMR32, SH-SY5Y, and GOTO) and S-type (SH-EP1, SK-N-AS, and LA1-5S) cell lines were treated with 1 μM TSA for 24 h before immunoblotting with anti-CLU antibody. β-Tubulin was used as a loading control. b SH-SY5Y and SH-EP1 cell lines were treated with 1 μM TSA and cell lysates were collected at different time points (4, 8,16, 20, and 24 h), and the expression of CLU was analyzed by immunoblotting. c mRNA level CLU is present in S-type SHEP1 cells whereas it is induced after 1 μM TSA treatment in N-type SH-SY5Y cells both after 16 and 24 h. d SH-SY5Y cells and SH-EP1 cell lines were treated with 1 μM of TSA, SAHA, or MS-275 for 24 h, and the expression of CLU was analyzed by immunoblotting

Fig. 4

CLU is induced by HDACI but not by other stressors in NB cells. Both N-type SH-SY5Y (a) and S-type SH-EP1 (b) cell lines were treated for 24 h with TSA (1 μM), cisplatin (10 μg/ml), doxorubicin (Dox) (0.5 μg/ml), etopicide (VP16) (10 μg/ml), or radiation (15 Gy for 1 or 17 h as indicated). The presence of CLU was analyzed by immunoblot using CLU-specific antibodies

Fig. 5

Knocking down CLU sensitizes NB cells to TSA-induced cell death. a Various amounts of CLU expression vectors (as indicated) were transfected into SH-SY5Y cells. The cells were treated with different concentration of TSA for 24 h as shown. Cell viability is determined by MTT assay. Results are expressed as the percentage of viable cells compared with vehicle-treated controls (mean ± SD; n = 3). b Stable SH-SY5Y cell line expressing full-length CLU or vector were treated with TSA (1 μM) for 0, 4, 8, and 16 h as indicated. Mitochondria were isolated; Bax level was determined by immunoblotting. VDCA1 is used as the loading control for mitochondria fraction. c SH-SY5Y cells or SH-EP1 cells were transfected with scrambled siRNA or CLU-specific siRNA. Forty-eight hours after transfection, cells were treated with TSA (1 μM, 24 h). Cells in mock sample received no transfection. Immunoblot shows effective knockdown of CLU. d SH-SY5Y cells or SH-EP1 cells were transfected with CLU-specific siRNA as shown in (c). The cells were treated with varying concentrations of TSA for 24 h as shown. Cell viability was determined by MTT assay. Results are expressed as the percentage of viable cells compared with vehicle-treated controls (mean ± SD; n = 3)

Fig. 6

CLU does not affect Ku70 acetylation or Ku70–Bax binding in NB cells. a Stable SH-SY5Y cell line expressing full-length CLU or vector were treated with TSA (1 μM, 24 h) and cytoplasmic extracts were immunoprecipitated using Bax-specific antibodies or normal rabbit serum (NRS) as control. The immunoprecipitates were separated by SDS–PAGE and probed with Ku70, CLU, or Bax antibodies. b SH-SY5Y cells were treated with vehicle or TSA (1 μM, 24 h). CLU was immunoprecipitated using CLU-specific antibodies or normal mouse serum (NMS). The immunoprecipitates were separated by SDS–PAGE and probed with Ku70 or CLU antibodies. c Stable SH-SY5Y cells expressing full-length CLU or vector were treated with TSA (1 μM, 24 h). Cytoplasmic extracts were immunoprecipitated using an anti-acetyl-lysine antibody (K103). The immunoblot was probed with Ku70 as shown. d CLU siRNA or scramble siRNA-treated SH-SY5Y cells were treated with TSA (1 μM, 24 h). Cytoplasmic extracts were immunoprecipitated using anti-acetyl-lysine antibody (K103). The immunoblot was probed with Ku70 antibody