HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

Abstract

Development of chemoresistance, especially to docetaxel (DTX), is the primary barrier to the cure of castration-resistant prostate cancer but its mechanism is obscure. Here, we report a seminal crosstalk between dying and residual live tumor cells during treatment with DTX that can result in outgrowth of a chemoresistant population. Survival was due to the induction of secretory/cytoplasmic clusterin (sCLU), which is a potent anti-apoptotic protein known to bind and sequester Bax from mitochondria, to prevent caspase 3 activation. sCLU induction in live cells depended on HMGB1 release from dying cells. Supernatants from DTX-treated DU145 tumor cells, which were shown to contain HMGB1, effectively induced sCLU from newly-plated DU145 tumor cells and protected them from DTX toxicity. Addition of anti-HMBG1 to the supernatant or pretreatment of newly-plated DU145 tumor cells with anti-TLR4 or anti-RAGE markedly abrogated sCLU induction and protective effect of the supernatant. Mechanistically, HMGB1 activated NFκB to promote sCLU gene expression and prevented the translocation of activated Bax to mitochondria to block cell death. Importantly, multiple currently-used chemotherapeutic drugs could release HMGB1 from tumor cells. These results suggest that acquisition of chemoresistance may involve the HMGB1/TLR4-RAGE/sCLU pathway triggered by dying cells to provide survival advantage to remnant live tumor cells.

Figure 1. HMGB1 induces clusterin from tumor cells and is released by dying tumor cells.

(A) DU145 prostate tumor cells, incubated with recombinant human HMGB1 (rhHMGB1) for 24–48 h, were lysed and analyzed by western blot for presence of clusterin, and for β-actin for equal loading. (B) DU145 tumor cells were treated with docetaxel (DTX) for 1–4 days and their supernatants were evaluated for HMGB1 by ELISA. (C) DU145 tumor cells were treated with the indicated chemotherapeutic agents for 24 h and the supernatants analyzed for HMGB1.

Figure 2. HMGB1 induces clusterin via TLR4 and RAGE.

(A) DU145 tumor cells, pretreated with DTX for 6 h, were washed and then reincubated in fresh medium for another 24 h. Supernatants were collected and added to freshly-plated DU145 tumor cells for 24 h, either untreated or pretreated with anti-HMGB1 for 30 min. (B) Flow cytometric analysis of DU145 tumor cells treated with DTX for 24 h demonstrated no change for RAGE and TLR4 expression. (C) DU145 tumor cells, untreated or pretreated with anti-TLR4, anti-RAGE or both antibodies for 30 min, were exposed to recombinant HMGB1 or supernatants from DTX-pretreated tumor cells. After 24 h, the cells were lysed and analyzed for presence for clusterin by western blot.

Figure 3. Down-regulation of clusterin expression after inhibition of NFκB-p65 phosphorylation.

(A) Recombinant HMGB1, added to DU145 tumor cells, enhanced NFκB-p65 phosphorylation within 1–5 min, as analyzed by western blot. (B,C) DU145 tumor cells, pretreated with medium, 20 μM PS1145 or 10 μM BAY11-7082 for 1 h, were cultured with HMGB1 for another 24 h. Cells were then lysed for analysis of clusterin mRNA expression by Q-PCR or clusterin protein expression by western blot. Analysis of phosphorylated NFκB-p65 was included to check for effectiveness of the inhibitors.

Figure 4. HMGB1 confers chemoresistance via TLR4 and RAGE.

(A) DU145 tumor cells, untreated or pretreated with control IgG, anti-TLR4, anti-RAGE, or both antibodies for 30 min, were exposed to recombinant HMGB1 for 24 h. Then, DTX was added for another 48 h prior to staining with methylene blue for visualization of live tumor cells. (B) The same experiment was conducted with recombinant HMGB1 substituted for supernatants from DTX-treated tumor cells. (C) DU145 tumor cells pretreated with recombinant HMGB1 were transfected with antisense-scramble or antisense-clusterin for 24 h, and then cells were cultured in medium with or without DTX for another 48 h prior to staining with methylene blue.

Figure 5. Dying tumor cells release HMGB1 which induces clusterin from neighboring live tumor cells.

DU145 tumor cells were untreated or treated with DTX for 24 h and then dually stained for HMGB1 (green) and clusterin (red), as well as DAPI (blue) to visualize the nucleus. Live tumor cells (indicated by white arrows) in medium culture have intact round blue nucleus (A), intranuclear green speckled HMGB1 (B) and little cytoplasmic red clusterin (C). This separation of HMGB1 and clusterin is cleary seen in the merged picture (D). However, DTX treatment causes a change in the dynamics of the tumor cells. A DTX-treated dying tumor cell, indicated by a red arrow in the center of a cell cluster, has an irregular blue nuclei (E), and has released its green HMGB1 from the nucleus into the cytoplasm and surrounding medium (F). The dying cell is surrounded by 4 live tumor cells (white arrows), which still have intact nuclei with intranuclear speckled green HMGB1. More remarkably, these 4 live tumor cells now express high levels of cytoplasmic red clusterin. Thus, the HMGB1 released from the central dying tumor cells appear to have induced clusterin from the 4 neighboring tumor cells.

Figure 6. Clusterin blocks DTX-mediated apoptosis by sequestering Bax from mitochondria.

DU145 tumor cells, pretreated with or without recombinant HMGB1 for 24 h, were treated with medium or DTX for 4 h. Mitochondria was visualized with MitoTracker Red, and activated Bax with 6A7 monoclonal antibody (green). Cells were examined by confocal microscopy (magnification, ×630) for colocalization of activated Bax with mitochondria. One representative image of three independent experiments is shown.