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HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guérin Functions as a Paracrine Factor to Potentiate the Direct Cellular Effects of Bacillus Calmette-Guérin
HMGB1 Release by Urothelial Carcinoma Cells in Response to Bacillus Calmette-Guérin Functions as a Paracrine Factor to Potentiate the Direct Cellular Effects of Bacillus Calmette-Guérin Guangjian Zhang,* Fanghong Chen,* Yanli Cao, Jonathan V. Amos, Gopit Shah and William A. See† From the Department of Urology, Medical College of Wisconsin, Milwaukee, Wisconsin
Abbreviations and Acronyms BCG ⫽ bacillus Calmette-Guérin CCL20 ⫽ chemokine (C-C motif) ligand 20 CD54 ⫽ intercellular adhesion molecule 1 CXCL ⫽ chemokine (C-X-C motif) ligand HMGB1 ⫽ high molecular group box protein 1 IL ⫽ interleukin NFB ⫽ nuclear factor-B NRF2 ⫽ nuclear factor (erythroidderived 2)-like 2 qrtPCR ⫽ quantitative rtPCR RAGE ⫽ receptor for advanced glycation end products rtPCR ⫽ reverse transcriptasepolymerase chain reaction UC ⫽ urothelial carcinoma Accepted for publication January 16, 2013. Supported by a grant from the Department of Veterans Affairs and the Milwaukee Veterans Affairs Medical Center. * Equal study contribution. † Correspondence: Department of Urology, Medical College of Wisconsin, 9200 West Wisconsin Ave., Milwaukee, Wisconsin 53226 (telephone: 414-805-0787; FAX: 414-805-0771; e-mail: [email protected]).
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Purpose: Prior study demonstrated that HMGB1 release by urothelial carcinoma cells in response to bacillus Calmette-Guérin is required for an in vivo antitumor effect. We evaluated the direct effects of HMGB1 on the in vitro response of urothelial carcinoma cells to bacillus Calmette-Guérin. Materials and Methods: Two human urothelial carcinoma cell lines were used to study the effect of exogenous HMGB1 alone and combined with bacillus Calmette-Guérin on the tumor cell response to bacillus Calmette-Guérin. Antibody mediated blockade of receptors for HMGB1 or HMGB1 protein was used to determine the contribution of paracrine HMGB1 release to bacillus CalmetteGuérin biological effects. Response end points evaluated included the activation of intracellular signaling pathways, gene transactivation and cytotoxicity. Results: Urothelial carcinoma cells expressed the receptor for HMGB1 signaling. Antibody blockade of the RAGE receptor confirmed the dependence of signaling in response to HMGB1 on RAGE function. Exogenous HMGB1 activated cell signaling pathways for NFB, NRF2 and CEBP. Quantitative reverse transcriptase-polymerase chain reaction on a panel of bacillus Calmette-Guérin responsive genes revealed peak expression resulting from the combination of bacillus Calmette-Guérin and HMGB1. Blockade of paracrine HMGB1 released in response to bacillus Calmette-Guérin using HMGB1 and/or RAGE receptor blocking antibodies showed a significant decrease in gene expression relative to that of bacillus Calmette-Guérin alone. HMGB1 potentiated the cytotoxic effects of bacillus Calmette-Guérin. Conclusions: HMGB1 released by urothelial carcinoma cells after bacillus Calmette-Guérin treatment functions as a paracrine factor to potentiate the urothelial carcinoma cell response to bacillus Calmette-Guérin. This paracrine activity likely contributes to the dependence of an in vivo tumor response on HMGB1 release. Key Words: urinary bladder, carcinoma, BCG vaccine, paracrine communication, HMGB1 protein INTRAVESICAL administration of attenuated Mycobacterium BCG as treatment for nonmuscle invasive bladder cancer elicits a complex response in the tumor and host. Recent studies demonstrated that HMGB1, a potent
chemokine and marker of nonapoptotic cell death, is released by a portion of UC tumor cells after BCG exposure.1 Increased HMGB1 levels were observed in the urine of patients after intravesical BCG therapy. Of
0022-5347/13/1903-1076/0 THE JOURNAL OF UROLOGY® © 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
http://dx.doi.org/10.1016/j.juro.2013.01.050 Vol. 190, 1076-1082, September 2013 RESEARCH, INC. Printed in U.S.A.
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HMGB1 FUNCTIONS AS PARACRINE FACTOR TO POTENTIATE BACILLUS CALMETTE-GUÉRIN
particular interest is that in vivo animal models showed that tumor cells engineered to have decreased HMGB1 release fail to respond to intravesical BCG.2 HMGB1 released by tumor cells after BCG treatment has the potential to influence the antitumor response at the level of the tumor and the host. In other systems HMGB1 signals through specific cell surface receptors to alter gene expression and the tumor phenotype. Paracrine release of HMGB1 in concert with the expression of tumor cell surface receptors for HMGB1 translates into local effects.3 As a powerful chemokine, HMGB1 can function to direct a host cellular immune response to the site of its release. Systemically, HMGB1 activates macrophages, induces neutrophil chemotaxis and alters vascular endothelial cell biology.4,5 To our knowledge it is unknown which, if either, of these mechanisms contributes to the HMGB1 dependent in vivo tumor response. Given the apparent importance of HMGB1 release for in vivo antitumor activity and the potential for it to exert a direct effect on tumor biology, we examined the effects of exogenous and endogenous HMGB1 on the in vitro biology of UC cells. Our results demonstrate that UC cells express functional receptors for HMGB1. Exogenous HMGB1 signals through these receptors to alter intracellular signaling, gene expression and cytotoxicity. Importantly, endogenous HMGB1 release in response to BCG acts as a paracrine factor to potentiate direct BCG effects on tumor biology. While not eliminating the possibility for a systemic effect, these findings support a direct effect of HMGB1 on tumor biology as a potential contributor to the antitumor response to BCG.
METHODS Cell Lines and BCG We used the human UC T24 (ATCC®) and 253J cell lines. Cells were maintained at 37C in 5% CO2 in RPMI 1640 (Gibco®) supplemented with 10% fetal bovine serum, penicillin and streptomycin (complete medium). TICE® BCG was used in these experiments. Freeze-dried BCG was reconstituted in complete medium at an estimated concentration of 4 ⫻ 108 viable organisms per ml.
Luciferase Reporter Assays UC cell exposure to BCG increases the activation of intracellular signaling pathways. We measured the effect of HMGB1 alone or combined with BCG on signaling pathway activation. In 24-well plates 253J and T24 cells were plated at 1 ⫻ 105 cells per well. At 24 hours the cells were transiently transfected with previously described nuclear factor-B, CEBP and NRF2 plasmid reporter constructs using Lipofectamine™ 2000 according to manufacturer instructions.6 Duplicate wells were set up for each group.
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At 24 hours after transfection, cells were left untreated or were treated with BCG (50:1 BCG to cells) and/or HMGB1. Six hours later the cells were washed with phosphate buffered saline and lysed with 1X Reporter Lysis Buffer (Promega, Madison, Wisconsin). Luciferase activity was measured using a Luciferase Assay System (Promega) according to manufacturer instructions. Luciferase activity was normalized to the protein concentration, as measured by the BCA™ Protein Assay Kit.
qrtPCR Cell and Proliferation Prior study demonstrated that UC cell exposure to BCG increases the expression of cell cycle regulatory and immune response genes. We measured the effect of HMGB1 alone and combined with BCG on gene expression. qrtPCR was done using the LightCycler® 480 Real-Time PCR System. On comparative analysis treatment groups included control cells and cells treated with BCG (50:1 BCG to cells) and/or HMGB1. The primer sequence was IL-6, 5=AGCCGCCCCACACAGA (upstream) and 5=-CCGTCGAGGATGTACCGAAT (downstream), IL-8, 5=-CTGGCCGTGGCTCTCTTG (upstream) and 5=-CCTTGGCAAAACTGCACCTT (downstream), CXCLI 5=-CCACTGCGCCCAAACC (upstream) and 5=-GCAGGATTGAGGCAAGCTTT (downstream), CXCL3, 5=-AATGGGAAGAAAGCTTGTCTCAA (upstream) and 5=-CCTTGTTCAGTATCTTTTCGA (downstream), CCL20, 5=-TCCTGGCTGCTTTGATGTCA (upstream) and 5=-AAAGTTGCTTGCTGCTTCTGAT (downstream), and -actin 5=-ACCGAGCGCGGCTACAG (upstream) and 5=-CTTAATGTCACGCACGATTTCC (downstream). Reactions were done using rtPCR Master Mix (Promega) composed of HotStarTaq® DNA polymerase, dinucleoside triphosphate, MgCl2 and ROX™ Dye. It was incubated with primers (0.4 M), probe (0.2 M), QuantiTect™ RT Mix and RNA template. The rtPCR program consisted of 1 cycle at 50C for 30 minutes for reverse transcription and 1 cycle at 95C with a 15-minute hold (hot start), followed by 40 cycles of denaturation at 94C for 15 seconds and annealing/extension at 60C for 60 seconds. Fluorescence data were collected at the end of each extension phase. -actin was used to normalize all other genes tested in the same RNA sample. Cell proliferation was measured using the MTT assay, as previously described.7
Statistical Analysis All experiments were done a minimum of 3 times. Data from individual experiments for assays using luciferase reporter constructs were subject to arithmetic normalization relative to the highest values of the corresponding replicate experiments. Unless otherwise noted, data were analyzed using 2-way ANOVA for repeated measures. Results were considered significant at p ⬍0.05. Graphic representation of the data is shown as the mean ⫹ SE.
RESULTS UC Cells Expressed Functional HMGB1 Receptors rtPCR on UC cell mRNA using RAGE specific primers revealed that the 253J and T24 cell lines expressed RAGE transcripts. Figure 1 shows a blot of the rtPCR reaction. Functional RAGE expression
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sponse to the 2.5 g/ml HMGB1 concentration was increased relative to untreated controls, representing 67% and 87% of the BCG induced response in 253J and T24 cells, respectively. HMGB1 combined with BCG demonstrated peak reporter activity at the 2.5 g/ml HMGB1 concentration with reporter activation representing 118% and 126%, respectively, of that observed in response to BCG alone. Adding BCG to HMGB1 significantly increased NRF2 activation relative to that of BCG alone across the range of HMGB1 concentrations. Figure 2 shows Figure 1. UC cell line RAGE expression. Blot of RAGE specific rtPCR products from 253J and T24 cells lines demonstrates size correct amplification product consistent with RAGE mRNA transcripts in each cell line.
was confirmed using RAGE specific blocking antibodies. RAGE blocking antibodies significantly inhibited NFB activation in response to exogenous HMGB1 in each cell line. NFB reporter activation in response to the combination of HMGB1 (5 g/ml) and anti-RAGE antibodies (5 g/ml) represented 32% and 23% of the response observed to HMGB1 alone in 253J and T24 cells, respectively (paired t test p ⬍0.01). HMGB1 Activated Multiple UC Cell Signaling Pathways Treatment of cells with HMGB1 increased the response of a luciferase reporter construct for NFB. Analysis of a dose-response relationship for the 2 cell lines showed a statistically significant relationship between the HMGB1 concentration and reporter activation for cells exposed to HMGB1 alone or HMGB1 combined with BCG (each group p ⬍0.00001). Reporter activation across the range of HMGB1 concentrations was significantly increased by adding BCG in the 253J and T24 cell lines (p ⬍0.0001 and ⬍0.001, respectively). Activation of a CEBP reporter construct by UC cells showed a significant dose-response relationship with HMGB1 concentration in the 2 cell lines (p ⫽ 0.001). Peak reporter activation in response to the 5.0 g/ml HMGB1 concentration represented 91% and 83% of the BCG induced response in 253J and T24 cells, respectively. CEBP reporter activation by the HMGB1 and BCG combination did not significantly correlate with the HMGB1 concentration in the 2 cell lines (p ⫽ 0.06). Reporter activation across the range of HMGB1 concentrations was significantly increased by adding BCG in the 253J and T24 cell lines (each p ⬍0.001). Activation of an NRF2 reporter construct by UC cells correlated with the HMGB1 concentration but failed to attain the level of statistical significance of HMGB1 alone or combined with BCG (p ⫽ 0.08 and 0.8, respectively). Peak reporter activation in re-
Figure 2. Mean ⫾ SE signaling pathway activation in response to HMGB1 and BCG alone or combined. Luciferase reporter constructs for NFB (A), CEBP (B) and NRF2 (C) were activated in response to BCG, HMGB1 or combination. NFB activation showed statistically significant dose-response relationship with HMGB1 alone and combined with BCG in each cell line. CEBP activation significantly correlated with HMGB1 concentration. For CEBP adding BCG to HMGB1 failed to show significant dose-response relationship with HMGB1. NRF2 activation did not significantly correlate with HMGB1 concentration for HMGB1 alone or combined with BCG. Activation of all reporters was significantly increased by BCG and HMGB1 combination relative to HMGB1 alone.
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Figure 3. Mean ⫾ SE gene expression in response to HMGB1 and/or BCG. UC cell exposure to exogenous HMGB1 resulted in significant dose dependent increase in expression of panel of 6 BCG responsive immune regulatory genes in 253J cell line (p ⬍0.001) (A). There was no significant relationship between HMGB1 concentration and gene expression in T24 cell line (p ⫽ 0.18) (B). In 253J and T24 cell lines BCG and HMGB1 combination significantly increased gene expression in HMGB1 dose dependent manner (p ⬍0.01 and ⬍0.05, respectively).
a graph of reporter activation (NFB, CEBP and NRF2) in response to HMGB1 alone or combined with BCG. HMGB1 Induced Gene Transcription HMGB1 showed a cell line dependent effect on the expression of a panel of 6 BCG activated immune response genes, as measured by qrtPCR. In the T24 cell line the expression of IL-6 and 8, CXCL1 and 3, CCL20 and CD54 failed to increase in response to HMGB1 and manifested no dose-response relationship (p ⫽ 0.18). In contrast, gene expression in the 253J cell line increased in response to HMGB1 and significantly correlated with the HMGB1 concentration (p ⬍0.001). HMGB1 combined with BCG significantly increased the expression of the 6 target genes in each cell line relative to that of BCG alone. Gene expres-
sion in response to the BCG and HMGB1 combination significantly correlated with the HMGB1 concentration in the 253J and T24 cell lines (p ⬍0.01 and p ⬍0.05, respectively). Figure 3 shows gene expression data on each cell line in response to HMGB1 alone or combined with BCG. Endogenous HMGB1 Released by UC Cells in Response to BCG Functioned as Paracrine Factor In this experiment we evaluated the role of HMGB1 release by UC cells in response to BCG treatment on direct BCG biological effects on gene expression. Blockade of HMGB1 signaling through a combination of RAGE receptor blocking and HMGB1 neutralizing antibodies significantly decreased gene expression in response to BCG alone in the 253J and T24 cell lines (p ⬍0.05 and ⬍0.001, respectively, fig. 4).
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Figure 4. Mean ⫾ SE paracrine HMGB1 contributed to UC cell gene expression in response to BCG. HMGB1 signaling blockade using RAGE receptor blocking and/or HMGB1 neutralizing antibodies inhibited BCG activated gene expression. Receptor blockade and HMGB1 neutralization combination significantly decreased expression of 6 BCG responsive genes in 253J (A) and T24 (B) cells (p ⬍0.05 and ⬍0.001, respectively).
HMGB1 Potentiated BCG Cytotoxicity UC cell exposure to BCG resulted in a direct cytotoxic effect. In this experiment we evaluated the effects of BCG and HMGB1 alone and in combination on cellular cytotoxicity. Metabolically active cell number as a function of time after treatment was measured using the MTT assay. Relative to untreated controls, BCG and HMGB1 significantly decreased the number of metabolically active 253J and T24 cells with time (BCG p ⬍0.001 and ⬍0.0001, respectively, and HMGB1 each p ⬍0.00001). Relative to BCG alone, exposure of cells to the BCG and HMGB1 combination resulted in a significant decrease in the number of metabolically active 253J and T24 cells (p ⬍0.001 and ⬍0.05, respectively, fig. 5).
DISCUSSION A growing body of evidence points to the interaction between the UC cell and BCG as having a central role in orchestrating the host immune response that culminates in an antitumor effect. BCG binding to the UC cell surface, followed by internalization,
stimulates a cellular response that is characterized by the expression of white blood cell receptors (CD54), and the activation of genes coding for chemokines (CXCL1 and 3, and CCL20) and cytokines (IL-6 and 8). In some cells BCG exposure results in necrotic cell death and release of the chemokine HMGB1 into the pericellular space. The resultant immune modulatory/inflammatory soup likely stimulates a host response targeting the site of release. While a specific role for many of these factors remains unproven, HMGB1 animal model data revealed that the release of this chemokine in response to BCG treatment is needed for an in vivo tumor response.1 BCG induced release of HMGB1 into the pericellular environment has the potential to act systemically and locally. Systemic responses may occur as the result of the recruitment and maturation of immune effector cell populations.8 Tumor expression of HMGB1 receptors could result in local responses, such as a direct cytotoxic effect on remaining tumor cells. Local effects could also include indirect pathways, such as HMGB1 stimulated, receptor medi-
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Figure 5. Mean ⫾ SE HMGB1 potentiated BCG cytotoxicity. UC cell exposure to BCG or HMGB1 significantly decreased number of metabolically active cells as function of time after treatment in 253J cells (p ⬍0.001 and ⬍0.0001, respectively) (A) and T24 cells (each p ⬍0.00001) (B). Relative to BCG alone, BCG and HMGB1 combination resulted in significant further decrease in number of metabolically active cells with time in 253J and T24 cells (p ⬍0.001 and ⬍0.05, respectively).
ated (paracrine) expression of immune modulatory genes by UC cells, which in turn potentiate the host immune reaction. Given the apparent importance of HMGB1, we evaluated the impact of HMGB1 on UC cell biology in the context of BCG treatment. The results of this study reveal that human UC cell lines express the principal receptor for HMGB1. RAGE is widely recognized as an important cell surface protein for HMGB1 mediated signaling.9 Antibody blocking studies targeting RAGE demonstrated the functionality of the HMGB1/RAGE axis in UC cells and its role in activating the NFB signaling pathway. UC cell exposure to exogenous HMGB1 also activated other known BCG responsive signaling pathways. CEBP and NRF2 pathways were activated to varying degrees in response to HMGB1. Not surprisingly, the transactivation of genes downstream of these signaling pathways in-
creased in response to HMGB1. In the context of modest increases in intracellular signaling in response to HMGB1 alone, the extent to which HMGB1 exerted a synergistic effect on gene expression in combination with BCG was striking. The table shows the effects of HMGB1 alone or combined with BCG on signaling pathway activation and gene expression. In the clinical setting HMGB1 release occurs in conjunction with BCG treatment. As a consequence, the simultaneous presence of the bacteria and the chemokine has the potential to function as a paracrine factor to alter the biology of adjacent UC cells. While it is interesting to note the influence of exogenous HMGB1, the most important finding in the current study is the role of endogenous HMGB1 in mediating the direct effects of BCG on UC cells. The data reveal that HMGB1 release by BCG exposed
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Table. T24 and 253J cell signaling pathway activation and gene expression after exposure to BCG and/or 5 g/ml HMGB1, and activation or expression in response to HMGB1 vs HMGB1 combined with blocking antibodies Fold Induction vs Controls BCG
BCG ⫹ HMGB1
HMGB1
% Response*
Signaling pathways NFB: 253J T24 CEBP: 253J T24 NRF2: 253J T24 CD54: 253J T24 IL-6: 253J T24 IL-8: 253J T24 CXCL1: 253J T24 CXCL3: 253J T24 CCL20: 253J T24
2.13 1.98
1.82 1.51
2.68 2.17
1.98 1.87
1.81 1.55
1.94 1.73
1.94 1.7
1.31 1.48 Gene expression
2.12 1.97
32 23 Not applicable
Not applicable
2.9 20.6
1.6 0.7
3.79 29.1
45 54
3.1 22.6
1.83 2
3.88 31.8
38 23
2.7 72.1
1.6 0.8
4.47 91
51 33
3.85 18.6
1.76 0.7
4.69 30.8
43 42
2 14.8
2.25 1.5
2.25 22.7
22 65
35 84
3.5 1.2
90 116
36 41
* [(BCG ⫹ antibody/BCG)] ⫻ 100.
cells, presumably those undergoing necrotic cell death, alters gene expression in adjacent viable cells. While these findings do not exclude a systemic effect, they suggest that the linkage between HMGB1 release and an in vivo antitumor response to BCG is in part due to the local/paracrine consequences of endogenous HMGB1. In conclusion, HMGB1 released by UC cells in response to BCG
exposure functions as a paracrine factor to potentiate the direct effects of BCG on UC biology.
ACKNOWLEDGMENTS Dr. Richard Williams, University of Iowa, provided the 253J cell line.
REFERENCES 1. See WA, Zhang G, Chen F et al: Bacille-Calmette Guerin induces caspase-independent cell death in urothelial carcinoma cells together with release of the necrosis-associated chemokine high molecular group box protein 1. BJU Int 2009; 103: 1714. 2. Zhang G, Chen F, Cao Y et al: HMGB1 release by urothelial carcinoma cells is required for the in vivo anti-tumor response to BCG. J Urol 2013; 189: 1541. 3. Ellerman JE, Brown CK and de Vera M: Masquerader: high mobility group box-1 and cancer. Clin Can Res 2007; 13: 2836.
4. Andersson U, Wang H and Palmblad K: High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 2000; 192: 565. 5. Treutiger CJ and Mullins GE: High mobility group 1 B-box mediates activation of human endothelium. J Immunol Methods 2003; 254: 375. 6. Chen F, Zhang G, Cao Y et al: MB49 murine urothelial carcinoma: molecular and phenotypic comparison to human cell lines as a model of the direct tumor response to bacillus Calmette-Guerin. J Urol 2009; 182: 2932.
7. Mossman T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55. 8. Rojas A, Figueroa H and Morales E: Fueling inflammation at tumor microenvironment: the role of multiligand/RAGE axis. Carcinogenesis 2010; 31: 334. 9. Sims GP, Rowe DC, Rietdijk ST et al: HMGB1 and RAGE in inflammation and cancer. Annu Rev Immunol 2010; 28: 367.
Abbreviations and Acronyms BCG ⫽ bacillus Calmette-Guérin CCL20 ⫽ chemokine (C-C motif) ligand 20 CD54 ⫽ intercellular adhesion molecule 1 CXCL ⫽ chemokine (C-X-C motif) ligand HMGB1 ⫽ high molecular group box protein 1 IL ⫽ interleukin NFB ⫽ nuclear factor-B NRF2 ⫽ nuclear factor (erythroidderived 2)-like 2 qrtPCR ⫽ quantitative rtPCR RAGE ⫽ receptor for advanced glycation end products rtPCR ⫽ reverse transcriptasepolymerase chain reaction UC ⫽ urothelial carcinoma Accepted for publication January 16, 2013. Supported by a grant from the Department of Veterans Affairs and the Milwaukee Veterans Affairs Medical Center. * Equal study contribution. † Correspondence: Department of Urology, Medical College of Wisconsin, 9200 West Wisconsin Ave., Milwaukee, Wisconsin 53226 (telephone: 414-805-0787; FAX: 414-805-0771; e-mail: [email protected]).
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Purpose: Prior study demonstrated that HMGB1 release by urothelial carcinoma cells in response to bacillus Calmette-Guérin is required for an in vivo antitumor effect. We evaluated the direct effects of HMGB1 on the in vitro response of urothelial carcinoma cells to bacillus Calmette-Guérin. Materials and Methods: Two human urothelial carcinoma cell lines were used to study the effect of exogenous HMGB1 alone and combined with bacillus Calmette-Guérin on the tumor cell response to bacillus Calmette-Guérin. Antibody mediated blockade of receptors for HMGB1 or HMGB1 protein was used to determine the contribution of paracrine HMGB1 release to bacillus CalmetteGuérin biological effects. Response end points evaluated included the activation of intracellular signaling pathways, gene transactivation and cytotoxicity. Results: Urothelial carcinoma cells expressed the receptor for HMGB1 signaling. Antibody blockade of the RAGE receptor confirmed the dependence of signaling in response to HMGB1 on RAGE function. Exogenous HMGB1 activated cell signaling pathways for NFB, NRF2 and CEBP. Quantitative reverse transcriptase-polymerase chain reaction on a panel of bacillus Calmette-Guérin responsive genes revealed peak expression resulting from the combination of bacillus Calmette-Guérin and HMGB1. Blockade of paracrine HMGB1 released in response to bacillus Calmette-Guérin using HMGB1 and/or RAGE receptor blocking antibodies showed a significant decrease in gene expression relative to that of bacillus Calmette-Guérin alone. HMGB1 potentiated the cytotoxic effects of bacillus Calmette-Guérin. Conclusions: HMGB1 released by urothelial carcinoma cells after bacillus Calmette-Guérin treatment functions as a paracrine factor to potentiate the urothelial carcinoma cell response to bacillus Calmette-Guérin. This paracrine activity likely contributes to the dependence of an in vivo tumor response on HMGB1 release. Key Words: urinary bladder, carcinoma, BCG vaccine, paracrine communication, HMGB1 protein INTRAVESICAL administration of attenuated Mycobacterium BCG as treatment for nonmuscle invasive bladder cancer elicits a complex response in the tumor and host. Recent studies demonstrated that HMGB1, a potent
chemokine and marker of nonapoptotic cell death, is released by a portion of UC tumor cells after BCG exposure.1 Increased HMGB1 levels were observed in the urine of patients after intravesical BCG therapy. Of
0022-5347/13/1903-1076/0 THE JOURNAL OF UROLOGY® © 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
http://dx.doi.org/10.1016/j.juro.2013.01.050 Vol. 190, 1076-1082, September 2013 RESEARCH, INC. Printed in U.S.A.
AND
HMGB1 FUNCTIONS AS PARACRINE FACTOR TO POTENTIATE BACILLUS CALMETTE-GUÉRIN
particular interest is that in vivo animal models showed that tumor cells engineered to have decreased HMGB1 release fail to respond to intravesical BCG.2 HMGB1 released by tumor cells after BCG treatment has the potential to influence the antitumor response at the level of the tumor and the host. In other systems HMGB1 signals through specific cell surface receptors to alter gene expression and the tumor phenotype. Paracrine release of HMGB1 in concert with the expression of tumor cell surface receptors for HMGB1 translates into local effects.3 As a powerful chemokine, HMGB1 can function to direct a host cellular immune response to the site of its release. Systemically, HMGB1 activates macrophages, induces neutrophil chemotaxis and alters vascular endothelial cell biology.4,5 To our knowledge it is unknown which, if either, of these mechanisms contributes to the HMGB1 dependent in vivo tumor response. Given the apparent importance of HMGB1 release for in vivo antitumor activity and the potential for it to exert a direct effect on tumor biology, we examined the effects of exogenous and endogenous HMGB1 on the in vitro biology of UC cells. Our results demonstrate that UC cells express functional receptors for HMGB1. Exogenous HMGB1 signals through these receptors to alter intracellular signaling, gene expression and cytotoxicity. Importantly, endogenous HMGB1 release in response to BCG acts as a paracrine factor to potentiate direct BCG effects on tumor biology. While not eliminating the possibility for a systemic effect, these findings support a direct effect of HMGB1 on tumor biology as a potential contributor to the antitumor response to BCG.
METHODS Cell Lines and BCG We used the human UC T24 (ATCC®) and 253J cell lines. Cells were maintained at 37C in 5% CO2 in RPMI 1640 (Gibco®) supplemented with 10% fetal bovine serum, penicillin and streptomycin (complete medium). TICE® BCG was used in these experiments. Freeze-dried BCG was reconstituted in complete medium at an estimated concentration of 4 ⫻ 108 viable organisms per ml.
Luciferase Reporter Assays UC cell exposure to BCG increases the activation of intracellular signaling pathways. We measured the effect of HMGB1 alone or combined with BCG on signaling pathway activation. In 24-well plates 253J and T24 cells were plated at 1 ⫻ 105 cells per well. At 24 hours the cells were transiently transfected with previously described nuclear factor-B, CEBP and NRF2 plasmid reporter constructs using Lipofectamine™ 2000 according to manufacturer instructions.6 Duplicate wells were set up for each group.
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At 24 hours after transfection, cells were left untreated or were treated with BCG (50:1 BCG to cells) and/or HMGB1. Six hours later the cells were washed with phosphate buffered saline and lysed with 1X Reporter Lysis Buffer (Promega, Madison, Wisconsin). Luciferase activity was measured using a Luciferase Assay System (Promega) according to manufacturer instructions. Luciferase activity was normalized to the protein concentration, as measured by the BCA™ Protein Assay Kit.
qrtPCR Cell and Proliferation Prior study demonstrated that UC cell exposure to BCG increases the expression of cell cycle regulatory and immune response genes. We measured the effect of HMGB1 alone and combined with BCG on gene expression. qrtPCR was done using the LightCycler® 480 Real-Time PCR System. On comparative analysis treatment groups included control cells and cells treated with BCG (50:1 BCG to cells) and/or HMGB1. The primer sequence was IL-6, 5=AGCCGCCCCACACAGA (upstream) and 5=-CCGTCGAGGATGTACCGAAT (downstream), IL-8, 5=-CTGGCCGTGGCTCTCTTG (upstream) and 5=-CCTTGGCAAAACTGCACCTT (downstream), CXCLI 5=-CCACTGCGCCCAAACC (upstream) and 5=-GCAGGATTGAGGCAAGCTTT (downstream), CXCL3, 5=-AATGGGAAGAAAGCTTGTCTCAA (upstream) and 5=-CCTTGTTCAGTATCTTTTCGA (downstream), CCL20, 5=-TCCTGGCTGCTTTGATGTCA (upstream) and 5=-AAAGTTGCTTGCTGCTTCTGAT (downstream), and -actin 5=-ACCGAGCGCGGCTACAG (upstream) and 5=-CTTAATGTCACGCACGATTTCC (downstream). Reactions were done using rtPCR Master Mix (Promega) composed of HotStarTaq® DNA polymerase, dinucleoside triphosphate, MgCl2 and ROX™ Dye. It was incubated with primers (0.4 M), probe (0.2 M), QuantiTect™ RT Mix and RNA template. The rtPCR program consisted of 1 cycle at 50C for 30 minutes for reverse transcription and 1 cycle at 95C with a 15-minute hold (hot start), followed by 40 cycles of denaturation at 94C for 15 seconds and annealing/extension at 60C for 60 seconds. Fluorescence data were collected at the end of each extension phase. -actin was used to normalize all other genes tested in the same RNA sample. Cell proliferation was measured using the MTT assay, as previously described.7
Statistical Analysis All experiments were done a minimum of 3 times. Data from individual experiments for assays using luciferase reporter constructs were subject to arithmetic normalization relative to the highest values of the corresponding replicate experiments. Unless otherwise noted, data were analyzed using 2-way ANOVA for repeated measures. Results were considered significant at p ⬍0.05. Graphic representation of the data is shown as the mean ⫹ SE.
RESULTS UC Cells Expressed Functional HMGB1 Receptors rtPCR on UC cell mRNA using RAGE specific primers revealed that the 253J and T24 cell lines expressed RAGE transcripts. Figure 1 shows a blot of the rtPCR reaction. Functional RAGE expression
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HMGB1 FUNCTIONS AS PARACRINE FACTOR TO POTENTIATE BACILLUS CALMETTE-GUÉRIN
sponse to the 2.5 g/ml HMGB1 concentration was increased relative to untreated controls, representing 67% and 87% of the BCG induced response in 253J and T24 cells, respectively. HMGB1 combined with BCG demonstrated peak reporter activity at the 2.5 g/ml HMGB1 concentration with reporter activation representing 118% and 126%, respectively, of that observed in response to BCG alone. Adding BCG to HMGB1 significantly increased NRF2 activation relative to that of BCG alone across the range of HMGB1 concentrations. Figure 2 shows Figure 1. UC cell line RAGE expression. Blot of RAGE specific rtPCR products from 253J and T24 cells lines demonstrates size correct amplification product consistent with RAGE mRNA transcripts in each cell line.
was confirmed using RAGE specific blocking antibodies. RAGE blocking antibodies significantly inhibited NFB activation in response to exogenous HMGB1 in each cell line. NFB reporter activation in response to the combination of HMGB1 (5 g/ml) and anti-RAGE antibodies (5 g/ml) represented 32% and 23% of the response observed to HMGB1 alone in 253J and T24 cells, respectively (paired t test p ⬍0.01). HMGB1 Activated Multiple UC Cell Signaling Pathways Treatment of cells with HMGB1 increased the response of a luciferase reporter construct for NFB. Analysis of a dose-response relationship for the 2 cell lines showed a statistically significant relationship between the HMGB1 concentration and reporter activation for cells exposed to HMGB1 alone or HMGB1 combined with BCG (each group p ⬍0.00001). Reporter activation across the range of HMGB1 concentrations was significantly increased by adding BCG in the 253J and T24 cell lines (p ⬍0.0001 and ⬍0.001, respectively). Activation of a CEBP reporter construct by UC cells showed a significant dose-response relationship with HMGB1 concentration in the 2 cell lines (p ⫽ 0.001). Peak reporter activation in response to the 5.0 g/ml HMGB1 concentration represented 91% and 83% of the BCG induced response in 253J and T24 cells, respectively. CEBP reporter activation by the HMGB1 and BCG combination did not significantly correlate with the HMGB1 concentration in the 2 cell lines (p ⫽ 0.06). Reporter activation across the range of HMGB1 concentrations was significantly increased by adding BCG in the 253J and T24 cell lines (each p ⬍0.001). Activation of an NRF2 reporter construct by UC cells correlated with the HMGB1 concentration but failed to attain the level of statistical significance of HMGB1 alone or combined with BCG (p ⫽ 0.08 and 0.8, respectively). Peak reporter activation in re-
Figure 2. Mean ⫾ SE signaling pathway activation in response to HMGB1 and BCG alone or combined. Luciferase reporter constructs for NFB (A), CEBP (B) and NRF2 (C) were activated in response to BCG, HMGB1 or combination. NFB activation showed statistically significant dose-response relationship with HMGB1 alone and combined with BCG in each cell line. CEBP activation significantly correlated with HMGB1 concentration. For CEBP adding BCG to HMGB1 failed to show significant dose-response relationship with HMGB1. NRF2 activation did not significantly correlate with HMGB1 concentration for HMGB1 alone or combined with BCG. Activation of all reporters was significantly increased by BCG and HMGB1 combination relative to HMGB1 alone.
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Figure 3. Mean ⫾ SE gene expression in response to HMGB1 and/or BCG. UC cell exposure to exogenous HMGB1 resulted in significant dose dependent increase in expression of panel of 6 BCG responsive immune regulatory genes in 253J cell line (p ⬍0.001) (A). There was no significant relationship between HMGB1 concentration and gene expression in T24 cell line (p ⫽ 0.18) (B). In 253J and T24 cell lines BCG and HMGB1 combination significantly increased gene expression in HMGB1 dose dependent manner (p ⬍0.01 and ⬍0.05, respectively).
a graph of reporter activation (NFB, CEBP and NRF2) in response to HMGB1 alone or combined with BCG. HMGB1 Induced Gene Transcription HMGB1 showed a cell line dependent effect on the expression of a panel of 6 BCG activated immune response genes, as measured by qrtPCR. In the T24 cell line the expression of IL-6 and 8, CXCL1 and 3, CCL20 and CD54 failed to increase in response to HMGB1 and manifested no dose-response relationship (p ⫽ 0.18). In contrast, gene expression in the 253J cell line increased in response to HMGB1 and significantly correlated with the HMGB1 concentration (p ⬍0.001). HMGB1 combined with BCG significantly increased the expression of the 6 target genes in each cell line relative to that of BCG alone. Gene expres-
sion in response to the BCG and HMGB1 combination significantly correlated with the HMGB1 concentration in the 253J and T24 cell lines (p ⬍0.01 and p ⬍0.05, respectively). Figure 3 shows gene expression data on each cell line in response to HMGB1 alone or combined with BCG. Endogenous HMGB1 Released by UC Cells in Response to BCG Functioned as Paracrine Factor In this experiment we evaluated the role of HMGB1 release by UC cells in response to BCG treatment on direct BCG biological effects on gene expression. Blockade of HMGB1 signaling through a combination of RAGE receptor blocking and HMGB1 neutralizing antibodies significantly decreased gene expression in response to BCG alone in the 253J and T24 cell lines (p ⬍0.05 and ⬍0.001, respectively, fig. 4).
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HMGB1 FUNCTIONS AS PARACRINE FACTOR TO POTENTIATE BACILLUS CALMETTE-GUÉRIN
Figure 4. Mean ⫾ SE paracrine HMGB1 contributed to UC cell gene expression in response to BCG. HMGB1 signaling blockade using RAGE receptor blocking and/or HMGB1 neutralizing antibodies inhibited BCG activated gene expression. Receptor blockade and HMGB1 neutralization combination significantly decreased expression of 6 BCG responsive genes in 253J (A) and T24 (B) cells (p ⬍0.05 and ⬍0.001, respectively).
HMGB1 Potentiated BCG Cytotoxicity UC cell exposure to BCG resulted in a direct cytotoxic effect. In this experiment we evaluated the effects of BCG and HMGB1 alone and in combination on cellular cytotoxicity. Metabolically active cell number as a function of time after treatment was measured using the MTT assay. Relative to untreated controls, BCG and HMGB1 significantly decreased the number of metabolically active 253J and T24 cells with time (BCG p ⬍0.001 and ⬍0.0001, respectively, and HMGB1 each p ⬍0.00001). Relative to BCG alone, exposure of cells to the BCG and HMGB1 combination resulted in a significant decrease in the number of metabolically active 253J and T24 cells (p ⬍0.001 and ⬍0.05, respectively, fig. 5).
DISCUSSION A growing body of evidence points to the interaction between the UC cell and BCG as having a central role in orchestrating the host immune response that culminates in an antitumor effect. BCG binding to the UC cell surface, followed by internalization,
stimulates a cellular response that is characterized by the expression of white blood cell receptors (CD54), and the activation of genes coding for chemokines (CXCL1 and 3, and CCL20) and cytokines (IL-6 and 8). In some cells BCG exposure results in necrotic cell death and release of the chemokine HMGB1 into the pericellular space. The resultant immune modulatory/inflammatory soup likely stimulates a host response targeting the site of release. While a specific role for many of these factors remains unproven, HMGB1 animal model data revealed that the release of this chemokine in response to BCG treatment is needed for an in vivo tumor response.1 BCG induced release of HMGB1 into the pericellular environment has the potential to act systemically and locally. Systemic responses may occur as the result of the recruitment and maturation of immune effector cell populations.8 Tumor expression of HMGB1 receptors could result in local responses, such as a direct cytotoxic effect on remaining tumor cells. Local effects could also include indirect pathways, such as HMGB1 stimulated, receptor medi-
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Figure 5. Mean ⫾ SE HMGB1 potentiated BCG cytotoxicity. UC cell exposure to BCG or HMGB1 significantly decreased number of metabolically active cells as function of time after treatment in 253J cells (p ⬍0.001 and ⬍0.0001, respectively) (A) and T24 cells (each p ⬍0.00001) (B). Relative to BCG alone, BCG and HMGB1 combination resulted in significant further decrease in number of metabolically active cells with time in 253J and T24 cells (p ⬍0.001 and ⬍0.05, respectively).
ated (paracrine) expression of immune modulatory genes by UC cells, which in turn potentiate the host immune reaction. Given the apparent importance of HMGB1, we evaluated the impact of HMGB1 on UC cell biology in the context of BCG treatment. The results of this study reveal that human UC cell lines express the principal receptor for HMGB1. RAGE is widely recognized as an important cell surface protein for HMGB1 mediated signaling.9 Antibody blocking studies targeting RAGE demonstrated the functionality of the HMGB1/RAGE axis in UC cells and its role in activating the NFB signaling pathway. UC cell exposure to exogenous HMGB1 also activated other known BCG responsive signaling pathways. CEBP and NRF2 pathways were activated to varying degrees in response to HMGB1. Not surprisingly, the transactivation of genes downstream of these signaling pathways in-
creased in response to HMGB1. In the context of modest increases in intracellular signaling in response to HMGB1 alone, the extent to which HMGB1 exerted a synergistic effect on gene expression in combination with BCG was striking. The table shows the effects of HMGB1 alone or combined with BCG on signaling pathway activation and gene expression. In the clinical setting HMGB1 release occurs in conjunction with BCG treatment. As a consequence, the simultaneous presence of the bacteria and the chemokine has the potential to function as a paracrine factor to alter the biology of adjacent UC cells. While it is interesting to note the influence of exogenous HMGB1, the most important finding in the current study is the role of endogenous HMGB1 in mediating the direct effects of BCG on UC cells. The data reveal that HMGB1 release by BCG exposed
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HMGB1 FUNCTIONS AS PARACRINE FACTOR TO POTENTIATE BACILLUS CALMETTE-GUÉRIN
Table. T24 and 253J cell signaling pathway activation and gene expression after exposure to BCG and/or 5 g/ml HMGB1, and activation or expression in response to HMGB1 vs HMGB1 combined with blocking antibodies Fold Induction vs Controls BCG
BCG ⫹ HMGB1
HMGB1
% Response*
Signaling pathways NFB: 253J T24 CEBP: 253J T24 NRF2: 253J T24 CD54: 253J T24 IL-6: 253J T24 IL-8: 253J T24 CXCL1: 253J T24 CXCL3: 253J T24 CCL20: 253J T24
2.13 1.98
1.82 1.51
2.68 2.17
1.98 1.87
1.81 1.55
1.94 1.73
1.94 1.7
1.31 1.48 Gene expression
2.12 1.97
32 23 Not applicable
Not applicable
2.9 20.6
1.6 0.7
3.79 29.1
45 54
3.1 22.6
1.83 2
3.88 31.8
38 23
2.7 72.1
1.6 0.8
4.47 91
51 33
3.85 18.6
1.76 0.7
4.69 30.8
43 42
2 14.8
2.25 1.5
2.25 22.7
22 65
35 84
3.5 1.2
90 116
36 41
* [(BCG ⫹ antibody/BCG)] ⫻ 100.
cells, presumably those undergoing necrotic cell death, alters gene expression in adjacent viable cells. While these findings do not exclude a systemic effect, they suggest that the linkage between HMGB1 release and an in vivo antitumor response to BCG is in part due to the local/paracrine consequences of endogenous HMGB1. In conclusion, HMGB1 released by UC cells in response to BCG
exposure functions as a paracrine factor to potentiate the direct effects of BCG on UC biology.
ACKNOWLEDGMENTS Dr. Richard Williams, University of Iowa, provided the 253J cell line.
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4. Andersson U, Wang H and Palmblad K: High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 2000; 192: 565. 5. Treutiger CJ and Mullins GE: High mobility group 1 B-box mediates activation of human endothelium. J Immunol Methods 2003; 254: 375. 6. Chen F, Zhang G, Cao Y et al: MB49 murine urothelial carcinoma: molecular and phenotypic comparison to human cell lines as a model of the direct tumor response to bacillus Calmette-Guerin. J Urol 2009; 182: 2932.
7. Mossman T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55. 8. Rojas A, Figueroa H and Morales E: Fueling inflammation at tumor microenvironment: the role of multiligand/RAGE axis. Carcinogenesis 2010; 31: 334. 9. Sims GP, Rowe DC, Rietdijk ST et al: HMGB1 and RAGE in inflammation and cancer. Annu Rev Immunol 2010; 28: 367.