Cisplatin Dependent Secretion of Immunomodulatory High Mobility Group Box 1 (HMGB1) Protein from Lung Cancer Cells

Abstract

High mobility group box 1 (HMGB1) is secreted from activated immune cells, necrotic cells, and certain cancers. Previous studies have reported that different patterns of post-translational modification, particularly acetylation and oxidation, mediate HMGB1 release and confer distinct extracellular HMGB1 signaling activity. Here we report that cisplatin but not carboplatin induces secretion of HMGB1 from human A549 non-small cell lung cancer (NSCLC) cells. Cisplatin-mediated HMGB1 secretion was dose-dependent and was regulated by nuclear exportin 1 (XPO1) also known as chromosomal maintenance 1 (CRM1) rather than adenosine diphosphate (ADP)-ribosylation, acetylation, or oxidation. HMGB1, as well as lysine acetylation and cysteine disulfide oxidation of secreted HMGB1, were monitored by sensitive and specific assays using immunoprecipitation, stable isotope dilution, differential alkylation, and nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (nano-LC-PRM/HRMS). A major fraction of the HMGB1 secreted by low-dose cisplatin treatment of A549 NSCLC cells was found to be in the fully reduced form. In contrast, mainly oxidized forms of HMGB1 were secreted by dimethyl sulfoxide (DMSO)-mediated apoptosis. These findings suggest that inhibition of XPO1 could potentiate the anti-tumor activity of cisplatin by increasing the nuclear accumulation of HMGB1 protein, an inhibitor of cisplatin DNA-adduct repair. Furthermore, low-dose cisplatin therapy could modulate the immune response in NSCLC through the established chemokine activity of extracellular reduced HMGB1. This could potentially enhance the efficacy of subsequent immunotherapy treatment.

Keywords: acetylation; chromatography; immune response; mass spectrometry; oxidation; platinum drugs.

Figure 1

Amino Acid Sequence of HMGB1. The 43 lysine residues are labeled in red and the 3 cysteine residues (Cys-23, Cys-45, and Cys-106) are labeled in green. NLS1 consists of residues 28–44 and NLS2 consists of residues 179–185. The acidic tail (residues 186–215) is labeled in brown.

Figure 2

Cisplatin induces HMGB1 secretion from lung cancer cells. Assays were performed following 24 h incubation A549 cells with cisplatin (Cisp, 20 μM), Cisp (100 μM, transplatin (Transp, 20 μM), Transp (100 μM), carboplatin (Carbo, 100 μM) and Carbo (300 μM). (A) Representative HMGB1 immunoblots of biological replicates (n = 3) comparing A549 cell culture media to media from untreated malignant mesothelioma cell lines REN and EMMESO. An anti-HMGB1 rabbit pAb against the C-terminal acidic tail of HMGB1 was used. (B) absolute quantification of secreted HMGB1 by nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry. Significant differences compared to PBS were determined using unpaired Student’s t-tests. Error bars show ± SD.

Scheme 1

Procedure for preparation and analysis of acetylation in secreted HMGB1. A549 cell culture media was collected and spiked with K*Y*-stable isotope labeling by amino acids in cell culture (SILAC) high mobility group box 1 (HMGB1) internal standard. HMGB1 proteoforms were isolated by overnight IP and eluted with glycine-HCl before CD₃-acetylation (Ac*) with D6-acetic anhydride in ammonium bicarbonate/acetonitrile buffer. Samples were subjected to either Glu-C, chymotrypsin, or Asp-N protease digests, and acetylated peptides were analyzed by nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry with acetylation status determined by differences in m/z.

Figure 3

Cisplatin-induced secretion of unacetylated HMGB1. Lysine-containing peptides from cisplatin-treated A549 media were analyzed for acetylation by nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (nanoLC-PRM/HRMS). Chromatograms show light (black) and heavy (red) transitions with CD₃ (*) or endogenous CH₃-acetylation. (A) NanoLC-PRM/HRMS chromatograms for NLS1 peptide HK²⁸K²⁹K³⁰HPDASVNFSE comparing b11 transitions for triply (first panel), doubly (second panel), and singly (third panel) acetylated NLS1 compared to unacetylated NLS1 peptide (fourth panel) and heavy NLS1 peptide (fifth panel). (B) NanoLC-PRM/HRMS chromatograms for NLS2 peptide K¹⁸⁰SK¹⁸¹K¹⁸²K¹⁸³K¹⁸⁴ comparing y8 transitions of different possible acetylation states. (C) NanoLC-PRM/HRMS chromatograms for N-terminal peptide GK³GDPK⁷K⁸PRGK¹²M(O)SSY comparing b4 transitions of different possible acetylation states.

Scheme 2

Procedure for preparation and redox analysis of secreted HMGB1. A549 cell culture media samples are incubated with iodoacetamide (IAA) to carbamidomethylate free (reduced) Cys residues. tris(2-carboxyethyl)-phosphine (TCEP)-reduced (SILAC) HMGB1 is added both before and after the reaction to act as an internal standard for both free (reduced) and disulfide (oxidized) HMGB1. HMGB1 is immunoprecipitated, eluted, and reduced with TCEP prior to N-ethylmaleimide derivatization. Tryptic peptides for PRM analysis are prepared by overnight trypsin digestion, whereas Glu-C peptides are prepared by overnight Glu-C digestion following in-solution CD₃-acetylation of the lysine residues with D6-acetic anhydride.

Figure 4

Cisplatin mediated the release of reduced and oxidized HMGB1 from lung cancer cells. (A) absolute quantification of reduced HMGB1 released by A549 cells (n = 3) determined by nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (nanoLC-PRM/HRMS) analysis. An unpaired Student’s t-test was used to compare cisplatin with PBS. (B) Site-specific cysteine (Cys) oxidation percentages calculated from nanoLC-PRM/HRMS analysis of carbamidomethyl (CAM)- and N-ethyl maleimide (NEM)-modified peptides in HMGB1 secreted from A549 cells (n = 3). (a) Cys-23. (b) Cys-45 (c) Cys-106. An unpaired Student’s t-test was used to compare cisplatin with DMSO. Error bars show ± SD.

Figure 5

Nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry analysis of Cys-containing peptides from cisplatin-treated A549 media. Chromatograms show corresponding light (black) and heavy (red) transitions. Carbamidomethyl (CAM)-modified transitions indicate free (reduced) Cys proteoforms and N-ethyl maleimide (NEM)-modified transitions indicate disulfide (oxidized) Cys proteoforms. (A) tryptic peptide M(O)SSYAFFQTC²³R with CAM-modified transitions (left) and NEM-modified transitions (right). (B) Glu-C peptide FSK(Ac*)K(Ac*)C⁴⁵SE with CAM-modified transitions (left) and NEM-modified transitions (right). (C) tryptic peptide RPPSAFFLFC¹⁰⁶SEYRPK with CAM-modified transitions (left) and NEM-modified transitions (right).

Figure 6

Cisplatin-mediated high mobility group box 1 (HMGB1) secretion is regulated by nuclear exportin 1 (XPO1). Representative anti-HMGB1 immunoblots from biological replicates (n = 3) of A549 cell culture media using anti-HMGB1 rabbit pAb against the C-terminal acidic tail of HMGB1. (A) Olaparib (50 µM) (B) GO-6983 (120 nM), and (C) Rottlerin (100 µM) treatments showed no effect on cisplatin (50 µM)-mediated HMGB1 secretion from A549 cells. Conversely, XPO1 (chromosomal maintenance 1; CRM1) inhibitors (D) leptomycin B (LMB) (4.5 nM) and (E) KPT-330 (75 nM) significantly attenuated cisplatin-mediated HMGB1 secretion. Normalized immunoblot quantification of HMGB1 in A549 cell culture media comparing effects of XPO1 inhibitors on cisplatin-mediated HMGB1 secretion. (F) LMB (4.5 nM). (G) KPT-330 (75 nM). Significant differences were determined using an unpaired Student’s t-test). Error bars show ± SD.

Figure 7

Increased cisplatin-mediated nuclear HMGB1 accumulation and decreased secretion after inhibition of nuclear transporter 1 (XPO1). HMGB1 levels were quantified in A549 cells by nano liquid chromatography-parallel reaction monitoring/high resolution/mass spectrometry in A549 subcellular fractions and A549 cell media after treatment with 50 µM cisplatin and a combination of 50 µM cisplatin with 4.5 nM leptomycin B (LMB). (A). Nuclear fraction. (B) Cytosolic fraction. (C) Cell media. Values are mean ± standard deviation. Statistical analysis was conducted using an unpaired Student’s t-test.

Figure 8

Cisplatin and leptomycin B (LMB) attenuate A549 cell growth and increase A549 cell death. Trypan Blue exclusion assay on biological replicates (n = 5) after cisplatin (50 µM), LMB (16 nM) and a combination of cisplatin (50 µM) and LMB (16 nM) treatment after 24-h. (A) Cell count after 24 h (upper) and 48 h (lower). (B) Cell viability (%) after 24 h and 48 h. (C) Dead cell count after 24 h and 48 h.

Figure 9

Cisplatin-mediated high mobility group box (HMGB1) secretion is regulated by nuclear exportin 1 (XPO1). HMGB1 contains two reported nuclear localization sequences (NLS1 and NLS2) and a potential nuclear export sequence (NES). Upon treatment of lung cancer cells with cisplatin, XPO1 (also known as chromosomal maintenance 1; CRM1) facilitates the nuclear export of unacetylated HMGB1 through the nuclear pore complexes (NPCs) into the cytosol (indicated by arrows). This translocation allows HMGB1 to be subsequently secreted into the extracellular space. Inhibitors of XPO1 such as leptomycin B (LMB) inhibit the binding of XPO1 to HMGB1, thereby preventing XPO1-regulated secretion of HMGB1 in the presence of cisplatin (indicated by cross signs on arrows). Figure created in Biorender.