HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress.

High mobility group box-1 (HMGB1) is involved in various diseases and is associated with the resistance of many types of human cancers to chemotherapy; however, its role in cancer metastasis remains unexplored. This study examined the HMGB1 status of both highly and poorly metastatic cancer cells in response to genotoxic stress. The weakly and highly metastatic mouse melanoma cell lines (B16 vs. B16-F10), human melanoma cell lines (SK-MEL-28 vs. SK-MEL-24), colon cancer cell lines (DLD-1 vs. LS174T), and wild-type (WT) vs. HMGB1 knockout (KO) mouse embryonic fibroblasts (MEFs) were treated with doxorubicin (Dox) and camptothecin (CPT), and then cellular morphology, senescence-associated ß-galactosidase staining, lactate dehydrogenase release, and caspase-3 activation were used to assess cell fate. To investigate the role of HMGB1 in p21 expression, HMGB1 and p21 expressions were examined by Western blotting, and the HMGB1-mediated p21 promoter luciferase assay was performed after small interfering RNA or overexpression of HMGB1 prior to Dox treatment. Although highly metastatic mouse melanoma B16-F10 cells preferred senescence, with persistent HMGB1 expression, poorly metastatic B16 cells entered apoptosis, with decreasing HMGB1 levels via cleavage under Dox treatment. Similarly, more metastatic human melanoma SK-MEL-24 and human colon cancer LS174T cells underwent senescence, whereas fewer metastatic melanoma SK-MEL-28 and DLD-1 cells exhibited apoptosis under Dox stimulation. In senescent B16-F10, SK-MEL-24, and LS174T cells treated with Dox, p21 levels were increased by persistent HMGB1 expression. Furthermore, HMGB1 depletion caused a senescence-apoptosis shift with p21 down-regulation in B16-F10 cells, and HMGB1 overexpression switched from apoptosis to senescence concomitantly with increased p21 expression in B16 cells after Dox treatment. The same effects were observed in both cell pairs of mouse melanoma and human colon cancer cells treated with CPT, another genotoxic stressor. Indeed, although WT MEF entered senescence accompanied by p21 increase, HMGB1 KO underwent apoptosis with p21 decrease by Dox treatment. In our cell model system, we demonstrated that highly metastatic cancer cells preferentially enter senescence, whereas apoptosis predominates in weakly metastatic cancer cells under genotoxic stress, which depends on the presence or absence of HMGB1, suggesting that the HMGB1-p21 axis is required for genotoxic stress-induced senescence. These findings suggest that HMGB1 modulation of cancers with different metastatic status could be a strategy for selectively enforcing tumor suppression.-Lee, J.-J., Park, I. H., Rhee, W. J., Kim, H. S., Shin, J.-S. HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress.

Risk stratification for locally advanced hepatocellular carcinoma using pretreatment alpha-foetoprotein and 18 F-fluoro-2-deoxyglucose positron emission tomography.

BACKGROUND & AIMS: We investigated the significance of 18 F-fluoro-2-deoxyglucose positron emission tomography (18 F-FDG PET) parameters and alpha-foetoprotein (AFP) levels in patients with locally advanced hepatocellular carcinoma (LA-HCC). METHODS: We retrospectively analysed data of 228 patients with LA-HCC who underwent pretreatment 18 F-FDG PET between January 2003 and December 2013. All patients were treated using liver-directed therapy involving radiotherapy. The maximum standardized uptake values (SUVs) and tumour-to-extratumoural liver SUV ratios were calculated, and pretreatment AFP values were obtained. RESULTS: Patients were divided into high and low maximum SUV (SUVmax) groups according to a SUV cut-off of 4.825 determined via receiver-operating characteristic analysis. High AFP level (>550 ng/mL) and high SUVmax were significant predictors of overall and progression-free survival. Better treatment responses and longer median progression-free and overall survival were observed in the low SUVmax group, compared to the high SUVmax group. Similar results were obtained for SUV ratio-based (cut-off value: 2.355) and AFP-based analyses (cut-off value: 550 ng/mL). Three risk groups were identified using the double biomarkers of SUVmax and AFP value as strong prognosticators predictive of survival outcomes. This risk stratification was identified as a prognosticator of survival outcomes, even after subgroup analyses. Furthermore, in high risk group, significantly high extrahepatic failure was shown while in low risk group, significantly low intrahepatic failure. CONCLUSIONS: Clinical significance of double biomarkers, SUV and AFP, could be translated into risk stratification for LA-HCC. It could be a valuable tool for survival outcome prediction.

Canagliflozin protects against cisplatin-induced acute kidney injury by AMPK-mediated autophagy in renal proximal tubular cells.

Sodium-glucose cotransporter 2 inhibitors, which are recently introduced as glucose-lowering agents, improve cardiovascular and renal outcomes in patients with diabetes mellitus. These drugs also have beneficial effects in various kidney disease models. However, the effect of SGLT2 inhibitors on cisplatin-induced acute kidney injury (AKI) and their mechanism of action need to be elucidated. In this study, we investigated whether canagliflozin protects against cisplatin-induced AKI, depending on adenosine monophosphate-activated protein kinase (AMPK) activation and following induction of autophagy. In the experiments using the HK-2 cell line, cell viability assay and molecular analysis revealed that canagliflozin protected renal proximal tubular cells from cisplatin, whereas addition of chloroquine or compound C abolished the protective effect of canagliflozin. In the mouse model of cisplatin-induced AKI, canagliflozin protected mice from cisplatin-induced AKI. However, treatment with chloroquine or compound C in addition to administration of cisplatin and canagliflozin eliminated the protective effect of canagliflozin. Collectively, these findings indicate that canagliflozin protects against cisplatin-induced AKI by activating AMPK and autophagy in renal proximal tubular cells.

Reactive oxygen species induce Cys106-mediated anti-parallel HMGB1 dimerization that protects against DNA damage.

Oxidative stress can induce covalent disulfide bond formation between protein-protein thiol groups and generate hydroxyl free radicals that damage DNA. HMGB1 is a DNA chaperone and damage-associated molecular pattern molecule. As a redox-sensitive protein, HMGB1 contains three cysteine residues: Cys23, Cys45, and Cys106. In this study, we focused on the relationship between HMGB1 dimerization and DNA stabilization under oxidative stress conditions. HMGB1 dimerization was positively modulated by CuCl2 and H2O2. Mutation of the Cys106 residue blocked dimer formation. Treatment of HEK293T cells with CuCl2 and H2O2 enhanced the oxidative self-dimerization of HMGB1, whereas this dimerization was inhibited in mutant HMGB1C106A cells. Furthermore, we performed a bimolecular fluorescence complementation assay to visualize Cys106 oxidation-induced HMGB1 dimerization in live cells exposed to oxidative stress and were able to reproduce the dimerization effect of HMGB1 in fluorescence resonance energy transfer analysis. Interestingly, dimerized HMGB1 bound to DNA with higher affinity than monomeric HMGB1. Dimerized HMGB1 protected DNA from damage due to hydroxyl free radicals and prevented cell death. In conclusion, dimerized HMGB1 may play a regulatory role in DNA stabilization under oxidative stress.

Plasmon-enhanced fluorescence correlation spectroscopy for super-localized detection of nanoscale subcellular dynamics.

In this report, we investigate plasmon-enhanced imaging fluorescence correlation spectroscopy (p-FCS). p-FCS takes advantage of extreme light confinement by localization at nanogap-based plasmonic nanodimer arrays (PNAs) for enhanced signal-to-noise ratio (SNR) and improved precision by registration with surface plasmon microscopy images. Theoretical results corroborate the enhancement by PNAs in the far-field. Near-field scanning optical microscopy was used to confirm near-field localization experimentally. Experimental confirmation was also conducted with fluorescent nanobeads. The concept was further applied to studying the diffusion dynamics of lysosomes in HEK293T cells stimulated by phorbol 12-myristate 13-acetate treatment. It was found that lysosomes demonstrate stronger super-diffusive behavior with relatively weaker sub-diffusion after stimulation. SNR measured of p-FCS was improved by 9.77 times over conventional FCS. This report is expected to serve as the foundation for an enhanced analytical tool to explore subcellular dynamics.

HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

Although cellular senescence has emerged as a novel therapeutic concept in cancer, its underlying mechanisms remain unclear. High mobility group box 1 (HMGB1) and stimulator of interferon genes (STING) are involved in senescence. However, their interactions in senescence have not been reported. Therefore, in this study, we investigated the relationships between HMGB1 and STING in senescence in cancer and other cells. In mouse melanoma cells and several other cell lines, doxorubicin treatment induced senescence in an HMGB1-dependent manner. These responses were mediated by STING, and this function of STING was negatively regulated by the E3 ligase tripartite motif protein 30α (TRIM30α). We also found that HMGB1 bound to the TRIM30α promoter and then suppressed its expression by inhibiting its transcription, which enhanced STING-induced senescence. This mechanism was further mediated by signal transducer and activator of transcription 6 (STAT6) and p21. Overall, our findings demonstrated that HMGB1 orchestrated STING-STAT6-p21-mediated senescence by regulating TRIM30α as an alternative anticancer mechanism.

Peroxiredoxin-mediated disulfide bond formation is required for nucleocytoplasmic translocation and secretion of HMGB1 in response to inflammatory stimuli.

The nuclear protein HMGB1 (high mobility group box 1) is secreted by monocytes-macrophages in response to inflammatory stimuli and serves as a danger-associated molecular pattern. Acetylation and phosphorylation of HMGB1 are implicated in the regulation of its nucleocytoplasmic translocation for secretion, although inflammatory stimuli are known to induce H2O2 production. Here we show that H2O2-induced oxidation of HMGB1, which results in the formation of an intramolecular disulfide bond between Cys23 and Cys45, is necessary and sufficient for its nucleocytoplasmic translocation and secretion. The oxidation is catalyzed by peroxiredoxin I (PrxI) and PrxII, which are first oxidized by H2O2 and then transfer their disulfide oxidation state to HMGB1. The disulfide form of HMGB1 showed higher affinity for nuclear exportin CRM1 compared with the reduced form. Lipopolysaccharide (LPS)-induced HMGB1 secretion was greatly attenuated in macrophages derived from PrxI or PrxII knockout mice, as was the LPS-induced increase in serum HMGB1 levels.

Evaluation of predictive factors of vertebral compression fracture after conventional palliative radiotherapy for spinal metastasis from colorectal cancer.

OBJECTIVE Delayed consequences of spinal radiotherapy (RT), including vertebral compression fracture (VCF), are critical complications. However, the predisposing factors that contribute to VCF after conventional RT are unclear. The aim of this study was to assess the incidence of VCF and to determine the predictors of VCF following conventional spinal RT specific to colorectal cancer (CRC). METHODS The authors retrospectively reviewed 237 spinal segments (147 metastatic and 90 nonmetastatic) in 53 patients with CRC who underwent RT with a median total dose of 30 Gy in 10 fractions between January 2007 and December 2014. The primary end point was the development of a VCF following RT, either de novo VCF or the progression of a baseline VCF. VCFs were assessed using the spinal instability neoplastic score (SINS) criteria. RESULTS Among all 237 spinal segments, 22 VCFs (9.3%) were observed following RT, including 13 de novo and 9 progressive fractures, and the median time to VCF was 4 months. All VCFs developed in metastatic spines. Among 147 metastatic spinal segments, 22 fractures were observed, with a 12-month cumulative incidence of VCF of 14.8%. Results of multivariable analysis indicated sex (p = 0.023) and SINS class II/III (p < 0.001) as risk factors related to development of a VCF in metastatic spinal segments. Among the SINS criteria, a lytic tumor and the presence of a baseline VCF were identified as predictors of VCF in metastatic spinal segments. CONCLUSIONS In osteolytic or mixed lesions that were predominant in spinal metastases of CRC, the incidence of VCF was not negligible, even in patients treated with conventional spinal RT. This was especially evident in patients with spinal metastases with a SINS score ≥ 7. Presence of a baseline VCF after spinal RT is a predictor of VCF development and should be observed carefully.

Treatment outcome of radiation therapy and concurrent targeted molecular therapy in spinal metastasis from renal cell carcinoma.

PURPOSE: To evaluate the clinical outcomes of patients who underwent radiation therapy with or without targeted molecular therapy for the treatment of spinal metastasis from renal cell carcinoma (RCC). MATERIALS AND METHODS: A total of 28 spinal metastatic lesions from RCC patients treated with radiotherapy between June 2009 and June 2015 were retrospectively reviewed. Thirteen lesions were treated concurrently with targeted molecular therapy (concurrent group) and 15 lesions were not (nonconcurrent group). Local control was defined as lack of radiographically evident local progression and neurological deterioration. RESULTS: At a median follow-up of 11 months (range, 2 to 58 months), the 1-year local progression-free rate (LPFR) was 67.0%. The patients with concurrent targeted molecular therapy showed significantly higher LPFR than those without (p = 0.019). After multivariate analysis, use of concurrent targeted molecular therapy showed a tendency towards improved LPFR (hazard ratio, 0.13; 95% confidence interval, 0.01 to 1.16). There was no difference in the incidence of systemic progression between concurrent and nonconcurrent groups. No grade ≥2 toxicities were observed during or after radiotherapy. CONCLUSION: Our study suggests the possibility that concurrent use of targeted molecular therapy during radiotherapy may improve LPFR. Further study with a large population is required to confirm these results.

Clinical outcomes of adjuvant radiation therapy and prognostic factors in early stage uterine cervical cancer.

PURPOSE: To evaluate the outcomes of adjuvant radiotherapy (RT) and to analyze prognostic factors of survival in the International Federation of Gynecology and Obstetrics (FIGO) IB-IIA uterine cervical cancer. MATERIALS AND METHODS: We retrospectively reviewed the medical records of 148 patients with FIGO IB-IIA uterine cervical cancer who underwent surgery followed by adjuvant RT at the Yonsei Cancer Center between June 1997 and December 2011. Adjuvant radiotherapy was delivered to the whole pelvis or an extended field with or without brachytherapy. Among all patients, 57 (38.5%) received adjuvant chemotherapy either concurrently or sequentially. To analyze prognostic factors, we assessed clinicopathologic variables and metabolic parameters measured on preoperative 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT). To evaluate the predictive performance of metabolic parameters, receiver operating characteristic curve analysis was used. Overall survival (OS) and disease-free survival (DFS) were analyzed by the Kaplan-Meier method. RESULTS: The median follow-up period was 63.2 months (range, 2.7 to 206.8 months). Locoregional recurrence alone occurred in 6 patients, while distant metastasis was present in 16 patients, including 2 patients with simultaneous regional failure. The 5-year and 10-year OSs were 87.0% and 85.4%, respectively. The 5-year and 10-year DFSs were 83.8% and 82.5%, respectively. In multivariate analysis, pathologic type and tumor size were shown to be significant prognostic factors associated with both DFS and OS. In subset analysis of 40 patients who underwent preoperative PET/CT, total lesion glycolysis was shown to be the most significant prognostic factor among the clinicopathologic variables and metabolic parameters for DFS. CONCLUSION: Our results demonstrated that adjuvant RT following hysterectomy effectively improves local control. From the subset analysis of preoperative PET/CT, we can consider that metabolic parameters may hold prognostic significance in early uterine cervical cancer patients. More effective systemic treatments might be needed to reduce distant metastasis in these patients.

Vertebral compression fractures after spine irradiation using conventional fractionation in patients with metastatic colorectal cancer.

PURPOSE: To evaluate the risk of vertebral compression fracture (VCF) after conventional radiotherapy (RT) for colorectal cancer (CRC) with spine metastasis and to identify risk factors for VCF in metastatic and non-metastatic irradiated spines. MATERIALS AND METHODS: We retrospectively reviewed 68 spinal segments in 16 patients who received conventional RT between 2009 and 2012. Fracture was defined as a newly developed VCF or progression of an existing fracture. The target volume included all metastatic spinal segments and one additional non-metastatic vertebra adjacent to the tumor-involved spines. RESULTS: The median follow-up was 7.8 months. Among all 68 spinal segments, there were six fracture events (8.8%) including three new VCFs and three fracture progressions. Observed VCF rates in vertebral segments with prior irradiation or pre-existing compression fracture were 30.0% and 75.0% respectively, compared with 5.2% and 4.7% for segments without prior irradiation or pre-existing compression fracture, respectively (both p < 0.05). The 1-year fracture-free probability was 87.8% (95% CI, 78.2-97.4). On multivariate analysis, prior irradiation (HR, 7.30; 95% CI, 1.31-40.86) and pre-existing compression fracture (HR, 18.45; 95% CI, 3.42-99.52) were independent risk factors for VCF. CONCLUSION: The incidence of VCF following conventional RT to the spine is not particularly high, regardless of metastatic tumor involvement. Spines that received irradiation and/or have pre-existing compression fracture before RT have an increased risk of VCF and require close observation.

The location of locoregional recurrence in pathologic T3N0, non-irradiated lower rectal cancer.

PURPOSE: To investigate the patterns of locoregional recurrence of pathologic T3N0 (pT3N0) lower rectal cancer omitting postoperative radiotherapy (RT) and explore the potential of modification of a RT field. MATERIALS AND METHODS: From Jan 2003 to Nov 2011, 35 patients omitting preoperative or postoperative RT for pT3N0 lower rectal cancer were included. We defined the lower rectal cancer as the tumor with the inferior margin located below the virtual line-a convergent level between rectal wall and levator ani muscle. All patients had radiologic examinations for recurrence evaluation during the follow-up duration. RESULTS: The median follow-up duration was 66.4 months (range, 1.4 to 126.1 months). Eight (22.9%) of the 35 patients had recurrence. Three (8.6%) was local recurrence (LR) only, 3 (8.6%) was distant metastasis (DM) only, and 2 (5.7%) was LR with DM. All LR were located at primary tumor sites. The overall survival rate, LR-free survival rate, and DM-free survival rate at 5 years was 79.8%, 83%, and 87%, respectively. All LR developed from tumors over 5 cm. However, there was no statistical significance (p = 0.065). There was no other risk factor for LR. CONCLUSION: Even though the patients included in this study had pathologically favorable pT3N0 rectal cancer, LR developed in 14.3% of patients. Most of the LR was located at primary tumor sites prior to surgery. Based on these findings, it might seem reasonable to consider postoperative RT with a smaller radiation field to the primary tumor site rather than the conventional whole pelvic irradiation.