Nonhistone Proteins HMGB1 and HMGB2 Differentially Modulate the Response of Human Embryonic Stem Cells and the Progenitor Cells to the Anticancer Drug Etoposide.

HMGB1 and HMGB2 proteins are abundantly expressed in human embryonic stem cells (hESCs) and hESC-derived progenitor cells (neuroectodermal cells, hNECs), though their functional roles in pluripotency and the mechanisms underlying their differentiation in response to the anticancer drug etoposide remain to be elucidated. Here, we show that HMGB1 and/or HMGB2 knockdown (KD) by shRNA in hESCs did not affect the cell stemness/pluripotency regardless of etoposide treatments, while in hESC-derived neuroectodermal cells, treatment resulted in differential effects on cell survival and the generation of rosette structures. The objective of this work was to determine whether HMGB1/2 proteins could modulate the sensitivity of hESCs and hESC-derived progenitor cells (hNECs) to etoposide. We observed that HMGB1 KD knockdown (KD) and, to a lesser extent, HMGB2 KD enhanced the sensitivity of hESCs to etoposide. Enhanced accumulation of 53BP1 on telomeres was detected by confocal microscopy in both untreated and etoposide-treated HMGB1 KD hESCs and hNECs, indicating that the loss of HMGB1 could destabilize telomeres. On the other hand, decreased accumulation of 53BP1 on telomeres in etoposide-treated HMGB2 KD hESCs (but not in HMGB2 KD hNECs) suggested that the loss of HMGB2 promoted the stability of telomeres. Etoposide treatment of hESCs resulted in a significant enhancement of telomerase activity, with the highest increase observed in the HMGB2 KD cells. Interestingly, no changes in telomerase activity were found in etoposide-treated control hNECs, but HMGB2 KD (unlike HMGB1 KD) markedly decreased telomerase activity in these cells. Changes in telomerase activity in the etoposide-treated HMGB2 KD hESCs or hNECs coincided with the appearance of DNA damage markers and could already be observed before the onset of apoptosis. Collectively, we have demonstrated that HMGB1 or HMGB2 differentially modulate the impact of etoposide treatment on human embryonic stem cells and their progenitor cells, suggesting possible strategies for the enhancement of the efficacy of this anticancer drug.

Knockdown of HMGB2 inhibits proliferation and invasion of renal tumor cells via the p-38MAPK pathway.

OBJECTIVE: To investigate the function of HMGB2 in renal tumor ACHN cells in vitro and in vivo and to study the underlying molecular mechanisms. PATIENTS AND METHODS: Kaplan-Meier analysis was used to study the relationship between expression of HMGB2 and prognosis of renal tumor. MTT assay was employed to examine cell proliferation and flow cytometry analysis was used to study the role of HMGB2 in cell apoptosis in ACHN cells. Transwell assays were used to explore the migration and invasion of ACHN cells. The effect of HMGB2 on tumor growth was investigated in vivo. Western blot was performed to evaluate the expression levels of p-JNK, p-ERK and p-p38MAPK. RESULTS: HMGB2 was upregulated in renal tumor and correlated with worse overall survival in renal tumor patients. Down-regulation of HMGB2 suppressed ACHN cells proliferation, invasion and migration in vitro. Moreover, down-regulation of HMGB2 inhibited tumor growth in vivo and HMGB2 exerts the oncogene function partly via the inhibition of p-p38MAPK activation. CONCLUSIONS: Our results provide novel insights into neuropathic pain and help to explore therapeutic targets in the treatment.

HMGB2 is associated with malignancy and regulates Warburg effect by targeting LDHB and FBP1 in breast cancer.

BACKGROUND: High-mobility group box 2 (HMGB2) is implicated in tumorigenesis in various cancers. However, the clinical significance of HMGB2 signaling in human breast cancer progression remains unknown. METHODS: We investigated HMGB2 expression in 185 cases of primary breast cancer and matched normal breast tissue specimens, and explored the underlying mechanisms of altered HMGB2 expression as well as the impact of this altered expression on breast cancer growth and on aerobic glycolysis using in vitro and animal models of breast cancer. RESULTS: HMGB2 was more highly expressed in tumor-cell nuclei of breast cancer cells than in the adjacent normal breast tissues (P < 0.05). Higher HMGB2 expression correlated with larger tumor size (P = 0.003) and advanced tumor stage (P = 0.033). A Cox proportional hazards model revealed that HMGB2 expression was an independent prognostic factor for breast cancer after radical resection (P < 0.05). Experimentally, knockdown of HMGB2 expression by stable transfected shRNA significantly decreased the growth and glycolysis of breast cancer cells both in vitro and in mouse models. Mechanically, promotion of breast cancer progression by HMGB2 directly and significantly correlated with activation of LDHB expression and inactivation of FBP1 expression. CONCLUSIONS: These results disclose a novel role for HMGB2 in reprogramming the metabolic process in breast cancer cells by targeting LDHB and FBP1 and provide potential prognostic predictors for breast cancer patients.

High-mobility group protein HMGB2 regulates human erythroid differentiation through trans-activation of GFI1B transcription.

Gfi-1B is a transcriptional repressor that is crucial for erythroid differentiation: inactivation of the GFI1B gene in mice leads to embryonic death due to failure to produce differentiated red cells. Accordingly, GFI1B expression is tightly regulated during erythropoiesis, but the mechanisms involved in such regulation remain partially understood. We here identify HMGB2, a high-mobility group HMG protein, as a key regulator of GFI1B transcription. HMGB2 binds to the GFI1B promoter in vivo and up-regulates its trans-activation most likely by enhancing the binding of Oct-1 and, to a lesser extent, of GATA-1 and NF-Y to the GFI1B promoter. HMGB2 expression increases during erythroid differentiation concomitantly to the increase of GfI1B transcription. Importantly, knockdown of HMGB2 in immature hematopoietic progenitor cells leads to decreased Gfi-1B expression and impairs their erythroid differentiation. We propose that HMGB2 potentiates GATA-1-dependent transcription of GFI1B by Oct-1 and thereby controls erythroid differentiation.

HMGB2 stabilizes p53 by interfering with E6/E6AP-mediated p53 degradation in human papillomavirus-positive HeLa cells.

We investigated the effect of HMGB2 on the stability of p53 protein in HeLa cells. Overexpression of HMGB2 led to accumulation of the p53 protein, whereas HMGB2 knockdown with siRNA resulted in a substantial decrease in the p53 protein level. The HMGB2-dependent increase of p53 stability was specific for HPV-positive HeLa cells as HCT116 and MCF7 cell lines did not demonstrate this response. Co-expression of HMGB2 and HPV E6 prevented HPV E6 protein-mediated ubiquitination and degradation of p53. FACS analysis exhibited that HeLa cells transfected with HMGB2 displayed decreased cell proliferation, with a concomitant increase of the p53 protein and arrest of the cell cycle, predominantly in G1 phase. Our findings collectively suggest that HMGB2 could stabilize p53 by interfering with E6/E6AP-mediated p53 degradation in HPV-positive HeLa cells.

Chromatin-associated proteins HMGB1/2 and PDIA3 trigger cellular response to chemotherapy-induced DNA damage.

The identification of new molecular components of the DNA damage signaling cascade opens novel avenues to enhance the efficacy of chemotherapeutic drugs. High-mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to the incorporation of nonnatural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high-mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; and heat shock 70 kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-fold to 50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked-down cancer cells after 24 to 72 hours of incubation with 1 micromol/L of cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress, and PDIA3 has been found essential for H2AX phosphorylation (no gamma-H2AX accumulated after 24-72 hours of incubation with 1 micromol/L of cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention.