HBXIP induces PARP1 via WTAP-mediated m6A modification and CEBPA-activated transcription in cisplatin resistance to hepatoma.

Poly (ADP-ribose) polymerase 1 (PARP1) is a DNA-binding protein that is involved in various biological functions, including DNA damage repair and transcription regulation. It plays a crucial role in cisplatin resistance. Nevertheless, the exact regulatory pathways governing PARP1 have not yet been fully elucidated. In this study, we present evidence suggesting that the hepatitis B X-interacting protein (HBXIP) may exert regulatory control over PARP1. HBXIP functions as a transcriptional coactivator and is positively associated with PARP1 expression in tissues obtained from hepatoma patients in clinical settings, and its high expression promotes cisplatin resistance in hepatoma. We discovered that the oncogene HBXIP increases the level of PARP1 m6A modification by upregulating the RNA methyltransferase WTAP, leading to the accumulation of the PARP1 protein. In this process, on the one hand, HBXIP jointly activates the transcription factor ETV5, promoting the activation of the WTAP promoter and further facilitating the promotion of the m6A modification of PARP1 by WTAP methyltransferase, enhancing the RNA stability of PARP1. On the other hand, HBXIP can also jointly activate the transcription factor CEBPA, enhance the activity of the PARP1 promoter, and promote the upregulation of PARP1 expression, ultimately leading to enhanced DNA damage repair capability and promoting cisplatin resistance in hepatoma. Notably, aspirin inhibits HBXIP, thereby reducing the expression of PARP1. Overall, our research revealed a novel mechanism for increasing PARP1 abundance, and aspirin therapy could overcome cisplatin resistance in hepatoma.

HBXIP blocks myosin-IIA assembly by phosphorylating and interacting with NMHC-IIA in breast cancer metastasis.

Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCßII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCßII, by coactivating Sp1, and triggered PKCßII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCßII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.

Upregulation of HBXIP contributed to the anti-DND by ginsenoside Rg1 after global cerebral ischemia-reperfusion.

OBJECTIVES: Ginsenoside Rg1 (Rg1) has been well-documented to be effective against ischemic/reperfusion (I/R) injury. However, whether it has therapeutic effect on delayed neuronal death is still unclear. The aim of this study is to investigate the effect of Rg1 on delayed neuronal death and elucidate its underlying mechanism. METHODS: Delayed neuronal death model was prepared by global cerebral ischemia-reperfusion in rats, Rg1 was intravenously administered once a day. Nissl and Fluoro Jade B staining were carried out to evaluate the effect of Rg1 on delayed neuronal death. Western blot and qPCR were used to investigate the levels of HBXIP and Survivin. HBXIP/Survivin complex was observed by co-immunoprecipitation. AAV-CMV-shRNA (HBXIP) was used to observe the role of HBXIP on delayed neuronal death improved by Rg1. KEY FINDINGS: Rg1 attenuated delayed neuronal death at the dose of 20 mg/kg, which also improved the mRNA and protein levels of HBXIP, as well as Survivin. Moreover, administration of Rg1 promoted the formation of HBXIP/Survivin complex, which contributed to the reduction of caspases signaling pathway. Knockdown of HBXIP abolished the alleviation of DND and inhibition of caspase cascade induced by Rg1. CONCLUSIONS: Rg1 alleviated delayed neuronal death by promoting anti-apoptosis effect by HBXIP/Survivin complex.

Sorafenib triggers ferroptosis via inhibition of HBXIP/SCD axis in hepatocellular carcinoma.

Sorafenib, which inhibits multiple kinases, is an effective frontline therapy for hepatocellular carcinoma (HCC). Ferroptosis is a form of iron-dependent programmed cell death regulated by lipid peroxidation, which can be induced by sorafenib treatment. Oncoprotein hepatitis B X-interacting protein (HBXIP) participates in multiple biological pro-tumor processes, including growth, metastasis, drug resistance, and metabolic reprogramming. However, the role of HBXIP in sorafenib-induced ferroptotic cell death remains unclear. In this study, we demonstrated that HBXIP prevents sorafenib-induced ferroptosis in HCC cells. Sorafenib decreased HBXIP expression, and overexpression of HBXIP blocked sorafenib-induced HCC cell death. Interestingly, suppression of HBXIP increased malondialdehyde (MDA) production and glutathione (GSH) depletion to promote sorafenib-mediated ferroptosis and cell death. Ferrostatin-1, a ferroptosis inhibitor, reversed the enhanced anticancer effect of sorafenib caused by HBXIP silencing in HCC cells. Regarding the molecular mechanism, HBXIP transcriptionally induced the expression of stearoyl-CoA desaturase (SCD) via coactivating the transcriptional factor ZNF263, resulting in the accumulation of free fatty acids and suppression of ferroptosis. Functionally, activation of the HBXIP/SCD axis reduced the anticancer activity of sorafenib and suppressed ferroptotic cell death in vivo and in vitro. HBXIP/SCD axis-mediated ferroptosis can serve as a novel downstream effector of sorafenib. Our results provide new evidence for clinical decisions in HCC therapy.

HBXIP blocks myosin-IIA assembly by phosphorylating and interacting with NMHC-IIA in breast cancer metastasis

Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCβII, by coactivating Sp1, and triggered PKCβII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCβII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.

Hbxip is essential for murine embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1.

HBXIP, also named LAMTOR5, has been well characterized as a transcriptional co-activator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5, and that depletion of Hbxip compromises the self-renewal of embryonic stem cells (ESCs), with reduced expression of pluripotency genes, reduced cell proliferation and decreased colony-forming capacity. In addition, both Hbxip-/- ESCs and E7.5 embryos displayed defects in ectodermal and mesodermal differentiation. Mechanistically, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, displayed differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/- and p18-/- mice, lacking subunits of the Ragulator complex, also shared similar phenotypes, embryonic lethality and retarded growth around E7-E8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

HBXIP is a novel regulator of the unfolded protein response that sustains tamoxifen resistance in ER+ breast cancer.

Endocrine-therapy-resistant estrogen receptor-positive (ER+) breast cancer cells often exhibit an augmented capacity to maintain endoplasmic reticulum (EnR) homeostasis under adverse conditions. Oncoprotein hepatitis B X-interacting protein (HBXIP) is a known transcriptional coactivator that promotes cancer development. However, it is unclear whether HBXIP participates in maintaining EnR homeostasis and promoting drug resistance in ER+ breast cancer. Here, we report that tamoxifen-resistant (TmaR) breast cancer cells exhibit increased expression of HBXIP, which acts as an inactivator of the unfolded protein response (UPR) to diminish tamoxifen-induced EnR stress. We show that HBXIP deficiency promotes EnR-associated degradation, enhances UPR-element reporter activity and cellular oxidative stress, and ultimately attenuates the growth of TmaR cells in vitro and in vivo. Mechanistically, we demonstrate that HBXIP acts as a chaperone of UPR transducer inositol-requiring enzyme 1a and diminishes production of reactive oxygen species (ROS) in TamR breast cancer cells. Upon loss of HBXIP expression, tamoxifen treatment hyperactivates IRE1α and its downstream proapoptotic pathways and simultaneously induces accumulation of intracellular ROS. This elevated ROS programmatically activates the other two branches of the UPR, mediated by PKR-like ER kinase and activating transcription factor 6α. Clinical investigations and Kaplan-Meier plotter analysis revealed that HBXIP is highly expressed in TamR breast cancer tissues. Furthermore, reinforced HBXIP expression is associated with a high recurrence and poor relapse-free survival rates in tamoxifen monotherapy ER+ breast cancer patients. These findings indicate that HBXIP is a regulator of EnR homeostasis and a potential target for TamR breast cancer therapy.

The modulation of PD-L1 induced by the oncogenic HBXIP for breast cancer growth.

Programmed death ligand-1 (PD-L1)/PD-1 checkpoint extensively serves as a central mediator of immunosuppression. A tumor-promoting role for abundant PD-L1 in several cancers is revealed. However, the importance of PD-L1 and how the PD-L1 expression is controlled in breast cancer remains obscure. Here, the mechanisms of controlling PD-L1 at the transcription and protein acetylation levels in promoting breast cancer growth are presented. Overexpressed PD-L1 accelerates breast cancer growth in vitro and in vivo. RNA-seq uncovers that PD-L1 can induce some target genes affecting many cellular processes, especially cancer development. In clinical breast cancer tissues and cells, PD-L1 and HBXIP are both increased, and their expressions are positively correlated. Mechanistic exploration identifies that HBXIP stimulates the transcription of PD-L1 through co-activating ETS2. Specifically, HBXIP induces PD-L1 acetylation at K270 site through interacting with acetyltransferase p300, leading to the stability of PD-L1 protein. Functionally, depletion of HBXIP attenuates PD-L1-accelerated breast tumor growth. Aspirin alleviates breast cancer via targeting PD-L1 and HBXIP. Collectively, the findings display new light into the mechanisms of controlling tumor PD-L1 and broaden the utility for PD-L1 as a target in breast cancer therapy.

Down-Regulation of HBXIP Inhibits Non-Small Cell Lung Cancer Growth and Enhances the Anti-Tumor Immunity of Mice by Reducing NRP-1.

OBJECTIVE: Hepatitis B X-interacting protein (HBXIP) interacts with hepatitis B virus X protein to participate in the replication of the hepatitis B virus and carcinogenesis. Cellular growth and metastasis of non-small-cell lung cancer (NSCLC) are repressed by HBXIP inhibition. However, the role and mechanism of HBXIP on NSCLC cell growth remain unknown. MATERIALS: Expression of HBXIP was assessed by qRT-PCR and Western blot. siRNA targeting HBXIP was applied to detect cell viability and proliferation by MTT and colony formation assays. In vivo tumor growth was assessed, and anti-tumor immunity was determined by flow cytometry. The downstream partners involved in HBXIP-mediated tumorigenesis were detected by Western blot. RESULTS: Expression of HBXIP and neuropilin1-1 (NRP-1) was higher in NSCLC tissues and cells than in paracancerous tissues and human lung epithelial cells. siRNA-mediated knockdown of HBXIP decreased the cell viability of NSCLC and suppressed proliferation. Protein expression of Lin28B and NRP-1 was reduced by the knockdown of HBXIP, and over-expression of Lin28B attenuated the HBXIP silence-induced decrease of NRP-1. In vivo tumor growth was suppressed by HBXIP silencing, and the knockdown of HBXIP enhanced anti-tumor immunity through the increase of CD4+ and CD8+ T lymphocytes. CONCLUSION: Down-regulation of HBXIP reduced Lin28B-mediated NRP-1 to suppress NSCLC cell growth and enhance anti-tumor immunity.

HBXIP accelerates glycolysis and promotes cancer angiogenesis via AKT/mTOR pathway in bladder cancer.

Abnormal metabolism and uncontrolled angiogenesis are two important characteristics of malignant tumors. Although HBXIP is known to be associated with a poor prognosis for bladder cancer (BC), its effects on glycolysis and angiogenesis in BC have not been investigated. BC prognosis and relative gene expression of HBXIP were analyzed using the GEPIA, UALCAN, and STRING databases. BC cell angiogenesis and glycolysis were assessed by vasculogenic mimicry and glycolysis assay. Human umbilical vein endothelial cell (HUVEC) viability, migration, and angiogenesis were assessed by CCK8, transwell, wound healing, and tube formation assays. The results showed that HBXIP was highly expressed in BC tissues and cells. Knockdown of HBXIP expression decreased the levels of glucose uptake, lactate production, and glycolytic enzyme expression in BC cells, and decreased cell viability and migration of HUVECs. Additionally, silencing HBXIP reduced the total length of tubes and number of intersections, and EPO and VEGF protein expression in BC cells and HUVECs. Furthermore, knockdown of HBXIP expression reversed cell viability, migration, tube formation, and vasculogenic mimicry under high glucose and lactate conditions. Mechanistically, silencing of HBXIP reduced the protein expression levels of pAKT-ser473 and pmTOR, and inhibition of HBXIP, AKT, and mTOR expression decreased glycolytic enzyme protein expression. Our findings suggest that HBXIP reduces glycolysis in BC cells via regulation of AKT/mTOR signaling, thereby blocking BC angiogenesis. Collectively, this study provides a potential strategy to target HBXIP and AKT/mTOR for regulating glycolysis progression concurrently with anti-angiogenesis effects, and thereby develop novel therapeutics for the treatment of BC.

Oncoprotein HBXIP promotes tumorigenesis through MAPK/ERK pathway activation in non-small cell lung cancer.

Objective: The oncoprotein, hepatitis B X-interacting protein (HBXIP), has been reported to play an important role in human malignancies. However, its functions in non-small cell lung cancer (NSCLC) are poorly understood. The goal of the present study was to identify the role of HBXIP in the regulation of NSCLC development. Methods: The level of HBXIP expression in NSCLC tissue was assessed by immunohistochemical and Western blot analyses, and its relationships with clinicopathological features and outcomes were statistically evaluated. The effects of HBXIP on NSCLC cell progression were assessed through cell viability, colony formation, and flow cytometry analyses in vitro. The mechanism by which HBXIP regulated the MAPK pathway was studied by Western blot, immunofluorescence, and immunoprecipitation assays. In addition, in vivo experiments were performed to evaluate the progression of NSCLC and ERK signaling pathway activation after HBXIP knockdown. Results: HBXIP was overexpressed in human NSCLC and was correlated with the invasiveness of NSCLC. The high expression of HBXIP in NSCLC was significantly correlated with gender (P = 0.033), N stage (P = 0.002), and tumor-node-metastasis stage (P = 0.008). In vitro experiments using an NSCLC cell line revealed that HBXIP knockdown resulted in the suppression of cell proliferation and colony formation, which was consistent with the enhanced cell cycle arrest in G1 phase. The results of a mechanistic investigation suggested that binding of HBXIP to MEK1 protein promoted MAPK/ERK signaling pathway activation in NSCLC by preventing the proteasome-mediated degradation of MEK1. In addition, the results obtained using in vivo subcutaneous tumor xenografts confirmed that HBXIP deficiency decreased MEK1 protein levels and NSCLC tumor growth. Conclusions: Taken together, our results showed that the HBXIP-MEK interaction promoted oncogenesis via the MAPK/ERK pathway, which may serve as a novel therapeutic target for cancers in which MAPK/ERK signaling is a dominant feature.

The oncogenic role of HBXIP.

Hepatitis B X-interacting protein (HBXIP) is a conserved protein of 19 kDa that was originally identified as a binding partner of hepatitis B virus X protein. Emerging evidence indicates that HBXIP is highly expressed in a variety of cancers and is correlated with poor clinical outcomes in cancer patients. HBXIP plays a critical role in cancer progression, but the underlying mechanisms are still unclear. In this review, we primarily focus on publications investigating HBXIP in cancer research, including its expression and clinical significance in cancer patients, its role as a coactivator of transcription factors in cancer cells, its inhibitory effects on the mitochondrial cytochrome c-caspase apoptotic pathway, as well as its roles in promoting mitosis and drug resistance in cancer cells, its regulatory effects on cancer metabolism, and its relationships with other signaling pathways or microRNAs in cancer. This review aims to compile and summarize existing knowledge of the functions of HBXIP in cancer, which provides a comprehensive reference for future studies on the oncogenic mechanisms of HBXIP.

HBXIP drives metabolic reprogramming in hepatocellular carcinoma cells via METTL3-mediated m6A modification of HIF-1α.

Cancer cells sustain high levels of glycolysis and glutaminolysis via reprogramming of intracellular metabolism, which represents a driver of hepatocellular carcinoma (HCC) progression. Understanding the mechanisms of cell metabolic reprogramming may present a new basis for liver cancer treatment. Herein, we collected HCC tissues and noncancerous liver tissues and found hepatitis B virus X-interacting protein (HBXIP) was found to be upregulated in HCC tissues and associated with poor prognosis. The N6-methyladenosine (m6A) level of hypoxia-inducible factor-1α (HIF-1α) in HCC cells was evaluated after the intervention of METTL3. The possible m6A site of HIF-1α was queried and the binding relationship between METTL3 and HIF-1α was verified. The interference of HBXIP suppressed HCC malignant behaviors and inhibited the Warburg effect in HCC cells. METTL3 was upregulated in HCC tissues and positively regulated by HBXIP. Overexpression of METTL3 restored cell metabolic reprogramming in HCC cells with partial loss of HBXIP. HBXIP mediated METTL3 to promote the metabolic reprogramming and malignant biological behaviors of HCC cells. The levels of total m6A in HCC cells and m6A in HIF-1α were increased. METTL3 had a binding relationship with HIF-1α and mediated the m6A modification of HIF-1α. In conclusion, HBXIP drives metabolic reprogramming in HCC cells via METTL3-mediated m6A modification of HIF-1α.

HBXIP Regulates Gastric Cancer Glucose Metabolism and Malignancy Through PI3K/AKT and p53 Signaling.

INTRODUCTION: Hepatitis B X-interacting protein (HBXIP) overexpression is related to the progression of multiple cancers. However, its role in gastric cancer (GC) remains unclear. MATERIALS AND METHODS: HBXIP expression was determined in human GC specimens and cell lines by quantitative polymerase chain reaction (qRT-PCR) and Western blot. The effects of HBXIP depletion or ectopic expression on GC proliferation were evaluated in vitro using the cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation, colony formation, and cell cycle assays. The in vivo effects were investigated using a mouse xenograft model. Apoptosis was evaluated by flow cytometry (in vitro) and immunohistochemistry (IHC; in vivo). Cell migration and invasion were evaluated in vitro using wound healing, transwell migration, and matrigel invasion assays; and in vivo by quantifying distant metastases from injection of GC cells in the lateral tail vein. RESULTS: Herein, we reported that HBXIP expression was higher in GC than in normal tissues, and this high expression indicated a poorer prognosis. Gain- and loss-of-function assays showed that HBXIP promoted GC proliferation, migration, and invasion, and inhibited apoptosis. High-performance liquid chromatography (HPLC) quantification of glycolytic metabolites revealed that HBXIP promoted glucose metabolic reprogramming. Investigation of the PI3K/AKT and p53 pathways highlighted their role in this HBXIP-mediated metabolic reprogramming. CONCLUSION: Our results indicate that the up-regulation of HBXIP leads to GC progression by positively regulating glucose metabolism. Therefore, HBXIP is a potential target for the treatment of GC.

HBXIP: a potential prognosis biomarker of colorectal cancer which promotes invasion and migration via epithelial-mesenchymal transition.

Hepatitis B X-interacting protein (HBXIP) is highly expressed in many cancers, but the correlation between the expression of HBXIP and the clinical significance and underlying molecular mechanisms in colorectal cancer (CRC) is still unclear. We selected 186 specimens from CRC patients for analyzing the relationship between the expression of HBXIP and the clinical-pathological features by immunohistochemistry. Migration and invasion experiments were performed to examine the effect of HBXIP on CRC cell metastasis. Besides, we also explored the possible molecular mechanism of HBXIP regulation of CRC cell metastasis by Western blot. Our data indicated that the HBXIP was overexpressed in CRC tissues. High HBXIP expression was correlated with metastasis and shorter survival times in patients with CRC and served as an independent factor for poor prognosis. Moreover, HBXIP promotes CRC metastasis by enhancing the epithelial-mesenchymal transition (EMT) process. Our findings provide the first evidence that HBXIP induces EMT to promote metastasis and predicts the poor prognosis of CRC. Therefore, HBXIP may become a new target for CRC treatment.

The effects of HBXIP on the biological functions of tongue squamous cell carcinoma cells and correlation with PI3K/Akt.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is the most malignant oral cancer, having a high mortality rate. METHODS: The effects of hepatitis B X-interacting protein (HBXIP) overexpression on the proliferation, migration, and invasion of TSCC cells were measured by micro-culture tetrazolium assay (MTT) assay, transwell assay and scratch test, respectively, and the effects of HBXIP mRNA overexpression on the protein expression levels of AKT, p-AKT, PI3K, p-PI3K and S100A4 were detected by western blotting. RESULTS: MTT assay showed that there were significantly more proliferating cells than in the experimental group. In the scratch test and transwell assay, the migration rate and the number of invading cells were remarkably greater in the experimental group. The expression levels of p-AKT, p-PI3K and S100A4 were increased in the experimental group after HBXIP overexpression. CONCLUSIONS: HBXIP mRNA overexpression can influence the proliferation, invasion, and migration of TSCC cells and promote their proliferation and migration by increasing the protein expression levels of p-AKT, p-PI3K and S100A4.

The oncoprotein HBXIP facilitates metastasis of hepatocellular carcinoma cells by activation of MMP15 expression.

Background: Due to the high recurrence and metastasis rate, the clinical outcomes of patients with hepatocellular carcinoma (HCC) are still unsatisfactory. Hepatitis B virus X-interacting protein (HBXIP) has been reported to play crucial roles in carcinogenesis. Purpose: We aimed to reveal the functional significance and underlying mechanism of HBXIP in HCC metastasis. Methods: Cell transwell assay, in vivo metastasis model, real-time PCR, western blot analysis, luciferase reporter and chromatin immunoprecipitation assays were applied. Results: Here, we detected the HBXIP expression level and determined its clinical significance in HCC. We found that HBXIP was significantly upregulated in HCC tissues, and correlated with vascular invasion, tumor metastasis and worse prognosis of HCC patients. HBXIP enhanced cell migration and invasion in vitro, and promoted the metastasis of HCC in vivo. Furthermore, we confirmed that HBXIP increased MMP15 expression through association with proto-oncogene c-myc. Depletion of c-myc abolished HBXIP-mediated MMP-15 upregulation. We also observed a positive correlation between HBXIP and MMP15 expression in HCC tissues. Conclusion: Our results establish a novel function for HBXIP-MMP15 regulation in HCC metastasis and suggest its candidacy as a new prognostic biomarker and therapeutic target for HCC metastasis.

Oncoprotein HBXIP induces PKM2 via transcription factor E2F1 to promote cell proliferation in ER-positive breast cancer.

We have reported that hepatitis B X-interacting protein (HBXIP, also termed LAMTOR5) can act as an oncogenic transcriptional co-activator to modulate gene expression, promoting breast cancer development. Pyruvate kinase muscle isozyme M2 (PKM2), encoded by PKM gene, has emerged as a key oncoprotein in breast cancer. Yet, the regulatory mechanism of PKM2 is still unexplored. Here, we report that HBXIP can upregulate PKM2 to accelerate proliferation of estrogen receptor positive (ER+) breast cancer. Immunohistochemistry analysis using breast cancer tissue microarray uncovered a positive association between the expression of HBXIP and PKM2. We also discovered that PKM2 expression was positively related with HBXIP expression in clinical breast cancer patients by real-time PCR assay. Interestingly, in ER+ breast cancer cells, HBXIP was capable of upregulating PKM2 expression at mRNA and protein levels in a dose-dependent manner, as well as increasing the activity of PKM promoter. Mechanistically, HBXIP could stimulate PKM promoter through binding to the -779/-579 promoter region involving co-activation of E2F transcription factor 1 (E2F1). In function, cell viability, EdU, colony formation, and xenograft tumor growth assays showed that HBXIP contributed to accelerating cell proliferation through PKM2 in ER+ breast cancer. Collectively, we conclude that HBXIP induces PKM2 through transcription factor E2F1 to facilitate ER+ breast cancer cell proliferation. We provide new evidence for the mechanism of transcription regulation of PKM2 in promotion of breast cancer progression.

MiR-145-targeted HBXIP modulates human breast cancer cell proliferation.

BACKGROUND: MiR-145 has been identified as a tumor suppressive microRNA in multiple cancers. In this current investigation, we searched for new direct targets of miR-145 and evaluated their effect on breast cancer development. METHODS: Targetscan was used to predict the target genes of miR-145. The targeting of miR-145 on oncogenic HBXIP was verified by luciferase reporter gene analysis. The effect of miR-145 on the level of messenger RNA and protein of HBXIP was evaluated by quantitative real-time PCR and immunoblotting. Correlations between miR-145 and HBXIP, as well as miR-145 expression, were analyzed in 30 paired breast cancer and noncancerous tissues by quantitative real-time PCR. Methyl thiazol tetrazolium and colony formation assays were applied to determine the cell proliferation ability. RESULTS: HBXIP was identified as a novel target gene of miR-145 in breast cancer. MiR-145 was found to dose-dependently decrease messenger RNA and protein expression of HBXIP in breast cancer MCF-7 cells. Notably, miR-145 expression was negatively related to HBXIP expression and was obviously reduced in breast cancer samples. Finally, miR-145 suppressed cell proliferation while its inhibitor, anti-miR-145, accelerated cell proliferation. Interestingly, silencing of HBXIP reversed the acceleration of cell proliferation induced by anti-miR-145 in breast cancer. CONCLUSION: Oncogenic HBXIP is a new direct target of tumor suppressive miR-145. Our findings reveal that miR-145-targeting HBXIP could be a potential therapeutic target in breast cancer.

Oncogenic HBXIP enhances ZEB1 through Sp1 to accelerate breast cancer growth.

BACKGROUND: There is abundant evidence to indicate that HBXIP functions as an oncoprotein and transcription co-activator during the development and promotion of cancers. In multiple cancers, ZEB1 serves as a transcription activator to regulate gene expression. We explored the roles of ZEB1 in HBXIP-induced breast cancer growth. METHODS: HBXIP regulation of ZEB1 was evaluated by reverse transcription PCR and immunoblotting. The stimulation of ZEB1 promoter by HBXIP and/or Sp1 was tested using luciferase reporter gene analysis. The alteration of cell proliferation mediated by HBXIP-induced ZEB1 was tested using methyl-thiazolyl-tetrazolium and 5-Ethynyl-2'-deoxyuridine (EdU) incorporation analysis. ZEB1 and HBXIP expression in human breast cancer tissues was analyzed using quantitative real-time PCR. The relationship between HBXIP and ZEB1 was confirmed by Pearson's correlation coefficient. RESULTS: We observed dose-dependent upregulation of ZEB1 by HBXIP in breast cancer cells. HBXIP can activate the ZEB1 promoter by interacting with transcription factor Sp1. Cell viability and EdU incorporation analysis showed that HBXIP could drive cell proliferation by enhancing ZEB1 in breast cancer. Using quantitative real-time PCR, ZEB1 overexpression and a positive relationship between ZEB1 and HBXIP were observed in clinical breast cancer samples. CONCLUSION: Oncogenic HBXIP controls the transcription regulation of ZEB1 by co-activating Sp1, thereby accelerating breast cancer growth.