Overexpression Of HAX-1 Research Articles

The Role of the Hematopoietic Cell-Specific Protein 1-Associated Protein X-1 in Human Papillomavirus 16 E2-Induced Human Cervical Squamous Carcinoma Cell Apoptosis via a Mitochondria-Dependent Pathway

Objectives: Human papillomavirus 16 (HPV 16) E2 is a transcriptional regulator that plays a key role in regulating a variety of biological responses. Hematopoietic cell-specific protein 1-related protein X-1 (HAX-1) is a mitochondrial membrane protein, and the HAX-1 gene is involved in the occurrence, growth, invasion, and metastasis of various human malignant tumors. The purpose of this study was to investigate the relationships among HPV 16 E2, the role of HAX-1 gene, and the underlying intracellular apoptotic mechanism of human cervical squamous carcinoma cells (C33a and SiHa). Methods: In this study, HAX-1 expression was examined using real-time polymerase chain reaction, Western blot, and immunohistochemical staining analysis. Apoptosis of cells was assessed by flow cytometry. The mitochondrial function was assessed by the mitochondrial copy number, reactive oxygen species (ROS) generation, the mitochondrial membrane potential (ΔΨm), and mitochondrial morphology. Results: Our study demonstrated that the expression of the HAX-1 gene was significantly increased in human cervical carcinoma tissues relative to noncancerous cervix tissues. HPV 16 E2 inhibited HAX-1 protein expression. Overexpression of HAX-1 increased the mitochondrial copy number, decreased the production of ROS, and maintained the integrity of the mitochondrial membrane and morphology. So, enhanced expression of the HAX-1 gene could abrogate the HPV 16 E2-induced cell apoptosis. Conclusion: Therefore, these data support a mechanism that HAX-1 plays a crucial role in HPV 16 E2-induced human cervical squamous carcinoma cell apoptosis in a mitochondrial-dependent manner.

Sanguisorba parviflora (Maxim.) Takeda alleviates cyclophosphamide-induced leukopenia by regulating haematopoietic cell-specific protein 1-associated protein X-1 gene expression.

We have previously shown that the saponins of Sanguisorba parviflora (Maxim.) Takeda (Sp. T) relieved cyclophosphamide-induced myelosuppression in leukopenic mice. Haematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) participated in the survival of neutrophils through the regulation of mitochondrial function. The aim of the present study was to comprehensively identify the role of HAX-1 in the mechanism of leukopenia alleviation by Sp.T. HAX-1gene and protein expression levels in peripheral blood neutrophils were examined using real-time quantitative reverse transcription-polymerase chain reaction, western blot and immunohistochemical assays. Neutrophil apoptosis was measured using flow cytometry. Mitochondrial function was determined via assessments of the reactive oxygen species (ROS) generation and mitochondrial membrane potential (ΔΨm) integrity levels. The HAX-1gene expression level in the peripheral blood neutrophils was significantly lower in patients with leukopenia than in healthy donors. The saponins of Sp.T induced HAX-1 expression and promoted myeloid progenitor cell (mEB8-ER cell) viability. HAX-1 overexpression reduced the production of ROS and maintained ΔΨm integrity. Cyclophosphamide-induced mitochondrial dysfunction and apoptosis could be abrogated by treatment with Sp.T or metformin. Our data suggest a mechanism through which Sp.T protects against chemotherapy-induced leukopenia by regulating HAX-1gene expression in a mitochondrial-dependent manner.

Sanguisorba parviflora (Maxim) Takeda alleviates cyclophosphamide-induced leukopenia via regulating the hematopoietic cell-specific protein 1-associated protein X-1 gene.

Our previous studies have shown that saponins of Sanguisorba parviflora (Maxim) Takeda (Sp.T) relieved cyclophosphamide-induced myelosuppression in mice with leukopenia. The hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) participated in the survival of neutrophils through the regulation of mitochondrial function. This study aimed to comprehensively identify the role of HAX-1 in Sp.T to alleviate leukopenia. HAX-1 expression was examined in the peripheral blood neutrophils using real-time polymerase chain reaction (PCR), Western blot analysis and immunohistochemical staining. Neutrophil apoptosis was measured by flow cytometry. Mitochondrial function was evaluated via reactive oxygen species (ROS) generation and mitochondrial membrane potential (ΔΨm) integrity. Our study indicated that the expression of the HAX-1 gene was significantly decreased in the peripheral blood neutrophils of leukopenia patients compared with healthy donors. The saponins of Sp.T induced HAX-1 expression and promoted myeloid progenitor cell (mEB8-ER cell) viability, while overexpression of HAX-1 reduced the production of reactive oxygen species (ROS) and maintained the integrity of the mitochondrial membrane potential. Cyclophosphamide-induced mitochondrial dysfunction and apoptosis could be abrogated by treatment with Sp.T or the addition of metformin. Our data support a mechanism where Sp.T protects against chemotherapy-induced leukopenia by regulating HAX-1 gene expression in a mitochondrial-dependent manner.

Novel Role of HAX-1 in Neurons Protection After Spinal Cord Injury Involvement of IRE-1.

Spinal cord injury (SCI) is one of the diseases with high probability of causing disability in human beings, and there is no reliable treatment at present. Neuronal apoptosis is a vital component of secondary injury and plays a critical role in the development of neurological dysfunction after spinal cord injury. In this study, we found that the expression and distribution of HAX-1 in neurons increased 1day after SCI. PC12 cells overexpressing HAX-1 showed decreased apoptosis and PC12 cells are more likely to undergo apoptosis after down-regulating HAX-1, which was confirmed via TUNEL experiments. We found GRP94 showed the same trend as HAX-1 in expression and interacted with HAX-1 and IRE-1 in both spinal cord tissue and PC12 cells, and this interaction seems to be enhanced after SCI. When the expression of HAX-1 was up-regulated, GRP94 also increased, but IRE-1 did not change at all. Further studies showed that overexpression of HAX-1 decreased the expression of pIRE-1, rather than IRE-1, and downstream proteins of the IRE signaling pathway (Caspase12, pJNK and CHOP) were significantly reduced, and vice versa. In animals treated with HAX-1 expressing adenovirus there are more neuronal cells remaining in the damaged spinal cord tissue, and hindlimb motor function of rats was significantly improved. So, we speculate that HAX-1 might play a role in protecting neurons from apoptosis after SCI by regulating the IRE-1 signaling pathway via promoting the dissociation of GRP94 from IRE-1. This may provide a theoretical basis and a potential therapeutic target for clinical improvement of neural function recovery after SCI.

HAX-1 overexpression in gastric cancer promotes cell proliferation.

BackgroundHAX-1 is involved in the regulation of cellular processes such as apoptosis, proliferation and migration and is closely related to tumorigenesis and tumor metastasis. However, expression of HAX-1 in gastric cancer and its role in tumor development and progression remain unclear.MethodsQuantitative polymerase chain reaction was used to detect the expression of HAX-1 mRNA in gastric cancer tissues and adjacent non-tumorous tissues. Expression of HAX-1, caspase-3 and caspase-9 was detected by immunohistochemistry in gastric cancer. Small hairpin RNA (shRNA) plasmid was employed to establish SGC-7901 cell lines that expressed HAX-1 at low levels. The effect of HAX-1 expression on cell proliferation will be studied at the cell level.ResultsQuantitative polymerase chain reaction (qPCR) showed HAX-1 mRNA expression to be significantly upregulated in gastric cancer tissues. Based on immunohistochemical analysis, upregulation of HAX-1 protein expression correlates positively with the degree of tumor differentiation, vascular tumor thrombus, tumor-node-metastasis stage and lymph node metastatic status and negatively with expression of the apoptosis-related proteins caspase-3 and caspase-9. In addition, high HAX-1 protein expression indicates a poor prognosis. Serum starvation-release experiments revealed that HAX-1 promotes the proliferation of SGC-7901 gastric cancer cells; as cell proliferation increased, expression of HAX-1 also increased, whereas the expression levels of the apoptosis-related proteins caspase-3 and caspase-9 decreased. HAX-1 siRNA transfection experiments demonstrated that HAX-1 gene knockdown causes cell cycle arrest at the G0/G1 phase, inhibits proliferation, and downregulates HAX-1 expression while enhancing expression of apoptosis-related proteins.ConclusionsHAX-1 might exert its proliferation-promoting and anti-apoptotic effects by inhibiting expression of the apoptosis-related proteins caspase-3 and caspase-9.HAX-1 may be a potential target for the treatment of gastric cancer.

MiR-654-5p Targets HAX-1 to Regulate the Malignancy Behaviors of Colorectal Cancer Cells.

Introduction. The biological roles of microRNA-654-5p (miR-654-5p) in cancers have been previously reported. However, its role in colorectal cancer (CRC) remains largely unknown. The purpose of this work was to investigate the roles and associated mechanisms in CRC. Methods Quantitative Real-Time PCR (qRT-PCR) was utilized to explore the expression pattern of miR-654-5p in CRC cells. Cell Counting Kit-8 (CCK-8) assay, wound-healing assay, and transwell invasion assay were conducted to investigate the effects of miR-654-5p on CRC cell proliferation, migration, and invasion, respectively. Moreover, the mechanisms behind miR-654-5p regulates CRC progression were investigated. Results Compared with normal cell line, miR-654-5p expression level was significantly suppressed in CRC cells. After overexpression of miR-654-5p, the malignancy behaviors of CRC cells including cell proliferation, migration, and invasion were remarkably decreased. Subsequently, we found hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) was a putative target for miR-654-5p. Rescue experiments showed overexpression of HAX-1 could partially reversed the effects of miR-654-4p on CRC cell events. Conclusion miR-654-5p was reduced expression in CRC cells and could regulate CRC progression via targeting HAX-1.

Cytoplasmic HAX1 Is an Independent Risk Factor for Breast Cancer Metastasis.

HAX1 is an antiapoptotic factor involved in the regulation of cell migration and calcium homeostasis, overexpressed in several cancers, including breast cancer. It has been suggested that HAX1 is also implicated in metastasis. Herein we report the results of meta-analysis of HAX1 expression, based on publicly available data, which confirms its significant overexpression in breast cancer and demonstrates copy number gain and prognostic value of HAX1 overexpression for metastatic relapse in ER+ tumors. IHC analysis reported here also reveals its significant overexpression in breast cancer samples from primary tumors, indicating significantly higher HAX1 protein levels in a group of patients who developed distant metastases in a disease course. Moreover, we demonstrate that HAX1 localization is important for the prediction of metastatic relapse and that cytoplasmic but not nuclear HAX1 is an independent risk factor for breast cancer metastasis.

Multimeric Protein Complexes in Regulation of Cardiomyocyte Calcium Cycling and Survival

Depressed sarcoplasmic reticulum (SR) Ca-cycling, leading to decreased function is a clinical hallmark of human and experimental heart failure. Thus, efforts have concentrated on elucidating the mechanisms contributing to aberrant SR Ca-homeostasis to uncover new therapeutic targets. Studies indicate that both SERCA and phospholamban (PLN) may represent attractive targets. However, the initial simple view of the PLN/SERCA regulatory complex has been modified by our identification of new proteins associated with regulation of SR Ca-transport and overall Ca-homeostasis in the cardiomyocyte. One of them is HAX-1, the anti-apoptotic protein, which interacts with PLN and alters the PLN inhibitory function. Acute or chronic overexpression of HAX-1 promoted PLN inhibition on the Ca-ATPase and decreased cardiomyocyte calcium kinetics and contractile parameters. Accordingly, ablation of HAX-1 significantly enhanced SERCA activity and calcium kinetics. Furthermore, the HAX-1/PLN interaction appeared to also regulate cardiomyocyte survival. The other regulatory protein is the SR intraluminal histidine-rich Ca-binding protein (HRC), which interacts with SERCA and regulates the enzyme's maximal Ca-transport velocity. Thus, HAX-1 and HRC may represent potential therapeutic targets in correcting the depressed SR Ca-cycling in heart failure. Furthermore, PLN activity is regulated by two phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which affect overall Ca-cycling and contractility. Interestingly, recent studies indicate that this multimeric SERCA/PLN-ensemble is involved in heart failure and arrhythmias, as well as apoptosis and cell death. Thus, targeted approaches to correct abnormalities at the SERCA/PLN complex may hold therapeutic promise in heart failure. In addition, we have identified human variants in these calcium-cycling genes that affect their “activity”, reflecting aberrant Ca-handling and increased cell death. Therefore, these mutations may be used as prognostic or diagnostic markers for heart failure and arrhythmia development.

Role of the tumour protein P53 gene in human cervical squamous carcinoma cells: Discussing haematopoietic cell-specific protein 1-associated protein X-1-induced survival, migration and proliferation.

The haematopoietic cell-specific protein 1-associated protein X-1 (HAX-1), as a mitochondrial membrane protein, induces cancer progression and metastasis. The present study aimed to investigate the role of HAX-1-induced survival, migration and proliferation of human cervical squamous carcinoma cells and to elucidate its potential molecular mechanisms. The level of HAX-1 was examined by quantitative polymerase chain reaction and western blot analyses. The survival, migration and proliferation of the human cervical squamous carcinoma SiHa cell line were measured by the water-soluble tetrazolium salt (WST-1) assay, Transwell assay and 3H-thymidine incorporation into DNA (3H-TdR) assay, respectively. The intracellular reactive oxygen species (ROS) was estimated by the fluorescence of H2DCFDA, and the mitochondrial membrane potential was tested using a JC-1 probe. The expression of the HAX-1 gene was significantly increased in human cervical carcinoma tissues relative to non-cancerous cervix tissues. Overexpression of HAX-1 increased the survival, migration and proliferation ability of SiHa cells, decreased the production of ROS, and maintained the integrity of the mitochondrial membrane and morphology. The effect brought on these cells could be abrogated by the addition of wild-type tumour protein P53 (p53) or carbonyl cyanide-p-trifluoro methoxyphenylhydrazone-induced mitochondrial dysfunction. In summary, these data support the notion that HAX-1 induced the survival, migration and proliferation of human cervical squamous carcinoma cells by inhibiting its downstream regulatory factor p53 in SiHa cells.

A long noncoding RNA promotes cellulase expression in Trichoderma reesei

BackgroundDue to its capability to secrete large quantities of plant biomass degrading enzymes (PBDE), Trichoderma reesei is widely applied for industrial purposes. In nature, expression of PBDE is efficiently regulated in this fungus. Several factors involved in this regulatory network have been identified. However, most of them are transcription factors. Long noncoding RNAs (lncRNAs) emerged as common players acting on epigenetic or transcriptional regulation in several eukaryotic organisms. To date, no lncRNA has been described in filamentous fungi.ResultsA lncRNA termed HAX1 was identified in T. reesei QM9414. In this study, it was characterized and evidence for its regulatory impact on cellulase expression was provided. Interestingly, different versions of HAX1 were identified in different strains (namely, QM6a, QM9414, and Rut-C30), varying in terms of RNA length. Remarkably, considerable longer variants of this lncRNA are present in hypercellulolytic strains compared to the wild-type strain QM6a. Based on these results, a correlation between RNA length and the functional impact of HAX1 on PBDE expression was supposed. This assumption was verified by overexpressing the most abundant HAX1 versions identified in QM6a, QM9414, and Rut-C30. Such HAX1 overexpression on the one hand was suitable for regaining the function in hax1 disruption strains, and on the other hand resulted in notably higher cellulase activities in QM6a, especially by the expression of longer HAX1 versions.ConclusionWith HAX1, for the first time the regulatory role of a lncRNA in filamentous fungi was uncovered. Besides this, a new player involved in the complex regulation of PBDE expression in T. reesei was identified. Due to its enhancing effect on cellulase activity, HAX1 was shown to be not only interesting for basic research, but also a promising candidate for expanding the set of biotechnological tools for industrial application of T. reesei.

Analysis of the expression of HAX-1 gene in human glioma.

Glioma, as the most common aggressive malignant tumor in the central nervous system, is still an insurmountable issue in neural diseases. The proliferation and survival mechanism of glioma cells need to be explored further for the development of glioma treatment. Hematopoietic cell-specific protein 1 associated protein X-1 (HAX-1) is well known for its anti-apoptotic effect. It was reported to play an important role in several malignant tumors. However, the effect of HAX-1 in glioma still remains unknown. This study aimed to investigate the expression of HAX-1 in glioma and the correlation between HAX-1 and the clinicopathological characteristics and prognosis of glioma. Quantitative reverse transcription polymerase chain reaction and Western blot analysis showed that HAX-1 was overexpressed in glioma cell lines compared with normal human astrocytes. This trend was confirmed by comparing the expression of HAX-1 in glioma tissues and nontumorous tissues. The study also analyzed the correlation between the expression of HAX-1 and clinicopathological characteristics of glioma and found the expression of HAX-1 to be highly related to the differentiation and World Health Organization stage of glioma tissues. The survival analysis revealed that HAX-1 was an independent prognostic factor. In conclusion, this novel study suggested that the overexpression of HAX-1 might contribute to the malignant progression of glioma.

Chemoresistance is associated with overexpression of HAX-1, inhibition of which resensitizes drug-resistant breast cancer cells to chemotherapy.

Acquired resistance to standard chemotherapy is the common and critical limitation for cancer therapy. Hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) has been reported to be upregulated in numerous cancers. However, the role of HAX-1 in oncotherapy remains unclear. In this study, we established MDA-MB-231 cell lines which were resistant to cisplatin (MDA-MB-231/CR) or doxorubicin (MDA-MB-231/DR) to study the chemoresistance in breast cancer. As a result, the HAX-1 which is an apoptosis-associated protein was observed to be overexpressed in both MDA-MB-231/CR and MDA-MB-231/DR compared with the routine MDA-MB-231 cells. Moreover, knockdown of HAX-1 via RNA interference decreased IC50 level of cisplatin by 70.91% in MDA-MB-231/CR cells, and the IC50 level of doxorubicin was decreased by 76.46% in MDA-MB-231/DR cells when the HAX-1 was downregulated. Additionally, we found that the knockdown of HAX-1 induced the release of cytochrome C from mitochondria, resulting in the activation of caspases. Taken together, our study indicates that the overexpression of HAX-1 is essential in the development of chemoresistance in breast cancer. Furthermore, we identify that HAX-1 may become the target for cancer therapy.

Endoplasmic reticulum (ER) stress triggers Hax1-dependent mitochondrial apoptotic events in cardiac cells.

Cardiomyocyte apoptosis is a major process in pathogenesis of a number of heart diseases, including ischemic heart diseases and cardiac failure. Ensuring survival of cardiac cells by blocking apoptotic events is an important strategy to improve cardiac function. Although the role of ER disruption in inducing apoptosis has been demonstrated, we do not yet fully understand how it influences the mitochondrial apoptotic machinery in cardiac cell models. Recent investigations have provided evidences that the prosurvival protein HCLS1-associated protein X-1 (Hax1) protein is intimately associated with the pathogenesis of heart disease, mitochondrial biology, and protection from apoptotic cell death. To study the role of Hax1 upon ER stress induction, Hax1 was overexpressed in cardiac cells subjected to ER stress, and cell death parameters as well as mitochondrial alterations were examined. Our results demonstrated that the Hax1 is significantly downregulated in cardiac cells upon ER stress induction. Moreover, overexpression of Hax1 protected from apoptotic events triggered by Tunicamycin-induced ER stress. Upon treatment with Tunicamycin, Hax1 protected from mitochondrial fission, downregulation of mitofusins 1 and 2 (MFN1 and MFN2), loss of mitochondrial membrane potential (∆Ψm), production of reactive oxygen species (ROS) and apoptotic cell death. Taken together, our results suggest that Hax1 inhibits ER stress-induced apoptosis at both the pre- and post-mitochondrial levels. These findings may offer an opportunity to develop new agents that inhibit cell death in the diseased heart.

Abstract 13122: Hax1 Mediates Hippo Pathway to Enhance Therapeutic Effect of Sca1 + Cardiac Stem Cell Against Myocardial Ischemia

Background: The Hippo pathway can regulate the fate and metabolic function of stem/progenitor cells through a kinase cascade that impinges on the transcription of growth-regulatory genes. This study investigates the role of HCLS-associated protein X-1 (HAX1) on the Hippo pathway with a goal of enhancing the therapeutic effects of sca1 + cardiac stem cells (CSCs sca1+ ) in mouse ischemic hearts. Methods and Results: Western Blot assessment showed that phosphorylation levels of Yes-associated protein (Yap), the terminal effector of Hippo pathway, was increased in CSCs sca1+ when endogenous HAX1 gene expression was silenced, whereas lentiviral over-expression of HAX1 maintained Yap activation by inhibiting its phosphorylation at Serine-397. ImageStream flow cytometry was then used to observe the enhanced nuclear translocation of Yap. Hypoxia exposure promoted this translocation to a greater extent in HAX1-overexpressing CSCs sca1+ (CSCs HAX1 ) than non-encoding DNA sequence-transduced CSCs sca1+ (CSCs Null ) ( Fig 1A ). HAX-1 over-expression enhanced cardioprotective molecular mechanisms by restraining the dissipation of mitochondrial membrane potential (Fig 1B) , inhibiting endoplasmic reticulum stress response, promoting HIF-1α expression, and upregulating anti-fibrotic gene expression in CSCs sca1+ response to hypoxia injury. Echocardiography and histological assessment demonstrated that contractile function was significantly improved and cardiac fibrosis was decreased in ischemic mouse hearts post engraftment of CSCs HAX1 when compared with those of the CSCs Null -transplanted group ( Fig 1C ). Conclusion: HAX1 over-expression can regulate Hippo signal and promote nuclear translocation of Yap, upregulating intrinsic protective mechanisms of CSCs sca1+ . This results in greater improvement of cardiac function in ischemic hearts post transplantation of HAX1 over-expressing CSCs sca1+ .

Protease Omi cleaving Hax-1 protein contributes to OGD/R-induced mitochondrial damage in neuroblastoma N2a cells and cerebral injury in MCAO mice.

In the penumbra after focal cerebral ischemia, an increase of protease Omi is linked to a decrease of Hs1-associated protein X-1 (Hax-1), a protein belonging to the Bcl-2 family. In this study we investigated the mechanisms underlying the regulation of Hax-1 by protease Omi in cerebral ischemia/reperfusion (I/R) injury. Mouse neuroblastoma N2a cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R); cell viability was assessed with MTT assay. Mice underwent 2-h middle cerebral artery occlusion (MCAO) and reperfusion, and the infarct volume was determined with TTC staining. The expression of Omi and Hax-1 was detected using immunoblot and immunofluorescence assays. The mitochondrial membrane potential was measured using TMRM staining. In the brains of MCAO mice, the protein level of Omi was significantly increased, while the protein level of Hax-1 was decreased. Similar changes were observed in OGD/R-treated N2a cells, but the mRNA level of Hax-1 was not changed. Furthermore, in OGD/R-treated N2a cells, knockdown of Omi significantly increased Hax-1 protein level. Immunofluorescence assay showed that Omi and Hax-1 were co-localized in mitochondria of N2a cells. OGD/R caused marked mitochondrial damage and apoptosis in N2a cells, while inhibition of Omi protease activity with UCF-101 (10 μmol/L) or overexpression of Hax-1 could restore the mitochondrial membrane potential and attenuate cell apoptosis. Moreover, pretreatment of MCAO mice with UCF-101 (7.15 mg/kg, ip) could restore Hax-1 expression, inhibit caspase activation, and significantly reduce the infarct volume. Protease Omi impairs mitochondrial function by cleaving Hax-1, which induces apoptosis in OGD/R-treated N2a cells and causes I/R injury in MCAO mice.

Analysis of HAX-1 gene expression in esophageal squamous cell carcinoma

ObjectiveTo explore the expression of HAX-1 mRNA and protein in esophageal squamous cell carcinoma (ESCC) and its relation with the prognosis of patients with ESCC.MethodsThe expression of HAX-1 mRNA and protein were detected with quantitative real-time RT-PCR and immunohistochemical method in 112 ESCC samples and 112 corresponding non-neoplastic samples. Survival curves were made with follow-up data. The relations of the prognosis with clinical and pathological characteristics were analyzed.ResultsThe expression level of HAX-1 mRNA and the strong positive rate of HAX-1 protein were significantly higher in ESCC samples (0.527 ± 0.060 and 45.54%) than that in non-neoplastic samples (0.121 ± 0.017 and 0.00%), and in ESCC samples with lymph node metastasis (0.554 ± 0.054 and 71.11%) than that in ESCC samples without lymph node metastasis (0.509 ± 0.058 and 28.36%) (all P < 0.01). HAX-1 mRNA expression level was a risk factor of lymph node metastasis in patients with ESCC (P = 0.000). There were significant differences in survival curves between lymph node metastatic group and non-metastatic group (P = 0.000), and among groups of HAX-1 protein expression +, ++and +++(,P = 0.000); but no statistical significance between male patients and female patients (P = 0.119), and between ≥60 years old patients and <60 years old patients (P = 0.705). The level of HAX-1 mRNA (P = 0.000) and protein (P = 0.005) were risk factors of survival, but lymph node metastasis (P = 0.477) was not.ConclusionThere is HAX-1 over-expression in ESCC tissue and HAX-1 mRNA level is a risk factor of lymph node metastasis. The level of HAX-1 mRNA and protein were risk factors of survival in patients with ESCC. HAX-1 may be a novel therapeutic target for ESCC treatment.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5130393079296037

Comment on “HAX1 Augments Cell Proliferation, Migration, Adhesion, and Invasion Induced by Urokinase-Type Plasminogen Activator Receptor”

Multifunctional HAX-1 protein is involved in the regulation of several key processes like calcium homeostasis, apoptosis, and cell migration. Clarification of its role in these or other processes and its molecular mechanisms of function remains to be established. Mekkawy et al. [1] report HAX-1 influence on cell proliferation, migration, adhesion, and invasion induced by the urokinase-type plasminogen activator receptor (uPAR), as a followup of the previous study, in which the authors demonstrated uPAR-HAX-1 interaction [2]. We would like to report our serious concerns about the HAX-1 expression system used in the study of Mekkawy et al., 2012 [1]. The authors use either pGEM-3Zf(+)HAX1 or pGEM-3Zf(+) as an empty vector to transfect human cell lines. These vectors are intended for in vitro transcription and propagation in bacteria; they are not mammalian expression vectors and do not possess the appropriate promoter sequences. pGEM-HAX-1 used in this study was obtained from Maria Olsson and is described in the work of Dufva et al. [3] as a vector generated for in vitro transcription by cloning HAX-1 coding region into pGEM-3Zf(+). Unless the vector was modified, we do not see the possibility of HAX-1 expression, other than endogenous, in the studied cell lines. Furthermore, no evidence of HAX-1 overexpression was provided (e.g., Western blot). Regarding these concerns, we consider the results indicating HAX-1 influence meaningless.

Novel Role of HAX-1 in Ischemic Injury Protection Involvement of Heat Shock Protein 90

Ischemic heart disease is characterized by contractile dysfunction and increased cardiomyocyte death, induced by necrosis and apoptosis. Increased cell survival after an ischemic insult is critical and depends on several cellular pathways, which have not been fully elucidated. To test the hypothesis that the anti-apoptotic hematopoietic lineage substrate-1-associated protein X-1 (HAX-1), recently identified as regulator of cardiac Ca cycling, also may ameliorate cellular injury with an ischemic insult. We report that cardiac ischemia/reperfusion injury is associated with significant decreases in HAX-1 levels ex vivo and in vivo. Accordingly, overexpression of HAX-1 improved contractile recovery, coupled with reduced infarct size, plasma troponin I level, and apoptosis. The beneficial effects were associated with decreased endoplasmic reticulum (ER) stress response through specific inhibition of the inositol-requiring enzyme (IRE-1) signaling pathway, including its downstream effectors caspase-12 and the transcription factor C/EBP homologous protein. Conversely, HAX-1 heterozygous-deficient hearts exhibited increases in infarct size and IRE-1 activity. The inhibitory effects of HAX-1 were mediated by its binding to the N-terminal fragment of the heat shock protein 90 (Hsp90). Moreover, HAX-1 sequestered Hsp90 from IRE-1 to the phospholamban-sarcoplasmic/endoplasmic reticulum calcium ATPase complex. The HAX-1 regulation was further supported by loss of IRE-1 inhibition in presence of the Hsp90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin. Cardiac ischemia-reperfusion injury is associated with decreases in HAX-1 levels. Consequently, overexpression of HAX-1 promotes cardiomyocyte survival, mediated by its interaction with Hsp90 and specific inhibition of IRE-1 signaling at the ER/sarcoplasmic reticulum.

RETRACTED ARTICLE: HAX-1 Promotes the Chemoresistance, Invasion, and Tumorigenicity of Esophageal Squamous Carcinoma Cells

HAX-1 is an anti-apoptotic factor and regulates the expression of DNA pol β. Interestingly, DNA polymerase pol β is overexpressed in esophageal squamous cell carcinoma (ESCC). However, the functional role of HAX-1 in ESCC remains unclear. To investigate the role of HAX-1 in chemoresistance, invasion, and tumorigenicity of ESCC. Lentivirus-mediated overexpression or knockdown of HAX-1 was employed to establish ESCC EC9706 cell lines that expressed HAX-1 at different levels. The biological behaviors of these engineered cells were characterized in vitro and in vivo using a xenograft nude mice model. In addition, HAX-1 and pol β expression in the tumor tissues was detected by RT-PCR and immunohistochemistry. HAX-1 overexpression promoted cell proliferation and resistance against cisplatin, increased cell invasion and suppressed apoptosis along with increased pol β expression. Conversely, HAX-1 knockdown inhibited the malignant phenotypes of EC9706 cells. The xenograft nude mice model demonstrated that HAX-1 overexpression or depletion led to increased or decreased tumor growth in vivo, respectively. Furthermore, a positive correlation of HAX-1 and pol β expression in the tumor tissues was observed. HAX-1 promotes the proliferation, chemoresistance, invasion, and tumorigenicity of ESCC, and this is correlated with increased poly β expression. HAX-1 may represent a potential target to overcome the resistance and metastasis of ESCC.

HAX1 Augments Cell Proliferation, Migration, Adhesion, and Invasion Induced by Urokinase-Type Plasminogen Activator Receptor

The urokinase-type plasminogen activator receptor (uPAR) is a cell surface receptor which has a multifunctional task in the process of tumorigenesis including cell proliferation, adhesion, migration, and invasion. Many of the biological functions of uPAR necessitate interactions with other proteins. We have shown previously that uPAR interacts with HAX1 protein (HS-1-associated protein X-1). In the current study, to gain insight into the possible role of HAX1 overexpression in regulation of uPAR signal transduction pathway, several function assays were used. We found that, upon stimulation of uPAR, HAX1 colocalizes with uPAR suggesting a physiological role for HAX1 in the regulation of uPAR signal transduction. HAX1 overexpression augments cell proliferation and migration in uPAR-stimulated cells. Moreover, HAX1 over-expression augmented uPAR-induced cell adhesion to vitronectin as well as cellular invasion. Our results suggest that HAX1 over-expression may underlay a novel mechanism to regulate uPAR-induced functions in cancer cells.