HS1-associated Protein X-1 Research Articles

Ring Finger Protein 217 Inhibits Ovarian Cancer Progression by Down‐Regulating HAX1 Expression

AbstractRing finger protein 217 (RNF217) has been found to interact with the antiapoptotic protein HS‐1‐associated protein X‐1 (HAX‐1) in myeloid leukemia cells. However, the understanding of RNF217 in ovarian cancer progression remains limited. The relative expression of RNF217 is screened in ovarian cancer using the GEPIA database and calculated its correlation with MKI67, CCNB1, and CDK4. OVCAR‐3 and SK‐OV‐3 cells are transfected with RNF217‐overexpression plasmids. Cell‐counting kit‐8 assay is utilized to assess proliferation. Immunoprecipitation is performed to reveal the interaction between RNF217 and HAX‐1, and a cycloheximide chase assay is performed to analyze HAX‐1 degradation. The GEPIA database indicated down‐regulated expression of RNF217 in ovarian cancer, negatively correlated with MKI67 (R = ‐0.26, P = 1.8e‐09), CCNB1 (R = ‐0.37, P = 3.2e‐18), and CDK4 expression (R = ‐0.24, P = 3.4e‐08). RNF217 overexpression down‐regulated the relative expression of MKI67, CCNB1, and CDK4 in OVCAR‐3 and SK‐OV‐3 cells, resulting in diminished proliferation. In vivo studies using OVCAR‐3 and SK‐OV‐3 cell line‐derived xenograft models also showed that RNF217 overexpression reduced ovarian cancer volume and weight. Furthermore, RNF217 overexpression in SK‐OV‐3 cells inhibited the protein expression of HAX1 by reducing its stability. In conclusion, RNF217 inhibits ovarian cancer progression by down‐regulating HS‐1‐associated protein X‐1 expression.

African swine fever virus MGF360-9L promotes viral replication by degrading the host protein HAX1

African swine fever virus (ASFV) infection causes African swine fever (ASF), a virulent infectious disease that threatens the safety of livestock worldwide. Studies have shown that MGF360–9 L is important for the virulence of ASFV and the host protein HS1-associated protein X-1 (HAX1) plays an important role in viral pathogenesis. This study aimed to clarify the mechanism by which HAX1 mediates ASFV replication through interactions with MGF360–9 L. The regions of interaction between MGF360–9 L and HAX1 were predicted and validated. HAX1 overexpression and RNA interference studies revealed that HAX1 is a host restriction factor that suppresses ASFV replication. Moreover, HAX1 expression was inhibited in ASFV-infected mature bone marrow–derived macrophages, and infection with the virulent MGF360–9 L gene deletion strain (∆MGF360–9 L) attenuated the inhibitory effect of the wild-type strain (WT) on HAX1 expression, suggesting a complex regulatory relationship between MGF360–9 L and HAX1. Furthermore, the E3 ubiquitin ligase RNF114 interacted with MGF360–9 L and HAX1, MGF360–9 L degraded HAX1 through the ubiquitin–proteasome pathway, and RNF114 facilitated the degradation of HAX1 by MGF360–9L-linked K48 ubiquitin chains through the ubiquitin–proteasome pathway, thereby facilitating ASFV replication. In conclusion, this study has enriched our understanding of the regulatory networks between ASFV proteins and host proteins and provided a reference for investigation into the pathogenesis and immune escape mechanism of ASFV.

CircGFPT1 regulates the growth and apoptosis of esophageal squamous cell carcinoma through miR-142-5p/HAX1 axis.

Currently, multiple circular RNAs (circRNAs) have been verified to act as essential regulators in the progression of esophageal squamous cell carcinoma (ESCC). However, there is no study regarding the role of circGFPT1 in the progression of cancers including ESCC. We aimed to investigate the role of circGFPT1 in ESCC progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to measure the expression of circGFPT1, miR-142-5p and HS1-associated protein X-1 (HAX1). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and 5-ethynyl-2'-deoxyuridine (EdU) assays were employed to evaluate cell proliferation. Cell migration and invasion were detected by wound-healing and transwell assays. Flow cytometry analysis was conducted to assess cell apoptosis. The protein expression of E-cadherin, N-cadherin, Vimentin, C-caspase3, HAX1 and nuclear proliferation marker (Ki67) was analyzed by western blot or immunohistochemistry assay. CircGFPT1 was up-regulated in ESCC tissues and cells. Silencing of circGFPT1 repressed cell proliferation and induced cell apoptosis in ESCC cells. CircGFPT1 acted as a sponge of miR-142-5p. The effects of circGFPT1 knockdown on ESCC cell proliferation and apoptosis were reversed by miR-142-5p inhibition. HAX1 was confirmed to be a target gene of miR-142-5p. CircGFPT1 knockdown inhibited HAX1 expression by targeting miR-142-5p. Additionally, circGFPT1 knockdown hampered tumorigenesis in vivo. CircGFPT1 promoted ESCC cell growth and repressed apoptosis by up-regulating HAX1 through sponging miR-142-5p.

Long Noncoding RNA Cytoskeleton Regulator RNA Suppresses Apoptosis in Hepatoma Cells by Modulating the miR-125a-5p/HS1-Associated Protein X-1 Axis to Induce Caspase-9 Inactivation.

The involvement of long noncoding RNAs in the carcinogenesis of hepatocellular carcinoma (HCC) has been well documented by substantial evidence. However, whether cytoskeleton regulator RNA (CYTOR) could affect the progression of HCC remains unclear. The relative expression of CYTOR, miR-125a-5p and HS1-associated protein X-1 (HAX-1) mRNA in HCC cells were determined via quantitative real-time polymerase chain reaction. The viability of treated HCC cells was measured by Cell Counting Kit-8 assay. Cell apoptosis was estimated by flow cytometry analysis, assessment of caspase-9 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, and Western blot of apoptosis-related proteins. The interplay between CYTOR or HAX-1 and miR-125a-5p was validated by dual-luciferase reporter assay. CYTOR was upregulated and miR-125a-5p was downregulated in HCC cells. CYTOR silencing inhibited cell proliferation and promoted cell apoptosis in HepG2 and SMMC-7721 cells. miR-125a-5p was sponged and negatively regulated by CYTOR, and HAX-1 was directly targeted and negatively modulated by miR-125a-5p. Overexpression of miR-125a-5p enhanced the repressive effects of CYTOR knockdown on HCC cells, and knockdown of HAX-1 enhanced the inhibitory effects of miR-125a-5p mimics on HCC cells. CYTOR silencing facilitates HCC cell apoptosis in vitro via the miR-125a-5p/HAX-1 axis.

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca2+ Handling and Arrhythmogenesis.

Phospholamban (PLN), a key modulator of Ca2+-homeostasis, inhibits sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) and regulates cardiac contractility. The human PLN mutation R14del has been identified in arrhythmogenic cardiomyopathy patients worldwide and is currently extensively investigated. In search of the molecular mechanisms mediating the pathological phenotype, we examined PLN-R14del associations to known PLN-interacting partners. We determined that PLN-R14del interactions to key Ca2+-handling proteins SERCA2a and HS-1-associated protein X-1 (HAX-1) were enhanced, indicating the super-inhibition of SERCA2a’s Ca2+-affinity. Additionally, histidine-rich calcium binding protein (HRC) binding to SERCA2a was increased, suggesting the inhibition of SERCA2a maximal velocity. As phosphorylation relieves the inhibitory effect of PLN on SERCA2a activity, we examined the impact of phosphorylation on the PLN-R14del/SERCA2a interaction. Contrary to PLN-WT, phosphorylation did not affect PLN-R14del binding to SERCA2a, due to a lack of Ser-16 phosphorylation in PLN-R14del. No changes were observed in the subcellular distribution of PLN-R14del or its co-localization to SERCA2a. However, in silico predictions suggest structural perturbations in PLN-R14del that could impact its binding and function. Our findings reveal for the first time that by increased binding to SERCA2a and HAX-1, PLN-R14del acts as an enhanced inhibitor of SERCA2a, causing a cascade of molecular events contributing to impaired Ca2+-homeostasis and arrhythmogenesis. Relieving SERCA2a super-inhibition could offer a promising therapeutic approach for PLN-R14del patients.

Long chain noncoding RNA-ROR promotes hypoxic injury of cardiomyocytes by targeting the miR-145/HAX-1 axis.

BackgroundLong non-coding RNAs (lncRNAs) are a class of non-protein coding RNAs greater than 200 nucleotides (nt) in length which have been shown to be significantly highly expressed in the heart tissue of mice undergoing thoracic aortic arch constriction (TAC). Micro RNAs (miRNAs) are a class of non-protein-coding RNAs. Many miRNAs have been reported to play a key role in the progression of myocardial hypertrophy. In this study, we aimed to investigate whether lncRNA reprogramming regulators (ROR) promotes hypoxic injury in cardiomyocytes by targeting and regulating the miR-145/HS1-associated protein X-1 (HAX-1) axis.MethodsA mouse model of myocardial hypertrophy was established by conventional TAC method, and the cardiomyocytes were isolated. We transfected pcDNA3.0-ROR vector, pcDNA3.0-HAX-1 vector plasmid, and miR-145 simulant into cardiomyocytes with Lipofectamine 2000. Luciferase reporter gene was used to analyze the targeting relationship between genes.ResultsThe expression of ROR in hypertrophic myocardium was significantly increased after phenylephrine (PE) intervention. After transfection with si-ROR, the ROR expression in hypertrophic cardiomyocytes treated with PE decreased significantly. Levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), creatine kinase (CK) decreased and superoxide dismutase (SOD) increased. The expression of miR-145 in cardiomyocytes was significantly down-regulated after PE treatment. In hypertrophic cardiomyocytes, after up-regulating the expression of miR-145, the relative messenger ribonucleic acid (mRNA) and protein expressions of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) induced by PE decreased. Compared with the miR-NC group, wild type (WT)-ROR activity in the miR-145 group was significantly inhibited (P<0.05), and mutant (MUT)-ROR activity had no significant change (P>0.05). When cardiomyocytes were transfected with HAX-1 3'URT WT vector along with miR-145 simulant, miR-145 inhibitor, and their respective controls. Compared with the control groups, the luciferase activity of cells transfected with simulant was significantly decreased (P<0.05), and increased in inhibitor group (P<0.05). Transfection of HAX-1 3'URT mutant vector did not show this phenomenon. ROR was negatively correlated with miR-145 expression and positively correlated with HAX-1 mRNA.ConclusionsThe lncRNA ROR can promote the expression of HAX-1 by competitive binding with miR-145, so as to promote the pathophysiological process of myocardial hypertrophy.

Effect of different expression patterns of HAX-1 on the proliferation and apoptosis of human astrocyte

Spinal cord injury (SCI), a serious damage of the central nervous system, has become an extremely important issue that threatens the health of people worldwide. The proliferation of astrocytes plays an important role in the repair of SCI, which has typical two-sided effects. The HS1-associated protein X-1 (HAX-1), plays an important role in the physiological and pathological processes of cell apoptosis, proliferation, migration, and invasion. However, the specific role and mechanism of HAX-1 in human astrocyte HA1800 are still unclear.To explore the effect of HAX-1 on the proliferation and apoptosis of HA1800 cells and preliminarily explore its possible underlying mechanism.The HA1800 cell lines with highand low-expression levels of HAX-1 were established using lentiviral vector pcDNA3.1. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were employed to determine the expression of HAX-1 after transfection. Cell viability and proliferation ability were estimated using MTT and 5-Ethynyl-2'deoxyuridine (EdU) assay. The effects of HAX-1 on the HA1800 cell cycle and apoptosis were determined using flow cytometry. The BCL-2/BAX ratio and the expression of Ki67 and c-Myc in the transfected cells were detected using qRT-PCR. The Gene Expression Profiling Interactive Analysis (GEPIA) database was used to determine the relationships of HAX-1, BAX and BCL-2.The HA1800 cell lines with high and low expression of HAX-1 were obtained. The MTT, EdU and flow cytometry showed that elevated HAX-1 could inhibit the proliferation, reduce the viability and promote the apoptosis of HA1800 cells. The qRT-PCR showed that the mRNA levels of Ki67, c-Myc and the BCL-2/BAX ratio were significantly decreased in the HAX-1 high-expression group, but increased in the HAX-1 low-expression group. The results from the GEPIA database showed that HAX-1 was positively correlated with BAX and BCL-2 in the spinal cord.The HAX-1 may influence the biological behavior of human HA1800 cells due to the progression of cell cycle and apoptosis associated with BCL-2/BAX.

The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1.

Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells.

Expression of Integrin β6 and HAX-1 Correlates with Aggressive Features and Poor Prognosis in Esophageal Squamous Cell Carcinoma.

PurposeThe development of esophageal squamous cell carcinoma (ESCC) is a complicated process in which cell adhesion and motility, mediated by integrins, are involved through connecting the cytoskeleton to extracellular matrix. Different mechanisms via which integrin β6 participates in cancer invasion and metastasis have been described by numerous studies; however, the expression and clinical significance of integrin β6 in ESCC remain unknown.MethodsTo investigate the differential expression of integrin β6 in ESCC, qPCR and immunohistochemistry assays were performed in 10 paired human samples. A total of 137 ESCC samples were further enrolled to evaluate the expression levels of integrin β6 and its endocytic trafficking regulator HS1-associated protein X-1 (HAX-1), followed by the evaluation of their correlation with clinicopathological parameters. The overall survival was analyzed using the Kaplan–Meier method, with significant variables further evaluated by multivariate Cox regression analyses.ResultsThe expression of integrin β6 was markedly increased in ESCC compared with matched adjacent normal tissues. Among the ESCC samples, positive expression of integrin β6 was observed in 41.6% tumors, which was associated with histological differentiation, lymph node metastasis and TNM stage. High expression of HAX-1 was detected in 47.4% tumors, and there was a positive relationship between the expression levels of integrin β6 and HAX-1. Furthermore, the expression of integrin β6 and HAX-1 were independent unfavorable indicators for prognosis. Patients with positive integrin β6 and high HAX-1 expression demonstrated worst outcomes.ConclusionThe present findings suggested the predictive value of integrin β6 and HAX-1 as independent indicators of poor prognosis for patients with ESCC, both of which may contribute to the tumor proliferation and metastasis, leading to ESCC progression. Therefore, combined targeting of integrin β6 and HAX-1 may provide a potential novel approach for the treatment of ESCC.

HTLV-1 viral oncoprotein HBZ contributes to the enhancement of HAX-1 stability by impairing the ubiquitination pathway.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia (ATL). The viral protein HTLV-1 basic leucine-zipper factor (HBZ), which is constitutively expressed in all ATL patient cells, contributes toward the development of ATL; however, the underlying mechanism has not been elucidated yet. Here, we identified HS-1-associated protein X-1 (HAX-1) as a novel binding partner of HBZ. Interestingly, HAX-1 specifically associated with HBZ-US, but not HBZ-SI, in the cytoplasm. HBZ suppressed the polyubiquitination levels of HAX-1 protein by inhibiting the association HAX-1 with F-box protein 25 (FBXO25), which is a member of the SCF E3 ubiquitin ligase complex, and promoted the stabilization of HAX-1 levels. In fact, the protein levels of HAX-1 were significantly increased in HTLV-1 infected and the overexpressing HBZ in uninfected T-cell lines. Enhanced HAX-1 correlated well to suppression of caspase 9 processing, suggesting that HBZ may contribute to the enhancement of antiapoptotic function for HAX-1. Our results revealed a role for HBZ on HAX-1 stabilization by abrogating the ubiquitination-mediated degradation pathway, which may play an important role in understanding the potential mechanisms of HTLV-1 related pathogenesis.

Accumulation of HAX-1 and PARL in brainstem- and cortical-type Lewy bodies in Parkinson's disease and dementia with Lewy bodies

HS1-associated protein X-1 (HAX-1) and presenilin-associated rhomboid-like protein (PALR) were reported to play an important role in the activation of HtrA2/Omi, which is also designated PARK13, in the mitochondria. To elucidate the role of HAX-1 and PARL in patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB), we performed immunohistochemical studies on HtrA2/Omi, HAX-1 and PARL using autopsied brains from 8 normal subjects, 10 patients with PD and 5 patients with DLB. In accordance with our previous report, brainstem-type and cortical Lewy bodies were strongly immunopositive for HtrA2/Omi. In the normal brains, HAX-1 and PARL immunoreactivities were observed in various types of neurons in the cerebral cortex, midbrain, and upper pons. HAX-1 and PARL immunoreactivities were also observed in the remaining neurons, and brainstem-type and cortical Lewy bodies were intensely immunoreactive for HAX-1 and PARL. Both immunoreactivities were localized to the halo or core of brainstem-type Lewy bodies. Our results suggest that brainstem-type and cortical Lewy bodies may contain HAX-1 and PARL as well as HtrA2/Omi, and that these proteins may partially contribute to the formation of Lewy bodies and may be associated with the pathogenesis of PD and DLB.

Frataxin deficiency in Friedreich's ataxia is associated with reduced levels of HAX-1, a regulator of cardiomyocyte death and survival.

Frataxin deficiency, responsible for Friedreich's ataxia (FRDA), is crucial for cell survival since it critically affects viability of neurons, pancreatic beta cells and cardiomyocytes. In FRDA, the heart is frequently affected with typical manifestation of hypertrophic cardiomyopathy, which can progress to heart failure and cause premature death. A microarray analysis performed on FRDA patient's lymphoblastoid cells stably reconstituted with frataxin, indicated HS-1-associated protein X-1 (HAX-1) as the most significantly upregulated transcript (FC = +2, P < 0.0006). quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and western blot analysis performed on (I) HEK293 stably transfected with empty vector compared to wild-type frataxin and (II) lymphoblasts from FRDA patients show that low frataxin mRNA and protein expression correspond to reduced levels of HAX-1. Frataxin overexpression and silencing were also performed in the AC16 human cardiomyocyte cell line. HAX-1 protein levels are indeed regulated through frataxin modulation. Moreover, correlation between frataxin and HAX-1 was further evaluated in peripheral blood mononuclear cells (PBMCs) from FRDA patients and from non-related healthy controls. A regression model for frataxin which included HAX-1, group membership and group* HAX-1 interaction revealed that frataxin and HAX-1 are associated both at mRNA and protein levels. Additionally, a linked expression of FXN, HAX-1 and antioxidant defence proteins MnSOD and Nrf2 was observed both in PBMCs and AC16 cardiomyocytes. Our results suggest that HAX-1 could be considered as a potential biomarker of cardiac disease in FRDA and the evaluation of its expression might provide insights into its pathogenesis as well as improving risk stratification strategies.

HAX-1 promotes the migration and invasion of hepatocellular carcinoma cells through the induction of epithelial–mesenchymal transition via the NF-κB pathway

The expression of HS-1-associated protein X-1 (HAX-1) plays a major role in the development of hepatocellular carcinoma (HCC). However, the function of HAX-1 in HCC metastasis is unclear. Quantitative real-time PCR and western blotting were used to examine HAX-1 expression in HCC cell lines with different metastatic potential, and in tumor tissues with or without intrahepatic metastasis. HCC tissue arrays (n = 144) were used to assess correlations between clinicopathological parameters and HAX-1 expression. We also examined the effect of HAX-1 on promoting HCC cell metastasis in vivo and in vitro. The results showed that the expression levels of HAX-1 were higher in metastatic HCC cell lines than in non-metastatic HCC cell lines. HAX-1 was also significantly upregulated in primary HCC tissues with intrahepatic metastasis compared with those without intrahepatic metastasis. HCC in patients with high HAX-1 expression is more likely to metastasize. HAX-1 expression was associated with malignant progression and poor prognosis, and HAX1 silencing inhibited HCC cell migration and invasion in vitro and decreased HCC cell lung metastasis in vivo, whereas HAX-1 overexpression had the inverse effect. Moreover, HAX-1 increased HCC cell metastasis by promoting the epithelial–mesenchymal transition (EMT) process. Finally, we revealed that HAX-1 modulated EMT in HCC cells by increasing NF-κB/p65 nuclear translocation. In conclusion, HAX-1 promotes HCC metastasis by EMT through activating the NF-κB pathway, suggesting that HAX-1 could be a potential therapeutic target for HCC treatment.

HAX1 is associated with neuronal apoptosis and astrocyte proliferation after spinal cord injury.

HS1-associated protein X-1 (HAX1) is a class of multifunctional protein, participated in various physiological processes such as cell apoptosis, proliferation and motility. However, the HAX1 expression and function in the spinal cord injury (SCI) pathological process have not been investigated. In our current research, the rat model of SCI was established, and then we explored the possible role of HAX1 after SCI. The results of western blot indicated that HAX1 was present in sham operated control group and significantly elevated at 3 days post SCI, then declined gradually. Immunohistochemical studies indicated HAX1 expression was enhanced significantly in white and gray matter at 3 days post SCI compared with sham operated group. Double immunofluorescence staining showed the proportion of cells, double-labeled HAX1 and neurons, astrocytes, increased significantly at 3 days post SCI. In addition, co-localization of HAX1/active caspase-3 and HAX1/PCNA was tested in cells. Furthermore, over-expression of HAX1 inhibited neuronal apoptosis in vitro, and in astrocytes HAX1 silencing could down-regulate PCNA expression post LPS treatment. Meanwhile, CCK8 assay showed that knockdown of HAX1 could inhibit the astrocyte proliferation. In summary, our data indicated that HAX1 might play significant roles in pathological process of neuronal apoptosis and astrocyte proliferation during SCI.

MLF1 is a proapoptotic antagonist of HOP complex-mediated survival

In the HAX1/HtrA2-OMI/PARL (HOP) mitochondrial protein complex, anti-apoptotic signals are generated by cleavage and activation of the serine protease HtrA2/OMI by the rhomboid protease PARL upon recruitment of both proteases to inner mitochondrial membrane protein HAX1 (HS1-associated protein X-1). Here we report the negative regulation of the HOP complex by human leukemia-associated myeloid leukemia factor 1 (MLF1). We demonstrate that MLF1 physically and functionally associates with HAX1 and HtrA2. Increased interaction of MLF1 with HAX1 and HtrA2 displaces HtrA2 from the HOP complex and inhibits HtrA2 cleavage and activation, resulting in the apoptotic cell death. Conversely, over-expressed HAX1 neutralizes MLF1's effect and inhibits MLF1-induced apoptosis. Importantly, Mlf1 deletion reverses B- and T-cell lymphopenia and significantly ameliorates the progressive striatal and cerebellar neurodegeneration observed in Hax1−/− mice, with a doubling of the lifespan of Mlf1−/−/Hax1−/− animals compared to Hax1−/− animals. Collectively, these data indicate that MLF1 serves as a proapoptotic antagonist that interacts with the HOP mitochondrial complex to modulate cell survival.

Grb7 and Hax1 may colocalize partially to mitochondria in EGF-treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1.

Growth factor receptor bound protein 7 (Grb7) is a signal-transducing adaptor protein that mediates specific protein-protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein-binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras-GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti-apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor-treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time-dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd.

Clinical and biological significance of HAX-1 overexpression in nasopharyngeal carcinoma.

HS1-associated protein X-1 (HAX-1) is an important marker in many types of cancers and contributes to cancer progression and metastasis. We examined the expression of HAX-1 in nasopharyngeal carcinoma (NPC) and experimentally manipulated its expression. We observed that HAX-1 expression is elevated in NPC and is correlated with lymph node metastasis, M classification, clinical stage, and poor prognosis. In addition, overexpression of HAX-1 promoted NPC proliferation both in vitro and in vivo. Exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. We found that NPC-derived exosomes are enriched in HAX-1 and accelerate NPC tumor growth and angiogenesis in vitro and in vivo. Furthermore, we demonstrated that oncogenic HAX-1 facilitates the growth of NPC when it is transferred via exosomes to recipient human umbilical vein endothelial cells (HUVECs). Oncogenic HAX-1 also increases the proliferation, migration, and angiogenic activity of HUVECs. Our findings provide unique insight into the pathogenesis of NPC and underscore the need to explore novel therapeutic targets such as HAX-1 to improve NPC treatment.

HAX-1 inhibits apoptosis in prostate cancer through the suppression of caspase-9 activation.

HS1 associated protein X-1 (HAX-1), a substrate of Src family tyrosine kinases, plays a critical role in cell apoptosis. However, its functions in prostate cancer remains unclear. The present study explored the role and mechanism of HAX-1 in cancer cell apoptosis. The mRNA and protein levels of HAX-1 in the prostate cancer cell lines PC-3, VCaP and DU145 were assessed. Cell proliferation, apoptosis and caspase-9 activities were assessed in DU145 after HAX-1 siRNA treatment. The mRNA and protein levels of HAX-1 in prostate cancer cell lines PC-3, VCaP and DU145 were significantly higher than those in the primary prostate epithelial cells, and DU145 possess the highest mRNA and protein levels compared to PC-3 and VCaP. When HAX-1 was knocked down in DU145, cell proliferation was significantly decreased, accompanied by a decrease in Ki67 protein expression. Compared with the control and control siRNA groups, HAX-1 siRNA promoted cell apoptosis and caspase-9 activation in DU145. Furthermore, prostate cancer cells co-transfected with HAX-1 and caspase-9 promoted viability and reduced apoptosis. In contract, co-transfection of caspase-9 and HAX-1 siRNA suppressed the cell viability and enhanced apoptosis. In summary, the present study demonstrated that HAX-1 inhibits cell apoptosis through caspase-9 inactivation.

Clinical significance of HAX-1 expression in laryngeal carcinoma.

HS1-associated protein X-1 (HAX-1) is a multifunctional protein that has been highlighted as an important marker in many types of cancers. However, little is known about the role of HAX-1 in laryngeal carcinoma. The purpose of the present study is to explore HAX-1 expression status and its associations with clinicopathologic features and survival in a well-defined cohort of laryngeal carcinoma. We examined the expression of HAX-1 at protein and mRNA levels in laryngeal carcinoma tissues and adjacent non-tumor tissues by immunohistochemistry, Western blotting and two-step quantitative real-time PCR analysis, respectively. We observed that HAX-1 was significantly elevated in laryngeal carcinoma. The relationship between the levels of HAX-1 expression and clinicopathologic characteristics was then analyzed. Overexpression of HAX-1 was significantly correlated with T classification, lymph node metastasis, clinical stage, and pathology. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. We find that patients with overexpression of HAX-1 had shorter overall survival rates. Finally, the significance of various survival variables was analyzed using multivariate Cox proportional hazards model. We found that overexpression of HAX-1 was an independent prognostic factor for patients with laryngeal carcinoma. Our findings hinted that overexpression of HAX-1 was a potentially unfavorable factor in the progression and prognosis of laryngeal carcinoma.

HAX1 regulates E3 ubiquitin ligase activity of cIAPs by promoting their dimerization.

HS-1-associated protein X-1 (HAX1) is a multi-functional protein which was first identified as a Hematopoietic cell specific Lyn Substrate 1 (HS1)-binding protein. Although the roles of HAX1 in apoptosis have been unraveled and HAX1 has been proposed to be involved in several diseases, additional roles of HAX1 are still being identified. Here, we demonstrated that HAX1 directly interacted with cellular Inhibitor of Apoptosis Proteins (cIAPs), ubiquitin E3 ligases which regulate the abundance of cellular proteins, via ubiquitin-dependent proteasomal degradation. We showed that HAX1 promotes auto-ubiquitination and degradation of cIAPs by facilitating the intermolecular homodimerization of RING finger domain. Moreover, HAX1 regulates the non-canonical Nuclear Factor-κB (NF-κB) signaling pathway by modulating the stability of NF-κB-Inducing Kinase (NIK), which is one of the substrates of cIAPs. Taken together, these results unveil a novel role of HAX1 in the non-canonical NF-κB pathway, and provide an important clue that HAX1 is a potential therapeutic target for the treatment of cancer.