BackgroundHAX-1 has been described as a protein potentially involved in carcinogenesis and especially metastasis. Its involvement in regulation of apoptosis and cell migration along with some data indicating its overexpression in cancer cell lines and tumors suggests that HAX-1 may play a role in neoplastic transformation. Here we present the first systematic analysis of HAX-1 expression in several solid tumors.MethodsUsing quantitative RT-PCR, we have determined the mRNA levels of HAX1 splice variant I in several solid tumors. We have also analyzed by semiquantitative and quantitative RT-PCR the expression of five HAX-1 splice variants in breast cancer samples and in normal tissue from the same individuals. Quantitative PCR was also employed to analyze the effect of estrogen on HAX1 expression in breast cancer cell line. Immunohistochemical analysis of HAX-1 was performed on normal and breast cancer samples.ResultsThe results reveal statistically important HAX1 up-regulation in breast cancer, lung cancer and melanoma, along with some minor variations in the splicing pattern. HAX-1 up-regulation in breast cancer samples was confirmed by immunohistochemical analysis, which also revealed an intriguing HAX-1 localization in the nuclei of the tumor cells, associated with strong ER status.ConclusionHAX-1 elevated levels in cancer tissues point to its involvement in neoplastic transformation, especially in breast cancer. The connection between HAX-1 nuclear location and ER status in breast cancer samples remains to be clarified.
HAX-1 is a multi-functional protein that is involved in the regulation of apoptosis, cell motility and calcium homeostasis. It is also reported to bind RNA: it associates with structural motifs present in the 3′ untranslated regions of at least two transcripts, but the functional significance of this binding remains unknown. Although HAX-1 has been detected in various cellular compartments, it is predominantly cytoplasmic. Our detailed localization studies of HAX-1 isoforms revealed partial nuclear localization, the extent of which depends on the protein isoform. Further studies demonstrated that HAX-1 is in fact a nucleocytoplasmic shuttling protein, dependent on the exportin 1 nuclear export receptor. Systematic mutagenesis allowed identification of the two nuclear export signals in the HAX-1 sequence. HAX-1 nuclear accumulation was observed after inhibition of nuclear export by leptomycin B, but also after specific cellular stress. The biological role of HAX-1 nuclear localization and shuttling remains to be established, but the HAX-1 transcript-binding properties suggest that it may be connected to mRNA processing and surveillance. In this study, HAX-1 status was shown to influence mRNA levels of DNA polymerase b, one of the HAX-1 mRNA targets, although this effect becomes pronounced only after specific stress is applied. Moreover, HAX-1 tethering to the reporter transcript caused a significant decrease in its expression. Additionally, the HAX-1 co-localization with P-body markers, reported here, implies a role in mRNA processing. These results suggest that HAX-1 may be involved in the regulation of expression of bound transcripts, possibly as part of the stress response. Structured digital abstract• HAX1 and DCP1A, colocalize by fluorescence microscopy (View Interaction: 1, 2) • HAX1 physically interacts with XPO1 by anti tagcoimmunoprecipitation (View interaction) Abbreviations CPEB, cytoplasmic polyadenylation element-binding protein; Crm1, chromosome region maintenance 1; Dcp1a, mRNA-decapping enzyme 1A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HuR, human antigen R, ELAV-like protein 1; LMB, leptomycin B; NES, nuclear export signal; POLB, DNA polymerase b; Pat1b, protein PAT1 homolog 1; rck/p54, ATP dependent RNA helicase DDX6; TG, thapsigargin; XPO1, exportin1.
was originally synthesized as a specific inhibitor of chymotrypsin-like serine proteases (1) and has been widely used to investigate signal transduction pathways that are involved in gene expression and cell survival/cell death. However, contradictory effects of TPCK on apoptosis have been reported. Hence, TPCK was shown to prevent apoptosis in a cell death stimulus-dependent manner in some model systems (2-4), and a recent study also suggested that TPCK may diminish apoptosis by direct and nonspecific inhibition of active caspases (5). On the other hand, TPCK was also reported to potentiate apoptosis induced by certain apoptotic stimuli, and this was suggested to occur, at least in some cases, through activation of cell cycle checkpoints via inhibition of the proteasome (3, 6). TPCK alone may also induce apoptosis. TPCK thus caused a reduction in activity of the transcription factor, NF-B, in a murine B lymphoma cell line resulting in a decrease in c-myc and activation of apoptosis (7,8). Subsequent studies have shown that TPCK can induce apoptosis in a human acute monocytic leukemia cell line through activation of mitochondria-dependent apoptosis signaling (4, 9). Studies in transformed human T cells also indicated that TPCK can induce apoptosis through inhibition of constitutive NF-B activation, and it was proposed that TPCK might affect sulfhydryl groups on proteins involved in regulating cell survival and NF-B acti- 4 The abbreviations used are: TPCK, N ␣ -tosyl-L-phenylalanine chloromethyl ketone; z, N-benzyloxycarbonyl; fmk, fluoromethyl ketone; AIF, apoptosisinducing factor; cIAP1, cellular inhibitor of apoptosis protein-1; cIAP2, cellular inhibitor of apoptosis protein-2; AMC, 7-amino-4-methylcoumarin; DHE, dihydroethidium; ⌬ m , mitochondrial transmembrane potential; EBV, Epstein-Barr virus; HAX-1, HS1-associated protein X-1; IB, inhibitor of NF-B; NAC, N-acetylcysteine; NF-B, nuclear factor B; PARP, poly(ADPribose) polymerase; PI, propidium iodide; ROS, reactive oxygen species; SCN, severe congenital neutropenia; TMRE, tetramethylrhodamine ethyl ester; XIAP, X chromosome-linked inhibitor of apoptosis protein.
Kostmann disease or severe congenital neutropenia (SCN) is an autosomal recessive disorder of neutrophil production. Homozygous HAX1 mutations were recently identified in SCN patients belonging to the original family in northern Sweden described by Kostmann. Moreover, recent studies have suggested an association between neurological dysfunction and HAX1 deficiency. Here we describe a patient with a compound heterozygous HAX1 mutation consisting of a nonsense mutation (c.568C > T, p.Glu190X) and a frame-shift mutation (c.91delG, p.Glu31LysfsX54) resulting in a premature stop codon. The patient has a history of neutropenia and a propensity for infections, but has shown no signs of neurodevelopmental abnormalities.
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