Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Mar 2:10:76.
doi: 10.1186/1471-2407-10-76.

HAX-1 overexpression, splicing and cellular localization in tumors

Alicja Trebinska  1 , Alina Rembiszewska, Karolina Ciosek, Konrad Ptaszynski, Sebastian Rowinski, Jolanta Kupryjanczyk, Janusz A Siedlecki, Ewa A Grzybowska
Affiliations

HAX-1 overexpression, splicing and cellular localization in tumors

Alicja Trebinska et al. BMC Cancer. .

Abstract

Background: HAX-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.

Methods: Using 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.

Results: The 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.

Conclusion: HAX-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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
HAX1 (splice variant I) expression levels in eight different solid tumors. HAX1 expression is significantly elevated in breast (A), lung (B) and colon (C) cancers, while in kidney (D), liver (E), ovary (F), prostate (G) and thyroid (H) cancers up-regulation was not detected. Quantitative data from cDNA panels were analyzed using the nonparametric Mann-Whitney test. Statistical significance was denoted under the box-whiskers plots for values of P < 0.05. In breast and lung cancers significant overexpression was observed for the advanced stages (III-IV), while in colon cancer for early stages (I-II).
Figure 2
Figure 2
HAX1 (splice variant I) expression analysis in disease-focused panels representing breast cancer (A), lung cancer (B) and melanoma (C). Data were analyzed using non-parametric Mann-Whitney test and statistical significance for values of P < 0.05 was denoted under the plots. For breast cancer samples significant overexpression level was detected in all stages (I, II, III and IV). Analysis of the lung cancer samples show significant overexpression for stages I and IV. For melanoma, HAX1 expression in all analyzed stages was significantly up-regulated (only stages III and IV were analyzed).
Figure 3
Figure 3
Alternative splicing of the human HAX1. Only five splice variants, generating putative protein product maintained in the same reading frame are depicted. Variants are named as in Carlsson et al., 2008 [25], nomenclature from Lees et al., 2008 [26] in brackets. The location of the variant-specific primers is depicted by arrows.
Figure 4
Figure 4
Expression of the five HAX1 splice variants detected by semi-quantitative PCR. A. Expression of the five HAX1 splice variants detected in the normal breast tissue reveals significant differences in expression levels, showing the prevalence of splice variant I. Normal breast tissue cDNA, splice variants I-V, 30 and 40 cycles. B. HAX1 splice variants expression in all 15 patients. The most marked difference was detected for variant III, the amplification of which was observed only in tumors in eight cases. In the remaining 7 cases no amplification was detected, except for patient 13, where a weak band was detected in the normal tissue sample, but not in the tumor sample. Higher amplification in tumors can be also observed in some patients for variant V (patients 1, 5, 7, 8, 11,13).
Figure 5
Figure 5
Quantitative analysis of the expression of HAX1 variants I and II. A. Expression of variant I is significantly higher than variant II, in both, normal and tumor tissues, but variant I/variant II expression ratio is higher in normal tissues (median 15) than in tumors (median 9), due to a relatively higher overexpression of variant II in tumors. B. Relative overexpression measured for variant I and variant II, calculated as a tumor/normal ratio. Overexpression, (expression higher than 1.5 fold) was found in 47% and 73% of cases for variants I and II, respectively. Dashed line indicates the level of expression in normal tissues. Order of patients according to the stage of the disease.
Figure 6
Figure 6
Estrogen has no effect on HAX1 expression. Preconditioned cells were treated with the increasing concentrations of beta-estradiol for 48 h. mRNA levels of HAX1 and estrogen-dependent cathepsin D mRNA were measured by quantitative PCR. A. Estrogen-dependent breast cancer cell line MCF-7. B. HeLa cell line.
Figure 7
Figure 7
Representative immunohistochemical staining of the analyzed breast cancer samples shows elevated levels of HAX-1 in tumor tissues. Each column represents samples from one patient. HAX-1 staining was detected in the nuclei (A) and the cytoplasm (B) of tumor cells (magnification × 400). Nuclear HAX-1 localization was observed only in the strongly ER-positive samples (C), while in the samples with cytoplasmic HAX-1 staining ER expression was weak (D). Control samples incubated with mouse IgG of the same subclasses and concentrations as the primary antibody (E and F) were negative. Normal samples from the analyzed two patients (G and H) do not show visible HAX-1 staining.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sharp TV, Wang HW, Koumi A, Hollyman D, Endo Y, Ye H, Du MQ, Boshoff C. K15 protein of Kaposi's sarcoma-associated herpesvirus is latently expressed and binds to HAX-1, a protein with antiapoptotic function. Journal of virology. 2002;76(2):802–816. doi: 10.1128/JVI.76.2.802-816.2002. - DOI - PMC - PubMed
    1. Han Y, Chen YS, Liu Z, Bodyak N, Rigor D, Bisping E, Pu WT, Kang PM. Overexpression of HAX-1 protects cardiac myocytes from apoptosis through caspase-9 inhibition. Circulation research. 2006;99(4):415–423. doi: 10.1161/01.RES.0000237387.05259.a5. - DOI - PubMed
    1. Chao JR, Parganas E, Boyd K, Hong CY, Opferman JT, Ihle JN. Hax1-mediated processing of HtrA2 by Parl allows survival of lymphocytes and neurons. Nature. 2008;452(7183):98–102. doi: 10.1038/nature06604. - DOI - PubMed
    1. Matsuda G, Nakajima K, Kawaguchi Y, Yamanashi Y, Hirai K. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) forms complexes with a cellular anti-apoptosis protein Bcl-2 or its EBV counterpart BHRF1 through HS1-associated protein X-1. Microbiology and immunology. 2003;47(1):91–99. - PubMed
    1. Kasashima K, Ohta E, Kagawa Y, Endo H. Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2. The Journal of biological chemistry. 2006;281(47):36401–36410. doi: 10.1074/jbc.M605260200. - DOI - PubMed

Publication types

Substances

LinkOut - more resources