HEX expression and localization in normal mammary gland and breast carcinoma

Abstract

Background: The homeobox gene HEX is expressed in several cell types during different phases of animal development. It encodes for a protein localized in both the nucleus and the cytoplasm. During early mouse development, HEX is expressed in the primitive endoderm of blastocyst. Later, HEX is expressed in developing thyroid, liver, lung, as well as in haematopoietic progenitors and endothelial cells. Absence of nuclear expression has been observed during neoplastic transformation of the thyroid follicular cells. Aim of the present study was to evaluate the localization and the function of the protein HEX in normal and tumoral breast tissues and in breast cancer cell lines.

Methods: HEX expression and nuclear localization were investigated by immunohistochemistry in normal and cancerous breast tissue, as well as in breast cancer cell lines. HEX mRNA levels were evaluated by real-time PCR. Effects of HEX expression on Sodium Iodide Symporter (NIS) gene promoter activity was investigated by HeLa cell transfection.

Results: In normal breast HEX was detected both in the nucleus and in the cytoplasm. In both ductal and lobular breast carcinomas, a great reduction of nuclear HEX was observed. In several cells from normal breast tissue as well as in MCF-7 and T47D cell line, HEX was observed in the nucleolus. MCF-7 treatment with all-trans retinoic acid enhanced HEX expression and induced a diffuse nuclear localization. Enhanced HEX expression and diffuse nuclear localization were also obtained when MCF-7 cells were treated with inhibitors of histone deacetylases such as sodium butyrate and trichostatin A. With respect to normal non-lactating breast, the amount of nuclear HEX was greatly increased in lactating tissue. Transfection experiments demonstrated that HEX is able to up-regulate the activity of NIS promoter.

Conclusion: Our data indicate that localization of HEX is regulated in epithelial breast cells. Since modification of localization occurs during lactation and tumorigenesis, we suggest that HEX may play a role in differentiation of the epithelial breast cell.

Figure 1

HEX expression and localization in normal tissues, breast carcinomas and cell lines. Immunohistochemical analysis was performed as described in the methods section. A and B: normal breast tissue. C: ductal carcinoma. D: lobular carcinoma. E: MCF-7 cells. F: T47D cells. G: HBL100 cells stained with p63 and CD10. H: HBL100 cells stained with HEX.

Figure 2

Effects of ATRA on HEX expression and localization in MCF-7 cells. HEX protein and HEX mRNA levels were evaluated by immunohistochemistry and real time PCR, respectively. A: untreated MCF-7 cells. B: MCF-7 cells treated for 15 hours with ATRA 1 μM. C: time-course of ATRA effect on HEX localization. Each square represents the mean value ± standard deviation of four 100 cells counts in different slides. D: time-course of ATRA effect on HEX mRNA levels. Each dot represents the mean value ± standard deviation of three independent determinations.

Figure 3

HEX nuclear localization in lactating breast. Panel A: non-lactating tissue. Panel B: lactating tissue. Panel C: quantification of the HEX nuclear positivity in non-lactating and lactating breast tissues. HEX nuclear positivity was evaluated by calculating separately nuclear and cytoplasmic reactivity in 1000 cells. Each bar represents the mean value ± standard deviation of nuclear HEX, expressed as percentage of stained cells in the nucleus.

Figure 4

HEX effects on NIS promoter. HeLa cells were transfected with 1 and 2 μg of the HEX or PAX8 expression vector together with plasmids containing the NIS and the CMV promoters linked to the LUC and the βGAL reporter genes, respectively. The CMV-βGAL plasmid was used to normalize for efficiency of transfection. Each dot represents the mean value ± standard deviation of four independent transfections. Results are expressed as percentage of the values obtained in cells not transfected with HEX or PAX8 expression vectors.

Figure 5

Effects of HDAC inhibitors on HEX expression and nuclear localization in MCF-7 cells. Cells were treated for 30 hours with NaB (3 mM) and TSA (300 nM). Then, HEX mRNA levels were evaluated by real-time PCR and HEX localization by immunohistochemistry. Panel A: effect of NaB and TSA on HEX mRNA levels. Panle B: effect of NaB and TSA on the HEX diffuse nuclear localization. In both panels each bar represents the mean value ± standard deviation of three independent measures. In both panels, according the paired Student's T test, the differences between treated and untreated cells were highly significant (at least: p < 0.01).