Gene: ITGA6 maps to 2q (Hogervorst F. et al., 1991). The alpha
6 promoter directs transcription initiation from a primary site 202 nucleotides from the translation initiation codon. Unlike most other integrin gene promoters, the alpha
6 promoter has a TATA box (GATAAA), which is located 22 nucleotides upstream from the primary transcription initiation site. A 190-bp region upstream from the TATA box is highly rich (78%) in C and G nucleotides and contains several Sp1 and AP2 binding sequences. However, full promoter activity (in the presence of the SV40 enhancer) requires only 78 bp of this C/G-rich sequence upstream from the TATA box. Slightly upstream from the C/G-rich region are a steroid receptor binding homolog and an epithelial-cell-specific E-pal sequence. Another possible epithelial cell-specific binding sequence (Ker1) is found immediately downstream from the TATA box. Cell-type-specific activities of the promoter paralleled the alpha
6 mRNA levels in four tested cell lines. In the presence of the SV40 enhancer, alpha
6 promoter activity increased approximately four-fold in primary keratinocytes and in HT1080 fibrosarcoma cells and 30-fold in T47D breast carcinoma cells, but remained undetectable in K562 leukemia cells. Genomic analysis that compared alpha
6-expressing with non-alpha
6-expressing cells suggested that DNA methylation is not involved in the silencing of the alpha
6 gene in alpha
6-negative cells. DNase I footprint analysis confirmed the binding of Sp1 and AP2 to their cognate sequences. A nuclear extract of high-alpha
6-expressing HBL-100 cells also produced significant binding to these sites, suggesting that the two transcription factors are probably involved in the positive regulation of the alpha
6 promoter (Lin C.S. et al., 1997).
mRNA: sizes: two alpha
6 mRNAs of around 6 kb have been found. They encodes two alpha
6 subunits (alpha
6A and alpha
6B), the cytoplasmic domain of alpha
6B being 18 amino acids longer than that of alpha
6A (Hogervorst F. et al., 1991).
Protein: the cDNA encodes a 1,050-amino acids (aa) protein with a 991-aa extracellular domain, a 23-aa transmembrane domain, and a 36-aa acid cytoplasmic domain. The alpha
6 subunit most closely resembles the alpha
3 subunit, which shows 40% identity (Hogervorst F. et al., 1991). This high degree of similarity may be the basis for their functional resemblance, since both alpha
3 and alpha
6 subunits, when associated with
beta1, function as laminin receptors and bind to the long arm of laminin. Alpha
6 may also form a laminin receptor by coupling with the beta
4 subunit. Alpha6 expression is cell-type-specific and generally is present at high levels in epithelial and endothelial cells.
Cell lines:
- Expression levels of alpha
6,
beta1 and beta
4 integrin sub-units were measured in a panel of human breast cancer cell lines by RT/PCR, immunoprecipitation and flow cytometry. All the lines expressed alpha
6, with the highest levels in the MDA-MB-231 and MDA-MB-435 cells. These grew the most aggressively and were metastastic in nude mice. Low levels of alpha
6 protein were measured in breast cancer cells that were poorly tumorigenic and non-metastatic in nude mice, and there was an inverse relationship between
ER and alpha
6 expression. RT/PCR revealed that all lines expressed the 2 isoforms of alpha
6, with the alpha
6A isoform generally more abundant than alpha
6B isoform. Clones of MDA-MB-435 were isolated by sterile sorting for cells with high or low alpha
6 expression, and two variants established from metastases in nude mice were found to differ in alpha
6 expression. When injected into nude mice. the alpha
6-high variants produced significantly more lung metastases than the alpha
6-low variants.
beta1 was abundant in all lines, while beta
4 was not detected in MDA-MB-134 cells, and in the MDA-MB-435 cells an alternately spliced variant of beta
4 was identified. Sequencing of the alternate variant revealed a novel sequence from a splicing event in the cytoplasmic tail of beta
4. None of the cells with this variant mRNA expressed detectable levels of beta
4 protein (Mukhopadhyay R. et al., 1999).
Tumors:
- Alpha
6 expression was investigated by immunohistology in normal breast tissue and 26 breast carcinomas. In normal tissues, there was weak staining of epithelial cytoplasm with alpha
6 antibody and moderate cell membrane staining. Strongest staining was present in a basement membrane distribution. In carcinomas, loss of cytoplasmic and cell membrane staining was variable, but basal membrane staining was diminished or absent in all tumours. Loss of basal membrane staining for alpha
6 integrin corresponded closely to loss of immunoreactivity for its ligand laminin in invasive breast cancer (D'Ardenne A.J. et al., 1991).
- By affinity chromatography and Western blot analyses of purified immune complexes, it was shown that the alpha
6beta
4 and alpha
6beta1 integrins coprecipitated with ErbB-2 in lysates from carcinoma or NIH3T3 cells overexpressing ErbB-2. Integrin-mediated activation of ErbB-2 receptors suggested that this association is functionally meaningful. Indeed, carcinoma cells treated with a monoclonal antibody to the alpha
6 integrin subunit showed a ligand-dependent increase of ErbB-2-phosphorylated molecules coprecipitated with integrins and an increased DNA synthesis (Falcioni R. et al., 1997).
- The expression of human alpha
6 integrin was investigated in invasive breast carcinomas of 119 women. In 50% of the tumors alpha
6 integrin was expressed in the majority of the cells, and this expression was correlated with reduced survival time. By contrast, the 24% of patients with breast tumors devoid of alpha
6 integrin expression all survived. The tumors were also evaluated for clinical risk factors including histological grading and steroid receptor level. The combination of these factors with alpha
6 integrin expression was superior in predicting overall survival than considering the other factors alone. The correlation with decreased survival time was consistent, regardless of whether the tumors expressed the alpha
6 integrin A or B forms (Friedrichs K. et al., 1995).
D'Ardenne A.J. et al. (1991) Co-ordinate expression of the alpha-6 integrin laminin receptor sub-unit and laminin in breast cancer. J. Pathol. 165, 213-220.
(PubMed)
Falcioni R. et al. (1997) Alpha 6 beta 4 and alpha 6
beta 1 integrins associate with ErbB-2 in human carcinoma cell lines. Exp. Cell. Res. 236, 76-85.
(PubMed)
Friedrichs K. et al. (1995) High expression level of alpha 6 integrin in human breast carcinoma is correlated with reduced survival. Cancer Res. 55, 901-906.
(PubMed)
Hogervorst F. et al. (1991) Molecular cloning of the human alpha-6 integrin subunit: alternative splicing of alpha-6 mRNA and chromosomal localization of the alpha-6 and beta-4 genes. Eur. J. Biochem. 199, 425-433.
(PubMed)
Lin C.S. et al. (1997) Identification of the human alpha6 integrin gene promoter. DNA Cell. Biol. 16, 929-937.
(PubMed)
Mukhopadhyay R. et al. (1999) Increased levels of alpha6 integrins are associated with the metastatic phenotype of human breast cancer cells. Clin. Exp. Metastasis 17, 325-332.
(PubMed)
