MeCP2 and MBD2 expression in human neoplastic and non-neoplastic breast tissue and its association with oestrogen receptor status

This study analysed mRNA expression of two members of the methyl-CpG-binding protein family - MeCP2 and MBD2 - in human non-neoplastic (n=11) and neoplastic (n=57) breast tissue specimens using a quantitative real-time PCR method. We observed higher expression levels of MeCP2 mRNA in neoplastic tissues than in non-neoplastic tissues (P=0.001), whereas no significant differences for MBD2 were detected. When studying the relations between the most important clinicopathologic features of breast cancer and the mRNA expression level of both MBDs, we found that oestrogen receptor (OR)-positive breast cancer specimens contained higher levels of MeCP2 mRNA than did OR-negative cancers (P=0.005). Furthermore, we observed statistically significantly higher levels of MeCP2 in non-neoplastic tissues expressing high levels of OR as compared to those expressing low levels (P=0.017). Finally, using a linear regression model, we identified a statistically significant association between OR expression and MeCP2 mRNA expression in neoplastic and non-neoplastic breast tissue specimens (P=0.003). In conclusion, we were able to demonstrate for the first time that there exists a strong association between OR status and MeCP2 mRNA expression. Furthermore, we speculate that MeCP2, regulated by OR, plays a key role in the differentiation processes in human breast tissues.     

Expression of the NF-kappaB-responsive gene BTG2 is aberrantly regulated in breast cancer

BTG2, a p53-inducible antiproliferative gene, is stimulated in breast cancer cells by activation of nuclear factor kappa B (NF-kappaB). In rat mammary glands, BTG2 is expressed in epithelial cells and levels decreased during pregnancy and lactation but recovered during involution. Estrogen and progestin suppress BTG2 expression, suggesting that these steroids, which stimulate proliferation and lobuloalveolar development of mammary epithelial cells, may downregulate BTG2 in the mammary gland during pregnancy. Consistent with the report that BTG2 inhibits cyclin D1 expression, suppression of BTG2 mRNA in the mammary gland during gestation, and by estrogen and progestin, correlated with stimulation of cyclin D1. Ectopic expression of BTG2 inhibited breast cancer cell growth by arresting cells in the G1 phase, an effect reversed by cyclin D1. BTG2 expression was very low or undetectable in human breast cancer cell lines compared with nontumorigenic mammary epithelial cells, and nuclear expression of BTG2 was absent in 65% of human breast tumors compared with adjacent matched normal glands. Spontaneous mammary tumors arising in a mouse model with targeted expression of the early region of the SV40 large tumor Ag demonstrated loss of BTG2 protein very early during the tumorigenic process. Thus deregulation of BTG2 may be an important step in the development of mammary tumors.     

Sulf-2, a proangiogenic heparan sulfate endosulfatase, is upregulated in breast cancer

Sulf-2 is an endosulfatase with activity against glucosamine-6-sulfate modifications within subregions of intact heparin. The enzyme has the potential to modify the sulfation status of extracellular heparan sulfate proteoglycan (HSPG) glycosaminoglycan chains and thereby to regulate interactions with HSPG-binding proteins. In the present investigation, data mining from published studies was employed to establish Sulf-2 mRNA upregulation in human breast cancer. We further found that cultured breast carcinoma cells expressed Sulf-2 mRNA and released enzymatically active proteins into conditioned medium. In two mouse models of mammary carcinoma, Sulf-2 mRNA was upregulated in comparison to its expression in normal mammary gland. Although mRNA was present in normal tissues, Sulf-2 protein was undetectable; it was, however, detected in some premalignant lesions and in tumors. The protein was localized to the epithelial cells of the tumors. In support of the possible mechanistic relevance of Sulf-2 upregulation in tumors, purified recombinant Sulf-2 promoted angiogenesis in the chick chorioallantoic membrane assay.     

Fog2 excision in mice leads to premature mammary gland involution and reduced Esr1 gene expression

The critical role for GATA family proteins in maintaining the normal (non-transformed) cell state is corroborated by the recent findings of mutations or methylation in GATA genes both in primary cancers and tumor lines including breast. Previously, microarray profiling studies determined that the highest expression of both GATA3 and ESR1 (estrogen receptor alpha) is seen in tumors associated with the most favorable survival outcomes, whereas the lowest expression of GATA3 is detected in tumor subtypes showing the worst outcomes. At this time, genes and pathways that are regulated by GATA3 in the mammary gland are not well defined. We have previously established a requirement for FOG (Friend Of GATA) cofactors during mouse development. Here we report that in the murine mammary gland Fog2 gene expression is upregulated upon pregnancy and lactation with prominent expression in the epithelial cells of the gland during post-lactational regression. Mammary-specific deletion of Fog2 identified a role for this gene during gland involution; excision of the Fog2 gene leads to the accelerated involution of the gland despite diminished levels of the remodeling enzymes. Importantly, the levels of several genes linked to the control of cancerous transformation in the breast (Esr1, Prg and Foxa1) are significantly reduced upon Fog2 excision. This implicates FOG2 in the maintenance of epithelial cell differentiation in the mammary gland and in performing a protective role in breast cancer.     

Deregulated expression of the PCPH proto-oncogene in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene

The PCPH proto-oncogene was identified by its frequent activation in Syrian hamster fetal cells exposed to 3-methylcholanthrene. We previously isolated human PCPH cDNA and studied its expression in normal human tissues. We report herein the pattern of PCPH expression in normal rat tissues. Each tissue expressed one major PCPH polypeptide that varied in molecular mass in different tissues. Normal mammary gland expressed a single PCPH polypeptide of 27 kDa. This PCPH form also was expressed in lactating mammary glands but at significantly greater levels. These results suggest the existence of tissue-specific regulatory mechanisms for PCPH expression that may be influenced by the differentiation stage. Our previous studies on the involvement of PCPH in human cancer showed that human breast tumor cell lines have frequent alterations in PCPH, including multiple PCPH polypeptide forms that are not expressed in normal cells. These cell lines also have frequent loss of a 27-kDa form identified as the only PCPH polypeptide expressed by normal human breast epithelial cells. In this study, we found that these same alterations occurred in vivo during mammary carcinogenesis in Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene, in both benign and malignant tumors, indicating that stable changes in PCPH expression took place early in the neoplastic process. Results showed that this experimental system is relevant to human breast carcinogenesis and provides an excellent model to study the molecular basis of the regulation of PCPH expression during normal differentiation and pathologic stages of neoplasia of the mammary gland and to analyze the role of PCPH in the carcinogenic process. Furthermore, the detection of atypical PCPH polypeptides in tumors suggests that PCPH immunodetection may be applied as a diagnostic tool for the early identification of neoplastic breast epithelial cells.     

Tyrosine kinase activity in breast cancer, benign breast disease, and normal breast tissue

Tyrosine specific protein kinase activity was determined in 70 specimens of the human mammary gland. These included 28 cancers of the breast, 21 benign breast diseases, and 21 normal breast tissues. We measured tyrosine kinase activity in the cytosol fraction and in the membrane fraction of the homogenates. In addition cytosolic aldolase activity was measured. Tyrosine kinase activity was determined using poly(glutamic acid:tyrosine = 4:1) as an artificial substrate. Cancers of the breast exhibited considerable higher tyrosine kinase activities in both cytosol and membrane fractions, compared to benign breast tumors (P less than or equal to 0.001). Benign tumors demonstrated increased activities in cytosol in comparison to normal breast tissues (P less than 0.001). Furthermore, there appears to be a strong association of an enhanced expression of activity of tyrosine kinase in cytosol of primary carcinomas and early systemic relapse. In combination with aldolase activity a nearly complete discrimination is achieved between malignant specimens on one hand and benign and normal tissues on the other.     

Protein kinase CK2 in mammary gland tumorigenesis.

Protein kinase CK2 is a ubiquitous and evolutionarily conserved serine/threonine kinase that is upregulated in many human cancers and can serve as an oncogene in lymphocytes. Recently, we have demonstrated that CK2 potentiates Wnt/beta-catenin signaling in mammary epithelial cells. To determine whether CK2 overexpression contributes to mammary tumorigenesis, we have performed comparative studies of human and rat breast cancer specimens and we have engineered transgenic mice with dysregulated expression of CK2alpha in the mammary gland. We find that CK2 is highly expressed in human breast tumor specimens and in carcinogen-induced rat mammary tumors. Overexpression of CK2alpha in the mammary gland of transgenic mice, under control of the MMTV-LTR, causes hyperplasia and dysplasia of the female mammary gland. Thirty per cent of the female MMTV-CK2alpha transgenic mice develop mammary adenocarcinomas at a median of 23 months of age, often associated with Wnt pathway activation, as evidenced by upregulation of beta-catenin protein. NF-kappaB activation and upregulation of c-Myc also occur frequently. Thus, in mice, rats, and humans, dysregulated expression of CK2 is associated with and is capable of contributing to mammary tumorigenesis. Targeted inhibition of CK2 could be useful in the treatment of breast cancer.     

PAK1 hyperactivation is sufficient for mammary gland tumor formation

Emerging data suggest that p21-activated kinase 1 (Pak1), a downstream signaling molecule of the small GTPases, growth factors, and lipid signaling, is upregulated or hyperactivated in human breast cancer. Until now, however, no direct causative role had been found for Pak1 in mammary tumor formation. We therefore sought to identify the role that Pak1 plays in mammary gland tumorigenesis. Our results showed that in a transgenic mouse model, overexpression of catalytically active Pak1 leads to the development of malignant mammary tumors and to a variety of other breast lesions, including focal solid nodules, ductal hyperplasia, and mini-intraductal neoplasm and adenoma. We also found that Pak1 hyperactivation increases the stimulation of downstream proliferative signaling effectors MEK1/2 and p38-MAPK in mammary tumor epithelial cells. Moreover, in our study, we detected expression of estrogen receptor-alpha expression and progesterone receptor expression during early stages of the lesions, but their expression was lost during the cells' transition to malignant invasive tumors. Finally, we found that consistent with a role in breast tumor progression, Pak1 expression and its nuclear accumulation was increased progressively during the transition from ductal hyperplasia to ductal carcinoma in situ to adenocarcinoma in widely used multistep polyoma-middle T-antigen transgenic mice. Together, these findings provide the first direct evidence that Pak1 deregulation may be sufficient for the formation of mammary gland tumors.     

Loss of expression of HDAC-recruiting methyl-CpG-binding domain proteins in human cancer.

Dysregulation of CpG-methylation is a common feature of many human cancers and tumour suppressor genes can be silenced by hypermethylation. Recently, 2 methyl-CpG-binding domain proteins have been linked to gene inactivation by their ability to recruit co-repressors and HDAC-activity to methylated gene promoters. Here, we have analysed mRNA expression of these genes, MeCP2 and MBD2, in a wide variety of primary human tumours. In solid tumours, expression levels of MBD2 (57/71) and MeCP2 (64/71) were significantly reduced in the majority of primary tumours as detected by quantitative real-time RT-PCR. Western blot analyses of MeCP2 in matched tumour-normal samples of patients with non-small-cell lung cancer (NSCLC) indicated reduced protein in a significant percentage of patients. In acute myelogenous leukaemia (n = 26), expression levels were only slightly reduced and did not differ between samples analysed at diagnosis or at the time of relapse. In early-stage NSCLC (n = 70) expression of MeCP2 and MBD2 was significantly lower in squamous cell carcinoma than in adenocarcinoma or large cell carcinoma (P = 0.03 and P = 0.01). To further elucidate the mechanisms of gene regulation, we analysed MeCP2 and MBD2 regulation during haematopoietic differentiation. No significant changes in MeCP2 or MBD2 expression were found when NB4 cells were differentiated toward granulocytes suggesting that neither differentiation nor cell cycle status were relevant for the reduced expression of these genes in human cancer. In conclusion, the significant loss of MeCP2 and MBD2 expression in human cancers suggests a potential role of this phenomenon in the development of solid human tumours.     

PTK6/BRK is expressed in the normal mammary gland and activated at the plasma membrane in breast tumors

Protein Tyrosine kinase 6 (PTK6/BRK) is overexpressed in the majority of human breast tumors and breast tumor cell lines. It is also expressed in normal epithelial linings of the gastrointestinal tract, skin, and prostate. To date, expression of PTK6 has not been extensively examined in the normal human mammary gland. We detected PTK6 mRNA and protein expression in the immortalized normal MCF-10A human mammary gland epithelial cell line, and examined PTK6 expression and activation in a normal human breast tissue microarray, as well as in human breast tumors. Phosphorylation of tyrosine residue 342 in the PTK6 activation loop corresponds with its activation. Similar to findings in the prostate, we detect nuclear and cytoplasmic PTK6 in normal mammary gland epithelial cells, but no phosphorylation of tyrosine residue 342. However, in human breast tumors, striking PTK6 expression and phosphorylation of tyrosine 342 is observed at the plasma membrane. PTK6 is expressed in the normal human mammary gland, but does not appear to be active and may have kinase-independent functions that are distinct from its cancer promoting activities at the membrane. Understanding consequences of PTK6 activation at the plasma membrane may have implications for developing novel targeted therapies against this kinase.