indicate a role for hormonal factors and in particular of estrogens in breast tumorigenesis. The incidence of sporadic breast cancer increases with age, reaches a peak between 45 and 50 years, and then declines slowly after menopause, a behavior reflecting the involvement of reproductive hormones (81). Moreover early age at menarche, late age at first birth, low parity, late menopause and hormone replacement therapies increase the risk of developing breast cancer, all these conditions reflecting hormonal patterns that lead to high levels of endogenous or exogenous estrogens (82). Interestingly several studies have reported complex crosstalk between estrogen modulated genes and peptide growth factors signaling cascades such as the Epidermal Growth Factor, (EGF), Insuline like Growth Factor (IGF) and Fibroblast Growth Factor (FGF) pathways (83-86).

2.1.1 Loss of Hormonal Control by Silencing of Nuclear Receptors

Many of the hormones implicated in maintaining mammary gland homeostasis act through members of the nuclear receptor superfamily, a large class of ligand-dependent transcription factors (87). Three classes of nuclear receptor have been identified: Type 1 or steroid receptor that include those for estrogens (ER), progesterone (PR); androgens (AR) glucorticoids (GR) and mineral corticoids (MR); Type II that includes those for all-trans-retinoic acid (RAR), thyroid hormone (TR) and vitamin D (VDR); Type III includes the so called orphan receptors for which an endogenous ligands have not been identified (64). Type I receptor, in absence of the ligand, are localized into the cytoplasm coupled with heath-shock protein. In presence of the agonist hormone they homodimerize and translocate to the nucleus were bind to palindromic response element. Type 2 receptors are localized in the nucleus and form heterodimer with the receptor for the 9-cis retinoic acid (RXR) binding constitutively to the response elements consisting of direct repeats where, in the absence of the ligand repress transcription (64). The activity of nuclear receptor is influenced by factors able to enhance (co-activators) or repress (co-repressor) the transcriptional activity (88).

Two Estrogen Receptors (ER) genes have been identified ESR1 encoding for ERa, and ESR2 encoding for ERP (89-91). In the normal human breast ERa is expressed approximately by 10-30% of the luminal cells but not expressed by the myoepithelial cells (92, 93). In contrast, ERP is expressed in both luminal and mioepithelial cells (94). The two estrogen receptors are highly conserved in their central DNA binding domain (C-terminus) but diverge in the amino-terminus (95), thus ErP can activate the same genes regulated by Era although in a less efficient manner. In cell that co expresses both receptors, ERP act as an efficient inhibitor of the ERa transcriptional activity (63). Despite the clear role of estrogens in mammary cell proliferation, the majority of proliferating cells in the adult breast do not express neither ERa nor ERP (96). The prevailing concept is that estrogens regulate cell growth indirectly by inducing Era positive cells to produce growth factor able to regulate proliferation in ERa negative cells (85, 97, 98). The ESR1 gene has a complex genomic organization, with at least eight promoters (Figure 3), whose utilization varies between different cell types (99). Therefore ERa expression results from the interplay between all the promoters, and their transcriptional regulators (76, 100-104). In vitro studies on breast cancer cell lines have demonstrated that the treatment with demethylating agents (i.e. 5-Azacytidine), can restore ERa expression. Hypermethylation of the first identified promoter, now named promoter A (Figure 3) (105), was investigated in several studies by methyl sensitive PCR or methylation specific restriction landmarks. Some levels of promoter hypermethylation were identified in 25% to 70% of the tumor analyzed (75, 106-108). Aberrant methylation of the promoter B, localized immediately upstream to promoter A, was also reported in breast cancer cell lines and primary tumors (109, 110), where correlated negatively with ERa expression (109). Recently hypermethylation of the distal F promoter, responsible for ERa transcription in bone tissues, was detected in ERa negative but not in ERa positive breast cancer cell lines (111). Transcription of the human ERP gene also occurs from at least two different promoters 0K and 0N (Figure 3) generating two mRNA that differ at the 5'-untraslated regions. Zhao et al. (112) reported extensive methylation of the 0N promoter in breast cancer cell lines and primary tumors in contrast to promoter 0K that was unmethylated. The treatment of cell lines with demethylating agents was able to reactivate expression of the ERP mRNA (112).

Figure 3. Genomic organization of 5'-untraslated regions (5'UTR) of human estrogen receptors ESR1 (ERa) and ESR2 (ERP). Promoters are identified as gray boxes, and relative exons as white boxes. ESR1 has at least seven more promoters beside the first identified, and now named A. All mRNA isoforms have a common acceptor splice site in exon 1 at approximately 70 bp from the ATG, broken lines are observed splicing (105). ESR2 has at least two different promoters encoding mRNA isoforms that diverge in their 5'-UTR (112).

Figure 3. Genomic organization of 5'-untraslated regions (5'UTR) of human estrogen receptors ESR1 (ERa) and ESR2 (ERP). Promoters are identified as gray boxes, and relative exons as white boxes. ESR1 has at least seven more promoters beside the first identified, and now named A. All mRNA isoforms have a common acceptor splice site in exon 1 at approximately 70 bp from the ATG, broken lines are observed splicing (105). ESR2 has at least two different promoters encoding mRNA isoforms that diverge in their 5'-UTR (112).

Conflicting data have been published regarding the correlation between ESR1 promoter methylation and receptor status determined by routine immunoistochemestry. In studies using MSP as well as those using methylation-sensitive restriction landmarks, aberrant methylation of ESR1 promoter A was found in only a proportion of tumors ER negative, as well as ESR1 promoter hypermethylation was detected in a significant number of ER positive breast cancers (75, 107-109, 113-115). A better correlation was found when ERa levels are determined by quantitative methods (107, 108). A recent study using microarray-based DNA methylation analysis, also indicate that methylation levels of ESR1 promoter A are not predictors of hormonal status. Surprising, in this study higher level of ESR1 methylation correlated with better prognosis in patients treated with tamoxifene (116). However, only one study has analyzed simultaneously two ESR1 promoters (A and B) finding complete ER negative status only when methylation was present in both (109).

The Progesterone Receptor (PR) gene encodes for two isoforms, PR-A and PR-B, both expressed in the luminal epithelial cells. PR-B is preferentially induced by ligand-bound Era, and presence of progesterone receptor in cancer cells is indicative of a functional estrogen receptor (92, 104, 117, 118). Progesterone receptor isoforms are transcribed from two promoters and translated from two alternative ATG. They only differ for a stretch of about 165 aminoacids in amino terminus of PR-B, a region encoding a transactivating domain required for target genes that can be activated by PR-B but not by PR-A (118). In cell where PR-A is inactive, PR-B function as a strong transactivator of PR-dependent genes, whereas the binding of PR-A to the target gene can repress transcriptional activity of PRB and other nuclear receptor including ERa (118, 119). Methylation status of the PR promoter was investigated by Lapidus et al. using methylation sensitive restriction analysis, they found three sites in the PR gene promoter that were unmethylated in normal breast and hypermethylated in 40% of breast tumors (107, 120).

Retinoids are dietary factors that possess antiproliferative, differentiative, immunomodulatory and apoptosis inducing properties, which act through Retinoic Acid Receptors (RARa, RARP and RARy) and Retinoids X Receptor (RXRa, RXRP, and RXRy) (121, 122). Both types of receptors are bound as RAR/RXR heterodimers or RXR homodimers to specific retinoic response elements (RAREs) located on the promoter region of target genes (123). In the absence of ligands, corepressor complexes interact with RAR-RXR heterodimes resulting in histone deacetylation, chromatine condensation and silencing of target genes. Agonist binding destabilizes the complex, and with the help of coactivators induces chromatin decondensation and receptor dependent transcription initiation (124). The

RARP gene encodes for four different transcripts, among those the RARP2 isoform transcribed from a promoter upstream to exon 3 is frequently lost in breast cancer, whereas RARa and RARy as well as RXRP are expressed in both normal breast and cancer cells (125, 126). The RARP2 promoter region contains a RARE element, thus its expression can be induced by retinoic acid. (124). RARP2 promoter methylation was found in 20 to 40% of primary breast cancer, and correlated with gene expression (75, 77, 127129). In RARP2 negative cell lines re-expression of the gene was demonstrated after treatment with demethylating agents and Histone DeACetylates (HDAC) inhibitors (127, 129-131). Interestingly, the frequency of RARP2 hypermethylation was significantly higher in breast cancer metastasis to bone, brain and lung (~85%) (132). An interesting mechanism for RARP epigenetic modulation was described in promielocytic leukemia. This neoplasm is characterized by the expression of the oncogenic fusion protein PML-RARa, which is able to induce hypermethylation of the RARE elements in the RARP promoter, this effect is reversed by the treatment with retinoic acid (133).

Gene silencing by promoter hypermethylation was also detected in breast cancer for thyroid hormone receptors (TRs). TRs are type II nuclear receptors, thus in absence of the ligand they repress transcription, the binding with the ligand triodiothyronine (T3) destabilize the receptor and activates transcription of target genes. There are eight TRs isoforms encoded by two genes TRa and TRP (134). In particular the isoform TRpi encoded by a gene located on chromosome 3p in a region frequently deleted in breast cancer, is hypermethylated in approximately 25% of the primary breast cancer (75, 135).

2.1.2 Self-activation of Peptide Growth Factors Signaling Pathways

In normal condition cell proliferation is finely regulated by secreted peptide with proliferative or growth inhibitory capability. Growth promoting factors interact with specific transmembrane receptors that initiate a cascade of signals leading ultimately to transcriptional activation of the cell cycle machinery. Self-activation of these transduction signals allows cancer cells to replicate in the absence of the proper mitogenic stimulus and regarding to the presence of inhibitory factors. The mitogen activated protein kinase (MAPK), and in particular, the ERK (extracellular-signal-regulated kinase) cascade is the most commonly involved in human cancers. The major activators of the ERK -pathway are peptide (i.e. EGF, IGF1, prolactin) that bind a tyrosine kinase transmembrane receptor leading to the activation of the G-protein Ras. Activated Ras interacts with a MAPK kinase kinase (Raf), that phosporylates a MAPK kinase (MEK), which finally activates

ERK (136, 137). Induction of the ERK pathway is usually associated with growth proliferation, but it can also determine inhibitory effects manifested by senescence, or apoptosis (137-139). In breast cancer the ERK-pathway is usually up regulated by over expression of the transmembrane epidermal growth factor receptor 2 (EGFR2 or HER2/neu or c-erB-2) as consequence of genomic amplification. Several data, however, indicates that promoter hypermethylation may also play a role in the deregulation of the ERK pathway in mammary tumors. The Ras Association domain Family 1A (RASSF1A) gene is methylated in 42-65% of breast cancer (136, 140-144). The function of this gene at the present is not known, but its homologue RASSF1C bind RAS in a GTP-dependent manner (145). It is possible that RASSF1A will interact with RAS in the same mode, mediating the inhibitory effects on the cell cycle. Loss of RASSF1A expression by methylation in human cancer may modify the balance of RAS activities toward a growth-promoting effect. Recently promoter hypermethylation was demonstrated for a novel member of the Ras GTPase activating family named DOC-2/DAB2 interacting protein (hDAB2IP) (146, 147). Aberrant methylation was detected in two CpG reach regions in approximately 50% of the cell lines and 40% of the primary tumors. For one of the two CpG rich regions examined a correlation with lymph node status was observed (148).

Cytokines are known to play an important role in breast cell functions, as trophic hormones and as mediators of host defense mechanisms (149). Like other cytokines IL-6 binds to a specific membrane receptor (IL6R) with activation of the Janus kinase (JAK) family leading ultimately to the phosphorylation of members of the STAT (Signal Transducer and Activators of Transcription) family of transcription factors, After phosphorylation STAT proteins can dimerize and translocate to the nucleus where they regulate transcription of several genes involved in cell growth and differentiation (150). The SOCS (Suppressor of Cytokine Signaling) family of proteins function as negative JAK/STAT regulator. Among the members of the SOCS family SOCS1 was found methylated in a subset of primary breast cancers. Aberrant methylation correlated with transcriptional silencing in breast cancer cell lines and treatment with 5-azacytidine restored expression (149).

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