The work reported here demonstrates that differentiation of the breast induced by an early pregnancy imprints a specific genomic signature that can be detected in postmenopausal women. Using bioinformatics methods we found transcriptomic differences between the breasts of parous and nulliparous women. These differentially expressed genes were used to identify enriched biological processes and pathways. Enriched biological processes related to up-regulated genes included RNA related processes, differentiation and development of epidermis and ectoderm, and cell-substrate junction assembly; whereas in the case of down-regulated genes the biological processes that were enriched included IGF-like growth factor signaling, somatic stem cell maintenance and apoptosis. Pathways that were enriched by up-regulated genes included breast cancer estrogen signaling, cell communication and mRNA processing machinery. Numerous pathways were enriched by down-regulated genes; the most significant ones were the insulin, Wnt and integrins signaling pathways, MAPK, cytokine-cytokine receptor interaction, tight junction and focal adhesion, all representing proteins that are highly expressed in malignancies.
The main components of the spliceosome machinery, including RNA and proteins that undergo dynamic changes during the splicing reaction, were up-regulated in the parous breast. Among them were the heterogeneous nuclear ribonucleoproteins (HNRPs) that include HNRPA3, HNRPA2B1, HNRPD and HNRPU
], which are implicated in the regulation of mRNA stability, as well as other functions, such as mammary gland involution
], negative regulation of telomere length maintenance
], and regulation of mRNA trafficking from the nucleus to distal processes in neural cells
]. Although further studies are needed to define their precise functional role in the postmenopausal breast, we postulate that they may play an important regulatory function as transcriptional regulators. In addition, post-transcriptional methylation of internal adenosine residues in eukaryotic mRNAs by METTL3 (methyltransferase like 3), which is up-regulated in the parous breast, could play a role in the efficiency of mRNA splicing, transport or translation in the differentiated breast epithelium. Other members of the spliceosome complex are the proteins encoded by the genes SF3B1, SFRS2, SFRS7, SFRS8, SFRS14, SFRS16, SNRP70, SNRPB, SNRPA1, PRF3 and PHF5A, all of which are overexpressed in the parous breast. In the case of the small nuclear ribonucleoproteins (snRNPs), there is evidence that they suppress tumor cell growth and may have major implications as cancer therapeutic targets. The pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. Among the members of the splicing factor compartment are CCNL1 and CCNL2 that participate in the pre-mRNA splicing process and are located in the nuclear speckles
]. These two genes are up-regulated in the parous breast and the CCNL2 protein is also overexpressed in the nucleus of breast epithelial cells. CCNL1 and CCNL2 are transcriptional regulators
] that modulate the expression of critical factors leading to cell apoptosis, possibly through the Wnt signal transduction pathway
], a signaling pathway that is enriched by down-regulated genes in the parous breast (Table
). In our previously published preclinical and clinical studies
], we have reported that pregnancy confers protection from breast cancer development by inducing gland differentiation, which imprints a specific and permanent genomic signature in this organ. A similar phenomenon was demonstrated in the breast of postmenopausal parous women characterized by fatty involution
]. We previously described a small case–control study of transcriptomic analysis of normal breast tissues obtained from parous and nulliparous women free of breast pathology and parous and nulliparous women with history of breast cancer that served as controls and cases, respectively
]. In order to investigate the degree of commonality between the previous case–control and the present study, we applied GO enrichment analysis to the gene lists generated from both studies. We found that processes involved in RNA metabolism and RNA processing were similar in both studies (see Additional file
: Table S4).
A number of non-coding RNAs that included XIST, MALAT-1 (also called NEAT2) and NEAT1 were up-regulated in the parous breast. XIST, which inactivates X chromosome as an early developmental process, plays an essential role in female mammals by providing dosage equivalence between males and females. Up-regulation of XIST occurs upon differentiation, whereas failure to express XIST is often seen in malignancies and in early embryogenesis
]. Our findings are supported by recent reports that suggest that XIST is expressed in adult well-differentiated cells in order to maintain gene repression
]. Oxytocin, a neurotransmitter that acts through it specific receptor OXTR and is overexpressed during lactation, up-regulates the expression of MALAT-1, a highly conserved non-coding RNA
]. Interestingly, both MALAT1 and OXTR remain overexpressed in the breast of postmenopausal parous women. NEAT1 and NEAT2 localize to the periphery and to the interior the spliceosome assembly factor SC35 domains or speckles. Our observation that in breast epithelial cells CCNL2 is highly enriched in nuclear speckles (Figure
) indicates that CCNL2 might colocalize with NEAT1 and NEAT2. The down-regulation of NEAT1, NEAT2 and XIST in the breast of nulliparous women, in whom this organ never reached a stage of complete differentiation similar to that achieved after completion of pregnancy and lactation
], suggests that the undifferentiated breast is not actively involved in the RNA metabolism that is necessary for maintaining a state of differentiation.
Although in this study we did not observe differential expression in estrogen receptor between parous and nulliparous breasts, several genes that are directly or indirectly regulated by estrogen receptor were up- or down-regulated in the parous breast and were found to be enriched in the breast cancer estrogen signaling gene set. Among them, GATA3, an important component of this gene set, is crucial to mammary gland morphogenesis and differentiation of progenitor cells. GATA3 has been suggested to be a tumor suppressor
], a fact supported by the observations that induction of its expression in GATA3-negative undifferentiated carcinoma cells is sufficient to induce tumor differentiation and inhibition of tumor dissemination
]. The down-regulation of RASD1 (RAS, dexamethasone-induced 1), a potential miR-375 target that negatively regulates ER alpha expression in breast cancer further confirms that the genes involved in the estrogen receptor regulated pathways could be under permanent transcriptional modification as a manifestation of a higher degree of cell differentiation of the parous breast, in spite of the lack of transcriptomic differences in the levels of the receptor between parous and nulliparous breast tissues.
Cell communication, which is a key element in the process of cell and organ differentiation, is well represented in the breast of parous women. The parous breast exhibits up-regulation of desmocollin (DSC3), a calcium-dependent glycoprotein that is a member of the desmocollin subfamily of the cadherin superfamily. Members of this desmosomal family, along with the desmogleins, are found primarily in epithelial cells where they constitute the adhesive proteins of the desmosome cell-cell junction and are required for cell adhesion and desmosome formation. In addition, the up-regulation of matrix Gla proteins (MGP), laminins (LAMA3 and LAMC2) and keratin 5 (KRT5) in the parous breast reflect the greater differentiated state of the breast epithelial cells
]. This concept is supported by the observation that the loss of Matrix Gla protein expression may be associated with tumor progression and metastasis
Our findings that insulin-like growth factor 1 (IGF-1) is down-regulated in the parous breast is consistent with published data reporting overall lower levels of IGF-1 in parous than in nulliparous women
] and support the association of IGF1 with increased breast cancer risk
]. It is known that IGF-1 stimulates mitosis and inhibits apoptosis, playing a significant role in signaling pathways involved in the pathogenesis of breast cancer. The down regulation of IGF-1 in the parous breast, in association with the significant down-regulation of SOX6, EBF1 (early B-cell factor 1), ABHD5, RASD1, a potential miR-375 target that negatively regulates ER alpha expression in breast cancer
], and RALGAPA2, could represent a significant driving force in the reduction of breast cancer risk conferred by pregnancy.