Progesterone downregulation of miR-141 contributes to expansion of stem-like breast cancer cells through maintenance of progesterone receptor and Stat5a.

Progesterone (P4) has emerged as an important hormone-regulating mammary stem cell (MaSC) populations. In breast cancer, P4 and synthetic analogs increase the number of stem-like cells within luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancers. These cells gain expression of de-differentiated cell markers CD44 and cytokeratin 5 (CK5), lose luminal markers ER and PR, and are more therapy resistant. We previously described that P4 downregulation of microRNA (miR)-29a contributes to the expansion of CD44(high) and CK5(+) cells. Here we investigated P4 downregulation of miR-141, a member of the miR-200 family of tumor suppressors, in facilitating an increase in stem-like breast cancer cells. miR-141 was the sole member of the miR-200 family P4-downregulated at the mature miRNA level in luminal breast cancer cell lines. Stable inhibition of miR-141 alone increased the CD44(high) population, and potentiated P4-mediated increases in both CD44(high) and CK5(+) cells. Loss of miR-141 enhanced both mammosphere formation and tumor initiation. miR-141 directly targeted both PR and signal transducer and activator of transcription 5A (Stat5a), transcription factors important for MaSC expansion. miR-141 depletion increased PR protein levels, even in cell lines where PR expression is estrogen dependent. Stat5a suppression via small interfering RNA or a small-molecule inhibitor reduced the P4-dependent increase in CK5(+) and CD44(high) cells. These data support a mechanism by which P4-triggered loss of miR-141 facilitates breast cancer cell de-differentiation through deregulation of PR and Stat5a, two transcription factors important for controlling mammary cell fate.

Progestin suppression of miR-29 potentiates dedifferentiation of breast cancer cells via KLF4.

The female hormone progesterone (P4) promotes the expansion of stem-like cancer cells in estrogen receptor (ER)- and progesterone receptor (PR)-positive breast tumors. The expanded tumor cells lose expression of ER and PR, express the tumor-initiating marker CD44, the progenitor marker cytokeratin 5 (CK5) and are more resistant to standard endocrine and chemotherapies. The mechanisms underlying this hormone-stimulated reprogramming have remained largely unknown. In the present study, we investigated the role of microRNAs in progestin-mediated expansion of this dedifferentiated tumor cell population. We demonstrate that P4 rapidly downregulates miR-29 family members, particularly in the CD44(+) cell population. Downregulation of miR-29 members potentiates the expansion of CK5(+) and CD44(+) cells in response to progestins, and results in increased stem-like properties in vitro and in vivo. We demonstrate that miR-29 directly targets Krüppel-like factor 4 (KLF4), a transcription factor required for the reprogramming of differentiated cells to pluripotent stem cells, and for the maintenance of breast cancer stem cells. These results reveal a novel mechanism, whereby progestins increase the stem cell-like population in hormone-responsive breast cancers, by decreasing miR-29 to augment PR-mediated upregulation of KLF4. Elucidating the mechanisms whereby hormones mediate the expansion of stem-like cells furthers our understanding of the progression of hormone-responsive breast cancers.

Cloning of a mammalian transcriptional activator that binds unmethylated CpG motifs and shares a CXXC domain with DNA methyltransferase, human trithorax, and methyl-CpG binding domain protein 1.

Ligand screening was utilized to isolate a human cDNA that encodes a novel CpG binding protein, human CpG binding protein (hCGBP). This factor contains three cysteine-rich domains, two of which exhibit homology to the plant homeodomain finger domain. A third cysteine-rich domain conforms to the CXXC motif identified in DNA methyltransferase, human trithorax, and methyl-CpG binding domain protein 1. A fragment of hCGBP that contains the CXXC domain binds to an oligonucleotide probe containing a single CpG site, and this complex is disrupted by distinct oligonucleotide competitors that also contain a CpG motif(s). However, hCGBP fails to bind oligonucleotides in which the CpG motif is either mutated or methylated, and it does not bind to single-stranded DNA or RNA probes. Furthermore, the introduction of a CpG dinucleotide into an unrelated oligonucleotide sequence is sufficient to produce a binding site for hCGBP. Native hCGBP is detected as an 88-kDa protein by Western analysis and is ubiquitously expressed. The DNA-binding activity of native hCGBP is apparent in electrophoretic mobility shift assays, and hCGBP trans-activates promoters that contain CpG motifs but not promoters in which the CpG is ablated. These data indicate that hCGBP is a transcriptional activator that recognizes unmethylated CpG dinucleotides, suggesting a role in modulating the expression of genes located within CpG islands.

YY1 binds five cis-elements and trans-activates the myeloid cell-restricted gp91(phox) promoter.

Four transcriptional activating cis-elements within the gp91(phox) promoter bind a protein complex of similar mobility and binding specificity, denoted BID (binding increased during differentiation). The intensity of BID complexes increases upon myeloid cell differentiation, coincident with induction of gp91(phox) expression, and BID competes with the transcriptional repressor CDP for binding to each of these promoter elements. To determine the identity of BID, an expression library was ligand screened with the BID-binding site that surrounds the -145-base pair (bp) region of the gp91(phox) promoter. One recovered factor that exhibits the expected binding specificity is YY1, a ubiquitous multifunctional transcription factor. BID complexes that form with the four binding sites within the gp91(phox) promoter are disrupted by YY1 antiserum, and a fifth YY1-binding site was detected in the -412-bp promoter region. Overexpression of YY1 in transient co-transfection assays trans-activates a minimal promoter containing two copies of the -145-bp binding site from the gp91(phox) promoter. Neither the level of YY1 protein nor DNA binding activity increases during myeloid cell differentiation. These studies identify a target gene of YY1 function in mature myeloid cells, and demonstrate that YY1 function can be controlled during myeloid development by the modulation of a competing DNA-binding factor.

Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression?

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.

Structure of the distal human gonadotropin releasing hormone (hGnrh) gene promoter and functional analysis in Gt1-7 neuronal cells.

To assess potential species-specific expression of gonadotropin releasing hormone (GnRH), the distal human (h) GnRH promoter was cloned, characterized and tested in gene transfer studies. The nucleotide sequence of approximately 3.8 kb of 5'-flanking region was determined. Homology to the rat (r) GnRH sequence was observed in the proximal promoter region between -551 h (-424 r) and the transcriptional start site and within multiple distal promoter regions. In contrast, there was little similarity in the sequences between -1131/-551 h and -1031/-424 r. A deletion panel of 5'-flanking hGnRH promoter constructs was made and tested in transient transfection assays in GnRH-producing mouse GT1-7 neuronal cells. The largest hGnRH promoter construct (-3832/+5 h) exhibited high levels of reporter activity, similar to that observed with the largest rGnRH construct (-3026/+116 r). However, in contrast to the rat gene, deletion of distal promoter sequences of the hGnRH promoter to -1971, -1131 or -551 did not result in a decrease in luciferase reporter activity. Further truncation to -350 resulted in a 3-fold decrease in luciferase activity. There was no preferential use of the putative upstream hGnRH start site in neuronal cells. DNase I protection assays showed unique protection patterns with nuclear extracts from GT1-7 and Gn10 neuronal cells and the hGnRH and rGnRH promoter fragments. These data suggest the presence of different cis-acting elements and transacting factors that mediate species-specific neuronal GnRH expression.

Direct binding of progesterone receptor to nonconsensus DNA sequences represses rat GnRH.

The mechanisms by which steroid receptors repress gene expression are not well understood. In this report, we show that progesterone receptor (PR), in the presence of progesterone (P) directly represses rat gonadotropin releasing hormone (rGnRH) gene transcription. Deletion analysis studies using transient transfection assays in GT1-7 neuronal cells mapped the effects of P to sequences in the proximal rGnRH promoter between -171 and -73. This DNA sequence lacks any consensus steroid response element binding sites. Cotransfection of a mutant progesterone receptor that lacks a functional DNA binding region (hPRcys) abolished repression of the rGnRH promoter by P. Gel mobility shift assays confirmed that PR directly binds to the DNA fragments -171/-126, -126/-73, and -111/-73, which encompass the negative progesterone response element (nPRE) of the rGnRH promoter. Mutagenesis of the rGnRH nPRE -171/-126 DNA fragment resulted in a loss of PR binding. Thus, direct DNA binding of PR to nonconsensus elements in the proximal rGnRH promoter inhibits rGnRH gene expression.