Cross talk between progesterone receptors and retinoic acid receptors in regulation of cytokeratin 5-positive breast cancer cells.

Half of estrogen receptor-positive breast cancers contain a subpopulation of cytokeratin 5 (CK5)-expressing cells that are therapy resistant and exhibit increased cancer stem cell (CSC) properties. We and others have demonstrated that progesterone (P4) increases CK5+ breast cancer cells. We previously discovered that retinoids block P4 induction of CK5+ cells. Here we investigated the mechanisms by which progesterone receptors (PR) and retinoic acid receptors (RAR) regulate CK5 expression and breast CSC activity. After P4 treatment, sorted CK5+ compared to CK5- cells were more tumorigenic in vivo. In vitro, P4-treated breast cancer cells formed larger mammospheres and silencing of CK5 using small hairpin RNA abolished this P4-dependent increase in mammosphere size. Retinoic acid (RA) treatment blocked the P4 increase in CK5+ cells and prevented the P4 increase in mammosphere size. Dual small interfering RNA (siRNA) silencing of RARα and RARγ reversed RA blockade of P4-induced CK5. Using promoter deletion analysis, we identified a region 1.1 kb upstream of the CK5 transcriptional start site that is necessary for P4 activation and contains a putative progesterone response element (PRE). We confirmed by chromatin immunoprecipitation that P4 recruits PR to the CK5 promoter near the -1.1 kb essential PRE, and also to a proximal region near -130 bp that contains PRE half-sites and a RA response element (RARE). RA induced loss of PR binding only at the proximal site. Interestingly, RARα was recruited to the -1.1 kb PRE and the -130 bp PRE/RARE regions with P4, but not RA alone or RA plus P4. Treatment of breast cancer xenografts in vivo with the retinoid fenretinide reduced the accumulation of CK5+ cells during estrogen depletion. This reduction, together with the inhibition of CK5+ cell expansion through RAR/PR cross talk, may explain the efficacy of retinoids in prevention of some breast cancer recurrences.

Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02-fold, P<0.01 compared with vehicle control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. Short hairpin RNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.

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.

Mapping the unique activation function 3 in the progesterone B-receptor upstream segment. Two LXXLL motifs and a tryptophan residue are required for activity.

Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription. AF1 and AF2 are common to the two isoforms of PR, PR-A and PR-B, whereas AF3 lies within the N-terminal 164 amino acids unique to PR-B, termed the "B-upstream segment" (BUS). To define the BUS regions that contribute to AF3 function, we generated a series of deletion and amino acid substitution mutants and tested them in three backgrounds as follows: BUS alone fused to the PR DNA binding domain (BUS-DBD), the entire PR-B N terminus linked to its DBD (NT-B), and full-length PR-B. Analyses of these mutants identified two regions in BUS whose loss reduces AF3 activity by more than 90%. These are associated with amino acids 54-90 (R1) and 120-154 (R2). R1 contains a consensus (55)LXXLL(59) motif (L1) identical to ones found in nuclear receptor co-activators. R2 is adjacent to a second nuclear receptor box (L2) at (115)LXXLL(119) and contains a conserved tryptophan (Trp-140). Their mutation completely disrupts AF3 activity in a promoter and cell type-independent manner. Critical mutations elicited similar effects on all three B-receptor backgrounds. This underscores the probability that these mutations alter a process linking BUS structure to the function of full-length PR-B in a fundamental way.

Different pathways regulate expression of the skeletal myosin heavy chain genes.

Mammalian skeletal muscles are a mosaic of different fiber types largely defined by differential myosin heavy chain (MyHC) expression. Little is known about the molecular mechanisms regulating expression of the MyHC gene family members in different fiber types. In this work, we identified several cis- and trans-elements that regulate expression of the three adult fast MyHC genes. Despite multiple DNA-binding motifs for well characterized muscle transcription factors upstream of all three fast MyHC genes, expression of MyoD/Myf-5, calcineurin, or NFAT3 had different effects on the three promoters. MyoD or Myf-5 overexpression preferentially activated the IIb promoter, whereas NFAT or activated calcineurin overexpression preferentially activated the IIa promoter. Calcineurin had a 50-100-fold stimulatory effect on the IIa promoter, and the known downstream effectors of calcineurin (myocyte enhancer factor-2 and NFAT) cannot completely account for this activation. Finally, we identified two elements critical for regulating MyHC-IId/x expression: a 130-base pair enhancer element and a CArG-like element that inhibited IId/x promoter activity in vitro. Thus, we have found specific regulatory pathways that are distinct for the three adult fast MyHC genes. These elements are logical candidates for fiber-specific control of skeletal muscle gene expression in vivo.

Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors.

Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBD(PR)) containing two highly conserved zinc fingers. DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors. We have now identified four HeLa cell nuclear proteins that copurify with a glutathionine-S-transferase-human DBD(PR )fusion protein. Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADP-ribose) polymerase. The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PK(CS). DNA-PK that was 'pulled-down' by DBD(PR) on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PK(CS), (2) transphosphorylate DBD(PR), and (3) phosphorylate a DNA-PK-specific p53 peptide substrate. DNA-PK was also able to associate with the DBD of the yeast activator GAL4. However, neither a PR DBD mutant lacking a structured first zinc finger (DBD(CYS)) nor the core DBD of the estrogen receptor (DBD(ER)) copurified DNA-PK, suggesting the interaction is not non-specific for DBDs. Lastly, we found that DNA-PK copurified with full-length human PR transiently expressed in HeLa cells, suggesting that the human PR/DNA-PK complex can assemble in vivo. These data show that DNA-PK and DBD(PR) interact, that DBD(PR) is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription.

Myosin heavy chains IIa and IId are functionally distinct in the mouse.

Myosin in adult murine skeletal muscle is composed primarily of three adult fast myosin heavy chain (MyHC) isoforms. These isoforms, MyHC-IIa, -IId, and -IIb, are >93% identical at the amino acid level and are broadly expressed in numerous muscles, and their genes are tightly linked. Mice with a null mutation in the MyHC-IId gene have phenotypes that include growth inhibition, muscle weakness, histological abnormalities, kyphosis (spinal curvature), and aberrant kinetics of muscle contraction and relaxation. Despite the lack of MyHC-IId, IId null mice have normal amounts of myosin in their muscles because of compensation by the MyHC-IIa gene. In each muscle examined from IId null mice, there was an increase in MyHC-IIa- containing fibers. MyHC-IIb content was unaffected in all muscles except the masseter, where its expression was extinguished in the IId null mice. Cross-sectional fiber areas, total muscle cross-sectional area, and total fiber number were affected in ways particular to each muscle. Developmental expression of adult MyHC genes remained unchanged in IId null mice. Despite this universal compensation of MyHC-IIa expression, IId null mice have severe phenotypes. We conclude that despite the similarity in sequence, MyHC-IIa and -IId have unique roles in the development and function of skeletal muscle.

Surprises with antiprogestins: novel mechanisms of progesterone receptor action.

When hormone antagonists have inappropriate agonist-like effects, the clinical consequences are grave. We describe novel molecular mechanisms by which antiprogestin-occupied progesterone receptors behave like agonists. These mechanisms include agonist-like transcriptional effects that do not require receptor binding to DNA at progesterone response elements, or that result from cross-talk between progesterone receptors and other signalling pathways. We discuss the complex structural organization of progesterone receptors, and demonstrate that the B receptor isoform has a unique third activation domain that may confer agonist-like properties in the presence of antiprogestins, whereas the A receptor isoform is a dominant-negative inhibitor. We argue that these novel mechanisms play a role in the apparent hormone resistance of breast cancers and the variable tissue-specific responses to antagonists.

A third transactivation function (AF3) of human progesterone receptors located in the unique N-terminal segment of the B-isoform.

Human progesterone target tissues contain two progesterone receptors: B-receptors (hPRB), which are 933 amino acids in length, and A-receptors (hPRA), which lack the N-terminal 164 amino acids. The two isoforms differ functionally when they are occupied by agonists or antagonists. We postulated that the unique 164-amino acid, B-upstream segment (BUS) is in part responsible for the functional differences between the two isoforms and have constructed a series of hPR expression vectors encoding BUS fused to isolated down-stream functional domains of the receptors. These include the two transactivation domains: activation function-1 (AF1), located in a 90-amino acid segment just up-stream of the DNA-binding domain (DBD) and nuclear localization signal (NLS), and AF2, located in the hormone-binding domain. BUS is a highly phosphorylated domain, and contains the serine residues responsible for the hPRB triplet protein structure. The construct containing BUS-DBD-NLS binds tightly to DNA when aided by accessory nuclear factors. In HeLa cells, BUS-DBD-NLS strongly and autonomously activates transcription of chloramphenicol acetyltransferase (CAT) from a promoter containing two progesterone response elements (PRE2-TATAtk-CAT). Transcription levels with BUS-DBD-NLS are equivalent to those seen with full-length hPRB, and are higher than those seen with hPRA. BUS specifically requires an intact hPR DBD to be transcriptionally active. DBD mutants that cannot bind DNA or whose DNA binding specificity has been switched to an estrogen response element cannot cooperate in BUS transcriptional activity. The function of BUS-DBD-NLS is promoter and cell specific. It does not transactivate a CAT reporter driven by the mouse mammary tumor virus promoter in HeLa cells and poorly transactivates PRE2-TATAtk-CAT in PR-negative T47D breast cancer cells. However, in the breast cancer cells, BUS-DBD-NLS transactivation of PRE2-TATAtk-CAT can be reconstituted by either elevating cellular levels of cAMP or linking BUS and DBD to AF1 or AF2 of hPR, each of which alone is also inactive in these cells. We conclude that hPRB contains a unique third activation function (AF3) located within BUS and requiring the functional DBD of hPR. Depending on the promoter or cell tested, AF3 can activate transcription autonomously, or it can functionally synergize with AF1 or AF2. Autonomous AF3 function may explain the unexpected transactivating actions of antiprogestin-occupied hPRB, an issue of importance in hormone-resistant breast cancers and in tissue-specific agonist-like effects of hormone antagonists.

New T47D breast cancer cell lines for the independent study of progesterone B- and A-receptors: only antiprogestin-occupied B-receptors are switched to transcriptional agonists by cAMP.

Because progesterone antagonists are growth inhibitors, they are in Phase III clinical trials for the treatment of breast cancer. However, when cellular cAMP levels are elevated, some antiprogestins inappropriately activate transcription. We have proposed that hormone "resistance" may result from such unintended stimulation of breast cancer by antagonists. In transient expression systems, the two natural isoforms of human progesterone receptors (PR), B-receptors and truncated A-receptors, have dissimilar effects on agonist-mediated transcription. We show here that in the presence of 8-Br-cAMP, antiprogestin-occupied B-receptors but not A-receptors become transcriptional activators. Therefore, we developed new model systems to study each PR isoform independently in a breast cancer setting: (a) a stable PR-negative monoclonal subline (T47D-Y) of PR-positive T47D breast cancer cells was selected by flow cytometric PR screening. T47D-Y cells are PR-negative by immunoassays, by ligand binding assay, by growth resistance to progestins, by failure to bind a progesterone response element (PRE) in vitro, and by failure to transactivate PRE-regulated promoters; and (b) T47D-Y cells were stably transfected with expression vectors encoding one or the other PR isoform, and two monoclonal cell lines were selected that express either B-receptors (T47D-YB) or A-receptors (T47D-YA) at levels equal to those seen in natural T47D cells. The ectopically expressed receptors are properly phosphorylated, and like endogenously expressed receptors, they undergo ligand-dependent down-regulation. The expected B:B or A:A homodimers are present in cell extracts from each cell line, but A:B heterodimers are missing in both. In the presence of agonists, cAMP-dependent, transcriptional synergism of PRE-regulated promoters is seen in both cell lines. By contrast, in the presence of the antiprogestins RU486 or ZK112993, inappropriate transactivation occurs in YB cells but not in YA cells. The class of antiprogestins represented by ZK98299, which blocks PR binding to DNA, does not activate transcription in either cell line. We propose that these new cell lines are physiological models for the study of PR isoform-specific antiprogestin resistance in breast cancer.

Antagonist-occupied human progesterone receptors bound to DNA are functionally switched to transcriptional agonists by cAMP.

When steroid hormone antagonists have inappropriate agonist effects, the clinical consequences are grave. Progesterone antagonists bind to two naturally occurring isoforms of human progesterone receptors (hPR), hPRB and the NH2-terminally truncated hPRA, and usually inhibit agonist-stimulated transcription. It is shown here that elevation of cAMP levels in a human breast cancer cell line leads to the functional reversal of progesterone antagonist action. While hPR occupied by the antagonists RU486 and ZK112993 are transcriptionally inactive, the antagonist-occupied receptors become strong activators of transcription in the presence of 8-Br-cAMP. However, this functional switch does not occur with the progesterone antagonist ZK98299, which, unlike RU486 and ZK112993, is unable to induce hPR binding to DNA. This suggests that the 8-Br-cAMP-induced transcriptional reversal requires that the antagonist-occupied receptors be bound to DNA. Even with agonist-occupied hPR, addition of 8-Br-cAMP results in a synergistic increase in transcriptional activity. When hPRA alone are transiently expressed in COS-1 cells, transcription of a reporter gene is stimulated by the agonist R5020 and by 8-Br-cAMP and is synergistic when both are present; but the 8-Br-cAMP-dependent component of transcription proceeds in the absence of hPRA, in the absence of the progesterone response element, and in the presence of a DNA-binding domain mutant of hPRA that cannot bind to the progesterone response element. Additionally, under the intracellular conditions in which 8-Br-cAMP activates antagonist-hPR complexes, there is no protein kinase A-mediated phosphorylation of the receptors. We discuss a model in which a gene that is independently transcribed by cAMP-responsive factors and by hPR can be selected for positive or negative regulation on the transcription complex due to additive or cooperative interactions between the two DNA-bound factors.