Luminal Epithelial Cell Differentiation Research Articles

The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer

BackgroundThe androgen receptor (AR) is a tumor suppressor in estrogen receptor (ER) positive breast cancer, a role sustained in some ER negative breast cancers. Key factors dictating AR genomic activity in a breast context are largely unknown. Herein, we employ an unbiased chromatin immunoprecipitation-based proteomic technique to identify endogenous AR interacting co-regulatory proteins in ER positive and negative models of breast cancer to gain new insight into mechanisms of AR signaling in this disease.ResultsThe DNA-binding factor GATA3 is identified and validated as a novel AR interacting protein in breast cancer cells irrespective of ER status. AR activation by the natural ligand 5α-dihydrotestosterone (DHT) increases nuclear AR-GATA3 interactions, resulting in AR-dependent enrichment of GATA3 chromatin binding at a sub-set of genomic loci. Silencing GATA3 reduces but does not prevent AR DNA binding and transactivation of genes associated with AR/GATA3 co-occupied loci, indicating a co-regulatory role for GATA3 in AR signaling. DHT-induced AR/GATA3 binding coincides with upregulation of luminal differentiation genes, including EHF and KDM4B, established master regulators of a breast epithelial cell lineage. These findings are validated in a patient-derived xenograft model of breast cancer. Interaction between AR and GATA3 is also associated with AR-mediated growth inhibition in ER positive and ER negative breast cancer.ConclusionsAR and GATA3 interact to transcriptionally regulate luminal epithelial cell differentiation in breast cancer regardless of ER status. This interaction facilitates the tumor suppressor function of AR and mechanistically explains why AR expression is associated with less proliferative, more differentiated breast tumors and better overall survival in breast cancer.

Maternal and embryonic signals cause functional differentiation of luminal epithelial cells and receptivity establishment.

Embryo implantation requires temporospatial maternal-embryonic dialog. Using single-cell RNA sequencing for the uterus from 2.5 to 4.5days post-coitum (DPC) and bulk sequencing for the corresponding embryos of 3.5 and 4.0 DPC pregnant mice, we found that estrogen-responsive luminal epithelial cells (EECs) functionally differentiated into adhesive epithelial cells (AECs) and supporting epithelial cells (SECs), promoted by progesterone. Along with maternal signals, embryonic Pdgfa and Efna3/4 signaling activated AECs and SECs, respectively, enhancing the attachment of embryos to the endometrium and furthering embryo development. This differentiation process was largely conserved between humans and mice. Notably, the developmental defects of SOX9-positive human endometrial epithelial cells (similar to mouse EEC) were related to thin endometrium, whereas functional defects of SEC-similar unciliated epithelial cells were related to recurrent implantation failure (RIF). Our findings provide insights into endometrial luminal epithelial cell development directed by maternal and embryonic signaling, which is crucial for endometrial receptivity.

Abstract 54: Regulation of luminal epithelial cell identity in the pre-tumorigenic mammary gland

Abstract The transcription factor MYC is frequently altered in a large portion of human breast cancers; the gene is amplified in ~15% of breast cancers and overexpressed in another 30-50%. Mouse mammary tumor models driven by overexpression of MYC, such as MMTV-Myc, also exhibit a rapid onset and high frequency of mammary tumors indicating that MYC overexpression plays an important role in driving breast carcinogenesis. However, the role of MYC in breast cancer initiation and subtype determination is not well understood. In the normal mammary gland, MYC is a critical regulator of and is essential for alveolar luminal epithelial cell differentiation during pregnancy. Aberrant MYC expression can promote improper alveolar development and milk production and is a potent inducer of cellular proliferation. Based on these known functions of MYC during development, we hypothesized that its overexpression may induce early changes in epithelial cell differentiation leading to pre-tumorigenic shifts in cell lineages that contribute to the formation of luminal breast tumors. To investigate this, we analyzed the mammary glands of MMTV-Myc (MYC) mice prior to any signs of tumor formation. As early as 5 weeks of age, MYC overexpression led to alterations at both the histological and cellular levels. Interestingly, pre-tumorigenic MYC glands phenotypically resembled mid-pregnant glands of WT mice. Specifically, pre-tumorigenic MYC glands demonstrated budding along the mammary ducts and expansion of a Sca1−/CD49b− luminal epithelial cell subset not typically seen in nulliparous mice. RNA-seq was performed on luminal epithelial cells isolated from WT or pre-cancerous MYC mammary glands to define the luminal differentiation states upon MYC overexpression. While there was an overlap in twenty-nine genes that control milk production and cellular metabolism between pre-cancerous MYC luminal cells and those from mid-pregnant WT mice, luminal cells from MYC mice also exhibited a unique fingerprint of forty-one genes that overlap with cell states found in breast cancer. These uniquely expressed luminal cell genes may be important for MYC-regulated tumorigenesis. Ongoing and future experiments will investigate the role of specific MYC-target genes in mammary gland development versus tumor formation to clarify the role of MYC in these divergent processes. Citation Format: Jerrica Breindel, Kayla M. Gross, Charlotte Kuperwasser. Regulation of luminal epithelial cell identity in the pre-tumorigenic mammary gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 54.

Functional Differentiation of Luminal Epithelial Cells Regulated by Maternal and Embryonic Signaling and Its Relationship With Receptivity Establishment

Functional Differentiation of Luminal Epithelial Cells Regulated by Maternal and Embryonic Signaling and Its Relationship With Receptivity Establishment

Loss of function of GATA3 induces basal-like mammary tumors.

Purpose: GATA3 is a transcription factor essential for mammary luminal epithelial cell differentiation. Expression of GATA3 is absent or significantly reduced in basal-like breast cancers. Gata3 loss-of-function impairs cell proliferation, making it difficult to investigate the role of GATA3 deficiency in vivo. We previously demonstrated that CDK inhibitor p18INK4c (p18) is a downstream target of GATA3 and restrains mammary epithelial cell proliferation and tumorigenesis. Whether and how loss-of-function of GATA3 results in basal-like breast cancers remains elusive.Methods: We generated mutant mouse strains with heterozygous germline deletion of Gata3 in p18 deficient backgrounds and developed a Gata3 depleted mammary tumor model system to determine the role of Gata3 loss in controlling cell proliferation and aberrant differentiation in mammary tumor development and progression.Results: Haploid loss of Gata3 reduced mammary epithelial cell proliferation with induction of p18, impaired luminal differentiation, and promoted basal differentiation in mammary glands. p18 deficiency induced luminal type mammary tumors and rescued the proliferative defect caused by haploid loss of Gata3. Haploid loss of Gata3 accelerated p18 deficient mammary tumor development and changed the properties of these tumors, resulting in their malignant and luminal-to-basal transformation. Expression of Gata3 negatively correlated with basal differentiation markers in MMTV-PyMT mammary tumor cells. Depletion of Gata3 in luminal tumor cells also reduced cell proliferation with induction of p18 and promoted basal differentiation. We confirmed that expression of GATA3 and basal markers are inversely correlated in human basal-like breast cancers.Conclusions: This study provides the first genetic evidence demonstrating that loss-of-function of GATA3 directly induces basal-like breast cancer. Our finding suggests that basal-like breast cancer may also originate from luminal type cancer.

The helix-loop-helix transcriptional regulator Id4 is required for terminal differentiation of luminal epithelial cells in the prostate.

Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. In this study we investigated the effect of loss of Id4 (Id4-/-) on mouse prostate development. Histological analysis was performed on prostates from 25 days, 3 months and 6 months old Id4-/- mice. Expression of Amacr, Ck8, Ck18, Fkbp51, Fkbp52, androgen receptor, Pten, sca-1 and Nkx3.1 was investigated by immunohistochemistry. Results were compared to the prostates from Nkx3.1-/- mice. Id4-/- mice had smaller prostates with fewer and smaller tubules. Subtle PIN like lesions were observed at 6mo. Decreased Nkx3.1 and Pten and increased stem cell marker sca-1, PIN marker Amacr and basal cell marker p63 was observed at all ages. Persistent Ck8 and Ck18 expression suggested that loss of Id4 results in epithelial commitment but not terminal differentiation in spite of active Ar. Loss of Id4 attenuates normal prostate development and promotes hyperplasia/ dysplasia with PIN like lesions. The results suggest that loss of Id4 maintains stem cell phenotype of “luminal committed basal cells”, identifying a unique prostate developmental pathway regulated by Id4.

Cysteine Dioxygenase Regulates the Epithelial Morphogenesis of Mammary Gland via Cysteine Sulfinic Acid

SummaryEpithelial morphogenesis is a common feature in various organs and contributes to functional formation. However, the molecular mechanisms behind epithelial morphogenesis remain largely unknown. Mammary gland is an excellent model system to investigate the molecular mechanisms of epithelial morphogenesis. In this study, we found that cysteine dioxygenase (CDO), a key enzyme in cysteine oxidative metabolism, was involved in mammary epithelial morphogenesis. CDO knockout (KO) females exhibited severe defects in mammary branching morphogenesis and ductal elongation, resulting in poor lactation. CDO contributes to the luminal epithelial cell differentiation, proliferation, and apoptosis mainly through its downstream product cysteine sulfinic acid (CSA). Exogenous supplementation of CSA not only rescued the defects in CDO KO mouse but also enhanced ductal growth in wild-type mouse. It suggests that CDO regulates luminal epithelial differentiation and regeneration via CSA and consequently contributes to mammary development, which raises important implications for epithelial morphogenesis and pathogenesis of breast cancer.

Prognostic implication of GATA3 gene mutation on survival in invasive ductal carcinoma of the breast.

e13114 Background: GATA3 encodes a transcription factor, which is involved in activation and suppression of genes involved in cell maturation. GATA3 is necessary in the adult mammary gland to maintain the integrity and function of the luminal epithelium. Methods: METABRIC project funded by cancer research UK, the British Columbia cancer foundation and the Canadian breast cancer foundation mapped 173 gene mutations and amplifications in 2,433 primary breast tumors. Retrospective analysis was done for patients with invasive ductal carcinoma of the breast in the age group 30-60; to study the effect of GATA3 mutation on survival. Median survival was obtained from Kaplan-Meier plot, and mortality between groups was compared by Odds ratio (OR). Results: A total of 1500 patients across all age groups had invasive ductal carcinoma. 650 were within the age group 30-60; of which 234 died due to the disease, 360 were alive and 55 died due to other causes. 398 patients (61.2%) tested positive for estrogen receptor (ER) and 521 patients (80.2%) were negative for HER2 (human epidermal growth factor receptor 2). TP53 (50%) and PIK3CA (36%) mutations were more prevalent. GATA3 mutation was found in 79 patients (12.34%); among which, all 79 patients tested positive for ER (100%) and 74 patients (94.9%) negative for HER2. 10 patients (12.7%) died due to the disease, 62 patients (78.5%) were alive and 7 patients (8.9%) died due to other causes. Hence, patients with GATA3 mutations were more likely to survive (OR 4.66; CI 2.33-9.29 p < 0.0001) than patients without the mutation. The median survival for patients with GATA3 mutation (300 months) was also greater than patients without the mutation (219 months). In addition, patients with GATA3 mutation were more likely to be ER positive and HER2 negative. Conclusions: In the mammary gland, GATA3 is required for luminal epithelial cell differentiation. Loss of GATA3 results in de-differentiation to stem cell phenotype. It is found that GATA3 mutation correlates with a better prognosis compared to more common TP53 and PIK3CA gene mutations.

Miz1, a Novel Target of ING4, Can Drive Prostate Luminal Epithelial Cell Differentiation.

How prostate epithelial cells differentiate and how dysregulation of this process contributes to prostate tumorigenesis remain unclear. We recently identified a Myc target and chromatin reader protein, ING4, as a necessary component of human prostate luminal epithelial cell differentiation, which is often lost in primary prostate tumors. Furthermore, loss of ING4 in the context of oncogenic mutations is required for prostate tumorigenesis. Identifying the gene targets of ING4 can provide insight into how its loss disrupts differentiation and leads to prostate cancer. Using a combination of RNA-Seq, a best candidate approach, and chromatin immunoprecipitation (ChIP), we identified Miz1 as a new ING4 target. ING4 or Miz1 overexpression, shRNA knock-down, and a Myc-binding mutant were used in a human in vitro differentiation assay to assess the role of Miz1 in luminal cell differentiation. ING4 directly binds the Miz1 promoter and is required to induce Miz1 mRNA and protein expression during luminal cell differentiation. Miz1 mRNA was not induced in shING4 expressing cells or tumorigenic cells in which ING4 is not expressed. Miz1 dependency on ING4 was unique to differentiating luminal cells; Miz1 mRNA expression was not induced in basal cells. Although Miz1 is a direct target of ING4, and its overexpression can drive luminal cell differentiation, Miz1 was not required for differentiation. Miz1 is a newly identified ING4-induced target gene which can drive prostate luminal epithelial cell differentiation although it is not absolutely required. Prostate 77:49-59, 2017. © 2016 Wiley Periodicals, Inc.

Androgen-Sensitized Apoptosis of HPr-1AR Human Prostate Epithelial Cells.

Androgen receptor (AR) signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. The mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5α-dihydrotestosterone (DHT) treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS), TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP) in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2) were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1) were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins, such that androgen signaling sensitizes mitochondria to apoptotic signaling, thus rendering HPr-1AR more vulnerable to cell death signals. Our study offers insight into AR-mediated regulation of prostate epithelial cell death signaling.

Abstract B16: Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. Cancer genome sequencing studies focusing on TNBC failed to identify novel recurrently mutated cancer-driving genes, obviating immediate opportunities for targeted therapeutic development. Here we have identified BET bromodomain inhibitors (BBDIs) as promising novel therapeutic agents in TNBC. Specifically, we have found that TNBC cells are significantly and preferentially growth inhibited by BBDIs (e.g., JQ1) with IC50s in the low nM range compared to luminal breast cancer cells. The growth of established xenografts derived from TNBC cell lines and primary patient samples were also efficiently inhibited by BBDIs treatment. BRD4, a target of BBDIs, is highly expressed in TNBCs compared to luminal cell lines and its downregulation using shRNAs inhibits TNBC cell growth in cell culture and induces tumor regression in vivo. In line with the role of BRD4 in transcription restart after G2/M, BBDI treatment prevented cell cycle re-entry, arrested TNBC cells in early G1, apoptosis, and induced luminal epithelial cell differentiation. Using integrated epigenomic analysis, we have identified the direct transcriptional targets of BBDI in TNBC. BBDI was found to efficiently displace chromatin-bound BRD4 in sensitive TNBC cells. This effect was more pronounced at genomic regions exhibiting elevated Bio-JQ1 binding by Chem-seq, implying strong association between BRD4 and JQ1 genomic binding patterns. By genome-wide study with ChIP-seq and RNA-seq, we have also found that selective disruption of super-enhancers associated genes by JQ1, leading to deregulation of coordinated transcriptional pathways involved in cell proliferation, invasion, and survival. The sensitivity of TNBC and other tumor types to BET inhibition establishes a rationale for clinical investigation and a motivation to understand mechanisms of resistance. After selecting derivatives with acquired resistance to BET inhibition in previously sensitive TNBCs, we identified a unique mechanism of epigenomic resistance to this epigenetic therapy. Resistant cells remained dependent on BRD4. However, TNBC cells adapt to BET bromodomain inhibition by maintaining recruitment of unmutated BRD4 to super-enhancers in a bromodomain-independent manner. Differential super-enhancer analysis found a significant gain in the number of SEs in resistant cells, and a less pronounced loss of fewer SEs. The gain of Bio-JQ1 SEs was associated with gain of BRD4 binding to these genomic loci and also with increased transcription of the associated genes. Among the top gained super-enhancers in resistant cells was an upstream and intragenic region of H3K27ac enrichment at the BCL-xL locus. Notably, BCL-xL was among the few highly up-regulated genes in resistant cells by expression profiling. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 due to decreased activity of PP2A that we identified as a BRD4 phosphatase. We have defined that hyper-phosphorylation of BRD4 increases the recruitment of BRD4 to MED1 and contributes to BBDI resistance. To explore the translational relevance of our findings, we conducted synergy screens with JQ1 and molecules targeting BCL-XL (ABT737), a gained super-enhancer in resistant cells, and modulators of BRD4 phosphorylation, a CK2 inhibitor CX-4945, and PP2A activator perphenazine (PPZ). We observed significant synergy between JQ1 and ABT737, CX-4945, and also PPZ implying that these drug combinations will likely achieve even higher efficacy in TNBCs than BBDIs alone. Together, these studies provide a rationale for BET inhibition in TNBC and argue for combination strategies to anticipate clinical drug resistance. Citation Format: Shaokun Shu, Charles Lin, Housheng Hansen He, Robert Witwicki, Justin Roberts, Doris Tabassum, Yi Liang, Muhammad Ekram, Ernest Doherty, Jonathan Brown, Hisham Mohammed, Clive D'Santos, Michael McKeown, Christopher Ott, Jun Qi, Min Ni, Prakash Rao, Melissa Duarte, Shwu-Yuan Wu, Cheng-Ming Chiang, Richard Young, Jason Carroll, Henry Long, Myles Brown, Shirley X. Liu, Clifford Meyer, James Bradner, Kornelia Polyak. Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B16.

Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells.

The androgen receptor (AR) mediates the developmental, physiologic, and pathologic effects of androgens including 5α-dihydrotestosterone (DHT). However, the mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells are not well understood, though they are central to prostate development, homeostasis, and neoplasia. Here, we identify androgen-responsive genes that restrain cell cycle progression and proliferation of human prostate epithelial cell lines (HPr-1AR and PC3-Lenti-AR), and we investigate the mechanisms through which AR regulates their expression. DHT inhibited proliferation of HPr-1AR and PC3-Lenti-AR, and cell cycle analysis revealed a prolonged G1 interval. In the cell cycle, the G1/S-phase transition is initiated by the activity of cyclin D and cyclin-dependent kinase (CDK) complexes, which relieve growth suppression. In HPr-1AR, cyclin D1/2 and CDK4/6 mRNAs were androgen-repressed, whereas CDK inhibitor, CDKN1A, mRNA was androgen-induced. The regulation of these transcripts was AR-dependent, and involved multiple mechanisms. Similar AR-mediated down-regulation of CDK4/6 mRNAs and up-regulation of CDKN1A mRNA occurred in PC3-Lenti-AR. Further, CDK4/6 overexpression suppressed DHT-inhibited cell cycle progression and proliferation of HPr-1AR and PC3-Lenti-AR, whereas CDKN1A overexpression induced cell cycle arrest. We therefore propose that AR-mediated growth suppression of HPr-1AR involves cyclin D1 mRNA decay, transcriptional repression of cyclin D2 and CDK4/6, and transcriptional activation of CDKN1A, which serve to decrease CDK4/6 activity. AR-mediated inhibition of PC3-Lenti-AR proliferation occurs through a similar mechanism, albeit without down-regulation of cyclin D. Our findings provide insight into AR-mediated regulation of prostate epithelial cell proliferation.

Abstract 964: GATA3 modulates chromatin structure to establish active enhancers in breast cancer cells

Abstract Master transcription factors regulate cell-type-specific gene expression to define cellular identities. One such gene, GATA3, is a key regulator of multiple cellular programs, including T lymphocyte development, mammary luminal epithelial cell differentiation and trophoblast development. Recently, comprehensive genomic analysis has identified GATA3 as one of the most frequently mutated genes in breast cancer. It is also known that GATA3 expression levels directly correlate with favorable prognosis. These findings strongly suggest that GATA3 plays a critical role in tumorigenesis. However, the molecular mechanism(s) underlying GATA3-mediated gene regulation in breast cancer cells is not clearly defined. GATA3 participates in a complicated regulatory network with FOXA1 and ER-alpha, governing the transcriptional program in luminal tumors. Biochemical analyses indicate that: (1) GATA3 binds to chromatin in an estrogen-independent manner, (2) GATA3 acts upstream of FOXA1. These studies suggest GATA3 may act as a pioneer factor, which is capable of independently associating with closed chromatin and modulating chromatin structure to establish an active enhancer. In order to investigate GATA3 function as a pioneer transcription factor, we chose the MDA-MB-231 breast cancer cell line, which is GATA3, FOXA1 and ER-alpha negative, and established stable cell lines expressing wild-type GATA3 or GFP as a control. Consistent with previous results, GATA3-expressing cells represented an epithelial phenotype at the cellular and molecular level. To determine whether GATA3 can direct reprogramming of chromatin conformation, we performed genome-wide analyses of the chromatin binding activity of GATA3 and its impact on histone modifications and chromatin structure. We will present recent results describing how GATA3 licenses enhancer function to direct the luminal transcriptional program. Citation Format: Motoki Takaku, Sara A. Grimm, Takashi Shimbo, Lalith Perera, Shinichi Machida, Hitoshi Kurumizaka, Paul A. Wade. GATA3 modulates chromatin structure to establish active enhancers in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 964. doi:10.1158/1538-7445.AM2015-964

Canonical Wnt signaling regulates Nkx3.1 expression and luminal epithelial differentiation during prostate organogenesis

The formation of the prostate gland requires reciprocal interactions between the epithelial and mesenchymal components of the embryonic urogenital sinus. However, the identity of the signaling factors that mediate these interactions is largely unknown. Our studies show that expression of the prostate-specific transcription factor Nkx3.1 is regulated by the canonical Wnt signaling pathway. Using mice carrying a targeted lacZ knock-in allele of Nkx3.1, we find that Nkx3.1 is expressed in all epithelial cells of ductal buds during prostate organogenesis. Addition of Wnt inhibitors to urogenital sinus explant culture greatly reduces prostate budding and inhibits Nkx3.1 expression as well as differentiation of luminal epithelial cells. Analyses of a TCF/Lef:H2B-GFP transgene reporter show that canonical Wnt signaling activity is found in urogenital mesenchyme but not urogenital sinus epithelium before prostate formation, and is later observed in the mesenchyme and epithelium of prostate ductal tips. Furthermore, TCF/Lef:H2B-GFP reporter activity is reduced in epithelial cells of Nkx3.1 null neonatal prostates, suggesting that Nkx3.1 functions to maintain canonical Wnt signaling activity in developing prostate bud tips. We propose that activated canonical Wnt signals and Nkx3.1 function in a positive feedback loop to regulate prostate bud growth and luminal epithelial differentiation.

Protective Effects of Prepubertal Genistein Exposure on Mammary Tumorigenesis Are Dependent on BRCA1 Expression

This study investigated whether prepubertal dietary exposure to genistein reduces mammary tumorigenesis by upregulating Brca1 expression in mice. Heterozygous Brca1(+/-) mice and their wild-type (WT) littermates were fed control AIN93G diet or 500 ppm genistein-supplemented AIN93G diet from postnatal day (PND) 15 to PND30 and then switched to AIN93G diet. Prepubertal dietary exposure to genistein reduced 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary incidence (P = 0.029) and aggressiveness of the tumors (P < 0.001) in the WT mice and upregulated the expression of Brca1 in their mammary glands (P = 0.04). In contrast, prepubertal genistein diet neither significantly reduced mammary tumorigenesis or tumor aggressivity nor increased Brca1 mRNA expression in the Brca1(+/-) mice. These results may be related to the opposing effects of prepubertal genistein diet on the expression of Rankl and CK5/CK18 ratio (marker of luminal epithelial cell differentiation) in the mammary gland and estrogen receptor (ER-α) and progesterone receptor (PgR) protein levels in the mammary tumor: these all were reduced in the WT mice or increased in Brca1(+/-) mice. Both the WT and Brca1(+/-) mice exhibited reduced levels of amphiregulin, CK5, and CK18, delayed ductal elongation and a reduction in terminal end bud number in the normal mammary gland, and reduced HER-2 protein levels in the mammary tumors; however, these effects were not sufficient to significantly reduce mammary tumorigenesis in Brca1(+/-) mice. Our results show that upregulation of Brca1 may be required for prepubertal dietary genistein exposure to reduce later mammary tumorigenesis, perhaps because in the absence of this upregulation, mice do not exhibit genistein-induced downregulation of ER-α, PgR, and Rankl.

Tissue‐Specific Roles of MED1/TRAP220 Nuclear Receptor‐Interacting LxxLL Motifs in vivo

Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand‐dependent interactions of the MED1 (a.k.a. TRAP220) subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is no information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Towards this end, we have generated MED1 LxxLL motif‐mutant knockin mice. Interestingly, these mice are both viable and fertile, and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. This was manifested as a significant impairment in duct elongation and branching morphogenesis and a decrease in mammary epithelial cell proliferation in the mutant mice. Consistent with this phenotype, as well as a loss of the strong ligand‐dependent estrogen receptor (ER)α‐Mediator interaction, expression of a number of known ERα‐regulated genes was down‐regulated in MED1‐mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen‐stimulated mammary duct growth in MED1‐mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor‐ and cell‐ specific in vivo role for MED1 LxxLL motifs, through Mediator‐ERα interactions, in mammary gland development.

Key roles for MED1 LxxLL motifs in pubertal mammary gland development and luminal-cell differentiation

Mediator recently has emerged as a central player in the direct transduction of signals from transcription factors to the general transcriptional machinery. In the case of nuclear receptors, in vitro studies have shown that the transcriptional coactivator function of the Mediator involves direct ligand-dependent interactions of the MED1 subunit, through its two classical LxxLL motifs, with the receptor AF2 domain. However, despite the strong in vitro evidence, there currently is little information regarding in vivo functions of the LxxLL motifs either in MED1 or in other coactivators. Toward this end, we have generated MED1 LxxLL motif-mutant knockin mice. Interestingly, these mice are both viable and fertile and do not exhibit any apparent gross abnormalities. However, they do exhibit severe defects in pubertal mammary gland development. Consistent with this phenotype, as well as loss of the strong ligand-dependent estrogen receptor (ER)alpha-Mediator interaction, expression of a number of known ERalpha-regulated genes was down-regulated in MED1-mutant mammary epithelial cells and could no longer respond to estrogen stimulation. Related, estrogen-stimulated mammary duct growth in MED1-mutant mice was also greatly diminished. Finally, additional studies show that MED1 is differentially expressed in different types of mammary epithelial cells and that its LxxLL motifs play a role in mammary luminal epithelial cell differentiation and progenitor/stem cell determination. Our results establish a key nuclear receptor- and cell-specific in vivo role for MED1 LxxLL motifs, through Mediator-ERalpha interactions, in mammary gland development.

A 'Field Effect' in Cancer Containing Breasts Results in an Altered Expression Profile of Myoepithelial Cells.

Abstract Myoepithelial cells have an important role in controlling growth and differentiation of luminal epithelial cells and the myoepithelial cell has been described as a tumour suppressor, inhibiting proliferation and invasion. We have previously showed that non-involved tissue from cancer-containing breasts (NTCCB) has lower apoptotic indices and altered expression of myoepithelial proteins such as epidermal growth factor receptor and β4-integrin when compared to normal breast tissue from age matched women without cancer. In addition, using immunohistochemistry, we have found a significant difference in the expression of fibroblast growth factor-2 (FGF-2), a modulator of cell survival and inducer of mitogenic activity in epithelial cells, with greater staining observed in myoepithelial cells from NTCCB.The hypothesis tested is that due to a 'field effect' occurring in 'normal' tissue of breasts in which a cancer develops, the expression profile of myoepithelial cells is altered resulting in loss of their natural function and tumour suppressor properties.For this study, myoepithelial cells were isolated using positive selection from reduction mammoplasties and non-involved tissue from mastectomy/wide local excision specimens. Cells were cultured for up to 3 passages, purity assessed and RNA extracted. In order to compare the expression profiles between the two groups, 5 myoepithelial cell samples from NTCCB and 6 age-matching samples from normal breast were processed using the Illumina Whole-Genome Expression cDNA Microarray BeadChips. Results were analysed using a stepwise forward selection artificial neural network (ANN) modelling approach and validated using real time qPCR TaqMan assays.The ANN algorithm ranked the genes based on the difference of their expression levels and their predictive value on accurately differentiating samples from the two groups. The two top ranked genes were matrix metallopeptidase 23B (MMP23B) that was upregulated in myoepithelial cells from NTCCB and sprouty homolog 1 (SPRY1), a FGF signalling antagonist and inhibitor of the Ras/MAP kinase pathway, that was downregulated. Among the rest of the genes that showed differences in expression were proteins of the extracellular matrix, members of the tumour necrosis factor superfamily and RAB38 a GTPase, member of the ras oncogene family. Data from the real time qPCR showed a lesser degree of significance for the differences of MMP23B and SPRY1 expression but confirmed the higher expression of FGF-2 in myoepithelial cells from NTCCB (p=0.021). qPCR testing for the extended subset of genes identified by the microarray in conjunction with western blotting is underway to further evaluate these differences.In conclusion, these data confirm the existence of field changes in normal breast in which a cancer develops, where differences in expression could alter the natural properties and functions of myoepithelial cells. They also suggest FGF-2 as a potential molecular target for further study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6120.

GATA3 in development and cancer differentiation: Cells GATA have it!

There is increasing evidence that the numerous mechanisms that regulate cell differentiation during normal development are also involved in tumorigenesis. In breast cancer, differentiation markers expressed by the primary tumor are routinely profiled to guide clinical decisions. Indeed, numerous studies have shown that the differentiation profile correlates with the metastatic potential of tumors. The transcription factor GATA3 has emerged recently as a strong predictor of clinical outcome in human luminal breast cancer. In the mammary gland, GATA3 is required for luminal epithelial cell differentiation and commitment, and its expression is progressively lost during luminal breast cancer progression as cancer cells acquire a stem cell-like phenotype. Importantly, expression of GATA3 in GATA3-negative, undifferentiated breast carcinoma cells is sufficient to induce tumor differentiation and inhibits tumor dissemination in a mouse model. These findings demonstrate the exquisite ability of a differentiation factor to affect malignant properties, and raise the possibility that GATA3 or its downstream genes could be used in treating luminal breast cancer. This review highlights our recent understanding of GATA3 in both normal mammary development and tumor differentiation.

Oct4A is expressed by a subpopulation of prostate neuroendocrine cells

Cancer stem cells are defined by their self-renewal and multi-potential capabilities and are hypothesized to be the source of primary and recurrent cancers. The stem cell properties of self-renewal and pluripotency in embryonic stem cells and germ cells are regulated by Oct4A, a splice variant of the POU5F1 (Oct3/4) gene, while the function of the alternative splice variant, Oct4B, is unknown. Rare cells that express Oct4 were identified in several somatic cancers, however, the differential contributions of the Oct4A and Oct4B variants were not determined. Oct4A expression and co-localization with lineage markers was performed with PCR and immunohistochemistry. Rare Oct4A expressing cells are present in human benign and malignant prostate glands and the number of Oct4A expressing cells increases in prostate cancers with high Gleason scores. Oct4A expressing cells were non-proliferative, and did not co-express markers of basal epithelial cell or luminal epithelial cell differentiation, or AMACR, a marker of prostate cancer epithelial cells. A subpopulation of the Oct4A expressing cells co-expressed Sox2, an embryonic stem cell marker, but did not express other putative stem cell markers, such as ABCG2, NANOG or CD133. The majority of Oct4A expressing cells co-expressed chromogranin A, and a subset of Oct4A expressing cells co-expressed synaptophysin, both markers of neuroendocrine differentiation. The increased number of cells that expressed Oct4A in prostate cancer compared to benign prostate, and in cancers of increasing grade, suggests that Oct4A/Chromogranin A co-expressing cells represent neuroendocrine cells in prostate cancer.