Effects of prenatal exposure to hexafluoropropylene oxide dimer acid on rat and offspring mammary gland development and associated hormone levels.

Androgen receptor DNA methylation regulates the timing and androgen sensitivity of mouse prostate ductal development

We observed a newborn boy with urorectal septum malformation sequence. Anomalies of the genitalia and rectum were present. He expired on the first day of life, due to severe lung hypoplasia. Autopsy showed a colon that ended in a blind sac, an enlarged bladder with no grossly visible urethra, and dysplastic kidneys. A cone-shaped tissue at the usual site of the bladder outlet contained tortuous and slit-like lumina, suggesting an undeveloped proximal urethra. The urethral structure was lined by transitional epithelium with squamous metaplasia. Many small buds-lined with columnar epithelium-branched from the urethral structure. These ductal buds lined with columnar epithelium stained for prostatic acid phosphatase. Basal cells surrounding the ductal buds stained for p63 and high molecular weight cytokeratin-supporting an interpretation that the buds were early prostatic ducts with normal histology. To our knowledge, these are the first histological images of an undeveloped, obstructed urethra associated with the urorectal septum malformation sequence.

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.

Abstract The GATA-3 transcription factor is a master regulator of cell fate determination in the mammary gland. Mice lacking expression of GATA-3 in the mammary gland fail to develop complete ductal branching or terminal end buds, while depletion of GATA-3 from adult mammary glands leads to loss of markers for luminal cell differentiation and to caspase-mediated death of luminal cells. In human breast cancer, GATA-3 expression is found in highly differentiated, estrogen receptor positive tumors with good prognosis, while loss correlates with highly metastatic tumors resistant to endocrine therapy. Regulation of GATA-3 expression is incompletely understood, though a positive-cross regulatory loop with the estrogen receptor alpha has been defined. The zinc finger protein Fliz-1 represses GATA-3 transcription in the T cell lineage, but its role in mammary cells is unknown. We hypothesize that Fliz-1 also can repress GATA-3 expression in mammary cells, leading to loss of estrogen response. To test this hypothesis, we identified a mouse mammary tumor cell line expressing high levels of Fliz1, and transfected the cells with a Fliz-1 silencing vector or negative control. Successful knockdown was confirmed by RT/PCR. Cells with decreased Fliz-1 expression showed increased expression of GATA-3 and estrogen receptor alpha mRNA relative to controls. The Fliz-1-expressing cells formed tumors that were insensitive to tamoxifen treatment when implanted in syngeneic BALB/c mice, while cells with decreased Fliz-1 expression exhibited decreased tumor growth in response to endocrine therapy. As a reciprocal experiment, we constructed a fusion gene linking Fliz-1 with a C-terminal green fluorescent protein tag. The fusion protein properly localizes to the nucleus of transfected MCF-7 (human breast cancer) and COMMA-D (mouse mammary epithelial) cells. Chromatin immunoprecipitation was used to show that Fliz1-GFP binds to an intronic regulatory region within the GATA-3 gene in COMMA-D cells. These data provide evidence supporting a role for Fliz-1 in control of GATA-3 expression in mammary cells and tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2197. doi:1538-7445.AM2012-2197

Abstract The classification of patient tumors by clinical subtype has gained great interest due to the implications for prognosis and treatment. For example, it is known that the five-year survival rate of patients diagnosed with the basal subtype of breast cancer is considerably lower than those with luminal subtypes because they lack expression of common drug targets. For this reason, it is important to elucidate the molecular mechanisms responsible for differentiation of mammary stem cell populations into basal and luminal cell lineages. Data presented in this poster outline a novel pathway whereby a ubiquitously expressed protein kinase, JNK2, influences the differentiation of basal, myoepithelial cells (MEps) while suppressing luminal cell (LEp) populations. Gross anatomical observation of whole-mounted mammary glands from wildtype (jnk2wt) and jnk2-/- (jnk2ko) mice revealed that JNK2 increases the rate of pubertal development. Loss of jnk2 increased the rate of ductal invasion, branching, and terminal end bud (TEB) count. Although a slight increase in proliferation was seen in jnk2ko glands, data suggested that it was not due to growth factor signaling. Instead, it was noticed that jnk2ko glands possess 35% fewer MEps (p=0.0078) with a corresponding increase in LEp populations (p=0.100). LEps are known to proliferate at a higher rate than MEps. The differentiation phenotype was corroborated through 3D culture of primarymammary epithelial cells (MECs). The rate of growth of 3D MEC acini was 31.5% higher in jnk2ko cultures than in jnk2wt (P<0.0001). Notch-1, a well-known regulator of MEC difFerentiation, is present in its active form in a greater proportion of cells within jnk2ko TEBs than jnk2wt (p=0.0118, 0.0209). Further, jnk2ko glands were found to possess greater amounts of Notch-1 protein as well as 10.5 times more expression of the Notch target gene, Hes-1 (p=0.005). These results were corroborated with 3D culture of primary MECs as well as in mammary tumor cells. Inhibition of Notch signaling with GSI, in 3D cultures of primary MECs, normalized growth (p=0.8992) and differentiation to jnk2wt control levels. QPCR results showed that jnk2ko glands possess 6.4 times more notch-1 transcript in vivo (P<0.0001). Luciferase assays comparing activity of the notch-1 wildtype promoter to a notch-1 promoter with mutated p53 response elements revealed a dependence of increased notch-1 promoter activity in jnk2ko cells on the p53 response element. QPCR showed 2.78 times more p53 transcript in jnk2ko mammary glands as compared to jnk2wt. These data suggest that JNK2 regulates MEC differentiation through p53 and its target, Notch-1. These findings are significant because they reveal JNK2 as a possible drug target for novel differentiation therapy of basal type breast tumors. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-15.

Abstract ETS proteins represent one of the largest families of transcription factors with diverse functions that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. The ETS family gene, PDEF (prostate derived ets factor), is expressed in normal epithelial tissues including prostate, breast, colon and bladder. Significantly, PDEF protein is present in non-invasive cancers but reduced or absent in most invasive cancers. By re-expression and knock-down of PDEF in cancer cell lines, we and others have shown that PDEF target genes control aspects of the metastatic process, including cell growth, migration and invasion. We hypothesize that PDEF is important for normal developmental processes and that loss of PDEF regulatory networks is a key event in the development of invasive cancer. To study the function of Pdef in vivo, we generated mice with constitutive Pdef knockout (Pdef −/−). Specific stages of mammary development were examined, including early-pubescent (5 week), virgin adult (8 week), pregnancy (days 7.5 and 13.5), lactation (days 1 and 7) and involution (day 7). Inhibition of the development of the ductal tree and an increase in ductal side branching and terminal end bud proliferation was observed in 5-8 week old virgin Pdef −/− mice. TGFβ1 and ERα are required for development of the ductal tree during puberty and mRNA levels for both are reduced in the Pdef −/− mice at 5 and 8 weeks of age. Incomplete lobuloalveoli development was observed in Pdef −/− pregnant and lactating mice, although no phenotypic effects were observed in the resultant pups. To explore the role of Pdef in mammary tumor progression and metastasis, we generated mice expressing the MMTV-PyMT or MMTV-Neu oncogene in wild type and Pdef −/− backgrounds. No impact of Pdef dosage upon tumor incidence or growth was observed in the PyMT model; however, initial tumor formation was delayed by 4 weeks (8.9 vs. 4.8) and time from tumor initiation to sacrifice was 3 weeks longer (29.4 vs 26.4) in the Pdef −/− Neu model. Significantly, an increase in lung metastasis was observed in the Pdef −/− mice in both the PyMT and Neu models. Thus, loss of Pdef in vivo is associated with defects in mammary development and increased metastasis in mouse models of breast cancer, suggesting Pdef is a metastasis suppressor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3397.

Mass spectrometry-based tissue profiling and imaging are technologies that allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1-5 weeks of age), at sexual maturation (6 weeks of age), and in adult prostate (at 10, 15, or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared with 15-week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore expression of some of these proteins, for example probasin and spermine-binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially alpha-acetylated on the N terminus, and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins that could potentially serve as biomarkers for prostate cancer.

AP2alpha (TFAP2A) and AP2gamma (TFAP2G) transcription factors have been implicated in the control of proliferation, differentiation and apoptosis of normal breast epithelium and in breast cancer. The aim of this study was to provide a comprehensive analysis of the expression patterns of TFAP2A and TFAP2G in the developing fetal breast anlage with other relevant markers. Sixty fetal and one infant human breast specimens from 14 weeks of gestational age to 5 months old were examined. The primary breast outgrowth/nipple showed TFAP2A expression by the basal cells (week 14), followed later by cytokeratin (CK) 5 co-expression (week 17). Sprouting of the secondary outgrowths was characterized by HER-2+ invading cells. Preliminary ductal buds were lined by TFAP2G/HER-1-expressing myoepithelial precursors (week 19). Maturation of TFAP2A/CK18+ epithelia and TFAP2G/smooth muscle actin-positive myoepithelia proceeded in a distal-to-proximal manner beginning in the terminal end buds (week 22). CK5+ progenitor cells and CK5/TFAP2A or CK5/TFAP2G co-expressing intermediary glandular or myoepithelial cells were found in the terminal end buds of neonatal fetal breast tissue. AP2 transcription factors may play decisive pacemaker roles in initiating and coordinating budding and branching processes during formation of the fetal breast anlage, possibly via modulation of an epidermal growth factor receptor.

Noggin is required for normal lobe patterning and ductal budding in the mouse prostate

Hedgehog (Hh) signaling has long been recognized for its role in axial patterning, mesenchymal-epithelial inductive signaling, and growth regulation during fetal development. In many embryonic tissues, Hh functions as a proliferative stimulus. Sonic hedgehog and Indian hedgehog are both expressed by the urothelium of the fetal prostate anlage, where they regulate cell proliferation and differentiation and play a role in prostate ductal budding. Whereas Hh signaling in mouse prostate diminishes during adolescence and is maintained at a low level in the adult, robust Hh signaling is commonly found in the adult human prostate. The reason(s) for robust Hh signaling in the adult human prostate and the actions of Hh signaling on growth and differentiation in the adult are not well understood. However, increased Hh signaling has been associated with prostate cancer and has been shown to accelerate prostate cancer growth. These observations suggest that inappropriate reawakening of this developmental growth signal may play a pivotal role in prostate neoplasia. This review examines the role of Hh signaling during early prostate growth and in its corollary actions during prostate disease, including benign prostate hyperplasia and prostate cancer. The use of Hh inhibitors as a therapeutic modality for androgen-independent treatment of prostate disease is also discussed.

The onset of prostate morphogenesis is involved in the interaction between mesenchyme and epithelium. Proprotein convertases (PCs) activate a variety of growth and differentiation factors including mesenchymal and epithelial factors, such as insulin-like growth factor (IGF) and transforming growth factor-beta (TGF-beta), which induce ductal budding and branching. In this study, we provide evidence that PCs play a critical role in prostatic budding from the urogenital sinus (UGS) and ductal branching morphogenesis of the neonatal rat ventral prostate. PCs were expressed only in the epithelial cells of neonatal rat prostate. PC activity in the ventral prostate was modulated by endogenous androgen. PC inhibition suppressed prostatic budding and branching. Taken together, our data indicates that androgen-induced PCs initiate the development of the prostate.

Functional compensation in Hedgehog signaling during mouse prostate development

BMP7 inhibits branching morphogenesis in the prostate gland and interferes with Notch signaling

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.

Immunohistochemical expression of progesterone receptors, growth hormone and insulin growth factor-I in feline fibroadenomatous change

Sonic hedgehog signalling and Gli gene activation is essential for prostate ductal bud formation

The Human Prostate Expresses Sonic Hedgehog During Fetal Development

Sonic Hedgehog Activates Mesenchymal Gli1 Expression during Prostate Ductal Bud Formation

Mesenchymal Factor Bone Morphogenetic Protein 4 Restricts Ductal Budding and Branching Morphogenesis in the Developing Prostate