Pathology

Smoothelin is a cytoplasmic protein expressed in differentiated smooth muscle cells. Immunohistochemical evaluation of smoothelin has previously been reported in gastrointestinal (GI) smooth muscle tumors, but has yet to be studied in smooth muscle tumors of uterine and other soft tissue origin. DOG1 expression is reported to be specific for GI stromal tumors; however, variable expression has been reported in leiomyosarcomas (LMS) depending on site of origin. Overexpression of p16 is common in LMS of uterine and other sites of origin, but has not been correlated with tumor grade. This study explores the differential expression of these markers, as well as caldesmon, in LMS cases to assess diagnostic utility. Using tissue microarrays and cases from Tulane Medical Center and Medical College of Wisconsin, expression of smoothelin, DOG1, caldesmon, and p16 was evaluated by immunohistochemistry in 87 cases of LMS. The cases were subdivided by location of origin into uterine (N=31) and no...

Breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) have been reported to have higher proliferative and metastatic activity in the presence of autocrine prolactin (PRL), indicating potential cooperation between HER2 and the PRL receptor (PRLR) during breast cancer progression. PRL can induce the tyrosine phosphorylation of HER2 which stimulates mitogen-activated protein kinase (MAPK) activity. To determine if this transactivation of HER2 by PRL contributes to anti-HER2 therapy resistance we examined the potential of combining Herceptin with a PRLR antagonist, G129R, which inhibits PRL-induced signaling, as a novel therapeutic strategy. Two PRL-expressing human breast cancer cell lines (T-47D and BT-474) that overexpress PRLR and HER2 to different degrees were chosen for this study. The phosphorylation status of HER2 and activation of MAPK, signal transducers and activators of transcription (STAT), as well as phosphatidylinositol-3 kinase (PI3K) signaling cascades were examined in response to Herceptin, G129R or a combination of the two in either the absence or presence of exogenous PRL. As a single agent, Herceptin was more effective than G129R at inhibiting AKT phosphorylation; whereas, G129R was superior at blocking STAT3 and STAT5 activation. G129R was also able to directly inhibit the HER2 phosphorylation. The combination of Herceptin and G129R had an additive inhibitory effect on HER2 and MAPK phosphorylation, confirming that the MAPK signaling is a converging pathway shared by both HER2 and the PRLR. Combination of Herceptin and G129R also additively inhibited cell proliferation in vitro and in vivo as measured by inhibition of the growth of T-47D and BT-474 xenografts in athymic nude mice. We conclude that an anti-HER2 and anti-PRLR regimen may offer a new approach to treat HER2-overexpressing breast cancers.

Background: Prolactin (PRL), a polypeptide hormone, has been implicated in breast tumorigenesis via promoting cell proliferation and survival. One of the mechanisms by which PRL is linked to breast cancer is through cross-talk with other oncogenic growth factors including human epidermal growth factor receptor 2 (HER2/neu). Studies have demonstrated that PRL is able to cross-activate HER2/neu and that a PRL receptor (PRLR) antagonist, G129R, is able to inhibit HER2/neu phosphorylation (pHER2/neu) in established breast cancer cell lines. In this study, we investigate the role of tumor associated fibroblasts (stromal cells) in this molecular cross-talk process, using natural tumors and tumor epithelial and fibroblast cell lines derived from female MMTV/neu transgenic mice. Methods: (1) Primary tumor studies. Mammary tumors from MMTV/neu transgenic mice were surgically removed and divided into two portions. One portion was cut into small chunks (3 mm3 cubes) and cultured three-dimensio...

To prolong the circulation half-life of human prolactin (hPRL), human GH (hGH), and their competitive antagonists, hPRL-G129R and hGH-G120R, we examined the effects of fusing a serum albumin-binding peptide (SA20) to their amino- or carboxyl-terminus. Fusion of the SA20 peptide to the amino-terminus of the ligands was less detrimental upon their ability to induce or inhibit signal transduction and cell proliferation in vitro than fusion to the carboxyl-terminus. Pharmacokinetic (PK) studies in mice revealed that the half-life of SA20-hPRL and SA20-hGH was prolonged and their clearance was reduced in comparison with hPRL and hGH. Pharmacodynamic (PD) studies in 8-week-old female mice revealed that lobuloalveolar development in mammary glands was greater in all three groups (daily, every 2 days, or every third day over a 12-day period) of mice treated with SA20-hPRL (4 mg/kg) compared with hPRL (3.59 mg/kg). Similarly, daily administration (i.p.) of SA20-hGH (8 mg/kg) or hGH (7.15 mg/...

The objective of this study was to determine if a functional heterodimer of prolactin receptor (PRLR) and growth hormone receptor (GHR) can be formed in humans. A novel ligand was designed that is composed of a GHR antagonist (B2036) and a PRLR antagonist (G129R) fused in tandem (B2036-G129R). Because both B2036 and G129R are binding site 2 inactive antagonists, the B2036-G129R fusion protein, in theory contains only two functional binding site 1s: one for GHR and one for PRLR. We examined the behavior of this chimeric ligand in cell lines known to express GHR, PRLR, or both receptors. The data presented show that B2036-G129R is inactive in IM-9 cells that express only GHR or Nb2 cells that express PRLR. In T-47D cells that coexpress PRLR and GHR, B2036-G129R activates JAK2/STAT5 signaling. These findings provide evidence that B2036-G129R is able to activate signal transduction through a heterodimer of PRLR and GHR in humans.

The transmembrane GH receptor (GHR) exists at least in part as a preformed homodimer on the cell surface. Structural and biochemical studies suggest that GH binds GHR in a 1:2 stoichiometry to effect acute GHR conformational changes that trigger the activation of the receptor-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signaling. Despite information about GHR-GHR association derived from elegant fluorescence resonance energy transfer/bioluminescence resonance energy transfer studies, an assessment of the dynamics of GH-induced GHR conformational changes has been lacking. To this end, we used a split luciferase complementation assay that allowed detection in living cells of specific ligand-independent GHR-GHR interaction. Furthermore, GH treatment acutely augmented complementation of enzyme activity between GHRs fused, respectively, to N- and C-terminal fragments of firefly luciferase. Analysis of the temporal pattern of GH-induced complementation changes, pharm...

Are STAT3 signaling molecules differentially expressed in endometriosis? Levels of phospho-STAT3 and HIF1A, its downstream signaling molecule, are significantly higher in eutopic endometrium from women with endometriosis when compared with women without the disease. Endometriosis is an estrogen-dependent inflammatory condition. Interleukin 6 (IL-6) is an inflammatory survival cytokine known to induce prolonged activation of STAT3 via association with the IL-6 receptor. Cross-sectional measurements of STAT3 and HIF1A protein levels in eutopic endometrium from women with endometriosis versus those without. Levels of phospho-STAT3 (pSTAT3) and HIF1A were examined in the endometrium of patients with and without endometriosis as well as in a non-human primate animal model using western blot and immunohistochemical analysis. Levels of pSTAT3 were significantly higher in the eutopic endometrium from women with endometriosis when compared with women without the disease in both the prolifera...

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.

Background: Better models of the tumor microenvironment (TME) are needed in order to reduce the number of anticancer drug failures in clinical trials and to reduce animal use. Current models fail because of 1) reliance on single cell systems; 2) reliance on artificial 2 dimensional (2D) monolayers; and 3) a focus on high throughput assays that pre-select drugs affecting growth and apoptosis. To more accurately model the TME, we developed a 3D perfusion co-culture system for purposes of anti-cancer testing. Materials & Methods: The system consisted of 3DKUBE™ 3D Cell Culture Plasticware, membranes, closed flow circuits, connectors, syringes and gas exchange reservoir bags. 3D static culture experiments combining a variety of cell types, scaffolds, media and reagent conditions, and analytical techniques were performed to define the co-culture conditions. Flow rates and cycling times are used to demonstrate: 1) fibroblast priming, 2) drug effect, 3) intra-spheroid cell viability and 4)...

Growth hormone Receptor (GHR) and prolactin receptor (PRLR) are homologous transmembrane cytokine receptors. Each pre-homodimerizes and ligand binding activates JAK2-STAT signaling pathways by inducing conformational changes within receptor homodimers. In humans, GHR is activated by growth hormone (GH), while PRLR is activated by both GH and prolactin (PRL). We previously devised a split luciferase complementation assay, in which one receptor is fused to an N-terminal luciferase fragment (Nluc) and the other receptor is fused to a C-terminal luciferase fragment (Cluc). When receptors approximate, luciferase activity (complementation) results. Using this assay, we reported ligand-independent GHR-GHR complementation and GH-induced complementation changes characterized by acute augmentation above basal signal, consistent with induction of conformational changes that bring GHR cytoplasmic tails closer. We also demonstrated association between GHR and PRLR in T47D human breast cancer cel...

695 It is well documented that mouse mammary tumor virus (MMTV)-HER2/neu transgenic (TG) mice have high mammary tumor incidence (t50=205 days). Overexpression of rat PRL within the mammary epithelium in TG mice has also been reported to induce mammary tumors and potentiate the actions of other mammary oncogenes. In our previous studies, we have shown that the hPRL antagonist, G129R, is effective as an anti-breast cancer agent both in vitro and in vivo. To investigate the role of hPRL and G129R in HER2/neu mammary tumorigenesis, we generated four lines of bi-transgenic (BiTG) mice that co-express MMTV-HER2/neu and hPRL or G129R under transcriptional control of either the metallothionein (MT, a systemic expression model) or MMTV promoter (a mammary specific expression model). Our unexpected results demonstrated that co-expression of low levels of hPRL in HER2/neu TG mice drastically reduced breast tumor incidence in both hPRL BiTG lines. The total tumor incidence in the MT-hPRLxHER2/neu BiTG line was 25/56 (t50=438 days); in the MMTV-hPRLxHER2/neu BiTG line, it was 2/36 (at the current age of 300 days). In contrast, co-expression of G129R in HER2/neu TG mice had no effect on overall tumor incidence [MT-G129RxHER2/neu, 50/53; MMTV-G129RxHER2/neu, 33/36]. One hypothesis that may help to explain the reduction in tumor incidence in the hPRL BiTG mice is that early exposure of the mammary gland to hPRL induces alterations that afford lifetime protection against the development of HER2/neu breast cancer. Our results further show that the mammary glands of female hPRL BiTG mice (1): undergo early completion of differentiation as indicated by ductal branching and terminal end bud change based on mammary gland whole mount comparisons; and (2): have both increased total ductal area (H&E staining) and decreased proliferative activity (Ki-67 IHC detection). Further, there was a significant difference in the tumor biomarkers between hPRL BiTG and HER/neu tumors. Phosphorylated HER2 was absent whereas higher levels of phosphorylated STAT-5 were seen in the hPRL BiTG tumors examined by western blot analyses, which suggests a less aggressive tumor type. In summary, expression of physiological concentrations of hPRL in HER2/neu TG mice induces early differentiation in the mammary gland, which leads to a drastic reduction in HER2/neu breast tumor incidence. This finding may open a new avenue for using hPRL as a chemopreventive agent specifically in HER2/neu initiated breast cancer.