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PDF file, 48K, PCR primers used in study.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a dual-function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two nonmalignant human mammary epithelial cell lines that form polarized, growth-arrested structures (acini) when cultured in three-dimensional laminin-rich extracellular matrix gels (lrECM). As acini begin to form, PTEN accumulates both in the cytoplasm and at cell-cell contacts where it colocalizes with the E-cadherin/β-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function–blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in Skbr-3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in three-dimensional lrECM, indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus seems to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells. [Cancer Res 2009;69(10):4545–52]

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PDF file, 3359K, Effect of Epim on ductal length, terminal endbuds, and lobuloalveolar structure formation.

PDF file, 845K, Epim overexpression leads to enlargement of organoid cyst and upregulation of Pgr during bud formation.

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Supplementary Figure 1 from Interaction of E-cadherin and PTEN Regulates Morphogenesis and Growth Arrest in Human Mammary Epithelial Cells

Supplementary Figure Legend from Interaction of E-cadherin and PTEN Regulates Morphogenesis and Growth Arrest in Human Mammary Epithelial Cells

Phytoestrogens, a class of plant-derived compounds that are structural mimics of estrogen, can bind to estrogen receptors, acting as either agonists or antagonists. They have been implicated in estrogen-mediated physiology, which makes them interesting targets of study, especially for biomedical applications in woman's health. The 1998 Women's Health Initiative sparked considerable interest in natural alternatives to hormone replacement therapy, thereby triggering many additional studies on phytoestrogens. In this review, key advancements in dietary phytoestrogens are addressed, emphasizing their relation to breast pathophysiology. Recent developments such as clinical trials, precise bioassays for screening and selection of potential phytoestrogens, drug delivery systems to enhance bioavailability of therapeutically favorable phytoestrogens, regulatory guidelines on phytoestrogen-based supplements, and avenues that need further improvement are also discussed.

// Sumit Mukherjee 1, 3, 5, * , Priya Ranjan Debata 5, 8, * , Rahman Hussaini 5 , Kaushiki Chatterjee 2, 4 , Juliet N.E. Baidoo 1, 3 , Samay Sampat 5 , Anita Szerszen 6 , Joseph P. Navarra 7 , Jimmie Fata 2 , Elena Severinova 6, 9 , Probal Banerjee 1, 5 and Mario R. Castellanos 6 1 Department of Chemistry, The College of Staten Island (CUNY), New York, NY, USA 2 CUNY Doctoral Program in Biology, CUNY Graduate Center, New York, NY, USA 3 CUNY Doctoral Program In Biochemistry, CUNY Graduate Center, New York, NY, USA 4 Department of Biology, The College of Staten Island (CUNY), New York, NY, USA 5 Center of Developmental Neuroscience, The College of Staten Island (CUNY), New York, NY, USA 6 Division of Research, Department of Medicine, Staten Island University Hospital (Northwell Health), New York, NY, USA 7 College of Pharmacy and Health Sciences, St. John’s University, New York, NY, USA 8 Current Address: Department of Zoology North Orissa University Baripada, Mayurbhanj, Odisha, India 9 Current Address: Cell Biology and Molecular Medicine, Rutgers University, Newark, NJ, USA * These authors contributed equally to this work Correspondence to: Probal Banerjee, email: probal.banerjee@csi.cuny.edu , probalbanerjee@yahoo.com Keywords: curcumin, cervical cancer, human papillomavirus, synergism, combination index Received: May 31, 2016 Accepted: September 02, 2016 Published: March 29, 2017 ABSTRACT Curcumin (from curry) (C) is highly potent against cervical cancer cells (CCC), but poor bioavailability has limited its clinical use. Similar natural polyphenols resveratrol (from grapes) (R), and epicatechin gallate (from green tea) (E) also display activity against CCC. By treating CCC (HeLa) with C, E, or R, or combinations of these compounds, we computed combination indices and observed a strong synergism among C, E, and R at the unique molar ratio 4:1:12.5. This combination, named as TriCurin, rapidly down regulated HPV18 E6 and NF-kB expression while concomitantly inducing the tumor suppressor protein p53 in HeLa cells. In the mouse c-Ha-ras and HPV16 E6, E7-expressing TC-1 CCC, both C and TriCurin elicited suppression of E6, induction of both p53 and acetyl-p53 (activated p53), and activation of caspase-3, but the TriCurin-evoked changes were several-fold greater than that produced by curcumin (4.7-fold for E6 inhibition, and 2-fold, 6-fold, and 1.7-fold for the induction of p53, acetyl-p53, and active caspase-3, respectively). Consequently, TriCurin was more potent in killing TC-1 and HeLa cells. Intralesional TriCurin treatment of tumors generated in mice by subcutaneously implanting the TC-1 CCC caused an 80–90% decrease in tumor growth. The ability of C to eliminate HeLa cells was significantly stabilized when delivered as TriCurin than when delivered alone. Topical application of TriCurin dispersed in a cream base afforded efficient transfer of C across the skin. Subcutaneous TriCurin injection yielded no adverse effect in tumor-naive healthy mice. Thus, TriCurin is a safe and promising therapeutic agent against HPV-associated disease.

The global health threat of antimicrobial resistance has created a pressing need to develop practical alternatives to conventional antibiotic agents. Peptide mimetic synthetic amphiphilic polymers are known to non-specifically disrupt the bacterial cell surface thus leading to highly hindered bacterial resistance development. We investigated the antibacterial activities of a terpolymer macromolecular architecture with a combination of 6-carbon and 2-carbon spacer arms (distance from polymer backbone to pendent cationic center) interspersed with counits with hydrophobic side groups. A random copolymer with a combination of 6-carbon spacer arm repeat units (60 mol%) and 2-carbon spacer arm (40 mol%) units is moderately active against bacteria and shows very low hemolytic activity. Incorporation of comonomer units with alkyl side groups, by replacing different levels of 2-carbon spacer arm counit, led to substantial increments in antibacterial activities without detrimental effects on hemolytic activities leading to highly selective (bacteria over red blood cells) antibacterial activity. Time-kill studies revealed rapid bactericidal activity of the terpolymer against both Staphylococcus aureus and Escherichia coli with 100% killing efficiency achieved within 1 h of polymer treatment, corresponding to a 5-log reduction of bacterial colony forming units. These results indicate the high potential of this amphiphilic terpolymer architecture in the development of alternatives to antibiotics.

Cervical cancer is one of the most common cancers in women living in developing countries. Due to a lack of affordable effective therapy, research into alternative anticancer compounds with low toxicity such as dietary polyphenols has continued. Our aim is to determine whether two structurally similar plant polyphenols, resveratrol and pterostilbene, exhibit anticancer and anti-HPV (Human papillomavirus) activity against cervical cancer cells. To determine anticancer activity, extensive in vitro analyses were performed. Anti-HPV activity, through measuring E6 protein levels, subsequent downstream p53 effects, and caspase-3 activation, were studied to understand a possible mechanism of action. Both polyphenols are effective agents in targeting cervical cancer cells, having low IC50 values in the µM range. They decrease clonogenic survival, reduce cell migration, arrest cells at the S-phase, and reduce the number of mitotic cells. These findings were significant, with pterostilbene often being more effective than resveratrol. Resveratrol and to a greater extent pterostilbene downregulates the HPV oncoprotein E6, induces caspase-3 activation, and upregulates p53 protein levels. Results point to a mechanism that may involve the downregulation of the HPV E6 oncoprotein, activation of apoptotic pathways, and re-establishment of functional p53 protein, with pterostilbene showing greater efficacy than resveratrol.

Phytoestrogens, a class of plant-derived compounds that are structural mimics of estrogen, can bind to estrogen receptors, acting as either agonists or antagonists. They have been implicated in estrogen-mediated physiology, which makes them interesting targets of study, especially for biomedical applications in woman's health. The 1998 Women's Health Initiative sparked considerable interest in natural alternatives to hormone replacement therapy, thereby triggering many additional studies on phytoestrogens. In this review, key advancements in dietary phytoestrogens are addressed, emphasizing their relation to breast pathophysiology. Recent developments such as clinical trials, precise bioassays for screening and selection of potential phytoestrogens, drug delivery systems to enhance bioavailability of therapeutically favorable phytoestrogens, regulatory guidelines on phytoestrogen-based supplements, and avenues that need further improvement are also discussed.

The efficient synthesis of curcumin-diesters, which serve as green plasticizers for poly (vinyl) chloride (PVC), is reported. The plasticizers lower the glass transition temperature of PVC, and their cytotoxicity and leaching-resistance properties are significantly better than commercially used phthalate plasticizers which have several limitations.

Background: The mammary gland is an ideal model to study the link between form and function in normal tissue. Perhaps as interesting as the cues necessary to generate this structure are the signals required to maintain its branched architecture over the lifetime of the organism, since likely these pathways are de-regulated in malignancies. Previously, we have shown that the Na+/H+ exchanger 1 (NHE1), a critical regulator of intracellular pH, was necessary for mammary branching morphogenesis. Here we provide strong evidence that NHE1 function is also necessary for maintaining mammary branched architecture. Results: Inhibition of NHE1 with 5-N-Methy-N-isobutyl amiloride (MIA) on branched structures resulted in a rapid (within 24 hr) and reversible loss of branched architecture that was not accompanied by any overt changes in cell proliferation or cell death. NHE1 inhibition led to a significant acidification of intracellular pH in the branched end buds that preceded a number of events, including altered tissue polarity of myoepithelial cells, loss of NHE1 basal polarity, F-actin rearrangements, and decreased E-cadherin expression. Conclusions: Our results implicate NHE1 function and intracellular pH homeostasis as key factors that maintain mammary tissue architecture, thus, indirectly allowing for mammary function as a milk-providing (form) and -producing (function) gland. Developmental Dynamics 243:229–242, 2014. © 2013 Wiley Periodicals, Inc.

Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intracellularly (Stx-2). The extracellular form Epim stimulates morphogenic processes in a range of tissues, including in murine mammary glands where its overexpression in luminal epithelial cells is sufficient to drive hyperplasia and neoplasia. We analyzed WAP-Epim transgenic mice to gain insight into how Epim promotes malignancy. Ectopic overexpression of Epim during postnatal mammary gland development led to early side-branching onset, precocious bud formation, and increased proliferation of mammary epithelial cells. Conversely, peptide-based inhibition of Epim function reduced side branching. Because increased side branching and hyperplasia occurs similarly in mice upon overexpression of the progesterone receptor isoform-a (Pgr-a), we investigated whether Epim exhibits these phenotypes through Pgr modulation. Epim overexpression indeed led to a steep upregulation of both total Pgr mRNA and Pgr-a protein levels. Notably, the Pgr antagonist RU486 abrogated Epim-induced ductal side branching, mammary epithelial cell proliferation, and bud formation. Evaluation of Epim signaling in a three-dimensional ex vivo culture system showed that its action was dependent on binding to its extracellular receptor, integrin-αV, and on matrix metalloproteinase 3 activity downstream of Pgr-a. These findings elucidate a hitherto unknown transcriptional regulator of Pgr-a, and shed light on how overexpression of Epim leads to malignancy. Cancer Res; 73(18); 5719–29. ©2013 AACR.

A number of Garcinia species accumulate benzophenone derivatives that may be useful for the treatment of breast cancer. The dereplication of new benzophenone derivatives from Garcinia species is challenging due to the occurrence of multiple isomers and the known compounds found in their extracts. In the current study, a strategy is described using the UPLC-QTOFMS(E) technique to identify tentatively the known and uncharacterized benzophenones of interest based upon the characteristic fragmentation ions. Several UPLC-QTOFMS peaks (a-ee) appeared to contain benzophenone derivatives, and 12 of these peaks contained compounds with MS ionization profiles not consistent with previously identified compounds from the seeds of Garcinia paucinervis, an endangered Chinese species. The targeted isolation of unidentified compounds of interest afforded five new benzophenones, paucinones E-I (1-5), which were determined by MS and NMR analysis and ECD spectroscopy. These compounds were evaluated for cytotoxicity against three breast cancer cell lines inclusive of MDA-MB-231, SKBR3, and MCF-7. These results indicate that the UPLC-QTOFMS(E)-guided isolation procedure is an efficient strategy for isolating new benzophenones from Garcinia species.

Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H + exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H +) across plasma membranes in exchange for extracellular sodium ions (Na +). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 μM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα > PI3K > NHE1 > pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis.

The molecule responsible for the enzyme activity plasma membrane (PM) aminophospholipid translocase (PM-APLT), which catalyzes phosphatidylserine (PS) translocation from the outer to the inner leaflet of the plasma membrane, is unknown in mammals. A C.elegans study has shown that ablation of Tat-1, which is an ortholog of a mammalian P-type ATPase, Atp8a1, causes PS externalization in the germ cells. We demonstrate here that the hippocampal cells of the dentate gyrus (DG), and Cornu Ammonis (CA1, CA3) in mice lacking Atp8a1 exhibit a dramatic increase in PS externalization. Although their hippocampi showed no abnormal morphology or heightened apoptosis, these mice displayed increased activity and a marked deficiency in hippocampusdependent learning, but no hyper-anxiety. Such observations indicate that Atp8a1 plays a crucial role in PM-APLT activity in the neuronal cells. In corroboration, ectopic expression of Atp8a1 but not its close homolog, Atp8a2, caused an increase in the population (Vmax) of PM-APLT without any change in its signature parameter Km in the neuronal N18 cells. Conversely, expression of a Ptype phosphorylation-site mutant of Atp8a1 (Atp8a1*) caused a decrease in Vmax of PM-APLT without significantly altering its Km. The ATp8a1*-expressing N18 cells also exhibited PS externalization without apoptosis. Together, our data strongly indicate that Atp8a1 plays a central

Receptors for basement membrane (BM) proteins, including dystroglycan (DG), coordinate tissue development and function by mechanisms that are only partially defined. To further elucidate these mechanisms, we generated a conditional knockout of DG in the epithelial compartment of the mouse mammary gland. Deletion of DG caused an inhibition of mammary epithelial outgrowth and a failure of lactation. Surprisingly, loss of DG in vivo did not disrupt normal tissue architecture or BM formation, even though cultured Dag1-null epithelial cells failed to assemble laminin-111 at the cell surface. The absence of DG was, however, associated with a marked loss in activity of signal transducer and activator of transcription 5 (STAT5). Loss of DG perturbed STAT5 signaling induced by either prolactin or growth hormone. We found that DG regulates signaling by both hormones in a manner that is dependent on laminin-111 binding, but independent of the DG cytoplasmic domain, suggesting that it acts via a co-receptor mechanism reliant on DG-mediated laminin assembly. These results demonstrate a requirement for DG in the growth and function of a mammalian epithelial tissue in vivo. Moreover, we reveal a selective role for DG in the control of multiple STAT5-dependent hormone signaling pathways, with implications for numerous diseases in which DG function is compromised.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a dual-function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two nonmalignant human mammary epithelial cell lines that form polarized, growth-arrested structures (acini) when cultured in three-dimensional laminin-rich extracellular matrix gels (lrECM). As acini begin to form, PTEN accumulates both in the cytoplasm and at cell-cell contacts where it colocalizes with the E-cadherin/β-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function–blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in Skbr-3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in three-dimensional lrECM, indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus seems to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells. [Cancer Res 2009;69(10):4545–52]

Dystroglycan (DG) is an extracellular matrix receptor implicated in muscular dystrophies and cancers. DG belongs to the membrane-tethered mucin family and is composed of extracellular (alpha-DG) and transmembrane (beta-DG) subunits stably coupled at the cell surface. These two subunits are generated by autoproteolysis of a monomeric precursor within a distinctive protein motif called sea urchin-enterokinase-agrin (SEA) domain, yet the purpose of this cleavage and heterodimer creation is uncertain. In this study, we identify a functional nuclear localization signal within beta-DG and show that, in addition to associating with alpha-DG at the cell surface, the full-length and glycosylated beta-DG autonomously traffics to the cytoplasm and nucleoplasm in a process that occurs independent of alpha-DG ligand binding. The trafficking pattern of beta-DG mirrors that of MUC1-C, the transmembrane subunit of the related MUC1 oncoprotein, also a heterodimeric membrane-tethered mucin created by SEA autoproteolysis. We show that the transmembrane subunits of both MUC1 and DG transit the secretory pathway prior to nuclear targeting and that their monomeric precursors maintain the capacity for nuclear trafficking. A screen of breast carcinoma cell lines of distinct pathophysiological origins revealed considerable variability in the nuclear partitioning of beta-DG, indicating that nuclear localization of beta-DG is regulated, albeit independent of extracellular ligand binding. These findings point to novel intracellular functions for beta-DG, with possible disease implications. They also reveal an evolutionarily conserved role for SEA autoproteolysis, serving to enable independent functions of mucin transmembrane subunits, enacted by a shared and poorly understood pathway of segregated subunit trafficking.

Transforming growth factor-alpha (TGFalpha) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPK(ERK1,2)); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPK(ERK1,2) for 1 h, induced by TGFalpha, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (15 min) of MAPK(ERK1,2), induced by FGF7, led to growth without branching. Unlike TGFalpha, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. The response of the explants to FGF10 was similar to that to FGF7. Simultaneous stimulation by FGF7 and TGFalpha indicated that the FGF7-induced MAPK(ERK1,2) signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGFalpha-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1), and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary organoid morphogenesis are intimately dependent on the duration of activation of MAPK(ERK1,2) activation.

Garcinia oblongifolia Champ. ex Benth. (Clusiaceae) is a well-known medicinal plant from southern China, with edible fruits. However, the phytochemistry and bioactivity of the different plant parts of G. oblongifolia have not been studied extensively. Comparative metabolic profiling and bioactivities of the leaf, branch, and fruit of G. oblongifolia were investigated. A total of 40 compounds such as biflavonoids, xanthones, and benzophenones were identified using UPLC-QTOF-MS and MS(E), including 15 compounds reported for the first time from this species. Heatmap analyses found that benzophenones, xanthones, and biflavonoids were predominately found in branches, with benzophenones present in relatively high concentrations in all three plant parts. Xanthones were found to have limited distribution in fruit while biflavonoids were present at only low levels in leaves. In addition, the cytotoxic (MCF-7 breast cancer cell line) and antioxidant (ABTS and DPPH chemical tests) activities of the crude extracts of G. oblongifolia indicate that the branch extract exhibits greater bioactivity than either the leaf or the fruit extracts. Orthogonal partial least squares discriminate analysis was used to find 12 marker compounds, mainly xanthones, from the branches, including well-known antioxidants and cytotoxic agents. These G. oblongifolia results revealed that the variation in metabolite profiles can be correlated to the differences in bioactivity of the three plant parts investigated. This UPLC-QTOF-MS strategy can be useful to identify bioactive constituents expressed differentially in the various plant parts of a single species.

Background Pathological classification of cervical intraepithelial neoplasia (CIN) is problematic as it relies on subjective criteria. We developed an imaging method that uses spectroscopy to assess the fluorescent intensity of cervical biopsies derived directly from hematoxylin and eosin (H&E) stained tissues. Methods Archived H&E slides were identified containing normal cervical tissue, CIN I, and CIN III cases, from a Community Hospital and an Academic Medical Center. Cases were obtained by consensus review of at least 2 senior pathologists. Images from H&E slides were captured first with bright field illumination and then with fluorescent illumination. We used a Zeiss Axio Observer Z1 microscope and an AxioVision 4.6.3-AP1 camera at excitation wavelength of 450–490 nm with emission captured at 515–565 nm. The 32-bit grayscale fluorescence images were used for image analysis. Results We reviewed 108 slides: 46 normal, 33 CIN I and 29 CIN III. Fluorescent intensity increased progressively in normal epithelial tissue as cells matured and advanced from the basal to superficial regions of the epithelium. In CIN I cases this change was less prominent as compared to normal. In high grade CIN lesions, there was a slight or no increase in fluorescent intensity. All groups examined were statistically different. Conclusion Presently, there are no markers to help in classification of CIN I-III lesions. Our imaging method may complement standard H&E pathological review and provide objective criteria to support the CIN diagnosis.

Epimorphin/syntaxin-2 (EPM) is a plasma membrane–anchored protein that has at least two distinct functions depending on its membrane topology: vesicle fusion when localized to the cytoplasmic surface and morphogenic signaling when localized to the extracellular surface. Transgenic mice that express full-length extracellular EPM fused to the NH2-terminal signal sequence of interleukin-2, under the control of the whey acidic protein (WAP) gene promoter, exhibit aberrant mammary gland morphogenesis associated with increased expression of CCAAT enhancer binding protein β (C/EBPβ). Here we report that aged nulliparous and uniparous female WAP-EPM transgenic mice develop alveolar hyperplasias and well-differentiated adenocarcinomas that express high levels of C/EBPβ, keratin-14, matrix metalloproteinase-3, and β-catenin. This study reveals another pathway in which overexpression and alteration of a normal morphogenic process promote the development of cancer in the mammary gland.

5 páginas, 4 figuras.-- Letter., The tumour microenvironment can be a potent carcinogen, not only by facilitating cancer progression and activating dormant cancer cells, but also by stimulating tumour formation1. We have previously investigated stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme upregulated in many breast tumours2, and found that MMP-3 can cause epithelial–mesenchymal transition (EMT) and malignant transformation in cultured cells3, 4, 5, and genomically unstable mammary carcinomas in transgenic mice3. Here we explain the molecular pathways by which MMP-3 exerts these effects: exposure of mouse mammary epithelial cells to MMP-3 induces the expression of an alternatively spliced form of Rac1, which causes an increase in cellular reactive oxygen species (ROS). The ROS stimulate the expression of the transcription factor Snail and EMT, and cause oxidative damage to DNA and genomic instability. These findings identify a previously undescribed pathway in which a component of the breast tumour microenvironment alters cellular structure in culture and tissue structure in vivo, leading to malignant transformation., This work was supported by grants from the OBER office of the Department of Energy and an Innovator award from the Department of Defense (to M.J.B.) and from the National Institutes of Health (to M.J.B. and Z.W.), and by fellowships from the American Cancer Society (D.C.R.), the National Cancer Institute (L.E.L.), the Department of Defense (H.L. and C.M.N.) and the California Breast Cancer Research Program (J.E.F).

The tumor suppressor PTEN is altered in many cancers, including breast cancer, but only a handful of factors are known to control its expression. PTEN plays a vital role in cell survival and proliferation by regulating Akt phosphorylation, a key component of the phosphatidylinositol 3 kinase (PI3K) pathway. Here we show that insulin-like growth factor-II (IGF-II), which signals through PI3K, regulates PTEN expression in the mammary gland. IGF-II injection into mouse mammary gland significantly increased PTEN expression. Transgenic IGF-II expression also increased mammary PTEN protein, leading to reductions in Akt phosphorylation, epithelial proliferation, and mammary morphogenesis. IGF-II induced PTEN promoter activity and protein levels and this involved the immediate early gene egr-1. Thus, we have identified a novel negative feedback loop within the PI3K pathway where IGF-II induces PTEN expression to modulate its physiologic effects.

A considerable body of research indicates that mammary gland branching morphogenesis is dependent, in part, on the extracellular matrix (ECM), ECM-receptors, such as integrins and other ECM receptors, and ECM-degrading enzymes, including matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). There is some evidence that these ECM cues affect one or more of the following processes: cell survival, polarity, proliferation, differentiation, adhesion, and migration. Both three-dimensional culture models and genetic manipulations of the mouse mammary gland have been used to study the signaling pathways that affect these processes. However, the precise mechanisms of ECM-directed mammary morphogenesis are not well understood. Mammary morphogenesis involves epithelial 'invasion' of adipose tissue, a process akin to invasion by breast cancer cells, although the former is a highly regulated developmental process. How these morphogenic pathways are integrated in the normal gland and how they become dysregulated and subverted in the progression of breast cancer also remain largely unanswered questions.

The dynamic interactions between cells and basement membranes serve as essential regulators of tissue architecture and function in metazoans, and perturbation of these interactions contributes to the progression of a wide range of human diseases, including cancers. Here, we reveal the pathway and mechanism for the endocytic trafficking of a prominent basement membrane protein, laminin-111 (referred to here as laminin), and their disruption in disease. Live-cell imaging of epithelial cells revealed pronounced internalization of laminin into endocytic vesicles. Laminin internalization was receptor mediated and dynamin dependent, and laminin proceeded to the lysosome through the late endosome. Manipulation of laminin receptor expression revealed that the dominant regulator of laminin internalization is dystroglycan, a laminin receptor that is functionally perturbed in muscular dystrophies and in many cancers. Correspondingly, laminin internalization was found to be deficient in aggressive cancer cells displaying non-functional dystroglycan, and restoration of dystroglycan function strongly enhanced the endocytosis of laminin in both breast cancer and glioblastoma cells. These results establish previously unrecognized mechanisms for the modulation of cell–basement-membrane communication in normal cells and identify a profound disruption of endocytic laminin trafficking in aggressive cancer subtypes.

Adult mammary tissue has been considered "resting" with minimal morphological change. Here, we reveal the dynamic nature of the nulliparous murine mammary gland. We demonstrate specific changes at the morphological and cellular levels, and uncover their relationship with the murine estrous cycle and physiological levels of steroid hormones. Differences in the numbers of higher-order epithelial branches and alveolar development led to extensive mouse-to-mouse mammary variations. Morphology (assigned grades 0-3) ranged from a complete lack of alveoli to the presence of numerous alveoli emanating from branches. Morphological changes were driven by epithelial proliferation and apoptosis, which differed between ductal versus alveolar structures. Proliferation within alveolar epithelium increased as morphological grade increased. Extensive alveolar apoptosis was restricted to tissue exhibiting grade 3 morphology, and was approximately 14-fold higher than at all other grades. Epithelial proliferation and apoptosis exhibited a positive relationship with serum levels of progesterone, but not with 17beta-estradiol. Compared with other estrous stages, diestrus was unique in that the morphological grade, epithelial proliferation, apoptosis, and progesterone levels all peaked at this stage. The regulated tissue remodeling of the mammary gland was orchestrated with mRNA changes in specific matrix metalloproteinases (MMP-9 and MMP-13) and specific tissue inhibitors of metalloproteinases (TIMP-3 and TIMP-4). We propose that the cyclical turnover of epithelial cells within the adult mammary tissue is a sum of spatial and functional coordination of hormonal and matrix regulatory factors.

IGF-II is a growth factor implicated in human cancers and animal tumor models. While the mitogenic properties of IGF-II are well documented, its ability to suppress apoptosis in vivo has never been proven. We generated independent MMTV-IGF-II transgenic mice to examine the control of epithelial apoptosis at the morphological, cellular and molecular levels during the physiological event of postlactation mammary involution. Transgenic IGF-II expression was achieved in mammary epithelium and increased IGF-II bioactivity was confirmed by phosphorylation of the insulin receptor substrate-1, a signaling molecule downstream of the type I IGF receptor. IGF-II overexpression induced a delay in mammary involution, as evident by increased mammary gland to body weight ratios and persistence of both functionally intact lobulo-alveoli and mammary epithelial cellularity. The delayed mammary involution resulted from a significant reduction in mammary epithelial apoptosis, and not from increased epithelial proliferation. Recombinant IGF-II pellets implanted into involuting mammary glands of wild-type mice provided further evidence that IGF-II protein inhibited local epithelial apoptosis. At the molecular level, phosphorylated Akt/PKB, but not Erk1 or Erk2, persisted in IGF-II overexpressors and temporally correlated with reduced epithelial apoptosis. Levels of the phosphatase PTEN were unaltered in the transgenic tissue suggesting that the maintenance of Akt/PKB phosphorylation resulted from sustained phosphorylation rather than altered dephosphorylation of PIP-3. Together, this data reveal that IGF-II inhibits apoptosis in vivo and this effect correlates with prolonged phosphorylation of Akt/PKB

Osteoprotegerin-ligand (OPGL) is a key osteoclast differentiation/activation factor essential for bone remodeling. We report that mice lacking OPGL or its receptor RANK fail to form lobulo-alveolar mammary structures during pregnancy, resulting in death of newborns. Transplantation and OPGL-rescue experiments in opgl−/− and rank−/− pregnant females showed that OPGL acts directly on RANK-expressing mammary epithelial cells. The effects of OPGL are autonomous to epithelial cells. The mammary gland defect in female opgl−/− mice is characterized by enhanced apoptosis and failures in proliferation and PKB activation in lobulo-alveolar buds that can be reversed by recombinant OPGL treatment. These data provide a novel paradigm in mammary gland development and an evolutionary rationale for hormonal regulation and gender bias of osteoporosis in females.

Phosphoinositide-3-OH kinases (PI(3)Ks) constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, transformation, differentiation and protection from apoptosis. PI(3)K-mediated activation of the cell survival kinase PKB/Akt, and negative regulation of PI(3)K signalling by the tumour suppressor PTEN (refs 3, 4) are key regulatory events in tumorigenesis. Thus, a model has arisen that PI(3)Ks promote development of cancers. Here we report that genetic inactivation of the p110gamma catalytic subunit of PI(3)Kgamma (ref. 8) leads to development of invasive colorectal adenocarcinomas in mice. In humans, p110gamma protein expression is lost in primary colorectal adenocarcinomas from patients and in colon cancer cell lines. Overexpression of wild-type or kinase-dead p110gamma in human colon cancer cells with mutations of the tumour suppressors APC and p53, or the oncogenes beta-catenin and Ki-ras, suppressed tumorigenesis. Thus, loss of p110gamma in mice leads to spontaneous, malignant epithelial tumours in the colorectum and p110gamma can block the growth of human colon cancer cells.

The concept of tumor suppression by tissue inhibitor of metalloproteinases (TIMPs) has evolved primarily from studies of genetically modulated tumor cells. The next step is to focus on the host and assess the protective potential of host TIMP-1 on primary tumor growth and metastasis. We generated two transgenic mouse lines with altered Timp-1 expression in skin and liver: one overexpressed Timp-1 (Timp-1high), and the other had antisense RNA–mediated Timp-1 reduction (Timp-1low). ESbL-lacZ T-lymphoma cells provided the tumor challenge, as they form primary tumors upon intradermal injection with spontaneous metastasis to liver. Metastases were examined in X-Gal–stained whole-organ mounts. Timp-1 overexpression inhibited intradermal tumor growth and spontaneous metastasis, leading to prolonged survival of the mice. The opposite effects occurred in Timp-1low mice, leading to shorter host survival. Experimental metastasis assays showed that Timp-1–compromised livers in Timp-1low mice showed at least a doubling of metastatic foci and numerous additional micrometastases, indicative of increased host susceptibility. However, Timp-1high mouse livers showed an unaltered metastatic load in the experimental metastasis assay. In conclusion, these data demonstrate that Timp-1 levels within a tissue predetermine the development and progression of T-cell lymphoma.

The mammary gland is an ideal model to study the link between form and function in normal tissue. Perhaps as interesting as the cues necessary to generate this structure are the signals required to maintain its branched architecture over the lifetime of the organism, since likely these pathways are de-regulated in malignancies. Previously, we have shown that the Na(+) /H(+) exchanger 1 (NHE1), a critical regulator of intracellular pH, was necessary for mammary branching morphogenesis. Here we provide strong evidence that NHE1 function is also necessary for maintaining mammary branched architecture.Inhibition of NHE1 with 5-N-Methy-N-isobutyl amiloride (MIA) on branched structures resulted in a rapid (within 24 hr) and reversible loss of branched architecture that was not accompanied by any overt changes in cell proliferation or cell death. NHE1 inhibition led to a significant acidification of intracellular pH in the branched end buds that preceded a number of events, including altered tissue polarity of myoepithelial cells, loss of NHE1 basal polarity, F-actin rearrangements, and decreased E-cadherin expression.Our results implicate NHE1 function and intracellular pH homeostasis as key factors that maintain mammary tissue architecture, thus, indirectly allowing for mammary function as a milk-providing (form) and -producing (function) gland.

Objective Human papillomavirus (HPV) infections remain a leading cause of mortality worldwide. In the U.S. strategies via screening and vaccination prevent HPV-associated cervical neoplasms, but consume immense healthcare costs. The spice component curcumin has potent anticancer and antiviral properties, which have been difficult to harness as a treatment, due to its poor systemic bioavailability. This project tests the possibility of developing a curcumin-based therapy for cervical cancer. Methods Using four HPV(+) cervical cancer cell lines and normal fibroblasts we first tested the selectivity and potency of curcumin in eliminating HPV(+) cells. Subsequently, we developed a curcumin-based cervical cream and tested its efficacy in eliminating apposed HPV(+) cells and also its possible side effects on the vaginal epithelium of healthy mice. Results Curcumin selectively eliminates a variety of HPV(+) cervical cancer cells (HeLa, ME-180, SiHa, and SW756), suppresses the transforming antigen E6, dramatically inhibits the expression of the pro-cancer protein epidermal growth factor receptor (EGFR), and concomitantly induces p53. Additionally, Vacurin, a uniform colloidal solution of curcumin in a clinically used amphipathic vaginal cream, eliminates apposed HeLa cells while suppressing the expression of EGFR. In mice, daily intravaginal application of Vacurin for three weeks produced no change in body weight and when the mice were sacrificed, the vaginal tract epithelium showed no Vacurin-evoked adverse effects. Conclusion We have developed a curcumin-based vaginal cream, which effectively eradicates HPV(+) cancer cells and does not affect non-cancerous tissue. Our preclinical data support a novel approach for the treatment of cervical HPV infection.

Curcumin, which is derived from the plant Curcuma longa, has received considerable attention as a possible anti-cancer agent. In cell culture, curcumin is capable of inducing apoptosis in cancer cells at concentrations that do not affect normal cells. One draw-back holding curcumin back from being an effective anti-cancer agent in humans is that it is almost completely insoluble in water and therefore has poor absorption and subsequently poor bioavailability. Here we have generated a number of curcumin derivatives (tetrahydro-curcumin, curcumin mono-carboxylic acid, curcumin mono-galactose, curcumin mono-alkyne and dendrimer-curcumin conjugate) to test whether any of them display both cytotoxicity and water solubility. Of those tested only dendrimer-curcumin conjugate exhibited both water solubility and cytotoxicity against SKBr3 and BT549 breast cancer cells. When compared to curcumin dissolved in DMSO, dendrimer-curcumin conjugate dissolved in water was significantly more effective in inducing cytotoxicity, as measured by the MTT assay and effectively induced cellular apoptosis measured by caspase-3 activation. Since dendrimer-curcumin conjugate is water soluble and capable of inducing potent cytotoxic effects on breast cancer cell lines, it may prove to be an effective anti-cancer therapy to be used in humans.

The concept of Ayurvedic expert guided drug discovery and development is defined and put to test systematically for the first time in literature. Western Science has explored only ~5% of the approximately 25,000 species of higher plants for drug leads. The ancient medical science of Ayurveda has however employed a much larger spectrum of plants for clinical treatment. Clerodendrum viscosum (CV), a commonly growing weed in the Indian subcontinent has been employed by S. Nirmalananda (Ayurvedic expert) for the treatment of cervical cancer. Here we isolate and characterize a water extract fraction (Cv-AP) from the root of CV and evaluate its anticervical cancer cell bioactivity. Our results indicate that Cv-AP possesses pro-apoptotic, anti-proliferative, and anti-migratory activity in a dose-dependent fashion against cervical cancer cell lines. In contrast, primary fibroblasts (control healthy cells), when exposed to similar concentrations of this extract, fail to undergo apoptosis and remain relatively unaffected. These findings suggest that Clerodendrum viscosum (CV) is a readily available source of components with potent anti-cancer activity and selective bioactivity against cervical cancer cells. The major component in CV-AP was identified as a glycoprotein via SDS Page and Concanavalin-A binding studies. This study serves to illustrate that systematic collaboration with Ayurveda is a practical and powerful strategy in drug discovery and development.

High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression.

The understanding of how estrogen and progesterone (P4) drive uterine remodeling in rodents has largely been based on studies involving administration of exogenous hormones, using steroid receptor-deficient mice, or relying on vaginal smears. In all these cases, the actual serum levels of 17β-estradiol (E2) and P4are not directly measured, and the relationship between physiological levels of female sex hormones and uterine remodeling in cycling mice has not been fully explored. Here, we measured the circulating levels of E2and P4in cycling mice and performed correlation analysis between hormone levels and epithelial and stromal uterine parameters, irrespective of the estrous stage. In parallel, these parameters were analyzed in relation to the more conventional method of vaginal smear classification of estrous stage. We found that circulating P4inversely correlated with uterine width, luminal epithelial proliferation, stromal apoptosis, and degradation of luminal epithelial basement membrane collagen type-IV. Circulating E2positively correlated with uterine width, stromal cell proliferation, and collagen type-I content, while it negatively correlated with glandular epithelial proliferation, loss of collagen type-IV surrounding glandular epithelium, and apoptosis in luminal, glandular, and stromal compartments. Our findings indicate that measuring P4or E2levels can predict many concurrent cellular and stromal events in the mouse uterus, suggesting that in naturally cycling mice cellular responses to hormone changes are not delayed, but occur very rapidly.

Multiple cancers exhibit mutations in beta-catenin that lead to increased stability, altered localization or amplified activity. beta-catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter beta-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased beta-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3(-/-) MEFs being more sensitive and Timp3(-/-) MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3(-/-) MECs had higher dephosphorylated beta-catenin levels and increased beta-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of beta-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated beta-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3(-/-) mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.

Mammary ducts are formed through a process of branching morphogenesis. We present results of experiments using a simulation model of this process, and discuss their implications for understanding mammary duct extension and bifurcation. The model is a cellular automaton approximation of a reaction-diffusion process in which matrix metalloproteinases represent the activator, inhibitors of matrix metalloproteinases represent the inhibitor, and growth factors serve as a substrate. We compare results from the simulation model with those from in-vivo experiments as part of an assessment of whether duct extension and bifurcation during morphogenesis may be a consequence of a reaction-diffusion mechanism mediated by MMPs and TIMPs.

Cell contraction and membrane blebbing are evolutionarily conserved events that occur during the execution phase of apoptosis. Several members of the TNF-ligand superfamily, which are associated with the promotion of a number of pathological processes, including inflammation and cancer are also capable of inducing membrane blebbing in some cell types. Most of these ligands are transmembrane bound but can be shed from the cell surface through proteolytic processing where soluble ligands can act as antagonists, as in the case of FAS ligand, or agonists, as with TNF-a. Here we provide evidence that the matrix metalloproteinase, MMP-3/stromelysin-1 induces rapid membrane blebbing in serum starved or cyclohexamide-treated MCF1OA human breast epithelial cells. MMP-3-mediated membrane blebbing is associated with reorganization of the actin cytoskeleton, upregulation of both p53 and p38 MAP kinase activity, and loss of cell surface E-cadherin. A broad-spectrum MMP inhibitor completely abolishes these reactions. To understand the signaling cascade initiated by MMP-3, we asked whether factors downstream of TNF-superfamily signaling were involved. We show that inhibitors against JNK and caspase-3, and RNAi reduction of MKK7, a known activator of JNK inhibit membrane blebbing. Moreover, stable expression of a dominant negative FADD (dnFADD), a downstream effector of several TNF superfamily ligands, renders MCF1OA cells resistant to membrane blebbing. Together these findings indicate that MMP-3 induces cell membrane blebbing through a TNF-superfamily signaling pathway and provides an impetus to further explore this protease in inflammation and cancer.

1085 Background: When compared to normal breast epithelial cells, breast cancer cells exhibit increased metabolic activity, which produces excessive intracellular hydrogen ions (H+). This increase ...