Normal Murine Mammary Gland Cells Research Articles

Abstract 3080: ILEI regulates LIFR expression and CSC self-renewal

Abstract Our lab has previously identified a mechanism of transforming growth factor beta (TGFβ)-mediated epithelial-to-mesenchymal transition (EMT) in normal murine mammary gland (NMuMG) cells. Reduced expression of heterogenous nuclear ribonucleoprotein E1 (hnRNP E1, hereafter “E1”) in NMuMG cells induces EMT, tumorigenesis, and metastasis. Our experimental data has revealed that increased expression of interleukin-like EMT inducer (ILEI) and of leukemia inhibitory factor receptor (LIFR) proteins occurs following E1 knockdown (E1KD). ILEI and LIFR have been shown to interact by coimmunoprecipitation/radiography experiments, and there is a correlation between ILEI expression and phosphorylation of the LIFR effector STAT3. ILEI is necessary to induce EMT in NMuMG cells, and spheroid formation capacity in non-adherent serum-depleted cell culture conditions is attenuated following either ILEI or LIFR knockdown. Loss of spheroid formation following ILEI knockdown is rescued by treatment with exogenous recombinant ILEI protein (rILEI); however, spheroid formation is not rescued by rILEI following LIFR knockdown. In immunocompromised mice, orthotopic grafts of NMuMG cells with E1KD (E1KD cells) with additional knockdown of either ILEI or LIFR display a decrease in tumor initiation, growth, and metastasis relative to control cells. These data suggest that ILEI-induced spheroid formation capacity involves LIFR, and that an ILEI/LIFR signaling axis promotes breast cancer stem cell (BCSC) phenotype. Characterization of the mechanisms that allow BCSCs to persist during chemotherapeutic interventions will advance our understanding of drug resistant and dormant breast cancer. The precise mechanisms involved in ILEI-mediated EMT and ILEI/LIFR-mediated BCSC induction are unknown. Semi-quantitative PCR data demonstrates that the increase in LIFR expression in E1KD cells is regulated at the transcriptional level. Additionally, E1KD cells with CRISPR-Cas9-mediated ILEI knockout (E1KD-ILEI-KO cells) show decreased luciferase signal relative to control cells following transfection of LIFR promoter DNA sequences placed upstream of a luciferase gene. Further, E1KD-ILEI-KO cells show attenuation of spheroid formation when compared to unaltered E1KD cells. Our data allow us to conclude that ILEI participates in the regulation of LIFR gene transcription, which plays a role in ILEI-induced maintenance of spheroid formation and self-renewal. Continued investigation will allow development of studies aimed at revealing the mechanisms involved in ILEI/LIFR axis-mediated disease progression in vivo. Citation Format: William S. Streitfeld, Annamarie C. Dalton, Philip H. Howe. ILEI regulates LIFR expression and CSC self-renewal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3080.

Design, synthesis and cytotoxic evaluation of peptoid analogs of an anticancer active triazolylpeptidyl penicillin.

Aim: Encouraged by the antitumor activity exhibited by triazolylpeptidyl penicillins, we decided to synthesize and evaluate a library of peptoid analogs. Results: The replacement of the dipeptide unit of the reference compound, TAP7f, was investigated. In addition, the effect of the triazole linking group on the biological activity of these new derivatives was evaluated, exchanging it with a glycine spacer. The cytotoxic effect of the library compounds was determined in the B16-F0 cell line and compared with the effects on normal murine mammary glandcells. Conclusion: Among the tested compounds, peptoid 4e exhibited the highest antiproliferative activity.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

A Hierarchical Regulatory Landscape during the Multiple Stages of EMT

Epithelial-mesenchymal transition (EMT) enables cells to gain migratory and invasive features underlined by major transcriptional and epigenetic reprogramming. However, most studies have focused on the endpoints of the EMT process, and the epistatic hierarchy of the transcriptional networks underlying EMT has remained elusive. We have used a siRNA-based, functional high-content microscopy screen to identify 46 (co)transcription factors ((co)TFs) and 13 miRNAs critically required for EMT in normal murine mammary gland (NMuMG) cells. We compared dynamic gene expression during EMT kinetics and used functional perturbation of critical (co)TFs and miRNAs to identify groups and networks of EMT genes. Computational analysis as well as functional validation experiments revealed interaction networks between TFs and miRNAs and delineated the hierarchical and functional interactions of multiple EMT regulatory networks in NMuMG cells.

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

Differential effects of Pax3 on expression of polysialyltransferases STX and PST in TGF-β-treated normal murine mammary gland cells.

Glycosylation of certain proteins at the mammalian cell surface is an essential event in carcinogenesis. Sialylation, one type of glycosylation, can act on multiple cell-behaviors, such as migration, growth, and malignant invasion. Two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), are responsible for synthesis of polysialic acid on neural cell adhesion molecule. We showed previously that STX and PST are oppositely expressed in normal murine mammary gland cells undergoing transforming growth factor-β-induced epithelial-mesenchymal transition. The molecular basis for regulation of STX and PST remained unclear. In the present study, we observed that transcription factor Pax3 upregulates STX expression, downregulates PST expression, and modulates upregulated expression of PSA, which attaches primarily to neural cell adhesion molecule to form PSA-NCAM. Overexpression of Pax3 in normal murine mammary gland cells transformed the expression of epithelial-mesenchymal transition markers E-cadherin and N-cadherin, and significantly promoted cell migration, but had no effect on cell proliferation.

Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells

Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long‐term exposure to concentrations of aluminium—in the form of aluminium chloride (AlCl3)—in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl3 form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl3. Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well‐established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens.

Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays.

Morphological changes in mitochondria induced by X-irradiation in normal murine mammary gland cells were studied with a live-cell microscopic imaging technique. Mitochondria were visualised by staining with a specific fluorescent probe in the cells, which express fluorescent ubiquitination-based cell-cycle indicator 2 (Fucci2) probes to visualise cell cycle. In unirradiated cells, the number of cells with fragmented mitochondria was about 20 % of the total cells through observation period (96 h). In irradiated cells, the population with fragmented mitochondria significantly increased depending on the absorbed dose. Particularly, for 8 Gy irradiation, the accumulation of fragmentation persists even in the cells whose cell cycle came to a stand (80 % in G1 (G0-like) phase). The fraction reached to a maximum at 96 h after irradiation. The kinetics of the fraction with fragmented mitochondria was similar to that for cells in S/G2/M phase (20 %) through the observation period (120 h). The evidences show that, in irradiated cells, some signals are continually released from a nucleus or cytoplasm even in the G0-like cells to operate some sort of protein machineries involved in mitochondrial fission. It is inferred that this delayed mitochondrial fragmentation is strongly related to their dysfunction, and hence might modulate radiobiological effects such as mutation or cell death.

GAL3ST2 from mammary gland epithelial cells affects differentiation of 3T3-L1 preadipocytes.

In the mammary gland, the involution that occurs when lactation ends is an important period for cancer development. We have previously demonstrated stromal-epithelium interactions evaluating conditioned medium of adipose tissue on breast epithelial metalloproteases activity (Creydt et al., Clin Transl Oncol 15:124-131, 2013). Here, we evaluated the effects of conditioned medium of breast epithelial mammary cells on stromal cells. Conditioned medium from normal murine mammary gland cell line (NMuMG) and conditioned medium proteins were obtained. Then, they were evaluated on modulation of adipocyte differentiation, using 3T3-L1 cell line. We described, for the first time, that breast epithelial mammary cells could produce the enzyme galactose 3-O-sulfotransferase 2 (GAL3ST2). Importantly, GAL3ST2 is present in NMMuMG and two human breast cancer cell lines, and it is more strongly expressed in more metastatic tumors. When 3T3-L1 preadipocyte differentiation was triggered in the presence of conditioned medium from NMuMG or GAL3ST2, triglyceride accumulation was decreased by 40% and C/EBPβ expression by 80% in adipocytes. In addition, the expression of FABP4 (aP2), another marker of adipocyte differentiation, was inhibited by 40% in GAL3ST2-treated cells. Taken together, these results suggest that GAL3ST2 would interfere with normal differentiation of 3T3-L1 preadipocytes; raising the possibility that it may affect normal differentiation of stromal preadipocytes and be a link to tumor metastatic capacity.

Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition.

The process of epithelial‑mesenchymal transition (EMT) is essential for the proliferation and metastasis of tumor cells. Our previous study demonstrated that the expression of gangliotetraosylceramide (Gg4) and the transcription of UDP‑Gal: β1,3-galactosyltransferase‑4 (β3GalT4), a gene which controls the expression of Gg4, are reduced during transforming growth factor (TGF)‑β‑induced EMT in normal murine mammary gland (NMuMG) cells. The present study revealed that the transcription level of β3GalT4 in patients with breast cancer was decreased compared with healthy control subjects. However, the molecular basis underlying these effects remains to be elucidated. Analysis of the β3GalT4 promoter sequence revealed a putative Smad‑binding element (SBE) of Smad4, which is a transcriptional factor in TGFβ responses and forms a complex with Smad3. To clarify the association between the Smad3/4 complex and the β3GalT4 gene during EMT in NMuMG cells, an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay were performed. The Smad3/4 complex was found to bind directly to the SBE of the β3GalT4 promoter. Overexpression of Smad3 and Smad4 through stable transfection had no notable effect on cell phenotype, but did alter the expression of various EMT protein markers. Treatment with TGFβ reduced the expression of Gg4 and the mRNA levels of the β3GalT4 gene in Smad3‑ and Smad4‑overexpressing cells compared with vector‑transfected cells. Expression of the epithelial markers E‑cadherin and β‑catenin decreased in parallel with the reduction in Gg4. These findings suggested that the activated Smad3/4 complex downregulated the expression of Gg4 and the β3GalT4 gene through translocation into the nucleus and binding to the β3GalT4 promoter.

Breast tumor cell TACE-shed MCSF promotes pro-angiogenic macrophages through NF-κB signaling

Most deaths associated with breast cancer, the most common malignancy in women, are caused by metastasis. Tumor associated macrophages significantly contribute to breast cancer progression and development of metastasis through the promotion of angiogenesis which involves a central regulator of macrophage functions: nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Macrophages are activated by macrophage colony stimulating factor (MCSF) and chemokine (C-C motif) ligand 2 (CCL2) to secrete angiogenic factors including vascular endothelial growth factor (VEGF). The release of MCSF from tumor cells is mediated by ectodomain shedding through tumor necrosis factor alpha converting enzyme activation (TACE). Here we determined whether tumor cells TACE-shed MCSF promotes angiogenesis through activation of the NF-κB pathway in macrophages and the subsequent release of VEGF. These interactions were modeled in vitro using a panel of mammary cells mimicking the breast cancer progression from normal murine mammary gland cells to metastatic 4T1 cells along with J774 macrophages, all derived from BALB/c mice. TACE and MCSF expressions were higher in metastatic cells compared to epithelial cells (p < 0.05). Tumor conditioned medias activated the expression of VEGF by macrophages through stimulation of the NF-κB pathway and resulting macrophage secretions that promoted high levels of endothelial cell tubes. Furthermore, the combinations of CCL2, also highly expressed by tumor cells, and MCSF promoted pro-angiogenic macrophages. These results highlight the key role of tumor cell TACE-shed MCSF and secreted CCL2 in stimulating pro-angiogenic macrophages.

595 GOLD NANOPARTICLE CAN SERVE AS AGENTS FOR IMMUNOMODULATION IN MURINE BLADDER TUMOR MODELS BY CAUSING THE CONFORMATIONAL CHANGES AND ATTENUATING THE BIOLOGICAL FUNCTION OF TGF-β1 PROTEIN

You have accessJournal of UrologyBladder Cancer: Basic Research (I)1 Apr 2013595 GOLD NANOPARTICLE CAN SERVE AS AGENTS FOR IMMUNOMODULATION IN MURINE BLADDER TUMOR MODELS BY CAUSING THE CONFORMATIONAL CHANGES AND ATTENUATING THE BIOLOGICAL FUNCTION OF TGF-β1 PROTEIN Yuh-Shyan Tsai, Yu-Hung Chen, Pai-Chiao Cheng, Hsin-Tzu Tsai, Ai-Li Shiau, Tzong-Shin Tzai, and Chao-Liang Wu Yuh-Shyan TsaiYuh-Shyan Tsai Tainan, Taiwan More articles by this author , Yu-Hung ChenYu-Hung Chen Tainan, Taiwan More articles by this author , Pai-Chiao ChengPai-Chiao Cheng Tainan, Taiwan More articles by this author , Hsin-Tzu TsaiHsin-Tzu Tsai Tainan, Taiwan More articles by this author , Ai-Li ShiauAi-Li Shiau Tainan, Taiwan More articles by this author , Tzong-Shin TzaiTzong-Shin Tzai Tainan, Taiwan More articles by this author , and Chao-Liang WuChao-Liang Wu Tainan, Taiwan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1991AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Gold nanoparticles (AuNPs) have been widely used as carriers due to their great bio-compatibility and unique physical properties and potentially as therapeutic agents for inactivating vascular endothelial growth factor (VEGF). Like VEGF, transforming growth factor-beta 1 (TGF-β1) is a member of the cysteine-knot structural superfamily and plays a pivotal role in many diseases and is known as an immunosuppressive agent. The study is aimed to investigate whether AuNPs can serve as an intratumoral immunomodulation and its mechanism. METHODS Murine bladder tumor-2 cells (MBT2), normal murine mammary gland cells (NMuMg), C3H/HeN and NOD-SCID mice were used for in vitro and in vivo studies, including cell growth assay and tumor growth assay. X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy were used for exploring the mechanism of AuNP-TGFβ1 conjugation. RESULTS The results reflected strong interactions between TGF-β1 and the surface of AuNPs when incubated with serum-containing medium, demonstrating a time- and dose-dependent pattern from both immunoassays and Western blotting studies. Compared with other serum proteins which also can bind to AuNP surface, AuNP-TGFβ1 conjugate is a thermodynamically favored compound. Epithelial cells underwent epithelial-mesenchymal transition (EMT) upon treatment with TGF-β1; however, treatment with AuNPs reversed this effect, as detected by cell morphology and expression levels of EMT markers. TGF-β1 was found to bind to AuNPs through S-Au bonds by X-ray photoelectron spectroscopy. To understand why the activity of TGF-β1 was attenuated after conjugating to AuNPs, Fourier transform infrared spectroscopy was employed to analyze the conformational changes of TGF-β1 on the surface of AuNPs. The results indicated that TGF-β1 underwent significant conformational changes at both secondary and tertiary structural levels after conjugation to the AuNP surface, resulting in the deactivation of TGF-β1 protein. An in vivo experiment also showed that addition of AuNPs attenuated the growth of TGF-β1-secreting murine bladder tumor-2 cells in syngeneic C3H/HeN mice, but not in immunocompromised NOD-SCID mice, and this was associated with an increase in the number of tumor-infiltrating CD4+ and CD8+ T lymphocytes, and a decrease in the number of intrasplenic Foxp3(+) lymphocytes. CONCLUSIONS AuNPs may be a promising agent for modulating tumor immunity through inhibiting immunosuppressive TGF-β1 signaling. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e243 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuh-Shyan Tsai Tainan, Taiwan More articles by this author Yu-Hung Chen Tainan, Taiwan More articles by this author Pai-Chiao Cheng Tainan, Taiwan More articles by this author Hsin-Tzu Tsai Tainan, Taiwan More articles by this author Ai-Li Shiau Tainan, Taiwan More articles by this author Tzong-Shin Tzai Tainan, Taiwan More articles by this author Chao-Liang Wu Tainan, Taiwan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Overall Sulfation of Heparan Sulfate from Pancreatic Islet β-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.

MIR-99a and MIR-99b Modulate TGF-β Induced Epithelial to Mesenchymal Plasticity in Normal Murine Mammary Gland Cells

Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-β pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs.Herein, we identified mir-99a and mir-99b as two novel TGF-β target miRNA genes, the expression of which increased during TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b inhibition decreased TGF-β activity by inhibiting SMAD3 phosphorylation, resulting in decreased migration and increased proliferation in response to TGF-β. However, mir-99a and mir-99b inhibition was insufficient to block TGF-β induced EMT of NMUMG cells.Mir-99a and mir-99b over-expression in epithelial NMUMG cells resulted in increased proliferation, migration and fibronectin expression, while E-cadherin and ZO-1 expression were negatively regulated.In conclusion, we identified mir-99a and mir-99b as two novel modulators of TGF-β pathway that alter SMAD3 phosphorylation, in turn altering cell migration and adhesion of mesenchymal NMUMG cells. The effect of mir-99a and mir-99b over-expression on NMUMUG proliferation is dependent upon the epithelial or mesenchymal status of the cells. Our study suggests that mir-99a and mir-99b may function as modulators within a complex network of factors regulating TGF-β induced breast epithelial to mesenchymal transition, as well as proliferation and migration of breast cancer cells, providing a possible target for future translationally oriented studies in this area.

Matrix rigidity regulates a switch between TGF-β1–induced apoptosis and epithelial–mesenchymal transition

The transforming growth factor-β (TGF-β) signaling pathway is often misregulated during cancer progression. In early stages of tumorigenesis, TGF-β acts as a tumor suppressor by inhibiting proliferation and inducing apoptosis. However, as the disease progresses, TGF-β switches to promote tumorigenic cell functions, such as epithelial-mesenchymal transition (EMT) and increased cell motility. Dramatic changes in the cellular microenvironment are also correlated with tumor progression, including an increase in tissue stiffness. However, it is unknown whether these changes in tissue stiffness can regulate the effects of TGF-β. To this end, we examined normal murine mammary gland cells and Madin-Darby canine kidney epithelial cells cultured on polyacrylamide gels with varying rigidity and treated with TGF-β1. Varying matrix rigidity switched the functional response to TGF-β1. Decreasing rigidity increased TGF-β1-induced apoptosis, whereas increasing rigidity resulted in EMT. Matrix rigidity did not change Smad signaling, but instead regulated the PI3K/Akt signaling pathway. Direct genetic and pharmacologic manipulations further demonstrated a role for PI3K/Akt signaling in the apoptotic and EMT responses. These findings demonstrate that matrix rigidity regulates a previously undescribed switch in TGF-β-induced cell functions and provide insight into how changes in tissue mechanics during disease might contribute to the cellular response to TGF-β.

Novel common integration sites targeted by mouse mammary tumor virus insertion in mammary tumors have oncogenic activity.

Non-acute transforming retroviruses like mouse mammary tumor virus (MMTV) cause cancer, at least in part, through integration near cellular genes involved in growth control, thereby de-regulating their expression. It is well-established that MMTV commonly integrates near and activates expression of members of the Wnt and Fgf pathways in mammary tumors. However, there are a significant number of tumors for which the proviral integration sites have not been identified. Here, we used high through-put screening to identify common integration sites (CISs) in MMTV-induced tumors from C3H/HeN and BALB/c mice. As expected, members of both the Wnt and Fgf families were identified in this screen. In addition, a number of novel CISs were found, including Tcf7l2, Antxr1/Tem8, and Arhgap18. We show here that expression of these three putative oncogenes in normal murine mammary gland cells altered their growth kinetics and caused their morphological transformation when grown in three dimensional cultures. Additionally, expression of Tcf7l2 and Antxr1/Tem8 sensitized cells to exogenous WNT ligand. As Tcf7l2, Antxr1/Tem8, and Arhgap18 have been associated with human breast and other cancers, these data demonstrate that MMTV-induced insertional mutation remains an important means for identifying genes involved in breast cancer.

Protein Kinase D Regulates Cofilin Activity through p21-activated Kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.

Lysophosphatidic acid (LPA) stimulates mouse mammary epithelial cell growth

LPA (lysophosphatidic acid) is a bioactive phospholipid having diverse effects on various types of tissues. When NMuMG (normal murine mammary gland) cells were cultured in the presence of 0-10 μM LPA, cell numbers were increased by dose dependency for the 6-day culture periods (P<0.05). In DNA synthesis assay, 10 μM LPA induced 4.5-fold more DNA synthesis compared with control (P<0.05). In addition, the cultured cell density in the given area was increased by LPA treatment. MMP (matrix metalloproteinase) inhibitor GM6001 and EGFR [EGF (epidermal growth factor) receptor] tyrosine kinase inhibitor AG1478 [tyrphostin AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline] significantly decreased LPA-induced DNA synthesis and cell growth without cell death (P<0.05). To test the hypothesis that LPA-induced cell growth is mediated through LPA subtype receptors, LPA subtype receptor gene expressions were amplified by PCR. NMuMG cells expressed LPA1 and LPA2 receptor genes in the presence of 10% FBS (fetal bovine serum). LPA treatments increased ERK1/2 (extracellular-signal-regulated kinase) phosphorylation at 30 min and then dephosphorylated at 2 h after treatment. LPA treatment phosphorylated at tyrosine residues on a variety of Gi and PI3-dependent signal transducers in NMuMG cells. These results suggest that LPA subtype receptors play a role as the active transactivator of EGFR-associated kinases as well as direct growth regulator in mammary tissues.

Abstract 3105: Extracellular matrix density and the development of breast acini and ducts in 3D cultures

Abstract Breast epithelial cells develop into polarized and highly organized acinar and ductal structures in response to stromal cues, including extracellular matrix composition and density, which can in part be reproduced in 3D culture conditions. Here we present the effects of increasing density of stroma composition on the ability of heterotypic cultures of epithelial and mesenchymal stem cells to organize into acinar and tubular structures in in vitro 3D cultures. Normal murine mammary gland (NMuMG) cells were cultured in combination (30:70) with mouse mesenchymal stem cells (D1), in 3D matrices composed of increasing concentrations (0.8 to 8 mg/ml) of collagen I and growth-factor reduced Matrigel® (1:1). The organization of the breast-like structures was assessed by immunohistochemistry. After 3-5 days in culture, the organization and the presence of acinus-like and tubule-like structures were determined. By immunohistochemical analysis, NMuMG cells co-cultured with D1 cells formed acinar and tubular structures with the NMuMG epithelial cells surrounding a lumen composed of dead cells while the D1 cells were mostly peripheral and separated by laminin-positive basement membrane. Collagen I /Matrigel® matrices with density of 1.6 and 2.4 mg/ml were associated with significantly higher concentrations of acinus-like structures compared to other densities (p&amp;lt;0.05). In contrast, the high concentration (8mg/ml) matrix prevented the development of acinus-like structures (p&amp;lt;0.05). The number of tube-like structures was drastically reduced as the density of the collagen I / Matrigel matrix increased from 0.8 to 3.2 mg/ml (p&amp;lt;0.05). These results indicate that the density of the matrix influences the organization and the generation of acinus-like and duct-like structures in the mammary tissues. Whether the biomechanical and density properties of the breast extracellular matrix can be harnessed to build improved tissue culture systems to benefit breast cancer patients remains to be determined. This work was supported by grants from the Department of Defense Era of Hope program (BC044778) and the National Science Foundation EFRI program (CBE0736007). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3105. doi:10.1158/1538-7445.AM2011-3105

Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-β

The epithelial-to-mesenchymal transition (EMT) is a basic cellular process that plays a key role in normal embryonic development and in cancer progression/metastasis. Our previous study indicated that EMT processes of mouse and human epithelial cells induced by TGF-β display clear reduction of gangliotetraosylceramide (Gg4) and ganglioside GM2, suggesting a close association of glycosphingolipids (GSLs) with EMT. In the present study, using normal murine mammary gland (NMuMG) cells, we found that levels of Gg4 and of mRNA for the UDP-Gal:β1-3galactosyltransferase-4 (β3GalT4) gene, responsible for reduction of Gg4, were reduced in EMT induced by hypoxia (∼1% O(2)) or CoCl(2) (hypoxia mimic), similarly to that for TGF-β-induced EMT. An increase in the Gg4 level by its exogenous addition or by transfection of the β3GalT4 gene inhibited the hypoxia-induced or TGF-β-induced EMT process, including changes in epithelial cell morphology, enhanced motility, and associated changes in epithelial vs. mesenchymal molecules. We also found that Gg4 is closely associated with E-cadherin and β-catenin. These results suggest that the β3GalT4 gene, responsible for Gg4 expression, is down-regulated in EMT; and Gg4 has a regulatory function in the EMT process in NMuMG cells, possibly through interaction with epithelial molecules important to maintain epithelial cell membrane organization.