Normal Mammary Gland Epithelial Cells Research Articles

Estimation of Toll-like Receptors- 9 Levels in Iraqi Breast Cancer Patients

Background: Innate receptor called toll-like receptor 9 is capable of identifying both microbial and vertebrate DNA.It has been found in both normal mammary gland epithelial cells and breast milk cells.TLR-9was estimated in serum of patients and breast tissue for breast disease. Methods: A 100 women (17–60 years old) were determined undergoing breast surgery at Babylon Province's Al-Fayhaa Al Ahly Hospital and AL-Hilla Teaching Hospital provided blood and breast tissue samples. Hospital histology lab and clinical surgery performed histological confirmation of breast diseases( benign and malignant tumors). Twenty blood samples were taken as controls from women who appeared to be in good health. TLR9 was measured in the serum of patients with breast diseases as well as the control group using ELIZA. Results: The mean level of TLR9 in serum of patient was 339.4667 ng/l while control was 316.9929 ng/l with found significant differences where P_value was (0.05). while tissue of patient was 276.1064 ng/l. Concentrations of TLR9 were significantly higher in serum compared with tissues at (P≥0.05).The results found no significantly differences in concentrations of TLR9 among types of diseases in sera of patients. This study found concentration of TLR9 increased with primary breast diseases and it might be prognostic marker Conclusion : This study found concentration of TLR 9 increased with primary breast diseases and it might be prognostic marker

Methodological and Cellular Factors Affecting the Magnitude of Breast Cancer and Normal Cell Radiosensitization Using Gold Nanoparticles.

Breast cancer (BC) is the most common malignant tumor in women, which most often originates from the epithelial tissue of the breast gland. One of the most recommended kinds of treatment is radiotherapy (RT), but irradiation (IR) can affect not only the cancer tumor but also the healthy tissue around it. Au nanoparticles (AuNPs) were proposed as a radiosensitizing agent for RT which would allow for lower radiation doses, reducing the negative radiation effects on healthy tissues. The main objective of the study is to assess the dependence on the radiosensitivity of BC (MDA-MB-231) and normal mammary gland epithelial cells (MCF12A) to ionizing radiation, caused by functionalized AuNPs under diverse conditions. The viability, uptake, reactive oxygen species induction, and mitochondrial membrane potential in cells were analyzed applying a time and concentration-dependent manner. After different incubation times with AuNPs, cells were exposed to 2 Gy. The determination of radiation effect in combination with AuNPs was investigated using the clonogenic assay, p53, and γH2AX level, as well as, Annexin V staining. Our results highlighted the strong need for assessing the experimental conditions' optimization before the AuNPs will be implemented with IR. Moreover, results indicated that AuNPs did not act universally in cells. AuNPs could be a promising tool as a radiotherapy sensitizing agent, but it should be specified and deeply investigated under what conditions it will be applied taking into consideration not only AuNPs modifications but also the model and experimental conditions.

Glucose-sensitive acetylation of Seryl tRNA synthetase regulates lipid synthesis in breast cancer

Abnormally enhanced de novo lipid biosynthesis has been increasingly realized to play crucial roles in the initiation and progression of varieties of cancers including breast cancer. However, the mechanisms underlying the dysregulation of lipid biosynthesis in breast cancer remain largely unknown. Here, we reported that seryl tRNA synthetase (SerRS), a key enzyme for protein biosynthesis, could translocate into the nucleus in a glucose-dependent manner to suppress key genes involved in the de novo lipid biosynthesis. In normal mammary gland epithelial cells glucose can promote the nuclear translocation of SerRS by increasing the acetylation of SerRS at lysine 323. In SerRS knock-in mice bearing acetylation-defective lysine to arginine mutation, we observed increased body weight and adipose tissue mass. In breast cancer cells the acetylation and nuclear translocation of SerRS are greatly inhibited. Overexpression of SerRS, in particularly the acetylation-mimetic lysine to glutamine mutant, dramatically inhibits the de novo lipid synthesis and hence greatly suppresses the proliferation of breast cancer cells and the growth of breast cancer xenografts in mice. We further identified that HDAC4 and HDAC5 regulated the acetylation and nuclear translocation of SerRS. Thus, we identified a SerRS-meditated inhibitory pathway in glucose-induced lipid biosynthesis, which is dysregulated in breast cancer.

Long noncoding RNA Linc00339 promotes triple-negative breast cancer progression through miR-377-3p/HOXC6 signaling pathway.

Recently, long noncoding RNAs (lncRNAs) have become the key gene regulators and prognostic biomarkers in various cancers. Through microarray data, Linc00339 was identified as a candidate oncogenic lncRNA. We compared the expression levels of Linc00339 in several breast cancer cell lines and normal mammary gland epithelial cell line. The effects of Linc00339 on tumor progression were examined both in vitro and in vivo. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were applied to evaluate the functions of Linc00339, miR-377-3p, and HOXC6 on cell proliferation. Flow cytometry analysis was used to detect apoptosis and cell cycle distribution. Overall survival(OS) was analyzed using data from The Cancer Genome Atlas and molecular taxonomy of breast cancer international consortium (METABRIC). Dual luciferase assay and RNA immunoprecipitation were performed to confirm the interaction between Linc003339 and miR-377-3p. Linc00339 was increased in breast cancer cell lines compared with the normal epithelial cell. Through in vitro and in vivo experiments, Linc00339 overexpression promoted triple-negative breast cancer (TNBC) proliferation, inhibited cell cycle arrest, and suppressed apoptosis. Silencing of Linc00339 obtained the opposite effects. Mechanistic investigations demonstrated that Linc00339 could sponge miR-377-3p and regulate its expression. Higher expression of miR-377-3p indicated longer OS in breast cancer patients, especially in TNBC patients. Overexpression of miR-377-3p retarded TNBC cell growth through regulating cell cycle distribution and apoptosis. And miR-377-3p was involved in Linc00339-mediated TNBC proliferation through regulating HOXC6 expression. Knockdown of HOXC6 inhibited TNBC progression. In conclusion, our results illuminated that the novel Linc00339/miR-377-3p/HOXC6 axis played a critical role in TNBC progression and might be a promising therapeutic target for TNBC treatment.

Parathyroid hormone-related protein overexpression protects goat mammary gland epithelial cells from calcium-sensing receptor activation-induced apoptosis.

Normal mammary gland epithelial cells and breast cancer cells express the calcium-sensing receptor (CaSR), which is the master regulator of systemic calcium metabolism. During lactation, activation of the CaSR in mammary epithelial cells downregulates parathyroid hormone-related protein (PTHrP) levels in milk and in the circulation, and increases calcium transport into milk. However, very little information is available on the role of CaSR in goat mammary gland epithelial cells (GMECs) apoptosis. In this investigation, the full-length cDNA of CaSR from Xinong Saanen dairy goats was cloned, which contains an open-reading frame of 3,258 bp encoding 1,085 amino acids with a predicted molecular weight of 121.0 kDa and an isoelectric point of 5.65. The amino acid sequence is highly homologous with sheep, and the goat CaSR gene is mapped to chromosome 1. Quantitative real-time PCR suggested that CaSR was predominantly expressed in the heart, kidney and mammary gland. Then, we found the stimulation of CaSR with its activator gadolinium chloride (GdCl3) contributed to increase CaSR mRNA levels in GMECs and simultaneously promoted cell apoptosis, and these effects were abrogated partially by NPS2390 which is an inhibitor of CaSR. We also demonstrated that Ca(2+) increased CaSR mRNA levels and induced GMECs apoptosis and restrained cell proliferation. In contrast, PTHrP overexpression protected GMECs from calcium-induced apoptosis, and promoted cell proliferation. In conclusion, these results suggest that PTHrP overexpression protects GMECs from CaSR activation-induced apoptosis.

PTK6/BRK is expressed in the normal mammary gland and activated at the plasma membrane in breast tumors.

Protein Tyrosine kinase 6 (PTK6/BRK) is overexpressed in the majority of human breast tumors and breast tumor cell lines. It is also expressed in normal epithelial linings of the gastrointestinal tract, skin, and prostate. To date, expression of PTK6 has not been extensively examined in the normal human mammary gland. We detected PTK6 mRNA and protein expression in the immortalized normal MCF-10A human mammary gland epithelial cell line, and examined PTK6 expression and activation in a normal human breast tissue microarray, as well as in human breast tumors. Phosphorylation of tyrosine residue 342 in the PTK6 activation loop corresponds with its activation. Similar to findings in the prostate, we detect nuclear and cytoplasmic PTK6 in normal mammary gland epithelial cells, but no phosphorylation of tyrosine residue 342. However, in human breast tumors, striking PTK6 expression and phosphorylation of tyrosine 342 is observed at the plasma membrane. PTK6 is expressed in the normal human mammary gland, but does not appear to be active and may have kinase-independent functions that are distinct from its cancer promoting activities at the membrane. Understanding consequences of PTK6 activation at the plasma membrane may have implications for developing novel targeted therapies against this kinase.

Fibroblast Phenotype Transformation by Cocultured Cancer Epithelial Cells

Cancer-associated fibroblasts (CAFs) play a critical role in tumor progression and are associated with high-grade malignancy and hindrance of anti-cancer pharmacotherapy. While it is observed that CAFs exhibit myofibroblast-like phenotypes and are highly contractile, the process by which normal fibroblasts (NFs) are transformed into CAFs is yet to be elucidated. To gain insight about this process, we constructed a coculture system where GFP-labeled NIH3T3 fibroblasts were cocultured with cancer or normal mammary gland epithelial cells, respectively. The dynamic interactions between fibroblasts and epithelial cells were recorded by timelapse microscopy for 20 hours upon seeding and tracked using image correlation analysis. The results showed that fibroblasts cocultured with cancer epithelial cells exhibited different cell mobility profiles compared to normal controls. Furthermore, pattern recognition-based image analysis revealed that fibroblasts/cancer epithelial cell coculture produced lesion-like structures whereas the control coculture produced gland-like structures. Fibroblasts were then microdissected from the cocultures to examine the expression level of various CAF-related proteins in Western blot. The results showed that myosin II phosphorylation is significantly upregulated in fibroblasts cocultured with cancer epithelial cells, indicating NF-CAF transformation can occur within 20 hours after NFs are in contact with cancer cells.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Quantitative Proteomics Profiling of Murine Mammary Gland Cells Unravels Impact of Annexin-1 on DNA Damage Response, Cell Adhesion, and Migration

Annexin 1 (ANXA1), the first characterized member of the annexin superfamily, is known to bind or annex to cellular membranes in a calcium-dependent manner. Besides mediating inflammation, ANXA1 has also been reported to be involved in important physiopathological implications including cell proliferation, differentiation, apoptosis, cancer, and metastasis. However, with controversies in ANXA1 expression in breast carcinomas, its role in breast cancer initiation and progression remains unclear. To elucidate how ANXA1 plays a role in breast cancer initiation, we performed stable isotope labeling of amino acids in cell culture analysis on normal mammary gland epithelial cells from ANXA1-heterozygous (ANXA1(+/-)) and ANXA1-null (ANXA1(-/-)) mice. Among over 4000 quantified proteins, we observed 214 up-regulated and 169 down-regulated with ANXA1(-/-). Bioinformatics analysis of the down-regulated proteins revealed that ANXA1 is potentially implicated in DNA damage response, whereas the analysis of up-regulated proteins showed the possible roles of ANXA1 in cell adhesion and migration pathways. These observations were supported by relevant functional assays. The assays for DNA damage response demonstrated an accumulation of more DNA damage with slower recovery on heat stress and an impaired oxidative damage response in ANXA1(-/-) cells in comparison with ANXA1(+/-) cells. Overexpressing Yes-associated protein 1 or Yap1, the most down-regulated protein in DNA damage response pathway cluster, rescued the proliferative response in ANXA1(-/-) cells exposed to oxidative damage. Both migration and wound healing assays showed that ANXA1(+/-) cells possess higher motility with better wound closure capability than ANXA1(-/-) cells. Knocking down of β-parvin, the protein with the highest fold change in the cell adhesion protein cluster, indicated an increased cell migration in ANXA1(-/-) cells. Altogether our quantitative proteomics study on ANXA1 suggests that ANXA1 plays a protective role in DNA damage and modulates cell adhesion and motility, indicating its potential role in cancer initiation as well as progression in breast carcinoma.

Abstract 985: Dissecting PI3K-AKT pathway by phosphoproteomic profiling of PIK3CA knock-in mutants

Abstract The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. PIK3CA mutations are observed in about 30% of breast cancers. Three recurrent oncogenic “hotspot” mutations comprise the majority of somatic PIK3CA mutations. Two of these mutations, E542K and E545K, occur in the helical domain found in exon 9, and the third mutation, H1047R, affects the kinase domain located within exon 20. Although many studies have implicated PIK3CA mutations with features of transformation, definitive mechanisms describing how these mutations promote cell growth and proliferation have not been fully elucidated. Two MCF10A (a normal mammary gland epithelial cell line) knock-in cell lines with E545K or H1047R hotspot mutations were used for our phosphoproteome study. We employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry to study phosphoproteomic alterations caused by PIK3CA onogenic mutations. In order to identify signaling proteins directly involved in PI3K-AKT pathway, a novel PIK3CA specific inhibitor (J124) was used to suppress PIK3CA activity. Phosphopeptides were enriched by TiO2-based strategy after strong cation exchange (SCX) fractionation and analyzed on a high resolution Fourier transform LTQ-Orbitrap Velos mass spectrometer. We identified 5,491 unique phosphopeptides from 1,546 unique proteins - of these, 2,239 peptides were hyperphosphorylated in MCF10A cell with PIK3CA mutants. Notably, the PIK3CA inhibitor, J124, could specifically suppress the phosphorylation levels of 1,187 hyperphosphorylated peptides. These are likely to be effectors of PI3Kalthough only 34 of them were previously known AKT1 substrates. By integrating data from protein microarray-based phosphorylation experiments with AKT1, we found 16 novel hyperphosphorylated proteins that were phosphorylated in vitro by AKT1. In addition to identifying novel AKT substrates, we also identified multiple receptor tyrosine kinases including EGFR, FGFR and EPHA2 to be hyperphosphorylated in PIK3CA mutant knockin cells. This novel discovery of the reverse crosstalk initiated from PI3K-AKT to RTKs could explain oncogenic consequences of cells with PIK3CA mutants such as growth factor-independent growth and invasive ability. In summary, our comprehensive phosphoproteomic study reveals that oncogenic activation of the PI3K-AKT pathway can activate a broad spectrum of signaling molecules. By integrating data from large-scale protein microarray studies, we were able to identify novel AKT substrates. The knowledge gained from these studiescan greatly enhance our understanding of the PI3K-AKT pathway and provide potential therapeutic targets for attenuating oncogenic effects of PIK3CA mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 985. doi:1538-7445.AM2012-985

Tumor-secreted PGE2 Inhibits CCL5 Production in Activated Macrophages through cAMP/PKA Signaling Pathway

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Efficacy of oncolytic reovirus against human breast cancer cells

Human reovirus can replicate and induce tumor cell lysis in several cancer types with an activated Ras signaling pathway. The aim of this study was to investigate the oncolytic effect of reovirus against breast cancer cells, and clarify the relationship between the susceptibility to reovirus and HER2 expression, which is associated with the Ras signaling pathway. Reovirus (serotype 3), 6 human breast cancer cell lines and normal mammary gland epithelial cell line were used in this study. The mRNA expression of HER2 receptor was examined by RT-PCR, and the protein of HER2 and activated Ras protein were examined by a Western blot analysis. In vitro, the cytopathic effects, viral protein synthesis, and cell viability on infection by reovirus were examined. Reovirus can infect all the 6 examined cancer cell lines, but the control cell line was not susceptible to the reovirus infection. The cytopathic effect appeared from day 1 after infection, and a 50% or greater cytotolysis was demonstrated at day 7 after infection. The Ras activities in all examined cell lines were higher than those in the control cell line. No relationship was observed in the susceptibility to the reovirus and HER2 expression. The increased Ras activity itself, regardless of the HER2 expression, may therefore play an important role in the susceptibility to reovirus of breast cancer cells. As a result, breast cancer may thus become a candidate target for oncolytic reovirus therapy.

Understanding microchannel culture: parameters involved in soluble factor signaling

While the importance of autocrine-paracrine signaling in vivo is clear, the ability to study the effects of secreted endogenous factors in vitro is hampered by canonical culture platforms. In multi-well plates, the large air-liquid interface gives rise to convective flows that continually mix the fluid disrupting the local diffusion-based accumulation. Simple microchannels provide a more controlled microenvironment that can be used to study secreted factor effects. Here, we utilize microchannel culture to examine basic culture parameters and their interactions using normal mammary gland epithelial cells (NMuMG). The following parameters were studied: (1) cell density (80 vs. 240 cells mm(-2)), (2) exogenous growth factors (epidermal growth factor [EGF] vs. fetal bovine serum), (3) medium change frequency (1 h, 4 h, 12 h), and (4) culture platform (microchannels vs. 96-well plates). The cells exhibited increased growth rates in microchannels as compared to 96-well plates. Cell proliferation increased as the frequency of media change decreased. For the microchannel geometries used, important threshold concentrations were reached in a few hours. In aggregate, the results indicate that the function of the four factors and their interactions on NMuMG growth are spatially and temporally related by molecular diffusion in the controlled microchannel space. The convective-free microchannel environment may prove useful for studying soluble factor signaling in vitro, and to test models and predictions of autocrine-paracrine signaling.

Synthetic analogues of migrastatin that inhibit mammary tumor metastasis in mice

Tumor metastasis is the most common cause of death in cancer patients. Here, we show that two, fully synthetic migrastatin analogues, core macroketone and core macrolactam, are potent inhibitors of metastasis in a murine breast tumor model. Administration of these readily accessible compounds nearly completely inhibits lung metastasis of highly metastatic mammary carcinoma cells. Treatment of tumor cells with core macroketone and core macrolactam blocks Rac activation, lamellipodia formation, and cell migration, suggesting that these chemical compounds interfere with the invasion step of the metastatic process. These compounds also inhibit the migration of human metastatic breast cancer cells, prostate cancer cells, and colon cancer cells but not normal mammary-gland epithelial cells, fibroblasts, and leukocytes. These data demonstrate that the macroketone and macrolactam core structures are specific small-molecule inhibitors of tumor metastasis. These compounds or their analogues could potentially be used in cancer-therapy strategies.