Wnt Pathway Activity in Breast Cancer Sub-Types and Stem-Like Cells

Introduction

Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear.

Methods

We used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro.

Results

Wnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres.

Conclusions

Collectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers.     

Intracellular Mechanics and Activity of Breast Cancer Cells Correlate with Metastatic Potential

Mechanics of cancer cells are directly linked to their metastatic potential, or ability to produce a secondary tumor at a distant site. Metastatic cells survive in the circulatory system in a non-adherent state, and can squeeze through barriers in the body. Such considerable structural changes in cells rely on rapid remodeling of internal structure and mechanics. While external mechanical measurements have demonstrated enhanced pliability of cancer cells with increased metastatic potential, little is known about dynamics of their interior and we expect that to change significantly in metastatic cells. We perform a comparative study, using particle-tracking to evaluate the intracellular mechanics of living epithelial breast cells with varying invasiveness. Particles in all examined cell lines exhibit super-diffusion with a scaling exponent of 1.4 at short lag times, likely related to active transport by fluctuating microtubules and their associated molecular motors. Specifics of probe-particle transport differ between the cell types, depending on the cytoskeleton network-structure and interactions with it. Our study shows that the internal microenvironment of the highly metastatic cells evaluated here is more pliable and their cytoskeleton is less dense than the poorly metastatic and benign cells. We thus reveal intracellular structure and mechanics that can support the unique function and invasive capabilities of highly metastatic cells.     

A New Signaling Pathway (JAK-Fes-phospholipase D) That Is Enhanced in Highly Proliferative Breast Cancer Cells

The products of the oncogene Fes and JAK3 are tyrosine kinases, whose expressions are elevated in tumor growth, angiogenesis, and metastasis. Phosphatidic acid, as synthesized by phospholipase D (PLD), enhances cancer cell survival. We report a new signaling pathway that integrates the two kinases with the lipase. A new JAK3-Fes-PLD2 axis is responsible for the highly proliferative phenotype of MDA-MB-231 breast cancer cells. Conversely, this pathway is maintained at a low rate of expression and activity levels in untransformed cells such as MCF10A. We also deciphered the inter-regulation that exists between the two kinases (JAK3 and the oncogene Fes) and between these two kinases and the lipase (PLD2). Whereas JAK3 and Fes marginally activate PLD2 in non-transformed cells, these kinases greatly enhance (>200%) PLD activity following protein-protein interaction through the SH2 domain and the Tyr-415 residue of PLD2. We also found that phosphatidic acid enhances Fes activity in MDA-MB-231 cells providing a positive activation loop between Fes and PLD2. In summary, the JAK3, Fes and PLD2 interactions in transformed cells maintain PLD2 at an enhanced level that leads to abnormal cell growth. Modulating this new JAK3-Fes-PLD2 pathway could be important to control the highly invasive phenotype of breast cancer cells.     

Comparative Evaluation of Nimesulide-Loaded Nanoparticles for Anticancer Activity Against Breast Cancer Cells

Recent clinical and epidemiological researches have declared that non-steroidal anti-inflammatory agents may display as antineoplastic agents and indicate pro-apoptotic and antiproliferative effects on cancer cells. The major purpose of this research was to develop a novel poly(ethyleneglycol)-block-poly(ε-caprolactone) (PEG-b-PCL) nano-sized particles encapsulated with nimesulide (NMS), a selective COX-2 inhibitor, and to evaluate its anticancer activity against MCF-7 breast cancer cells. NMS-encapsulated PEG-b-PCL nanoparticles were fabricated using three different production techniques: (i) by emulsion-solvent evaporation using a high shear homogenizer, (ii) by emulsion-solvent evaporation using an ultrasonicator, and (iii) by nanoprecipitation. Nanoparticles were evaluated with respect to the entrapment efficiency, size characteristics, drug release rates, thermal behavior, cell viability assays, and apoptosis. The resulting nanoparticles were found to be spherical shapes with negative surface charges. The average diameter of all nanoparticles ranged between 148.5 and 307.2 nm. In vitro release profiles showed that all nanoparticles exhibited a biphasic release pattern. NMS-loaded PEG-b-PCL nanoparticles demonstrated significant anticancer activity against MCF-7 breast cancer cells in a dose-dependent manner, and the effects of nanoparticles on cell proliferation were significantly affected by the preparation techniques. The nanoparticles developed in this work displayed higher potential for the NMS delivery against breast cancer treatment for the future.     

Global Analysis of Transcriptional Regulation by Poly(ADP-ribose) Polymerase-1 and Poly(ADP-ribose) Glycohydrolase in MCF-7 Human Breast Cancer Cells

Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs to stably knock down PARP-1 or PARG in MCF-7 cells followed by expression microarray analyses. Correlation analyses showed that the majority of genes affected by the knockdown of one factor were similarly affected by the knockdown of the other factor. The most robustly regulated common genes were enriched for stress-response and metabolic functions. In chromatin immunoprecipitation assays, PARP-1 and PARG localized to the promoters of positively and negatively regulated target genes. The levels of chromatin-bound PARG at a given promoter generally correlated with the levels of PARP-1 across the subset of promoters tested. For about half of the genes tested, the binding of PARP-1 at the promoter was dependent on the binding of PARG. Experiments using stable re-expression of short hairpin RNA-resistant catalytic mutants showed that PARP-1 and PARG enzymatic activities are required for some, but not all, target genes. Collectively, our results indicate that PARP-1 and PARG, nuclear enzymes with opposing enzymatic activities, localize to target promoters and act in a similar, rather than antagonistic, manner to regulate gene expression.     

齐墩果醇-龙胆三糖苷雌激素样作用及其对肝癌HepG2细胞增殖的影响

肝癌目前已经成为世界第三大癌症,临床数据显示雌激素及其受体与肝癌关系密切.苯并呋喃类化合物有报道具有雌激素样作用.所以,作为苯并呋喃的一种,研究齐墩果醇-龙胆三糖苷雌激素样作用及其对HepG2细胞增殖的影响显得尤为重要.在瞬时转染有ERE(雌激素作用元件)报告基因的HepG2中,齐墩果醇-龙胆三糖苷显示了同17β-雌二醇一样激活ERE报告基因的作用;而在瞬时转染有CRE(cAMP 作用元件)报告基因的HepG2中,齐墩果醇-龙胆三糖苷也显示了升高cAMP浓度的作用,进一步提示齐墩果醇-龙胆三糖苷的类雌激素样作用.齐墩果醇-龙胆三糖苷对HepG2细胞增殖实验显示,30 μmol/L浓度时该化合物的雌激素样作用同17β-雌二醇一样有相似的促HepG2细胞增殖作用.     

Misbehaviour of XIST RNA in Breast Cancer Cells

A role of X chromosome inactivation process in the development of breast cancer have been suggested. In particular, the relationship between the breast cancer predisposing gene BRCA1 and XIST, the main mediator of X chromosome inactivation, has been intensely investigated, but still remains controversial. We investigated this topic by assessing XIST behaviour in different groups of breast carcinomas and in a panel of breast cancer cell lines both BRCA1 mutant and wild type. In addition, we evaluated the occurrence of broader defects of heterochromatin in relation to BRCA1 status in breast cancer cells. We provide evidence that in breast cancer cells BRCA1 is involved in XIST regulation on the active X chromosome, but not in its localization as previously suggested, and that XIST can be unusually expressed by an active X and can decorate it. This indicates that the detection of XIST cloud in cancer cell is not synonymous of the presence of an inactive X chromosome. Moreover, we show that global heterochromatin defects observed in breast tumor cells are independent of BRCA1 status. Our observations sheds light on a possible previously uncharacterized mechanism of breast carcinogenesis mediated by XIST misbehaviour, particularly in BRCA1-related cancers. Moreover, the significant higher levels of XIST-RNA detected in BRCA1-associated respect to sporadic basal-like cancers, opens the possibility to use XIST expression as a marker to discriminate between the two groups of tumors.     

SAICAR Induces Protein Kinase Activity of PKM2 that Is Necessary for Sustained Proliferative Signaling of Cancer Cells

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Lung Epithelial Cells Induce Both Phenotype Alteration and Senescence in Breast Cancer Cells

Purpose

The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells.

Methods

Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype.

Results

Co-culture of the breast cancer cells with the normal lung cells had different effects on the epithelial and mesenchymal carcinoma cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slightly more mesenchymal-spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin expressed in MDA-MB-231 using shRNA prevented this phenotypic reversion in co-culture. Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture.

Conclusions

Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells coincident with changing their phenotypes.     

Apoptosis and Autophagy in Breast Cancer Cells following Exemestane Treatment

Aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are used for hormone-dependent breast cancer treatment. Exemestane, a steroidal that belongs to the third-generation of AIs, is a mechanism-based inhibitor that binds covalently and irreversibly, inactivating and destabilizing aromatase. Since the biological effects of exemestane in breast cancer cells are not totally understood, its effects on cell viability, cell proliferation and mechanisms of cell death were studied in an ER-positive aromatase-overexpressing breast cancer cell line (MCF-7aro). The effects of 3-methyladenine (3-MA), an inhibitor of autophagy and of ZVAD-FMK, an apoptotic inhibitor, in exemestane treated cells were also investigated. Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G(0)/G1 or in G(2)/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis.     

乳腺癌患者循环肿瘤细胞与癌干细胞相关性的初步研究

目的：检测乳腺癌患者外周血单核细胞（PBMC）中循环肿瘤细胞（CTC）和具有癌干细胞（CSC）标志的CTC（CSC-CTC）,探讨患者外周血微转移与CSC的相关性。方法：患者和健康者PBMC与磁珠偶联上皮细胞黏附分子单抗孵育后,用磁性分离法富集PBMC中的上皮细胞。以CK＋为患者PBMC中CTC标志,用流式细胞术（FCM）检测健康者和患者的PBMC中CK＋细胞及CK＋/CD44＋/CD24-细胞含量,并比较各组间CTC、CSC-CTC含量的差异。结果：用FCM在73.07%的患者中检测到CTC,在19例检测到CTC的患者中18例有CSC-CTC（94.74%）,CTC中CSC数量比例平均为19.01%,且患者PBMC中CTC和CSC-CTC比例与临床TNM分期相关。结论：初步建立了患者外周血CSC-CTC的检测方法,结果显示乳腺癌患者外周血微转移中有CSC-CTC的参与,临床分期越晚的患者CTC和CSC-CTC的数量越多。