Project description:Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions, including actin cytoskeleton remodeling, cell migration, gene transcription, and cell proliferation. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably used immortal or tumorigenic cell lines to study Rho GTPase functions and there are no studies on the potential of Rho small GTPase to overcome senescence checkpoints and induce preneoplastic transformation of human mammary epithelial cells (hMEC). We show here that ectopic expression of wild-type (WT) RhoA as well as a constitutively active RhoA mutant (G14V) in two independent primary hMEC strains led to their immortalization and preneoplastic transformation. These cells have continued to grow over 300 population doublings (PD) with no signs of senescence, whereas cells expressing the vector or dominant-negative RhoA mutant (T19N) senesced after 20 PDs. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well-known Rho effectors including Rho kinase, PKN, mDia1, and mDia2, was also capable of immortalizing hMECs. Notably, similar to parental normal cells, Rho-immortalized cells have WT p53 and intact G(1) cell cycle arrest on Adriamycin treatment. Rho-immortalized cells were anchorage dependent and were unable to form tumors when implanted in nude mice. Lastly, microarray expression profiling of Rho-immortalized versus parental cells showed altered expression of several genes previously implicated in immortalization and breast cancer progression. Taken together, these results show that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked to cellular transformation and breast cancer.
Project description:Cyclin E, a key cell cycle regulatory protein, has been linked to oncogenesis when dysregulated. We have previously shown that overexpression of cyclin E causes replication stress, leading to failure to complete replication at specific chromosomal loci during S phase of the cell cycle. This in turn promotes chromosomal damage during anaphase. Here we show that non-transformed human mammary epithelial cell clones that survive such aberrant mitoses have a specific and reproducible pattern of chromosomal Copy Number Alterations (CNAs) that we have characterized and termed the cyclin E CNA signature. Using a number of computational approaches, we show that this signature resembles one specific CNA pattern enriched in differentiated epithelial-like tumors of the breast and ovary. Analysis of the CNA profile of these clones provides a potential mechanism for cyclin E-mediated oncogenesis.
Project description:The function of long non-coding RNA H19 (H19) on cell proliferation has been observed in various cell types, and the increased expression of H19 was also found in the lipopolysaccharide (LPS)-induced inflammatory bovine mammary epithelial cells (MAC-T). However, the roles of H19 in the inflammatory response and physiological functions of bovine mammary epithelial cell are not clear. In the present study, we found that overexpression of H19 in MAC-T cells significantly promoted cell proliferation, increased the protein and mRNA level of β-casein, and enhanced the expression of tight junction (TJ)-related proteins while inhibited staphylococcus aureus adhesion to cells. In addition, results demonstrated that overexpression of H19 affected the LPS-induced immune response of MAC-T cells by promoting expressions of inflammatory factors, including TNF-α, IL-6, CXCL2 and CCL5, and activating the NF-κB signal pathway. Our findings indicate that H19 is likely to play an important role in maintaining normal functions and regulating immune response of bovine mammary epithelial cells.
Project description:Members of the phosphatidylinositol 3-kinase-related kinase family, in particular the ataxia-telangiectasia mutated (ATM) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), regulate cellular responses to DNA double-strand breaks. Increased sensitivity to ionizing radiation (IR) in DNA-PKcs- or ATM-deficient cells emphasizes their important roles in maintaining genome stability. Furthermore, combined knockout of both kinases is synthetically lethal, suggesting functional complementarity. In the current study, using human mammary epithelial cells with ATM levels stably knocked down by >90%, we observed an IR-induced G(2) checkpoint that was only slightly attenuated. In marked contrast, this G(2) checkpoint was significantly attenuated with either DNA-PK inhibitor treatment or RNA interference knockdown of DNA-PKcs, the catalytic subunit of DNA-PK, indicating that DNA-PK contributes to the G(2) checkpoint in these cells. Furthermore, in agreement with the checkpoint attenuation, DNA-PK inhibition in ATM-knockdown cells resulted in reduced signaling of the checkpoint kinase CHK1 as evidenced by reduced CHK1 phosphorylation. Taken together, these results show a DNA-PK-dependent component to the IR-induced G(2) checkpoint, in addition to the well-defined ATM-dependent component. This may have important implications for chemotherapeutic strategies for breast cancers.
Project description:Molecular-level analyses of breast carcinogenesis benefit from vivo disease models. Estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) overexpression targeted to mammary epithelial cells in genetically engineered mouse models induces largely similar rates of proliferative mammary disease in prereproductive senescent mice. Herein, with natural reproductive senescence, Esr1 overexpression compared with CYP19A1 overexpression resulted in significantly higher rates of preneoplasia and cancer. Before reproductive senescence, Esr1, but not CYP19A1, overexpressing mice are tamoxifen resistant. However, during reproductive senescence, Esr1 mice exhibited responsiveness. Both Esr1 and CYP19A1 are responsive to letrozole before and after reproductive senescence. Gene Set Enrichment Analyses of RNA-sequencing data sets showed that higher disease rates in Esr1 mice were accompanied by significantly higher expression of cell proliferation genes, including members of prognostic platforms for women with early-stage hormone receptor-positive disease. Tamoxifen and letrozole exposure induced down-regulation of these genes and resolved differences between the two models. Both Esr1 and CYP19A1 overexpression induced abnormal developmental patterns of pregnancy-like gene expression. This resolved with progression through reproductive senescence in CYP19A1 mice, but was more persistent in Esr1 mice, resolving only with tamoxifen and letrozole exposure. In summary, genetically engineered mouse models of Esr1 and CYP19A1 overexpression revealed a diversion of disease processes resulting from the two distinct molecular pathophysiological mammary gland-targeted intrusions into estrogen signaling during reproductive senescence.
Project description:Vaccinia-related kinase 1 (VRK1) is a pro-proliferative nuclear kinase. Mice engrafted with VRK1-depleted MDA-MB-231 breast cancer cells have been shown to develop fewer distal metastases than controls, suggesting VRK1 might play a role in cell migration, invasion, and/or colonization. In work described herein, we investigated the impact of VRK1 overexpression on human mammary epithelial cells. In 2D culture, VRK1 overexpression diminishes cell migration and invasion and impairs the migration-associated processes of cell spreading and cytoskeletal rearrangement. VRK1-overexpressing cells show reduced accumulation of the mesenchymal marker vimentin and increased accumulation of the epithelial markers E-cadherin and claudin-1. VRK1 overexpression also leads to reduced levels of the transcriptional repressors snail, slug, and twist1. Cumulatively, these data indicate that VRK1 overexpression augments the epithelial properties of both MCF10a and MDA-MB-231 cells. We further studied the impact of VRK1 on the epithelial properties of MCF10a cells in 3D matrigel culture, in which cells proliferate and form epithelial sheets that mature into hollow spherical acini. VRK1 overexpression significantly accelerates the initial stages of cell proliferation, leading to larger acini that nevertheless differentiate and mature. Our analysis of human tumor tissue microarrays (TMAs) revealed that VRK1 protein levels are higher in lymph node metastases than in patient-matched mammary tumors. Using public databases, we determined that VRK1 is among the top 10% of overexpressed transcripts in multiple subtypes of invasive breast cancer, and that high levels of VRK1 expression are correlated with decreased relapse-free survival. In sum, overexpression of VRK1, by regulating the transcription repressors snail, slug, and twist1, can promote a mesenchymal-to-epithelial transition (MET) in cell culture. VRK1-mediated MET might facilitate the colonization of distal sites by metastatic breast cancer cells, providing some insight into the frequent association of VRK1 overexpression with breast malignancies and the correlation between VRK1 overexpression and poor clinical outcome.
Project description:Overexpression of the ERBB2 receptor in transfectants of a human mammary epithelial cell line (MTSV1-7) is associated with a reduced ability to undergo morphogenesis in vitro and with a decreased level of expression of the E-cadherin and alpha 2 integrin genes. The inhibition of expression of the adhesion molecules has been shown to be at the level of transcription by using nuclear run-on assays and by following transcription of a reporter gene fused to 5' sequences of the E-cadherin gene. To relate the effects on gene transcription to a functional ERBB2 protein, signaling from the receptor was inhibited by the antibody 4D5, which blocks phosphorylation of ERBB2 on tyrosine residues and association of the protein with the GRB2/Sem5 protein. After treatment with the antibody 4D5, the ERBB2 transfectants regain the ability to form three-dimensional structures in collagen gels and the rates of transcription of the genes encoding the E-cadherin and the alpha 2 integrin subunit are restored to the levels seen in MTSV1-7neo cells. These results demonstrate that the inhibition of morphogenesis and transcription of specific adhesion molecules in human mammary epithelial cells can be affected by signals generated by the ERBB2 receptor and suggest a role for ERBB2 overexpression in tumor progression and metastasis.
Project description:Most breast cancers arise from luminal epithelial cells, and 25-30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1β treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis.