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CRISPR deletion of MIEN1 in breast cancer cells.

ABSTRACT: Migration and Invasion Enhancer (MIEN1) is an oncogene which is involved in facilitating motility of cancer cells through actin dynamics and gene expression. Increased MIEN1 expression in many types of tumors leads to disease progression and metastatic propensity. It is unclear precisely how MIEN1 is involved in this process and more studies are required to tease out the mechanisms. Here we show that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genome editing effectively produced specific genomic deletions in the MIEN1 gene which led to the abrogation of its expression in breast cancer cells. The single guide RNAs (sgRNAs) mediated targeting of MIEN1 was specific and none of the clones screened for off-target cleavage revealed any insertions or deletions (indels). Additionally, disruption of the MIEN1 gene did not alter the cell morphology, growth, proliferation or survival. Knocking out MIEN1 in these breast cancer cells will allow future studies to determine the exact role MIEN1 plays in breast tumor metastasis, which might lead to production of novel therapeutics to treat this and other cancers.

SUBMITTER: Van Treuren T

PROVIDER: S-EPMC6171864 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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CRISPR deletion of MIEN1 in breast cancer cells.

Van Treuren Timothy T Vishwanatha Jamboor K JK

PloS one 20181004 10

Migration and Invasion Enhancer (MIEN1) is an oncogene which is involved in facilitating motility of cancer cells through actin dynamics and gene expression. Increased MIEN1 expression in many types of tumors leads to disease progression and metastatic propensity. It is unclear precisely how MIEN1 is involved in this process and more studies are required to tease out the mechanisms. Here we show that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genome editing effectively pro ...[more]

PMID: 30286132

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Project description:Background Breast cancer is the leading cause of cancer related deaths in women. Triple negative breast cancers are highly aggressive, have an overall poor prognosis and has limited therapeutic options. Previously, we have identified an environmental induced gene mdig that predicted the overall survival in breast cancer patients and regulated breast cancer cell growth, motility and invasion partially through DNA and histone methylation. Mdig’s role in breast cancer growth and motility has been revealed however a comprehensive analysis of the proteomic profile of mdig affected cells in triple negative breast cancer has not been attempted yet. Methods We applied mass spectrometry technology featuring global proteomics and post translational modifications in triple negative breast cancer cells MDA-MB-231 that were deleted for mdig via CRISPR Cas 9 gene editing. Using label-free bottom up quantitative proteomics, we compared the proteomes of wildtype control (WT) and mdig knockout (KO) MDA-MB-231 cells and identified the proteins and pathways that are significantly altered with mdig deletion. Ingenuity Pathway Analysis (IPA) platform was further used to explore the signaling pathway networks with differentially expressed proteins and identified top canonical pathways, upstream regulators, and causal disease networks. Results 904 differentially expressed (p < 0.005) proteins were identified in MDA-MB-231 cells knocked out for mdig. Post-translational modification (PTM) analysis indicated that mdig loss reduced the abundance of oxidized histidine 39 of 60S ribosomal protein L27a (q = 0.00030) whereas global abundance of di-methylated lysine containing peptides was increased (p = 0.02). Relevance Our data provides the first insight towards the protein expression pattern in breast cancer associated with a complete disruption of the mdig gene. Differentially expressed proteins between WT and KO MDA-MB-231 triple negative breast cancer cells provided substantial information regarding the key proteins, biological process and pathways that are specifically upregulated in breast cancer tumorigenicity and invasiveness. Mdig affected signaling pathways and hub molecules will benefit for the development of novel treatment strategies for the development of breast cancer therapies.

Project description:BackgroundThe tumor microenvironment within the breast is rich in adipose elements. The interaction between adipose cells and breast cancer is poorly understood, particularly as it pertains to patients with genetic susceptibility to breast cancer. This study focuses on the phenotype of human adipose-derived stem cells with the BRCA1 mutation and the effect they may have on breast cancer cell behavior.MethodsCRISPR/Cas9 was used to generate de novo BRCA1-knockdown human adipose-derived stem cells. The effect of the BRCA1 knockdown on the adipose-derived stem cell phenotype was compared to wild-type adipose-derived stem cells and patient-derived breast adipose-derived stem cells with known BRCA1 mutations. Interactions between adipose-derived stem cells and the MDA-MB-231 breast cancer cell line were evaluated.ResultsBRCA1-knockdown adipose-derived stem cells stimulated MDA-MB-231 proliferation (1.4-fold increase on day 4; p = 0.0074) and invasion (2.3-fold increase on day 2; p = 0.0171) compared to wild-type cells. Immunofluorescence staining revealed higher levels of phosphorylated ataxia telangiectasia-mutated activation in BRCA1-knockdown cells (72.9 ± 5.32 percent versus 42.9 ± 4.97 percent; p = 0.0147), indicating higher levels of DNA damage. Beta-galactosidase staining demonstrated a significantly higher level of senescence in BRCA1-knockdown cells compared with wild-type cells (7.9 ± 0.25 percent versus 0.17 ± 0.17 percent; p < 0.0001). Using quantitative enzyme-linked immunosorbent assay to evaluate conditioned media, the authors found significantly higher levels of interleukin-8 in BRCA1-knockdown cells (2.57 ± 0.32-fold; p = 0.0049).ConclusionsThe authors show for the first time that the BRCA1 mutation affects the adipose-derived stem cell phenotype. Moreover, CRISPR/Cas9-generated BRCA1-knockdown adipose-derived stem cells stimulate a more aggressive behavior in breast cancer cells than wild-type adipose-derived stem cells. This appears to be related to increased inflammatory cytokine production by means of a DNA damage-mediated cell senescence pathway.

Project description:BackgroundMicroRNAs (miRNAs) are crucial molecules that regulate gene expression and hence pathways that are key to prostate cancer progression. These non-coding RNAs are highly deregulated in prostate cancer thus facilitating progression of the disease. Among the many genes that have gained importance in this disease, Migration and invasion enhancer 1 (MIEN1), a novel gene located next to HER2/neu in the 17q12 amplicon of the human chromosome, has been shown to enhance prostate cancer cell migration and invasion, two key processes in cancer progression. MIEN1 is differentially expressed between normal and cancer cells and tissues. Understanding the regulation of MIEN1 by microRNA may enable development of better targeting strategies.MethodsThe miRNAs that could target MIEN1 were predicted by in silico algorithms and microarray analysis. The validation for miRNA expression was performed by qPCR and northern blotting in cells and by in situ hybridization in tissues. MIEN1 and levels of other molecules upon miRNA regulation was determined by Western blotting, qPCR, and immunofluorescence. The functional effects of miRNA on cells were determined by wound healing cell migration, Boyden chamber cell invasion, clonal and colony formation assays. For knockdown or overexpression of the miRNA or overexpression of MIEN1 3'UTR, cells were transfected with the oligomiRs and plasmids, respectively.ResultsA novel miRNA, hsa-miR-940 (miR-940), identified and validated to be highly expressed in immortalized normal cells compared to cancer cells, is a regulator of MIEN1. Analysis of human prostate tumors and their matched normal tissues confirmed that miR-940 is highly expressed in the normal tissues compared to its low to negligible expression in the tumors. While MIEN1 is a direct target of miR-940, miR-940 alters MIEN1 RNA, in a quantity as well as cell dependent context, along with altering its downstream effectors. The miR-940 inhibited migratory and invasive potential of cells, attenuated their anchorage-independent growth ability, and increased E-cadherin expression, implicating its role in mesenchymal-to-epithelial transition (MET).ConclusionsThese results, for the first time, implicate miR-940, a regulator of MIEN1, as a promising novel diagnostic and prognostic tool for prostate cancer.

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CRISPR deletion of MIEN1 in breast cancer cells.

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CRISPR deletion of MIEN1 in breast cancer cells.

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