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Impact of CXCR4 and CXCR7 knockout by CRISPR/Cas9 on the function of triple-negative breast cancer cells.

ABSTRACT: Background:Breast cancer is one of the most common malignancies threatening women's health. Triple-negative breast cancer (TNBC) is a special type of breast cancer with high invasion and metastasis. CXCL12 and its receptors CXCR4 and CXCR7 play a crucial role in the progress of breast cancer. The aim of this study was to investigate the effect of CXCR4 and CXCR7 on the function of TNBC. Materials and methods:We used the CRISPR/Cas9 technique to carry out a single knockout of the CXCR4 or CXCR7 gene and co-knockout of CXCR4 and CXCR7 genes in the TNBC cell line (MDA-MB-231). The single knockout and co-knockout cells were screened and verified by PCR sequencing and Western blot assay, the effect of single knockout and co-knockout on the proliferation of TNBC cells was examined using the Cell Counting Kit-8 and colony formation assays, the migration and invasion of TNBC cells were examined by the transwell and wound-healing assays, the changes in the cell cycle distribution after knockout were detected by flow cytometry, and the difference in the migration and invasion of single knockout and co-knockout induced by CXCL12 was observed by adding CXCL12 in the experimental group. Results:The single knockout of the CXCR4 or CXCR7 gene significantly reduced the cell proliferation, growth, migration, and invasion and delayed the conversion of the G1/S cycle, while the co-knockout inhibited these biological abilities more significantly. In both the knockout and control groups, the migration and invasion of CXCL12-added cells were significantly stronger than those of the non-CXCL12-added cells, and CXCL12 induced lesser migration and invasion in the CXCR4 and CXCR7 co-knockout group than in the single knockout groups. Conclusion:The knockout of the CXCR4 and CXCR7 genes affects the binding capacity and functions of CXCL12, inhibits the malignant progression of TNBC cells significantly, and may become a potential target for the treatment of TNBC.

SUBMITTER: Yang M

PROVIDER: S-EPMC6527053 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Impact of CXCR4 and CXCR7 knockout by CRISPR/Cas9 on the function of triple-negative breast cancer cells.

Yang Meng M Zeng Chen C Li Peiting P Qian Liyuan L Ding Boni B Huang Lihua L Li Gang G Jiang Han H Gong Ni N Wu Wei W

OncoTargets and therapy 20190517

<h4>Background</h4>Breast cancer is one of the most common malignancies threatening women's health. Triple-negative breast cancer (TNBC) is a special type of breast cancer with high invasion and metastasis. CXCL12 and its receptors CXCR4 and CXCR7 play a crucial role in the progress of breast cancer. The aim of this study was to investigate the effect of CXCR4 and CXCR7 on the function of TNBC.<h4>Materials and methods</h4>We used the CRISPR/Cas9 technique to carry out a single knockout of the C ...[more]

PMID: 31190884

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Project description:BackgroundTriple negative breast cancer (TNBC) accounts for 16% of breast cancers and represents an aggressive subtype that lacks targeted therapeutic options. In this study, mass spectrometry (MS)-based tyrosine phosphorylation profiling identified aberrant FGFR3 activation in a subset of TNBC cell lines. This kinase was therefore evaluated as a potential therapeutic target.MethodsMS-based tyrosine phosphorylation profiling was undertaken across a panel of 24 TNBC cell lines. Immunoprecipitation and Western blot were used to further characterize FGFR3 phosphorylation. Indirect immunofluorescence and confocal microscopy were used to determine FGFR3 localization. The selective FGFR1-3 inhibitor, PD173074 and siRNA knockdowns were used to characterize the functional role of FGFR3 in vitro. The TCGA and Metabric breast cancer datasets were interrogated to identify FGFR3 alterations and how they relate to breast cancer subtype and overall patient survival.ResultsHigh FGFR3 expression and phosphorylation were detected in SUM185PE cells, which harbor a FGFR3-TACC3 gene fusion. Low FGFR3 phosphorylation was detected in CAL51, MFM-223 and MDA-MB-231 cells. In SUM185PE cells, the FGFR3-TACC3 fusion protein contributed the majority of phosphorylated FGFR3, and largely localized to the cytoplasm and plasma membrane, with staining at the mitotic spindle in a small subset of cells. Knockdown of the FGFR3-TACC3 fusion and wildtype FGFR3 in SUM185PE cells decreased FRS2, AKT and ERK phosphorylation, and induced cell death. Knockdown of wildtype FGFR3 resulted in only a trend for decreased proliferation. PD173074 significantly decreased FRS2, AKT and ERK activation, and reduced SUM185PE cell proliferation. Cyclin A and pRb were also decreased in the presence of PD173074, while cleaved PARP was increased, indicating cell cycle arrest in G1 phase and apoptosis. Knockdown of FGFR3 in CAL51, MFM-223 and MDA-MB-231 cells had no significant effect on cell proliferation. Interrogation of public datasets revealed that increased FGFR3 expression in breast cancer was significantly associated with reduced overall survival, and that potentially oncogenic FGFR3 alterations (eg mutation and amplification) occur in the TNBC/basal, luminal A and luminal B subtypes, but are rare.ConclusionsThese results indicate that targeting FGFR3 may represent a therapeutic option for TNBC, but only for patients with oncogenic FGFR3 alterations, such as the FGFR3-TACC3 fusion. Video abstract.

Dataset Information

Impact of CXCR4 and CXCR7 knockout by CRISPR/Cas9 on the function of triple-negative breast cancer cells.

Publications

Impact of CXCR4 and CXCR7 knockout by CRISPR/Cas9 on the function of triple-negative breast cancer cells.

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