N. Novikov (Tomsk, Russian Federation)
Author Of 1 Presentation
37P - Genetic alteration in KLK5 associated with individual mode of breast cancer invasion
Abstract
Background
Increasing data demonstrate that various genetic alterations are involved in the process of breast cancer invasion; however, their contribution to the individual mode of breast cancer invasion, which is associated with distant metastasis, remains unclear.
Methods
Samples of a tumor and normal breast tissue obtained during surgery from patients with breast cancer (n=10) were used. Type of the invasive component (collective or individual) was determined using morphological analysis of hematoxylin and eosin-stained sections of tumor tissue. DNA isolated from samples was used to prepare exome libraries (SureSelect XT HS, Agilent, USA). Sequencing was performed on the NextSeq500 platform in the PE75 mode (Illumina, USA). Data processing was carried out using the GATK pipeline. Selection of genetic variants found only in cases with an individual mode of breast cancer invasion (compared to the collective mode of invasion) and potentially associated with cell migration and invasion on the basis of literature data was carried out. A stable cell line MCF10A with KLK5 knockout (MCF10A KLK5 KO) was established using CRISPR/Cas9 genetic editing system. Stable cell lines MDA-MB-231 with KLK5 variants (MDA-MB-231 KLK5 WT, MDA-MB-231 KLK5 H142P) were established using a site-directed mutagenesis and plasmid transfection. Cell migration analysis was performed using videomicroscopy. Statistical analysis was carried out using the DiPer program.
Results
Based on the results of whole-exome sequencing and subsequent selection of genetic variants, PIK3CA H1047R, KLK5 H142P and other genetic alterations potentially associated with individual mode of breast cancer invasion were found. Genetic variant KLK5 H142P was selected for in vitro analysis. Analysis of the migration of MCF10A KLK5 KO cells demonstrated that they migrate faster than MCF10A KLK5 WT cells (p<0.01). Analysis of migration of MDA-MB-231 cells with KLK5 variants showed that MDA-MB-231 KLK5 WT cells migrate slower than MDA-MB-231 WT cells and MDA-MB-231 KLK5 H142P (p<0.01).
Conclusions
Genetic variant KLK5 H142P is associated with individual mode of breast cancer invasion and affects the migratory behavior of tumor cells.
Legal entity responsible for the study
The authors.
Funding
Russian Foundation for Basic Research, French National Centre for Scientific Research.
Disclosure
All authors have declared no conflicts of interest.
Presenter Of 1 Presentation
37P - Genetic alteration in KLK5 associated with individual mode of breast cancer invasion
Abstract
Background
Increasing data demonstrate that various genetic alterations are involved in the process of breast cancer invasion; however, their contribution to the individual mode of breast cancer invasion, which is associated with distant metastasis, remains unclear.
Methods
Samples of a tumor and normal breast tissue obtained during surgery from patients with breast cancer (n=10) were used. Type of the invasive component (collective or individual) was determined using morphological analysis of hematoxylin and eosin-stained sections of tumor tissue. DNA isolated from samples was used to prepare exome libraries (SureSelect XT HS, Agilent, USA). Sequencing was performed on the NextSeq500 platform in the PE75 mode (Illumina, USA). Data processing was carried out using the GATK pipeline. Selection of genetic variants found only in cases with an individual mode of breast cancer invasion (compared to the collective mode of invasion) and potentially associated with cell migration and invasion on the basis of literature data was carried out. A stable cell line MCF10A with KLK5 knockout (MCF10A KLK5 KO) was established using CRISPR/Cas9 genetic editing system. Stable cell lines MDA-MB-231 with KLK5 variants (MDA-MB-231 KLK5 WT, MDA-MB-231 KLK5 H142P) were established using a site-directed mutagenesis and plasmid transfection. Cell migration analysis was performed using videomicroscopy. Statistical analysis was carried out using the DiPer program.
Results
Based on the results of whole-exome sequencing and subsequent selection of genetic variants, PIK3CA H1047R, KLK5 H142P and other genetic alterations potentially associated with individual mode of breast cancer invasion were found. Genetic variant KLK5 H142P was selected for in vitro analysis. Analysis of the migration of MCF10A KLK5 KO cells demonstrated that they migrate faster than MCF10A KLK5 WT cells (p<0.01). Analysis of migration of MDA-MB-231 cells with KLK5 variants showed that MDA-MB-231 KLK5 WT cells migrate slower than MDA-MB-231 WT cells and MDA-MB-231 KLK5 H142P (p<0.01).
Conclusions
Genetic variant KLK5 H142P is associated with individual mode of breast cancer invasion and affects the migratory behavior of tumor cells.
Legal entity responsible for the study
The authors.
Funding
Russian Foundation for Basic Research, French National Centre for Scientific Research.
Disclosure
All authors have declared no conflicts of interest.