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Expression profiling of migrated and invaded breast cancer cells predicts early metastatic relapse and reveals Krüppel-like factor 9 as a potential suppressor of invasive growth in breast cancer.

Limame R, de Beeck KO, Van Laere S, Croes L, De Wilde A, Dirix L, Van Camp G, Peeters M, De Wever O, Lardon F, Pauwels P - Oncoscience (2013)

Bottom Line: Next to loss in invasive vs non-invasive cell lines, KLF9 also showed significantly lower expression levels in the "early" invasive cell population, in several public expression data sets and in clinical breast cancer samples when compared to normal tissue.Overexpression of EGFP-KLF9 fusion protein significantly altered morphology and blocked invasion and growth of MDA-MB-231 cells in vitro.In addition, KLF9 expression correlated inversely with mitotic activity in clinical samples, indicating anti-proliferative effects.

View Article: PubMed Central - PubMed

Affiliation: Center for Oncological Research (CORE), University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium ; These authors equally contributed to this work.

ABSTRACT
Cell motility and invasion initiate metastasis. However, only a subpopulation of cancer cells within a tumor will ultimately become invasive. Due to this stochastic and transient nature, in an experimental setting, migrating and invading cells need to be isolated from the general population in order to study the gene expression profiles linked to these processes. This report describes microarray analysis on RNA derived from migrated or invaded subpopulations of triple negative breast cancer cells in a Transwell set-up, at two different time points during motility and invasion, pre-determined as "early" and "late" in real-time kinetic assessments. Invasion- and migration-related gene expression signatures were generated through comparison with non-invasive cells, remaining at the upper side of the Transwell membranes. Late-phase signatures of both invasion and migration indicated poor prognosis in a series of breast cancer data sets. Furthermore, evaluation of the genes constituting the prognostic invasion-related gene signature revealed Krüppel-like factor 9 (KLF9) as a putative suppressor of invasive growth in breast cancer. Next to loss in invasive vs non-invasive cell lines, KLF9 also showed significantly lower expression levels in the "early" invasive cell population, in several public expression data sets and in clinical breast cancer samples when compared to normal tissue. Overexpression of EGFP-KLF9 fusion protein significantly altered morphology and blocked invasion and growth of MDA-MB-231 cells in vitro. In addition, KLF9 expression correlated inversely with mitotic activity in clinical samples, indicating anti-proliferative effects.

No MeSH data available.


Related in: MedlinePlus

KLF9 is downregulated in invasive cell linesNon-invasive breast cancer cell lines show higher expression levels of KLF9 mRNA than invasive cell lines. Results from Transwell Matrigel invasion experiments are shown in red (left axis), accompanied by micrographs of representative crystal violet-stained membranes (obj. 20X) per cell line mentioned below. All experiments have been performed in technical and biological triplicates. Normalized relative expression levels for KLF9 as measured by RT-qPCR are shown in green (right axis). All RT-qPCR experiments have been performed in at least technical duplicates and biological triplicates. All results shown are means + SD.
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Figure 3: KLF9 is downregulated in invasive cell linesNon-invasive breast cancer cell lines show higher expression levels of KLF9 mRNA than invasive cell lines. Results from Transwell Matrigel invasion experiments are shown in red (left axis), accompanied by micrographs of representative crystal violet-stained membranes (obj. 20X) per cell line mentioned below. All experiments have been performed in technical and biological triplicates. Normalized relative expression levels for KLF9 as measured by RT-qPCR are shown in green (right axis). All RT-qPCR experiments have been performed in at least technical duplicates and biological triplicates. All results shown are means + SD.

Mentions: The microarray-based gene expression studies revealed that KLF9 (also known as BTEB1) was significantly downregulated in the invasive subpopulation of MDA-MB-231 cells. This finding has been validated by RT-qPCR (Supplementary Fig 2) and possibly implies a suppressing capacity on breast cancer cell invasiveness. Therefore, we screened a panel of breast cancer cell lines for KLF9-expression using RT-qPCR. It was found that non-invasive cell lines (MCF-7, SKBR-3, T47D, ZR-75-1, CAMA-1 and MDA-MB-361) showed significantly higher levels of KLF9 mRNA when compared to invasive cell lines (MDA-MB-231 and MDA-MB-468) (Fig 3).


Expression profiling of migrated and invaded breast cancer cells predicts early metastatic relapse and reveals Krüppel-like factor 9 as a potential suppressor of invasive growth in breast cancer.

Limame R, de Beeck KO, Van Laere S, Croes L, De Wilde A, Dirix L, Van Camp G, Peeters M, De Wever O, Lardon F, Pauwels P - Oncoscience (2013)

KLF9 is downregulated in invasive cell linesNon-invasive breast cancer cell lines show higher expression levels of KLF9 mRNA than invasive cell lines. Results from Transwell Matrigel invasion experiments are shown in red (left axis), accompanied by micrographs of representative crystal violet-stained membranes (obj. 20X) per cell line mentioned below. All experiments have been performed in technical and biological triplicates. Normalized relative expression levels for KLF9 as measured by RT-qPCR are shown in green (right axis). All RT-qPCR experiments have been performed in at least technical duplicates and biological triplicates. All results shown are means + SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295756&req=5

Figure 3: KLF9 is downregulated in invasive cell linesNon-invasive breast cancer cell lines show higher expression levels of KLF9 mRNA than invasive cell lines. Results from Transwell Matrigel invasion experiments are shown in red (left axis), accompanied by micrographs of representative crystal violet-stained membranes (obj. 20X) per cell line mentioned below. All experiments have been performed in technical and biological triplicates. Normalized relative expression levels for KLF9 as measured by RT-qPCR are shown in green (right axis). All RT-qPCR experiments have been performed in at least technical duplicates and biological triplicates. All results shown are means + SD.
Mentions: The microarray-based gene expression studies revealed that KLF9 (also known as BTEB1) was significantly downregulated in the invasive subpopulation of MDA-MB-231 cells. This finding has been validated by RT-qPCR (Supplementary Fig 2) and possibly implies a suppressing capacity on breast cancer cell invasiveness. Therefore, we screened a panel of breast cancer cell lines for KLF9-expression using RT-qPCR. It was found that non-invasive cell lines (MCF-7, SKBR-3, T47D, ZR-75-1, CAMA-1 and MDA-MB-361) showed significantly higher levels of KLF9 mRNA when compared to invasive cell lines (MDA-MB-231 and MDA-MB-468) (Fig 3).

Bottom Line: Next to loss in invasive vs non-invasive cell lines, KLF9 also showed significantly lower expression levels in the "early" invasive cell population, in several public expression data sets and in clinical breast cancer samples when compared to normal tissue.Overexpression of EGFP-KLF9 fusion protein significantly altered morphology and blocked invasion and growth of MDA-MB-231 cells in vitro.In addition, KLF9 expression correlated inversely with mitotic activity in clinical samples, indicating anti-proliferative effects.

View Article: PubMed Central - PubMed

Affiliation: Center for Oncological Research (CORE), University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium ; These authors equally contributed to this work.

ABSTRACT
Cell motility and invasion initiate metastasis. However, only a subpopulation of cancer cells within a tumor will ultimately become invasive. Due to this stochastic and transient nature, in an experimental setting, migrating and invading cells need to be isolated from the general population in order to study the gene expression profiles linked to these processes. This report describes microarray analysis on RNA derived from migrated or invaded subpopulations of triple negative breast cancer cells in a Transwell set-up, at two different time points during motility and invasion, pre-determined as "early" and "late" in real-time kinetic assessments. Invasion- and migration-related gene expression signatures were generated through comparison with non-invasive cells, remaining at the upper side of the Transwell membranes. Late-phase signatures of both invasion and migration indicated poor prognosis in a series of breast cancer data sets. Furthermore, evaluation of the genes constituting the prognostic invasion-related gene signature revealed Krüppel-like factor 9 (KLF9) as a putative suppressor of invasive growth in breast cancer. Next to loss in invasive vs non-invasive cell lines, KLF9 also showed significantly lower expression levels in the "early" invasive cell population, in several public expression data sets and in clinical breast cancer samples when compared to normal tissue. Overexpression of EGFP-KLF9 fusion protein significantly altered morphology and blocked invasion and growth of MDA-MB-231 cells in vitro. In addition, KLF9 expression correlated inversely with mitotic activity in clinical samples, indicating anti-proliferative effects.

No MeSH data available.


Related in: MedlinePlus