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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: 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.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

Experimental design for in vitro isolation of RNA from migratory/invasive and reference populationsA. After 24h of serum starvation, MDA-MB-231 cells were seeded into Transwell inserts with (invasion) or without a layer of Matrigel (migration). At the indicated early and late time points, total RNA was isolated after direct lysis of the respective cell populations on the membranes. This procedure was performed in triplicate for each condition. B. Time point selection for RNA-isolation from migratory and invasive cells. Dynamic migration (left) and invasion (right) profiles of MDA-MB-231 cells have been generated by xCELLigence RTCA in correlating conditions with the Transwell experiments (panel A). Arrowheads indicate selected time points for RNA-isolation as described in panel A. Inset shows an integrated plot of the migration (blue graph) and invasion (red graph) patterns.
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Figure 1: Experimental design for in vitro isolation of RNA from migratory/invasive and reference populationsA. After 24h of serum starvation, MDA-MB-231 cells were seeded into Transwell inserts with (invasion) or without a layer of Matrigel (migration). At the indicated early and late time points, total RNA was isolated after direct lysis of the respective cell populations on the membranes. This procedure was performed in triplicate for each condition. B. Time point selection for RNA-isolation from migratory and invasive cells. Dynamic migration (left) and invasion (right) profiles of MDA-MB-231 cells have been generated by xCELLigence RTCA in correlating conditions with the Transwell experiments (panel A). Arrowheads indicate selected time points for RNA-isolation as described in panel A. Inset shows an integrated plot of the migration (blue graph) and invasion (red graph) patterns.

Mentions: Genome-wide approaches to elucidate gene expression patterns can be applied to identify genes essentially associated with specific biological phenotypes. As cell motility and invasion have a 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 [8]. This report describes microarray analysis on minute RNA-quantities derived from migrated or invaded subpopulations of triple negative MDA-MB-231 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 (Fig 1). 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 a differential expression of members of the Krüppel-like transcription factor family (KLFs) and more specifically, Krüppel-like factor 9 (KLF9) was identified as a potential key player in invasive growth of breast cancer.


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)

Experimental design for in vitro isolation of RNA from migratory/invasive and reference populationsA. After 24h of serum starvation, MDA-MB-231 cells were seeded into Transwell inserts with (invasion) or without a layer of Matrigel (migration). At the indicated early and late time points, total RNA was isolated after direct lysis of the respective cell populations on the membranes. This procedure was performed in triplicate for each condition. B. Time point selection for RNA-isolation from migratory and invasive cells. Dynamic migration (left) and invasion (right) profiles of MDA-MB-231 cells have been generated by xCELLigence RTCA in correlating conditions with the Transwell experiments (panel A). Arrowheads indicate selected time points for RNA-isolation as described in panel A. Inset shows an integrated plot of the migration (blue graph) and invasion (red graph) patterns.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Experimental design for in vitro isolation of RNA from migratory/invasive and reference populationsA. After 24h of serum starvation, MDA-MB-231 cells were seeded into Transwell inserts with (invasion) or without a layer of Matrigel (migration). At the indicated early and late time points, total RNA was isolated after direct lysis of the respective cell populations on the membranes. This procedure was performed in triplicate for each condition. B. Time point selection for RNA-isolation from migratory and invasive cells. Dynamic migration (left) and invasion (right) profiles of MDA-MB-231 cells have been generated by xCELLigence RTCA in correlating conditions with the Transwell experiments (panel A). Arrowheads indicate selected time points for RNA-isolation as described in panel A. Inset shows an integrated plot of the migration (blue graph) and invasion (red graph) patterns.
Mentions: Genome-wide approaches to elucidate gene expression patterns can be applied to identify genes essentially associated with specific biological phenotypes. As cell motility and invasion have a 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 [8]. This report describes microarray analysis on minute RNA-quantities derived from migrated or invaded subpopulations of triple negative MDA-MB-231 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 (Fig 1). 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 a differential expression of members of the Krüppel-like transcription factor family (KLFs) and more specifically, Krüppel-like factor 9 (KLF9) was identified as a potential key player in invasive growth of breast cancer.

Bottom Line: 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.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