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MDA-MB-231 breast cancer cells overexpressing single VEGF isoforms display distinct colonisation characteristics

View Article: PubMed Central - PubMed

ABSTRACT

Background:: Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that has important roles in angiogenesis. Our knowledge of the significance of VEGF isoforms in human cancer remains incomplete.

Methods:: Bioluminescence imaging and transcriptomic analysis were used to study the colonisation capacity of the human breast cancer cells MDA-MB-231 controlling or overexpressing the VEGF165 or VEGF189 isoform (named cV-B, V165-B and V189-B, respectively) in nude mice.

Results:: When injected into the bloodstream, V189-B cells induced less metastasis in the lungs and bone than V165-B and cV-B control cells, consistent with longer survival of these mice and delay in tumour uptake in the mice injected with a V189-B clone. Histological analysis confirmed that there were less αSMA-positive cells in the lungs of the mice injected with V189-B. In vitro V189-B cells decreased both cell invasion and survival. Using transcriptomic analysis, we identified a subset of 18 genes expressed differentially between V189 and V165 cell lines and in 120 human breast tumours. V165 was associated with poor prognosis, whereas V189 was not, suggesting a complex regulation by VEGF isoforms. Our results showed a negative correlation between the expression pattern of VEGF189 and the levels of expression of seven genes that influence metastasis.

Conclusion:: Our findings provide the first evidence that VEGF isoforms have different effects on breast cancer cell line colonisation in vivo.

No MeSH data available.


Related in: MedlinePlus

Lower invasive capacity and survival of V189-B clones in limiting serum conditions. (A) Histogram showing invasive capacities of cV-B, V189-B and V165-B clones and corresponding sh-NRP-1. Results are presented as % invasion with respect to control cells, for which the value was set at 100% (means of four independent experiments ±s.d.). ***P<0.05. Significant values were shown for V189 vs cV or V165 vs cV. **P<0.05. Significant values were shown for V189 vs sh-NRP-1 of VEGF189 clones (B and C). Survival/apoptotic assay. (B) Histogram showing the mean±s.d. of apoptotic cells in the cV-B, V189-B and V165-B clones. *P<0.01. Significant values were shown for V189 and V165 vs cV. (C) Representative flow cytometry profiles for cV-B, V189-B and V165-B clones incubated in medium supplemented with 0.5% FBS.
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fig2: Lower invasive capacity and survival of V189-B clones in limiting serum conditions. (A) Histogram showing invasive capacities of cV-B, V189-B and V165-B clones and corresponding sh-NRP-1. Results are presented as % invasion with respect to control cells, for which the value was set at 100% (means of four independent experiments ±s.d.). ***P<0.05. Significant values were shown for V189 vs cV or V165 vs cV. **P<0.05. Significant values were shown for V189 vs sh-NRP-1 of VEGF189 clones (B and C). Survival/apoptotic assay. (B) Histogram showing the mean±s.d. of apoptotic cells in the cV-B, V189-B and V165-B clones. *P<0.01. Significant values were shown for V189 and V165 vs cV. (C) Representative flow cytometry profiles for cV-B, V189-B and V165-B clones incubated in medium supplemented with 0.5% FBS.

Mentions: We previously demonstrated that VEGF can affect non-angiogenic aspects of tumour cell progression, through autocrine loop acting on tumour cells (Bachelder et al, 2001, 2003; Lee et al, 2007; Hervé et al, 2008; Vintonenko et al, 2011; Perrot-Applanat and Di Benedetto, 2012). VEGF165 has been shown to increase the invasiveness and survival of breast cancer cells (Orimo et al, 2005, Oskarsson et al, 2011). We further analysed the invasiveness of VEGF189-overexpressing clones, in the Matrigel invasion chamber assays. As NRP-1 was demonstrated to interact with VEGF189 and to modulate VEGF effect on cell survival (Vintonenko et al, 2011), we performed the silencing of NRP-1 to investigate the modulation of invasion induced by VEGF 189 and 165 isoforms (Figure 2A). The number of V189-overexpressing MDA-MB-231 cells invading the Matrigel was significantly decreased by half compared with the more invasive control cV-B and V165-B clones (Figure 2A). We observed that silencing the expression of NRP-1 had no effect on VEGF165-B clones or the cV-B control invasion, whereas it reversed the decrease of VEGF189-expressing cell invasion. In addition, the clones were analysed in cell culture in limiting conditions (0.5% FBS and 1 g l−i glucose) by FACS analysis (Figure 2B and C). As expected, the number of apoptotic V189-B cells (AnnV+/PI and AnnV+/PI+) was increased about two-fold as compared with cV-B and V165-B cells (Figure 2B and C). Thus, VEGF189 may act as an autocrine factor via NRP-1, resulting in lower levels of cell invasion than with the VEGF165 isoform.


MDA-MB-231 breast cancer cells overexpressing single VEGF isoforms display distinct colonisation characteristics
Lower invasive capacity and survival of V189-B clones in limiting serum conditions. (A) Histogram showing invasive capacities of cV-B, V189-B and V165-B clones and corresponding sh-NRP-1. Results are presented as % invasion with respect to control cells, for which the value was set at 100% (means of four independent experiments ±s.d.). ***P<0.05. Significant values were shown for V189 vs cV or V165 vs cV. **P<0.05. Significant values were shown for V189 vs sh-NRP-1 of VEGF189 clones (B and C). Survival/apoptotic assay. (B) Histogram showing the mean±s.d. of apoptotic cells in the cV-B, V189-B and V165-B clones. *P<0.01. Significant values were shown for V189 and V165 vs cV. (C) Representative flow cytometry profiles for cV-B, V189-B and V165-B clones incubated in medium supplemented with 0.5% FBS.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559830&req=5

fig2: Lower invasive capacity and survival of V189-B clones in limiting serum conditions. (A) Histogram showing invasive capacities of cV-B, V189-B and V165-B clones and corresponding sh-NRP-1. Results are presented as % invasion with respect to control cells, for which the value was set at 100% (means of four independent experiments ±s.d.). ***P<0.05. Significant values were shown for V189 vs cV or V165 vs cV. **P<0.05. Significant values were shown for V189 vs sh-NRP-1 of VEGF189 clones (B and C). Survival/apoptotic assay. (B) Histogram showing the mean±s.d. of apoptotic cells in the cV-B, V189-B and V165-B clones. *P<0.01. Significant values were shown for V189 and V165 vs cV. (C) Representative flow cytometry profiles for cV-B, V189-B and V165-B clones incubated in medium supplemented with 0.5% FBS.
Mentions: We previously demonstrated that VEGF can affect non-angiogenic aspects of tumour cell progression, through autocrine loop acting on tumour cells (Bachelder et al, 2001, 2003; Lee et al, 2007; Hervé et al, 2008; Vintonenko et al, 2011; Perrot-Applanat and Di Benedetto, 2012). VEGF165 has been shown to increase the invasiveness and survival of breast cancer cells (Orimo et al, 2005, Oskarsson et al, 2011). We further analysed the invasiveness of VEGF189-overexpressing clones, in the Matrigel invasion chamber assays. As NRP-1 was demonstrated to interact with VEGF189 and to modulate VEGF effect on cell survival (Vintonenko et al, 2011), we performed the silencing of NRP-1 to investigate the modulation of invasion induced by VEGF 189 and 165 isoforms (Figure 2A). The number of V189-overexpressing MDA-MB-231 cells invading the Matrigel was significantly decreased by half compared with the more invasive control cV-B and V165-B clones (Figure 2A). We observed that silencing the expression of NRP-1 had no effect on VEGF165-B clones or the cV-B control invasion, whereas it reversed the decrease of VEGF189-expressing cell invasion. In addition, the clones were analysed in cell culture in limiting conditions (0.5% FBS and 1 g l−i glucose) by FACS analysis (Figure 2B and C). As expected, the number of apoptotic V189-B cells (AnnV+/PI and AnnV+/PI+) was increased about two-fold as compared with cV-B and V165-B cells (Figure 2B and C). Thus, VEGF189 may act as an autocrine factor via NRP-1, resulting in lower levels of cell invasion than with the VEGF165 isoform.

View Article: PubMed Central - PubMed

ABSTRACT

Background:: Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that has important roles in angiogenesis. Our knowledge of the significance of VEGF isoforms in human cancer remains incomplete.

Methods:: Bioluminescence imaging and transcriptomic analysis were used to study the colonisation capacity of the human breast cancer cells MDA-MB-231 controlling or overexpressing the VEGF165 or VEGF189 isoform (named cV-B, V165-B and V189-B, respectively) in nude mice.

Results:: When injected into the bloodstream, V189-B cells induced less metastasis in the lungs and bone than V165-B and cV-B control cells, consistent with longer survival of these mice and delay in tumour uptake in the mice injected with a V189-B clone. Histological analysis confirmed that there were less &alpha;SMA-positive cells in the lungs of the mice injected with V189-B. In vitro V189-B cells decreased both cell invasion and survival. Using transcriptomic analysis, we identified a subset of 18 genes expressed differentially between V189 and V165 cell lines and in 120 human breast tumours. V165 was associated with poor prognosis, whereas V189 was not, suggesting a complex regulation by VEGF isoforms. Our results showed a negative correlation between the expression pattern of VEGF189 and the levels of expression of seven genes that influence metastasis.

Conclusion:: Our findings provide the first evidence that VEGF isoforms have different effects on breast cancer cell line colonisation in vivo.

No MeSH data available.


Related in: MedlinePlus