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Functional analysis of prognostic gene expression network genes in metastatic breast cancer models.

Geiger TR, Ha NH, Faraji F, Michael HT, Rodriguez L, Walker RC, Green JE, Simpson RM, Hunter KW - PLoS ONE (2014)

Bottom Line: The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2].Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer.Here, we validate one of the highly connected genes as a metastasis associated gene.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Identification of conserved co-expression networks is a useful tool for clustering groups of genes enriched for common molecular or cellular functions [1]. The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2]. Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer. Here, we validate one of the highly connected genes as a metastasis associated gene. Tpx2, the most highly connected gene within a proliferation network specifically prognostic for estrogen receptor positive (ER+) breast cancers, enhances metastatic disease, but in a tumor autonomous, proliferation-independent manner. Histologic analysis suggests instead that variation of TPX2 levels within disseminated tumor cells may influence the transition between dormant to actively proliferating cells in the secondary site. These results support the co-expression network approach for identification of new metastasis-associated genes to provide new information regarding the etiology of breast cancer progression and metastatic disease.

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Knockdown of Tpx2 does not induce prototypical MET in 6DT1 cells.A) E-cadherin mRNA levels in 6DT1 shCtrl, 6DT1-shTpx2#1, and 6DT1-shTpx2#2 cells were measured by qRT-PCR and displayed relative to levels in shRNA control cells. Error bars represent standard deviations. B) Western blot analysis for E-cadherin, beta-catenin, N-cadherin, and vimentin of cell lines described in a). Beta-actin serves as loading control. 67NR and 4T1 cells serve as positive and negative controls for the different EMT markers. C) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells (and 67NR and 4T1 cells as controls) were immunofluorescently stained for E-cadherin and actin cytoskeleton was labeled with phalloidin (green). DAPI was used for nuclear staining. Confocal images are shown at 63x magnification.
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pone-0111813-g007: Knockdown of Tpx2 does not induce prototypical MET in 6DT1 cells.A) E-cadherin mRNA levels in 6DT1 shCtrl, 6DT1-shTpx2#1, and 6DT1-shTpx2#2 cells were measured by qRT-PCR and displayed relative to levels in shRNA control cells. Error bars represent standard deviations. B) Western blot analysis for E-cadherin, beta-catenin, N-cadherin, and vimentin of cell lines described in a). Beta-actin serves as loading control. 67NR and 4T1 cells serve as positive and negative controls for the different EMT markers. C) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells (and 67NR and 4T1 cells as controls) were immunofluorescently stained for E-cadherin and actin cytoskeleton was labeled with phalloidin (green). DAPI was used for nuclear staining. Confocal images are shown at 63x magnification.

Mentions: It has recently been shown that activated Aurora kinase can induce epithelial-mesenchymal transition (EMT) [20]. Since EMT is known to facilitate metastasis [21] and TPX2 has previously been shown to activate AURK [22], we speculated that Tpx2 depletion may impair metastasis through the reverse process, mesenchymal-epithelial transition (MET). To this end, we analyzed the expression levels of several epithelial and mesenchymal markers in 6DT1 shTpx2 and 6DT1 shRNA control cells. The mRNA levels of the epithelial marker E-cadherin were slightly elevated in 6DT1 shTpx2 cells (Figure 7A), however this did not translate into increased E-cadherin protein levels by either western blot analysis (Figure 7B) or immunofluorescence and confocal microscopy analysis (Figure 7C). Similarly, beta-catenin, another epithelial marker, was unchanged and the mesenchymal markers N-cadherin and vimentin were expressed below detection levels in both 6DT1 shTpx2 and 6DT1 shRNA control cells (Figure 7B). It thus appears that in the 6DT1 mammary carcinoma cells, Tpx2 functionally contributes to metastasis through an unknown mechanism, but independent of cell proliferation or EMT.


Functional analysis of prognostic gene expression network genes in metastatic breast cancer models.

Geiger TR, Ha NH, Faraji F, Michael HT, Rodriguez L, Walker RC, Green JE, Simpson RM, Hunter KW - PLoS ONE (2014)

Knockdown of Tpx2 does not induce prototypical MET in 6DT1 cells.A) E-cadherin mRNA levels in 6DT1 shCtrl, 6DT1-shTpx2#1, and 6DT1-shTpx2#2 cells were measured by qRT-PCR and displayed relative to levels in shRNA control cells. Error bars represent standard deviations. B) Western blot analysis for E-cadherin, beta-catenin, N-cadherin, and vimentin of cell lines described in a). Beta-actin serves as loading control. 67NR and 4T1 cells serve as positive and negative controls for the different EMT markers. C) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells (and 67NR and 4T1 cells as controls) were immunofluorescently stained for E-cadherin and actin cytoskeleton was labeled with phalloidin (green). DAPI was used for nuclear staining. Confocal images are shown at 63x magnification.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219783&req=5

pone-0111813-g007: Knockdown of Tpx2 does not induce prototypical MET in 6DT1 cells.A) E-cadherin mRNA levels in 6DT1 shCtrl, 6DT1-shTpx2#1, and 6DT1-shTpx2#2 cells were measured by qRT-PCR and displayed relative to levels in shRNA control cells. Error bars represent standard deviations. B) Western blot analysis for E-cadherin, beta-catenin, N-cadherin, and vimentin of cell lines described in a). Beta-actin serves as loading control. 67NR and 4T1 cells serve as positive and negative controls for the different EMT markers. C) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells (and 67NR and 4T1 cells as controls) were immunofluorescently stained for E-cadherin and actin cytoskeleton was labeled with phalloidin (green). DAPI was used for nuclear staining. Confocal images are shown at 63x magnification.
Mentions: It has recently been shown that activated Aurora kinase can induce epithelial-mesenchymal transition (EMT) [20]. Since EMT is known to facilitate metastasis [21] and TPX2 has previously been shown to activate AURK [22], we speculated that Tpx2 depletion may impair metastasis through the reverse process, mesenchymal-epithelial transition (MET). To this end, we analyzed the expression levels of several epithelial and mesenchymal markers in 6DT1 shTpx2 and 6DT1 shRNA control cells. The mRNA levels of the epithelial marker E-cadherin were slightly elevated in 6DT1 shTpx2 cells (Figure 7A), however this did not translate into increased E-cadherin protein levels by either western blot analysis (Figure 7B) or immunofluorescence and confocal microscopy analysis (Figure 7C). Similarly, beta-catenin, another epithelial marker, was unchanged and the mesenchymal markers N-cadherin and vimentin were expressed below detection levels in both 6DT1 shTpx2 and 6DT1 shRNA control cells (Figure 7B). It thus appears that in the 6DT1 mammary carcinoma cells, Tpx2 functionally contributes to metastasis through an unknown mechanism, but independent of cell proliferation or EMT.

Bottom Line: The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2].Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer.Here, we validate one of the highly connected genes as a metastasis associated gene.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Identification of conserved co-expression networks is a useful tool for clustering groups of genes enriched for common molecular or cellular functions [1]. The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2]. Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer. Here, we validate one of the highly connected genes as a metastasis associated gene. Tpx2, the most highly connected gene within a proliferation network specifically prognostic for estrogen receptor positive (ER+) breast cancers, enhances metastatic disease, but in a tumor autonomous, proliferation-independent manner. Histologic analysis suggests instead that variation of TPX2 levels within disseminated tumor cells may influence the transition between dormant to actively proliferating cells in the secondary site. These results support the co-expression network approach for identification of new metastasis-associated genes to provide new information regarding the etiology of breast cancer progression and metastatic disease.

Show MeSH
Related in: MedlinePlus