Limits...
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.

Show MeSH

Related in: MedlinePlus

Knockdown of Tpx2 does not impair 6DT1 cell proliferation in vitro.A) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells were seeded in triplicate at equal densities and passaged every 3–4 days for 17 days. Cumulative cell numbers were determined at each passage. B) 2000 cells were seeded in quadruplicates into 48-well cell culture plates and imaged for >80h. Average cell density during logarithmic growth from 20 h–80 h after seeding is displayed and exponential trend lines are shown in black. C) Exponentially growing cells were pulsed with 10 µM BrdU for 25 min, stained for DNA and BrdU content and analyzed by FACS. Percentage of cells in G1, S, and G2/M phase are indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4219783&req=5

pone-0111813-g004: Knockdown of Tpx2 does not impair 6DT1 cell proliferation in vitro.A) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells were seeded in triplicate at equal densities and passaged every 3–4 days for 17 days. Cumulative cell numbers were determined at each passage. B) 2000 cells were seeded in quadruplicates into 48-well cell culture plates and imaged for >80h. Average cell density during logarithmic growth from 20 h–80 h after seeding is displayed and exponential trend lines are shown in black. C) Exponentially growing cells were pulsed with 10 µM BrdU for 25 min, stained for DNA and BrdU content and analyzed by FACS. Percentage of cells in G1, S, and G2/M phase are indicated.

Mentions: Next we investigated whether knockdown of Tpx2 in 6DT1 cells affects proliferation in vitro. In vitro growth curve analysis revealed no difference in the proliferation rate of 6DT1 shTpx2 and 6DT1 shRNA control cells (shCtrl) (Figure 4A), while Tpx2 expression levels remained suppressed in 6DT1 shTpx2 cells throughout the 17-day time course of the experiment (data not shown). We also measured 6DT1 shTpx2 and 6DT1 shRNA control cell proliferation in a short-term growth assay and, again, failed to measure any significant difference in cell proliferation rates (Figure 4B). Likewise, a BrdU incorporation assay did not show significant differences in the percentile distribution of cells in G1, S, or G2/M phase of the cell cycle (Figure 4C).


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 impair 6DT1 cell proliferation in vitro.A) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells were seeded in triplicate at equal densities and passaged every 3–4 days for 17 days. Cumulative cell numbers were determined at each passage. B) 2000 cells were seeded in quadruplicates into 48-well cell culture plates and imaged for >80h. Average cell density during logarithmic growth from 20 h–80 h after seeding is displayed and exponential trend lines are shown in black. C) Exponentially growing cells were pulsed with 10 µM BrdU for 25 min, stained for DNA and BrdU content and analyzed by FACS. Percentage of cells in G1, S, and G2/M phase are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4219783&req=5

pone-0111813-g004: Knockdown of Tpx2 does not impair 6DT1 cell proliferation in vitro.A) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells were seeded in triplicate at equal densities and passaged every 3–4 days for 17 days. Cumulative cell numbers were determined at each passage. B) 2000 cells were seeded in quadruplicates into 48-well cell culture plates and imaged for >80h. Average cell density during logarithmic growth from 20 h–80 h after seeding is displayed and exponential trend lines are shown in black. C) Exponentially growing cells were pulsed with 10 µM BrdU for 25 min, stained for DNA and BrdU content and analyzed by FACS. Percentage of cells in G1, S, and G2/M phase are indicated.
Mentions: Next we investigated whether knockdown of Tpx2 in 6DT1 cells affects proliferation in vitro. In vitro growth curve analysis revealed no difference in the proliferation rate of 6DT1 shTpx2 and 6DT1 shRNA control cells (shCtrl) (Figure 4A), while Tpx2 expression levels remained suppressed in 6DT1 shTpx2 cells throughout the 17-day time course of the experiment (data not shown). We also measured 6DT1 shTpx2 and 6DT1 shRNA control cell proliferation in a short-term growth assay and, again, failed to measure any significant difference in cell proliferation rates (Figure 4B). Likewise, a BrdU incorporation assay did not show significant differences in the percentile distribution of cells in G1, S, or G2/M phase of the cell cycle (Figure 4C).

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