image_5_Analysis of Hierarchical Organization in Gene Expression Networks Reveals Underlying Principles of Collective Tumor Cell Dissemination and Metastatic Aggressiveness of Inflammatory Breast Cancer.tif

<p>Clusters of circulating tumor cells (CTCs), despite being rare, may account for more than 90% of metastases. Cells in these clusters do not undergo a complete epithelial-to-mesenchymal transition (EMT), but retain some epithelial traits as compared to individually disseminating tumor cells. Determinants of single cell dissemination versus collective dissemination remain elusive. Inflammatory breast cancer (IBC), a highly aggressive breast cancer subtype that chiefly metastasizes via CTC clusters, is a promising model for studying mechanisms of collective tumor cell dissemination. Previous studies, motivated by a theory that suggests physical systems with hierarchical organization tend to be more adaptable, have found that the expression of metastasis-associated genes is more hierarchically organized in cases of successful metastases. Here, we used the cophenetic correlation coefficient (CCC) to quantify the hierarchical organization in the expression of two distinct gene sets, collective dissemination-associated genes and IBC-associated genes, in cancer cell lines and in tumor samples from breast cancer patients. Hypothesizing that a higher CCC for collective dissemination-associated genes and for IBC-associated genes would be associated with retention of epithelial traits enabling collective dissemination and with worse disease progression in breast cancer patients, we evaluated the correlation of CCC with different phenotypic groups. The CCC of both the abovementioned gene sets, the collective dissemination-associated genes and the IBC-associated genes, was higher in (a) epithelial cell lines as compared to mesenchymal cell lines and (b) tumor samples from IBC patients as compared to samples from non-IBC breast cancer patients. A higher CCC of both gene sets was also correlated with a higher rate of metastatic relapse in breast cancer patients. In contrast, neither the levels of CDH1 gene expression nor gene set enrichment analysis (GSEA) of the abovementioned gene sets could provide similar insights. These results suggest that retention of some epithelial traits in disseminating tumor cells as IBC progresses promotes successful breast cancer metastasis. The CCC provides additional information regarding the organizational complexity of gene expression in comparison to GSEA. We have shown that the CCC may be a useful metric for investigating the collective dissemination phenotype and a prognostic factor for IBC.</p

image_2_Analysis of Hierarchical Organization in Gene Expression Networks Reveals Underlying Principles of Collective Tumor Cell Dissemination and Metastatic Aggressiveness of Inflammatory Breast Cancer.tif

<p>Clusters of circulating tumor cells (CTCs), despite being rare, may account for more than 90% of metastases. Cells in these clusters do not undergo a complete epithelial-to-mesenchymal transition (EMT), but retain some epithelial traits as compared to individually disseminating tumor cells. Determinants of single cell dissemination versus collective dissemination remain elusive. Inflammatory breast cancer (IBC), a highly aggressive breast cancer subtype that chiefly metastasizes via CTC clusters, is a promising model for studying mechanisms of collective tumor cell dissemination. Previous studies, motivated by a theory that suggests physical systems with hierarchical organization tend to be more adaptable, have found that the expression of metastasis-associated genes is more hierarchically organized in cases of successful metastases. Here, we used the cophenetic correlation coefficient (CCC) to quantify the hierarchical organization in the expression of two distinct gene sets, collective dissemination-associated genes and IBC-associated genes, in cancer cell lines and in tumor samples from breast cancer patients. Hypothesizing that a higher CCC for collective dissemination-associated genes and for IBC-associated genes would be associated with retention of epithelial traits enabling collective dissemination and with worse disease progression in breast cancer patients, we evaluated the correlation of CCC with different phenotypic groups. The CCC of both the abovementioned gene sets, the collective dissemination-associated genes and the IBC-associated genes, was higher in (a) epithelial cell lines as compared to mesenchymal cell lines and (b) tumor samples from IBC patients as compared to samples from non-IBC breast cancer patients. A higher CCC of both gene sets was also correlated with a higher rate of metastatic relapse in breast cancer patients. In contrast, neither the levels of CDH1 gene expression nor gene set enrichment analysis (GSEA) of the abovementioned gene sets could provide similar insights. These results suggest that retention of some epithelial traits in disseminating tumor cells as IBC progresses promotes successful breast cancer metastasis. The CCC provides additional information regarding the organizational complexity of gene expression in comparison to GSEA. We have shown that the CCC may be a useful metric for investigating the collective dissemination phenotype and a prognostic factor for IBC.</p

data_sheet_1_Analysis of Hierarchical Organization in Gene Expression Networks Reveals Underlying Principles of Collective Tumor Cell Dissemination and Metastatic Aggressiveness of Inflammatory Breast Cancer.docx

<p>Clusters of circulating tumor cells (CTCs), despite being rare, may account for more than 90% of metastases. Cells in these clusters do not undergo a complete epithelial-to-mesenchymal transition (EMT), but retain some epithelial traits as compared to individually disseminating tumor cells. Determinants of single cell dissemination versus collective dissemination remain elusive. Inflammatory breast cancer (IBC), a highly aggressive breast cancer subtype that chiefly metastasizes via CTC clusters, is a promising model for studying mechanisms of collective tumor cell dissemination. Previous studies, motivated by a theory that suggests physical systems with hierarchical organization tend to be more adaptable, have found that the expression of metastasis-associated genes is more hierarchically organized in cases of successful metastases. Here, we used the cophenetic correlation coefficient (CCC) to quantify the hierarchical organization in the expression of two distinct gene sets, collective dissemination-associated genes and IBC-associated genes, in cancer cell lines and in tumor samples from breast cancer patients. Hypothesizing that a higher CCC for collective dissemination-associated genes and for IBC-associated genes would be associated with retention of epithelial traits enabling collective dissemination and with worse disease progression in breast cancer patients, we evaluated the correlation of CCC with different phenotypic groups. The CCC of both the abovementioned gene sets, the collective dissemination-associated genes and the IBC-associated genes, was higher in (a) epithelial cell lines as compared to mesenchymal cell lines and (b) tumor samples from IBC patients as compared to samples from non-IBC breast cancer patients. A higher CCC of both gene sets was also correlated with a higher rate of metastatic relapse in breast cancer patients. In contrast, neither the levels of CDH1 gene expression nor gene set enrichment analysis (GSEA) of the abovementioned gene sets could provide similar insights. These results suggest that retention of some epithelial traits in disseminating tumor cells as IBC progresses promotes successful breast cancer metastasis. The CCC provides additional information regarding the organizational complexity of gene expression in comparison to GSEA. We have shown that the CCC may be a useful metric for investigating the collective dissemination phenotype and a prognostic factor for IBC.</p

Survivin expression in breast cancer from public databases.

<p><b>A)</b> Survivin expression is approximately seven-fold higher in invasive breast carcinoma compared to normal breast (p = 5.5e-31) from the TCGA data set in the Oncomine public database. <b>B)</b> From the GOBO public dataset, survivin expression increases with breast cancer stage (p < 0.00001). <b>C)</b> Survivin is expressed 2.3-fold higher in triple-negative breast cancer compared to all other molecular subtypes (p = 3.5e-8) in the Bittner breast data set in Oncomine. <b>D)</b> Similarly, survivin expression is 2.3-fold higher in estrogen receptor-negative compared to estrogen receptor-positive breast cancers (p = 9e-46) in the Curtis breast data set [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120719#pone.0120719.ref024" target="_blank">24</a>] in Oncomine.</p

Mammosphere-formation efficiency in MCF7 and SUM149 when selected drugs are administered to survivin-DN cells.

<p><b>A,C)</b> Neither Taxol nor gamma secretase inhibitor decrease mammosphere-formation efficiency in MCF7 control or survivin-DN cells. <b>B)</b> SUM149 survivin-DN cells are sensitized by treatment with 10 nM Taxol (p < 0.001). <b>D)</b> Gamma secretase inhibitor shows no effect on mammosphere formation in SUM149 control or survivin-DN cells. Error bars indicate standard deviation.</p

Survival Fraction at 2 Gy for survivin-DN in MCF7 and SUM149.

<p>Survival Fraction at 2 Gy for survivin-DN in MCF7 and SUM149.</p

Representative figures for monolayer and mammosphere clonogenic assays in MCF7 and SUM149.

<p><b>A,C,E)</b> MCF7 survivin-DN cells are radio-protective in monolayer cultures, mammosphere cultures, and also mammosphere cultures undergoing a fractionated regimen. <b>B)</b> SUM149 survivin-DN cells are radiosensitive compared to the control in monolayer cultures. <b>D,F)</b> SUM149 survivin-DN cells show no statistical difference in response to radiation, for both mammosphere cultures and mammosphere cultures exposed to a fractionated regiment. Error bars indicate standard deviation.</p

Colony- and mammosphere-formation efficiency in MCF7 and SUM149 breast cancer cell lines.

<p><b>A)</b> In MCF7, the FUGW control forms significantly more colonies than the survivin-DN-transfected cells (p < 0.01). <b>B)</b> In SUM149, the survivin-DN cells form significantly more colonies than the control (p < 0.01). <b>C,D)</b> In both MCF7 and SUM149, there is no statistical difference in mammosphere-formation efficiency between the control and survivin-DN clone. Error bars indicate standard deviation.</p

Cells transduced with the survivin dominant-negative construct display higher levels of apoptotic markers.

<p><b>A)</b> MCF7 and SUM149 breast cancer cell lines were successfully transduced with the survivin dominant-negative construct, as shown by Western blot. <b>B)</b> Survivin-DN cells display greater levels of cleaved caspase 3 compared to the control; MCF7 shows no expression of caspase 3, consistent with the literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120719#pone.0120719.ref032" target="_blank">32</a>]. <b>C)</b> Survivin-DN cells have a greater fraction of sub-G1 (i.e. apoptotic) cells compared to the control, when stained with Propidium iodide.</p

Overall survival in breast cancer stratified by survivin expression using two public databases.

<p>Kaplan-Meier Plotter (A,C,E) and Gene Expression-Based Outcome for Breast Cancer Online (B,D,F) data are shown. Red = high survivin expression at selected cutoff expression. <b>A,B)</b> High survivin expression is prognostic for poor outcome in all breast cancer patients. <b>C,D)</b> Likewise, high survivin expression predicts for poor outcome in patients with estrogen receptor-positive breast cancer. <b>E,F)</b> In patients with estrogen receptor-negative breast cancer, survivin expression is not associated with clinical outcome.</p