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Anoikis-resistant subpopulations of human osteosarcoma display significant chemoresistance and are sensitive to targeted epigenetic therapies predicted by expression profiling.

Foley JM, Scholten DJ, Monks NR, Cherba D, Monsma DJ, Davidson P, Dylewski D, Dykema K, Winn ME, Steensma MR - J Transl Med (2015)

Bottom Line: We show that AI growth results in a global gene expression profile change accompanied by significant chemoresistance (up to 75 fold, p<0.05).AI cells were equally tumorigenic as their adherent counterparts, but showed a significantly decreased rate of growth in-vitro and in-vivo (p<0.05).Targeting epigenetic regulation of this process may be a viable therapeutic strategy.

View Article: PubMed Central - PubMed

Affiliation: Helen DeVos Children's Hospital, Spectrum Health System, Grand Rapids, MI, USA. jessica.foley@vai.org.

ABSTRACT

Background: Osteosarcoma (OS) is the most common type of solid bone cancer, with latent metastasis being a typical mode of disease progression and a major contributor to poor prognosis. For this to occur, cells must resist anoikis and be able to recapitulate tumorigenesis in a foreign microenvironment. Finding novel approaches to treat osteosarcoma and target those cell subpopulations that possess the ability to resist anoikis and contribute to metastatic disease is imperative. Here we investigate anchorage-independent (AI) cell growth as a model to better characterize anoikis resistance in human osteosarcoma while using an expression profiling approach to identify and test targetable signaling pathways.

Methods: Established human OS cell lines and patient-derived human OS cell isolates were subjected to growth in either adherent or AI conditions using Ultra-Low Attachment plates in identical media conditions. Growth rate was assessed using cell doubling times and chemoresistance was assessed by determining cell viability in response to a serial dilution of either doxorubicin or cisplatin. Gene expression differences were examined using quantitative reverse-transcription PCR and microarray with principal component and pathway analysis. In-vivo OS xenografts were generated by either subcutaneous or intratibial injection of adherent or AI human OS cells into athymic nude mice. Statistical significance was determined using student's t-tests with significance set at α=0.05.

Results: We show that AI growth results in a global gene expression profile change accompanied by significant chemoresistance (up to 75 fold, p<0.05). AI cells demonstrate alteration of key mediators of mesenchymal differentiation (β-catenin, Runx2), stemness (Sox2), proliferation (c-myc, Akt), and epigenetic regulation (HDAC class 1). AI cells were equally tumorigenic as their adherent counterparts, but showed a significantly decreased rate of growth in-vitro and in-vivo (p<0.05). Treatment with the pan-histone deacetylase inhibitor vorinostat and the DNA methyltransferase inhibitor 5-azacytidine mitigated AI growth, while 5-azacytidine sensitized anoikis-resistant cells to doxorubicin (p<0.05).

Conclusions: These data demonstrate remarkable plasticity in anoikis-resistant human osteosarcoma subpopulations accompanied by a rapid development of chemoresistance and altered growth rates mirroring the early stages of latent metastasis. Targeting epigenetic regulation of this process may be a viable therapeutic strategy.

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Anoikis-resistant OS cells demonstrate distinct patterns of altered gene expression. A: mRNA expression of the target genes Oct4, Axin2, and Sox2 in AI-grown OS cells after the second passage (p2) was determined using qrt-PCR normalized to adherent cells. Asterisks indicate statistical significance (**p < 0.01). B: Principal component analysis of gene expression using microarray. Gene expression differences resulted in clustering of the adherent cells (squares) compared to the AI cells (triangles). This was more apparent for the patient-derived OS cells than the established OS cell lines. Blue color indicates established OS cell line, green color indicates pre-chemotherapy patient-derived OS cell line, red color indicates post-chemotherapy patient-derived OS cell line, black color indicates pulmonary metastasis patient-derived OS cell line, dotted lines connect paired AI/adherent samples. C: Shortest pathway analysis of the top 300 differentially expressed genes identifies class I HDACs, Runx2, and Akt as central contributors.
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Fig3: Anoikis-resistant OS cells demonstrate distinct patterns of altered gene expression. A: mRNA expression of the target genes Oct4, Axin2, and Sox2 in AI-grown OS cells after the second passage (p2) was determined using qrt-PCR normalized to adherent cells. Asterisks indicate statistical significance (**p < 0.01). B: Principal component analysis of gene expression using microarray. Gene expression differences resulted in clustering of the adherent cells (squares) compared to the AI cells (triangles). This was more apparent for the patient-derived OS cells than the established OS cell lines. Blue color indicates established OS cell line, green color indicates pre-chemotherapy patient-derived OS cell line, red color indicates post-chemotherapy patient-derived OS cell line, black color indicates pulmonary metastasis patient-derived OS cell line, dotted lines connect paired AI/adherent samples. C: Shortest pathway analysis of the top 300 differentially expressed genes identifies class I HDACs, Runx2, and Akt as central contributors.

Mentions: We assessed gene expression differences that may be important for OS cell anoikis resistance using both qrt-PCR and microarray gene expression profiling. Expression of genes involved in developmental programs including Sox2, Oct4, and Wnt signaling were increased in the AI-grown cells relative to adherent cells (Figure 3A). This finding was statistically significant for the mRNA expression of Sox2. The gene expression of established and patient-derived human OS cells cultured under AI conditions to at least the first passage was compared with that of cells cultured in adherent conditions using gene expression profiling with principal component analysis (PCA). There were clear differences in the molecular signatures between adherent and AI cells among established and patient-derived OS cells, although this was less obvious in the established cell lines (Figure 3B). A likely explanation is that the established OS cell lines used are clonally evolved and chemically transformed versions of primary tumor cells, whereas the patient-derived cell lines were grown in culture briefly (<5 passages) prior to analysis. Shortest pathway analysis of the top 300 differentially expressed genes confirmed significant alteration of Runx2, class I HDACs, and Akt (Figure 3C). GeneGo de-novo network generation identified convergence on Wnt/β-catenin, c-myc, and alpha family G-proteins in the molecular signature of AI growth (Additional file 1: Figure S1a and b). These findings pertaining to Runx2 and Wnt/β-catenin signaling are supported through western blot analysis (Additional file 1: Figure S1c). We identified 19 genes that passed false discovery with a corrected p-value less than 0.05 (Table 2). Upregulated genes included those involved in the DNA repair, oncogenic signaling, osteosarcoma pathogenesis, and differentiation, including those specific to mesenchymal lineages [23-26]. Downregulated genes were involved in processes such as proliferation and tumor suppressive functions [27,28]. These results suggest that anoikis-resistant OS cells capable of AI growth have expression changes in key genes that are necessary for cell growth and survival under these conditions.Figure 3


Anoikis-resistant subpopulations of human osteosarcoma display significant chemoresistance and are sensitive to targeted epigenetic therapies predicted by expression profiling.

Foley JM, Scholten DJ, Monks NR, Cherba D, Monsma DJ, Davidson P, Dylewski D, Dykema K, Winn ME, Steensma MR - J Transl Med (2015)

Anoikis-resistant OS cells demonstrate distinct patterns of altered gene expression. A: mRNA expression of the target genes Oct4, Axin2, and Sox2 in AI-grown OS cells after the second passage (p2) was determined using qrt-PCR normalized to adherent cells. Asterisks indicate statistical significance (**p < 0.01). B: Principal component analysis of gene expression using microarray. Gene expression differences resulted in clustering of the adherent cells (squares) compared to the AI cells (triangles). This was more apparent for the patient-derived OS cells than the established OS cell lines. Blue color indicates established OS cell line, green color indicates pre-chemotherapy patient-derived OS cell line, red color indicates post-chemotherapy patient-derived OS cell line, black color indicates pulmonary metastasis patient-derived OS cell line, dotted lines connect paired AI/adherent samples. C: Shortest pathway analysis of the top 300 differentially expressed genes identifies class I HDACs, Runx2, and Akt as central contributors.
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Related In: Results  -  Collection

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Fig3: Anoikis-resistant OS cells demonstrate distinct patterns of altered gene expression. A: mRNA expression of the target genes Oct4, Axin2, and Sox2 in AI-grown OS cells after the second passage (p2) was determined using qrt-PCR normalized to adherent cells. Asterisks indicate statistical significance (**p < 0.01). B: Principal component analysis of gene expression using microarray. Gene expression differences resulted in clustering of the adherent cells (squares) compared to the AI cells (triangles). This was more apparent for the patient-derived OS cells than the established OS cell lines. Blue color indicates established OS cell line, green color indicates pre-chemotherapy patient-derived OS cell line, red color indicates post-chemotherapy patient-derived OS cell line, black color indicates pulmonary metastasis patient-derived OS cell line, dotted lines connect paired AI/adherent samples. C: Shortest pathway analysis of the top 300 differentially expressed genes identifies class I HDACs, Runx2, and Akt as central contributors.
Mentions: We assessed gene expression differences that may be important for OS cell anoikis resistance using both qrt-PCR and microarray gene expression profiling. Expression of genes involved in developmental programs including Sox2, Oct4, and Wnt signaling were increased in the AI-grown cells relative to adherent cells (Figure 3A). This finding was statistically significant for the mRNA expression of Sox2. The gene expression of established and patient-derived human OS cells cultured under AI conditions to at least the first passage was compared with that of cells cultured in adherent conditions using gene expression profiling with principal component analysis (PCA). There were clear differences in the molecular signatures between adherent and AI cells among established and patient-derived OS cells, although this was less obvious in the established cell lines (Figure 3B). A likely explanation is that the established OS cell lines used are clonally evolved and chemically transformed versions of primary tumor cells, whereas the patient-derived cell lines were grown in culture briefly (<5 passages) prior to analysis. Shortest pathway analysis of the top 300 differentially expressed genes confirmed significant alteration of Runx2, class I HDACs, and Akt (Figure 3C). GeneGo de-novo network generation identified convergence on Wnt/β-catenin, c-myc, and alpha family G-proteins in the molecular signature of AI growth (Additional file 1: Figure S1a and b). These findings pertaining to Runx2 and Wnt/β-catenin signaling are supported through western blot analysis (Additional file 1: Figure S1c). We identified 19 genes that passed false discovery with a corrected p-value less than 0.05 (Table 2). Upregulated genes included those involved in the DNA repair, oncogenic signaling, osteosarcoma pathogenesis, and differentiation, including those specific to mesenchymal lineages [23-26]. Downregulated genes were involved in processes such as proliferation and tumor suppressive functions [27,28]. These results suggest that anoikis-resistant OS cells capable of AI growth have expression changes in key genes that are necessary for cell growth and survival under these conditions.Figure 3

Bottom Line: We show that AI growth results in a global gene expression profile change accompanied by significant chemoresistance (up to 75 fold, p<0.05).AI cells were equally tumorigenic as their adherent counterparts, but showed a significantly decreased rate of growth in-vitro and in-vivo (p<0.05).Targeting epigenetic regulation of this process may be a viable therapeutic strategy.

View Article: PubMed Central - PubMed

Affiliation: Helen DeVos Children's Hospital, Spectrum Health System, Grand Rapids, MI, USA. jessica.foley@vai.org.

ABSTRACT

Background: Osteosarcoma (OS) is the most common type of solid bone cancer, with latent metastasis being a typical mode of disease progression and a major contributor to poor prognosis. For this to occur, cells must resist anoikis and be able to recapitulate tumorigenesis in a foreign microenvironment. Finding novel approaches to treat osteosarcoma and target those cell subpopulations that possess the ability to resist anoikis and contribute to metastatic disease is imperative. Here we investigate anchorage-independent (AI) cell growth as a model to better characterize anoikis resistance in human osteosarcoma while using an expression profiling approach to identify and test targetable signaling pathways.

Methods: Established human OS cell lines and patient-derived human OS cell isolates were subjected to growth in either adherent or AI conditions using Ultra-Low Attachment plates in identical media conditions. Growth rate was assessed using cell doubling times and chemoresistance was assessed by determining cell viability in response to a serial dilution of either doxorubicin or cisplatin. Gene expression differences were examined using quantitative reverse-transcription PCR and microarray with principal component and pathway analysis. In-vivo OS xenografts were generated by either subcutaneous or intratibial injection of adherent or AI human OS cells into athymic nude mice. Statistical significance was determined using student's t-tests with significance set at α=0.05.

Results: We show that AI growth results in a global gene expression profile change accompanied by significant chemoresistance (up to 75 fold, p<0.05). AI cells demonstrate alteration of key mediators of mesenchymal differentiation (β-catenin, Runx2), stemness (Sox2), proliferation (c-myc, Akt), and epigenetic regulation (HDAC class 1). AI cells were equally tumorigenic as their adherent counterparts, but showed a significantly decreased rate of growth in-vitro and in-vivo (p<0.05). Treatment with the pan-histone deacetylase inhibitor vorinostat and the DNA methyltransferase inhibitor 5-azacytidine mitigated AI growth, while 5-azacytidine sensitized anoikis-resistant cells to doxorubicin (p<0.05).

Conclusions: These data demonstrate remarkable plasticity in anoikis-resistant human osteosarcoma subpopulations accompanied by a rapid development of chemoresistance and altered growth rates mirroring the early stages of latent metastasis. Targeting epigenetic regulation of this process may be a viable therapeutic strategy.

Show MeSH
Related in: MedlinePlus