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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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Related in: MedlinePlus

Anoikis-resistant OS are viable and exhibit lower growth rates. A-C: Representative AI-grown OS cells generated from mHOS (A), 143B (B), and a patient derived OS cell line, MS206-2 (C). D: Growth rates measured as cell doublings per day. Results shown are average ± standard error. Asterisks indicate statistical significance (**p < 0.01, ***p < 0.001). E: Western blot analysis of 143B and MG-63 OS cells shows decreased Ki67 protein levels in AI grown cells (P2 spheres) compared to adherent cells.
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Fig1: Anoikis-resistant OS are viable and exhibit lower growth rates. A-C: Representative AI-grown OS cells generated from mHOS (A), 143B (B), and a patient derived OS cell line, MS206-2 (C). D: Growth rates measured as cell doublings per day. Results shown are average ± standard error. Asterisks indicate statistical significance (**p < 0.01, ***p < 0.001). E: Western blot analysis of 143B and MG-63 OS cells shows decreased Ki67 protein levels in AI grown cells (P2 spheres) compared to adherent cells.

Mentions: When plated in ultra-low attachment plates, subpopulations of established and patient-derived human OS single cell suspensions generated spherical colonies within 4 days (Figure 1A-C). The viability of OS cells after continued growth in AI conditions remained high, comparable to that of adherent cells (data not shown). When we compared the growth rate of cells cultured in adherent conditions relative to those in AI conditions, cell doublings per day was 2-3 times greater for the adherent cells (Figure 1D). Prior studies have observed an increase in OS cell death in response to AI growth, which we also observed in some of our cell lines (data not shown) [22]. In cells surviving AI growth, we observed decreased Ki67 protein levels, indicating that proliferation differences can also contribute to these growth rate alterations (Figure 1E). Thus, subpopulations of human OS cells are capable of resisting anoikis and surviving AI growth, but at a lower growth rate.Figure 1


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 are viable and exhibit lower growth rates. A-C: Representative AI-grown OS cells generated from mHOS (A), 143B (B), and a patient derived OS cell line, MS206-2 (C). D: Growth rates measured as cell doublings per day. Results shown are average ± standard error. Asterisks indicate statistical significance (**p < 0.01, ***p < 0.001). E: Western blot analysis of 143B and MG-63 OS cells shows decreased Ki67 protein levels in AI grown cells (P2 spheres) compared to adherent cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419490&req=5

Fig1: Anoikis-resistant OS are viable and exhibit lower growth rates. A-C: Representative AI-grown OS cells generated from mHOS (A), 143B (B), and a patient derived OS cell line, MS206-2 (C). D: Growth rates measured as cell doublings per day. Results shown are average ± standard error. Asterisks indicate statistical significance (**p < 0.01, ***p < 0.001). E: Western blot analysis of 143B and MG-63 OS cells shows decreased Ki67 protein levels in AI grown cells (P2 spheres) compared to adherent cells.
Mentions: When plated in ultra-low attachment plates, subpopulations of established and patient-derived human OS single cell suspensions generated spherical colonies within 4 days (Figure 1A-C). The viability of OS cells after continued growth in AI conditions remained high, comparable to that of adherent cells (data not shown). When we compared the growth rate of cells cultured in adherent conditions relative to those in AI conditions, cell doublings per day was 2-3 times greater for the adherent cells (Figure 1D). Prior studies have observed an increase in OS cell death in response to AI growth, which we also observed in some of our cell lines (data not shown) [22]. In cells surviving AI growth, we observed decreased Ki67 protein levels, indicating that proliferation differences can also contribute to these growth rate alterations (Figure 1E). Thus, subpopulations of human OS cells are capable of resisting anoikis and surviving AI growth, but at a lower growth rate.Figure 1

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