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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

5-azaC treatment inhibits anchorage-independent OS cell growth while sensitizing to doxorubicin. A/B: Adherent mHOS cells were treated with either 2 μM 5-azaC or vehicle alone for 24 hours before being plated in AI conditions in the absence of drug and allowed to grow for 4 days. 5-azaC treatment decreased the cells ability to form spheres (A) and resulted in a significant decrease in cell viability (B). C/D: Adherent mHOS cells were treated with either 2 μM 5-azaC or vehicle alone for 24 hours before being plated in AI conditions in the absence of drug. 24 hours later the cells were treated with one concentration from a serial dilution of doxorubicin, with cell viability being measured after an additional 72 hours. 5-azaC treatment resulted in a left-shift in the doxorubicin dose-response curve (C) and a significant decrease in the percent IC50 relative to vehicle alone (D). Asterisks indicate statistical significance (*p < 0.05).
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Fig6: 5-azaC treatment inhibits anchorage-independent OS cell growth while sensitizing to doxorubicin. A/B: Adherent mHOS cells were treated with either 2 μM 5-azaC or vehicle alone for 24 hours before being plated in AI conditions in the absence of drug and allowed to grow for 4 days. 5-azaC treatment decreased the cells ability to form spheres (A) and resulted in a significant decrease in cell viability (B). C/D: Adherent mHOS cells were treated with either 2 μM 5-azaC or vehicle alone for 24 hours before being plated in AI conditions in the absence of drug. 24 hours later the cells were treated with one concentration from a serial dilution of doxorubicin, with cell viability being measured after an additional 72 hours. 5-azaC treatment resulted in a left-shift in the doxorubicin dose-response curve (C) and a significant decrease in the percent IC50 relative to vehicle alone (D). Asterisks indicate statistical significance (*p < 0.05).

Mentions: We next tested the effect of 5-azaC treatment on OS cell growth and the response to doxorubicin under AI conditions. We treated adherent mHOS cells with 2 μM 5-azaC or vehicle alone for 24 hours before plating in ultra-low attachment plates in the absence of drug. Treatment with 5-azaC decreased the ability of the mHOS cells to grow and survive in AI conditions (Figure 6A, B). Treatment with 5-azaC also resulted in a left-shift in the 72 hr dose-response curve of AI mHOS cells to doxorubicin, indicating that these cells were more sensitive to doxorubicin-mediated toxicity (Figure 6C). This was reflected in a statistically significant decrease in the 72 hr doxorubicin IC50 of 5-azaC treated AI cells compared to vehicle treated cells (Figure 6D). Treatment of adherent mHOS cells with 5-azaC trended towards inhibiting cell growth, although this was not statistically significant after the same number of controlled replicates (Additional file 3: Figure S2b). However, no clear difference in the response to doxorubicin was observed in adherent cells treated with 5-azaC (Additional file 3: Figure S2b). These results suggest that 5-azaC treatment can effectively inhibit the growth of anoikis-resistant OS cells while simultaneously sensitizing them to chemotherapy.Figure 6


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)

5-azaC treatment inhibits anchorage-independent OS cell growth while sensitizing to doxorubicin. A/B: Adherent mHOS cells were treated with either 2 μM 5-azaC or vehicle alone for 24 hours before being plated in AI conditions in the absence of drug and allowed to grow for 4 days. 5-azaC treatment decreased the cells ability to form spheres (A) and resulted in a significant decrease in cell viability (B). C/D: Adherent mHOS cells were treated with either 2 μM 5-azaC or vehicle alone for 24 hours before being plated in AI conditions in the absence of drug. 24 hours later the cells were treated with one concentration from a serial dilution of doxorubicin, with cell viability being measured after an additional 72 hours. 5-azaC treatment resulted in a left-shift in the doxorubicin dose-response curve (C) and a significant decrease in the percent IC50 relative to vehicle alone (D). Asterisks indicate statistical significance (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: 5-azaC treatment inhibits anchorage-independent OS cell growth while sensitizing to doxorubicin. A/B: Adherent mHOS cells were treated with either 2 μM 5-azaC or vehicle alone for 24 hours before being plated in AI conditions in the absence of drug and allowed to grow for 4 days. 5-azaC treatment decreased the cells ability to form spheres (A) and resulted in a significant decrease in cell viability (B). C/D: Adherent mHOS cells were treated with either 2 μM 5-azaC or vehicle alone for 24 hours before being plated in AI conditions in the absence of drug. 24 hours later the cells were treated with one concentration from a serial dilution of doxorubicin, with cell viability being measured after an additional 72 hours. 5-azaC treatment resulted in a left-shift in the doxorubicin dose-response curve (C) and a significant decrease in the percent IC50 relative to vehicle alone (D). Asterisks indicate statistical significance (*p < 0.05).
Mentions: We next tested the effect of 5-azaC treatment on OS cell growth and the response to doxorubicin under AI conditions. We treated adherent mHOS cells with 2 μM 5-azaC or vehicle alone for 24 hours before plating in ultra-low attachment plates in the absence of drug. Treatment with 5-azaC decreased the ability of the mHOS cells to grow and survive in AI conditions (Figure 6A, B). Treatment with 5-azaC also resulted in a left-shift in the 72 hr dose-response curve of AI mHOS cells to doxorubicin, indicating that these cells were more sensitive to doxorubicin-mediated toxicity (Figure 6C). This was reflected in a statistically significant decrease in the 72 hr doxorubicin IC50 of 5-azaC treated AI cells compared to vehicle treated cells (Figure 6D). Treatment of adherent mHOS cells with 5-azaC trended towards inhibiting cell growth, although this was not statistically significant after the same number of controlled replicates (Additional file 3: Figure S2b). However, no clear difference in the response to doxorubicin was observed in adherent cells treated with 5-azaC (Additional file 3: Figure S2b). These results suggest that 5-azaC treatment can effectively inhibit the growth of anoikis-resistant OS cells while simultaneously sensitizing them to chemotherapy.Figure 6

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