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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

Altered growth rates of anoikis-resistant AI-grown cells are maintainedin-vivo. mHOS (A) and MS206-2 (B) cells were under AI (red) and adherent conditions (blue) and were injected intratibially into athymic nude mice. Data shown are trend lines fit to the growth data, with standard deviations for the average tumor volume for that time point. Asterisks indicate statistical significance (**p < 0.01).
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Fig4: Altered growth rates of anoikis-resistant AI-grown cells are maintainedin-vivo. mHOS (A) and MS206-2 (B) cells were under AI (red) and adherent conditions (blue) and were injected intratibially into athymic nude mice. Data shown are trend lines fit to the growth data, with standard deviations for the average tumor volume for that time point. Asterisks indicate statistical significance (**p < 0.01).

Mentions: To validate whether this alteration in growth rate would hold true in an in-vivo setting, 2×105 AI and adherent mHOS cells were injected intratibially into athymic nude mice. Although no differences were noted in tumor initiation rates, there were significant differences in the growth rates once tumors were detected. The tumors derived from AI-grown cells grew more slowly than those from adherent cells (Figure 4). AI-grown patient-derived MS206-2 cells also grew more slowly, although the results were not statistically significant. These results indicate that the slow-growth phenotype of cultured OS cells grown under AI conditions is maintained in-vivo.Figure 4


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)

Altered growth rates of anoikis-resistant AI-grown cells are maintainedin-vivo. mHOS (A) and MS206-2 (B) cells were under AI (red) and adherent conditions (blue) and were injected intratibially into athymic nude mice. Data shown are trend lines fit to the growth data, with standard deviations for the average tumor volume for that time point. Asterisks indicate statistical significance (**p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Altered growth rates of anoikis-resistant AI-grown cells are maintainedin-vivo. mHOS (A) and MS206-2 (B) cells were under AI (red) and adherent conditions (blue) and were injected intratibially into athymic nude mice. Data shown are trend lines fit to the growth data, with standard deviations for the average tumor volume for that time point. Asterisks indicate statistical significance (**p < 0.01).
Mentions: To validate whether this alteration in growth rate would hold true in an in-vivo setting, 2×105 AI and adherent mHOS cells were injected intratibially into athymic nude mice. Although no differences were noted in tumor initiation rates, there were significant differences in the growth rates once tumors were detected. The tumors derived from AI-grown cells grew more slowly than those from adherent cells (Figure 4). AI-grown patient-derived MS206-2 cells also grew more slowly, although the results were not statistically significant. These results indicate that the slow-growth phenotype of cultured OS cells grown under AI conditions is maintained in-vivo.Figure 4

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