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

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.

Figure 1

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.

Figure 2

Anoikis-resistant OS cells show resistance to standard OS chemotherapies. Dose-response curves for cells grown under AI or adherent conditions treated with doxorubicin (top panel) or cisplatin (bottom panel) in a set of serial dilutions for 72 hours. Cell viability was determined as a percentage of the untreated control (0 μM doxorubicin or cisplatin).

Figure 3

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.

Figure 4

Altered growth rates of anoikis-resistant AI-grown cells are maintained in-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).

Figure 5

Vorinostat treatment and preconditioning inhibits anchorage-independent OS cell growth. A/B: mHOS cells were treated with either 2 μM vorinostat or DMSO alone and allowed to grow for 4 days in AI conditions. Vorinostat treatment decreased the number of spheres (A), and resulted in a statistically significant decrease in cell viability (B). C/D: Adherent mHOS cells were pretreated for 12 days with either 2 μM vorinostat or DMSO alone and subsequently plated and allowed to grow for 4 days in AI conditions in the absence of drug. Vorinostat pretreatment decreased the number of spheres (C) and resulted in a significant decrease in cell viability (D). Asterisks indicate statistical significance (*p < 0.05, ***p < 0.001).

Figure 6

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).