Hes4: A potential prognostic biomarker for newly diagnosed patients with high-grade osteosarcoma

Abstract

Background: Prognostic biomarkers for osteosarcoma (OS) at the time of diagnosis are lacking. Necrotic response of OS to preoperative chemotherapy correlates with survival and is determined 3-4 months after diagnosis. The purpose of this study is to identify biomarkers that will stratify patients into good or poor responders to chemotherapy at diagnosis and determine the role of potential biomarkers in OS pathogenesis.

Procedure: Because OS may be caused by disruptions of osteogenic differentiation, and the Notch pathway is one regulator of bone development, we examined the link between Notch effectors, OS differentiation, and OS outcome. We probed the R2: Genomics Analysis and Visualization Platform for RNA expression levels of Notch targets in mixed high-grade OS pretreatment biopsies. We used human OS cell lines in vitro and in mice to determine the role of the Notch target hairy/enhancer of split 4 (Hes4) in OS.

Results: We found that in OS patients, high expression of Hes4 is correlated with decreased metastasis-free and overall survival. Human OS cells that overexpress Hes4 are more immature and have an increased invasive capacity in vitro. This was not universal to all Notch effectors, as Hes1 overexpression induced opposing effects. When injected into NSG mice, Hes4-overexpressing OS cells produced significantly larger, more lytic tumors and significantly more metastases than did control cells.

Conclusions: Hes4 overexpression promotes a more aggressive tumor phenotype by preventing osteoblastic differentiation of OS cells. Hes4 expression may allow for the stratification of patients into good or poor responders to chemotherapy at diagnosis.

Keywords: Hes4; Notch; osteogenic differentiation; osteosarcoma; prognostic factor.

Figure 1.. High Hes4 expression in pretreatment high grade OS tumor biopsies correlates with decreased metastasis free and overall survival outcomes.

The R2: Genomics Analysis and Visualization Platform was used to generate Kaplan-Meier metastasis-free and overall survival curves using the Mixed Osteosarcoma - Kuijjer - 127 - vst - ilmnhwg6v2 data set. Genome-wide gene expression analysis was performed using 84 pretreatment high-grade diagnostic OS biopsy samples. Two different sets of control samples were used for comparison: osteoblasts (n = 3) and mesenchymal stem cells (n = 12; GEO accession number GSE28974). High Hes4 expression correlated with significantly lower probabilities of both overall and metastasis-free survival than low Hes4 expression (overall survival, P < 0.01; metastasis-free survival, P < 0.05).

Figure 2.. Hes4 overexpression increases OS cell invasion but not proliferation.

(A) Hes4 overexpression does not affect proliferation. The percentages of GFP-positive CCHD and HOS cells over time after stable retroviral transduction with GFP or GFP-Hes4 (normalized to day 5 after transduction) were quantified at various time points and expressed as the mean cell number (± SEM; n = 3). (B)Hes4 overexpression increases OS in vitro invasion. CCHD-GFP, CCHD-GFP-Hes4, HOS-GFP and HOS-GFP-Hes4 cells were plated on a 24-well BioCoat Matrigel invasion chamber with an 8-μm pore size. Medium with 10% fetal bovine serum was used in the bottom well of the chamber. At 24 (HOS) or 48 (CCHD) hours, migrated cells were counted. The graph shows the mean number of migrated cells per field (±SEM; n = 3). *P ≤ 0.05; ***P ≤ 0.001.

Figure 3.. Hes4 expression decreases osteoblastic differentiation.

Alizarin Red S staining of HOS-GFP and HOS-GFP-Hes4 cells was performed after 21 days in differentiation media. (A) Representative images of Alizarin Red S staining within 24 well plates. (B) Mean number of foci of calcium deposition per 24-well plate well (± SEM; n = 3). *P < 0.05.

Figure 4.. Overexpression of Hes4 increases the expression of RunX2 and osterix and decreases alkaline phosphatase.

(A) Hes4 overexpression increases RunX2 and osterix expression, and decreases alkaline phosphatase expression. RNA was harvested from CCHD and HOS cells 3–5 days after transduction with GFP or GFP-Hes4. RT-qPCR for RunX2, osterix, and alkaline phosphatase expression (as indicated) was normalized according to GAPDH expression relative to that in GFP-transduced control cells. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Bars, mean ± SEM (n = 3). (B) Even during forced differentiation, Hes4 prevents increased alkaline phosphatase expression. RNA harvested from CCHD and HOS cells 10–15 days after transduction with GFP or GFP-Hes4 and after 3–9 days of incubation with differentiation media. RT-qPCR was performed for alkaline phosphatase expression, normalized according to GAPDH expression relative to that in GFP-transduced control cells. **P ≤ 0.01. Bars, mean ± SEM (n = 3).

Figure 5.. Hes4 overexpression increases OS tumor aggressiveness.

Mice were injected with CCHD-GFP or CCHD-GFP-Hes4 cells into the tibia. Six-weeks post injection, all mice were sacrificed and processed for analysis. (A) CCHD-GFP-Hes4 cells formed significantly larger primary tumors than did CCHD-GFP. Each dot represents one tumor (n = 15). ***P < 0.001. (B) Mice injected with Hes4-overexpressing OS cells had significantly more lytic primary tumors than did control mice. a grading system (0–4, with 4 indicating the most lytic destruction) was used to quantify the extent of bone destruction in mice with primary CCHD-GFP or CCHD-GFP-Hes4 tumors. Each dot represents the lytic score for one tumor (mean ± SEM, n = 15). ***P < 0.001. (C) Mice injected with Hes4-overexpressing OS cells had significantly more metastases than did control mice. Metastatic lesions were quantified by counting vimentin-positive nodules on five sections per lung. Each dot represents one mouse (n = 15 per group). ***P < 0.0001. Black bars indicate 200μm, orange bars indicate 20 μm.

Figure 6.. Expression of osteogenic commitment markers in human OS samples correlates with significantly decreased overall and metastasis-free survival outcomes.

The R2: Genomics Analysis and Visualization Platform was used to generate Kaplan-Meier metastasis-free and overall survival curves using the Mixed Osteosarcoma - Kuijjer - 127 - vst - ilmnhwg6v2 data set. Genome-wide gene expression analysis was performed using 84 pretreatment high-grade diagnostic OS biopsy samples. Two different sets of control samples were used for comparison: osteoblasts (n = 3) and mesenchymal stem cells (n = 12; GEO accession number GSE28974). Patients with high RunX2 or osterix expression had significantly lower probabilities of both overall and metastasis-free survival than did those with low RunX2 or osterix expression. *P < 0.01; **P < 0.05.