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This article has been retractedRetraction in: Clin Cancer Res. 2013 September 15; 19(18): 5256    See also: PMC Retraction Policy

A critical role of Notch signaling in osteosarcoma invasion and metastasis

Abstract

Purpose

Notch signaling is an important mediator of growth and survival in several cancer types, with Notch pathway genes functioning as oncogenes or tumor suppressors in different cancers. However, the role of Notch in osteosarcoma is unknown.

Experimental Design

We assessed the expression of Notch pathway genes in human osteosarcoma cell lines and patient samples. We then employed pharmacologic and retroviral manipulation of the Notch pathway and studied the impact on osteosarcoma cell proliferation, survival, anchorage-independent growth, invasion and metastasis in vitro and in vivo.

Results

Notch pathway genes, including Notch ligand DLL1, Notch 1 and 2, and the Notch target gene HES1 were expressed in osteosarcoma cells, and expression of HES1 was associated with invasive and metastatic potential. Blockade of Notch pathway signaling with a small molecule inhibitor of gamma secretase eliminated invasion in matrigel without affecting cell proliferation, survival, or anchorage-independent growth. Manipulation of Notch and HES1 signaling demonstrated a crucial role for HES1 in osteosarcoma invasiveness and metastasis in vivo.

Conclusion

These studies identify a new invasion and metastasis-regulating pathway in osteosarcoma and define a novel function for the Notch pathway: regulation of metastasis. Since the Notch pathway can be inhibited pharmacologically, these findings point toward possible new treatments to reduce invasion and metastasis in osteosarcoma.

Introduction

Osteosarcoma is the third most common cancer in childhood and adolescents and the most common cancer of bone (1). With combination treatment (neoadjuvant chemotherapy, surgery and adjuvant chemotherapy), the 5-year survival for patients who do not have metastatic disease at diagnosis is 60 to 70% (2-4). However, for patients who present with metastatic disease or whose tumor recurs, outcomes are far worse: less than 30% and less than 20% survival, respectively (5). Pulmonary metastasis is the predominant site of osteosarcoma recurrence and the most common cause of death. Unfortunately, survival has not improved for twenty years despite multiple clinical trails with increased intensity, and further gains with refinements in cytotoxic chemotherapy regimens alone are unlikely. Thus new therapeutic targets and approaches must be sought.

Our knowledge of the mechanistic control of invasion and metastasis in osteosarcoma is limited. Only Ezrin and Fas have been linked mechanistically to osteosarcoma metastasis. Ezrin is a cell membrane-cytoskeleton linking protein that allows the cell to interact with the microevironment and facilitates signal transduction (6). Impaired Fas signaling may allow osteosarcoma cells to evade host resistance in the lung (7). Thus far neither Fas nor Ezrin has been a likely target for drug therapy, illustrating the need for new therapeutic targets for osteosarcoma. ERBB2 (Her-2) has been shown to associate with poor clinical outcome of osteosarcoma (8). Other ERBB family member proteins, EGFR and nuclear Her4, are also expressed in osteosarcoma (9). Inhibition of ERBB family signaling by CI-1033 (pan-ERBB inhibitor) induces cell growth inhibition and apoptosis (10), suggesting that ERBB signaling is a potential therapeutic target for osteosarcoma.

Notch signaling plays a key role in the normal development of many tissues and cell types through diverse effects on cell fate decision, stem cell renewal, differentiation, survival and proliferation (11). The Notch signaling pathway includes Notch ligands, receptors, negative and positive modifiers, and Notch target transcription factors. The Notch genes (Notch1- Notch4), originally identified by homology to a single Notch gene from Drosophila, encode highly conserved cell surface receptors (12, 13). After activation by ligand binding, the Notch proteins are proteolytically cleaved in two steps by ADAM10 and γ–secretase, after which the intracellular domain of Notch (ICN) is translocated to the nucleus (14). Nuclear ICN interacts with the transcription factor CSL (15), also known as RBP-Jκ, and the mastermind-like (MAML) protein, which leads to transcriptional activation of CSL target genes (16). These include the basic helix-loop-helix (bHLH) transcription factors of the Hes family and Hes-related repressor proteins (Herp), e.g. HES1, HES5, HERP2 (15). Hes and Herp family proteins are transcriptional repressors. Independent of CSL activity, intracellular Notch receptors can interact with the Notch target protein DELTEX1 (DTX1) (17), which modulates Notch-mediated transcription and promotes feedback inhibition of Notch pathway signaling.

Notch signaling is aberrantly activated in a variety of human cancers, including T-cell acute lymphoblastic leukemia (T-ALL), lung, colorectal, prostate and breast carcinomas (18-21). More recently, a tumor suppressor role for Notch signaling has been identified in B cell malignancies (22), neural crest tumors (23) and skin cancer (24). Therefore, Notch signaling appears to function as an oncogene or a tumor suppressor, depending on the cellular context. Pharmacologic manipulation of Notch signaling is becoming a new strategy for human cancers. γ–Secretase inhibitors (GSI), originally developed for Alzheimer’s disease, can inhibit the proteolytic processing of Notch receptors by γ–secretase, which is essential for Notch activation (25), and are being investigated clinically in T cell leukemia and breast cancer.

Notch signaling is also involved in bone development. Notch-deficient mice have severe skeletal abnormalities (26-28). Strikingly, mutations in the Notch ligand DLL3 are responsible for “pudgy” mice and spondylocostal dysostosis in humans (29, 30). Furthermore, overexpression of Notch ligand and receptors impairs osteoblastic and osteoclastic cell differentiation from precursor cells (31, 32). Notch receptor expression has been reported in osteosarcoma (33, 34). However, the function of Notch pathway in osteosarcoma has not been established.

To determine the role of Notch signaling in osteosarcoma, we measured the impact of Notch pathway expression on cell proliferation, transformation and invasion in human osteosarcoma models. Through pharmacologic and direct retroviral modulation of Notch pathway, we found that Notch signaling induces invasiveness and metastasis of osteosarcoma in vitro and in vivo, but does not affect cell proliferation, survival or tumorigenesis. More importantly, the Notch target gene HES1 is sufficient to induce an invasive and metastatic phenotype in osteosarcoma.

Materials and Methods

Patients

Human tumor cell samples were obtained from malignant effusions removed from patients with widely disseminated osteosarcoma. Both patients consented to have tumor tissue banked for research purposes under a protocol approved by the Institutional Review Board of U. T. M. D. Anderson Cancer Center.

Cell culture and experimental reagents

Human osteosarcoma cell lines OS 187, COL and KRIB were described previously (9, 35). Human osteosarcoma cell line SAOS2 was purchased from ATCC (American Type Culture Collection) and its metastatic subline LM7 was derived at our institution from SAOS2 cells by seven serial passages as metastatic pulmonary nodules in immunodeficient mice (36). Normal human osteoblast cells (hOSB) were purchased from Cambrex Bio Science and cultured in osteoblast basal medium (OBM) (Cambrex) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 1% osteoblast growth supplement (Cambrex) and 1% penicillin/streptomycin. Osteosarcoma cell lines OS187, SAOS2, LM7 and KRIB were cultured in complete DMEM medium [DMEM (Invitrogen) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin]. COL cells were cultured in complete DMEM medium containing 1% Insulin-Transferrin-Selenium-A (ITS) (Invitrogen). All cells were incubated in a 5% CO2 atmosphere at 37°C. γ–Secretase inhibitor XXI (Compound E, (S,S)-2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide) was purchased from Calbiochem. All other chemicals and solutions were from Sigma-Aldrich unless otherwise indicated.

RT-PCR and quantitative realtime PCR analyis

TRIzol Reagent (Invitrogen) method was used for RNA isolation. First strand cDNA was synthesized from total RNA with oligo(dT) primers (Invitrogen) as described (37). The cDNA products were used for RT-PCR and quantitative PCR analysis. The primer sequences are listed in Supplemental Table 1. Human actin was used as an internal control. Real-time PCR analysis was performed using the iCycler iQ quantitative PCR system (Bio-rad) using 2×SYBR Green PCR Master Mix (Bio-rad) following manufacturer’s protocol. Data were analyzed according to the comparative Ct method and were normalized to actin expression in each sample (9).

Cell proliferation, anchorage-independent growth and cell cycle analysis

Cell growth was measured daily with Vi-Cell cell viability analyzer (Beckman Coulter). Anchorage-independent growth was examined by colony formation in soft agar. Briefly, cells were suspended in DMEM containing 0.4% agar and 10% FBS, and plated onto a bottom layer containing 0.8% agar. The cells were plated at a density of 1×103 per well in a 24-well plate, and colonies were counted 14 days later. Each condition was analyzed in triplicate, and all experiments were repeated three times. For cell cycle analysis, cells were fixed in ethanol and stained with 50 μg/ml propidium iodide (PI) in saline overnight. Alternatively, DRAQ5 (Alexis biochemicals) was added to live cells to a final concentration of 5 μM for 10 minutes. PI and DRAQ5 were measured using a FACScaliber flow cytometer (BD) and analyzed with FlowJo software (Tree Star Inc).

Invasion assay

The invasion ability of osteosarcoma cells with or without treatment was tested by BD Matrigel™ Invasion Chamber (8 micron pore size) (BD biosciences) according to manufacturer’s protocol. Briefly, 5×104 osteosarcoma cells in 0.5ml of serum-free medium were seeded into the upper chamber of the system. Bottom wells in the system were filled with DMEM medium with 10% FBS as a chemoattractant. After incubation, the cells in the upper chamber were removed, and the cells outside of the bottom membrane were stained with HEMA3 stain set (Fisher Diagnostics). Preliminary studies determined 48 hours incubation optimal, while too few cells had migrated by 24 hours. Therefore, all studies presented here were assessed at 48 hours, as recommended by the manufacturer. Cell migration was quantified by direct microscopic visualization and counting.

Western blot analysis

Cells were lysed in lysis buffer (1% Triton X-100, 150mM NaCl, and 20mM Na2PO4) with Complete Protease Inhibitor Cocktail (Roche Diagnostics). The protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA). The total cell lysates were separated using SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked with 4% BSA in 1× TBS-T buffer for 30 minutes and then incubated with rabbit anti-HES1 antibody (1:1000) (Chemicon) for 2 hours. HRP-conjugated anti-rabbit IgG (1:3000) (GE Healthcare) was used as the secondary antibody and the chemiluminescent signals were detected by Immobilon Western detection system (Millipore).

Retroviral Transduction

To modulate Notch signaling in tumor cells, the intracellular domain of human Notch1 (ICN1, aa1760-2555), dominant negative mastermind (dnMAM, aa13-74), or full length HES1 was inserted into MSCV-based retroviral vector MigR1 co-expressing GFP as an expression marker (kind gifts from Jon Aster and Warren Pear) (22, 38). Retroviral transduction procedures were described previously (22). After transduction, cells were either analyzed for cell cycle profiles of GFP+/− cells in the whole population, or sorted for >99% GFP+ cells for subsequent assays as indicated in individual experiments.

In vivo orthotopic mouse model of osteosarcoma lung metastasis, radiographic and histological analysis

Animal experiments were approved by the University of Texas MD Anderson Cancer Center Committee on Use and Care of Animals. Human osteosarcoma cells (2×105 in 20μl of the cell suspension) were injected into left tibia of 5-week-old NOD/SCID/IL2Rgamma-deficient mice (Jackson Laboratories). Tumor size was measured with calipers weekly. To examine the osteolytic lesions, radiographs were taken with a Faxitron MX-20 X-ray machine (Faxitron X-ray). Six weeks after injection, the mice were sacrificed. Serial sections of lung were prepared and stained with hematoxylin and eosin (H&E). Metastatic nodules in lungs were quantified by direct microscopic visualization and counting of a single lung section.

Statistics and supplemental data

Triplicate samples were analyzed in each experiment, and experiments were repeated three times, unless otherwise indicated. Mean, standard deviation (SD) and P values base on the 2-tailed t test were calculated with Excel X (Microsoft). The supplemental data, including seven supplemental figures and one supplemental table, can be found with this article online.

Results

The Notch pathway is present in osteosarcoma cells and correlates with tumor metastatic phenotype in osteosarcoma cells

To assess the status of the Notch pathway in osteosarcoma, we examined Notch pathway gene expression by semi-quantitative and quantitative PCR in normal human osteoblasts (hOSB) and four human osteosarcoma cell lines: OS187, COL, SAOS2 and its metastatic subline SAOS2-LM7 (LM7). We measured expression of human Notch ligand DLL1, receptors Notch1-4, and downstream targets HES1, HES5, HERP2 and DTX1 genes (Figure 1A and 1B). Notch pathway genes are present in normal osteoblasts and osteosarcoma cells. OS187, COL and LM7 cells have strong expression of DLL1, Notch1-3 and downstream target genes HES1, and HERP2. The expression of Notch signaling members shows correlation with metastatic phenotype in SAOS2 and its metastatic cell line LM7 (Figure 1A and 1B). The metastatic subline LM7 has strong DLL1, Notch1-2 and HES1, weak HERP2 expression, but no Notch4 expression, while parental SAOS2 has only Notch4, but not DLL1 and Notch1-3 expression. We also assessed tumor samples from two patients with widely metastatic osteosarcoma and malignant effusions. Notch1, 2 and 4, HES1 and HERP2 were expressed in metastatic patient samples. These results suggested that Notch may be active and associated with metastasis in osteosarcoma.

Figure 1
Notch and HES1 expression are correlated with metastatic phenotype in osteosarcoma cells and tumors of osteosarcoma patients. A: RT-PCR analysis of Notch pathway genes (ligand DLL1, receptors NOTCH1-4, and downstream targets HES1, HES5, HERP2 and DTX1 ...

Downregulation of Notch signaling by γ–secretase inhibition suppresses osteosarcoma cell invasion, but has no effect on cell proliferation and tumorigenesis

To test if the Notch pathway contributes to osteosarcoma pathogenesis, a small molecule inhibitor of γ-secretase (GSI) was used to inhibit Notch signaling pharmacologically in osteosarcoma cells. In OS187 cells, the Notch target gene HES1 was downregulated at both the mRNA and protein levels by GSI at 0.1 and 0.3 nM concentrations, and abolished at 1 nM concentration (Figure 2A), suggesting that Notch signaling is blocked by GSI at sub-nanomolar concentrations, as expected with this GSI.

Figure 2
Effects of Gamma-secretase inhibitor (GSI) on the cell proliferation and in vitro invasiveness of osteosarcoma cells. A: Notch signaling inhibition by GSI at the indicated concentrations was inferred by HES1 expression at mRNA and protein level. OS187 ...

We then assessed proliferation, tumorigenesis and invasiveness in vitro under GSI treatment. GSI did not affect proliferation in any osteosarcoma cell lines tested (Figure S1A-D). Nor did it affect in vitro tumorigenesis of these cell lines as measured by colony formation in soft agar (Figure S2A-D).

We also measured the effects of GSI on the tumor cell invasion in vitro. We seeded the cells in the matrigel transwells for 24 hour and 48 hour. We found that very few cells can invade through the matrigel at 24 hours. After 48 hours, tumor cells can invade through the matrigel well. We also assessed the growth of cells on matrigel for 24 and 48 hours, and found no difference in total cell number between the GSI treatment and control group (data not shown). Therefore all matrigel experiments were assessed at 48 hours. Our results show that GSI significantly suppressed the invasiveness of osteosarcoma cell line OS187 in a dose-dependent manner (Figure 2B). SAOS2 cells have low metastatic potential and weak invasiveness. In contrast, LM7, the metastatic subline of SAOS2, showed strong invasiveness in vitro, compared to parental SAOS2 cells, and GSI reduced the invasiveness of LM7 to levels similar to SAOS2 (Figure 2C). In one cell line, COL, GSI treatment did not affect invasiveness (Figure 2D). However, HES1 expression in this line also did not change with GSI treatment, even up to 20 μM (data not shown), suggesting that HES1 expression is sustained in the absence of Notch receptor cleavage in this particular cell line.

Manipulation of Notch signaling controls the invasiveness of osteosarcoma cell lines

γ–Secretase can regulate multiple proteins, including Notch receptors, ERBB4, CD44 and cadherins (39), by cleaving the transmembrane domain of these proteins. To confirm that the effects of GSI on tumor cell invasion result specifically from Notch inhibition, we manipulated Notch signaling activity in osteosarcoma cells using Notch pathway gene expression constructs. Osteosarcoma cells were transduced with retroviral constructs expressing the constitutively active intracellular domain of Notch1 (ICN1), the Notch target gene HES1, the Notch pathway inhibitor dominant negative Mastermind-like (dnMAM) or the empty vector (MigR1) to upregulate or downregulate Notch signaling. In osteosarcoma cells, manipulation of Notch signaling had no effect on the cell proliferation (Figure S3A-D), cell cycle (Figure S4A-D) or in vitro tumorigenesis (Figure S5A-D).

However, upregulation of Notch signaling by ICN1 increased the in vitro invasiveness of OS187 and SAOS2 (Figure 3A and 3B) cell lines. Importantly, expression of HES1 was sufficient to induce this invasiveness. Consistent with these findings, down-regulation of Notch signaling by dnMAM decreased the invasion ability of OS187 (Figure 3A) and LM7 cell lines (Figure 3C), similar to the effects of GSI. RT-PCR showed that HES1 expression was upregulated by ICN1 and downregulated by dnMAM in OS187, SAOS2 and LM7 (Figure 3A-C) cells. LM7 cells transduced with ICN1 or HES1 did not survive after cell sorting in three separate experiments, presumably due to toxicity in these particular cell lines. We also measured additional Notch downstream targets HES5, HERP2 and DTX1 in OS187 cells transduced with ICN1, dnMAM, HES1 or vector. We found that neither HES5, HERP2 nor DTX1 expression correlated with the invasiveness of OS187 cell line (data not shown). These data suggest that the impact of Notch signaling on osteosarcoma invasiveness is likely mediated by the expression of HES1.

Figure 3
Effects of manipulation of Notch signaling on osteosarcoma invasiveness in vitro. A-C: Top, relative invasiveness in vitro of OS187 cells (1×104 cells/well) (A), SAOS2 cells (1.5×105 cells/well) (B) and LM7 cells (5×104 cells/well) ...

Transduction with constitutively active Notch1 rescues osteosarcoma cells from GSI-mediated inhibition of invasion

We have shown that inhibition of γ-secretase, an enzyme necessary for Notch activation, impeded the invasiveness of osteosarcoma cells in vitro (Figure 2). Retroviral transduction of Notch pathway genes also demonstrated that Notch mediated HES1 expression was sufficient to enhance osteosarcoma invasion. To confirm that the GSI effect is due to its impact on the Notch pathway, we assessed the ability of ICN1 and HES1 to rescue osteosarcoma invasiveness from GSI treatment. OS187 cells transduced with constitutively active ICN1 or HES1 retained invasive potential even in 1nM GSI, while transduction with the empty vector conferred no resistance (Figure 4A and B). These data demonstrate that the loss of osteosarcoma invasiveness induced by GSI can be rescued by Notch-mediated HES1 expression.

Figure 4
Effects of GSI (1nM) on in vitro invasiveness of OS187 cells transduced with empty vector, ICN1 (*P<0.05) and HES1 (*P<0.05). GSI (1nM) significantly suppressed invasion of OS187 cells transduced with empty vector. However, OS187 cells ...

Inhibition of Notch/HES1 signaling suppresses osteosarcoma metastasis in vivo

To investigate the effect of Notch signaling inhibition on osteosarcoma tumor metastasis in vivo, we developed a novel orthotopic osteosarcoma murine xenograft model using OS187 cells. Intra-tibial injection of unmodified human OS187 cells in NOD/SCID/IL2Rgamma mice induced primary osteosarcoma tumor formation two weeks after inoculation. These primary tumors gave rise to microscopically detectible micrometastases in the lungs within 4-6 weeks after injection, mimicking the malignant process from early tumor growth to development of lung metastasis. Using this OS187 orthotopic xenograft model, we examined the effects on metastasis of inhibition of Notch signaling and HES1 expression by dnMAM. OS187 cells were transduced with either dnMAM or the empty vector prior to intratibilial injection, and primary tumors were allowed to grow for six weeks. Radiographs of the primary tumors demonstrated no significant difference between dnMAM-transduced and empty vector-transduced cells in the osteolytic primary lesions formed (Figure 5A and B). The primary tumors from dnMAM and empty vector control cells had similar tumor latency and growth rates (Figure 5C). However, inhibition of Notch signaling and HES1 expression by dnMAM significantly decreased the number of lung metastases observed (P<0.005, Figure 5A, B and D). We counted all visible micrometastases (tumor cluster with > 10 cells) in a single 5 micron thick coronal section from the middle of the lungs. The vector control group had an average of 15 metastases per slice of lung, while the dnMAM group averaged only one small metastatic focus. These results demonstrate that blocking Notch and HES1 signaling prevented osteosarcoma invasion and metastasis in vivo.

Figure 5
Effects of manipulation of Notch signaling on osteosarcoma invasiveness and metastasis in vivo. A and B: Representative X-ray images (left) show the osteolytic lesion in the primary tumor site (tibia) of both vector and dnMAM group. Two representative ...

Discussion

Metastasis causes 90% of human cancer deaths (40) and defining the mechanisms controlling metastasis is essential to improving cancer survival. For osteosarcoma, lung metastasis is the major cause of death, since the primary tumor usually can be treated effectively with surgery and chemotherapy. If the mechanisms regulating invasion and metastasis of osteosarcoma can be clearly defined, there are likely to be key elements that can be exploited therapeutically, reducing metastasis and improving survival. The Notch pathway may yield treatment targets for many, but not all, cancers, since Notch can act as an oncogene in some malignancies and a tumor suppressor in others (21). Identifying the Notch pathway components that control metastasis is likely to be key in targeting this pathway for osteosarcoma and other solid tumors.

Our report specifically identifies Hes1 as the Notch pathway gene that is critical for osteosarcoma invasion. Recently, Notch signaling was suggested to promote the invasive ability of pancreatic cancer cells through upregulating VEGF and MMPs expression in vitro (41). The functions defined here are distinct, since Hes1 signaling is not involved in anchorage independent growth and cell proliferation in vitro or tumor latency and growth in vivo of osteosarcoma, and VEGF and MMPs appear unaffected by Hes1 in osteosarcoma (data not shown). Therefore, our results demonstrate that invasive behavior can be separated from other cancer characteristics, and that HES1 signaling causes invasion and may be responsible for the Notch-mediated metastatic phenotype in osteosarcoma. This essential and specific role of Notch signaling on the invasion and metastasis has not been described previously in any tumor types.

Notch signaling can induce expression of several downstream target genes, including HES1, HES5, and HERP2 (20). Other Notch downstream targets include pre-T alpha, cyclin D1, NF-κB and p21Cip1 (20). The diversity of Notch functions in different cell lineages is achieved through tissues-specific activation of particular downstream target genes. Our data demonstrate that Notch signaling controls invasion and metastasis, and that the Notch downstream target HES1 is the effector. Because HES1 is a transcriptional regulator, other functional genes related to invasion and metastasis should be regulated by HES1. There are several known classes of proteins involved in the cell-to-microenvironment interaction and invasive or metastatic process, such as cell-cell adhesion molecules, extracellular proteases and angiogenesis promoting factors (42). Our efforts to identify HES1 regulated targets have excluded several known metastatic genes, such as cell scaffolding protein Ezrin, cell adhesion molecule ICAM1, matrix-degrading protease MMP9, and VEGF (data not shown). But our screening for HES1 target genes is still limited. An unbiased high-throughput screening, such as cDNA microarray analysis, will likely be necessary to identify all the relevant genes regulated by HES1 in osteosarcoma.

Invasiveness of cell line COL is resistant to GSI treatment, because GSI cannot suppress HES1 expression in this cell line. This result suggests either that Hes1 expression is Notch-independent in COL cells, or that these cells have an as-yet uncharacterized activating mutation in the Notch pathway. This mechanism remains to be elucidated.

To study the mechanisms involved in the pathogenesis of human osteosarcoma and evaluate the drug efficacy in preclinical studies, animal models are critical. The KRIB model is a well-established orthotopic osteosarcoma model, in which intra-osseous implantation forms local tumor within 4 weeks and subsequently metastasize to the lung in 6 weeks (35). However, KRIB cells were derived from human osteosarcoma cells transformed with oncogenic Ras, a mutation never found in spontaneous osteosarcoma samples (35). The LM7 metastatic model requires intravenous administration, and does not form orthotopic tumors (36). Thus LM7 is unable to examine mechanisms of metastasis relating to release of cancer cells from the primary tumor. The OS 187 orthotopic tumor xenograft model is an improved model for several reasons. OS187 cells are human osteosarcoma tumor cells without any experimentally-induced genetic modification. This orthotopic model can reproduce all aspects of malignant behavior, from the establishment and growth of primary tumors to the subsequent micrometastatic hematogenous spread to the lungs. It is amenable to genetic and pharmacologic manipulation, which should prove useful both for mechanistic studies and preclinical testing of promising new therapies. Using this model, we demonstrated the importance of Notch pathway signaling for osteosarcoma metastasis.

Therapeutic agents that target Notch signaling are being developed for antitumor effects. Originally, GSIs were developed for potential use in prevention of Alzheimer’s disease, in which the proteolytic cleavage of the β-amyloid precursor protein (APP) by β-secretase and γ-secretase leads to the accumulation of neurotoxic Aβ peptide in the brain (39). However, the clinical trails demonstrated potent Notch pathway inhibition. With the discovery of aberrant Notch signaling in human cancer, the potential use of GSI for various human malignancies has been explored. Currently, GSI (γ-secretase inhibitor) small molecules that target proteolytic step of Notch activation is being evaluated for advanced breast cancer and relapsed or refractory T-ALL (NCI Clinical Trial Protocol ID: 2004_97NCT00100152, http://www.cancer.gov/clinicaltrials). Our results suggest that Notch and HES1 signaling may be a rationale therapeutic target to prevent metastasis for human osteosarcoma patients, and using GSI or ADAMS inhibitors may reduce the rate at which metastatic disease develops. Our data also indicate that Notch pathway activation, especially HES1 gene expression, should be evaluated as a novel marker for the metastatic potential of osteosarcoma in patients.

In conclusion, we reveal a specific and essential role of Notch signaling in promoting osteosarcoma invasiveness and metastasis in vitro and in vivo. Our report also suggests that HES1 merits investigation as a prognostic marker for osteosarcoma. Furthermore, inhibition of Notch signaling by small molecular inhibitors may have important therapeutic applications for treating and preventing osteosarcoma metastasis.

Supplementary Material

Supplemental data

Supplementary Figure 1. Cell proliferation rate of osteosarcoma cells with GSI treatment. Cell proliferation rates are not affected by GSI in OS187 (A), COL (B), SAOS2 (C) and LM7 (D) cells. Cell proliferation rate with GSI treatment was accessed by counting cell yield for 5 days.

Supplementary Figure 2. Anchorage-independent growth of osteosarcoma cells with GSI treatment. Soft agar colony formation is not affected significantly by GSI in OS187 (A), COL (B), SAOS2 (C) and LM7 (D) cells.

Supplementary Figure 3. Cell proliferation rate of osteosarcoma cells with manipulation of Notch signaling. Cell proliferation is not affected by ICN1, dnMAM or HES1 in OS187 (A), COL (B) SAOS2 (C) and LM7 (D) cells. Cells were transduced with vector, ICN1, dnMAM or HES1, and analyzed by flow cytometry. The percentage of GFP+ cells (transduced) was measured 24 (day 0), 48 (day1), 72 (day 2) and 96 hours (day3) after transduction, then normalized to day 0.

Supplementary Figure 4. Cell cycle profiles of GFP+ cells (transduced with empty vector, ICN1, dnMAM or HES1 of OS187 (A), COL (B) SAOS2 (C) and LM7 (D). GFP+ cells are sorted and analyzed by flow cytometry.

Supplementary Figure 5. Anchorage-independent growth of osteosarcoma cells with manipulation of Notch signaling. Soft agar colony formation is not affected significantly by ICN1, dnMAM or HES1 in OS187 (A), COL (B) SAOS2 (C) and LM7 (D).

Supplementary Table 1 Primers used for RT-PCR and quantitative PCR in the study.

Acknowledgements

The authors thank Dr. Eugenie Kleinermen and Dr. Douglas D. Boyd for helpful discussions and Dr. Shufang Jia and Ms. Wendy Fang for technical help.

Grant Support: This work is supported by Physician Scientist Awards from U.T. M.D. Anderson Cancer Center to Dr. D. Hughes and Dr. P. Zweidler-McKay. Dr. Hughes also is supported in part by NIH grant #5K08CA118730-02. Dr. P. Zhang is supported by the Jori Zemel Children’s Bone Cancer Foundation (www.JoriZemel.com) and Hope Street Kids Foundation (www.hopestreetkids.org). The flow cytometry core laboratory is supported in part by NIH grant #5P30CA016672-32.

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