Abstract C96: The effect of differential expression of HES1 on the metastatic dormancy of osteosarcoma
Authors: McManus Madonna , Yang Yanwen , Hughes Dennis 
Published: August 2014

Osteosarcoma (OS) is the most common primary bone cancer in childhood. The majority of patients diagnosed with osteosarcoma will present with concurrent micro-metastatic disease. Interestingly, micro-metastatic lesions will typically remain dormant for anywhere from 6 months to 3 years after the surgical resection of the primary site. The Notch signaling pathway, a key component in normal bone development that is implicated as a key mediator in a number of various cancers, is initiated when membrane-bound ligands (Jagged 1 or 2, Delta-like 1, 3, or 4) on the surface of a cell interact with a membrane-bound Notch receptor (Notch 1, 2, 3, or 4) on another cell. This interaction induces the release of the intracellular domain of Notch (ICN). Once activated, ICN enters the nucleus where it forms a transcriptional complex with Mastermind-like 1 (MAM1) and promotes transcription of a number of CSL target genes including Hairy/Enhancer of Split 1 (HES1).

The effects of Notch1 and HES1 on OS are still not well understood. To alter Notch pathway member expression, OS cells (HOS, CCHD, CCHO) were transduced with GFP labeled retroviral constructs containing ICN1, the constitutively active intracellular domain of the Notch1 receptor, HES1, a constitutively active form of the transcription factor and downstream target of Notch activation, dnMAM, a truncated form of MAM that inhibits ICN induced transcription by binding to ICN1 but not CSL, and dnHES1, a dominant negative form of HES1 that does not allow HES1 to bind to its downstream targets. Cell proliferation was characterized using colony formation and competitive proliferation assays. Colony formation assays measure the ability for a cancer cell to produce a colony after any given treatment, while the competitive proliferation assay monitors GFP positivity in a mixed population by flow cytometry over a period of up to 50 days to compare changes in positive and negative populations. PARP cleavage and caspase activity were monitored to determine if changes in proliferation were due to apoptotic death.

Transduction of ICN1 and HES1 in HOS, CCHD, and CCHO incurs a proliferative disadvantage relative to non-transduced cells and cells transduced with dnMAM and dnHES1. HOS cells transduced with HES1 differentiate into two distinct populations: intermediate positive HES1 (HES1++) and high positive HES1 (HES1+++). HES1+++ expression increases caspase activity within 48 hours after transduction, while HES1++ did not incur an observed effect on the cell cycle throughout the 40 days of the experiment. Interestingly, however, when HES1++ cells are sorted from the GFP negative population, they fail to form colonies. This suggests that the GFP negative HOS population may provide growth stimulating signals to HES1++ HOS cells which are necessary for proliferation. This presents a potential mechanism that may explain clinical metastatic dormancy; as cells detach from the primary tumor, they are no longer exposed to the stimulatory effect of the tumor population, and are able to remain dormant and resistant to chemotherapeutics.

Citation Format: Madonna McManus, Yanwen Yang, Dennis Hughes. The effect of differential expression of HES1 on the metastatic dormancy of osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C96.