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Effect of hypoxia-inducible secreted protein, tenascin c, on 4T1 tumour cells in vitro and in vivo

University of British Columbia

Introduction: Metastatic cancer is responsible for 90% of cancer related deaths. Past research focused mainly on the primary tumour, leaving the process of metastasis poorly understood. Poorly oxygenated (hypoxic) tumour cells express hypoxia inducible proteins and have a more aggressive and invasive phenotype correlated with poorer prognosis. Hypoxic tumour cells are responsible for increased angiogenesis, invasion, matrix deposition and remodelling along with many other functions. We hypothesize hypoxic tumour secreted proteins are responsible for promoting metastasis. Our aim is to identify these hypoxia inducible secreted proteins and determine mechanisms promoting metastasis. Methods: Mammary carcinoma cells (4T1 – metastatic and 67NR – non-metastatic) were placed in 1% O₂ (hypoxic) or 21% O₂ (normoxic) for 24 hours. Stable Isotope Labelling of Amino acids in Cell culture (SILAC) and mass spectrometry were used to perform a quantitative proteomic screen of conditioned medium. Proteins in 4T1 conditioned media, up-regulated in hypoxia and absent from the 67NR results represented candidate secreted proteins in metastasis. Tenascin C (TNC), a candidate protein identified from the proteomic screen was stably knocked down and over-expressed. In vitro, the Boyden chamber and wound healing assay were used to study invasion and migration. In vivo, metastasis was assessed using flow cytometry-based quantification of metastasized tumour cells in the murine lungs. Results: (TNC) was identified as a secreted protein with a role promoting metastasis in vivo through enhanced migratory ability. In vitro, knockdown of TNC in 4T1 enhanced migratory ability whereas over-expression decreased migratory ability. These results were contradictory to the expected results based on the hypothesized in vivo role. However, in vivo knockdown of TNC in 4T1 tumour cells resulted in a significant decrease in lung metastases. These results are consistent with the expected role of TNC in vivo. Conclusions: Despite the contradictory results in vitro, TNC had a positive metastatic role potentially through a migratory mechanism. TNC represents a potential new therapeutic drug target. Given the 4T1 cell line results, these data support further examination of the migratory role of TNC and how it promotes metastasis. In addition, TNC expression in other tumour cell lines including human breast cancer should be examined.

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2014-02-06

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/45992

Pathology and Laboratory Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/