Identification of C1orf106 as a TGF-β target gene which promotes clonogenicity and anchorage-independent growth


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Lauren Strathearn1,Susan Mason2,Lindsay Spender1,Karen Blyth2,Gareth Inman1
1University of Dundee,2CRUK Beatson Institute

Abstract

Background

The dual and opposing roles of Transforming Growth Factor-β signalling (TGF-β) in tumorigenesis are well recognized.  However the exact mechanisms behind this switch in biology from tumour suppressive to tumour promoting are complex and lesser understood.  Microarray analysis identified the uncharacterised open reading frame C1orf106 as a novel TGF-β target gene required for TGF-β induced anchorage-independent growth of vulval squamous cell carcinoma cells.  Here we further investigate TGF-β regulation of C1orf106 and its potential role in tumorigenesis.

 

Method

C1orf106 gene and protein expression was assayed by qRT-PCR and western blotting following exogenous stimulation or inhibition of TGF-β signalling.  The role of the canonical TGF-β pathway in regulating C1orf106 transcriptional induction was explored by siRNA-mediated gene silencing.  C1orf106 mRNA expression levels and influence on clinical outcomes were assessed in the Oncomine™ and KM Plotter platforms.  The phenotypic effects of stable overexpression or shRNA mediated knockdown of C1orf106 expression was investigated in breast cancer cell lines.  Anchorage-independent growth and colony-forming assays, and a limiting dilution xenograft were carried out to assess tumourigenicity in vitro and in vivo, respectively.

Results

C1orf106 mRNA and protein induction in response to TGF-β stimulation was observed in a plethora of cell types and determined to be SMAD3-dependent.  Analysis of publically available datasets indicated that elevated C1orf106 mRNA expression is associated with poor clinical outcomes in breast cancer. C1orf106 expression correlated with metastatic progression in the 4T1 murine mammary carcinoma model.  High C1orf106 was associated with enhanced anchorage-independent growth ability, colony forming capacity and clonogenicity in vitro in this model system and trended towards increased tumour initiation frequency in vivo.

Conclusion

We have identified C1orf106 as a novel and robust TGF-β target gene correlating with poor prognosis in breast cancer and shown it to enhance tumourigenicity in a series of breast cancer cell lines.