PHLDA1 pleckstrin homology like domain family. A member 1. Cell movement of CRC cell lines (2.49E-07). Invasion of carcinoma cell lines (3.89E-06).
The influence of upregulated transcription factor SOX9 in endometrial endometrioid carcinoma Liam Greene1, Dharani Hapangama2, Olga Vasieva1 1. Institute of Integrative Biology, University of Liverpool, Liverpool, UK 2. Institute of Translational Medicine, University of Liverpool, Liverpool, UK
ABSTRACT Sex-determining region Y-box 9 (SOX9) is a master regulator of developmental organogenesis, and a regulator of adult stem cell populations. A growing body of research observes aberrant SOX9 expression in a range of cancers, with a developing pattern suggesting a role facilitating malignant transformation and tumourigenesis. Upregulated SOX9 is observed in hyperplastic and programmed endometrial proliferation, as well as in endometrial endometrioid carcinoma, the most common subtype of endometrial cancer. Using a bioinformatic approach we investigated the perturbations in transcriptome associated with and controlled by upregulated SOX9 in EEC patients; observing considerable evidence that SOX9 can facilitate malignant transformation by activating genes and gene networks involved in deregulating tissue architecture and driving epithelial-mesenchymal transition. Ets variant 5 (ETV5) was uncovered as a potential endometrium-specific effector of SOX9’s malignant potential.
INTRODUCTION The transcription factor sex-determining region Y-box 9 (SOX9) plays a master role regulating body-wide tissue organisation and cell lineage determination during foetal organogenesis, controlling programs such as epithelial-mesenchymal transition (EMT), invasion, migration, proliferation and differentiation 1-3. A versatile sensitivity to local signalling, with a broad range of upstream regulators and downstream has been observed, placing SOX9 at a hub of morphogenic control of tissue architecture4-6. In the mature organism this functional profile is contracted to a core role in maintaining the potency and mediating the differentiation of adult stem cells (ASCs), particularly those in epithelial crypts4, 5, 7-9. Unsurprisingly, aberrant SOX9 expression is observed in a range of cancers, often in association with malignancy, in a manner which suggests recapitulation of its developmental program and/or deregulation of its mature functions. SOX9 is attributed some tumour suppressive function10, 11, but the dominant trend in the literature describes a pattern of oncogenicity in association with upregulated SOX9. Some examples include increased tumour growth and EMT in cancer of the prostate5, 12, 13, lung14 and breast15, and tumour growth facilitation in cancer of the skin16 and pancreas4, 17. Furthermore SOX9 expression is positively correlated with pre-cancerous hyperplastic development in pancreas18 and prostate19. This oncogenic potential is more common in cancers of organs where SOX9 is involved in ASC maintenance20. Implicated in many of these scenarios is SOX9’s complex, mutual inter-regulatory relationship with Wnt signalling4, 5, 16. In addition to Wnt, research reveals a range of more or less context- or methodology-specific cofactors and signalling pathways working with oncogenic SOX9, including androgen in prostate13, 21, Notch, Slug and SOX10 in breast22, K-ras in pancreas23, and Hedgehog in basal cell carcinoma16, 24. This variation echoes the multifunctionality inherent to SOX9 due to its
broad spectrum of transcriptional targets; and argues in favour of employing a broad scope when researching functional implications of aberrant SOX9 in cancer. Upregulated SOX9 is also implicated in endometrial carcinoma (EC)25, 26 the most common gynaecological malignancy in western populations, and the second most lethal cancer in females27, 28. The endometrioid endometrial carcinoma (EEC) subtype accounts for 70-80% EC diagnoses29, and is characterised by development from hyperplastic precursors26, 29, 30, which SOX9 overexpression has been shown to drive in mouse models31. Beyond this direct observation, SOX9’s possible role in EEC is poorly understood, yet there is a set of suggestive observations: SOX9 expression is localised to endometrial ASCs26; SOX9 is upregulated in proliferative stages of endometrial fertility cycles25, 26, in the proliferative disorder endometriosis, and in the post-menopausal endometrium26 (from which the vast majority of EEC originate); Wnt signalling is heavily implicated in the endometrial proliferative regimen and EEC development32, 33, and SOX9 has been observed co-localised with primary Wnt effector beta-catenin in the nucleus of endometrial epithelial cells34. Furthermore, a subset of 10-20% of EEC cases develop an unpredictably aggressive phenotype, with higher rates of metastasis35. It is possible the morphogenic potential of upregulated SOX9, observed in other cancers, may play a role in this transformation The single targeted functional analysis of SOX9’s role in EC performed so far described a tumour suppressing interaction with p53 via upregulation of p21 and inhibition of cycle progression25, however we argue the scale of focus is not sufficient to infer the full potential of SOX9’s influence, given its master regulator capacity. As such, the current research was designed to investigate the broad transcriptomic changes associated with upregulated SOX9, and the phenotypic implications thereof, in the context of EEC. The primary goal was to elucidate gene regulatory networks (GRNs) under SOX9’s direct control and the downstream effects of their regulation. Using transcriptomic data from Illumina Base Space, a bioinformatic analysis of differentially expressed genes (DEGs) correlating to SOX9 dysregulation in EEC patients was carried out, with collated chromatin immunoprecipitation (ChIP) data used to infer SOX9’s regulatory influence within the dataset. Given the established and putative roles of SOX9 in other cancers, it was hypothesised SOX9 upregulation would be associated with active EMT and malignancy-related pathways, evidence of increased tumour growth and activated Wnt signalling. METHODS Differentially expressed gene lists: Using the EEC cohort in the Base Space: Cohort Analyser (Illumina, Inc., San Diego, CA.) platform (n=360), groups of patients with upregulated SOX9 (fold change (f.c) >+2, n=219) and SOX9 unchanged/downregulated (f.c. 2cm from tumour36. Using Cohort Analyser’s Group Comparison: RNA Analysis tool, a list of genes with significantly perturbed expression between these groups was created (p=+2) or unchanged/downregulated (f.c.
