Probes for INS

The transcription factor SOX9 is critical for prostate development, and dysregulation of SOX9 is implicated in prostate cancer (PCa). However, the SOX9-dependent genes and pathways involved in both normal and neoplastic prostate epithelium are largely unknown. Here, we performed SOX9 ChIP sequencing analysis and transcriptome profiling of PCa cells and determined that SOX9 positively regulates multiple WNT pathway genes, including those encoding WNT receptors (frizzled [FZD] and lipoprotein receptor-related protein [LRP] family members) and the downstream β-catenin effector TCF4. Analyses of PCa xenografts and clinical samples both revealed an association between the expression of SOX9 and WNT pathway components in PCa. Finally, treatment of SOX9-expressing PCa cells with a WNT synthesis inhibitor (LGK974) reduced WNT pathway signaling in vitro and tumor growth in murine xenograft models. Together, our data indicate that SOX9 expression drives PCa by reactivating the WNT/β-catenin signaling that mediates ductal morphogenesis in fetal prostate and define a subgroup of patients who would benefit from WNT-targeted therapy.

SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7α Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.

Abstract

PURPOSE:

Understanding tumour heterogeneity is an important challenge in current cancer research. Transcription and epigenetic profiling of cultured melanoma cells have defined at least two distinct cell phenotypes characterised by distinctive gene expression signatures associated with high or low/absent expression of Microphthalmia-associated transcription factor (MITF). Nevertheless, heterogeneity of cellpopulations and gene expression in primary human tumours is much less well characterised.

EXPERIMENTAL DESIGN:

We performed single cell gene expression analyses on 472 cells isolated from needle biopsies of 5 primary human melanomas, 4 superficial spreading and one acral melanoma. The expression of MITF-high and MITF-low signature genes was assessed and compared to investigate intra and inter-tumoural heterogeneity and correlated gene expression profiles.

RESULTS:

Single cell gene expression analyses revealed varying degrees of intra and inter-tumour heterogeneity conferred by the variable expression of distinct sets of genes in different tumours. Expression of MITF partially correlated with that of its known target genes while SOX10 expression correlated best with PAX3 and ZEB2. Nevertheless, cells simultaneously expressing MITF-high and MITF-low signature genes were observed both by single cell analyses and RNAscope.

CONCLUSIONS:

Single cell analyses can be performed on limiting numbers of cells from primary human melanomas revealing their heterogeneity. While tumours comprised variable proportions of cells with the MITF-high and MITF-low gene expression signatures characteristic of melanoma cultures, primary tumours also comprised cells expressing markers of both signatures defining a novel cell state in tumours in vivo.