Identification and characterisation of novel SMAD2/3 binding partners underlying pancreatic cancer cell plasticity

Pancreatic cancer is one of the deadliest malignancies in human with a 5-year survival rate of less than 10% and median survival time of 7-11 months. The most common type of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC) which accounts for more than 90% of cases, and is associated with poor prognosis due to late diagnosis, aggressive growth and therapy resistance. Accumulating evidence shows that PDAC cell is able to successfully complete metastatic cascade and survive therapeutic stress due to extraordinary cellular plasticity which is defined as a cell ability to change its phenotype. The molecular mechanisms underlying PDAC cell phenotypic plasticity are not completely understood but it is well-established that cell phenotype is largely determined by its transcriptional program. Elevated levels of transforming growth factor β (TGF-β) have been detected in PDAC tissue and intracellular effector molecules of canonical TGF-β pathway, namely SMAD2 and SMAD3, have been shown to enhance transcriptional reprogramming. Since the transcriptional program activated by SMAD2/3 is determined by the sequence-specific transcription factors that SMAD2/3 interact with, we hypothesised that the phenotype switching of PDAC cell may be mediated by SMAD2/3 switching binding partners.
In the current thesis, SMAD2/3 co-immunoprecipitation followed by western blot and mass spectrometry analysis was used and novel SMAD2/3 transcription factor binding partners were identified. In particular, SOX13 was shown to interact with SMAD2/3 in SMAD4-positive and SMAD4-negative PDAC cell lines, and several shared genomic binding sites of SOX13 and SMAD2/3 were detected by chromatin immunoprecipitation followed by sequencing (ChIP-seq). To uncover the target genes of SOX13 and investigate the role of SOX13 in modulating transcriptional response to TGF-β signalling, SOX13 knockdown cell line was generated, and transcriptomes of control and knockdown cell lines were analysed using bulk RNA sequencing (RNA-seq). The transcriptomic analysis revealed that SOX13 controls the expression of ECM-related genes and promotes activation of hypoxia response signature in normoxic conditions. Importantly, SOX13 was demonstrated to be required for transcriptional activation of key hypoxia-associated gene CA9 by TGF-β/SMAD pathway. Several pieces of evidence indicated that the regulation of hypoxia response signature by SOX13 and SMAD2/3 relies on cooperation with HIF-1α. In addition, mass cytometry panel for studying pancreatic cancer heterogeneity was developed and validated. By combining the novel mass cytometry panel, knockdown cell lines, non-adherent culture system and RNA-seq data, involvement of SOX13 in transcriptional networks that promote acquisition of stemness(high) phenotype characterised by high expression of Nestin, DCLK1, CXCR4 and Musashi-1 was described. In vitro functional assays revealed that depletion of SOX13 reduces proliferation and impairs clonogenicity.
In addition, weak interaction between SMAD2/3 and FOSL2 was detected. SMAD2/3 and FOSL2 co-bind to a number of genomic loci and cooperate to induce expression of MMP7 in response to TGF-β pathway activation. To describe FOSL2 regulon, FOSL2 knockdown cell line was generated. RNA-seq analysis highlighted FOSL2 role in transcriptional control of lipid metabolism and cholesterol homeostasis. Mass cytometry analysis suggested that FOSL2 may promote acquisition of a SSEA-1(low), Nestin(low) and CD24(high) phenotype. Therefore, FOSL2 appears to contribute to transcriptional networks governing classical, stemness(medium/low) phenotype. FOSL2 knockdown cells proliferate slower than control cells and exhibit diminished clonogenic potential.
In summary, transcription factor partners of SMAD2/3 play an important role in transcriptional regulation of pancreatic cancer cell phenotype. The nuanced control of cellular phenotype is achieved through combinatorial gene regulation mediated by cooperation between diverse transcription factors (e.g., SMAD2/3, SOX13 and HIF-1α).

Keywords: pancreatic cancer
SMAD
stemness
SOX13
cancer cell plasticity
Advisor: Pauklin, S
Hulley, P