The Heat Shock Transcription Factor HSF1 Induces Ovarian Cancer Epithelial-Mesenchymal Transition in a 3D Spheroid Growth Model.

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. The proteotoxic stress-responsive transcription factor HSF1 is frequently overexpressed in a variety of cancers and is vital to cellular proliferation and invasion in some cancers. Upon analysis of various patient data sets, we find that HSF1 is frequently overexpressed in ovarian tumor samples. In order to determine the role of HSF1 in ovarian cancer, inducible HSF1 knockdown cell lines were created. Knockdown of HSF1 in SKOV3 and HEY ovarian cancer cell lines attenuates the epithelial-to-mesenchymal transition (EMT) in cells treated with TGFβ, as determined by western blot and quantitative RT-PCR analysis of multiple EMT markers. To further explore the role of HSF1 in ovarian cancer EMT, we cultured multicellular spheroids in a non-adherent environment to simulate early avascular tumors. In the spheroid model, cells more readily undergo EMT; however, EMT inhibition by HSF1 becomes more pronounced in the spheroid model. These findings suggest that HSF1 is important in the ovarian cancer TGFβ response and in EMT

Spheroid growth in ovarian cancer alters transcriptome responses for stress pathways and epigenetic responses

<div><p>Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. These poor statistics are related to a lack of early symptoms and inadequate screening techniques. This results in the cancer going undetected until later stages when the tumor has metastasized through a process that requires the epithelial to mesenchymal transition (EMT). In lieu of traditional monolayer cell culture, EMT and cancer progression in general is best characterized through the use of 3D spheroid models. In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFβ to induce EMT. Transcripts that included Coiled-Coil Domain Containing 80 (CCDC80), Solute Carrier Family 6 (Neutral Amino Acid Transporter), Member 15 (SLC6A15), Semaphorin 3E (SEMA3E) and PIF1 5'-To-3' DNA Helicase (PIF1) were downregulated more than 10-fold in the 3D cells while Inhibitor Of DNA Binding 2, HLH Protein (ID2), Regulator Of Cell Cycle (RGCC), Protease, Serine 35 (PRSS35), and Aldo-Keto Reductase Family 1, Member C1 (AKR1C1) were increased more than 50-fold. Interestingly, EMT factors, stress responses and epigenetic processes were significantly affected by 3D growth. The heat shock response and the oxidative stress response were also identified as transcriptome responses that showed significant changes upon 3D growth. Subnetwork enrichment analysis revealed that DNA integrity (e.g. DNA damage, genetic instability, nucleotide excision repair, and the DNA damage checkpoint pathway) were altered in the 3D spheroid model. In addition, two epigenetic processes, DNA methylation and histone acetylation, were increased with 3D growth. These findings support the hypothesis that three dimensional ovarian cell culturing is physiologically different from its monolayer counterpart.</p></div

The most differentially expressed transcripts in the HEY 3D compared to HEY 2D.

<p>The most differentially expressed transcripts in the HEY 3D compared to HEY 2D.</p

DNA methylation was significantly enriched following sub-network enrichment analysis when comparing 3D vs. 2D growth.

<p>DNA methylation was preferentially increased approximately 4% (197 genes measured, P = 0.006) in the 3D group at the level of the transcriptome based on the sub-network enrichment analysis. Red indicates the gene is up-regulated and blue indicates the gene is down-regulated. All genes for DNA methylation are listed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182930#pone.0182930.s005" target="_blank">S4 Table</a>.</p

HEY cells treated with TGFβ have distinct gene expression profiles when grown as 3D spheroids vs. 2D monolayers.

<p>A) Hierarchical clustering of expression profiles. Clustering revealed that each of the two groups (4 biological replicates per group) form into distinct clades based on expression. B) Principle component analysis for expression profiles. Variability in transcriptome response separates strongly along the first PCA1. Red color is HEY 3D biological replicates and green color is HEY 2D biological replicates. The four biological replicates for HEY 2D are more closely related in expression as compared to HEY 3D.</p