Artur Mayerhofer

The impact of hypoxia on the cellular phenotype and proteome of KGN cells

Graphical Abstract Abstract Human KGN cells are a widely studied cellular model for human granulosa cell tumors (GCTs), as well as for non-tumorous human granulosa cells. Such studies are typically performed at oxygen (O 2 ) levels that are equivalent to the atmospheric concentration for practical reasons. However, in most tissues of the human body, as well as in tumors, much lower O 2 levels exist. Hypoxic conditions can regulate the phenotype and the behavior of cells in a cell-type-specific way. However, how they may impact KGN cells was not well known. The aim of this study was therefore to evaluate the consequences of a defined hypoxic condition (1% O 2 ; 10 days) in KGN cells. Compared to cells cultured in atmospheric conditions, hypoxia reduced the numbers and increased the size of KGN cells but did not alter cell velocity in cell migration studies. Hypoxia affected the cellular proteome of KGN cells (57 more and 75 less abundant proteins). The associated pathways indicate, among others, metabolic adaptations and mitochondrial changes evoked by hypoxia. The cellular responses may be related, in part, to the increased nuclear expression of hypoxia-inducible factor 1α (HIF1α). The results provide a detailed picture of hypoxia-induced changes in KGN cells and are a resource for future studies. If transferable to the in situ situation, they shed light on how variable O 2 levels within the GCT microenvironment may regulate tumor cells and thereby may contribute to tumor cell heterogeneity. Lay summary Human cells can be isolated from the body and kept alive in incubators, in which, mainly for practical reasons, the oxygen (O 2 ) levels typically correspond to the surrounding atmosphere. This does not reflect the conditions within the human body, where much lower O 2 levels exist. This study shows the importance of low O 2 levels on the cellular make-up and behavior of KGN cells, which are accepted models for a group of ovarian tumors (GCTs), as well as normal, non-tumorous granulosa cells (from which GCTs originate). We found that when KGN cells were exposed to low O 2 levels, cell numbers and cell size, as well as cellular proteins and RNA, changed. The experiments, revealing adaptations associated with hypoxia, may contribute to a better understanding of the normal ovary and ovarian tumors.

Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors

Sirtuins (SIRTs) are NAD+-dependent deacetylases that regulate proliferation and cell death. In the human ovary, granulosa cells express sirtuin 1 (SIRT1), which has also been detected in human tumors derived from granulosa cells, i.e., granulosa cell tumors (GCTs), and in KGN cells. KGN cells are an established cellular model for the majority of GCTs and were used to explore the role of SIRT1. The SIRT1 activator SRT2104 increased cell proliferation. By contrast, the inhibitor EX527 reduced cell numbers, without inducing apoptosis. These results were supported by the outcome of siRNA-mediated silencing studies. A tissue microarray containing 92 GCTs revealed nuclear and/or cytoplasmic SIRT1 staining in the majority of the samples, and also, SIRT2-7 were detected in most samples. The expression of SIRT1–7 was not correlated with the survival of the patients; however, SIRT3 and SIRT7 expression was significantly correlated with the proliferation marker Ki-67, implying roles in tumor cell proliferation. SIRT3 was identified by a proteomic analysis as the most abundant SIRT in KGN. The results of the siRNA-silencing experiments indicate involvement of SIRT3 in proliferation. Thus, several SIRTs are expressed by GCTs, and SIRT1 and SIRT3 are involved in the growth regulation of KGN. If transferable to GCTs, these SIRTs may represent novel drug targets.