Luteinizing Hormone–Induced Up-Regulation of ErbB-2 Is Insufficient Stimulant of Growth and Invasion in Ovarian Cancer Cells

Abstract

The effects of luteinizing hormone (LH), a gonadotropic hormone implicated in the development of ovarian cancer, are mediated by specific binding to its G protein -coupled receptor, the LH receptor (LHR). Activated LHR initiates second messenger responses, including cyclic AMP (cAMP) and inositol phosphate. Because cAMP increases expression of ErbB-2, a receptor tyrosine kinase whose overexpression in cancers correlates with poor survival, we hypothesized that LH may regulate ErbB-2 expression. Cell surface LHR expression in stable transformants of the ErbB-2 -overexpressing ovarian cancer cell line SKOV3 was confirmed by PCR and whole-cell ligand binding studies. Second messenger accumulation in the LHR-expressing cells confirmed signaling through Gs and Gq. Western blots of total protein revealed that LHR introduction up-regulated ErbB-2 protein expression 2-fold and this was further up-regulated in a time-and dose-dependent manner in response to LH. Forskolin and 8Br-cAMP also up-regulated ErbB-2 in both LHR-expressing and mock-transfected cells, indicating that regulation of ErbB-2 is a cAMP-mediated event. Kinase inhibitor studies indicated the involvement of protein kinase A -mediated, protein kinase C -mediated, epidermal growth factor receptor -mediated, and ErbB-2 -mediated mechanisms. The LH-induced up-regulation of ErbB-2 was insufficient to overcome the negative effects of LH on proliferation, invasion, and migration. A molecular signature for this nonaggressive phenotype was determined by Taqman array to include increased and decreased expression of genes encoding adhesion proteins and metalloproteinases, respectively. These data establish a role for LH and LHR in the regulation of ErbB-2 expression and suggest that, in some systems, ErbB-2 up-regulation alone is insufficient in producing a more aggressive phenotype.

Transcriptomic Analysis of Human Ovarian Cancer Cells: Changes Mediated by Luteinizing Hormone Receptor Activation

“…It did, however, reduce the invasive index of the cells by a small margin. LH was found to reduce migration and proliferation of LHR+ cells but not of LHR-cells, while the invasiveness was not altered (Warrenfeltz et al, 2008).…”

“…The SKOV-3 human ovarian cancer cell line was selected since it does not express LHR (Parrott et al, 2001;Mandai et al, 2007;Warrenfeltz et al, 2008) and can serve as a negative control. Some lines do, however, appear to express LHR and respond to LH (Lau et al, 2010).…”

“…Following transfection with a full-length human LHR-pcDNA3 or an empty vector, two sub-lines were generated, one expressing about 12,000 receptors per cell (LHR+) and the other, i.e. the mock-transfected cells, that does not express LHR (LHR-) (Warrenfeltz et al, 2008). The LHR+ cells bound radio-labeled human chorionic gonadotropin with a Kd of 0.3 nM in saturation binding assays and an IC50 of 0.8 nM in competition binding assays.…”

“…RNA was extracted from SKOV-3 cells (Warrenfeltz et al, 2008), and the resulting cDNAs were analyzed by Almac Diagnostics (Durham, NC, USA) using the Affymetrix Human U133 Plus2 Arrays (Cui et al, 2011c). In a parallel study on microRNA expression, the ovarian cancer DSA TM array (Almac Diagnostics) was used (Cui et al, 2011b).…”

“…In addition, considerable effort has also been invested into the use of gene expression profiling techniques by others to elucidate their utility in predicting metastasis and survivability in ovarian cancer (Lancaster et al, 2006;Sabatier et al, 2009). Herein we focus our discussion on the in vitro use of an ovarian carcinoma cell line, SKOV-3 cells (Warrenfeltz et al, 2008;Puett et al, 2010;Cui et al, 2011b,c), rather than tumor tissues, in part because of the desire to work with an established cell line in which continuing studies can be performed in a controlled and reproducible manner. Such cell lines have provided an enormous wealth of information on the characteristics of ovarian cells and their responses to various inhibitors or growth factors Mandai et al, 2007).…”