Hypoxia Regulates DPP4 Expression, Proteolytic Inactivation, and Shedding from Ovarian Cancer Cells

Abstract

The treatment of ovarian cancer has not significantly changed in decades and it remains one of the most lethal malignancies in women. The serine protease dipeptidyl peptidase 4 (DPP4) plays key roles in metabolism and immunity, and its expression has been associated with either pro- or anti-tumour effects in multiple tumour types. In this study, we provide the first evidence that DPP4 expression and enzyme activity are uncoupled under hypoxic conditions in ovarian cancer cells. Whilst we identified strong up-regulation of DPP4 mRNA expression under hypoxic growth, the specific activity of secreted DPP4 was paradoxically decreased. Further investigation revealed matrix metalloproteinases (MMP)-dependent inactivation and proteolytic shedding of DPP4 from the cell surface, mediated by at least MMP10 and MMP13. This is the first report of uncoupled DPP4 expression and activity in ovarian cancer cells, and suggests a previously unrecognized, cell- and tissue-type-dependent mechanism for the regulation of DPP4 in solid tumours. Further studies are necessary to identify the functional consequences of DPP4 processing and its potential prognostic or therapeutic value.

Keywords: DPP4; MMP; hypoxia; ovarian cancer; tumour microenvironment.

Figure 1

The effect of hypoxia on cell growth, HIF-1α and DPP4 expression in ovarian cancer cells. Ovarian cancer cell lines OVCAR4, SKOV3, and CaOV3 were seeded at ~10⁶ cells/25 cm² flask and cultured under normoxic or hypoxic conditions for up to 48 h. (A) Cell density and viability at 48 h. (B) HIF-1α mRNA expression and (C) protein abundance were measured by qRT-PCR and ELISA respectively. (D) DPP4 mRNA expression was measured by qRT-PCR. Data represent the mean ± SD, n = 3/group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2

Hypoxia induces shedding of inactive DPP4 from ovarian cancer cells. OVCAR4 cells were cultured under normoxia or hypoxia for 48 h. ELISA for quantitation of DPP4 protein abundance and enzyme assays for DPP4 activity were performed on (A) cell lysates and (B) conditioned media. Enzyme activity is expressed as specific activity relative to the measured abundance of DPP4. The data represent the mean ± SD, n = 3/group. *p < 0.05; **p < 0.01.

Figure 3

Hypoxia regulates protease expression in ovarian cancer cells. Cell lysates and conditioned media from OVCAR4 cells grown under normoxia and hypoxia were analysed for abundance of 27 different human proteases (Supplementary Table S1) using antibody arrays. (A) Normalized detection intensities for cellular and secreted proteases (MMP1, MMP10, MMP13, CTSB, CTSD, CTSZ, and CTSV) that were significantly different following hypoxic incubation of OVCAR4 cells. (B) Expression levels of MMP1, MMP10, and MMP13 were validated by qRT-PCR. The data represent the mean ± SD, n = 3/group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Matrix metalloproteinases (MMP) activity influences DPP4 expression and release in ovarian cancer cells. OVCAR4 cells were treated with the pan-MMP inhibitor GM6001 (25 µM) and cultured under normoxia or hypoxia for 48 h. Cells were harvested and analysed for (A) DPP4 mRNA expression; and conditioned media were collected and analysed for (B) soluble DPP4 protein abundance and (C) DPP4 enzyme activity. The data represent the mean ± SD, n = 3/group. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

MMP10 and MMP13 affect DPP4 expression and release from ovarian cancer cells. OVCAR4 cells with stable shRNA knockdown of MMP10 or MMP13 were cultured under normoxia or hypoxia for 48 h. Total RNA was extracted, and qRT-PCR performed to confirm (A) knockdown of MMP10 and MMP13 relative to controls, and (B) examine the effects of MMP10 and MMP13 on DPP4 mRNA expression. DPP4 ELISA and enzyme assay were performed on conditioned media of cells to assess changes in (C) sDPP4 release and (D) specific activity induced by the downregulation of MMP10 and MMP13. The data represent the mean ± SD, n = 3/group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.