The Olfactory Receptor Gene Product, OR5H2, Modulates Endometrial Cancer Cells Proliferation via Interaction with the IGF1 Signaling Pathway - PubMed (original) (raw)
The Olfactory Receptor Gene Product, OR5H2, Modulates Endometrial Cancer Cells Proliferation via Interaction with the IGF1 Signaling Pathway
Rand Shibel et al. Cells. 2021.
Abstract
Endometrial cancer is the most common gynecologic malignancy in Western countries. The insulin-like growth factor-1 (IGF1) axis has an important role in endometrial cancer biology and emerged as a promising therapeutic target in oncology. However, there is an urgent need to identify biomarkers that may help in patient stratification and prognosis. Laron syndrome (LS) is a type of dwarfism that results from the mutation of the growth hormone receptor (GHR) gene, leading to congenital IGF1 deficiency. While high circulating IGF1 is regarded as a risk factor in cancer, epidemiological studies have shown that LS patients are protected from cancer development. Recent genome-wide profilings conducted on LS-derived lymphoblastoid cells led to the identification of a series of genes whose over- or under-representation in this condition might be mechanistically linked to cancer protection. The olfactory receptor 5 subfamily H member 2 (OR5H2) was the top downregulated gene in LS, its expression level being 5.8-fold lower than in the control cells. In addition to their typical role in the olfactory epithelium, olfactory receptors (ORs) are expressed in multiple tissues and play non-classical roles in various pathologies, including cancer. The aim of our study was to investigate the regulation of OR5H2 gene expression by IGF1 in endometrial cancer. Data showed that IGF1 and insulin stimulate OR5H2 mRNA and the protein levels in uterine cancer cell lines expressing either a wild-type or a mutant p53. OR5H2 silencing led to IGF1R downregulation, with ensuing reductions in the downstream cytoplasmic mediators. In addition, OR5H2 knockdown reduced the proliferation rate and cell cycle progression. Analyses of olfr196 (the mouse orthologue of OR5H2) mRNA expression in animal models of GHR deficiency or GH overexpression corroborated the human data. In summary, OR5H2 emerged as a novel target for positive regulation by IGF1, with potential relevance in endometrial cancer.
Keywords: IGF1 receptor; Laron syndrome; OR5H2; endometrial cancer; insulin-like growth factor-1 (IGF1); olfactory receptors.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Figure 1
Expression of OR5H2 mRNA in the LS- and control-derived lymphoblastoid cell lines. (A) Dot-plot analysis of OR5H2 mRNA in lymphoblastoid cultures was conducted as described in Lapkina-Gendler et al. [19]. Dots denote individual LS patients (blue dots) and age-, gender- and ethnic-origin-matched controls (red dots). Numbers near each dot are code numbers. The y-axis denotes arbitrary gene expression values as measured in gene arrays. (B) Total RNA was obtained from four individual LS and four control lymphoblastoid cell lines and OR5H2 mRNA levels were measured by qPCR. The y-axis denotes the fold-change in gene expression between individual LS patients (blue bars) and matching controls (red bars). The bars represent the mean ± SEM of three independent cultures.
Figure 2
Effect of IGF1 and insulin on OR5H2 mRNA levels in USPC1 and USPC2 endometrial cancer cell lines. (A) USPC1 and USPC2 cells were kept in serum-free (starvation) or serum-containing (full) media for 24 h, after which total RNA was extracted and steady-state OR5H2 mRNA levels were measured by qPCR. (B) USPC1 and USPC2 cells were serum-starved for 24 h, after which they were treated with IGF1 or insulin (50 ng/mL) for 24 h. At the end of the incubation period, cells were harvested and the OR5H2 and GAPDH mRNA levels were quantitated by qPCR. A value of 1 was given to the normalized value in control cells. Bars are mean ± SEM of three independent experiments, each performed in triplicate.
Figure 3
Effect of insulin and IGF1 on OR5H2 protein levels in USPC1 and USPC2 cell lines. USPC1 and USPC2 cell lines were starved overnight and then incubated with IGF1 or insulin (50 ng/mL) for an additional 24 h. Cells were lysed and levels of OR5H2 were measured by Western blots. Levels of HSP70 were measured as a loading control. Bars represent densitometric values of OR5H2 levels normalized to the corresponding HSP70 band. A value of 100% was given to the normalized value in untreated cells. Results of a representative experiment, repeated three times with similar results, are shown.
Figure 4
Effect of OR5H2 knockdown on downstream signaling proteins in USPC1 cells. USPC1 cells were transfected with 7.5 nM of OR5H2 siRNA (or non-targeting siRNA) for 72 h. At the end of the incubation period cells were harvested and lysates (130 mg) were analyzed by Western blots for IGF1R (A), total and phospho-AKT and ERK (B), and total and phospho-p53 (C) levels. Scanning densitometric analyses are presented for each protein. *, p < 0.01 versus respective control.
Figure 5
Effect of OR5H2 knockdown on downstream signaling proteins in USPC2 cells. USPC2 cells were transfected with 10 nM of OR5H2 siRNA (or non-targeting siRNA) for 48 h, after which cells were harvested and evaluated for IGF1R (A), phospho- and total-AKT and ERK (B), and total and phospho-p53 (C) levels. *, p < 0.01 versus respective control. Ψ p < 0.05 versus respective control.
Figure 6
Analysis of physical interactions between IGF1R and OR5H2. USPC1 and USPC2 cells were lysed and immunoprecipitated with anti-IGF1R. Precipitates were electrophoresed through 10% SDS-PAGE, blotted onto nitrocellulose filters, and immunoblotted with anti-OR5H2, anti-IGF1R or anti-Sumo1. IP, immunoprecipitation; WB, Western blotting; TL, total lysate. Results of a typical experiment repeated twice with similar results are shown.
Figure 7
Effect of OR5H2 knockdown on cell proliferation. USPC1 and USPC2 cells (1 × 104 cells/well) were plated on 6-cm plates in serum-containing media and, after 24 h, OR5H2 gene expression was abolished as described in Materials and Methods. Cell proliferation was measured after 72 h (USPC1) or 48 h (USPC2) using a Cellometer cell counter. Results of a representative experiment repeated three times with similar results are shown. *, p < 0.01 versus respective control.
Figure 8
Effect of OR5H2 knockdown on cell cycle progression. USPC1 (A) and USPC2 (B) cells were seeded in quadruplicate dishes and transfected with OR5H2 siRNA or non-targeting siRNA for 72 h or 48 h, respectively. Cell cycle distribution was assessed by FACS analysis. The figure shows the results of a representative experiment repeated three times with similar results.
Figure 9
Expression of olfr196 mRNA in GHRKO and GH transgenic mice. Total RNA was prepared from the kidneys (A), ovaries (B) and uteri (C) of GHRKO (A,B) or GH transgenic (C) mice, or control littermates, and the olfr196 mRNA levels were measured by qPCR. Bars represent mean ± SD of 3–6 independent samples. *, p < 0.05 versus respective control.
References
- LeRoith D. Clinical relevance of systemic and local IGF-I: Lessons from animal models. Pediatr. Endocrinol. Rev. 2008;5:739–743. - PubMed
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