Ghrelin Upregulates Oncogenic Aurora A to Promote Renal Cell Carcinoma Invasion

Abstract

Ghrelin is a peptide hormone, originally identified from the stomach, that functions as an endogenous ligand of the growth hormone secretagogue receptor (GHSR) and promotes growth hormone (GH) release and food intake. Increasing reports point out ghrelin's role in cancer progression. We previously characterized ghrelin's prognostic significance in the clear cell subtype of renal cell carcinoma (ccRCC), and its pro-metastatic ability via Snail-dependent cell migration. However, ghrelin's activity in promoting cell invasion remains obscure. In this study, an Ingenuity Pathway Analysis (IPA)-based investigation of differentially expressed genes in Cancer Cell Line Encyclopedia (CCLE) dataset indicated the potential association of Aurora A with ghrelin in ccRCC metastasis. In addition, a significant correlation between ghrelin and Aurora A expression level in 15 ccRCC cell line was confirmed by variant probes. ccRCC patients with high ghrelin and Aurora A status were clinically associated with poor outcome. We further observed that ghrelin upregulated Aurora A at the protein and RNA levels and that ghrelin-induced ccRCC in vitro invasion and in vivo metastasis occurred in an Aurora A-dependent manner. Furthermore, MMP1, 2, 9 and 10 expressions are associated with poor outcome. In particular, MMP10 is significantly upregulated and required for the ghrelin-Aurora A axis to promote ccRCC invasion. The results of this study indicated a novel signaling mechanism in ccRCC metastasis.

Keywords: MMP10; aurora A; ghrelin; invasion.

Figure 1

The analysis of Cancer Cell Line Encyclopedia (CCLE) dataset via Ingenuity Pathway Analysis (IPA) points out Aurora A is potentially involved in ghrelin-mediated ccRCC metastasis. (A) RCC cell lines and relative ghrelin expressions retrieved from CCLE dataset were listed. (B) 15 ccRCC cell lines were separated into high and low ghrelin groups. The differentially expressed genes between two groups were identified (threshold: >1.5 fold change and p < 0.05). Canonical pathway analysis was performed to rank the matched (A) Diseases and Disorders and (C) Molecular and Cellular Function in IPA database. (D) Statistical P values and log2 transformed expressions of Aurora A (AURKA) and its interactive network were shown. The red and green circles represent upregulation and downregulation, respectively.

Figure 1

The analysis of Cancer Cell Line Encyclopedia (CCLE) dataset via Ingenuity Pathway Analysis (IPA) points out Aurora A is potentially involved in ghrelin-mediated ccRCC metastasis. (A) RCC cell lines and relative ghrelin expressions retrieved from CCLE dataset were listed. (B) 15 ccRCC cell lines were separated into high and low ghrelin groups. The differentially expressed genes between two groups were identified (threshold: >1.5 fold change and p < 0.05). Canonical pathway analysis was performed to rank the matched (A) Diseases and Disorders and (C) Molecular and Cellular Function in IPA database. (D) Statistical P values and log2 transformed expressions of Aurora A (AURKA) and its interactive network were shown. The red and green circles represent upregulation and downregulation, respectively.

Figure 2

ccRCC patients with high ghrelin and Aurora A expression status correlates with poor outcome. Kaplan–Meier plot of cancer patients divided into high and low expression of Aurora A (A) and Ghrelin (B) were shown. (C) Kaplan–Meier plot of combining Aurora A and Ghrelin expression levels was analyzed. Data of 562 clear cell type RCC cases were retrieved from TCGA (KIRC gene expression (IlluminaHiSeq) dataset). The dataset showed the gene level transcription estimates, as in log2(x + 1) transformed RSEM normalized count. Subgroup was determined according to the ranking in expression level of indicated genes. (D) ccRCC patients of high GHRL (D) or AURKA (E) expression level correlates with poor disease-free survival. Data was analyzed using dataset (Wuttig Wirth Renal Kidney GSE22541), and was retrieved from the SurvExpress database.

Figure 2

ccRCC patients with high ghrelin and Aurora A expression status correlates with poor outcome. Kaplan–Meier plot of cancer patients divided into high and low expression of Aurora A (A) and Ghrelin (B) were shown. (C) Kaplan–Meier plot of combining Aurora A and Ghrelin expression levels was analyzed. Data of 562 clear cell type RCC cases were retrieved from TCGA (KIRC gene expression (IlluminaHiSeq) dataset). The dataset showed the gene level transcription estimates, as in log2(x + 1) transformed RSEM normalized count. Subgroup was determined according to the ranking in expression level of indicated genes. (D) ccRCC patients of high GHRL (D) or AURKA (E) expression level correlates with poor disease-free survival. Data was analyzed using dataset (Wuttig Wirth Renal Kidney GSE22541), and was retrieved from the SurvExpress database.

Figure 3

Ghrelin expression correlates with Aurora A in RCC cell lines. (A,B) Correlations in expression level of ghrelin and Aurora-A in ccRCC cell lines were respectively analyzed using different probes. Raw data was retrieved from CCLE dataset. (C) Endogenous Aurora A and ghrelin expression at protein level in ccRCC cell panel was determined by western blot method. Relative protein levels and statistical correlation were analyzed and shown after normalizing with β-actin internal control.

Figure 4

Ghrelin upregulates Aurora A in ccRCC. (A) RNA expression levels of indicated molecules were examined upon lentiviral-based ghrelin overexpression in Caki-1 cell clone 1 and clone2. (B) Regulation of ghrelin to Aurora A was investigated in ACHN cells using lentiviral-based overexpression method. (C) The modulation at RNA level was further examined by QPCR method. (D) The regulation of ghrelin overexpression to Aurora A protein was investigated by western blot in Caki-1 (D) and in ACHN cells (E). Figures were represented from the results of three repeated experiments with similar pattern. * p < 0.05, ** p < 0.01.

Figure 5

Aurora A is required in ghrelin-mediated RCC metastasis in vitro and in vivo. (A) Cell migration assay was performed by transwell devices using stable clones of ACHN cell. Numbers of cell migration in each group were counted after 5h of incubation. (B) Knockdown efficacy of Aurora A by specific siRNA in 786-0 cells was examined. Cells were treated with 100 or 200 nM of control siRNA or siRNA specific to Aurora A (C) Relative cell invasion ability in 786-0 cells upon Aurora A knockdown was studied. (D) RCC metastasis was investigated in ACHN cells (D) and in 786-0 cells (E) overexpressing ghrelin and combined stable Aurora A silencing. Representative images of lung surface nodule in indicated groups were showed (left). Numbers of lung nodule in each group were quantified 8 weeks after cell injection. n = 7 per each group (right). Figures were represented from the results of three repeated experiments with similar pattern. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

MMP10 is required in the ghrelin-Aurora A signaling axis to promote ccRCC invasion. (A) Kaplan–Meier plot showing the association of indicated MMP with RCC (TCGA) patient survival was represented. Data were retrieved from Human Protein Atlas website. (B) Representative Aurora A and MMP10 expression pattern in ACHN cells overexpressing ghrelin combined with Aurora A or MMP10 knockdown respectively. (C) Alternations in relative ACHN cell invasion ability were shown. Magnification: 100× (D) The regulation of ghrelin toward MMP10 expression was further studied in ACHN cells silencing GHS-R1a. Figures were represented from the results of three repeated experiments with similar pattern. ** p < 0.01, *** p < 0.001.