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Abstract

Postoperative recurrence for papillary renal cell carcinoma (PRCC) remains a tough problem in clinic. Previous studies have shown that general control nonderepressible kinase 2 (GCN2) was critically involved in tumour development. However, its function and clinical significance in renal cancer remain unknown. In this study, we investigated the role of GCN2 in PRCC. GCN2 silencing suppressed the viability and proliferation, promoted apoptosis of renal cancer cells. We found that the protein level of GCN2 was increased in PRCC tissues. Immunohistochemistry was performed in 84 patients with PRCC to explore the association of GCN2 level with clinical significance. High GCN2 protein level was observed to be significantly correlated with adverse clinicopathological parameters, such as larger tumor size, higher TNM stage, higher Fuhurman Grade, and lymph node metastasis. We evaluated patient outcomes according to various clinical parameters as well as GCN2 expression by Kaplan-Meier curves. Multivariate analysis revealed that GCN2 overexpression can be a predictive factor correlated with reduced OS and PFS of postoperative PRCC patients. Collectively, GCN2 is potentially to play crucial roles in PRCC progression, and its overexpression may be used to predict poor prognosis and promising therapeutic strategy for PRCC patients.

Keywords

GCN2 PRCC prognosis overall survival progression free survival

1. Introduction

Renal cell carcinoma (RCC) is the common urological tumor leading to significant mortality, and the morbidity has risen steadily [1]. Papillary renal cell carcinoma (PRCC) is the second prevalent phenotype of RCC, accounting for 10–15% [2]. Due to recurrence, postoperative prognosis of PRCC patients were more unfavourable than other subtype of RCC [3]. PRCC is a heterogeneous histologic subtype of renal cancer consisting of two main subtypes [4]. Type II carcinomas, characterized by high nuclear grade and accompanied by aggressive behaviour, usually experience worse prognosis compared to type I carcinomas [5]. Moreover, the genomic analysis data of PRCC in the TCGA has shown more complexity [6]. Recently, improved understanding of RCC molecular biology led to targeted agents and better treatment outcomes [7]. However, the related study and progress in PRCC is rare.

Figure 1.

Knockdown of GCN2 inhibits renal cancer cell viability. (A) The expression of GCN2 protein level in three renal cancer cell lines (ACHN, 786O and Caki-1) and normal human renal tubular epithelial cell (HK-2). (B) Western blot result of GCN2 expression in ACHN and 786O cells transfected with negative control (N.C.) siRNA or GCN2 siRNAs. (C–F) Cells were also analyzed by CCK-8 assays (C–E), EdU assay (F). ${}^{**}p<$ 0.01, error bars indicate mean $\pm$ SD, t test.

General control nonderepressible kinase 2 (GCN2), a stress-activated protein kinase, remains inactive in normal conditions and modulates the expression of amino acid biosynthetic genes by phosphorylation of translation initiation factor eIF2 $\alpha$ when encountering amino acid deficiency in tumor microenvironment [8, 9, 10]. GCN2 pathway is one of the key mechanisms in maintaining metabolic homeostasis to promote tumor survival and aggressiveness [11, 12, 13]. It was also reported to evaluate stress resistance in kidney ischemia reperfusion injury models and control intestinal inflammation by suppressing inflammasome activation [14, 15]. Recently, GCN2 has been shown to be involved in cancer cell apoptosis in various cancer cells [16]. The crucial role of GCN2 in tumorigenesis has recently been demonstrated in several human malignant tumors, including breast, liver, and lung cancer [17, 18, 19, 20]. Nevertheless, the functional and prognostic role of GCN2 in PRCC remain unknown. Further, there is no biological marker for early diagnosis of recurrence and prognostic factor in PRCC until now.

Here, we show that GCN2 silencing decrease survival and promote apoptosis of renal cancer cells. We further evaluate the relationship between GCN2 expressions and clinical prognosis in PRCC patients.

Figure 2.

Knockdown of GCN2 induces renal cancer cell apoptosis. (A) Annexin V-PI flow cytometric were used to analysis ACHN and 786O cells transfected with negative control (N.C.) siRNA or GCN2 siRNAs. (B) Cell apoptosis percentages indicated that the cell viability decreased in GCN2-silenced cells. ${}^{**}p<$ 0.01, ${}^{***}p<$ 0.001. Error bars indicate mean $\pm$ SD, t test.

Figure 3.

Expression of GCN2 in renal cancer cell lines and PRCC tissue samples. (A) Expression of GCN2 protein in 12 pairs of tumor tissues and adjacent normal tissues. N: adjacent normal tissues; T: tumor tissues. (B) The relative protein level of GCN2 was higher in tumor tissues than adjacent normal tissues. ${}^{**}p<$ 0.01, error bars indicate mean $\pm$ SD, t test.

2. Results

2.1 GCN2 knockdown inhibited cell viability and promoted apoptosis in vitro

Growing evidences indicated that GCN2 is involved in tumor survival [13, 21]. By examining the protein level, we found GCN2 was differentially expressed in three renal cancer cell lines, ACHN, 786O and Caki-1 (Fig. 1A). To identify the function of GCN2 in renal cancer cells, RNA interferences were used to knock down GCN2 expression in ACHN and 786O cells. After knocking down of GCN2 (Fig. 1B), transfected cells were subjected to CCK8 assays to evaluate cell viability and proliferation rates at specific periods (0, 24, 48 and 72 h). In both cell lines, GCN2 silencing significantly reduced the cell viability (Fig. 1C) and proliferation rate (Fig. 1D and E). Also, we use EdU to label proliferating cells. EdU is a thymidine analog that is incorporated into replicated chromosomal DNA during the S phase of the cell cycle. In both cell lines, less transfected cells labelled with EdU were observed comparing with control group (Fig. 1F). Furthermore, results of flow cytometry indicated that percentage of apoptotic cells detected in silencing group were significant higher than control group (Fig. 2A and B). Taken together, our data indicate that the knocking down GCN2 inhibited renal cancer cell viability by inducing cell apoptosis.

Table 1
82 patients characteristics of clinical informations

Characteristics	Patients ( $n=$ 82)
Median age	54.6 years (22–80 years)
Gender
Male	62
Female	20
Tumor stage
Low (I, II)	60
High (III, IV)	22
Fuhrman grade
Low (I, II)	47
High (III, IV)	35
Lymph invasion	15
Mean tumor size	5.5 cm (1–13 cm)
Median follow-up	61.2 months (16–173 months)

Figure 4.

GCN2 expression in PRCC tissues. Low GCN2 immunoexpression (Patient A) and high GCN2 immunoexpression (Patient B) in neoplastic cells of PRCC I. Low GCN2 immunoexpression (Patient C) and high GCN2 immunoexpression (Patient D) in neoplastic cells of PRCC II. Reduced from 200 $\times$ and 400 $\times$ respectively.

Table 2

Association of GCN2 expression and clinicopathological variables of PRCC

	GCN2
	Low (%)	High (%)	p Value
No.pts	48 (58.5)	34 (41.5)
Gender			0.438
Male	38 (79.2)	24 (70.6)
Female	10 (20.8)	10 (2.4)
Age			0.823
$\leqslant$ 55	26 (54.2)	17 (50.0)
$>$ 55	22 (45.8)	17 (50.0)
Type			0.004
I	22 (45.8)	5 (14.7)
II	26 (54.2)	29 (85.3)
Size			$<$ 0.001
$\leqslant$ 7 cm	43 (89.6)	18 (52.9)
$>$ 7 cm	5 (10.4)	16 (47.1)
TNM stage			$<$ 0.001
Low (I, II)	45 (93.8)	15 (44.1)
High (III, IV)	3 (6.2)	19 (55.9)
Fuhrman grade			$<$ 0.001
Low (I, II)	38 (79.2)	9 (26.5)
High (III, IV)	10 (20.8)	25 (73.5)
Lymph invasion			$<$ 0.001
Neg	46 (95.8)	21 (61.8)
Pos	2 (4.2)	13 (38.2)

2.2 Clinical significance of GCN2 expression in PRCC specimens

Western blotting analyses revealed that the GCN2 expression level in human PRCC tissues was higher than normal kidney tissues (Fig. 3A and B). GCN2 expression was higher in PRCC. To investigate and confirm the correlation between GCN2 level and the clinicopathologic variables of PRCC, GCN2 expression was evaluated in 84 PRCC tissues by immunohistochemical (IHC) staining. Summary of clinical parameters of patients with PRCC are shown in Table 1. Positive GCN2 expression was found in 82 of 84 cases consisting of 48 low expression and 34 high expression (Fig. 4). Elevated GCN2 level was significantly correlated with adverse clinicopathological parameters such as tumor subtype, size, Fuhrman grade, TNM stage, and lymph node invasion (Table 2). Noticeable, elevated GCN2 expression was more prevalent in cases of subtype 2 (85.3% vs 54.2%, $p=$ 0.004). Elevated GCN2 expression is associated with higher Fuhrman grade (III, IV) (73.5% vs 20.8%, $p<$ 0.001), higher TNM stage (III, IV) (55.9% vs 6.2%, $p<$ 0.001), lymph node invasion (38.2% vs 4.2%, $p<$ 0.001) and tumor size greater than 7 cm (47.1% vs 10.4%, $p<$ 0.001). Our results suggest that GCN2 expression is increased in PRCC, and upregulated expression of GCN2 is associated with poor clinical features.

2.3 High expression of GCN2 correlates with poor prognosis in PRCC

The median follow-up of this cohort is 61.2 months (range 16 to 173). Kaplan-Meier survival curves generated for GCN2 in the whole cohort showed that patient with high GCN2 expression had significantly worsen outcomes.(OS: $p<$ 0.001, Fig. 5A; PFS: $p<$ 0.001, Fig. 5B).

Table 3
Overall survival and PFS survival in PRCC by COX survival analysis

	Univariable analyses			Multivariable analysis
	HR	95% CI	p Value	HR	95% CI	p Value
Overall survival
Gender (F vs M)	0.555	0.252–1.223	0.144	–
Age 55 or greater vs less than 55	1.042	0.502–2.163	0.912	–
Type (2 vs 1)	1.857	0.756–4.565	0.177	–
Fuhrman (high vs low)	2.303	1.104–4.802	0.026	0.801	0.358–1.793	0.589
Size ( $\geqslant$ 7 cm vs $<$ 7)	3.155	1.501–6.633	0.002	1.134	0.454–2.833	0.787
TNM stage (high vs low)	11.447	4.879–26.853	$<$ 0.001	4.364	1.457–13.071	0.008
Lymph (pos vs neg)	7.181	3.361–15.346	$<$ 0.001	3.385	1.047–10.948	0.042
GCN2 (high vs low)	9.384	3.727–23.631	$<$ 0.001	9.250	2.956–28.945	$<$ 0.001
Progress-free survival
Gender (F vs M)	0.577	0.282–1.180	0.132	–
Age 55 or greater vs less than 55	1.061	0.546–2.060	0.862	–
Type (2 vs 1)	2.046	0.893–4.686	0.09	–
Fuhrman (high vs low)	2.504	1.278–4.908	0.008	0.599	0.271–1.325	0.205
Size ( $\geqslant$ 7 cm vs $<$ 7)	2.615	1.337–5.116	0.005	0.932	0.414–2.097	0.865
TNM stage (high vs low)	6.677	3.386–13.168	$<$ 0.001	2.869	1.156–7.120	0.023
Lymph (pos vs neg)	11.193	4.790–26.158	$<$ 0.001	3.951	1.274–12.250	0.017
GCN2 (high vs low)	8.897	3.993–19.825	$<$ 0.001	9.180	3.229–26.099	$<$ 0.001

HR, hazard ratio; CI, confidence interval.

Figure 5.

Comparison of overall survival (A) curves and progression free survival (B) of PRCC patients in relation to the expression of the GCN2 in cancer cell cytoplasm.

To investigate whether GCN2 could be predictive for clinical prognosis of PRCC, Cox regression model was performed. Multivariate analyses revealed that TNM stage (III, IV), lymph node invasion and high GCN2 expression were considered of independent prognostic markers of overall survival (Table 3, HR $=$ 4.364, $p=$ 0.008; HR $=$ 3.385, $p=$ 0.042;HR $=$ 9.250, $p<$ 0.001).

On univariate survival analyses for PFS, tumor size $>$ 7 cm, Fuhrman grade (III, IV), TNM stage (III, IV), lymph node invasion and high GCN2 expression were correlated with reduced PFS. Multivariate analyses revealed that high expression level of GCN2 remained significantly powerful indicator of poor PFS (HR $=$ 9.180, $p<$ 0.001). Apart from GCN2 expression, high TNM stage and lymph node invasion were selected as important prognostic indicators (HR $=$ 2.869, $p=$ 0.023; HR $=$ 3.951, $p=$ 0.017).

3. Discussion

Renal cell carcinoma (RCC) has a higher morbidity and mortality in China [22]. PRCC is a less understood subtype of renal cancer. Traditionally, outcomes for PRCC patients were much more favourable compared with ccRCC [23, 24]. However, recurrence progress after nephrectomy remains a significant clinical problem and has a worse prognosis for PRCC than ccRCC [25, 26]. So far, there are no standards or effective forms of therapy for recurrence of PRCC patients. Given these facts, prognostic markers and therapeutic agents are needed for this disease. These findings may attribute to the heterogeneous feature of PRCC which characterized by variations in both recrudescent possibility and patient outcomes.

The outcome for ccRCC, which is the most prevalent phenotype, has been dramatically improved by targeted therapy [27, 28, 29]. Tyrosine kinase inhibitor is the main standard therapeutic option for advanced RCC patients. It has antitumor effect due to the inhibition of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). VEGF expression is also significantly increased in PRCC, and associated with tumor stage [30]. However, although the targeted drugs show activity in treatment of advanced PRCC, the clinical responses remain overall lower than in ccRCC [31, 32]. This is probably because PRCC is a heterogeneous disease that consists of aggressive phenotype, and little is known about the genetic basis. The discovery at the molecular level of PRCC is currently being studied [6]. Recently, MET has been found participate in mechanism of resistance of VEGFR inhibitors and seems to be critical in the tumorigenesis of PRCC. Regrettably, clinical trails demonstrated the antitumor activity of MET inhibitors with an response rate less than 20% in PRCC, such as Foretinib [33] and Savolitinib [34]. It is reasonable to provide molecular insights into PRCC reclassification, inform clinical recommendations to the development of early diagnosis and accurate treatment.

Studies have demonstrated that GCN2 activation led to VEGF expression in cancer cells, thereby enhancing the angiogenesis and aggressiveness of neoplastic cells [35, 36]. GCN2 and the downstream target active transcription factor 4 (ATF4) were important for tumor survival in nutrient deprivation microenvironment [37, 38]. We observed that GCN2 was significantly elevated in PRCC tissues and the upregulated GCN2 level was linked to an aggressive phenotype. GCN2 may be a biomarker of poor prognosis with PRCC.

For PRCC patients, lymph node invasion, metastasis and higher Fuhrman Grade were considered independent predictors of PFS [39, 40]. Nevertheless, even within patients of the same pathological parameters, there are variations in patient survival. Consequently, there is a need to explore other predictors for PRCC patients. In present study, we observed that PRCC patients with high GCN2 levels exhibited worse survival and increased risk of recurrence. Univariate analyses and multivariate analyses verified this result was statistically significant (Table 3, $p<$ 0.001; $p<$ 0.001) (Table 3, $p=$ 0.017; $p=$ 0.002). Although it is reported that Type2 papillary histology predicts poor outcome in patients [41], in our study phenotype was not an independent predictor of patients’ prognosis, possibly due to the small amount of patients ( $n=$ 17, 43 for Types 1 and 2 respectively). Some limitations of this study should be noted. Our study was carried out mainly on retrospective observation with small number of samples. To increase the predictive accuracy, further studies of larger scale prospective studies are needed to verify our conclusion.

To conclude, our study illustrated that GCN2 level was closely associateed with PRCC clinical parameters, larger tumor size, higher TNM stage, higher Fuhurman Grade, and lymph node metastasis. GCN2 overexpression is a prognostic biomarker linked to decreased OS and PFS of PRCC patients. GCN2 may be used as an indicator of prognosis and a molecular target for anticancer therapies in PRCC.

4. Materials and methods

4.1 Study subjects

Eighty-four consecutive PRCC patients with PRCC underwent surgical therapy from January 2003 to April 2015 at Nanjing University Affiliated Drum Tower Hospital. All subjects signed written informed consent. The study was approved by the Hospital Ethics Boards prior to initiation. Patients received chemotherapy or molecular targeted therapy were excluded from this study. All PRCC tumors were re-examined and graded, according to Fuhrman and 7th American Joint Committee Cancer (AJCC) Staging Manual grading system [42].

4.2 Cell culture and transfections

The human renal cancer cell line ACHN, 786O, Caki-1 and human kidney proximal tubule epithelial cell line HK2 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained according to the distributor’s recommendations. Small-interfering RNA (siRNA) targeting GCN2 were designed using siDirect software. GCN2 siRNA (h) (sc-45644) and negative control siRNA were transfected into cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA).

4.3 Immunohistochemistry

Paraffin sections (3 $\mu$ m) from samples were deparaf-finized and rehydrated in descending ethanol series and water. After heat-induced antigen retrieval and endogenous peroxidase blocing, the slides were incubated with GCN2 polyclonal antibody (1:100) (PA5-13924, Thermo Fisher Scientific, USA) overnight at 4 ${{}^{\circ}}$ C.

For the immunohistochemical assessment, staining was evaluated independently by 2 trained uropathologists. Immunohistochemical score (IHC) was caculated by multiplying the intensity score and proportion scores. IHC score system: 0-negative, 1–4 low and 5–12 high. Intensity of staining system: negative: 0, weak: 1, moderate: 2, and strong: 3. Area of staining positivity system: 0 (less than 10%), 1 (11% to 25%), 2 (26% to 50%), 3 (51% to 75%) and 4 (greater than 75%).

4.4 Western blotting

Total protein was isolated from RCC tissues, adjacent normal tissues or RCC cells using RIPA lysis buffer (Vazyme, Piscataway, NJ, USA). 20 $\mu$ g proteins were loaded on a 3% stacking/10% separating SDS–PAGE and run for 3.5 hours at 100 V. Following electrophoresis, protein was transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with anti-GCN2 (Cell Signaling Technology, Danvers, MA), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 ${}^{\circ}$ C overnight. Following incubation with secondary antibody at room temperature for 1 h, membranes were detected by ECL solution and visualized by ChemiScope Mini Imaging System (Clinx Science Instruments Co.Ltd, China).

4.5 RT-qPCR

Total RNAs were extracted from cell cultures using Trizol (Invitrogen, Carlsbad, CA, USA). RNA was transcribed into cDNA and amplified with PrimeScript RT Reagent Kit (Takara, Otsu, Japan). RT-qPCR were performed in accordance with manufacturer’s instructions (Takara, Otsu, Japan). The primers used for PCR as listed as below:

GCN2: 5’-GCAGGTACACAGCTCAGCTC-3’ (upstream) and 5’-CAACAACTGCTGGGGACTC C-3’ (downstream), GAPDH: 5’-AGCCACATCGCTCAGACAC-3’ (upstream) and 5’-GCCCAATACGACCAAATCC-3’ (downstream).

Comparative threshold cycle 2 ${}^{-\Delta\Delta\text{Ct}}$ method was used to calculate gene expression.

4.6 Cell viability and apoptosis detection

For viability detection, transfected cells were measured according to manufacturer’s protocol. CCK8 assay purchased from Vazyme (Piscataway, NJ, USA). Edu assay purchased from (Ribobio, Guangzhou, China).

For apoptosis detection, transfected cells were washed for 3 times and suspended after centrifugal and detected by Annexin V-FITC assay (Abcam, Cambridge, UK) according to the manufacture’s protocol.

JC-1 staining of ACHN and 786O were performed according to manufacturer’s protocol (Solarbio tech, Beijing, China).

4.7 Statistical analyses

Correlations between GCN2 expression and clinic prognostic variables were studied using the $\chi$ 2 test. The endpoints of this cohort were overall survival (OS) and progression free survival (PFS). OS was calculated from the date of surgical excision of the primary tumor to the date of death. PFS was defined as the interval between the date of surgery and the date of the first identification of recurrence or death. OS and PFS curves were constructed by the Kaplan-Meier method. Univariate analyses of patient outcomes were performed by two-sided long-rank test. Cox regression model was generated for multivariate analysis to remove nonsignificant parameters. Two-sided $p$ -values were calculated. Statistical calculation were carried out using Prism 6.05 and SPSS ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ 22.0.

Footnotes

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81502203, 81572519), the National Natural Science Foundation of Jiangsu Province (BK20150097), the “Summit of the Six Top Talents” Program of Jiangsu Province (SWYY-084), the General Financial Grant from the China Postdoctoral Foundation (2017M621729), the Postdoctoral Research Foundation of Jiangsu Province (1701021B) and Nanjing Medical Science and technique Development Foundation (QRX17139).

Conflict of interest

We have no financial disclosures to declare and no conflicts of interest to report.

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GCN2 is a potential prognostic biomarker for human papillary renal cell carcinoma

Abstract

Keywords

1. Introduction

2.1 GCN2 knockdown inhibited cell viability and promoted apoptosis in vitro

Table 1 82 patients characteristics of clinical informations

2.3 High expression of GCN2 correlates with poor prognosis in PRCC

Table 3 Overall survival and PFS survival in PRCC by COX survival analysis

4. Materials and methods

4.1 Study subjects

4.2 Cell culture and transfections

4.3 Immunohistochemistry

4.4 Western blotting

4.5 RT-qPCR

4.6 Cell viability and apoptosis detection

4.7 Statistical analyses

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
82 patients characteristics of clinical informations

Table 3
Overall survival and PFS survival in PRCC by COX survival analysis