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. 2016 Apr 19;7(16):22385-96.
doi: 10.18632/oncotarget.8107.

Reduced cytosolic carboxypeptidase 6 (CCP6) level leads to accumulation of serum polyglutamylated DNAJC7 protein: A potential biomarker for renal cell carcinoma early detection

Chong Li  1   2   3 , Jihan Wang  4 , Junfeng Hao  2 , Baijun Dong  5 , Yi Li  6 , Xiaoxiao Zhu  2 , Juan Ding  1 , Shuangchun Ren  1 , Heping Zhao  4 , Song Wu  3 , Yong Tian  2 , Guo-Qing Wang  1
Affiliations

Reduced cytosolic carboxypeptidase 6 (CCP6) level leads to accumulation of serum polyglutamylated DNAJC7 protein: A potential biomarker for renal cell carcinoma early detection

Chong Li et al. Oncotarget. .

Abstract

Renal cell carcinoma (RCC) is frequently diagnosed at advanced stages of disease, although early diagnosis has much favorable prognosis. This study assessed aberrant expression of cytosolic carboxypeptidase 6 (CCP6) leading to accumulation of serum polyglutamylated DNAJC7 as a biomarker for early RCC detection. A total of 835 RCCs, 143 chronic nephritis, 170 kidney stones and 415 health controls were collected for qRT-PCR, immunohistochemistry and Western blot analysis of CCP6 expression and mass spectrometry of DNAJC7 and polyglutamylated DNAJC7. The data showed that CCP6 expression was significantly decreased in 30 RCC tissues and that mass spectrometric and pull-down analysis identified DNAJC7 as a substrate of CCP6 and showed upregulated polyglutamylated-DNAJC7 (polyE-DNAJC7) in sera of RCC patients. The electrochemiluminescence immunoassay of large-scale serum samples from multi-institutes further confirmed the remarkable increase of polyE-DNAJC7 in 805 RCCs compared to that of 385 healthy controls (p < 0.001), 128 patients with chronic nephritis (p < 0.001), and 153 with kidney stone (p < 0.001). Serum level of DNAJC7-polyE protein was also associated with advanced RCC stage and grade in 805 patients. The data from the current study for the first time demonstrated increased serum polyglutamylated DNAJC7 as a potential biomarker for RCC early detection and association with advanced tumor stages and grade, which provides support of further polyglutamylation research in RCC.

Keywords: CCP6; diagnosis biomarker; polyE-DNAJC7; polyglutamylation; renal cell carcinoma.

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Conflict of interest statement

The authors declare no conflicts of interest in this work.

Figures

Figure 1
Figure 1. Downregulated CCP6 expression in RCC tissues
(A) RT-PCR. Expression of CCP6 and β-actin mRNA was measured using RT-PCR in RCC cancer tissues (CT) and pericancerous tissues (PT). **P < 0.01. (B) Western blot analysis of CCP6 protein (58 kD) and β-actin (43 kD) expression in RCC cancer tissues (CT) and pericancerous tissues (PT). The graph is the density comparison of CCP6 protein expression (CCP6/β-actin) of RCC cancer tissues vs. pericancerous tissues. **P < 0.01. (C) Immunohistochemical staining of CCP6 expression in RCC cancer tissues and pericancerous tissues. (×200).
Figure 2
Figure 2. Glutamylated GST-DNAJC7 protein binding to CCP6 protein in RCC tissues and sera
(A) Protein polyglutamylation was analyzed by immunoblotting with the GT335 antibody. Protein lysates from CCP6-deficient and the control mouse kidneys were analyzed. (B) CCP6-wt and CCP6-mut were immobilized with Affi-gel10 resin to go through kidney lysates for affinity chromatography. The eluted fractions were visualized by SDS-PAGE followed by silver staining. (C) Immunoprecipitation assay. The pull-down assays using GST-DNAJC7 incubated with the lysates from Myc-tagged CCP6-wt and Myc-tagged CCP6-mut 293T cells. Data were repeated for three times. (D) The pull-down/input ratios of CCP6 protein density in CCP6-wt and CCP6-mut 293 T cells. **P < 0.01. (E) Immunoprecipitation-Western blot. DNAJC7 protein with a glutamylated modification status in RCC sera. Immunoblotting analysis of GT335 signal in sera from RCC patients and health controls was immunoprecipitated with the anti-DNAJC7 antibody.
Figure 3
Figure 3. Consort diagram of the experimental design
Figure 4
Figure 4. Serum concentration (RLU) and ROC analysis of polyglutamylated-DNAJC7 protein
(A) Concentration (RLU) of serum polyglutamylated DNAJC7 protein in chronic nephritis, kidney stone patients, health controls, early stage (I) RCC and all RCC patients in the test cohort. (B) ROC analysis of polyglutamylated DNAJC7 protein as a discriminative biomarker between early stage (I) RCC and non-tumor samples (chronic nephritis & kidney stone patients & health controls) in the cohort. (C) Concentration (RLU) of serum polyglutamylated DNAJC7 protein in chronic nephritis, kidney stone patients, health controls, early stage (I) RCC and all RCC patients in validation cohort. (D) ROC analysis of polyglutamylated DNAJC7 protein as a discriminative biomarker between early stage (I) RCC patients and non-tumor samples (chronic nephritis & kidney stone patients & health controls) in validation cohort. (E) Concentration (RLU) of serum polyglutamylated DNAJC7 protein in different stages of RCC patients in validation cohort. (F) ROC analysis of polyglutamylated DNAJC7 protein as a discriminative biomarker between III–IV RCC and I-II RCC patients in validation cohort. (G) Concentration (RLU) of serum polyglutamylated DNAJC7 protein in different grades of RCC patients in validation cohort. (H) ROC analysis of polyglutamylated DNAJC7 protein as a discriminative biomarker between Grade3 and Grade1-2 RCC patients in validation cohort.
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