Yonsei Med J.  2019 Apr;60(4):326-335. 10.3349/ymj.2019.60.4.326.

Potential Oncogenic Role of the Papillary Renal Cell Carcinoma Gene in Non-Small Cell Lung Cancers

• Jang SH
• Jiang Y
• Shin S
• Jung SH
• Jung CK
• Chung YJ

• KMID: 2450187
• DOI: http://doi.org/10.3349/ymj.2019.60.4.326

Abstract

PURPOSE
Papillary renal cell carcinoma (PRCC) gene, which located in 1q23.1, is recurrently amplified in non-small cell lung cancer (NSCLC). However, it is unknown whether PRCC is overexpressed in primary NSCLCs and whether PRCC overexpression contributes to lung tumorigenesis. In this study, we aimed to identify the profiles of PRCC expression in Korean NSCLC patients and to elucidate the role of PRCC overexpression on lung tumorigenesis.
MATERIALS AND METHODS
We performed immunohistochemistry analysis with a tissue array containing 161 primary NSCLCs. Small interfering RNA targeting PRCC (siPRCC) was transfected into two lung cancer cell lines (NCI-H358 and A549), after which tumor growth, migration, and invasion were observed. Expressions of cell proliferation-, cell cycle-, and metastasis-related molecules were examined by Western blot analysis. We also explored the in vivo effect of PRCC silencing.
RESULTS
PRCC overexpression was recurrently observed in NSCLCs (95/161, 59%). After siPRCC treatment, tumor cell proliferation, colony formation, and anchorage independent growth were significantly reduced (p < 0.001 for all three effects). Migration and invasiveness were also significantly repressed (p < 0.001 for both effects). Reflecting cell proliferation, cell cycle, and metastasis, the expressions of Ki67, cyclin D1, AKT-1, pAKT, NF-kB p65, vimentin and CXCL-12 were found to be downregulated. Through mouse xenograft analysis, we confirmed that PRCC silencing significantly repressed a xenograft tumor mass in vivo (p < 0.001).
CONCLUSION
The present data provide evidence that PRCC overexpression is involved in the tumorigenesis and progression of lung cancer.

Keyword

Lung cancer; PRCC; overexpression; siRNA

MeSH Terms

Animals
Blotting, Western
Carcinogenesis
Carcinoma, Non-Small-Cell Lung
Carcinoma, Renal Cell*
Cell Cycle
Cell Line
Cell Proliferation
Cyclin D1
Heterografts
Humans
Immunohistochemistry
Lung Neoplasms*
Lung*
Mice
Neoplasm Metastasis
NF-kappa B
RNA, Small Interfering
Vimentin
Cyclin D1
NF-kappa B
RNA, Small Interfering
Vimentin

Figure

• 

  Fig. 1 Immunohistochemistry for papillary renal cell carcinoma (PRCC) in lung adenocarcinoma (AC) and squamous cell carcinoma (SCC). Nuclear staining intensity was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Grade 2–3 was considered as overexpression. Original magnification ×400.

• 

  Fig. 2 Baseline expression levels of PRCC in four lung cancer cell lines (NCI-H23, NCI-H358, NCI-H460 and A549) and normal lung cells (CCD25-LU) by Western blot analysis. PRCC expression in Western blot analysis was measured by densitometry. α-tubulin was used as an internal control. Relative signal intensity ratios of PRCC are presented in between the two plots. PRCC, papillary renal cell carcinoma.

• 

  Fig. 3 siPRCC-mediated silencing of PRCC expression in lung cancer cell lines. After siPRCC transfection (siPRCC-1, siPRCC-2, and siPRCC-3) into NCI-H358 and A549 cells, PRCC expression was measured by real-time quantitative reverse transcriptase polymerase chain reaction (A) and Western blot analysis (B). Relative signal intensity ratios of PRCC are presented in between the two plots. PRCC, papillary renal cell carcinoma.

• 

  Fig. 4 Effect of silencing overexpressed PRCC on lung cancer cell growth. (A) The effect of PRCC silencing on the proliferation of NCI-H358 and A549 cells was measured by BrdU incorporation assay. The proliferation of both PRCC-silenced cells was significantly repressed, compared with that of non-silenced control cells. All measurements were repeated three times, and the mean optical density values with SEM were plotted for each case. (B) Colony formation assays for the effect of PRCC silencing (×1). Cells were transfected with siNEG and siPRCC, respectively, and incubated in for 7 days. Colonies larger than 1 mm in diameter were counted. Bar chart in the right box represents the average colony numbers in siPRCC-treated cells compared to those in siNEG-treated cells. (C) Effect of PRCC silencing on anchorage independent growth (×100). Bar chart in the right-side plot represents the average colony numbers. *p<0.001. PRCC, papillary renal cell carcinoma.

• 

  Fig. 5 Effects of PRCC knockdown on migration and invasion. (A) Motility potential of siPRCC- and siNEG-transfected cells was examined using transwell chambers. After crystal violet staining, the numbers of colonies in the five microscopic fields (×200) were counted. Bar chart in the right box represents the average number of migrated cells in siPRCC-treated cells and in siNEG-treated cells. (B) Invasive potential of siPRCC- and siNEG-transfected cells was examined using Matrigel-coated transwell chambers. After crystal violet staining, the numbers of colonies in the five microscopic fields (×200) were counted. Bar chart in the right box represents the average number of migrated cells in PRCC-treated cells and in siNEG-treated cells. *p<0.001. PRCC, papillary renal cell carcinoma.

• 

  Fig. 6 Protein expressions of cell proliferation-, cell cycle-, and metastasis-related molecules in siPRCC- and siNEG-treated lung cancer cells. Expressions of Ki67, cyclin D1, AKT-1, pAKT, NF-kB p65, vimentin, and CXCL-12 were observed by Western blot analysis. β-actin was used as loading control.

• 

  Fig. 7 siPRCC-mediated regression of established tumor growth in vivo. (A) Photograph of EGFP stable expressing NCI-H358 cells (H358-pEGFP-N1). (B) Whole-animal fluorescent signal images of H358-pEGFP-N1 tumor in nude mice. H358-pEGFP-N1 cells were transplanted into both flanks of immunodeficient nude mice. After 15 days, the mice were given intratumoral injections of siPRCC (left) or siNEG (right). Fluorescence is presented as a pseudocolor scale: red, the highest photon flux; blue, the lowest photon flux. (C) Monitoring of tumor growth by fluorescence. To investigate the tumor cell growth kinetics, injected H358-pEGFP-N1 cells were monitored for their GFP fluorescence. The total flux is expressed as the mean±SEM (n=6). (D) Monitoring of tumor growth by tumor weight. Tumor weights were measured at sacrifice and were compared between xenograft mice administered with PRCC siRNA and sterile PBS (n=6). *p<0.05, †p<0.001. PRCC, papillary renal cell carcinoma; EGFP, enhanced green fluorescent protein.

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