Parathyroid hormone-related protein serves as a prognostic indicator in oral squamous cell carcinoma

doi:10.1186/s13046-014-0100-y

. 2014 Dec 18;33(1):100.

doi: 10.1186/s13046-014-0100-y.

Parathyroid hormone-related protein serves as a prognostic indicator in oral squamous cell carcinoma

Zhongjing Lv^{1

2}, Xiangbing Wu^{3

4}, Wei Cao^{5

6}, ZongZe Shen^{7

8}, Lizhen Wang^{9

10}, FuRong Xie^{11

12}, JianJun Zhang^{13

14}, Tong Ji^{15

16}, Ming Yan^{17

18}, WanTao Chen^{19

20}

Affiliations

¹ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. zhongjing_lv2012@163.com.
² Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. zhongjing_lv2012@163.com.
³ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. wuxiangbing2007@163.com.
⁴ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. wuxiangbing2007@163.com.
⁵ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. caowei561521@hotmail.com.
⁶ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. caowei561521@hotmail.com.
⁷ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. johnnyshen7603@163.com.
⁸ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. johnnyshen7603@163.com.
⁹ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. lizhenwang535@hotmail.com.
¹⁰ Department of Oral Pathology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. lizhenwang535@hotmail.com.
¹¹ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. xfr1988@163.com.
¹² Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. xfr1988@163.com.
¹³ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. zjjshuobo@163.com.
¹⁴ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. zjjshuobo@163.com.
¹⁵ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. jitong70@hotmail.com.
¹⁶ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. jitong70@hotmail.com.
¹⁷ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. yanming8012@126.com.
¹⁸ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. yanming8012@126.com.
¹⁹ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. chenwantao2002@hotmail.com.
²⁰ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. chenwantao2002@hotmail.com.

PMID: 25539663
PMCID: PMC4393566
DOI: 10.1186/s13046-014-0100-y

Parathyroid hormone-related protein serves as a prognostic indicator in oral squamous cell carcinoma

Zhongjing Lv et al. J Exp Clin Cancer Res. 2014.

. 2014 Dec 18;33(1):100.

doi: 10.1186/s13046-014-0100-y.

Authors

Affiliations

¹ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. zhongjing_lv2012@163.com.
² Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. zhongjing_lv2012@163.com.
³ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. wuxiangbing2007@163.com.
⁴ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. wuxiangbing2007@163.com.
⁵ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. caowei561521@hotmail.com.
⁶ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. caowei561521@hotmail.com.
⁷ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. johnnyshen7603@163.com.
⁸ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. johnnyshen7603@163.com.
⁹ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. lizhenwang535@hotmail.com.
¹⁰ Department of Oral Pathology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. lizhenwang535@hotmail.com.
¹¹ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. xfr1988@163.com.
¹² Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. xfr1988@163.com.
¹³ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. zjjshuobo@163.com.
¹⁴ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. zjjshuobo@163.com.
¹⁵ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. jitong70@hotmail.com.
¹⁶ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. jitong70@hotmail.com.
¹⁷ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. yanming8012@126.com.
¹⁸ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. yanming8012@126.com.
¹⁹ Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. chenwantao2002@hotmail.com.
²⁰ Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology, Shanghai, China. chenwantao2002@hotmail.com.

PMID: 25539663
PMCID: PMC4393566
DOI: 10.1186/s13046-014-0100-y

Abstract

Background: In our previous study, parathyroid hormone-like hormone (PTHLH) which encodes parathyroid hormone-related protein (PTHrP) was revealed to be up-regulated in oral squamous cell carcinoma (OSCC) compared with paired apparently normal surgical margins using microarray method. However, the function and prognostic indicators of PTHLH/PTHrP in OSCC remain obscure.

Methods: The mRNA levels of PTHLH and its protein levels were investigated in 9 OSCC cell lines and in 36 paired OSCC specimens by real-time PCR and western blotting. The biological function of PTHLH/PTHrP was investigated using small interfering RNA (siRNA) in 3 OSCC cell lines, and immunohistochemistry was used to estimate the prognostic value of PTHrP in 101 patients with head and neck squamous cell carcinoma (HNSCC), including OSCC and oropharyngeal squamous cell carcinoma. Cell cycle was tested by flow cytometry and cell cycle related genes were investigated by western blotting and immunocytochemistry assay.

Results: This study showed that the mRNA and protein levels of PTHLH in 9 OSCC cell lines were much higher than that in normal epithelial cells (P < 0.0001). In 36 paired OSCC tissues, PTHLH mRNA expressions were found higher in 32 OSCC tissues than that of paired apparently normal surgical margins (P = 0.0001). The results revealed that the down-regulation of PTHLH/PTHrP by siRNAs could reduce cell proliferation and inhibit plate and soft agar colony formation as well as affect the cell cycle of OSCC cells. The key proteins related to the cell cycle were changed by anti-PTHLH siRNA. The results showed that cyclin D1 and CDK4 expressions were significantly reduced in the cells transfected with anti-PTHLH siRNA. On the other hand, the expression of p21 was increased. The results also showed that high PTHrP level was associated with poor pathologic differentiation (P = 0.0001) and poor prognosis (P = 0.0003) in patients with HNSCC.

Conclusions: This study suggests that PTHLH/PTHrP is up-regulated in OSCCs. Therefore, PTHLH/PTHrP could play a role in the pathogenesis of OSCC by affecting cell proliferation and cell cycle, and the protein levels of PTHrP might serve as a prognostic indicator for evaluating patients with HNSCCs.

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Figures

**Figure 1**
**PTHLH mRNA and PTHrP protein expression in OSCC specimens and cell lines. (A)** PTHLH mRNA expression was analyzed in 36 paired OSCC specimens including cancerous tissues and apparently normal surgical margins using real-time PCR (Mean ± SE: 2.618 ± 0.495, rang: 0.0817 ± 12.010 vs. Mean ± SE: 7.376 ± 1.040, rang: 0.988 ± 20.873; P = 0.0001; two-tailed Student’s t-test). **(B)** PTHrP protein expression was determined in 5 representative paired OSCC specimens by western blotting analysis (N, apparently normal surgical margin; C, cancerous tissue). **(C)** PTHLH mRNA levels were determined in 9 representative OSCC cell lines, primary normal epithelial cells and normal tissues showing in Figure 1A using real-time PCR (P < 0.0001; One-way ANOVA). **(D)** PTHrP protein levels were determined in 9 representative OSCC cell lines and primary normal epithelial cells using western blotting.

**Figure 2**
**The effect of PHTLH/PTHrP on cell proliferation and colony formation. (A)** PTHrP protein expression was confirmed in WSU-HN6, HN13 and CAL-27 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA using western blotting. **(B)** Cell proliferation of WSU-HN6, HN13 and CAL-27 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA were analyzed using the CCK-8 kit, each point showed the mean values of triplicate wells (Mean ± SE). **(C)** Plate colony formation of WSU-HN6, HN13 and CAL-27 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA was analyzed. **(D)** Soft agar colony formation assay was analyzed between the cell lines transfected with anti-PTHLH P-si and cell lines transfected with scrambled siRNA.

**Figure 3**
**The impact of PTHLH/PTHrP on the cell cycle. (A)** Cell cycle was analyzed in WSU-HN6, HN13 and CAL-27 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA. **(B)** The proteins of Cyclin D1, CDK4 and p21 which related to cell cycle were determined in WSU-HN6 and HN13 cell lines transfected with anti-PTHLH siRNA P-si or scrambled siRNA. **(C)** The fold change in western blot (Figure B) was quantified and analyzed. **(D)** Immunocytochemistry staining of cells transfected with PTHLH-siRNA was used to analyze the expression of PTHLH, Cyclin D1, CDK4 and p21.

**Figure 4**
**The clinical significance of PTHrP overexpression was analyzed in 93 patients with HNSCCs. (A)** PTHrP expression was detected in poorly differentiated, moderately differentiated and well differentiated specimens and in normal epithelial specimens, as shown by immunohistochemistry. **(B)** Kaplan-Meier survival curves illustrating the overall survival of 93 patients with HNSCC according to the PTHrP protein levels. **(C)** Two patients with base of tongue squamous cell carcinoma in this study were positive for HPV16/18 and p16 expression using ISH and IHC methods.

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References

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[1] Gaykalova DA, Mambo E, Choudhary A, Houghton J, Buddavarapu K, Sanford T, Darden W, Adai A, Hadd A, Latham G, Danilova LV, Bishop J, Li RJ, Westra WH, Hennessey P, Koch WM, Ochs MF, Califano JA, Sun W. Novel insight into mutational landscape of head and neck squamous cell carcinoma. PLoS One. 2014;9:e93102. doi: 10.1371/journal.pone.0093102. - DOI - PMC - PubMed

[2] Gaykalova DA, Mambo E, Choudhary A, Houghton J, Buddavarapu K, Sanford T, Darden W, Adai A, Hadd A, Latham G, Danilova LV, Bishop J, Li RJ, Westra WH, Hennessey P, Koch WM, Ochs MF, Califano JA, Sun W. Novel insight into mutational landscape of head and neck squamous cell carcinoma. PLoS One. 2014;9:e93102. doi: 10.1371/journal.pone.0093102. - DOI - PMC - PubMed

[3] Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333:1157–1160. doi: 10.1126/science.1208130. - DOI - PMC - PubMed

[4] Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333:1157–1160. doi: 10.1126/science.1208130. - DOI - PMC - PubMed

[5] Parkin D, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74–108. doi: 10.3322/canjclin.55.2.74. - DOI - PubMed

[6] Parkin D, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74–108. doi: 10.3322/canjclin.55.2.74. - DOI - PubMed

[7] Pries R, Hogrefe L, Xie L, Frenzel H, Brocks C, Ditz C, Wollenberg B. Induction of c-Myc-dependent cell proliferation through toll-like receptor 3 in head and neck cancer. Int J Mol Med. 2008;21:209–215. - PubMed

[8] Pries R, Hogrefe L, Xie L, Frenzel H, Brocks C, Ditz C, Wollenberg B. Induction of c-Myc-dependent cell proliferation through toll-like receptor 3 in head and neck cancer. Int J Mol Med. 2008;21:209–215. - PubMed

[9] Yan B, Li H, Yang X, Shao J, Jang M, Guan D, Zuo S, Van Waes C, Chen Z, Zhan M. Unraveling regulatory programs for NF-kappaB, p53 and microRNAs in head and neck squamous cell carcinoma. PLoS One. 2013;8:e73656. doi: 10.1371/journal.pone.0073656. - DOI - PMC - PubMed

[10] Yan B, Li H, Yang X, Shao J, Jang M, Guan D, Zuo S, Van Waes C, Chen Z, Zhan M. Unraveling regulatory programs for NF-kappaB, p53 and microRNAs in head and neck squamous cell carcinoma. PLoS One. 2013;8:e73656. doi: 10.1371/journal.pone.0073656. - DOI - PMC - PubMed

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Parathyroid hormone-related protein serves as a prognostic indicator in oral squamous cell carcinoma

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Parathyroid hormone-related protein serves as a prognostic indicator in oral squamous cell carcinoma

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