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Clinical significance of PHPT1 protein expression in lung cancer

XU, An-jian; XIA, Xiang-hou; DU, Song-tao; GU, Jun-chao

doi: 10.3760/cma.j.issn.0366-6999.2010.22.014
Original article
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Background In our previous studies, we found the expression of 14-kD phosphohistidine phosphatase (PHPT1) was associated with lung cancer cells migration and invasion, and PHPT1 mRNA expression level in lung cancer tissues clinically correlated with lymph node metastasis. In the present study, we aimed to further investigate the expression of PHPT1 protein in lung cancer.

Methods Expression of PHPT1 protein in tissue samples from 146 lung cancers and 30 normal tissues adjacent to lung cancers was assessed using immunohistochemical method. Fisher's exact test was used to analyze expression patterns of PHPT1 protein in these tissue types. Meanwhile, we studied the correlation between expression of PHPT1 protein and clinicopathological features in lung cancer.

Results Significantly higher expression levels of PHPT1 protein were found in lung cancer samples ( 53.42%) than in normal tissues adjacent to lung cancer (23.33%) (P=0.003). Fisher's exact test showed that lung cancer stage positively correlated with expression of PHPT1 protein (P=0.02), and lung cancer samples with lymph node metastasis showed higher PHPT1 protein expression (P=0.016) than the samples without lymph node metastasis.

Conclusions The results of this study agree with findings from our previous study of PHPT1 mRNA expression in lung cancer tissues, and strongly suggest that PHPT1 protein is closely associated with the carcinogenesis and metastasis of lung cancer. Thus, therapy targeting PHPT1 (inhibition or silencing) could be potentially benefited for lung cancer patients.

Chin Med J 2010;123(22):3247–3251

Beijing Tropical Disease Institute (Xu AJ and Gu JC), Department of General Surgery (Xia XH, Du ST and Gu JC), Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

Correspondence to: GU Jun-chao, Beijing Tropical Disease Institute, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China (Tel: 86–10–63138570. Fax: 86–10–63139265. Email: gujunchao668@163.com)

This work was supported by the grants from Capital Foundation for Development of Medical Science (No. 2005–2031) and National Natural Science Foundation of China (No. 30772121). Conflict of interest statement: none.

(Received February 29, 2010)

Edited by WANG Mou-yue

Lung cancer is the leading cause of cancer-related mortality worldwide. In some countries, it has become the number one cancer killer, accounting for more deaths than prostate cancer, breast cancer, and colorectal cancer combined.1 The estimated new lung cancer deaths for 2007 were 31% and 26% of all male and female cancer deaths, respectively.2 This is largely due to the late stage of diagnosis and the lack of effective treatments. Although established biomarkers such as carcinoembryonic antigen (CEA), neurone specific enolase (NSE), p53, carbohydrate antigen 19–9 (CA19–9) and cytokeratin 19 fragment (CYFRA21–1) are commonly used as lung cancer biomarkers,3,4 several potential biomarkers, such as S100 calcium-binding protein All (S100A11) and ubiquitin-conjugating enzyme E2 T (UBE2T),5,6 have been investigated recently, and the precise mechanisms of those biomarkers leading to lung cancer have not been well elucidated.

The 14-kD phosphohistidine phosphatase (PHPT1), also known as PHP14, is similar to the janus proteins of drosophila and was the first protein histidine phosphatase discovered in vertebrates.7,8 Unlike Ser/Thr phosphatase, phosphohistidine phosphatase is a specific phosphatase family mainly existed in prokaryotes. However, among the total phosphorylations in eukaryotes, 7% of phosphorylations are histidine phosphorylations and they may involve in signal transduction and cancer.9,10 As reported, PHPT1 could dephosphorylate ATP-citrate lyase11 and β-subunit of G protein12in vitro and might take part in neuronal signaling transduction. Furthermore, the structure study against PHPT1 had revealed that the histidine 53 was its active site.13 In our previous studies, we found that the expression of PHPT1 was associated with tumor invasion as its expression is increased in high metastasis cell line;5 furthermore, in vivo and in vitro studies had revealed the great roles of PHPT1 in lung cancer cells migration and invasion.14 However, the expression of PHPT1 protein in lung cancer patients' tissues has rarely been reported. To further address the relationship between PHPT1 expression and lung cancer, we examined the protein expression of PHPT1 in lung cancer patients’ tissues and investigated its correlation with clinicopathlogical features of lung cancer using immunohistochemical approach.

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METHODS

Patients and materials

A total of 146 formalin-fixed and paraffin-embedded lung cancer tissues and 30 normal tissues adjacent to lung cancers were obtained from three microarrays (lung cancer tissues No. CC04–11–005, No. NC04–03–001 and No. CC04–21–001) purchased from Cybrdi Co., Ltd (Xi'an, China) (Tables 1 and 2). All lung cancer patients had a pathological diagnosis and none of them had received any chemotherapy, radiotherapy or endocrine therapy prior to operation. All samples under the study were collected between December 2005 and July 2006 and informed consent was signed by each donor. All of the resected primary tumors and regional lymph nodes were stained with hematoxylin and eosin and histologically examined according to the International Union Against Cancer Tumor-Node-Metastasis classi-fication (2003 revised edition).

Table 1

Table 1

Table 2

Table 2

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Expression of PHPT1 recombinant protein and preparation of PHPT1 polyclonal antibody

The prokaryote expression vector pET-30a (+)-PHPT1 was constructed by cloning the full-length gene of PHPT1 into the pET-30a (+) (Novagen, Madison, WI, USA) empty vector using primers as following: sense: 5'-TTACATATGGCGGTGGCGGACCT-3'; antisense: 5'-ACTCTCGAGGTAGCCGTCGTTAGCC-3'. Then, the pET-30a(+)-PHPT1 vector was transformed into the E. coli BL21 cells to obtain the N-His-tagged recombinant PHPT1 protein. The purification of the His-PHPT1 fusion protein in transformed cell extracts was performed by affinity chromatography on Ni-NTA affinity column (QIAGEN GmbH, Hilden, Germany) using native protocol according to the manufacturer's instructions. Protein purity and quantity were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie blue and confirmation of the protein identified by mass spectrometry. Five BALB/c mice were immunized with 150 μg purified His-PHPT1 recombinant proteins in complete Freund's adjuvant, followed by 3 booster injections in incomplete Freund's adjuvant at 2-week intervals. Immune serum was collected and analyzed 1 week after the final booster injection by Western blotting analysis. Immune serum was collected and analyzed 1 week after the final booster injection by Western blotting analysis.

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Immunohistochemistry

Paraffin-embedded sections were dewaxed at 60°C for 3 hours, deparaffinized in xylene and rehydrated in graded alcohols. Antigen retrieval was carried out in an antigen retrieval buffer (10 mmol/L citrated buffer (pH 6.0)) at 95°C in a microwave cooker for 10 minutes. The activation of endogenous peroxidase was blocked with 3% H2O2 and followed by blocking with 3% normal goat serum for 30 minutes in room temperature. After washing in phosphate buffered solution (PBS), the sections were incubated with the primary antibody (mouse anti-PHPT1 serum, 1:100 dilution, prepared as described above) overnight at 4°C, and the PowerVisionTM two-step histostaining reagent and 3,3'-diaminobenzidine tetrahydrochloride substrate kit (Zhongshan Golden Bridge Biotechnology Co., China) were used to visualize the localization of the antigen according to the manufacturer's instructions. Finally, sections were counterstained with hematoxylin and mounted in neutral balsam. Preimmune sera were used as a negative control. Immunohistochemistry (IHC) staining was evaluated semiquantitatively by a pathologist and a biologist respectively. PHPT1 expression was classified using a 3-point scale: +, 10%—50% positive cells with weak staining; ++, 10%—50% positive cells with strong staining; +++, > 50% positive cells with strong staining. The percentage of positive tumor cells in 10 visual fields selected randomly served as the evaluation rule, and all of the staining results were categorized as follows: +, week staining; ++, middle staining; +++, strong staining. Preimmune sera were used as a negative control.

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Statistical analysis

All statistical calculations were carried out using SPSS 11.5 statistical software (SPSS Co., USA). The relationship between the expression of PHPT1 and clinicopathological features was analyzed with Student's t test and Fisher's exact test. P <0.05 was considered statistically significant.

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RESULTS

Preparation of PHPT1 polyclonal antibody

For preparation of PHPT1 polyclonal antibody, we expressed His-PHPT1 fusion protein in pET-30a (+)-PHPT1 vector transformed E. coli BL21 cell and following purification using affinity chromatography on Ni-NTA affinity column. As a result, we successfully obtained the native His-PHPT1 fusion protein in supernantant of E. coli BL21 cell lysis (Figure 1A) and the purified His-PHPT1 fusion protein reached at least to 90% purity after affinity chromatography (Figure 1B). The identification of PHPT1 was performed by mass spectrometry (Figure 1C). After three times of immunization with BALB/c mice using the purified His-PHPT1 fusion protein, the specificity of mouse anti-human PHPT1 antibody was confirmed by Western blotting analysis (Figure 1D).

Figure 1.

Figure 1.

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Higher expression of PHPT1 protein in lung cancer tissues than that in normal lung tissues

In this study, the protein expression of histidine phosphatase, PHPT1 in various types of lung cancer tissues and normal lung tissues was investigated (Table 1). Positive staining of PHPT1 protein was identified as brownish-yellow granules in cytoplasm and nuclear. The protein expression of PHPT1 was rather week in normal lung tissues compared to lung cancer tissues (Figure 2A and 2B). In various types of lung cancer tissues including adenocarcinoma, squamous cell carcinoma and small cell lung carcinoma tissues, most of the tissues exhibited the strong protein expression of PHPT1 (53.42%, 78 of 146 patients’ tissues. Figure 2C-2F).

Figure 2.

Figure 2.

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PHPT1 protein expression correlated with stage, lymph node metastasis in lung cancer patients

As listed in Table 2, among 146 lung cancer tissues, the increasing expression of PHPT1 protein was found significantly associated with clinicopathological features. In stages I and II, strong positive expression of PHPT1 protein was 42.65% (29 of 68 patients’ tissues); however, in stages III and IV, strong positive expression increased to 62.82% (49 of 78 patients’ tissues) (P=0.02). Furthermore, strong positive staining of PHPT1 protein was found in 63 of 105 (60%) cases, in the lymph node metastasis group (N1–3); while among the 41 cases without lymph node metastasis (N0), only 15 had strong positive immunostaining for PHPT1 (36.58%) (P=0.016). As shown in Figure 3, significantly higher PHPT1 protein expression was detected in the lymph node metastasis group than in the group without lymph node metastasis.

Figure 3.

Figure 3.

Figure 3.

Figure 3.

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DISCUSSION

In recent years, the interest of studying the role of impaired signalling for diseases15,16 has grown and a prime target is enzymes that are regulated with the help of kinases and dephosphorylation occurring only in bacteria vs. serine/threonine and tyrosine phosphorylation/dephosphorylation in vertebrate has been shown to be no longer correct.9 PHPT1, the first protein histidine phosphatase discovered in vertebrates,7,8 had been found its great roles in lung cancer migration and invasion in our previous in vitro studies.5,14 Based on the investigations in cell lines, we performed immunohistochemistry to analyze the protein expression of PHPT1 in lung cancer patients’ tissues and the relationship of its expression with clinicopathological features.

Immunohistochemistry is a very good method to investigate the location of a protein thereby revealing its function. It provides morphology of the examined tissue, unlike for example ELISA-techniques; immuno-histochemistry is relatively easy to perform and is a sensitive method. The method can be used as a complement to further confirm other methods results. To this point, we compared the results of PHPT1 mRNA expression in lung cancer tissues in our previous study,14 in which 57.1% of lung cancer patients had higher PHPT1 mRNA expression than that in their adjacent noncancerous tissues and 75.0% of lung cancer patients with lymph nodule metastasis had higher PHPT1 mRNA expression than that in their adjacent noncancerous tissues; while the results in this study further confirmed the positive correlation of PHPT1 expression with 53.42% of strong PHPT1 protein staining in lung cancer patients’ tissues compared to 23.33% of strong PHPT1 protein staining in normal tissues to lung cancer. Furthermore, the expression of PHPT1 protein also correlated with the lung cancer patients' clinicopathological features with 60% of strong PHPT1 protein staining in lung cancer patients’ tissues with lymph node metastasis. The decrease of positive samples is mainly due to the outside of the moderate staining samples in positive group in order to facilitate the clinical assessment.

As reported in our previous studies, PHPT1 was differentially expressed in different metastasis potential of lung cancer cell lines,5 and silencing or overexpression of PHPT1 might affect the lung cancer cells migration and invasion.14 Those results suggested that the expression of PHPT1 might vary in different lung cancer patients’ tissues with different clinicopathological features. Interestingly, the results in this study supported the postulate and confirmed that PHPT1 was not only taking part in lung cancer cell migration and invasion but also could be a sign of lung cancer and its development.

In conclusion, we show that PHPT1 protein is overexpressed in lung cancer tissues and this overexpression also positively correlates with lung cancer stage and lymph node metastasis. These data strongly confirmed that PHPT1 closely associated with the carcinogenesis and metastasis of lung cancer. Thus, therapy targeting PHPT1 (inhibition or silencing) could potentially benefit for lung cancer treatment and prevention.

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Keywords:

PHPT1 protein; lung cancer; immunohistochemistry

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