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G Model LUNG-3075; No. of Pages 6 ARTICLE IN PRESS 4 S. Zhang et al. / Lung Cancer xxx (2008) xxx–xxx Fig. 3. PYK2, p-Tyr402, p-ERK1/2 level in lung cell lines with different metastatic potentials by Western blotting analysis. The ratio between the optical density of specific bands and �-actin of the same cells was calculated (i.e. O.D.P/a, O.D.p-Y/a and O.D.p-E/a) and expressed graphically. (A) Human bronchial epithelial (HBE) cells expressed the minimal level of PYK2, and the higher metastatic BE1 cells showed much higher level of PYK2 compared with the lower metastatic LH7 and A549 cells. (B) p-Tyr402 is scarcely detected in HBE cells, and BE1 cells showed higher level of p-Tyr402 compared with LH7 and A549 cells. (C) HBE cells showed a basal level of p-ERK1/2, and BE1 cells exhibited higher level of p-ERK1/2 than LH7 and A549 cells. �-Actin was used as a loading control to assure equal amounts of protein in all lanes. The experiments for each cell line were repeated at least three times. The results are represented as mean ± S.D. of three independent experiments. antibody, and results for normal and tumor samples were shown in Fig. 1I and J. 3.2. Comparative analysis and clinicopathological correlation As shown in Table 1, no statistical difference was found between the higher PYK2 expression and the characteristics of tumor size (T1 + T2 versus T3 + T4, p = 0.102) as well as differentiation (high versus low, p = 0.605). However, patients with higher PYK2 expression had advanced stage of NSCLC (I + II versus III + IV, p = 0.012). In addition to promotion of cell proliferation, PYK2 also increased the tumor cells invasiveness and migration [20]. Therefore, the association between PYK2 expression revealed by immunohistochemistry and the presence of lymph node metastasis at the time of resection was analyzed statistically. Comparison of PYK2 expression was made between the NSCLCs with different lymph node status. PYK2 immunoreactivity was stronger in the NSCLCs with lymph node metastasis compared with the node negative cases, with moderate–strong immunostaining. Immunohistochemistry showed a statistically significant correlation between higher protein expression and a positive node (p = 0.007). 3.3. PYK2 expression in NSCLCs by Western blotting Western blotting was used to evaluate PYK2 expression in 30 NSCLCs and paired non-tumorous lung tissues distant from the primary tumor of the same case. The increased PYK2 expression was found in 27 NSCLC samples in comparison with the nontumorous counterparts. The western blotting of eight samples is shown in Fig. 2A, and the optical density of the tumorous (T) and non-tumorous (N) tissues of the same patient was measured and expressed graphically (Fig. 2B). Previous researches have demonstrated that PYK2 overexpression enhanced cell mobility [8], thus comparison of PYK2 expression was made between NSCLCs with and without lymph node metastasis statistically. PYK2 expression was significant higher in node-positive NSCLCs (Fig. 2B). 3.4. PYK2 expression and activity in lung cell lines As we expected, expression of PYK2 was found quite weak in human bronchial epithelial (HBE) cells, and higher metastatic BE1 cells have intense PYK2 expression compared with the lower metastatic LH7 and A549 cells (Fig. 3A). PYK2 activity is regulated by tyrosine phosphorylation [17], especially the autophosphorylation site Tyr402, so the phosphorylation status of Tyr402 measured by western blotting was examined to evaluate the activation and possible function of PYK2 in different lung cell lines. The p-Tyr402 was detected in all lung tumor cell lines examined, however scarcely in HBE cells. Moreover, the level of p-Tyr402 in the NSCLC cell lines was also various, with a significant difference between BE1 and LH7 or A549 cells, and much stronger p-Tyr402 was observed in BE1 cells, as shown in Fig. 3B. These results suggest that up-regulation of PYK2 and increased activity are correlated with the metastatic potential of NSCLC cell lines. 3.5. ERK1/2 activation in lung cell lines ERK1/2 activity linked to the phosphorylated PYK2 has been demonstrated previously [7], thus to further investigate the correlation between the activation of ERK1/2 and metastatic potential of lung tumor, we examined the p-ERK1/2 in various lung cell lines. Elevated p-ERK1/2 was associated with the higher metastatic BE1 cells, similar to the intense p-Tyr402, while the p-ERK1/2 was also quite low in PYK2-inactive HBE cells, as shown in Fig. 3C. These findings provide a possibility that PYK2/ERK signaling plays a role in promoting tumor cells more aggressive in NSCLC. 4. Discussion PYK2 is a non-receptor tyrosine kinase, mediates various biological processes, such as cell proliferation [18] and migration [19], all of which are critical to tumorigenesis, invasion and metastasis, and suggest a significant role of PYK2 in the development and progression of cancer [20]. PYK2 expression is up-regulated in a variety of human tumors including liver and brain tumors. Aiming at interfering PYK2 expression or blocking PYK2 activation may be helpful in the progression of effective anticancer therapies. Recent researches have been shown that inhibited PYK2 activity was correlated with the inactivation of ERK1/2, and reduced the adhesive ability of prostate cancer cells [7]. This study evaluated PYK2 expression in NSCLCs, with regard to the tumor size, histological type, differentiation, stage and lymph node status of NSCLCs, to determine the clinical significance of PYK2 for the advanced NSCLCs. We examined 90 tumors by means of immunohistochemistry, 30 of which were also analyzed by western blotting, and found a statistical evidence of PYK2 up-regulation in NSCLCs. Weak–moderate PYK2 immunostaining was observed in ciliated epithelial cells in bronchus, the serous cells of the submucosal glands, as well as normal alveoli. While PYK2 immunoreactivity was detected extensively in every neoplastic tissue, supporting the potential role in tumorigenesis, promoting proliferation and survival of tumor cells [5]. We also found that PYK2 overexpression was common in NSCLCs, regardless of the histological type and differentiation. However, patients with higher PYK2 expression had a significant metastatic phenotype. The immunohistochemical observations may be further supported by our semi-quantitative western blotting evaluations of Please cite this article in press as: Zhang S, et al., Up-regulation of proline-rich tyrosine kinase 2 in non-small cell lung cancer, Lung Cancer (2008), doi:10.1016/j.lungcan.2008.05.008
G Model LUNG-3075; No. of Pages 6 PYK2 expression in 30 tumorous and paired non-tumorous counterparts. PYK2 expression were significantly higher in the neoplastic than the non-neoplastic tissues, which was consistent with previous studies of PYK2 overexpression in human malignant tumors [4]. Moreover, tumorous samples in which PYK2 higher expression was determined by western blotting came from patients with lymph node metastasis at the time of resection. Taken together, these findings suggest that PYK2 up-regulation stimulated cell proliferation, and migration as well, possibly by attenuating the adhesive ability of tumor cells in NSCLCs [6]. Despite that further research is required, these results suggest a crucial role of PYK2 in affecting tumorigenesis and aggression of NSCLC. Studies have been reported that PYK2 activity especially in Tyr402 in human malignant tumors has been correlated with outgrowth and invasiveness of breast cancer cells [9], and active ERK1/2 was included in LPA-induced PC12 cell migration mediated by PYK2 [3]. So we also examined the expression and p-Tyr402 of PYK2 and the p-ERK1/2 in the human bronchial epithelial cells (HBE) and different NSCLC cell lines. HBE cells as normal control, lower metastatic A549 cells as we confirmed previously, as well as lower metastatic LH7 cells and higher metastatic BE1 cells were included in this study. We found that the expression of PYK2 was quite weak in HBE cells, while the higher metastatic BE1 cells expressed the maximal level of PYK2, compared with the lower metastatic LH7 and A549 cells. So it is acceptable that PYK2 overexpression stimulated cell proliferation and more aggressive. Furthermore, PYK2 up-regulation was accompanied by the increasing PYK2 activity as evaluated by the level of p-Tyr402 [21], and ERK1/2 also proved to be more activated in higher metastatic BE1 cells. However, HBE cells expressed a low level of PYK2 with a scarcely detectable p-Tyr402 and a low p-ERK1/2. It was further indicated that PYK2 expression and activation might play a role in promoting a more malignant phenotype by regulating the activity of ERK1/2 in NSCLC. Previous studies have been shown that PYK2 was essential for the pulmonary vascular endothelial cell spreading and migration [22], besides PYK2 kinase activity enhanced migration of endothelial cells [23], which resulted in vasculogenesis and angiogenesis. We also found PYK2 immunostaining in the endothelial cells of neoplastic stroma in several cases of NSCLCs, which showed lymph node positive. This pattern of PYK2 expression may indicate the invasive and metastatic malignant phenotype of NSCLCs. Therefore, the PYK2 mediated signaling pathway may become a promising target for tumor inhibition. It has recently been shown that inhibiting PYK2 activity by the expression of kinase inactive PYK2 mutant attenuated the adhesive ability of prostate cancer cells [7]. The similar report has been found in glioma cells, in which the suppression of PYK2 activity within the FERM domain reduced the capacity of PYK2 to stimulate glioma cell migration [24]. As far as lung cancer is concerned, PYK2 appears to be the key interactive effector of Src in mediating the survival signals in lung adenocarcinoma cells upon detachment, which may contribute to the metastasis of malignant lung tumors [6]. Moreover, inhibition of PYK2 expression by siRNA, attenuated anchorage-independent survival and proliferation of SCLC cells [25]. In conclusion, our study demonstrates the facts that upregulation of PYK2 is common in NSCLC tissues, increased expression and activity of PYK2 in lung cell lines are correlated with higher metastatic potential, possibly by the activation of ERK1/2. The detection of higher PYK2 expression by western blotting in 27 of the 30 NSCLCs examined suggests that PYK2 may play a role in promoting malignant transformation, which is crucial to the tumorigenesis of NSCLC. The suppression of PYK2 expression and phosphorylation in Tyr402 may provide a helpful target for inhibitory therapies of metastasis in NSCLC. The regulatory ARTICLE IN PRESS S. Zhang et al. / Lung Cancer xxx (2008) xxx–xxx 5 significance of PYK2 in NSCLC therefore requires further investigation. Conflict of interest None declared. Acknowledgements We are grateful to Prof. Jie Zheng for offering the cell lines used in this study. We also thank teacher Chengyao Xie and Nan Liu for technical assistance, and the members of our department for useful suggestions. References [1] Massa A, Casagrande S, Bajetto A, Porcile C, Barbieri F, Thellung S, et al. SDF-1 controls pituitary cell proliferation through the activation of ERK1/2 and the Ca 2+ -dependent, cytosolic tyrosine kinase PYK2. Ann N Y Acad Sci 2006;1090:385–98. [2] Schindler EM, Baumgartner M, Gribben EM, Li L, Efimova T. The role of prolinerich protein tyrosine kinase 2 in differentiation-dependent signaling in human epidermal keratinocytes. J Invest Dermatol 2007;127:1094–106. [3] Park SY, Li H, Avraham S. RAFTK/PYK2 regulates EGF-induced PC12 cell spreading and movement. Cell Signal 2007;19:289–300. [4] Gutenberg A, Bruck W, Buchfelder M, Ludwig HC. Expression of tyrosine kinases FAK and PYK2 in 331 human astrocytomas. Acta Neuropathol (Berl) 2004;108:224–30. [5] Sun CK, Ng KT, Sun BS, Ho JW, Lee TK, Ng I, et al. The significance of prolinerich tyrosine kinase 2 (PYK2) on hepatocellular carcinoma progression and recurrence. Br J Cancer 2007;97:50–7. [6] Wei L, Yang Y, Zhang X, Yu Q. Altered regulation of Src upon cell detachment protects human lung adenocarcinoma cells from anoikis. Oncogene 2004;23:9052–61. [7] Yuan TC, Lin FF, Veeramani S, Chen SJ, Earp HS 3rd, Lin MF. ErbB-2 via PYK2 upregulates the adhesive ability of androgen receptor-positive human prostate cancer cells. Oncogene 2007;26:7552–9. [8] Lipinski CA, Tran NL, Menashi E, Rohl C, Kloss J, Bay RC, et al. The tyrosine kinase PYK2 promotes migration and invasion of glioma cells. Neoplasia 2005;7:435–45. [9] Fernandis AZ, Prasad A, Band H, Klösel R, Ganju RK. Regulation of CXCR4mediated chemotaxis and chemoinvasion of breast cancer cells. Oncogene 2004;23:157–67. [10] Zrihan-Licht S, Fu Y, Settleman J, Schinkmann K, Shaw L, Keydar I, et al. RAFTK/PYK2 tyrosine kinase mediates the association of p190 RhoGAP with RasGAP and is involved in breast cancer cell invasion. Oncogene 2000;19:1318–28. [11] McShan GD, Zagozdzon R, Park SY, Zrihan-Licht S, Fu Y, Avraham S, et al. Csk homologous kinase associates with RAFTK/PYK2 in breast cancer cells and negatively regulates its activation and breast cancer cell migration. Int J Oncol 2000;21:197–205. [12] Boutahar N, Guignandon A, Vico L, Lafage-Proust MH. Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and prolinerich tyrosine kinase 2 tyrosine sites involved in ERK activation. J Biol Chem 2004;279:30588–99. [13] Basile JR, Gavard J, Gutkind JS. Plexin-B1 utilizes RHOA and ROK to promote the integrin-dependent activation of AKT and ERK, and endothelial cell motility. J Biol Chem 2007;282:34888–95. [14] Rocic P, Govindarajan G, Sabri A, Lucchesi PA. A role for PYK2 in regulation of ERK1/2 MAP kinases and PI3-kinase by ANG II in vascular smooth muscle. Am J Physiol Cell Physiol 2001;280:C90–9. [15] Zrihan-Licht S, Avraham S, Jiang S, Fu Y, Avraham HK. Coupling of RAFTK/PYK2 kinase with c-Abl and their role in the migration of breast cancer cells. Int J Oncol 2004;24:153–9. [16] Zhu W, Zheng J, Fang W. Isolation and charaterization of human lung cancer cell subline with different metastatic potential. Chin J Pathol 1995;24: 136–8. [17] Park SY, Avraham HK, Avraham S. RAFTK/PYK2 activation is mediated by trans-acting autophosphorylation in a Src-dependent manner. J Biol Chem 2004;279:33315–22. [18] Picascia A, Stanzione R, Chieffi P, Kisslinger A, Dikic I, Tramontano D. Prolinerich tyrosine kinase 2 regulates proliferation and differentiation of prostate cells. Mol Cell Endocrinol 2002;186:81–7. [19] Lipinski CA, Tran NL, Bay C, Kloss J, McDonough WS, Beaudry C, et al. Differential role of proline-rich tyrosine kinase 2 and focal adhesion kinase in determining glioblastoma migration and proliferation. Mol Cancer Res 2003;1:323–32. [20] Rucci N, Ricevuto E, Ficorella C, Longo M, Perez M, Di Giacinto C, et al. In vivo bone metastases, osteoclastogenic ability, and phenotypic characterization of human breast cancer cells. Bone 2004;34:697–709. Please cite this article in press as: Zhang S, et al., Up-regulation of proline-rich tyrosine kinase 2 in non-small cell lung cancer, Lung Cancer (2008), doi:10.1016/j.lungcan.2008.05.008
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