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Carcinogenesis Advance Access originally published online on September 1, 2008
Carcinogenesis 2008 29(11):2096-2105; doi:10.1093/carcin/bgn203
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Proline-rich tyrosine kinase 2 (Pyk2) promotes proliferation and invasiveness of hepatocellular carcinoma cells through c-Src/ERK activation

Chris K. Sun, Kwan Man*, Kevin T. Ng, Joanna W. Ho, Zophia X. Lim, Qiao Cheng, Chung-Mau Lo, Ronnie T. Poon and Sheung-Tat Fan

Department of Surgery and Centre for Cancer Research, The University of Hong Kong, Pokfulam, Hong Kong, China

* To whom correspondence should be addressed. Department of Surgery and Centre for Cancer Research, The University of Hong Kong, L9-55, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China. Tel: +852 28199646; Fax: +852 28199634; Email: kwanman{at}hkucc.hku.hk Correspondence may also be addressed to Ronnie T.Poon. Tel: +852 28553641; Fax: +852 28175475; Email: poontp{at}hkucc.hku.hk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The aim of the current study is to elucidate the mechanism of proline-rich tyrosine kinase 2 (Pyk2)-mediated cell proliferation and invasiveness in hepatocellular carcinoma (HCC) cells. Human HCC cell lines PLC and MHCC97L were stably transfected with either full-length Pyk2 or C-terminal non-kinase region of Pyk2 (PRNK). Functional studies on cell proliferation and invasion were conducted in vitro by colony formation assay, adhesion assay, migration assay and wound-healing assay. For the in vivo study, an orthotopic nude mice liver tumor model was applied to investigate the effects of Pyk2 overexpression on tumor growth and metastasis. Overexpression of Pyk2 in PLC cells resulted in an upregulation of colony formation (P = 0.021) and adhesion toward laminin (P = 0.018). Pyk2 promoted wound recovery by stimulation of actin stress fiber polymerization. In the in vivo study, transfection of PRNK in MHCC97L cells significantly decreased tumor volume (P = 0.001) and the incidence of lung metastasis (P = 0.014). Overexpression of Pyk2 promoted the activation of c-Src, formation of Pyk2/c-Src complex and activated the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-signaling pathway. Pyk2 upregulated the activation of ERK1/2 that is insensitive to MAPK/ERK kinase (MEK)1/2 inhibition. On the contrary, PRNK overexpression downregulated the activation of c-Src and ERK/MAPK-signaling pathways. Immunofluorescence staining showed that the focal adhesion localization of Pyk2 is a major determinant for c-Src and ERK/MAPK activation. In conclusion, our results showed that Pyk2 promoted cell proliferation and invasiveness by upregulation of the c-Src and ERK/MAPK-signaling pathways.

Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FAT, focal adhesion targeting; FBS, fetal bovine serum; HCC, hepatocellular carcinoma; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; PRNK, C-terminal non-kinase region of Pyk2; Pyk2, proline-rich tyrosine kinase 2


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Hepatocellular carcinoma (HCC) is the primary malignancy of the liver. It is the fifth most common cancer in the world and ranks third in cancer-related deaths (1). Long-term prognosis remains unsatisfactory due to tumor recurrence and limited response to chemotherapy and radiotherapy (2–5). Therefore, understanding the mechanism of HCC proliferation and metastasis may improve the efficacy of the current treatment modalities against this disease.

Proline-rich tyrosine kinase 2 (Pyk2), also known as cell adhesion kinase β, is a non-receptor tyrosine kinase that belongs to the focal adhesion kinase (FAK) family. Unlike FAK, Pyk2 is mainly localized at the focal adhesion and nucleus of HCC cells (6). Pyk2 is activated by autophosphorylation at tyrosine residue Y402 that functions as a docking site for the SH2 domain of Src (7). In rat vascular smooth muscle cells, Pyk2 is coprecipitated with catalytically active c-Src after angiotensin II stimulation, suggesting that Pyk2 may link Ca2+ signaling to c-Src activation (8).

PRNK is the C-terminal non-kinase region of Pyk2. It is characterized as a splice variant form of Pyk2. Similar to Pyk2, it contains the focal adhesion targeting (FAT) domain for focal adhesion translocation after its translation. It is present in brain and spleen by alternative splicing (9). PRNK will interact with some of the Pyk2-binding partners due to sequence similarity with Pyk2. However, it may not interact with Graf and p130Cas. Therefore, it has been reported as dominant-negative regulator to prevent Pyk2 binding with its partners for signaling pathways activation (9).

c-Src expression and kinase activity were upregulated in various cancers including lung, breast, colon, pancreatic, ovarian and neural cancers (10–12). In addition, there is evidence suggesting that Src is involved in the transformation of metastatic phenotype by activation of signaling pathways associated with cell motility and invasion (13–15). Regulation of c-Src tyrosine kinase activity is through its phosphorylation status. There are two major phosphorylation sites for c-Src, namely Y419 and Y530 (Y416 and Y527 in chicken) (16). Y527/530 is the negative regulatory site for c-Src. When Y416/419 is phosphorylated, it is displaced from the substrate-binding pocket, allowing kinase access to the substrate and thus making c-Src catalytically active (17,18). Up till now, the interaction between Pyk2 and c-Src in cell proliferation and invasiveness of HCC is still unclear.

The ERK/mitogen-activated protein kinases (MAPKs) pathway is one of the most important pathways that controls cell survival, proliferation and invasion in various cancers (19–21). Activated ERK1/2 may regulate the activation of >160 downstream signaling molecules including transcription factors (22). There is increased evidence of the ERK/MAPK-signaling pathway in the progression of cancer but the role of Pyk2 in its activation is still unknown in HCC.

Our previous study showed that Pyk2 is overexpressed in HCC and contributed to poor prognosis. Transfection of full-length Pyk2 in PLC cells resulted in an increase in proliferation and invasiveness in vitro (6). However, the underlying mechanism is still unknown. We hypothesize that Pyk2 may interact with c-Src and ERK/MAPK pathway to upregulate proliferation and invasiveness of HCC cells. The purpose of the current study is to investigate the role of Pyk2 in proliferation and invasiveness of HCC cells and its correlation with c-Src and ERK/MAPK activation. PLC and MHCC97L cells were chosen for this study due to their respective low- and high-baseline Pyk2 expressions. PLC cells were forced to express the full-length Pyk2 and PRNK by transfection. MHCC97L cells were transfected with PRNK to suppress Pyk2 activation. Functional studies on cell motility, cell invasion and proliferation in vitro and in vivo were performed.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Plasmids and antibodies
Plasmids pCDNA3-Pyk2 and pCDNA3-PRNK were the gifts from Dr Joseph Loftus, Mayo Clinic Scottsdale. pCDNA 3.1 (+) vector was purchased from Invitrogen (Carlsbad, CA). pGL3 control vector was purchased from Promega (Madison, WI).

Monoclonal antibodies against Pyk2 (clone 11), FAK, paxillin and Ki67 were purchased from BD Transduction Laboratories (San Jose, CA). Monoclonal antibodies against Pyk2 Y402, total c-Src, phospho-Src Y416, non-phospho-Src Y527, total MEK, phospho-MEK, total ERK, phospho-ERK and MEK inhibitor U0126 were purchased from Cell signaling (Danvers, MA). c-Src inhibitor, Lavendustin C was purchased from Calbiochem (Darmstadt, Germany). Rhodamine phalloidin, rhodamine anti-rabbit IgG and Alexa fluor 488 anti-rabbit IgG were purchased from Molecular Probes (Carlsbad, CA).

Cell culture, transfection and stable cell lines
Human HCC cell line PLC was purchased from the American Type Culture Collection (Manassas, VA) and was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/ml streptomycin (Life Technologies, Carlsbad, CA). Human metastatic HCC cell line MHCC97L was a gift from Prof. Z.Y.Tang, Fudan University, Shanghai, China.

Stable transfection of PLC cells has been reported previously (6). Briefly, PLC cells were transfected with pCDNA 3.1 (+), pCDNA3-Pyk2 and pCDNA3-PRNK vectors, respectively. MHCC97L cells were transfected with either pCDNA 3.1 (+) or pCDNA-PRNK. Resistant clones were spread on 24-well plates using cloning cylinders and maintained in 0.6 mg/ml G418 for PLC cells and 0.2 mg/ml for MHCC97L cells. For confirmation of transfection efficiency, total RNA was extracted from the clones as reported previously (23). Reverse transcription–polymerase chain reaction was performed using primers flanking the C-terminal region of Pyk2 (PRNK-sense 5'-AGAAGCCACCACGGCTCGGT-3' and PRNK-antisense 5'-GGTAGGAGATCGTCCACGCT-3') with glyceraldehyde 3-phosphate dehydrogenase primers used for normalization. For in vivo imaging, cells were further transfected with a pGL3 control vector and positive clones were selected according to the luciferase activity with the IVIS® imaging system (Xenogen Corporation, Alameda, CA) (24–26).

Immunofluorescence and localization of F-actin
For the immunofluorescence assay, cells were labeled with antibodies against Pyk2 Y402, total Pyk2, FAK and paxillin in blocking buffer for 1 h at 37°C. The cells were then labeled with Alexa fluor 488 goat anti-rabbit IgG or rhodamine goat anti-rabbit IgG for 30 min at 37°C. For localization of F-actin, cells were again labeled with antibodies against rhodamine phalloidin. The slides were viewed under an Eclipse E600 image analysis system (Nikon, Tokyo, Japan).

Wound-healing assay
Cells were seeded onto 4 cm dishes with 2 mm grid. A linear wound was made by scratching the monolayer cell culture with pipette tips after cell confluency. DMEM with G418 and FBS (5 or 0.5%) was added for cell growth. Wound healing of the cells was observed under a light microscope after 24 and 48 h. The experiment was conducted in triplicate and photographic images were shown based on the experimental results.

Soft agar assay
Cells (3 x 104) were trypsinized, counted and resuspended in 2 ml of DMEM supplemented with 10% FBS and 0.4% agar. The cell mixture was seeded on a layer of 0.8% bottom agar in six-well plates and allowed to grow for 3 weeks. The assay was performed in triplicate for each stable transfectant. After 3 weeks of incubation, colonies with >15 cells were counted. A total of five fields were counted for each plate.

Colony formation assay
Cells were seeded onto six-well plates at a density of 5 x 103 cells per well, with G418 at the concentration of 0.6 mg/ml for PLC transfectants and 0.2 mg/ml for MHCC97L transfectants. Culture medium was changed at regular time intervals. The growth of the colonies was examined 2 weeks later with 0.1% crystal violet stain. Colonies were reported as the average ±SD 2 weeks after plating.

Migration assay
Cells were trypsinized, counted and resuspended in 2 ml of serum-free DMEM. Around 50 000 cells were seeded into the upper chamber, with the lower chamber supplemented with DMEM and with 10% FBS. The cells were allowed to incubate at 37°C for 36 h with 5% CO2. Afterward, cells on the inner surface of the upper chamber were removed. Cells that had penetrated through the chamber were fixed, stained with 1% crystal violet and counted using a light microscope. The experiment was performed in triplicate.

Adhesion assay
Adhesion assay was performed with monolayer cell cultures grown to 70% confluency in T75 flasks. Before assay, cells were serum starved overnight. The cells were washed with phosphate-buffered saline, trypsinized and resuspended in the serum-free medium for 30 min. Diluted cells (2.0 x 105 cells/ml) were added to 96-well culture plates precoated with laminin (5 µg/ml) and incubated for 45 min. The cells were washed twice with serum-free medium, fixed in 1% glutaraldehyde in phosphate-buffered saline and stained with 0.1% crystal violet. The stains were solubilized in 0.5% Triton X-100. The number of adhesive cells was measured in terms of quantity of stain uptake by each individual set. Optical densities were recorded by using a plate reader (Bio-Rad, Hercules, CA).

Immunoprecipitation and immunoblotting
Immunoprecipitation was performed on the whole-cell lysates using antibodies against c-Src and Pyk2 Y402. The cells were serum starved for 24 h before stimulating with 20% serum for 30 min. After washing with ice-cold phosphate-buffered saline, the cells were lysed on plate with ice-cold cell lysis buffer. After incubation for 2 h at 4°C with protein G-agarose (Sigma, St Louis, MO), cell lysates were incubated with corresponding primary antibodies for 4 h at 4°C. Immunoprecipitates were washed twice with ice-cold lysis buffer. Immunoblotting was done as reported previously (6).

Orthotopic nude mice liver tumor model
An orthotopic nude mice liver tumor model with PLC and MHCC97L cells was established (27). Briefly, ~1 x 107 cells in 0.2 ml of culture medium were injected subcutaneously into the right flank of Balb/c nude mice (4 weeks old) that were then observed daily for signs of tumor development. Once the subcutaneous tumor reached 1 cm in diameter, it was removed and cut into ~1–2 mm cubes, which were implanted into the left liver of another group of nude mice (6 weeks old). Tumor growths were monitored once a week for 7 weeks by the Xenogen IVIS® in vivo imaging system (24–26). The mice were anesthetized by intraperitoneal injection of pentobarbital (Abbott Laboratories, North Chicago, IL) at a dose of 50 mg/kg using a 25 x 5/8'' gauge needle. Afterward, the mice were administrated with firefly luciferin (150 mg/kg, Xenogen Corporation) by intraperitoneal injection for image taking. Mice implanted with tumors generated from PLC transfectants were killed at day 30 or day 48. Mice implanted with tumors generated from MHCC97L transfectants were killed 5 or 7 weeks after tumor implantation for harvesting of the liver tumor and lung. The volume of liver tumors was determined according to methods described by Janik et al. (28). Liver tumors or lung metastatic nodules were confirmed by hematoxylin and eosin staining. The protocol for the in vivo animal study, meeting the standards required by the United Kingdom Co-ordinating Committee on Cancer Research guidelines, had been approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong. The project number is CULATR-1133-05.

Immunohistochemistry assay for cell proliferation and apoptosis
Immunostaining was performed following the protocols reported previously with some modifications (29). Briefly, the cells were stained with anti-Ki-67 monoclonal antibody at 4°C overnight followed by standard avidin–biotin-peroxidase complex technique. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay was performed as reported previously (30). All paraffin sections were analyzed by two independent investigators with knowledge in histopathology. All relevant information about the samples was excluded to eliminate selection bias. Pictures were taken of five fields of view in each section of tumor harvested. Representative pictures truly presenting the experimental results were shown.

Statistics and data analyses
Unless stated, all in vitro experiments were repeated three times. For in vivo experiments, all experimental groups had no less than eight animals per group. The {chi}2 test was performed to compare discrete variables. Mann–Whitney U-test was employed for statistical comparison of continuous variables. P < 0.05 was considered statistically significant. Calculations were performed by using the SPSS computer software version 12 (SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
PRNK significantly affected the distribution of total Pyk2 in cultured cells
To investigate whether Pyk2 or PRNK will affect the expression of FAK, western blotting was performed to determine the expression of FAK in PLC and MHCC97L transfectants. There was no significant difference between the levels of total and phosphorylated FAK (FAK Y397) in each transfectant, indicating that both Pyk2 and PRNK did not affect the expression of FAK (Figure 1B). Transfection of PRNK in both cell lines (PLC and MHCC97L) resulted in the dephosphorylation of Pyk2 Y402 as compared with their corresponding empty vector controls. To investigate the effects of PRNK expression on the distribution of Pyk2 and FAK in MHCC97L cells (with high expression level of Pyk2 and FAK), immunofluorescence staining was performed to determine the localizations of Pyk2, FAK and paxillin in MHCC97L-PRNK cells. Transfection of PRNK in MHCC97L cells resulted in the diffuse cytoplasmic distribution of Pyk2, compared with the directional focal adhesion and perinuclear localization of Pyk2 in MHCC97L-vector cells (Figure 1C). The cells were further stained with paxillin to detect the localization of focal adhesions (31). Transfection of PRNK in MHCC97L cells resulted in the redistribution of paxillin inside the cell as well. Diffuse cytoplasmic staining was observed in MHCC97L-PRNK cells as compared with the focal adhesion and perinuclear staining of the MHCC97L-vector cells. For the distribution of FAK, there was no observable difference in the staining pattern of total FAK of the MHCC97L-vector and MHCC97L-PRNK cells, suggesting that PRNK selectively affected the distribution of Pyk2 rather than FAK (Figure 1D). In summary, PRNK downregulated the phosphorylation and distribution of Pyk2 without affecting the expression and distribution of FAK.


Figure 1
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  		Fig. 1. PRNK expression in MHCC97L cells and PLC cells affected the distribution of Pyk2 in the cytoplasm with no effects on the expression, phosphorylation and distribution of FAK. (A) Confirmation of plasmid insert by reverse transcription–polymerase chain reaction using housekeeping gene glyceraldehyde 3-phosphate dehydrogenase as control. (B) Western blotting was performed to detect the levels of Pyk2, Pyk2 Y402, FAK and FAK Y397 in PLC stable transfectants. Transfection of PRNK in PLC-PRNK cells resulted in decreased levels of Pyk2 Y402. (C) Transfection of PRNK in MHCC97L cells altered the distribution of Pyk2 and paxillin. Positive staining of Pyk2 (red) in MHCC97L-vector cells was present in the focal adhesions and perinuclear region. Transfection of PRNK resulted in the diffuse cytoplasmic distribution of total Pyk2 and paxillin (green) in MHCC97L-PRNK cells. (D) Transfection of PRNK in MHCC97L cells did not affect the distribution of total FAK.

 
Pyk2 promoted colony formation and adhesiveness toward laminin
As shown in Figure 2A, overexpression of Pyk2 in PLC cells significantly increased adhesion-dependent colony formation. There was a 2.5-fold increase of colony-forming ability as compared with the vector control (P = 0.021). In MHCC97L cells, which overexpressed Pyk2, transfection of PRNK significantly downregulated colony-forming ability (P = 0.026). In terms of anchorage-independent growth, transfection of PRNK significantly suppressed the growth of both PLC and MHCC97L cells in soft agar (P = 0.018 for PLC cells and P = 0.002 for MHCC97L cells). To determine whether this difference was caused by the altered adhesiveness toward the extracellular matrix protein, an adhesion assay was performed to quantify the cell’s adhesiveness toward laminin. As shown in Figure 2B, Pyk2 transfectants had increased ability to adhere to the precoated plates with laminin. Pyk2 full-length transfectants (PLC-Pyk2) had a 2-fold increased adhesion ability when compared with the vector control, which was statistically significant (P = 0.018). Transfection of PRNK in MHCC97L cells dramatically reduced cell adhesiveness to laminin (P = 0.017). These results showed that Pyk2 upregulated cell adhesiveness toward laminin and adhesion-dependent colony formation.


Figure 2
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  		Fig. 2. Pyk2 promoted colony formation and adhesiveness toward laminin. (A) Effects of Pyk2 overexpression on colony-forming ability of (a) PLC-vector, (b) PLC-Pyk2, (c) PLC-PRNK, (d) MHCC97L-vector and (e) MHCC97L-PRNK cells. Transfection of Pyk2 in PLC-Pyk2 cells increased the colony-forming ability on culture plate as compared with PLC-vector cells. Transfection of PRNK in PLC-PRNK cells and MHCC97L cells significantly decreased the colony-forming ability of the cells as compared with PLC-vector cells and MHCC97L-vector cells, respectively. (f and g) PRNK transfection in PLC-PRNK cells and MHCC97L-PRNK cells significantly decreased the anchorage-independent growth in soft agar. (B) Pyk2 regulated the adhesiveness of the cells toward laminin. Transfection of Pyk2 in PLC cells resulted in increased adhesiveness toward laminin. Transfection of PRNK in MHCC97L cells resulted in decreased adhesiveness. **PLC-Pyk2 versus PLC-vector: P < 0.05. ***MHCC97L-vector versus MHCC97L-PRNK: P < 0.05.

 
Pyk2 expression promoted cell motility and actin stress fiber polymerization
We investigated the effect of Pyk2 in cell migration using a monolayer wound-healing assay (Figure 3A). Overexpression of Pyk2 upregulated HCC cell motility as compared with the vector control. Wound recovery was enhanced upon forced expression of Pyk2. Similar results were observed in the presence of high (5%) or low (0.5%) levels of serum (data not shown), indicating that the difference in cell motility was not affected by serum concentration. Transfection of PRNK in PLC cells reduced wound recovery, as compared with PLC-vector cells. To verify the effects of Pyk2 on cell motility, a cell migration assay was performed (Figure 3B). Overexpression of Pyk2 promoted cell migration as compared with the vector control. These results suggested that Pyk2 promoted cell motility. To further confirm that the difference in wound recovery was caused by the increased actin stress fiber polymerization, immunofluorescence staining was performed to study the effects of Pyk2 activation on stress fiber polymerization (Figure 3C). For the vector control, positive staining of activated Pyk2 was observed in the lamellipodial structures colocalized with the stress fibers. For PLC-Pyk2 transfectants, more green positive signals of phosphorylated Pyk2 Y402 sites were observed at the lamellipodia positions with more polymerization of actin stress fibers. Suppression of Pyk2 by PRNK decreased the number and the intensity of phosphorylated Pyk2 Y402 in the lamellipodial structures with less stress fibers being observed. These results showed that Pyk2 overexpression might upregulate cell motility together with an increase of actin stress fiber polymerization.


Figure 3
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  		Fig. 3. Pyk2 upregulated cell motility and actin stress fiber polymerization. (A) Wound-healing assay using PLC-vector, PLC-Pyk2 and PLC-PRNK cells was performed as described in Materials and Methods. (B) Effects of Pyk2 overexpression on cell motility as shown by a transwell migration assay. **PLC-Pyk2 versus PLC-vector and PLC-PRNK: P < 0.05. (C) Effects of Pyk2 overexpression on actin stress fiber polymerization. Cells were stained with Pyk2 Y402 monoclonal antibody (green) and rhodamine phalloidin (red). Transfection of Pyk2 in PLC cells resulted in more positive Pyk2 Y402 signals (green) at the focal adhesions with more polymerized actin stress fibers (red). Transfection of PRNK in PLC-PRNK cells resulted in the decreased number of positive Pyk2 Y402 signals (green) at the focal adhesions together with less polymerized stress fibers (red).

 
Pyk2 promoted c-Src, MEK1/2 and ERK1/2 activation
To determine whether Pyk2 regulated c-Src activation in PLC cells, coprecipitation was performed to determine the presence of the Pyk2/c-Src signaling complex. Pyk2 was found to form a signaling complex with c-Src in vitro (Figure 4A). Overexpression of Pyk2 (PLC-Pyk2) increased the level of Pyk2/c-Src signaling complexes as compared with the vector control. Transfection of PRNK significantly decreased the level of Pyk2/c-Src signaling complexes. To confirm that Pyk2 was in its activated state upon the formation of the signaling complex, coprecipitation of Pyk2 Y402/c-Src antibodies was performed. Pyk2 overexpression upregulated the formation of Pyk2 Y402/c-Src complex (Figure 4A). Transfection of PRNK downregulated the formation of Pyk2 Y402/c-Src complex. These results demonstrated that activated Pyk2 bound with c-Src at focal adhesions and upregulated the formation of the Pyk2/c-Src complex.


Figure 4
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  		Fig. 4. Pyk2 overexpression in PLC cells stimulated the formation of Pyk2/c-Src complex, activation of c-Src and ERK/MAPK pathways and insensitivity to MEK1/2 inhibition. (A) Pyk2 promoted the formation of Pyk2/c-Src complex. Pyk2 overexpression in PLC cells stimulated the formation of Pyk2/c-Src complex as compared with the vector control. The transfection of PRNK in PLC cell downregulated the formation of Pyk2/c-Src complex as compared with the PLC-vector control. Another set of precipitation was done by using anti-Pyk2 Y402. Pyk2 Y402/c-Src complex was then detected by western blot using anti-c-Src polyclonal antibody to confirm that Pyk2 was phosphorylated upon binding to c-Src. The levels of Pyk2 Y402–c-Src complex was in accordance with the levels of Pyk2/c-Src complex. It confirmed that Pyk2 is activated when binding to c-Src. Pyk2 overexpression in PLC-Pyk2 cells enhanced the activation of c-Src as compared with PLC-vector cells. (B) Overexpression of Pyk2 in PLC-Pyk2 cells resulted in upregulation of MEK1/2 and ERK. (C) Overexpression of Pyk2 in PLC-Pyk2 cells resulted in insensitivity to MEK1/2 inhibition. (D) Combination treatment of c-Src inhibitor Lavendustin C and MEK1/2 inhibitor U0126 in PLC-Pyk2 cells attenuated the colony formation. Cells were grown in cultured medium supplemented with (a) dimethyl sulfoxide control, (b) U0126 (1 µM), (c) U0126 (10 µM), (d) Lavendustin C (200 nM), (e) Lavendustin C (200 nM) with U0126 (1 µM) and (f) Lavendustin C with U0126 (10 µM). *, ** and *** (a) versus (c), (e) and (f), respectively, P < 0.05.

 
To study whether c-Src was activated upon Pyk2 activation, western blotting was performed to determine the levels of activated c-Src in cell lysates (Figure 4A). Overexpression of Pyk2 upregulated the phosphorylation of c-Src at tyrosine residue 419 (Y419) and non-phosphorylation at tyrosine residue 527 (Y527). There was a remarkable increase in c-Src Y419 for the PLC-Pyk2 cells. These results confirmed that Pyk2 upregulates c-Src activation.

To determine whether the activation of signaling molecules is as a result of c-Src activation, western blot was performed to determine the levels of phosphorylated MEK1/2 and ERK1/2 in cultured cells. As shown in Figure 4B, overexpression of Pyk2 increased MEK1/2 phosphorylation in both serine residue 217 and 221 as compared with the PLC-vector control. Similar results were observed in the activation of ERK1/2. Taken together, these results suggested that Pyk2 activated c-Src by formation of Pyk2–c-Src signaling complex and contribute to activation of MAPK pathway signaling molecules.

Pyk2 increased insensitivity to MEK1/2 inhibition in vitro
To determine the effects of MEK1/2 inhibition on the phosphorylation of ERK1/2, MEK1/2 inhibitor U0126 was used to inhibit MEK1/2 phosphorylation in vitro (Figure 4C). PLC cells were serum starved overnight and left untreated (dimethyl sulfoxide control) or treated with U0126 at 1 or 10 µM for 2 h. Levels of ERK1/2 phosphorylation were determined by western blotting using total ERK1/2 as the loading control. Treatment of PLC-vector cells with U0126 significantly attenuated ERK1/2 phosphorylation (Figure 4C). PLC-Pyk2 cells were insensitive to MEK1/2 inhibition in short exposure time with U0126. Addition of U0126 had no effects on ERK1/2 phosphorylation between the treatment and control groups. There was no difference in ERK1/2 phosphorylation in presence or absence of serum, suggesting that the phosphorylation of ERK1/2 in PLC-Pyk2 cells was MEK independent. Transfection of PRNK suppressed the phosphorylation of ERK1/2 in both the control and treatment groups, suggesting that the inhibition of Pyk2 activation by PRNK downregulated the activation of ERK1/2. These results indicated that Pyk2 was involved in the regulation of ERK1/2 activation.

To further investigate the effects of long-term MEK1/2 and c-Src inhibition on adhesion-dependent cell proliferation, a colony formation assay was performed in presence of both U0126 and c-Src inhibitor Lavendustin C. There was a dose-dependent decrease in the colony-forming ability when MEK inhibitor U0126 was added in high dosage (10 µM). There was a further decrease in the colony-forming ability when both U0126 and Lavendustin C were added, showing that both MEK1/2 and c-Src inhibition were involved in colony formation of PLC-Pyk2 cells.

Pyk2 promoted tumor growth and inhibited tumor apoptosis in vivo
Subsequently, the growths of cell transfectants were investigated in vivo. PLC-Pyk2 transfectants generated a significantly larger tumor as compared with the vector control (Figure 5A). For PLC-vector transfectants, a clear demarcation between tumor and neighboring liver tissues was observed. For the PLC-Pyk2 tumors, there was no clear demarcation between the tumor and neighboring liver tissues. Vascular invasion by tumor cells was observed in the hematoxylin and eosin staining (Figure 5).


Figure 5
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  		Fig. 5. Pyk2 promoted tumor growth and prevented apoptosis in vivo. (A) Orthotopic nude mice liver tumor model was applied as described in Materials and Methods. (I) Tumors generated from the PLC-Pyk2, PLC-PRNK and PLC-vector transfectants. (II) Tumors generated from the MHCC97L-vector and MHCC97L-PRNK transfectants. Eight mice were included in each experimental group. Representative tumor photos were shown. (B) Tumors generated from PLC-Pyk2 cells contained more proliferating cells and less apoptotic nuclei. Immunohistochemistry showed the expression of Ki67 and apoptotic nuclei in tumors generated from PLC-vector, PLC-Pyk2 and PLC-PRNK. **PLC-Pyk2 versus PLC-vector: P < 0.05. ***MHCC97L-vector versus MHCC97L-PRNK: P < 0.05.

 
To investigate the cell proliferation and apoptosis of the cell transfectants on the tumors, Ki-67 staining and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining were performed, respectively. PLC-Pyk2 tumors had significantly higher numbers of positively stained proliferating nucleus, as compared with the PLC-vector control (Figure 5B). Tumors generated from PLC-PRNK cells had significantly less proliferating nuclei and more apoptotic nuclei, as compared with the PLC-vector and PLC-Pyk2 tumors. These results demonstrated that Pyk2 overexpression in PLC cells promoted the cell proliferation and prevented apoptosis in vivo.

PRNK expression in metastatic cell line suppressed invasive growth pattern and lung metastasis in vivo
Finally, we investigated the effects of PRNK overexpression on tumor growth and the incidence of lung metastasis in MHCC97L cells. Tumor samples were collected 5 and 7 weeks after the tumor implantation. For the MHCC97L-vector control, a large tumor was observed at the left lobe of the liver (Figure 5AII). Transfection of PRNK in MHCC97L cells significantly reduced tumor size (P = 0.001), as compared with the vector control in all time points. Transfection of PRNK significantly reduced the tumor growth (Figure 6A and B) and venous invasion (Figure 6C) as well as the incidence of lung metastasis (Figure 6A). Hematoxylin and eosin staining of the liver tumor showed that the vector transfectants (control group) displayed a more invasive growth pattern (Figure 6C). Invasion of the vessels was also observed. For the vector control, lung metastasis was present in 50% of the cases 5 weeks post-implantation and 100% of the cases 7 weeks post-implantation. For MHCC97L-PRNK transfectants, no lung metastasis was observed in all mice at 5 week post-implantation (P = 0.014) and 50% of the mice 7 weeks post-implantation (P = 0.014). Venous invasion in the liver tumor was less prominent in the PRNK clones (Figure 6C). These results showed that suppression of Pyk2 activation by PRNK decreased the incidence of lung metastasis in nude mice.


Figure 6
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  		Fig. 6. PRNK suppressed tumor growth and lung metastasis in vivo. MHCC97L-vector and MHCC97L-PRNK cells were implanted onto the mice liver as described in Materials and Methods. Tumor growth was monitored every week for 7 weeks by using the IVIS® imaging system. Lung metastasis was observed in weeks 5 and 7 post-operation for MHCC97L-vector tumors. (A) Representative images showing the growth and distant metastasis of the tumor by in vivo imaging. In order to clearly demonstrate the luciferase signal of distant metastases, the abdomens of the mice were covered at week 7 after tumor implantation to prevent the disturbance of strong positive signals from the primary liver tumors. (B). Comparison of the signal density of the tumor under Xenogen in vivo imaging system. *P < 0.05. (C) Confirmation of the venous invasion and lung metastasis by hematoxylin and eosin staining. Lung metastasis (arrow) and venous tumor thrombus (arrow head) and invasive tumor growth pattern (intrahepatic metastasis) in liver was demonstrated in the group with MHCC97L-vector tumor cells. On the contrary, less lung metastasis and clear margins between liver tumor and non-tumor were found in the group with MHCC97L-PRNK.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Based on the findings in the current study, our hypothesis has been validated that Pyk2 is involved in the process of cell proliferation and invasion in HCC. A positive correlation between Pyk2 expression and rate of cell proliferation, cell motility and invasion was observed. In the current study, we have shown that transfection of PRNK in PLC and MHCC97L cells downregulated the phosphorylation of Pyk2 at Y402, without affecting its expression. The intensity of Pyk2 phosphorylation at Y402 is positively correlated with its localization at focal adhesions. The altered distribution of Pyk2 by PRNK provided the evidence that the localization of Pyk2 affected its functional role as an adapter for transduction of extracellular signals, through its FAT and subsequent phosphorylation state. The FAT of Pyk2 is necessary for its autophosphorylation, interaction with other focal adhesion proteins and activation of the MAPK pathway. Other investigators have reported similar findings about the FAT and its functional role on FAK to carry out its optimal function and phosphorylation (32). Moreover, our results have shown that the altered localization of Pyk2 does not affect the localization of FAK. This excluded the possibility that the altered localization of Pyk2 was the effect of Pyk2 displacement in focal adhesions by FAK. In fact, PRNK also contain the FAT domain, which is the binding site for paxillin (33). Currently, we cannot rule out the possibility that PRNK altered the distribution of Pyk2 by competitive binding with paxillin. However, it does not affect our conclusion that the disruption of Pyk2 localization to the focal adhesions is positively correlated with downregulation of Pyk2 phosphorylation, cell proliferation and invasion.

The intracellular linkage between focal adhesions and cytoskeleton acts as an adapter for transduction of cell signals from the extracellular matrix to the polymerization of actin stress fibers and nuclei. The role of FAK has been reported in the modulation of cell motility and invasion in normal cells and malignant cells (7,34). In this study, we demonstrated that the recruitment of Pyk2 to the focal adhesions is critical for modulation of liver cancer cell motility. We have shown that Pyk2 overexpression and phosphorylation at Y402 is positively correlated with actin stress fiber polymerization. The difference in actin stress fiber polymerization is further confirmed by a linear wound-healing assay. Dynamic remodeling of the cytoskeleton by Pyk2 truly accounted for the difference in cell migration and distant metastasis in vitro and in vivo. The overexpression of Pyk2 in PLC cells resulted in an invasive growth pattern, as compared with the PLC-vector tumors. Although overexpression of Pyk2 in PLC cells could not upregulate the incidence of lung metastasis in vivo due to its independence on Pyk2-mediated cell signaling, transfection of PRNK in MHCC97L cells decreased the incidence of lung metastasis and inhibited tumor growth in vivo. This is because the MHCC97L cells are more functionally dependent on Pyk2 than PLC cells, given their higher baseline level of Pyk2 expression. As a result, the suppression of Pyk2 activation by PRNK in MHCC97L cells showed a more profound downregulation of metastasis than PLC-PRNK cells.

The interactions between cancer cells and laminin have been reported as a key event in tumor invasion and metastasis progression (35–37). Adherence of tumor cells to laminin may also promote cell proliferation and prevent cells from apoptosis (38). The interaction of extracellular matrix molecules with integrins may activate many other signals and mediators such as FAK, protein kinase C, Rho small guanosine triphosphatases, phosphoinositide 3-kinase and MAPK, resulting in cytoskeleton rearrangement and activation of cell invasion signals (39). In our study, overexpression of Pyk2 in PLC cells resulted in upregulation of adhesiveness. Transfection of PRNK in MHCC97L cell, which has high baseline level of Pyk2, resulted in downregulation of adhesiveness toward laminin and colony formation in soft agar. These results showed that Pyk2 regulates adhesion toward the extracellular matrix laminin, which is governed by its focal adhesion localization.

The interaction of Src kinase and FAK has been reported previously as an important step in cancer progression (40). FAK may integrate signals from integrins and growth factor receptors to control and coordinate cell survival and cell migration (41). The autophosphorylation of FAK at tyrosine residue 397 created a binding site for Src. Upon formation of FAK–Src complex, Src will upregulate the catalytic activity of FAK by phosphorylating FAK on tyrosine residues on Y407, Y861 and Y925 (34). Our functional studies clearly showed that overexpression of Pyk2 upregulated the formation of Pyk2/c-Src complex and activation of c-Src. Pyk2 is autophosphorylated at Y402 upon binding with c-Src, indicating that the Pyk2 in the signaling complex is in an activated form. Src kinase overexpression has been associated with the progression of many cancers (13,14). Activation of Src is obviously associated with anchorage-independent growth in lung adenocarcinoma (15). In this study, formation of the Pyk2/c-Src complex resulted in an increase in anchorage-dependent proliferation and anchorage-independent growth. These results agree with a previous report that Pyk2 and Src kinases compensated for the loss of FAK activity in FAK– cells (42). The inhibition of c-Src by Lavendustin C further suppressed the colony formation of the cells upon MEK1/2 inhibition, suggesting that both c-Src and Pyk2 are involved in the process of colony formation.

Our study demonstrated that the targeting of Pyk2 at focal adhesions is positively correlated with the activation of the ERK/MAPK-signaling pathway. The activation of ERK/MAPK pathway may enhance proliferation and protect cells from apoptosis (43). Our results indicated that Pyk2 can activate MAPK signaling through multiple signaling cascades. A previous report suggested that FAK directly activated ERK by forming a FAK–ERK–Pax complex, upon proepithelin stimulation in bladder cancer (44). In addition to these results, we have shown that colony formation of PLC-Pyk2 cells is MEK- and c-Src-dependent, as shown by the colony formation assay using MEK and c-Src inhibitors. These results showed that Pyk2 regulates the colony formation of PLC cells through c-Src and ERK1/2 activation.

There are a few limitations to this study. Firstly, the baseline level of Pyk2 in PLC cells is so low that the forced expression of Pyk2 or PRNK may not truly represent the situation where cells depend on Pyk2 for proliferation and invasion. As shown by the adhesion assays and soft agar assays, the transfection of PRNK in PLC slightly upregulated the colony-forming ability and adhesiveness toward laminin, as compared with the vector control. This could be explained by the low baseline level of Pyk2 in PLC cells. In MHCC97L cells, which have high baseline level of Pyk2, transfection of PRNK significantly downregulated both the colony-forming ability and cell adhesiveness. Secondly, the in vitro and in vivo study may not truly mimic the clinical situation in HCC patients. The use of athymic nude mice may exclude the effects of host immunity on the proliferation and metastasis of cancer cells. The use of immortalized cell lines could only represent part of the situation in HCC due to its heterogeneity. Apart from these limitations, this study provides valuable insight into the mechanism of Pyk2-mediated cell proliferation and invasion in HCC cell lines.

The interaction of Pyk2 with other signaling molecules in control of cell motility is worthwhile for further study. The possible involvement of epithelial to mesenchymal transition may help to explain the difference in cell motility. In conclusion, Pyk2 plays an important role in the regulation of cell proliferation, cell motility and invasion by the activation of c-Src and ERK/MAPK pathways in HCC cell lines. A subcellular localization of Pyk2 is a major determinant to carry out its function.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Research Grant Council General Research Funding (757406), Hong Kong.


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received April 26, 2008; revised July 29, 2008; accepted August 23, 2008.


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Focal Adhesion Kinase as a Therapeutic Target in Cancer
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