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Abstract

Periostin (POSTN) is an extracellular matrix protein which is overexpressed in a variety of cancers and has been related to tumorigenesis of renal cell carcinoma. However, the involvement of POSTN in renal cell carcinoma migration, invasion, and their underlying mechanisms has not been established. In this study, renal cell carcinoma cell lines stably overexpressing POSTN were established using a lentiviral vector, and the effects of POSTN on renal cell carcinoma cell migration and invasion were investigated. POSTN overexpression increased the migration and invasion capabilities of renal cell carcinoma cell lines as well as activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. Integrin α_vβ₃ and α_vβ₅ antibodies inhibited POSTN overexpression or recombinant POSTN-induced focal adhesion kinase activation, cell migration, and invasion. Furthermore, lentivirus-mediated focal adhesion kinase knockdown and c-Jun N-terminal kinase inhibitor reduced POSTN-enhanced phosphorylation of c-Jun N-terminal kinase, matrix metalloproteinase-9 and matrix metalloproteinase-2 expressions, cell migration, and invasion. Our research thus indicates that POSTN promotes renal cell carcinoma cell migration and invasion through interaction with integrins α_vβ₃ and α_vβ₅ and subsequent activation of the focal adhesion kinase/c-Jun N-terminal kinase pathway. These results suggest that POSTN plays a critical role in renal cell carcinoma metastasis and may represent a potential target for novel therapeutic approaches against renal cell carcinoma.

Keywords

Periostin renal cell carcinoma cell migration cell invasion integrin focal adhesion kinase/c-Jun N-terminal kinase pathway

Introduction

In recent years, because of an increase in the application of ultrasound in physical examinations, the number of incidentally diagnosed renal cell carcinomas (RCC) has increased in China.^1,2 The clear cell subtype of renal cell carcinomas (ccRCC) is the most frequent and aggressive subtype of all RCC cases, accounting for more than 50% of cases in China.¹ Surgery remains the main treatment for ccRCC; however, it is not sufficiently effective for patients who present with advanced and metastatic disease relative to the patients who present with localized disease.³ Therefore, a better understanding of the mechanisms involved in RCC metastasis is urgently required for the development of more effective therapeutic approaches.

Periostin (POSTN), also known as osteoblast-specific factor 2, is a 93-kDa-secreted protein and was originally found to promote cell adhesion and motility in osteoblastic cells.⁴ The highly conserved N-terminal region of POSTN regulates cell function by binding to integrins while the variable C-terminal region regulates cell-matrix organization and interactions by binding extracellular matrix (ECM) proteins.⁵ POSTN is present in many normal tissues and tissues under stress conditions and has been shown to be essential for the functioning of bone, tooth, and heart.^6–8

Recently, overexpression of POSTN has been observed in a wide variety of cancers including colon cancer,⁹ non-small cell lung cancer,¹⁰ pancreatic cancer,¹¹ breast cancer,¹² gastric cancer,¹³ and prostate cancer.^14,15 POSTN is thought to increase cell survival, angiogenesis, epithelial-mesenchymal transition (EMT), invasion, and metastasis of carcinoma cells by binding to integrins, including α_vβ₃, α_vβ₅, and α₆β₄.² Depending on context, POSTN can activate specific integrin-mediated signaling pathways such as Akt/protein kinase B and focal adhesion kinase (FAK) signaling pathways, inducing different biological responses.² Our previous study showed that POSTN could induce EMT of prostate cancer cells through PI3K/Akt/GSK-3 beta signaling pathways.¹⁶ In RCC, POSTN was elevated in stromal fibroblasts and enhanced ccRCC cell attachment.¹⁷ However, the involvement of POSTN in other biological processes such as migration and invasion of RCC is still unclear. In this study, our goal was to clarify the effect of POSTN on migration and invasion of RCC cells and explore the underlying mechanism.

Materials and methods

Cell culture

The human RCC cell lines 786-O and ACHN were obtained from American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco’s Modified Eagle’s Media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C in 5% CO₂.

Lentiviral infections and creation of stable cell lines

Lentiviral particles encoding POSTN cDNA (Lv-POSTN) were constructed as described previously.¹⁶ For stable overexpression of POSTN, 786-O and ACHN cells were transfected with Lv-POSTN for 24 h, and complete medium containing 5 mg/mL puromycin was then used to select stably transfected cells. The empty lentiviral vector was used as negative control (Lv-NC). Stable cells overexpressing POSTN were obtained after 3 weeks, and the expression level of POSTN was determined by western blotting and real-time polymerase chain reaction (PCR).

For knockdown of FAK, cells were transiently transfected with FAK short hairpin RNA (shRNA) (human) lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA) according to the manufacturer’s protocol.

Real-time PCR

Total RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcription was performed using SuperScript III First-strand Synthesis SuperMix (Invitrogen) according to the manufacturer’s instructions. Real-time PCR was performed on the Applied Biosystems StepOnePlus system (Foster City, CA, USA) using the following primers: POSTN, 5′-GAGCTTTACAACGGGCAAATAC-3′ (sense) and 5′-CTCCCTTGCTTACTCCCTTTC-3′ (antisense); β-actin, 5′-CACTCTTCCAGCCTTCCTTC-3′(sense) and 5′-GTACAGGTCTTTGCGGATGT-3′ (antisense). Each measurement was repeated at least three times, and all measurements were normalized to the corresponding β-actin content values using the 2^−ΔΔCt method.

Western blotting

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer and 10 µg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis as described.¹⁶ After transfer to polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) and incubation with appropriate primary and secondary antibodies, protein bands were visualized using enhanced chemiluminescence reagents (Pierce Biotechnology, Rockford, IL, USA). The following primary antibodies were used: anti-POSTN (Santa Cruz), anti-p-FAK, anti-FAK, anti-p-JNK, anti-JNK, anti-p-AKT, anti-AKT (all from Cell Signaling Technology, Danvers, MA, USA), anti-MMP-2 (Novus Biologicals, Littleton, CO, USA), anti-MMP-9 (Abcam plc., Cambridge, MA, USA), and anti-β-actin (Santa Cruz).

Wound-healing migration assay

RCC cells were plated on six-well tissue culture plates and grown to subconfluence (90%) in serum-containing medium. A scratch wound was then made in the monolayer using a pipette tip. The debris was removed and the cells were cultured in serum-free media. After incubation for an additional 24 h, cell migration was analyzed in six different microscopic fields and calculated as the percentage of wound healing.

Cell invasion assay

The invasion capability of RCC cells was determined using transwell assays. In brief, 5 × 10⁴ cells were harvested in serum-free media and plated into the upper chamber of Matrigel invasion chambers (BD Biocoat, Bedford, MA, USA). Medium containing 10% FBS was applied to the lower chamber as chemoattractant. In some experiments, cells were treated with antibodies against α_Vβ₃ and α_Vβ₅ (10 µg/mL; Millipore), and inhibitor of c-Jun N-terminal kinase (JNK; SP600125, 10 µM; Santa Cruz). After 24-h incubation, the invading cells were fixed in 4% paraformaldehyde and stained with 1% toluidine blue. Cell numbers were quantified by counting in 10 random microscopic fields.

Zymography assay

Matrix metalloproteinase (MMP)-2 and MMP-9 activities were detected using zymography assays. Cells were incubated with different treatments in serum-free media for 24 h, and the proteins in the conditioned medium were concentrated. Proteins (10 µg) were then loaded on a 10% polyacrylamide gel containing 0.1% gelatin (Sigma-Aldrich). After staining with 0.5% Coomassie blue R250 (Sigma-Aldrich, St. Louis, MO, USA) for 3 h, gels were destained overnight in 30% methanol and 10% glacial acetic acid.

Statistical analysis

Significance of differences was analyzed using Student’s t test. Results are presented as mean ± standard error of mean (SEM) from at least three independent experiments. A value of p < 0.05 was considered statistically significant.

Results

Creation of stable RCC cell lines overexpressing POSTN

RCC 786-O and ACHN cells were transfected with lentivirus expressing POSTN complementary DNA (cDNA) and then selected by puromycin. Real-time PCR assays showed that the mRNA expression levels of POSTN in transfected 786-O and ACHN cells were significantly higher than in cells without lentivirus transfection or transfected with Lv-NC (Figure 1(a)). Similar results were also found by western blotting assay, in which POSTN protein levels were significantly increased after Lv-POSTN transfection (Figure 1(b)). These results indicated that RCC cell lines stably overexpressing POSTN had been established.

Figure 1.

Creation of renal cell carcinoma (RCC) cell lines stably overexpressing POSTN. RCC 786-O and ACHN cells were transfected with lentivirus encoding POSTN cDNA and selected by puromycin exposure. (a) mRNA expression of POSTN was detected by real-time PCR. (b) Protein levels of POSTN were detected by western blotting. Values are mean ± SD of three independent experiments.

POSTN overexpression promoted RCC cell migration and invasion

Cell migration capacity of 786-O and ACHN cells transfected with or without POSTN were analyzed by wound-healing assays (Figure 2(a) and (b)). The result showing that POSTN-overexpressing RCC cells exhibited higher wound closure activity than control cells suggesting that POSTN enhanced the motility of RCC cells.

Figure 2.

The effect of POSTN overexpression on migration of RCC cells. Migration of (a) 786-O and (b) ACHN cells transfected with or without POSTN were identified by wound-healing assays. Representative images are shown at the top. Values are mean ± SD of three independent experiments.

The invasion capacity of 786-O and ACHN cells transfected with or without POSTN was then examined by transwell assay. As shown in Figure 3(a) and (b), invasive cells were increased after POSTN overexpression compared with the normal and Lv-NC transfection groups, indicating that POSTN promoted the invasive capacity of RCC cells. Moreover, we detected an effect of POSTN overexpression on MMP activity which has been closely correlated with degradation of basement membrane and invasion of cancer cells.¹⁸ The results from zymography assays showed that activity of MMP-2 and MMP-9 was significantly increased compared with control cells (Figure 3(c) and (d)). This result suggested that enhanced activity of MMP may contribute to POSTN-induced RCC invasiveness.

Figure 3.

The effects of POSTN overexpression on the invasion and MMP activity of RCC cells. Invasion by (a) 786-O and (b) ACHN cells transfected with or without POSTN was detected by transwell assay. (c and d) MMP activity was demonstrated by zymography assay. Values are mean ± SD of at least three independent experiments.

α_Vβ₃- and α_Vβ₅-mediated POSTN-induced RCC cell migration and invasion

It has been reported that POSTN regulates smooth muscle cell migration through activation of the FAK pathway mediated by interaction with alphaV-integrins.¹⁹ Here, we first determined whether the FAK pathway was activated by POSTN in RCC cells. As shown in Figure 4(a), low levels of FAK phosphorylation (p-FAK) were detected in both wild type and Lv-NC transfected 786-O cells. POSTN overexpression increased the phosphorylation level of FAK while not markedly affecting total levels of FAK. Integrins α_Vβ₃ and α_Vβ₅ have been identified as major potential receptors of POSTN in multiple cell types,^20–22 but the interaction between POSTN and integrins in RCC cells has not yet been elucidated. To analyze the roles of integrins α_Vβ₃ and α_Vβ₅ on POSTN-mediated RCC cell migration and invasion, specific neutralizing antibodies were used to block both integrins. As shown in Figure 4(b), POSTN-induced upregulation of FAK phosphorylation was inhibited by treatment with either alone or combined α_Vβ₃ and α_Vβ₅ antibody. Moreover, MMP-2 and MMP-9 activity in 786-O cells was also significantly inhibited by α_Vβ₃ or α_Vβ₅ blocking antibody (Figure 4(c)). Migration and invasion abilities of 786-O cells treated with α_Vβ₃ or α_Vβ₅ antibody were also significantly lower than that in cells without antibody blocking conditions (Figure 4(d) and (e)).

Figure 4.

POSTN activation of FAK and promotion of cell invasion through α_Vβ₃ and α_Vβ₅. (a) The effects of POSTN overexpression on FAK phosphorylation were demonstrated by western blotting. (b) 786-O cells transfected with or without POSTN were incubated with antibodies against integrins α_Vβ₃ and/or α_Vβ₅. FAK phosphorylation was detected by western blotting. (c) MMP activity was demonstrated by zymography assay. (d) Cell migration was identified by wound-healing assays. (e) Invasion ability was detected by transwell assay. (f) 786-O cells were incubated with 10 µg/mL recombinant human Periostin (rPOSTN) and antibodies against integrins α_Vβ₃ and/or α_Vβ₅. FAK phosphorylation was detected by western blotting. (g) Cell migration and (h) invasion ability were identified by wound-healing assays and transwell assay. Values are mean ± SD of at least three independent experiments.

To directly confirm that integrin α_Vβ₃ or α_Vβ₅ mediated the function of POSTN, recombinant human POSTN was used to treat 786-O cells. As shown in Figure 4(f)–(h), exogenous POSTN also induced upregulation of FAK phosphorylation and enhanced the motility and invasion capacity of RCC cells. These functions of POSTN were inhibited by either alone or combining α_Vβ₃ and α_Vβ₅ blocking antibody treatment. These results indicated that integrins α_Vβ₃ and α_Vβ_5.mediated POSTN-induced activation of FAK and MMPs and subsequently cell migration and invasion.

POSTN promoted RCC cell migration and invasion through the FAK/JNK pathway

FAK is linked with cell migration and invasion by activated multiple downstream signaling factors including JNK and Akt.^23,24 To determine whether the FAK/JNK pathway was also involved in POSTN-induced RCC cell migration and invasion, we examined the effects of lentiviral-mediated FAK interference and JNK inhibitor SP600125 on POSTN function. As shown in Figure 5(a), POSTN overexpression increased phosphorylated JNK levels, whereas FAK knockdown attenuated the increase, which suggested that POSTN activated JNK signaling through FAK. Meanwhile, we found no significant change in phosphorylated Akt levels, suggesting that Akt signaling was not a major downstream signal of POSTN and FAK in RCC cells. We also detected the effect of POSTN on the protein levels of MMP-2 and MMP-9, and found that POSTN overexpression increased these two MMPs levels. Furthermore, FAK knockdown or JNK inhibitor attenuated the MMP levels, abrogated MMP activities (Figure 5(b)), cell migration (Figure 5(c)), and invasion (Figure 5(d)), which were all enhanced by POSTN. These results indicated that POSTN promoted RCC cell migration and invasion through the FAK/JNK pathway.

Figure 5.

POSTN promotion of RCC cell invasion through the FAK/JNK pathway. The 786-O cells stably expressing POSTN or control vector were transfected with FAK shRNA or incubated with JNK inhibitor SP600125. (a) Western blotting shows the levels of phosphorylated and total FAK, JNK, and AKT, and the levels of MMP-9 and MMP-2. (b) MMP activity was demonstrated by zymography assay. (c) Cell migration was identified by wound-healing assay. (d) Cell invasion was detected by transwell assay. Values are mean ± SD of at least three independent experiments.

Discussion

POSTN is an ECM protein which is involved in a variety of biological processes including tumorigenesis by interacting with other ECM proteins or binding to cellular integrins.² POSTN upregulation was observed in high-grade RCC tumors and was significantly associated with poor overall survival.^25,26 However, the relationship between POSTN and RCC metastasis, as well as the underlying mechanism, has not been clearly identified. In this study, we found that lentiviral-mediated POSTN overexpression activated the FAK/JNK signaling pathway via interaction with integrins α_Vβ₃ and α_Vβ₅, further increasing MMP activity and promoting RCC cell migration and invasion.

Consistent with previous studies in other cancers,^20,27,28 our results showed that POSTN was also involved in motility and invasion of RCC. These data, combined with a recent report that extracellular POSTN could enhance A498 ccRCC cell attachment,¹⁷ suggest that POSTN plays a critical role in the multistep cascade process of cancer metastasis. Invasion of basement membrane and ECM is necessary for metastasis of RCC, which depends on degradation of these components mainly by MMP. In this study, we found that POSTN overexpression significantly increased the enzyme activity of MMP-2 and MMP-9, which likely contributes to RCC cells migration and invasion.

Binding to integrins and subsequent activation of intracellular pathways is an important mechanism by which POSTN exerts its pro-tumorigenic effect.² Multiple integrins, including α_vβ₃, α_vβ₅, α₆β₄, and α₅β₁, have been reported to interact with POSTN in different cell types. Our results showed that α_Vβ₃ or α_Vβ₅ antibody blocked POSTN-promoted MMP activity, migration, and invasion abilities of RCC cells, demonstrating that α_Vβ₃ or α_Vβ₅ was involved in the effect of POSTN in RCC. These integrins mediate POSTN functions in epithelial ovarian carcinoma²⁰ and were also previously observed to be expressed in RCC.²⁹ Meanwhile, we found that α_Vβ₃ or α_Vβ₅ antibody inhibited POSTN-induced phosphorylation of FAK, which is a tyrosine kinase always activated by signaling from integrins.³⁰ Similar to POSTN overexpression, exogenous POSTN also promoted cell migration and invasion, and induced phosphorylation of FAK, which were inhibited by α_Vβ₃ or α_Vβ₅ antibody. These results verified the interaction of POSTN and these integrins more directly.

FAK is a nonreceptor tyrosine kinase and is one of most prominent components of integrin signaling.³⁰ Integrin activates FAK by inducing conformational changes and auto-phosphorylation of FAK. Our results showed that POSTN was unable to significantly enhance cell migration and invasion when FAK was downregulated. These data indicated that an integrin (α_Vβ₃/α_Vβ₅)/FAK pathway was required for POSTN to promote migration and invasion of RCC.

FAK contributes to cancer development through diverse molecular signaling pathways. For example, the extracellular signal–regulated kinases (ERK) pathway is required for FAK to maintain the growth or cell motility of at least some tumor cells.^31,32 FAK suppresses apoptosis by binding to a receptor-interacting protein, which is then sequestered from the death-receptor complex.³³ FAK also regulates cell invasion through JNK signaling and MMP-mediated matrix degradation.³² Although FAK may mediate cell migration and invasion through different signaling pathways, here, JNK activity was shown to be involved in both POSTN-induced cell migration and invasion in RCC cells. FAK knockdown or JNK inhibitor attenuated the MMP levels, which were enhanced by POSTN, suggesting that MMP-9 and MMP-2 are downstream factors of POSTN/FAK/JNK signaling, and POSTN may promote migration and invasion in RCC cells through upregulation of MMP expression. In addition, our previous study¹⁶ showed that POSTN could induce EMT and cell invasion through PI3K/Akt signaling in prostate cancer cells; however, POSTN-activated Akt was not observed in RCC cells, suggesting that the function of POSTN is dependent on cell type.

In conclusion, this study demonstrates that POSTN promoted migration and invasion of RCC cells through interaction with integrins α_vβ₃ and α_vβ₅ and subsequent activation of the FAK/JNK pathway. This finding may provide new prospects for developing novel therapeutic approaches against RCC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was funded by the National Natural Science Foundation of China (Nos 81201620 and 81372316) and ChenGuang Program of Shanghai Municipal Education Commission (N158554).

References

Pang

Guan

. Urologic cancer in China. Jpn J Clin Oncol 2016; 46(6): 497–501.

Morra

Moch

Periostin expression and epithelial-mesenchymal transition in cancer: a review and an update. Virchows Arch 2011; 459(5): 465–475.

Haddad

Rini

BI.

Current treatment considerations in metastatic renal cell carcinoma. Curr Treat Options Oncol 2012; 13(2): 212–229.

Takeshita

Kikuno

Tezuka

. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Biochem J 1993; 294(Pt 1): 271–278.

Norris

Damon

Mironov

. Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues. J Cell Biochem 2007; 101(3): 695–711.

Conway

Molkentin

JD.

Periostin as a heterofunctional regulator of cardiac development and disease. Curr Genomics 2008; 9(8): 548–555.

Bonnet

Garnero

Ferrari

Periostin action in bone. Mol Cell Endocrinol 2015; 432: 75–82.

Romanos

Asnani

Hingorani

. PERIOSTIN: role in formation and maintenance of dental tissues. J Cell Physiol 2014; 229(1): 1–5.

Bao

Ouyang

Bai

. Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway. Cancer Cell 2004; 5(4): 329–339.

10.

Takanami

Abiko

Koizumi

Expression of periostin in patients with non-small cell lung cancer: correlation with angiogenesis and lymphangiogenesis. Int J Biol Markers 2008; 23(3): 182–186.

11.

Fukushima

Kikuchi

Nishiyama

. Periostin deposition in the stroma of invasive and intraductal neoplasms of the pancreas. Mod Pathol 2008; 21(8): 1044–1053.

12.

Puglisi

Puppin

Pegolo

. Expression of periostin in human breast cancer. J Clin Pathol 2008; 61(4): 494–498.

13.

Sun

Wei

. Expression of periostin and its clinicopathological relevance in gastric cancer. World J Gastroenterol 2007; 13(39): 5261–5266.

14.

Sun

Zhao

. Periostin: a promising target of therapeutical intervention for prostate cancer. J Transl Med 2011; 9: 99.

15.

Sun

Song

. Periostin identified as a potential biomarker of prostate cancer by iTRAQ-proteomics analysis of prostate biopsy. Proteome Sci 2011; 9: 22.

16.

Tong

Zhao

. Periostin mediates TGF-beta-induced epithelial mesenchymal transition in prostate cancer cells. Cell Physiol Biochem 2015; 36(2): 799–809.

17.

Bakhtyar

Wong

Kapoor

. Clear cell renal cell carcinoma induces fibroblast-mediated production of stromal periostin. Eur J Cancer 2013; 49(16): 3537–3546.

18.

John

Tuszynski

The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathol Oncol Res 2001; 7(1): 14–23.

19.

Jin

Norris

. Periostin mediates vascular smooth muscle cell migration through the integrins alphavbeta3 and alphavbeta5 and focal adhesion kinase (FAK) pathway. Atherosclerosis 2010; 208(2): 358–365.

20.

Gillan

Matei

Fishman

. Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrins and promotes cell motility. Cancer Res 2002; 62(18): 5358–5364.

21.

Matsuzawa

Arai

Nomura

. Periostin of human periodontal ligament fibroblasts promotes migration of human mesenchymal stem cell through the αvβ3 integrin/FAK/PI3K/Akt pathway. J Periodontal Res 2015; 50(6): 855–863.

22.

Sugiyama

Kanno

Nishimichi

. Periostin promotes hepatic fibrosis in mice by modulating hepatic stellate cell activation via αv integrin interaction. J Gastroenterol 2016; 51(12): 1161–1174.

23.

Lin

Shih

YW.

Antitumor effects of the flavone chalcone: inhibition of invasion and migration through the FAK/JNK signaling pathway in human gastric adenocarcinoma AGS cells. Mol Cell Biochem 2014; 391(1–2): 47–58.

24.

Chen

Huang

. MicroRNA-379-5p inhibits tumor invasion and metastasis by targeting FAK/AKT signaling in hepatocellular carcinoma. Cancer Lett 2016; 375(1): 73–83.

25.

Dahinden

Ingold

Wild

. Mining tissue microarray data to uncover combinations of biomarker expression patterns that improve intermediate staging and grading of clear cell renal cell cancer. Clin Cancer Res 2010; 16(1): 88–98.

26.

Morra

Rechsteiner

Casagrande

Relevance of periostin splice variants in renal cell carcinoma. Am J Pathol 2011; 179(3): 1513–1521.

27.

Baril

Gangeswaran

Mahon

. Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the beta4 integrin and the PI3k pathway. Oncogene 2007; 26(14): 2082–2094.

28.

Utispan

Sonongbua

Thuwajit

. Periostin activates integrin α5β1 through a PI3K/AKT-dependent pathway in invasion of cholangiocarcinoma. Int J Oncol 2012; 41(3): 1110–1118.

29.

Rabb

Barroso-Vicens

Adams

. Alpha-V/beta-3 and alpha-V/beta-5 integrin distribution in neoplastic kidney. Am J Nephrol 1996; 16(5): 402–408.

30.

McLean

Carragher

Avizienyte

. The role of focal-adhesion kinase in cancer—a new therapeutic opportunity. Nat Rev Cancer 2005; 5(7): 505–515.

31.

Aguirre Ghiso

. Inhibition of FAK signaling activated by urokinase receptor induces dormancy in human carcinoma cells in vivo. Oncogene 2002; 21(16): 2513–2524.

32.

Hsia

Mitra

Hauck

. Differential regulation of cell motility and invasion by FAK. J Cell Biol 2003; 160(5): 753–767.

33.

Kurenova

Yang

. Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein. Mol Cell Biol 2004; 24(10): 4361–4371.