An integrin beta4-EGFR unit promotes hepatocellular carcinoma lung metastases by enhancing anchorage independence through activation of FAK–AKT pathway

Cancer Letters 376 (2016) 188–196

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

An integrin beta4-EGFR unit promotes hepatocellular carcinoma lung metastases by enhancing anchorage independence through activation of FAK–AKT pathway Chao Leng, Zhan-guo Zhang, Wei-xun Chen, Hong-ping Luo, Jia Song, Wei Dong, Xuan-ru Zhu, Xiao-ping Chen, Hui-fang Liang *, Bi-xiang Zhang ** Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan, Hubei, China

A R T I C L E

I N F O

Article history: Received 1 December 2015 Received in revised form 9 February 2016 Accepted 11 March 2016 Keywords: Hepatocellular carcinoma Anoikis Integrin beta4 Metastases EGFR

A B S T R A C T

Anoikis, a form of programmed cell death, occurs when the cells are detached from the appropriate extracellular matrix. Anoikis resistance or anchorage independence is necessary for distant metastases of cancer. The mechanisms by which hepatocellular carcinoma (HCC) cells become resistant to anoikis are not fully understood. Integrin beta4 (ITGB4, also known as CD104) is associated with progression of many human cancers. In this study, we demonstrate that ITGB4 is over-expressed in HCC tissues and aggressive HCC cell lines. To explore the role of ITGB4 in HCC, we inhibited its expression using small interfering RNA in two HCC cell lines: HCCLM3 and HLF. We show that knockdown of ITGB4 significantly enhanced susceptibility to anoikis through inhibition of AKT/PKB signaling. Moreover, ITGB4 interacts with epidermal growth factor receptor (EGFR) in a ligand independent manner. Inactivation of EGFR inhibits the anchorage independence and AKT pathway promoted by ITGB4. Further investigation proved that the ITGB4–EGFR unit triggers the focal adhesion kinase (FAK) to activate the AKT signaling pathway. Finally, we demonstrate that over-expression of ITGB4 is positively associated with tumor growth and lung metastases of HCC in vivo. Collectively, we demonstrate for the first time that ITGB4 is overexpressed in HCC tissues and promotes metastases of HCC by conferring anchorage independence through EGFRdependent FAK–AKT activation. © 2016 Elsevier Ireland Ltd. All rights reserved.

Introduction Hepatocellular carcinoma (HCC) is responsible for the third largest number of cancer-associated deaths worldwide [1]. Although there have been great advances in the treatment of HCC, the prognosis is poor because of a high incidence of metastases and recurrence after surgical resection [2–4]. Tumor metastasis is a multistep, multifactorial process, an essential characteristic of which is anchorage independence [5]. Tumor cells must overcome anoikis induced by detachment from the primary tumor and associated extracellular matrix [6,7]. The cancer cells exploit many mechanisms to confer anoikis resistance, includ-

Abbreviations: HCC, hepatocellular carcinoma; EMT, epithelial–mesenchymal transition; ITGB4, integrin beta4; ANLT, adjacent nontumorous liver tissue; Dox, doxycycline; Co-IP, co-immunoprecipitation; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; H&E, hematoxylin and eosin; PBS, phosphatebuffered saline; PKB, protein kinase B; EGFR, epidermal growth factor receptor; FAK, focal adhesion kinase. * Corresponding author. Tel.: +86 27 83662599; fax: +86 27 83662851. E-mail address: [email protected] (H. Liang). ** Corresponding author. Tel.: +86 27 83662599; fax: +86 27 83662851. E-mail address: [email protected] (B. Zhang). http://dx.doi.org/10.1016/j.canlet.2016.03.023 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

ing changes in expression of integrins, undergoing epithelial– mesenchymal transition (EMT) and the constitutive activation of prosurvival signaling pathways and altered metabolism [8]. In the past decades, many molecules have been reported to play a vital role in regulating anchorage independence of HCC such as androgen receptor (AR) [9], microRNA-26a [10] and maspin [11]. Although these significant advances have been made, mechanisms governing anoikis resistance of HCC remain poorly understood and have yet to be translated into effective remedies for patients. Integrins play a central role in regulating anchorage independence [8]. Integrin beta4 (ITGB4), a member of the integrin family, has been reported to regulate cancer progression [12]. Its role in modulating anoikis in HCC remains unclear. To address this, we investigated the role of ITGB4 in anoikis and metastases of HCC and found that it was a critical factor in the patho-physiology of the disease. Materials and methods Patients and HCC tissue specimens Specimens of 68 HCC tissues were collected from patients who performed hepatic resection at the Hepatic Surgery Center, Tongji Hospital of Huazhong University of Science and Technology (HUST) (Wuhan, China). These patients included 7 women

C. Leng et al./Cancer Letters 376 (2016) 188–196

and 61 men with a median age of 48 years, range 26–79 years. Matched fresh specimens of adjacent non-tumorous liver tissue (ANLT) and HCC were lysed separately for western-blot detection. Clinico-pathological data of these patients were obtained from the medical and pathological records. Prior written informed consent was obtained from each patient and the study procedure was approved by the Ethics Committee of Tongji Hospital. Cell lines and cell culture MHCC97-L, MHCC97-H and HCCLM3 were purchased from the Liver Cancer Institute of Fudan University. 293T, Huh7, Hep3B and HepG2 were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). HLF cells were deposited in the Hepatic Surgery Center, Tongji Hospital. These cell lines were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and penicillin/streptomycin and incubated in 5% CO2 at 37 °C. Chemicals, inhibitors and antibodies Puromycin and G-418 were purchased from Cayman Chemical. Doxycycline (Dox) was purchased from MedChem express. AG1478, Erlotinib, PF573228, PF562271, Dasatinib and MK2206 were purchased from Selleck. Antibodies used were as follows: Integrin α6 was from Abcam. Integrin β4 (C-20), Integrin β4 (H-101), GAPDH (0411) and β-actin (ACTBD11B7) were from Santa Cruz. DAPI, Cy3-conjugated donkey antigoat IgG and FITC-conjugated donkey anti-rabbit IgG were from Goodbio Technology Co., Ltd (Wuhan, China). EGF Receptor (D38B1), Met (D1C2), SRC (36D10), PhosphoSRC Family (Tyr416) (D49G4), HER2/ErbB2 (29D8), Phospho-FAK (Tyr397), PhosphoFAK (Tyr925), FAK (D2R2E), Akt (pan) (C67E7), Phospho-Akt (Thr308) (D25E6) and Phospho-Akt (Ser473) (D9E) were from Cell Signaling. Plasmids, lentivirus and clones selection Tet-pLKO-neo was a gift from Dmitri Wiederschain (Addgene plasmid # 21916) [13]. pMD2.G and psPAX2 were a gift from Didier Trono (Addgene plasmid # 12259 and 12260). The ITGB4 shRNA lentivirus was purchased from GeneChem Co., Ltd, (Shanghai, China). The inducible AKT1 shRNA lentivirus was produced as described previously [14]. The target sequences relating to ITGB4 and AKT1 are TRCN0000057771 and TRCN0000039793, respectively (Open Biosystems) [15,16]. The nonspecific control target sequence is TTCTCCGAACGTGTCACGT [17]. To obtain stable cell clones, HCC cells were infected with lentivirus for 24 h and selected with growth medium containing 3 μg/ml puromycin for 3 days and/or 400 μg/ml G418 for 7 days. Stable transfected clones were validated by western blot. Anchorage-independent growth and anoikis assay The anchorage-independent growth ability was evaluated by soft agarose colony formation assay as previously described [18]. Briefly, HCC cells (2 × 104, counted by Cellometer Mini, Nexcelom Bioscience, Massachusetts, USA) were suspended in 1 ml of 0.3% agarose containing 10% fetal bovine serum and then plated on the top of 1 ml 0.8% agarose in 6-well cell culture plate. Two weeks later, three random fields of each well were pictured and colonies over 50 μm were counted. Anoikis was assessed by Cellular DNA Fragmentation Elisa (Roche) after 24 h of culture on ultralow attachment plates (Corning) [19]. Co-Immunoprecipitation (Co-IP) and Western blot analysis For Co-Immunoprecipitation assays, cells were cultured attached or floated for 24 h, then the cells were lysed, precleared with protein-A agarose (Roche) and incubated with anti-ITGB4 or EGFR antibody overnight. The immune complex was precipitated by protein-A agarose, washed and analyzed by western blot. The western blot assay was performed as previously reported [20]. Briefly, total cell and tissue lysates (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with primary antibody overnight at 4 °C on a rocking platform. Then the membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized by the ChemiDoc™ MP Imaging System (Bio-Rad). Immunohistochemistry and immunofluorescence The immunohistochemistry was performed as previously described [21]. ITGB4 primary antibody was diluted at 1:100. The immunofluorescent staining was carried out according to the method previously reported [22]. Briefly, cells were cultured in suspension for the indicated times and fixed with 2% paraformaldehyde/0.1% Triton X-100 for 30 min on ice. Then cells were pipetted onto poly-L-lysine coated slides, incubated with primary antibody at 4 °C overnight and secondary antibody at 37 °C for 1 h. For immunofluorescent double-labeling, mixtures of two primary and two secondary antibodies were used. Nuclei were labeled with DAPI.

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Tumorigenicity assay Five-week-old male BALB/c nu/nu mice were bred under specific pathogenfree (SPF) conditions. All animals were cared for according to the Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Committee on the Ethics of Animal Experiments of the Tongji Medical College, HUST. For tumorigenicity assay, 1 × 106 tumor cells suspended in phosphate-buffered saline (PBS) were injected subcutaneously into the flanks of the nude mice (n = 6 per group). Doxycycline (2 mg/ml) was administrated in the drinking water when necessary. All mice were sacrificed five weeks later and tumor volumes were calculated. Lung metastases assay in vivo In vivo lung metastases assay was performed as previously reported [23]. Briefly, 1 × 106 tumor cells suspended in PBS were injected intravenously via lateral tail veins of the mice. Six weeks later, all of the mice were sacrificed and lung tissues were removed and fixed in 4% phosphate-buffered neutral formalin for 72 h. Lung metastases were analyzed by hematoxylin and eosin (H&E) staining. The number of lung metastases was quantified as formerly described [24]. Statistical analysis All of the data were presented as mean ± SEM. Statistical analysis was conducted using Prism 5.0 (GraphPad Software, La Jolla, CA, USA) software. Categorical data were analyzed by Chi-square test or Fisher’s exact test. Quantitative data were compared with two-tailed Student t test, analysis of variance (ANOVA) with Tukey– Kramer multiple comparisons test or Wilcoxon signed-rank test. A two-tailed value of P < 0.05 was considered statistically significant for all tests.

Results ITGB4 is up-regulated in human HCC tissues and invasive HCC cell lines To explore the role of ITGB4 in HCC, the expression level of ITGB4 in 68 matched HCC and ANLT was determined by western blotting. The results showed that the average expression level of ITGB4 protein in HCC was 6.29 times greater than that in ANLT (0.44 ± 0.12 versus 0.07 ± 0.01, P < 0.001; Fig. 1A and B and Fig. S1). To figure out in which kind of cells in tumor tissue expressed ITGB4, we performed immunohistochemistry staining for ITGB4 on 31 ITGB4 positive tumors identified by western blotting. The data demonstrated that ITGB4 mainly expressed in the tumor cells but not the infiltrated stromal cells and the invasion front had an increased ITGB4 expression (Fig. 1C). We further explored the ITGB4 expression in 7 HCC cell lines. High ITGB4 expression was found in those cells with high aggressive behavior (HLF, MHCC97L, MHCC97H, HCCLM3), while the less aggressive HCC cell lines (Hep3B, HepG2, Huh7) had low or no ITGB4 expression (Fig. 1D and E) [25,26]. The expression level of integrin α6, with which ITGB4 forms a heterodimer [27], was very low and not correlated to that of ITGB4 (Fig. 1D and F). Therefore, we proposed that ITGB4 plays a role as a tumor promoter in HCC progression. Overexpressed ITGB4 is associated with malignant clinic-pathological characteristics In our cohort, 45.6% of HCC tissues (31/68) expressed ITGB4. To illustrate the relevance of ITGB4 expression to the clinic-pathological characteristics of HCC, patients were divided according to the expression of ITGB4 (positive or negative). Nine established factors of HCC malignancy were analyzed and the results demonstrated that high ITGB4 expression is significantly associated with the aggressive tumor phenotypes of local invasion (P = 0.0386) and poor differentiation (P = 0.0446) (Table 1). These results suggested that ITGB4 may promote HCC metastases. ITGB4 enhances anchorage independence To scrutinize how ITGB4 promotes the progression of HCC in vitro, we infected HCCLM3 and HLF cells with lentivirus containing an

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A

B

N T NTNTNT NT ITGB4 GAPDH N T NTNT NT NT ITGB4 GAPDH

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Case 2 HCC

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200 SCs

400

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HCC

SCs

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SCs HCC

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F Integrin α6 ITGB4 beta-actin

Fig. 1. HCC tissues and invasive HCC cell lines over-expressed ITGB4. (A) Representative western blot results of ITGB4 expression in human ANLTs and HCC tissues. (B) Statistical analyses showed that the expression level of ITGB4 in HCC tissues was significantly higher than that in ANLTs. ***P < 0.001. N, ANLTs; T, HCC tissues. (C) Four representative cases of immunohistochemistry staining for ITGB4. ITGB4 mainly expressed in the tumor cells. Infiltrated stromal cells had no ITGB4 expression. SCs, stromal cells. (D) Western blot results showed that ITGB4 was down-regulated in low aggressive HCC cell lines Hep3B, HepG2 and Huh7 compared with high aggressive HCC cell lines HLF, MHCC97L, MHCC97H and HCCLM3. The integrin α6, with which ITGB4 forms a heterodimer, was overexpressed only in MHCC97H cells. (E) Western blot densitometry graph showing significant difference in ITGB4 expression in HCC cells. These cell lines were categorized according to their aggressiveness. *P < 0.05. (F) Densitometry graph showing relative expression level of integrin α6 and ITGB4 in HLF and HCCLM3 cells. Relative to beta-actin in these two cell lines, expression level of integrin α6 was much lower than that of ITGB4 * P < 0.05.

inhibitory ITGB4 shRNA sequence or a scramble sequence, termed HCCLM3-Vec and HCCLM3-shB4 or HLF-Vec and HLF-shB4, respectively. The ability of the shRNA to effectively and specifically inhibit the ITGB4 expression in both HCCCLM3 and HLF cells was determined by western blotting (Fig. 2A). Anchorage independence or anoikis resistance has been shown to be an integrin-regulated process [5,28]. We next performed soft agarose colony formation and anoikis assay to evaluate the role of ITGB4 in anchorage independence. Inhibition of ITGB4 expression in HCCLM3 and HLF cells significantly suppressed anchorageindependent proliferation in soft agarose (P < 0.001) (Fig. 2B and C). After culture in suspension for 24 h, anoikis was assessed by a cell death ELISA kit. This demonstrated that HCCLM3-shB4 and HLFshB4 cells were significantly more susceptible to anoikis than their control cells respectively (P < 0.001) (Fig. 2D). AKT/PKB acts downstream of ITGB4 to promote anchorage independence AKT/PKB signaling has been shown to promote multiple aspects of tumor development including cell survival. We next

determined whether the AKT signaling plays a role in the ITGB4regulated anoikis resistance. First, the HCCLM3-Vec, HCCLM3shB4, HLF-Vec and HLF-shB4 cells were cultured in suspension for 24 h and subsequent western blotting showed that inhibition of ITGB4 expression resulted in a reduction in the proportion of both Ser473 and Thr308 phosphorylated AKT (Fig. 3A). Second, using the AKT inhibitor MK-2206 (Fig. S2), we inquired whether AKT is necessary for anchorage-independent growth of HCC. Using the soft agarose colony formation assay, we found that ITGB4-induced anchorageindependent growth was significantly reversed by the addition of MK-2206 (P < 0.001) (Fig. 3B). To further confirm the effect of AKT in anchorage independence, we generated doxycycline-inducible AKT1 shRNA lentivirus (shAKT1(i)). All four HCC cell lines were stably infected with this lentivirus, which efficiently inhibited AKT1 expression (Fig. S3). We next investigated the effect of AKT1 knockdown to disrupt anchorage independence using the soft agarose colony formation and anoikis assays. Consistent with our original findings, inducible knockdown of AKT1 alone could significantly reverse ITGB4 promoted anchorage independence compared with control cells (Fig. 3C and D). We conclude from these data that AKT signaling is necessary for ITGB4-induced anchorage independence.

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Table 1 Correlations between ITGB4 expression and clinic-pathological features of HCC. Clinic-pathological features

Gender Male Female Age (years) ≤65 >65 Tumor size ≤5 cm >5 cm Liver cirrhosis Negative Positive Tumor nodule number Solitary Multiple (≥2) AFP (ng/ml) ≤20 >20 HBV Negative Positive Edmondson–Steiner grade I, II III, IV Local invasion* Absence Presence

n

ITGB4 expression

P value

Negative

Positive

61 7

32 5

29 2

0.4416

58 10

32 5

26 5

0.7617

26 42

15 22

11 20

0.6691

10 58

7 30

3 28

0.3264

62 6

35 2

27 4

0.4003

22 46

13 24

9 22

0.5921

7 61

6 31

1 30

0.1158

44 24

20 17

24 7

0.0446**

44 24

28 9

16 15

0.0386**

* It means that HCC nodules had no intact capsule and invaded into neighboring vessels or bile ducts. ** P < 0.05. Overexpressed ITGB4 was associated with poor differentiation and local invasion.

A

191

ITGB4 interacts with EGFR in a ligand-independent manner It has been reported that integrins cooperate with receptor tyrosine kinases (RTK) to promote tumor progression [12]. We hypothesized that ITGB4 would interact with an RTK to exert its role in HCC cells. We examined the expression of a number of RTKs including ErbB2, c-Met and EGFR in the HCCLM3 and HLF cells and found that only EGFR is over-expressed in both cell lines (Fig. 4A). Indeed, Co-IP results showed that ITGB4 could interact with EGFR in both attached cells and cells in suspension (Fig. 4B). To further confirm the interaction between ITGB4 and EGFR, we performed immunofluorescent double-labeling analysis. The results showed that ITGB4 and EGFR colocalized in the cell membrane of HLF and HCCLM3 cells (Fig. 4C). We examined EGF by ELISA in the culture media and cannot detect EGF produced by HLF and HCCLM3 cells (data not shown). Next we checked whether EGFR could be triggered by ITGB4 binding in suspension. Using western-blotting for phosphorylated receptor, we found that EGFR activated in substratum detachment was suppressed by ITGB4 down-regulation (Fig. 4D). To reveal the effect of EGFR on AKT signaling, HLF-Vec, HLF-shB4, HCCLM3-Vec and HCCLM3-shB4 cells were cultured in suspension and treated with two EGFR inhibitors, AG1478 and erlotinib, respectively. Western blot results demonstrated that ITGB4induced AKT activation in anchorage independence was inhibited in the presence of EGFR inhibitors (Fig. 4E). It was reported that integrin heterodimers can be internalized by clathrin-mediated endocytosis [29], so we examined if this was the case in the HCC cells cultured in suspension. Immunofluorescent staining experiment showed that ITGB4 was located on the cell membrane and the

B

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Fig. 2. Knockdown of ITGB4 expression and its role in anchorage independence of HCC cells. (A) Lentivirus-mediated shRNA could markedly decrease the expression of ITGB4 in HLF and HCCLM3 cells. (B, C) Downregulation of the expression of ITGB4 significantly inhibited anchorage-independent growth of HCC cells. ***P < 0.001. (D) Anoikis resistance of HCC cells was reversed efficiently by knockdown of ITGB4 expression. ***P < 0.001. Scale bars represent 100 μm.

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Fig. 3. ITGB4 enhanced anchorage independence by activating AKT pathway. (A) In HLF and HCCLM3 cells, AKT phosphorylation was induced by substratum detachment, which was partially reversed by down-regulation of ITGB4 expression. Densitometry graphs of P-AKT Thr308/Ser473 of each cell line were shown. Sus., cells cultured in suspension. *P < 0.05. (B) Inhibition of AKT activation by AKT specific inhibitor MK2206 suppressed ITGB4 promoted anchorage-independent proliferation of HLF-Vec and HCCLM3-Vec cells, which was similar to the effect of down-regulation of ITGB4 expression. Culture media was supplemented with 5 μM MK2206 or equivalent DMSO and changed every two days. ***P < 0.001. (C) Doxycycline inducible shRNA was employed to suppress the expression of AKT. The results showed that ITGB4 enhanced anchorageindependent growth of HCC cells was significantly decreased by knockdown of AKT expression. Culture media was added with doxycycline (100 ng/ml) and changed every two days. ***P < 0.001. (D) A Cellular Death Elisa was performed to investigate the effect of AKT on ITGB4 induced anoikis resistance. These data indicated that suppression of AKT expression increased the susceptibility of HCC cells to anoikis, which was the same as knockdown of ITGB4 did. Cells were pretreated with doxycycline (100 ng/ml) for 7 days to substantially inhibit the AKT expression. *P < 0.05, ***P < 0.001. NS, non-significant. Scale bars represent 100 μm.

ITGB4–EGFR complex was not internalized over time when cultured in suspension (Fig. 4C). We next measured the effect of AG1478 and erlotinib in the soft agarose colony formation assay and found that these inhibitors significantly suppressed anchorage-independent proliferation (Fig. 4F). Our findings confirmed that ITGB4 interacts with EGFR in a ligand-independent manner to confer anoikis resistance to HCC by activating AKT signaling.

the activation of FAK but had no effect on SRC. Consistent with this, we found that inhibitors of FAK (PF573228 or PF562271) resulted in inactivation of AKT, while the inhibitors of SRC (Dasatinib) could not (Fig. 5C and D). Finally we found that the FAK inhibitors, PF573228 and PF562271, significantly inhibited the anchorage independence of HCC cells (Fig. 5E). Taken together, these findings suggest that the ITGB4–EGFR unit activate the FAK–AKT axis to confer anchorage independence.

ITGB4 and EGFR coordinately activate AKT by FAK rather than SRC Integrins, without intrinsic tyrosine kinase activity, signal through the recruitment and activation of non-receptor tyrosine kinases, such as FAK and SRC [30,31]. We next investigated how the ITGB4– EGFR unit activates AKT signaling to confer anoikis resistance in HCC. HCCLM3-Vec, HCCLM3-shB4, HLF-Vec and HLF-shB4 cells were cultured in suspension and subjected to western-blotting. This demonstrated that FAK and SRC were phosphorylated in suspension, which was reversed by knockdown of ITGB4 (Fig. 5A). Next the experiment was repeated in the presence of EGFR inhibitors. As shown in Fig. 5B, the addition of AG1478 and erlotinib suppressed

The ITGB4–EGFR–FAK–AKT axis promote tumorigenicity and pulmonary metastases of HCC in vivo To validate our findings in vivo, we performed a tumorigenicity assay in Balb/c nude mice. 1 × 106 HCCLM3-Vec, HCCLM3shB4, HCCLM3-shAKT1(i) and HCCLM3-shB4+AKT1(i) cells were injected subcutaneously into the left flanks of mice (n = 6). Doxycycline (2 mg/ml in the drinking water) was added when necessary. Five weeks later the mice were sacrificed and tumor volumes (V) were calculated by the formula: V = W2 × L × 0.5, where W represents width and L represents length of the tumor. We found that

C. Leng et al./Cancer Letters 376 (2016) 188–196

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Fig. 4. ITGB4 interacted with EGFR and regulated anchorage independence. (A) Western blot results demonstrated that only EGFR was commonly expressed in HLF and HCCLM3 cells. (B) Interaction between ITGB4 and EGFR was assessed by Co-immunoprecipitation. These results showed that ITGB4 formed a complex with EGFR no matter how the cells were cultured in attachment or suspension. The interaction between ITGB4 and EGFR was EGF-independent. A, cells cultured in attachment. WCL, whole cell lysate. (C) Immunofluorescent double-labeling for ITGB4 and EGFR. Cells were cultured in suspension for the indicated times, merged pictures indicated that ITGB4 interacted with EGFR in the cell membrane and the ITGB4–EGFR complex was not internalized over time. (D) Western blot results showed that ITGB4 affected EGFR phosphorylation. Down-regulation of ITGB4 partially inhibited EGFR phosphorylation induced by substratum detachment. Densitometry graphs of P-EGFR Tyr1068/1173 were shown. *P < 0.05. (E) EGFR inhibitors blocked ITGB4 promoted AKT phosphorylation. Western blot results showed that AKT phosphorylation level in HCCLM3-Vec and HLF-Vec cells treated with EGFR inhibitors was much lower than that in untreated cells. The HLF and HCCLM3 cells were cultured in suspension and treated for 24 h with EGFR inhibitors AG1478 and erlotinib (10 μM), respectively. Densitometry graphs representing P-EGFR Tyr1068/1173 and P-AKT Thr308 were shown. *P < 0.05. (F) EGFR inhibitors reversed ITGB4 enhanced anchorage independent growth. Soft agarose colony formation assay was performed and these results showed that EGFR inhibitors suppressed ITGB4 conferred anchorage independent proliferation. HLF and HCCLM3 cells were treated with AG1478 and erlotinib (10 μM) respectively and culture media was changed every two days. ***P < 0.001. NS, non-significant. Scale bars represent 100 μm.

knockdown of either ITGB4 or AKT1 resulted in a significant reduction in tumor growth, although the combination of the two had no additional effect over ITGB4 knockdown alone (Fig. 6A). These data suggest that AKT acted downstream of ITGB4 in vivo. Finally, we examined the role of anchorage independence induced by ITGB4 in lung metastases. The above four cell lines (1 × 10 6 ) were

injected intravenously via lateral tail veins. Compared with the control cells, knockdown of ITGB4 or AKT1 significantly inhibited HCC lung metastases and knockdown of the two had no additional effect over ITGB4 knockdown alone (Fig. 6B). In conclusion, these data confirmed that the ITGB4–EGFR–FAK–AKT axis enhanced tumor growth and lung metastases of HCC.

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sus(h) 0 1 3 6 9 12 24 0 1 3 6 9 12 24 P-FAK Tyr397 P-FAK Tyr925 FAK P-Src Tyr416 Src beta-actin

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HLF-shB4

Fig. 5. The ITGB4–EGFR unit activated FAK–AKT pathway to promote anchorage independence. (A) Phosphorylation of FAK and Src induced by suspension was reversed by knockdown of ITGB4 expression. Cells were cultured in suspension for different times. The phosphorylation level of FAK and Src was presented by densitometry graphs. *P < 0.05. (B) EGFR was involved in phosphorylation of FAK rather than Src. EGFR inhibitors blocked FAK phosphorylation in suspension but had no effect on Src phosphorylation. HLF and HCCLM3 cells were cultured in suspension and treated for 24 h with AG1478 and erlotinib (10 μM), respectively. Densitometry graphs of the western blot results were shown. *P < 0.05, NS, non-significant. (C) FAK inhibitors decreased AKT phosphorylation. Western blot results revealed that AKT phosphorylation level decreased significantly in HLF-Vec and HCCLM3-Vec cells treated with FAK inhibitors. HLF and HCCLM3 cells were cultured in suspension and treated for 24 h with FAK inhibitors PF573228 and PF562271 (10 μM), respectively. Graphs showing the densitometry results of the western blot were presented. *P < 0.05. (D) Src inhibitors had no effect on AKT phosphorylation. Cells were cultured in suspension and treated for 24 h with Src inhibitors dasatinib (10 μM). Densitometry graphs representing the western blot results were shown. *P < 0.05, NS, non-significant. (E) FAK inhibitors suppressed anchorage independent growth. HLF and HCCLM3 cells were treated with PF573228 and PF562271 (10 μM) respectively, these data proved that FAK inhibitors efficiently suppressed colony formation of HCC cells. ***P < 0.001. NS, non-significant. Scale bars represent 100 μm.

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A

B HCCLM3 Vec

shAKT1(i)

shB4

shB4+AKT1(i)

100 400 Fig. 6. The ITGB4–EGFR unit enhanced tumor growth and lung metastases of HCC in vivo. (A) HCCLM3-Vec, HCCLM3-shB4, HCCLM3-shAKT1(i) and HCCLM3-shB4+AKT1(i) cells (1 × 106) were injected subcutaneously into the left flanks of Balb/c nude mice (n = 6). Doxycycline was added in the drinking water (2 mg/ml). These results demonstrated that knockdown of ITGB4 and (or) AKT1 expression inhibited tumorigenicity in vivo. **P < 0.01. (B) The above four cells were injected intravenously by lateral tail veins. Lung metastases were validated by H&E staining. These data proved that down-regulation of ITGB4 and (or) AKT1 significantly inhibited pulmonary metastases of HCCLM3 cells. *P < 0.05, ***P < 0.001. NS, non-significant. Scale bars represent 100 μm.

Discussion To form metastases, tumor cells complete a series of cellbiological events, which is referred to as the invasion-metastases cascade. To reach the distant organ sites, tumor cells must disseminate from the primary tumor and intravasate into the vessels, then only the cells that survive in the circulation can reach the secondary organ [5]. Anchorage independence or anoikis resistance is a critical mechanism for tumor cells to overcome various stresses encountered in this process [8]. Integrin signaling can promote migration, proliferation and survival of tumor cells, thus overexpression of particular integrins in the primary tumor are often associated with increased metastases and poor prognosis [32]. Integrins such as integrin beta1 [33] and integrin αvβ3 [34] have been reported to confer anchorage independence. Previous studies showed that ITGB4 cooperate with RTK to promote tumorigenesis of mammary and prostate cancer cells [15,35]. In the present study, we demonstrate that ITGB4 was commonly over-expressed in HCC tissues and aggressive HCC cell lines, indicating an association of ITGB4 with metastasis in HCC. ITGB4 promotes tumor progression in various cancers by different mechanisms, such as promoting epithelial–mesenchymal transition, proliferation and tumor progenitor cells self-renew [15,35,36]. We evaluated the role of ITGB4 in HCC metastases. The results show that ITGB4 promoted anchorage independence in HCC cancer cells. Further molecular studies demonstrate that AKT was activated by ITGB4 and enhanced anchorage independent growth in HCC cells. Signal pathway is a complicated network, our results in Fig. 3C indicated that in HCC cells AKT could be activated by other

molecules in addition to ITGB4, such as growth factors [37], to regulate anchorage independence. Thus, ITGB4 confers anchorage independence partially by activating the AKT signaling pathway. Integrins transmit signals in both a ligand dependent and independent manner, where they can form a complex with RTKs to regulate cell proliferation, migration and survival [38,39]. But the associations between ITGB4 and RTKs remain unclear in HCC. In this study, we demonstrate that ITGB4 interacted with EGFR to activate the AKT signaling and regulate anoikis. Many kinases act downstream of integrins to protect tumor cells against anoikis, including FAK and SRC [40]. Our results show that the ITGB4– EGFR unit triggered FAK rather than SRC to activate the AKT signaling. Inactivation of EGFR and FAK by their specific inhibitors respectively both inhibited the anchorage independent proliferation. Animal models further validate that ITGB4 promoted HCC tumor growth and lung metastases dependent of AKT pathway. There are many signal pathways regulated by integrins to promote tumor progression, such as NF-κB and ERK/MAPK [41]. Our data in Fig. 6 also indicated this possibility. Therefore, we could conclude that ITGB4 cooperated with EGFR to activate the FAK–AKT pathway in HCC to enhance anchorage independence and pulmonary metastases. In conclusion, our data have demonstrated that ITGB4 is overexpressed in HCC and its overexpression is associated with local invasion and poor differentiation. Furthermore, we have proved that ITGB4 enhance HCC metastases through promoting anchorage independence by interacting with EGFR to trigger the FAK–AKT pathway. Collectively, our results support ITGB4 as a potential target for treatment of HCC metastases.

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