Deletion of TRIB3 disrupts the tumor progression induced by integrin αvβ3 in lung cancer

Human lung cancer cells A549 (established in 1972 by D.J. Giard, et al., through an explant culture of adenocarcinomic lung tissue of a 58-year-old Caucasian male, belonging to hypotriploid alveolar basal epithelial cells) and PC-9 (a human non-small cell lung cancer (adenocarcinoma) with EGFR mutation) were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 (Gibico, MA, USA) supplemented with 10% fetal bovine serum (Gibco, MA, USA). Integrin αvβ3 positive/negative cells were isolated using fluorescence-activated cell sorting. Tumor cells were labeled with 5 μl anti-integrin αvβ3 antibody (ab190147, Abcam, Cambridge, UK) per 10⁶ cells. Integrin αvβ3 positive/negative populations were sorted using a FACSAria machine (BD, CA, USA). FAK inhibitor Y15 and AKT inhibitor 3CAI were purchased from MCM (NJ, USA). Pep2-Ae was purchased from Solarbio (Beijing, China). Cisplatin (Cis) and paclitaxel (PTX) were purchased from Sigma (NJ, USA).

Cell proliferation was determined using the CCK8 kit (Biyuntian, Beijing, China). Briefly, 1 × 10³ treated A549 or PC-9 cells were seeded into 96-well culture plates. 20 μl of CCK-8 solution was added into the 96 wells in determined time points. After 37 °C incubation of 2 h, absorbance was measured at 450 nm on a microplate reader (Bio-Rad, MA, USA). Each experiment was performed for independent three times.

Colony formation assay was conducted to evaluate the tumorigenic potential of cancer cells. Briefly, A549 or PC-9 cells (200 cells per well) were seeded into the 6-well plates and cultured at 37 °C for 14 days. After that, the colonies were fixed by 4% paraformaldehyde and stained by crystal violet. Colonies were pictured and counted. Each experiment was repeated independently in triplicate.

Transwell analysis was conducted to evaluate cell migration of cancer cells. A549 or PC-9 cells (1 × 10⁵ cells) were seeded in the upper transwell chamber (8 μm, Corning, CA, USA). The bottom chamber was filled with 0.5 ml medium containing 20% FBS. After 24 h, cells were fixed with 4% paraformaldehyde, and then stained with 0.05% crystal violet. The cells numbers were count. Each experiment was repeated independently in triplicate.

Western blotting was performed to examine the protein level of targeted signaling molecule. The protein lysates from A549 and PC-9 cells were separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membrane was incubated with the primary antibodies against to anti-p-FAK (ab81298, 1:1000, Abcam, Cambridge, UK), anti-t-FAK (ab40794, 1:1000, Abcam, Cambridge, UK), anti-p-AKT (ab38449, 1:1000, Abcam, Cambridge, UK), anti-t-AKT (ab8805, 1:1000, Abcam, Cambridge, UK), anti-FOXO1 (ab179450, 1:1000, Abcam, Cambridge, UK), anti-SOX2 (ab92494, 1:1000, Abcam, Cambridge, UK), anti-TRIB3 (ab75846, 1:1000, Abcam, Cambridge, UK) and anti-β-actin (ab8226, 1:1000, Abcam, Cambridge, UK), followed by incubation with an HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK).

Sorted tumor cells were lysed with coimmunoprecipitation buffer (25 mM Tri-cl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 2.5 mM MgCl, 0.5 mM EDTA, 5% Glycerol). Samples were then incubated with IP antibodies overnight at 4 °C. After that, samples were incubated with Protein A/G Plus-Agarose (Thermo, MA, USA) for 2 h at 4 °C. AKT-TRIB3 interaction complexes were separated from the beads by boiling and subjected to SDS-PAGE, detected using immunoblotting.

The cytotoxicity of A549 or PC-9 cells to chemotherapy or inhibitor was analyzed using the FITC-Annexin V/ PE-PI apoptosis detection kit (BD, NJ, USA). Briefly, agents treated tumor cells were resuspended and stained with FITC-Annexin V and PE-PI staining solution for 15 min. Then cells apoptosis was detected by flow cytometry on a C6 flow cytometer (BD, NJ, USA). Each experiment was repeated for three independent times.

For small interfering RNA (siRNA) inhibition of TRIB3, human TRIB3 siRNA (5′-GCGGUUGGAGUUGGAUGACAACUUA-3′ and 5′-GCGUGAUCUCAAGCUGUGUCGCUUU-3′) were obtained from Qingke Co (Beijing, China). A549 and PC-9 cells were transfected with siRNA at a concentration of 20 μmol/ ml using lipofectamine RNAiMAX (Thermo, MA, USA). The TRIB3 silence efficiency was determined using quantified polymerase chain reaction (qPCR) or western blotting.

Female NOD-SCID mice (6 ~ 8 weeks) were purchased from Huafukang (Beijing, China). All mice were housed in a specific pathogen-free facility. All animal experiments were performed according to the guidelines approved by the Institute Ethics Committee of Tianjin Medical University General Hospital. To explore the anticancer effects of chemotherapy combining molecule inhibitor, subcutaneous lung cancer model was established. 10⁶ A549 cells (50 μl PBS) were subcutaneously injected into NOD-SCID mice. After two weeks, mice were treated with PBS, PTX (5 mg/kg), Cis (5 mg/kg) and Pep2-Ae (10 mg/kg) by tail vein injection every two days. The tumor volumes were of mice were recorded every day (n = 6). Survival was recorded on a daily basis (n = 6). The calculation formula of tumor volume: tumor volume = length × width ²/2. For tumorigenesis analysis, 10⁵ A549 cells (50 μl PBS) or PC-3 cells (50 μl PBS) were subcutaneously injected into NOD-SCID mice. After 30 days, the tumor-bearing mice were counted. Each experiment was repeated independently in triplicate.

The TCGA data were downloaded from http://​ualcan.​path.​uab.​edu/​index.​html and https://​www.​cbioportal.​org/​. Each experiment was performed for at least three independent times. Results were presented as the mean ± SEM and the statistical significance was analyzed using GraphPad 6.0 software (La Jolla, CA, USA). Statistical significance between groups was calculated by Student’s t test for two groups or by one-way ANOVA for more than two groups. The survival rates were determined by Kaplan–Meier survival analysis, *p < 0.05; **p < 0.01; ns, no significant difference.

As reported in previous studies, cancer cells with aberrant integrin expression exhibited enhanced stem-like phenotypes and migratory properties [14]. Our aim was to elucidate the potential role of integrin αvβ3 in driving NSCLC progression. To do this, we used fluorescence-activated cell sorting to isolated integrin αvβ3 positive cells from NSCLC cell lines A549 and PC-9. The cell proliferation and colony formation were examined ex vivo, and enhanced capability of proliferation (Fig. 1A) or colony formation (Fig. 1B) was found in αvβ3 positive cells compared to unsorted or αvβ3 negative cells. In consistent, αvβ3 positive A549 cells also revealed strengthened tumor growth (Fig. 1C) and tumorigenesis (Fig. 1D) in immunodeficient mice, indicating that integrin αvβ3 promoted cells proliferation and stem-like phenotypes in NSCLC cells. Tumor cells with stem-like phenotypes frequently showed migratory and invasive properties. Herein, to assess the influence of integrin αvβ3 in cell migration, transwell analysis were conducted in the A549 and PC-9 cells. As a result, integrin αvβ3 significantly promoted A549 and PC-9 cells migration (Fig. 1E). We next explored the role of integrin αvβ3 in NSCLC patient prognosis. However, no significantly difference of integrin αvβ3 expression was observed in the tumor tissues or para-carcinoma tissues (Fig. 1F). And patients with low αvβ3 expression possessed no advantages in overall survival compared with the high αvβ3 expression group (Fig. 1G). Those results suggested that integrin αvβ3 promoted tumor progression of NSCLC in vitro, whereas no positive correlation between integrin αvβ3 and NSCLC patients prognosis was found.

Given that integrin αvβ3 have no influence on NSCLC prognosis, whereas exhibiting pro-tumor effects in vitro, we sought to explore the underlying mechanism of tumor progression induced by integrin αvβ3. Activation of integrins downstream signaling pathways, such as AKT signaling pathway for promoting cell survival, was dependent on the phosphorylation of FAK [15]. Here, the expression of FAK and AKT was examined in A549 and PC-3 cells. Both phosphorylated FAK and phosphorylated AKT were upregulated in αvβ3 positive A549/PC-9 cells (Fig. 2A). We next further suppressed the FAK/AKT signals in NSCLC cells by using FAK inhibitor Y15 and AKT inhibitor 3CAI to treat integrin αvβ3 positive A549/PC-9 cells. Intriguingly, blockade of FAK/AKT signals efficiently suppressed the cells proliferation (Fig. 2B) and colony formation (Fig. 2C) induced by integrin αvβ3. Meanwhile, integrin αvβ3 positive A549/PC-9 cells revealed weakened capability of migration after Y15 and 3CAI treatment (Fig. 2D), suggesting that integrin αvβ3 promoted NSCLC cells proliferation through FAK/AKT signaling pathway. Subsequently, we continued to evaluate the influence of FAK/AKT on prognosis of NSCLC patients. However, no direct correlation was observed between FAK/AKT expression and overall survival in NSCLC patients (Fig. 2E and F). Those results suggested that additional signaling molecular might participate in the FAK/AKT associated NSCLC progression.

Compelling findings provided evidence that TRIB3 linked stress signals are capable of promoting tumor initiation and progression by supporting cancer stemness, which is coordinated with elevated FOXO1 and AKT1 axis [16]. Herein, we analyzed TRIB3 expression and overall survival in NSCLC patients from TCGA database. Intriguingly, elevated expression of TRIB3 was observed in tumor tissues in comparison with the para-carcinoma tissues (Fig. 3A). And a high correlation was found in TRIB expression and overall survival in NSCLC patients (Fig. 3B), indicating that TRIB3 served as the major regulator in NSCLC progression. Thus, we silenced TRIB3 in A549 and PC-9 cells (Fig. 3C), and further sorted integrin αvβ3 positive NSCLC cells to examine the cell proliferation. Notably, suppression of TRIB3 retarded the proliferative effects induced by integrin αvβ3 in A549/PC-9 cells (Fig. 3D), whereas no significant difference was found in integrin αvβ3 negative NSCLC cells (Fig. 3E). Also, the strengthened capability of colony formation (Fig. 3F) and cell migration (Fig. 3G) was aborted when silencing TRIB3 in αvβ3 positive A549 and PC-9 cells. Collectively, those results suggested that integrin αvβ3 induced tumor progression was TRIB3 dependent.

Previous documents have proved that TRIB3 could interact with AKT1 to abrogate FOXO1 degradation, resulting in the up-regulating of FOXO1 and downstream transcription factor SOX2 activation [12]. Firstly, we examined the expression FOXO1 and SOX2 in A549 and PC-3 cells. Notably, enhanced expression of FOXO1 and SOX2 was found in integrin αvβ3 positive A549 and PC-9, and suppression of AKT or TRIB3 retarded up-regulation of FOXO1/SOX2 (Fig. 4A). Next, we wondered whether TRIB3 mediated FOXO1 upregulation by interacting with AKT1. The endogenous AKT1 was co-immunoprecipitated with TRIB3. And obvious interaction between AKT1 and TRIB3 was found in integrin αvβ3 positive A549 and PC-9 cells (Fig. 4B). To further confirm the role of TRIB3-AKT1 axis in NSCLC progression, we used Pep2–Ae to interrupt the contact between TRIB3 and AKT1 and examined the cell proliferation of integrin αvβ3 positive cells. Intriguingly, Pep2–Ae treatment significantly suppressed the cell proliferation (Fig. 4C) and colony formation (Fig. 4D) of integrin αvβ3 positive A549 and PC-9 cells. Meanwhile, interruption of TRIB3-AKT1 axis retarded the cell migration in NSCLC cells (Fig. 4E). Together, those results suggested that TRIB3 contacted with AKT1 and upregulated FOXO1 expression, resulting in the SOX2 activation and NSCLC progression.

Given the importance of TRIB3-AKT axis in NSCLC development, we examined whether disruption of TRIB3-AKT interaction could improve the anticancer effects in NSCLC. Firstly, we combined Pep2–Ae with chemotherapeutic PTX/Cis to treat A549 and PC-9. Intriguingly, Pep2-Ae treatment revealed no influence on the integrin αvβ3 negative cells (Fig. 5A and B), however, significantly strengthened the cytotoxicity of PTX (Fig. 5C) and Cis (Fig. 5D) to integrin αvβ3 positive A549 and PC-9. Those results implicated that blockade of TRIB3-AKT interaction efficiently suppressed the tumor progression induced by integrin αvβ3 and improved the outcome of chemotherapy in NSCLC. Next, tumors arising from the subcutaneous implication of A549 cells in mice was established for anticancer effects analysis in vivo. Using this model, subcutaneous A549 tumors were established in mice and, then treated with chemotherapeutic Cis, PTX and Pep2–Ae. Notably, Pep2–Ae treatment resulted in the suppression of tumor growth, and strengthened the anticancer effects of Cis (Fig. 5E and F) and PTX (Fig. 5G and H) in A549 bearing mice. On the basis of our results, we suggested that suppression of TRIB3-AKT axis could efficiently impair tumor growth and improve the outcome of chemotherapy in NSCLC.

Previous in vitro and in vivo studies have suggested the association between integrin αvβ3 and tumor progression [6, 17]. Our observation that integrin αvβ3 could efficiently promote lung cancer cells progression in vitro, which is in consistent with previous reports. However, the TCGA database analysis implicated that no significant difference was found between the patients with high/low expression of integrin αvβ3. Here, we reported in the present study that integrin αvβ3 facilitated the lung cancer proliferation and invasion through FAK/AKT signaling pathway, which was dependent on the collaboration with TRIB3. Depletion of TRIB3 resulted in the inactivation of AKT signals, the downstream pro-survival signals of integrin αvβ3 (Fig. 6). To our knowledge, these data firstly provided evidence that tumor integrin αvβ3 contributed to the tumor progression through an TRIB3 dependent manner.

Increasing evidence suggested that differential expression cancer stem cell markers, such as ALDH1, CD133 and CD44, was tightly correlated with the pathological subtypes and tumor development of lung cancer [18, 19]. Importantly, there is recent evidence that integrins also potentiate cancer stem cell function, especially in lung cancer. In agreement with previous reports that integrin αvβ3 correlated with the metastasis of lung cancer cells [20], we demonstrated that integrin αvβ3 efficiently promoted the cells invasion in lung cancer. Furthermore, we provided evidence that integrin αvβ3 promoted the stem-like phenotypes and proliferation of A549 and PC-9. Accordingly, compelling findings suggested that integrin αvβ3 served as novel cancer stem cells marker, that contributed the stemness up-regulation in melanoma and breast cancer [21, 22].

In mechanism, C Chetty and his colleagues suggested that MMP-2 could up-regulate VEGF expression via integrin αvβ3/PI3K/AKT signaling in lung cancer cells [8]. And previous reported indicated the activation of integrin αvβ3 and FAK/NF-kB signals was correlated with the cancer metastasis [23]. Our study further demonstrated integrin αvβ3 mediated FAK/AKT signaling pathway activation to promote lung cancer cells proliferation. More importantly, we further demonstrated that TRIB3 interacted with AKT1 to up-regulate FOXO1 and SOX2 expression, which was indispensable for the pro-tumor effects induced by integrin αvβ3.

Overexpression of pluripotency genes, including c-Myc, SOX2 and Oct3/4, could promote the cancer stem cells and contribute to the tumor progression [24‐26]. Additionally, clusters of mutations occurring at certain regions of the genome are a previously unrecognized key player in regulating tumor progression [27]. In our study, we demonstrated the aberrant upregulation of TRIB3 correlated with the lung cancer progression, which was associated with the SOX2 transcription factor activation. SOX2 played a crucial role, by not only promoting pluripotency but also facilitating self-renewal and differentiation [28]. SOX2 was overexpressed in several tumor types, including lung and prostate cancer [29, 30]. However, the underlying mechanism by which SOX2 was upregulated in tumor tissues remained unclear. Compelling reports have suggested that specific cellular receptors, such as integrins and EGFR could mediate the pro-survival signaling pathways and SOX2 activation [31]. However, increasing evidence implicated that blockade of upstream integrins receptors by inhibitors was insufficient to suppress the tumor progression induced by those pro-survival signals [32]. More importantly, the expression of integrin αvβ3 was found in abundant tumor cells/tissues, whereas the expression of SOX2 was only found in those cancer stem cells, which accounted for a little proportion in tumor cells [33, 34]. In our study, no significant correlation was found in integrin αvβ3 expression and overall survival in lung cancer patients, despite the pro-tumor effects of integrin αvβ3 in vitro. We demonstrated that the pro-tumor effects and the activation of SOX2 was dependent on a TRIB3 manner, and the patient prognosis was influence by the TRIB3 expression. In mechanism, the interaction between AKT and TRIB3 was necessary for the FOXO1/AKT/SOX2 activation in lung cancer. Our results further described the underlying mechanism of integrin αvβ3 and SOX2 in regulating lung cancer development, which provided new sight for target therapy in lung cancer.

In conclusion, our study suggested that TRIB3 links integrin αvβ3 signals to induce lung cancer progression, which was coordinated with AKT/SOX2 signals. Interrupt of TRIB3/AKT axis by Pep2–Ae efficiently improved the outcome of chemotherapy, which describing an innovative approach for lung cancer treatment.