SH2B1 promotes NSCLC cell proliferation through PI3K/Akt/mTOR signaling cascade

Ethical approval and Informed consent statements

Written informed consent was obtained from 104 participants before the study. The clinical protocol for this study was approved by the Medical Research Ethics Committee of Xiangya Hospital, Central South University (CSU) (#201403216). All mice experiments were approved by the Animal Ethics Committee and conducted by the official recommendations for the Care and Use Laboratory Animals of Xiangya Hospital, CSU (#201403217).

Tissue specimens

A total of 104 fresh NACLC tumor specimens were archived by radical resection from those NSCLC patients without any chemotherapy and radiotherapy intervention at the Department of Thoracic Surgery of Xiangya Hospital from January 2010 to December 2011. The tumor-node-metastasis was classified according to the criteria of the 8th AJCC/UICC (American Joint Committee on Cancer/International Union Against Cancer) TNM staging system. There were 29 cases in Stage I, 33 cases in Stage II and 42 cases in Stage III. All freshly collected NSCLC tumor tissues were frozen and stored in liquid nitrogen until required. All surgical resection samples were evaluated by a board-certified pathologist.

Cell culture

The human NSCLC cell lines A549 and H1299 (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Gibco, Gaithersburg, VA, USA) supplemented with 10% fetal bovin serum (FBS) and penicillin/streptomycin and then incubated at 37 °C in a humidified 5% CO₂ atmosphere.

RNA extraction and qRT-PCR analyses

Total RNA from cultured cells was extracted using the TRIzol reagent (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer’s manual. The expression level was quantified using SYBR Green Assay Kit (Takara, Japan). Real-time PCR was performed in triplicate using the Applied Biosystems ViiA 7 Sequence Detection system (Life technologies, CA, USA). GAPDH was used to normalize mRNA and calculate the relative expression of each transcript. The following pairs of primers were synthesized as: C-Myc (F): 5′-CCTCCACTCGGAAGGACTATC-3′, (R): 5′-TTCGCCTCTTGACATTCTCC-3′; GAPDH (F): 5′-ACCCACTCCTCCACCTTTGACG-3′, (R): 5′- TCTCTTCCTCTT GTGCTCTTG-3′.

Immunohistochemistry (IHC)

IHC analysis was performed on 4 µm-thick formalin-fixed and paraffin-embedded NSCLC tissue sections from each tumor sample, using the PV-60001/PV-6002 Two-Step IHC Detection Reagent (ZhongShan Golden Bridge Bio, Beijing, China) according to the manufacturer’s protocol. In brief, antigen retrieval was performed in buffer for 10 min in a microwave oven. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 min at room temperature. Slides were incubated with

the following primary antibodies, anti-SH2B1 (1:100, ab196575, Abcam), anti-Akt (1:500, ab8805, Abcam), anti-Akt (phospho S473) (1:200, ab8932, Abcam), anti-mTOR (1:1000, ab2732, Abcam), anti-mTOR (phosphor S2448) (1:50, ab109268, Abcam), anti-PTEN (1:100, ab31392, Abcam, Cambridge, MA, USA), anti-Ki67 antibody (1:200, Cell signaling, Danvers, MA, USA) at 4 °C overnight. Following washing with PBS, slides were incubated with HRP-labeled secondary antibody for 50 min at room temperature. Immunoreactive products were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and counterstained with Mayer’s hematoxylin. For negative controls, slides were treated with 0.01 mol/L PBS instead of primary antibodies under the same conditions.

The IHC staining scores were determined by combining the intensity of staining and the proportions of positively stained cells. The standard of staining intensity was graded that: 1, no staining; 2, weak staining (light yellow); 3, moderate staining (yellow brown); and 4, strong staining (brown). The positive cell proportions were scored according to the following standard: 0, < 5% positive cell; 1, 6–25% positive cells; 2, 26–50% positive cells; 3, 51–75% positive cells; 4, 76–100% positive cells. We evaluated protein expression using staining index (SI), which was calculated of the proportion of positive cells and the staining intensity score as possible total scores of 0, 1, 2, 3, 4, 5, 6, 7, and 8. Samples with SI ≤ 4 were defined as low (negative) expression and SI > 4 as high (positive) expression. The degree of SI was reviewed and scored separately by two independent pathologists.

Xenografted tumor model and H&E staining

Male BALB/c-nu mice (age of 4–5 weeks, 16–18 g) were purchased from the Hunan Slack King of Laboratory Animal Co., Ltd (Changsha, China). Mice were randomly divided into groups (n = 6 per group) before injection. A total of 1 × 10⁷ cells were subcutaneously injected into the left dorsal flank per mouse. Measure tumors length and width and then calculate tumor volumes (L × W²/2) with calipers on alternate days. On day 30, the mice were euthanized and tumors were excised to paraffin embedded. Serial 4.0 μm sections were cut and subjected to haematoxylin and eosin (H&E) staining and IHC analyzed using an anti-Ki67 (1:200, Cell signaling, Danvers, MA, USA). The proportion of Ki67 positive cells were counted as proliferation index. All mice experiments were approved by the Animal Ethics Committee and conducted by the official recommendations for the Care and Use Laboratory Animals of Xiangya Hospital, CSU (#201403217).

Western blotting

Whole-cell lysates were prepared from human NSCLC cell lines after harvesting and centrifuging. Protein concentrations were quantified using BCA kit (Beyotime Bio, Shanghai, China). The proteins were denatured, electrophoresed (SDS-PAGE gel Kit, Beyotime Bio, Shanghai, China), and then transferred to PVDF membranes (Millipore Co., MA, USA). Subsequently, the PVDF membranes were blocked with non-fat milk powder, incubated with primary and secondary (HRP-labeled goat anti-rabbit IgG) antibodies, and visualized (Bio-Rad Image Lab Software).

Western blotting was performed using the primary antibodies, anti-SH2B1 (1:1000, ab196575, Abcam), anti-Akt (1:600, ab8805, Abcam), anti-Akt (phospho S473) (1:300, ab8932, Abcam), anti-mTOR (1:1500, ab2732, Abcam), anti-mTOR (phosphor S2448) (1:1000, ab109268, Abcam), anti-PTEN (1:600, ab31392, Abcam), anti-C-Myc (1:1000, ab32072, Abcam), antibodies (Abcam, Cambridge, MA, USA), and anti-GAPDH antiboty (1:3000. D110016, Sangon Biotech, Shanghai, China). Following the initial western blotting assay, the membranes were stripped and the band intensities were relative to GAPDH. The quantification of protein bands was performed using Image J software.

Cell proliferation assay

2 × 10³ cells were seeded and cultured in triplicates in 96 well plates. Cell viability was measured every day over a 5-day period using cell count kit-8 (CCK8, #CK04, Dojindo Molecular Technologies, Rockville, MD 20850, USA). Cells were incubated with 10 μl CCK8 for 1 h at 37 °C, and absorbance was measured at 450 nm using the Elx808 Absorbance Microplate Reader (BioTek Instruments, USA).

Colony formation assays

Cells were suspended in 0.6% agarose and placed in 24-well plates at low density (2.5 × 10² cells per plate). After 2 weeks, the colonies were fixed with methanol and counted.

Cell cycle analysis

Cells were trypsinized, collected and rinsed twice with PBS and fixed with cold absolute alcohol at 4 °C overnight. After fixation, cells were incubated with 0.5 ml PBS, 5 μl propidium iodide (20×) and 10 μl RNase A (50×) for 30 min at 37 °C without light before analysis. The cell cycle was analyzed by a flow cytometry (BD, San Jose, CA, USA).

Flow cytometry detection

Cells were collected and stained using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA). Cell apoptosis were quantified using a flow cytometry (BD, San Jose, CA, USA).

Gene knockdown and gene overexpression

shRNA against SH2B1 and control shRNA were constructed into the lentiviral vector pHY-LV-KD5.1 (GFP-lentivirus, Hanyin Co. Shanghai, China). The recombinant SH2B1-shRNA lentivirus (target sequence: 5′-TAACAACCAGTACTCCTTCGTGTGAGC-3′) and the negative control lentivirus (target sequence: 5′-TTCTCCGAACGTGTCACGT -3′) were prepared and titered to 10⁹ TU/ml (transfection unit). To obtained a stable SH2B1-knockdown cell line, 2 × 10⁵ A549 cells seeded in per well of six-well dishes. The cells were then infected with the same titer virus with 8 μg/ml polybrene on the following day. Approximately 72 h after viral infection, the culture medium was replaced with selection medium containing 4 μg/ml puromycin after GFP expression was confirmed under a fluorescence microscope. The cells were cultured for at least 14 days. The puromycin-resistant cell clones were isolated, amplified in medium containing 2 μg/ml puromycin for 7–9 days, and then transferred to a medium without puromycin. The clones were designated shSH2B1 (knock down) or shCtrl (negative control) cells.

Lentiviral vectors for human SH2B1 expression sequence were constructed by Hanyin Co. (Shanghai, China). The cDNA of SH2B1 with a C-terminal Flag-tag were cloned into the lentiviral vector pHY-LV-OE1.6. Stable cell lines expressing SH2B1 were generated by retroviral infection using H1299 cells, and selected with 2 μg/ml puromycin for 7–9 days. Further details of methods were carried out as gene knockdown described. The clones were designated H1299-SH2B1 (overexpression) or H1299-vector (negative control) cells. Transfection was conducted using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions [10].

Statistical data analysis

All statistical analyses in this study were carried out using SPSS 22.0 statistical software. Data were shown as mean ± SD, and 2-tailed paired Student’s t-test was used for comparing the difference between groups unless otherwise stated. P < 0.05 was considered statistically significant.

Functional significance of SH2B1 in NSCLC cells in vitro

In a previous study, we had observed that SH2B1 was significantly upregulated in NSCLC tissues and cell lines, and high SH2B1 expression was associated with tumor size [4]. We next explored its functional significance in NSCLC. Lentivirus-mediated knockdown of endogenous SH2B1 (in A549) and upregulation of SH2B1 (in H1299) were performed to evaluated the cellular outcomes using several assays [11]. To investigate whether SH2B1 affects cell cycle in NSCLC cells, we performed FACS cells cycle distribution analysis. Knockdown of SH2B1 in A549 cell lines, which had high level of SH2B1, resulted in decreased percentage of S phase (P = 0.0023) cells and increased cells residing at the G1 phase (P = 0.0002, Additional file 1: Figure S1A), reflecting that fewer A549-shSH2B1 cells were cycling and further indicating a slowed cell proliferation rate. While cyclin D1 was required for overcoming the G1/S checkpoint, so we performed the RT-qPCR and Western blot. And the data revealed that cyclin D1 in A549 cells were significantly decreased after transfection of SH2B1-shRNA compared with control shRNA (Fig. 1c). Meanwhile, flow cytometry analysis revealed that overexpression of SH2B1 had a significant shift from G1 to S phase (P = 0.0110, Additional file 1: Figure S1B), and this effect was associated with induction of the expression of cyclin D1 (Fig. 1d), and so accelerated the cell cycle progression.

In addition, increased cells’ G1-S phase propagation in NSCLC was linked to reduced apoptosis. To further test the impact of SH2B1 on the apoptosis in NSCLC cells, we performed an apoptosis assay by FACS analysis. Transfection of SH2B1-shRNA resulted in a significant increase in the percentage of apoptotic cells, compared with the control shRNA, in A549 cells (P = 0.0007, Fig. 1a). Moreover, knockdown of SH2B1 dramatically increased the level of caspase-3 activation, which play important role in apoptosis, compared with A549-shCtrl (Fig. 1c). In line with this, decreased apoptotic cells (P = 0.0209, Fig. 1b) and caspase-3 activation (Fig. 1d) were seen in H1299-SH2B1 cells compared with control H1299-vector.

SH2B1 promotes NSCLC cell proliferation in vitro

The above data indicated that SH2B1 was involved in the regulation of NSCLC cell cycle progression and cell apoptosis. We speculated that SH2B1 may play an essential role in NSCLC cell proliferation. To investigate this hypothesis, we performed CCK8 assay in A549 and H1299 cell line. Knockdown of SH2B1 in A549 cell significantly inhibited cell proliferation compared to control A549-shCtrl cells (P < 0.001, Fig. 2a). Similarly, the proliferation rate of H1299 cells were significantly increased after overexpression of SH2B1 and the number of H1299-SH2B1 cells was more than that of control H1299-vector cell (P < 0.001, Fig. 2b).

Similar results obtained from soft agar colony formation assay. SH2B1 knockdown attenuated the colony formation of A549 cells, as the number (P = 0.0322) and mean size (P = 0.0215) of single-cell-derived soft agar colonies were both markedly decreased (Fig. 2c). Moreover, over-expression of SH2B1 caused to significantly elevated colony and sphere forming capacity of H1299 cells (Fig. 2d).

SH2B1 promotes NSCLC cell proliferation in vivo

To verify the positive role of SH2B1 in NSCLC cells growth, in vivo nude mice (age, 4 weeks) were performed xenograft tumor assays using A549 cells stably transfected with either SH21-shRNA (abbreviated as shSH2B1 hereafter simplicity) or negative control lentiviruses (shCtrl). Subcutaneous tumor xenografts were monitored on alternate days (Fig. 3a, c). For A549 cell lines, the shSH2B1 cells grew tumors at a much slower rate compared to shCtrl cells (Fig. 3a). At the time of sacrifice, shSH2B1 cell tumors were significantly smaller compared with those of shCtrl cells (Fig. 3a). Furthermore, xenograft tumors cell proliferation was assessed by Ki-67 immunostaining. The estimated result obtained from Ki-67 staining sowed SH2B1-knockdown (shSH2B1) tumors displayed significantly less proliferative cells (Fig. 3b). Most notably, overexpression of SH2B1 in H1299 cells (H1299-SH2B1) significantly increased the tumor growth rate and tumor mass in vivo (Fig. 3c). In addition, the number of Ki-67-posive cells were high in SH2B1-overexpression tumors compared with control cells containing vector control (Fig. 3d).

SH2B1 regulates NSCLC cell proliferation through Akt/mTOR pathway in vitro

The above data indicated that SH2B1 was involved in the regulation of NSCLC cell proliferation. To elucidate the molecular mechanisms by which SH2B1 modulates NSCLC cell proliferation, we investigated the effects of knock-down and over-express SH2B1 on the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of Rapamycin (mTOR) signaling, which is an important regulator of cell proliferation and frequently aberrantly activated in human cancers [12, 13]. Western blot showed that transfection with SH2B1 shRNA significantly decreased Akt (Ser 473) and mTOR (Ser2448) phosphorylation, but increased PTEN expression in A549 cells (Fig. 4a). On the other hand, overexpression of SH2B1 induced activation of Akt and mTOR, but decreased PTEN expression in H1299 cells (Fig. 4a). Furthermore, we examined the effect of SH2B1 on Akt/mTOR signaling by using Akt specific inhibitor deguelin (Fig. 4b). We observed that the protein changes in Akt pathway targets (p-Akt, p-mTOR and PTEN) induced by SH2B1 over-expression reversed by deguelin (Fig. 4b). Importantly, NSCLC cells proliferation was increased in S2B1 over-expression cell (H1299-SH2B1), while the proliferation effect was dramatically impaired by deguelin (Fig. 4c) and mTORC1 inhibitor rapamycin (Additional file 1: Figure S2). Taken together, these data suggested that the Akt/mTOR signaling cascade might participate in SH2B1-induced cell proliferation in NSCLC cells.

Clinical relevance of SH2B1 and its targets in NSCLC

Finally, we examined whether SH2B1 mediated Akt and mTOR activity, and PTEN suppression in NSCLC are clinically relevant. In 104 human NSCLC samples obtained from Xiangya hospital Thoracic Tissue Bank, IHC assay was performed. Representative pictures were depicted in Fig. 5a. IHC analysis revealed there was a significant correlation between SH2B1 expression and expression of p-AKT, p-mTOR and PTEN. In the Fig. 5b and Additional file 1: Figure S3, SH2B1 expression strongly correlated with p-Akt (Ser 473) and p-mTOR (Ser 2448) expression (P < 0.001), but inversely correlated with PTEN expression (P < 0.001). Collectively, these results further support the notion that SH2B1 overexpression activates Akt/mTOR signaling cascade leading to NSCLC progression.