Numb inhibits epithelial-mesenchymal transition via RBP-Jκ-dependent Notch1/PTEN/FAK signaling pathway in tongue cancer

Tongue squamous cancer cell lines (SCC-9 and CAL-27) and 293 T cell were from American Tissue Culture Collection (ATCC, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin at 37 °C, 5% CO₂. Of which, SCC-9 cells were grown in 1:1 Hams F-12, DMEM (modified). Primary normal human oral keratinocytes (NHOK), purchased from PriCells, were isolated from human gingival tissues and cultured as described previously [17]. PTEN inhibitor VO-Ohpic trihydrate was purchased from MedChemExpress (USA) and worked at the concentration of 35 nM.

Numb cDNA sequence was cloned and ligated into pCDH-MCS-EF1-puro vector. RBP-Jκ short hairpin RNA (shRNA-RBP-Jκ) or negative control shRNA (shRNA-NC) were inserted into PLKO.1 vector. In order to make lentiviruses, pPAX2 and pVSVG vectors plus transfer vector were introduced into 293 T cells. Then, we harvested the supernatant at 48 h post transfection, which was filtered with 0.45 μm membrane and concentrated by a centrifugal filter (EMD Millipore, Amicon Ultra 100 k). When subject to virus infection, the cells medium was treated with virus supernatant at 1:5 ratios, post 24 h, we used ~ 2 μg/mL puromycin to select the Numb-overexpressed stable cell lines. The shRNA sequences are synthesized as previously described [18].

We performed this experiment as standard procedure in accordance with previous descriptions [8]. The following antibodies were used: anti-E-cadherin (cell signaling technology, USA), anti-N-cadherin (cell signaling technology, USA), anti-Snail (cell signaling technology, USA), anti-MMP-9 (cell signaling technology, USA), anti-Numb (cell signaling technology, USA), anti-Notch1 (cell signaling technology, USA), anti-FAK (cell signaling technology, USA), anti-p-FAK (cell signaling technology, USA), anti-PTEN (cell signaling technology, USA), anti-RBP-Jκ (millipore, USA), anti-GAPDH (Proteintech, USA).

To perform this assay, TSCC (1 × 10⁵cells) were mixed with about 200 μL of FBS-free medium. Chambers containing 8.0 μm pore membranes (Millipore) with matrix was used in this assay. The TSCC cells were seeded into the top chamber. Afterwards, about 500 μL complete medium was added to bottom chamber. Roughly 48 h post incubation, the invaded cells were fixed and stained with crystal violet, and finally were counted using the inverted microscope.

We measured migration of cells in vitro through wound healing assay. In brief, 2 × 10⁵ SCC-9 and CAL-27 cells were passaged to 6-well plates. The cells were incubated in the complete culture medium under normal conditions for 16 h. then, we scratched the monolayer and incubated cells in fresh medium without FBS for 24 h. Eventually, we measured the wound width. we visualized and photographed 3 different locations under the inverted microscope.

We harvested cells and extracted total RNAs by Trizol method. Then, chloroform was added to the solution. The sample was subject to centrifuge at 12,000 rpm for 10 min, and was transferred to new RNase-free EP tube. Resulting solution was mixed with equal volume of isopropanol and subject to centrifuge at 12,000 rpm for 10 min. After that, removing the supernatant and adding 70% ethanol to wash pellet. Eventually, discarding the ethanol and drying the RNA pellet. And, we used 35 μL Rnase-free H₂O to dissolve RNA.

We employed ~ 1 μg of RNA for reverse transcription. SYBR dye was used to detect signaling, the GAPDH serves as internal control. The primers used in this study as follows:

All experiments were conducted for three replicates, all values were represented as mean ± SD, comparisons of two groups were done using two-tailed unpaired student’s t-test. *P < 0.05 was considered statistically significant.

Recently, it has been studied that Notch1 signaling is implicated in progression of tongue cancer [8]. To validate, we examined the expression of Numb (Notch1 regulator) and Notch1 in tongue squamous cancer cell lines (SCC-9 and CAL-27) and normal human oral keratinocytes (NHOK) by RT-qPCR method. We found that Numb expression level was significantly decreased in SCC-9 and CAL-27 cells compared with that in NHOK cells (Fig. 1a). By contrast, the level of Notch1 was increased by about 2 folds in SCC-9 and CAL-27 cells (Fig. 1b). These data suggested that, indeed, Numb and Notch1 signaling were implicated into tongue cancer progression.

To further confirm the anti-tumor role of Numb in tongue cancer, we constructed Numb-overexpressed SCC-9 and CAL-27 cell lines. First, we used MTT assay to evaluate SCC-9 and CAL-27 cells growth and found that cell proliferation ability of cells were markedly suppressed upon Numb overexpression compared to negative control (NC) (Fig. 2a).

Next, we assessed cell migration by wound healing assay when the cells transfected with empty vector or Numb at the time points of 0, 24, 48 h. The results demonstrated that Numb-overexpressed SCC-9 and CAL-27 cells displayed impaired migration ability relative to NC group cells (Fig. 2b and c). In addition, we also investigated the role of Numb in invasion of tongue cancer cells (SCC-9 and CAL-27) using transwell invasion experiment at 24 h. Our data revealed that the number of invaded tongue cancer cells expressing Numb was remarkably lower than NC group cells (Fig. 2d and e). Collectively, these results indicated that Numb had an inhibitory role in tongue cancer proliferation and metastasis.

In order to dissect the molecular mechanism Numb-suppressed proliferation and tongue cancer cells EMT, we probed expression of Notch1 and EMT-associated genes (Fig. 3a and b). Western blot analyses demonstrated that Notch1 expression level was decreased in Numb-overexpressed SCC-9 and CAL-27 cells. More importantly, we observed that EMT-inhibited genes (E-cadherin) expression level was upregulated, whereas RBP-Jκ and EMT-promoted genes (N-cadherin, Snail, MMP-9) expression level was downregulated. These data showed Numb had a critical role in regulation of Notch1 signaling, RBP-Jκ and EMT-associated genes expression, which is more likely to mediate Numb-inhibited EMT of tongue cancer cells.

To explore the role of PTEN in regulation of EMT-associated genes expression in tongue cancer cell lines (SCC-9 and CAL-27), we utilized PTEN inhibitor VO-Ohpic trihydrate to abolish the function of PTEN and then detected expression level of EMT-associated genes (Fig. 4a and b). As shown in the western blot, PTEN expression was suppressed by VO-Ohpic trihydrate in Numb-overexpressed SCC-9 and CAL-27 cells. However, phosphorylated form of FAK was elevated in the presence of VO-Ohpic trihydrate. In addition, the result showed VO-Ohpic trihydrate treatment disrupted EMT-related genes expression level modulated by Numb. Together, our data suggested that PTEN inhibitor VO-Ohpic trihydrate could effectively block Numb-inhibited EMT genes expression in SCC-9 and CAL-27 cells.

Next, we attempted to assess whether RBP-Jκ was key for proliferation and metastasis of tongue cancer cells. We generated RBP-Jκ-depleted tongue cancer cell lines (SCC-9 and CAL-27) using short hairpin RNA approach. Then, MTT experiment was carried out to detect cell proliferation of SCC-9 and CAL-27 cells. We found that RBP-Jκ knockdown SCC-9 and CAL-27 cells grew more slowly than NC cells (Fig. 5a).

To test whether RBP-Jκ had a role in invasion and migration of tongue cancer cells, we carried out transwell invasion assay and wound healing assay to evaluate the invasion and migration ability of the cells, respectively. We observed that RBP-Jκ knockdown resulted in impaired migration capability of SCC-9 and CAL-27 cells (Fig. 5b and c). In parallel, fewer numbers of invaded cells (decreased by 30–40%) were stained by crystal violet when the cells were transfected with RBP-Jκ shRNA compared to NC cells at 24 h (Fig. 5d and e). In summary, all these results revealed that RBP-Jκ was important for proliferation, migration and invasion of tongue cancer cells (SCC-9 and CAL-27).

To clarify the underlying molecular mechanism of RBP-Jκ knockdown-caused proliferation and metastasis change of tongue cancer cells, we attempted to probe PTEN, FAK and EMT-related genes expression after RBP-Jκ knockdown in SCC-9 and CAL-27 cells (Fig. 6a and b). Western blot analyses demonstrated that PTEN and EMT-inhibited gene (E-cadherin) expression level were upregulated in RBP-Jκ knockdown cells, on the other hand, p-FAK and EMT-promoted genes (N-cadherin, Snail, MMP-9) expression level was downregulated upon RBP-Jκ depletion. Therefore, we concluded that RBP-Jκ was a key regulator of PTEN/FAK/EMT-related genes pathway.

We next attempted to figure out whether PTEN inhibitor also influenced expression of p-FAK and EMT-related genes in the tongue cancer cells with RBP-Jκ knockdown (Fig. 7a and b). Western blot analyses showed PTEN and E-cadherin was modestly downregulated in RBP-Jκ-knockdown cells treated with VO-Ohpic trihydrate. Nonetheless, p-FAK and EMT-promoted genes (N-cadherin, Snail, MMP-9) expression level were modestly upregulated in response to VO-Ohpic trihydrate treatment. Our data indicated that PTEN inhibition had the ability to partially rescue RBP-Jκ knockdown-induced changes in expression of p-FAK and EMT-regulated genes.