Oncogenic effect of PHLDB2 is associated with epithelial–mesenchymal transition and E-cadherin regulation in colorectal cancer

The colon cancer data were downloaded from cBioportal (TCGA, Provisional) [7, 8]. Analyses were performed on the patients/samples with available data corresponding to each aspect. The Log-rank test was used to determine the disease-free survival. The Pearson correlation coefficient was calculated between PHLDB2 and the EMT markers.

Human colon cancer cell line SW620 was purchased from the American Type Culture Collection (ATCC), and HCT116 p53^−/− cells were generous gifts from Dr. Bert Vogelstein at the John Hopkins Medical Institutes. The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen), at 37 °C, 5% CO₂. Human recombinant TGF-β1 was purchased from R&D Systems (Minneapolis, MN). The antibodies were purchased from Santa Cruz (anti-E-cadherin, and anti-Vimentin), Sigma-Aldrich (anti-PHLDB2), Thermo Fisher (anti-MDM2) and Millipore (anti-glyceraldehyde-3-phosphate dehydrogenase, GAPDH). PHLDB2 siRNAs and non-specific control oligo were purchased from Ambion. Transfection was performed using Lipofectamine 2000 (Invitrogen) per manufacture’s instruction.

Cells were transfected with non-specific control or PHLDB2 siRNA and 24 h after transfection, the cells were replated into Matrigel-coated upper champers (24-well transwell inserts, BD Bioscience) for invasion assay, or into 6-well plates for wound healing migration assay as described previously [6]. Briefly, for the invasion assay, the upper champers were refreshed with serum free medium while the lower champers filled with culture medium containing reconstitution buffer or 5 ng/ml TGF-β at 6 h after replating. The cells were allowed to invade for 48 h, and non-invasive cells were removed with cotton swab and invasive cells were fixed with methanol and stained with crystal violet. Cell numbers were quantitated from 3 different wells. Wound healing assay was performed by comparing the gaps generated with P200 tip scratching between 0 h and 24 h time points.

Studies suggest that PHLDB2 is a potential oncogene [1‐3, 6], but its clinical implication remains less understood. In order to address this question, we analyzed a set of colorectal adenocarcinoma data deposited by the Cancer Genome Atlas (TCGA) in the cBioportal website [7, 8]. This set included the largest number of patients with RNA-seq data available from TCGA. Consistent with our previous report [6], the disease-free survival of the patients with low PHLDB2 expression (median survival undefined) is significantly longer from those with high PHLDB2 expression (median survival 71.48 months), as shown in Fig. 1a. In line with this result, the expression level of PHLDB2 is associated with pathologic tumor stage (Fig. 1b) and regional lymph nodes pathology (Fig. 1c) defined by American Joint Committee on Cancer (AJCC), as the PHLDB2 levels in Stage III and Stage IV or in N2 are significantly higher than in Stage I or N0, respectively. In the attempt to find association between PHLDB2 and tumor invasiveness, we found that patients with either lymphovascular invasion or vascular invasion also have significantly higher expression of PHLDB2 than those without these indicators (Fig. 2a, b). Although the same analysis does not show significant difference in perineural invasion, there is still a trend of elevated expression of PHLDB2 in perineural invasion indicator positive patients (Fig. 2c). Together, these results indicate that high level of PHLDB2 is associated with poorer clinical outcomes and tumor invasiveness in colorectal cancer patients.

The epithelial–mesenchymal transition (EMT) is a program activated in certain population of cancer cells to confer metastasis by facilitating cell migration and invasion [4, 5]. While PHLDB2 is an important protein involved in focal adhesion disassembly in migrating cells, we next sought to investigate if PHLDB2 is correlated with any EMT markers in the same set of colorectal cancer patients. Surprisingly, among the well-known EMT markers [5], at least nine of them are highly correlated with PHLDB2 expression. As shown in Fig. 3, the Pearson correlation analysis demonstrates that these EMT markers include cytoskeletal markers [α-SMA (ACTA2) and Vimentin (VIM)], cell surface proteins [N-cadherin (CDH2) and OB-Cadherin (CDH11)], extracellular matrix (ECM) proteins [Laminin 5 (LAMA5) and Fibronectin (FN1)], and transcription factors [Snail2 (SNAI2), ZEB1, and Ets-1 (ETS1)]. The correlation was especially high with cytoskeletal markers (Fig. 3a, b), cell surface proteins (Fig. 3c, d), and transcription factors (Fig. 3g–i). These findings indicate that PHLDB2 highly likely plays an important role in mediating EMT process.

As we have demonstrated the correlation of PHLDB2 with multiple EMT markers in clinical data, we next sought to further validate the role of PHLDB2 in EMT in cell-based assays. TGF-β signaling is an important pathway in inducing EMT phenotype [9]. To test the function of PHLDB2 in TGF-β-induced EMT, we treated HCT116 p53^−/− cells with TGF-β while knocking down PHLDB2 using siRNA and subjected the cells to wound healing assay and transwell invasion assay. As shown in Fig. 4a, c, at 24 h after scratching, TGF-β treatment promoted the wound gap to a full closure, while the control cells were only half-way closed. Markedly, PHLDB2 knockdown resulted in ~ 80% heal in the presence of TGF-β, and significantly mitigated TGF-β effect. To confirm that this effect was achieved specifically by PHLDB2 knockdown, we employed another siRNA against the non-coding region of PHLDB2 (si-PHLDB2x) and re-introduced Flag-PHLDB2 into cells. The results shown in Additional file 1: Figure S1 clearly demonstrated that si-PHLDB2x also significantly attenuated TGF-β induced cell migration, which was rescued by re-introducing exogenous PHLDB2. Similar results were observed in another colon cancer cell line SW620 (Additional file 1: Figure S2). In line with the wound healing assay, TGF-β treatment induced ~ 58% more invasive cells compared to control group, while this induction was significantly attenuated by PHLDB2 depletion (Fig. 4b, d). Interestingly, while TGF-β treatment clearly suppressed E-cadherin expression, and induced Vimentin expression, it also caused a significant increase in PHLDB2 protein level (Fig. 4e). Meanwhile, PHLDB2 knockdown resulted in less effective reduction of E-cadherin by TGF-β (Fig. 4e). Collectively, these data suggest that PHLDB2 is indeed an important mediator of EMT, likely in the downstream of TGF-β pathway, as PHLDB2 depletion at least partially compromised TGF-β-induced EMT phenotypes as well as E-cadherin inhibition.

The observation from Fig. 4e led us to further evaluate the change of PHLDB2 expression in response to EMT induction. We treated HCT116 p53^−/− cells with increasing concentrations of TGF-β or epidermal growth factor (EGF) for 24 h and collected cell lysates for Western Blot and qRT-PCR analysis. As shown in Fig. 5a, in addition to reducing E-cadherin and inducing Vimentin protein expression, TGF-β also up-regulated PHLDB2 protein level in a dose-dependent manner. Up-regulation of PHLDB2 by TGF-β also occurred at mRNA level (Fig. 5b). Similarly, EGF treatment also resulted in a dose-dependent induction of PHLDB2 protein and mRNA expression (Fig. 5c, d). In line with our previous finding, these data suggest that in colon cancer cells, EMT induction by two important pathways, TGF-β and EGF, transcriptionally activates PHLDB2 expression.

Mouse double minute 2 (MDM2) has been reported to promote EMT by negatively regulating E-cadherin expression through ubiquitination and degradation [10]. Considering our observation in Fig. 4e that PHLDB2 knockdown attenuated the reduction of E-cadherin by TGF-β, we next sought to test if PHLDB2 had any function in regulating E-cadherin expression involving MDM2. We transfected HCT116 p53^−/− cells with HA-MDM2 in the absence or the presence of Flag-PHLDB2 and performed co-immunoprecipitation assay using anti-Flag antibody. Interestingly, the result in Fig. 6a showed that these two exogenous proteins indeed bound to each other. We further used anti-MDM2 antibody for endogenous protein and the result confirmed the interaction between PHLDB2 and MDM2 (Fig. 6b). In order to test the effect of PHLDB2 on MDM2-mediated E-cadherin expression, we then transfected HCT116 cells with HA-MDM2 or Flag-PHLDB2 either alone or together, and found that E-cadherin protein was reduced by HA-MDM2 or Flag-PHLDB2 alone and the reduction was more profound when both HA-MDM2 and Flag-PHLDB2 were present (Fig. 6c). Together, these observations indicate that by interacting with MDM2, PHLDB2 could facilitate MDM2-mediated E-cadherin down-regulation.