PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

To investigate PinX1 expression in breast cancer, immunohistochemistry was carried out in TMA slides (Figure 1A). Samples with IRS 0–3 and IRS 4–12 were classified as low and high expression of PinX1. Of the 405 breast cancer analyzed, low and high expression of PinX1 staining were 52.3% (212/405) and 47.7% (193/405), respectively (Table 1). We then analyzed the correlations between PinX1 expression and characteristics of the breast carcinomas, and found that PinX1 staining was dramatically decreased in histology grade II and III compared with histology grade I (P = 0.001, χ² test, Table 1). We also found that PinX1 expression is significantly correlated with lymph node metastasis (P = 0.002, χ² test, Table 1). However, we did not find any significant correlations between PinX1 expression and other clinicopathologic variables, including patient age, tumor size, ER status, PR status, HER2 status and p53 status.

To further study whether PinX1 expression correlates with the survival of patients, Kaplan-Meier survival curves were constructed using 5-year overall and disease-specific cumulative survival to compare the patients with high PinX1 staining to those with low PinX1 staining (n = 236, follow-up time, 60 months). Our data revealed that low PinX1 staining correlated with both poorer overall and disease-specific patient survival (P = 0.010 and P = 0.003, respectively, log-rank test; Figure 1B & C). The 5-year overall cumulative survival rate dropped from 65.3% in patients with high PinX1 expression to 48.0% in those with low PinX1 expression, and the 5-year disease-specific cumulative survival rate dropped from 78.4% in patients with high PinX1 expression to 57.5% in those with low PinX1 expression.

Moreover, we examined whether PinX1 expression was an independent prognostic factor for breast cancer. The univariate Cox regression analyses revealed that PinX1 expression was an independent prognostic marker for breast cancer patients overall survival (hazard ratio, 0.573; 95% CI, 0.371-0.884; P = 0.012; Table  2 ) and disease-specific survival (hazard ratio, 0.417; 95% CI, 0.230-0.755; P = 0.004; Table  2 ). In multivariate Cox regression analysis, we found that PinX1 expression was also an independent prognostic marker for both 5-year overall survival (hazard ratio, 0.527; 95% CI, 0.404-0.656; P = 0.027; Table 3) and disease-specific survival (hazard ratio, 0.429; 95% CI, 0.222-0.630; P = 0.012; Table 3). Our results definitely confirmed that low PinX1 expression is associated with poor prognosis, suggesting that PinX1 may function as a prognostic marker for breast cancer.

Because low PinX1 expression is associated with poor prognosis, supporting PinX1 may play important roles in one or more steps of tumor metastasis. We investigated the involvement of PinX1 in breast cancer cells migration and invasion. We transiently transfected MDA-MB-231 and BT-549 cells with control siRNA and PinX1 siRNA or pEGFP-C3 and pEGFP-C3-PinX1 plasmids, respectively. Forty-eight or twenty-four hours after transfection, PinX1 protein was significantly knockdown or overexpressed in cancer cells, respectively (Figure 2A & B). In cell migration assay, we found that the ability of cell migration was drastically increased after PinX1 knockdown in both MDA-MB-231 and BT-549 cell line (Figure 2C). In contrast, overexpression of PinX1 inhibited cell migration (Figure 2D). Meanwhile, the results of the cell invasion assay corresponded with the cell migration assay (Figure 2E & F). However, overexpression or knockdown of PinX1 had no detectable effect on the proliferation of breast cancer cells under normal culture conditions (data not shown).

To investigate the mechanisms of PinX1 regulating migration and invasion in breast cancer cells, we performed western blot to detect the MMPs protein levels and gelatin zymography to observe the MMPs activity. Our result showed that the MMP-9 expression and activity were negatively regulated by PinX1 in MDA-MB-231 and BT-549 cells,but not MMP-2 (Figure 3A & B & C). So we supposed PinX1 suppress migration and invasion of breast cancer cells by regulating MMP-9 expression and activity. To further validate our assumption, we added MMP-9 inhibitor I (sc-311437, Santa Cruz) at the same time of PinX1 siRNA transfecting into cells. As expected, the upregulation of MMP-9 was blocked by MMP-9 inhibitor I (Figure 3D & E). We also validated this hypothesis by migration and invasion analysis, the migration and invasion ability can be enhanced by knocking down PinX1 in MDA-MB-231 and BT-549 cells, nevertheless, these regulations were blocked by MMP-9 inhibitor I (Figure 3F & G).

As we know TIMP-1 and TIMP-2 is the tissue inhibitor of MMP-9 and MMP-2, so we detected the expression of TIMP protein. Our data showed that MMP-9 expression was up-regulated or down-regulated corresponded with PinX1 knockdown or overexpression, however, the expression of TIMP-1 had not changed correspondingly. Moreover, neither overexpressing nor silencing PinX1 had any effect on the protein levels of MMP-2 and TIMP-2 (Figure 3A & B).

Furthermore, increasing evidence demonstrate that several MMPs (including MMP-9) expression and activation were regulated by NF-κB activation in many human cancers [27,28]. G-patch domain of PinX1 is also an important nucleic acids binding domain which could combine with the C-terminus of the NF-κB-repression factor (NRF) [29]. Thus, PinX1 may also inhibit the transcriptional activity of NF-κB proteins by direct protein-protein interaction with its G-patch domain. Western blot results showed that the level of NF-κB-p65 protein was dramatically increased in PinX1^KD-MDA-MB-231 cells and PinX1^KD-BT-549 cells. In contrast, NF-κB-p65 expression was down-regulated sharply in PinX1^OE-MDA-MB-231 cells and PinX1^OE-BT-549 cells (Figure 4A & B). To further confirm whether PinX1 regulated MMP-9 expression via the NF-κB signaling pathway in human breast cancer cells, we transfected NF-κB-p65 siRNA (Santa Cruz) into PinX1^KD-MDA-MB-231 cells and PinX1^KD-BT-549 cells. Our result indicated that the MMP-9 expression was up-regulated in PinX1^KD-MDA-MB-231 cells and PinX1^KD-BT-549 cells, but these effects were further blocked by silencing NF-κB-p65 with the specific siRNA (Figure 4A & B). These data provide definite evidence that PinX1 may modulate MMP-9 expression by NF-κB transcription factor.

We also validated the mechanism by migration and invasion analysis. The migration and invasion ability can be enhanced in PinX1^KD-MDA-MB-231 cells and PinX1^KD-BT-549 cells, however, these effects were dramatically reversed by treatment with NF-κB-p65 siRNA (Figure 4C & D). These data suggested that PinX1 may regulate the migration and invasion via the NF-κB/MMP-9 signaling pathway.

Lastly, we examined whether PinX1 suppressed breast cancer metastasis in vivo. PinX1^OE-MDA-MB-231, PinX1^KD-MDA-MB-231 and Ctrl-MDA-MB-231 cell lines were established previously. After 3 weeks selection following with lentivirus infection, the PinX1 protein levels of these cell lines were confirmed by western blot. Then continuing to incubate them without adding puromycin for 2 months, we determined that the PinX1 protein expression levels of three stable cell lines had not been changed (Figure 5A).

The BALB/c nude mice were randomly divided into three groups: PinX1^OE, PinX1^KD and Control group, each group consisted of 10 mice. Three groups of nude mice were injected through tail vein with PinX1^OE-MDA-MB-231, PinX1^KD-MDA-MB-231 and Ctrl-MDA-MB-231 cells respectively. After 2 months, three groups of mice were sacrificed and their lungs were resected and fixed in 10% buffered formalin for metastatic nodules counting and further histopathological analysis. Randomly selected metastatic nodules had been validated by H&E staining (Figure 5B). Extensive tumor formation was found in PinX1^KD group. In contrast, the lungs in PinX1^OE group had fewer and smaller detectable tumor nodules (Figure 5C). Compared with PinX1^OE group, the dramatic increase in the number of the tumor nodules was observed in PinX1^KD group, meanwhile, PinX1^OE group and PinX1^KD group had significant differences compared with Control group respectively (Figure 5D).