022, respectively, Fisher’s exact test) (Fig. 7). There was, however, no association between SP1 and MMP2 expression using immunohistochemistry (P = 0.740). No association between the clinicopathologic features and SP1 and

MMP2 overexpression was found. Despite the fact that PTEN has been extensively studied and is implicated in cell migration,15-19 its underlying molecular mechanisms in HCC progression and metastasis have not been clearly TSA HDAC order elucidated. Therefore, it is of strategic importance to characterize the functional consequence of PTEN underexpression and the deregulated downstream signaling. In line with our clinical findings, the metastatic cell line H2M had a lower PTEN protein expression compared with its primary HCC counterpart cell line H2P.10 To study the functional consequences of PTEN loss in HCC, two HCC cell lines, SMMC-7721 and BEL-7402, with relatively higher endogenous PTEN levels were used to establish the stable PTEN-knockdown clones. ACP-196 concentration Because more than one stable knockdown clone was used, this likely eliminated clonal effect in the assays. We documented that knockdown of PTEN significantly promoted cell migration and invasion

in vitro, suggesting its possible role in HCC metastasis. Similar results were obtained with both cell lines, which suggests that the enhanced cell migration and invasion associated with loss of PTEN expression was not cell line–specific. In addition, our PTEN-deficient MEFs also exhibited increased cell migration and invasion compared with wild-type

MEFs. This indicates that the association of PTEN loss with metastasis is not cell type–specific. Overall, our in vitro results were consistent with our clinical observation that PTEN underexpression was significantly associated with more frequent tumor microsatellite formation in human HCCs. Tumor microsatellite formation in human HCCs is one of the histologic features of tumor metastasis. Furthermore, we observed that the PTEN−/− MEFs also possessed enhanced PIK3C2G proliferation rate (Supporting Fig. 2), and this was consistent with our finding of p-AKT activation, thereby triggering the prosurvival pathways. The enhanced cell proliferation is in agreement with our finding of an association of PTEN underexpression with increased tumor size in human HCCs. To eliminate the additive effect or cell proliferation in contribution to the enhanced cell migration and invasion observed, mitomycin C, a drug that blocks cell proliferation, was added in these assays. To move across the Matrigel layer in the cell invasion assay, cells need to secrete certain enzymes to degrade the extracellular matrix. Indeed, the Matrigel coated onto the invasion chamber in the cell invasion assay comprise three main substances: 56% laminin, 31% collagen IV, and 8% entactin, all by weight. MMP2 and MMP9 gelatinases primarily act to destroy the major constituent collagen IV.