Pdcd4 suppresses colon carcinoma cell invasion by a novel mechanism. The suppression occurs at least in part through inhibition of MAP4K1 expression. Pdcd4 suppresses transformation and tumor phenotype by inhibiting c-Jun and c-Fos transactivation and, consequently, attenuating AP-1-dependent transcription (48
). The present study extends this mechanistic understanding to now implicate the JNK kinase activation pathway as a Pdcd4 target (Fig. ). Inhibition of JNK kinase activity by Pdcd4 is attributable to attenuated expression of MAP4K1, a kinase three steps upstream of JNK (Fig. and ). Because JNK is the major kinase responsible for activating phosphorylation of c-Jun, these findings provide a mechanistic explanation for the inhibition by Pdcd4 of c-Jun transactivation and consequently of the AP-1 transactivation important to tumor progression.
Pdcd4 decreases the level of endogenous phospho-c-Jun but not of c-Jun protein in colon carcinoma RKO cells (Fig. ). This contrasts with the observations of others (5
) who reported attenuated levels of ectopically introduced c-Jun protein in quail fibroblast QT6 cells expressing Pdcd4. It is possible that QT6 cells may have higher protease activity than RKO cells or that ectopic and endogenous c-Jun are expressed or activated differently.
Elevation of the Pdcd4 protein level in RKO cells is not sufficient to induce apoptosis or to alter the cell cycle (Fig. ). Moreover, transgenic expression of Pdcd4 in mouse epidermis does not induce apoptosis in vivo (22
). Although Pdcd4 was initially identified as being up-regulated in response to apoptosis inducers (40
), cytotoxic chemotherapeutics have been found to inhibit Pdcd4 expression (3
). Recently, Afonja et al. (1
) showed that transient transfection of Pdcd4 cDNA induces apoptosis in breast cancer T-47D and MCF-7 cells (1
). Our results dissociating Pdcd4 expression from apoptosis are in agreement with previous findings that overexpression of Pdcd4 cDNA does not induce apoptosis in NIH 3T3 cells (40
). A possible explanation for the discrepancies is that an additional factor present in breast cancer cell lines may be required for inducing apoptosis by Pdcd4, while this factor is deficient in RKO and NIH 3T3 cells as well as mouse epidermis.
Elevation of Pdcd4 protein in RKO cells inhibits Matrigel invasion, cell motility, and ECM protease activity (Fig. ). The suppression of RKO cell invasion by Pdcd4 appears to be attributable at least in part to inhibition of AP-1-dependent transcription (Fig. ). AP-1 is a transcription factor complex comprised of Jun-Jun homodimers or Jun-Fos heterodimers. The Jun protein family includes c-Jun, JunB, and JunD. The Fos protein family contains c-Fos, Fra-1, Fra-2, and FosB. AP-1 has been shown to regulate several events required for cell invasion, including expression of MMPs (4
) and cell motility (35
). Increased expression or activation of AP-1 component proteins enhances invasion (20
). Cells transformed by oncogenic forms of Fos or Jun proteins are invasive (20
). In addition, inhibition of AP-1 activity by dominant negative c-Jun, TAM67, suppresses invasive ability in keratinocytes (17
), fibroblasts (26
), and squamous carcinomas (49
) and blocks papilloma-to-carcinoma conversion (12
). The inhibition of AP-1-dependent transcription by Pdcd4 (Fig. ) is likely to contribute to suppression of RKO cell invasion (Fig. ). Transactivation of the AP-1 protein complex occurs principally through the MAPK cascade (38
). The MAPK signaling cascade includes three signal transduction pathways, i.e., JNK, ERK, and p38 signaling. JNK regulates the activation of c-Jun by phosphorylating it at Ser-63 and Ser-73. Suppression of JNK activity by blockade of the receptor for advanced glycation end products decreases tumor metastasis (43
). Moreover, the JNK inhibitor SP600125 represses c-Jun activation and type IV collagenase expression, required events for invasion. These findings indicate that suppression of JNK activity resulting in inhibition of AP-1 transactivation is an important mechanism for suppression of tumor cell invasion (42
The finding that Pdcd4 inhibits the expression of MAP4K1 mRNA and protein (Fig. ) indicates that MAP4K1 is a molecular target of Pdcd4. MAP4K1 is a mammalian STE20-like protein serine/threonine kinase which activates the JNK signaling pathway (31
). MAP4K1 activates JNK through the signaling pathway MAP4K1 → TAK1 → MKK4 → JNK (52
) and does not affect other MAPK signaling pathways, including ERK and p38 signaling (25
). Therefore, MAP4K1 specifically enhances AP-1-dependent transcription through activation of c-Jun (29
). Some reports suggest that MAP4K1 may inhibit AP-1 activity (30
). It is possible that, by binding to different associated proteins, MAP4K1 may become a positive or a negative regulator of AP-1-dependent transcription (6
). MAP4K1 has been shown to play a critical role in T-cell activation (28
). Although MAP4K1 is constitutively expressed in several tissues (21
), the function of MAP4K1 is still unclear. The observation that down-regulation of MAP4K1 suppresses invasive ability in RKO cells may suggest a motility-related function of MAP4K1 in normal tissues.
Previously, we have demonstrated that Pdcd4 is a binding partner of eIF4A that inhibits eIF4A's helicase activity (46
). Pdcd4 preferentially inhibits translation of mRNAs having secondary structure in the 5′UTR (45
). So-called “translationally repressed” mRNAs have been described which contain G- or C-rich regions in the 5′UTR with potential to form a stable secondary structure(s) in the 5′UTR (8
). Typically, the products of such “translationally repressed” mRNAs are transcription factors, growth factors, or proto-oncogenes (15
). The mechanism by which Pdcd4 inhibits transcription from the MAP4K1 promoter (Fig. ) may involve inhibiting translation of a transcription factor(s) or of a transcription factor activator(s) that is required for MAP4K1 transcription. Lankat-Buttgereit et al. (27
) have recently reported that overexpression of Pdcd4 cDNA in HEK293 cells decreases the protein but not the mRNA level of carbonic anhydrase, suggesting that carbonic anhydrase may be a candidate translational target of Pdcd4. Direct translational targets of Pdcd4 have not yet been demonstrated.
In conclusion, in addition to inhibiting tumor promoter-induced transformation and tumor phenotype (47
) as well as tumorigenesis (22
), Pdcd4 suppresses human tumor cell invasion. MAP4K1 appears to be a critical target of Pdcd4 when it inhibits invasion. Inhibited MAP4K1 expression leads to inhibition of JNK activation and consequently to inhibition of AP-1 activity and invasion. Elevating or mimicking Pdcd4 expression may be a promising therapeutic strategy.