Accumulating evidence shows that Pin1 is upregulated in many human cancers including breast, prostate, lung, hepatic, cervical and colon cancer, and that increased expression of Pin1 is correlated with poor prognosis in prostate cancer (13
). Since ablation of Pin1 could prevent development of mammary carcinoma induced by oncogenic Neu or Ras in mice, this suggests an important role for Pin1 in cancer (43
). Others suggested that it plays a tumor suppressor role (25
). Indeed loss of Pin1 can reportedly lead to deregulation of cyclin E and c-Myc thereby increasing genomic instability, and is believed to sensitize cells to oncogenic transformation (44
). Here we provide evidence that Pin1 functionally associates with the oncogenic Rel subunits of NF-κB and that this interaction plays an important role in promoting the nuclear localization and the transcriptional, pro-proliferative and transforming properties of Rel proteins. We show that Pin1 markedly enhances the weak transforming activity of hc-Rel in primary lymphocytes. We also demonstrate that inhibition of Pin1 severely compromises proliferation of Rel/NF-κB dependent human lymphoma cells and is accompanied by suppression of Rel/NF-κB-dependent gene expression. These findings are consistent with accumulating evidence supporting an important role for Pin1 deregulation during tumorigenesis and the pro-proliferative capacity of tumor cells (46
), and emphasize an important role in Rel’s oncogenic activity. This may be particularly relevant since upregulation of Pin1 is seen in many human leukemia and lymphoma specimens in which Rel/NF-κB is known to be constitutively activated (Supplementary Fig. 1
While Pin1 failed to transform primary lymphocytes on its own, it significantly enhanced Rel’s transforming activity dependent on its ability to alter the dynamics of Rel protein nuclear import/export to tip the equilibrium in favor of increased nuclear accumulation. This most likely results from Pin1’s ability to induce proline isomerization, thereby preventing Rel inhibition by IκBα (12
).In this regard it is not surprising that Pin1 had a more dramatic effect in enhancing the transforming activity of the weakly transforming hc-Rel protein compared to the potently oncogenic v-Rel, since v-Rel is known to be significantly more resistant to inhibition by IκBα than c-Rel (47
). Upregulation of Pin1 may thus emerge as novel means to enhance the contribution of hc-Rel in human cancer by helping to dampen its negative feedback inhibition. Additionally since the 14-3-3 proteins can facilitate efficient nuclear export of IκBα-p65 complexes by binding to both RelA/p65 and IκBα, and 14-3-3 binds to RelA (amino acids 278–283) in close proximity of the Thr254-Pro recognition motif for Pin1 (48
), this raises the possibility that Pin1 might also preclude export of NF-κB/IκB complexes by interfering with NF-κB regulation by 14-3-3. In both scenarios, altered nucleo-cytoplasmic shuttling of Rel/NF-κB following upregulation Pin1 would contribute to sustained activation of Rel/NF-κB signaling and oncogenesis.
The N-terminal sequences that flank RelA’s Thr254-Pro motif are highly conserved among Rel/NF-κB family members, but greater variability is seen in those that flank its C-terminus. Although the kinase responsible for phosphorylation of Thr254 in RelA’s Thr-Pro motif has yet to be identified, efficient association of RelA with Pin1 is dependent on cell stimulation with cytokine TNFα (12 and right panel). In contrast, TNF had little effect on the interaction of v-Rel or hc-Rel with Pin1 (). This, together with the sequence divergence at the C-terminus of the Pin1 recognition motif in Rel/NF-κB proteins, suggests that different regulatory mechanisms may dictate interaction of Pin-1 with Rel and RelA proteins. Further studies will be needed to address this issue.
Our finding that Pin1 knockdown compromised the growth of Rel-dependent lymphoma cells and the expression Rel-dependent target genes points to Pin1 as a possible therapeutic target in these and other Rel-dependent tumors. However since pharmacological inhibition of Pin1 with juglone compromised both the growth and the survival of lymphoma cells, it appears that juglone shows some off-target effects. Indeed juglone has been shown to affect the activity of RNA polymerase II through Pin1 and to also prevent postmitotic protein dephosphorylation (49
), although its effects on the growth and survival of lymphoma cells was observed at a concentration 10- to 75-fold lower than those reported to inhibit the activity of RNA polymerase II (49
) or to block dephosphorylation of mitotic phosphoproteins (50
). Nevertheless our results with Pin1 shRNA revealed off-target effects for juglone, and suggest that more specific inhibitors of Pin1 will need to be identified for possible therapeutic application. Overall, our studies demonstrate that Pin1 plays a crucial role in promoting Rel-mediated transcription and transformation, and that inhibition of Pin1 can significantly compromise the proliferation of Rel-dependent lymphoma cells. Thus Pin1 may be an attractive therapeutic target in Rel/NF-κB-dependent leukemia/lymphomas.