The previous findings supported a pivotal role for p38 in the induction and/or maintenance of dormancy. The fact that a transient inhibition of p38 with SB203580, which causes a burst in ERK activation,22,49
or siRNA (our unpublished results) interrupts dormancy suggested that p38 activates a dominant program that maintains the phenotype. However, the downstream mechanism that p38 engages to maintain dormancy was poorly understood. A proteomic search for p38 regulated genes comparing tumorigenic versus dormant D-HEp3 cells in which p38 signaling was high or inhibited with SB20358064
revealed that in D-HEp3 cells p38 upregulated four endoplasmic reticulum (ER)-resident chaperones (BiP, ER60, HSP47 and cyclophilin B). Expression of these proteins is normally induced during adaptation to stress signaling caused by unfolding of proteins in the ER (), a response termed unfolded protein response (UPR).64
Figure 3 Regulation of ER-stress by p38 and tumor dormancy. (A) Simplified overview of pathways of the UPR relevant to this review. ER-stress induces dissociation of BiP from PERK and IRE1 and their subsequent activation via trans-phosphorylation. IRE1 activation (more ...)
In normal conditions, secreted proteins are folded in the ER and the folding “capacity” of this organelle depends on molecular chaperones that coordinate this process in steady-state conditions. However, several pathophysiological conditions can compromise the folding capacity of the ER resulting in protein aggregation and induction of signals that activate the UPR. For reviews see refs. 65–67. The UPR serves as a stress-induced checkpoint by concomitantly (1) inducing the expression of ER chaperones to cope with the misfolded proteins and (2) decreasing rate of protein synthesis to relieve the protein load in the ER. While ER stress forces the cells into growth arrest (G0
) to pause and restore homeostasis, chronic ER stress can result in apoptosis (). The ability of ER-stress to concomitantly induce survival and growth arrest prompted us to study its link to dormancy of HEp3 cells, because these are the two parameters that define the dormancy of these cells. A critical pathway in the UPR is the one regulated by protein kinase/RNase and RNA-dependent protein kinase-like ER kinase (PERK). PERK is maintained in an inactive state by the association of BiP with its lumenal domains. Upon protein unfolding, BiP dissociates from PERK and preferentially binds to the unfolded proteins resulting in PERK activation and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). This event leads to protein synthesis repression and induction of G0
arrest (). The UPR also induces GADD34, a regulatory subunit of the eIF2α phosphatase PP1 that relieves translation repression,68,69
allowing translation of the UPR response genes to favor proper protein folding and return to homeostasis.
Most of the published data (reviewed in refs. 25, 70-73
) suggested that p38 mainly functions in promoting cell cycle arrest and apoptosis. Although this might be true, p38 may also signal to promote cell survival.64,74-77
In our model it appears that the level of p38 signaling that is achieved is coupled to survival and not apoptosis. Mechanistic analysis revealed that in dormant HEp3 cells p38-dependent upregulation of BiP correlated with constitutive activation PERK and phosphorylation of eIF2α.64
Activation of PERK signaling during ER stress was shown to induce G0
arrest and cell survival,78
the two components of dormancy. BiP has also been shown to promote survival following ER stress79
as well as in response to DNA-damaging agents and other stresses.80,81
Thus, we further hypothesized that PERK and BiP may provide a survival advantage to D-HEp3 cells (). We found64
that p38 activation of PERK and upregulation of BiP in D-HEp3 cells renders them highly resistant to ER stress-inducing agents such as tunicamycin but also to TOPOII inhibitors such as doxorubicin. Further, the BiP-mediated resistance of D-HEp3 cells to doxorubicin treatment was also evidenced in vivo, where the cells are not dividing (our unpublished results). In addition, inhibition of PERK using a dominant negative PERKΔC-term mutant or overexpression of GADD34, increased the cell death of DHEp3 cells after etoposide treatments in vitro. Our results have identified two pathways that control survival of dormant, but not proliferative tumor cells. We have also determined that eIF2α phosphorylation by PERK, but not BiP expression, might be involved in the induction of growth arrest and dormancy in vivo (Ref. 64
and our unpublished results). This may be linked to the finding that PERK mediated growth arrest results from reduced translation of cyclin D1 ().82-84
The ability of cells to co-opt stress signaling pathways, such as the p38SAPK
and/or PERK to cope with stress, may be a trait that allows tumor cells to proliferate in adverse environments or to enter a state of dormancy that protects them from deleterious conditions. Translational repression is a theme in tumor cell adaptation to stress,85
as pathways that are inactivated upon nutritional stress, such as the AKT->mTOR pathway, or classical pathways that sense genotoxic damage such as p5386
will reduce CAP-dependent translation through the modulation of 4E-BP and eIF4E.
These findings may help to unravel an unexplored link between p38, translational control and the regulation of cell cycle and survival and may serve as a starting point to the understanding of cell cycle-independent mechanisms that make dormant metastasis resistant to chemotherapy. Further, that BiP inhibition renders dormant cells susceptible to TOPOII inhibition without interrupting dormancy provides a novel conceptual framework to design strategies to induce killing and to eradicate the residual dormant tumor cells after surgery and/or radio- or chemotherapy without interrupting their growth arrest.