Bortezomib inhibits the proteasome, a universal pathway essential for all cells. To improve therapeutic strategies it is crucial to understand the underlying mechanisms for its selective cytotoxicity against tumor cells. Here we used gene expression profiling to characterize the stress response of MCL cells during bortezomib treatment in vitro and in vivo. We discovered a prominent transcriptional response indicating activation of the UPR and oxidative stress pathways. Unexpectedly, genes encoding detoxifying and antioxidant molecules were strongly upregulated in sensitive but not in resistant cells. Contrary to our expectation we could not detect a reactive homeostatic response in resistant cells that might enable these cells to tolerate proteasome inhibition. However, we discovered that high baseline expression of NRF2 target genes in untreated cells correlated with drug resistance in vitro and in vivo. Thus, our data implicate high cellular antioxidant capacity as a mechanism of bortezomib resistance. In contrast, acute upregulation of the antioxidant response in sensitive cells was not sufficient to restore homeostasis. The strong reactive upregulation of NRF2 target genes then appears as a correlate of an overwhelming cytotoxic insult. However, we cannot rule out that under certain circumstances NRF2 may contribute to the induction of apoptosis. ATF4 that can activate antioxidant genes as well as pro-apoptotic mechanisms serves as an example of such a dual role.15,37
The BH3-only protein NOXA is induced in response to bortezomib in many cell types in vitro. Here, we show for the first time NOXA upregulation in primary tumor cells in MCL patients undergoing treatment with bortezomib. In keeping with its function as a terminal effector of bortezomib cytotoxicity,22,31
NOXA was upregulated only in cells that also demonstrated a stress response by gene expression profiling. While originally described as a p53 target gene, p53 is not necessary for NOXA induction in response to proteasome inhibition.22
Recently, Wang and colleagues demonstrated an alternative pathway of NOXA activation involving epigenetic modification of the promoter combined with transcriptional activation by ATF3 and ATF4 dimerization.15
As predicted by these studies, we found ATF3 and ATF4 upregulation in sensitive but not in resistant cells. Whereas ATF3 mRNA increased as part of the bortezomib-induced transcriptional response, ATF4 mRNA was largely unchanged, in keeping with its regulation through a translational mechanism downstream of PERK.17
For many cell types disruption of protein and ER homeostasis has been linked to induction of the UPR and bortezomib-induced cytotoxicity in vitro.18
This effect may be particularly important in secretory cells and has been proposed as an explanation for the high sensitivity of plasma cells to proteasome inhibition.4,14
In vitro we observed bortezomib-induced upregulation of ATF6, XBP1, and NRF2 gene signatures indicating activation of all three arms of the UPR. This was confirmed by the demonstration of IRE1 phosphorylation, XBP1 splicing, and increased protein levels of ATF4 in bortezomib-treated cell lines. In contrast, in vivo we observed primarily an NRF2 gene expression response and no activation of the IRE1/XBP1 arm of the UPR. Thus, while bortezomib induces both UPR and oxidative stress responses in vitro, our results suggest that in vivo oxidative stress may dominate, at least in some settings. Given that UPR and oxidative stress response are modulated by several cellular factors including biosynthetic load, amino acid starvation, and redox homeostasis,38
all of which can differ significantly between in vitro and in vivo environments, such differences are certainly plausible. It is also notable that proteasome inhibition with bortezomib consistently induces NOXA but not BIM,22,31
whereas the apoptotic response of the UPR in response to classic pharmacologic or pathophysiologic ER stressors has been associated with BIM upregulation.21
Taken together, these data indicate that bortezomib-induced cytotoxicity differs in several aspects from classic UPR-associated mechanisms.
We found a prominent antioxidant response in response to bortezomib both in vitro and in vivo. Oxidative stress is closely linked with ER stress. On one hand, ROS induce protein modifications that may lead to disruption of protein homeostasis and increased demands on protein degradation, on the other hand protein folding is one of the major sources of intracellular ROS.19,25
ER and oxidative stress also overlap in the activation of NRF2; ROS directly inactivate KEAP1, which binds and sequesters NRF2 in the cytoplasm, while PERK-mediated phosphorylation of NRF2 inhibits KEAP1 binding.37
Inhibition of KEAP1 binding stabilizes NRF2 and allows its translocation to the nucleus, where it regulates genes involved in detoxification, redox homeostasis, and proteome maintenance. NRF2 thereby promotes cellular homeostasis in the face of oxidative and ER stress. In vitro, bortezomib increased ROS levels significantly in sensitive cells, but only minimally in resistant cells. We were unable to show a bortezomib-dependent increase in ROS in primary tumor cells because fresh cells were not prospectively analyzed for ROS and the freeze–thaw process in DMSO interfered with the analysis. However, we detected increased NRF2 protein expression in tumor cells isolated directly from patients.
In cancer biology NRF2 has many at times even contradictory functions.39,40
NRF2 can protect cells from the carcinogenic effects of ROS. Natural compounds that increase NRF2 are therefore being studied as cancer preventing agents. On the other hand, high NRF2 expression is associated with more aggressive tumors, higher proliferation rate, and even resistance to chemotherapy. Here, we describe that high baseline activity of NRF2 in MCL cell lines as well as in primary tumors cell is associated with bortezomib resistance. In support of a protective role of NRF2 against bortezomib-induced cytotoxicity a genome wide RNAi screen in colon cancer identified knockdown of NRF2 as a synthetic lethal hit.41
NRF2 expression has also been linked with in vitro sensitivity to other chemotherapeutic agents in solid tumor cell lines.42,43
Whether treatment resistance is due to the overall enhanced ability of cells to deal with toxic insults or one distinct NRF2 target gene is undefined. While NRF2 upregulates proteasome subunits,44
we have previously shown that proteasome capacity and the degree of inhibition of the chymotrypsin-like activity by bortezomib does not differ between sensitive and resistant cells.31
The basis for increased NRF2 activity in a subset of MCL is unclear but could reflect differences in cellular differentiation. We recently reported that partial plasma cell differentiation of MCL cells is associated with bortezomib resistance.45
Given that increased expression of genes involved in protein folding and redox homeostasis are critical elements of plasma cell differentiation,19,25,46
this could explain an increased ability to cope with oxidative stress and account for reduced sensitivity to bortezomib.
This study is limited to the analysis of patients with leukemic disease supported by analysis of cell line models. Our findings thus await confirmation in nodal disease, and in a larger group of patients. Nevertheless, we have made several novel observations that direct further studies. The correlation of increased cellular antioxidant capacity with bortezomib resistance could yield predictive markers of treatment success and identifies avenues for synergistic treatment approaches. The relative contribution of NRF2 to bortezomib resistance, a possible proapoptotic role, and the mechanism of its upregulation in a subset of MCL require further study.