Prior in vitro
studies from our laboratories in chronic myelogenous leukemia (CML) cells have noted that inhibitors of MEK1/2 enhanced geldanamycin lethality by promoting mitochondrial dysfunction (28
). The present studies focused more precisely on defining the mechanism(s) by which these agents altered cell survival in hepatoma and pancreatic cancer cells in vitro.
Our findings demonstrated that combined exposure of tumor cells to 17AAG and MEK1/2 inhibitors (PD98059; PD184352; AZD6244) promoted inhibition of the ERK1/2 and AKT pathways and activation of the p38 MAPK pathway. The reduced activity within the ERK1/2 and AKT pathways lowered the cell death threshold of hepatoma cells at multiple points within the extrinsic and intrinsic apoptosis pathways as judged by suppressed protein levels of c-FLIP-s, BCL-XL and XIAP, whose reduced levels of expression could be rescued by molecular activation of AKT and MEK1. Drug-induced activation within the p38 MAPK pathway was a pro-apoptotic stimulus as judged by p38 MAPK-dependent: CD95 localization in the plasma membrane; CD95 association with pro-caspase 8; and activation of BAX and BAK. Loss of MEK1/2 and AKT pathway function reduced c-FLIP-s expression and in parallel facilitated activation of p38 MAPK. Without suppression of c-FLIP-s levels activation of CD95 was incapable of promoting caspase 8 activation/tumor cell killing, regardless of downstream BAX and BAK activation and inhibition of BCL-XL and XIAP expression. This argues that modulation of c-FLIP-s levels represented a key nodal point proximal to CD95 death receptor activation for the manifestation of 17AAG and MEK1/2 inhibitor toxicity in tumor cells (Figure S8
HSP90 antagonists, of which the ansamycin analogue geldanamycin and its less toxic derivatives, 17AAG and 17DMAG, represent the prototypes, have become a focus of considerable interest as anti-neoplastic agents, and clinical trials involving 17AAG and 17DMAG have been initiated over the last 5–10 years (e.g. 21). These agents act by disrupting the chaperone function of HSP90, leading to the ultimate proteasomal degradation of diverse signal transduction regulatory proteins implicated in the neoplastic cell survival, including Raf-1, B-Raf, AKT, and ERBB family receptors. Mutant active kinase proteins, including activated B-Raf and Bcr-Abl have been noted to be particularly susceptible to agents that disrupt HSP90 function (e.g. 20). The basis for the tumor cell selectivity of 17AAG is not definitively known however there is evidence that HSP90 derived from tumor cells has an increased affinity for geldanamycins compared with HSP90 protein obtained from normal cells (32
). One difficulty with the development of 17AAG has been the limited water solubility of this drug and an analogue of 17AAG, 17DMAG, which is considerably more water-soluble than 17AAG, has been synthesized. MEK1/2 inhibitors were previously shown to enhance the lethality of DMAG in CML cells and evidence from our present analyses indicates that PD184352 also enhances 17DMAG lethality in human hepatoma cells (28
Whilst some hepatoma tumors have been noted to express mutated active forms of Ras and B-Raf proteins, the penetrance of such mutations within the hepatoma patient population as a whole has not been noted to be as prevalent as the well described high mutational rate of these proteins found in other G.I. malignancies such as pancreatic adenocarcinoma or colorectal carcinoma (33
). Of note, however, is that 17AAG and MEK1/2 inhibitors interact to kill pancreatic carcinoma cells. Mutations in PI3 kinase and loss of PTEN function/expression in hepatoma have also been noted (35
). These findings would suggest that the lethal interaction of 17AAG with MEK1/2 inhibitors we observe in HuH7, HEPG2 and HEP3B hepatoma cells or in other unrelated epithelial tumor cell types is unlikely to be due to a simple suppression of a small subset of hyper-activated HSP90 client proteins as would be predicted based on expression of, for example, mutated active B-Raf or K-RAS. In contrast to pancreatic or colorectal malignancies, virally induced cancers e.g. by hepatitis B virus, the HEP3B cell line is an example, are more prevalent in liver cancers and the key transforming protein of HBV, pX, has been shown by many groups, including this laboratory, to increase the activities of the ERK1/2, AKT and JNK1/2 pathways and enhance the expression of cell cycle regulatory proteins such as p16, p21 and p27 in primary hepatocytes in a dose-dependent manner (37
). At present there are no published studies indicating whether pX is an HSP90 client protein. Based on the concept of oncogene addiction, however, hepatoma cells such as HEP3B expressing pX could in theory have higher basal levels of ERK1/2 and AKT activity which would in turn make them more susceptible to cell death processes following inhibition of these signal transduction pathways by 17AAG and MEK1/2 inhibitor exposure. Further studies will be required to determine definitively whether HBV infected hepatoma isolates are more sensitive to the 17AAG and MEK1/2 inhibitor drug combination than those lacking transforming HBV proteins.
The Raf-MEKl/2-ERKl/2 pathway exerts cytoprotective actions in a wide variety of transformed cell types which has lead to the development of multiple pharmacologic inhibitors of the pathway, including inhibitors of Ras farnesylation and geranylgeranylation, the multi-kinase and Raf inhibitor Sorafenib and the MEK1/2 inhibitors PD184352, PD0325901 and AZD6244 (40
). PD184352 has undergone clinical evaluation in phase I and phase II trials involving patients with advanced malignancies and inhibition of ERK1/2 phosphorylation in tumor tissues and peripheral blood mononuclear cells was observed at higher drug doses indicating that achieving desired pharmacodynamic effects in vivo
was feasible. However, the relative pharmacodynamic profile of PD1843 52 was not considered to be optimal and as a single agent the drug did not generate any objective tumor growth delay responses in a phase II trial (43
). More potent MEK1/2 inhibitors with superior pharmacokinetic characteristics (PD0325901, AZD6244) are currently undergoing clinical evaluation and encouragingly our present studies demonstrated that AZD6244 and 17AAG were competent to interact in a synergistic fashion to kill tumor cells via an extrinsic pathway-dependent mechanism. Studies beyond the scope of the present manuscript will be required to determine whether PD0325901 and AZD6244 can interact with DMAG in vitro and in vivo to kill human hepatoma and other carcinoma cell types.
We noted that administration of low concentrations of PD184352 or of 17AAG in hepatoma cells resulted in an initial abrogation of ERK1/2 phosphorylation, followed by a gradual recovery towards vehicle control treated levels. On the other hand, co-administration of PD184352 and 17AAG resulted in the profound and sustained dephosphorylation of ERK1/2 throughout the entire measured 24h exposure interval. Similarly, only under conditions of drug co-administration was a more modest AKT (S473) dephosphorylation observed. In view of evidence that the duration of MEK/ERK and AKT signaling plays a critical role in the biological consequences of activation of these pathways it is tempting to speculate that sustained inactivation of both ERK1/2 and AKT signaling partially contributes to the lethality of the PD184352 and 17AAG drug regimen in these cells.
The relative roles of ERK1/2 versus AKT inactivation in the promotion of cell killing by 17AAG and MEK1/2 inhibitor treatment were also noted to be slightly different comparing HEPG2 and HEP3B cells. In HEPG2 cells, expression of constitutively active MEK1 did not significantly protect cells from 17AAG and MEK1/2 inhibitor toxicity whereas expression of activated AKT reduced toxicity by ~50%. In HEPG2 cells expression of activated MEK1 in the presence of activated AKT, however, abolished 17AAG and MEK1/2 inhibitor toxicity. In HEP3B cells, both activated MEK1 and activated AKT each approximately equally contributed to suppressing cell killing induced by17AAG and MEK1/2 inhibitor exposure. There are many examples of this form of cell behavior where in some cell types survival is mediated primarily by the actions of one pathway with a secondary or non-existent protective role for other pathways, and in others where survival is shared between many pathways. In hepatocytes/hepatoma cells, the regulation of c-FLIP protein expression has been linked to both the ERK1/2 and AKT pathways (e.g. 11, 44). Thus in the majority of malignancies, based on tumor cell heterogeneity within the tumor, the likelihood that specific inhibition of only one signaling module will achieve a measurable prolonged therapeutic effect will probably be small, which may explain why even when ERK1/2 phosphorylation was significantly suppressed in patient tumors in the presence of PD184352, little benefit was clinically observed. As 17AAG will inhibit not only the ERK1/2 and AKT pathways, and in the presence of a MEK1/2 inhibitor act to cause prolonged suppression of pathway function, but will, furthermore, also reduce the stability of additional cytoprotective HSP90 client proteins such as HIE la, our data argue that the simultaneous targeting of multiple protective pathways by 17AAG and MEK1/2 inhibitors may represent a ubiquitous and better approach to kill cancer cells (45
). In a similar vein to reliance on one pathway for a major cellular effect, resistance to 17AAG and MEK1/2 inhibitor exposure could in theory be mediated by reduced expression levels of the death receptor CD95; indeed, HuH7 cells, which have very low expression of CD95 and were relatively resistant to drug exposure killing, compared to HEPG2 and HEP3B cells (46
Geldanamycins are known to have the capacity to generate reactive oxygen species in G.I. tumor cells (47
); prior studies from our laboratory have also shown 17AAG to induce ROS in primary hepatocytes and hepatoma cells (3
). Our data argued that ROS production was a key component in p38 MAPK activation after 17AAG and MEK1/2 inhibitor exposure, together with suppression of ERK1/2 and AKT activity. As AZD6244 has recently been shown to reduce hepatoma growth in vivo, collectively, with our present findings, including our in vivo data using HEP3B, and in Mia Paca2 cells (Unpublished findings), it is tempting to speculate that the 17AAG and MEK1/2 inhibitors could have in vivo potential as a therapeutic tool in the treatment of hepatoma and pancreatic cancer (49
). Additional studies of will be required to determine whether and how 17AAG and/or 17DMAG and MEK1/2 inhibitors interact in vivo to suppress tumor cell viability and growth.