Treatment of pancreatic and liver cancer cells that express mutant active RAS proteins and other membrane-proximal oncogenic signaling proteins with the geldanamycin HSP90 inhibitor 17AAG and the MEK1/2 inhibitor PD184352 resulted in greater than additive increases in tumor cell killing within 48h (). PD184352 and 17AAG were shown to synergistically interact to kill multiple cancer cell types in long-term colony formation assays (Table S1
). As MiaPaca2, PANC-1 and HEPG2 cells express mutated active K-RAS, K-RAS and N-RAS proteins, respectively, and we next examined whether other GI tumor cell types that express mutated active RAS proteins, or activated downstream effectors of RAS such as B-RAF and PI3K, also present with a greater than additive toxic interaction between 17AAG and MEK1/2 inhibitor.
PD184352 and 17AAG interact to kill multiple human GI tumor cell lines
HT-29 colon cancer cells express a mutated active B-RAF protein, but do not express a mutated RAS allele. SW620 and SW480 cells are patient matched colon cancer lines that express two alleles of mutated active K-RAS: SW620 has also been noted to be refractory to death receptor-induced lethality in part by lacking ceramide synthase 6 (LASS6) expression (33
). HCT116 cells express a single mutated active allele of K-RAS as well as a mutated active PI3K p110α protein (34
). In HCT116, SW480 and HT29 cells, PD184352 and 17AAG interacted in an additive to greater than additive manner to cause tumor cell death (). However, SW620 cells were refractory to drug-induced killing.
To determine the most important pathways downstream of activated RAS proteins in GI tumor cells that define 17AAG+PD184352 toxicity, we made use of HCT116 cells, and variants of HCT116 cells that are genetically deleted for K-RAS D13 and transfected with H-RAS V12 and with effector mutants of H-RAS V12 (27
). In short term viability assays, PD184352 and 17AAG interacted in a greater than additive manner to kill parental HCT116 cells (). Knock out of K-RAS D13 significantly suppressed drug toxicity and transfection of H-RAS V12 into these cells restored drug lethality. Of note, HCT116 cells lacking K-RAS D13 expression are non-tumorigenic (35
). Transfection of H-RAS V12 G37 into tumor cells lacking K-RAS D13; a RAS effector domain mutant that specifically activates the RAL-GDS pathway, profoundly enhanced the toxicity of 17AAG, and of 17AAG+MEK1/2 inhibitor treatment ( and Figure S1
Expression of H-RAS V12 G37 enhanced 17AAG- and 17AAG+PD184352 – induced activation of JNK1/2 and inactivation of ERK1/2 (, upper blots). Inhibition of JNK1/2 signaling suppressed the toxicity of 17AAG+PD184352 in cells expressing H-RAS V12 G37, but not in parental HCT116 cells (, lower graph). This is of particular note because in hepatoma cells inhibition of p38 MAPK and not that of JNK1/2 was responsible for drug combination lethality (27
Signaling by RAL-GDS downstream of H-RAS V12 promotes 17AAG toxicity
Expression of activated MEK1 significantly suppressed the toxicity of 17AAG and the MEK1/2 inhibitor PD98059 (that does not inhibit MEK1 EE) in both parental cells and in cells expressing H-RAS V12 G37 (). Knock down of CD95 in parental HCT116 cells blocked the potentiation of 17AAG lethality by PD184352 however knock down of CD95 in cells expressing H-RAS V12 G37 had no effect on the toxicity of the drug interaction (). In a similar manner to parental HCT116 cells, inhibition of CD95/extrinsic pathway signaling blocked 17AAG+PD184352 – induced cell killing in pancreatic and liver cancer cells, and in these cells the drug combination also caused CD95 activation (; Figure S2
). CD95 activation could be mediated by either FAS-L or by ligand independent processes e.g. increased ROS or ceramide levels (36
). Use of a neutralizing anti-FAS ligand antibody CD95 did not block either drug-induced CD95 activation or drug-induced cell killing ().
MEK1/2 inhibitor and 17AAG toxicity in GI cancer cells is mediated by CD95-caspase 8 signaling and does not require FAS-L
Prior studies from this group have demonstrated in hepatoma cells that agents which quench ROS block 17AAG + MEK1/2 inhibitor toxicity, though in those analyses we did not measure actual alterations in ROS generation (27
). Treatment of hepatoma cells with 17AAG weakly increased ROS levels that were significantly enhanced by inhibition of MEK1/2 (). As CD95 signaling was not responsible for enhanced morbidity of HCT116 cells expressing H-RAS V12 G37, we determined whether a differential induction of reactive oxygen species comparing parental and H-RAS V12 G37 HCT116 cells could explain the altered levels of cell survival after drug exposure and the CD95 –independent nature of drug-induced killing in cells expressing H-RAS V12 G37. As was noted in hepatoma cells, in parental HCT116 cells PD184352 rapidly enhanced 17AAG -induced ROS levels (). In cells expressing H-RAS V12 RAL 37, however, both 17AAG and 17AAG+PD184352 treatment enhanced ROS production, ~2.5-fold above that observed in parental cells, and in addition prolonged drug-induced ROS generation. Quenching of ROS production blocked CD95 activation and drug –induced cell killing (, data not shown). Molecular quenching of ROS production in cells expressing H-RAS V12 RAL 37, via over-expression of thioredoxin (TRX), suppressed drug-induced JNK pathway activation; JNK pathway signaling playing a key role in drug-induced killing (Figure S3
Mitochondrial-dependent generation of ROS plays a key role in CD95 activation
A significant source of ROS generation induced by this drug combination was dependent on functioning mitochondria, as judged by use of mitochondria deficient Rho zero HuH7 cells (). Similar data arguing for mitochondrial-dependent induction of ROS by this drug combination was also obtained in HEPG2 cells (Figure S4
). It is known that activation of CD95 can generate ROS via activation of NADPH oxidases; as HuH7 cells lack CD95 our data with 17AAG and PD184352 thus tend to support an NADPH oxidase independent form of ROS generation (37
). As previously stated, the small GTP-binding protein RAL is activated by RAL-GDS, one of the effector molecules for RAS. Active RAL binds to a GTPase activating protein for CDC42 and RAC, and the activity of NADPH oxidase is regulated by RAC. Thus of note was that while inhibition of p47phox
–dependent NADPH oxidase in parental HCT116 cells did not alter drug-induced ROS levels, knock down of p47phox
in HCT116 cells expressing H-RAS V12 RAL 37 significantly reduced the induction of ROS and the enhanced levels of tumor cell killing (Figure S5
). Thus enhanced drug toxicity in H-RAS V12 G37 cells was due to high levels of NADPH oxidase –dependent ROS generation.
In prior studies we have noted in a stimulus specific manner that ligand-independent activation of CD95 could be dependent on the actions of acidic sphingomyelinase and/or the de novo ceramide synthesis pathway (29
). Knock down of acidic sphingomyelinase expression or treatment with an inhibitor of the de novo ceramide synthesis pathway blocked 17AAG + PD184352 –induced CD95 activation (, upper IHC). Inhibition of acidic sphingomyelinase or the de novo ceramide synthesis pathway significantly reduced 17AAG + MEK1/2 inhibitor –induced ROS levels (, lower graph). SW620 cells, that lack LASS6 expression, were resistant to 17AAG + MEK1/2 inhibitor toxicity however stable expression of LASS6 in SW620 cells significantly enhanced the lethality of 17AAG and of 17AAG + MEK1/2 inhibitor treatment, and facilitated drug-induced CD95 activation ().
Ceramide-dependent generation of ROS plays a key role in CD95 activation
Treatment with 17AAG causes mitochondrial-dependent ROS generation and also promotes an unfolded protein response/endoplasmic reticulum stress. As Ca2+
homeostasis plays a central role in the functions of both organelles, we next investigated whether drug-induced ROS generation was dependent on changes in the levels of cytosolic Ca2+
. Knock down of acidic sphingomyelinase or inhibition of de novo ceramide synthesis in hepatoma cells did not alter the induction of cytosolic Ca2+
levels by 17AAG+PD184352 treatment (, left panel). However, quenching of cytosolic Ca2+
using Calbindin D28 suppressed drug-induced ROS (, right panel). In SW620 cells regardless of LASS6 expression 17AAG+PD184352 treatment increased cytosolic Ca2+
levels however in vector control transfected SW620 cells, lacking LASS6 expression, drug exposure only weakly increased ROS levels (Figures S6 and S7
). Expression of LASS6 restored drug-induced ROS generation in SW620 cells. Hence, 17AAG and MEK1/2 inhibitors increase cytosolic Ca2+
levels that activate ceramide synthetic/regulatory pathways which in turn lead to the generation of ROS. Increased Ceramide and ROS levels play an essential role in promoting mitochondrial dysfunction and CD95 activation.
In addition to linking ceramide in CD95 signaling, we have also shown in several prior studies that activation of CD95 increased autophagic vesicle formation, an event that was “protective” against CD95-induced apoptosis (29
). Treatment of hepatoma cells with 17AAG + MEK1/2 inhibitor enhanced autophagic vesicle formation 6h-24h after exposure that was blocked by knock down of ATG5 or CD95, or by expression of dominant negative PERK (). HuH7 hepatoma cells do not express CD95 and did not produce a significant autophagic response following 17AAG and MEK1/2 inhibitor exposure (). However, expression of CD95-YFP in HuH7 cells increased the number of drug-induced puncate LC3-GFP vesicles, and in agreement with CD95 mediating the toxic actions of this drug combination. Knock down of ATG5 or Beclin1 enhanced 17AAG+MEK1/2 inhibitor toxicity whereas expression of dominant negative PERK reduced drug lethality (). Similar data were obtained in ATG5 null SV40 transformed MEFs (Figure S8
). Deletion of PERK or expression of dominant negative eIF2α (S51A) in SV40 transformed MEFs suppressed the toxicity of 17AAG+MEK1/2 inhibitor exposure ().
MEK1/2 inhibitor + 17AAG treatment increases the levels of protective autophagic vesicles in a CD95-, PERK- and ATG5-dependent fashion
In addition to promoting phosphorylation of PERK and eIF2α, treatment with 17AAG+MEK1/2 inhibitor increased the levels of CHOP and of IRE1 without altering the levels of ATF6 and, surprisingly, decreased the levels of the ER resident HSP70 family chaperone GRP78/BiP (, upper inset panel). Over-expression of GRP78/BiP protected cells from drug-induced killing (Figure S9
). Over-expression of GRP78/BiP suppressed drug induced autophagy and CD95 activation, and drug-induced ROS generation (Figures S10 and S11
). Molecular quenching of Ca2+
signaling suppressed basal levels of GRP78/BiP but also inhibited drug-induced loss of GRP78/BiP levels (Figure S12
). In contrast, over-expression of GRP78/BiP suppressed drug-induced Ca2+
signaling (Figure S13
). Collectively these findings argue that the primary increase in Ca2+
levels following geldanamycin + MEK1/2 inhibitor exposure is responsible for loss of protective HSP70 family chaperone function facilitating ROS generation and CD95 activation.