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Tumor Biology |
Program in Molecular Biology [M. S., F. L.], Program in Cell Biology and Genetics [N. R.], and Department of Medicine [R. T., N. R.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ansamycins were originally isolated on the basis of their ability to revert v-src-transformed fibroblasts (17) . Subsequently, they were shown to have antiproliferative effects on cells transformed with a number of oncogenes, particularly those encoding tyrosine kinases (18) . Inhibition of cell growth is associated with apoptosis and, in certain cellular systems, with induction of differentiation (19) .5 These findings have led to the development of a GM derivative currently in Phase I clinical trials. The spectrum of ansamycin targets suggests that these drugs disrupt multiple key regulatory pathways. However, the induction of apparently complete reversion of the transformed phenotype in certain malignant cells (20) and the occasional profound differentiation of treated tumor cell lines (19) 5 made us question whether the effects of ansamycins should be attributed to global inhibition of Hsp90 housekeeping function. Instead, we considered whether occupancy of the pocket exposed a regulatory function of this chaperone.
The ansamycin-binding pocket in the NH2-terminus of Hsp90 is highly conserved and has weak homology to the ATP-binding site of DNA gyrase (1, 21) . This pocket has been shown to bind ATP and ADP with low affinity and to have weak ATPase activity (22, 23) . Studies on the effects of binding to the pocket have led to different conclusions regarding the effects of ansamycins on Hsp90 function. High concentrations of drug were shown to prevent the binding of Hsp90 to several of its protein partners (7, 10, 24) . However, in other systems, the drug has been shown to stabilize the Hsp90-refolded protein complex and prevent ATP-dependent release of the mature protein. The stable complex is subsequently degraded in the proteasome (25) . Occupancy of the pocket could then have two potential consequences: (a) it could result in global pharmacological inhibition of Hsp90 functions; and (b) it could determine whether Hsp90 protein partners are folded or degraded, depending on the nature of the ligand.
To address the possibility that the binding of ansamycins to Hsp90 regulates specific signaling pathways, we investigated the mechanism whereby they inhibit cellular proliferation. We found that HA effectively arrests the growth of a variety of tumor cell lines in G1 phase. G1 block is associated with accumulation of hypophosphorylated RB and loss of G1 cyclin-associated kinase activities. This inhibition involved a rapid decrease in the expression of cyclin D, cyclin E, cdk4, and cdk6. In addition, a late induction of p27Kip1 was also seen. However, the decline in cyclin D1 preceded other observed cell cycle effects, suggesting that the cyclin D-RB pathway is the primary target of the drug. Indeed, treatment of Rb-negative tumor cell lines with HA failed to induce G1 arrest, demonstrating that the effects of ansamycins on G1 are RB dependent. These results suggest that ansamycins selectively affect mitogenic signaling pathways upstream of RB, including those that control the levels of cyclin D. Furthermore, Hsp90 may play a role in regulating these pathways under certain physiologic conditions.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flow Cytometry.
Nuclei were isolated for flow cytometry assays as described previously
(13)
, stained with ethidium bromide, and analyzed using a
Becton Dickinson fluorescence-activated cell sorter. Statistical data
were obtained using Multicycle program software.
Western Blot Analysis.
Treated cells were harvested, washed with PBS, and lysed in NP40 lysis
buffer [50 mM Tris (pH 7.4), 1% NP40, 150 mM NaCl, 40 mM NaF, 1
mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin,
aprotinin, and soybean trypsin inhibitor] for 30 min on ice. Lysates
were centrifuged at 15,000 x g for 10 min to
pellet debris, and the protein concentration of the supernatant was
determined by bicinchoninic acid protein assay (Pierce).
Equal amounts of total protein were resolved by SDS-PAGE and transferred onto Immobilon polyvinylidene difluoride membranes (Millipore) by electroblotting. Blots were blocked overnight in 5% nonfat milk in TBS-T [0.1% Tween-20 TBS, 10 mM Tris (pH 7.4), and 150 mM NaCl] at 4°C and subsequently probed with antibody raised against the protein of interest. Anti-cyclin D1, cyclin D3, cyclin E, cyclin A, p27Kip1, cdk2, cdk4, and cdk6 antibodies were obtained from Santa Cruz Biotechnology and anti-RB antibodies purchased from Pharmingen. After incubation with horseradish peroxidase-conjugated secondary antibodies, proteins were detected using chemiluminescence (Amersham). All quantitations were performed using the Gel Doc 1000 and Molecular Analyst software from Bio-Rad.
Immune Complex in vitro Kinase Assays.
For cdk4- and cdk6-associated in vitro kinase assays,
treated cells were resuspended in lysis buffer [50 mM
HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5
mM EGTA, 1 mM DTT, 0.1% Tween-20, 10%
glycerol, 10 mM ß-glycerophosphate, 1 mM NaF,
0.1 mM Na3VO4, 0.2 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin and
aprotinin], sonicated and centrifuged at 15,000 x g
for 10 min. Anti-cdk4 or anti-cdk6 antibodies preincubated with protein
G-Sepharose (Pharmacia) were then incubated with 450 µg of total
lysate for 1 h at 4°C. Immune complexes were washed four times
with lysis buffer, two times with kinase buffer (250 mM
HEPES-KOH, 50 mM MgCl2, 5 mM DTT,
12.5 mM EGTA, 50 mM ß-glycerophosphate, 5
mM NaF, and 0.5 mM
Na3VO4) and incubated in 30 µl of kinase
buffer containing 0.2 µg of full-length GST-RB (QED Bioscience,
Inc.), 10 µCi [
-32P]ATP, and 300 µM
Li-ATP for 10 min at 30°C. The reaction was stopped by the addition
of SDS-PAGE sample buffer and boiled for 5 min. Proteins were resolved
on SDS-PAGE, transferred onto nitrocellulose membrane, and exposed to
autoradiography film.
For cyclin E in vitro kinase assay, 100 µg of NP40 lysate
were incubated for 1 h at 4°C with cyclin E antibodies
preincubated with protein A-Sepharose (Pharmacia). Immune complexes
were washed four times with lysis buffer, two times with kinase buffer
[20 mM Tris (pH 7.4), 7.5 mM MgCl2, and 1 mM
DTT] and incubated in 40 µl of kinase buffer containing 2 µg of
histone H1, 10 µCi of [-32P]ATP, and 300
µM Li-ATP for 10 min at 37°C. The reaction was stopped
by the addition of SDS-PAGE sample buffer and boiled for 5 min.
Proteins were resolved on SDS-PAGE, transferred onto Immobilon, and
exposed to autoradiography film.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This HA-induced G1 arrest was also observed in 10
additional cell lines tested, demonstrating the generality of the
effect (data not shown). An increase in cells with 4N DNA content was
also observed after drug treatment. This accumulation, however, was
lost by longer treatment
times6
and likely represents a lagging population.
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). Cyclin E-associated kinase activity was down-regulated later, declining
30% within 2 h after drug addition (Fig. 3B
). Thus, HA
appears to induce accumulation of hypophosphorylated RB by inhibiting
G1 cyclin-associated kinase activities.
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). Cyclin D3 and cyclin E protein expression were also down-regulated,
with lowered levels initially observed at 2 h after addition of HA
(Fig. 4A
). A reduction of 50% was seen for both cyclins D3
and E by 4 h. In contrast to its effects on the G1
cyclins, HA did not detectably inhibit expression of cyclin A (Fig. 4A
).
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). cdk4 protein levels were reduced by 50% in 6 h,
whereas cdk6 protein levels fell to 50% by 4 h. The total level
of cdk2 protein, however, remained unchanged, and instead, HA caused an
accumulation of the faster migrating, active form of the protein by
12 h after drug treatment (Fig. 4B
). Despite this
accumulation of the active form of cdk2, E/cdk2-associated kinase
activity is reduced, indicating that the cyclin E subunit is the
rate-limiting component.
Induction of cdk inhibitors has been shown to accompany cell cycle
arrest in response to various antiproliferative stimuli. As such, the
level of p27Kip1 was examined in HA-treated cells. An
initial increase in the expression of p27Kip1 was observed
by 24 h after HA treatment. Further induction of the protein was
detected when treatment was extended, with a 7-fold increase seen by
48 h (Fig. 4C
). p21Cip1 protein expression
was undetectable in Colo 205 cells by Western blot analysis. Together,
these results demonstrate that HA inhibits G1
cyclin-associated kinase activities by down-regulating expression of
the G1 cyclins and cdk4 and cdk6. Induction of
p27Kip1 occurs after the observed declines in kinase
activity and is likely a consequence and not a cause of kinase
inhibition and G1 arrest.
HA Fails to Induce G1 Arrest in Rb-negative Carcinoma
Cell Lines.
The observed loss of cyclin D1 protein occurred rapidly and preceded
that of cyclin E. To determine whether this loss is a primary cause or
result of G1 arrest, HA was added to Colo 205 cells
released from aphidicolin block. Cyclin D1 expression declined within
30 min, suggesting that HA down-regulates cyclin D1 independently of
G1 arrest (Fig. 4D
). Together, these findings
raise the possibility that the primary downstream target of HA is
cyclin D. Several studies have shown that cyclin D regulates
G1 progression by inducing the phosphorylation of RB
(27–33)
, and that, in the absence of RB, cyclin
D-associated kinase activity is dispensable (34–39)
.
Inhibition of cyclin E-associated kinase, however, has been shown to
cause G1 arrest independently of RB status
(40)
. Thus, if HA works by selectively inhibiting pathways
that affect cyclin D-associated kinase activity, it would not affect
G1 progression in cells lacking functional RB. In four such
cell lines, MB-MDA 468, BT-549, DU145, and DU4475, HA treatment
inhibited cell growth but failed to induce a G1 block and
instead, caused an accumulation of cells with 4N DNA content
(Fig. 5
& data not shown). This is not attributable to a general defect in
G1-S regulation in Rb-deficient cells because rapamycin
induced a G1 arrest in MDA 468 cells (Fig. 6)
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). In contrast, the Rb-negative MB-MDA 468 cells progressed through
G1, and the majority of cells were in S and
G2-M 24 h after release (Fig. 7B
). These
results demonstrate that HA induces G1 arrest in an
RB-dependent manner. Furthermore, these findings imply that the protein
targets of HA affect G1 progression solely by regulating
pathways upstream of RB.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The NH2-terminal of Hsp90 contains a highly conserved pocket that has homology to the DNA gyrase ATPase (1, 21) and binds ATP and ADP with low affinity (22, 23) . Several natural products derived from microorganisms, including the ansamycins and radicicol, have apparently evolved as high-affinity ligands for this pocket (1) . Occupancy of this pocket by these compounds prevents maturation of Hsp90 protein substrates and leads to their proteasome-dependent degradation (9, 10) . The function of these molecules in the microorganism is unknown, but they have been speculated to protect the host from other microorganisms.
In cancer cells, ansamycins induce the degradation of several proteins important for maintaining the transformed phenotype. These include ErbB family and insulin-like growth factor receptor tyrosine kinases (9, 13–16) , Raf (7, 11) , and the steroid receptors (4, 10, 12) . Treatment of various tumor cell lines with ansamycins results in growth inhibition and subsequently, in cell death. However, a drug that inhibits general chaperone housekeeping function and causes the destruction of so many key signaling proteins would be expected to cause rapid, extensive cytotoxicity. This is in apparent contradiction to the induction of differentiation in tumor cell lines and selective toxicity for tumor cells observed in animal models (44) .
In this study, we set out to examine whether ansamycins inhibit cell growth by disrupting specific signaling pathways or general chaperone function. We found that HA caused most tumor cell lines to undergo growth arrest in G1 phase of the cell cycle. This inhibition was accompanied by hypophosphorylation of RB and correlated with decreases in D-type cyclins and cyclin E-associated kinase activities. The mechanism of inhibition of G1 cyclin-associated kinase activities appeared multifactorial, consistent with disruption of multiple pathways responsible for mitogenic signaling. HA treatment was found to be accompanied by down-regulation of D-type cyclins, cdk4, cdk6, and cyclin E as well as a late induction in p27Kip1.
However, the decrease in cyclin D1 preceded that of other G1 regulators. In addition, loss of cyclin D1 was the cell cycle effect most consistently observed in cell lines tested. This decrease was not a result of G1 arrest, occurring with rapid kinetics even when HA was added to cells synchronized at the G1-S interface. Furthermore, down-regulation of cyclin D was also observed in Rb-negative cells that do not arrest in G1 in response to HA.6 These data, instead, suggest that ansamycins cause G1 arrest by selectively inhibiting pathways required for D-type cyclin-associated kinase activity. This proved to be the case. The most recognized substrate of D-type cyclin-associated kinases is RB (26–33, 45) . We show that when treated with HA, tumor cells that lack functional RB failed to arrest in G1. Furthermore, RB-negative cells synchronized with nocodazole were able to traverse G1 and progressed into S phase in the presence of ansamycins.
Two surprising conclusions are implied by these data. The results suggest that the signaling proteins targeted by HA, including steroid receptors, many transmembrane tyrosine kinases, and Raf, affect G1 progression solely by regulating pathways upstream of RB. Inhibition of cyclin E-associated kinase activity and other late G1 targets, as well as induction of p27Kip1, would be expected to cause G1 arrest irrespective of RB status (40, 46, 47) . Thus, rapamycin, lovastatin, and overexpression of p27Kip1 induce G1 block in cells lacking RB, in contrast to HA (46, 48) .
The data also imply that Hsp90 specifically regulates elements of pathways responsible for induction of cyclin D-associated kinase activity. One may speculate that under certain environmental conditions or stresses, occupancy of the Hsp90 pocket leads to degradation of these elements, hypophosphorylation of RB, and G1 arrest. Recently, certain stresses have been shown to cause a G1 block mediated by reduction in cyclin D expression (49, 50) . Moreover, the mitogenic effects of growth factor-receptor tyrosine kinases and estradiol activation of estrogen receptor have been shown to converge at the level of induction of cyclin D (51) . These findings together further suggest that the effects of multiple environmental stimuli are integrated at the level of cyclin D expression.
The reduction of cyclin D expression by ansamycins would seem sufficient to cause G1 block. However, it is not necessarily the only consequence of inhibition of these Hsp90-dependent pathways. Our data does not rule out the possibility that these pathways regulate other determinants of cyclin D-associated kinase activity, such as the expression of cdk4, cdk6, and members of the p16INK4a family. In fact, Hsp90 is involved in the maturation of cdk4 (42, 43) , and under certain conditions it may be a direct target of ansamycins. The inhibition of Hsp90 function, thus, may result in the coordinate down-regulation of several pathways required for cyclin D kinase activity. The reduction in cyclin D expression by ansamycins, nevertheless, appears to be one of their major modes of action. In previous work, we showed that the half-life of D-type cyclins is not affected by ansamycins, and therefore these proteins are unlikely to be direct targets of the drug (52) . Growth factor-stimulated translation of D-type cyclins has been shown to occur via a phosphatidylinositol-3-kinase/Akt pathway (52) . Ansamycins inhibit this pathway (52) , in part by degrading upstream transmembrane tyrosine kinases, and likely down-regulate D-type cyclin expression in this manner.
An ansamycin, 17-allylaminogeldanamycin, is currently in Phase I clinical trials in patients with advanced cancer. This drug may be most useful in cancers dependent on protein targets that are especially sensitive to its action, such as the HER2 tyrosine kinase in breast cancer and androgen receptor in prostate cancer. The data presented here suggest that a primary consequence of degradation of the targets is G1 arrest associated with decreased cyclin D expression and hypophosphorylation of RB. These may prove to be useful markers of drug effectiveness in patients whose tumors are accessible for analysis. Furthermore, G1 progression is unaffected by drug in tumors that lack wild-type RB. Instead, these cells accumulate in the G2-M phase of the cell cycle and undergo apoptosis. The mechanism of this phenomenon is under investigation, but it seems likely that the clinical response of tumors with wild-type RB will be fundamentally different from those that lack this protein.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by National
Cancer Institute Breast Specialized Program of Research Excellence
program Grant P50CA68425-02 (to N. R.). 
2 Present address: Memorial Sloan-Kettering Cancer
Center at St. Clare’s, 400 West Blackwell Street, Dover, NJ 07801.
3 To whom requests for reprints should be
addressed, Memorial Sloan-Kettering Cancer Center, at Box 271, 1275
York Ave., New York, NY 10021. Phone: (212) 639-2369; Fax: (212)
717-3627; E-mail: rosenn{at}mskcc.org
4 The abbreviations used are: HA, herbimycin A;
GM, geldanamycin; RB, retinoblastoma protein; cdk, cyclin-dependent
kinase; Hsp90, heat shock protein 90.
5 P. N. Munster and N. Rosen. Hsp90
regulation by ansamycins causes differentiation and apoptosis in
breast cancer cell lines, manuscript in preparation.
6 M. Srethapakdi and N. Rosen, unpublished
observations.
Received 10/22/99. Accepted 5/23/00.
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REFERENCES |
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