Abstract
Most anticancer drugs fail to eradicate tumors, leading to the development of drug resistance and disease recurrence. The Hedgehog signaling plays a crucial role during embryonic development, but is also involved in cancer development, progression, and metastasis. The Hedgehog receptor Patched (Ptc) is a Hedgehog signaling target gene that is overexpressed in many cancer cells. Here, we show a link between Ptc and resistance to chemotherapy, and provide new insight into Ptc function. Ptc is cleared from the plasma membrane upon interaction with its ligand Hedgehog, or upon treatment of cells with the Hedgehog signaling antagonist cyclopamine. In both cases, after incubation of cells with doxorubicin, a chemotherapeutic agent that is used for the clinical management of recurrent cancers, we observed an inhibition of the efflux of doxorubicin from Hedgehog-responding fibroblasts, and an increase of doxorubicin accumulation in two different cancer cell lines that are known to express aberrant levels of Hedgehog signaling components. Using heterologous expression system, we stringently showed that the expression of human Ptc conferred resistance to growth inhibition by several drugs from which chemotherapeutic agents such as doxorubicin, methotrexate, temozolomide, and 5-fluorouracil. Resistance to doxorubicin correlated with Ptc function, as shown using mutations from Gorlin's syndrome patients in which the Ptc-mediated effect on Hedgehog signaling is lost. Our results show that Ptc is involved in drug efflux and multidrug resistance, and suggest that Ptc contributes to chemotherapy resistance of cancer cells. Mol Cancer Res; 10(11); 1496–508. ©2012 AACR.
This article is featured in Highlights of This Issue, p. 1401
Introduction
The Hedgehog (Hh) signaling pathway plays a crucial role in growth and patterning during embryonic development. In adults, recent studies suggest a role of this pathway in stem cell self-renewal and in the mobilization of endogenous stem cells for tissue repair and regeneration after injury and disease (1, 2). After auto-processing and lipid modification, the secreted fully active N-terminal Hedgehog domain (HhN) initiates signaling by binding to its receptor Patched (Ptc), which induces the internalization and the degradation of Ptc, and relieves the inhibition of the signal transducer Smoothened (Smo). Smo is then relocalized at the plasma membrane and activated. This triggers a cascade of downstream events that culminates in the activation or derepression of Hh pathway target genes such as Ptc and others involved in different developmental fate responses through the zinc finger transcription factors Gli. In the absence of Hh, the pathway is turned off because of the inhibition exerted by Ptc on Smo (3). Mutations in Hh signaling components have been identified in basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma, and aberrant activity of the pathway has been shown to be involved in the development of many other tumors (lung, esophagus, stomach, pancreas, biliary tract, breast, prostate, and brain; refs. 4 and 5). Many of these tumors contain cancer stem cells (CSC) that are thought to be responsible for tumor maintenance, progression, and relapse of the disease because of high expression level of Hh signaling components (4, 6) and of multidrug resistance (MDR) transporters (7). For instance, Hh signaling plays a crucial role in controlling the self-renewal behavior of malignant mammary cells (8), glioma stem cells (9), myeloid leukemia cells (10), or cancer stem-like cells in gastric cancer (11). Moreover, many studies have reported a correlation between Hh signaling and treatment resistance of cancer cells (4, 12, 13). Indeed, activated Hh signaling upregulates the expression of the MDR transporters MDR1 (ABCB1 or P-glycoprotein) and BCRP (ABCG2); (12–14). These MDR transporters are ATP-binding cassette (ABC) transporters that mediate the energy-dependent transport of drugs out of the cells, and are one of the best known mechanisms of drug resistance in cancer (15). Furthermore, the Hh pathway inhibitor HhAntag691 (GDC-0449), which has been used to treat medulloblastoma in animal models and has recently entered clinical trials for a variety of solid tumors, has been shown to inhibit the expression of these ABC transporters (4, 16). Sims-Mourtada and colleagues (12) reported that while inhibition of ABC transporter expression using MDR1 and BCRP siRNAs significantly reduced the effect of Hh pathway activation on chemotherapy resistance, cells treated with exogenous Hh ligand still had a survival advantage over control cells suggesting that these ABC transporters are not the sole factors involved in chemotherapy resistance.
We recently showed that the Hh receptor Ptc contributed to cholesterol efflux from cells, and we proposed that this activity could be responsible for the inhibition of the relocalization of Smo in the plasma membrane, which is necessary for Hh signaling activation (17). Ptc has the same topology and presents sequence similarities with the resistance-nodulation-division (RND) family of prokaryotic permeases, which transport a large variety of molecules, and with the Niemann-Pick C1 protein (NPC1), which functions as a cholesterol transporter but also as a multidrug permease, and is considered as the first mammalian member of the RND family (18, 19). Ptc being a Hh target gene, the Ptc protein is overexpressed in many cancers presenting aberrant Hh signaling. We therefore thought that Ptc could also function as a multidrug transporter.
In this report, we studied the involvement of Ptc in drugs efflux using heterologous expression of human Ptc in yeast and Xenopus oocytes, Hh-responsive fibroblasts, and 2 different cancer cell lines overexpressing Hh signaling components. Our results show that Ptc participates to drug efflux and MDR, and suggest that Ptc contributes to chemotherapy resistance of cancer cells.
Materials and Methods
Plasmid construction
pYEP-hPtcG509VD513Y-MAP and pSP64-hPtc-MAP were obtained from the construct pYEP-hPtc-MAP (20) as described in the supporting information, and were verified by sequencing.
Cell cultures
Leukemia K562 cell line (obtained from Dr. S. Brown) was cultured in RPMI 1640 medium supplemented with 5% FBS, 1 mmol/L sodium pyruvate, 2 mmol/L l-glutamine, 50 units/mL penicillin, and 50 μg/mL streptomycin. Human melanoma cell line MDA-MB-435 (obtained from C. Vandier, initially purchased from the ATCC) and NIH 3T3 mouse fibroblast cells (ATCC CRL-1658, kindly donated by F. Delaunay) were grown in DMEM medium (Invitrogen) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37°C in a 5% CO2/95% air water-saturated atmosphere.
K699 Saccharomyces cerevisiae yeast strain (Mata, ura3, and leu 2–3, kindly donated by R. Arkowitz) were transformed with pYEP-hPtc-MAP, pYEP-mMyo-MAP, or pYEP-hPtcG509VD513Y-MAP expression vector and grown as described (20) at 18°C until OD600 = 5–7.
Yeast growth inhibition by drugs
S. cerevisiae expressing wild-type human Ptc (hPtc WT), hPtcG509VD513Y, or mouse Myodulin (mMyo) were precultured to OD600 nm = 2, diluted to OD600 nm = 0.2 in rich medium supplemented or not with 4.5, 9, 13.5, or 18 μmol/L of doxorubicin (Sigma), 190 μmol/L of hygromycin B (Invitrogen), 330 μmol/L of methotrexate (Fluka), 700 μmol/L of temozolomide (Sigma), 243 μmol/L of Hoechst 33342 (H33342; Invitrogen), 25 μmol/L of acriflavine (Sigma), 400 μmol/L of 5-fluorouracil (Sigma) or 423 μmol/L of rhodamine 123 (Invitrogen), and grown at 18°C in 48-well plates (BD Biosciences). Absorbance at 600 nm was recorded during growth.
Purification of ShhN protein
The active amino-terminal domain of the murine Sonic Hedgehog protein corresponding to amino acids 25 to 198 (ShhN) was expressed, purified, and shown to activate the Hh pathway as described previously (20).
Oocyte injection
Isolation and cRNA injection of oocytes were performed as described previously (21). Briefly, SapI linearized pSP64-hPtc-MAP plasmid was transcribed by SP6 RNA polymerase (Ambion transcription kit). Female Xenopus laevis were anesthetized with MS222 according to the procedure recommended by our ethics committee. Stage V-VI oocytes were injected with 10 or 20 ng of hPtc cRNA. Injected oocytes were maintained at 18°C in Modified Barth's Saline (MBS pH 7.4: 85 mmol/L NaCl, 1 mmol/L KCl, 2.4 mmol/L NaHCO3, 0.82 mmol/L MgSO4, 0.33 mmol/L Ca(NO3)2, 0.41 mmol/L CaCl2, 10 mmol/L HEPES, 4.5 mmol/L NaOH, supplemented with 10 U/mL penicillin and 10 μg/mL streptomycin) for 3 days before the experiments were run.
Protein quantification
Protein concentrations were determined by the Bradford method using a Bio-Rad kit.
SDS-PAGE and Western blotting
Membrane or total extracts from mammalian cells or yeasts were prepared according to (17). Oocyte membranes were prepared from 30 oocytes according to (21). Samples were separated on 8% SDS-PAGE and transferred to nitrocellulose membranes (Amersham) using standard techniques. After 1 hour at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 450 mmol/L NaCl, 0.1% Tween-20, and 4% non-fat milk), nitrocellulose membranes were incubated overnight at 4°C with mouse monoclonal anti-HA antibodies (dilution 1:20; 20), or for 2 hours at room temperature with rabbit anti-Ptc antiserum (Ab130; 1:1000) or rabbit polyclonal anti-Smo antibodies (1:3000; generous gift from M. Ruat), or rabbit polyclonal anti-Gli1 antibodies (C68H3; Cell Signaling Technology), and washed twice in blocking buffer before incubation for 2 hours at 4°C with polyclonal anti-mouse (1:5000) or polyclonal anti-rabbit (1:3000) immunoglobulin coupled to horseradish peroxidase (Dako). Detection was carried out with an ECL kit (Millipore) and the Fusion FX7 system® (Vilber-Lourmat).
Doxorubicin incorporation measurements
For doxorubicin incorporation in MDA-MB-435 cells, a protocol was adapted from (22). MDA-MB-435 cells were seeded on coverslips coated with rat collagen type I in 6-well plates and allowed to grow to 80% confluence. Coverslips were incubated for 1 hour at 37°C with 10 μmol/L doxorubicin in the presence or the absence of 30 nmol/L ShhN, rinsed with phosphate buffer (pH 7.4), fixed for 10 minutes with 4% paraformaldehyde (Sigma), and observed by microscopy using a Nikon Ti Eclipse coupled with a EMCCD camera (Andor Ixon 897) equipped with a 40×/1.25 WI Apochromat Lambda-S objective. Doxorubicin fluorescence was observed through the following filter combinations: Ex = 482 ± 35 nm (FITC)/DM506 (FITC)/Em = 593 ± 40 nm (TRITC). Deconvolution was conducted using the classic maximum likelihood estimation (CMLE) method: Huygens Professional 3.5.1p2 on Windows 64b. For quantification, cell fluorescence was analyzed using Image J software on 15 to 20 cells of 2 different fields.
K562 cells were grown to 0.6 × 106 to 0.8 × 106 before addition of 10 μmol/L cyclopamine (CPN, Jentaur) or 10 μmol/L lovastatin (Sigma) for 24 hours. Cells were rinsed twice with physiologic buffer (140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgSO4, 5 mmol/L glucose, 20 mmol/L HEPES, pH 7.4), resuspended to OD600 nm = 1 with the same buffer supplemented or not with 30 nmol/L of ShhN, and added to spectrometer cuvettes containing 1 μmol/L doxorubicin in physiologic buffer ± 30 nmol/L ShhN to OD600 nm = 0.1. Time-dependent doxorubicin fluorescence quenching was recorded simultaneously in the different cuvettes at 25°C using a SAFAS spectrometer (ex = 470 ± 10 nm, em = 590 ± 10 nm).
Drug influx measurements
K562 cells were grown to 0.6 × 106 − 0.8 × 106 cells/mL before addition of 10 μmol/L CPN, 30 nmol/L SAG (Enzo Life Sci), or 10 μmol/L lovastatin for 24 hours, rinsed twice with physiologic buffer, resuspended with the same buffer supplemented or not with 30 nmol/L of ShhN or 5 μmol/L Verapamil (Sigma) for 30 minutes, and incubated with 10 μmol/L doxorubicin for 30 minutes at 25°C on a rotating wheel protected from light.
Yeasts expressing hPtc or mMyo were grown at 18°C until an OD600 of 7, washed with cold water, resuspended at an OD600 of 10 in Hepes–NaOH buffer (pH 7.0) supplemented or not with 5 mmol/L of glucose (Sigma) or 2-deoxy-d-glucose, and incubated with 10 μmol/L doxorubicin or 0.5 μmol/L acriflavine for 5, 15, 60, or 120 minutes at 25°C on a rotating wheel protected from light.
For K562 cells and yeasts, after pulse centrifugation, cells were resuspended in 200 μL of buffer and placed in 96-well plates. The doxorubicin or the acriflavine fluorescence contained in cells was measured using a plate reader (Fluostar, Labtech) (ex = 490 ± 10 nm and em = 610 ± 10 nm for doxorubicin, ex = 430 ± 10 nm and em = 505 ± 10 nm for acriflavine). Results were analyzed using the Student t test, in which significance is attained at P < 0.05.
Drug efflux measurements
NIH 3T3 cells were cultured in 24-well plates and loaded with 10 μmol/L of doxorubicin in culture medium for 2 hours at 37°C. Either 10 μmol/L of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or DMSO was added to the wells 15 minutes before the end of incubation as described (23). Cells were rinsed twice with physiologic buffer, and incubated with the same buffer supplemented or not with 30 nmol/L of ShhN for 30, 60, or 90 minutes at 37°C under shaking at 50 rpm, while protected from light. The cell supernatant was centrifuged for 5 minutes at 6800 × g, and the doxorubicin fluorescence in the supernatants was measured.
Control oocytes and oocytes injected with hPtc RNAs were incubated overnight in MBS with 100 μmol/L doxorubicin. Oocytes were then quickly rinsed 3 times in cold MBS and doxorubicin loaded into oocytes was measured. Oocytes were placed in 250 μL of fresh MBS at 18°C. After 90 minutes, the fluorescence contained in 200 μL of supernatant was measured. Each experiment was carried out with 3 to 4 groups of 10 control oocytes or hPtc expressing oocytes.
Drug efflux experiments with yeasts were adapted from (24). Yeasts expressing hPtc, hPtcG509VD513Y, or mMyo were washed with cold water, resuspended at an OD600 of 10 in Hepes–NaOH buffer (pH 7.0) supplemented with 5 mmol/L of Glucose or 2-deoxy-d-glucose, and incubated with 10 μmol/L doxorubicin or 0.5 μmol/L acriflavine for 2 hours at 25°C on a rotating wheel protected from light. Either 10 μmol/L of CCCP or DMSO was added to the medium 15 minutes before harvesting. Yeasts were then washed quickly, resuspended in the same buffer, and incubated at 25°C on the rotating wheel for 7 minutes and centrifuged for 1 minute at 18,000 × g. The doxorubicin or acriflavine contained in the supernatants was measured.
The doxorubicin and acriflavine fluorescence were measured in the plate reader (ex = 490 ± 10 nm/em = 610 ± 10 nm and ex = 430 ± 10 nm/em = 505 ± 10 nm for doxorubicin and acriflavine, respectively). Doxorubicin or acriflavine fluorescence contained in cells after loading was measured and variations in the doxorubicin loading were taken into account in efflux data presented. The loading variation factor between samples was calculated and fluorescence intensities measured in supernatants were divided by this factor. The results were analyzed using the Student t test in which significance is attained at P < 0.05.
Drug binding
Doxorubicin or acriflavine binding experiments were adapted from (24). One microgram of membranes prepared from several cultures of yeasts expressing hPtc, hPtcG509VD513Y, or mMyo as previously described (20) were deposited in 96-well plates. Five hundred nanomolars of doxorubicin or acriflavine were added to the wells and the fluorescence quenching was recorded in the plate reader and analyzed.
Neutral Red fluorimetric detection for high-sensitivity cytotoxicity evaluation
MDA MB-435 cells were seeded on 96-well microplates (NUNC) to achieve a 40% to 50% confluence for 48 hours culture growth. Medium was then removed and replaced with 100 μL medium containing or not 30 nmol/L ShhN for 1 hour before adding 100 μL more medium containing increasing concentrations of doxorubicin. After 24 hours, microplates were incubated 3 hours at 37°C with 200 μL/wells neutral red (NR) solution (50 μg/mL in DMEM). After a rapid wash with PBS at 4°C, microplates were gently tapped several times on absorbent paper. Cells were solubilized with 200 μL of a solution containing 1% acetic acid, 49% H2O, 50% ethanol by vortexing 5 minutes at 700 rpm. A total of 170 μL/well was transferred into a black 96-well plate for fluorescent measurements (ex = 544 nm/em = 590 nm; Optima BMG Labtek).
Results
Expression of human Ptc protein in Xenopus oocytes increases doxorubicin efflux
Oocytes injected with human Ptc (hPtc) RNAs (20 or 10 ng) which express the hPtc protein in their membrane (Fig. 1A), or with water, were incubated with the chemotherapeutic agent doxorubicin. After quick rinsing and incubation in buffer for 90 minutes, we collected the supernatants and measured their doxorubicin-related fluorescence intensity. After correction for doxorubicin charge variations, we observed that doxorubicin yields in supernatants of oocytes injected with 10 ng of hPtc RNAs were not significantly different from that of control oocytes (not shown), but were more than 2 times higher in supernatants of oocytes injected with 20 ng of hPtc RNAs (Fig. 1B). High Ptc expression level increased doxorubicin efflux out of injected oocytes.
Human Ptc expressed in Xenopus oocytes enhances doxorubicin efflux. A, membrane from 30 oocytes injected with water (lane 1), or with hPtc cRNA (20 ng [lane 2] and 10 ng [lane 3]) were immunoblotted with antibodies against Ptc. B, oocytes (10 per experiments) injected with water (control) or hPtc cRNA (20 ng) were incubated for 16 hours with 100 μmol/L doxorubicin before rinsing and incubation in buffer. After 90 minutes, the doxorubicin fluorescence of the supernatants was measured. Shown are mean ± SEM (P = 0.0002).
Expression of human Ptc protein in S. cerevisiae confers resistance to several drugs and increases doxorubicin and acriflavine efflux
We expressed hPtc in the plasma membrane of the yeast S. cerevisiae as previously described (20), and we examined the drug sensitivity of hPtc expressing yeasts in comparison to yeasts expressing the nonrelevant membrane protein Myodulin (Myo) (25) used as control (Fig. 2A). We observed that hPtc conferred a resistance to growth inhibition by doxorubicin, methotrexate, temozolomide, 5-fluorouracil, hygromycin B, acriflavine, and H33342 (Fig. 2B). These drugs are antracyclin antibiotic, antimetabolites, or alkylating agents used to treat a wide range of cancers, but also antibiotics, antiseptics, or fluorescent dyes (Supplementary Fig. S1). The resistance to growth inhibition by doxorubicin depended on the doxorubicin concentration added to the culture medium (Supplementary Fig. S2). After loading yeast with doxorubicin, rinsing, and resuspension in buffer, we directly measured the fluorescence of extruded doxorubicin in the supernatants. After correction for doxorubicin charge variations, dye fluorescence measured in hPtc-expressing yeasts supernatants was found to be significantly higher than in control yeasts supernatants (1.2 times; Fig. 3A). Treatment of yeasts with 2-deoxy-d-glucose to inhibit glycolysis and de-energize cells enhanced this effect to 1.45 times (Fig. 3A), suggesting that doxorubicin efflux increase observed in hPtc-expressing yeasts was energy-independent (24). Moreover, we observed that treatment of yeasts with a proton motive force (PMF) disrupter such as CCCP (23) inhibited the increase of doxorubicin content in hPtc-expressing yeasts supernatants, suggesting that the doxorubicin efflux enhancement related to hPtc expression required an intact PMF (Fig. 3A). In de-energized conditions, the expression of hPtc also increased acriflavine and H33342 efflux (1.33 and 1.2 times, respectively), but did not significantly modify rhodamine 123 efflux (Fig. 3B). These results are supported by drug accumulation experiments, which showed that doxorubicin and acriflavine accumulated more in de-energized yeasts because of the inhibition of ABC transporters (Supplementary Fig. S3). In these conditions, the expression of hPtc decreased doxorubicin, acriflavine, and H33342 accumulation by 0.5, 0.5, and 0.2 times respectively, and did not significantly modify rhodamine 123 accumulation. These results are in full agreement with those obtained for growth inhibition, and indicate that hPtc facilitates the efflux of doxorubicin, acriflavine, and H33342. We then prepared membrane extracts to measure the binding of doxorubicin and acriflavine, looking at the fluorescence quenching caused by the incorporation of the dyes in a hydrophobic environment (26). Membrane prepared from yeasts expressing hPtc caused a faster fluorescence quenching than membrane prepared from control yeasts, suggesting that doxorubicin and acriflavine bound to hPtc (Fig. 3C).
hPtc confers resistance to growth inhibition by doxorubicin. A, membranes from yeasts overexpressing Myo (control) or hPtc were immunoblotted with anti-HA antibodies. B, yeasts overexpressing Myo (control) or hPtc were grown in normal culture medium, or in medium supplemented with doxorubicin, hygromycin B, methotrexate, temozolomide, H33342, acriflavine, 5-fluorouracil, or rhodamine 123. Shown are mean ± SEM (n = 3).
hPtc expression in yeast enhances efflux and binding of doxorubicin. A, hPtc enhances doxorubicin efflux in an energy-independent way and by means of a proton motive force. Yeasts expressing Myo (control) or hPtc were incubated for 2 hours with 10 μmol/L doxorubicin in buffer supplemented with 5 mmol/L of glucose or with 5 mmol/L 2-deoxy-d-glucose, 10 μmol/L of CCCP (or DMSO) was added to the medium 15 minutes before harvesting. Yeasts were washed, resuspended in buffer, and centrifuged after 7 minutes. The doxorubicin fluorescence of the supernatants was measured. Shown are mean ± SEM (n = 8, **P < 0.005, ***P < 0.0005). B, hPtc expression enhances H33342 and acriflavine efflux. Same experiments as (A) were carried out incubating yeasts with 10 μmol/L rhodamine, H33342, or acriflavine in the presence of 5 mmol/L 2-deoxy-d-glucose. Data presented were corrected for dye loading variations and are mean ± SEM (n = 15, P < 0.0005). C, doxorubicin and acriflavine bind to hPtc. Membranes prepared from yeasts expressing Myo (control) or hPtc were added in 96-well plates containing 500 nmol/L doxorubicin or 500 nmol/L acriflavine, and time-dependent fluorescence quenching was measured. Shown are mean ± SEM (n = 3, *P < 0.05, **P < 0.005, ***P < 0.0005).
Ptc mutations inhibit doxorubicin resistance and efflux
Ptc possesses in its putative fourth transmembrane segment a motif GXXXD, which is highly conserved in Niemann-Pick disease protein (NPC1), and many bacterial transporters of the RND family such as the MDR protein from Pseudomonas aeroginosa MexB, the Escherichia coli MDR efflux pump AcrB, and the cation/proton antiporter involved in MDR in Gram-negative bacteria Ralstonia sp. CzcA (27) (Fig. 4A and B). We realized the double mutation hPtcVXXXY in which the glycine in position 509 was replaced by a valine and the aspartic acid in position 513 by a tyrosine (Fig. 4B). The yields of mutant proteins expressed in the yeast plasma membrane are comparable to the wild-type (WT) protein (Fig. 4C). Interestingly, yeasts expressing the mutant protein hPtcVXXXY transported significantly less doxorubicin (Fig. 4D) and showed less resistance to growth inhibition by doxorubicin (Fig. 4F) than yeasts expressing WT protein, but showed no significant difference in doxorubicin binding (Fig. 4E). These results strongly support a role of Ptc in doxorubicin efflux and resistance.
Ptc mutations inhibit yeast resistance to doxorubicin and doxorubicin efflux. A, schematic representation of the topologies of Ptc, the Niemann-Pick disease protein (NPC1), and the MDR protein from the Pseudomonas aeroginosa MexD showing a repeated domain composed of 6 transmembrane domains and a hydrophilic loop. B, the GXXXD motif of the putative transmembrane segment 4 is highly conserved in human and drosophila Ptc, in NPC1, and in bacterial transporters of the RND family such as MexB, AcrB, and CzcA. The glycine residue in position 509 and the aspartic acid residue in position 513 were replaced by a valine and a tyrosine, respectively, to produce the mutant protein hPtcG509VD513Y named hPtcVXXXY. C, the mutant protein hPtcVXXXY is fully expressed in yeast. Membranes were prepared from yeasts expressing hPtcWT or hPtcVXXXY, and immunoblotted with antibodies against HA or actin (loading control). D, mutations G509VD513Y inhibit doxorubicin efflux. Yeasts expressing Myo (control), hPtcWT, or hPtcVXXXY were incubated for 2 hours with 10 μmol/L doxorubicin, washed, resuspended in buffer, and centrifuged after 7 minutes. The fluorescence intensity of the supernatants was measured and corrected for loading differences. Shown are mean ± SEM (n = 15, ***P < 0.0005). E, mutations G509VD513Y do not significantly inhibit doxorubicin binding. Membranes prepared from yeasts expressing Myo (control), hPtc or hPtcVXXXY were added in 96-well plates containing 500 nmol/L doxorubicin, and time-dependent fluorescence quenching was measured. Shown are mean ± SEM (n = 10). F, mutations G509VD513Y inhibit resistance to doxorubicin. Yeasts expressing Myo (control), hPtcWT, or hPtcVXXXY were grown in normal culture medium or in medium supplemented with 9 or 18 μmol/L doxorubicin. The growth curves reported are representative of 3 independent experiments.
Modulation of Hh signaling affects doxorubicin efflux from fibroblasts
To investigate the involvement of Ptc in doxorubicin efflux in endogenous systems, we first used mouse fibroblasts NIH 3T3. These cells are highly responsive to the morphogene Sonic Hedgehog (Shh), and have been widely used to study the Hh pathway (3, 28–30). When added to the fibroblasts culture medium, purified N-terminal domain of Shh (ShhN) binds to its receptor Ptc and induces Ptc internalization and degradation. This releases Smo inhibition and activates Shh signaling (28, 31). As expected, we observed that the presence of purified ShhN in the culture medium decreased the amount of Ptc protein in membrane preparations to 44 ± 4% of Ptc signal obtained in the absence of ShhN (Fig. 5A). Fibroblasts were grown in 24-well plates, loaded with doxorubicin, rinsed, and incubated with physiologic buffer supplemented or not with purified ShhN. The doxorubicin fluorescence measured in the supernatants after 30, 60, and 90 minutes was significantly lower in wells containing ShhN compared with wells that did not contain ShhN (Fig. 5B). ShhN inhibited doxorubicin efflux to about two third of the efflux measured at 30 minutes. When cells were treated with CCCP, doxorubicin efflux was reduced to two third of the efflux measured in control, and ShhN treatment had no effect on doxorubicin efflux (Fig. 5C), suggesting that the doxorubicin efflux related to Ptc required intact PMF. Treatment of fibroblasts for 48 hours with the Hh signaling antagonist cyclopamine (CPN) reduced Ptc expression to 45 ± 12% (Fig. 5D). Indeed, the binding of CPN to the receptor Smo inhibits Hh signal transduction and represses the expression of Hh target genes such as Ptc (32). Fibroblasts grown in 24-well plates were treated or not with CPN before incubation with doxorubicin, rinsed, and incubated for 30 minutes in physiologic buffer. We observed that the doxorubicin fluorescence of the supernatants was significantly lower in wells containing cells treated with CPN compared with wells containing untreated cells (about two third of the control) indicating that treatment with CPN decreased doxorubicin efflux (Fig. 5E). We observed a time dependence of the effect of CPN pretreatment on doxorubicin efflux (Fig. 5F). These results show that modulating endogenous Ptc protein levels in fibroblasts affected doxorubicin efflux.
Modulation of Hh signaling affects doxorubicin efflux from fibroblast cells. A, ShhN decreases the level of Ptc protein. Enriched plasma membrane fractions from NIH 3T3 cells incubated or not for 3 hours with 30 nmol/L ShhN were immunoblotted with antibodies against Ptc. The Ptc signal was quantified using Image J software from 3 independent experiments. B, ShhN inhibits doxorubicin efflux from fibroblasts. NIH 3T3 were cultured in 24-well plates and incubated for 2 hours with 10 μmol/L doxorubicin, and rinsed. Physiologic buffer supplemented or not with 30 nmol/L ShhN was added to the wells, and the doxorubicin fluorescence of the supernatants was measured after 30, 60, and 90 minutes and corrected for loading differences. The mean ± SEM of at least 3 independent experiments are presented (*P < 0.05). C, the doxorubicin efflux mediated by Ptc depends on the proton gradient. Ten micromolars of CCCP (or DMSO) was added to the wells 15 minutes before the end of incubation with doxorubicin, and the doxorubicin fluorescence of the supernatants was measured 60 minutes after rinsing. Mean values ± SEM are presented (n = 4, **P < 0.005). D, treatment of fibroblasts with CPN inhibits Ptc protein expression. NIH 3T3 fibroblasts were treated or not for 48 hours with 10 μmol/L CPN before membrane preparation and immunoblotting. The signals of Ptc and loading control were quantified using Image J software. The histogram is the mean ± SEM of 3 independent experiments. E, CPN treatment decreases doxorubicin efflux. NIH 3T3 fibroblasts were cultured in 24-well plates, treated for 48 hours with 10 μmol/L CPN before incubation with 10 μmol/L doxorubicin for 2 hours, and rinsed. Physiologic buffer was added to the wells, and the doxorubicin fluorescence of the supernatants was measured after 30 minutes and corrected for loading differences. Mean values ± SEM are presented (n = 7, *P < 0.05). F, doxorubicin efflux inhibition increases with the duration of CPN treatment. NIH 3T3 fibroblasts were treated for 18, 36, and 48 hours with 10 μmol/L CPN before incubation with doxorubicin. Fluorescence intensity of the supernatants was measured 30 minutes after rinsing. Data were corrected for loading differences and mean ± SEM are presented.
Modulation of Hh signaling affects doxorubicin accumulation in 2 different cancer cell lines
MDA-MB-435 cells are derived from the M14 melanoma cell line and used to study cancer metastasis (33). These cells have been shown to overexpress the Hh target genes Ptc and Gli (34). Indeed, Western blots on MDA-MB-435 extracts revealed that Ptc was relatively highly expressed in these cells and that ShhN treatment for 3 hours reduced the levels of Ptc as it was already observed with the fibroblasts NIH 3T3 (Fig. 6A). MDA-MB-435 cells were incubated with doxorubicin in the presence or absence of ShhN, and were analyzed using cell imaging (Fig. 6B). The image analysis shows that the presence of ShhN significantly increased intracellular doxorubicin fluorescent intensity (1.25 times), suggesting that ShhN also inhibited doxorubicin efflux from melanoma cells. Neutral Red assays showed that MDA-MB-435 mortality induced by doxorubicin is dose-dependent and reached between 65% and 85% with 2.5 μmol/L doxorubicin (Fig. 6C left). Interestingly, the addition of ShhN 1 hour before doxorubicin significantly increased cell mortality (Fig. 6C, right), which is in good agreement with the doxorubicin accumulation enhancement observed in these cells in the presence of ShhN (Fig. 6B).
Hh signaling and doxorubicin accumulation in melanoma cells. A, Ptc is expressed in melanoma cells. Total protein extracts from MDA-MB-435 cells were immunoblotted. The signals of Ptc and loading control were quantified using Image J software. The histogram is the mean ± SEM of 3 independent experiments (***P < 0.0005). B, ShhN enhances doxorubicin accumulation in melanoma cells. MDA-MB-435 cells were incubated for 1 hour at 37°C with 10 μmol/L doxorubicin in the presence (right) or the absence (left) of 30 nmol/L ShhN, and observed by microscopy. Reflecting (top) and fluorescence (bottom) images are presented. The histogram presents the quantification of the fluorescence of 20 cells in 2 different fields analyzed using Image J software (P = 0.008). C, ShhN increases doxorubicin cytotoxicity. Neutral Red assays were carried out on cells incubated with increasing concentrations of doxorubicin (left). Effect of ShhN treatment on doxorubicin cytotoxicity is reported (right). Mean ± SEM are presented (n = 4 to 6; *P < 0.05, **P < 0.005).
Hh signaling has an important role also in hematopoietic stem cell self-renewal and in maintenance of cancer stem cells in leukemia (10, 13). Shh, Smo, and the Shh target genes Gli1 and Ptc are expressed in the myeloid line K562 (13, 35). Treatment of K562 cells for 3 hours with ShhN or for 48 hours with the Smo antagonist CPN reduced Ptc levels by about 30% or 50%, respectively (Fig. 7A). We also observed that CPN treatment reduced the expression of the Hh transcription factor Gli1 by a mean of 50% (Supplementary Fig. S4). We observed that leukemia cells pretreated with CPN accumulated significantly more doxorubicin than untreated cells, and in contrast, cells pretreated with the Smo agonist SAG accumulated significantly less doxorubicin than untreated cells. The presence of ShhN during doxorubicin incubation increased doxorubicin accumulation and reversed the effect of SAG. As expected, inhibition of multidrug transporters with verapamil increased dye accumulation in these cells (Fig. 7B). We confirmed these results by looking at the doxorubicin fluorescence quenching in solution, which is another way to measure doxorubicin incorporation in cells. Indeed, the intercalation of the chromophoric rings of doxorubicin between adjacent DNA base pairs has been shown to cause significant reduction in the fluorescence of the drug (26). We observed that the addition of K562 cells pretreated with CPN to a doxorubicin solution induced more fluorescence quenching than the addition of untreated cells. Furthermore, the presence of ShhN in the doxorubicin solution increased the fluorescence quenching of the drug (Fig. 7C). These results show that CPN and ShhN increased doxorubicin incorporation in K562, which is in full agreement with results obtained from accumulation experiments.
Modulation of Hh signaling modifies doxorubicin incorporation in leukemia cells. A, Ptc is expressed in K562 cells. Total protein extracts (200 μg) from K562 cells pretreated or not with 30 nmol/L ShhN (3 hours), 10 μmol/L CPN (48 hours) or 10 μg/mL lovastatin (24 hours) were immunoblotted. The signals of Ptc and loading control were quantified using Image J software. The histogram is the mean ± SEM of 3 independent experiments (***P < 0.0005). B, modulators of Hh signaling act on doxorubicin accumulation in K562 cells. K562 cells pretreated or not with the Hh signaling agonist SAG (30 nmol/L), the Hh signaling antagonist CPN (10 μmol/L), the ABC transporters inhibitor Verapamil (5 μmol/L) were incubated for 30 minutes in physiologic buffer containing 10 μmol/L doxorubicin supplemented or not with 30 nmol/L ShhN before rinsing and fluorescence measurement. Values reported are the mean ± SEM of 3 independent experiments. C, CPN pretreatment or ShhN presence increases doxorubicin incorporation in K562 cells. K562 cells pretreated or not with CPN (10 μmol/L) were added to spectrofluorimeter cuvettes containing 1 μmol/L doxorubicin in physiologic buffer supplemented or not with ShhN protein (30 nmol/L). Time-dependent doxorubicin fluorescence quenching was recorded simultaneously in the 3 cuvettes. The quenching slopes were calculated for 3 independent experiments and mean ± SEM were reported (*P < 0.05). D, lovastatin treatment increases doxorubicin incorporation in K562 cells. K562 cells pretreated or not with lovastatin (10 μg/mL) were added to spectrofluorimeter cuvettes containing 1 μmol/L doxorubicin in physiologic buffer. Time-dependent doxorubicin fluorescence quenching was recorded simultaneously in both cuvettes. The quenching slopes were calculated for 3 independent experiments and mean ± SEM were reported (*P < 0.05).
Martirossyan and colleagues showed that treatment of human ovarian carcinoma cells with lovastatin resulted in more intracellular doxorubicin accumulation and increased the drug efficacy (36). Remarkably, we observed that lovastatin treatment reduced the expression of the Shh target genes Ptc (Fig. 7A) and Gli1 (Supplementary Fig. S4), and enhanced doxorubicin incorporation (Fig. 7D) in leukemia cells, suggesting a link between Hh signaling and lovastatin effect on doxorubicin treatment of certain cancer cells.
Discussion
In this study, we show that the expression of the human Hh receptor Patched (hPtc) in Xenopus oocytes and in the yeast S. cerevisiae increased the efflux of doxorubicin, a chemotherapeutic agent used to treat recurrent cases of cancers (36). This doxorubicin efflux enhancement correlated with a decrease of doxorubicin accumulation in yeasts expressing hPtc, was energy-independent indicating that this effect was not because of ABC transporters, and was dependent of the proton motive force. These results are in full agreement with growth inhibition experiments, which showed that hPtc expression conferred a resistance of yeasts to doxorubicin, and strongly suggest that the Hh receptor Ptc is involved in doxorubicin efflux and resistance when expressed in the yeast S. cerevisiae.
To find out if Ptc also plays a role in drug efflux when endogenously expressed in cells, we studied doxorubicin efflux from the highly Shh-responsive mouse fibroblasts NIH 3T3. ShhN ligand addition during efflux measurements, or pretreatment of fibroblasts with the Hh signaling antagonist cyclopamine (CPN), significantly decreased the doxorubicin efflux from fibroblasts and the amount of Ptc protein at the fibroblast plasma membrane. Indeed, CPN inhibits the expression of the Hh signaling target genes such as Ptc, and ShhN binds to Ptc receptor and induces its internalization and degradation (28, 37–39). Therefore, the decrease of doxorubicin efflux observed in the presence of ShhN ligand or after treatment with CPN could be because of the clearance of Ptc from the fibroblast plasma membrane (Supplementary Fig. S3A and S3B). We also observed that ShhN presence or CPN treatment increased doxorubicin accumulation in 2 different cancer cell lines known to aberrantly express Hh signaling components (13, 34, 35, 40). Furthermore, Neutral Red assays showed that ShhN increased the cytotoxicity of doxorubicin on melanoma cells. The results obtained with mammalian cells expressing endogenous Ptc protein are in perfect agreements with results obtained with hPtc-expressing yeasts, and strengthen the hypothesis that Ptc protein participated to doxorubicin efflux and resistance. We observed that yeasts expressing hPtc protein carrying the double mutation G509VD513Y significantly inhibited resistance to doxorubicin and efflux without significantly affecting doxorubicin binding in comparison with yeasts expressing WT hPtc. These mutations, which are part of the 6 missense mutations of Ptc found in Gorlin's syndrome patients, were shown to inhibit Ptc suppression activity on the Hh pathway (27) and are localized at the motif GXXXD. This motif is highly conserved in the RND family of prokaryotic permeases and is associated with the transport activity of these proteins which efflux lipophilic drugs, detergents, bile salts, fatty acids, metal ions, and dyes from the cytosol of gram-negative bacteria (18, 41). Finally, we showed that the expression of hPtc in yeasts conferred resistance to other molecules used in chemotherapy such as methotrexate, temozolomide, or 5-fluorouracil, to antibiotics such as hygromycin B, to antiseptics such as acriflavine, and to dyes such as H33342, and that Ptc expression allowed yeasts to efflux the antiseptic acriflavine as efficiently as doxorubicin.
Taken together, our results show that Ptc participates to multidrug efflux, and suggest that Ptc contributes to chemotherapy resistance of cancer cells. However, as transport measurements using purified Ptc protein have not been done, we cannot conclude that Ptc is itself a multidrug transporter and the second eukaryotic member of the RND permease family with the Niemann-Pick C1 protein (42).
This study provides a new insight into the Hh receptor function, which is in good agreement with our recent findings showing that Ptc contributes to cholesterol efflux from cells (17). Indeed, NPC1 protein functions both as a cholesterol transporter and a multidrug permease (18, 19), and ABC transporters are key regulators of cellular cholesterol export from macrophages and other cell types (43). Sims-Mourtada and colleagues findings suggest that Hh signaling promotes chemotherapy resistance in part by enhancing expression of ABC transporters (12). Our results suggest that Ptc, which expression is also enhanced in many cancers, is another pathway by which Hh signaling induces drug resistance, and that the use of Ptc inhibitors could mitigate chemotherapy resistance of cancer cells (Supplementary Fig. S5A and S5B).
Several studies have reported that statins inhibits proliferation and cell division of cancer cells (44, 45), and clinical trials have shown that the addition of statins to traditional chemotherapeutic strategies can increase the efficacy of chemotherapy (46, 47). In particular, Martirosyan and colleagues (36) recently showed that lovastatin induces apoptosis of ovarian cancer cells and synergizes with doxorubicin, which is used in the treatment of primary and relapsed drug-resistant ovarian cancer. Moreover, lovastatin has been shown to bind directly to the P-glycoprotein and to decrease doxorubicin accumulation in MDR tumor cells (48). Statins such as lovastatin inhibit cholesterol biosynthesis (49), and, therefore, decrease cholesterol intracellular concentration. We recently showed that intracellular cholesterol decrease inhibits Hh signaling and Ptc expression (17). It probably also inhibits the expression of ABC transporters MDR1 and BCRP, which are Hh target genes such as Ptc (12, 14, 50). Our present results show that lovastatin treatment of leukemia cells enhanced the incorporation of doxorubicin and inhibited the expression of Ptc and Gli1 as did the Hh signaling inhibitor CPN. Therefore, the inhibition of the Hh signaling and of the expression of proteins involved in multidrug transport such as P-glycoprotein, MRP1, and Ptc could be another mechanism by which statins such as lovastatin increase chemotherapy efficiency (see scheme in Supplementary Fig. S5C and S5D). This study suggests that the use of Ptc inhibitors in combination with statins and chemotherapy should be a promising therapeutic strategy to overcome the resistance of cancer cells to chemotherapeutic treatment.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
This work was supported by grants from the CNRS, the Conseil Général des Alpes Maritimes and the foundation France Cancer 06. A. Tomico is supported by a CNRS grant.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Acknowledgments
The authors thank Drs Olivier Soriani, Franck Borgese, and Ludger Johannes for helpful discussions and critical reading of the manuscript. The authors also thank Maëlle Ogier and Sébastien Schaub for providing access to the PRISM microscopy platform.
Footnotes
Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).
- Received November 29, 2011.
- Revision received May 21, 2012.
- Accepted June 5, 2012.
- ©2012 American Association for Cancer Research.
References
Volume 10, Issue 11
