ABCMdb

PMID: 22472121

Basseville A, Tamaki A, Ierano C, Trostel S, Ward Y, Robey RW, Hegde RS, Bates SE
Histone deacetylase inhibitors influence chemotherapy transport by modulating expression and trafficking of a common polymorphic variant of the ABCG2 efflux transporter.
Cancer Res. 2012 Jul 15;72(14):3642-51. Epub 2012 Apr 3., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCG2 p.Gln141Lys As one of the most frequent variants in human ABCG2, the polymorphism Q141K impairs expression, localization, and function, thereby reducing drug clearance and increasing chemotherapy toxicity. Login to comment 3 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys As one of the most frequent variants in human ABCG2, the polymorphism Q141K impairs expression, localization, and function, thereby reducing drug clearance and increasing chemotherapy toxicity. Login to comment 4 ABCG2 p.Gln141Lys Mechanistic investigations revealed that the ABCG2 Q141K variant was fully processed but retained in the aggresome, a perinuclear structure, where misfolded proteins aggregate. Login to comment 7 ABCG2 p.Gln141Lys Together, these results showed how HDIs are able to restore wild-type functions of the common Q141K polymorphic isoform of ABCG2. Login to comment 8 ABCG2 p.Gln141Lys Together, these results showed how HDIs are able to restore wild-type functions of the common Q141K polymorphic isoform of ABCG2. Login to comment 16 ABCG2 p.Gln141Lys The most studied ABCG2 polymorphism is the nonsynonymous single-nucleotide polymorphism (SNP) C421A, which results in a glutamic acid to lysine substitution at amino acid 141 (Q141K), localized in the ATP-binding domain. Login to comment 17 ABCG2 p.Gln141Lys The most studied ABCG2 polymorphism is the nonsynonymous single-nucleotide polymorphism (SNP) C421A, which results in a glutamic acid to lysine substitution at amino acid 141 (Q141K), localized in the ATP-binding domain. Login to comment 18 ABCG2 p.Gln141Lys ABCG2 harboring Q141K has impaired protein expression, incomplete trafficking to the plasma membrane, and decreased function. Login to comment 19 ABCG2 p.Gln141Lys ABCG2 harboring Q141K has impaired protein expression, incomplete trafficking to the plasma membrane, and decreased function. Login to comment 20 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Clinically, the Q141K polymorphism has been linked to reduced clearance of some ABCG2 substrate drugs including diflomotecan, irinotecan, topotecan, gefitinib, rosuvastatin, atorvastatin, fluvastatin, simvastatin, and sulfasalazine (6). Login to comment 21 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys In addition to its impact on pharmacokinetics, the Q141K SNP has recently been linked to at least 10% of all gout cases, as the decreased ABCG2 function reduces urate elimination (7). Login to comment 22 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Because of the clinical significance of the Q141K polymorphism in anticancer drug pharmacokinetics and potential involvement in carcinogenesis, we studied processing and trafficking of the intracellular trapped variant protein and ways of rescuing Q141K ABCG2 to restore it to its wild-type (WT) phenotype. Login to comment 34 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys WT and Q141K ABCG2 sublines were created by transfection of the pcDNA5/FRT/V5-His-TOPO vector (Invitrogen) expressing WT or Q141K ABCG2 into Flp-In-293 cells, an HEK293 subline bearing a single integrated Flp Recombination Target site (Invitrogen), according to the manufacturer's instructions. Login to comment 35 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys WT and Q141K ABCG2 sublines were created by transfection of the pcDNA5/FRT/V5-His-TOPO vector (Invitrogen) expressing WT or Q141K ABCG2 into Flp-In-293 cells, an HEK293 subline bearing a single integrated Flp Recombination Target site (Invitrogen), according to the manufacturer's instructions. Login to comment 57 ABCG2 p.Gln141Lys Results Determination of Q141K ABCG2 trafficking Flp-In-293 cells were selected for this study, as one copy of the vector is known to insert at a unique specific site, resulting in comparable RNA levels. Login to comment 58 ABCG2 p.Gln141Lys To evaluate ABCG2 WT and Q141K variant localization in cells, immunofluorescent experiments were carried out. Login to comment 59 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Results Determination of Q141K ABCG2 trafficking Flp-In-293 cells were selected for this study, as one copy of the vector is known to insert at a unique specific site, resulting in comparable RNA levels. Login to comment 60 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys To evaluate ABCG2 WT and Q141K variant localization in cells, immunofluorescent experiments were carried out. Login to comment 61 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys As previously reported (15-17), we saw that Q141K ABCG2 proteins had incomplete trafficking and cytoplasmic retention, whereas WT proteins were mainly localized HDI Impact on the Expression and Trafficking of Q141K ABCG2 www.aacrjournals.org Cancer Res; 72(14) July 15, 2012 3643 American Association for Cancer ResearchCopyright (c) 2012 on October 25, 2012cancerres.aacrjournals.orgDownloaded from Published OnlineFirst April , in the cell membrane (Fig. 1A). Login to comment 62 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Also noted was staining of large aggregates of proteins, suggesting that the Q141K ABCG2 variant was accumulated in a localized cellular area. Login to comment 63 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Although Q141K and WT ABCG2 mRNA levels were equal, variant protein levels were 4-fold lower, suggesting that a posttranslational mechanism regulates expression (Fig. 1B). Login to comment 64 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Accordingly, the Q141K variant showed 5-fold decreased surface expression compared with WT. Login to comment 65 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys We hypothesized that Q141K variant underexpression was due to protein degradation. Login to comment 66 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys To explore whether Q141K ABCG2 was degraded by the ubiquitin-proteasome, the endosome-lysosome, or the autophagic pathways, we treated thecells with the proteasome inhibitor MG132; bafilomycin A1, which inhibits lysosomal degradation; and 3-methyladenine, which inhibits autophagosome formation. Login to comment 67 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Bafilomycin and MG132 treatment increased total protein expression (between 150% and 180%) for WT and Q141K protein (Fig. 1C). Login to comment 68 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys These data suggest that in normal processing, a small portion of WT and Q141K ABCG2 are degraded via the proteasomal pathway, whereas the fully processed proteins thatreach thesurface aredegraded by the lysosome, as observed for other transmembrane proteins (18). Login to comment 69 ABCG2 p.Gln141Lys Interestingly, the inhibition of the autophagic pathway induced a 3-fold increase in Q141K ABCG2 levels but barely affected WT protein expression. Login to comment 70 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys This indicates that autophagy is the main posttranslational mechanism, leading to a loss of Q141K variant expression. Login to comment 72 ABCG2 p.Gln141Lys Determination of Q141K ABCG2 localization, processing, and degradation pathways. Login to comment 79 ABCG2 p.Gln141Lys We also observed that a higher proportion of Q141K variant is ubiquitinated than in the WT protein (Supplementary Fig. S2). Login to comment 80 ABCG2 p.Gln141Lys We also observed that a higher proportion of Q141K variant is ubiquitinated than in the WT protein (Supplementary Fig. S2). Login to comment 81 ABCG2 p.Gln141Lys To examine the processing of Q141K ABCG2, whole-cell lysates from Flp-In-293 cells were deglycosylated with PNGase F and Endo H. Login to comment 82 ABCG2 p.Gln141Lys To examine the processing of Q141K ABCG2, whole-cell lysates from Flp-In-293 cells were deglycosylated with PNGase F and Endo H. ABCG2 contains a polysaccharide chain on asparagine 596 (19). Login to comment 84 ABCG2 p.Gln141Lys No change in molecular mass was observed after Endo H digestion (Fig. 1D), suggesting that both WT and Q141K ABCG2 were completely processed and not retained in the endoplasmic reticulum. Login to comment 87 ABCG2 p.Gln141Lys Results showed that Q141K was not retained in Golgi (Fig. 1E). Login to comment 88 ABCG2 p.Gln141Lys We then asked whether Q141K ABCG2 proteins were retained in aggresomes. Login to comment 91 ABCG2 p.Gln141Lys Thus, the major fraction of Q141K variant accumulated in the aggresomes. Login to comment 94 ABCG2 p.Gln141Lys The Q141K ABCG2 variant reached its mature form but was mainly sequestered in the aggresome and degraded via autophagy. Login to comment 98 ABCG2 p.Gln141Lys The effects of mitoxantrone were thus assayed in Q141K ABCG2 variant trafficking. Login to comment 100 ABCG2 p.Gln141Lys Treatment with 5 mmol/L mitoxantrone for 24 hours caused a small increase in WT and Q141K total ABCG2 expression (around 125% for mRNA and 120% for protein compared with untreated cells). Login to comment 101 ABCG2 p.Gln141Lys Surface expression showed a 160% induction in WT cells and a 250% induction in Q141K cells (Fig. 2A). Login to comment 103 ABCG2 p.Gln141Lys Q141K variant showed a marked homogeneous staining pattern, with loss of aggresome localization, whereas Flp-In-293 WT cells showed no change (Fig. 2B). Login to comment 104 ABCG2 p.Gln141Lys Finally, ABCG2 net efflux was determined by fluorescence-activated cell sorting (FACS) in Flp-In-293 WT and Q141K cells after mitoxantrone treatment (Fig. 2C). Login to comment 105 ABCG2 p.Gln141Lys Pretreatment with mitoxantrone for 24 hours caused a weak increase of substrate net efflux (113% and 128% for WT and Q141K ABCG2, respectively). Login to comment 112 ABCG2 p.Gln141Lys Total and surface protein expressions were similarly increased (210%-310% for WT ABCG2 and 260%-375% for Q141K ABCG2). Login to comment 116 ABCG2 p.Gln141Lys Again, basal plasma membrane localization of WT far exceeded that in Flp-In-293 Q141K cells, where ABCG2 was localized in aggresomes (Fig. 3B). Login to comment 117 ABCG2 p.Gln141Lys After exposure to HDIs, WT ABCG2 showed a minimal change in localization, whereas a dramatic change was seen for Q141K ABCG2. Login to comment 119 ABCG2 p.Gln141Lys In Fig. 3C, we observed in detail that in romidepsin-treated Flp-In-293 Q141K cells, the aggresome-localized ABCG2 had almost disappeared in favor of a strong surface localization. Login to comment 120 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2- specific efflux was then determined by FACS after HDI treatment in Flp-In-293 WT and Q141K cells. Login to comment 122 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys However, the change in transport ability following exposure to the HDIs in Flp-In-293 Q141K cells was notably higher: 200% for romidepsin, 163% for panobinostat, and 210% for vorinostat, compared with untreated Flp-In-293 Q141K cells (Fig. 3D). Login to comment 123 ABCG2 p.Gln141Lys Increased Q141K ABCG2 function was confirmed by a cell death assay using the ABCG2 substrate, pheophorbide A (Fig. 3E). Login to comment 124 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Indeed, Flp-In-293 Q141K cells pretreated for 24 hours with HDI Impact on the Expression and Trafficking of Q141K ABCG2 www.aacrjournals.org Cancer Res; 72(14) July 15, 2012 3645 American Association for Cancer ResearchCopyright (c) 2012 on October 25, 2012cancerres.aacrjournals.orgDownloaded from Published OnlineFirst April 3, romidepsin showed a decrease in celldeath after pheophorbide A treatment compared with cells not pretreated. Login to comment 126 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Altogether, these results indicate that romidepsin, vorinostat, and panobinostat induced an increase in WT and Q141K ABCG2 expression that was associated with an improved trafficking to the cell surface in the Q141K variant. Login to comment 127 ABCG2 p.Gln141Lys The increase also came with a gain of Q141K ABCG2 function, indicating that the surface variant was functional. Login to comment 128 ABCG2 p.Gln141Lys Transcriptional effects of HDIs on Q141K ABCG2 rescue Our goal was to determine the mechanism by which HDI treatment leads to improvement of ABCG2 mutant trafficking. Login to comment 132 ABCG2 p.Gln141Lys We then observed, in both Flp-In-293 WT and Q141K cells, that the mitoxantrone- and HDI-induced increases in surface expression were abolished by inhibition of protein synthesis (Fig. 4A), and the same results were obtained with the transcription inhibitor actinomycin D (Supplementary Fig. S5). Login to comment 135 ABCG2 p.Gln141Lys This suggests that mitoxantrone only modified the trafficking of the neosynthesized ABCG2 and would not act on Q141K ABCG2 already trapped in the aggresome. Login to comment 139 ABCG2 p.Gln141Lys We compared the data from mitoxantrone and HDI effect on Q141K ABCG2 localization and function. Login to comment 141 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys HDIs A Surface B C ProteinANRm WT Q141K %Comparedwithuntreatedcells %Comparedwithuntreatedcells WT Q141K Flp-ln-293 WT Flp-ln-293 Q141K WT Q141K WT Q141K * 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Compared with WT Compared with WT Compared with WT Ctl MX Ctl MX ABCG2 relative efflux ABCG2 expression * * 0 50 100 150 200 250 300 350 Ctl MX Figure 2. Login to comment 144 ABCG2 p.Gln141Lys Q141K mRNA, total protein, and surface protein expression was 93%, 28%, and 21%, respectively, of WT ABCG2. Login to comment 145 ABCG2 p.Gln141Lys B, ABCG2 was shown by confocal microscopy in Flp-In-293 WT and Q141K cells after 24-hour treatment with mitoxantrone. Login to comment 147 ABCG2 p.Gln141Lys Q141K relative efflux was 39.5% of WT ABCG2. Login to comment 148 ABCG2 p.Gln141Lys Ctl, control. Basseville et al. Cancer Res; 72(14) July 15, 2012 Cancer Research3646 induced mainly Q141K ABCG2 trafficking to the membrane, accompanied by a greater increase in protein function. Login to comment 149 ABCG2 p.Gln141Lys (c) 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst April 3, induced mainly Q141K ABCG2 trafficking to the membrane, accompanied by a greater increase in protein function. Login to comment 152 ABCG2 p.Gln141Lys Nontranscriptional mechanisms of HDIs inducing Q141K ABCG2 rescue Considering ABCG2 acetylation as a possible mechanism of rescue, we immunoprecipitated ABCG2 and immunoblotted for acetylated lysine but saw no effect of HDIs on the transporter acetylation state (data not shown). Login to comment 153 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys We observed that Q141K ABCG2 was retained in aggresomes. Login to comment 154 ABCG2 p.Gln141Lys We observed that Q141K ABCG2 was retained in aggresomes. Login to comment 160 ABCG2 p.Gln141Lys Impact of HDIs on WT and Q141K ABCG2 protein expression, localization, and function. Login to comment 161 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys A, ABCG2 mRNA, total and surface protein were, respectively, quantified by qPCR, immunoblotting, and FACS analysis in Flp-In-293 WT and Q141K cells following 24-hour exposure to 46 nmol/L romidepsin (RD), 100 nmol/L panobinostat (PN), 10 mmol/L vorinostat (VR), or 1 mmol/L valproic acid (VA). Login to comment 162 ABCG2 p.Gln141Lys A, ABCG2 mRNA, total and surface protein were, respectively, quantified by qPCR, immunoblotting, and FACS analysis in Flp-In-293 WT and Q141K cells following 24-hour exposure to 46 nmol/L romidepsin (RD), 100 nmol/L panobinostat (PN), 10 mmol/L vorinostat (VR), or 1 mmol/L valproic acid (VA). Login to comment 163 ABCG2 p.Gln141Lys C, ABCG2 and g-tubulin were observed in Q141K cells after 24-hour RD treatment (magnified example). Login to comment 164 ABCG2 p.Gln141Lys C, ABCG2 and g-tubulin were observed in Q141K cells after 24-hour RD treatment (magnified example). Login to comment 166 ABCG2 p.Gln141Lys E, cells were pretreated or not with RD dilutions for 24 hours, then treated for 24 hours with pheophorbide A (pheo A; 1 mmol/L for WT and 0.5 mmol/L for Q141K cells), and cell death was measured by flow cytometry. Login to comment 167 ABCG2 p.Gln141Lys E, cells were pretreated or not with RD dilutions for 24 hours, then treated for 24 hours with pheophorbide A (pheo A; 1 mmol/L for WT and 0.5 mmol/L for Q141K cells), and cell death was measured by flow cytometry. Login to comment 168 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Ctl RD PN VR VA Flp-In-293 WT Flp-In-293 Q141K ABCG2/γ-tub/nucleus in RD-treated Q141K cells %Comparedwithuntreatedcells %Comparedwithuntreatedcells Protein SurfaceAN A Rm C B WT Q141K Ctl RD PN VR VA D ABCG2 relative efflux 0 50 100 150 200 250 300 350 400 450 500 0 50 100 150 200 250 300 350 400 450 500 0 50 100 150 200 250 300 350 400 450 500 WT Q141K WT Q141K WT Q141K E Pheo A-induced cell death %Celldeathcomparedwith pheophorbideAuntreatedcells ** compared with WT compared with WT compared with WT * ** * ** * ** * ** * ** * ** * ** * ** * ** * 0 100 200 300 400 500 600 700 WT Q141K Ctl RD PN VR VA not pretreat. 24-h RD pretreat.: 9 nmol/L 18 nmol/L 46 nmol/L 0 50 100 150 200 250 300 350 400 450 HDI Impact on the Expression and Trafficking of Q141K ABCG2 www.aacrjournals.org Cancer Res; 72(14) July 15, 2012 3647 American Association for Cancer ResearchCopyright (c) 2012 on October 25, 2012cancerres.aacrjournals.orgDownloaded from Published OnlineFirst April 3, We next examined the role of microtubules, known to be involved in retrograde transport, in ABCG2 localization. Login to comment 169 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Ctl RD PN VR VA Flp-In-293 WT Flp-In-293 Q141K ABCG2/b3;-tub/nucleus in RD-treated Q141K cells % Compared with untreated cells % Compared with untreated cells Protein Surface A N A R m C B WT Q141K Ctl RD PN VR VA D ABCG2 relative efflux 0 50 100 150 200 250 300 350 400 450 500 0 50 100 150 200 250 300 350 400 450 500 0 50 100 150 200 250 300 350 400 450 500 WT Q141K WT Q141K WT Q141K E Pheo A-induced cell death % Cell death compared with pheophorbide A untreated cells * * compared with WT compared with WT compared with WT * ** * ** * ** * ** * ** * ** * ** * ** * ** * 0 100 200 300 400 500 600 700 WT Q141K Ctl RD PN VR VA not pretreat. 24-h RD pretreat.: 9 nmol/L 18 nmol/L 46 nmol/L 0 50 100 150 200 250 300 350 400 450 HDI Impact on the Expression and Trafficking of Q141K ABCG2 www.aacrjournals.org Cancer Res; 72(14) July 15, 2012 3647 We next examined the role of microtubules, known to be involved in retrograde transport, in ABCG2 localization. Login to comment 175 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys To evaluate HDAC6 involvement in Q141K ABCG2 rescue, we treated Flp-In-293 Q141K cells with the HDAC6 inhibitor tubastatin (28). Login to comment 176 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys To evaluate HDAC6 involvement in Q141K ABCG2 rescue, we treated Flp-In-293 Q141K cells with the HDAC6 inhibitor tubastatin (28). Login to comment 184 ABCG2 p.Gln141Lys To confirm these data, we conducted an siRNA assay to inhibit the expression of dynamitin, and we assessed the effect of HDIs on ABCG2 Q141K rescue after dynamitin knockdown. Login to comment 185 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys As shown in Fig. 5F, dynamitin knockdown blunted the effect of HDIs on Q141K ABCG2 rescue with a significant effect for romidepsin. Login to comment 186 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Discussion We saw that the Q141K variant was retained in aggresomes and then degraded by the autophagic pathway. Login to comment 187 ABCG2 p.Gln141Lys Discussion We saw that the Q141K variant was retained in aggresomes and then degraded by the autophagic pathway. Login to comment 189 ABCG2 p.Gln141Lys Aggresome formation facilitates the capture of aggregated proteins by the autophagic pathway (30-33), which we observed to be the case for Q141K ABCG2. Login to comment 190 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys A previous study had shown in the same Flp-In-293 cell model that Q141K ABCG2 was subjected to ubiquitin-mediated proteasomal and lysosomal degradation, with a 2-fold increase in expression after treatment with both inhibitors (17). Login to comment 191 ABCG2 p.Gln141Lys A previous study had shown in the same Flp-In-293 cell model that Q141K ABCG2 was subjected to ubiquitin-mediated proteasomal and lysosomal degradation, with a 2-fold increase in expression after treatment with both inhibitors (17). Login to comment 193 ABCG2 p.Gln141Lys It is interesting to note that the expressed WT and Q141K ABCG2 variants are found in a fully mature form, contrary to what has been observed with other ABC transporters. Login to comment 194 ABCG2 p.Gln141Lys It is interesting to note that the expressed WT and Q141K ABCG2 variants are found in a fully mature form, contrary to what has been observed with other ABC transporters. Login to comment 196 ABCG2 p.Gln141Lys We sought to rescue Q141K ABCG2 trafficking using HDIs. Login to comment 197 ABCG2 p.Gln141Lys We sought to rescue Q141K ABCG2 trafficking using HDIs. Login to comment 198 ABCG2 p.Gln141Lys Transcriptional effects of HDIs on ABCG2 Q141K rescue. Login to comment 199 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys A, quantification of ABCG2 surface expression by FACS in Flp-In-293 WT and Q141K cells after 24-hour exposure to mitoxantrone (MX) and HDIs in presence or absence of cycloheximide (CHX). Login to comment 200 ABCG2 p.Gln141Lys A, quantification of ABCG2 surface expression by FACS in Flp-In-293 WT and Q141K cells after 24-hour exposure to mitoxantrone (MX) and HDIs in presence or absence of cycloheximide (CHX). Login to comment 202 ABCG2 p.Gln141Lys Basseville et al. Cancer Res; 72(14) July 15, 2012 Cancer Research3648 expression with a conserved ratio for WT as for Q141K variant. Login to comment 203 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys More importantly, HDIs caused a strong relocalization of the Q141K variant from aggresome to cell surface and increased Q141K-mediated efflux. Login to comment 204 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys We sought to understand why these HDI-induced Q141K proteins, instead of accumulating in the aggresomes, were able to traffic to the cell surface. Login to comment 205 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys More importantly, HDIs caused a strong relocalization of the Q141K variant from aggresome to cell surface and increased Q141K-mediated efflux. Login to comment 206 ABCG2 p.Gln141Lys We sought to understand why these HDI-induced Q141K proteins, instead of accumulating in the aggresomes, were able to traffic to the cell surface. Login to comment 215 ABCG2 p.Gln141Lys We observed that colchicine was also able to rescue Q141K ABCG2 trafficking. Login to comment 217 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys We deduced that HDIs similarly inhibit Q141K retrograde transport toward the aggresome, likely via dynamitin overexpression. Login to comment 219 ABCG2 p.Gln141Lys We deduced that HDIs similarly inhibit Q141K retrograde transport toward the aggresome, likely via dynamitin overexpression. Login to comment 220 ABCG2 p.Gln141Lys Nontranscriptional effects of HDIs on ABCG2 Q141K rescue. Login to comment 221 ABCG2 p.Gln141Lys A, vimentin and g-tubulin localization were observed by confocal microscopy in Flp-in-293 Q141K cells after a 24-hour treatment with 46 nmol/L romidepsin (RD). Login to comment 222 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys B, the effect of colchicine (colch; 1 mmol/L, 24 hours) on ABCG2 localization was determined by immunofluorescence in Flp-In-293 Q141K ABCG2 cells. Login to comment 223 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys C, ABCG2 total expression was quantified by immunoblot in Flp-In-293 WT and Q141K cells after 24-hour exposure to 1 mmol/L colchicine. Login to comment 224 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys D, quantification of ABCG2 surface expression by FACS in Flp-In-293 Q141K cells after 24-hour exposure to HDIs in presence or absence of 2.5 mmol/L tubastatin (tuba). Login to comment 225 ABCG2 p.Gln141Lys C, ABCG2 total expression was quantified by immunoblot in Flp-In-293 WT and Q141K cells after 24-hour exposure to 1 mmol/L colchicine. Login to comment 226 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys F, the knockdown of dynamitin was observed after 48-hour transfection in Q141K cells by immunoblotting (left). Login to comment 227 ABCG2 p.Gln141Lys The effect of dynamitin knockdown was then assessed on ABCG2 Q141K protein expression (surface and total) after a subsequent 24-hour HDI treatment. Login to comment 228 ABCG2 p.Gln141Lys F, the knockdown of dynamitin was observed after 48-hour transfection in Q141K cells by immunoblotting (left). Login to comment 229 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys HDI Impact on the Expression and Trafficking of Q141K ABCG2 www.aacrjournals.org Cancer Res; 72(14) July 15, 2012 3649 American Association for Cancer ResearchCopyright (c) 2012 on October 25, 2012cancerres.aacrjournals.orgDownloaded from Published OnlineFirst April 3, an inflammatory stimulus, HDAC1 competed with the adaptor proteins for binding to motor proteins that travel along the microtubules, impairing axonal transport in neurons. Login to comment 231 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Nawrocki and colleagues (40) also showed that inhibition of HDAC6 led to disruption of the aggresomes, but our experiments suggested that this deacetylase was not involved in the Q141K ABCG2 rescue. Login to comment 232 ABCG2 p.Gln141Lys On the basis of our results, we propose a multipathway mechanism for HDI-induced Q141K ABCG2 rescue to surface (Fig. 6). Login to comment 233 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys HDIs induced Q141K ABCG2 surface localization by an increase in ABCG2 transcription coupled to dynamitin overexpression, which reduced aggresome targeting by inhibition of dynein/microtubule retrograde transport. Login to comment 234 ABCG2 p.Gln141Lys On the basis of our results, we propose a multipathway mechanism for HDI-induced Q141K ABCG2 rescue to surface (Fig. 6). Login to comment 235 ABCG2 p.Gln141Lys HDIs induced Q141K ABCG2 surface localization by an increase in ABCG2 transcription coupled to dynamitin overexpression, which reduced aggresome targeting by inhibition of dynein/microtubule retrograde transport. Login to comment 253 ABCG2 p.Gln141Lys Proposed mechanism of multiple target HDI-mediated Q141K ABCG2 rescue. Login to comment 254 ABCG2 p.Gln141Lys HDIs act on Q141K ABCG2 trafficking by inducing ABCG2 overexpression (1) and likely a trafficking/folding partner overexpression (3), as well as by inhibition of retrograde transport from cytoplasm to aggresome (2). Login to comment 274 ABCC7 p.Gly551Asp Pedemonte N, Sonawane ND, Taddei A, Hu J, Zegarra-Moran O, Suen YF, et al. Phenylglycine and sulfonamide correctors of defective delta F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating. Login to comment 294 ABCG2 p.Gln141Lys Major SNP (Q141K) variant of human ABC transporter ABCG2 undergoes lysosomal and proteasomal degradations. Login to comment 307 ABCG2 p.Gln141Lys Furukawa T, Wakabayashi K, Tamura A, Nakagawa H, Morishima Y, Osawa Y, et al. Major SNP (Q141K) variant of human ABC transporter ABCG2 undergoes lysosomal and proteasomal degradations. Login to comment 347 ABCC7 p.Gly551Asp Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, Drevinek P, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. Login to comment 349 ABCG2 p.Gln141Lys HDI Impact on the Expression and Trafficking of Q141K ABCG2 www.aacrjournals.org Cancer Res; 72(14) July 15, 2012 3651 American Association for Cancer ResearchCopyright (c) 2012 on October 25, 2012cancerres.aacrjournals.orgDownloaded from Published OnlineFirst April 3, Login to comment 384 ABCG2 p.Gln141Lys HDI Impact on the Expression and Trafficking of Q141K ABCG2 www.aacrjournals.org Cancer Res; 72(14) July 15, 2012 3651 2012;72:3642-3651. Login to comment