ABCC3 p.Val1322Phe
| Predicted by SNAP2: | A: D (75%), C: N (66%), D: D (95%), E: D (91%), F: D (91%), G: D (91%), H: D (91%), I: D (63%), K: D (91%), L: D (85%), M: D (85%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (85%), T: D (80%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, W: D, Y: D, |
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[hide] ABCC multidrug transporters in childhood neuroblas... J Natl Cancer Inst. 2011 Aug 17;103(16):1236-51. Epub 2011 Jul 28. Henderson MJ, Haber M, Porro A, Munoz MA, Iraci N, Xue C, Murray J, Flemming CL, Smith J, Fletcher JI, Gherardi S, Kwek CK, Russell AJ, Valli E, London WB, Buxton AB, Ashton LJ, Sartorelli AC, Cohn SL, Schwab M, Marshall GM, Perini G, Norris MD
ABCC multidrug transporters in childhood neuroblastoma: clinical and biological effects independent of cytotoxic drug Efflux.
J Natl Cancer Inst. 2011 Aug 17;103(16):1236-51. Epub 2011 Jul 28., 2011-08-17 [PMID:21799180]
Abstract [show]
BACKGROUND: Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux. METHODS: A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)-driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN(1/-), 205 hMYCN(+/1) mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan-Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided. RESULTS: Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the "poor prognosis" expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001). CONCLUSION: ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.
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No. Sentence Comment
69 For stable clones expressing ABCC3, BE(2)-C cells were transduced with retroviral pCMV14-3xFLAG-ABCC3 or the ATP-binding site mutant, pCMV14-3xFLAG-ABCC3-V1322F.
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ABCC3 p.Val1322Phe 21799180:69:154
status: NEW70 The V1322F mutation was predicted to abolish transporter activity while still being localized in the plasma membrane and is based on studies involving the closely related transporter, ABCC6, in which a single amino acid change in a region N-terminal to the Walker A motif in the second ABC domain abolishes catalytic activity (20) while retaining correct membrane localization in mammalian cell lines (Andras Varadi, personal communication).
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ABCC3 p.Val1322Phe 21799180:70:4
status: NEW75 Generation of ATP-binding site mutants was carried out accordingtotheQuikChangeLightningSite-DirectedMutagenesis Kit (Stratagene, La Jolla, CA) using forward primer 5'-ATC CTT GTG TTG AAT GAG GCC ACG GCA G-3' for ABCC1-D1454N (single mutant), forward primer 5'-ATC CTT GTG TTG CTT CTG GCC ACG GCA G-3' for ABCC1-DE1454/1455LL (double mutant) and forward primer 5'-AGG TGG GGA TCT TCG GCC GCA CTG G-3' for ABCC3-V1322F.
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ABCC3 p.Val1322Phe 21799180:75:411
status: NEW281 ABCC3 transport activity is necessary for the observed effects on cell motility and clonogenicity, because the wound closure and colony-forming ability of cells expressing catalytically inactive ABCC3 (ABCC3-V1322F) were indistinguishable from cells transfected with empty vector (mean % of wound open ± 95% CI: ABCC3 C4, 5.4 ± 4.3 vs ABCC3 D1, 3.0 ± 2.4 vs empty vector, 1.5 ± 2.0, P = .34, one-way ANOVA, three independent experiments; Figure 6, C and mean numberof colonies ± 95% CI: ABCC3 C4, 91.1 ± 6.5 vs ABCC3 D1, 92.0 ± 7.4 vs empty vector, 90.9 ± 9.4, P = .98, one-way ANOVA, three independent experiments; Figure 6, D).
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ABCC3 p.Val1322Phe 21799180:281:208
status: NEW283 A) Western blot analysis of ABCC3 protein expression following stable transduction of BE(2)-C cells with either empty vector, wild-type (wt) ABCC3 (clones A12, B12) or ABCC3 V1322F mutant (clones C4, D1) constructs.
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ABCC3 p.Val1322Phe 21799180:283:174
status: NEW[hide] Multidrug resistance protein 3 loss promotes tumor... Oncogene. 2015 Jun 15. doi: 10.1038/onc.2015.218. Wiel C, Gras B, Vindrieux D, Warnier M, Gitenay D, Le Calve B, Ferrand M, Augert A, Bernard D
Multidrug resistance protein 3 loss promotes tumor formation by inducing senescence escape.
Oncogene. 2015 Jun 15. doi: 10.1038/onc.2015.218., [PMID:26073088]
Abstract [show]
Oncogenic-stress-induced senescence (OIS) is a stress response allowing normal cells, when receiving oncogenic signals, to stably arrest their proliferation. OIS thus acts to prevent aberrant cell proliferation and tumor formation. To identify novel tumor suppressive pathways, we have recently completed a loss-of-function genetic screen to identify novel genes promoting escape from OIS and thus, potentially, tumor formation when their functions are lost. Using this approach, we unexpectedly found that loss of function of the multidrug resistance protein 3 (MRP3 or ABCC3) promotes escape from OIS in human epithelial cells. Importantly, ABCC3 expression is reduced in human skin tumors, and ABCC3-knockout mice display increased sensitivity to RAS-induced skin carcinogenesis, concomitantly with decreased OIS. ABCC3 participates in resistance to chemotherapy via its transporter activity. Our data show that this transporter activity is involved in ABCC3-induced senescence, demonstrating that this protein has a complex role in cancer, since its loss of function may promote escape from OIS and tumor formation whereas its gain of function promotes resistance to chemotherapy.Oncogene advance online publication, 15 June 2015; doi:10.1038/onc.2015.218.
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62 The pro-senescence effect of ABCC3 requires its transport activity ABCC3 is well known to act as a transporter of multiple macromolecules.17-19 To establish whether the pro-senescence effect of ABCC3 relies on its transporter activity, we generated a V1322F ABCC3 mutant.
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ABCC3 p.Val1322Phe 26073088:62:251
status: NEW63 The V1322F mutation has been shown to inhibit the transport activity of ABCC proteins without altering its Figure 2.
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ABCC3 p.Val1322Phe 26073088:63:4
status: NEW85 membrane localization.26 We infected HEC-TR cells with a control, ABCC3, or V1322F ABCC3 expression vector and checked for constitutive expression by immunoblotting (Figure 3a), RTqPCR (Figure 3b) and immunofluorescence (Supplementary Figure 2a).
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ABCC3 p.Val1322Phe 26073088:85:76
status: NEW90 HEC-TR cells were infected with an empty control vector (ctrl), an ABCC3-encoding vector (ABCC3), or a vector encoding a transport-deficient ABCC3 mutant (ABCC3 V1322F) and neomycin selected.
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ABCC3 p.Val1322Phe 26073088:90:161
status: NEW91 Generation of the ABCC3-V1322F mutant (ATP-binding site mutant) was carried out with and as recommended for the Quik Change Lightning Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA).
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ABCC3 p.Val1322Phe 26073088:91:24
status: NEW97 (c) MTT assay to measure etoposide efflux in HECs expressing an empty plasmid (ctrl) or a plasmid encoding wild-type ABCC3 or a mutated version thereof (V1322F).
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ABCC3 p.Val1322Phe 26073088:97:153
status: NEW113 This confirms that ABCC3 V1322F has lost its transporter activity (Figure 3c).
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ABCC3 p.Val1322Phe 26073088:113:25
status: NEW114 Strikingly, expression of the ABCC3 V1322F was unable to promote premature senescence: the results of the proliferation, SA-b2;-Gal activity, and IL8 expression assays showed no early effect after infection alone (Figure 3d, upper panel, 3e and f, left panel) and no effect at p6 in infected cells subjected to mild oncogenic stress (Figure 3d, lower panel, 3e and f, right panel).
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ABCC3 p.Val1322Phe 26073088:114:36
status: NEW