Human copper transporter 1 (hCTR1) is the high-affinity copper influx transporter in mammalian cells that also mediates the influx of cisplatin. Loss of hCTR1 expression has been implicated in the development of resistance to this cancer chemotherapeutic agent. It has turned out to be very difficult to develop antibodies to hCTR1 and polyclonal antibodies produced by different laboratories have yielded conflicting results. We have characterized a newly-available rabbit monoclonal antibody that reacts with an epitope on the N-terminal end of hCTR1 that now permits rigorous identification and quantification of hCTR1 using Western blot analysis. Postnuclear membrane (PNM) preparations made from cells engineered to express high levels of myc-tagged hCTR1, and cells in which the expression of hCTR1 was knocked down, were used to characterize the antibody. The identity of the bands detected was confirmed by immunoprecipitation, surface biotinylation and deglycosylation of myc-tagged hCTR1. Despite the specificity expected of a monoclonal antibody, the anti-hCTR1 detected a variety of bands in whole cell lysates (WCL), which made it difficult to quantify hCTR1. This problem was overcome by isolating post-nuclear membranes and using these for further analysis. Three bands were identified using this antibody in PNM preparations that migrated at 28, 33-35 and 62-64kDa. Multiple lines of evidence presented here suggest that the 33-35 and 62-64kDa bands are hCTR1 whereas the 28kDa band is a cross-reacting protein of unknown identify. The 33-35kDa band is consistent with the expected MW of the glycosylated hCTR1 monomer. This analysis now permits rigorous identification and quantification of hCTR1.

The copper influx transporter CTR1 is also a major influx transporter for cisplatin (cDDP) in tumor cells. It influences the cytotoxicity of cDDP both in vivo and in vitro. Whereas Cu triggers internalization of CTR1 from the plasma membrane, cDDP does not. To investigate the mechanisms of these effects, myc-tagged forms of wild type hCTR1 and variants in which Y103 was converted to alanine, C189 was converted to serine, or the K178/K179 dilysine motif was converted to alanines were re-expressed in mouse embryo cells in which both alleles of CTR1 had been knocked out and also in HEK293T cells. The Y103A mutation and to a lesser extent the C189S mutation reduced internalization of CTR1 induced by Cu while the K178A/K179A had little effect. Both Y103 and C189 were required for Cu and cDDP transport whereas the K178/K179 motif was not. While Y103 lies in an YXXM motif that, when phosphorylated, is a potential docking site for phosphatidylinositol 3-kinase and other proteins involved in endocytosis, Western blot analysis of immunoprecipitated myc-CTR1, and proteomic analysis of peptides derived from CTR1, failed to identify any basal or Cu-induced phosphorylation. However, proteomic analysis did identify an interaction of CTR1 with IRS-4 and this was confirmed by co-immunoprecipitation from HEK cells expressing either FLAG-CTR1 or myc-CTR1. The interaction was greater in the Y103A-expressing cells. We conclude that Y103 is required for the internalization of hCTR1 in response to Cu, that this occurs by a mechanism other than phosphorylation and that mutation of Y103 modulates the interaction with IRS-4.

The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus.

Mammalian cells have two influx Cu transporters that form trimers in membranes. CTR1 is the high affinity transporter that resides largely in the plasma membrane, and CTR2 is the low affinity transporter that is primarily associated with vesicular structures inside the cell. The major differences between CTR1 and CTR2 are that CTR1 contains a HIS/MET-rich domain N-terminal of the METS that participate in the first two stacked rings that form the pore, and a longer C-terminal tail that includes a Cu binding HIS-CYS-HIS (HCH) motif right at the end. It has been reported that CTR1 and CTR2 are physically associated with each other in the cell. We used the CRISPR-Cas9 technology to knock out either CTR1 or CTR2 in fully malignant HEK293T and OVCAR8 human ovarian cancer cells to investigate the interaction of CTR1 and CTR2. We report here that the level of CTR2 protein is markedly decreased in CTR1 knockout clones while the CTR2 transcript level remains unchanged. CTR2 was found to be highly ubiquitinated in the CTR1 knock out cells, and inhibition of the proteasome prevented the degradation of CTR2 when CTR1 was not present while inhibition of autophagy had no effect. Re-expression of CTR1 rescued CTR2 from degradation in the CTR1 knockout cells. We conclude that CTR1 is essential to maintain the stability of CTR2 and that in the absence of CTR1 CTR2 is degraded by the proteasome. This reinforces the concept that the functions of CTR1 and CTR2 are inter-dependent within the Cu homeostasis system.

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G0-G1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253)3]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253)3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253)3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177-86. ©2018 AACR.

APTO-253 is a small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies. We sought to determine the mechanisms of action and basis for resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination studies. The cellular pharmacology of APTO-253 was analyzed in Raji lymphoma cells and a subline selected for resistance (Raji/253R). Using LC/MS/ESI analysis, APTO-253 was found to convert intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)₃]. The intracellular content of Fe(253)₃ exceeded that of the native drug by approximately 18-fold, and Fe(253)₃ appears to be the most active form. Treatment of cells with APTO-253 caused DNA damage, which led us to ask whether cells deficient in homologous recombination (i.e., loss of BRCA1/2 function) were hypersensitive to this drug. It was found that loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 of a magnitude similar to the effects of PARP inhibitors, olaparib. Raji cells selected for 16-fold acquired resistance had 16-fold reduced accumulation of Fe(253)₃ RNA-seq analysis revealed that overexpression of the ABCG2 drug efflux pump is a key mechanism of resistance. ABCG2-overexpressed HEK-293 cells were resistant to APTO-253, and inhibition of ABCG2 reversed resistance to APTO-253 in Raji/253R. APTO-253 joins the limited repertoire of drugs that can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. Mol Cancer Ther; 17(6); 1167-76. ©2018 AACR.

The molecular underpinnings of acquired resistance to carboplatin are poorly understood and often inconsistent between in vitro modeling studies. After sequential treatment cycles, multiple isogenic clones reached similar levels of resistance, but significant transcriptional heterogeneity. Gene-expression based virtual synchronization of 26,772 single cells from 2 treatment steps and 4 resistant clones was used to evaluate the activity of Hallmark gene sets in proliferative (P) and quiescent (Q) phases. Two behaviors were associated with resistance: (1) broad repression in the P phase observed in all clones in early resistant steps and (2) prevalent induction in Q phase observed in the late treatment step of one clone. Furthermore, the induction of IFNα response in P phase or Wnt-signaling in Q phase were observed in distinct resistant clones. These observations suggest a model of resistance hysteresis, where functional alterations of the P and Q phase states affect the dynamics of the successive transitions between drug exposure and recovery, and prompts for a precise monitoring of single-cell states to develop more effective schedules for, or combination of, chemotherapy treatments.