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Editorials |
Medical Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania
As a relatively polar molecule, CDDP1 is thought to use specific plasma membrane systems for passage into cells, although entry by passive diffusion is also likely to occur. Alterations in these plasma membrane systems have been considered to be important resistance factors because one of the most consistent features of CDDP-resistant cell lines is decreased intracellular drug levels (1) . Whereas the membrane determinants of accumulation have therefore been of keen interest, their molecular identities have gone undefined for many years. An interesting and noteworthy advance in this area has been the recent determination that the same proteins that mediate transport of copper are also capable of transporting CDDP. In this issue of Clinical Cancer Research an analysis of the potential for a copper transporter to impact treatment of ovarian cancer patients with platinum-based regimens is reported (2) .
Copper is an essential metal that functions as a cofactor for enzymes involved in diverse metabolic pathways, including cytochrome c oxidase (the terminal enzyme of the mitochondrial respiratory chain), superoxide dismutase (an antioxidant that represents 
1% of cellular protein), and dopamine-ß-monoxygenase (catecholamine biosynthesis). Although copper is a required cofactor, it is also highly reactive in the cell and, when present in excess, exerts significant toxicity consequent to the generation of free radicals. Thus, copper is subject to complex homeostatic mechanisms that maintain appropriate intracellular concentrations and restrict its ability to inflict intracellular damage. This process is accomplished by the action of specific copper transporters and chaperones (3
, 4)
. Copper influx into the cell is mediated by Ctr1, a high affinity copper transporter. Specific chaperones, known as COX17, CCS, and HAH1, respectively, then deliver copper from the plasma membrane to the mitochondria, to cytoplasmic superoxide dismutase, and to two copper transporters located in the trans-Golgi apparatus. These two copper transporters, ATP7A and ATP7B, are members of the P-type ATPase family of cation transporters and are the products of the genes affected in two disorders of copper accumulation in humans, Menkes disease and Wilson disease, respectively (5
, 6)
. In the liver, ATP7B transports copper into the trans-Golgi either for incorporation into copper-requiring proteins, or for extrusion into the bile, the latter of which is accomplished by vesicular trafficking from the trans-Golgi to the canalicular (apical) surface of hepatocytes and represents a major route of copper elimination from the body. In most other tissues, copper transport into the trans-Golgi is accomplished by ATP7A. Like ATP7B, ATP7A shuttles between the trans-Golgi and the plasma membrane in a process that mediates copper extrusion from the cell. Trafficking of ATP7A and ATP7B to the plasma membrane is thought to be regulated by intracellular copper levels (7)
. Copper uptake is also regulated by copper levels, at least as determined for Ctr1p, a yeast homologue of Ctr1, in that both CTR1 transcription and Ctr1p degradation are regulated by an usual mechanism involving a copper-sensing transcription factor (8
, 9)
.
The first insight into the involvement of copper transporters in the cellular pharmacology of CDDP was provided by Komatsu et al. (10)
, who found that ectopic expression of ATP7B in KB-3-1 cells conferred CDDP resistance associated with decreased accumulation of this agent (Fig. 1)
. In addition, they reported increased expression of ATP7B in a CDDP-resistant prostate cancer cell line. This study thus indicated that ATP7B had the facility for extruding CDDP from the cell and that the pump could be induced as a resistance factor. The involvement of copper transporters in CDDP resistance was extended to copper uptake systems by Ishida et al. (11)
who used transposon mutagenesis to identify CDDP resistance factors in yeast and found that inactivation of CTR1 was able to confer resistance to CDDP. In accord with the notion that Ctr1p is able to mediate uptake of CDDP, Ctr1p-deficient yeast exhibited decreased levels of CDDP bound to DNA and decreased CDDP accumulation. A functional link between CDDP and copper transport in the context of Ctr1p was further elucidated in wild-type yeast by experiments showing that copper and CDDP behaved as mutual competitive inhibitors, as would be expected were the two compounds common substrates for Ctr1p. That is, copper was able to reduce CDDP accumulation and attenuate CDDP toxicity, and conversely, CDDP could reduce copper accumulation. It was also demonstrated that, like copper, CDDP is able to stimulate degradation of Ctr1p. Finally, Ishida et al. (11)
went on to demonstrate, by analyzing the CDDP sensitivity of embryonic stem cells that are homozygous for deletion of Ctr1, that mammalian Ctr1 is capable of mediating the uptake of CDDP and thereby functioning as a sensitivity factor for this agent.
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Several studies have now evaluated expression of ATP7B in human cancers. To date, significant levels of expression have been reported for cancers of the esophagus, stomach, breast, ovary, and oral mucosa, suggesting that ATP7B may be a factor that contributes to inherent CDDP chemoresistance (15, 16, 17, 18, 19, 20) . In addition, the absence of expression in adjacent normal tissues, a finding that is in accord with prior studies showing that ATP7B is largely restricted to liver, suggests that ATP7B is induced in certain cancers and also raises the possibility that cancer cells may have altered copper metabolism and/or requirements. CDDP resistance mechanisms in ovarian cancer have been intensely studied because of the striking inherent sensitivity of this cancer to CDDP-based regimens, and the fact that the majority of patients with advanced disease nevertheless relapse. This situation indicates that CDDP resistance is acquired during treatment. With respect to ovarian cancer, a study in which ATP7B expression was examined in 82 specimens is of interest, in that expression was found in 44% of samples, and high levels correlated with less differentiated cancers and with higher risk of recurrence (17) . In addition, within a group of patients with moderately/poorly differentiated specimens, high ATP7B expression levels correlated with decreased survival.
In this issue of Clinical Cancer Research, the first description of ATP7A expression in human cancers is reported. Samimi et al. (2) report that ATP7A is widely expressed in human cancers, including breast, stomach, colon, ovary, lung, and prostate. Similar to the case with ATP7B, expression of ATP7A in several types of cancers was increased by comparison with expression in normal adjacent tissue. In addition to surveying a variety of cancers, a more detailed evaluation of ovarian cancer samples was undertaken by the retrospective analysis of 54 paired samples obtained before initial treatment and after treatment with at least two cycles of a CDDP- or carboplatin-based regimen. This analysis showed that ATP7A was expressed in 54% of pretreatment samples (28 samples). In addition, patients for whom expression in posttreatment samples was increased in comparison with pretreatment levels had decreased survival. Although the percentage of samples that expressed ATP7A after treatment (46%) was somewhat lower than the percentage in pretreatment samples, and the number of patients for whom expression decreased from a detectable level to undetectable (13 patients) was greater than the reverse situation (10 patients), overall this analysis suggests that induction of ATP7A expression could negatively impact the effectiveness of platinum-based chemotherapy.
These reports showing that cells can deploy energy-dependent copper pumps for defense against CDDP and carboplatin, that Ctr1 is able to mediate influx of platinum agents, and that expression of ATP7A and ATP7B correlates with worse prognosis in ovarian cancer provide intriguing insights into the cellular pharmacology of platinum compounds and into potential resistance factors associated with these agents. In addition to indicating that further investigation into the relationship between expression of copper transporters and clinical outcomes in patients treated with platinum-based chemotherapy regimens is needed, these advances also raise many important questions. Among the most important are the extent to which CDDP accumulation in the cell is attributable to copper transporters, and the extent to which alterations in the expression levels of these transporters account for the accumulation deficit that has been consistently documented in CDDP-resistant cellular models. Most studies of CDDP-resistant cell lines indicate that reduced accumulation is consequent to decreased influx, as opposed to increased efflux (1) . For this reason, evaluation of Ctr1 expression in addition to ATP7A and ATP7B in patient samples will be of particular interest. Also, the mechanism by which ATP7B and presumably ATP7A depress intracellular levels of platinum compounds is unclear; like normal tissues, the pumps appear to be localized in the trans-Golgi apparatus in cancer cells. This suggests that the pumps may operate to sequester platinum compounds in intracellular vesicles, which may itself provide some degree of cellular protection, and that cellular efflux is accomplished by vesicular trafficking to the plasma. How CDDP affects transcriptional and posttranscriptional regulation of copper pumps and whether copper chaperones are able to interact with CDDP are also open questions. Finally, it should be borne in mind that in addition to copper transporters, other membrane systems, such as certain members of the MRP family (MRP2 and possibly MRP6; Ref. 21 ), have the ability to affect CDDP accumulation and that the molecular identification of other proteins involved in this process remains an important goal.
ACKNOWLEDGMENTS
I thank Steven Johnson, Kathleen Scotto, and James Gallo for critically reviewing this editorial.
FOOTNOTES
Grant support: NIH Grant CA73728, National Cancer Institute Core Grant CA06927, and an appropriation from the Commonwealth of Pennsylvania to the Fox Chase Cancer Center.
Requests for reprints: Gary D. Kruh, Medical Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
1 The abbreviations used are: CDDP, cisplatin; MRP, multidrug resistance protein. 
Received 8/29/03; accepted 9/11/03.
REFERENCES
Key Article
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  I.-S. Song, N. Savaraj, Z. H. Siddik, P. Liu, Y. Wei, C. J. Wu, and M. T. Kuo Role of human copper transporter Ctr1 in the transport of platinum-based antitumor agents in cisplatin-sensitive and cisplatin-resistant cells Mol. Cancer Ther., December 1, 2004; 3(12): 1543 - 1549. [Abstract] [Full Text] [PDF]
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(GST) may enhance complex formation. CDDP damages DNA by the formation of adducts, principally at the N7 sites of purine bases. The most toxic adducts appear to be intrastrand cross-links. Damaged DNA can be repaired, primarily by the nucleotide excision repair (NER) pathway, or lead to cell death by apoptosis. Mismatch repair (MMR) system competency and tumor suppressors facilitate apoptosis, whereas oncogenes block this process. For simplicity, ATP7A and ATP7B are shown at the plasma membrane but are localized in the trans-Golgi network in cells. By analogy with the mechanism by which ATP7B functions to extrude copper into bile, CDDP extrusion by ATP7B, and possibly by ATP7A, may involve vesicle trafficking to the plasma membrane.