We show here that in vitro ATP7B gene silencing leads to increased sensitivity to cisplatin in both parental and cisplatin-resistant cell lines. ATP7B gene silencing using a highly efficient method for systemic siRNA delivery resulted in antitumor efficacy in a platinum-resistant model of ovarian cancer in mice. Addition of cisplatin to ATP7B siRNA-DOPC, but not control siRNA-DOPC, further reduced the tumor burden in these mice, indicating that the effects were not due to nonspecific siRNA toxicity. These findings may be explained based on a benefit resulting from the coadministration of both drugs working independently of each other and not necessarily in vivo sensitization. The antitumor activity was accomplished through decreased tumor cell proliferation and MVD and increased apoptosis. To the best of our knowledge, this study provides the first in vivo evidence about the therapeutic efficacy of targeting ATP7B in combination with cisplatin in platinum-resistant ovarian cancer.
Given the central role of platinum-based chemotherapy in the treatment of many cancers, including ovarian cancer, strategies to circumvent inherent or acquired resistance are highly desirable. Death of tumor cells in response to chemotherapy is dependent on several factors, including the amount of drug that enters the cell and the nucleus, amount of DNA adduct formation, cell tolerability to DNA adducts, and the ability to repair DNA damage (43
). Previously, Nakayama and associates (21
) observed that the levels of ATP7B inversely correlated with cisplatin sensitivity in nine gynecologic cancer cell lines and that ovarian cancer patients with ATP7B expression had significantly poorer response to cisplatin-based chemotherapy than patients lacking detectable ATP7B expression. The contribution of ATP7B to cisplatin resistance is evident from the results of ATP7B down-regulation. Reduced expression of ATP7B achieved by siRNA knockdown resulted in a 2.5-fold enhancement of sensitivity of these cells to cisplatin. Significantly, the down-regulation of another Cu-ATPase ATP7A had no effect, emphasizing the important and specific role of ATP7B in resistance of A2780-CP20 cells to cisplatin.
The different effects of ATP7A and ATP7B silencing on cisplatin resistance may be linked to their different levels of expression and/or their distinct trafficking properties. In A2780-CP20 cells, ATP7A traffics toward the plasma membrane in response to copper elevation (data not shown), presumably to export excess metal out of the cell. In contrast, ATP7B shows no relocalization in response to either copper or cisplatin and is likely to sequester metals in the intracellular compartments. If in A2780-CP20 cells the ATP7A-mediated export of cisplatin is much slower than cisplatin uptake, then ATP7A inactivation would not have a significant effect on cisplatin resistance. In contrast, the concept that ATP7B controls intracellular distribution of cisplatin is supported by our observation that down-regulation of ATP7B does not significantly alter the cellular content of cisplatin, and yet, a 30% increase in DNA adduct formation is observed. This result reflects the increased nuclear availability of cisplatin in response to ATP7B down-regulation. Therefore, the major mechanism through which overexpression of ATP7B increases resistance to cisplatin seems to involve blockade of drug access to the nucleus. This may be due either to ATP7B pumping cisplatin into the lumen of the TGN or to simple binding of the drug by multiple metal-binding sites in the NH2
-terminal domain (N-ATP7B) of the transporter or both. The latter possibility is supported by our finding that the recombinant N-ATP7B alone increased cell resistance to cisplatin. While this work was in preparation, new in vitro
data (showing that cisplatin stimulates the catalytic activity of ATP7B and that this stimulation requires the NH2
-terminal region) by Leonhardt and colleagues (46
) provided additional evidence for the role of the NH2
-terminal metal-binding domain in functional interactions between cisplatin and ATP7B.
Despite efficient down-regulation of ATP7B in A2780-CP20 cells, reversal of the resistant phenotype was not complete, indicating possible involvement of other components of cellular copper handling machinery (such as high-affinity copper transporter hCTR1, metallochaperones, and ATP7B regulator COMMD1; ref. 47
). Furthermore, changes in expression of Cu-ATPases alter the intracellular copper balance (17
) and this in turn induces changes in the cell transcriptome (48
). However, these additional mechanisms do not diminish the major contribution of ATP7B. The in vivo
therapy with combination of ATP7B siRNA-DOPC and cisplatin significantly reduced tumor burden, decreased cell proliferation and MVD, and increased apoptosis, suggesting that this approach might be useful in patients with platinum-resistant ovarian carcinoma. Copper plays an important role in angiogenesis and it might be a required cofactor of VEGF-mediated angiogenesis (42
). Copper stimulates the proliferation and migration of endothelial cells and is required for the secretion of several angiogenic factors by tumor cells. Copper may bind to angiogenic growth factors or regulate the production of angiogenic growth factors such as VEGF (42
). Thus, therapy aimed at depleting copper levels through silencing the transporters may have an additional benefit by reducing angiogenesis. In summary, our data indicate that ATP7B-targeted therapy may represent a novel therapeutic approach for platinum-resistant human ovarian cancer.