Our studies in a mouse model of HPV16-induced cervical carcinoma demonstrate that the copper chelator tetrathiomolybdate (TM) has a synergistic antitumor effect when combined with cisplatin treatment, by increasing uptake of cisplatin into tumors but not normal tissues while concomitantly inhibiting tumor angiogenesis. We show that cisplatin adduct levels in various mouse organs correlate with mRNA levels of Ctr1, supporting its in vivo involvement in cisplatin uptake. Pretreatment of cultured human cervical and ovarian cancer cells with TM resulted in increased cisplatin sensitivity, encouraging applicability to human cancers. TM-enhanced cisplatin killing was dependent on Ctr1, as mouse cells deleted for the Ctr1 gene were not sensitized to cisplatin by TM, in comparison to isogenic cells with wild-type Ctr1. Notably, low levels of Ctr1 mRNA in human ovarian tumors are associated with decreased disease-free survival after platinum-based therapy in patients, suggesting that Ctr1 may serve as a clinical predictor of response to platinum agents.
Previous genetic experiments with yeast and mouse cells demonstrated that the copper transporter Ctr1 is a major determinant of cisplatin uptake and sensitivity, which could be modulated by copper levels. These in vitro
results suggested a strategy to increase cisplatin uptake, but a challenge was to find a clinically feasible method to do so selectively in tumors, without affecting normal organs to avoid untoward toxicities of cisplatin. Surprisingly, we were able to achieve this goal by reducing systemic copper levels with a copper chelator in a mouse model of cervical cancer. We do not fully understand the mechanism by which reduced systemic copper leads to increased cisplatin uptake only in tumors. Copper is essential for a variety of key cellular processes such as respiration, free radical detoxification, and iron uptake (Kim et al., 2008
). Cancer cells may have a greater demand for copper than normal cells for proliferation and survival. Indeed, the copper transporter Ctr1 was more highly expressed in cervical carcinoma of the HPV16/E2
mice than in the wild-type cervix. Concordant with our observation is a recent demonstration using micro-beam synchrotron X-ray fluorescence that copper is concentrated in tumor regions of tissue specimens obtained from invasive ductal carcinoma of the breast (Farquharson et al., 2008
Although pretreatment of mice with the copper chelator TM resulted in increased cisplatin sensitivity and adducts in tumors, we were unable to detect any increase in the levels of Ctr1 mRNA or protein. In vitro
, elevated extracellular copper causes endocytosis and degradation of human Ctr1 protein (Petris et al., 2003
). We did not, however, observe any change in Ctr1 protein localization in the TM-treated cervix of HPV16/E2
mice. It is possible that copper starvation enhances cisplatin transport activity by changing the conformation of Ctr1, allowing more cisplatin to be transported into cells. Exogenous copper has been shown to induce structural rearrangements in yeast Ctr1 (Sinani et al., 2007
). Further biochemical studies will be required to elucidate the mechanism by which copper chelation causes increased cisplatin uptake.
Drug-induced toxicity is a common cause for discontinuation or dose reduction of chemotherapeutic drugs during cancer treatment. Cancers that are deemed “resistant” to a chemotherapeutic drug fail to respond to the dosage tolerated by patients without untoward side effects. These “resistant” cancers might in principle still respond to a higher dosage if toxicities were not manifest. By reducing bioavailable copper with the copper chelator tetrathiomolybdate (TM), we were able to increase cisplatin activity in highly metabolic tumors while comparatively sparing normal organs. TM was developed for the treatment of patients with Wilson’s disease, an autosomal recessive disorder of copper transport that results in excessive accumulation of copper and toxicitiy. Phase II and III trials with Wilson’s disease patients have demonstrated that TM is a fast-acting and well-tolerated drug (Brewer 2009
). The major side effect is anemia and leukopenia due to copper depletion, which can be reversed with a drug holiday or dose reduction.
Additionally, the independent anti-angiogenic effects of the copper chelator TM present it as a drug that targets both tumor parenchyma and stroma, by enhancing cisplatin efficacy against the cancer cells whilst inhibiting angiogenesis in the tumor microenvironment. Combination regimens involving copper chelating and platinum-containing drugs may improve the treatment of cervical, ovarian, and other cancers for which cisplatin is currently in use, and for cancers that are treated with carboplatin, whose uptake is also mediated by Ctr1 (Holzer et al., 2006
). Such a therapeutic strategy may even prove effective in treating cancers that are inherently resistant to cisplatin or have developed resistance. With the development of a second-generation TM analog that depletes copper more quickly and is more stable (Lowndes et al, 2008
), it may be possible to manipulate the activity of Ctr1 even more effectively. The mechanistic principles and results elaborated in this report should motivate discussion of analogous clinical trials in patients with cervical, ovarian, and other platinum-responsive cancers.