The data presented in this report should not be taken as - nor was our experimental approach intended to generate - prescriptive guidance for the clinical application of statins as adjuvants to conventional chemotherapeutic agents. Rather, our objective was to illuminate the confounding literature bearing on the adjunctive role of statins in chemotherapy. To that end, we assayed a single statin, lovastatin, paired with each of ten commonly prescribed chemotherapeutic agents, in highly reproducible dilution assays; the data from the assays were submitted to rigorous statistical analysis to identify synergistic and antagonistic interactions, according to the Loewe additivity model. Because the ten chemotherapeutic agents have different mechanisms of action (Table 1), it was not surprising that in combination with lovastatin, they exhibited a spectrum of global interactions, ranging from synergism to neutrality to antagonism (Fig. 1a). In considering the Loewe synergy scores for the various drug interactions, we concur with the view “that any synergy model should be treated as an exploratory ranking statistic for prioritization of the most potent combinations for further evaluation…” [36]. In this context, we identified four chemotherapeutic drugs – tamoxifen, doxorubicin, methotrexate and rapamycin – which from our dual drug studies merit further investigation.
The global metric SUM_SYN_ANT proved useful in identifying the four dual-drug combinations which exhibited strongly synergistic interactions within their concentration spaces. However, undue reliance upon global metrics risks overlooking potentially useful synergistic interactions which are confined to portions of the concentration space, sometimes with coexisting antagonism (see Fig. 4d,e and Fig. 5c,f). Lovastatin in combination with rapamycin proffers a case in point: lower concentrations of rapamycin interact synergistically with higher concentrations of lovastatin yet the converse yields significant antagonism (Fig. 5c,f). A comparison of the interactions of lovastatin with doxorubicin versus methotrexate further illustrate pitfalls of global metrics. The SUM_SYN_ANT score for doxorubicin is higher (+ 5.43) than for methotrexate (+ 2.93). However, the synergistic interactions for doxorubicin occur only with the highest two concentrations tested (Fig. 3d), a clinically problematic pattern. By contrast, the synergistic interactions of methotrexate are found with lower concentrations of the chemotherapeutic drug (Fig. 4d,e), an observation suggesting the possibility that adding a statin might not only improve the efficacy of methotrexate but also allow a reduction in its dosage, with attendant mitigation of methotrexate’s substantial toxicity.
Together these observations lead to a sobering conclusion: the therapeutic consequences – be they advantageous or detrimental – of a statin/chemotherapeutic drug combination may hinge upon the concentrations of each achieved at the tumor site. Consequently, targeted delivery strategies [38] with precise control of concentration ratios of the two drugs may merit consideration.
Cognizant of the limitations of global metrics, we gave greater weight in our assessments to the synergy distribution matrices (see, for example, Fig. 2d), in which the statistically significant values are colored for clarity. It should be emphasized that the displayed values – the synergy scores – represent mathematical calculations derived from a comparison of the experimental dose-response space to the reference dose-response space, in accordance with the Loewe model. On the other hand, the experimental dose-response surface (see, for example, Fig. 2c) portrays the efficacy of the various concentration pairs normalized to the drug-free controls. Both are informative.
Two features of the experimental design merit further discussion. Earlier we set forth our rationale for utilizing the model organism Saccharomyces cerevisiae. Our primary consideration was that the cellular target be an indifferent one, free of the biologic and genetic biases inherent in every cancer cell line; choosing any one human cancer cell line for the assays risked biasing the assays for or against a particular drug. The balanced pool of heterozygously-deleted essential gene strains, which we created in order to have a cellular substrate with consistent genetic diversity, proved to be only modestly more sensitive to the various drugs than the cognate wildtype strain. However, because each deletion is bar coded, the pool provides a useful resource for the analysis of genetic resistance to drug treatments, a study beyond the scope of this report.
A second important consideration in experimental design was the choice of synergy model. The Combenefit software package renders three models (Bliss, HSA and Loewe). The Bliss and Loewe model are arguably the most popular synergy models, but all synergy models have inherent flaws [36, 39]. The probabilistic Bliss model assumes independent but competing drug actions whereas the Loewe additivity model assumes nonindependence; that is, the two drugs may interact with the same targets or pathways [36]. Because of the remarkable pleiotropy of statins (reviewed in detail in [40]), including a number of interactions with a variety of signaling pathways, we posited nonindependence of lovastatin and the individual chemotherapeutic drugs and therefore chose the Loewe model as more appropriate. .
Having demonstrated net synergism of four drug combinations in the yeast model, we selected one – tamoxifen and lovastatin - for additional study in human cancer cell lines. This combination exhibited the strongest synergy on day two but also was intriguing because of its unanticipated substantial efficacy in S. cerevisiae, which is devoid of estrogen receptors. (Although an estrogen binding protein has been identified in budding yeast [41, 42], the protein demonstrated negligible binding of tamoxifen [41].) The efficacy of tamoxifen alone (Fig. 2b) or paired with lovastatin (Fig. 2c,d) therefore likely results from activation of one or more of tamoxifen’s known off-target pathways (reviewed in [43]).
Of the three human breast cancer cell lines assayed, MCF-7 – which possesses estrogen receptors – demonstrated strong synergy with lovastatin (Fig. 7c), in a pattern resembling that seen with yeast (Fig. 2d). The other two breast cancer cell lines tested (see Fig. 7f,i), consistently achieved synergy with only the highest concentrations of lovastatin. Together, these observations frame a paradox: the combination of tamoxifen plus lovastatin was strongly synergistic in an organism, S. cerevisiae, devoid of estrogen receptors, yet displayed a similar pattern of strong synergism only in a breast cancer cell line, MCF7, which possesses estrogen receptors. Perhaps the simplest resolution of this seeming contradiction is that lovastatin interacts synergistically with tamoxifen only when tamoxifen alone effectively inhibits cell growth, regardless of the cellular mechanism by which that occurs. It therefore seems unlikely – to our disappointment - that a statin will lend efficacy to tamoxifen in triple-negative breast cancer, for example, in which tamoxifen is impotent.
We sought further proof of principle in support of our S. cerevisiae-based experimental design by testing the combination of cisplatin and lovastatin in three human cell lines: A549 (lung adenocarcinoma); HT29 (colon adenocarcinoma) and MCF7 (breast carcinoma). This combination was chosen as a surrogate for the six neutral or antagonistic pairings because it so commonly prescribed for a variety of malignancies. In the yeast model, the combination exhibited strong antagonism (Table 2, Fig. 1a,d; Additional file 2: Fig. S6d). Congruent with our S. cerevisiae data, the metrics for the three cell lines were antagonistic, even more so than those calculated from the yeast assays (Table 2), lending further support for the validity of our model.
Does our study illuminate the contradictory landscape of chemotherapeutic regimens incorporating statins, our purpose as set forth earlier? Perhaps so. In the Background we cited the systemic review and meta-analysis by Farooqui et al., demonstrating that the addition of a statin to conventional therapy failed to improve progression-free or overall survival. Of the ten studies included in their analysis, six incorporated - either as the sole chemotherapeutic drug or as a component of a multi-drug regimen - agents which we found to be either neutral or antagonistic: etoposide, cisplatin, gemcitabine, and 5-FU (one protocol included both cisplatin and epirubicin, which is related to doxorubicin). Of the remaining four studies, one specified whole brain irradiation, and three incorporated drugs (afatinib, thalidomide and gefitinib) which we have not assayed. Thus, our data and the Farooqi meta-analysis provide mutually supportive, albeit circumstantial, evidence affirming the lack of efficacy of at least four statin plus chemotherapeutic drug combinations. Unfortunately, the larger systemic review by Mei et al. [23], which demonstrated beneficial effects of statins upon survival in cancer patients, did not specify the individual chemotherapeutic drugs used in the 95 studies included in the analysis.