In-depth analysis shows synergy between erlotinib and miR-34a

Abstract

Tyrosine kinase inhibitors directed against epidermal growth factor receptor (EGFR-TKI), such as erlotinib, are effective in a limited fraction of non-small cell lung cancer (NSCLC). However, the majority of NSCLC and other cancer types remain resistant. Therapeutic miRNA mimics modeled after endogenous tumor suppressor miRNAs inhibit tumor growth by repressing multiple oncogenes at once and, therefore, may be used to augment drug sensitivity. Here, we investigated the relationship of miR-34a and erlotinib and determined the therapeutic activity of the combination in NSCLC cells with primary and acquired erlotinib resistance. The drug combination was also tested in a panel of hepatocellular carcinoma cells (HCC), a cancer type known to be refractory to erlotinib. Using multiple analytical approaches, drug-induced inhibition of cancer cell proliferation was determined to reveal additive, antagonistic or synergistic effects. Our data show a strong synergistic interaction between erlotinib and miR-34a mimics in all cancer cells tested. Synergy was observed across a range of different dose levels and drug ratios, reducing IC50 dose requirements for erlotinib and miR-34a by up to 46-fold and 13-fold, respectively. Maximal synergy was detected at dosages that provide a high level of cancer cell inhibition beyond the one that is induced by the single agents alone and, thus, is of clinical relevance. The data suggest that a majority of NSCLC and other cancers previously not suited for erlotinib may prove sensitive to the drug when used in combination with a miR-34a-based therapy.

Figure 1. miR-34a restores sensitivity to erlotinib in non-small cell lung cancer cells.

(A) Dose-dependent effect of erlotinib in parental HCC827 cells. Cells were treated with erlotinib in a serial dilution for 3 days, and cellular proliferation was determined by AlarmaBlue. (B) HCC827 cells resistant to erlotinib (HCC827^res) were developed by incubating cells with increasing erlotinib concentrations over the course of 10 weeks until cells grew normally at concentrations equal to IC₉₀ in parental HCC827. (C, D) HCC827^res and H1299 cells were reverse-transfected with 0.3 nM miR-34a or miR-NC, and incubated in media supplemented with erlotinib in a serial dilution. After 3 days, cellular proliferation was determined. IC₅₀ values of erlotinib alone or in combination with miRNA are shown in the graphs.

Figure 2. Synergistic effects between miR-34a and erlotinib in NSCLC cells.

(A) Combination index (CI) analysis. CI values were generated by linear regression (red) and non-linear regression methods (blue). Trendlines indicate CI values at any given effect (Fa, fraction affected, % inhibition), and symbols represent CI values derived from actual data points. CI = 1, additivity; CI>1, antagonism; CI<1, synergy. (B) Isobologram analysis. The diagonal, dotted line indicates additivity, and the red symbol shows dose requirements to achieve 50% and 80% (A549, H1299, H460) or 30% and 50% (H226) cancer cell inhibition, respectively. Data points below the line of additivity indicate synergy, data points above denote antagonism. (C) Curve shift analysis. Data derived from non-linear regression trendlines were normalized to IC₅₀ values of the single agents (IC₅₀ eq) and plotted in the same graph. Left and right shifts of the dose-response curves of the combination (red dotted line) relative to the dose-response curves of the single agents (grey, black) indicate synergy or antagonism, respectively. Actual experimental data points are shown (symbols).

Figure 3. Multiple ratios of erlotinib and miR-34a cooperate synergistically in A549 cells.

(A) Summary table showing potency (Fa), CI and DRI values of erlotinib and miR-34a combined at various concentrations and ratios. The molar miR-34-erlotinib ratios 1∶533, 1∶1333, 1∶3333 (IC₅₀:IC₅₀ ratio), 1∶8333, and 1∶20833 are indicated in yellow, green, red (IC₅₀:IC₅₀ ratio), blue and maroon. (B) Combination index plot of various drug ratios. CI values from actual data points are indicated by symbols. (C) Isobologram at 80% cancer cell inhibition. Colored symbols represent the 80% isobole of various ratios. The black line represents the isobole derived from actual erlotinib-miR-34a combinations that produced 80% (±2%) inhibition. (D) Curve shift analysis of various drug ratios.

Figure 4. Erlotinib and miR-34a synergize in HCC cells.

(A) Combination index analysis. (B) Isobologram analysis. (C) Curve shift analysis. See legend to Figure 2 for explanation of graphs.