Profiling SLCO and SLC22 genes in the NCI-60 cancer cell lines to identify drug uptake transporters

Abstract

Molecular and pharmacologic profiling of the NCI-60 cell panel offers the possibility of identifying pathways involved in drug resistance or sensitivity. Of these, decreased uptake of anticancer drugs mediated by efflux transporters represents one of the best studied mechanisms. Previous studies have also shown that uptake transporters can influence cytotoxicity by altering the cellular uptake of anticancer drugs. Using quantitative real-time PCR, we measured the mRNA expression of two solute carrier (SLC) families, the organic cation/zwitterion transporters (SLC22 family) and the organic anion transporters (SLCO family), totaling 23 genes in normal tissues and the NCI-60 cell panel. By correlating the mRNA expression pattern of the SLCO and SLC22 family member gene products with the growth-inhibitory profiles of 1,429 anticancer drugs and drug candidate compounds tested on the NCI-60 cell lines, we identified SLC proteins that are likely to play a dominant role in drug sensitivity. To substantiate some of the SLC-drug pairs for which the SLC member was predicted to be sensitizing, follow-up experiments were performed using engineered and characterized cell lines overexpressing SLC22A4 (OCTN1). As predicted by the statistical correlations, expression of SLC22A4 resulted in increased cellular uptake and heightened sensitivity to mitoxantrone and doxorubicin. Our results indicate that the gene expression database can be used to identify SLCO and SLC22 family members that confer sensitivity to cancer cells.

Figure 1

mRNA expression of SLCO and SLC22 family members in normal tissue samples (A) and the NCI-60 cancer cell line panel (B). These clustered image maps show patterns of gene expression assessed by real-time quantitative RT-PCR. Red and blue indicate high and low expression, respectively. The hierarchical clustering on each axis was done using the average-linkage algorithm with 1−r as the distance metric, where r is the Pearson's correlation coefficient, after subtracting column means. Included among the NCI-60 cancer cell lines are leukemias (LE), melanomas (ME), and cancers of breast (BR), central nervous system (CNS), colon (CO), lung (LC), ovarian (OV), prostate (PR), and renal (RE) origin. Three independent real-time quantitative RT-PCR measurements were performed. For more details, see Supporting Information Table S1 and Supporting Information Table S2.

Figure 2

Distribution of r values for the SLC22 and SLCO drug-gene pairs (white bars). The distribution is overlaid with the r value distributions for the same compound drug-gene pairs set with ABC transporters (gray bars), showing the overall similarity in correlation distribution for the two transporter families. The Pearson correlation coefficient r values for ABC transporters are weighted slightly to the negative, as a negative r value is predictive for compounds that are substrates for the efflux transporters, whereas the SLC/SLCO r values are skewed slightly to the positive as substrates of SLC/SLCO transporters would have enhanced cellular accumulation and therefore increased cytotoxicity when a given SLC/SLCO transporter is over-expressed.

Figure 3

Prediction of substrates for an SLC (SLC22A4 (OCTN1)) from correlation analysis. Scatter plot showing the correlation (r) of SLC22A4 mRNA expression with sensitivity of the NCI-60 cancer cell lines to mitoxantrone (A) and doxorubicin (B). C and D, Chemical structures of mitoxantrone and doxorubicin. (NSC numbers of drugs are shown in parentheses.) Both the GI₅₀ and crossing point (CP) values across the NCI-60 panel were mean-centered and multiplied by −1 (dlogGI₅₀ and dCP respectively) to indicate activity and expression relative to the mean.

Figure 4

Confirmation of protein expression and function of SLC22A4 (OCTN1) in established stable transfectants used in all follow-up experiments to evaluate whether correlations reveal functional interaction. A, Crude membrane fraction from cells stably transfected with mock vector (Mock/KB-3-1) or SLC22A4 (OCTN1) was subjected to Western blot analysis with anti-V5-HRP antibody. Two clones (Clone #1 and Clone #2) of SLC22A4 (OCTN1)/KB-3-1 in which protein expression was confirmed are shown. B, Cellular localization of SLC22A4 (OCTN1) in the SLC22A4 (OCTN1)/KB-3-1 and Mock/KB-3-1 cells was assessed by immunocytochemical analysis using anti-V5-FITC antibody. Result of SLC22A4 (OCTN1)/KB-3-1 Clone #1 is shown as a representative. C, Uptake of a known substrate ([¹⁴C]TEA, 60 µM) for SLC22A4 (OCTN1) by SLC22A4 (OCTN1)/KB-3-1 and Mock/KB-3-1 cells. †P < 0.005, Mock/KB-3-1 vs. SLC22A4 (OCTN1)/KB-3-1.

Figure 5

Validation of the prediction by drug sensitivity assay. Growth inhibition of the SLC22A4 (OCTN1)/KB-3-1 (◆) and Mock/KB-3-1 (○) cells treated with either mitoxantrone (A) or doxorubicin (B) for 72 hrs was evaluated by CCK-8 assay. Result of SLC22A4 (OCTN1)/KB-3-1 Clone #1 is shown as a representative. Each experiment was performed independently at least three times. Note that error bars are mostly obscured by the data points, and that differences are statistically significant (as shown in Table 1).

Figure 6

Uptake of mitoxantrone and doxorubicin by the SLC22A4 (OCTN1)/KB-3-1 and the Mock/KB-3-1 cells. A and B, Time course of uptake of [³H]mitoxantrone (3 µM) and [¹⁴C]doxorubicin (3 µM) by the SLC22A4 (OCTN1)/KB-3-1 (◆) and the Mock/KB-3-1 (○) cells. C and D, Uptake of [³H]mitoxantrone (3 µM) and [¹⁴C]doxorubicin (3 µM) by the SLC22A4 (OCTN1)/KB-3-1 (black bars) and the Mock/KB-3-1 cells (gray bars) in the absence (control) or presence of 30 µM of non-radiolabeled TEA, mitoxantrone, or doxorubicin. Uptake values are shown in fold change to that of the Mock/KB-3-1 cells incubated in the absence of non-radiolabeled drugs. Data are the means ± SD of values from three independent experiments, each done in duplicate. †P < 0.005, ‡P < 0.05, SLC22A4 (OCTN1)/KB-3-1 vs. Mock/KB-3-1 without non-radiolabeled compounds. *P < 0.005, **P < 0.05, SLC22A4 (OCTN1)/KB-3-1 without non-radiolabeled compounds vs. SLC22A4 (OCTN1)/KB-3-1 with non-radiolabeled compounds.