Genetically defined subsets of human pancreatic cancer show unique in vitro chemosensitivity

Abstract

Purpose: Pancreatic cancer is the fourth cause of death from cancer in the western world. Majority of patients present with advanced unresectable disease responding poorly to most chemotherapeutic agents. Chemotherapy for pancreatic cancer might be improved by adjusting it to individual genetic profiles. We attempt to identify genetic predictors of chemosensitivity to broad classes of anticancer drugs.

Experimental design: Using a panel of genetically defined human pancreatic cancer cell lines, we tested gemcitabine (antimetabolite), docetaxel (antimicrotubule), mitomycin C (MMC; alkylating), irinotecan (topoisomerase I inhibitor), cisplatin (crosslinking), KU0058948 (Parp1 inhibitor), triptolide (terpenoid drug), and artemisinin (control).

Results: All pancreatic cancer cell lines were sensitive to triptolide and docetaxel. Most pancreatic cancer cells were also sensitive to gemcitabine and MMC. The vast majority of pancreatic cancer cell lines were insensitive to cisplatin, irinotecan, and a Parp1 inhibitor. However, individual cell lines were often sensitive to these compounds in unique ways. We found that DPC4/SMAD4 inactivation sensitized pancreatic cancer cells to cisplatin and irinotecan by 2- to 4-fold, but they were modestly less sensitive to gemcitabine. Pancreatic cancer cells were all sensitive to triptolide and 18% were sensitive to the Parp1 inhibitor. P16/CDKN2A-inactivated pancreatic cancer cells were 3- to 4-fold less sensitive to gemcitabine and MMC.

Conclusions: Chemosensitivity of pancreatic cancer cells correlated with some specific genetic profiles. These results support the hypothesis that genetic subsets of pancreatic cancer exist, and these genetic backgrounds may permit one to personalize the chemotherapy of pancreatic cancer in the future. Further work will need to confirm these responses and determine their magnitude in vivo.

Figure 1. Chemosensitivity of PC cell lines to broad classes of chemotherapeutic agents

Drug responses (Log₁₀ of the IC50 values) of 34 PC cell lines tested with 7 chemotherapeutic drugs representing different mechanisms of action, and the control drug (artemisinin). Each cell line result is represented by a single circle. Horizontal lines indicate the median IC50 value for each drug for all cell lines. The four drugs on the left are generally sensitive drugs, and the four drugs on the right are generally inactive.

Figure 2. Reponses of individual PC cell lines to the 4 sensitive drugs (note Y axis scale, 0 to 120nM)

Cytotoxic effects of gemcitabine (A), triptolide (B), docetaxel (C) and MMC (D) on PC cells. Gemcitabine (A) demonstrated generally good responses, although unique cell lines showed more than 100 fold less sensitivities (* indicates outlier cell lines). Triptolide (B) and docetaxel (C) demonstrated nearly universal toxicities at doses in the single digit nanomolar concentration. MMC (D) showed more variation in response, and unique cell lines (*) showed more than 40 fold less sensitivity.

Figure 3. Reponses of individual PC cell lines to the 4 insensitive drugs (note Y axis scale, 0 to 10,000nM)

Cytotoxic effects of cisplatin (A), irinotecan (B) and Parp1 inhibitor(C) on pancreatic cancer cell viability demonstrated variable drug responses, and unique cell lines showed more than 200 fold variation in their sensitivities to these drugs (*indicates uniquely sensitive cell line). Artemisinin (D) showed the nearly invariant resistance at doses in greater than 10,000 nanomolar, although unique cell lines (*) were sensitive.

Figure 4. Correlations of drug response to genotype

Cytotoxic effect of irinotecan (A) was related to DPC4/SMAD4 inactivation, those of triptolide (B) and Parp1 inhibitor (C) were related to TP53 inactivation, and that of MMC (D) was related to P16 inactivation. Wilcoxon rank sum tests were used to compare differences in the median IC50 by mutation status. “md” indicates either mutation or deletion and the “wt” indicates wildtype. For TP53.md group in panel C, solid circles indicate TP53 mutated PC cell lines, and triangles indicate TP53 deleted PC cell lines.

Figure 5. Reponses of isogenic cancer cell lines to anticancer drugs

Two pairs of isogenic pancreatic cancer cell lines for DPC4/SMAD4 were tested with cisplatin (A), irinotecan (B) and gemcitabine (C). Results for Pa01C isogenic cells are shown, as they demonstrate higher TGFβ functional complementation. Three pairs of isogenic colon cancer cell lines for TP53 demonstrated differences in response for triptolide (D). Differences of IC50 value for isogenic pairs were compared with paired t-test analysis and significant P values indicated. IC50s were calculated for each experiment using 6 replicates per dose, and three separate experiments were used to calculate the mean IC50 for each drug and cell line combination (18 replicate wells totally per dose).

Figure 6. Chemosensitivity correlation analysis of PC cell lines using evolutionary trees and cluster analysis

Cluster analysis was employed to analyze chemosensitivity correlation of PC cell lines, and a threshold of 0.65 (spearman correlation) indicates a positive correlation. Panel A is a clustered heatmap and panel B is a network map for PC cell lines. Different drugs clustered because of similar drug response profiles for PC cell lines (A). In panel B, the dots represent individual cell lines. Cell lines are connected by a line to every other sample with which it was correlated by at least 0.65. Related cancer cells are demonstrated by “boxes”(thin arrows) with “Xs” in the center for 4 cell lines and triangles between 3 cell lines (thick arrows). The line thickness indicates the strength of the correlation. Individual points mean that sample does not correlate with anyone else.