Sensitivity of Colorectal Cancer to Arginine Deprivation Therapy is Shaped by Differential Expression of Urea Cycle Enzymes

Abstract

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

Figure 1

Arginine auxotrophy in CRC. (A) Growth curves of the indicated cell lines with or without arginine supplementation. Data are presented as mean ± SEM of three independent experiments. (B) Representative flow cytometry scatterplots of EdU incorporation in HCT116, RKO, HT29 and SW480 CRC cell lines grown in control medium or arginine-free medium for 24 h. EdU was measured using the Click-iT® EdU Alexa Fluor® kit and total DNA stained using FxCycle™ Violet Stain. (C) Quantification of EdU incorporation from three independent experiments. Data are presented as mean ± SEM, two-tailed t-test. *P < 0.05, ***P < 0.001. (D) Western blot analysis of Cyclin D1 (CycD1) protein expression in HCT116, RKO and SW480 CRC cell lines following arginine deprivation for the indicated times. Actin was used as endogenous loading control. Original western blots are reported in Supplementary Fig. S15. (E) Graph showing the growth of xenografted HCT116 cells in immunocompromised mice fed a control diet or an arginine-free diet. Data are plotted as mean ± SEM and were analyzed using mixed linear regression analysis (P = 0.03; P < 0.05 indicates a statistically significant difference in tumor growth rate between control and treated animals over time). (F) Weight of excised tumor measured at endpoint. Data are plotted as mean ± SEM. *P < 0.05, two-tailed t-test (n = 4 animal per group). (G) Animal body weights at endpoint plotted as mean ± SEM. No statistical differences were detected between the two diet groups, two-tailed t-test (n = 4 animals per group).

Figure 2

Reduced expression of OTC and ASS1 in CRC. (A) OTC and (B) ASS1 H-score were assessed on CRC TMA. Representative histo-spots are shown on the left. The bar graphs indicate H-score distribution according to Duke’s stage, whereas the distribution of protein expression within the whole TMA cohort is reported in the pie chart. *P < 0.05 (C) Western blot analysis of OTC and ASS1 after 72 h treatment with 5 µM 5-Azacytidine (5-AZA). Human liver extract was used as positive control for urea cycle enzymes, actin was used as endogenous loading control. Original western blots are reported in Supplementary Fig. S15.

Figure 3

CRC cells are sensitive to arginase treatment. (A) Dose-Response Non-Linear Regression Curves and IC₅₀ values of the indicated CRC cell lines treated with the rhArg1peg5000. The percentage (%) of cell growth was calculated relative to the cell numbers in corresponding PBS-treated control samples, which was selected as 100%. IC₅₀ values were obtained from non-linear regression analysis of concentration of the drug vs response curves. The results were obtained from three independent experiments. Quadruplicate samples were assessed for cell growth after a 6-day period of treatment by cell counting for each individual experiment. The error bars represent ± SEM. (B) Representative flow cytometry scatterplots of EdU incorporation in HCT116, RKO, HT29 and SW480 CRC cell lines after 72 h treatment with rhArg1peg5000 (0.5 μg/mL) or PBS-vehicle control. EdU was measured using the Click-iT® EdU Alexa Fluor® kit and total DNA stained using FxCycle™ Violet Stain. (C) Quantification of EdU incorporation from three independent experiments. Data are presented as mean ± SEM. *P < 0.05, ****P < 0.0001, two-tailed t-test. (D) Western blot analysis of the cell cycle markers Cyclin D1 and D3 in cells treated for the indicated time with rhArg1peg5000 (0.5 μg/mL). Actin was used as endogenous loading control. (E) Quantification of Cyclin D1 and D3 protein expression from triplicate experiments. Original western blots are reported in Supplementary Fig. S15. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA.

Figure 4

Pharmacological depletion of arginine using rhArg1peg5000 reduces tumor growth in vivo. (A) Tumor growth in athymic nude mice subcutaneously injected with 10⁶ RKO or SW480 CRC cells. Mice were randomized into Control (n = 8) and Treatment (n = 8) groups. Treated mice were administered intraperitoneally with 0.5 mg of rhArg1peg5000/animal twice a week, while control mice were injected with an equal volume of PBS. Data are plotted as mean ± SEM and were analyzed using mixed linear regression analysis (P = 0.012 for RKO and P = 0.03 for SW480, P < 0.05 indicates a statistically significant difference in tumor growth rate between control and treated animals over time). (B) Western blotting of lysates from tumor xenografts for assessment of urea cycle enzymes OTC and ASS1. Human liver lysate was used as a positive control and actin as endogenous loading control. Original western blots are reported in Supplementary Fig. S15.

Figure 5

CRC cells are sensitive to arginine deiminase treatment. (A) Dose-Response Non-Linear Regression Curves and IC₅₀ values of the indicated CRC cell lines treated with the ADI-PEG20. The percentage (%) of cell growth was calculated relative to the cell numbers in corresponding PBS-treated control samples, which was selected as 100%. IC₅₀ values were obtained from non-linear regression analysis of concentration of the drug vs response curves. The results were obtained from three independent experiments. Quadruplicate samples were assessed for cell growth after a 6-day period of treatment by cell counting for each individual experiment. The error bars represent ±SEM. (B) Representative flow cytometry scatterplots of EdU incorporation in HCT116, RKO, HT29 and SW480 CRC cell lines after 72 h treatment with ADI-PEG20 (1 μg/mL) or PBS-vehicle control. EdU was measured using the using Click-iT® EdU Alexa Fluor® kit and total DNA stained using FxCycle™ Violet Stain. (C) Quantification of EdU incorporation from three independent experiments. Data are presented as mean ± SEM. *P < 0.05, ****P < 0.0001, two-tailed t-test. (D) Western blot analysis of the cell cycle markers Cyclin D1 and D3 and the urea cycle enzyme ASS1 in cells treated for the indicated time with ADI-PEG20 (1 μg/mL). Actin was used as endogenous loading control. Original western blots are reported in Supplementary Fig. S15. (E) Quantification of Cyclin D1 and D3 protein expression from triplicate experiments. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA.

Figure 6

Pharmacological depletion of arginine using ADI-PEG20 reduces tumor growth in vivo. (A) Tumor xenografts growth in athymic nude mice subcutaneously injected with 10⁶ RKO CRC cells. Mice were randomized into Control (n = 8) and Treatment (n = 8) groups. Treated mice were administered intraperitoneally (IP) with 5IU of ADI-PEG20 per animal per week, while control mice were injected with equal volume of PBS. Data are plotted as mean ± SEM and were analyzed using mixed linear regression analysis (P = 0.045, P < 0.05 indicates a statistically significant difference in tumor growth rate between control and treated animals over time). (B) Representative images of ki67-stained xenograft tumors and ki67 proliferation index of tumors from vehicle-treated and ADI-PEG20-injected animals. Data are presented as mean ± SEM (n = 7 animals per group). *P < 0.05, two-tailed t-test. Size bars = 100 μm. (C) Western blot analysis of the cell cycle markers Cyclin D1 and D3 and urea cycle enzyme ASS1 in xenograft tumors isolated from PBS-injected controls and ADI-PEG20-treated animals. Actin was used as endogenous loading control. Original western blots are reported in Supplementary Fig. S15. (D) Graph bars show quantification of Cyclins levels using Image J software. Data are presented as mean ± SEM (n = 7 animals per group). *P < 0.05, two-tailed t-test. (E) Representative images of ASS1-stained xenograft tumors and ASS1 H-score index of tumors from vehicle-treated and ADI-PEG20-injected animals. Data are presented as mean ± SEM (n = 7 animals per group). **P < 0.01, two-tailed t-test. Size bars = 100 μm.