Development of a resistance-like phenotype to sorafenib by human hepatocellular carcinoma cells is reversible and can be delayed by metronomic UFT chemotherapy

Abstract

Acquired resistance to antiangiogenic drugs, such as sorafenib, is a major clinical problem. We studied development of a resistance to sorafenib in new preclinical models of human hepatocellular carcinoma (HCC) along with a strategy to delay such resistance--combination with metronomic chemotherapy. Three different xenograft models were studied using human Hep3B HCC cells, which are highly responsive to sorafenib, namely, orthotopic and subcutaneous transplant in severe combined immunodeficient mice, and an orthotopic transplant in nude mice. The complementary DNA for the β-subunit of human choriogonadotropin was transfected into HCC cells, and urine levels of the protein were monitored as a surrogate of tumor burden. Extended daily treatments, sometimes interrupted by a break period of 3 to 7 days to allow recovery from toxicity at sorafenib doses of 30 to 60 mg/kg, were maintained until and after evidence of tumor relapse. Initially responsive tumors seemed to develop a resistance-like phenotype after long-term daily treatment (e.g., >42 days) at doses of 30 to 60 mg/kg. Transplantation of cell lines established from progressing tumors into new hosts showed that the resistant phenotype was not propagated. Furthermore, a regimen of daily metronomic uracil + tegafur (UFT, an oral 5-fluorouracil prodrug) chemotherapy with a less toxic regimen of sorafenib (15 mg/kg per day) significantly delayed the onset of resistance (>91 days). In conclusion, development of a resistance-like phenotype to sorafenib is reversible, and metronomic UFT plus sorafenib may be a promising and well-tolerated treatment for increasing efficacy by delaying emergence of such resistance.

Figure 1

Effect of sorafenib regimens on orthotopically implanted human Hep3B HCC cells in SCID mice. Hep3B cells were tagged with β-hCG and implanted into the left lobe of the liver (n = 5 per treatment group). (A) Urine β-hCG levels of pooled samples, normalized to urine creatinine levels, of mice treated with sorafenib monotherapies (at 15, 30, or 60 mg/kg per day by gavage) or sorafenib (at 30 mg/kg per day) combined with eithermetronomic CTX (20mg/kg per day, per os) ormetronomic UFT (15 mg/kg per day by gavage). Treatments were carried out during the periods indicated with breaks in sorafenib dosing starting when toxicity (i.e., > 10% average body weight loss) was observed in each treatment group. The graph shows the beneficial effect of the sorafenib regimens compared with controls treated by gavage of vehicle alone, on the relative tumor burden. Green asterisk indicates amouse treated with 15 mg/kg per day of sorafenib that died at that time point. Red asterisk indicates a mouse treated with 60 mg/kg per day of sorafenib that died at that time point. (B) Corresponding weight measurements of the tumor mass in the liver of mice in each treatment group at the time of sacrifice (note that because of the different treatment efficacies, mice fromthe groupswere killed at differentweeks after the initiation of treatment, as indicated). (C) Correspondingmouse weight curves as a measure of relative toxicity of the different sorafenib treatment regimens. Note that for individual groups, sorafenib treatment was suspended for the indicated times whenever toxicity was observed in each group.

Figure 2

Effect of sorafenib regimens on subcutaneously implanted Hep3B cells in SCID mice. Hep3B cells were tagged with β-hCG and implanted into the left flank of SCID mice (n = 5 per treatment group). (A) Urine β-hCG levels of pooled samples, normalized to urine creatinine levels, of mice treated with sorafenib monotherapies (at 15, 30, or 60 mg/kg per day by gavage). Treatments were carried out during the periods indicated with breaks in sorafenib dosing starting when toxicity (i.e., > 10% average body weight loss) was observed in each treatment group. The graph shows the beneficial effect of the sorafenib regimens, compared with controls treated by gavage of vehicle alone, on the relative tumor burden. (B) Corresponding tumor volume curvesmirror the beneficial effect of the sorafenib regimens compared with controls. (C) Corresponding weightmeasurements of the tumormass of mice in each treatment group at the time of sacrifice (note that because of the different treatment efficacies, mice from the groups were killed at different weeks after the initiation of treatment, as indicated). (D) Corresponding mouse weight curves as a measure of relative toxicity of the different sorafenib regimens. Note that for individual groups, sorafenib treatment was suspended for the indicated times whenever toxicity was observed in each group.

Figure 3

Comparison of slopes of early phase responding tumors and during the later relapse phase of tumor “relapse” of Hep3B subcutaneous tumors in SCID mice. Upper panel shows the slopes of tumor growth curves based on tumor volume measurements. (A) The slope of the early phase tumor volume growth curve from day 7 to 38 in the 30-mg/kg per day of sorafenib-treated mice. (B) Slope of the relapse phase of the tumor volume growth curve, fromday 42 to 84 in 30mg/kg per day of sorafenib-treated mice. (C) Slope of the early phase of the tumor volume growth curve from day 7 to 38 in the 60-mg/kg per day of sorafenib-treated mice. (D) Slope of the relapse phase of the tumor volume growth curve fromday 42 to 84 in 60mg/kg per day of sorafenib-treated mice. Lower panel shows the slopes, based on β-hCG growth curves. (E) Slope of the early phase of the β-hCG curve fromday 7 to 35 in the 30-mg/kg per day of sorafenib-treated mice. (F) Slope of the relapse phase of the β-hCG curve from day 42 to 84 in the 30-mg/kg per day sorafenib-treated mice. (G) Slope of the early phase of the β-hCG growth curve from day 7 to 35 in the 60-mg/kg per day of sorafenib-treated mice. (H) Slope of the relapse phase of the β-hCG growth curve from day 42 to 84 in the 60-mg/kg per day of sorafenib-treated mice.

Figure 4

Effect of sorafenib regimens on orthotopically implanted Hep3B variants derived from tumors that developed resistance to sorafenib. The cell lines shown in this figure are from a single tumor from each treatment group, and these were implanted into the left lobe of the liver of SCID mice (n = 5 per treatment group). The variants tested are (i and ii) Hep3B parental cells, (iii and iv) Nex30-OT1 (a cell line obtained from an orthotopic tumor that relapsed on 30 mg/kg per day sorafenib), (v and vi) Nex60-OT (a cell line obtained from an orthotopic tumor that relapsed on 60 mg/kg per day of sorafenib), (vii and viii) Nex30-SC (a cell line obtained from a subcutaneous tumor that relapsed on 30 mg/kg per day of sorafenib), and (xi and x) Nex60-SC (a cell line obtained from a subcutaneous tumor that relapsed on 30 mg/kg per day of sorafenib). Left panels: Urine hCG levels of pooled samples, normalized to urine creatinine levels, of mice treated with sorafenib monotherapies (at 30 or 60 mg/kg per day by gavage). Compared with control, both sorafenib regimens impaired the growth of the various Hep3B variants comparable to parental Hep3B. Right panels: Corresponding mouse weight curves as a measure of relative toxicity of the different sorafenib regimens.

Figure 5

Effect of sorafenib regimens on orthotopically implanted Hep3B-hCG cells in nude mice. Green asterisk indicates a mouse treated with 30 mg/kg per day of sorafenib that died at that time point. Red asterisk indicates a mouse treated with 60 mg/kg per day sorafenib that died at that time point. (A) Urine hCG levels of pooled samples, normalized to urine creatinine levels, of mice treated with sorafenib monotherapies (at 30 or 60mg/kg per day by gavage). Hep3-hCG tumor growth impairment by sorafenib is similar to the effects seen in SCID mice. (B) However, corresponding mouse weight curves as a measure of relative toxicity reveal a lower degree of toxicity in nude compared with SCID mice.

Figure 6

Effect of reduced-dose sorafenib combined with metronomic UFT on orthotopically implanted Hep3B-β-hCG cells in SCID mice (n = 5 per treatment group). (A) Urine β-hCG levels of individual mice, normalized to urine creatinine levels, treated with sorafenib alone (15 mg/kg per day), metronomic UFT alone (15mg/kg per day), or sorafenib andmetronomicUFT combination therapy (15mg/kg per day each). Although the benefit of the corresponding monotherapies is modest, sorafenib and metronomic UFT combination therapy delays the occurrence of treatment resistance. (B) Kaplan-Meier survival curves confirm the superior treatment benefit of the combination regimen, whereas no significant weight loss is observed. (C) MS indicates median survival.