Hiroyuki Okabe

Efficacy of combination chemotherapy using a novel oral chemotherapeutic agent, TAS-102, together with bevacizumab, cetuximab, or panitumumab on human colorectal cancer xenografts

TAS-102 is a novel oral nucleoside antitumor agent that consists of trifluridine (FTD) and tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5, and was approved in Japan in March 2014 for the treatment of patients with unresectable advanced or recurrent colorectal cancer that is refractory to standard therapies. In the present study, we used colorectal cancer xenografts to assess whether the efficacy of TAS-102 could be improved by combining it with bevacizumab, cetuximab or panitumumab. TAS-102 was orally administered twice a day from day 1 to 14, and bevacizumab, cetuximab and panitumumab were administered intraperitoneally twice a week for 2 weeks. Growth inhibitory activity was evaluated based on the relative tumor volume (RTV) after 2 weeks of drug administration and time taken for the relative tumor volume to increase five-fold (RTV5). Tumor growth inhibition and RTV5 with TAS-102 and bevacizumab combination treatment were significantly better than those with TAS-102 or bevacizumab alone in the SW48 and HCT116 tumor models, and the concentration of phosphorylated FTD in tumors determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was higher in the TAS-102 and bevacizumab combination group than in the TAS-102 monotherapy group. The combination of TAS-102 and cetuximab or panitumumab was also significantly more effective than either monotherapy in the SW48 tumor model. There was no significant difference in the body weight between the mice treated with TAS-102 monotherapy and any of the combination therapies on day 29. Our preclinical findings indicate that the combination therapy of TAS-102, bevacizumab and cetuximab or panitumumab is a promising treatment option for colorectal cancer.

Induction of thymidine phosphorylase by interferon and taxanes occurs only in human cancer cells with low thymidine phosphorylase activity

Thymidine phosphorylase (TP) regulates intracellular thymidine metabolism. It has been reported to be a prognostic factor for tumor angiogenesis and to activate some prodrugs of 5-fluorouracil (5-FU) to 5-FU. There is also evidence that TP is induced by interferons (IFNs) and xenobiotics, such as cyclophosphamide and taxanes, in experimental human cancer cells and xenografts. We investigated the induction of TP expression by IFNalpha and Paclitaxel in vitro and in vivo in human tumor cells with low and with high TP activity. TP activity in KB, NUGC-3, and KOC2S cells, which had low TP activity, was increased 2 to 4 fold by IFNalpha, but was still lower than in non-treated SHIN-3 and HRA cells, which have high TP activity. IFNalpha did not promote TP activity in SHIN-3 and HRA cells, but expression of TP mRNA increased 2 to 4 fold in response to IFNalpha in all cells tested. These results suggest that the expression of TP protein would be regulated post-transcriptionally by another factor after IFN-induced amplification of TP mRNA. A single dose of Paclitaxel to nude mice xenografted with KB and KM20C tumors, expressing low TP activity, increased TP activity about 4 to 7 fold compared to non-treated tumors. In contrast, TP expression in MX-1 and H-31 tumors was originally high and did not change by the treatment of Paclitaxel. The activities of uridine phosphorylase in all tumors used showed no changes in response to IFNalpha or Paclitaxel. We determined the level of STAT1alpha, an IFN-inducible transcription factor of the TP gene, and found that it was low in low TP expressing tumor cells and markedly increased to about 4 fold by IFN, almost reaching the level in high TP expressing cells whose STAT1alpha level was unchanged by IFN. When TP activity and STAT1alpha expression in clinically resected colorectal cancers were simultaneously measured, almost all tumors had high expression of both TP and STAT1alpha. In conclusion, our results suggest that IFN and Paclitaxel affect human cancer cells with low TP activity but not those with high TP activity and that the STAT1alpha expression may reflect TP activity, at least in experimental human cancer cells.

Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA

Trifluridine (FTD) and 2′-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA.

Abstract 1783: Trifluorothymidine incorporation into DNA strongly enforces the potential of TAS-102 and leads to remarkably prolonged survival of cancer patients

TAS-102 is a novel oral antitumor agent, consisting of trifluorothymidine (FTD) as the active component and thymidine phosphorylase inhibitor (TPI) which prevents degradation of FTD. In a double-blind randomized phase II study conducted in Japan, TAS-102 significantly improved overall survival of metastatic colorectal cancer patients, compared with placebo (TAS-102, n = 112; Placebo, n = 57; median OS, 9.0 vs. 6.6 months; HR, 0.56; p = 0.001). We investigated mechanisms of action of TAS-102 to understand how prognosis of cancer patients was improved. In vitro study, FTD was previously reported to have two mechanisms, FTD incorporation into DNA and thymidylate synthase (TS) inhibition. In this study, we show that FTD incorporation into DNA is the main mechanism of TAS-102 and the key to potentiate anti-tumor efficacy of FTD. We found that although TS inhibition of FTD reduced rapidly after drug removal, the cytotoxicity of FTD did not diminish. On the other hand, the cytotoxicity of FdUrd diminished after drug removal. As compared to FdUrd, FTD was incorporated into DNA to a greater extent by the time drug was removed. It is thought that the FTD retained in DNA contributes to sustained growth inhibition. Therefore, FTD incorporation into DNA is considered to be essential for enforcing the potential of FTD. Additionally, we investigated the in vivo potency of TAS-102 by using daily divided oral administration since this regimen was the most optimal to maximize FTD incorporation into DNA. Interestingly, TAS-102 showed marked tumor growth delay even after end of the administration and remarkable prolonged survival of mice implanted with KM20C derived from human colon cancer. Collectively, these data suggest that TAS-102 improved survival of cancer patients primarily due to FTD incorporation into DNA. FTD is previously reported to affect DNA structure, and we speculate that TAS-102 exerts its anti-tumor effects through interfering with DNA function once adequate amounts of FTD have been accumulated in the DNA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1783. doi:1538-7445.AM2012-1783

Effective Sequential Combined Chemotherapy with Trifluridine/Tipiracil and Regorafenib in Human Colorectal Cancer Cells

Salvage chemotherapy for refractory metastatic colorectal cancer using trifluridine/tipiracil (FTD/TPI) and regorafenib has shown survival benefits. We evaluated the antitumor effects of FTD or FTD/TPI combined with regorafenib in vitro and in vivo. SW620, HCT 116, and HT-29 human colorectal cancer cell lines were treated with FTD and regorafenib simultaneously and sequentially. Cell death, incorporation of FTD into DNA, and molecules related to FTD and regorafenib-associated cell death were investigated. The antitumor effects of FTD combined with regorafenib in SW620 and COLO205 xenografts were also evaluated. Cell death was greater after sequential treatment with FTD followed by regorafenib in SW620 cells, but not in HCT 116 and HT-29 cells, than after treatment with FTD alone, which was attributable to thymidylate synthase reduction with the induction of apoptosis. In contrast, simultaneous and sequential exposure to regorafenib followed by FTD, but not FTD alone, attenuated the cell death effect. Furthermore, combined FTD/TPI treatment followed by regorafenib had greater antitumor activity than either monotherapy in SW620 and COLO205 xenograft models. Treatment results following regorafenib administration subsequent to FTD or FTD/TPI suggest that sequential therapy with FTD/TPI prior to regorafenib may be effective in a clinical setting.