Acquired resistance to trastuzumab/pertuzumab or to T-DM1 in vivo can be overcome by HER2 kinase inhibition with TAS0728.

HER2-targeting antibodies (trastuzumab, pertuzumab) and a HER2-directed antibody-drug conjugate (trastuzumab emtansine: T-DM1) are used for the treatment of HER2-overexpressing breast cancer. However, these treatments eventually become ineffective due to acquired resistance and there is an urgent need for alternative therapies. TAS0728 is a small-molecule, irreversible selective HER2 kinase inhibitor. In the present study, we established new in vivo models of cancer resistance by continuous exposure to a combination of trastuzumab and pertuzumab or to T-DM1 for evaluating the effect of TAS0728 on HER2 antibody-resistant populations. Treatment with trastuzumab and pertuzumab or with T-DM1 initially induced tumor regression in NCI-N87 xenografts. However, tumor regrowth during treatment indicated loss of drug effectiveness. In tumors with acquired resistance to trastuzumab and pertuzumab or to T-DM1, HER2-HER3 phosphorylation was retained. Switching to TAS0728 resulted in a significant anti-tumor effect associated with HER2-HER3 signal inhibition. No alternative receptor tyrosine kinase activation was observed in these resistant tumors. Furthermore, in a patient-derived xenograft model derived from breast cancer refractory to both trastuzumab/pertuzumab and T-DM1, TAS0728 exerted a potent anti-tumor effect. These results suggest that tumors with acquired resistance to trastuzumab and pertuzumab and to T-DM1 are still dependent on oncogenic HER2-HER3 signaling and are vulnerable to HER2 signal inhibition by TAS0728. These results provide a rationale for TAS0728 therapy for breast cancers that are refractory to established anti-HER2 therapies.

Activity of TAS4464, a novel NEDD8 activating enzyme E1 inhibitor, against multiple myeloma via inactivation of nuclear factor κB pathways.

The ubiquitin proteasome pathway is essential for the proliferation and survival of multiple myeloma (MM) cells. TAS4464, a novel highly potent inhibitor of NEDD8 activating enzyme, selectively inactivates cullin-RING ubiquitin E3 ligases, resulting in accumulation of their substrates. Here, we examined 14 MM cell lines treated with TAS4464. TAS4464 induced growth arrest and cell death in the MM cell lines even in the presence of bone marrow stromal cells. It also induced the accumulation of phospho-inhibitor of κBα and phospho-p100, impaired the activities of nuclear factor κB (NF-κB) transcription factors p65 and RelB, and decreased the expression of NF-κB target genes, suggesting that TAS4464 inhibits both the canonical and non-canonical NF-κB pathways. TAS4464 had similar effects in an in vivo human-MM xenograft mouse model in which it was also observed to have strong antitumor effects. TAS4464 synergistically enhanced the antitumor activities of the standard MM chemotherapies bortezomib, lenalidomide/dexamethasone, daratumumab and elotuzumab. Together, these results suggest that the anti-MM activity of TAS4464 occurs via inhibition of the NF-κB pathways, and that treatment with TAS4464 is a potential approach for treating MM by single and combination therapies.

Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS) expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU) treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.

Substitution of anti-androgens and tegafur-uracil combination therapy for castration-resistant prostate cancer: results of a multi-center randomized phase II study.

We conducted this study to determine whether substitution with anti-androgen (SOA) and tegafur-uracil (a pro­drug of 5-FU) combination therapy is more effective than SOA alone after relapse from initial hormonal therapy. Patients who were histologically confirmed and relapsed after initial hormonal therapy were included. All patients were randomly allocated into two groups: SOA alone (group A) or SOA combined with tegafur-uracil (group B). The mRNA expression of four enzymes, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phospho-ribosyltransferase (OPRT) and thymidine phosphorylase (TP), in prostate cancer cells was analyzed by quantitative reverse-transcription polymerase chain reaction. Fifty-two patients were enrolled in this study. The median age was 77 (range: 47-92) years. The PSA response rate in group B (61.5%) tended to be higher compared to that in group A (34.6%) (p=0.095). Group B (median: 15.9 months) had a significantly longer time to PSA progression (TTP) compared to group A (6.4 months) (p=0.014). In patients with a lower TS expression or a higher OPRT expression, group B demonstrated a higher PSA response rate compared to group A (p=0.019 and p=0.041, respectively). In addition, in the patients with a lower TS expression, group B demonstrated a significantly longer TTP compared to group A (p=0.018). There were no severe adverse events in either treatment group. After relapse from initial hormonal therapy, SOA combined with tegafur-uracil is effective and well tolerated. The TS mRNA expression level may be a predictive factor for this combination therapy.

Up-regulation of insulin-like growth factor-binding protein 3 by 5-fluorouracil (5-FU) leads to the potent anti-proliferative effect of androgen deprivation therapy combined with 5-FU in human prostate cancer cell lines.

In this study, we investigated the synergistic mechanism of anti-androgen and 5-fluorouracil (5-FU) combination therapy against castration-resistant prostate cancer (CRPC). Four prostate cancer cell lines, LNCaP, 22Rv1, DU145 and PC3, were examined for their growth dependency on androgens and the insulin-like growth factor 1 (IGF1). We assessed the expression changes of certain growth factor receptors and regulating proteins when treated with 5-FU, and found that 5-FU increased the expression of the IGF-binding protein 3 (IGFBP3). Furthermore, 5-FU inhibited the phosphorylation of Akt and p70 S6K, while the knockdown of IGFBP3 reduced the levels of poly (ADP-ribose) polymerase cleaved by 5-FU in PC3 cells. Therefore, the up-regulation of IGFBP3 by 5-FU not only inhibits cell growth by reducing the IGF1 signal but also induces apoptosis in PC3 cells. The synergistic effect of bicalutamide and 5-FU on 22Rv1 cells was reduced by IGFBP3 gene silencing using small-interfering RNA. These results suggest that the up-regulation of IGFBP3 induced by 5-FU plays an important role in the potent anti-tumor effect of 5-FU combined with anti-androgens on CRPC. Androgen-deprivation therapy combined with 5-FU could therefore be an appropriate therapy for CRPC patients.

Hydroxyflutamide enhances cellular sensitivity to 5-fluorouracil by suppressing thymidylate synthase expression in bicalutamide-resistant human prostate cancer cells.

We investigated the antitumor effects of combination therapy with anti-androgens and 5-fluorouracil (5-FU), and examined the underlying mechanism of the treatment. Initially, we established the bicalutamide-resistant subline CDX25R from the androgen receptor (AR)-positive human prostate cancer cell line LNCaP through continuous exposure to bicalutamide. CDX25R cells lost the ability to respond to androgens, but still expressed AR. They showed significant resistance to bicalutamide, but had high sensitivity to hydroxyflutamide (OH-flutamide) compared with LNCaP cells. The CDX25R subline was thus considered to be a suitable model for prostate cancer that has developed resistance to first-line hormonal therapy but shows sensitivity to an alternative approach. Combined treatment with 5-FU and OH-flutamide had a synergistic effect on CDX25R cells. OH-flutamide decreased expression of the transcription factor E2F1, and subsequently of thymidylate synthase (TS), in CDX25R cells but not in AR-negative DU145 cells. This suggested that OH-flutamide enhanced the growth-inhibitory activity of 5-FU in CDX25R cells by reducing TS expression through the AR pathway. Combined therapy with 5-FU and OH-flutamide may, therefore, be appropriate for patients with prostate cancer that has acquired resistance to initial hormone therapy including bicalutamide.

[DLST as a method for detecting TS-1-induced allergy].

Drug-induced allergic adverse events including rash, interstitial pneumonia and hepatic injury are often observed in a few patients treated with anticancer drugs that are 5-FU derivatives, including TS-1. In patients suspected to be liable to develop allergic reactions, the drug-induced lymphocyte stimulation test (DLST), based on the (3)H-thymidine incorporation ratio into the DNA of lymphocytes derived from the patients, is generally employed to identify drugs that could induce allergy. In this case report, we conducted the DLST on 20 healthy volunteers without TS-1 treatment in order to obtain reference data on the evaluation of TS-1-induced allergy. Even though all 20 volunteers were healthy, there were 6 positive responses to the DLST. In view of the positive response to TS-1 in healthy volunteers undergoing the DLST, it is suggested that the DLST could show a false positive response through an intracellular function that accelerates incorporation of thymidine in the lymphocytes by the salvage pathway after inhibition of DNA de novo synthesis caused by 5-FU derivative anticancer, including TS-1. Therefore, such a positive response to the drugs is considered, in fact, to be false-positive in the DLST. In view of the occurrence of false-positive results, the possibility of drug-induced allergy in patients receiving TS-1 should be carefully evaluated using a combination of other clinical examinations.