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Tumors consist of heterogeneous cell populations that contain cancer cell subpopulations with anticancer drug‐resistant properties called “persister” cells. While this early‐phase drug tolerance is known to be related to the stem cell‐like characteristic of persister cells, how the stem cell‐related pathways contribute to drug resistance has remained elusive. Here, we conducted a single‐cell analysis based on the stem cell lineage‐related and gastric cell lineage‐related gene expression in patient‐derived gastric cancer cell models. The analyses revealed that 5‐fluorouracil (5‐FU) induces a dynamic change in the cell heterogeneity. In particular, cells highly expressing stem cell‐related genes were enriched in the residual cancer cells after 5‐FU treatment. Subsequent functional screening identified aldehyde dehydrogenase 1A3 (ALDH1A3) as a specific marker and potential therapeutic target of persister cells. ALDH1A3 was selectively overexpressed among the ALDH isozymes after treatment with 5‐FU or SN38, a DNA topoisomerase I inhibitor. Attenuation of ALDH1A3 expression by RNA interference significantly suppressed cell proliferation, reduced the number of persister cells after anticancer drug treatment and interfered with tumor growth in a mouse xenograft model. Mechanistically, ALDH1A3 depletion affected gene expression of the mammalian target of rapamycin (mTOR) cell survival pathway, which coincided with a decrease in the activating phosphorylation of S6 kinase. Temsirolimus, an mTOR inhibitor, reduced the number of 5FU‐tolerant persister cells. High ALDH1A3 expression correlated with worse prognosis of gastric cancer patients. These observations indicate that the ALDH1A3‐mTOR axis could be a novel therapeutic target to eradicate drug‐tolerant gastric cancer cells. Using a single‐cell analysis of patient‐derived gastric cancer cells, we identified aldehyde dehydrogenase 1A3 (ALDH1A3) as a therapeutic target of anticancer drug‐tolerant persister cells. ALDH1A3 knockdown significantly suppressed cell proliferation and reduced the number of persister cells after treatment with 5‐fluorouracil (5‐FU) and SN38. Mechanistically, ALDH1A3 depletion downregulated the mTOR cell survival pathway, and an mTOR inhibitor reduced the number of drug‐tolerant persister cells.

Cancer cells adopt multiple strategies to escape tumor surveillance by the host immune system and aberrant amino acid metabolism in the tumor microenvironment suppresses the immune system. Among the amino acid‐metabolizing enzymes is an L‐amino‐acid oxidase called interleukin‐4 induced 1 (IL4I1), which depletes essential amino acids in immune cells and is associated with a poor prognosis in various cancer types. Although IL4I1 is involved in immune metabolism abnormalities, its effect on the therapeutic efficacy of immune checkpoint inhibitors is unknown. In this study, we established murine melanoma cells overexpressing IL4I1 and investigated their effects on the intratumor immune microenvironment and the antitumor efficacy of anti‐programmed death‐ligand 1 (PD‐L1) antibodies (Abs) in a syngeneic mouse model. As a result, we found that IL4I1‐overexpressing B16‐F10‐derived tumors showed resistance to anti‐PD‐L1 Ab therapy. Transcriptome analysis revealed that immunosuppressive genes were globally upregulated in the IL4I1‐overexpressing tumors. Consistently, we showed that IL4I1‐overexpressing tumors exhibited an altered subset of lymphoid cells and particularly significant suppression of cytotoxic T cell infiltration compared to mock‐infected B16‐F10‐derived tumors. After treatment with anti‐PD‐L1 Abs, we also found a more prominent elevation of tumor‐associated macrophage (TAM) marker, CD68, in the IL4I1‐overexpressing tumors than in the mock tumors. Consistently, we confirmed an enhanced TAM infiltration in the IL4I1‐overexpressing tumors and a functional involvement of TAMs in the tumor growth. These observations indicate that IL4I1 reprograms the tumor microenvironment into an immunosuppressive state and thereby confers resistance to anti‐PD‐L1 Abs. Interleukin‐4 induced 1 overexpression upregulates immunosuppressive gene expression, decreases CD8+ T cell infiltration, and causes resistance to the anti‐ programmed death‐ligand‐1 Ab therapy in a mouse melanoma model.

The vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis is an essential regulator of angiogenesis and important therapeutic target in cancer. Ramucirumab is an anti-VEGFR2 monoclonal antibody used for the treatment of several cancers. Increased circulating VEGF-A levels after ramucirumab administration are associated with a worse prognosis, suggesting that excess VEGF-A induced by ramucirumab negatively affects treatment efficacy and that neutralizing VEGF-A may improve treatment outcomes. Here, we evaluated the effect of combination treatment with an anti-VEGFR2 antibody and anti-VEGF-A antibody on gastric tumor progression and normal tissues using a preclinical BALB/c-nu/nu mouse xenograft model. After anti-VEGFR2 antibody treatment in mice, a significant increase in plasma VEGF-A levels was observed, mirroring the clinical response. The elevated VEGF-A was host-derived. Anti-VEGF-A antibody co-administration enhanced the anti-tumor effect of the anti-VEGFR2-antibody without exacerbating the toxicity. Mechanistically, the combination treatment induced intra-tumor molecular changes closely related to angiogenesis inhibition and abolished the gene expression changes specifically induced by anti-VEGFR2 antibody treatment alone. We particularly identified the dual treatment-selective downregulation of ZEB1 expression, which was critical for gastric cancer cell proliferation. These data indicate that the dual blockade of VEGF-A and VEGFR2 is a rational strategy to ensure the anti-tumor effect of angiogenesis-targeting therapy.

Recent progress in the development of molecular cancer therapeutics has revealed new types of antitumor drugs, such as Herceptin, Gleevec, and Iressa, as potent therapeutics for specific tumors. Our work has focused on molecular cancer therapeutics, mainly in the areas of drug resistance, apoptosis and apoptosis resistance, and survival‐signaling, which is related to drug resistance. In this review, we describe our research on molecular cancer therapeutics, including molecular mechanisms and therapeutic approaches. Resistance to chemotherapeutic drugs is a principal problem in the treatment of cancer. P‐Glycoprotein (P‐gp), encoded by the MDR1 gene, is a multidrug transporter and has a major role in multidrug resistance (MDR). Targeting of P‐gp by small‐molecular compounds and/or antibodies is an effective strategy to overcome MDR in cancer, especially hematologic malignancies. Several P‐gp inhibitors have been developed and are currently under clinical phased studies. In addition to the multi‐drug transporter proteins, cancer cells have several drug resistance mechanisms. Solid tumors are often placed under stress conditions, such as glucose starvation and hypoxia. These conditions result in topo II poison resistance that is due to proteasome‐mediated degradation of DNA topoisomerases. Proteasome inhibitors effectively prevent this stress‐induced drug resistance. Glyoxalase I, which is often elevated in drug‐ and apoptosis‐resistant cancers, offers another possibility for overcoming drug resistance. It plays a role in detoxification of methylglioxal, a side product of glycolysis, which is highly reactive with DNA and proteins. Inhibitors of glyoxalase I selectively kill drug‐resistant tumors that express glyoxalase I. Finally, the susceptibility of tumor cells to apoptosis induced by antitumor drugs appears to depend on the balance between pro‐apoptotic and survival (anti‐apoptotic) signals. PI3K‐Akt is an important survival signal pathway, that has been shown to be the target of various antitumor drugs, including UCN‐01 and geldanamycin, new anticancer drugs under clinical evaluation. Our present studies provide novel targets for future effective molecular cancer therapeutics. (Cancer Sci 2003; 94: 15–21)

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third‐generation EGFR‐tyrosine kinase inhibitor (EGFR‐TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR‐TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor‐like biological activity of lamellarin N into kinase inhibitor‐like activity. Ba/F3 and PC‐9 cells expressing the EGFR in‐frame deletion within exon 19 (del ex19)/T790M/C797S triple‐mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple‐mutant EGFR PC‐9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple‐mutant EGFR PC‐9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR‐TKI that prevents the development of cross‐resistance against known drugs in this class. The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs). One of these EGFR‐TKIs, lamellarin 14, is effective against the C797S mutant EGFR.

Background Tumours consist of heterogeneous cancer cells and are likely to contain drug-tolerant cell subpopulations, causing early relapse. However, treatment strategies to eliminate these cells have not been established. Methods We established gastric cancer patient-derived cells (PDCs) to examine the contribution of CD44 splicing variant 9 (CD44v9)-positive cells in gastric cancer drug tolerance. We performed gene expression signature-based in silico screening using JFCR_LinCAGE, our anticancer compound gene expression database and subsequent validation in BALB/c-nu/nu mouse xenograft to identify agents targeting the drug-tolerant cancer cells. Results CD44v9-positive cancer cells were enriched among residual cancer cells after treatment with SN-38, an active metabolic of irinotecan. CD44v9 protein was responsible for this drug resistance. We identified epidermal growth factor receptor (EGFR) inhibitors as agents that can target CD44v9-positive cell populations in gastric cancer PDCs. CD44v9 promoted cell proliferation, and EGFR inhibition attenuated CD44v9 protein expression through downregulation of the AKT and the ERK signalling pathways, leading to preferential suppression of CD44v9-positive cells. Importantly, EGFR inhibitors significantly reduced the number of residual cancer cells after cytotoxic anticancer drug treatment and enhanced the antitumor effect of irinotecan in vivo. Conclusions EGFR inhibitors could be potential agents to eradicate cytotoxic anticancer drug-tolerant gastric cancer cell populations.

Purpose Liver metastasis (LM) is associated with poor prognosis in patients with metastatic colorectal cancer (mCRC). Here, we investigated the prognostic utility of several serum factors in mCRC patients with or without LM who were treated with anti-angiogenic agents in first-line (FL) or salvage-line (SL) settings. Methods A combined cohort of 125 patients was analyzed in this single institute pooled analysis: FL cohort receiving bevacizumab ( n = 71) and SL cohort receiving regorafenib ( n = 54). Blood samples were obtained at baseline (BL) and during treatment, and serum factors were measured by ELISA. Overall survival (OS) was analyzed using Kaplan–Meier curves, the log-rank test, and Cox proportional hazard regression methods. Results In univariate analysis of the combined cohort, right-sided CRC, primary unresected tumor, wild-type KRAS , LM, ≥ 2 metastatic sites, and SL were associated with shorter OS; in multivariable analysis, LM and SL remained significant. Serum angiopoietin-2 (Ang-2) levels ≥ 2190.3 pg/ml and interleukin (IL)-8 levels ≥ 15.1 pg/ml at BL were significantly associated with LM. Using these cut-off values, patients with higher Ang-2 or IL-8 levels at BL had shorter OS than those with lower BL levels (Ang-2: hazard ratio [HR] 2.57, 95% confidence interval [CI] 1.47–4.51, P = 0.001; IL-8: HR 4.31, 95%CI 2.11–8.79, P < 0.001). High serum IL-8 level remained a significant predictor of shorter OS in multivariable analysis (HR 3.24, 95%CI 1.47–7.16, P = 0.004). Conclusion Circulating IL-8 and Ang-2 levels are associated with LM in mCRC patients. IL-8 may be a prognostic marker of response to anti-angiogenic therapy, regardless of the treatment timing.

In identifying biomarkers for anticancer drugs, the lack of objectivity in selecting candidate factors makes interpretation difficult. We performed preclinical analysis and a translational validation study to identify candidate biomarkers for regorafenib efficacy in metastatic colorectal cancer (mCRC). Using in silico COMPARE analysis with a human cancer cell line panel, JFCR39, we selected candidate biomarkers whose expression correlates with regorafenib sensitivity. We validated predictive values in mCRC patients receiving regorafenib (discovery, n = 53) and FTD/TPI (control, n = 16). Blood samples were obtained at baseline (BL), before the second cycle (2nd), and at progressive disease (PD), and biomarker levels were measured using ELISA. Our analysis showed that high matrix metalloproteinase (MMP)-14 expression was associated with a high sensitivity to regorafenib. In the discovery cohort, high MMP-14 levels at BL and PD were correlated with tumor shrinkage and longer progression-free survival (PFS). A subsequent analysis of other related factors further indicated that the patients with decreased MMP-9 levels at the 2nd had higher disease control rates, tumor shrinkage, longer PFS, and overall survival than those with increased changes. These findings were not observed in the control cohort. Our study suggests MMP-14 and MMP-9 may serve as prognostic markers for regorafenib and provide insights into novel combination therapies with anti-MMP-9 agents or FTD/TPI.

Lipid metabolism is often elevated in cancer cells and plays an important role in their growth and malignancy. Acyl‐CoA synthetase (ACS), which converts long‐chain fatty acids to acyl‐CoA, is overexpressed in various types of cancer. However, the role of ACS in cancer remains unknown. Here, we found that ACS enzyme activity is required for cancer cell survival. Namely, the ACS inhibitor Triacsin c induced massive apoptosis in glioma cells while this cell death was completely suppressed by overexpression of ACSL5, the Triacsin c–resistant ACS isozyme, but not by overexpression of a catalytically inactive ACSL5 mutant. ACS inhibition by Triacsin c markedly potentiated the Bax‐induced intrinsic apoptotic pathway by promoting cytochrome c release and subsequent caspase activation. These effects were abrogated by ACSL5 overexpression. Correspondingly, ACS inhibition synergistically potentiated the glioma cell death induced by etoposide, a well‐known activator of apoptosis. Furthermore, in a nude mouse xenograft model, Triacsin c at a non‐toxic dose enhanced the antitumor efficacy of a low‐dose chemotherapy with etoposide. These results indicate that ACS is an apoptosis suppressor and that ACS inhibition could be a rational strategy to amplify the antitumor effect of etoposide. (Cancer Sci 2009)

Efficacy and safety of biweekly cetuximab plus irinotecan were evaluated to provide guidance for its use in Japan as third‐line treatment for pretreated metastatic colorectal cancer (mCRC) patients harboring wild‐type KRAS exon 2. Objective response rate (ORR) was used as primary endpoint based on an expected proportion of 0.23 with confidence width of 0.298 (95% CI, 0.105‐0.403), which showed 35 to be the minimal participant number. Forty patients, refractory to first‐ and second‐line chemotherapy containing irinotecan, oxaliplatin, and fluoropyrimidine, were enrolled. ORR and disease control rate were 25.0% (95% CI: 11.5‐38.4) and 72.5% (95% CI: 56.8‐86.4), respectively. Median progression‐free survival (PFS), overall survival (OS), and number of courses were 5.70 months (95% CI: 2.7‐7.9), 15.1 months (95% CI: 11.8‐19.0), and 10.5 (range: 3.0‐31.0), respectively. Grade 3 adverse events were skin toxicity (12.5%), diarrhea (10.0%), neutropenia (5.0%), febrile neutropenia (5.0%), nausea (5.0%), anorexia (5.0%), and fatigue (2.5%). Cmax mean was 723.2 μg/mL after first dose. High area under the curve (AUC)last variance was associated with t1/2 range of 131.2‐1209.6 hours (median, 174.4 hours). Early tumor shrinkage (ETS) and median depth of response were 25.0% and 13.0%, respectively. Mutation frequencies in KRAS exon 3 or 4, NRAS, BRAF, and PIK3CA were 5.5%, 2.7%, 8.3%, and 5.5%, respectively. Multivariate Cox regression analysis assessed whether any gene mutations and ETS are predictors for PFS, and whether performance status, synchronous metastasis, and ETS are predictors for OS. Importantly, the data provide guidance for a biweekly cetuximab plus irinotecan regimen in mCRC patients. Efficacy and safety of biweekly cetuximab plus irinotecan treatment were similar in patients from Japan and in a western population.