Interleukin-4 induced 1-mediated resistance to an immune checkpoint inhibitor through suppression of CD8+ T cell infiltration in melanoma.

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.

APC/PIK3CA mutations and ß-catenin status predict tankyrase inhibitor sensitivity of patient-derived colorectal cancer cells.

BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.

Intratumor transforming growth factor-ß signaling with extracellular matrix-related gene regulation marks chemotherapy-resistant gastric cancer.

Drug-tolerant persister (DTP) cells remain following chemotherapy and can cause cancer relapse. However, it is unclear when acquired resistance to chemotherapy emerges. Here, we compared the gene expression profiles of gastric cancer patient-derived cells (GC PDCs) and their respective xenograft tumors with different sensitivities to 5-fluorouracil (5-FU) by using immunodeficient female BALB/c-nu mice. RNA sequencing analysis of 5-FU-treated PDCs demonstrated that DNA replication/cell cycle-related genes were transiently induced in the earlier phase of DTP cell emergence, while extracellular matrix (ECM)-related genes were sustainably upregulated during long-term cell survival in 5-FU-resistant residual tumors. NicheNet analysis, which uncovers cell-cell signal interactions, indicated the transforming growth factor-ß (TGF-ß) pathway as the upstream regulator in response to 5-FU treatment. This induced ECM-related gene expression in the 5-FU-resistant tumor model. In the 5-FU-resistant residual tumors, there was a marked upregulation of cancer cell-derived TGF-ß1 expression and increased phosphorylation of SMAD3, a downstream regulator of the TGF-ß receptor. By contrast, these responses were not observed in a 5-FU-sensitive tumor model. We further found that TGF-ß-related upregulation of ECM genes was preferentially observed in non-responders to chemotherapy with 5-FU and/or oxaliplatin among 22 patient-derived xenograft tumors. These observations suggest that chemotherapy-induced activation of the TGF-ß1/SMAD3/ECM-related gene axis is a potential biomarker for the emergence of drug resistance in GCs.

Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor.

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.

Serum IL-8 level as a candidate prognostic marker of response to anti-angiogenic therapy for metastatic colorectal cancer.

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.

ALDH1A3-mTOR axis as a therapeutic target for anticancer drug-tolerant persister cells in gastric cancer.

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.

Epidermal growth factor receptor mRNA expression: A potential molecular escape mechanism from regorafenib.

Regorafenib has improved the survival of patients with refractory metastatic colorectal cancer (mCRC), yet the mechanisms of inherited or acquired resistance are not well understood. A total of 50 patients with refractory mCRC were enrolled. Circulating tumor cell (CTC) enumeration was carried out at baseline, day 21 after initiation of regorafenib, and at the time of progression of disease (PD) using the CellSearch System (Veridex LLC, NJ, USA). Poly(A) mRNA was extracted from CTCs, and gene expression of epithelial and epithelial-mesenchymal transition markers was analyzed by a multiplex-PCR based DNA Chip. Patients with fewer than 3 CTCs at baseline and day 21 had a longer progression-free survival than those with 3 or more CTCs (3.3 vs 2.0 months, P = .008 and 3.3 vs 2.0 months, P = .004, respectively). Patients with fewer than 3 CTCs at baseline and day 21 had a longer overall survival (OS) than those with 3 or more CTCs (10.0 vs 4.6 months, P < .001 and 8.7 vs 3.8 months, P = .003, respectively). In multivariable analysis, CTC counts remained significantly associated with OS at baseline and day 21 (P = .019 and P = .028). Circulating tumor cell EGFR gene expression was upregulated at day 21 and/or PD in 64% of patients. Patients had significantly increased EGFR expression at PD compared to baseline (P = .041) and at day 21 and/or PD compared to baseline (P = .004). Our findings suggest that CTC count and EGFR expression could be useful markers of regorafenib efficacy and outcomes. Upregulation of CTC EGFR expression might be a molecular escape mechanism under regorafenib therapy.

c-KIT regulates stability of cancer stemness in CD44-positive colorectal cancer cells.

Cancer stem cells (CSCs) are subpopulations of cancer cells with high self-renewal potential that are involved in tumor progression and recurrence. It has been postulated that CSCs and non-stem cancer cells are inter-convertible. However, precise mechanisms for the plasticity and stability of cancer stemness remain elusive. Here, we demonstrate that CD44-positive colorectal CSC fractions contain two types of cancer cells: "CD44-stable" cells, in which CD44 expression is stably sustained, and "CD44-trasnsient" cells, which are rapidly converted to CD44-negative cells. CD44-stable cells expressed higher levels of c-KIT tyrosine kinase than CD44-transient cells. c-KIT knockdown by siRNAs converted the CD44-positive cells to CD44-negative cells, which expressed lower levels of stem cell markers such as ASCL2 and EPCAM. In the CD44-positive cells, c-KIT phosphorylation level was very low whereas stem cell factor, a c-KIT ligand, elevated c-KIT phosphorylation without affecting stem cell marker expression. CRISPR-Cas9-mediated knockout of the c-KIT gene in CD44 stable cells attenuated the CSC properties including expression of CD44 and other stem cell markers, clonogenicity and in vivo tumorigenic potential in a mouse xenograft model. These observations suggest that the colorectal CSC fractions contain cancer cells with differential plasticity, which is determined by c-KIT.

A phase I study to determine the maximum tolerated dose of trifluridine/tipiracil and oxaliplatin in patients with refractory metastatic colorectal cancer: LUPIN study.

Background The effectiveness of reintroducing oxaliplatin for metastatic colorectal cancer (mCRC) refractory to both oxaliplatin and irinotecan was previously reported in a phase II study (RE-OPEN). We conducted a phase I study to determine the maximum tolerated dose of oxaliplatin plus trifluridine/tipiracil (FTD/TPI) in patients with refractory mCRC. Patients and Methods Three dosages of intravenous oxaliplatin (50, 65 and 85 mg/m2) on days 1 and 15 and a fixed dose of FTD/TPI 35 mg/m2 twice daily (bid) on days 1-5 and 15-19 every 4 weeks were investigated in patients with refractory mCRC using a 3 + 3 design. Eligible patients had received prior oxaliplatin-based treatment that achieved a response or stable disease followed by confirmed disease progression at least 6 months before entering the study. Results Twelve patients were enrolled in the study. Three of six patients in the oxaliplatin 85 mg/m2 cohort had dose-limiting toxicities (DLTs) with treatment delays during the second cycle at ≥8 days due to grade ≥ 2 neutropenia or grade 2 AST/ALT increased. No DLTs were observed in the other cohorts. Grade ≥ 3 AEs were neutropenia (n = 3), thrombocytopenia (n = 1), anorexia (n = 1), and nausea (n = 1). There was no evidence of allergic reaction to oxaliplatin or severe peripheral sensory neuropathy. Conclusions A combination of FTD/TPI 35 mg/m2 bid on days 1-5 and 15-19 and oxaliplatin 85 mg/m2 on days 1 and 15 every 4 weeks could be a suitable regimen for the recommended dose of FTD/TPI plus oxaliplatin in patients with refractory mCRC.

In silico chemical screening identifies epidermal growth factor receptor as a therapeutic target of drug-tolerant CD44v9-positive gastric cancer cells.

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.

Phase II trial of biweekly cetuximab and irinotecan as third-line therapy for pretreated KRAS exon 2 wild-type colorectal cancer.

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.

Comprehensive transcriptomic analysis of molecularly targeted drugs in cancer for target pathway evaluation.

Targeted therapy is a rational and promising strategy for the treatment of advanced cancer. For the development of clinical agents targeting oncogenic signaling pathways, it is important to define the specificity of compounds to the target molecular pathway. Genome-wide transcriptomic analysis is an unbiased approach to evaluate the compound mode of action, but it is still unknown whether the analysis could be widely applicable to classify molecularly targeted anticancer agents. We comprehensively obtained and analyzed 129 transcriptomic datasets of cancer cells treated with 83 anticancer drugs or related agents, covering most clinically used, molecularly targeted drugs alongside promising inhibitors of molecular cancer targets. Hierarchical clustering and principal component analysis revealed that compounds targeting similar target molecules or pathways were clustered together. These results confirmed that the gene signatures of these drugs reflected their modes of action. Of note, inhibitors of oncogenic kinase pathways formed a large unique cluster, showing that these agents affect a shared molecular pathway distinct from classical antitumor agents and other classes of agents. The gene signature analysis further classified kinome-targeting agents depending on their target signaling pathways, and we identified target pathway-selective signature gene sets. The gene expression analysis was also valuable in uncovering unexpected target pathways of some anticancer agents. These results indicate that comprehensive transcriptomic analysis with our database (http://scads.jfcr.or.jp/db/cs/) is a powerful strategy to validate and re-evaluate the target pathways of anticancer compounds.

BET protein-dependent E2F pathway activity confers bell-shaped type resistance to tankyrase inhibitors in APC-mutated colorectal cancer.

WNT/ß-catenin signaling is aberrantly activated in colorectal cancer (CRC) mainly by loss-of-function mutations in adenomatous polyposis coli (APC) and is involved in tumor progression. Tankyrase inhibitors, which suppress WNT/ß-catenin signaling, are currently in pre-clinical and clinical trials. However, the mechanisms of resistance to tankyrase inhibitors remain unclear. In this study, we established tankyrase inhibitor-resistant CRC cells, JC73-RK100, from APC-mutated patient-derived CRC cells. JC73-RK100 cells and several CRC cell lines were sensitive to tankyrase inhibitors at low concentrations but were resistant at high concentrations, showing an intrinsic/acquired bell-shaped dose response. Mechanistically, tankyrase inhibitors at high concentrations promoted BRD3/4-dependent E2F target gene transcription and over-activated cell cycle progression in these cells. BET inhibitors canceled the bell-shaped dose response to tankyrase inhibitors. Combination of tankyrase and BET inhibitors significantly suppressed tumor growth in a mouse xenograft model. These observations suggest that the combination of tankyrase and BET inhibitors may be a useful therapeutic approach to overcome the resistance of a subset of CRCs to tankyrase inhibitors.

Pharmacologic Targeting of Histone H3K27 Acetylation/BRD4-dependent Induction of ALDH1A3 for Early-phase Drug Tolerance of Gastric Cancer.

Anticancer drug-tolerant persister (DTP) cells at an early phase of chemotherapy reshape refractory tumors. Aldehyde dehydrogenase 1 family member A3 (ALDH1A3) is commonly upregulated by various anticancer drugs in gastric cancer patient-derived cells (PDC) and promotes tumor growth. However, the mechanism underlying the generation of ALDH1A3-positive DTP cells remains elusive. Here, we investigated the mechanism of ALDH1A3 expression and a combination therapy targeting gastric cancer DTP cells. We found that gastric cancer tissues treated with neoadjuvant chemotherapy showed high ALDH1A3 expression. Chromatin immunoprecipitation (ChIP)-PCR and ChIP sequencing analyses revealed that histone H3 lysine 27 acetylation was enriched in the ALDH1A3 promoter in 5-fluorouracil (5-FU)-tolerant persister PDCs. By chemical library screening, we found that the bromodomain and extraterminal (BET) inhibitors OTX015/birabresib and I-BET-762/molibresib suppressed DTP-related ALDH1A3 expression and preferentially inhibited DTP cell growth. In DTP cells, BRD4, but not BRD2/3, was recruited to the ALDH1A3 promoter and BRD4 knockdown decreased drug-induced ALDH1A3 upregulation. Combination therapy with 5-FU and OTX015 significantly suppressed in vivo tumor growth. These observations suggest that BET inhibitors are efficient DTP cell-targeting agents for gastric cancer treatment. SIGNIFICANCE: Drug resistance hampers the cure of patients with cancer. To prevent stable drug resistance, DTP cancer cells are rational therapeutic targets that emerge during the early phase of chemotherapy. This study proposes that the epigenetic regulation by BET inhibitors may be a rational therapeutic strategy to eliminate DTP cells.

Amino acid influx via LAT1 regulates iron demand and sensitivity to PPMX-T003 of aggressive natural killer cell leukemia.

Aggressive natural killer cell leukemia (ANKL) is a rare hematological malignancy with a fulminant clinical course. Our previous study revealed that ANKL cells proliferate predominantly in the liver sinusoids and strongly depend on transferrin supplementation. In addition, we demonstrated that liver-resident ANKL cells are sensitive to PPMX-T003, an anti-human transferrin receptor 1 inhibitory antibody, whereas spleen-resident ANKL cells are resistant to transferrin receptor 1 inhibition. However, the microenvironmental factors that regulate the iron dependency of ANKL cells remain unclear. In this study, we first revealed that the anti-neoplastic effect of PPMX-T003 was characterized by DNA double-strand breaks in a DNA replication-dependent manner, similar to conventional cytotoxic agents. We also found that the influx of extracellular amino acids via LAT1 stimulated sensitivity to PPMX-T003. Taken together, we discovered that the amount of extracellular amino acid influx through LAT1 was the key environmental factor determining the iron dependency of ANKL cells via adjustment of their mTOR/Myc activity, which provides a good explanation for the different sensitivity to PPMX-T003 between liver- and spleen-resident ANKL cells, as the liver sinusoid contains abundant amino acids absorbed from the gut.

Development of a gene expression database and related analysis programs for evaluation of anticancer compounds.

Genome-wide transcriptional expression analysis is a powerful strategy for characterizing the biological activity of anticancer compounds. It is often instructive to identify gene sets involved in the activity of a given drug compound for comparison with different compounds. Currently, however, there is no comprehensive gene expression database and related application system that is; (i) specialized in anticancer agents; (ii) easy to use; and (iii) open to the public. To develop a public gene expression database of antitumor agents, we first examined gene expression profiles in human cancer cells after exposure to 35 compounds including 25 clinically used anticancer agents. Gene signatures were extracted that were classified as upregulated or downregulated after exposure to the drug. Hierarchical clustering showed that drugs with similar mechanisms of action, such as genotoxic drugs, were clustered. Connectivity map analysis further revealed that our gene signature data reflected modes of action of the respective agents. Together with the database, we developed analysis programs that calculate scores for ranking changes in gene expression and for searching statistically significant pathways from the Kyoto Encyclopedia of Genes and Genomes database in order to analyze the datasets more easily. Our database and the analysis programs are available online at our website (http://scads.jfcr.or.jp/db/cs/). Using these systems, we successfully showed that proteasome inhibitors are selectively classified as endoplasmic reticulum stress inducers and induce atypical endoplasmic reticulum stress. Thus, our public access database and related analysis programs constitute a set of efficient tools to evaluate the mode of action of novel compounds and identify promising anticancer lead compounds.

Neutralization of the induced VEGF-A potentiates the therapeutic effect of an anti-VEGFR2 antibody on gastric cancer in vivo.

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.

Potential Molecular Cross Talk Among CCR5 Pathway Predicts Regorafenib Responsiveness in Metastatic Colorectal Cancer Patients.

BACKGROUND: Genetic variants in the CCL5/CCR5 pathway have been shown to predict regorafenib efficacy in patients with metastatic colorectal cancer (mCRC). This study investigated the biological role of CCL4 and CCL3 gene polymorphisms in patients with refractory mCRC treated using regorafenib. PATIENTS AND METHODS: We analyzed the genomic DNA extracted from mCRC patients receiving regorafenib. Serum factor levels at baseline, day 21, and progressive disease (PD) were measured using ELISA. RESULTS: Decreased CCL4 levels at day 21 or increased CCL3 levels at PD were associated with better clinical outcomes. In patients with any CCL5 rs2280789 G allele, CCL3 significantly increased between BL and day 21 compared with the A/A variant (72.7% vs. 23.1%, p=0.006), but CCL4 decreased (31.8% vs. 69.2%, p=0.043). CONCLUSION: Increased CCL3 and decreased CCL4 seen in specific genotypes may serve as potential biomarkers of regorafenib in mCRC patients.

Pharmacological targeting of constitutively active truncated androgen receptor by nigericin and suppression of hormone-refractory prostate cancer cell growth.

In prostate cancer, blockade of androgen receptor (AR) signaling confers a therapeutic benefit. Nevertheless, this standard therapy allows relapse of hormone-refractory prostate cancer (HRPC) with a poor prognosis. HRPC cells often express variant ARs, such as point-mutated alleles and splicing isoforms, resulting in androgen-independent cell growth and resistance to antiandrogen (e.g., flutamide). However, a pharmacological strategy to block such aberrant ARs remains to be established. Here, we established a reporter system that monitors AR-mediated activation of a prostate-specific antigen (PSA) promoter. Our chemical library screening revealed that the antibiotic nigericin inhibits AR-mediated activation of the PSA promoter and PSA production in prostate cancer cells. Nigericin suppressed the androgen-dependent LNCaP cell growth even though the cells expressed a flutamide-resistant mutant AR. These effects were caused by AR suppression at the mRNA and post-translational levels. In HRPC 22Rv1 cells, which express the full-length AR and the constitutively active, truncated ARs lacking the carboxyl-terminal ligand-binding domain, small interfering RNA-mediated knockdown of both AR isoforms efficiently suppressed the androgen-independent cell growth, whereas knockdown of the full-length AR alone had no significant effect. It is noteworthy that nigericin was able to mimic the knockdown of both AR isoforms: it reduced the expression of the full-length and the truncated ARs, and it induced G(1) cell-cycle arrest and apoptosis of 22Rv1 cells. These observations suggest that nigericin-like compounds that suppress AR expression at the mRNA level could be applied as new-type therapeutic agents that inhibit a broad spectrum of AR variants in HRPC.

Tankyrase Inhibitors Target Colorectal Cancer Stem Cells via AXIN-Dependent Downregulation of c-KIT Tyrosine Kinase.

Cancer stem cells (CSC) constitute heterogeneous cell subpopulations of a tumor. Although targeting CSCs is important for cancer eradication, no clinically approved drugs that target CSCs have been established. Tankyrase poly(ADP-ribosyl)ates and destabilizes AXIN, a negative regulator of ß-catenin, and promotes ß-catenin signaling. Here, we report that tankyrase inhibitors downregulate c-KIT tyrosine kinase and inhibit the growth of CD44-positive colorectal CSCs. c-KIT expression in CD44-positive subpopulations of colorectal cancer COLO-320DM cells is associated with their tumor-initiating potential in vivo Tankyrase inhibitors downregulate c-KIT expression in established cell lines, such as COLO-320DM and DLD-1, and colorectal cancer patient-derived cells. These effects of tankyrase inhibitors are caused by reducing the recruitment of SP1 transcription factor to the c-KIT gene promoter and depend on AXIN2 stabilization but not ß-catenin downregulation. Whereas c-KIT knockdown inhibits the growth of CD44-positive COLO-320DM cells, c-KIT overexpression in DLD-1 cells confers resistance to tankyrase inhibitors. Combination of a low-dose tankyrase inhibitor and irinotecan significantly inhibited the growth of COLO-320DM tumors in a mouse xenograft model. These observations suggest that tankyrase inhibitors target c-KIT-positive colorectal CSCs and provide a novel therapeutic strategy for cancer.