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.

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer.

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

Glutamine deficiency drives transforming growth factor-ß signaling activation that gives rise to myofibroblastic carcinoma-associated fibroblasts.

Tumor-promoting carcinoma-associated fibroblasts (CAFs), abundant in the mammary tumor microenvironment (TME), maintain transforming growth factor-ß (TGF-ß)-Smad2/3 signaling activation and the myofibroblastic state, the hallmark of activated fibroblasts. How myofibroblastic CAFs (myCAFs) arise in the TME and which epigenetic and metabolic alterations underlie activated fibroblastic phenotypes remain, however, poorly understood. We herein show global histone deacetylation in myCAFs present in tumors to be significantly associated with poorer outcomes in breast cancer patients. As the TME is subject to glutamine (Gln) deficiency, human mammary fibroblasts (HMFs) were cultured in Gln-starved medium. Global histone deacetylation and TGF-ß-Smad2/3 signaling activation are induced in these cells, largely mediated by class I histone deacetylase (HDAC) activity. Additionally, mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is attenuated in Gln-starved HMFs, and mTORC1 inhibition in Gln-supplemented HMFs with rapamycin treatment boosts TGF-ß-Smad2/3 signaling activation. These data indicate that mTORC1 suppression mediates TGF-ß-Smad2/3 signaling activation in Gln-starved HMFs. Global histone deacetylation, class I HDAC activation, and mTORC1 suppression are also observed in cultured human breast CAFs. Class I HDAC inhibition or mTORC1 activation by high-dose Gln supplementation significantly attenuates TGF-ß-Smad2/3 signaling and the myofibroblastic state in these cells. These data indicate class I HDAC activation and mTORC1 suppression to be required for maintenance of myCAF traits. Taken together, these findings indicate that Gln starvation triggers TGF-ß signaling activation in HMFs through class I HDAC activity and mTORC1 suppression, presumably inducing myCAF conversion.

YBX1 Regulates Satellite II RNA Loading into Small Extracellular Vesicles and Promotes the Senescent Phenotype.

Senescent cells secrete inflammatory proteins and small extracellular vesicles (sEVs), collectively termed senescence-associated secretory phenotype (SASP), and promote age-related diseases. Epigenetic alteration in senescent cells induces the expression of satellite II (SATII) RNA, non-coding RNA transcribed from pericentromeric repetitive sequences in the genome, leading to the expression of inflammatory SASP genes. SATII RNA is contained in sEVs and functions as an SASP factor in recipient cells. However, the molecular mechanism of SATII RNA loading into sEVs is unclear. In this study, we identified Y-box binding protein 1 (YBX1) as a carrier of SATII RNA via mass spectrometry analysis after RNA pull-down. sEVs containing SATII RNA induced cellular senescence and promoted the expression of inflammatory SASP genes in recipient cells. YBX1 knockdown significantly reduced SATII RNA levels in sEVs and inhibited the propagation of SASP in recipient cells. The analysis of the clinical dataset revealed that YBX1 expression is higher in cancer stroma than in normal stroma of breast and ovarian cancer tissues. Furthermore, high YBX1 expression was correlated with poor prognosis in breast and ovarian cancers. This study demonstrated that SATII RNA loading into sEVs is regulated via YBX1 and that YBX1 is a promising target in novel cancer therapy.

Role of EMT in the DNA damage response, double-strand break repair pathway choice and its implications in cancer treatment.

Numerous epithelial-mesenchymal transition (EMT) characteristics have now been demonstrated to participate in tumor development. Indeed, EMT is involved in invasion, acquisition of stem cell properties, and therapy-associated resistance of cancer cells. Together, these mechanisms offer advantages in adapting to changes in the tumor microenvironment. However, recent findings have shown that EMT-associated transcription factors (EMT-TFs) may also be involved in DNA repair. A better understanding of the coordination between the DNA repair pathways and the role played by some EMT-TFs in the DNA damage response (DDR) should pave the way for new treatments targeting tumor-specific molecular vulnerabilities, which result in selective destruction of cancer cells. Here we review recent advances, providing novel insights into the role of EMT in the DDR and repair pathways, with a particular focus on the influence of EMT on cellular sensitivity to damage, as well as the implications of these relationships for improving the efficacy of cancer treatments.

G-quadruplex-forming nucleic acids interact with splicing factor 3B subunit 2 and suppress innate immune gene expression.

G-quadruplex (G4), a non-canonical higher-order structure formed by guanine-rich nucleic acid sequences, affects various genetic events in cis, including replication, transcription and translation. Whereas up-regulation of innate immune/interferon-stimulated genes (ISGs) is implicated in cancer progression, G4-forming oligonucleotides that mimic telomeric repeat-containing RNA suppress ISG induction in three-dimensional (3D) culture of cancer cells. However, it is unclear how G4 suppresses ISG expression in trans. In this study, we found that G4 binding to splicing factor 3B subunit 2 (SF3B2) down-regulated STAT1 phosphorylation and ISG expression in 3D-cultured cancer cells. Liquid chromatography-tandem mass spectrometry analysis identified SF3B2 as a G4-binding protein. Either G4-forming oligonucleotides or SF3B2 knockdown suppressed ISG induction, whereas Phen-DC3, a G4-stabilizing compound, reversed the inhibitory effect of G4-forming oligonucleotides on ISG induction. Phen-DC3 inhibited SF3B2 binding to G4 in vitro. SF3B2-mediated ISG induction appeared to occur independently of RNA splicing because SF3B2 knockdown did not affect pre-mRNA splicing under the experimental conditions, and pharmacological inhibition of splicing by pladienolide B did not repress ISG induction. These observations suggest that G4 disrupts the ability of SF3B2 to induce ISGs in cancer. We propose a new mode for gene regulation, which employs G4 as an inhibitory trans-element.

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.

Novel tankyrase inhibitors suppress TDP-43 aggregate formation.

Transactive response DNA-binding protein of 43 kDa (TDP-43) abnormally forms aggregates in certain subtypes of frontotemporal lobar degeneration (FTLD) and in amyotrophic lateral sclerosis (ALS). The pathological forms of TDP-43 have reported to be associated with poly(ADP-ribose) (PAR), which regulates the properties of these aggregates. A recent study has indicated that tankyrase, a member of the PAR polymerase (PARP) family, regulates pathological TDP-43 formation under conditions of stress, and tankyrase inhibitors suppress TDP-43 aggregate formation and cytotoxicity. Since we reported the development of tankyrase inhibitors that are more specific than conventional inhibitors, in this study, we examined their effects on the formation of TDP-43 aggregates in cultured cells. Time-lapse imaging showed that TDP-43 aggregates appeared in the nucleus within 30 min of treatment with sodium arsenite. Several tankyrase inhibitors suppressed the formation of aggregates and decreased the levels of the tankyrase protein. Immunohistochemical studies demonstrated that tankyrase was localized to neuronal cytoplasmic inclusions in the spinal cords of patients with ALS. Moreover, the tankyrase protein levels were significantly higher in the brains of patients with FTLD than in the brains of control subjects. These findings suggest that the inhibition of tankyrase activity protects against TDP-43 toxicity. Tankyrase inhibitors may be a potential treatment to suppress the progression of TDP-43 proteinopathies.

Design and synthesis of 14 and 15-membered macrocyclic scaffolds exhibiting inhibitory activities of hypoxia-inducible factor 1α.

Inspired by the privileged molecular skeletons of 14- and 15-membered antibiotics, we adopted a relatively unexplored synthetic approach that exploits alkaloidal macrocyclic scaffolds to generate modulators of protein-protein interactions (PPIs). As mimetics of hot-spot residues in the α-helices responsible for the transcriptional regulation, three hydrophobic sidechains were displayed on each of the four distinct macrocyclic scaffolds generating diversity of their spatial arrangements. Modular assembly of the building blocks followed by ring-closing olefin metathesis reaction and subsequent hydrogenation allowed concise and divergent synthesis of scaffolds 1-4. The 14-membered alkaloidal macrocycles 2-4 demonstrated similar inhibition of hypoxia-inducible factor (HIF)-1α transcriptional activities (IC50 between 8.7 and 10 µM), and 4 demonstrated the most potent inhibition of cell proliferation in vitro (IC50 = 12 µM against HTC116 colon cancer cell line). A docking model suggested that 4 could mimic the LLxxL motif in HIF-1α, in which the three sidechains are capable of matching the spatial arrangements of the protein hot-spot residues. Unlike most of the stapled peptides, the 14-membered alkaloidal scaffold has a similar size to the α-helix backbone and does not require additional atoms to induce α-helix mimetic structure. These experimental results underscore the potential of alkaloidal macrocyclic scaffolds featuring flexibly customizable skeletal, stereochemical, substitutional, and conformational properties for the development of non-peptidyl PPI modulators targeting α-helix-forming consensus sequences responsible for the transcriptional regulation.

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.

From the wings to the center stage of chromosomes.

Telomere-binding protein TRF2 protects the linear chromosome ends, telomeres, from being recognized as damaged DNA. TRF2 also regulates gene expression outside telomeres, but the detailed mechanism has not been fully understood. Mukherjee and colleagues have employed ChIP-Seq and biochemical analyses to identify G-quadruplexes at gene promoters across the genome as nontelomeric TRF2-binding sites. TRF2 occupancy on such target sites leads to epigenetic gene repression, implicating TRF2-G-quadruplex interaction as a sophisticated regulator of gene expression.

Crossroads of telomere biology and anticancer drug discovery.

The telomere is the specialized nucleoprotein complex at the end of the chromosome. Its highly conserved 5'-TTAGGG-3' repeats and shelterin protein complexes form a protective loop structure to maintain the integrity and stability of linear chromosomes. Although human somatic cells gradually shorten telomeres to undergo senescence or crisis, cancer cells activate telomerase, or the recombination-based mechanism to maintain telomeres and exhibit immortality. As the most frequent non-coding mutations in cancer, gain-of-function mutations in the promoter region of the telomerase catalytic subunit, TERT, trigger telomerase activation. Promoter methylation and copy number gain are also associated with the enhanced TERT expression. Although telomerase inhibitors were pioneered from telomere-directed therapeutics, their efficacies are limited to cancer with short telomeres and some hematological malignancies. Other therapeutic approaches include a nucleoside analog incorporated to telomeres and TERT promoter-driven oncolytic adenoviruses. Tankyrase poly(ADP-ribose) polymerase, a positive regulator of telomerase, has been rediscovered as a target for Wnt-driven cancer. Meanwhile, telomeric nucleic acids form a higher-order structure called a G-quadruplex (G4). G4s are formed genome-wide and their dynamics affect various events, including replication, transcription, and translation. G4-stabilizing compounds (G4 ligands) exert anticancer effects and are in clinical investigations. Collectively, telomere biology has provided clues for deeper understanding of cancer, which expands opportunities to discover innovative anticancer drugs.

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.

G-quadruplex in cancer biology and drug discovery.

G-quadruplex (G4) is a non-canonical nucleic acid structure formed in guanine-rich DNA or RNA. G4s are formed not only in vitro but also in vivo and are attracting considerable interest owing to their potential involvement in biological processes, including replication, transcription, mRNA splicing, translation and epigenetic regulation of the genome. In this review, we outline the functions of G4 in cellular biology and their implication in human pathogenesis, especially in cancer. Furthermore, we describe the properties of G4-stabilizing chemical compounds, G4 ligands, and their application for cancer therapeutics.

Tankyrase promotes primary precursor miRNA processing to precursor miRNA.

Tankyrases (TNKS and TNKS2) are members of poly(ADP-ribose) polymerase (PARP) family proteins. Tankyrase has multiple ankyrin repeat cluster (ARC) domains, which recognize the tankyrase-binding motifs in proteins including the telomeric protein, TRF1 and Wnt signal regulators, AXINs. However, the functional significance of tankyrase interaction with many other putative binding proteins remains unknown. Here, we found that several proteins involved in microRNA (miRNA) processing have putative tankyrase-binding motifs and their functions are regulated by tankyrase. First, chemical inhibition of tankyrase PARP activity downregulated the expression levels of precursor miRNAs (pre-miRNAs) but not primary precursor miRNAs (pri-miRNAs). A subsequent reporter assay revealed that tankyrase inhibitors or PARP-dead mutant tankyrase overexpression repress pri-miRNA processing to pre-miRNA. Conversely, a PARP-1/2 inhibitor, olaparib, did not affect pri-miRNA processing. Tankyrase ARCs bound to DGCR8 and DROSHA, which are essential components for pri-miRNA processing and have putative tankyrase-binding motifs. These observations indicate that tankyrase binds to Microprocessor, DGCR8 and DROSHA complex and modulates pri-miRNA processing to pre-miRNA.

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.