Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture.

Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.

PRMT1 Sustains De Novo Fatty Acid Synthesis by Methylating PHGDH to Drive Chemoresistance in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) chemoresistance hampers the ability to effectively treat patients. Identification of mechanisms driving chemoresistance can lead to strategies to improve treatment. Here, we revealed that protein arginine methyltransferase-1 (PRMT1) simultaneously methylates D-3-phosphoglycerate dehydrogenase (PHGDH), a critical enzyme in serine synthesis, and the glycolytic enzymes PFKFB3 and PKM2 in TNBC cells. 13C metabolic flux analyses showed that PRMT1-dependent methylation of these three enzymes diverts glucose toward intermediates in the serine-synthesizing and serine/glycine cleavage pathways, thereby accelerating the production of methyl donors in TNBC cells. Mechanistically, PRMT1-dependent methylation of PHGDH at R54 or R20 activated its enzymatic activity by stabilizing 3-phosphoglycerate binding and suppressing polyubiquitination. PRMT1-mediated PHGDH methylation drove chemoresistance independently of glutathione synthesis. Rather, activation of the serine synthesis pathway supplied α-ketoglutarate and citrate to increase palmitate levels through activation of fatty acid synthase (FASN). Increased palmitate induced protein S-palmitoylation of PHGDH and FASN to further enhance fatty acid synthesis in a PRMT1-dependent manner. Loss of PRMT1 or pharmacologic inhibition of FASN or protein S-palmitoyltransferase reversed chemoresistance in TNBC. Furthermore, IHC coupled with imaging MS in clinical TNBC specimens substantiated that PRMT1-mediated methylation of PHGDH, PFKFB3, and PKM2 correlates with chemoresistance and that metabolites required for methylation and fatty acid synthesis are enriched in TNBC. Together, these results suggest that enhanced de novo fatty acid synthesis mediated by coordinated protein arginine methylation and protein S-palmitoylation is a therapeutic target for overcoming chemoresistance in TNBC. SIGNIFICANCE: PRMT1 promotes chemoresistance in TNBC by methylating metabolic enzymes PFKFB3, PKM2, and PHGDH to augment de novo fatty acid synthesis, indicating that targeting this axis is a potential treatment strategy.

Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature.

Direct reprogramming, inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors, is a technology with anticipated medical applications. However, due to the many unresolved aspects of the induction mechanisms, it is essential to thoroughly analyze the epigenomic state of the generated cells. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs. We found that the CpG methylation state was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, leading to the medical applications of this technology.

The role of pioneer transcription factors in the induction of direct cellular reprogramming.

Regenerative medicine is a highly advanced medical field that aims to restore tissues and organs lost due to diseases and injury using a person's own cells or those of others. Direct cellular reprogramming is a promising technology that can directly induce cell-fate conversion from terminally differentiated cells to other cell types and is expected to play a pivotal role in applications in regenerative medicine. The induction of direct cellular reprogramming requires one or more master transcription factors with the potential to reconstitute cell type-specific transcription factor networks. The set of master transcription factors may contain unique transcription factors called pioneer factors that can open compacted chromatin structures and drive the transcriptional activation of target genes. Therefore, pioneer factors may play a central role in direct cellular reprogramming. However, our understanding of the molecular mechanisms by which pioneer factors induce cell-fate conversion is still limited. This review briefly summarizes the outcomes of recent findings and discusses future perspectives, focusing on the role of pioneer factors in direct cellular reprogramming.

Direct Conversion of Human Endothelial Cells Into Liver Cancer-Forming Cells Using Nonintegrative Episomal Vectors.

Liver cancer is an aggressive cancer associated with a poor prognosis. Development of therapeutic strategies for liver cancer requires fundamental research using suitable experimental models. Recent progress in direct reprogramming technology has enabled the generation of many types of cells that are difficult to obtain and provide a cellular resource in experimental models of human diseases. In this study, we aimed to establish a simple one-step method for inducing cells that can form malignant human liver tumors directly from healthy endothelial cells using nonintegrating episomal vectors. To screen for factors capable of inducing liver cancer-forming cells (LCCs), we selected nine genes and one short hairpin RNA that suppresses tumor protein p53 (TP53) expression and introduced them into human umbilical vein endothelial cells (HUVECs), using episomal vectors. To identify the essential factors, we examined the effect of changing the amounts and withdrawing individual factors. We then analyzed the proliferation, gene and protein expression, morphologic and chromosomal abnormality, transcriptome, and tumor formation ability of the induced cells. We found that a set of six factors, forkhead box A3 (FOXA3), hepatocyte nuclear factor homeobox 1A (HNF1A), HNF1B, lin-28 homolog B (LIN28B), MYCL proto-oncogene, bHLH transcription factor (L-MYC), and Kruppel-like factor 5 (KLF5), induced direct conversion of HUVECs into LCCs. The gene expression profile of these induced LCCs (iLCCs) was similar to that of human liver cancer cells, and these cells effectively formed tumors that resembled human combined hepatocellular-cholangiocarcinoma following transplantation into immunodeficient mice. Conclusion: We succeeded in the direct induction of iLCCs from HUVECs by using nonintegrating episomal vectors. iLCCs generated from patients with cancer and healthy volunteers will be useful for further advancements in cancer research and for developing methods for the diagnosis, treatment, and prognosis of liver cancer.

Meiosis-specific ZFP541 repressor complex promotes developmental progression of meiotic prophase towards completion during mouse spermatogenesis.

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development.

Direct reprogramming of human umbilical vein- and peripheral blood-derived endothelial cells into hepatic progenitor cells.

Recent advances have enabled the direct induction of human tissue-specific stem and progenitor cells from differentiated somatic cells. However, it is not known whether human hepatic progenitor cells (hHepPCs) can be generated from other cell types by direct lineage reprogramming with defined transcription factors. Here, we show that a set of three transcription factors, FOXA3, HNF1A, and HNF6, can induce human umbilical vein endothelial cells to directly acquire the properties of hHepPCs. These induced hHepPCs (hiHepPCs) propagate in long-term monolayer culture and differentiate into functional hepatocytes and cholangiocytes by forming cell aggregates and cystic epithelial spheroids, respectively, under three-dimensional culture conditions. After transplantation, hiHepPC-derived hepatocytes and cholangiocytes reconstitute damaged liver tissues and support hepatic function. The defined transcription factors also induce hiHepPCs from endothelial cells circulating in adult human peripheral blood. These expandable and bipotential hiHepPCs may be useful in the study and treatment of human liver diseases.

The Dynamics of Transcriptional Activation by Hepatic Reprogramming Factors.

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.

Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries.

Cells of multicellular organisms have diverse characteristics despite having the same genetic identity. The distinctive phenotype of each cell is determined by molecular mechanisms such as epigenetic changes that occur throughout the lifetime of an individual. Recently, technologies that enable modification of the fate of somatic cells have been developed, and the number of studies using these technologies has increased drastically in the last decade. Various cell types, including neuronal cells, cardiomyocytes, and hepatocytes, have been generated using these technologies. Although most direct reprogramming methods employ forced transduction of a defined sets of transcription factors to reprogram cells in a manner similar to induced pluripotent cell technology, many other strategies, such as methods utilizing chemical compounds and microRNAs to change the fate of somatic cells, have also been developed. In this review, we summarize transcription factor-based reprogramming and various other reprogramming methods. Additionally, we describe the various industrial applications of direct reprogramming technologies.

Generation of Nanog reporter mice that distinguish pluripotent stem cells from unipotent primordial germ cells.

Nanog is a core transcription factor specifically expressed not only in the pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced PSCs (iPSCs), but also in the unipotent primordial germ cells (PGCs). Although Nanog promoter/enhancer regions are well characterized by in vitro analyses, direct correlations between the regulatory elements for Nanog expression and in vivo expression patterns of Nanog have not been fully clarified. In this study, we generated Nanog-RFP transgenic (Tg) mice in which expression of red fluorescent protein (RFP) is driven by a 5.2 kb Nanog promoter/enhancer region. As expected, RFP was expressed in the inner cell mass of blastocysts, ESCs, and iPSCs. However, RFP fluorescence was not observed in PGCs, although Nanog was expressed in PGCs. Because RFP fluorescence was visible in the PGC-derived pluripotent EGCs in culture, it was suggested that the reporter gene expression was specifically activated in PSCs. In conclusion, we have generated a novel Nanog-RFP Tg mouse line that can selectively tag PSCs over unipotent PGCs.

Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors.

Hepatocellular carcinoma (HCC) accounts for a large proportion of liver cancer cases and has an extremely poor prognosis. Therefore, novel innovative therapies for HCC are strongly desired. As gene therapy tools for HCC, 2 hepatic transcription factors (TF), HNF4A and HNF1A, have been used to suppress proliferation and to extinguish cancer-specific characteristics of target cells. However, our present data demonstrated that single transduction of HNF4A or HNF1A had only a limited effect on suppression of HCC cell proliferation. Thus, in this study, we examined whether combinations of TF could show more effective antitumor activity, and found that combinatorial transduction of 3 hepatic TF, HNF4A, HNF1A and FOXA3, suppressed HCC cell proliferation more stably than single transduction of these TF. The combinatorial transduction also suppressed cancer-specific phenotypes, such as anchorage-independent growth in culture and tumorigenicity after transplantation into mice. HCC cell lines transduced with the 3 TF did not recover their proliferative property after withdrawal of anticancer drugs, indicating that combinatorial expression of the 3 TF suppressed the growth of all cell subtypes within the HCC cell lines, including cancer stem-like cells. Transcriptome analyses revealed that the expression levels of a specific gene set involved in cell proliferation were only decreased in HCC cells overexpressing all 3 TF. Moreover, combined transduction of the 3 TF could facilitate hepatic differentiation of HCC cell lines. Our strategy for inducing stable inhibition and functional differentiation of tumor cells using a defined set of TF will become an effective therapeutic strategy for various types of cancers.

Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a malignant epithelial neoplasm composed of cells resembling cholangiocytes that line the intrahepatic bile ducts in portal areas of the hepatic lobule. Although ICC has been defined as a tumor arising from cholangiocyte transformation, recent evidence from genetic lineage-tracing experiments has indicated that hepatocytes can be a cellular origin of ICC by directly changing their fate to that of biliary lineage cells. Notch signaling has been identified as an essential factor for hepatocyte conversion into biliary lineage cells at the onset of ICC. However, the mechanisms underlying Notch signal activation in hepatocytes remain unclear. Here, using a mouse model of ICC, we found that hepatic macrophages called Kupffer cells transiently congregate around the central veins in the liver and express the Notch ligand Jagged-1 coincident with Notch activation in pericentral hepatocytes. Depletion of Kupffer cells prevents the Notch-mediated cell-fate conversion of hepatocytes to biliary lineage cells, inducing hepatocyte apoptosis and increasing mortality in mice. These findings will be useful for uncovering the pathogenic mechanism of ICC and developing prevenient and therapeutic strategies for this refractory disease.

In vitro selection of bispecific diabody fragments using covalent bicistronic DNA display.

Bispecific antibodies with two different antigen-binding sites have been widely used for a variety of medical applications. The activity and stability of antibody fragments can be improved by in vitro evolution. Although the affinity and stability of small bispecific antibody fragments such as diabodies can be further optimized by in vitro display technologies, cell-free display of bispecific antibody fragments has not been reported. In this study, we applied a covalent bicistronic DNA display for the in vitro selection of heterodimeric diabodies. First, we confirmed the antigen-binding activities of a diabody synthesized by an in vitro transcription and translation system. However, when we performed DNA-display selection of a model diabody library in a proof-of-principle experiment, no enrichment of the diabody gene was observed, likely due to a low yield of the diabody heterodimer. To overcome this issue, we introduced cysteine residues at the VH-VL interface of the diabody heterodimer. Using the disulfide-stabilized diabodies, we successfully enriched the diabody gene from a model library. Our results indicate that the covalent bicistronic DNA display technique could be useful for improving the stability and affinity of bispecific diabody fragments.

Endosomal escape efficiency of fusogenic B18 and B55 peptides fused with anti-EGFR single chain Fv as estimated by nuclear translocation.

UNLABELLED: Although monoclonal antibodies have been used not only as analytical tools but also as biologic therapeutics, they cannot target intracellular proteins due to their large molecular size and low membrane permeability, which limit their applications. During previous attempts to delivery antibodies intracellularly, the low efficiency of escape from endosomes to the cytosol reduced the bioavailability of antibodies or antibody-conjugated effectors. Recently, we found that the fusogenic peptides (FPs) B18 and B55 from bindin, a sea urchin gamete recognition protein, facilitated the endosomal escape of FP-fused enhanced green fluorescent protein (eGFP) and/or of co-administered cargos such as dextrans [Niikura et al. A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape. J. CONTROL: Release 2015;212:85-93]. In this study, we constructed FP-fused anti-epidermal growth factor receptor (EGFR) single-chain Fv (αEGFR[scFv]) proteins and evaluated their endosomal escape efficiency by utilizing a nuclear localization signal). When the FP-fused αEGFR[scFv] proteins were incubated with A431 cells, the estimated endosomal escape efficiency of αEGFR[scFv]-B18 was significantly higher than that of αEGFR[scFv] alone, suggesting that the B18 peptide facilitates endosomal escape of the conjugated scFv in cis. Moreover, αEGFR[scFv]-B55 promoted the intracellular uptake of co-administered eGFP and dextrans in trans. These results imply that B18- and B55-fused antibodies may be useful for the cell-specific intracellular delivery of biomacromolecules.

PURE mRNA display for in vitro selection of single-chain antibodies.

mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low. To establish an efficient platform for the PURE mRNA display of scFv, we performed affinity selection of a library of scFv antibodies with a C-terminal random sequence and obtained C-terminal sequences that increased the formation of mRNA-protein conjugates. We also identified unexpected common substitution mutations around the start codon of scFv antibodies, which were inferred to destabilize the mRNA secondary structure. This destabilization causes an increase in protein expression and the efficiency of the formation of mRNA-protein conjugates. We believe these improvements should make the PURE mRNA display more efficient for selecting antibodies for diagnostic and therapeutic applications.

A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape.

The low efficiency of endosomal escape has been considered a bottleneck for the cytosolic delivery of biomacromolecules such as proteins and DNA. Although fusogenic peptides (FPs) such as HA2 have been employed to improve the intracellular delivery of biomacromolecules, the FPs studied thus far are not adequately efficient in enabling endosomal escape; therefore, novel FPs with higher activity are required. In this context, we focused on FPs derived from a sea urchin fertilization protein, bindin, which is involved in gamete recognition (B18, residues 103-120 and B55, residues 83-137 of mature bindin). We show that enhanced green fluorescent protein (EGFP)-fused B55 peptide binds to plasma membranes more strongly than EGFP-B18 and promotes the intracellular delivery of dextrans, which were co-administered using the trans method in a pH-dependent manner without affecting cell viability and proliferation, whereas conventional EGFP-HA2 did not affect dextran internalization. Furthermore, EGFP-B55 promoted the intracellular delivery of biomacromolecules such as antibodies, ribonuclease and plasmidic DNA using the trans method. Because the promotion of intracellular delivery by EGFP-B55 was suppressed by endocytosis inhibitors, EGFP-B55 is considered to have facilitated the endosomal escape of co-administered cargos. These results suggested that an FP that promotes the intracellular delivery of a variety of biomacromolecules with no detectable cytotoxicity should be useful for the cytosolic delivery of membrane-impermeable molecules for biomedical and biotechnological applications.

Specific and quantitative labeling of biomolecules using click chemistry.

Specific and highly efficient fluorescent labeling techniques for biomolecules, especially for proteins, are required for the quantitative analyses of bio-phenomena and for subsequent systems biology. Although expression of exogenous proteins fused with fluorescent tags, such as green fluorescent protein, is the most widely used method for quantitative bio-analysis, the following problems need to be considered carefully: (1) precise stoichiometric control in living cells is difficult, and (2) the bulkiness of the fluorescent tags restricts analysis of the inherent physical and biological properties of the proteins. Therefore, novel techniques to specifically and stoichiometrically label intrinsic proteins or other biomolecules in living cells should be developed. Click chemistry reactions (e.g., Huisgen cycloaddition and Staudinger ligation) are the most promising approaches for this purpose, because these chemical reactions have following advantages: (1) bioorthogonal reactions; (2) mild reaction conditions suitable for fragile biomolecules, cells, and tissues; (3) extremely high reaction ratio; (4) small size of the functional groups for the cross-coupling reactions; (5) stable covalent bonding; and (6) simple metabolic labeling procedures in living cells, using various biomolecular analogs. Diverse quantitative biological studies have been carried out using this technology (e.g., quantification of novel synthesized proteins and observation of post-translational modifications). In this review, I explain the basics of chemical probing with click chemistry, and discuss its recent applications in the field of quantitative biology. Furthermore, I discuss the capability, significance, and future of the chemical probing of proteins, with an emphasis on the use of click chemistry in the field of the quantitative biology.

Structural basis for inhibition of the MDM2:p53 interaction by an optimized MDM2-binding peptide selected with mRNA display.

The oncoprotein MDM2 binds to tumor suppressor protein p53 and inhibits its anticancer activity, which leads to promotion of tumor cell growth and tumor survival. Abrogation of the p53:MDM2 interaction reportedly results in reactivation of the p53 pathway and inhibition of tumor cell proliferation. We recently performed rigorous selection of MDM2-binding peptides by means of mRNA display and identified an optimal 12-mer peptide (PRFWEYWLRLME), named MDM2 Inhibitory Peptide (MIP), which shows higher affinity for MDM2 (and also its homolog, MDMX) and higher tumor cell proliferation suppression activity than known peptides. Here we determined the NMR solution structure of a MIP-MDM2 fusion protein to elucidate the structural basis of the tight binding of MIP to MDM2. A region spanning from Phe3 to Met11 of MIP forms a single α-helix, which is longer than those of the other MDM2-binding peptides. MIP shares a conserved Phe3-Trp7-Leu10 triad, whose side chains are oriented towards and fit into the hydrophobic pockets of MDM2. Additionally, hydrophobic surface patches that surround the hydrophobic pockets of MDM2 are covered by solvent-exposed MIP residues, Trp4, Tyr6, and Met11. Their hydrophobic interactions extend the interface of the two molecules and contribute to the strong binding. The potential MDM2 inhibition activity observed for MIP turned out to originate from its enlarged binding interface. The structural information obtained in the present study provides a road map for the rational design of strong inhibitors of MDM2:p53 binding.

Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices.

Although oxidoreductases are widely used in many applications, such as biosensors and biofuel cells, improvements in the function of existing oxidoreductases or the discovery of novel oxidoreductases with greater activities is desired. To increase the activity of oxidoreductases by directed evolution, a powerful screening technique for oxidoreductases is required. In this study, we demonstrate the utility of boron-doped diamond (BDD) microelectrodes for quantitative and potentially high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. We first confirmed that BDD microelectrodes can quantify the activity of low concentrations (10-100 pM) of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase with a measuring time of 1 ms per sample. In addition, we found that poisoning of BDD microelectrodes can be repressed by optimizing the pH and by adding l-arginine to the enzyme solution as an antiaggregation agent. Finally, we fabricated a microfluidic device containing a BDD electrode for the first time and observed the elevation of the oxidation current of NADH with increasing flow rate. These results imply that the combination of a BDD microelectrode and microfluidics can be used for high-throughput screening of an oxidoreductase library containing a large number (>10(6)) of samples, each with a small (nanoliter) sample volume.

MIP-2A is a novel target of an anilinoquinazoline derivative for inhibition of tumour cell proliferation.

We recently identified a novel anilinoquinazoline derivative, Q15, as a potent apoptosis inducer in a panel of human cancer cell lines and determined that Q15 targets hCAP-G2, a subunit of condensin II complex, leading to abnormal cell division. However, whether the defect in normal cell division directly results in cell death remains unclear. Here, we used an mRNA display method on a microfluidic chip to search for other Q15-binding proteins. We identified an additional Q15-binding protein, MIP-2A (MBP-1 interacting protein-2A), which has been reported to interact with MBP-1, a repressor of the c-Myc promoter. Our results indicate that Q15 inhibits the interaction between MIP-2A and MBP-1 as well as the expression of c-Myc protein, thereby inducing cell death. This study suggests that the simultaneous targeting of hCAP-G2 and MIP-2A is a promising strategy for the development of antitumor drugs as a treatment for intractable tumours.