CYB5R3 functions as a tumor suppressor by inducing ER stress-mediated apoptosis in lung cancer cells via the PERK-ATF4 and IRE1α-JNK pathways.

Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomic analysis revealed that CYB5R3 overexpression increased the production of nicotinamide adenine dinucleotide (NAD+) and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress signaling is induced via activation of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD+ activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent intrinsic cell death. Our findings highlight the importance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.

Design, Synthesis and Biological Evaluation of Novel MDH Inhibitors Targeting Tumor Microenvironment.

MDH1 and MDH2 enzymes play an important role in the survival of lung cancer. In this study, a novel series of dual MDH1/2 inhibitors for lung cancer was rationally designed and synthesized, and their SAR was carefully investigated. Among the tested compounds, compound 50 containing a piperidine ring displayed an improved growth inhibition of A549 and H460 lung cancer cell lines compared with LW1497. Compound 50 reduced the total ATP content in A549 cells in a dose-dependent manner; it also significantly suppressed the accumulation of hypoxia-inducible factor 1-alpha (HIF-1α) and the expression of HIF-1α target genes such as GLUT1 and pyruvate dehydrogenase kinase 1 (PDK1) in a dose-dependent manner. Furthermore, compound 50 inhibited HIF-1α-regulated CD73 expression under hypoxia in A549 lung cancer cells. Collectively, these results indicate that compound 50 may pave the way for the development of next-generation dual MDH1/2 inhibitors to target lung cancer.

DDIAS, DNA damage-induced apoptosis suppressor, is a potential therapeutic target in cancer.

Increasing evidence indicates that DNA damage-induced apoptosis suppressor (DDIAS) is an oncogenic protein that is highly expressed in a variety of cancers, including colorectal cancer, lung cancer, breast cancer, and hepatocellular carcinoma (HCC). The discovery of DDIAS as a novel therapeutic target and its role in human cancer biology is fascinating and noteworthy. Recent studies have shown that DDIAS is involved in tumorigenesis, metastasis, DNA repair and synthesis, and drug resistance and that it plays multiple roles with distinct binding partners in several human cancers. This review focuses on the function of DDIAS and its regulatory proteins in human cancer as potential targets for cancer therapy, as well as the development and future prospects of DDIAS inhibitors.

DNA methylome and single-cell transcriptome analyses reveal CDA as a potential druggable target for ALK inhibitor-resistant lung cancer therapy.

Acquired resistance to inhibitors of anaplastic lymphoma kinase (ALK) is a major clinical challenge for ALK fusion-positive non-small-cell lung cancer (NSCLC). In the absence of secondary ALK mutations, epigenetic reprogramming is one of the main mechanisms of drug resistance, as it leads to phenotype switching that occurs during the epithelial-to-mesenchymal transition (EMT). Although drug-induced epigenetic reprogramming is believed to alter the sensitivity of cancer cells to anticancer treatments, there is still much to learn about overcoming drug resistance. In this study, we used an in vitro model of ceritinib-resistant NSCLC and employed genome-wide DNA methylation analysis in combination with single-cell (sc) RNA-seq to identify cytidine deaminase (CDA), a pyrimidine salvage pathway enzyme, as a candidate drug target. CDA was hypomethylated and upregulated in ceritinib-resistant cells. CDA-overexpressing cells were rarely but definitively detected in the naïve cell population by scRNA-seq, and their abundance was increased in the acquired-resistance population. Knockdown of CDA had antiproliferative effects on resistant cells and reversed the EMT phenotype. Treatment with epigenome-related nucleosides such as 5-formyl-2'-deoxycytidine selectively ablated CDA-overexpressing resistant cells via accumulation of DNA damage. Collectively, our data suggest that targeting CDA metabolism using epigenome-related nucleosides represents a potential new therapeutic strategy for overcoming ALK inhibitor resistance in NSCLC.

Synaptotagmin 11 scaffolds MKK7-JNK signaling process to promote stem-like molecular subtype gastric cancer oncogenesis.

BACKGROUND: Identifying biomarkers related to the diagnosis and treatment of gastric cancer (GC) has not made significant progress due to the heterogeneity of tumors. Genes involved in histological classification and genetic correlation studies are essential to develop an appropriate treatment for GC. METHODS: In vitro and in vivo lentiviral shRNA library screening was performed. The expression of Synaptotagmin (SYT11) in the tumor tissues of patients with GC was confirmed by performing Immunohistochemistry, and the correlation between the expression level and the patient's survival rate was analyzed. Phospho-kinase array was performed to detect Jun N-terminal kinase (JNK) phosphorylation. SYT11, JNK, and MKK7 complex formation was confirmed by western blot and immunoprecipitation assays. We studied the effects of SYT11 on GC proliferation and metastasis, real-time cell image analysis, adhesion assay, invasion assay, spheroid formation, mouse xenograft assay, and liver metastasis. RESULTS: SYT11 is highly expressed in the stem-like molecular subtype of GC in transcriptome analysis of 527 patients with GC. Moreover, SYT11 is a potential prognostic biomarker for histologically classified diffuse-type GC. SYT11 functions as a scaffold protein, binding both MKK7 and JNK1 signaling molecules that play a role in JNK1 phosphorylation. In turn, JNK activation leads to a signaling cascade resulting in cJun activation and expression of downstream genes angiopoietin-like 2 (ANGPTL2), thrombospondin 4 (THBS4), Vimentin, and junctional adhesion molecule 3 (JAM3), which play a role in epithelial-mesenchymal transition (EMT). SNU484 cells infected with SYT11 shRNA (shSYT11) exhibited reduced spheroid formation, mouse tumor formation, and liver metastasis, suggesting a pro-oncogenic role of SYT11. Furthermore, SYT11-antisense oligonucleotide (ASO) displayed antitumor activity in our mouse xenograft model and was conferred an anti-proliferative effect in SNU484 and MKN1 cells. CONCLUSION: SYT11 could be a potential therapeutic target as well as a prognostic biomarker in patients with diffuse-type GC, and SYT11-ASO could be used in therapeutic agent development for stem-like molecular subtype diffuse GC.

TNF-α Secreted from Macrophages Increases the Expression of Prometastatic Integrin αV in Gastric Cancer.

The tumor microenvironment comprising blood vessels, fibroblasts, immune cells, and the extracellular matrix surrounding cancer cells, has recently been targeted for research in cancer therapy. We aimed to investigate the effect of macrophages on the invasive ability of gastric cancer cells, and studied their potential mechanism. In transcriptome analysis, integrin αV was identified as a gene increased in AGS cells cocultured with RAW264.7 cells. AGS cells cocultured with RAW264.7 cells displayed increased adhesion to the extracellular matrix and greater invasiveness compared with AGS cells cultured alone. This increased invasion of AGS cells cocultured with RAW264.7 cells was inhibited by integrin αV knockdown. In addition, the increase in integrin αV expression induced by tumor necrosis factor-α (TNF-α) or by coculture with RAW264.7 cells was inhibited by TNF receptor 1 (TNFR1) knockdown. The increase in integrin αV expression induced by TNF-α was inhibited by both Mitogen-activated protein kinase (MEK) inhibitor and VGLL1 S84 peptide treatment. Finally, transcription of integrin αV was shown to be regulated through the binding of VGLL1 and TEAD4 to the promoter of integrin αV. In conclusion, our study demonstrated that TNFR1-ERK-VGLL1 signaling activated by TNF-α secreted from RAW264.7 cells increased integrin αV expression, thereby increasing the adhesion and invasive ability of gastric cancer cells.

LW1497, an Inhibitor of Malate Dehydrogenase, Suppresses TGF-ß1-Induced Epithelial-Mesenchymal Transition in Lung Cancer Cells by Downregulating Slug.

LW1497 suppresses the expression of the hypoxia-inducing factor (HIF)-1α inhibiting malate dehydrogenase. Although hypoxia and HIF-1α are known to be important in cancer, LW1497 has not been therapeutically applied to cancer yet. Thus, we investigated the effect of LW1497 on the epithelial-mesenchymal transition (EMT) of lung cancer cells. A549 and H1299 lung cancer cells were induced to undergo via TGF-ß1 treatment, resulting in the downregulation of E-cadherin and upregulation of N-cadherin and Vimentin concurrently with increases in the migration and invasion capacities of the cells. These effects of TGF-ß1 were suppressed upon co-treatment of the cells with LW1497. An RNA-seq analysis revealed that LW1497 induced differential expression of genes related to hypoxia, RNA splicing, angiogenesis, cell migration, and metastasis in the A549 lung cancer cell lines. We confirmed the differential expression of Slug, an EMT-related transcription factor. Results from Western blotting and RT-PCR confirmed that LW1497 inhibited the expression of EMT markers and Slug. After orthotopically transplanting A549 cancer cells into mice, LW1497 was administered to examine whether the lung cancer progression was inhibited. We observed that LW1497 reduced the area of cancer. In addition, the results from immunohistochemical analyses showed that LW1497 downregulated EMT markers and Slug. In conclusion, LW1497 suppresses cancer progression through the inhibition of EMT by downregulating Slug.

DGG-100629 inhibits lung cancer growth by suppressing the NFATc1/DDIAS/STAT3 pathway.

DNA damage-induced apoptosis suppressor (DDIAS) promotes the progression of lung cancer and hepatocellular carcinoma through the regulation of multiple pathways. We screened a chemical library for anticancer agent(s) capable of inhibiting DDIAS transcription. DGG-100629 was found to suppress lung cancer cell growth through the inhibition of DDIAS expression. DGG-100629 induced c-Jun NH(2)-terminal kinase (JNK) activation and inhibited NFATc1 nuclear translocation. Treatment with SP600125 (a JNK inhibitor) or knockdown of JNK1 restored DDIAS expression and reversed DGG-100629-induced cell death. In addition, DGG-100629 suppressed the signal transducer and activator of transcription (STAT3) signaling pathway. DDIAS or STAT3 overexpression restored lung cancer cell growth in the presence of DGG-100629. In a xenograft assay, DGG-100629 inhibited tumor growth by reducing the level of phosphorylated STAT3 and the expression of STAT3 target genes. Moreover, DGG-100629 inhibited the growth of lung cancer patient-derived gefitinib-resistant cells expressing NFATc1 and DDIAS. Our findings emphasize the potential of DDIAS blockade as a therapeutic approach and suggest a novel strategy for the treatment of gefitinib-resistant lung cancer.

VGLL1 phosphorylation and activation promotes gastric cancer malignancy via TGF-ß/ERK/RSK2 signaling.

We previously reported that vestigial-like 1 (VGLL1), a cofactor of transcriptional enhanced associate domain 4 (TEAD4), is transcriptionally regulated by PI3K and ß-catenin signaling and is involved in gastric cancer malignancy. However, the precise mechanism underlying the regulation of VGLL1 activation remains unknown. Therefore, we aimed to investigate the molecular mechanism underlying the transforming growth factor-ß (TGF-ß)-mediated activation of VGLL1 and the VGLL1-TEAD4 interaction in gastric cancer cells. We showed that TGF-ß enhanced VGLL1 phosphorylation and that this phosphorylated VGLL1 functioned as a transcription cofactor of TEAD4 in NUGC3 cells. TGF-ß also increased the phosphorylation of ERK and ribosomal S6 kinase 2 (RSK2) in NUGC3 cells, thereby triggering the translocation of phosphorylated RSK2 to the nucleus. Site-directed mutagenesis and immunoprecipitation experiments revealed that RSK2 phosphorylated VGLL1 at S84 in the presence of TGF-ß. Mutation of VGLL1 at S84 suppressed VGLL1-TEAD4 binding and the subsequent transcriptional activation of matrix metalloprotease 9 (MMP9). Moreover, VGLL1 peptide containing S84 suppressed the TGF-ß-induced MMP9 expression and reduced the invasion and proliferation of gastric cancer cells, whereas VGLL1 peptide containing S84A did not. Furthermore, suppression of expression or activation of VGLL1 enhances the therapeutic effects of lapatinib. Collectively, these results indicate that VGLL1 phosphorylation via TGF-ß/ERK/RSK2 signaling plays a crucial role in MMP9-mediated malignancy of gastric cancer. In addition, our study highlights the therapeutic potential of the peptide containing VGLL1 S84 for the treatment of gastric cancer.

The disubstituted adamantyl derivative LW1564 inhibits the growth of cancer cells by targeting mitochondrial respiration and reducing hypoxia-inducible factor (HIF)-1α accumulation.

Targeting cancer metabolism has emerged as an important cancer therapeutic strategy. Here, we describe the synthesis and biological evaluation of a novel class of hypoxia-inducible factor (HIF)-1α inhibitors, disubstituted adamantyl derivatives. One such compound, LW1564, significantly suppressed HIF-1α accumulation and inhibited the growth of various cancer cell lines, including HepG2, A549, and HCT116. Measurements of the oxygen consumption rate (OCR) and ATP production rate revealed that LW1564 suppressed mitochondrial respiration, thereby increasing the intracellular oxygen concentration to stimulate HIF-1α degradation. LW1564 also significantly decreased overall ATP levels by inhibiting mitochondrial electron transport chain (ETC) complex I and downregulated mammalian target of rapamycin (mTOR) signaling by increasing the AMP/ATP ratio, which increased AMP-activated protein kinase (AMPK) phosphorylation. Consequently, LW1564 promoted the phosphorylation of acetyl-CoA carboxylase, which inhibited lipid synthesis. In addition, LW1564 significantly inhibited tumor growth in a HepG2 mouse xenograft model. Taken together, the results indicate that LW1564 inhibits the growth of cancer cells by targeting mitochondrial ETC complex I and impairing cancer cell metabolism. We, therefore, suggest that LW1564 may be a potent therapeutic agent for a subset of cancers that rely on oxidative phosphorylation for ATP generation.

Miconazole inhibits signal transducer and activator of transcription 3 signaling by preventing its interaction with DNA damage-induced apoptosis suppressor.

DNA damage-induced apoptosis suppressor (DDIAS) facilitates the survival of lung cancer by suppressing apoptosis. Moreover, DDIAS promotes tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) via their interaction. Here, we identified miconazole as an inhibitor of DDIAS/STAT3 interaction by screening a chemical library using a yeast two-hybrid assay. Miconazole inhibited growth, migration and invasion of lung cancer cells. Furthermore, miconazole suppressed STAT3 tyrosine Y705 phosphorylation and the expression of its target genes, such as cyclin D1, survivin and snail but had no suppressive effect on the activation of ERK1/2 or AKT, which is involved in the survival of lung cancer. As expected, no interaction between DDIAS and STAT3 occurred in the presence of miconazole, as confirmed by immunoprecipitation assays. Mouse xenograft experiments showed that miconazole significantly suppressed both tumor size and weight in an NCI-H1703 mouse model. Tyrosine phosphorylation of STAT3 at Y705 and expression of its targets, such as cyclin D1, survivin and snail, were decreased in miconazole-treated tumor tissues, as compared with those in vehicle-treated tumor tissues. These data suggest that miconazole exerts an anti-cancer effect by suppressing STAT3 activation through inhibiting DDIAS/STAT3 binding.

DDIAS promotes STAT3 activation by preventing STAT3 recruitment to PTPRM in lung cancer cells.

DNA damage-induced apoptosis suppressor (DDIAS) regulates cancer cell survival. Here we investigated the involvement of DDIAS in IL-6-mediated signaling to understand the mechanism underlying the role of DDIAS in lung cancer malignancy. We showed that DDIAS promotes tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is constitutively activated in malignant cancers. Interestingly, siRNA protein tyrosine phosphatase (PTP) library screening revealed protein tyrosine phosphatase receptor mu (PTPRM) as a novel STAT3 PTP. PTPRM knockdown rescued the DDIAS-knockdown-mediated decrease in STAT3 Y705 phosphorylation in the presence of IL-6. However, PTPRM overexpression decreased STAT3 Y705 phosphorylation. Moreover, endogenous PTPRM interacted with endogenous STAT3 for dephosphorylation at Y705 following IL-6 treatment. As expected, PTPRM bound to wild-type STAT3 but not the STAT3 Y705F mutant. PTPRM dephosphorylated STAT3 in the absence of DDIAS, suggesting that DDIAS hampers PTPRM/STAT3 interaction. In fact, DDIAS bound to the STAT3 transactivation domain (TAD), which competes with PTPRM to recruit STAT3 for dephosphorylation. Thus we show that DDIAS prevents PTPRM/STAT3 binding and blocks STAT3 Y705 dephosphorylation, thereby sustaining STAT3 activation in lung cancer. DDIAS expression strongly correlates with STAT3 phosphorylation in human lung cancer cell lines and tissues. Thus DDIAS may be considered as a potential biomarker and therapeutic target in malignant lung cancer cells with aberrant STAT3 activation.

PI3K/AKT/ß-Catenin Signaling Regulates Vestigial-Like 1 Which Predicts Poor Prognosis and Enhances Malignant Phenotype in Gastric Cancer.

Although gastric cancer is a common cause of cancer mortality worldwide, its biological heterogeneity limits the available therapeutic options. Therefore, identifying novel therapeutic targets for developing effective targeted therapy of gastric cancer is a pressing need. Here, we investigate molecular function and regulatory mechanisms of Vestigial-like 1 (VGLL1) in gastric cancer. Microarray analysis of 556 gastric cancer tissues revealed that VGLL1 was a prognostic biomarker that correlated with PI3KCA and PI3KCB. VGLL1 regulates the proliferation of gastric cancer cells, as shown in live cell imaging, sphere formation, and in vivo xenograft model. Tail vein injection of NUGC3 cells expressing shVGLL1 resulted in less lung metastasis occurring when compared to the control. In contrast, larger metastatic lesions in lung and liver were detected in the VGLL1-overexpressing NUGC3 cell xenograft excision mouse model. Importantly, VGLL1 expression is transcriptionally regulated by the PI3K-AKT-ß-catenin pathway. Subsequently, MMP9, a key molecule in gastric cancer, was explored as one of target genes that were transcribed by VGLL1-TEAD4 complex, a component of the transcription factor. Taken together, PI3K/AKT/ß-catenin signaling regulates the transcription of VGLL1, which promotes the proliferation and metastasis in gastric cancer. This finding suggests VGLL1 as a novel prognostic biomarker and a potential therapeutic target.

Bcl-2-dependent synthetic lethal interaction of the IDF-11774 with the V0 subunit C of vacuolar ATPase (ATP6V0C) in colorectal cancer.

BACKGROUND: The IDF-11774, a novel clinical candidate for cancer therapy, targets HSP70 and inhibits mitochondrial respiration, resulting in the activation of AMPK and reduction in HIF-1α accumulation. METHODS: To identify genes that have synthetic lethality to IDF-11774, RNA interference screening was conducted, using pooled lentiviruses expressing a short hairpin RNA library. RESULTS: We identified ATP6V0C, encoding the V0 subunit C of lysosomal V-ATPase, knockdown of which induced a synergistic growth-inhibitory effect in HCT116 cells in the presence of IDF-11774. The synthetic lethality of IDF-11774 with ATP6V0C possibly correlates with IDF-11774-mediated autolysosome formation. Notably, the synergistic effect of IDF-11774 and the ATP6V0C inhibitor, bafilomycin A1, depended on the PIK3CA genetic status and Bcl-2 expression, which regulates autolysosome formation and apoptosis. Similarly, in an experiment using conditionally reprogramed cells derived from colorectal cancer patients, synergistic growth inhibition was observed in cells with low Bcl-2 expression. CONCLUSIONS: Bcl-2 is a biomarker for the synthetic lethal interaction of IDF-11774 with ATP6V0C, which is clinically applicable for the treatment of cancer patients with IDF-11774 or autophagy-inducing anti-cancer drugs.

RNF25 promotes gefitinib resistance in EGFR-mutant NSCLC cells by inducing NF-κB-mediated ERK reactivation.

Non-small cell lung cancer (NSCLC) patients with EGFR mutations initially respond well to EGFR tyrosine kinase inhibitors (TKIs) but eventually exhibit acquired or innate resistance to the therapies typically due to gene mutations, such as EGFR T790M mutation or a second mutation in the downstream pathways of EGFR. Importantly, a significant portion of NSCLC patients shows TKI resistance without any known mechanisms, calling more comprehensive studies to reveal the underlying mechanisms. Here, we investigated a synthetic lethality with gefitinib using a genome-wide RNAi screen in TKI-resistant EGFR-mutant NSCLC cells, and identified RNF25 as a novel factor related to gefitinib resistance. Depletion of RNF25 expression substantially sensitized NSCLC cells to gefitinib treatment, while forced expression of RNF25 augmented gefitinib resistance in sensitive cells. We demonstrated that RNF25 mediates NF-κB activation in gefitinib-treated cells, which, in turn, induces reactivation of ERK signal to cause the drug resistance. We identified that the ERK reactivation occurs via the function of cytokines, such as IL-6, whose expression is transcriptionally induced in a gefitinib-dependent manner by RNF25-mediated NF-κB signals. These results suggest that RNF25 plays an essential role in gefitinib resistance of NSCLC by mediating cross-talk between NF-κB and ERK pathways, and provide a novel target for the combination therapy to overcome TKI resistance of NSCLC.

DDIAS suppresses TRAIL-mediated apoptosis by inhibiting DISC formation and destabilizing caspase-8 in cancer cells.

DNA damage-induced apoptosis suppressor (DDIAS) has an anti-apoptotic function during DNA damage in lung cancer. However, the anti-apoptotic mechanism of DDIAS in cancer cells under other conditions has not been reported. We report here that DDIAS protects cancer cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by two distinct mechanisms in non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC) cells. DDIAS depletion sensitized NSCLC and HCC cells to TRAIL-mediated apoptosis, an effect that was abrogated by pharmacological or genetic inhibition of caspase-8 and was independent of caspase-9, p53, or mitogen-activated protein kinase signaling. Interestingly, we found that the N terminus of DDIAS interacted with the death effector domain of Fas-associated protein death domain (FADD) and prevented its recruitment to the death-inducing signaling complex (DISC), thereby blocking caspase-8 activation. DDIAS knockdown also suppressed epidermal growth factor-induced phosphorylation of p90 ribosomal S6 kinase (RSK) 2 and stabilized caspase-8 by preventing its ubiquitination and proteasomal degradation. This effect was abolished by RSK2 overexpression. Taken together, DDIAS has dual functions in inhibiting DISC formation as well as in destabilizing caspase-8, thereby suppressing TRAIL-mediated apoptosis of cancer cells. Thus, we suggest that DDIAS can serve as an effective therapeutic target in the treatment of NSCLC and HCC.

Elasticity-based development of functionally enhanced multicellular 3D liver encapsulated in hybrid hydrogel.

Current in vitro liver models provide three-dimensional (3-D) microenvironments in combination with tissue engineering technology and can perform more accurate in vivo mimicry than two-dimensional models. However, a human cell-based, functionally mature liver model is still desired, which would provide an alternative to animal experiments and resolve low-prediction issues on species differences. Here, we prepared hybrid hydrogels of varying elasticity and compared them with a normal liver, to develop a more mature liver model that preserves liver properties in vitro. We encapsulated HepaRG cells, either alone or with supporting cells, in a biodegradable hybrid hydrogel. The elastic modulus of the 3D liver dynamically changed during culture due to the combined effects of prolonged degradation of hydrogel and extracellular matrix formation provided by the supporting cells. As a result, when the elastic modulus of the 3D liver model converges close to that of the in vivo liver (≅ 2.3 to 5.9 kPa), both phenotypic and functional maturation of the 3D liver were realized, while hepatic gene expression, albumin secretion, cytochrome p450-3A4 activity, and drug metabolism were enhanced. Finally, the 3D liver model was expanded to applications with embryonic stem cell-derived hepatocytes and primary human hepatocytes, and it supported prolonged hepatocyte survival and functionality in long-term culture. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. STATEMENT OF SIGNIFICANCE: We provide a functionally improved 3D liver model that recapitulates in vivo liver stiffness. We have experimentally addressed the issues of orchestrated effects of mechanical compliance, controlled matrix formation by stromal cells in conjunction with hepatic differentiation, and functional maturation of hepatocytes in a dynamic 3D microenvironment. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. Additionally, recent advances in the stem-cell technologies have made the development of 3D organoid possible, and thus, our study also provides further contribution to the development of physiologically relevant stem-cell-based 3D tissues that provide an elasticity-based predefined biomimetic 3D microenvironment.

Methyl 3-(3-(4-(2,4,4-Trimethylpentan-2-yl)phenoxy)-propanamido)benzoate as a Novel and Dual Malate Dehydrogenase (MDH) 1/2 Inhibitor Targeting Cancer Metabolism.

Previously, we reported a hypoxia-inducible factor (HIF)-1 inhibitor LW6 containing an (aryloxyacetylamino)benzoic acid moiety inhibits malate dehydrogenase 2 (MDH2) using a chemical biology approach. Structure-activity relationship studies on a series of (aryloxyacetylamino)benzoic acids identified selective MDH1, MDH2, and dual inhibitors, which were used to study the relationship between MDH enzyme activity and HIF-1 inhibition. We hypothesized that dual inhibition of MDH1 and MDH2 might be a powerful approach to target cancer metabolism and selected methyl-3-(3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propanamido)-benzoate (16c) as the most potent dual inhibitor. Kinetic studies revealed that compound 16c competitively inhibited MDH1 and MDH2. Compound 16c inhibited mitochondrial respiration and hypoxia-induced HIF-1α accumulation. In xenograft assays using HCT116 cells, compound 16c demonstrated significant in vivo antitumor efficacy. This finding provides concrete evidence that inhibition of both MDH1 and MDH2 may provide a valuable platform for developing novel therapeutics that target cancer metabolism and tumor growth.

The novel hypoxia-inducible factor-1α inhibitor IDF-11774 regulates cancer metabolism, thereby suppressing tumor growth.

HIF-1 is associated with poor prognoses and therapeutic resistance in cancer patients. We previously developed a novel hypoxia-inducible factor (HIF)-1 inhibitor, IDF-11774, a clinical candidate for cancer therapy. We also reported that IDF-1174 inhibited HSP70 chaperone activity and suppressed accumulation of HIF-1α. In this study, IDF-11774 inhibited the accumulation of HIF-1α in vitro and in vivo in colorectal carcinoma HCT116 cells under hypoxic conditions. Moreover, IDF-11774 treatment suppressed angiogenesis of cancer cells by reducing the expression of HIF-1 target genes, reduced glucose uptake, thereby sensitizing cells to growth under low glucose conditions, and decreased the extracellular acidification rate (ECAR) and oxygen consumption rate of cancer cells. Metabolic profiling of IDF-11774-treated cells revealed low levels of NAD+, NADP+, and lactate, as well as of intermediates in glycolysis and the tricarboxylic acid cycle. In addition, we observed elevated AMP and diminished ATP levels, resulting in a high AMP/ATP ratio. The level of AMP-activated protein kinase phosphorylation also increased, leading to inhibition of mTOR signaling in treated cells. In vivo xenograft assays demonstrated that IDF-11774 exhibited substantial anticancer efficacy in mouse models containing KRAS, PTEN, or VHL mutations, which often occur in malignant cancers. Collectively, our data indicate that IDF-11774 suppressed hypoxia-induced HIF-1α accumulation and repressed tumor growth by targeting energy production-related cancer metabolism.

Synthesis and biological evaluation of kresoxim-methyl analogues as novel inhibitors of hypoxia-inducible factor (HIF)-1 accumulation in cancer cells.

We designed and synthesized strobilurin analogues as hypoxia-inducible factor (HIF) inhibitors based on the molecular structure of kresoxim-methyl. Biological evaluation in human colorectal cancer HCT116 cells showed that most of the synthesized kresoxim-methyl analogues possessed moderate to potent inhibitory activity against hypoxia-induced HIF-1 transcriptional activation. Three candidates, compounds 11b, 11c, and 11d were identified as potent inhibitors against HIF-1 activation with IC50 values of 0.60-0.94µM. Under hypoxic condition, compounds 11b, 11c, and 11d increased the intracellular oxygen contents, thereby attenuating the hypoxia-induced accumulation of HIF-1α protein.