MYC/Glutamine Dependency Is a Therapeutic Vulnerability in Pancreatic Cancer with Deoxycytidine Kinase Inactivation-Induced Gemcitabine Resistance.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most life-threatening malignancies. Although the deoxycytidine analog gemcitabine has been used as the first-line treatment for PDAC, the primary clinical challenge arises because of an eventual acquisition of resistance. Therefore, it is crucial to elucidate the mechanisms underlying gemcitabine resistance to improve treatment efficacy. To investigate potential genes whose inactivation confers gemcitabine resistance, we performed CRISPR knockout (KO) library screening. We found that deoxycytidine kinase (DCK) deficiency is the primary mechanism of gemcitabine resistance, and the inactivation of CRYBA2, DMBX1, CROT, and CD36 slightly conferred gemcitabine resistance. In particular, gene expression analysis revealed that DCK KO cells displayed a significant enrichment of genes associated with MYC targets, folate/one-carbon metabolism and glutamine metabolism pathways. Evidently, chemically targeting each of these pathways significantly reduced the survival of DCK KO cells. Moreover, the pathways enriched in DCK KO cells represented a trend similar to those in PDAC cell lines and samples of patients with PDAC with low DCK expression. We further observed that short-term treatment of parental CFPAC-1 cells with gemcitabine induces the expression of several genes, which promote synthesis and transport of glutamine in a dose-dependent manner, which suggests glutamine availability as a potential mechanism of escaping drug toxicity in an initial response for survival. Thus, our findings provide insights into novel therapeutic approaches for gemcitabine-resistant PDAC and emphasize the involvement of glutamine metabolism in drug-tolerant persister cells. IMPLICATIONS: Our study revealed the key pathways involved in gemcitabine resistance in PDAC, thus providing potential therapeutic strategies.

KDM4B promotes acute myeloid leukemia associated with AML1-ETO by regulating chromatin accessibility.

Epigenetic alterations of chromatin structure affect chromatin accessibility and collaborate with genetic alterations in the development of cancer. Lysine demethylase 4B (KDM4B) has been identified as a JmjC domain-containing epigenetic modifier that possesses histone demethylase activity. Although recent studies have demonstrated that KDM4B positively regulates the pathogenesis of multiple types of solid tumors, the tissue specificity and context dependency have not been fully elucidated. In this study, we investigated gene expression profiles established from clinical samples and found that KDM4B is elevated specifically in acute myeloid leukemia (AML) associated with chromosomal translocation 8;21 [t(8;21)], which results in a fusion of the AML1 and the eight-twenty-one (ETO) genes to generate a leukemia oncogene, AML1-ETO fusion transcription factor. Short hairpin RNA-mediated KDM4B silencing significantly reduced cell proliferation in t(8;21)-positive AML cell lines. Meanwhile, KDM4B silencing suppressed the expression of AML1-ETO-inducible genes, and consistently perturbed chromatin accessibility of AML1-ETO-binding sites involving altered active enhancer marks and functional cis-regulatory elements. Notably, transduction of murine KDM4B orthologue mutants followed by KDM4B silencing demonstrated a requirement of methylated-histone binding modules for a proliferative surge. To address the role of KDM4B in leukemia development, we further generated and analyzed Kdm4b conditional knockout mice. As a result, Kdm4b deficiency attenuated clonogenic potential mediated by AML1-ETO and delayed leukemia progression in vivo. Thus, our results highlight a tumor-promoting role of KDM4B in AML associated with t(8;21).

Ascorbate sensitizes human osteosarcoma cells to the cytostatic effects of cisplatin.

Osteosarcoma (OS) is the most common malignant bone tumor and a leading cause of cancer-related deaths in children and adolescents. Current standard treatments for OS are a combination of preoperative chemotherapy, surgical resection, and adjuvant chemotherapy. Cisplatin is used as the standard chemotherapeutic for OS treatment, but it induces various adverse effects, limiting its clinical application. Improving treatment efficacy without increasing the cisplatin dosage is desirable. In the present study, we assessed the combined effect of ascorbate on cisplatin treatment using cultured human OS cells. Co-treatment with ascorbate induced greater suppression of OS cell but not nonmalignant cell proliferation. The chemosensitizing effect of ascorbate on cisplatin treatment was tightly linked to ROS production. Altered cellular redox state due to increased ROS production modified glycolysis and mitochondrial function in OS cells. In addition, OS cell sphere formation was markedly decreased, suggesting that ascorbate increased the treatment efficacy of cisplatin against stem-like cells in the cancer cell population. We also found that enhanced MYC signaling, ribosomal biogenesis, glycolysis, and mitochondrial respiration are key signatures in OS cells with cisplatin resistance. Furthermore, cisplatin resistance was reversed by ascorbate. Taken together, our findings provide a rationale for combining cisplatin with ascorbate in therapeutic strategies against OS.

Telomeres and sirtuins: at the end we meet again.

Telomeres and sirtuins are independently implicated in causing disease and aging, but how they cooperate is not well understood. A recent study demonstrates that telomere shortening represses sirtuins and increasing sirtuin activity stabilizes telomeres and improves telomere-dependent disease, suggesting that these two pathways are tightly intertwined.

High Fat Diet Triggers a Reduction in Body Fat Mass in Female Mice Deficient for Utx demethylase.

Obesity increases the risk of metabolic disorders like diabetes mellitus and dyslipidemia. However, how metabolic status is sensed and regulates cellular behavior is unclear. Utx is an H3K27 demethylase that influences adipocyte function in vitro. To examine its role in vivo, we generated mice lacking Utx in adipocytes (UtxAKO). Although all UtxAKO mice grew normally on a normal chow diet (NCD), female UtxAKO mice on a high fat diet (HFD) showed striking reductions in body fat compared to control mice (Ctrl). Gene expression profiling of adipose tissues of HFD-fed UtxAKO female mice revealed decreased expression of rate-limiting enzymes of triacylglycerol synthesis but increased expression of those of cholesterol/steroid hormone synthesis. Moreover, these animals resisted adiposity induced by ovariectomy and exhibited increased estrogen in visceral adipose tissues. Thus, upon HFD feeding, Utx regulates lipid metabolism in adipose tissues by influencing the local hormonal microenvironment. Conversely, Utx deficiency skews lipid catabolism to enhance cholesterol/steroid hormone production and repress obesity.

Necl-5/poliovirus receptor interacts with VEGFR2 and regulates VEGF-induced angiogenesis.

RATIONALE: Vascular endothelial growth factor (VEGF), a major proangiogenic agent, exerts its proangiogenic action by binding to VEGF receptor 2 (VEGFR2), the activity of which is regulated by direct interactions with other cell surface proteins, including integrin α(V)ß(3). However, how the interaction between VEGFR2 and integrin α(V)ß(3) is regulated is not clear. OBJECTIVE: To investigate whether Necl-5/poliovirus receptor, an immunoglobulin-like molecule that is known to bind integrin α(V)ß(3), regulates the interaction between VEGFR2 and integrin α(V)ß(3), and to clarify the role of Necl-5 in the VEGF-induced angiogenesis. METHODS AND RESULTS: Necl-5-knockout mice displayed no obvious defect in vascular development; however, recovery of blood flow after hindlimb ischemia and the VEGF-induced neovascularization in implanted Matrigel plugs were impaired in Necl-5-knockout mice. To clarify the mechanism of the regulation of angiogenesis by Necl-5, we investigated the roles of Necl-5 in the VEGF-induced angiogenic responses in vitro. Knockdown of Necl-5 by siRNAs in human umbilical vein endothelial cells (HUVECs) inhibited the VEGF-induced capillary-like network formation on Matrigel, migration, and proliferation, and conversely, enhanced apoptosis. Coimmunoprecipitation assays showed the interaction of Necl-5 with VEGFR2, and knockdown of Necl-5 prevented the VEGF-induced interaction of integrin α(V)ß(3) with VEGFR2. Knockdown of Necl-5 suppressed the VEGFR2-mediated activation of downstream proangiogenic and survival signals, including Rap1, Akt, and endothelial nitric oxide synthase. CONCLUSIONS: These results demonstrate the critical role of Necl-5 in angiogenesis and suggest that Necl-5 may regulate the VEGF-induced angiogenesis by controlling the interaction of VEGFR2 with integrin α(v)ß(3), and the VEGFR2-mediated Rap1-Akt signaling pathway.

Role of afadin in vascular endothelial growth factor- and sphingosine 1-phosphate-induced angiogenesis.

RATIONALE: Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis. OBJECTIVE: We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism. METHODS AND RESULTS: Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell-cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin-PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K-Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis. CONCLUSIONS: These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.

Interaction and localization of Necl-5 and PDGF receptor beta at the leading edges of moving NIH3T3 cells: Implications for directional cell movement.

It was previously shown that platelet-derived growth factor (PDGF) receptor physically and functionally interacts with integrin alpha(v)beta(3), effectively inducing cell movement. We previously showed that Necl-5, originally identified as a poliovirus receptor, interacts with integrin alpha(v)beta(3) and enhances its clustering and the formation of focal complexes at the leading edges of moving cells, resulting in an enhancement of cell movement. We showed here that Necl-5 additionally interacts with PDGF receptor in NIH3T3 cells and regulates the interaction between PDGF receptor and integrin alpha(v)beta(3), effectively inducing directional cell movement. PDGF receptor co-localized with Necl-5 and integrin alpha(v)beta(3) at peripheral ruffles over lamellipodia, which were formed at the leading edges of moving cells in response to PDGF, but not at the focal complexes under these ruffles, whereas Necl-5 and integrin alpha(v)beta(3) co-localized at these focal complexes. The clustering of these three molecules at peripheral ruffles required the activation of integrin alpha(v)beta(3) by vitronectin and the PDGF-induced activation of the small G protein Rac and subsequent re-organization of the actin cytoskeleton. These results indicate a key role of Necl-5 in directional cell movement by physically and functionally interacting with both integrin alpha(v)beta(3) and PDGF receptor.

Necl-5/poliovirus receptor interacts in cis with integrin alphaVbeta3 and regulates its clustering and focal complex formation.

Integrin alphavbeta3, which forms focal complexes at leading edges in moving cells, is up-regulated in cancer cells and so is implicated in their invasiveness. Necl-5, originally identified as a poliovirus receptor and also up-regulated in cancer cells, colocalizes with integrin alphavbeta3 at leading edges in moving cells and enhances growth factor-induced cell movement. Here, we show that Necl-5 interacts directly, in cis, with integrin alphavbeta3, and enhances integrin alphavbeta3 clustering and focal complex formation at leading edges in NIH3T3 cells. The extracellular region of Necl-5, but not the cytoplasmic region, is necessary for its interaction with integrin alphavbeta3; however, both regions are necessary for its action. An interaction between integrin alphavbeta3 and vitronectin and PDGF-induced activation of Rac are also necessary for integrin alphavbeta3 clustering. The interaction between Necl-5 and integrin alphavbeta3 enhances PDGF-induced Rac activation, facilitating integrin alphavbeta3 clustering presumably in a feedback amplification manner. Thus, Necl-5 has a critical role in integrin alphavbeta3 clustering and focal complex formation.

Nerve growth factor attenuates endoplasmic reticulum stress-mediated apoptosis via suppression of caspase-12 activity.

Following endoplasmic reticulum (ER) stress, which occurs via inhibition of the glycosylation of newly synthesized proteins, caspase family proteins are activated to promote ER stress-mediated apoptosis. Here we report that nerve growth factor (NGF) suppressed the ER stress-mediated apoptosis in tunicamycin-treated PC12 cells through an extensive decrease of the caspase-3/-9/-12 activity. Detailed analysis of the mechanism underlying the NGF-mediated cell survival revealed that the activities of all seriate caspases were reduced through the phosphatidylinositol 3-kinase (PI3-K) signaling pathway induced by NGF. Moreover, we found that the activity of c-Jun N-terminal kinase (JNK) was not essential for the tunicamycin-induced apoptosis of PC12 cells. These results demonstrate that the inactivation of caspase-12 via the NGF-mediated PI3-K signaling pathway leads to inactivation of the caspase cascade including caspase-3 and -9.