The membrane-linked adaptor FRS2ß fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis.

Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2ß, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2ß deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2ß-deficient premalignant tissues. We found that colocalization of FRS2ß and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2ß contain more stroma. The elucidation of the FRS2ß-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.

Mitochondrial one-carbon metabolic enzyme MTHFD2 facilitates mammary gland development during pregnancy.

Mitochondrial one-carbon metabolism is crucial for embryonic development and tumorigenesis, as it supplies one-carbon units necessary for nucleotide synthesis and rapid cell proliferation. However, its contribution to adult tissue homeostasis remains largely unknown. To examine its role in adult tissue homeostasis, we specifically investigated mammary gland development during pregnancy, as it involves heightened cell proliferation. We discovered that MTHFD2, a mitochondrial one-carbon metabolic enzyme, is expressed in both luminal and basal/myoepithelial cell layers, with upregulated expression during pregnancy. Using the mouse mammary tumor virus (MMTV)-Cre recombinase system, we generated mice with a specific mutation of Mthfd2 in mammary epithelial cells. While the mutant mice were capable of properly nurturing their offspring, the pregnancy-induced expansion of mammary glands was significantly delayed. This indicates that MTHFD2 contributes to the rapid development of mammary glands during pregnancy. Our findings shed light on the role of mitochondrial one-carbon metabolism in facilitating rapid cell proliferation, even in the context of the adult tissue homeostasis.

Semaphorin signaling via MICAL3 induces symmetric cell division to expand breast cancer stem-like cells.

Cancer stem-like cells (CSCs) are expanded in the CSC niche by increased frequency of symmetric cell divisions at the expense of asymmetric cell divisions. The symmetric division of CSCs is important for the malignant properties of cancer; however, underlying molecular mechanisms remain largely elusive. Here, we show a cytokine, semaphorin 3 (Sema3), produced from the CSC niche, induces symmetric divisions of CSCs to expand the CSC population. Our findings indicate that stimulation with Sema3 induced sphere formation in breast cancer cells through neuropilin 1 (NP1) receptor that was specifically expressed in breast CSCs (BCSCs). Knockdown of MICAL3, a cytoplasmic Sema3 signal transducer, greatly decreased tumor sphere formation and tumor-initiating activity. Mechanistically, Sema3 induced interaction among MICAL3, collapsin response mediator protein 2 (CRMP2), and Numb. It appears that activity of MICAL3 monooxygenase (MO) stimulated by Sema3 is required for tumor sphere formation, interaction between CRMP2 and Numb, and accumulation of Numb protein. We found that knockdown of CRMP2 or Numb significantly decreased tumor sphere formation. Moreover, MICAL3 knockdown significantly decreased Sema3-induced symmetric divisions in NP1/Numb-positive BCSCs and increased asymmetric division that produces NP1/Numb negative cells without stem-like properties. In addition, breast cancer patients with NP1-positive cancer tissues show poor prognosis. Therefore, the niche factor Sema3-stimulated NP1/MICAL3/CRMP2/Numb axis appears to expand CSCs at least partly through increased frequency of MICAL3-mediated symmetric division of CSCs.

MUSASHI-2 confers resistance to third-generation EGFR-tyrosine kinase inhibitor osimertinib in lung adenocarcinoma.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in patients with non-small-cell lung cancer (NSCLC) harboring EGFR mutations. However, due to acquired resistance to EGFR-TKIs, even patients on third-generation osimertinib have a poor prognosis. Resistance mechanisms are still not fully understood. Here, we demonstrate that the increased expression of MUSASHI-2 (MSI2), an RNA-binding protein, is a novel mechanism for resistance to EGFR-TKIs. We found that after a long-term exposure to gefitinib, the first-generation EGFR-TKI lung cancer cells harboring the EGFR-TKI-sensitive mutations became resistant to both gefitinib and osimertinib. Although other mutations in EGFR were not found, expression levels of Nanog, a stemness core protein, and activities of aldehyde dehydrogenase (ALDH) were increased, suggesting that cancer stem-like properties were increased. Transcriptome analysis revealed that MSI2 was among the stemness-related genes highly upregulated in EGFR-TKI-resistant cells. Knockdown of MSI2 reduced cancer stem-like properties, including the expression levels of Nanog, a core stemness factor. We demonstrated that knockdown of MSI2 restored sensitivity to osimertinib or gefitinib in EGFR-TKI-resistant cells to levels similar to those of parental cells in vitro. An RNA immunoprecipitation (RIP) assay revealed that antibodies against MSI2 were bound to Nanog mRNA, suggesting that MSI2 increases Nanog expression by binding to Nanog mRNA. Moreover, overexpression of MSI2 or Nanog conferred resistance to osimertinib or gefitinib in parental cells. Finally, MSI2 knockdown greatly increased the sensitivity to osimertinib in vivo. Collectively, our findings provide proof of principle that targeting the MSI2-Nanog axis in combination with EGFR-TKIs would effectively prevent the emergence of acquired resistance.

MCM10 compensates for Myc-induced DNA replication stress in breast cancer stem-like cells.

Cancer stem-like cells (CSCs) induce drug resistance and recurrence of tumors when they experience DNA replication stress. However, the mechanisms underlying DNA replication stress in CSCs and its compensation remain unclear. Here, we demonstrate that upregulated c-Myc expression induces stronger DNA replication stress in patient-derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini-chromosome maintenance protein 10 (MCM10), a firing (activating) factor of DNA replication origins, to compensate for DNA replication stress in CSCs. MCM10 expression is upregulated in CSCs and is maintained by c-Myc. c-Myc-dependent collisions between RNA transcription and DNA replication machinery may occur in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to these collisions to ensure the progression of replication. Moreover, patient-derived breast CSCs were found to be dependent on MCM10 for their maintenance, even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. Further, MCM10 depletion decreased the growth of cancer cells, but not of normal cells. Therefore, MCM10 may robustly compensate for DNA replication stress and facilitate genome duplication in cancer cells in the S-phase, which is more pronounced in CSCs. Overall, we provide a preclinical rationale to target the c-Myc-MCM10 axis for preventing drug resistance and recurrence of tumors.

A novel oral inhibitor for one-carbon metabolism and checkpoint kinase 1 inhibitor as a rational combination treatment for breast cancer.

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.

Upregulation of S100A10 in metastasized breast cancer stem cells.

Metastatic progression remains the major cause of death in human breast cancer. Cancer cells with cancer stem cell (CSC) properties drive initiation and growth of metastases at distant sites. We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, the expression levels of S100A10 and its family proteins were much higher in the CD44+ cancer cells metastasized to the liver than those at the primary site. Knockdown of S100A10 in breast cancer cells suppressed and overexpression of S100A10 in breast cancer PDX cells enhanced their invasion abilities and 3D organoid formation capacities in vitro. Mechanistically, S100A10 regulated the matrix metalloproteinase activity and the expression levels of stem cell-related genes. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers.

Alveolar Macrophages Drive Hepatocellular Carcinoma Lung Metastasis by Generating Leukotriene B4.

Macrophages in lungs can be classified into two subpopulations, alveolar macrophages (AMs) and interstitial macrophages (IMs), which reside in the alveolar and interstitial spaces, respectively. Accumulating evidence indicates the involvement of IMs in lung metastasis, but the roles of AMs in lung metastasis still remain elusive. An i.v. injection of a mouse hepatocellular carcinoma (HCC) cell line, BNL, caused lung metastasis foci with infiltration of AMs and IMs. Comprehensive determination of arachidonic acid metabolite levels revealed increases in leukotrienes and PGs in lungs in this metastasis model. A 5-lipoxygenase (LOX) inhibitor but not a cyclooxygenase inhibitor reduced the numbers of metastatic foci, particularly those of a larger size. A major 5-LOX metabolite, LTB4, augmented in vitro cell proliferation of human HCC cell lines as well as BNL cells. Moreover, in this lung metastasis course, AMs exhibited higher expression levels of the 5-LOX and LTB4 than IMs. Consistently, 5-LOX-expressing AMs increased in the lungs of human HCC patients with lung metastasis, compared with those without lung metastasis. Furthermore, intratracheal clodronate liposome injection selectively depleted AMs but not IMs, together with reduced LTB4 content and metastatic foci numbers in this lung metastasis process. Finally, IMs in mouse metastatic foci produced CCL2, thereby recruiting blood-borne, CCR2-expressing AMs into lungs. Thus, AMs can be recruited under the guidance of IM-derived CCL2 into metastatic lungs and can eventually contribute to the progression of lung metastasis by providing a potent arachidonic acid-derived tumor growth promoting mediator, LTB4.

A cyclic peptide-grafted Fc with hepatocyte growth factor functionality ameliorates hepatic fibrosis in a non-alcoholic steatohepatitis mouse model.

The regenerative functions associated with cytokines and growth factors have immense therapeutic potential; however, their poor pharmacokinetics, resulting from structural features, hinder their effectiveness. In this study, we aimed to enhance the pharmacokinetics of growth factors by designing receptor-binding macrocyclic peptides through in vitro mRNA display and grafting them into loops of immunoglobulin's crystallizable region (Fc). As a model, we developed peptide-grafted Fc proteins with hepatocyte growth factor (HGF) functionality that exhibited a prolonged circulation half-life and could be administered subcutaneously. The Fc-based HGF mimetic alleviated liver fibrosis in a mouse model fed a choline-deficient high-fat diet, which induces hepatic features of non-alcoholic steatohepatitis, including fibrosis, showcasing its potential as a therapeutic intervention. This study provides a basis for developing growth factor and cytokine mimetics with improved pharmacokinetics, expanding their therapeutic applications.

The acquisition of malignant potential in colon cancer is regulated by the stabilization of Atonal homolog 1 protein.

The transcription factor Atonal homolog 1 (Atoh1) plays crucial roles in the differentiation of intestinal epithelium cells. Although we have reported that the Atoh1 protein was degraded in colon cancer by aberrant Wnt signaling, a recent study has indicated that the Atoh1 protein is expressed in mucinous colon cancer (MC) and signet ring cell carcinoma (SRCC). However, the roles of the Atoh1 protein in MC are unknown. To mimic MC, a mutated Atoh1 protein was stably expressed in undifferentiated colon cancer cells. Microarray analysis revealed the acquisition of not only the differentiated cell form, but also malignant potential by Atoh1 protein stabilization. In particular, Atoh1 enhanced Wnt signaling, resulting in the induction of Lgr5 as a representative stem cell marker with the enrichment of cancer stem cells. Moreover, the fluorescent ubiquitination-based cell cycle indicator system with time-lapse live imaging demonstrated cell cycle arrest in the G0/G1 phase by Atoh1 protein stabilization. In conclusion, the Atoh1 protein regulates malignant potential rather than the differentiation phenotype of MC, suggesting the mechanism by which MC and SRCC are more malignant than non-mucinous adenocarcinoma.

TUG1-mediated R-loop resolution at microsatellite loci as a prerequisite for cancer cell proliferation.

Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.

FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters.

The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain-containing ion transport regulator 3 (FXYD3), a component of the Na+/K+ pump. Accordingly, FXYD3+ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3+ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3+ CSCs were sensitive to senolytic Na+/K+ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3+ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na+/K+ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.

Cytoplasmic DNA accumulation preferentially triggers cell death of myeloid leukemia cells by interacting with intracellular DNA sensing pathway.

Accumulating evidence indicates the presence of cytoplasmic DNAs in various types of malignant cells, and its involvement in anti-cancer drug- or radiotherapy-mediated DNA damage response and replication stress. However, the pathophysiological roles of cytoplasmic DNAs in leukemias remain largely unknown. We observed that during hematopoietic stem cell transplantation (HSCT) in mouse myeloid leukemia models, double-stranded (ds)DNAs were constitutively secreted in the form of extracellular vesicles (EVs) from myeloid leukemia cells and were transferred to the donor cells to dampen their hematopoietic capabilities. Subsequent analysis of cytoplasmic DNA dynamics in leukemia cells revealed that autophagy regulated cytoplasmic dsDNA accumulation and subsequent redistribution into EVs. Moreover, accumulated cytoplasmic dsDNAs activated STING pathway, thereby reducing leukemia cell viability through reactive oxygen species (ROS) generation. Pharmaceutical inhibition of autophagosome formation induced cytoplasmic DNA accumulation, eventually triggering cytoplasmic DNA sensing pathways to exert cytotoxicity, preferentially in leukemia cells. Thus, manipulation of cytoplasmic dsDNA dynamics can be a novel and potent therapeutic strategy for myeloid leukemias.

Cancer stem-like properties and gefitinib resistance are dependent on purine synthetic metabolism mediated by the mitochondrial enzyme MTHFD2.

Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)-the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib, caused by elevated expression of components of the ß-catenin pathway, exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in these gefitinib-resistant cancer cells. Moreover, overexpression of MTHFD2 in gefitinib-sensitive lung cancer cells conferred resistance to gefitinib. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.

Drug discovery of anticancer drugs targeting methylenetetrahydrofolate dehydrogenase 2.

Many anticancer drugs have serious adverse effects; therefore, it is necessary to target features specific to cancer cells to minimize the effects on healthy cells. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was reported to be specifically enhanced in cancer. We confirmed the validity of MTHFD2 as a drug discovery target using clinical data. In addition, we performed in silico screening to design an anticancer drug specifically targeting MTHFD2. Analysis of the clinical data indicated that MTHFD2 was enhanced in most cancers compared with normal tissues, and affected the prognosis in cancer patients. Candidate compounds for MTHFD2 inhibitors were identified using in silico drug discovery techniques, and the important interactions for MTHFD2 binding were determined. In addition, these candidate compounds decreased levels of MTHFD2 metabolites in cancer cells. The findings of the present study may help to develop anticancer drugs targeting MTHFD2, with a view to minimizing the adverse effects of anticancer drugs.

An autocrine/paracrine circuit of growth differentiation factor (GDF) 15 has a role for maintenance of breast cancer stem-like cells.

Cancer stem cells are thought to be responsible for tumor growth, recurrence, and resistance to conventional cancer therapy. However, it is still unclear how they are maintained in tumor tissues. Here, we show that the growth differentiation factor 15 (GDF15), a member of the TGFß family, may maintain cancer stem-like cells in breast cancer tissues by inducing its own expression in an autocrine/paracrine manner. We found that GDF15, but not TGFß, increased tumor sphere formation in several breast cancer cell lines and patient-derived primary breast cancer cells. As expected, TGFß strongly stimulated the phosphorylation of Smad2. GDF15 also stimulated the phosphorylation of Smad2, but the GDF15-induced tumor sphere forming efficiency was not significantly affected by treatment with SB431542, an inhibitor of the TGFß signaling. Although TGFß transiently activated ERK1/2, GDF15 induced prolonged activation of ERK1/2. Treatment with U0126, an inhibitor of the MEK-ERK1/2 signaling, greatly inhibited the GDF15-induced tumor sphere formation. Moreover, cytokine array experiments revealed that GDF15, but not TGFß, is able to induce its own expression; furthermore, it appears to form an autocrine/paracrine circuit to continuously produce GDF15. In addition, we found heterogeneous expression levels of GDF15 among cancer cells and in human breast cancer tissues using immunohistochemistry. This may reflect a heterogeneous cancer cell population, including cancer stem-like cells and other cancer cells. Our findings suggest that GDF15 induces tumor sphere formation through GDF15-ERK1/2-GDF15 circuits, leading to maintenance of GDF15high cancer stem-like cells. Targeting GDF15 to break these circuits should contribute to the eradication of tumors.

Long-term Inflammation Transforms Intestinal Epithelial Cells of Colonic Organoids.

BACKGROUND AND AIMS: Patients with ulcerative colitis [UC] are at an increased risk of developing colitis-associated cancer [CAC], suggesting that continuous inflammation in the colon promotes the transformation of colonic epithelial cells. However, the mechanisms underlying cell transformation in UC remain unknown. We therefore aimed to investigate the effect of long-term inflammation on intestinal epithelial cells [IECs] using organoid culture. METHODS: IECs were isolated from mouse colon, and were cultured according to a method for a three-dimensional [3D] organoid culture. To mimic chronic inflammation, a mixture of cytokines and bacterial components were added to the medium for over a year. Cell signal intensity was assessed by 3D immunofluorescence. Cell transformation was assessed by microarray with gene set enrichment analysis. RESULTS: Stimulation with cytokines resulted in a significant induction of target genes for the nuclear factor [NF]-κB pathway in colonic organoids. Following 60 weeks of continuous stimulation, cell differentiation was suppressed. Continuous stimulation also resulted in significant amplification of NF-κB signalling. Amplified NF-κB signalling by long-term stimulation remained in colonic organoids even 11 weeks after the removal of all cytokines. Some genes were specifically upregulated only in colonic organoids after the removal all cytokines following long-term stimulation. CONCLUSIONS: Colonic organoids stimulated with cytokines for a prolonged period were established as in vitro model to assess long-term epithelial responses to inflammatory cytokines. Chronic inflammation led to sustained NF-κB signalling activation in colonic organoids, resulting in cell transformation that might be related to the carcinogenesis of CAC in UC.

Essential roles of the interaction between cancer cell-derived chemokine, CCL4, and intra-bone CCR5-expressing fibroblasts in breast cancer bone metastasis.

From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3 clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth factor (CTGF)/CCN2. Tumor cell-derived CCL4 could induce fibroblasts to express CTGF/CCN2, which could support 4T1.3 clone proliferation under hypoxic culture conditions. Thus, the CCL4-CCR5 axis can contribute to breast cancer metastasis to bone by mediating the interaction between cancer cells and fibroblasts in bone cavity.

Oncogenic Fusion Gene CD74-NRG1 Confers Cancer Stem Cell-like Properties in Lung Cancer through a IGF2 Autocrine/Paracrine Circuit.

The CD74-Neuregulin1 (NRG1) fusion gene was recently identified as novel driver of invasive mucinous adenocarcinoma, a malignant form of lung cancer. However, the function of the CD74-NRG1 fusion gene in adenocarcinoma pathogenesis and the mechanisms by which it may impart protumorigenic characteristics to cancer stem cells (CSC) is still unclear. In this study, we found that the expression of the CD74-NRG1 fusion gene increased the population of lung cancer cells with CSC-like properties. CD74-NRG1 expression facilitated sphere formation not only of cancer cells, but also of nonmalignant lung epithelial cells. Using a limiting dilution assay in a xenograft model, we further show that the CD74-NRG1 fusion gene enhanced tumor initiation. Mechanistically, we found that CD74-NRG1 expression promoted the phosphorylation of ErbB2/3 and activated the PI3K/Akt/NF-κB signaling pathway. Furthermore, the expression of the secreted insulin-like growth factor 2 (IGF2) and phosphorylation of its receptor, IGF1R, were enhanced in an NF-κB-dependent manner in cells expressing CD74-NRG1. These findings suggest that CD74-NRG1-induced NF-κB activity promotes the IGF2 autocrine/paracrine circuit. Moreover, inhibition of ErbB2, PI3K, NF-κB, or IGF2 suppressed CD74-NRG1-induced tumor sphere formation. Therefore, our study provides a preclinical rationale for developing treatment approaches based on these identified pathways to suppress CSC properties that promote tumor progression and recurrence.

Pyruvate kinase M2, but not M1, allele maintains immature metabolic states of murine embryonic stem cells.

The M2 isoform of pyruvate kinase, the final rate-limiting enzyme of aerobic glycolysis, is expressed during embryonic development. In contrast, the M1 isoform is expressed in differentiated cells due to alternative splicing. Here we investigated murine embryonic stem cells (ESCs) with Pkm1 or Pkm2 knock-in alleles. Pkm1 allele knock-in resulted in excessive oxidative phosphorylation and induced the formation of cysteine-thiol disulfide-dependent complexes of forkhead box class-O (FOXO) transcription factors, which resulted in altered endoderm differentiation. In contrast, Pkm2 knock-in induced synthesis of a methylation-donor, S-adenosylmethionine, and increased unsaturated eicosanoid groups, which contributed to the redox control and maintenance of ESC undifferentiated status. Because PKM2 is also a critical enzyme for the cancer-specific Warburg effect, our results demonstrate an important role for the Pkm2 allele in establishing intracellular redox conditions and modulating PKM1-dependent oxidative phosphorylation events to achieve an appropriate ESC differentiation program.