The noncanonical function of borealin, a component of chromosome passenger complex, promotes glycolysis via stabilization of survivin in squamous cell carcinoma cells.

The chromosome passenger complex (CPC) is a kinase complex formed by Aurora B, borealin, survivin and inner centromere protein (INCENP). The CPC is active during mitosis and contributes to proper chromosome segregation via the phosphorylation of various substrates. Overexpression of each CPC component has been reported in most cancers. However, its significance remains unclear, as only survivin is known to confer chemoresistance. This study showed that the overexpression of borealin, a CPC component, stabilized survivin protein depending on its interaction with survivin. Unexpectedly, the accumulation of survivin by borealin overexpression did not affect the well-characterized functions of survivin, such as chemoresistance and cell proliferation. Interestingly, the overexpression of borealin promoted lactate production but not the overexpression of the deletion mutant that lacks the ability to bind to survivin. Consistent with these findings, the expression levels of glycolysis-related genes were enhanced in borealin-overexpressing cancer cells. Meanwhile, the overexpression of survivin alone did not promote lactate production. Overall, the accumulation of the borealin-survivin complex promoted glycolysis in squamous cell carcinoma cells. This mechanism may contribute to cancer progression via excessive lactate production.

Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via regulation of pyruvate dehydrogenase phosphorylation.

A high-salt diet significantly impacts various diseases, ilncluding cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, we determined that the pyruvate dehydrogenase phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmosensing mechanism. Additionally, we demonstrate that Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Collectively, our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of pyruvate dehydrogenase phosphorylation by direct osmosensing within mitochondria.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures.

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

A KDM6 inhibitor potently induces ATF4 and its target gene expression through HRI activation and by UTX inhibition.

UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.

Oncorequisite role of an aldehyde dehydrogenase in the pathogenesis of T-cell acute lymphoblastic leukemia.

Aldehyde dehydrogenases (ALDHs) are overexpressed in various types of cancers. One of the ALDH family genes, ALDH1A2, is aberrantly expressed in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases. However, its molecular function and role in T-ALL pathogenesis are largely unknown. ChIP-seq and RNA-seq analyses showed that the oncogenic transcription factor TAL1 and its regulatory partners bind to the intronic regulatory element of the ALDH1A2 gene, directly inducing a T-ALL-specific isoform with enzymatic activity. ALDH1A2 was preferentially expressed in the TAL1-positive T-ALL subgroup. In T-ALL cell lines, depletion of ALDH1A2 inhibited cell viability and induced apoptosis. Interestingly, gene expression and metabolomic profiling revealed that ALDH1A2 supported glycolysis and the TCA cycle, accompanied by NADH production, by affecting multiple metabolic enzymes to promote ATP production. Depletion of ALDH1A2 increased the levels of reactive oxygen species (ROS), while ROS levels were reduced by ALDH1A2 overexpression both in vitro and in vivo. Overexpression of ALDH1A2 accelerated tumor onset and increased tumor penetrance in a zebrafish T-ALL model. Taken together, our results indicate that ALDH1A2 protects against intracellular stress and promotes T-ALL cell metabolism and survival. ALDH1A2 overexpression enables leukemic clones to sustain a hyper-proliferative state driven by oncogenes.

Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization.

The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.

Controlling the Phenotype of Tumor-Infiltrating Macrophages via the PHD-HIF Axis Inhibits Tumor Growth in a Mouse Model.

The tumor microenvironment (TME) polarizes tumor-infiltrating macrophages toward tumor support. Macrophage-abundant tumors are highly malignant and are the cause of poor prognosis and therapeutic resistance. In this study, we show that the prolyl hydroxylase (PHD) inhibitor FG-4592 (FG) inhibits tumor growth of macrophage-abundant tumors and prolongs mouse survival. FG not only normalizes tumor vessels and improves tumor oxygenation but also directly affects macrophages and activates phagocytosis through the PHD-hypoxia-inducible factor (HIF) axis. Remarkably, FG can promote phagocytic ability of the Ly6Clo subset of tumor-infiltrating macrophages, leading to tumor growth inhibition. Moreover, Ly6Cneg macrophages contributed to blood vessel normalization. Using a malignant tumor mouse model, we characterized macrophage function and subsets. Altogether, our findings suggest that the PHD inhibitor can promote the anti-tumor potential of macrophages to improve cancer therapy.

A dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, attenuates cardiac dysfunction after myocardial infarction independently of DPP-4.

Dipeptidyl peptidase-4 (DPP-4) inhibitors not only improve impaired glucose tolerance in diabetes, but also have pleiotropic extra-pancreatic effects such as preconditioning effect for myocardial ischemia-reperfusion injury. Here, we investigated the anti-remodeling effects of linagliptin, a DPP-4 inhibitor, by use of DPP-4-deficient rats. After the induction of myocardial infarction (MI), Fischer 344 rats with inactivating mutation of DPP-4 were orally administrated with a DPP-4 inhibitor, linagliptin (5 mg kg-1·day-1), or vehicle in drinking water for 4 weeks. Linagliptin did not affect hemodynamic status, body weight, and infarct size. In echocardiography, linagliptin tended to improve left ventricular (LV) systolic function, and significantly improved LV diastolic function, surprisingly. Interstitial fibrosis in marginal region and macrophage infiltration were significantly lower in the linagliptin group than those in the vehicle group. Fibrosis-related gene expressions, such as collagen I and transforming growth factor-ß1 (TGF-ß1), and inflammation-related expressions, such as macrophage chemotactic protein 1 and matrix metalloproteinase-2 (MMP-2), were significantly suppressed in marginal area of the linagliptin-treated rats compared with the vehicle rats. The TGF-ß1 and MMP-2 protein levels were attenuated by linagliptin in DPP-4-deficient cardiac fibroblasts. Linagliptin can attenuate MI-induced cardiac remodeling via a DPP-4-independent pathway.

Author Correction: Interaction between von Hippel-Lindau Protein and Fatty Acid Synthase Modulates Hypoxia Target Gene Expression.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Hypoxia-inducible factor-2 alpha up-regulates CD70 under hypoxia and enhances anchorage-independent growth and aggressiveness in cancer cells.

Hypoxia-inducible factors (HIFs) facilitate cellular adaptation to environmental stress such as low oxygen conditions (hypoxia) and consequently promote tumor growth. While HIF-1α functions in cancer progression have been increasingly recognized, the contribution of HIF-2α remains widely unclear despite accumulating reports showing its overexpression in cancer cells. Here, we report that HIF-2α up-regulates the expression of CD70, a cancer-related surface antigen that improves anchorage-independent growth in cancer cells and is associated with poor clinical prognosis, which can be induced via epigenetic modifications mediated by DNMT1. The ablation of CD70 by RNAi led to decreased colony forming efficiency in soft agar. Most strikingly, we identified the emergence of CD70-expressing cells derived from CD70-negative cell lines upon prolonged hypoxia exposure or DNMT1 inhibition, both of which significantly reduced CpG-nucleotide methylations within CD70 promoter region. Interestingly, DNMT1 expression was decreased under hypoxia, which was rescued by HIF-2α knockdown. In addition, the expression of CD70 and colony forming efficiency in soft agar were decreased by knockdown of HIF-2α. These findings indicate that CD70 expression and an aggressive phenotype of cancer cells is driven under hypoxic conditions and mediated by HIF-2α functions and epigenetic modifications. This provides additional insights into the role of HIF-2α in coordinated regulation of stem-like functions and epigenetics that are important for cancer progression and may present additional targets for the development of novel combinatorial therapeutics.

Interaction between von Hippel-Lindau Protein and Fatty Acid Synthase Modulates Hypoxia Target Gene Expression.

Hypoxia-inducible factors (HIFs) play a central role in the transcriptional response to changes in oxygen availability. Stability of HIFs is regulated by multi-step reactions including recognition by the von Hippel-Lindau tumour suppressor protein (pVHL) in association with an E3 ligase complex. Here we show that pVHL physically interacts with fatty acid synthase (FASN), displacing the E3 ubiquitin ligase complex. This results in HIF-α protein stabilization and activation of HIF target genes even in normoxia such as during adipocyte differentiation. 25-hydroxycholesterol (25-OH), an inhibitor of FASN expression, also inhibited HIF target gene expression in cultured cells and in mouse liver. Clinically, FASN is frequently upregulated in a broad variety of cancers and has been reported to have an oncogenic function. We found that upregulation of FASN correlated with induction of many HIF target genes, notably in a malignant subtype of prostate tumours. Therefore, pVHL-FASN interaction plays a regulatory role for HIFs and their target gene expression.

Hypoxia-inducible factor-1α promotes cell survival during ammonia stress response in ovarian cancer stem-like cells.

Ammonia is a toxic by-product of metabolism that causes cellular stresses. Although a number of proteins are involved in adaptive stress response, specific factors that counteract ammonia-induced cellular stress and regulate cell metabolism to survive against its toxicity have yet to be identified. We demonstrated that the hypoxia-inducible factor-1α (HIF-1α) is stabilized and activated by ammonia stress. HIF-1α activated by ammonium chloride compromises ammonia-induced apoptosis. Furthermore, we identified glutamine synthetase (GS) as a key driver of cancer cell proliferation under ammonia stress and glutamine-dependent metabolism in ovarian cancer stem-like cells expressing CD90. Interestingly, activated HIF-1α counteracts glutamine synthetase function in glutamine metabolism by facilitating glycolysis and elevating glucose dependency. Our studies reveal the hitherto unknown functions of HIF-1α in a biphasic ammonia stress management in the cancer stem-like cells where GS facilitates cell proliferation and HIF-1α contributes to the metabolic remodeling in energy fuel usage resulting in attenuated proliferation but conversely promoting cell survival.

The hypoxia-inducible epigenetic regulators Jmjd1a and G9a provide a mechanistic link between angiogenesis and tumor growth.

Hypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression.

Selective inhibition of unfolded protein response induces apoptosis in pancreatic cancer cells.

Endoplasmic reticulum stress from unfolded proteins is associated with the proliferation of pancreatic tumor cells, making the many regulatory molecules of this pathway appealing targets for therapy. The objective of our study was to assess potential therapeutic efficacy of inhibitors of unfolded protein response (UPR) in pancreatic cancers focusing on IRE1α inhibitors. IRE1α-mediated XBP-1 mRNA splicing encodes a transcription factor that enhances transcription of chaperone proteins in order to reverse UPR. Proliferation assays using a panel of 14 pancreatic cancer cell lines showed a dose- and time-dependent growth inhibition by IRE1α-specific inhibitors (STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, toyocamycin). Growth inhibition was also noted using a clonogenic growth assay in soft agar, as well as a xenograft in vivo model of pancreatic cancer. Cell cycle analysis showed that these IRE1α inhibitors caused growth arrest at either the G1 or G2/M phases (SU8686, MiaPaCa2) and induced apoptosis (Panc0327, Panc0403). Western blot analysis showed cleavage of caspase 3 and PARP, and prominent induction of the apoptotic molecule BIM. In addition, synergistic effects were found between either STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, or toyocamycin and either gemcitabine or bortezomib. Our data suggest that use of an IRE1α inhibitor is a novel therapeutic approach for treatment of pancreatic cancers.

Oncogenic role of nuclear accumulated Aurora-A.

Aurora-A, also known as Aik, BTAK, or STK15, is a centrosomal serine/threonine protein kinase, which is proto-oncogenic and is overexpressed in a wide range of human cancers. Besides gene amplification and mRNA overexpression, proteolytic resistance mechanisms are thought to contribute to overexpression of Aurora-A. However, it is not yet clear how overexpressed Aurora-A affects the expression of transformed phenotype. Here, we found that nuclear accumulation of Aurora-A was critical for transformation activity. Cellular protein fractionation experiments and immunoblot analysis demonstrated a predominance of Aurora-A in the nuclear soluble fraction in head and neck cancer cells. Indirect immunofluorescence using confocal laser microscopy confirmed nuclear Aurora-A in head and neck cancer cells, while most oral keratinocytes exhibited only centrosomal localization. The expression of nuclear export signal-fused Aurora-A demonstrated that the oncogenic transformation activity was lost on disruption of the nuclear localization. Thus, the cytoplasmic localization of overexpressed Aurora-A previously demonstrated by immunohistochemical analysis is not likely to correspond to that in intact cancer cells. This study identifies an alternative mode of Aurora-A overexpression in cancer, through nuclear rather than cytoplasmic functions. We suggest that substrates of Aurora-A in the cell nuclear soluble fraction can represent a novel therapeutic target for cancer.

Constitutive phosphorylation of aurora-a on ser51 induces its stabilization and consequent overexpression in cancer.

BACKGROUND: The serine/threonine kinase Aurora-A (Aur-A) is a proto-oncoprotein overexpressed in a wide range of human cancers. Overexpression of Aur-A is thought to be caused by gene amplification or mRNA overexpression. However, recent evidence revealed that the discrepancies between amplification of Aur-A and overexpression rates of Aur-A mRNA were observed in breast cancer, gastric cancer, hepatocellular carcinoma, and ovarian cancer. We found that aggressive head and neck cancers exhibited overexpression and stabilization of Aur-A protein without gene amplification or mRNA overexpression. Here we tested the hypothesis that aberration of the protein destruction system induces accumulation and consequently overexpression of Aur-A in cancer. PRINCIPAL FINDINGS: Aur-A protein was ubiquitinylated by APC(Cdh1) and consequently degraded when cells exited mitosis, and phosphorylation of Aur-A on Ser51 was observed during mitosis. Phosphorylation of Aur-A on Ser51 inhibited its APC(Cdh1)-mediated ubiquitylation and consequent degradation. Interestingly, constitutive phosphorylation on Ser51 was observed in head and neck cancer cells with protein overexpression and stabilization. Indeed, phosphorylation on Ser51 was observed in head and neck cancer tissues with Aur-A protein overexpression. Moreover, an Aur-A Ser51 phospho-mimetic mutant displayed stabilization of protein during cell cycle progression and enhanced ability to cell transformation. CONCLUSIONS/SIGNIFICANCE: Broadly, this study identifies a new mode of Aur-A overexpression in cancer through phosphorylation-dependent inhibition of its proteolysis in addition to gene amplification and mRNA overexpression. We suggest that the inhibition of Aur-A phosphorylation can represent a novel way to decrease Aur-A levels in cancer therapy.

Establishment and characterization of a spindle cell squamous carcinoma cell line.

BACKGROUND: Spindle cell squamous carcinoma (SCSC) is a rare and peculiar biphasic malignant neoplasm that occurs mainly in the upper aerodigestive tract. It consists of sarcomatoid proliferation of pleomorphic spindle-shaped cells and squamous cell carcinoma. METHODS: Here, we established a SCSC cell line from a tumour arisen in gingiva. We characterized the feature of a SCSC cell line by immunohistochemistry. To know the biological feature, we examined the cell growth, invasiveness and epithelial-mesenchymal transition markers of a SCSC cell line in comparison with oral squamous cell carcinoma (OSCC) cell lines. RESULTS: By immunohistochemical analyses, the primary tumour expressed cytokeratin and vimentin, indicating carcinosarcoma-like characters. This tumour also showed overexpression of p53 protein. Cultured SCSC cells resulted in bypass of crisis and maintenance over passage 100. The established SCSC cell line was spindle-shaped and showed identical immunohistochemical characters to those of primary tumour cells. Similar to the primary tumour, the cell line showed p53 overexpression and had p53 mutation at codon 132: AAG (lys)-->AAT (asp). The SCSC cell line grew slower than two other OSCC cell lines (MSCC-1 and HSC-2), whereas SCSC cells had remarkable invasiveness in comparison with these cell lines. Moreover, SCSC cells expressed wnt-5a and vimentin mRNA at high levels, but did not express E-cadherin mRNA. This expression pattern of the markers was similar to that of mesenchymal cells, not of epithelial cells. CONCLUSION: In the present study, we newly established a SCSC cell line with strong invasiveness. This is the first report on the establishment of SCSC cell line. The SCSC cell line can be a useful cell model for the study to know the cytodifferentiation and nature of SCSC.

Periostin promotes invasion and anchorage-independent growth in the metastatic process of head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer. Typically, HNSCC cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. However, molecular mechanisms associated with the invasion and metastasis of HNSCC remain poorly understood. Here, we identified periostin as an invasion-promoting factor in HNSCC by comparing the gene expression profiles between parent HNSCC cells and a highly invasive clone. Indeed, periostin overexpression promoted invasion and anchorage-independent growth both in vitro and in vivo in HNSCC cells. Moreover, periostin-overexpressing cells spontaneously metastasized to cervical lymph nodes and to the lung through their aggressive invasiveness in an orthotopic mouse model of HNSCC. Interestingly, periostin was highly expressed in HNSCCs in comparison with normal tissues, and the level of periostin expression was well correlated with the invasiveness of HNSCC cases. In summary, these findings suggest that periostin plays an important role in the invasion and anchorage-independent growth of HNSCC.

Aurora-B/AIM-1 kinase activity is involved in Ras-mediated cell transformation.

Aurora-B, previously known as AIM-1, is a conserved eukaryotic mitotic protein kinase. In mammals, this kinase plays an essential role in chromosomal segregation processes, including chromosome condensation, alignment, control of spindle checkpoints, chromosome segregation, and cytokinesis. Aurora-B is overexpressed in various cancer cells, suggesting that the kinase activity perturbs chromosomal segregation processes. Its forced overexpression induces chromosomal number instability and progressive tumorigenicity in rodent cells in vitro and in vivo. Nevertheless, based on focus formation in BALB/c 3T3 A31-1-1 cells, Aurora-B is not oncogenic. Here, we show that Aurora-B kinase activity augments Ras-mediated cell transformation. RNA interference with short hairpin RNA inhibits transformation by Ras and its upstream oncogene Src, but not by the downstream oncogene Raf. In addition, the inner centromere protein, which is a passenger protein associated with Aurora-B, has a similar ability to potentiate the activity of oncogenic Ras. These data indicate that elevated Aurora-B activity promotes transformation by oncogenic Ras by enhancing oncogenic signaling and by converting chromosome number-stable cells to aneuploid cells.