S100A8 may govern hyper-inflammation in severe COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic threatens human species with mortality rate of roughly 2%. We can hardly predict the time of herd immunity against and end of COVID-19 with or without success of vaccine. One way to overcome the situation is to define what delineates disease severity and serves as a molecular target. The most successful analogy is found in BCR-ABL in chronic myeloid leukemia, which is the golden biomarker, and simultaneously, the most effective molecular target. We hypothesize that S100 calcium-binding protein A8 (S100A8) is one such molecule. The underlying evidence includes accumulating clinical information that S100A8 is upregulated in severe forms of COVID-19, pathological similarities of the affected lungs between COVID-19 and S100A8-induced acute respiratory distress syndrome (ARDS) model, homeostatic inflammation theory in which S100A8 is an endogenous ligand for endotoxin sensor Toll-like receptor 4/Myeloid differentiation protein-2 (TLR4/MD-2) and mediates hyper-inflammation even after elimination of endotoxin-producing extrinsic pathogens, analogous findings between COVID-19-associated ARDS and pre-metastatic lungs such as S100A8 upregulation, pulmonary recruitment of myeloid cells, increased vascular permeability, and activation coagulation cascade. A successful treatment in an animal COVID-19 model is given with a reagent capable of abrogating interaction between S100A8/S100A9 and TLR4. In this paper, we try to verify our hypothesis that S100A8 governs COVID-19-associated ARDS.

The Sympathetic Nervous System Contributes to the Establishment of Pre-Metastatic Pulmonary Microenvironments.

Emerging evidence suggests that neural activity contributes to tumor initiation and its acquisition of metastatic properties. More specifically, it has been reported that the sympathetic nervous system regulates tumor angiogenesis, tumor growth, and metastasis. The function of the sympathetic nervous system in primary tumors has been gradually elucidated. However, its functions in pre-metastatic environments and/or the preparation of metastatic environments far from the primary sites are still unknown. To investigate the role of the sympathetic nervous system in pre-metastatic environments, we performed chemical sympathectomy using 6-OHDA in mice and observed a decrease in lung metastasis by attenuating the recruitment of myeloid-derived suppressor cells. Furthermore, we note that neuro-immune cell interactions could be observed in tumor-bearing mouse lungs in conjunction with the decreased expression of Sema3A. These data indicate that the sympathetic nervous system contributes to the preparation of pre-metastatic microenvironments in the lungs, which are mediated by neuro-immune cell interactions.

Eph/Ephrin Signaling in the Tumor Microenvironment.

The Eph/ephrin system plays a vital role in diverse physiological events such as neurogenesis, vasculogenesis, and cell adhesion. Expression analysis of mRNA and protein in clinical samples revealed the involvement of the Eph/ephrin system in tumorigenesis, Alzheimer's disease, and atherosclerosis. Therefore, the Eph/ephrin system is considered a promising therapeutic target. However, no molecularly targeted drug against Ephs and ephrins is being used in the clinic thus far.Tumors are composed of various types of cells, including fibroblasts, immune cells, and endothelial cells. Recent studies showed the contribution of these cells to tumor growth, tumor progression, drug resistance, and metastasis. In this chapter, we discuss the role of Eph/ephrin system in the tumor microenvironment and describe its functions in tumor initiation, angiogenesis, cancer stem cell, tumor immunity, and also the metastatic environment.

Analysis of ADAM12-Mediated Ephrin-A1 Cleavage and Its Biological Functions.

Accumulating evidence indicates that an elevated ephrin-A1 expression is positively correlated with a worse prognosis in some cancers such as colon and liver cancer. The detailed mechanism of an elevated ephrin-A1 expression in a worse prognosis still remains to be fully elucidated. We previously reported that ADAM12-cleaved ephrin-A1 enhanced lung vascular permeability and thereby induced lung metastasis. However, it is still unclear whether or not cleaved forms of ephrin-A1 are derived from primary tumors and have biological activities. We identified the ADAM12-mediated cleavage site of ephrin-A1 by a Matrix-assisted laser desorption ionization mass spectrometry and checked levels of ephrin-A1 in the serum and the urine derived from the primary tumors by using a mouse model. We found elevated levels of tumor-derived ephrin-A1 in the serum and the urine in the tumor-bearing mice. Moreover, inhibition of ADAM-mediated cleavage of ephrin-A1 or antagonization of the EphA receptors resulted in a significant reduction of lung metastasis. The results suggest that tumor-derived ephrin-A1 is not only a potential biomarker to predict lung metastasis from the primary tumor highly expressing ephrin-A1 but also a therapeutic target of lung metastasis.

Roles of EphA1/A2 and ephrin-A1 in cancer.

The biological functions of the Eph/ephrin system have been intensively investigated and well documented so far since its discovery in 1987. Although the Eph/ephrin system has been implicated in pathological settings such as Alzheimer's disease and cancer, the molecular mechanism of the Eph/ephrin system in those diseases is not well understood. Especially in cancer, recent studies have demonstrated that most of Eph and ephrin are up- or down-regulated in various types of cancer, and have been implicated in tumor progression, tumor malignancy, and prognosis. However, they lack consistency and are in controversy. The localization patterns of EphA1 and EphA2 in mouse lungs are very similar, and both knockout mice showed similar phenotypes in the lungs. Ephrin-A1 that is a membrane-anchored ligand for EphAs was co-localized with EphA1 and EphA2 in lung vascular endothelial cells. We recently uncovered the molecular mechanism of ephrin-A1-induced lung metastasis by understanding the physiological function of ephrin-A1 in lungs. This review focuses on the function of EphA1, EphA2, and ephrin-A1 in tumors and an establishment of pre-metastatic microenvironment in the lungs.

Pleckstrin homology domain of p210 BCR-ABL interacts with cardiolipin to regulate its mitochondrial translocation and subsequent mitophagy.

Chronic myeloid leukemia (CML) is caused by the chimeric protein p210 BCR-ABL encoded by a gene on the Philadelphia chromosome. Although the kinase domain of p210 BCR-ABL is an active driver of CML, the pathological role of its pleckstrin homology (PH) domain remains unclear. Here, we carried out phospholipid vesicle-binding assays to show that cardiolipin (CL), a characteristic mitochondrial phospholipid, is a unique ligand of the PH domain. Arg726, a basic amino acid in the ligand-binding region, was crucial for ligand recognition. A subset of wild-type p210 BCR-ABL that was transiently expressed in HEK293 cells was dramatically translocated from the cytosol to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, which induces mitochondrial depolarization and subsequent externalization of CL to the organelle's outer membrane, whereas an R726A mutant of the protein was not translocated. Furthermore, only wild-type p210 BCR-ABL, but not the R726A mutant, suppressed CCCP-induced mitophagy and subsequently enhanced reactive oxygen species production. Thus, p210 BCR-ABL can change its intracellular localization via interactions between the PH domain and CL to cope with mitochondrial damage. This suggests that p210 BCR-ABL could have beneficial effects for cancer proliferation, providing new insight into the PH domain's contribution to CML pathogenesis.

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis.

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1(-/-)) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4(-/-) mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.

Action mechanisms of histone deacetylase inhibitors in the treatment of hematological malignancies.

Histone deacetylases (HDACs) critically regulate gene expression by determining the acetylation status of histones. Studies have increasingly focused on the activities of HDACs, especially involving non-histone proteins, and their various biological effects. Aberrant HDAC expression observed in several kinds of human tumors makes HDACs potential targets for cancer treatment. Several preclinical studies have suggested that HDAC inhibitors show some efficacy in the treatment of acute myelogenous leukemia with AML1-ETO, which mediates transcriptional repression through its interaction with a complex including HDAC1. Recurrent mutations in epigenetic regulators are found in T-cell lymphomas (TCLs), and HDAC inhibitors and hypomethylating agents were shown to act cooperatively in the treatment of TCLs. Preclinical modeling has suggested that persistent activation of the signal transducer and activator of transcription signaling pathway could serve as a useful biomarker of resistance to HDAC inhibitor in patients with cutaneous TCL. Panobinostat, a pan-HDAC inhibitor, in combination with bortezomib and dexamethasone, has achieved longer progression-free survival in patients with relapsed/refractory multiple myeloma (MM) than the placebo in combination with bortezomib and dexamethasone. Panobinostat inhibited MM cell growth by degrading protein phosphatase 3 catalytic subunit α (PPP3CA), a catalytic subunit of calcineurin. This degradation was suggested to be mediated by the blockade of the chaperone function of heat shock protein 90 due to HDAC6 inhibition. Aberrant PPP3CA expression in advanced MM indicated a possible correlation between high PPP3CA expression and the pathogenesis of MM. Furthermore, PPP3CA was suggested as a common target of panobinostat and bortezomib.

Serum amyloid A3 binds MD-2 to activate p38 and NF-κB pathways in a MyD88-dependent manner.

Serum amyloid A (SAA) 3 is a major component of the acute phase of inflammation. We previously reported that SAA3 served as an endogenous peptide ligand for TLR4 to facilitate lung metastasis. Because these experiments were performed with SAA3 recombinant proteins purified from Escherichia coli or mammalian cells, we could not rule out the possibility of LPS contamination. In this study, we used SAA3 synthetic peptides to eliminate the presence of LPS in SAA3. We found that the SAA3 synthetic peptide (aa 20-86) (20-86) stimulated cell migration and activated p38 in a manner dependent on TLR4, MD-2, and MyD88. SAA3 (20-86) also activated NF-κB and Rho small GTPase. Using surface plasmon resonance analysis, the binding constant KD values between SAA3 (20-86) or SAA3 (43-57) and TLR4/MD-2 protein highly purified by the baculovirus system were 2.2 and 30 µM, respectively. FLAG-tagged SAA3 tightly bound to protein A-tagged MD-2, but not to TLR4 in baculovirus coinfection experiments. Although SAA3 (20-86) caused a low, but appreciable level of endocytosis in TLR4, it induced the upregulation of both IL-6 and TNF-α, but not IFN-ß1. An i.v. injection of SAA3 (43-57) induced the lung recruitment of CD11b(+)Gr-1(+) cells at an estimated serum concentration around its KD value toward TLR4/MD-2. Taken together, these results suggest that SAA3 directly binds MD-2 and activates the MyD88-dependent TLR4/MD-2 pathway.

Endothelial focal adhesion kinase mediates cancer cell homing to discrete regions of the lungs via E-selectin up-regulation.

Primary tumors secrete factors that alter the microenvironment of distant organs, rendering those organs as fertile soil for subsequent metastatic cancer cell colonization. Although the lungs are exposed to these factors ubiquitously, lung metastases usually develop as a series of discrete lesions. The underlining molecular mechanisms of the formation of these discrete lesions are not understood. Here we show that primary tumors induce formation of discrete foci of vascular hyperpermeability in premetastatic lungs. This is mediated by endothelial cell-focal adhesion kinase (FAK), which up-regulates E-selectin, leading to preferential homing of metastatic cancer cells to these foci. Suppression of endothelial-FAK or E-selectin activity attenuates the number of cancer cells homing to these foci. Thus, localized activation of endothelial FAK and E-selectin in the lung vasculature mediates the initial homing of metastatic cancer cells to specific foci in the lungs.

Ephrin-A1 expression induced by S100A8 is mediated by the toll-like receptor 4.

The deregulation of Eph/ephrin protein expression has been shown to lead to tumor development and progression. Both mRNA and protein expression analyses using clinical samples have demonstrated that ephrin-A1 is over-expressed in various cancers and positively correlates with a poor prognosis for cancer patients. The prognosis of cancer patients depends on metastasis to distant organs. We previously demonstrated that ADAM12 metalloproteinase cleaved ephrin-A1 and ADAM12-cleaved ephrin-A1 enhanced vascular permeability by degrading VE-cadherin and the EphA2 receptor at the plasma membrane. An increase of soluble ephrin-A1 levels in the serum facilitated tumor cell recruitment to the lungs, which resulted in lung metastasis. We also found that ephrin-A1 was overexpressed in 3LL tumors, a highly metastatic tumor, in mice and TNFα, an authentic positive regulator of ephrin-A1, was not elevated in the tumors, whereas S100A8 was. Moreover, S100A8 induced ephrin-A1 expression mediated by the toll-like receptor 4 (TLR4). S100A8 is known to be an endogenous ligand for TLR4 and its expression was shown to be increased in the lungs at the premetastatic phase. Thus, S100A8 and ephrin-A1 contribute to lung metastasis. Therefore, elucidating the regulation mechanism of ephrin-A1 overexpression is of importance and may lead to the development of therapeutic drugs against tumor growth and metastasis.

Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis.

Primary tumours influence the environment in the lungs before metastasis. However, the mechanism of metastasis is not well understood. Here, we show that the inflammatory chemoattractants S100A8 and S100A9, whose expression is induced by distant primary tumours, attract Mac 1 (macrophage antigen 1)(+)-myeloid cells in the premetastatic lung. In addition, tumour cells use this mechanism, through activation of the mitogen-activated protein kinase (MAPK) p38, to acquire migration activity with pseudopodia for invasion (invadopodia). The expression of S100A8 and S100A9 was eliminated in lung Mac 1(+)-myeloid cells and endothelial cells deprived of soluble factors, such as vascular endothelial growth factor A (VEGF-A), tumour necrosis factor alpha (TNFalpha) and transforming growth factor beta (TGFbeta) both in vitro and in vivo. Neutralizing anti-S100A8 and anti-S100A9 antibodies blocked the morphological changes and migration of tumour cells and Mac 1(+)-myeloid cells. Thus, the S100A8 and S100A9 pathway may be common to both myeloid cell recruitment and tumour-cell invasion.

Novel multivalent S100A8 inhibitory peptides attenuate tumor progression and metastasis by inhibiting the TLR4-dependent pathway.

The tumor-elicited inflammation is closely related to tumor microenvironment during tumor progression. S100A8, an endogenous ligand of Toll-like receptor 4 (TLR4), is known as a key molecule in the tumor microenvironment and premetastatic niche formation. We firstly generated a novel multivalent S100A8 competitive inhibitory peptide (divalent peptide3A5) against TLR4/MD-2, using the alanine scanning. Divalent peptide3A5 suppressed S100A8-mediated interleukin-8 and vascular endothelial growth factor production in human colorectal tumor SW480 cells. Using SW480-transplanted xenograft models, divalent peptide3A5 suppressed tumor progression in a dose-dependent manner. We demonstrated that combination therapy with divalent peptide3A5 and bevacizumab synergistically suppressed tumor growth in SW480 xenograft models. Using syngeneic mouse models, we found that divalent peptide3A5 improved the efficacy of anti-programmed death (PD)1 antibody, and lung metastasis. In addition, by using multivalent peptide library screening based on peptide3A5, we then isolated two more candidates; divalent ILVIK, and tetravalent ILVIK. Of note, multivalent ILVIK, but not monovalent ILVIK showed competitive inhibitory activity against TLR4/MD-2 complex, and anti-tumoral activity in SW480 xenograft models. As most tumor cells including SW480 cells also express TLR4, S100A8 inhibitory peptides would target both the tumor microenvironment and tumor cells. Thus, multivalent S100A8 inhibitory peptides would provide new pharmaceutical options for aggressive cancers.

Variant spectrum of PIEZO1 and KCNN4 in Japanese patients with dehydrated hereditary stomatocytosis.

Hereditary stomatocytosis (HSt) is a type of congenital hemolytic anemia caused by abnormally increased cation permeability of erythrocyte membranes. Dehydrated HSt (DHSt) is the most common subtype of HSt and is diagnosed based on clinical and laboratory findings related to erythrocytes. PIEZO1 and KCNN4 have been recognized as causative genes, and many related variants have been reported. We analyzed the genomic background of 23 patients from 20 Japanese families suspected of having DHSt using a target capture sequence and identified pathogenic/likely pathogenic variants of PIEZO1 or KCNN4 in 12 families.

Molecular biology of chronic myeloid leukemia.

Detailed information on the crystal structure of the pharmacologically targeted domains of the BCR-ABL molecule and on its intracellular signaling, which are potentially involved in growth, anti-apoptosis, metabolism and stemness, has made the study of chronic myeloid leukemia the most successful field in tumor biology. However, we now face the issue of drug resistance due to deregulation in the quality control of both DNA and protein. BCR-ABL is basically a misfolded protein with intrinsically disordered regions, which not only produces endoplasmic reticulum stress followed by unfolded protein response in some settings, but also conformational plasticity that may affect the structure of the whole molecule. The intercellular signaling derived from the leukemic cell microenvironment may influence the intracellular responses that take place in a manner both dependent on and independent of BCR-ABL tyrosine kinase activity.

Bag1 directly routes immature BCR-ABL for proteasomal degradation.

Degradation of BCR-ABL oncoproteins by heat shock protein 90 (Hsp90) inhibitors in chronic myelogenous leukemia is expected to overcome resistance to ABL tyrosine kinase inhibitors. However, the precise mechanisms still remain to be uncovered. We found that while c-Cbl E3 ligase induced ubiquitin-dependent degradation of mature and phosphorylated BCR-ABL proteins, another E3 ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) degraded immature BCR-ABL proteins and efficiently suppressed BCR-ABL-dependent leukemic growth. Interestingly, Bag1 (Bcl-2-associated athanogene-1), a nucleotide exchange factor for Hsc70, directly bound BCR-ABL with a high affinity, which was enhanced by CHIP and Hsp90 inhibitors, inhibited by imatinib and competed with Hsc70. Bag1 knockdown abrogated Hsp90 inhibitor-induced BCR-ABL degradation. Bag1 induced binding of immature BCR-ABL to proteasome. Expression of Bag1 induced BCR-ABL degradation and growth suppression in Ba/F3 cells when Hsc70 was knocked down with or without CHIP induction. CHIP appears to sort newly synthesized Hsp90-unchaperoned BCR-ABL to the proteasome not only by inhibiting Hsc70 and thereby promoting Bag1 to bind BCR-ABL, but also by ubiquitinating BCR-ABL. Bag1 may direct CHIP/Hsc70-regulated protein triage decisions on BCR-ABL immediately after translation to the degradation pathway.

Inflammation-associated premetastatic niche formation.

Metastasis remains the leading cause of cancer-related death. In 1889, Stephen Paget originally proposed the theory "seed-and-soil." Both cancer cell-intrinsic properties ("seed") and fertile microenvironment ("soil") are essential for metastasis formation. To date, accumulating evidences supported the theory using mouse models. The formation of a premetastatic niche has been widely accepted as an accel for metastasis. Similar to tumor microenvironment, various types of cells, such as immune cells, endothelial cells, and fibroblasts are involved in premetastatic niche formation. We have discovered that primary tumors hijack Toll-like receptor 4 (TLR4) signaling to establish a premetastatic niche in the lung by utilizing the endogenous ligands. In this review, we discuss the mechanisms that underlie inflammation-associated premetastatic niche formation upon metastasis, focusing especially on myeloid cells and macrophages as the cells executing and mediating complicated processes.

FOXA1 plays a role in regulating secretoglobin 1a1 expression in the absence of CCAAT/enhancer binding protein activities in lung in vivo.

Secretoglobin (SCGB) 1A1, also called Clara cell secretor protein (CCSP) or Clara cell-specific 10-kDa protein (CC10), is a small molecular weight secreted protein mainly expressed in lung, with anti-inflammatory/immunomodulatory properties. Previous in vitro studies demonstrated that CCAAT/enhancer-binding proteins (C/EBPs) are the major transcription factors for the regulation of Scbg1a1 gene expression, whereas FOXA1 had a minimum effect on the transcription. To determine the in vivo role of C/EBPs in the regulation of SCGB1A1 expression, experiments were performed in which A-C/EBP, a dominant-negative form of C/EBP that interferes with DNA binding activities of all C/EBPs, was specifically expressed in lung. Surprisingly, despite the in vitro findings, expression of SCGB1A1 mRNA was not decreased in vivo in the absence of C/EBPs. This may be due to a compensatory role assumed by FOXA1 in the regulation of Scgb1a1 gene expression in lung in the absence of active C/EBPs. This disconnect between in vitro and in vivo results underscores the importance of studies using animal models to determine the role of specific transcription factors in the regulation of gene expression in intact multicellular complex organs such as lung.

[Encounter of cancer cells with bone. Cancer and inflammation--input and output of NF-κB - ].

NF-κB activation is frequently found in a variety of tumors, especially in those that are resistant to chemotherapy. The malignant phenotypes of tumor cells that are required for invasion and metastasis could be established by the output of NF-κB signaling including interactions with white blood cells, anti-apoptosis and angiogenesis. Consequently, by utilizing the similar mechanisms for immune cells to migrate through the body, tumor cells migrate via blood stream, survive against selective pressures in other organs they stop by, achieve re-growth as metastasis, or make a clinical relapse by traveling back to the primary site.

Extracellular mRNA transported to the nucleus exerts translation-independent function.

RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether "naked nonvesicular extracellular mRNA" (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1ß (IL1ß)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3'-untranslated region of nex-IL1ß-mRNA and transports it to the nucleus. The nex-IL1ß-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA.