Exosome proteomes reveal glycolysis-related enzyme enrichment in primary canine mammary gland tumor compared to metastases.

OBJECTIVE: Numerous evidence has highlighted the differences between primary tumors and metastases. Nonetheless, the differences in exosomal proteins derived from primary tumor and metastases remain elusive. Here, we aimed to identify differentially expressed exosomal proteins from primary canine mammary gland tumor and metastases to understand how they shape their own tumor microenvironment. METHODS: We clearly distinguished primary canine mammary gland tumors (CHMp) from metastases (CHMm) and profiled the proteins within their secreted exosomes using LC-MS/MS. Moreover, the abundance of glycolysis enzymes (GPI, LDHA) in CHMp exosome was verified with Western blotting, To broaden the scope, we extended to human colorectal cancer-derived exosomes (SW480 vs. SW620) for comparison. RESULTS: We identified significant differences in 87 and 65 proteins derived from CHMp and CHMm, respectively. Notably, glycolysis enzymes (GPI, LDHA, LDHB, TPI1, and ALDOA) showed specific enrichment in exosomes from the primary tumor. CONCLUSION: We observed significant differences in the cellular proteome between primary tumors and metastases, and intriguingly, we identified a parallel heterogeneity the protein composition of exosomes. Specifically, we reported that glycolysis enzymes were significantly enriched in CHMp exosomes compared to CHMm exosomes. We further demonstrated that this quantitative difference in glycolysis enzymes persisted across primary and metastases, extending to human colorectal cancer-derived exosomes (SW480 vs. SW620). Our findings of the specific enrichment of glycolysis enzymes in primary tumor-derived exosomes contribute to a better understanding of tumor microenvironment modulation and heterogeneity between primary tumors and metastases.

ARID3C Acts as a Regulator of Monocyte-to-Macrophage Differentiation Interacting with NPM1.

ARID3C is a protein located on human chromosome 9 and expressed at low levels in various organs, yet its biological function has not been elucidated. In this study, we investigated both the cellular localization and function of ARID3C. Employing a combination of LC-MS/MS and deep learning techniques, we identified NPM1 as a binding partner for ARID3C's nuclear shuttling. ARID3C was found to predominantly localize with the nucleus, where it functioned as a transcription factor for genes STAT3, STAT1, and JUNB, thereby facilitating monocyte-to-macrophage differentiation. The precise binding sites between ARID3C and NPM1 were predicted by AlphaFold2. Mutating this binding site prevented ARID3C from interacting with NPM1, resulting in its retention in the cytoplasm instead of translocation to the nucleus. Consequently, ARID3C lost its ability to bind to the promoters of target genes, leading to a loss of monocyte-to-macrophage differentiation. Collectively, our findings indicate that ARID3C forms a complex with NPM1 to translocate to the nucleus, acting as a transcription factor that promotes the expression of the genes involved in monocyte-to-macrophage differentiation.

Real-Time Longitudinal Evaluation of Tumor Blood Vessels Using a Compact Preclinical Fluorescence Imaging System.

Tumor angiogenesis is enhanced in all types of tumors to supply oxygen and nutrients for their growth and metastasis. With the development of anti-angiogenic drugs, the importance of technology that closely monitors tumor angiogenesis has also been emerging. However, to date, the technology for observing blood vessels requires specialized skills with expensive equipment, thereby limiting its applicability only to the laboratory setting. Here, we used a preclinical optical imaging system for small animals and, for the first time, observed, in real time, the entire process of blood vessel development in tumor-bearing mice injected with indocyanine green. Time-lapse sequential imaging revealed blood vessel volume and blood flow dynamics on a microscopic scale. Upon analyzing fluorescence dynamics at each stage of tumor progression, vessel volume and blood flow were found to increase as the tumor developed. Conversely, these vascular parameters decreased when the mice were treated with angiogenesis inhibitors, which suggests that the effects of drugs targeting angiogenesis can be rapidly and easily screened. The results of this study may help evaluate the efficacy of angiogenesis-targeting drugs by facilitating the observation of tumor blood vessels easily in a laboratory unit without large and complex equipment.

METTL8 mRNA Methyltransferase Enhances Cancer Cell Migration via Direct Binding to ARID1A.

The association of RNA modification in cancer has recently been highlighted. Methyltransferase like 8 (METTL8) is an enzyme and its role in mRNA m3C modification has barely been studied. In this study, we found that METTL8 expression was significantly up-regulated in canine mammary tumor and investigated its functional roles in the tumor process, including cancer cell proliferation and migration. METTL8 expression was up-regulated in most human breast cancer cell lines tested and decreased by Yin Yang 1 (YY1) transcription factor knockdown, suggesting that YY1 is a regulating transcription factor. The knockdown of METTL8 attenuated tumor cell growth and strongly blocked tumor cell migration. AT-rich interactive domain-containing protein 1A (ARID1A) was identified as a candidate mRNA by METTL8. ARID1A mRNA binds to METTL8 protein. ARID1A mRNA expression was not changed by METTL8 knockdown, but ARID1A protein level was significantly increased. Collectively, our study indicates that METTL8 up-regulated by YY1 in breast cancer plays an important role in cancer cell migration through the mRNA modification of ARID1A, resulting in the attenuation of its translation.

Common plasma protein marker LCAT in aggressive human breast cancer and canine mammary tumor.

Breast cancer is one of the most frequently diagnosed cancers. Although biomarkers are continuously being discovered, few specific markers, rather than classification markers, representing the aggressiveness and invasiveness of breast cancer are known. In this study, we used samples from canine mammary tumors in a comparative approach. We subjected 36 fractions of both canine normal and mammary tumor plasmas to highperformance quantitative proteomics analysis. Among the identified proteins, LCAT was selectively expressed in mixed tumor samples. With further MRM and Western blot validation, we discovered that the LCAT protein is an indicator of aggressive mammary tumors, an advanced stage of cancer, possibly highly metastatic. Interestingly, we also found that LCAT is overexpressed in high-grade and lymph-node-positive breast cancer in silico data. We also demonstrated that LCAT is highly expressed in the sera of advanced-stage human breast cancers within the same classification. In conclusion, we identified a possible common plasma protein biomarker, LCAT, that is highly expressed in aggressive human breast cancer and canine mammary tumor. [BMB Reports 2020; 53(12): 664-669].

Analysis of opposing histone modifications H3K4me3 and H3K27me3 reveals candidate diagnostic biomarkers for TNBC and gene set prediction combination.

Breast cancer encompasses a major portion of human cancers and must be carefully monitored for appropriate diagnoses and treatments. Among the many types of breast cancers, triple negative breast cancer (TNBC) has the worst prognosis and the least cases reported. To gain a better understanding and a more decisive precursor for TNBC, two major histone modifications, an activating modification H3K4me3 and a repressive modification H3K27me3, were analyzed using data from normal breast cell lines against TNBC cell lines. The combination of these two histone markers on the gene promoter regions showed a great correlation with gene expression. A list of signature genes was defined as active (highly enriched H3K4me3), including NOVA1, NAT8L, and MMP16, and repressive genes (highly enriched H3K27me3), IRX2 and ADRB2, according to the distribution of these histone modifications on the promoter regions. To further enhance the investigation, potential candidates were also compared with other types of breast cancer to identify signs specific to TNBC. RNA-seq data was implemented to confirm and verify gene regulation governed by the histone modifications. Combinations of the biomarkers based on H3K4me3 and H3K27me3 showed the diagnostic value AUC 93.28% with P-value of 1.16e-226. The results of this study suggest that histone modification analysis of opposing histone modifications may be valuable toward developing biomarkers and targets for TNBC. [BMB Reports 2020; 53(5): 266-271].

Exploring the key communicator role of exosomes in cancer microenvironment through proteomics.

There have been many attempts to fully understand the mechanism of cancer behavior. Yet, how cancers develop and metastasize still remain elusive. Emerging concepts of cancer biology in recent years have focused on the communication of cancer with its microenvironment, since cancer cannot grow and live alone. Cancer needs to communicate with other cells for survival, and thus they secrete various messengers, including exosomes that contain many proteins, miRNAs, mRNAs, etc., for construction of the tumor microenvironment. Moreover, these intercellular communications between cancer and its microenvironment, including stromal cells or distant cells, can promote tumor growth, metastasis, and escape from immune surveillance. In this review, we summarized the role of proteins in the exosome as communicators between cancer and its microenvironment. Consequently, we present cancer specific exosome proteins and their unique roles in the interaction between cancer and its microenvironment. Clinically, these exosomes might provide useful biomarkers for cancer diagnosis and therapeutic tools for cancer treatment.

Modification of the plasma complement protein profile by exogenous estrogens is indicative of a compromised immune competence in marine medaka (Oryzias melastigma).

Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17α-ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection-induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection-induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex-specific changes in the plasma proteome, with the male plasma composition clearly becoming "feminised". Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin-2-like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post-translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex-differences in fish immunity and how these differences might be modified by xenoestrogens.

Effects of water accommodated fractions (WAFs) of crude oil in two congeneric copepods Tigriopus sp.

Oil pollution has deleterious effects on marine ecosystems. However, the toxicity of crude oil towards Antarctic marine organisms has not been well studied. We compared the deleterious effects of water accommodated fractions (WAFs) of crude oil on reproduction, intracellular reactive oxygen species (ROS) levels, and antioxidant enzymatic activity in Antarctic (Tigriopus kingsejongensis) and temperate (Tigriopus japonicus) copepods. Reproductive rates of T. kingsejongensis and T. japonicus were significantly reduced (P < 0.05) in response to WAFs. Furthermore, T. kingsejongensis showed elevated levels of ROS and higher antioxidant enzyme (glutathione peroxidase [GPx]) activity than T. japonicus in response to WAFs. CYP genes from congeneric copepods were identified and annotated to better understand molecular detoxification mechanisms. We observed significant up-regulation (P < 0.05) of Tk-CYP3024A3 and Tj-CYP3024A2 in response to WAFs, suggesting that CYP genes may contribute to the detoxification mechanism in response to WAF exposure. These finding also suggest that WAFs may induce oxidative stress, leading to reproductive impairment in copepods. Furthermore, Tk-CYP3024A3 and Tj-CYP3024A2 genes can be considered as potential biomarkers of WAF toxicity in the congeneric copepods T. kingsejongensis and T. japonicus. This study will be helpful for enhancing our knowledge on the harmful effects of WAFs in Antarctic and temperate copepods and provides insight into the underlying molecular mechanisms.

Genome-wide identification of nuclear receptor (NR) genes and the evolutionary significance of the NR1O subfamily in the monogonont rotifer Brachionus spp.

Nuclear receptors (NRs) are a large family of transcription factors that are involved in many fundamental biological processes. NRs are considered to have originated from a common ancestor, and are highly conserved throughout the whole animal taxa. Therefore, the genome-wide identification of NR genes in an animal taxon can provide insight into the evolutionary tendencies of NRs. Here, we identified all the NR genes in the monogonont rotifer Brachionus spp., which are considered an ecologically key species due to their abundance and world-wide distribution. The NR family was composed of 40, 32, 29, and 32 genes in the genomes of the rotifers B. calyciflorus, B. koreanus, B. plicatilis, and B. rotundiformis, respectively, which were classified into seven distinct subfamilies. The composition of each subfamily was highly conserved between species, except for NR1O genes, suggesting that they have undergone sporadic evolutionary processes for adaptation to their different environmental pressures. In addition, despite the dynamics of NR evolution, the significance of the conserved endocrine system, particularly for estrogen receptor (ER)-signaling, in rotifers was discussed on the basis of phylogenetic analyses. The results of this study may help provide a better understanding the evolution of NRs, and expand our knowledge of rotifer endocrine systems.

Ultraviolet B radiation induces impaired lifecycle traits and modulates expression of cytochrome P450 (CYP) genes in the copepod Tigriopus japonicus.

To evaluate the effects of ultraviolet B (UV-B) radiation at the developmental, reproductive, and molecular levels in aquatic invertebrates, we measured UV-B-induced acute toxicity, impairments in developmental and reproductive traits, and UV-B interaction with the entire family of cytochrome P450 (CYP) genes in the intertidal benthic copepod Tigriopus japonicus. We found a significant, dose-dependent reduction (P<0.05) in the survival of T. japonicus that began as a developmental delay and decreased fecundity. The 48h LD10 and LD50 were 1.35 and 1.84kJ/m2, and the CYP inhibitor (PBO) elevated mortality, confirming the involvement of CYP genes in UV-B induced toxicity. Low-dose UV-B (1.5kJ/m2) induced developmental delays, and higher doses (6-18kJ/m2) caused reproductive impairments in ovigerous females. The significant up-regulation of CYP genes belonging to clans 2/3/MT/4/20 in T. japonicus exposed to UV-B (12kJ/m2) confirmed molecular interaction between UV-B and CYP genes. Moreover, orphan CYPs, such as CYP20A1, provide good insight on the deorphanization of invertebrate CYPs. Overall, these results demonstrate the involvement of UV-B radiation in the expression of all the CYP genes in T. japonicus and their susceptibility to UV-B radiation. This will provide a better understanding of the mechanistic effects of UV-B in copepods through the predicted AhR-mediated up-regulation of CYP genes.

Marine copepod cytochrome P450 genes and their applications for molecular ecotoxicological studies in response to oil pollution.

Recently, accidental spills of heavy oil have caused adverse effects in marine organisms. Oil pollution can induce damages on development and reproduction, linking with detrimental effects on diverse molecular levels of genes and proteins in plankton and fish. However, most information was mainly focused on marine vertebrates and consequently, limited information was available in marine invertebrates. Furthermore, there is still a lack of knowledge bridging in vivo endpoints with the functional regulation of cytochrome P450 (CYP) genes in response to oil spill pollution in marine invertebrates. In this paper, adverse effects of oil spill pollution in marine invertebrates are summarized with the importance of CYP genes as a potential biomarker, applying for environmental monitoring to detect oil spill using marine copepods.

Genome-wide identification of ATP-binding cassette (ABC) transporters and conservation of their xenobiotic transporter function in the monogonont rotifer (Brachionus koreanus).

The ATP-binding cassette (ABC) transporter family is one of the largest gene family in animals, and members of this family are known to be involved in various biological processes due to their ability to transport a wide range of substrates across membranes using ATP cleavage-derived energy. We identified 61 ABC transporters in the genome of the monogonont rotifer Brachionus koreanus, and classified these into eight distinct subfamilies (A-H) by phylogenetic analysis. ABC transporters in the rotifer B. koreanus are comprised of 11 ABCA genes, 19 ABCB genes, 14 ABCC genes, 3 ABCD genes, 1 ABCE gene, 3 ABCF genes, 8 ABCG genes, and 2 ABCH genes. Extensive gene duplication and loss events in synteny were observed in several subfamilies. In particular, massive gene duplications of P-glycoproteins (P-gps), multidrug resistance proteins (MRPs), and Bk-Abcg-like proteins were observed. The ability of these B. koreanus proteins to function as multixenobiotic resistance (MXR) ABC transporters was validated using specific fluorescence substrates/inhibitors. The ABC transporter superfamily members identified in this study will be useful in future toxicological studies, and will facilitate comparative studies of the evolution of the ABC transporter superfamily in invertebrates.

Identification of the Full 46 Cytochrome P450 (CYP) Complement and Modulation of CYP Expression in Response to Water-Accommodated Fractions of Crude Oil in the Cyclopoid Copepod Paracyclopina nana.

The 46 cytochrome P450 (CYP) gene superfamily was identified in the marine copepod Paracyclopina nana after searching an RNA-seq database and comparing it with other copepod CYP gene families. To annotate the 46 Pn-CYP genes, a phylogenetic analysis of CYP genes was performed using a Bayesian method. Pn-CYP genes were separated into five different clans: CYP2, CYP3, CYP20, CYP26, and mitochondrial. Among these, the principal Pn-CYP genes involved in detoxification were identified by comparing them with those of the copepod Tigriopus japonicus and were examined with respect to their responses to exposure to a water-accommodated fraction (WAF) of crude oil and to the alkylated forms of two polycyclic aromatic hydrocarbons (PAHs; phenanthrene and fluorene). The expression of two Pn-CYP3027 genes (CYP3027F1 and CYP3027F2) was increased in response to WAF exposure and also was upregulated in response to the two alkylated PAHs. In particular, Pn-CYP3027F2 showed the most notable increase in response to 80% WAF exposure. These two responsive CYP genes (Pn-CYP3027F1 and CYP3027F2) were also phylogenetically clustered into the same clade of the WAF- and alkylated PAH-specific CYP genes of the copepod T. japonicus, suggesting that these CYP genes would be those chiefly involved in detoxification in response to WAF exposure in copepods. In this paper, we provide information on the copepod P. nana CYP gene superfamily and also speculate on its potential role in the detoxification of PAHs in marine copepods. Despite the nonlethality of WAF, Pn-CYP3027F2 was rapidly and significantly upregulated in response to WAF that may serve as a useful biomarker of 40% or higher concentration of WAF exposure. This paper will be helpful to better understand the molecular mechanistic events underlying the metabolism of environmental toxicants in copepods.