AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

Evolution of the coronavirus spike protein in the full-length genome and defective viral genome under diverse selection pressures.

How coronaviruses evolve by altering the structures of their full-length genome and defective viral genome (DVG) under dynamic selection pressures has not been studied. In this study, we aimed to experimentally identify the dynamic evolutionary patterns of the S protein sequence in the full-length genome and DVG under diverse selection pressures, including persistence, innate immunity and antiviral drugs. The evolutionary features of the S protein sequence in the full-length genome and in the DVG under diverse selection pressures are as follows: (i) the number of nucleotide (nt) mutations does not necessarily increase with the number of selection pressures; (ii) certain types of selection pressure(s) can lead to specific nt mutations; (iii) the mutated nt sequence can be reverted to the wild-type nt sequence under the certain type of selection pressure(s); (iv) the DVG can also undergo mutations and evolve independently of the full-length genome; and (v) DVG species are regulated during evolution under diverse selection pressures. The various evolutionary patterns of the S protein sequence in the full-length genome and DVG identified in this study may contribute to coronaviral fitness under diverse selection pressures.

Feline mammary carcinoma-derived extracellular vesicle promotes liver metastasis via sphingosine kinase-1-mediated premetastatic niche formation.

BACKGROUND: Feline mammary carcinoma (FMC) is one of the most prevalent malignancies of female cats. FMC is highly metastatic and thus leads to poor disease outcomes. Among all metastases, liver metastasis occurs in about 25% of FMC patients. However, the mechanism underlying hepatic metastasis of FMC remains largely uncharacterized. RESULTS: Herein, we demonstrate that FMC-derived extracellular vesicles (FMC-EVs) promotes the liver metastasis of FMC by activating hepatic stellate cells (HSCs) to prime a hepatic premetastatic niche (PMN). Moreover, we provide evidence that sphingosine kinase 1 (SK1) delivered by FMC-EV was pivotal for the activation of HSC and the formation of hepatic PMN. Depletion of SK1 impaired cargo sorting in FMC-EV and the EV-potentiated HSC activation, and abolished hepatic colonization of FMC cells. CONCLUSIONS: Taken together, our findings uncover a previously uncharacterized mechanism underlying liver-metastasis of FMC and provide new insights into prognosis and treatment of this feline malignancy.

Identification of salivary autoantibodies as biomarkers of oral cancer with immunoglobulin A enrichment combined with affinity mass spectrometry.

Globally, oral cavity squamous cell carcinoma (OSCC) is one of the most common fatal illnesses. Its high mortality is ascribed to the fact that the disease is often diagnosed at a late stage, which indicates an urgent need for approaches for the early detection of OSCC. The use of salivary autoantibodies (autoAbs) as OSCC biomarkers has numerous advantages such as easy access to saliva samples and efficient detection of autoAbs using well-established secondary reagents. To improve OSCC screening, we identified OSCC-associated autoAbs with the enrichment of salivary autoAbs combined with affinity mass spectrometry (MS). The salivary IgA of healthy individuals and OSCC patients was purified with peptide M-conjugated beads and then applied to immunoprecipitated antigens (Ags) in OSCC cells. Using tandem MS analysis and spectral counting-based quantitation, the level of 10 Ags increased in the OSCC group compared with the control group. Moreover, salivary levels of autoAbs to the 10 Ags were determined by a multiplexed bead-based immunoassay. Among them, seven were significantly higher in early-stage OSCC patients than in healthy individuals. A marker panel consisting of autoAbs to LMAN2, PTGR1, RAB13, and UQCRC2 was further developed to improve the early diagnosis of OSCC.

Development of a salivary autoantibody biomarker panel for diagnosis of oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC) is a destructive disease with increasing incidence. OSCC is usually diagnosed at an advanced stage, which leads to poor outcomes of OSCC patients. Currently, there is a lack of biomarkers with sufficient effectiveness in early diagnosis of OSCC. To ameliorate OSCC screening, we evaluated the performances of salivary autoantibodies (auto-Abs) to nine proteins (ANXA2, CA2, ISG15, KNG1, MMP1, MMP3, PRDX2, SPARC, and HSPA5) as OSCC biomarkers. A multiplexed immunoassay using a fluorescence bead-based suspension array system was established for simultaneous assessment of the salivary levels of the above nine auto-Abs and a known OSCC-associated auto-Ab, anti-p53. Compared to healthy individuals (n = 140), the salivary levels of nine auto-Abs were significantly elevated in OSCC patients (n = 160). Notably, the salivary levels of the 10 auto-Abs in the early-stage OSCC patients (n = 102) were higher than that in the healthy group. Most importantly, utilizing a marker panel consisting of anti-MMP3, anti-PRDX2, anti-SPARC, and anti-HSPA5 for detection of early-stage OSCC achieved a sensitivity of 63.8% with a specificity of 90%. Collectively, herein we established a multiplex auto-Ab platform for OSCC screening, and demonstrated a four-auto-Ab panel which shows clinical applicability for early diagnosis of OSCC.

The Implication of Serum Autoantibodies in Prognosis of Canine Mammary Tumors.

Canine mammary tumor (CMT) is the most prevalent neoplasm in female dogs. Tumor recurrence and metastasis occur in malignant CMT (MMT) dogs after surgery. Identification of serum prognostic biomarkers holds the potential to facilitate prediction of disease outcomes. We have identified CMT-associated autoantibodies against thymidylate synthetase (TYMS), insulin-like growth factor-binding protein 5 (IGFBP5), hyaluronan and proteoglycan link protein 1 (HAPLN1), and anterior gradient 2 (AGR2), i.e., TYMS-AAb, IGFBP5-AAb, HAPLN1-AAb, and AGR2-AAb, respectively, by conducting serological enzyme-linked immunosorbent assays (ELISA). Herein we assessed serum AAb levels in 11 MMT dogs before and after surgery, demonstrating that IGFBP5-AAb and HAPLN1-AAb significantly decrease at 3- and 12-months post-surgery (p < 0.05). We evaluated the correlation between the presurgical AAb level and overall survival (OS) of 90 CMT dogs after surgery. Kaplan-Meier survival analysis reveals that IGFBP5-AAbHIgh and TYMS-AAbHigh are significantly correlated with worse OS (p = 0.017 and p = 0.029, respectively), while AGR2-AAbLow is correlated with somewhat poorer OS (p = 0.086). Areas under a time-dependent receiver operating characteristic curve (AUC) of IGFBP5-AAb and TYMS-AAb in predicting OS of MMT dogs are 0.611 and 0.616, respectively. Notably, MMT dogs presenting TYMS-AAbHigh/IGFBP5-AAbHigh/AGR2-AAbLow have worst OS (p = 0.0004). This study reveals an association between the serum AAb level and CMT prognosis.

Interactome Profiling of N-Terminus-Truncated NS1 Protein of Influenza A Virus Reveals Role of 14-3-3γ in Virus Replication.

Influenza A virus is transmitted through a respiratory route and has caused several pandemics throughout history. The NS1 protein of influenza A virus, which consists of an N-terminal RNA-binding domain and a C-terminal effector domain, is considered one of the critical virulence factors during influenza A virus infection because the viral protein can downregulate the antiviral response of the host cell and facilitate viral replication. Our previous study identified an N-terminus-truncated NS1 protein that covers the C-terminus effector domain. To comprehensively explore the role of the truncated NS1 in cells, we conducted immunoprecipitation coupled with LC-MS/MS to identify its interacting cellular proteins. There were 46 cellular proteins identified as the components of the truncated NS1 protein complex. As for our previous results for the identification of the full-length NS1-interacting host proteins, we discovered that the truncated NS1 protein interacts with the γ isoform of the 14-3-3 protein family. In addition, we found that the knockdown of 14-3-3γ in host cells reduced the replication of the influenza A/PR8 wild-type virus but not that of the PR8-NS1/1-98 mutant virus, which lacks most of the effector domain of NS1. This research highlights the role of 14-3-3γ, which interacts with the effector domain of NS1 protein, in influenza A viral replication.

Serum Level of Tumor-Overexpressed AGR2 Is Significantly Associated with Unfavorable Prognosis of Canine Malignant Mammary Tumors.

Canine malignant mammary tumors (MMTs) are prevalent malignancy in intact female dogs with a high incidence of metastasis and recurrence. A current lack of easily accessible tumor biomarkers hinders a timely assessment of the disease outcome. We previously identified anterior gradient protein 2 (AGR2) with higher protein abundance in canine MMT tissues compared with normal counterparts. AGR2 is an endoplasmic reticulum-resident protein disulfide isomerase involved in the regulation of protein processing and also exists extracellularly via secretion to exert pro-oncogenic functions. In the present study, we validated overexpression of AGR2 in canine MMT tissues from 45 dogs using immunohistochemistry and immunoblotting, and assessed serum AGR2 levels in 81 dogs with MMTs and 21 benign cases using a competitive enzyme-linked immunosorbent assay (ELISA). Our data revealed that serum eAGR2 levels are significantly correlated with MMT progression (p = 0.0007) and remote tumor metastasis (p = 0.002). Moreover, elevated levels of serum eAGR2 are associated with an unfavorable overall survival of MMT dogs in later stage (p = 0.0158). Area under the time-dependent ROC curve (AUC) of serum eAGR2 level as a prognostic indicator was 0.839. Collectively, this study uncovered that serum eAGR2 level is significantly associated with an adverse outcome of MMT dogs and holds a predictive potential in MMT prognosis.

K160 in the RNA-binding domain of the orf virus virulence factor OV20.0 is critical for its functions in counteracting host antiviral defense.

The OV20.0 virulence factor of orf virus antagonizes host antiviral responses. One mechanism through which it functions is by inhibiting activation of the dsRNA-activated protein kinase R (PKR) by sequestering dsRNA and by physically interacting with PKR. Sequence alignment indicated that several key residues critical for dsRNA binding were conserved in OV20.0, and their contribution to OV20.O function was investigated in this study. We found that residues F141, K160, and R164 were responsible for the dsRNA-binding ability of OV20.0. Interestingly, mutation at K160 (K160A) diminished the OV20.0-PKR interaction and further reduced the inhibitory effect of OV20.0 on PKR activation. Nevertheless, OV20.0 homodimerization was not influenced by K160A. The contribution of the dsRNA-binding domain and K160 to the suppression of RNA interference by OV20.0 was further demonstrated in plants. In summary, K160 is essential for the function of OV20.0, particularly its interaction with dsRNA and PKR that ultimately contributes to the suppression of PKR activation.

Significant association of serum autoantibodies to TYMS, HAPLN1 and IGFBP5 with early stage canine malignant mammary tumours.

Canine mammary tumours (CMTs) are the most prevalent neoplasms in female dogs. Despite the high incidence of such tumours, a lack of easily accessible biomarkers still impedes early diagnosis of malignant CMTs. Herein we identify thymidylate synthetase (TYMS), hyaluronan and proteoglycan link protein 1 (HAPLN1) and insulin-like growth factor-binding protein 5 (IGFBP5) as CMT antigens eliciting corresponding autoantibodies in CMT cases. We establish enzyme-linked immunosorbent assays (ELISAs) to detect autoantibodies to TYMS (TYMS-AAb), HAPLN1 (HAPLN1-AAb) and IGFBP5 (IGFBP5-AAb) in sera from 81 dogs with malignant CMTs (41 in Stage I), 24 with benign CMTs and 35 healthy controls. Levels of all the three autoantibodies are elevated in the malignant group compared with the healthy or the benign group; notably, the elevated autoantibody levels significantly correlate with the stage-I CMTs. For discriminating malignant CMTs from healthy control, the area under curve (AUC) of TYMS-AAb is 0.694 with specificity of 82.9% and sensitivity of 50.6%. The AUC of utilising HAPLN1-AAb for distinguishing the stage-I CMTs from healthy controls is 0.711 with specificity of 77.1% and sensitivity of 58.5%. In differentiating malignant CMTs from the benign, the AUC of IGFBP5-AAb reaches 0.696 with specificity of 70.8% and sensitivity of 67.9%, and a combination of IGFBP5-AAb and TYMS-AAb increases the AUC to 0.72. Finally, the AUC of combined HAPLN1-AAb and IGFBP5-AAb in discriminating the stage-I CMTs from the benign achieves 0.731. Collectively, this study highlights a significant association of the three serum autoantibodies with early stage malignant CMTs.

Proteomic Profiling of Paired Interstitial Fluids Reveals Dysregulated Pathways and Salivary NID1 as a Biomarker of Oral Cavity Squamous Cell Carcinoma.

Patients with oral cavity squamous cell carcinoma (OSCC) are frequently first diagnosed at an advanced stage, leading to poor prognosis and high mortality rates. Early detection of OSCC using body fluid-accessible biomarkers may improve the prognosis and survival rate of OSCC patients. As tumor interstitial fluid is a proximal fluid enriched with cancer-related proteins, it is a useful reservoir suitable for the discovery of cancer biomarkers and dysregulated biological pathways in tumor microenvironments. Thus, paired interstitial fluids of tumor (TIF) and adjacent noncancerous (NIF) tissues from 10 OSCC patients were harvested and analyzed using one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Using label-free spectral counting-based quantification, 113 proteins were found to be up-regulated in the TIFs compared with the NIFs. The gene set enrichment analysis (GSEA) revealed that the differentially expressed TIF proteins were highly associated with aminoacyl tRNA biosynthesis pathway. The elevated levels of 4 proteins (IARS, KARS, WARS, and YARS) involved in the aminoacyl tRNA biosynthesis were verified in the OSCC tissues with immunohistochemistry (IHC). In addition, nidogen-1 (NID1) was selected for verification as an OSCC biomarker. Salivary level of NID1 in OSCC patients (n = 48) was significantly higher than that in the healthy individuals (n = 51) and subjects with oral potentially malignant disorder (OPMD; n = 53). IHC analysis showed that NID1 level in OSCC tissues was increased compared with adjacent noncancerous epithelium (n = 222). Importantly, the elevated NID1 level was correlated with the advanced stages of OSCC, as well as the poor survival of OSCC patients. Collectively, the results suggested that TIF analysis facilitates understanding of the OSCC microenvironment and that salivary NID1 may be a useful biomarker for OSCC.

Identification of Salivary Biomarkers for Oral Cancer Detection with Untargeted and Targeted Quantitative Proteomics Approaches.

Oral cavity squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. In Taiwan, OSCC is the fifth leading cause of cancer-related mortality and leads to 2800 deaths per year. The poor outcome of OSCC patients is principally ascribed to the fact that this disease is often advanced at the time of diagnosis, suggesting that early detection of OSCC is urgently needed. Analysis of cancer-related body fluids is one promising approach to identify biomarker candidates of cancers. To identify OSCC biomarkers, salivary proteomes of OSCC patients, individuals with oral potentially malignant disorders (OPMDs), and healthy volunteers were comparatively profiled with isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry (MS). The salivary levels of 67 and 18 proteins in the OSCC group are elevated and decreased compared with that in the noncancerous group (OPMD and healthy groups), respectively. The candidate biomarkers were further selected using the multiple reaction monitoring (MRM)-MS and validated with the immunoassays. More importantly, the higher salivary level of three proteins, complement factor H (CFH), fibrinogen alpha chain (FGA), and alpha-1-antitrypsin (SERPINA1) was correlated with advanced stages of OSCC. Our results indicate that analysis of salivary proteome is a feasible strategy for biomarker discovery, and the three proteins are potential salivary markers for OSCC diagnosis.

Adenosine Deaminase Acting on RNA 1 Associates with Orf Virus OV20.0 and Enhances Viral Replication.

Orf virus (ORFV) infects sheep and goats and is also an important zoonotic pathogen. The viral protein OV20.0 has been shown to suppress innate immunity by targeting the double-stranded RNA (dsRNA)-activated protein kinase (PKR) by multiple mechanisms. These mechanisms include a direct interaction with PKR and binding with two PKR activators, dsRNA and the cellular PKR activator (PACT), which ultimately leads to the inhibition of PKR activation. In the present study, we identified a novel association between OV20.0 and adenosine deaminase acting on RNA 1 (ADAR1). OV20.0 bound directly to the dsRNA binding domains (RBDs) of ADAR1 in the absence of dsRNA. Additionally, OV20.0 preferentially interacted with RBD1 of ADAR1, which was essential for its dsRNA binding ability and for the homodimerization that is critical for intact adenosine-to-inosine (A-to-I)-editing activity. Finally, the association with OV20.0 suppressed the A-to-I-editing ability of ADAR1, while ADAR1 played a proviral role during ORFV infection by inhibiting PKR phosphorylation. These observations revealed a new strategy used by OV20.0 to evade antiviral responses via PKR.IMPORTANCE Viruses evolve specific strategies to counteract host innate immunity. ORFV, an important zoonotic pathogen, encodes OV20.0 to suppress PKR activation via multiple mechanisms, including interactions with PKR and two PKR activators. In this study, we demonstrated that OV20.0 interacts with ADAR1, a cellular enzyme responsible for converting adenosine (A) to inosine (I) in RNA. The RNA binding domains, but not the catalytic domain, of ADAR1 are required for this interaction. The OV20.0-ADAR1 association affects the functions of both proteins; OV20.0 suppressed the A-to-I editing of ADAR1, while ADAR1 elevated OV20.0 expression. The proviral role of ADAR1 is likely due to the inhibition of PKR phosphorylation. As RNA editing by ADAR1 contributes to the stability of the genetic code and the structure of RNA, these observations suggest that in addition to serving as a PKR inhibitor, OV20.0 might modulate ADAR1-dependent gene expression to combat antiviral responses or achieve efficient viral infection.

Proteome Analyses Reveal Positive Association of COL2A1, MPO, TYMS, and IGFBP5 with Canine Mammary Gland Malignancy.

PURPOSE: To identify aberrantly expressed proteins contributing to pathogenesis of canine mammary tumors (CMTs) which are the most prevalent neoplasms in female dogs and include different types. EXPERIMENTAL DESIGN: Frozen tissue specimens of normal mammary gland (n = 7), lobular hyperplasia (n = 6), simple carcinoma (n = 6), and complex carcinoma (n = 6) are collected from 11 CMT cases. Tissue homogenates are comparatively analyzed by the isobaric tags for relative and absolute quantification (iTRAQ) combined with LC-MS/MS to identify proteins differentially expressed in different-type CMT tissues. RESULTS: Among 3795 proteins identified and quantified among all groups, 133, 127, and 98 proteins are particularly overexpressed in simple carcinoma, complex carcinoma, and both types, respectively, compared with normal and hyperplastic tissues. Moreover, collagen type II alpha 1 chain (COL2A), myeloperoxidase (MPO), thymidylate synthetase (TYMS), and insulin-like growth factor-binding protein 5 (IGFBP5) are validated to be highly expressed in different-type CMT tissues using immunoblotting and immunohistochemistry. Notably, COL2A1 and IGFBP5 levels are correlated with clinical stages. CONCLUSIONS AND CLINICAL RELEVANCE: COL2A1, MPO, TYMS, and IGFBP5 protein levels are positively associated with CMT development. Data expedite further investigations to improve treatment regimens for CMT.

Detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in Taiwan.

Porcine deltacoronavirus (PDCoV) was initially documented in Hong Kong and later in the United States, South Korea, and Thailand. To investigate if PDCoV is also present in Taiwan, three swine coronaviruses-PDCoV, porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV)-were tested using real-time reverse transcription polymerase chain reaction (rRT-PCR) in 172 rectal swab samples from piglets exhibiting diarrhea between January 2016 and May 2017 on 68 pig farms in Taiwan. The rRT-PCR results were positive for PDCoV (29/172, 16.9%), PEDV (36/172, 20.9%), TGEV (2/172, 1.2%), and coinfections (16/172, 9.3%). After cloning and sequencing, PDCoV nucleocapsid genes were analyzed. Phylogeny results indicated that the nucleotide sequences of all isolates were like those reported in other countries. To further trace PDCoV in the period of 2011 to 2015, an enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against PDCoV. The results showed that 279 of 1,039 (26.9%) sera were positive for the PDCoV nucleocapsid protein, implying that PDCoV might have existed in Taiwan before 2011.

Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9 Virus Reveals an Inhibitory Role of NS1 in Host mRNA Maturation.

Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.

The role of SET/I2PP2A in canine mammary tumors.

Canine mammary tumor is the most common neoplasm in female dogs, and it has generated considerable attention as a translational model for human breast cancer. Ser/Thr protein phosphatase 2A (PP2A) plays a critical role as a tumor suppressor, and SET/I2PP2A, the endogenous inhibitory protein of PP2A, binds directly to PP2A and suppresses its phosphatase activity. Here, we investigated the role of SET in the tumorigenic growth in canine mammary tumor as well as in the sensitivity of tumors to existing therapeutics. Elevated protein levels of SET were observed in advanced-stage of canine mammary tumor tissues of dogs compared with paired normal tissues. Knockdown of SET expression in a canine mammary tumor cell line CIP-m led to increased PP2A activity and decreased cell proliferation, colony formation, and in vivo tumor growth. We observed suppression of mTOR, ß-catenin, and NFκB signaling by SET knockdown. The sensitivity of CIP-m cells to doxorubicin was decreased by SET knockdown, while SET knockdown in CIP-m cells did not affect sensitivity to 4-OH-tamoxifen, carboplatin, bortezomib, and X-ray radiation. These data suggest that SET plays important roles in the tumor progression of a subset of canine mammary tumor by suppressing PP2A activity and enhancing mTOR, ß-catenin, and NFκB signaling.

Multipoint Kras oncogene mutations potentially indicate mucinous carcinoma on the entire spectrum of mucinous ovarian neoplasms.

Kras mutation is a common phenomenon in many human neoplasms. We aimed to assess the Kras mutational status along the histological continuum from normal ovaries to the development of benign, borderline and malignant ovarian mucinous neoplasms. We analyzed 41 cases of malignant, 10 cases of borderline, 7 cases of benign mucinous ovarian tumors and 7 cases of normal ovarian tissue. The prevalence of Kras mutations in the normal ovary was 0.00% (n=0/7), while the prevalence in benign, borderline and malignant mucinous neoplasms was 57.14% (n=4/7), 90.00% (n=9/10) and 75.61% (n=31/41), respectively. Multiple Kras mutations were detected in 6 cases of mucinous carcinoma, including 5 double mutations with G13D/V14I (n=1), G12V/G13S (n=1), G12D/G13S (n=3) and one triple mutation with A11V/G13N/V14I (n=1). We identified six cases with 3 novel Kras mutations not previously described in the COSMIC database, which included A11V (n=3) and V14I (n=2) in mucinous carcinomas, and A11T (n=1) in a mucinous borderline tumor. In conclusion, Kras mutation appears to be one of the imperative events in the ovarian mucinous adenoma-borderline tumor-carcinoma sequence, as increased numbers of Kras mutations have been shown to be the strongest predictor of unequivocal malignancy in ovarian mucinous neoplasms.

Proteomics Analysis Reveals Involvement of Krt17 in Areca Nut-Induced Oral Carcinogenesis.

The areca nut is a known carcinogen that causes oral cancer in individuals in Southeast Asia, but the molecular mechanism that leads to this malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), which have been chronically exposed to areca nut extract (ANE). To elucidate the molecular basis of areca nut-induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE-treated sublines were determined using isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Over 1000 proteins were identified in four sublines, and 196 proteins were found to be differentially expressed in at least two ANE-treated sublines. A bioinformatic analysis revealed that these proteins participate in several pathways, and one of the most prominent pathways was the regulation of epithelial to mesenchymal transition (EMT). In all, 24 proteins including Krt17 were confirmed to be differentially expressed in the ANE-treated sublines. To reveal additional information on the mechanism of ANE-induced carcinogenesis, Krt17 was further investigated. Krt17 knockdown significantly suppressed ANE-induced cell migration and invasion and modulated the EMT process. Furthermore, in a murine model of carcinogen-induced (arecoline cocktail, an active compound of ANE) oral cancer, Krt17 was significantly up-regulated in all hyperplastic tissues and in carcinoma tissues (p < 0.001). In conclusion, we have identified a proteome of oral cancer cells that is associated with chronic areca nut exposure. Krt17 was demonstrated to contribute to areca nut-induced oral malignancy. The results of this study contribute to risk assessment, disease prevention and other clinical applications associated with areca nut-induced oral cancer.

Interactome analysis of the EV71 5' untranslated region in differentiated neuronal cells SH-SY5Y and regulatory role of FBP3 in viral replication.

Enterovirus 71 (EV71), a single-stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia-Pacific region. Through interactions with host proteins, the 5' untranslated region (5'UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5'UTR in neuronal cells, we performed a biotinylated RNA-protein pull-down assay in conjunction with LC-MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein-protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far-upstream element binding protein 3 (FBP3) was able to bind to the EV71 5'UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5'UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.