Acetylation regulates the nucleocytoplasmic distribution and oncogenic function of karyopherin alpha 2 in lung adenocarcinoma.

Karyopherin subunit alpha 2 (KPNA2, importin α1) is a nucleoplasmic protein responsible for the nuclear import of proteins with classical nuclear localization signals. Aberrant nuclear accumulation of KPNA2 has been observed in numerous cancer tissues. AMP-activated protein kinase (AMPK) is involved in the phosphorylation and acetylation of KPNA2 in enterocytes. However, the impact of these post-translational modifications on modulating the nucleocytoplasmic distribution of KPNA2 and its oncogenic role remain unclear. Unlike nuclear accumulation of wild-type KPNA2, which promoted lung cancer cell migration, KPNA2 Lys22 acetylation-mimicking mutations (K22Q and K22Q/S105A) prevented nuclear localization of KPNA2 and reduced the cell migration ability. Cytosolic KPNA2 K22Q interacted with and restricted the nuclear entry of E2F transcription factor 1 (E2F1), an oncogenic cargo protein of KPNA2, in lung cancer cells. Intriguingly, the AMPK activator EX229 promoted the nuclear export of KPNA2 S105A. However, the CBP/p300 inhibitor CCS-1477 abolished this phenomenon, suggesting that CBP/p300-mediated acetylation of KPNA2 promoted KPNA2 nuclear export in lung cancer cells. Collectively, our findings suggest that the CBP/p300 positively regulates KPNA2 acetylation, which enhances its cytosolic localization and suppresses its oncogenic activity in lung cancer.

Global Genome Diversity and Recombination in Mycoplasma pneumoniae.

Genomic changes in Mycoplasma pneumoniae caused by adaptation to environmental or ecologic pressures are poorly understood. We collected M. pneumoniae from children who had confirmed pneumonia in Taiwan during 2017-2020. We used whole-genome sequencing to compare these isolates with a worldwide collection of current and historical clinical strains for characterizing population structures. A phylogenetic tree for 284 strains showed that all sequenced strains consisted of 5 clades: T1-1 (sequence type [ST]1), T1-2 (mainly ST3), T1-3 (ST17), T2-1 (mainly ST2), and T2-2 (mainly ST14). We identified a putative recombination block containing 6 genes (MPN366‒371). Macrolide resistance involving 23S rRNA mutations was detected for each clade. Clonal expansion of macrolide resistance occurred mostly within subtype 1 strains, of which clade T1-2 showed the highest recombination rate and genome diversity. Functional characterization of recombined regions provided clarification of the biologic role of these recombination events in the evolution of M. pneumoniae.

Nuclear accumulation of KPNA2 impacts radioresistance through positive regulation of the PLSCR1-STAT1 loop in lung adenocarcinoma.

Lung adenocarcinoma (ADC) is the predominant histological type of lung cancer, and radiotherapy is one of the current therapeutic strategies for lung cancer treatment. Unfortunately, biological complexity and cancer heterogeneity contribute to radioresistance development. Karyopherin α2 (KPNA2) is a member of the importin α family that mediates the nucleocytoplasmic transport of cargo proteins. KPNA2 overexpression is observed across cancer tissues of diverse origins. However, the role of KPNA2 in lung cancer radioresistance is unclear. Herein, we demonstrated that high expression of KPNA2 is positively correlated with radioresistance and cancer stem cell (CSC) properties in lung ADC cells. Radioresistant cells exhibited nuclear accumulation of KPNA2 and its cargos (OCT4 and c-MYC). Additionally, KPNA2 knockdown regulated CSC-related gene expression in radioresistant cells. Next-generation sequencing and bioinformatic analysis revealed that STAT1 activation and nuclear phospholipid scramblase 1 (PLSCR1) are involved in KPNA2-mediated radioresistance. Endogenous PLSCR1 interacting with KPNA2 and PLSCR1 knockdown suppressed the radioresistance induced by KPNA2 expression. Both STAT1 and PLSCR1 were found to be positively correlated with dysregulated KPNA2 in radioresistant cells and ADC tissues. We further demonstrated a potential positive feedback loop between PLSCR1 and STAT1 in radioresistant cells, and this PLSCR1-STAT1 loop modulates CSC characteristics. In addition, AKT1 knockdown attenuated the nuclear accumulation of KPNA2 in radioresistant lung cancer cells. Our results collectively support a mechanistic understanding of a novel role for KPNA2 in promoting radioresistance in lung ADC cells.

Genomic Insight into the Spread of Meropenem-Resistant Streptococcus pneumoniae Spain23F-ST81, Taiwan.

Incidence of invasive pneumococcal disease caused by antimicrobial-resistant Streptococcus pneumoniae types not included in pneumococcal conjugate vaccines has increased, including a penicillin- and meropenem-resistant serotype 15A-ST63 clone in Japan. During 2013-2017, we collected 206 invasive pneumococcal isolates in Taiwan for penicillin and meropenem susceptibility testing. We found serotypes 15B/C-ST83 and 15A-ST63 were the most prevalent penicillin- and meropenem-resistant clones. A transformation study confirmed that penicillin-binding protein (PBP) 2b was the primary meropenem resistance determinant, and PBP1a was essential for high-level resistance. The rate of serotype 15B/C-ST83 increased during the study. All 15B/C-ST83 isolates showed an ermB macrolide resistance genotype. Prediction analysis of recombination sites revealed 12 recombination regions in 15B/C-ST83 compared with the S. pneumoniae Spain23F-ST81 genome. Pneumococcal clones rapidly recombine to acquire survival advantages and undergo local expansion under the selective pressure exerted by vaccines and antimicrobial drugs. The spread of 15B/C-ST83 is alarming for countries with high antimicrobial pressure.

Proteomic characterization of arsenic and cadmium exposure in bladder cells.

RATIONALE: Accumulating evidence has linked prolonged exposure to heavy metals to cancer occurrence in the urinary system. However, the specific biological mechanisms responsible for the association of heavy metals with the unusually high incidence of upper tract urothelial carcinoma in Taiwan are complex and incompletely understood. METHODS: To elucidate the specific biological mechanism and identify molecular indicators of the unusually high association of upper tract urothelial carcinoma with heavy metal exposure, protein expression following the treatment of T24 human bladder carcinoma and RT4 human bladder papilloma cell line models with arsenic (As) and cadmium (Cd) was studied. Proteomic changes in these cell models were integrated with data from a human bladder cancer (BLCA) tissue proteome to identify possible protein indicators of heavy metal exposure. RESULTS: After mass spectrometry based proteomic analysis and verification by Western blotting procedures, we identified 66 proteins that were up-regulated and 92 proteins that were down-regulated in RT4 cell extracts after treatment with As or Cd. Some 52 proteins were up-regulated and 136 proteins were down-regulated in T24 cell extracts after treatment with Cd. We further confirmed that down-expression of the PML (promyelocytic leukemia) protein was sustained for at least 75 days after exposure of bladder cells to As. Dysregulation of these cellular proteins by As was associated with three biological pathways. Immunohistochemical analyses of paraffin-embedded BLCA tissue slides confirmed that PML protein expression was decreased in BLCA tumor cells compared with adjacent noncancerous epithelial cells. CONCLUSIONS: These data suggest that PML may play an important role in the pathogenesis of BLCA and may be an indicator of heavy metal exposure in bladder cells.

Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan.

Most cases of oral squamous cell carcinoma (OSCC) develop from visible oral potentially malignant disorders (OPMDs). The latter exhibit heterogeneous subtypes with different transformation potentials, complicating the early detection of OSCC during routine visual oral cancer screenings. To develop clinically applicable biomarkers, we collected saliva samples from 96 healthy controls, 103 low-risk OPMDs, 130 high-risk OPMDs, and 131 OSCC subjects. These individuals were enrolled in Taiwan's Oral Cancer Screening Program. We identified 302 protein biomarkers reported in the literature and/or through in-house studies and prioritized 49 proteins for quantification in the saliva samples using multiple reaction monitoring-MS. Twenty-eight proteins were successfully quantified with high confidence. The quantification data from non-OSCC subjects (healthy controls + low-risk OPMDs) and OSCC subjects in the training set were subjected to classification and regression tree analyses, through which we generated a four-protein panel consisting of MMP1, KNG1, ANXA2, and HSPA5. A risk-score scheme was established, and the panel showed high sensitivity (87.5%) and specificity (80.5%) in the test set to distinguish OSCC samples from non-OSCC samples. The risk score >0.4 detected 84% (42/50) of the stage I OSCCs and a significant portion (42%) of the high-risk OPMDs. Moreover, among 88 high-risk OPMD patients with available follow-up results, 18 developed OSCC within 5 y; of them, 77.8% (14/18) had risk scores >0.4. Our four-protein panel may therefore offer a clinically effective tool for detecting OSCC and monitoring high-risk OPMDs through a readily available biofluid.

Golgi tethering factor golgin-97 suppresses breast cancer cell invasiveness by modulating NF-κB activity.

BACKGROUND: Golgin-97 is a tethering factor in the trans-Golgi network (TGN) and is crucial for vesicular trafficking and maintaining cell polarity. However, the significance of golgin-97 in human diseases such as cancer remains unclear. METHODS: We searched for a potential role of golgin-97 in cancers using Kaplan-Meier Plotter ( http://kmplot.com ) and Oncomine ( www.oncomine.org ) datasets. Specific functions of golgin-97 in migration and invasion were examined in golgin-97-knockdown and golgin-97-overexpressing cells. cDNA microarray, pathway analysis and qPCR were used to identify gene profiles regulated by golgin-97. The role of golgin-97 in NF-κB signaling pathway was examined by using subcellular fractionation, luciferase reporter assay, western blot analysis and immunofluorescence assay (IFA). RESULTS: We found that low expression of golgin-97 correlated with poor overall survival of cancer patients and was associated with invasiveness in breast cancer cells. Golgin-97 knockdown promoted cell migration and invasion, whereas re-expression of golgin-97 restored the above phenotypes in breast cancer cells. Microarray and pathway analyses revealed that golgin-97 knockdown induced the expression of several invasion-promoting genes that were transcriptionally regulated by NF-κB p65. Mechanistically, golgin-97 knockdown significantly reduced IκBα protein levels and activated NF-κB, whereas neither IκBα levels nor NF-κB activity was changed in TGN46- or GCC185-knockdown cells. Conversely, golgin-97 overexpression suppressed NF-κB activity and restored the levels of IκBα in golgin-97-knockdown cells. Interestingly, the results of Golgi-disturbing agent treatment revealed that the loss of Golgi integrity was not involved in the NF-κB activation induced by golgin-97 knockdown. Moreover, both TGN-bound and cytosolic golgin-97 inhibited NF-κB activation, indicating that golgin-97 functions as an NF-κB suppressor regardless of its subcellular localization. CONCLUSION: Our results collectively demonstrate a novel and suppressive role of golgin-97 in cancer invasiveness. We also provide a new avenue for exploring the relationship between the TGN, golgin-97 and NF-κB signaling in tumor progression.

Is the whole greater than the sum of its parts? De novo assembly strategies for bacterial genomes based on paired-end sequencing.

BACKGROUND: Whole genome sequence construction is becoming increasingly feasible because of advances in next generation sequencing (NGS), including increasing throughput and read length. By simply overlapping paired-end reads, we can obtain longer reads with higher accuracy, which can facilitate the assembly process. However, the influences of different library sizes and assembly methods on paired-end sequencing-based de novo assembly remain poorly understood. RESULTS: We used 250 bp Illumina Miseq paired-end reads of different library sizes generated from genomic DNA from Escherichia coli DH1 and Streptococcus parasanguinis FW213 to compare the assembly results of different library sizes and assembly approaches. Our data indicate that overlapping paired-end reads can increase read accuracy but sometimes cause insertion or deletions. Regarding genome assembly, merged reads only outcompete original paired-end reads when coverage depth is low, and larger libraries tend to yield better assembly results. These results imply that distance information is the most critical factor during assembly. Our results also indicate that when depth is sufficiently high, assembly from subsets can sometimes produce better results. CONCLUSIONS: In summary, this study provides systematic evaluations of de novo assembly from paired end sequencing data. Among the assembly strategies, we find that overlapping paired-end reads is not always beneficial for bacteria genome assembly and should be avoided or used with caution especially for genomes containing high fraction of repetitive sequences. Because increasing numbers of projects aim at bacteria genome sequencing, our study provides valuable suggestions for the field of genomic sequence construction.

[Everolimus combined with all-trans retinoid acid reverses drug resistance in acute promyelocytic leukemia NB4-R1 cells].

OBJECTIVE: To investigate the effect of everolimus(RAD001)combined with all-trans retinoid acid(ATRA) on drug resistance of ATRA-resistance acute promyelocytic leukemia(APL) cell line NB4-R1 and its molecular mechanism. METHODS: APL NB4-R1 cells were treated with different concentrations of RAD001(1 nmol/L, 10 nmol/L and 100 nmol/L) with ATRA(1µmol/L) for 24, 48 and 72 h, respectively. The differentiation of NB4-R1 cells was analyzed by flow cytometry with CD11b staining and nitro blue tetrozolium(NBT) reduction test. Cell cycle was detected by cell cycle staining kit and apoptosis was detected by flow cytometry with Annexin V/PI staining. Protein expressions of LC-3II, PML-RARα, P-P70S6K and P-4E-BP1 were determined by Western blotting. RESULTS: RAD001 combined with ATRA significantly induced NB4-R1 cells differentiation, but RAD001 or ATRA alone did not enhance NB4-R1 differentiation. The co-treatment induced accumulation of cells in G1 phase and decreased the proportion of cells in S phase. The combined treatment had no effect on cell apoptosis. The differentiation rate of NB4-R1 cells in 100 nmol/L RAD001, 1µmol/L ATRA, RAD001 combined with ATRA and control groups was(2.29±0.57)%,(17.06±2.65)%,(54.47±4.91)% and(2.54±0.53)%, respectively; the proportion of cells in G1 phase was(35.20±11.97)%,(33.54±6.25)%,(53.70±8.73)% and(27.40±6.01)%, respectively; cells apoptosis rate was(2.30±0.14)%,(2.25±0.21)%,(2.40±0.28)% and(1.95±0.07)%, respectively. The combination of RAD001 with ATRA significantly inhibited mTOR signaling downstream proteins P-P70S6K, P-4E-BP1 and enhanced autophagy-related protein LC3-II and Beclin 1. The co-treatment also induced degradation of fusion protein PML-RARα. CONCLUSION: RAD001 combined with ATRA can induce cell differentiation, inhibit cell cycle, resulting the reverse of drug resistance in NB4-R1 cells, which is associated with increase of autophagy level and degradation of PML-RARα.

Clinical validation of a saliva-based matrix metalloproteinase-1 rapid strip test for detection of oral cavity cancer.

BACKGROUND: We previously identified matrix metalloproteinase-1 (MMP-1) as one of the most promising salivary biomarkers for oral squamous cell carcinoma (OSCC) and developed a sensitive ELISA for MMP-1 with good performance in detection of OSCC using a cohort of 1160 saliva samples. METHODS: A time-saving rapid strip test (RST) for MMP-1 was developed in this study and its diagnostic performance compared with ELISA using saliva samples from a new cohort of 603 subjects (171 healthy controls, 236 patients with oral potentially malignant disorders, and 196 OSCC patients). RESULTS: Salivary MMP-1 levels measured using RST and ELISA were highly comparable and both assays could effectively distinguish between OSCC and non-cancerous groups. Similar to ELISA, receiver operating characteristic curve analysis of the MMP-1 RST was effective in identifying patients with OSCC at different oral cavity sites and stages. CONCLUSIONS: Salivary MMP-1 can be sensitively detected using both RST and ELISA methods. Our newly developed point-of-care MMP-1 RST is a promising in vitro diagnostic device (IVD) that may serve as a novel auxiliary tool in the routine clinical detection and monitoring of OSCC.