A Comparative Analysis of Data Analysis Tools for Data-Independent Acquisition Mass Spectrometry.

Data-independent acquisition (DIA) mass spectrometry-based proteomics generates reproducible proteome data. The complex processing of the DIA data has led to the development of multiple data analysis tools. In this study, we assessed the performance of five tools (OpenSWATH, EncyclopeDIA, Skyline, DIA-NN, and Spectronaut) using six DIA datasets obtained from TripleTOF, Orbitrap, and TimsTOF Pro instruments. By comparing identification and quantification metrics and examining shared and unique cross-tool identifications, we evaluated both library-based and library-free approaches. Our findings indicate that library-free approaches outperformed library-based methods when the spectral library had limited comprehensiveness. However, our results also suggest that constructing a comprehensive library still offers benefits for most DIA analyses. This study provides comprehensive guidance for DIA data analysis tools, benefiting both experienced and novice users of DIA-mass spectrometry technology.

A pathway activity-based proteomic classifier stratifies prostate tumors into two subtypes.

Prostate cancer (PCa) is the second most common cancer in males worldwide. The risk stratification of PCa is mainly based on morphological examination. Here we analyzed the proteome of 667 tumor samples from 487 Chinese PCa patients and characterized 9576 protein groups by PulseDIA mass spectrometry. Then we developed a pathway activity-based classifier concerning 13 proteins from seven pathways, and dichotomized the PCa patients into two subtypes, namely PPS1 and PPS2. PPS1 is featured with enhanced innate immunity, while PPS2 with suppressed innate immunity. This classifier exhibited a correlation with PCa progression in our cohort and was further validated by two published transcriptome datasets. Notably, PPS2 was significantly correlated with poor biochemical recurrence (BCR)/metastasis-free survival (log-rank P-value < 0.05). The PPS2 was also featured with cell proliferation activation. Together, our study presents a novel pathway activity-based stratification scheme for PCa.

AR imposes different effects on ZFHX3 transcription depending on androgen status in prostate cancer cells.

Both androgen receptor (AR) and the ZFHX3 transcription factor modulate prostate development. While AR drives prostatic carcinogenesis, ZFHX3 is a tumour suppressor whose loss activates the PI3K/AKT signalling in advanced prostate cancer (PCa). However, it is unknown whether ZFHX3 and AR are functionally related in PCa cells and, if so, how. Here, we report that in AR-positive LNCaP and C4-2B PCa cells, androgen upregulates ZFHX3 transcription via androgen-induced AR binding to the androgen-responsive elements (AREs) of the ZFHX3 promoter. Androgen also upregulated ZFHX3 transcription in vivo, as castration dramatically reduced Zfhx3 mRNA and protein levels in mouse prostates, and ZFHX3 mRNA levels correlated with AR activities in human PCa. Interestingly, the binding of AR to one ARE occurred in the absence of androgen, and the binding repressed ZFHX3 transcription as this repressive binding was interrupted by androgen treatment. The enzalutamide antiandrogen prevented androgen from inducing ZFHX3 transcription and caused excess ZFHX3 protein degradation. In human PCa, ZFHX3 was downregulated and the downregulation correlated with worse patient survival. These findings establish a regulatory relationship between AR and ZFHX3, suggest a role of ZFHX3 in AR function and implicate ZFHX3 loss in the antiandrogen therapies of PCa.

The transcription factor ZFHX3 is crucial for the angiogenic function of hypoxia-inducible factor 1α in liver cancer cells.

Angiogenesis is a hallmark of tumorigenesis, and hepatocellular carcinoma (HCC) is hypervascular and therefore very dependent on angiogenesis for tumor development and progression. Findings from previous studies suggest that in HCC cells, hypoxia-induced factor 1α (HIF1A) and zinc finger homeobox 3 (ZFHX3) transcription factors functionally interact in the regulation of genes in HCC cells. Here, we report that hypoxia increases the transcription of the ZFHX3 gene and enhances the binding of HIF1A to the ZFHX3 promoter in the HCC cell lines HepG2 and Huh-7. Moreover, ZFHX3, in turn, physically associated with and was functionally indispensable for HIF1A to exert its angiogenic activity, as indicated by in vitro migration and tube formation assays of human umbilical vein endothelial cells (HUVECs) and microvessel formation in xenograft tumors of HCC cells. Mechanistically, ZFHX3 was required for HIF1A to transcriptionally activate the vascular endothelial growth factor A (VEGFA) gene by binding to its promoter. Functionally, down-regulation of ZFHX3 in HCC cells slowed their tumor growth, and addition of VEGFA to conditioned medium from ZFHX3-silenced HCC cells partially rescued the inhibitory effect of this medium on HUVEC tube formation. In human HCC, ZFHX3 expression was up-regulated, and this up-regulation correlated with both HIF1A up-regulation and worse patient survival, confirming a functional association between ZFHX3 and HIF1A in human HCC. We conclude that ZFHX3 is an angiogenic transcription factor that is integral to the HIF1A/VEGFA signaling axis in HCC cells.

SUMOylation of the transcription factor ZFHX3 at Lys-2806 requires SAE1, UBC9, and PIAS2 and enhances its stability and function in cell proliferation.

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.

The association of marital status with cognitive function and the role of gender in Chinese community-dwelling older adults: a cross-sectional study.

BACKGROUND: Evidence of the association between marital status and cognitive function in Chinese older adults is limited. AIMS: To examine the relationship between marital status and cognitive function and to explore the role of gender amongst older adults from three Chinese communities. METHODS: A total of 1376 participants aged 60 years or over were included in this cross-sectional study. Cognitive function was assessed using the Chinese version of the mini-mental state examination (MMSE). Marital status and other variables were collected using a standardized questionnaire. Multiple linear regression models were used to examine associations between marital statuses and cognitive function amongst the target population. The moderating role of gender in these potential associations has also been explored. RESULTS: In univariate linear regression models, compared to being married, both being widowed (ß [95% CI]: -1.46[-2.78 to - 0.13]) and being single (ß [95% CI]: - 4.88[-6.43 to - 3.38]) were associated with lower MMSE scores. After adjustment for confounding factors, the significant association of being widowed with MMSE scores disappeared (ß [95% CI: - 0.08[- 1.04 to 0.86]), but the association of being single with MMSE scores still existed (ß [95% CI]: - 1.87[- 3.17 to - 0.58]). Furthermore, the association of being single with MMSE scores was statistically significant in men (ß [95% CI]: - 5.25[- 7.17 to - 3.33]) but not in women (ß [95% CI: 0.88[- 0.87 to 2.64]). DISCUSSION AND CONCLUSIONS: Being single was associated with poorer cognitive function compared with their married counterparts in older Chinese men but not in women. More preventive measurements should be implemented for single men to reduce or delay cognitive decline. This is particularly important in the context of an aging population in China.

CINP is a novel cofactor of KLF5 required for its role in the promotion of cell proliferation, survival and tumor growth.

Krüppel-like factor 5 (KLF5) both suppresses and promotes tumor growth depending on cellular context. The mechanisms underlying tumor promotion could be targetable for therapy. Although a number of transcriptional targets of KLF5 have been identified and implicated in KLF5-mediated tumor growth, how KLF5 regulates these genes remains to be addressed. Here we performed coimmunoprecipitation (co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the TSU-Pr1 bladder cancer cell line, in which KLF5 is shown to promote tumor growth, to identify KLF5-interacting nuclear proteins that are necessary for KLF5's tumor promoting function. LC-MS/MS revealed 122 potential KLF5 binding proteins in the nuclear proteins precipitated by the KLF5 antibody, and the top nine candidates included AHNAK, TFAM, HSDL2, HNRNPC, CINP, IST1, FBL, PABPC1 and SNRNP40. SRB assays of these nine proteins indicated that silencing CINP had the most potent inhibitory effect on cell growth in KLF5-expressing cells but did not affect parental TSU-Pr1 cells. Further analyses not only confirmed the physical interaction between KLF5 and CINP, also demonstrated that knockdown of CINP attenuated the effects of KLF5 on cell cycle progression, apoptosis and tumorigenesis. Silencing CINP also attenuated the effect of KLF5 on the expression of a number of genes and signaling pathways, including cell cycle regulator Cyclin D1 and apoptosis-related Caspase 7. These results suggest that CINP is a cofactor of KLF5 that is crucial for the promotion of tumor growth, and that the KLF5-CINP interaction could be a novel therapeutic target for inhibiting KLF5-promoted tumor growth.

Proteomic landscape profiling of primary prostate cancer reveals a 16-protein panel for prognosis prediction.

Prostate cancer (PCa) is the most common malignant tumor in men. Currently, there are few prognosis indicators for predicting PCa outcomes and guiding treatments. Here, we perform comprehensive proteomic profiling of 918 tissue specimens from 306 Chinese patients with PCa using data-independent acquisition mass spectrometry (DIA-MS). We identify over 10,000 proteins and define three molecular subtypes of PCa with significant clinical and proteomic differences. We develop a 16-protein panel that effectively predicts biochemical recurrence (BCR) for patients with PCa, which is validated in six published datasets and one additional 99-biopsy-sample cohort by targeted proteomics. Interestingly, this 16-protein panel effectively predicts BCR across different International Society of Urological Pathology (ISUP) grades and pathological stages and outperforms the D'Amico risk classification system in BCR prediction. Furthermore, double knockout of NUDT5 and SEPTIN8, two components from the 16-protein panel, significantly suppresses the PCa cells to proliferate, invade, and migrate, suggesting the combination of NUDT5 and SEPTIN8 may provide new approaches for PCa treatment.

DPHL v.2: An updated and comprehensive DIA pan-human assay library for quantifying more than 14,000 proteins.

A comprehensive pan-human spectral library is critical for biomarker discovery using mass spectrometry (MS)-based proteomics. DPHL v.1, a previous pan-human library built from 1,096 data-dependent acquisition (DDA) MS data of 16 human tissue types, allows quantifying of 10,943 proteins. Here, we generated DPHL v.2 from 1,608 DDA-MS data. The data included 586 DDA-MS data acquired from 18 tissue types, while 1,022 files were derived from DPHL v.1. DPHL v.2 thus comprises data from 24 sample types, including several cancer types (lung, breast, kidney, and prostate cancer, among others). We generated four variants of DPHL v.2 to include semi-tryptic peptides and protein isoforms. DPHL v.2 was then applied to two colorectal cancer cohorts. The numbers of identified and significantly dysregulated proteins increased by at least 21.7% and 14.2%, respectively, compared with DPHL v.1. Our findings show that the increased human proteome coverage of DPHL v.2 provides larger pools of potential protein biomarkers.

Establishment of a lncRNA-Based Prognostic Gene Signature Associated With Altered Immune Responses in HCC.

Hepatocellular carcinoma (HCC) is a common malignancy with higher mortality, and means are urgently needed to improve the prognosis. T cell exclusion (TCE) plays a pivotal role in immune evasion, and lncRNAs represent a large group of tumor development and progression modulators. Using the TCGA HCC dataset (n=374), we identified 2752 differentially expressed and 702 TCE-associated lncRNAs, of which 336 were in both groups. As identified using the univariate Cox regression analysis, those associated with overall survival (OS) were subjected to the LASSO-COX regression analysis to develop a prognosis signature. The model, which consisted of 11 lncRNAs and was named 11LNCPS for 11-lncRNA prognosis signature, was validated and performed better than two previous models. In addition to OS and TCE, higher 11LNCPS scores had a significant correlation with reduced infiltrations of CD8+ T cells and dendritic cells (DCs) and decreased infiltrations of Th1, Th2, and pro B cells. As expected, these infiltration alterations were significantly associated with worse OS in HCC. Analysis of published data indicates that HCCs with higher 11LNCPS scores were transcriptomically similar to those that responded better to PDL1 inhibitor. Of the 11LNCPS lncRNAs, LINC01134 and AC116025.2 seem more crucial, as their upregulations affected more immune cell types' infiltrations and were significantly associated with TCE, worse OS, and compromised immune responses in HCC. LncRNAs in the 11LNCPS impacted many cancer-associated biological processes and signaling pathways, particularly those involved in immune function and metabolism. The 11LNCPS should be useful for predicting prognosis and immune responses in HCC.

Novel Gene Signatures Predictive of Patient Recurrence-Free Survival and Castration Resistance in Prostate Cancer.

Molecular signatures predictive of recurrence-free survival (RFS) and castration resistance are critical for treatment decision-making in prostate cancer (PCa), but the robustness of current signatures is limited. Here, we applied the Robust Rank Aggregation (RRA) method to PCa transcriptome profiles and identified 287 genes differentially expressed between localized castration-resistant PCa (CRPC) and hormone-sensitive PCa (HSPC). Least absolute shrinkage and selection operator (LASSO) and stepwise Cox regression analyses of the 287 genes developed a 6-gene signature predictive of RFS in PCa. This signature included NPEPL1, VWF, LMO7, ALDH2, NUAK1, and TPT1, and was named CRPC-derived prognosis signature (CRPCPS). Interestingly, three of these 6 genes constituted another signature capable of distinguishing CRPC from HSPC. The CRPCPS predicted RFS in 5/9 cohorts in the multivariate analysis and remained valid in patients stratified by tumor stage, Gleason score, and lymph node status. The signature also predicted overall survival and metastasis-free survival. The signature's robustness was demonstrated by the C-index (0.55-0.74) and the calibration plot in all nine cohorts and the 3-, 5-, and 8-year area under the receiver operating characteristic curve (0.67-0.77) in three cohorts. The nomogram analyses demonstrated CRPCPS' clinical applicability. The CRPCPS thus appears useful for RFS prediction in PCa.

The Cardiac Glycoside Deslanoside Exerts Anticancer Activity in Prostate Cancer Cells by Modulating Multiple Signaling Pathways.

Prostate cancer (PCa) is a leading cause of cancer-related deaths among men worldwide, and novel therapies for advanced PCa are urgently needed. Cardiac glycosides represent an attractive group of candidates for anticancer repurposing, but the cardiac glycoside deslanoside has not been tested for potential anticancer activity so far. We found that deslanoside effectively inhibited colony formation in vitro and tumor growth in nude mice of PCa cell lines 22Rv1, PC-3, and DU 145. Such an anticancer activity was mediated by both the cell cycle arrest at G2/M and the induction of apoptosis, as demonstrated by different functional assays and the expression status of regulatory proteins of cell cycle and apoptosis in cultured cells. Moreover, deslanoside suppressed the invasion and migration of PCa cell lines. Genome-wide expression profiling and bioinformatic analyses revealed that 130 genes were either upregulated or downregulated by deslanoside in both 22Rv1 and PC-3 cell lines. These genes enriched multiple cellular processes, such as response to steroid hormones, regulation of lipid metabolism, epithelial cell proliferation and its regulation, and negative regulation of cell migration. They also enriched multiple signaling pathways, such as necroptosis, MAPK, NOD-like receptor, and focal adhesion. Survival analyses of the 130 genes in the TCGA PCa database revealed that 10 of the deslanoside-downregulated genes (ITG2B, CNIH2, FBF1, PABPC1L, MMP11, DUSP9, TMEM121, SOX18, CMPK2, and MAMDC4) inversely correlated, while one deslanoside-upregulated gene (RASD1) positively correlated, with disease-free survival in PCa patients. In addition, one deslanoside-downregulated gene (ENG) inversely correlated, while three upregulated genes (JUN, MXD1, and AQP3) positively correlated with overall survival in PCa patients. Some of the 15 genes have not been implicated in cancer before. These findings provide another candidate for repurposing cardiac glycosides for anticancer drugs. They also suggest that a diverse range of molecular events underlie deslanoside's anticancer activity in PCa cells.

ZFHX3 Promotes the Proliferation and Tumor Growth of ER-Positive Breast Cancer Cells Likely by Enhancing Stem-Like Features and MYC and TBX3 Transcription.

Breast cancer is a common malignancy, but the understanding of its cellular and molecular mechanisms is limited. ZFHX3, a transcription factor with many homeodomains and zinc fingers, suppresses prostatic carcinogenesis but promotes tumor growth of liver cancer cells. ZFHX3 regulates mammary epithelial cells' proliferation and differentiation by interacting with estrogen and progesterone receptors, potent breast cancer regulators. However, whether ZFHX3 plays a role in breast carcinogenesis is unknown. Here, we found that ZFHX3 promoted the proliferation and tumor growth of breast cancer cells in culture and nude mice; and higher expression of ZFHX3 in human breast cancer specimens was associated with poorer prognosis. The knockdown of ZFHX3 in ZFHX3-high MCF-7 cells decreased, and ZFHX3 overexpression in ZFHX3-low T-47D cells increased the proportion of breast cancer stem cells (BCSCs) defined by mammosphere formation and the expression of CD44, CD24, and/or aldehyde dehydrogenase 1. Among several transcription factors that have been implicated in BCSCs, MYC and TBX3 were transcriptionally activated by ZFHX3 via promoter binding, as demonstrated by luciferase-reporter and ChIP assays. These findings suggest that ZFHX3 promotes breast cancer cells' proliferation and tumor growth likely by enhancing BCSC features and upregulating MYC, TBX3, and others.

KLF5 Is Crucial for Androgen-AR Signaling to Transactivate Genes and Promote Cell Proliferation in Prostate Cancer Cells.

Androgen/androgen receptor (AR) signaling drives both the normal prostate development and prostatic carcinogenesis, and patients with advanced prostate cancer often develop resistance to androgen deprivation therapy. The transcription factor Krüppel-like factor 5 (KLF5) also regulates both normal and cancerous development of the prostate. In this study, we tested whether and how KLF5 plays a role in the function of AR signaling in prostate cancer cells. We found that KLF5 is upregulated by androgen depending on AR in LNCaP and C4-2B cells. Silencing KLF5, in turn, reduced AR transcriptional activity and inhibited androgen-induced cell proliferation and tumor growth in vitro and in vivo. Mechanistically, KLF5 occupied the promoter of AR, and silencing KLF5 repressed AR transcription. In addition, KLF5 and AR physically interacted with each other to regulate the expression of multiple genes (e.g., MYC, CCND1 and PSA) to promote cell proliferation. These findings indicate that, while transcriptionally upregulated by AR signaling, KLF5 also regulates the expression and transcriptional activity of AR in androgen-sensitive prostate cancer cells. The KLF5-AR interaction could provide a therapeutic opportunity for the treatment of prostate cancer.

ZFHX3 is indispensable for ERß to inhibit cell proliferation via MYC downregulation in prostate cancer cells.

Both estrogen receptor 2 (ESR2, also known as estrogen receptor beta (ERß)) and the zinc-finger homeobox 3 (ZFHX3, also known as ATBF1 for AT motif-binding factor 1) modulate prostate development and suppress prostatic tumorigenesis in mice. ZFHX3 is integral to proper functions of ESR1 (i.e., estrogen receptor alpha (ERα)), which belongs to the same family of proteins as ESR2, but is hardly expressed in prostate epithelial cells. It is not clear how ZFHX3 suppresses prostatic tumorigenesis. In this study, we investigated whether ZFHX3 and ERß functionally interact with each other in the suppression of prostatic tumorigenesis. In two androgen receptor (AR)-positive prostate cancer cell lines, C4-2B and LNCaP, we first validated ERß's tumor suppressor activity indicated by the inhibition of cell proliferation and repression of MYC expression. We found that loss of ZFHX3 increased cell proliferation and MYC expression, and downregulation of MYC was necessary for ZFHX3 to inhibit cell proliferation in the same cell lines. Importantly, loss of ZFHX3 prevented ERß from suppressing cell proliferation and repressing MYC transcription. Biochemically, ERß and ZFHX3 physically interacted with each other and they both occupied the same region of the common MYC promoter, even though ZFHX3 also bound to another region of the MYC promoter. Higher levels of ZFHX3 and ERß in human prostate cancer tissue samples correlated with better patient survival. These findings establish MYC repression as a mechanism for ZFHX3's tumor suppressor activity and ZFHX3 as an indispensable factor for ERß's tumor suppressor activity in prostate cancer cells. Our data also suggest that intact ZFHX3 function is required for using ERß-selective agonists to effectively treat prostate cancer.

A Complex Fluviolacustrine Environment on Early Mars and Its Astrobiological Potentials.

Chloride-bearing deposits and phyllosilicates-bearing units are widely distributed in the southern highlands of Mars, but these phases are rarely found together in fluviolacustrine environments. The study of the coexistence of these minerals can provide important insights into geochemistry, water activity, and ultimately the climate and habitability of early Mars. Here we use high-resolution compositional and morphological orbiter data to identify and characterize the context of diverse minerals in a Noachian fluviolacustrine environment west of Knobel crater (6.7°S, 226.8°W). The chlorides in this region are likely formed through the evaporation of brines in a closed topographic basin. The formation age of chlorides is older than 3.7 Ga, based on stratigraphic relationships identified and previously obtained crater retention ages. The timing of the alteration of basaltic materials to iron-magnesium smectites in relation to the chloride formation in this location is enigmatic and is unable to be resolved with currently available remote sensing data. Importantly, we find that this close relationship between these key minerals revealed by the currently available data details a complex and intimate history of aqueous activity in the region. Of critical importance are the evaporitic deposits as analogous terrestrial deposits have been shown to preserve ancient biosignatures and possibly even sustain microbial communities for hundreds of millions of years. These salts could have protected organic matter from ultraviolet radiation, or even allow modern habitable microenvironments in the shallow subsurface through periodic deliquescence. The high astrobiology potential of this site makes it a good candidate for future landed and sample return missions (e.g., the Chinese 2020 Mars mission).

Transmission electron microscopy and electron diffraction study of BSA-loaded quaternized chitosan nanoparticles.

Chitosan nanoparticles, O-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (O-HTCC) nanoparticles and bovine serum albumin (BSA) loaded chitosan and O-HTCC nanoparticles of a size (about 200-600 nm) were obtained through the process of ionic gelation between chitosan or O-HTCC and sodium tripolyphosphate (TPP). The physicochemical properties of nanoparticles made from chitosan, O-HTCC, BSA loaded chitosan, and BSA loaded O-HTCC were determined by transmission electron microscopy (TEM), polarized optical microscopy (POM), photon correlation spectroscopy (PCS), and X-ray diffraction (XRD) pattern. Zeta potential was also performed to understand the surface properties of nanoparticles and their ability to bind negatively charged BSA. TEM, POM, and XRD suggested that ionic-gelation process significantly influenced the crystallinity of BSA, and greater chain realignment in the BSA-loaded chitosan and O-HTCC nanoparticles. PCS revealed that BSA-loaded chitosan nanoparticles were bigger than chitosan nanoparticles in size and BSA-loaded O-HTCC nanoparticles were smaller than O-HTCC nanoparticles in size.