An 18-gene signature of recurrence-associated endothelial cells predicts tumor progression and castration resistance in prostate cancer.

BACKGROUND: The prognostic and therapeutic implications of endothelial cells (ECs) heterogeneity in prostate cancer (PCa) are poorly understood. METHODS: We investigated associations of EC heterogeneity with PCa recurrence and castration resistance in 8 bulk transcriptomic and 4 single-cell RNA-seq cohorts. A recurrence-associated EC (RAEC) signature was constructed by comparing 11 machine learning algorithms through nested cross-validation. Functional relevances of RAEC-specific genes were also tested. RESULTS: A subset of ECs was significantly associated with recurrence in primary PCa and named RAECs. RAECs were characteristic of tip and immature cells and were enriched in migration, angiogenesis, and collagen-related pathways. We then developed an 18-gene RAEC signature (RAECsig) representative of RAECs. Higher RAECsig scores independently predicted tumor recurrence and performed better or comparably compared to clinicopathological factors and commercial gene signatures in multiple PCa cohorts. Of the 18 RAECsig genes, FSCN1 was upregulated in ECs from PCa with higher Gleason scores; and the silencing of FSCN1, TMEME255B, or GABRD in ECs either attenuated tube formation or inhibited PCa cell proliferation. Finally, higher RAECsig scores predicted castration resistance in both primary and castration-resistant PCa. CONCLUSION: This study establishes an endothelial signature that links a subset of ECs to prostate cancer recurrence and castration resistance.

Interruption of KLF5 acetylation promotes PTEN-deficient prostate cancer progression by reprogramming cancer-associated fibroblasts.

Inactivation of phosphatase and tensin homolog (PTEN) is prevalent in human prostate cancer and causes high-grade adenocarcinoma with a long latency. Cancer-associated fibroblasts (CAFs) play a pivotal role in tumor progression, but it remains elusive whether and how PTEN-deficient prostate cancers reprogram CAFs to overcome the barriers for tumor progression. Here, we report that PTEN deficiency induced Krüppel-like factor 5 (KLF5) acetylation and that interruption of KLF5 acetylation orchestrated intricate interactions between cancer cells and CAFs that enhance FGF receptor 1 (FGFR1) signaling and promote tumor growth. Deacetylated KLF5 promoted tumor cells to secrete TNF-α, which stimulated inflammatory CAFs to release FGF9. CX3CR1 inhibition blocked FGFR1 activation triggered by FGF9 and sensitized PTEN-deficient prostate cancer to the AKT inhibitor capivasertib. This study reveals the role of KLF5 acetylation in reprogramming CAFs and provides a rationale for combined therapies using inhibitors of AKT and CX3CR1.

Opportunistic screening with multiphase contrast-enhanced dual-layer spectral CT for osteoblastic lesions in prostate cancer compared with bone scintigraphy.

Our study aimed to compare bone scintigraphy and dual-layer detector spectral CT (DLCT) with multiphase contrast enhancement for the diagnosis of osteoblastic bone lesions in patients with prostate cancer. The patients with prostate cancer and osteoblastic bone lesions detected on DLCT were divided into positive bone scintigraphy group (pBS) and negative bone scintigraphy group (nBS) based on bone scintigraphy. A total of 106 patients (57 nBS and 49 pBS) was included. The parameters of each lesion were measured from DLCT including Hounsfield unit (HU), 40-140 keV monochromatic HU, effective nuclear numbers (Zeff), and Iodine no water (InW) value in non-contrast phase (N), the arterial phase (A), and venous phase (V). The slope of the spectral curve at 40 and 100 keV, the different values of the parameters between A and N phase (A-N), V and N phase (V-N), and hybrid prediction model with multiparameters were used to differentiate pBS from nBS. Receiver operating characteristic analysis was performed to compare the area under the curve (AUC) for differentiating the pBS group from the nBS group. The value of conventional HU values, slope, and InW in A-N and V-N, and hybrid model were significantly higher in the pBS group than in the nBS group. The hybrid model of all significant parameters had the highest AUC of 0.988, with 95.5% sensitivity and 94.6% specificity. DLCT with arterial contrast enhancement phase has the potential to serve as an opportunistic screening tool for detecting positive osteoblastic bone lesions, corresponding to those identified in bone scintigraphy.

Multidisciplinary-derived clinical score for accurate prediction of long-term mortality in fibrotic lung disease patients.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) stands out as one of the most aggressive forms of interstitial lung diseases (ILDs), currently without a definitive cure. Multidisciplinary discussion (MDD) is now considered a cornerstone in diagnosing and differentiating ILD subtypes. The Gender-Age-Physiology (GAP) score, developed to assess IPF prognosis based on sex, age, forced vital capacity, and diffusion capacity for carbon monoxide (DLCO), is limited in not considering dyspnea and functional impairment during the walking test. We proposed a MDD-based clinical score for mortality prediction among those patients. METHODS: From December 2018 to December 2019, we enrolled ILD patients with IPF and non-IPF and followed-up them till December 2020. Based on DLCO, modified Medical Research Council (mMRC) Dyspnea Scale, and six-minute walking test (6MWT) distance, a functional score was developed for mortality prediction. RESULTS: We enrolled 104 ILD patients, 12 (11.5%) died by the one-year follow-up. In receiver operating characteristic (ROC) curve analysis, DLCO (% predicted) was the most accurate variable predicting one-year mortality with an area under curve (AUC) of 0.88 (95% confidence interval [CI] = 0.80-0.94), followed by mMRC Dyspnea Score (AUC = 0.82 [95% CI = 0.73-0.89]), 6MWT distance (AUC = 0.80 [95% CI = 0.71-0.88]), and GAP score (AUC = 0.77 [95% CI = 0.67-0.84]). Only the GAP score (hazard ratio [HR] = 1.55, 95% CI = 1.03-2.34, p = 0.0.37) and functional score (HR = 3.45, 95% CI = 1.11-10.73, p = 0.032) were significantly associated with one-year mortality in multivariable analysis. CONCLUSION: The clinical score composite of DLCO, mMRC Dyspnea Scale, and 6MWT distance could provide an accurate prediction for long-term mortality in ILD patients, laying out a helpful tool for managing and following these patients.

Factors influencing long-term outcomes in fibrotic interstitial lung disease (F-ILD) diagnosed through multidisciplinary discussion (MDD): a prospective cohort study.

BACKGROUND: The diagnostic process for fibrotic interstitial lung disease (F-ILD) is notably intricate, necessitating a multidisciplinary discussion to achieve consensus based on both clinical and radiological features. This study investigated the shared and distinctive long-term mortality predictors among the two primary phenotypes of F-ILD, namely idiopathic pulmonary fibrosis (IPF) and connective tissue disease-associated interstitial lung disease (CTD-ILD). METHODS: We included patients with F-ILD diagnosed from December 2018 to December 2019 and conducted follow-up assessments until February 2023. Age, gender, usual interstitial pneumonia (UIP) pattern, gender-age-physiology (GAP) score, modified Medical Research Council (mMRC) dyspnea score, antifibrotic agent use, pulmonary function test parameters, and six-minute walking test (6MWT) parameters were recorded at baseline and used as mortality predictors in a multivariate Cox regression model. RESULTS: We enrolled 104 ILD patients. The survival rate of non-IPF patients was more than twice that of IPF patients (78.9% vs. 34%, p < 0.001), and the survival rate of patients with a GAP score of 0-2 was more than twice that of patients with a score of > 2 (93.2% vs. 36.6%, p < 0.001). Older age, male gender, definite UIP pattern, higher GAP score, higher mMRC dyspnea score, lower forced expiratory volume in one second/forced vital capacity (FEV1/FVC), shorter 6MWT distance, and lower initial and final SpO2 were also associated with higher long-term mortality (p < 0.05). In multivariable analysis, only a GAP score of > 2 (hazard ratio [HR]:16.7; 95% confidence interval [CI] 3.28-85.14; p = 0.001) and definite UIP pattern (HR: 4.08; 95% CI 1.07-15.5; p = 0.039) were significantly associated with overall mortality. CONCLUSION: The long-term mortality rate of IPF patients was higher than that of CTD-ILD patients. The GAP score and UIP patterns were significant mortality predictors for both IPF and CTD-ILD patients.

Coexpression analysis of lncRNAs and mRNAs identifies potential regulatory long noncoding RNAs involved in the inflammatory effects of lipopolysaccharide on bovine mammary epithelial cells.

BACKGROUND: The infection of bovine mammary glands by pathogenic microorganisms not only causes animal distress but also greatly limits the development of the dairy industry and animal husbandry. A deeper understanding of the host's initial response to infection may increase the accuracy of selecting drug-resistant animals or facilitate the development of new preventive or therapeutic intervention strategies. In addition to their functions of milk synthesis and secretion, bovine mammary epithelial cells (BMECs) play an irreplaceable role in the innate immune response. To better understand this process, the current study identified differentially expressed long noncoding lncRNAs (DE lncRNAs) and mRNAs (DE mRNAs) in BMECs exposed to Escherichia coli lipopolysaccharide (LPS) and further explored the functions and interactions of these lncRNAs and mRNAs. RESULTS: In this study, transcriptome analysis was performed by RNA sequencing (RNA-seq), and the functions of the DE mRNAs and DE lncRNAs were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, we constructed a modulation network to gain a deeper understanding of the interactions and roles of these lncRNAs and mRNAs in the context of LPS-induced inflammation. A total of 231 DE lncRNAs and 892 DE mRNAs were identified. Functional enrichment analysis revealed that pathways related to inflammation and the immune response were markedly enriched in the DE genes. In addition, research results have shown that cell death mechanisms, such as necroptosis and pyroptosis, may play key roles in LPS-induced inflammation. CONCLUSIONS: In summary, the current study identified DE lncRNAs and mRNAs and predicted the signaling pathways and biological processes involved in the inflammatory response of BMECs that might become candidate therapeutic and prognostic targets for mastitis. This study also revealed several possible pathogenic mechanisms of mastitis.

Comprehensive Analysis of lncRNA and mRNA Expression Profile of Macrophage RAW264.7 Stimulated by Antimicrobial Peptide BSN-37.

BACKGROUND: BSN-37, a novel antimicrobial peptide (AMP) containing 37 amino acid residues isolated from the bovine spleen, has not only antibacterial activity but also immunomodulatory activity. Recent evidence shows that long non-coding RNAs (lncRNAs) play an important role in regulating the activation and function of immune cells. The purpose of this experiment was to investigate the lncRNA and mRNA expression profile of mouse macrophages RAW264.7 stimulated by bovine antimicrobial peptide BSN-37. METHODS: The whole gene expression microarray was used to detect the differentially expressed lncRNA and mRNA between antimicrobial peptide BSN-37 activated RAW264.7 cells and normal RAW264.7 cells. KEGG pathway analysis and GO function annotation analysis of differentially expressed lncRNAs and mRNA were carried out. Eight kinds of lncRNAs and nine kinds of mRNA with large differences were selected for qRT-PCR verification, respectively. RESULTS: In the current study, we found that 1294 lncRNAs and 260 mRNAs were differentially expressed between antibacterial peptide BSN-37 treatment and control groups. Among them, Bcl2l12, Rab44, C1s, Cd101 and other genes were associated with immune responses and were all significantly up-regulated. Mest and Prkcz are related to cell growth, and other genes are related to glucose metabolism and lipid metabolism. In addition, some immune-related terms were also found in the GO and KEGG analyses. At the same time, real-time quantitative PCR was used to verify selected lncRNA and mRNA with differential expression. The results of qRT-PCR verification were consistent with the sequencing results, indicating that our data were reliable. CONCLUSION: This study provides the lncRNA and mRNA expression profiles of RAW264.7 macrophages stimulated by antimicrobial peptide BSN-37 and helps to provide a reference value for subsequent studies on lncRNA regulation of antimicrobial peptide BSN-37 immune function.

Feasibility of Computed Tomography-guided Percutaneous Renal Cryoablation Under Local Anesthesia: A Single Center Experience in Taiwan.

BACKGROUND/AIM: To survey the safety and efficacy of percutaneous cryoablation for renal tumors under local anesthesia and pain control by using the -40°C lethal isotherm of the ice ball to cover the tumor margin as well as the coaxial cryoablation technique. PATIENTS AND METHODS: All procedures were performed between February 2014 and November 2021 with computed tomography (CT) guidance. All tumors were ablated by following the aforementioned plan, according to which tumor margins were covered by the -40°C lethal isotherm. Hydrodissection and coaxial cryoablation were performed in some cases to avoid organ injury and massive bleeding. 2% xylocaine was used for local anesthesia and 50 mg of pethidine (meperidine) was injected intramuscularly for pain control and sedation. The complications were evaluated and the Kaplan-Meier method was used to estimate local recurrence-free survival (LRFS). RESULTS: Sixty-five tumors [49 renal cell carcinomas (RCC) and 16 angiomyolipomas] were ablated in 55 patients (median Charlson Comorbidity Index=5.0). Local recurrence occurred in three of the 49 RCC cases. Two received a second cryoablation. LRFS at three and five years were both 91%. LRFS at three and five years reached 100% in tumors <3 cm. A large tumor (≥3 cm) was observed in the recurrence group. Hemorrhage was the most common complication (76.9%). Two patients who needed blood transfusion did not receive coaxial cryoablation. Three (4.6%) major complications (Clavien-Dindo grade ≥3) occurred. CONCLUSION: By using -40°C as the pre-plan tumor coverage, with the aid of coaxial cryoablation and multiplanar reconstruction method, CT-guided percutaneous renal cryoablation under local anesthesia is a safe and effective procedure in patients with many comorbidities.

Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer.

BACKGROUND: Castration-resistant prostate cancer often metastasizes to the bone, and such bone metastases eventually become resistant to available therapies, leading to the death of patients. Enriched in the bone, TGF-ß plays a pivotal role in bone metastasis development. However, directly targeting TGF-ß or its receptors has been challenging for the treatment of bone metastasis. We previously found that TGF-ß induces and then depends on the acetylation of transcription factor KLF5 at K369 to regulate multiple biological processes, including the induction of EMT, cellular invasiveness, and bone metastasis. Acetylated KLF5 (Ac-KLF5) and its downstream effectors are thus potential therapeutic targets for treating TGF-ß-induced bone metastasis in prostate cancer. METHODS: A spheroid invasion assay was applied to prostate cancer cells expressing KLF5K369Q, which mimics Ac-KLF5, to screen 1987 FDA-approved drugs for invasion suppression. Luciferase- and KLF5K369Q-expressing cells were injected into nude mice via the tail artery to model bone metastasis. Bioluminescence imaging, micro-CT), and histological analyses were applied to monitor and evaluate bone metastases. RNA-sequencing, bioinformatic, and biochemical analyses were used to understand nitazoxanide (NTZ)-regulated genes, signaling pathways, and the underlying mechanisms. The binding of NTZ to KLF5 proteins was evaluated using fluorescence titration, high-performance liquid chromatography (HPLC), and circular dichroism (CD) analysis. RESULTS: NTZ, an anthelmintic agent, was identified as a potent invasion inhibitor in the screening and validation assays. In KLF5K369Q-induced bone metastasis, NTZ exerted a potent inhibitory effect in preventive and therapeutic modes. NTZ also inhibited osteoclast differentiation, a cellular process responsible for bone metastasis induced by KLF5K369Q. NTZ attenuated the function of KLF5K369Q in 127 genes' upregulation and 114 genes' downregulation. Some genes' expression changes were significantly associated with worse overall survival in patients with prostate cancer. One such change was the upregulation of MYBL2, which functionally promotes bone metastasis in prostate cancer. Additional analyses demonstrated that NTZ bound to the KLF5 protein, KLF5K369Q bound to the promoter of MYBL2 to activate its transcription, and NTZ attenuated the binding of KLF5K369Q to the MYBL2 promoter. CONCLUSIONS: NTZ is a potential therapeutic agent for bone metastasis induced by the TGF-ß/Ac-KLF5 signaling axis in prostate cancer and likely other cancers.

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.

Regulated lytic cell death in breast cancer.

Breast cancer (BC) is a very common cancer among women and one of the primary causes of death in women worldwide. Because BC has different molecular subtypes, the challenges associated with targeted therapy have increased significantly, and the identification of new therapeutic targets has become increasingly urgent. Blocking apoptosis and inhibiting cell death are important characteristics of malignant tumours, including BC. Under adverse conditions, including exposure to antitumour therapy, inhibition of cell death programmes can promote cancerous transformation and the survival of cancer cells. Therefore, inducing cell death in cancer cells is fundamentally important and provides new opportunities for potential therapeutic interventions. Lytic forms of cell death, primarily pyroptosis, necroptosis and ferroptosis, are different from apoptosis owing to their characteristic lysis, that is, the production of cellular components, to guide beneficial immune responses, and the application of lytic cell death (LCD) in the field of tumour therapy has attracted considerable interest from researchers. The latest clinical research results confirm that lytic death signalling cascades involve the BC cell immune response and resistance to therapies used in clinical practice. In this review, we discuss the current knowledge regarding the various forms of LCD, placing a special emphasis on signalling pathways and their implications in BC, which may facilitate the development of novel and optimal strategies for the clinical treatment of BC.

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 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.

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.

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.

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.

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.

Aqp-1 Gene Knockout Attenuates Hypoxic Pulmonary Hypertension of Mice.

Objective- Hypoxic pulmonary hypertension (HPH) is characterized by proliferative vascular remodeling. Abnormal pulmonary artery smooth muscle cells proliferation and endothelial dysfunction are the primary cellular bases of vascular remodeling. AQP1 (aquaporin-1) is regulated by oxygen level and has been observed to play a role in the proliferation and migration of pulmonary artery smooth muscle cells. The role of AQP1 in HPH pathogenesis has not been directly determined to date. To determine the possible roles of AQP1 in the pathogenesis of HPH and explore its possible mechanisms. Approach and Results- Aqp1 knockout mice were used, and HPH model was established in this study. Primary pulmonary artery smooth muscle cells, primary mouse lung endothelial cells, and lung tissue sections from HPH model were used. Immunohistochemistry, immunofluorescence and Western blot, cell cycle, apoptosis, and migration analysis were performed in this study. AQP1 expression was upregulated by chronic hypoxia exposure, both in pulmonary artery endothelia and medial smooth muscle layer of mice. Aqp1 deficiency attenuated the elevation of right ventricular systolic pressures and mitigated pulmonary vascular structure remodeling. AQP1 deletion reduced abnormal cell proliferation in pulmonary artery and accompanied with accumulation of HIF (hypoxia-inducible factor). In vitro, Aqp1 deletion reduced hypoxia-induced proliferation, apoptosis resistance, and migration ability of primary cultured pulmonary artery smooth muscle cells and repressed HIF-1α protein stability. Furthermore, Aqp1 deficiency protected lung endothelial cells from apoptosis in response to hypoxic injury. Conclusions- Our data showed that Aqp1 deficiency could attenuate hypoxia-induced vascular remodeling in the development of HPH. AQP1 may be a potential target for pulmonary hypertension treatment.

Adult-to-adult living donor liver transplantation preoperative survey using MDCT, a single medical center experience in Taiwan.

This study evaluated multi-detector computed tomography (MDCT) scans performed on potential living donors for adult-to-adult liver transplantation (LDLT), with the aim of identifying significant findings that could be used to exclude potential transplantation donors. We retrospectively reviewed the medical records of 151 consecutive potential adult donors for LDLT from May 2007 to January 2015. Liver parenchyma steatosis, focal hepatic mass or intraabdominal malignancy, vascular variations, and donor liver volume were evaluated via MDCT. Grounds for excluding potential donors were also recorded and analyzed. Of the 151 potential donors, nine (6.0%) had moderate to severe fatty liver, 37 (24.5%) had hepatic arterial variants, 22 (14.6%) had significant portal venous variants, and more than half were found to have right accessory inferior hepatic vein. No intraabdominal malignancies were found. Eighty-eight potential donors were rejected, with the most common cause being insufficient recipient volume or remnant donor volume (47.7%), moderate to severe parenchymal steatosis (10.2%), and recipient expiration prior to transplantation (8.0%). An additional 16 potential donors were excluded by the surgical team due to the complexity of their portal venous variations. The rate of exclusion by pre-transplant imaging evaluation with MDCT was 33.8%. MDCT can provide accurate quantification of donor liver volume and steatosis severity along with precise demonstration of vascular variants, which are crucial for the preoperative evaluation of LDLT. However, MDCT may be ineffective for evaluating the biliary system without hepatobiliary-excreted contrast agent and has the disadvantage of ionizing radiation.