WNT2-SOX4 positive feedback loop promotes chemoresistance and tumorigenesis by inducing stem-cell like properties in gastric cancer.

Gastric cancer (GC) is characterized by its vigorous chemoresistance to current therapies, which is attributed to the highly heterogeneous and immature phenotype of cancer stem cells (CSCs) during tumor initiation and progression. The secretory WNT2 ligand regulates multiple cancer pathways and has been demonstrated to be a potential therapeutic target for gastrointestinal tumors; however, its role involved in gastric CSCs (GCSCs) remains unclear. Here, we found that overexpression of WNT2 enhanced stemness properties to promote chemoresistance and tumorigenicity in GCSCs. Mechanistically, WNT2 was positively regulated by its transcription factor SOX4, and in turn, SOX4 was upregulated by the canonical WNT2/FZD8/ß-catenin signaling pathway to form an auto-regulatory positive feedback loop, resulting in the maintenance of GCSCs self-renewal and tumorigenicity. Furthermore, simultaneous overexpression of both WNT2 and SOX4 was correlated with poor survival and reduced responsiveness to chemotherapy in clinical GC specimens. Blocking WNT2 using a specific monoclonal antibody significantly disrupted the WNT2-SOX4 positive feedback loop in GCSCs and enhanced the chemotherapeutic efficacy when synergized with the chemo-drugs 5-fluorouracil and oxaliplatin in a GCSC-derived mouse xenograft model. Overall, this study identified a novel WNT2-SOX4 positive feedback loop as a mechanism for GCSCs-induced chemo-drugs resistance and suggested that the WNT2-SOX4 axis may be a potential therapeutic target for gastric cancer treatment.

Targeting LGSN restores sensitivity to chemotherapy in gastric cancer stem cells by triggering pyroptosis.

Gastric cancer (GC) is notoriously resistant to current therapies due to tumor heterogeneity. Cancer stem cells (CSCs) possess infinite self-renewal potential and contribute to the inherent heterogeneity of GC. Despite its crucial role in chemoresistance, the mechanism of stemness maintenance of gastric cancer stem cells (GCSCs) remains largely unknown. Here, we present evidence that lengsin, lens protein with glutamine synthetase domain (LGSN), a vital cell fate determinant, is overexpressed in GCSCs and is highly correlated with malignant progression and poor survival in GC patients. Ectopic overexpression of LGSN in GCSC-derived differentiated cells facilitated their dedifferentiation and treatment resistance by interacting with vimentin and inducing an epithelial-to-mesenchymal transition. Notably, genetic interference of LGSN effectively suppressed tumor formation by inhibiting GCSC stemness maintenance and provoking gasdermin-D-mediated pyroptosis through vimentin degradation/NLRP3 signaling. Depletion of LGSN combined with the chemo-drugs 5-fluorouracil and oxaliplatin could offer a unique and promising approach to synergistically rendering this deadly cancer eradicable in vivo. Our data place focus on the role of LGSN in GCSC regeneration and emphasize the critical importance of pyroptosis in battling GCSC.

A novel endothelial-related prognostic index by integrating single-cell and bulk RNA sequencing data for patients with kidney renal clear cell carcinoma.

Background: Endothelial cells in the tumor microenvironment play an important role in the development of kidney renal clear cell carcinoma (KIRC). We wanted to further identify the function of endothelial cells in KIRC patients by integrating single-cell and bulk RNA sequencing data. Methods: Online databases provide the original data of this study. An endothelial-related prognostic index (ERPI) was constructed and validated by R version 3.6.3 and relative packages. Results: The ERPI consisted of three genes (CCND1, MALL, and VWF). Patients with high ERPI scores were significantly correlated with worse prognosis than those with low ERPI scores in the TCGA training group, TCGA test group, and GSE29609 group. A positive correlation was identified between the ERPI score and poor clinical features. The results of functional analysis indicated that ERPI was significantly associated with immune-related activities. We suggested that patients with high ERPI scores were more likely to benefit from immunotherapy based on the results of immune checkpoints, tumor microenvironment, stemness index, and TCIA, while patients with low ERPI scores were sensitive to gemcitabine, docetaxel, paclitaxel, axitinib, pazopanib, sorafenib, and temsirolimus according to the results of the "pRRophetic" algorithm. Therefore, this ERPI may help doctors choose the optimal treatment for patients with KIRC. Conclusion: By integrating single-cell and bulk RNA sequencing data from KIRC patients, we successfully identified the key genes from the perspective of endothelial cells in the tumor microenvironment and constructed ERPIs that had positive implications in precision medicine.

Modulating Lysine Crotonylation in Cardiomyocytes Improves Myocardial Outcomes.

BACKGROUND: Ischemic heart disease is a major global public health challenge, and its functional outcomes remain poor. Lysine crotonylation (Kcr) was recently identified as a post-translational histone modification that robustly indicates active promoters. However, the role of Kcr in myocardial injury is unknown. In this study, we aimed to clarify the pathophysiological significance of Kcr in cardiac injury and explore the underlying mechanism. METHODS: We investigated the dynamic change of both the Kcr sites and protein level in left ventricular tissues at 2 time points following sham or cardiac ischemia-reperfusion injury, followed by liquid chromatography-coupled tandem mass tag mass spectrometry. After validation of the enriched protein Kcr by immunoprecipitation and Western blot, the function and mechanism of specific Kcr sites were further investigated in vitro and in vivo by gain- or loss-of-function mutations targeting Kcr sites of selected proteins. RESULTS: We found that cardiac ischemia-reperfusion injury triggers preferential Kcr of proteins required for cardiomyocyte contractility, including mitochondrial and cytoskeleton proteins, which occurs largely independently of protein-level changes in the same proteins. Those exhibiting Kcr changes were associated not only with disruption of cardiomyocyte mitochondrial, sarcomere architecture, and gap junction but also with cardiomyocyte autophagy and apoptosis. Modulating site-specific Kcr of selected mitochondrial protein IDH3a (isocitrate dehydrogenase 3 [NAD+] alpha) at K199 and cytoskeletal protein TPM1 (tropomyosin alpha-1 chain) at K28/29 or enhancing general Kcr via sodium crotonate provision not only protects cardiomyocyte from apoptosis by inhibiting BNIP3 (Bcl-2 adenovirus E18 19-kDa-interacting protein 3)-mediated mitophagy or cytoskeleton structure rearrangement but also preserves postinjury myocardial function by inhibiting fibrosis and apoptosis. CONCLUSIONS: Our results indicate that Kcr modulation is a key response of cardiomyocytes to ischemia-reperfusion injury and may represent a novel therapeutic target in the context of ischemic heart disease.

Wnt4 is crucial for cardiac repair by regulating mesenchymal-endothelial transition via the phospho-JNK/JNK.

Rational: Wnt4 plays a critical role in development and is reactivated during fibrotic injury; however, the role of Wnt4 in cardiac repair remains unclear. In this study, our aim was to clarify the pathophysiological role and mechanisms of Wnt4 following acute cardiac ischemic reperfusion injury. Methods and results: We investigated the spatio-temporal expression of Wnt4 following acute cardiac ischemic reperfusion injury and found that Wnt4 was upregulated as an early injury response gene in cardiac fibroblasts near the injury border zone and associated with mesenchymal-endothelial transition (MEndoT), a beneficial process for revascularizing the damaged myocardium in cardiac repair. Using ChIP assay and in vitro and in vivo loss- and gain-of-function, we demonstrated that Wnt4 served as a crucial downstream target gene of p53 during MEndoT. Wnt4 knockdown in cardiac fibroblasts led to decreased MEndoT and worsened cardiac function. Conversely, Wnt4 overexpression in cardiac fibroblasts induced MEndoT in these cells via the phospho-JNK/JNK signaling pathway; however, both the p53 and Wnt4 protein levels were dependent on the ß-catenin signaling pathway. JNK activation plays a critical role in the induction of MEndoT and is crucial for Wnt4 regulated MEndoT. Moreover, Wnt4 overexpression specifically in cardiac fibroblasts rescued the cardiac function worsening due to genetic p53 deletion by decreasing fibrosis and increasing MEndoT and vascular density. Conclusion: Our study revealed that Wnt4 plays a pivotal role in cardiac repair with involvement of phospho-JNK mediated MEndoT and is a crucial gene for cardiac fibroblast-targeted therapy in heart disease.

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells.

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T overexpressing human NTCP, HNF4α, RXRα and PPARα) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines. HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.

Defined host factors support HBV infection in non-hepatic 293T cells.

Hepatitis B virus (HBV) is a human hepatotropic virus. However, HBV infection also occurs at extrahepatic sites, but the relevant host factors required for HBV infection in non-hepatic cells are only partially understood. In this article, a non-hepatic cell culture model is constructed by exogenous expression of four host genes (NTCP, HNF4α, RXRα and PPARα) in human non-hepatic 293T cells. This cell culture model supports HBV entry, transcription and replication, as evidenced by the detection of HBV pgRNA, HBV cccDNA, HBsAg, HBeAg, HBcAg and HBVDNA. Our results suggest that the above cellular factors may play a key role in HBV infection of non-hepatic cells. This model will facilitate the identification of host genes that support extrahepatic HBV infection.

Frizzled Receptors as Potential Therapeutic Targets in Human Cancers.

Frizzled receptors (FZDs) are a family of seven-span transmembrane receptors with hallmarks of G protein-coupled receptors (GPCRs) that serve as receptors for secreted Wingless-type (WNT) ligands in the WNT signaling pathway. Functionally, FZDs play crucial roles in regulating cell polarity, embryonic development, cell proliferation, formation of neural synapses, and many other processes in developing and adult organisms. In this review, we will introduce the basic structural features and review the biological function and mechanism of FZDs in the progression of human cancers, followed by an analysis of clinical relevance and therapeutic potential of FZDs. We will focus on the development of antibody-based and small molecule inhibitor-based therapeutic strategies by targeting FZDs for human cancers.

FZD7 is a novel prognostic marker and promotes tumor metastasis via WNT and EMT signaling pathways in esophageal squamous cell carcinoma.

Frizzled (FZD) proteins are receptors for secreted WNT proteins and play a critical role in the malignant progression of various cancers. However, the role of human FZD family members in esophageal squamous cell carcinoma (ESCC) was rarely investigated. In this study, we found that the FZD7 gene was the most commonly up-regulated FZD member in ESCC cell lines compared with other FZDs. TMA studies further validated that FZD7 protein was up-regulated in 165 of 252 (65.5%) informative ESCC patients and significantly correlated with poor overall survival (P=0.001). Additionally, multivariate Cox regression analysis showed that FZD7 overexpression was an independent prognostic factor for ESCC patients. Ectopic expression of FZD7 could promote ESCC cell metastasis both in vitro and in vivo. Under WNT3A stimulation, FZD7 was able to induce the nuclear translocation of ß-catenin and activate the downstream targets of WNT/ß-catenin signaling, as well as promote epithelial-mesenchymal transition (EMT) potential in ESCC cells. Our study demonstrated for the first time that FZD7 contributes to the malignant progression of ESCC and represents a novel prognostic marker and a potential therapeutic target for ESCC patients.

RNA editing of SLC22A3 drives early tumor invasion and metastasis in familial esophageal cancer.

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2, a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals.

Correlation between cognitive functions and syndromes of traditional Chinese medicine in amnestic mild cognitive impairment.

OBJECTIVE: To explore the correlation between the cognitive functions and syndromes of traditional Chinese medicine (TCM) in amnestic mild cognitive impairment (aMCI), and to provide evidence for clinical syndrome differentiation treatment. METHODS: Six hundred subjects from Dongzhimen Hospital and seven communities in Beijing, aged between 40 and 85 years, accepted neuropsychological assessments, imaging and biochemical examinations, and syndrome differentiation, from whom 159 aMCI patients, 213 normal control (NC) subjects and 171 Alzheimer's dementia (AD) patients were screened out. Correlation between the cognitive functions and TCM syndromes in aMCI patients was analyzed. RESULTS: Mini-Mental State Examination (MMSE) score in aMCI patients was closely correlated with kidney essence vacuity and deficiency of blood and qi (r = -0.11, r = -0.11; P = 0.003, P = 0.015). Delayed Word Recall (DWR) score was correlated with kidney essence vacuity (r = -0.20, P = 0.020). Instant Story Recall (ISR) and Delayed Story Recall (DSR) scores were respectively correlated with turbid phlegm blocking upper orifices (r = -0.11, r = -0.27; P = 0.021, P = 0.000). Language function was correlated with kidney essence vacuity and deficiency of blood and qi (r = -0.11, r = -0.13; P = 0.042, P = 0.007). Attention/calculation was also closely correlated with kidney essence vacuity and deficiency of blood and qi (r = -0.10, r = -0.21; P = 0.039, P = 0.010). Attention score of aMCI patients was correlated with excess of heat toxin syndrome (r = -0.29, P = 0.026). CONCLUSION: The memory decline of aMCI is correlated with kidney essence vacuity and turbid phlegm blocking upper orifices. Furthermore, turbid phlegm blocking upper orifices is correlated with episodic memory decline, which is closely related to AD. The aMCI patients with phlegm have the risk to progress into AD. Although other cognitive functions of aMCI remain relatively intact, the patients' language function, attention/calculation and the whole cognitive function may be worsen as the aggravation of kidney essence vacuity, deficiency of blood and qi, phlegm and heat toxin, and may eventually lead to multiple cognitive domains impairment, even dementia.