Conditional knockout of the NSD2 gene in mouse intestinal epithelial cells inhibits colorectal cancer progression.

BACKGROUND: Nuclear receptor-binding SET domain 2 (NSD2) is a histone methyltransferase, that catalyzes dimethylation of lysine 36 of histone 3 (H3K36me2) and is associated with active transcription of a series of genes. NSD2 is overexpressed in multiple types of solid human tumors and has been proven to be related to unfavorable prognosis in several types of tumors. METHODS: We established a mouse model in which the NSD2 gene was conditionally knocked out in intestinal epithelial cells. We used azoxymethane and dextran sodium sulfate to chemically induce murine colorectal cancer. The development of colorectal tumors were investigated using post-necropsy quantification, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with wild-type (WT) control mice, NSD2fl/fl -Vil1-Cre mice exhibited significantly decreased tumor numbers, histopathological changes, and cytokine expression in colorectal tumors. CONCLUSIONS: Conditional knockout of NSD2 in intestinal epithelial cells significantly inhibits colorectal cancer progression.

Anti-apoptotic protein BCL-XL as a therapeutic vulnerability in gastric cancer.

BACKGROUND: New therapeutic targets are needed to improve the outcomes for gastric cancer (GC) patients with advanced disease. Evasion of programmed cell death (apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro-survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. METHOD: Here we explored the role of BCL2L1 and the encoded anti-apoptotic BCL-XL in GC. Using Droplet Digital PCR (ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL-XL inhibitors A1155463 and A1331852, pan-inhibitor ABT-263, and VHL-based PROTAC-BCL-XL was analyzed using (CellTiter-Glo) CTG assay in vitro. Western Blot (WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC-BCL-XL kills GC cells. Co-immunoprecipitation (Co-IP) was used to investigate the mechanism of A1331852 and ABT-263 kills GC cell lines. DDPCR, WB, and real-time PCR (RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. RESULTS: The functional assay showed that a subset of GC cell lines relies on BCL-XL for survival. In gastric cancer cell lines, BCL-XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT-263, indicating that ABT-263 is not an optimal inhibitor of BCL-XL. VHL-based PROTAC-BCL-XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time- and dose-dependent manner through the proteasome pathway. Statistical analysis showed that the BCL-XL protein level predicts the response of GC cells to BCL-XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL-XL expression. CONCLUSION: We identified BCL-XL as a promising therapeutic target in a subset of GC cases with high levels of BCL-XL protein expression. Functionally, we demonstrated that both selective BCL-XL inhibitors and VHL-based PROTAC BCL-XL can potently kill GC cells that are reliant on BCL-XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL-XL expression, but the BCL-XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL-XL-targeting compounds. Taken together, our study pinpointed BCL-XL as potential druggable target for specific subsets of GC.

Signal Amplification Pretargeted PET/Fluorescence Imaging Based on Human Serum Albumin-Encapsulated GdF3 Nanoparticles for Diagnosis of Ovarian Cancer.

Different modal imaging techniques could be complementary in tumor diagnosis. Human serum albumin (HSA)-encapsulated GdF3 nanoparticles were developed as T1 magnetic resonance imaging (MRI) contrast agents. However, no significant T1 enhancement in the tumor site of the SKOV3 human ovarian cancer xenograft tumor model was observed within 3 h after injection of tetrazine-modified GdF3@HSA NPs through small-animal MRI. After intravenous injection of 18F (or Cy7)-labeled Reppe anhydride, pretargeted positron emission tomography (PET) (near-infrared (NIR) fluorescence) imaging was used to reveal the pharmacokinetics of GdF3@HSA NPs in the SKOV3 xenograft mouse model to locate the tumor. The probe based on Reppe anhydride achieved rapid ligation with tetrazine-modified GdF3@HSA nanoparticles (NPs), which accumulated in tumor through Reppe anhydride/tetrazine bioorthogonal chemistry. This pretargeting strategy enabled excellent tumor visualization and quantification at an early period after nanoparticle injection (3 h p.i.), while the MRI images with significant T1 enhancement could be obtained until 24 h after injection of Gd-based contrast agents only. In vivo pretargeted multimodal imaging based on the tetrazine/Reppe anhydride system using HSA-encapsulated GdF3 nanoparticles would be beneficial for amplification of the imaging signal in the disease site and enhancing diagnostic efficiency.

Loss of endothelial EMCN drives tumor lung metastasis through the premetastatic niche.

BACKGROUND: Metastasis is the primary cause of cancer-related mortality. Metastasis involves a complex multistep process during which individual tumor cells spread primarily through destruction of the endothelial barrier, entering the circulatory system to colonize distant organs. However, the role of the endothelial barrier as the rate-limiting process in tumor metastasis and how these processes affect the regulation of the host microenvironment at the molecular level are poorly understood. METHODS: Here, we analyzed differentially expressed genes in breast cancer and lung adenocarcinoma, including metastatic and recurrent specimens, using TCGA dataset. The effects of EMCN on endothelial cells in vitro and in vivo were analyzed by assessing angiogenesis and vascular permeability, respectively. We established a syngeneic mouse model of endothelial cell-specific knockout of EMCN (EMCNecko) to study the role of EMCN in tumor growth and metastasis. Transcriptome sequencing, Western blotting, qPCR and immunofluorescence confirmed important factors in the premetastatic niche. A mouse model of allograft tumor resection with lung metastasis was established to confirm the therapeutic effect of a notch inhibitor combined with an anti-TGF-ß antibody. RESULTS: We found a strong correlation of EMCN deficiency with tumor recurrence and metastasis. Comparative experiments in WT and EMCNecko mice revealed that endothelial EMCN deficiency did not affect primary tumor growth significantly but strongly promoted spontaneous metastasis. EMCN deficiency was associated with gene profiles that regulate cell junctions in vitro and enhance vascular permeability in vivo. Mechanistically, EMCN deficiency mainly affected the host microenvironment and led to the formation of a lung premetastatic niche by recruiting Ly6G+ neutrophils and upregulating MMP9, S100A8/A9 and TGF-ß expression. Anti-TGF-ß antibody effectively eliminated TGF-ß-induced neutrophil polarization, thereby reducing lung metastasis. Notably, the combination of a Notch inhibitor and an anti-TGF-ß antibody effectively inhibited tumor growth and lung metastasis and prolonged the survival time of mice. CONCLUSIONS: We present a new translational strategy of EMCN as a new key player in tumor lung metastasis by affecting the host microenvironment. These findings could provide a sound theoretical basis for clinical treatment.

Heterogeneous nuclear ribonucleoprotein K promotes the progression of lung cancer by inhibiting the p53-dependent signaling pathway.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein. Reportedly, hnRNPK is overexpressed in many human tumors, and such overexpression is associated with poor prognosis, implicating the role of hnRNPK as an oncogene during tumorigenesis. In this study, hnRNPK expression in lung cancer tissues was investigated. METHODS: Briefly, hnRNPK was knocked down in lung cancer cell lines, and effects of knockdown on the cell proliferation, migration, and cell cycle were assessed using a cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assay and flow cytometry. The effects of hnRNPK knockdown on the p53-dependent signaling pathway were examined using western blotting. Finally, the effect of hnRNPK knockdown on tumor growth was verified in vivo using a lung cancer xenograft mouse model. RESULTS: hnRNPK knockdown inhibited the cell proliferation, migration and cell cycle. In addition to phenotypic changes, hnRNPK knockdown upregulated expressions of pCHK1, pCHK2, and p53，p21，cyclin D1, thereby mediating the DNA damage response (DDR). The regulatory function of hnRNPK during p53/p21/cyclin D1 signaling in hnRNPK-knockdown A549 cells was confirmed by suppressed the protein expression of associated signaling pathways, which inhibited DDR. CONCLUSION: hnRNPK plays a crucial role in the progression of lung cancer, ultimately affecting survival rate. Inhibition of progression of lung cancer cells induced by hnRNPK-knockdown is dependent on activation of p53 by the p53/p21/cyclin D1 pathway.

Real-time in vivo imaging reveals specific nanoparticle target binding in a syngeneic glioma mouse model.

Nanomaterial-based drug delivery is a promising strategy for glioma treatment. However, the detailed dynamics of nanoparticles in solid glioma are still a mystery, including their intratumoral infiltration depth, penetration, retention time, and distribution. Revealing these processes in detail requires repeated intravital imaging of the corresponding brain tumor regions over time during glioma growth. Hereby, we established a syngeneic orthotopic cerebral glioma mouse model by combining the chronic cranial window and two-photon microscopy. Thus, we were able to investigate the dynamics of the nanoparticles during long-term glioma growth. Three hours after the intravenous (i.v.) injection of integrin αVß3 binding conjugated silicon nanoparticles (SNPs-PEG-RGD-FITC), green nanoparticles had already infiltrated the brain glioma, and then more nanoparticles penetrated into the solid brain tumor and were retained for at least 8 days. However, the amount of control SNPs-PEG-FITC that infiltrated into the solid brain tumor was very low. Moreover, we found that SNPs-PEG-RGD-FITC were not only located in the tumor border but could also infiltrate into the core region of the solid tumor. In vitro assay also confirmed the high binding affinity between GL-261-Tdtomato cells and SNPs-PEG-RGD-FITC. Our results indicate that SNPs-PEG-RGD-FITC has high penetration and retention in a solid glioma and our model provides novel ideas for the investigation of nanoparticle dynamics in brain tumors.

Uncovering the interplay between pH receptors and immune cells: Potential drug targets (Review).

Extracellular acidosis is associated with various immunopathological states. The microenvironment of numerous solid tumours and inflammatory responses during acute or chronic infection are all related to a pH range of 5.5­7.0. The relationship between inflammation and immune escape, cancer metabolism, and immunologic suppression drives researchers to focus on the effects of low pH on diverse components of disease immune monitoring. The potential effect of low extracellular pH on the immune function reveals the importance of pH in inflammatory and immunoreactive processes. In this review, the mechanism of how pH receptors, including monocarboxylate transporters (MCTs), Na+/H+ exchanger 1, carbonic anhydrases (CAs), vacuolar­ATPase, and proton­sensing G­protein coupled receptors (GPCRs), modulate the immune system in disease, especially in cancer, were studied. Their role in immunocyte growth and signal transduction as part of the immune response, as well as cytokine production, have been documented in great detail. Currently, immunotherapy strategies have positive therapeutic effects for patients. However, the acidic microenvironment may block the effect of immunotherapy through compensatory feedback mechanisms, leading to drug resistance. Therefore, we highlight promising therapeutic developments regarding pH manipulation and provide a framework for future research.

Inhibition of host Ogr1 enhances effector CD8+ T-cell function by modulating acidic microenvironment.

Immunotherapies for cancer, such as immune checkpoint blockade or adoptive T-cell transfer, can lead to a long-lasting clinical response. But the therapeutic response rate remains low on account of many tumors that have evolved sophisticated strategies to evade immune surveillance. Solid tumors are characterized by the highly acidic microenvironment, which may weaken the effectiveness of antitumor immunity. Here, we explored a promising therapeutic development deployed by pH manipulation for avoiding immunoevasion. The highly acidified microenvironment of melanoma induces the expression of G-protein-coupled receptor (Ogr1) in T cells, which weakened their effective function and promote tumor growth. Ogr1 inhibition reactivate CD8+ T cells and have a cytotoxic role by reducing the activity of high glycolysis, resulting in comparatively low acidification of the tumor microenvironment, and leads to tumor suppression. In addition, the adoptive transfer of Ogr1-/--CD8+ T cells enhanced the antitumor responses, with the potential for immediate clinical transformation.

Novel spontaneous myelodysplastic syndrome mouse model.

Background: Myelodysplastic syndrome (MDS) is a group of disorders involving hemopoietic dysfunction leading to leukemia. Although recently progress has been made in identifying underlying genetic mutations, many questions still remain. Animal models of MDS have been produced by introduction of specific mutations. However, there is no spontaneous mouse model of MDS, and an animal model to simulate natural MDS pathogenesis is urgently needed. Methods: In characterizing the genetically diverse mouse strains of the Collaborative Cross (CC) we observed that one, designated JUN, had abnormal hematological traits. This strain was thus further analyzed for phenotypic and pathological identification, comparing the changes in each cell population in peripheral blood and in bone marrow. Results: In a specific-pathogen free environment, mice of the JUN strain are relatively thin, with healthy appearance. However, in a conventional environment, they become lethargic, develop wrinkled yellow hair, have loose and light stools, and are prone to infections. We found that the mice were cytopenic, which was due to abnormal differentiation of multipotent bone marrow progenitor cells. These are common characteristics of MDS. Conclusions: A mouse strain, JUN, was found displaying spontaneous myelodysplastic syndrome. This strain has the advantage over existing models in that it develops MDS spontaneously and is more similar to human MDS than genetically modified mouse models. JUN mice will be an important tool for pathogenesis research of MDS and for evaluation of new drugs and treatments.

Summary of animal models of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a malignant tumor of the hematological system characterized by long-term, progressive refractory hemocytopenia. In addition, the risk of leukemia is high, and once it develops, the course of acute leukemia is short with poor curative effect. Animal models are powerful tools for studying human diseases and are highly effective preclinical platforms. Animal models of MDS can accurately show genetic aberrations and hematopoietic clone phenotypes with similar cellular features (such as impaired differentiation and increased apoptosis), and symptoms can be used to assess existing treatments. Animal models are also helpful for understanding the pathogenesis of MDS and its relationship with acute leukemia, which helps with the identification of candidate genes related to the MDS phenotype. This review summarizes the current status of animal models used to research myelodysplastic syndrome (MDS).

Involvement of the Wnt/ß-Catenin signaling pathway in the heterogenous nuclear ribonucleoprotein K-driven inhibition of proliferation and migration in head and neck squamous cell carcinoma.

The abnormal upregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) expression levels were reported to be involved in the progression of various types of cancer. Therefore, it is hypothesized that hnRNP K may serve as a useful diagnostic marker and antitumor target; however, only a few studies to date have investigated the exact role of hnRNP K in head and neck squamous cell carcinoma (HNSCC) and the potential downstream signaling pathway involved. The present study aimed to identify the roles of hnRNP K in the proliferation and migration of HNSCC, and the possible signaling pathways hnRNP K may be associated with in HNSCC. hnRNP K expression levels in clinical HNSCC samples were analyzed using the Oncomine and UALCAN databases, and its association with the survival of patients with HNSCC was analyzed using the tumor-immune system interactions database. Short hairpin RNA targeting hnRNP K was transfected into the CAL-27 cell line to establish HNSCC cells with stable hnRNP K-knockdown. Cell viability was analyzed using a Cell Counting Kit-8 assay and an absolute count assay, and cell proliferation was measured using 5-ethynyl-2'-deoxyuridine incorporation and colony formation assays. Migratory ability of cells was analyzed using wound healing assay and transwell assay. The growth of xenografts derived from hnRNP K-knockdown cells was also evaluated, and bioinformatics analyses were performed using the Gene Ontology and Kyoto Encyclopedia for Genes and Genomes databases to determine the possible downstream signaling pathways of hnRNP K. Furthermore, the status of the Wnt/ß-Catenin signaling pathway in hnRNP K-knockdown cells mediated by small interfering RNA was determined using reverse transcription-quantitative PCR and western blotting. The results revealed that the expression levels of hnRNP K were upregulated in HNSCC cell lines and tissues. Moreover, the upregulation of hnRNP K expression levels was associated with poor survival of patients with HNSCC. The knockdown of hnRNP K also decreased HNSCC cell proliferation and migration, and inhibited tumor growth in nude mice. Bioinformatics analyses identified the Wnt/ß-Catenin signaling pathway as a possible downstream signaling pathway of hnRNP K. Knockdown of hnRNP K significantly downregulated the expression levels of Wnt/ß-Catenin signaling pathway-related proteins; while with knockdown of hnRNP K and overexpression of ß-Catenin, the expression levels of Wnt/ß-Catenin signaling pathway-related proteins were partially rescued. In conclusion, the present findings indicated that hnRNP K may serve as a candidate diagnostic biomarker and a promising therapeutic target for HNSCC.

Research progress on the structure and function of endomucin.

Endomucin is a type I integral membrane glycoprotein, which is expressed in venous and capillary endothelial cells. It consists of 261 amino acids with an extracellular domain that is highly O-glycosylated at serine and threonine residues and has several potential N-glycosylation sites. Endomucin plays an important role in biological processes such as cell interaction, molecular cell signaling, angiogenesis and cell migration, and in recent years it has also been identified as an anti-adhesion molecule and a marker of endothelial cells. While it has been shown to be involved in a number of physiological and pathological mechanisms, many of its functions remain unknown, and further study is needed. This article reviews research progress on the function of endomucin to date, in order to provide guidance for future studies.

Downregulation of HNRNPK in human cancer cells inhibits lung metastasis.

BACKGROUND: Lung cancer frequently occurs in the clinic, leading to poor prognosis and high mortality. Markers for early diagnosis of lung cancer are scarce, and further potential therapeutic targets are also urgently needed. METHOD: We established a new mouse model in which the specific gene HNRNPK (heterogeneous nuclear ribonucleoprotein K) was downregulated after administration of doxycycline. The lung metastatic nodules were investigated using bioluminescence imaging, micro-CT, and autopsy quantification. RESULTS: Compared with the short hairpin negative control group, less lung metastatic nodules were formed in the short hairpin RNA group. CONCLUSION: Downregulation of HNRNPK in cancer cells can inhibit lung metastasis.

HNRNPK inhibits gastric cancer cell proliferation through p53/p21/CCND1 pathway.

Gastric cancer (GC) is one of the most common human cancers. The molecular mechanisms underlying GC carcinogenesis and progression are still not well understood. In this study, we showed that heterogeneous nuclear ribonucleoprotein K (HNRNPK) was an effective prognostic marker for GC patients especially in early stage. Overexpression of HNRNPK can retard tumor cell proliferation and colony formation in vitro and inhibit tumor growth in vivo through p53/p21/CCND1 axis. Bioinformatics analyses indicated that HNRNPK associated genes were enriched in cell cycle and DNA replication process. Protein-protein interaction network showed that HNRNPK was physically interacted with p53, p21 and other cancer related genes. Besides, GSEA showed that HNRNPK expression was positively correlated with GAMMA radiation response and DNA repair, while negatively correlated with angiogenesis, TGF-ß and Hedgehog pathway activation. Finally, several chemicals including Glycine that may repress GC progression through upregulating HNRNPK are suggested. Our study demonstrated that HNRNPK may play as a tumor suppressor in gastric cancer and could be a potential therapeutic target for GC.

Smurf1 targets Securin for ubiquitin-dependent degradation and regulates the metaphase-to-anaphase transition.

The HECT E3 ligase Smurf1 (Smad ubiquitination regulatory factor 1) plays a critical role in several important biological pathways by targeting many proteins for ubiquitination and degradation, such as Smad1/5, MEKK2 and RhoA. However, the function of Smurf1 in metaphase-to-anaphase transition remains unclear. Here, we show that Smurf1 interacts with and targets Securin, an inhibitor of sister-chromatid separation, for poly-ubiquitination and proteasomal degradation. Further results demonstrate that Securin is a physiological substrate of Smurf1 in MEF cells. Knockdown of Smurf1 results in sister-chromatid separation inhibition and delay of anaphase onset. This study provides the first evidence that Smurf1 functions as a novel regulator for the metaphase-to-anaphase transition.

Smurf1 controls S phase progression and tumorigenesis through Wee1 degradation.

Smad ubiquitination regulatory factor 1 (Smurf1) is a HECT-type E3 ubiquitin ligase that regulates several important signaling pathways, including the bone morphogenetic protein pathway and the transforming growth factor-beta (TGF-ß) signaling pathway. However, the function of Smurf1 in cell cycle progression remains unclear. Here, we demonstrate that silencing of Smurf1 results in S phase arrest, confirming that Smurf1 is required for S phase progression. Furthermore, we demonstrate that Smurf1-mediated S phase progression is largely dependent on the ubiquitination-dependent degradation of Wee1. This study defines a novel role for Smurf1 in controlling S phase progression by promoting Wee1 degradation.

Research Advances in the Mechanisms of Gastric Cancer Multidrug Resistance.

Gastric cancer is one of the most common human malignancies and the third cause of death from cancer in China and worldwide. Chemotherapy is still one of the major treatment options for advanced gastric cancer. However,the efficacy of chemotherapy for gastric cancer remains poor due to its insensitivity and the development of multidrug resistance (MDR). While many molecules and mechanisms have been found to be associated with the development of gastric cancer MDR,the specific mechanisms remains unclear. In our current article,we reviews the identification of MDR-related molecules and mechanisms,with an attempt to a better understand the specific mechanisms of gastric cancer MDR and thus provide new insights into the fight against gastric cancer MDR.