Role In Cell Invasion Research Articles

Engineered TIMP2 with narrow MMP-9 specificity is an effective inhibitor of invasion and proliferation of triple-negative breast cancer cells

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix (ECM) proteins, functioning in various physiological processes such as tissue remodeling, embryogenesis, and morphogenesis. Dysregulation of these enzymes is linked to multiple diseases. Specific inhibition of particular MMPs is crucial for anti-MMP drug development as some MMPs have shown anti-disease properties. In this study, we aimed to design a highly specific inhibitor of MMP-9, that plays a crucial role in cell invasion and metastasis, using tissue inhibitor of metalloproteinases 2 (TIMP2), an endogenous broad-family MMP inhibitor, as a prototype. In our earlier work, we were able to narrow down the specificity of the N-terminal domain of TIMP2 (N-TIMP2) toward MMP-9, yet at the expense of lowering its affinity to MMP-9. In this study, a library of N-TIMP2 mutants based on previous design with randomized additional positions was sorted for binding to MMP-9 using yeast surface display. Two selected N-TIMP2 mutants were expressed, purified and their inhibitory activity against a panel of MMPs was measured. The best engineered N-TIMP2 mutant (REY) exhibited a 2-fold higher affinity to MMP-9 compared to that of the WT N-TIMP2, and 6- to 1.1x104-fold increase in binding specificity toward MMP-9 compared to five alternative MMPs. Moreover, REY demonstrated a significant increase in inhibition of cell invasion and proliferation compared to the WT N-TIMP2 in MDA-MB-231 breast cancer cells. Therefore, our engineered N-TIMP2 mutant emerges as a promising candidate for future therapeutic development, offering precise targeting of MMP-9 in MMP-9-driven diseases.

Expression profile and function of secretogranin V, and its effects on the malignant behavior of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is recognized as one of the most aggressive cancers with a poor prognosis. Global expression profiling was conducted on primary ESCC tissues with distant metastases. We investigated the identification of secretogranin V (SCG5) as a promising biomarker for the detection and assessment of ESCC. SCG5 transcription levels were evaluated in 21 ESCC cell lines. Small interfering RNA-mediated knockdown experiments validated SCG5's roles in cell invasion, proliferation, and migration. We utilized a mouse subcutaneous xenograft model to assess tumor growth. SCG5 expression was measured in 164 ESCC tissues by quantitative reverse transcription quantitative polymerase chain reaction, and its association with clinicopathological parameters was investigated. SCG5 protein levels were assessed in surgically resected tissues from 177 patients with ESCC using a tissue microarray. The mRNA expression levels of SCG5 varied widely in ESCC cell lines. The in vitro cell invasion, proliferation, and migration of ESCC cells were suppressed by the knockdown of SCG5. Mouse xenograft models revealed that tumor growth was reduced by small interfering RNA-mediated SCG5 knockdown. Analysis of clinical samples demonstrated that SCG5 mRNA was expressed in ESCC compared to adjacent normal esophageal tissues. High SCG5 mRNA expression was linked to significant decreases in overall and disease-specific survival. Furthermore, SCG5 protein expression was linked to a decrease in disease-specific survival and disease-free survival. The expression of the SCG5 was significantly associated with disease-specific survival, suggesting that SCG5 may play a significant role as a diagnostic and prognostic biomarker for ESCC.

Marine derived macrolide bryostatin 4 inhibits the TGF-β signaling pathway against acute erythroleukemia.

Acute erythroleukemia (AEL) is a rare and highly aggressive subtype of acute myeloid leukemia (AML) with an extremely poor prognosis when treated with available drugs. Therefore, new investigational agents capable of inducing remission are urgently required. Bioinformatics analysis, western blot and qRT-PCR were used to reveal the potential biological mechanism of bryostatin 4 (B4), an antineoplastic macrolide derived from the marine bryozoan Bugula neritina. Then, in vivo experiments were conducted to evaluate the role of transforming growth factor (TGF)-β signaling in the progression of AEL. Our results revealed that the proliferation of K562 cells and TF-1 cells was significantly inhibited by B4 at IC50 values of 37 nM and 52 nM, respectively. B4 inhibited TGF-β signaling and its downstream pathway targets, particularly the phosphorylation of Smad2, Smad3, Ras, C-RAF, ERK1/2, and MEK. B4 also played an important role in cell invasion and migration in K562 cells and TF-1 cells by reducing the protein levels of the mesenchymal cell marker vimentin. Moreover, Flow cytometry and western blot analyses demonstrated that B4 induced apoptosis and initiated G0/G1 phase arrest by modulating mitochondrial dysfunction and cyclin-dependent kinase (CDK) expression. These findings indicated that B4 could inhibit the proliferation, migration, invasion, and TGF-β signaling pathways of AEL cells, thus suggesting that B4 possesses therapeutic potential as a treatment for AEL.

KIF1C and new Huntingtin-interacting protein 1 binding proteins regulate rheumatoid arthritis fibroblast-like synoviocytes' phenotypes.

Huntingtin-interacting protein-1 (HIP1) is a new arthritis severity gene implicated in the regulation of the invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). These invasive properties of FLS strongly correlate with radiographic and histology damage in patients with RA and rodent models of arthritis. While HIP1 has several intracellular functions, little is known about its binding proteins, and identifying them has the potential to expand our understanding of its role in cell invasion and other disease-contributing phenotypes, and potentially identify new targets for therapy. FLS cell lines from arthritic DA (highly invasive) and from arthritis-protected congenic rats R6 (minimally invasive), which differ in an amino-acid changing HIP1 SNP, were cultured and lysed, and proteins were immunoprecipitated with an anti-HIP1 antibody. Immunoprecipitates were analyzed by mass spectrometry. Differentially detected (bound) proteins were selected for functional experiments using siRNA knockdown in human RA FLS to examine their effect in cell invasiveness, adhesion, cell migration and proliferation, and immunofluorescence microscopy. Proteins detected included a few known HIP1-binding proteins and several new ones. Forty-five proteins differed in levels detected in the DA versus R6 congenic mass spectrometry analyses. Thirty-two of these proteins were knocked down and studied in vitro, with 10 inducing significant changes in RA FLS phenotypes. Specifically, knockdown of five HIP1-binding protein genes (CHMP4BL1, COPE, KIF1C, YWHAG, and YWHAH) significantly decreased FLS invasiveness. Knockdown of KIF1C also reduced RA FLS migration. The binding of four selected proteins to human HIP1 was confirmed. KIF1C colocalized with lamellipodia, and its knockdown prevented RA FLS from developing an elongated morphology with thick linearized actin fibers or forming polarized lamellipodia, all required for cell mobility and invasion. Unlike HIP1, KIF1C knockdown did not affect Rac1 signaling. We have identified new HIP1-binding proteins and demonstrate that 10 of them regulate key FLS phenotypes. These HIP1-binding proteins have the potential to become new therapeutic targets and help better understand the RA FLS pathogenic behavior. KIF1C knockdown recapitulated the morphologic changes previously seen in the absence of HIP1, but did not affect the same cell signaling pathway, suggesting involvement in the regulation of different processes.

Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2.

Convergent QSAR Models for the Prediction of Cruzain Inhibitors.

Chagas disease is a parasitosis caused by Trypanosoma cruzi. Cruzain, the major cysteine protease from T. cruzi, is an excellent therapeutic target in the search for antichagasic drugs. It is important in the role of cell invasion, replication, differentiation, and metabolism of the parasite. In this work, we developed and assessed multiple quantitative structure-activity relationship (QSAR) models for a set of 61 cruzain inhibitors. These models include two-dimensional (2D) QSAR, three-dimensional (3D) QSAR, such as comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), and Hologram QSAR (HQSAR). In total, we generated 10 major and 114 minor model variations. Molecular docking was used to successfully align the molecules. All CoMFA and CoMSIA models, which incorporate multiple fields, demonstrated robustness in our analysis. Steric fields exhibited satisfactory convergence in the contour maps, while the electrostatic field converged into a single small region. The HQSAR model taking into consideration only Atoms and Connectivity, with fragment sizes ranging from two to five atoms, was considered the best of the HQSAR variations, despite exhibiting a higher level of deviance. In total, 78 model variations meet the minimum requirements to be considered acceptable. We found that using as few as five descriptors it is possible to obtain robust results with 2D-QSAR. Models such as Random Forest, Tree Ensemble, Linear Regression, and HQSAR are recommended for working with large data sets, while the 3D-QSAR models are intended to study the geometry of the ligands, to optimize them into new and better performing antichagasics. Virtual Screening of a set of hydrazones, guided by the top-performing models, identified promising candidates for experimental validation. Among them, dv007 and dv015 exhibited consistently high predicted pIC50 values (7.26 and 7.24, respectively), making them compelling candidates for further drug development.

Expression of mammalian cell entry genes in clinical isolates of M. tuberculosis and the cell entry potential and immunological reactivity of the Rv0590A protein.

Mammalian cell entry (mce) operons play a vital role in cell invasion and survival of M. tuberculosis. Of the mce genes, the function of Rv0590A is still unknown. The present study was performed to investigate the function and immunogenic properties of the protein Rv0590A. Human leukemia monocytic cell line (THP-1) derived macrophages were infected with M. tuberculosis H37Rv at 3, 6, and 24h of infection. The maximum colony forming units (CFU) were observed at 6h (p < 0.005), followed by 3h after infection. M. tuberculosis H37Rv and clinical isolates representative of Delhi/CAS, EAI, Beijing, Haarlem and Euro-American-superlineage were included in the study for expression analysis of mce1A, mce2A, mce3A, mce4A, and Rv0590A genes. Maximum upregulation of all mce genes was observed at 3h of infection. All the five clinical isolates and H37Rv upregulated Rv0590A at various time points. Macrophage infection with M. tuberculosis H37Rv-overexpressing Rv0590A gene showed higher intracellular CFU as compared to that of wild-type H37Rv. Further, purified Rv0590A protein stimulated the production of TNFα, IFNγ, and IL-10 in macrophages. Thus, Rv0590A was found to be involved in cell invasion and showed good immunological response.

Luteolin-Zn Complex Inhibits Invasion and Migration of M2-Like TAMs via the Downregulation of AMPK/mTOR and PI3K/Akt/mTOR Signaling Pathway Under Hypoxia

Purpose: The high mortality rate of malignant tumors is often attributable to the loss of surgical opportunities due to late diagnosis when invasion and metastasis have significantly affected the patient. A hypoxic microenvironment can promote the progression of malignant tumors. This study explored the invasion resistance and migration ability of luteolin-Zn complexes. Methods: We created a low-oxygen environment using a 3-atmosphere incubator. The appropriate drug concentration was determined using the CCK8 experiment. We determined its role in cell invasion and migration through scratch and transwell experiments. Western blotting, polymerase chain reaction, and cellular immunity experiments were used to study the mechanism and its impact on the secretion of invasion and migration factors. Results: Our results indicated that the luteolin-Zn complex significantly reduced MMP2, MMP9, N-Ca, and HIF-1ɑ expression. It also upregulated TIMP1 and E-Ca expression. Moreover, its capabilities may be achieved by regulating the AMPK/mTOR and PI3K/Akt/mTOR signaling pathways. Conclusions: The luteolin-Zn complex was highly resistant to the invasion and migration of M2-like tumor-related macrophages. This may exert a unique influence on mTOR by integrating various signals. This study suggests that the luteolin-Zn complex has a strong anticancer effect under hypoxic conditions.

Pleckstrin-2-promoted PPM1B degradation plays an important role in transforming growth factor-β-induced breast cancer cell invasion and metastasis.

Transforming growth factor-β (TGF-β) is known to promote breast cancer cell migration, invasion, and dissemination; however, the underlying molecular mechanisms are not yet well characterized. Here, we report that TGF-β induces pleckstrin-2 (PLEK2) expression by Smad3 and signal transducer and activator of transcription 3 (STAT3) activating PLEK2 promoter activity. Higher PLEK2 expression is associated with poor prognosis in breast cancer patients. Overexpression and knockout experiments in MDA-MB-231 and MCF-7 breast cancer cells revealed that PLEK2 promotes cell migration, invasion, and dissemination in 2D and 3D cell culture. Moreover, PLEK2 promotes metastasis of breast cancer cells in vivo. Pleckstrin-2 localizes to the cell membrane and cell protrusions following TGF-β treatment. Furthermore, inhibition of PI3K phosphorylation abolishes TGF-β- and PLEK2-induced cell invasion. The carboxyl-terminal PH domain of PLEK2 is critical for TGF-β- and PLEK2-induced Akt activation and plays an important role in cell invasion. Pleckstrin-2 interacts with PPM1B and promotes its ubiquitin-dependent degradation. The PLEK2-PPM1B axis utilizes nuclear factor-κB signaling to promote cell migration and invasion. Our data implicate the TGF-β-STAT3/Smad3-PLEK2-PPM1B signaling cascade in TGF-β-induced breast cancer cell migration and invasion. These findings suggest that PLEK2/PPM1B could represent novel targets for the intervention of breast cancer metastasis.

Discovery of AXL Degraders with Improved Potencies in Triple-Negative Breast Cancer (TNBC) Cells.

AXL kinase is heavily involved in tumorigenesis, metastasis, and drug resistance of many cancers, and several AXL inhibitors are in clinical investigations. Recent studies demonstrated that the N-terminal distal region of AXL plays more important roles in cell invasiveness than its C-terminal kinase domain. Therefore, degradation of AXL may present a novel superior therapeutic approach than the kinase inhibitor therapy. Herein, we report the discovery of a series of new AXL PROTAC degraders. One representative compound 6n potently depletes AXL with a DC50 value of 5 nM in MDA-MB-231 TNBC cells. It also demonstrates significantly improved potencies against the AXL signaling activation, cell proliferation, migration and invasion of TNBC cells comparing with the corresponding kinase inhibitor. Moreover, the compound exhibits promising therapeutic potential both in patient-derived organoids and a xenograft mouse model of MDA-MB-231 cells.

(-)-Epigallocatechin-3-Gallate Prevents IL-1β-Induced uPAR Expression and Invasiveness via the Suppression of NF-κB and AP-1 in Human Bladder Cancer Cells.

(-)-Epigallocatechin-3-O-gallate (EGCG), a primary green tea polyphenol, has powerful iron scavengers, belongs to the family of flavonoids with antioxidant properties, and can be used to prevent cancer. Urokinase-type plasminogen activator receptors (uPARs) are glycosylphosphatidylinositol (GPI)-anchored cell membrane receptors that have crucial roles in cell invasion and metastasis of several cancers including bladder cancer. The mechanism of action of EGCG on uPAR expression has not been reported clearly yet. In this study, we investigated the effect of EGCG on interleukin (IL)-1β-induced cell invasion and uPAR activity in T24 human bladder cancer cells. Interestingly, nuclear factor (NF)-κB and activator protein (AP)-1 transcription factors were critically required for IL-1β-induced high uPAR expression, and EGCG suppressed the transcriptional activity of both the ERK1/2 and JNK signaling pathways with the AP-1 subunit c-Jun. EGCG blocked the IL-1β-stimulated reactive oxygen species (ROS) production, in turn suppressing NF-κB signaling and anti-invasion effects by inhibiting uPAR expression. These results suggest that EGCG may exert at least part of its anticancer effect by controlling uPAR expression through the suppression of ERK1/2, JNK, AP-1, and NF-κB.

Ultrasensitive Fibroblast Activation Protein-α-Activated Fluorogenic Probe Enables Selective Imaging and Killing of Melanoma In Vivo.

Melanoma is a malignant cancer with a high risk of metastasis and continued increase in death rates over the past decades, and its prognosis is highly related to the disease's stage, while early detection and treatment of melanoma are significant to the improvement of its therapy outcome. Different from the traditional methods for disease diagnosis, enzyme-activated fluorescent probes were developed rapidly due to their high sensitivity and temporal-spatial ratio and have been widely applied in tumor detection, surgical navigation, and cancer-related research. Fibroblast activation protein-α (FAPα), a serine-type cell surface protease that plays important roles in cell invasion and extracellular matrix degradation, is widely involved in tumor progression such as malignant melanoma, so developing a FAPα activity-based molecular tool would be of great potential for the early diagnosis and therapy of melanoma. However, few fluorescent probes targeting FAPα have been applied in melanoma-related studies, and thus, the construction of FAPα activity-based fluorescent probes for melanoma detection is in urgent need. By incorporating the selective recognition unit with a red-emission fluorophore, cresyl violet, we herein report an ultrasensitive (limit of detection = 5.3 ng/mL) fluorogenic probe for FAPα activity sensing, named CV-FAP; the acquired probe showed a significantly higher binding affinity (15.7-fold) and overall catalytic efficiency (2.6-fold) when compared with those of the best reported FAPα probes. The good performance of CV-FAP made it possible to discriminate malignant melanoma cells and tumor-bearing mice from normal cells and mice with high contrast. More importantly, CV-FAP showed significant antitumor activity toward melanoma in cultured cells and tumor-bearing nude mice (over 95% inhibited tumor growth) with good safety, which made it an ideal theranostic agent for melanoma.

Abstract 179: Tumor - driven extracellular matrix remodeling influences therapeutic response

Abstract Brain cancers remain some of the most challenging tumors to treat. The goal of our research is to use a three-dimensional in vitro model to acquire in-depth understanding of the unique environment of brain tumors to find new biomarkers of disease and develop more efficient therapeutic approaches. We are interested in revealing potential associations between common genetic tumor mutations and the capacity to remodel the extracellular microenvironment. The understanding of the multiple sources of tumor heterogeneity is crucial to improve current therapeutic approaches. Glioblastomas (GBM) present a very poor prognosis, they exhibit high infiltration into brain parenchyma, impairing surgical resection and leading to recurrence. Brain extracellular matrix (ECM) plays a key role in glioblastoma invasion and therapeutic resistance. In particular, the aberrant biosynthesis of the main component of brain ECM, hyaluronic acid (HA), has been associated to pathological conditions (1). In this study, we investigate the influence of tumor extracellular HA in glioblastoma progression and resistance to a targeted tyrosine kinase inhibitor (erlotinib). We have established engineered brain tumor biomaterials based on functionalized gelatin hydrogels decorated with covalently bound HA. This biomaterial approach can monitor the response of patient-derived xenograft cell populations with different molecular signatures in combination with microfluidic devices (2, 3). We have found that the combinatorial treatment of anti-CD44 and erlotinib show synergistic effect in some EGFR overexpressing tumors containing HA (2). Moreover, we can investigate the signaling mechanisms associated to these phenotypic alterations. Different signaling pathways are altered in the tumor cells depending on the microenvironment as they respond to an EGFR and CD44 inhibitor. While the decrease in invasion and proliferation in gelatin-only matrices comes from the negative regulation of ERK pathway, HA plays a favorable role in the inhibition of EGFR, through STAT3 deactivation (4). The MW and abundance of HA are mediated by the activity of HA synthases and hyaluronidases. HAS2 can produce very high molecular weight species and seems to play a significant role in cell invasion and cancer progression. In these HA-rich tumors we find a heterogeneous profile of MW distributions secreted by different tumor types, additionally we learnt that EGFR inhibition led to the secretion of high MW HA, associated to the upregulation of HAS2. We reveal here the importance of the tumor-associated HA biosynthesis as a mechanism of cancer cells to adapt to drug insults. The ability to manipulate tumor ECM can improve therapeutic outcomes and restrict glioblastoma growth and infiltration. (1) Monslow J. Front. Immunol. 2015; 6:231.(2) Pedron S. Adv. Healthcare Mater. 2017; 6:1700529.(3) Pedron S. MRS Commun. 2017; 7:442-449.(4) Pedron S. Biomaterials 2019; 219:119371. Citation Format: Edward R. Neves, Brendan A. Harley, Sara Pedron. Tumor - driven extracellular matrix remodeling influences therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 179.

Targeting MUCL1 protein inhibits cell proliferation and EMT by deregulating β‑catenin and increases irinotecan sensitivity in colorectal cancer.

With >1.85 million cases and 850,000 deaths annually, colorectal cancer (CRC) is the third most common cancer detected globally. CRC is an aggressive malignancy with metastasis and, in spite of advances in improved treatment regimen, distant disease failure rates remain disappointingly high. Mucin-like 1 (MUCL1) is a small glycoprotein highly expressed mainly in breast cancer. The involvement of the MUCL1 protein in CRC progression and the underlying mechanism have been largely unknown. The aim of the present study was to investigate the MUCL1 expression profile and its functional significance in CRC. The Cancer Genome Atlas dataset revealed that MUCL1 expression was higher in colorectal tumor compared with normal tissues. MUCL1 was also revealed to be expressed in human CRC cell lines. The results demonstrated that MUCL1 promoted cell proliferation and colony formation, confirming its oncogenic potential. Silencing MUCL1 with short interfering RNA inhibited the protein expression of Bcl2 family proteins, such as Bcl2 and BclxL. Targeting MUCL1 resulted in significant inhibition in cell invasive and migratory behavior of HT-29 and SW620 cells. In addition, the expression of E-cadherin increased whereas the expression of vimentin decreased in MUCL1-silenced cells, confirming inhibition of epithelial-mesenchymal transition (EMT) process. Thus, it was revealed that MUCL1 plays a notable role in cell invasion and migration by inhibiting EMT in CRC. Mechanistically, MUCL1 drives β-catenin activation by Ser-552 phosphorylation, nuclear accumulation and transcriptional activation. Targeting MUCL1 increases the drug sensitivity of CRC cells towards irinotecan. These findings thus demonstrated that MUCL1 acts as a modifier of other pathways that play an important role in CRC progression and MUCL1 was identified as a potential target for CRC therapeutics.

Testicular orphan receptor 4 (TR4) promotes papillary thyroid cancer invasion via activating circ-FNLA/miR-149-5p/MMP9 signaling

The incidence and mortality of papillary thyroid cancer (PTC) have steadily increased. Although conventional therapies are very effective toward differentiated PTC patients, very limited therapeutic options are applicable to those patients with distant metastases. Therefore, better understanding of the molecular biology of metastatic PTC helps identify novel targets and facilitates the development of new therapies. In this study, we first found that testicular orphan receptor 4 (TR4) was significantly increased in PTC tumors spreading to lymph nodes compared to the paired primary tumors. Experimental evidence suggested that TR4 drove PTC progression via promoting its cell invasion and cell migration. Mechanistically, TR4 transcriptionally regulated the expression level of circ-filamin A (FLNA), which competed with matrix metalloproteinase 9 (MMP9) for microRNA (miR)-149-5p binding and led to an increased protein level of MMP9. Interruption assays with various gene manipulations verified that the TR4/circ-FLNA/miR-149-5p/MMP9 signaling axis played a central role in cell invasion and cell migration of PTC cells. Moreover, a xenografted mouse model also confirmed that the TR4/circ-FLNA signal promoted PTC tumor growth. Overall, our study pinpoints the oncogenic role of TR4 in PTC development, and the targeting of TR4/circ-FLNA/miR-149-5p/MMP9 signaling may be an alternative option for metastatic PTC patients.

Repeat-Driven Generation of Antigenic Diversity in a Major Human Pathogen,Trypanosoma cruzi.

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan parasite, is the causative agent of American trypanosomiasis (Chagas disease). Having a very plastic, repetitive and complex genome, the parasite displays a highly diverse repertoire of surface molecules, with pivotal roles in cell invasion, immune evasion and pathogenesis. Before 2016, the complexity of the genomic regions containing these genes impaired the assembly of a genome at chromosomal level, making it impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here describe the genome assembly of the Sylvio X10/1 genome sequence, which since 2016 has been used as a reference genome sequence for T. cruzi clade I (TcI), produced using high coverage PacBio single-molecule sequencing. It was used to analyze deep Illumina sequence data from 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene distribution showed the unusual duality in the organization of the parasite genome, a synteny of the core genomic region with related protozoa flanked by unique and highly plastic multigene family clusters encoding surface antigens. The presence of abundant interspersed retrotransposons in these multigene family clusters suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response on these TcI strains. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure.

Cholic Acid Stimulates MMP-9 in Human Colon Cancer Cells via Activation of MAPK, AP-1, and NF-κB Activity

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in cell invasion and cancer metastasis. In this study, we showed that cholic acid (CA), a major primary bile acid, can induce MMP-9 expression in colon cancer HT29 and SW620 cells. CA increased reactive oxygen species (ROS) production and also activated phosphorylation of ERK1/2, JNK, and p38 MAPK. Specific inhibitors and mutagenesis studies showed that ERK1/2 and JNK functioned as upstream signals in the activation of AP-1, and p38 MAPK functioned as an upstream signal in the activation of NF-κB. N-acetyl-L-cysteine (NAC, an ROS scavenger) and diphenyleneiodonium chloride (DPI, an NADPH oxidase inhibitor) inhibited CA-induced activation of ERK1/2, JNK, and p38 MAPK, indicating that ROS production by NADPH oxidase could be the furthest upstream signal in MMP-9 expression. Colon cancer cells pretreated with CA showed remarkably enhanced invasiveness. Such enhancement was partially abrogated by MMP-9-neutralizing antibodies. These results demonstrate that CA could induce MMP-9 expression via ROS-dependent ERK1/2, JNK-activated AP-1, and p38-MAPK-activated NF-κB signaling pathways, which in turn stimulate cell invasion in human colon cancer cells.

Downregulated placental expression of linc00468 contributes to trophoblast dysfunction by inducing epithelial-mesenchymal transition.

BackgroundPreeclampsia (PE) is a widespread progressive condition that can occur pregnancy and is related to high maternal morbidity and fetal mortality in the perinatal period. However, the exact mechanism responsible has not been specific. Accumulating evidence has highlighted the prominent role of the epithelial‐mesenchymal transition (EMT) in the biological behaviors of PE.MethodsWe explored the role of a lincRNA in extravillous trophoblast (EVTs) cell viability, migration, invasion and apoptosis in vitro, along with the use of linc00468 knockdown or overexpression. Clinically, we discovered that the expression of linc00468 was frequently correlated with adverse clinical features and poor prognosis of PE patients.ResultsWe uncovered that linc00468 was downregulated in PE samples compared to in healthy tissues and in trophoblast cells. Functionally, gain and loss-of-function experiments demonstrated that linc00468 inhibited cell proliferation, migration, invasion and linc00468 accelerated apoptosis of the trophoblast phenotype in cell lines. Moreover, we demonstrated that downregulation of linc00468 promoted the expression of E-cadherin and β-catenin but reduced the expression of N-cadherin, Vimentin and Snail, resulting in progression of EMT.ConclusionsIn conclusion, linc00468 promoted EMT and a consequent increase in invasiveness in HTR-8/Svneo and JAR EVT cell lines. Our study provides the first evidence that linc00468 has a pivotal role in cell invasion and promotes intrinsic and extrinsic EMT ability of PE.

Potential biomarkers of ductal carcinoma in situ progression

BackgroundDuctal carcinoma in situ is a non-obligate precursor of invasive breast carcinoma and presents a potential risk of over or undertreatment. Finding molecular biomarkers of disease progression could allow for more adequate patient treatment. We aimed to identify potential biomarkers that can predict invasiveness risk.MethodsIn this epithelial cell-based study archival formalin-fixed paraffin-embedded blocks from six patients diagnosed with invasive lesions (pure invasive ductal carcinoma), six with in-situ lesions (pure ductal carcinoma in situ), six with synchronous lesions (invasive ductal carcinoma with an in-situ component) and three non-neoplastic breast epithelium tissues were analyzed by gene expression profiling of 770 genes, using the nCounter® PanCancer Pathways panel of NanoString Technologies.ResultsThe results showed that in comparison with non-neoplastic tissue the pure ductal carcinoma in situ was one with the most altered gene expression profile. Comparing pure ductal carcinoma in situ and in-situ component six differentially expressed genes were found, three of them (FGF2, GAS1, and SFRP1), play a role in cell invasiveness. Importantly, these genes were also differentially expressed between invasive and noninvasive groups and were negatively regulated in later stages of carcinogenesis.ConclusionsWe propose these three genes (FGF2, GAS1, and SFRP1) as potential biomarkers of ductal carcinoma in situ progression, suggesting that their downregulation may be involved in the transition of stationary to migrating invasive epithelial cells.

MicroRNA-296-5p Promotes Cell Invasion and Drug Resistance by Targeting Bcl2-Related Ovarian Killer, Leading to a Poor Prognosis in Pancreatic Cancer

Background/Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive invasion, early metastasis, and resistance to chemotherapy, leading to a poor prognosis. To clarify the molecular mechanism of these malignant characteristics, we performed a genome-wide microRNA (miRNA) array analysis utilizing micro-cancer tissues from patients with unresectable PDAC (stage IV), obtained by endoscopic ultrasound-fine needle aspiration (EUS-FNA). Methods: The expression profiles of 2,042 miRNAs were determined using micro-cancer tissues from 13 patients with unresectable PDAC obtained by EUS-FNA. The relationship between individual miRNA levels and overall survival (OS) was analyzed. Possible target genes for miRNAs were bioinformatically analyzed using the online database miRDB. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and PK-8 were transfected with miRNA mimic or small interfering RNA, and cell invasion, epithelial-mesenchymal transition (EMT), and apoptosis markers were examined. miRNA and mRNA expressions were examined by quantitative polymerase chain reaction. Results: Of 2,042 miRNAs, the 10 that exhibited the lowest correlation coefficient (p ≤ 0.005) between miRNA expression level and OS among the patients were identified. The miRDB and expression analysis in cancer cell lines for the 10 miRNAs identified miR-296-5p and miR-1207-5p as biomarkers predictive of shorter survival (p < 0.0005). Bioinformative target gene analysis and transfection experiments with miRNA mimics showed that Bcl2-related ovarian killer (BOK), a pro-apoptotic gene, is a target for miR296-5p in pancreatic cancer cells; transfection of miR-296-5p mimic into PANC-1, MIA PaCa-2, and PK-8 cells resulted in significant suppression of BOK mRNA and protein expression. These transfectants showed significantly higher invasion capability compared with control cells, and knock down of BOK in pancreatic cancer cells similarly enhanced invasion capability. Transfectants of miR-296-5p mimic also exhibited aberrant expression of EMT markers, including vimentin and N-cadherin. Moreover, these transfectants showed a significantly lower apoptosis rate in response to 5-fluorouracil and gemcitabine with a decrease of BOK expression, suggesting a role of miR-296-5p in drug resistance. Conclusion: These results suggest that miR-296-5p is a useful biomarker for a poor prognosis in patients with PDAC, and that the miR-296-5p/BOK signaling axis plays an important role in cell invasion, drug resistance, and EMT in PDACs.