Reduced Migration Capacity Research Articles

Bioinformatics analysis and experimental validation of C6orf120 as a potential prognostic marker and therapeutic target for liver hepatocellular carcinoma.

The C6orf120 gene is a novel gene whose function has not been fully defined. Previous studies have associated it with various liver pathologies, but its specific role in hepatocellular carcinoma (LIHC) remains unclear. This study aimed to investigate the diagnostic and prognostic value of C6orf120 in LIHC, as well as its potential biological functions. In this preliminary research, we utilized data from various databases and bioinformatics tools, including TCGA, GEO, TIMER2, HPA, GEPIA, Linkeomics, Metascape, CIBERSORT, TargetScan, DIANA-microT, RNAinter, and ENCORI, to analyze the expression patterns and mechanisms of C6orf120 in LIHC. Our bioinformatics analysis revealed that C6orf120 is upregulated in LIHC and may serve as a diagnostic and prognostic biomarker. The aberrant expression of C6orf120 in LIHC was further supported by clinical samples and cell lines. In vitro experiments demonstrated that the knockdown of C6orf120 in HepG2 cells significantly reduced migration capacity without affecting proliferation. Additionally, the downregulation of C6orf120 in LIHC cells appeared to inhibit endothelial cell migration and angiogenesis, which are critical in tumorigenesis and development. In conclusion, our findings suggest that C6orf120 could serve as a novel diagnostic and prognostic biomarker for LIHC and is expected to be a prognostic marker and a potential therapeutic target in the clinical management of LIHC.

In vitro blastocyst implantation and trophoblast migration are disrupted by the UV filter benzophenone-3 (BP3)

Benzophenone-3 (BP3) is a common ingredient in personal care products (PCPs) due to its well-established effectiveness in absorbing UV radiation. Sunscreen products are among the most widely used PCPs-containing BP3 applied to the skin, resulting in significant human exposure to BP3 primarily through a dermal application. In the present work, we have tested the action of three environmentally relevant concentrations of BP3 (2, 20 and 200 μg/L) on an in vitro model of implantation of murine blastocysts and on migration ability of the human trophoblast cell line Swan 71. We showed that BP3 caused a significant reduction of blastocyst expansion and a delayed hatching in a non-monotonic way. Besides, embryos displayed a delayed attachment in the three BP3 groups, resulting in a smaller implantation area on the 6th day of culture: BP3(2) (0.32 ± 0.07 mm2); BP3(20) (0.30 ± 0.08 mm2) and BP3(200) (0.25 ± 0.06 mm2) in comparison to the control (0.42 ± 0.07 mm2). We also found a reduced migration capacity of the human first-trimester trophoblast cell line Swan 71 in a scratch assay when exposed to BP3: the lowest dose displayed a higher uncovered area (UA) at 6h when compared to the control, whereas a higher UA of the wound was observed for the three BP3 concentrations at 18 and 24 h of exposure. The changes in UA provoked by BP3 restored to normal values in the presence of flutamide, an androgen receptor (AR) inhibitor. These results indicate that a direct impairment on early embryo implantation and a defective migration of extravillous trophoblast cells through the androgen receptor pathway can be postulated as mechanisms of BP3-action on early gestation with potential impact on fetal growth.

Adventitial delivery of miR-145 to treat intimal hyperplasia post vascular injuries through injectable and in-situ self-assembling peptide hydrogels

Intimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. To develop a vascular adventitial drug delivery system to treat intimal hyperplasia post vascular injuries, we loaded miR-145-5p-agomir (miR-145) into an injectable and in-situ self-assembling RAD peptide hydrogel. In vitro data showed that the miR-145 could be well incorporated into the RAD peptide hydrogels and released in a slow and controlled manner. The released miR-145 could transfect SMCs successfully, and the transfected SMCs exhibited a reduced migration capacity and higher expressions of SMC contractile biomarkers as compared to the non-transfected SMCs. In vivo data showed that the retention of the miR-145 was greatly elongated by the RAD peptide hydrogels. In addition, the application of the miR-145-loaded RAD peptide hydrogels surrounding injured arteries decreased the proliferative SMCs, promoted the regeneration of endothelium, reduced the macrophage infiltration, inhibited the neointimal formation and prevented adverse ECM remodeling via downregulation of KLF4 expression. The RAD peptide hydrogels loaded with miR-145 can successfully inhibit intimal hyperplasia after vascular injuries and thus hold great potential as an innovative extravascular drug delivery approach to treat vascular diseases. Statement of significanceIntimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. Our work here demonstrates that the RAD peptide hydrogels loaded with miR-145-5p-agomir (miR-145) can successfully reverse intimal hyperplasia after vascular injuries and thus hold great potential as an innovative vascular adventitial drug delivery approach to treat vascular diseases. Our work proposes a possible paradigm shift from endovascular drug delivery to extravascular drug delivery for vascular disorder treatment.

Cell consequences of loss of function of the epigenetic factor EHMT1

EHMT1 is an epigenetic factor with histone methyltransferase activity that appears mutated in Kleefstra syndrome, a neurodevelopmental genetic disorder characterized by developmental delay, intellectual disability, and autistic-like features. Despite recent progress in the study of the function of this gene and the molecular etiology of the disease, our knowledge of how EHMT1 haploinsufficiency causes Kleefstra syndrome is still very limited. Here, we show that EHMT1 depletion in RPE1 cells leads to alterations in the morphology and distribution of different subcellular structures, such as the Golgi apparatus, the lysosomes and different cell adhesion components. EHMT1 downregulation also increases centriolar satellites detection, which may indicate a role for EHMT1 in centrosome functioning. Furthermore, the migration process is also altered in EHMT1 depleted cells, which show reduced migration capacity. We consider that the described phenotypes could open new possibilities for understanding the functional impact of EHMT1 haploinsufficiency in Kleefstra syndrome, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in this neurodevelopmental disorder. This knowledge could be relevant not only for the treatment of this syndrome, but also for other neurodevelopmental conditions that could share similar deregulated cellular pathways.

Lysosomal Function and Intracellular Position Determine the Malignant Phenotype in Malignant Melanoma

Lysosomes are central in cell homeostasis and participate in macromolecular degradation, plasma membrane repair, exosome release, cell adhesion/migration, and apoptosis. In cancer, alterations in lysosomal function and spatial distribution may facilitate disease progression. In this study, we show enhanced lysosomal activity in malignant melanoma cells compared with that in normal human melanocytes. Most lysosomes show perinuclear location in melanocytes, while they are more dispersed in melanoma, with retained proteolytic activity and low pH also in the peripheral population. Rab7a expression is lower in melanoma cells than in melanocytes, and by increasing Rab7a, lysosomes are relocated to the perinuclear region in melanoma. Exposure to the lysosome-destabilizing drug L-leucyl-L-leucine methyl ester causes higher damage in the perinuclear subset of lysosomes in melanomas, whereas differences in subpopulation susceptibility cannot be found in melanocytes. Interestingly, melanoma cells recruit the endosomal sorting complex required for transport-III core protein CHMP4B, involved in lysosomal membrane repair, rather than initiate lysophagy. However, when the perinuclear lysosomal position is promoted by Rab7a overexpression or kinesore treatment, lysophagy is increased. In addition, Rab7a overexpression is accompanied by reduced migration capacity. Taken together, the study emphasizes that alterations in lysosomal properties facilitate the malignant phenotype and declares the targeting of lysosomal function as a future therapeutic approach.

Long-Term Treatment with Simvastatin Leads to Reduced Migration Capacity of Prostate Cancer Cells.

Statins have been shown to improve survival of metastatic prostate cancer (mPCa). Nevertheless, their therapeutic use is still under debate. In the present study, we investigated the short-term effects of three different statins (simvastatin, atorvastatin and rosuvastatin) in various PCa cell lines mimicking androgen-sensitive and -insensitive PCa. Moreover, we generated three new PCa cell lines (LNCaPsim, ABLsim, PC-3sim) that were cultured with simvastatin over several months. Our data showed that the three statins expressed highly diverse short-term effects, with the strongest growth-inhibitory effect from simvastatin in PC-3 cells and almost no effect from rosuvastatin in any of the cell lines. Long-term treatment with simvastatin resulted in a loss of response to statins in all three cell lines, which was associated with an upregulation of cholesterol and fatty acid pathways as revealed through RNA sequencing. Despite that, long-term treated cells exhibited diminished spheroid growth and significantly reduced migration capacity per se and to differentiated osteoclasts. These findings were strengthened by reduced expression of genes annotated to cell adhesion and migration after long-term simvastatin treatment. Notably, mPCa patients taking statins were found to have lower numbers of circulating tumor cells in their blood with reduced levels of PSA and alkaline phosphatase. Our data suggest that long-term usage of simvastatin hampers the metastatic potential of PCa cells and may therefore be a potential therapeutic drug for mPCa.

Blue light attenuates TGF-β2-induced epithelial-mesenchymal transition in human lens epithelial cells via autophagy impairment

BackgroundPathogenesis of posterior capsular opacification (PCO) was related to pathological epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). It has been reported that blue light could have an effect on EMT. This study aims to elucidate the role and potential mechanism of autophagy in EMT after blue light exposure in LECs.MethodsHLE-B3 cells were treated with TGF-β2 with different concentration and time to induce EMT as a model of PCO in vitro. Cells were exposed to blue light with or without TGF-β2. The expression levels of EMT-associated markers were analyzed by qRT-PCR, western blotting and cell migration ability was determined by transwell migration assay and wound healing assay. The expressions of autophagy-related proteins were analyzed by western blotting, immunofluorescence and transmission electron microscopy. Rapamycin and chloroquine were utilized in cells for autophagy activation and inhibition.ResultsTGF-β2 induced autophagy activation during EMT progression in HLE-B3 cells in a dose- and time-dependent manner. Blue light exposure inhibited TGF-β2-induced EMT characterized by inhibited expression of EMT related markers and reduced migration capacity. Meanwhile, blue light exposure impaired autophagy activated by TGF-β2. Furthermore, Autophagy activation with rapamycin rescued EMT attenuated by blue light. Autophagy inhibition with chloroquine reduced TGF-β2-induced EMT in HLE-B3 cells.ConclusionBlue light exposure had inhibited effects on TGF-β2-induced EMT in LECs through autophagy impairment, which provides a new insight on prevention and treatment of PCO.

Impaired repair properties of endothelial colony-forming cells in patients with granulomatosis with polyangiitis.

In patients with ANCA‐associated vasculitis, interactions between neutrophils and endothelial cells cause endothelial damage and imbalance. Endothelial colony‐forming cells (ECFCs) represent a cellular population of the endothelial lineage with proliferative capacity and vasoreparative properties. This study aimed to evaluate the angiogenic capacity of ECFCs of patients with granulomatosis with polyangiitis (GPA). The ECFCs of 13 patients with PR3‐positive GPA and 14 healthy controls were isolated and characterized using fluorescence‐activated cell sorting, capillary tube formation measurement, scratching assays and migration assays with and without plasma stimulation. Furthermore, three patients with active disease underwent post‐treatment recollection of ECFCs for longitudinal evaluation. The ECFCs from the patients and controls showed similar capillary structure formation. However, the ECFCs from the patients with inactive GPA exhibited early losses of angiogenic capacity. Impairments in the migration capacities of the ECFCs were also observed in patients with GPA and controls (12th h, p = 0.05). Incubation of ECFCs from patients with GPA in remission with plasma from healthy controls significantly decreased migration capacity (p = 0.0001). Longitudinal analysis revealed that treatment significantly lowered ECFC migration rates. This study revealed that ECFCs from the patients with PR3‐positive GPA in remission demonstrated early losses of tube formation and reduced migration capacity compared to those of the healthy controls, suggesting impairment of endothelial function.

Investigating wound healing characteristics of gingival and skin keratinocytes in organotypic cultures

ObjectivesThe gingiva heals at an accelerated rate with reduced scarring when compared to skin. Potential well-studied factors include immune cell number, angiogenesis disparities and fibroblast gene expression. Differential keratinocyte gene expression, however, remains relatively understudied. This study explored the contrasting healing efficiencies of gingival and skin keratinocytes, alongside their differential gene expression patterns. Methods3D organotypic culture models of human gingiva and skin were developed using temporarily immortalised primary keratinocytes. Models were wounded for visualisation of re-epithelialisation and analysis of keratinocyte migration to close the wound gap. Concurrently, differentially expressed genes between primary gingival and skin keratinocytes were identified, validated, and functionally assessed. ResultsCharacterisation of the 3D cultures of gingiva and skin showed differentiation markers that recapitulated organisation of the corresponding in vivo tissue. Upon wounding, gingival models displayed a significantly higher efficiency in re-epithelialisation and stratification versus skin, repopulating the wound gap within 24 hours. This difference was likely due to distinct patterns of migration, with gingival cells demonstrating a form of sheet migration, in contrast to skin, where the leading edge was typically 1-2 cells thick. A candidate approach was used to identify several genes that were differentially expressed between gingival and skin keratinocytes. Knockdown of PITX1 resulted in reduced migration capacity of gingival cells. ConclusionGingival keratinocytes retain in vivo superior wound healing capabilities in in vitro 2D and 3D environments. Intrinsic gene expression differences could result in gingival cells being ‘primed’ for healing and play a role in faster wound resolution. Clinical Significance StatementThe successful development of organotypic models, that recapitulate re-epithelialisation, will underpin further studies to analyse the oral response to wound stimuli, and potential therapeutic interventions, in an in vitro environment.

Nuclear Respiratory Factor 1 Overexpression Inhibits Proliferation and Migration of PC3 Prostate Cancer Cells.

The role of nuclear respiratory factor 1 (NRF1) on the prostate cancer progression is controversial. We aimed to investigate the effect of NRF1 overexpression on the metastasis potential of PC3 prostate cancer cells and the associated molecular mechanisms. Cell survival, migration capacity, mitochondrial biogenesis, the expression of TGF-β signaling and EMT markers were examined after overexpression and silencing of NRF1 in PC3 cells. We found that NRF1-overexpressing cells exhibited a decreased cell viability and proliferation ability as well as a reduced migration capacity compared to control cells. Moreover, ectopic expression of NRF1 increased the mitochondrial biogenesis and inhibited the EMT characteristics, including a decrease in the mesenchymal marker, α-SMA and an increase in the epithelial cell marker, E-cadherin. We also demonstrated that overexpression of NRF1 suppressed the expression of TGF-β signaling in PC3 cells. As expected, silencing of NRF1 reversed the abovementioned effects. This study demonstrated that upregulation of NRF1 holds the potential to inhibit the metastasis of prostate cancer, possibly through an elevation of mitochondrial biogenesis and the subsequent repression of TGF-β-associated EMT. Therapeutic avenues that increase NRF1 expression may serve as an adjunct to conventional treatments of prostate cancer.

EZH2 presents a therapeutic target for neuroendocrine tumors of the small intestine

Small intestinal neuroendocrine tumors (SI-NETs) are slow-growing tumors that seem genetically quite stable without highly recurrent mutations, but are epigenetically dysregulated. In contrast to the undetectable expression of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase in the enterochromaffin cells of the small intestine, we found high and differential expression of EZH2 in primary SI-NETs and corresponding metastases. Silencing EZH2 in the SI-NET cell line CNDT2.5 reduced cell proliferation and induced apoptosis. Furthermore, EZH2 knockout inhibited tumor progression in a CNDT2.5 SI-NET xenograft mouse model, and treatment of SI-NET cell lines CNDT2.5 and GOT1 with the EZH2-specific inhibitor CPI-1205 decreased cell viability and promoted apoptosis. Moreover, CPI-1205 treatment reduced migration capacity of CNDT2.5 cells. The EZH2 inhibitor GSK126 also repressed proliferation of CNDT2.5 cells. Recently, metformin has received wide attention as a therapeutic option in diverse cancers. In CNDT2.5 and GOT1 cells, metformin suppressed EZH2 expression, and inhibited cell proliferation. Exposure of GOT1 three-dimensional cell spheroids to CPI-1205 or metformin arrested cell proliferation and decreased spheroid size. These novel findings support a possible role of EZH2 as a candidate oncogene in SI-NETs, and suggest that CPI-1205 and metformin should be further evaluated as therapeutic options for patients with SI-NETs.

Comparative Cytotoxic Activity of Hydroxytyrosol and Its Semisynthetic Lipophilic Derivatives in Prostate Cancer Cells.

A high adherence to a Mediterranean diet has been related to numerous beneficial effects in human health, including a lower incidence and mortality of prostate cancer (PCa). Olive oil is an important source of phenolic bioactive compounds, mainly hydroxytyrosol (HT), of this diet. Because of the growing interest of this compound and its derivatives as a cancer chemopreventive agent, we aimed to compare the in vitro effect of HT isolated from olive mill wastewaters and five semisynthetic alkyl ether, ester, and nitro-derivatives against prostate cancer (PCa) cell lines. The effect in cell proliferation was determined in RWPE-1, LNCaP, 22Rv1, and PC-3 cells by resazurin assay, the effect in cell migration by wound healing assay, and tumorsphere and colony formation were evaluated. The changes in key signaling pathways involved in carcinogenesis were assessed by using a phosphorylation pathway profiling array and by Western blotting. Antiproliferative effects of HT and two lipophilic derivatives [hydroxytyrosyl acetate (HT-Ac)/ethyl hydroxytyrosyl ether (HT-Et)] were significantly higher in cancerous PC-3 and 22Rv1 cells than in non-malignant RWPE-1 cells. HT/HT-Ac/HT-Et significantly reduced migration capacity in RWPE-1 and PC-3 and prostatosphere size and colony formation in 22Rv1, whereas only HT-Ac and HT-Et reduced these functional parameters in PC-3. The cytotoxic effect in 22Rv1 cells was correlated with modifications in the phosphorylation pattern of key proteins, including ERK1/2 and AKT. Consistently, HT-Ac and HT-Et decreased p-AKT levels in PC-3. In sum, our results suggest that HT and its lipophilic derivatives could be considered as potential therapeutic tools in PCa.

Prevention of High Glucose-Mediated EMT by Inhibition of Hsp70 Chaperone.

Hyperglycemia may contribute to the progression of carcinomas by triggering epithelial-to-mesenchymal transition (EMT). Some proteostasis systems are involved in metastasis; in this paper, we sought to explore the mechanism of Hsp70 chaperone in EMT. We showed that knockdown of Hsp70 reduced cell migration capacity concomitantly with levels of mRNA of the Slug, Snail, and Twist markers of EMT, in colon cancer cells incubated in high glucose medium. Conversely, treatment of cells with Hsp70 inducer U-133 were found to elevate cell motility, along with the other EMT markers. To prove that inhibiting Hsp70 may reduce EMT efficiency, we treated cells with a CL-43 inhibitor of the HSF1 transcription factor, which lowered Hsp70 and HSF1 content in the control and induced EMT in carcinoma cells. Importantly, CL-43 reduced migration capacity, EMT-linked transcription factors, and increased content of epithelial marker E-cadherin in colon cancer cells of three lines, including one derived from a clinical sample. To prove that Hsp70 chaperone should be targeted when inhibiting the EMT pathway, we treated cancer cells with 2-phenylethynesulfonamide (PES) and demonstrated that the compound inhibited substrate-binding capacity of Hsp70. Furthermore, PES suppressed EMT features, cell motility, and expression of specific transcription factors. In conclusion, the Hsp70 chaperone machine efficiently protects mechanisms of the EMT, and the safe inhibitors of the chaperone are needed to hamper metastasis at its initial stage.

Targeting long noncoding RNA PMIF facilitates osteoprogenitor cells migrating to bone formation surface to promote bone formation during aging.

Rationale: The migration of mesenchymal osteoprogenitor cells (OPCs) to bone formation surface is the initial step of osteoblastogenesis before they undergo osteoblast differentiation and maturation for governing bone formation. However, whether the migration capacity of OPCs is compromised during aging and how it contributes to the aging-related bone formation reduction remain unexplored. In the present study, we identified a migration inhibitory factor (i.e., long noncoding RNA PMIF) and examined whether targeting lnc-PMIF could facilitate osteoprogenitor cells migrating to bone formation surface to promote bone formation during aging.Methods: Primary OPCs from young (6-momth-old) and aged (18-momth-old) C57BL/6 mice and stable lnc-PMIF knockdown/overexpression cell lines were used for in vitro and in vivo cell migration assay (i.e., wound healing assay, transwell assay and cell intratibial injection assay). RNA pulldown-MS/WB and RIP-qPCR were performed to identify the RNA binding proteins (RBPs) of lnc-PMIF. Truncations of lnc-PMIF and the identified RBP were engaged to determine the interaction motif between them by RNA pulldown-WB and EMSA. By cell-based therapy approach and by pharmacological approach, small interfering RNA (siRNA)-mediated lnc-PMIF knockdown were used in aged mice. The cell migration ability was evaluated by transwell assay and cell intratibial injection assay. The bone formation was evaluated by microCT analysis and bone morphometry analysis.Results: We reported that the decreased bone formation was accompanied by the reduced migration capacity of the bone marrow mesenchymal stem cells (BMSCs, the unique source of OPCs in bone marrow) in aged mice. We further identified that the long non-coding RNA PMIF (postulated migration inhibitory factor) (i.e., lnc-PMIF) was highly expressed in BMSCs from aged mice and responsible for the reduced migration capacity of aged OPCs to bone formation surface. Mechanistically, we found that lnc-PMIF could bind to human antigen R (HuR) for interrupting the HuR-β-actin mRNA interaction, therefore inhibit the expression of β-actin for suppressing the migration of aged OPCs. We also authenticated a functionally conserved human lncRNA ortholog of the murine lnc-PMIF. By cell-based therapy approach, we demonstrated that replenishing the aged BMSCs with small interfering RNA (siRNA)-mediated lnc-PMIF knockdown could promote bone formation in aged mice. By pharmacological approach, we showed that targeted delivery of lnc-PMIF siRNA approaching the OPCs around the bone formation surface could also promote bone formation in aged mice.Conclusion: Toward translational medicine, this study hints that targeting lnc-PMIF to facilitate aged OPCs migrating to bone formation surface could be a brand-new anabolic strategy for aging-related osteoporosis.

Inhibitory Effects of a Reengineered Anthrax Toxin on Canine and Human Osteosarcoma Cells.

Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA.

Abstract 2614: Elucidation of the molecular mechanisms mediating sorafenib-resistance in Hepatocellular carcinoma

Abstract Sorafenib is the only first line and FDA-approved drug available for the treatment of advanced hepatocellular carcinoma (HCC). Despite an increased overall patient survival of about 3-5 months with sorafenib, majority of the HCC patients acquire sorafenib-resistance, thus making sorafenib treatment ineffective in long-term. Therefore, there is an urgent need to explore the mechanisms underlying sorafenib-resistance to develop better treatment strategies to combat this deadly disease. To achieve this goal, we first established sorafenib-resistant cells by repeated treatment of different HCC cell lines (Huh7, Hep3B and HepG2) with increasing concentrations of sorafenib up to 6µM. MTT assays confirmed that all three cell types treated with sorafenib achieved resistance and were termed soraR to distinguish them from their sorafenib-sensitive (soraS) counterparts. Further characterization of the soraR cells showed increased expression of mesenchymal markers including Slug, Snail, Zeb2, vimentin, and reduced expression of epithelial marker (E-cadherin), indicative of Epithelial-Mesenchymal Transition (EMT). Moreover soraR cells were found to have high migratory potential in wound healing assay and higher sphere-forming capability compared to the soraS cells. To find out the signaling pathways which could be responsible for promoting sorafenib-resistance, RT2-profiler PCR Array analysis for cytokine and chemokines (PAHS-150Z from Qiagen) were performed, which revealed an increase in the expression of BMP6 and BMP7 mRNA levels in the soraR cells. The results were validated by qPCR. In addition, western blot analyses showed an increase in pSmad1, 5, 9 levels in the soraR cells, suggesting a potential activation of BMP6, 7 axis in sorafenib-resistance. Effect of inhibition of BMP6 and BMP7 signaling in re-sensitizing the SoraR cells towards sorafenib was determined next. Flow cytometric analysis of Hep3B-SoraR cells treated with DMH1, a small molecule inhibitor of BMP receptors in combination with sorafenib showed increased cell death which was accompanied with increased expression of cleaved PARP and cleaved caspase 3 in western blot. BMP6 and BMP7 inhibition also reduced migration capacity of soraR cell types as examined by wound healing assay. The results were further confirmed by treating soraR cells with recombinant BMP6 and BMP7 and examining their effect on migration and sorafenib-mediated apoptosis. Interestingly, exogenous BMP6 and BMP7 treatment was found to enhance migration capacity and rescue the soraS cells from sorafenib-mediated cell death. These findings indicated an important role of BMP6 and BMP7 pathways in mediating sorafenib-resistance in HCC and suggested that pharmacological targeting of BMP6 and BMP7 signaling could be an effective strategy to ameliorate sorafenib-resistance in advanced HCC. Citation Format: Kanchan Vishnoi, Rong Ke, Randhir Kumar, Navin Viswakarma, Ajay Rana, Basabi Rana. Elucidation of the molecular mechanisms mediating sorafenib-resistance in Hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2614.

CCDC88B is required for mobility and inflammatory functions of dendritic cells.

The Coiled Coil Domain Containing Protein 88B (CCDC88B) gene is associated with susceptibility to several inflammatory diseases in humans and its inactivation in mice protects against acute neuroinflammation and models of intestinal colitis. We report that mice lacking functional CCDC88B (Ccdc88bMut ) are defective in several dendritic cells (DCs)-dependent inflammatory and immune reactions in vivo. In these mice, an inflammatory stimulus (LPS) fails to induce the recruitment of DCs into the draining lymph nodes (LNs). In addition, OVA-pulsed Ccdc88bMut DCs injected in the footpad do not induce recruitment and activation of antigen-specific CD4+ and CD8+ T cells in their draining LN. Experiments in vitro indicate that this defect is independent of the ability of mutant DCs to capture and present peptide antigen to T cells. Rather, kinetic analyses in vivo of wild-type and Ccdc88bMut DCs indicate a reduced migration capacity in the absence of the CCDC88B protein expression. Moreover, using time-lapse light microscopy imaging, we show that Ccdc88bMut DCs have an intrinsic motility defect. Furthermore, in vivo studies reveal that these reduced migratory properties lead to dampened contact hypersensitivity reactions in Ccdc88b mutant mice. These findings establish a critical role of CCDC88B in regulating movement and migration of DCs. Thus, regulatory variants impacting Ccdc88b expression in myeloid cells may cause variable degrees of DC-dependent inflammatory response in situ, providing a rationale for the genetic association of CCDC88B with several inflammatory and autoimmune diseases in humans.

Two‐pore channel 2 (TPC2) Role in Melanoma

Two‐pore channels (TPC), a recently described class of NAADP‐ and PI(3,5)P2–sensitive calcium‐permeable cation channels in the endolysosomal system of cells have a crucial rule in cellular functions and have been linked to various type of diseases including cancer. Here, TPC2 protein stable silencing in melanoma cancer cell lines was developed using CRISPR/Cas9 genome editing technology. TPC2 knockout (KO) in B16 murine melanoma cancer cell was associated with higher viability and migration rates with higher melanin accumulation inside the cells compared to wild type controls. TPC2 KO CHL1 human melanoma cancer cells showed an increased level of proliferation with reduced migration capacity. Both KO cell lines had lower invasion rates and lacked capacity for angiogenesis compared to wild type controls. Proteomic analysis was used to identify novel targets which were affected by genetic modification of TPC2. Thus, the findings of the present study indicated that CRISPR/Cas9 model is an ideal and cost‐effective tool to study the role of TPC2 in melanoma cancer cells which can be further used as a target to understand the biological basis of this disease.Support or Funding InformationRoyal Embassy of Saudi Arabia Cultural Bureau

Long Noncoding RNA-Enriched Vesicles Secreted by Hypoxic Cardiomyocytes Drive Cardiac Fibrosis

Long non-coding RNAs (lncRNAs) have potential as novel therapeutic targets in cardiovascular diseases, but detailed information about the intercellular lncRNA shuttling mechanisms in the heart is lacking. Here, we report an important novel crosstalk between cardiomyocytes and fibroblasts mediated by the transfer of lncRNA-enriched extracellular vesicles (EVs) in the context of cardiac ischemia. lncRNA profiling identified two hypoxia-sensitive lncRNAs: ENSMUST00000122745 was predominantly found in small EVs, whereas lncRNA Neat1 was enriched in large EVs in vitro and in vivo. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. In addition, lncRNA Neat1 was transcriptionally regulated by P53 under basal conditions and by HIF2A during hypoxia. The function of Neat1 was further elucidated in vitro and in vivo. Silencing of Neat1 in vitro revealed that Neat1 was indispensable for fibroblast and cardiomyocyte survival and affected fibroblast functions (reduced migration capacity, stalled cell cycle, and decreased expression of fibrotic genes). Of translational importance, genetic loss of Neat1 in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance.

Disturbance of the Epithelial-Mesenchymal Transition as a Cause of the Defective Development of Human Spontaneous Abortions with Abnormal Karyotype.

Genetic program regulating epithelial-mesenchymal transition is activated at the early stages of embryogenesis. Abnormal karyotype of the embryo disrupts the fundamental process of epithelial-mesenchymal transition, which is seen from incomplete morphological rearrangement and reduced migration capacity of cells, as well as low level of expression of epithelial-mesenchymal transition markers (fibronectin, collagen, and glycosaminoglycans). Disturbances of the epithelial-mesenchymal transition affect the proliferative potential of cells with aberrant karyotype, which contributes to the delay of embryonic development and correlates with the formation of abnormal phenotype in carriers of chromosomal abnormalities.