Erianin Controls Collagen-Mediated Retinal Angiogenesis via the RhoA/ROCK1 Signaling Pathway Induced by the alpha2/beta1 Integrin-Collagen Interaction.

Purpose: Erianin has been reported to inhibit tumor activity by suppressing the expression of integrins. It is hypothesized that erianin can inhibit retinal neovascularization in collagen by suppressing the expression of integrins. With an aim to test this hypothesis, the regulation of erianin on collagen-mediated retinal angiogenesis via the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) signaling pathway induced by α2 and ß1 integrin-collagen interactions was investigated. Methods: The effects of erianin on human retinal vascular endothelial cells (HRVECs) were assessed in vitro using a hypoxia model in a three-dimensional cell culture induced by cobalt (II) chloride (CoCl2). A hypoxia-induced retinopathy model in adult zebrafish and zebrafish embryos was established to assess the antiangiogenic effect of erianin with and without vitreous collagen in vivo. The expression of α2 and ß1 integrin and RhoA/ROCK1 pathway in HRVECs and zebrafish retinas were analyzed. Results: In vitro, collagen improved the angiogenic potential of HRVECs, including migration, adhesion, and tube formation, in a three-dimensional cell culture model. Erianin suppressed the angiogenic processes of the CoCl2-induced hypoxia HRVEC model in a concentration-dependent manner. In vivo, erianin reduced retinal angiogenesis in the hypoxia-induced retinopathy model in adult and embryo zebrafish. Erianin inhibited the expression of α2 and ß1 integrin and RhoA/ROCK1 in a hypoxia-induced model in vitro in three-dimensional cell culture and in vivo in adult zebrafish. Conclusions: Collagen-mediated retinal angiogenesis may be regulated by erianin via the RhoA/ROCK1 signaling pathway induced by α2 and ß1 integrin-collagen interactions. These findings suggest that erianin has the therapeutic potential on intraocular collagen-mediated retinal angiogenesis.

Inhibited effect of an RGD peptide hydrogel on the expression of ß1-integrin, FAK, and Akt in Tenon's capsule fibroblasts.

Tenon's capsule fibroblasts are the main cellular components of filtration tract scar that limit the success rate of glaucoma filtration surgery. Scar formation results from infiltration and proliferation of fibroblasts into damaged areas, meanwhile synthesis of extracellular matrix glycoproteins. Integrins are cell surface receptors for extracellular molecules that mediate cell adhesion, spreading, migration, and invasion. They bind their ligands often through recognition of short amino-acid sequences-arginine-glycine-aspartic acid (RGD). Peptides that contain RGD sequence can compete with RGD containing insoluble matrix proteins for binding to the integrin receptor and thus prevent the downstream signaling pathway. Increasing evidence supports that ß1-integrin/focal adhesion kinase (FAK)/Akt signal pathway plays an important role in fibrogenesis and scar formation in different tissues. In consideration of advantages of peptide hydrogel, that is well biocompatibility, gel state, degradability, good drug loading, we designed and fabricated an RGD peptide hydrogel, and hypothesized that it could inhibit the expression of ß1-integrin, FAK, and Akt in Tenon's capsule fibroblasts. Rheology results showed that 1% wt Fmoc-FFGGRGD peptide solution could self-assemble into hydrogel. Western blot analysis revealed that there were statistical differences between control group and 1% wt group in ß1-integrin/ß-actin, FAK/ß-actin, Akt/ß-actin respectively (*p < .05). The relative mRNA expression of ß1-integrin, FAK, Akt in control group and 1% wt group were also statistically different respectively (*p < .05). We proved that 1% wt Fmoc-FFGGRGD self-assembly peptide hydrogel could inhibit the expression of ß1-integrin, FAK and Akt in Tenon's capsule fibroblasts. It is a promising way to solve scar formation of glaucoma filter channel.

BDNF promoted osteoblast migration and fracture healing by up-regulating integrin ß1 via TrkB-mediated ERK1/2 and AKT signalling.

Brain-derived neurotrophic factor (BDNF) has been reported to participate in fracture healing, whereas the mechanism is still unclear. Since osteoblast migration is important for fracture healing, investigating effects of BDNF on osteoblasts migration may help to reveal its mechanism. Here, MC3T3-E1 cells were used in vitro while closed femur fracture mice were applied in vivo. Cells migration was assessed with Transwell assay. The protein expression was analysed by immunoblotting. X-ray and Micro-CT were performed at different time after fracture. Our results showed that BDNF promoted MC3T3-E1 cells migration, integrin ß1 expression and ERK1/2 and AKT phosphorylation. K252a, a specific inhibitor for TrkB, suppressed BDNF-induced migration, integrin ß1 expression and activation of ERK1/2 and AKT. PD98059 (an ERK1/2 inhibitor) and LY294002 (an AKT inhibitor) both inhibited BDNF-induced migration and integrin ß1 expression while integrin ß1 blocking antibody only suppressed cell migration. X-ray and Micro-CT analyses showed that the adenoviral carried integrin ß1 shRNA group had slower fracture healing at 7 and 21 days, but not 35 days compared to the control group. Thus, we proposed that BDNF stimulated MC3T3-E1 cells migration by up-regulating integrin ß1 via TrkB mediated ERK1/2 and AKT signalling, and this may help to enhance the fracture healing.

Electrical Stimulation of pediatric cardiac-derived c-kit+ progenitor cells improves retention and cardiac function in right ventricular heart failure.

Nearly 1 in every 120 children born has a congenital heart defect. Although surgical therapy has improved survival, many of these children go on to develop right ventricular heart failure (RVHF). The emergence of cardiovascular regenerative medicine as a potential therapeutic strategy for pediatric HF has provided new avenues for treatment with a focus on repairing or regenerating the diseased myocardium to restore cardiac function. Although primarily tried using adult cells and adult disease models, stem cell therapy is relatively untested in the pediatric population. Here, we investigate the ability of electrical stimulation (ES) to enhance the retention and therapeutic function of pediatric cardiac-derived c-kit+ progenitor cells (CPCs) in an animal model of RVHF. Human CPCs isolated from pediatric patients were exposed to chronic ES and implanted into the RV myocardium of rats. Cardiac function and cellular retention analysis showed electrically stimulated CPCs (ES-CPCs) were retained in the heart at a significantly higher level and longer time than control CPCs and also significantly improved right ventricular functional parameters. ES also induced upregulation of extracellular matrix and adhesion genes and increased in vitro survival and adhesion of cells. Specifically, upregulation of ß1 and ß5 integrins contributed to the increased retention of ES-CPCs. Lastly, we show that ES induces CPCs to release higher levels of pro-reparative factors in vitro. These findings suggest that ES can be used to increase the retention, survival, and therapeutic effect of human c-kit+ progenitor cells and can have implications on a variety of cell-based therapies. Stem Cells 2019;37:1528-1541.

The intercellular expression of type-XVII collagen, laminin-332, and integrin-ß1 promote contact following during the collective invasion of a cancer cell population.

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-ß1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-ß1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-ß1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-ß1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

Dynamic expression of α6 integrin indicates epidermal cell behaviors.

Skin epidermis is a stratified epithelium that composed of interfollicular epidermis (IFE) and hair follicles (HFs). Integrins are cell-cell and cell-matrix adhesive ligands that play important roles in epidermal cell proliferation, migration and differentiation behaviors. Here, we analyzed the expression of both α6 and ß1 integrins. In vitro epidermal cell culture, both α6 and ß1 integrins displayed downregulation upon high Ca2+ induced differentiation. During wound healing (WH), α6 integrin showed dynamic expression, first greatly upregulated in unclosed wounds and then downregulated upon re-epithelialization. Further analysis of different wound regions confirmed α6 integrin significantly increased in migratory cells and migration was coupled with differentiation. However, expression level of ß1 integrin did not show significant correlation with migration. We discovered that α6 integrin directly indicates epidermal cell differentiation and wound directed migration behaviors with its expression level.

ß1 Integrin is essential for fascin-mediated breast cancer stem cell function and disease progression.

Breast cancer remains the second cause of tumor-related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem-like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin-bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, ß1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin-mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self-renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co-expression and short disease-free as well as overall survival in chemo-treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self-renewability and chemoresistance strongly encourages for dual targeting of fascin-ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root.

Gelatin nonwovens-based epithelial morphogenesis involves a signaling axis comprising EGF-receptor, MAP kinases ERK 1/2, and ß1 integrin.

In biomaterials research, biomechanics which support tissue regeneration steadily gains of importance. Hence, we have previously shown that gelatin-based electrospun nonwoven mats (NWMs) with a distinct modulus of elasticity (3.2 kPa) promotes epithelial morphogenesis. Since molecular mechanisms of this morphogenesis are still unknown, the present study aims at identifying molecules, involved herein. Epithelia established on the NMWs showed persistence of the activated state of the epidermal growth factor receptor (EGF-R), phosphorylated at the src-specific tyrosine 845 (EGF-RT845 ) throughout the observation period of 10 days. To elucidate whether the observed morphogenesis mechanistically involves EGF-R signaling, we inhibited EGF-R, by employing the EGF-RT845 specific inhibitor Gefitinib (IRESSA®). Gefitinib administration yielded a reduced expression of the ß1 integrin subunit, a well-known cell-matrix interaction receptor, concomitant with downregulation of p42/44 ERK1/2 MAP-kinase activity. To elucidate whether the observed downregulation of ß1 is EGF-RT845 -dependent or emerging from ERK1/2 signaling, we exposed epithelia, grown on the NWMs, with the ERK1/2-directed inhibitor U0126. In the absence of Gefitinib, inhibition of p42/44 MAP-kinase activity resulted in decreased ß1 integrin protein levels, thus indicating that ß1 expression is dependent on ERK1/2 and not EGF-RT845 . Our results showed the first time that an EGF-R-ß1 integrin-signaling axis, including ERK1/2, promotes NWM-elasticity-based epithelial morphogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 663-677, 2019.

Molecular analysis of biocompatibility of anodized titanium with deposited silver nanodendrites.

Titanium (>99.6% purity) and its anodically oxidized modifications, with and without deposited silver nanodendrites regarding its biocompatibility were evaluated. In human gingival fibroblasts and osteoblast cell lines grown on tested samples, the level of expression of genes encoding αV (ITGAV) and ß1 (ITGB1) integrin subunits also genes encoding focal adhesion (FAK) and extracellular-signal regulated (ERK) kinases was assessed. For this purpose, the qualitative and quantitative PCR technique was used. The expression of studied genes was dependent on the origin of cell lines and the type of evaluated material. The high expression of PBGD and ITGAV genes in fibroblasts grown on the surface of anodically modified titanium with deposited silver nanodendrites indicates potentially high biocompatibility of these samples for soft tissue cells. The high expression of the ITGB1 and ERK1 genes and the enhanced expression of the FAK gene in osteoblasts cells grown on the tested material was also observed. Summarizing, the nanocrystalline Ti modified with silver deposits showed higher biocompatibility in comparison with the conventional pure Ti samples.

TGF-ß1 modulates podocyte migration by regulating the expression of integrin-ß1 and -ß3 through different signaling pathways.

AIMS: Podocyte migration may lead foot process effacement and proteinuria. Transforming growth factor-ß1 (TGF-ß1) and integrins are involved in the adhesion and migration of cells. However, the crosstalk of TGF-ß1 and integrins is unclear. Here, we examined how TGF-ß1 regulates the expression of integrin-ß1 and -ß3 to modulate podocyte adhesion and migration. MAIN METHODS: Podocytes were exposed to TGF-ß1 and/or the inhibitors of Smad2/3, ERK and p38, then the expression of integrin-ß1 and -ß3 was assessed by Real-time PCR and western blot analyses. Podocyte adhesion and migration were measured under TGF-ß1 treatment and/or anti-integrin-ß3 antibody by cell adhesion assay and wound healing assay. KEY FINDINGS: TGF-ß1 had no effect on integrin-ß1 mRNA expression. In the analysis of protein expression, TGF-ß1 decreased the mature form of integrin-ß1, but increased both the precursor form and core peptide of integrin-ß1. The inhibitors of ERK and p38, but not Smad2/3, abrogated TGF-ß1-induced changes in integrin-ß1 protein expression. TGF-ß1 increased integrin-ß3 mRNA and protein levels. The inhibitors of Smad2/3, ERK and p38 attenuated the TGF-ß1-induced increase in integrin-ß3 mRNA and protein levels. Podocyte adhesion and migration were enhanced under the stimulation of TGF-ß1. The blockade of interactions between integrin-αvß3 and the extracellular matrix by the anti-integrin-ß3 antibody abrogated the TGF-ß1-induced enhancement in podocyte adhesion and migration. SIGNIFICANCE: Our results demonstrate that TGF-ß1up-regulates integrin-ß3 expression and down-regulates integrin-ß1 expression through different pathways. The up-regulation of integrin-ß3 expression enhances podocyte migration. This study provides a novel mechanism for TGF-ß1 signaling in regulating podocyte migration.

ß1-integrin-matrix interactions modulate cerebral microvessel endothelial cell tight junction expression and permeability.

Acutely following focal cerebral ischemia disruption of the microvessel blood-brain barrier allows transit of plasma proteins into the neuropil as edema formation that coincides with loss of microvessel endothelial ß1-integrins. We extend previous findings to show that interference with endothelial ß1-integrin-matrix adhesion by the monoclonal IgM Ha2/5 increases the permeability of primary cerebral microvascular endothelial cell monolayers through reorganization of claudin-5, occludin, and zonula occludens-1 (ZO-1) from inter-endothelial borders. Interference with ß1-integrin-matrix adhesion initiates F-actin conformational changes that coincide with claudin-5 redistribution. ß1-integrin-matrix interference simultaneously increases phosphorylation of myosin light chain (MLC), while inhibition of MLC kinase (MLCK) and Rho kinase (ROCK) abolishes the Ha2/5-dependent increased endothelial permeability by 6 h after ß1-integrin-matrix interference. These observations are supported by concordant observations in the cortex of a high-quality murine conditional ß1-integrin deletion construct. Together they support the hypothesis that detachment of ß1-integrins from abluminal matrix ligands increases vascular endothelial permeability through reorganization of tight junction (TJ) proteins via altered F-actin conformation, and indicate that the ß1-integrin-MLC signaling pathway is engaged when ß1-integrin detachment occurs. These findings provide a novel approach to the research and treatment of cerebral disorders where the breakdown of the blood-brain barrier accounts for their progression and complication.

PRL-3/PTP4A3 phosphatase regulates integrin ß1 in adhesion structures during migration of human ocular melanoma cells.

In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL-3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL-3 overexpression in OCM-1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL-3 in the metastatic spreading of uveal melanoma. Here, we aimed to identify PRL-3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin ß1, which is the most highly expressed integrin in our cohort of uveal melanomas. We show that preventing PRL-3 anchorage to the plasma membrane i) abolishes PRL-3-induced migration in OCM-1 cells, ii) specifically enhances the spreading of OCM-1 cells overexpressing PRL-3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin ß1 on collagen I. Knockdown experiments confirmed integrin ß1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin ß1, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin ß1 may be dephosphorylated by PRL-3 in its intracytoplasmic S/T region, an important motif for integrin-mediated cell adhesion. Finally, we observed that PRL-3 regulated the clustering of integrin ß1 in FAs on collagen I but not on fibronectin. This work identifies PRL-3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL-3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood.

[10]-Gingerol Reverts Malignant Phenotype of Breast Cancer Cells in 3D Culture.

Breast cancer is a complex and multifactorial disease. Tumors have a heterogeneous microenvironment, which have multiple interactions with other cell types, greatly influencing the behavior of tumor cells and response to therapy. The 3D culture mimics the microenvironment better found in vivo and is more appropriated than the traditional 2D culture made from plastic to test the cellular response to drugs. To investigate the effects of [10]-gingerol on breast tumor cells, we used physiologically relevant three-dimensional (3D) cultures of malignant and non-malignant human breast cells grown in laminin-rich extracellular matrix gels (lr-ECM). Our results showed selective cytotoxicity of [10]-gingerol against the malignant T4-2 breast cancer cell line compared to non-malignant S1 cells. The compound reverted the malignant phenotype of the cancer cells, downregulating the expression of epidermal growth factor receptor (EGFR) and ß1-integrin. Moreover, [10]-gingerol induced apoptosis in this cell line. These results suggest that [10]-gingerol may be an effective compound to use as adjuvant therapy in breast cancer treatment. J. Cell. Biochem. 118: 2693-2699, 2017. © 2017 Wiley Periodicals, Inc.

Mesothelial cells interact with tumor cells for the formation of ovarian cancer multicellular spheroids in peritoneal effusions.

Epithelial ovarian cancer (EOC) dissemination is primarily mediated by the shedding of tumor cells from the primary site into ascites where they form multicellular spheroids that rapidly lead to peritoneal carcinomatosis. While the clinical importance and fundamental role of multicellular spheroids in EOC is increasingly appreciated, the mechanisms that regulate their formation and dictate their cellular composition remain poorly characterized. To investigate these important questions, we characterized spheroids isolated from ascites of women with EOC. We found that in these spheroids, a core of mesothelial cells was encased in a shell of tumor cells. Analysis further revealed that EOC spheroids are dynamic structures of proliferating, non-proliferating and hypoxic regions. To recapitulate these in vivo findings, we developed a three-dimensional co-culture model of primary EOC and mesothelial cells. Our analysis indicated that, compared to the OVCAR3 cell line, primary EOC cells isolated from ascites as well as mesothelial cells formed compact spheroids. Analysis of heterotypic spheroid microarchitecture revealed a structure that grossly resembles the structure of spheroids isolated from ascites. Cells that formed compact spheroids had elevated expression of ß1 integrin and low expression of E-cadherin. Addition of ß1 integrin blocking antibody or siRNA-mediated downregulation of ß1 integrin resulted in reduced tightness of the spheroids. Interestingly, the loss of MUC16 and E-cadherin expression resulted in the formation of more compact spheroids. Therefore, our findings support the heterotypic nature of spheroids from malignant EOC ascites. In addition, our data describe an unusual link between E-cadherin expression and less compact spheroids. Our data also emphasize the role of MUC16 and ß1 integrin in EOC spheroid formation.

miR-17 inhibits ovarian cancer cell peritoneal metastasis by targeting ITGA5 and ITGB1.

An essential step in the peritoneal spread of ovarian cancer is the adhesion and implantation of tumor cells to the mesothelium layer. Integrin α5 and ß1 have been reported to mediate the initial adhesion process and to correlate with disease survival in ovarian cancer. However, the molecular mechanism of integrin α5ß1 dysregulation in tumorigenesis and metastasis remained enigmatic. In the present study, using the US NCI60 database, we identified miR-17 as a candidate regulator targeting both integrin α5 and ß1. The level of miR-17 was evidently inversely correlated with that of α5 and ß1 in ovarian cancer cell lines. Specifically, miR-17 bound directly to the 3' untranslated region (3'UTR) of α5 and ß1 and suppressed their expression. Forced expression of miR-17 led to markedly diminished adhesion and invasion of ovarian cancer cells in vitro, and notably reduced metastatic nodules inside the peritoneal cavity in in vivo SKOV3 xenografts model. Moreover, ectopic expression of miR-17 in ovarian cancer cells resulted in repressed ILK phosphorylation as well as decreased production of active matrix metalloproteinase-2 (MMP-2). Our results indicated that miR-17 hampered ovarian cancer peritoneal propagation by targeting integrin α5 and ß1. These findings supported the utility of miR-17/α5ß1 to be considered as valuable marker for metastatic potential of ovarian cancer cells, or a therapeutic target in ovarian cancer treatment.

Nitric oxide increases the migratory activity of non-small cell lung cancer cells via AKT-mediated integrin αv and ß1 upregulation.

BACKGROUND: Previously, nitric oxide (NO) has been found to affect the metastatic behavior of various types of cancer. In addition, it has been found that alterations in integrin expression may have profound effects on cancer cell survival and migration. Here, we aimed at assessing the effects of non-toxic concentrations of NO on human non-small cell lung cancer (NSCLC) cells, including the expression of integrins and the migration of these cells. METHODS: The cytotoxic and proliferative effects of NO on human NSCLC-derived H460, H292 and H23 cells were tested by MTT assay. The migration capacities of these cells was evaluated by wound healing and transwell migration assays. The expression of integrins and migration-associated proteins was determined by Western blot analyses. RESULTS: We found that NO treatment caused a significant increase in the expression of integrin αv and ß1 in all three NSCLC-derived cell lines tested. Known migration-associated proteins acting downstream of these integrins, including focal adhesion kinase (FAK), active RhoA (Rho-GTP) and active cell division control 42 (Cdc42-GTP), were found to be significantly activated in response to NO. In addition, we found that NO-treated cells showed an increased motility and that this motility was associated with a significant increase in the number of filopodia per cell. We also found that NO-treated cells exhibited increased active protein kinase G (PKG), protein kinase B (AKT) and FAK expression levels. Using a pharmacological approach, we found that the integrin-modulating effect of NO is most likely brought about by a PKG/AKT-dependent mechanism, since the observed changes in integrin expression were abolished by AKT inhibitors, but not by FAK inhibitors. CONCLUSION: Our data suggest a novel role of NO in the regulation of integrin expression and, concomitantly, the migratory capacity of NSCLC cells.

Conditional knockout mice demonstrate function of Klf5 as a myeloid transcription factor.

Krüppel-like factor 5 (Klf5) encodes a zinc-finger transcription factor and has been reported to be a direct target of C/EBPα, a master transcription factor critical for formation of granulocyte-macrophage progenitors (GMP) and leukemic GMP. Using an in vivo hematopoietic-specific gene ablation model, we demonstrate that loss of Klf5 function leads to a progressive increase in peripheral white blood cells, associated with increasing splenomegaly. Long-term hematopoietic stem cells (HSCs), short-term HSCs (ST-HSCs), and multipotent progenitors (MPPs) were all significantly reduced in Klf5(Δ/Δ) mice, and knockdown of KLF5 in human CD34(+) cells suppressed colony-forming potential. ST-HSCs, MPPs, and total numbers of committed progenitors were increased in the spleen of Klf5(Δ/Δ) mice, and reduced ß1- and ß2-integrin expression on hematopoietic progenitors suggests that increased splenic hematopoiesis results from increased stem and progenitor mobilization. Klf5(Δ/Δ) mice show a significant reduction in the fraction of Gr1(+)Mac1(+) cells (neutrophils) in peripheral blood and bone marrow and increased frequency of eosinophils in the peripheral blood, bone marrow, and lung. Thus, these studies demonstrate dual functions of Klf5 in regulating hematopoietic stem and progenitor proliferation and localization in the bone marrow, as well as lineage choice after GMP, promoting increased neutrophil output at the expense of eosinophil production.

Expression of transmembrane protein 26 (TMEM26) in breast cancer and its association with drug response.

We have previously shown that stromal cells desensitize breast cancer cells to the anti-estrogen fulvestrant and, along with it, downregulate the expression of TMEM26 (transmembrane protein 26). In an effort to study the function and regulation of TMEM26 in breast cancer cells, we found that breast cancer cells express non-glycosylated and N-glycosylated isoforms of the TMEM26 protein and demonstrate that N-glycosylation is important for its retention at the plasma membrane. Fulvestrant induced significant changes in expression and in the N-glycosylation status of TMEM26. In primary breast cancer, TMEM26 protein expression was higher in ERα (estrogen receptor α)/PR (progesterone receptor)-positive cancers. These data suggest that ERα is a major regulator of TMEM26. Significant changes in TMEM26 expression and N-glycosylation were also found, when MCF-7 and T47D cells acquired fulvestrant resistance. Furthermore, patients who received aromatase inhibitor treatment tend to have a higher risk of recurrence when tumoral TMEM26 protein expression is low. In addition, TMEM26 negatively regulates the expression of integrin ß1, an important factor involved in endocrine resistance. Data obtained by spheroid formation assays confirmed that TMEM26 and integrin ß1 can have opposite effects in breast cancer cells. These data are consistent with the hypothesis that, in ERα-positive breast cancer, TMEM26 may function as a tumor suppressor by impeding the acquisition of endocrine resistance. In contrast, in ERα-negative breast cancer, particularly triple-negative cancer, high TMEM26 expression was found to be associated with a higher risk of recurrence. This implies that TMEM26 has different functions in ERα-positive and -negative breast cancer.

The NF-κB subunit RelB regulates the migration and invasion abilities and the radio-sensitivity of prostate cancer cells.

NF-κB subunits play important roles in carcinogenesis of a variety of human malignancies and response to cancer therapy; however, the contribution of an individual subunit has not been thoroughly defined. Constitutive activation of the canonical NF-κB subunit is a critical event in prostate carcinogenesis. Recent findings point out that RelB, which contributes to the non-canonical NF-κB activity, functions importantly in the prostate cancer progression. Here, we investigated systemically the functional roles of RelB in prostate cancer and examine its significance as a therapeutic target. Targeting RelB using short hairpin RNA approach in androgen-independent DU145 prostate cancer cells interfered with various biological behaviors of cells. We observed that RelB knockdown inhibited prostate cancer cell growth, migration, and invasion, and enhanced proteasome inhibitor sensitivity. The altered expression of anti-apoptotic gene Bcl-2 played critical roles in regulating both spontaneous and radiation-induced apoptosis in the presence of RelB knockdown. For the first time, we showed that RelB knockdown significantly attenuated the migration and invasion of DU145 prostate cancer cells, due to the reduction of integrin ß-1. Collectively, we provided evidence that RelB functioned as an oncogene in prostate cancer. Developing a RelB-targeted therapeutic intervention, is valuable in treating advanced, metastatic prostate cancer.

miR-199a-5p regulates ß1 integrin through Ets-1 to suppress invasion in breast cancer.

Increasing evidence has revealed that miR-199a-5p is actively involved in tumor invasion and metastasis as well as in the decline of breast cancer tissues. In this research, overexpression of miR-199a-5p weakened motility and invasion of breast cancer cells MCF-7 and MDA-MB-231. Upregulation of Ets-1 increased breast cancer cell invasion, but the mechanism by which miR-199a-5p modulates activation of Ets-1 in breast cancer was not clarified. We investigated the relationship between miR-199a-5p and Ets-1 on the basis of 158 primary breast cancer case specimens, and the results showed that Ets-1 expression was inversely correlated with endogenous miR-199a-5p. Overexpression of miR-199a-5p reduced the mRNA and protein levels of Ets-1 in MCF-7 and MDA-MB-231 cells, whereas anti-miR-199a-5p elevated Ets-1. siRNA-mediated Ets-1 knockdown phenocopied the inhibition invasion of miR-199a-5p in vitro. Moreover, luciferase reporter assay revealed that miR-199a-5p directly targeted 3'-UTR of Ets-1 mRNA. This research revealed that miR-199a-5p could descend the levels of ß1 integrin by targeting 3'-UTR of Ets-1 to alleviate the invasion of breast cancer via FAK/Src/Akt/mTOR signaling pathway. Our results provide insight into the regulation of ß1 integrin through miR-199a-5p-mediated Ets-1 silence and will help in designing new therapeutic strategies to inhibit signal pathways induced by miR-199a-5p in breast cancer invasion.