Claudin-17 Deficiency in Mice Results in Kidney Injury Due to Electrolyte Imbalance and Oxidative Stress.

The multi-gene claudin (CLDN) family of tight junction proteins have isoform-specific roles in blood-tissue barrier regulation. CLDN17, a putative anion pore-forming CLDN based on its structural characterization, is assumed to regulate anion balance across the blood-tissue barriers. However, our knowledge about CLDN17 in physiology and pathology is limited. The current study investigated how Cldn17 deficiency in mice affects blood electrolytes and kidney structure. Cldn17-/- mice revealed no breeding abnormalities, but the newborn pups exhibited delayed growth. Adult Cldn17-/- mice displayed electrolyte imbalance, oxidative stress, and injury to the kidneys. Ingenuity pathway analysis followed by RNA-sequencing revealed hyperactivation of signaling pathways and downregulation of SOD1 expression in kidneys associated with inflammation and reactive oxygen species generation, demonstrating the importance of Cldn17 in the maintenance of electrolytes and reactive oxygen species across the blood-tissue barrier.

Regulation of Let-7a-5p and miR-199a-5p Expression by Akt1 Modulates Prostate Cancer Epithelial-to-Mesenchymal Transition via the Transforming Growth Factor-ß Pathway.

Akt1 suppression in advanced cancers has been indicated to promote metastasis. Our understanding of how Akt1 orchestrates this is incomplete. Using the NanoString®-based miRNA and mRNA profiling of PC3 and DU145 cells, and subsequent data analysis using the DIANA-mirPath, dbEMT, nCounter, and Ingenuity® databases, we identified the miRNAs and associated genes responsible for Akt1-mediated prostate cancer (PCa) epithelial-to-mesenchymal transition (EMT). Akt1 loss in PC3 and DU145 cells primarily induced changes in the miRNAs and mRNAs regulating EMT genes. These include increased miR-199a-5p and decreased let-7a-5p expression associated with increased TGFß-R1 expression. Treatment with locked nucleic acid (LNA) miR-199a-5p inhibitor and/or let-7a-5p mimic induced expression changes in EMT genes correlating to their anticipated effects on PC3 and DU145 cell motility, invasion, and TGFß-R1 expression. A correlation between increased miR-199a-5p and TGFß-R1 expression with reduced let-7a-5p was also observed in high Gleason score PCa patients in the cBioportal database analysis. Collectively, our studies show the effect of Akt1 suppression in advanced PCa on EMT modulating miRNA and mRNA expression changes and highlight the potential benefits of miR-199a-5p and let-7a-5p in therapy and/or early screening of mPCa.

Neuroprotective Effects of Fingolimod in a Cellular Model of Optic Neuritis.

Visual dysfunction resulting from optic neuritis (ON) is one of the most common clinical manifestations of multiple sclerosis (MS), characterized by loss of retinal ganglion cells, thinning of the nerve fiber layer, and inflammation to the optic nerve. Current treatments available for ON or MS are only partially effective, specifically target the inflammatory phase, and have limited effects on long-term disability. Fingolimod (FTY) is an FDA-approved immunomodulatory agent for MS therapy. The objective of the current study was to evaluate the neuroprotective properties of FTY in the cellular model of ON-associated neuronal damage. R28 retinal neuronal cell damage was induced through treatment with tumor necrosis factor-α (TNFα). In our cell viability analysis, FTY treatment showed significantly reduced TNFα-induced neuronal death. Treatment with FTY attenuated the TNFα-induced changes in cell survival and cell stress signaling molecules. Furthermore, immunofluorescence studies performed using various markers indicated that FTY treatment protects the R28 cells against the TNFα-induced neurodegenerative changes by suppressing reactive oxygen species generation and promoting the expression of neuronal markers. In conclusion, our study suggests neuroprotective effects of FTY in an in vitro model of optic neuritis.

Inhibition of glypican-1 expression induces an activated fibroblast phenotype in a human bone marrow-derived stromal cell-line.

Cancer-associated fibroblasts (CAFs) are the most abundant stromal cell type in the tumor microenvironment. CAFs orchestrate tumor-stromal interactions, and contribute to cancer cell growth, metastasis, extracellular matrix (ECM) remodeling, angiogenesis, immunomodulation, and chemoresistance. However, CAFs have not been successfully targeted for the treatment of cancer. The current study elucidates the significance of glypican-1 (GPC-1), a heparan sulfate proteoglycan, in regulating the activation of human bone marrow-derived stromal cells (BSCs) of fibroblast lineage (HS-5). GPC-1 inhibition changed HS-5 cellular and nuclear morphology, and increased cell migration and contractility. GPC-1 inhibition also increased pro-inflammatory signaling and CAF marker expression. GPC-1 induced an activated fibroblast phenotype when HS-5 cells were exposed to prostate cancer cell conditioned media (CCM). Further, treatment of human bone-derived prostate cancer cells (PC-3) with CCM from HS-5 cells exhibiting GPC-1 loss increased prostate cancer cell aggressiveness. Finally, GPC-1 was expressed in mouse tibia bone cells and present during bone loss induced by mouse prostate cancer cells in a murine prostate cancer bone model. These data demonstrate that GPC-1 partially regulates the intrinsic and extrinsic phenotype of human BSCs and transformation into activated fibroblasts, identify novel functions of GPC-1, and suggest that GPC-1 expression in BSCs exerts inhibitory paracrine effects on the prostate cancer cells. This supports the hypothesis that GPC-1 may be a novel pharmacological target for developing anti-CAF therapeutics to control cancer.

Akt-independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS-CoV2 infection.

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.

ALK-1 to ALK-5 ratio dictated by the Akt1-ß-catenin pathway regulates TGFß-induced endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) indispensable in embryogenesis also occurs in several human pathologies. Although transforming growth factor-ß (TGFß) has been demonstrated to induce EndMT, the type-I receptors (ALK-1 and ALK-5) responsible for TGFß-induced EndMT is unclear. In the current study, we investigated the role of the Akt1 pathway in ALK1 and ALK5 expression regulation in response to TGFß1 and TGFß2 in human microvascular endothelial cells (HMECs). Whereas treatment with TGFß1 and TGFß2 or Akt1 gene silencing promoted EndMT accompanied by increased ALK5 expression and reduced ALK1 expression accompanied by increased expression of N-cadherin and reduced expression of eNOS in HMECs, treatment with ALK-5 inhibitor (SB431542) blunted these effects. Importantly, the inhibitor of ß-catenin (ICG-001) suppressed TGFß1- and TGFß2-induced ALK5 expression in both normal and Akt1 deficient HMECs indicating the integral role of Akt1-ß-catenin pathway in the regulation of ALK5 expression promoting EndMT.

Distinct effects of pharmacological inhibition of stromelysin1 on endothelial-to-mesenchymal transition and myofibroblast differentiation.

Endothelial-to-mesenchymal transition (EndMT) and fibroblast-to-myofibroblast (FibroMF) differentiation are frequently reported in organ fibrosis. Stromelysin1, a matrix metalloprotease-3 (MMP3) has been indicated in vascular pathologies and organ injuries that often lead to fibrosis. In the current study, we investigated the role of stromelysin1 in EndMT and FibroMF differentiation, which is currently unknown. In our results, whereas TGFß2 treatment of endothelial cells (ECs) induced EndMT associated with increased expression of stromelysin1 and mesenchymal markers such as α-smooth muscle actin (αSMA), N-cadherin, and activin linked kinase-5 (ALK5), inhibition of stromelysin1 blunted TGFß2-induced EndMT. In contrast, treatment of NIH-3T3 fibroblasts with TGFß1 promoted FibroMF differentiation accompanied by increased expression of αSMA, N-cadherin, and ALK5. Intriguingly, stromelysin1 inhibition in TGFß1-stimulated myofibroblasts further exacerbated fibroproliferation with increased FibroMF marker expression. Gene Expression Omnibus (GEO) data analysis indicated increased stromelysin1 expression associated with EndMT and decreased stromelysin1 expression in human pulmonary fibrosis fibroblasts. In conclusion, our study has identified that EndMT and FibroMF differentiation are reciprocally regulated by stromelysin1.

Sterically stabilized liposomes targeting P21 (RAC1) activated kinase-1 and secreted phospholipase A2 suppress prostate cancer growth and metastasis.

Metastatic prostate cancer (PCa) has a very high mortality rate in men, in Western countries and lacks reliable treatment. The advanced-stage PCa cells overexpress P21 (RAC1) activated kinase-1 (PAK1) and secreted phospholipase A2 (sPLA2) suggesting the potential utility of pharmacologically targeting these molecules to treat metastatic PCa. The small molecule, inhibitor targeting PAK1 activation-3 (IPA3) is a highly specific allosteric inhibitor of PAK1; however, it is metabolically unstable once in the plasma thus, limiting its utility as a chemotherapeutic agent. In the present study, the efficacy and specificity of IPA3 were combined with the stability and the sPLA2-targeted delivery method of two sterically stabilized liposomes [sterically stabilized long-circulating liposomes (SSL)-IPA3 and sPLA2 responsive liposomes (SPRL)-IPA3, respectively] to inhibit PCa growth and metastasis. It was found that twice-a-week administration of either SSL-IPA3 or SPRL-IPA3 for 3 weeks effectively suppressed the growth of PC-3 cell tumor xenografts implanted in athymic nude mice. Both drug formulations also inhibited the metastasis of intravenously administered murine RM1 PCa cells to the lungs of C57BL/6 mice. Whereas the twice-a-week administration of SSL-IPA3 significantly inhibited the spontaneous PCa metastasis to the lungs in Transgenic Adenocarcinoma of the Mouse Prostate mice, the administration of free IPA3 had no significant therapeutic benefit. The results present two novel IPA3 encapsulated liposomes to treat metastatic PCa.

Differential regulation of TGFß type-I receptor expressions in TGFß1-induced myofibroblast differentiation.

Fibroblast-to-myofibroblast (FibroMF) differentiation is crucial for embryogenesis and organ fibrosis. Although transforming growth factor-ß (TGFß) is the primary mediator of FibroMF differentiation, the type-I receptor (TGFßRI) responsible for this has not yet been confirmed. In the current study, we investigated the ALK1 and ALK5 expressions in TGFß1-stimulated NIH 3T3 fibroblasts to compare with the data from the Gene Expression Omnibus (GEO) repository. In our results, whereas TGFß1 treatment promoted FibroMF differentiation accompanied by increased ALK5 expression and reduced ALK1 expression, TGFß1-induced FibroMF differentiation and increased α-smooth muscle actin (αSMA) and ALK5 expression were inhibited by co-treatment with ALK5 inhibitor SB431542. GEO database analysis indicated increased ALK5 expression and reduced ALK1 expression in fibrotic compared to normal mouse or human tissues correlating with organ fibrosis progression. Finally, the inhibitors of Akt, mTOR, and ß-catenin suppressed TGFß1-induced ALK5 expression, indicating that the Akt pathway promotes FibroMF differentiation via ALK5 expression and fibrosis.

Patients with acute respiratory distress syndrome exhibit increased stromelysin1 activity in the blood samples.

Enzyme activity analyses in the blood are expected to be reliable, non-invasive diagnostic as well as prognostic markers to reflect disease progression in acute lung injury (ALI). The objective of the current study was to evaluate the enzymatic activity of stromelysin1 (matrix metalloprotease-3) in the plasma/serum samples collected from ALI patients compared to the samples collected from healthy controls. Gene expression omnibus (GEO) database analysis indicated a correlation between increased stromelysin1 gene expression and the incidence of ALI in various animal models. Our analysis of patient plasma/serum samples from healthy controls and ALI patients revealed a significant, 3-fold increase in stromelysin1 activity in ALI plasma/serum compared to healthy subjects with no difference in stromelysin1 activity between the serum and plasma samples. Interestingly, no significant difference in stromelysin1 activity between non-smoking and smoking subjects was observed. These findings provide fundamental information on the potential reliability of stromelysin1 activity analysis, combined with other biomarkers in development, in blood samples for the early detection of ALI.

PAK1 inhibitor IPA-3 mitigates metastatic prostate cancer-induced bone remodeling.

Metastatic prostate cancer (PCa) has high mortality and a poor 5-year survival rate primarily due to the lack of effective treatments. Bone is the primary site of PCa metastasis in humans and the development of reliable therapeutic options for bone metastatic PCa will make a huge impact in reducing the mortality among these patients. Although P21 activated kinases (PAKs) have been studied in the past for their role in cancer, the efficacy of targeting PAKs to treat lung and bone metastatic PCa has not been tested yet. In the current study, we report that targeting PAK1 using IPA-3, an allosteric inhibitor of PAK1 kinase activity, significantly inhibits the murine metastatic PCa (RM1) cell proliferation and motility in vitro, and metastasis to the lungs in vivo. More importantly, we demonstrate for the first time that treatment with IPA-3 can blunt metastatic PCa-induced bone remodeling in vivo as analyzed by the 3-dimensional microcomputer tomography analysis. Our study has identified IPA-3 as a potential drug to treat bone metastatic PCa.

Delayed Akt suppression in the lipopolysaccharide-induced acute lung injury promotes resolution that is associated with enhanced effector regulatory T cells.

The adaptive immune response could play a major role in the resolution of lung injury. Although regulatory T cells (Tregs) have been implicated in promoting the resolution of lung injury, therapeutic strategies to enhance Treg quantity and activity at the site of injury need further exploration. In the current study, Akt inhibition using triciribine (TCBN), given 48 h after lipopolysaccharide (LPS) administration, increased Tregs-promoted resolution of acute lung injury (ALI). TCBN treatment enhanced the resolution of LPS-induced ALI on day 7 by reducing pulmonary edema and neutrophil activity associated with an increased number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs, specifically in the injured lungs and not in the spleen. Treatment of EL-4 T-lymphocytes with two Akt inhibitors (TCBN and MK-2206) for 72 h resulted in increased FoxP3 expression in vitro. On the other end, Treg-specific PTEN knockout (PTENTreg KO) mice that have a higher Akt activity in its Tregs exhibited a significant impairment in ALI resolution, increased edema, and neutrophil activity associated with a reduced number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs as compared with the control group. In conclusion, our study identifies a potential target for the treatment of late-stage ALI by promoting resolution through effector Treg-mediated suppression of inflammation.

Nodal pathway activation due to Akt1 suppression is a molecular switch for prostate cancer cell epithelial-to-mesenchymal transition and metastasis.

Several studies have unraveled the negative role of Akt1 in advanced cancers, including metastatic prostate cancer (mPCa). Hence, understanding the consequences of targeting Akt1 in the mPCa and identifying its downstream novel targets is essential. We studied how Akt1 deletion in PC3 and DU145 cells activates the Nodal pathway and promotes PCa epithelial-to-mesenchymal transition (EMT) and metastasis. Here we show that Akt1 loss increases Nodal expression in PCa cells accompanied by activation of FoxO1/3a, and EMT markers Snail and N-cadherin as well as loss of epithelial marker E-cadherin. Treatment with FoxO inhibitor AS1842856 abrogated the Nodal expression in Akt1 deleted PCa cells. Akt1 deficient PCa cells exhibited enhanced cell migration and invasion in vitro and lung metastasis in vivo, which were attenuated by treatment with Nodal pathway inhibitor SB505124. Interestingly, Nodal mRNA analysis from two genomic studies in cBioportal showed a positive correlation between Nodal expression and Gleason score indicating the positive role of Nodal in human mPCa. Collectively, our data demonstrate Akt1-FoxO3a-Nodal pathway as an important mediator of PCa metastasis and present Nodal as a potential target to treat mPCa patients.

The unconventional role of Akt1 in the advanced cancers and in diabetes-promoted carcinogenesis.

Decades of research have elucidated the critical role of Akt isoforms in cancer as pro-tumorigenic and metastatic regulators through their specific effects on the cancer cells, tumor endothelial cells and the stromal cells. The pro-cancerous role of Akt isoforms through enhanced cell proliferation and suppression of apoptosis in cancer cells and the cells in the tumor microenvironment is considered a dogma. Intriguingly, studies also indicate that the Akt pathway is essential to protect the endothelial-barrier and prevent aberrant vascular permeability, which is also integral to tumor perfusion and metastasis. To complicate this further, a flurry of recent reports strongly indicates the metastasis suppressive role of Akt, Akt1 in particular in various cancer types. These reports emanated from different laboratories have elegantly demonstrated the paradoxical effect of Akt1 on cancer cell epithelial-to-mesenchymal transition, invasion, tumor endothelial-barrier disruption, and cancer metastasis. Here, we emphasize on the specific role of Akt1 in mediating tumor cell-vasculature reciprocity during the advanced stages of cancers and discuss how Akt1 differentially regulates cancer metastasis through mechanisms distinct from its pro-tumorigenic effects. Since Akt is integral for insulin signaling, endothelial function, and metabolic regulation, we also attempt to shed some light on the specific effects of diabetes in modulating Akt pathway in the promotion of tumor growth and metastasis.

Pharmacological inhibition of ß-catenin prevents EndMT in vitro and vascular remodeling in vivo resulting from endothelial Akt1 suppression.

Endothelial to mesenchymal transition (EndMT), where endothelial cells acquire mesenchymal characteristics has been implicated in several cardiopulmonary, vascular and fibrotic diseases. The most commonly studied molecular mechanisms involved in EndMT include TGFß, Notch, interleukin, and interferon-γ signaling. As of today, the contributions of Akt1, an important mediator of TGFß signaling and a key regulator of endothelial barrier function to EndMT remains unclear. By using the ShRNA based gene silencing approach and endothelial-specific inducible Akt1 knockdown (ECKOAkt1) mice, we studied the role of Akt1 in EndMT in vitro and pathological vascular remodeling in vivo. Stable, Akt1 silenced (ShAkt1) human microvascular endothelial cells (HMECs) indicated increased expression of mesenchymal markers such as N-cadherin and α-SMA, phosphorylation of Smad2/3, cellular stress via activation of p38 MAP Kinase and the loss of endothelial nitric oxide synthase (eNOS) accompanied by a change in the morphology of HMECs in vitro and co-localization of endothelial and mesenchymal markers promoting EndMT in vivo. EndMT as a result of Akt1 loss was associated with increased expression of TGFß2, a potent inducer of EndMT and mesenchymal transcription factors Snail1, and FoxC2. We observed that hypoxia-induced lung vascular remodeling is exacerbated in ECKOAkt1 mice, which was reversed by pharmacological inhibition of ß-catenin. Thus, we provide novel insights into the role of Akt1-mediated ß-catenin signaling in EndMT and pathological vascular remodeling, and present ß-catenin as a potential target for therapy for various cardiopulmonary diseases involving vascular remodeling.

Estimation of genetic architecture of biochemical traits in mid-late cauliflower (Brassica oleracea L. var. botrytis) under sub-temperate conditions of north western Himalayas.

Understanding the genetic basis of biochemical traits of different cauliflower genotypes is essential for planning the effective breeding strategies in genetic improvement. To determine the mode of inheritance of dry matter content and biochemical traits, we made crosses using four genotypes of cauliflower, and obtained F1, F2, BC1 and BC2 populations. The six generations obtained were replicated thrice and evaluated in a randomized block design. The generation mean analysis of data showed the presence of duplicate epistasis in dry matter content which suggested the adoption of reciprocal recurrent selection and biparental mating for the improvement of the trait. However, in case of vitamin C, complementary type of epistasis was reported in three crosses, which indicated the exploitation of heterosis breeding of enhancing vitamin C. It can be concluded that the role of gene action was in general more complex for the traits studied. The nature and magnitude of gene effects varies character-wise as well as cross-wise. Hence, for the improvement of dry matter content and biochemical traits in a particular cross, a specific breeding strategy has to be implemented.

Endothelial stromelysin1 regulation by the forkhead box-O transcription factors is crucial in the exudative phase of acute lung injury.

Enhanced vascular permeability is associated with inflammation and edema in alveoli during the exudative phase of acute respiratory distress syndrome (ARDS). Mechanisms leading to the endothelial contribution on the early exudative stage of ARDS are not precise. We hypothesized that modulation of endothelial stromelysin1 expression and activity by Akt1-forkhead box-O transcription factors 1/3a (FoxO1/3a) pathway could play a significant role in regulating pulmonary edema during the initial stages of acute lung injury (ALI). We utilized lipopolysaccharide (LPS)-induced mouse ALI model in vivo and endothelial barrier resistance measurements in vitro to determine the specific role of the endothelial Akt1-FoxO1/3a-stromelysin1 pathway in ALI. LPS treatment of human pulmonary endothelial cells resulted in increased stromelysin1 and reduced tight junction claudin5 involving FoxO1/3a, associated with decreased trans-endothelial barrier resistance as determined by electric cell-substrate impedance sensing technology. In vivo, LPS-induced lung edema was significantly higher in endothelial Akt1 knockdown (EC-Akt1-/-) compared to wild-type mice, which was reversed upon treatment with FoxO inhibitor (AS1842856), stromelysin1 inhibitor (UK356618) or with shRNA-mediated FoxO1/3a depletion in the mouse lungs. Overall, our study provides the hope that targeting FoxO and styromelysin1 could be beneficial in the treatment of ALI.

Isoform-specific effects of transforming growth factor ß on endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) was first reported in the embryogenesis. Recent studies show that EndMT also occurs in the disease progression of atherosclerosis, cardiac and pulmonary fibrosis, pulmonary hypertension, diabetic nephropathy, and cancer. Although transforming growth factor ß (TGFß) is crucial for EndMT, it is not clear which isoform elicits a predominant effect. The current study aims to directly compare the dose-dependent effects of TGFß1, TGFß2, and TGFß3 on EndMT and characterize the underlying mechanisms. In our results, all three TGFß isoforms induced EndMT in human microvascular endothelial cells after 72 hr, as evidenced by the increased expression of mesenchymal markers N-cadherin and α-smooth muscle actin as well as the decreased expression of endothelial nitric oxide synthase. Interestingly, the effect of TGFß2 was the most pronounced. At 1 ng/ml, only TGFß2 treatment resulted in significantly increased phosphorylation (activation) of Smad2/3 and p38-MAPK and increased expression of mesenchymal transcription factors Snail and FoxC2. Intriguingly, we observed that treatment with 1 ng/ml TGFß1 and TGFß3, but not TGFß2, resulted in an increased expression of TGFß2, thus indicating that EndMT with TGFß1 and TGFß3 treatments was due to the secondary effects through TGFß2 secretion. Furthermore, silencing TGFß2 using small interfering RNA blunted the expression of EndMT markers in TGFß1- and TGFß3-treated cells. Together, our results indicate that TGFß2 is the most potent inducer of EndMT and that TGFß1- and TGFß3-induced EndMT necessitates a paracrine loop involving TGFß2.

Endothelial Akt1 loss promotes prostate cancer metastasis via ß-catenin-regulated tight-junction protein turnover.

BACKGROUND: Cancer research, in general, is focused on targeting tumour cells to limit tumour growth. These studies, however, do not account for the specific effects of chemotherapy on tumour endothelium, in turn, affecting metastasis. METHODS: We determined how endothelial deletion of Akt1 promotes prostate cancer cell invasion in vitro and metastasis to the lungs in vivo in endothelial-specific Akt1 knockdown mice. RESULTS: Here we show that metastatic human PC3 and DU145 prostate cancer cells invade through Akt1-deficient human lung endothelial cell (HLEC) monolayer with higher efficiency compared to control HLEC. Although the endothelial Akt1 loss in mice had no significant effect on RM1 tumour xenograft growth in vivo, it promoted metastasis to the lungs compared to the wild-type mice. Mechanistically, Akt1-deficient endothelial cells exhibited increased phosphorylation and nuclear translocation of phosphorylated ß-catenin, and reduced expression of tight-junction proteins claudin-5, ZO-1 and ZO-2. Pharmacological inhibition of ß-catenin nuclear translocation using compounds ICG001 and IWR-1 restored HLEC tight-junction integrity and inhibited prostate cancer cell transendothelial migration in vitro and lung metastasis in vivo. CONCLUSIONS: Here we show for the first time that endothelial-specific loss of Akt1 promotes cancer metastasis in vivo involving ß-catenin pathway.

Suppression of Akt1-ß-catenin pathway in advanced prostate cancer promotes TGFß1-mediated epithelial to mesenchymal transition and metastasis.

Akt1 is essential for the oncogenic transformation and tumor growth in various cancers. However, the precise role of Akt1 in advanced cancers is conflicting. Using a neuroendocrine TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model, we first show that the genetic ablation or pharmacological inhibition of Akt1 in mice blunts oncogenic transformation and prostate cancer (PCa) growth. Intriguingly, triciribine (TCBN)-mediated Akt inhibition in 25-week old, tumor-bearing TRAMP mice and Akt1 gene silencing in aggressive PCa cells enhanced epithelial to mesenchymal transition (EMT) and promoted metastasis to the lungs. Mechanistically, Akt1 suppression leads to increased expression of EMT markers such as Snail1 and N-cadherin and decreased expression of epithelial marker E-cadherin in TRAMP prostate, and in PC3 and DU145 cells. Next, we identified that Akt1 knockdown in PCa cells results in increased production of TGFß1 and its receptor TGFß RII, associated with a decreased expression of ß-catenin. Furthermore, treatment of PCa cells with ICG001 that blocks nuclear translocation of ß-catenin promoted EMT and N-cadherin expression. Together, our study demonstrates a novel role of the Akt1-ß-catenin-TGFß1 pathway in advanced PCa.