Activation Of Akt Signaling Pathways Research Articles

Lycium barbarum Polysaccharide Ameliorates Heat-Stress-Induced Impairment of Primary Sertoli Cells and the Blood-Testis Barrier in Rat via Androgen Receptor and Akt Phosphorylation.

Male infertility induced by heat stress has been attracting more and more attention. Heat stress not only causes apoptosis of spermatocytes but also has adverse effects on Sertoli cells, further damaging spermatogenesis. Lycium barbarum polysaccharide (LBP) is the main bioactive component of Lycium barbarum, which has a protective effect on male reproduction, but its mechanism is still unclear. In this study, our results proved that LBP blocked the inhibitory effect on the proliferation activity of Sertoli cells after heat stress, reversed the dedifferentiation of Sertoli cells induced by heat stress, and ameliorated the structural integrity of the blood-testis barrier. In addition, it increased the expression of the androgen receptor and activated Akt signaling pathway to resist heat-stress-induced injury of Sertoli cells.

NAT10 Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Mediating N4-Acetylcytidine Modification of Gremlin 1.

Objective To investigate the function of NAT10 in mesenchymal stem cell (MSC) osteogenic differentiation and study the mechanism by which NAT10 affects MSC osteogenesis by mediating Gremlin 1 N4-acetylcytidine (ac4C) modification. Methods Osteogenic differentiation of MSCs was induced, and the osteogenic ability was evaluated with alizarin red S (ARS) and alkaline phosphatase (ALP) assays. The NAT10 expression level during MSC osteogenesis was measured by western blot (WB). MSCs were transfected with lentiviruses to inhibit (Sh-NAT10) or overexpress NAT10 (Over-NAT10), and the osteogenic differentiation ability was assessed by ARS, ALP, and osteogenic gene marker assays. β-Catenin, Akt, and Smad signaling pathway component activation levels were assessed, and the expression levels of key Smad signaling pathway molecules were determined by PCR and WB. The Gremlin 1 mRNA ac4C levels were analyzed using RIP-PCR, and the Gremlin 1 mRNA degradation rate was determined. Sh-Gremlin 1 was transfected to further investigate the role of NAT10 and Gremlin 1 in MSC osteogenesis. Results During MSC osteogenesis, NAT10 expression, ARS staining, and the ALP level gradually increased. Decreasing NAT10 expression inhibited, and increasing NAT10 expression promoted MSC osteogenic differentiation. NAT10 affected the BMP/Smad rather than the Akt and β-Catenin signaling pathway activation by regulating Gremlin 1 expression. The Gremlin 1 mRNA ac4C level was positively regulated by NAT10, which accelerated Gremlin 1 degradation. Sh-Gremlin 1 abolished the promotive effect of NAT10 on MSC osteogenic differentiation. Conclusion NAT10 positively regulated MSC osteogenic differentiation through accelerating the Gremlin 1 mRNA degradation by increasing its ac4C level. These results may provide new mechanistic insight into MSC osteogenesis and bone metabolism in vivo.

Copper and Selenium stimulates CYP19A1 expression in caprine ovarian granulosa cells: possible involvement of AKT and WNT signalling pathways.

The role of copper and selenium on activation of estradiol synthesis pathways viz. PKA/AKT/WNT is not clearly elucidated. On this background we attempt to elcuiated the role of copper and selenium on mRNA expression of genes associated with estradiol synthesis in caprine ovarian granulose cell models. Ovarian granulosa cells from medium (3-5mm) sized follicles were aspirated and distributed separately to different groups. Group I: control, Group II: cupric chloride (Cu: 0.5mM), Group III: sodium selenite (Se: 100ng/ml), Group IV:Cu + Se. The cells (105/well) were cultured in 96 well plate in the base culture medium of MEMα comprising of nonessential amino acids (1.1mM), FSH (10ng/mL), transferrin (5µg/mL), IGF-I (2ng/mL), androstenedione (10-6M), penicillin (100IU/mL), streptomycin (0.1mg/mL) and fungizone (0.625µl/mL) and insulin (1ng/mL). The cells were incubated in a carbondioxide incubator (38 °C, 5% CO2, 95% RH). The medium was changed on alternate days and cells were harvested on day 6. Day 6 media was used for estimation of estradiol. The RNA isolated form harvested cells was used for qPCR assay. There was no significant (p > 0.05) difference in estradiol concentration between groups. The mRNA expression of AKT1, CYP19A1, WNT2 & 4, FZD6 and APC2 were significantly (p < 0.05) higher in Cu and Cu + Se groups compared to control. Whereas, the mRNA transcript of DVL1 and CSNK1 was significantly (p < 0.05) higher in Cu + Se group compared to control. Incontrast, no significant difference in mRNA expression of PRKAR1A and CTNNB1 was noticed. Our study support a key role of copper and selenium in activation of AKT and WNT signalling pathway that further lead to increase in the mRNA expression of CYP19A1.

Exosomal miR-222 from adriamycin-resistant MCF-7 breast cancer cells promote macrophages M2 polarization via PTEN/Akt to induce tumor progression.

Exosome-mediated intercellular communication is considered to be an effective mode for malignant cells to transform biological behaviors in stromal cells. However, the mechanisms by which exosomes modulate macrophages within tumor microenvironment remain largely unclear. In this study, we found that both adriamycin-resistant breast cancer (BCa) cells and the corresponding exosomes (A/exo) were capable of inducing macrophages M2 polarization, which promoted the mobility, proliferation, migration and invasion of BCa cells. Since exosomes deliver microRNAs to affect cellular functions in recipient cells, we confirmed that miR-222 was significantly enriched in A/exo and could be successfully transferred to macrophages. Increased miR-222 level was also detected in exosomes derived from plasma and tissues of chemoresistant patients. Moreover, exosomal miR-222 from A/exo polarized M2 macrophages by targeting PTEN and activating Akt signaling pathway, which promoted BCa cells progression in a feed back loop. Co-culture of adriamycin-resistant BCa cells with macrophages in which miR-222 was upregulated or treated with A/exo facilitated tumor growth in vivo. Collectively, our data demonstrated that chemoresistant BCa cells could remodel macrophages within tumor microenvironment by secreting exosomal miR-222, which directly targeted PTEN and caused Akt cascade activation and macrophages M2 polarization. Our findings may provide a foundation for a promising strategy of BCa treatment by targeting exosomes or exosomal miR-222.

Silencing of lncRNA UCA1 inhibited the pathological progression in PCOS mice through the regulation of PI3K/AKT signaling pathway

BackgroundPolycystic ovary syndrome (PCOS) is the most common hormonal disorder among reproductive-aged women worldwide, however, the mechanisms and progression of PCOS still unclear due to its heterogeneous nature. Using the human granulosa-like tumor cell line (KGN) and PCOS mice model, we explored the function of lncRNA UCA1 in the pathological progression of PCOS.ResultsCCK8 assay and Flow cytometry were used to do the cell cycle, apoptosis and proliferation analysis, the results showed that UCA1 knockdown in KGN cells inhibited cell proliferation by blocking cell cycle progression and promoted cell apoptosis. In the in vivo experiment, the ovary of PCOS mice was injected with lentivirus carrying sh-UCA1, the results showed that knockdown of lncRNA UCA1 attenuated the ovary structural damage, increased the number of granular cells, inhibited serum insulin and testosterone release, and reduced the pro-inflammatory cytokine production. Western blot also revealed that UCA1 knockdown in PCOS mice repressed AKT activation, inhibitor experiment demonstrated that suppression of AKT signaling pathway, inhibited the cell proliferation and promoted apoptosis.ConclusionsOur study revealed that, in vitro, UCA1 knockdown influenced the apoptosis and proliferation of KGN cells, in vivo, silencing of UCA1 regulated the ovary structural damage, serum insulin release, pro-inflammatory production, and AKT signaling pathway activation, suggesting lncRNA UCA1 plays an important role in the pathological progression of PCOS.

Phloridzin Acts as an Inhibitor of Protein-Tyrosine Phosphatase MEG2 Relevant to Insulin Resistance.

Inhibition of the megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2, also named PTPN9) activity has been shown to be a potential therapeutic strategy for the treatment of type 2 diabetes. Previously, we reported that PTP-MEG2 knockdown enhances adenosine monophosphate activated protein kinase (AMPK) phosphorylation, suggesting that PTP-MEG2 may be a potential antidiabetic target. In this study, we found that phloridzin, isolated from Ulmus davidiana var. japonica, inhibits the catalytic activity of PTP-MEG2 (half-inhibitory concentration, IC50 = 32 ± 1.06 μM) in vitro, indicating that it could be a potential antidiabetic drug candidate. Importantly, phloridzin stimulated glucose uptake by differentiated 3T3-L1 adipocytes and C2C12 muscle cells compared to that by the control cells. Moreover, phloridzin led to the enhanced phosphorylation of AMPK and Akt relevant to increased insulin sensitivity. Importantly, phloridzin attenuated palmitate-induced insulin resistance in C2C12 muscle cells. We also found that phloridzin did not accelerate adipocyte differentiation, suggesting that phloridzin improves insulin sensitivity without significant lipid accumulation. Taken together, our results demonstrate that phloridzin, an inhibitor of PTP-MEG2, stimulates glucose uptake through the activation of both AMPK and Akt signaling pathways. These results strongly suggest that phloridzin could be used as a potential therapeutic candidate for the treatment of type 2 diabetes.

Effects and Mechanisms of Tripartite Motif Containing 14 Downregulation on Proliferation, Migration, and Invasion of Cancerous Pancreatic PANC-1 Cells

This study aimed to investigate the effect and mechanism of TRIM14 downregulation on the apoptosis, migration, and invasion of cancerous pancreatic PANC-1 cells. PANC-1 cells cultured in vitrowere classified to a control (normal culture), negative (neutral siRNA transfection), and siTRIM14 group (TRIM14 siRNA transfection). RT-PCR was adopted to test TRIM14 mRNA expression. Cellular proliferation was determined by CCK-8, and transwell chamber invasion and apoptosis by flow cytometry. AKT signaling pathway related proteins CyclinD1, MMP-2, Bcl-2, and AKT phosphorylation, and TRIMI14 protein expression, were determined by western blotting. Compared with the control group, TRIMI14 expression, cellular proliferation ability, infiltration, transfer AKT phosphorylation, and TRIMI14, CyclinD1, MMP-2, and Bcl-2 protein expression were greatly reduced in siTRIM14 cells, and the apoptotic ability was significantly enhanced (P &lt; 0.05). However, no striking differences were detected between the negative and control groups (P &gt; 0.05). Downregulating TRIM14 expression can inhibit the proliferation, invasion, and migration of PANC-1 cells, and promote apoptosis. The mechanism may be associated with the inhibition of AKT signaling pathway activation.

Twisted gastrulation signaling modulator 1 promotes the ability of glioma cell through activating Akt pathway.

Glioblastoma is one of the most common primary nervous system tumors and has a high mortality rate. It is necessary to explore a novel biological target and treatment approach. Twisted gastrulation signaling modulator 1 (TWSG1) is expressed in many tumors and closely related to tumor growth and proliferation. However, there is almost no report about the mechanism of TWSG1 in glioma. We used a glioma chip to detect the expression level of TWSG1 by Immunohistochemistry. The overexpression and silence experiments of TWSG1 were performed to assay the biological function of TWSG1 in LN229 and U251 cells. Subcutaneous xenograft mouse model presented the effect of TWSG1 expression on the malignant behavior of tumor cells. Experimental results displayed that the expression level for TWSG1 was substantially elevated in gliomas compared to that in normal brain tissue. The expression knockdown of TWSG1 caused inhibition of glioma cell proliferation. Besides, TWSG1 overexpression enhanced proliferation in glioma cells, and the capacity of proliferation was partly abolished by the PI3K inhibitor LY294002. We found that TWSG1 affected the activity of Akt signaling pathway. In conclusion, TWSG1 is overexpressed in glioma tissue and promotes tumor proliferation through Akt signaling pathway, may serve as a potential target for glioma diagnosis and therapy.

Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells.

Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins.

Isoforms of MUC16 activate oncogenic signaling through EGF receptors to enhance the progression of pancreatic cancer

Aberrant expression of CA125/MUC16 is associated with pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. However, knowledge of the contribution of MUC16 to pancreatic tumorigenesis is limited. Here, we show that MUC16 expression is associated with disease progression, basal-like and squamous tumor subtypes, increased tumor metastasis, and short-term survival of PDAC patients. MUC16 enhanced tumor malignancy through the activation of AKT and GSK3β oncogenic signaling pathways. Activation of these oncogenic signaling pathways resulted in part from increased interactions between MUC16 and epidermal growth factor (EGF)-type receptors, which were enhanced for aberrant glycoforms of MUC16. Treatment of PDAC cells with monoclonal antibody (mAb) AR9.6 significantly reduced MUC16-induced oncogenic signaling. mAb AR9.6 binds to a unique conformational epitope on MUC16, which is influenced by O-glycosylation. Additionally, treatment of PDAC tumor-bearing mice with either mAb AR9.6 alone or in combination with gemcitabine significantly reduced tumor growth and metastasis. We conclude that the aberrant expression of MUC16 enhances PDAC progression to an aggressive phenotype by modulating oncogenic signaling through ErbB receptors. Anti-MUC16 mAb AR9.6 blocks oncogenic activities and tumor growth and could be a novel immunotherapeutic agent against MUC16-mediated PDAC tumor malignancy.

Abstract PO-054: A stromal PTEN/STAT3 signaling axis promotes immune evasion and tumor progression in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDAC) is among the most lethal cancer types, with more than 90% of patients succumbing to the disease within five years of diagnosis. The most notable hallmark of PDAC tumors is an inordinately dense stromal reaction comprised of extracellular matrix proteins, immune infiltrates, endothelial cells, and activated fibroblasts. The stroma experiences a dramatic expansion in parallel with the progression of the tumor, suggesting it is an active participant in PDAC development. However, key studies targeting stromal development through Hedgehog signaling revealed tumor-suppressive functions of the stroma, further complicating the strategy to target the stroma as a single component. Thus, our understanding of the complex relationship between the tumor and its microenvironment and how to effectively target the stroma itself remains incomplete. Previous work by our lab demonstrates that phosphatase and tensin homolog (PTEN) expression is lost in approximately 25% of patients in a PDAC cohort, and lower PTEN expression correlates with worsened patient outcome. Additionally, inhibition of Hedgehog signaling results in loss of PTEN expression and subsequent activation of Akt signaling pathways, and increased tumor growth. The purpose of the current study is to determine the effect of PTEN loss in PDAC cancer-associated fibroblasts (CAFs) on tumor progression and dissemination, and immune evasion. Using a combination of in vitro and in vivo experiments, we have found robust activation of Signal Transducer and Activator of Transcription 3 (STAT3) in PTEN-null pancreatic fibroblasts. We also see selective alterations in the secretome of pancreatic fibroblasts, including a marked increase in IL-6, CXCL1, and CXCL2. Our group has developed a genetically engineered mouse model of PDAC using a dual recombinase system in which the fibroblast-specific protein-1 (FspCre) transgene is used to conditionally delete genes of interest in the fibroblasts of Pdx1Flpki/+;FSF-KrasG12D/+;p53frt/+ (KPF) mice while driving tumorigenesis in the pancreatic epithelium. Conditional deletion of STAT3 in stromal fibroblasts of these mice results in increased overall survival, reduced tumor progression, and alterations within the immune landscape of the tumor microenvironment. Based on our current findings, we believe that a PTEN/STAT3/CXCL1/2 signaling axis within stromal CAFs produces an immunosuppressive tumor microenvironment that favors PDAC progression. Future experiments are needed to validate and confirm this conclusion, including defining the exact mechanisms by which PTEN loss leads to STAT3 activation and elevated secretion of CXCL1/2. This project further highlights the nuanced and complex relationship between PDAC tumor and stroma, and may serve to advance our understanding of how the stroma can be more precisely and effectively targeted to reduce tumor burden and increase the efficacy of immune therapy. Citation Format: Catherine B. MarElia-Bennett, Julia E. Lefler, Michael C. Ostrowski. A stromal PTEN/STAT3 signaling axis promotes immune evasion and tumor progression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-054.

Circular RNA circSEMA5A promotes bladder cancer progression by upregulating ENO1 and SEMA5A expression.

Bladder cancer (BC) is one of the most commonly diagnosed urologic carcinomas, with high recurrence and death rates. Circular RNAs (circRNAs) are a class of noncoding RNAs which are anomalously expressed in cancers and involved in the progression of cancers. In this study, we found that circSEMA5A was upregulated in BC tissues and cell lines. The overexpressed circSEMA5A was correlated with malignant characteristics of BC. In vitro data indicated that circSEMA5A promoted proliferation, suppressed apoptosis, facilitated migration, accelerated invasion, enhanced angiogenesis and promotes glycolysis of BC. Mechanistically, circSEMA5A served as a miRNA sponge for miR-330-5p to upregulates Enolase 1 (ENO1) expression and facilitated the activation of Akt and β-catenin signaling pathways. Then, we showed that circSEMA5A exerted its biological functions partially via miR-330-5p/ENO1 signaling. Moreover, circSEMA5A raised SEMA5A expression by recruiting EIF4A3 to enhance the mRNA stability of SEMA5A, and thereby accelerated BC angiogenesis. To sum up, circSEMA5A is upregulated in BC and facilitates BC progression by mediating miR-330-5p/ENO1 signaling and upregulating SEMA5A expression.

Pristimerin Inhibits Osteoclast Differentiation and Bone Resorption in vitro and Prevents Ovariectomy-Induced Bone Loss in vivo.

IntroductionOsteoporosis is a metabolic bone disease characterized by reduced bone quantity and microstructure, typically owing to increased osteoclastogenesis and/or enhanced osteoclastic bone resorption, resulting in uncontrolled bone loss, which primarily affects postmenopausal women. In consideration of the severe side effects of current drugs for osteoporosis, new safe and effective medications are necessary. Pristimerin (Pri), a quinone methide triterpene extracted from Celastraceae and Hippocrateaceae members, exhibits potent antineoplastic and anti-inflammatory effects. However, its effect on osteoclasts remains unknown.Materials and MethodsWe evaluated the anti-osteoclastogenic and anti-resorptive effect of Pri on bone marrow-derived osteoclasts and its underlying mechanism in vitro. In addition, the protective effect of Pri on ovariectomy model was also explored in vivo.ResultsIn vitro, Pri inhibited osteoclast differentiation and mature osteoclastic bone resorption in a time- and dose-dependent manner. Further, Pri suppressed the expression of osteoclast-related genes and the activation of key proteins. Pri also inhibited the early activation of ERK, JNK MAPK, and AKT signaling pathways in bone marrow-derived macrophages (BMMs), ultimately inhibiting the induction and activation of the crucial osteoclast transcriptional factor nuclear factor of activated T‐cell cytoplasmic 1 (NFATc1). In vivo, consistent with our in vitro data, Pri clearly prevented ovariectomy-induced bone loss.ConclusionOur data showed that Pri inhibits the differentiation and activation of osteoclasts in vitro and in vivo, and could be a promising candidate for treating osteoporosis.

Effect of shikonin on function of rheumatoid arthritis fibroblast like synoviocytes

To observe the effect of shikonin on the proliferation, migration, adhesion and invasion of rheumatoid arthritis(RA) fibroblast like synoviocytes induced by tumor necrosis factor-α(TNF-α), and to explore its mechanism of action from aspects of protein kinase B(Akt) and mitogen activated protein kinase(MAPK) signaling pathways. TNF-α(20 ng·mL~(-1)) was used in this experiment to induce human RA fibroblast like synovial cell line(MH7 A). After addition of different concentrations of shikonin(0.025, 0.05, 0.1 pmol·L~(-1)), the proliferation, migration, adhesion and invasion ability of MH7 A cells were detected by MTT test, scratch test, adhesion test, Transwell invasion test, respectively. Protein expression of Akt and MAPK signaling pathway molecules in MH7 A cells was detected by Western blot. The results showed that as compared with the control group, TNF-α could significantly induce the proliferation, migration, adhesion and invasion of MH7 A cells, and increase the phosphorylation level of Akt, JNK, p38 and extracellular regulatory protein kinase(ERK). As compared with the TNF-α group, shikonin had no significant effect on TNF-α-induced proliferation of MH7 A cells after 24 h treatment, and it could reduce the TNF-α-induced proliferation of MH7 A cells in a concentration dependent manner after 48 h treatment. Shikonin also significantly reduced the TNF-α-induced migration, adhesion, invasion and phosphorylation levels of Akt, JNK, p38, ERK in MH7 A cells within 24 h. These results suggested that shikonin could reduce the proliferation, migration, adhesion and invasion ability of MH7 A cells induced by TNF-α, and its mechanism may be related to the down-regulation of Akt and MAPK signaling pathway activation.

Abstract 5010: Targeting of AKT pathway inhibit high-risk neuroblastoma growth

Abstract High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology. Despite intensive genotoxic therapies, long-term survival for NB patients remains poor (&amp;lt;45%), and the disease accounts for almost 15% of all pediatric cancer deaths. Relapse of metastatic, drug-resistant disease, and treatment-related toxicities mandate the development of novel therapeutic strategies for NB patients. Oncogenic activation of AKT signaling pathway has been shown in different cancer types, including NB. Activation of AKT pathway induce cancer progression, proliferation, metabolism, angiogenesis, and resistance to drug-induced apoptosis. Activation of PI3K (phosphatidylinositol 3-kinase) and PDK1 (3-Phosphoinositide-dependent kinase 1) are the two major steps that mediate the AKT phosphorylation and activation. In the present study, we performed a custom low-density Human AKT pathway array and found that genes coding for PI3K, PIP3, PDK1, and AKT are overexpressed in NB cells in comparison to normal human fibroblast cells. Further, we analyzed a total of 1135 NB patient samples to determine the effect of PI3K and PDK1 overexpression on NB patient survival and correlation with disease stage. Kaplan-Meier analyses of patient datasets revealed that both PI3K and PDK1 high levels strongly correlate with poor overall and event-free survival of NB patients (p&amp;lt;0.0001). In addition, higher stage, more aggressive tumors have significantly higher PI3K and PDK1 expression, suggesting that gain of PI3K or PDK1 function may lead to de-differentiated invasive malignancy. To further analyze the effect of PI3K and PDK1 on NB cell proliferation, we used specific small molecule inhibitors that target PI3K and PDK1, and performed cell proliferation assays in NB cell lines. Results show that inhibition of either PI3K or PDK1 significantly inhibit NB cell proliferation (p&amp;lt;0.01) in a dose-dependent manner. Furthermore, both of the inhibitors induce apoptosis, block cell cycle progression, inhibit colony formation, and inhibit cell migration in NB cells, in comparison to control. Additionally, we performed in vitro 3D sphere formation assays that mimic in vivo tumor growth, and found that inhibition of AKT pathway by either inhibiting PI3K or PDK1 leads to significant reduction of sphere growth and size in contrast to control. Overall, these data highlight that: a) AKT signaling pathway is implicated in NB growth, b) PI3K and PDK1 upstream of the AKT are important regulators of NB proliferation, and c) direct targeting of these regulators is a novel therapeutic approach to high-risk NB. We will further explore these strategies and combine them with current therapies to develop effective therapeutic approaches for NB patients. Citation Format: Rameswari Chilamakuri, Saurabh Agarwal. Targeting of AKT pathway inhibit high-risk neuroblastoma growth [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 5010.

Long noncoding RNA LEF1‐AS1 acts as a microRNA‐10a‐5p regulator to enhance MSI1 expression and promote chemoresistance in hepatocellular carcinoma cells through activating AKT signaling pathway

Long noncoding RNAs (lncRNAs) contribute to the development of hepatocellular carcinoma (HCC), which could regulate various HCC biological characteristics. Here, the study seeks to investigate the role of lncRNA LEF1-AS1 in HCC cell chemoresistance by regulating microRNA (miR)-10a-5p and Musashi1 (MSI1). The microarray-based analysis was employed to identify the HCC-related lncRNA-miRNA-gene regulatory network. Expression patterns of LEF1-AS1, miR-10a-5p, and MSI1 in the HCC cell lines, tissues were accessed by means of reverse transcription-quantitative polymerase chain reaction. Next, the interaction among LEF1-AS1, miR-10a-5p, and MSI1 in HCC was accessed by bioinformatics and dual-luciferase reporter gene assay. Then, the cell line resistant to cisplatin was established, which was then treated with sh/oe-lncRNA LEF1-AS1, miR-10a-5p-mimic, and oe/sh-MSI1 vectors alone or in combination. Afterward, the effect of LEF1-AS1, miR-10a-5p, and MSI1 on HCC cell chemoresistance, proliferation, and apoptosis was assessed. At last, in vivo experiments confirmed the role of MSI1 in tumor growth and chemoresistance in HCC. LEF1-AS1 might potentially affect the growth and chemoresistance of HCC cells by regulating miR-10a-5p and MSI1. LEF1-AS1 and MSI1 expression patterns were elevated, while miR-10a-5p was repressed in HCC tissues and cell lines. LEF1-AS1 combined to miR-10a-5p and regulated MSI1, thereby activating the protein kinase B (AKT) signaling pathway. Knockdown of LEF1-AS1 and MSI1 or elevation of miR-10a-5p compromised the proliferation of Huh7 cell line resistant to DDP and promoted its chemosensitivity and apoptosis. At last, these in vitro findings were also confirmed in vivo. Our results unraveled LEF1-AS1 acts as a miR-10a-5p modulator to promote chemoresistance of HCC cells by stimulating MSI1 and activating the AKT signaling pathway, which might provide a novel therapeutic target for HCC.

USP18 promotes cell proliferation and suppressed apoptosis in cervical cancer cells via activating AKT signaling pathway

BackgroundThe deubiquitinating (DUB) enzyme ubiquitin-specific protease 18 (USP18), also known as UBP43, is an ubiquitin-specific protease linked to several human malignancies. However, USP18’s underlying function in human cervical cancer remains unclear. In the current study, we aimed to analyse the role of USP18 and its signalling pathways in cervical cancer.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining were performed to analyse USP18 levels in cervical cancer and matched to adjacent normal tissues. Moreover, RNA interference (RNAi) and lentiviral-mediated vector transfections were performed to silence and overexpress USP18, respectively, in cervical cancer cells. Further, Cell Counting Kit-8 (CCK-8) and Annexin V/PI staining assays were used to assess its biological function in cell proliferation and apoptosis, respectively. A xenograft model was used to examine USP18’s function in vivo.ResultsThe present findings demonstrated that USP18 was overexpressed in cervical cancer specimens and cell lines. Silencing USP18 in SiHa and Caski cervical cancer cell lines inhibited cell proliferation, induced apoptosis, and promoted cleaved caspase-3 expression. In contrast, USP18 overexpression showed the opposite effects in human HcerEpic cells. A Gene Set Enrichment Analysis revealed that USP18 was enriched in the PI3K/AKT signalling pathway in cervical cancer. Hence, the PI3K/AKT inhibitor LY294002 was used to determine the relationship between USP18 and AKT in cervical cancer cells. Importantly, LY294002 significantly abolished the effects of USP18 overexpression in cervical cancer cells. In vivo, USP18 silencing inhibited human cervical cancer cells’ tumorigenicity.ConclusionsThe current study indicates that USP18 is an oncogenic gene in cervical cancer. Our findings not only deepened the understanding of USP18’s biological function in cervical cancer pathogenesis, but we also provided novel insight for cervical cancer therapy.Trial registrationRetrospectively registered.

Long noncoding RNA HOTAIR promotes breast cancer development by targeting ZEB1 via sponging miR-601

BackgroundBreast cancer (BC) is a common malignancy worldwide. It has been reported that long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) is abnormally expressed in BC. However, the role of HOTAIR in the malignancy of BC is worth further discussion. This study aims to clarify the function and molecular mechanism of HOTAIR in BC.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression of HOTAIR, microRNA (miR)-601 and zinc finger E-box binding homeobox 1 (ZEB1). Cell counting kit-8 (CCK-8) and transwell assay were used to detect the proliferation, migration and invasion of cells. Further, the protein levels of AKT, phosphorylated-AKT (p-AKT), ZEB1 and Ki-67 were confirmed by western blot (WB) assay. Moreover, dual-luciferase reporter assay was applied to examine the targeting relationship between HOTAIR and miR-601 or miR-601 and ZEB1. In addition, animal experiments were conducted to verify the effect of HOTAIR on BC tumor growth in vivo.ResultsHOTAIR was upregulated in BC tissues and cells, and its knockdown suppressed the proliferation, migration, invasion and the activity of AKT signaling pathway of BC cells. HOTAIR could serve as a sponge of miR-601. Further experiments revealed that miR-601 inhibitor could reverse the inhibition effect of HOTAIR silencing on the progression of BC. Meanwhile, ZEB1 was a target of miR-601, and its overexpression could invert the suppression effect of miR-601 overexpression on the progression of BC. Additionally, ZEB1 expression was regulated by HOTAIR and miR-601. Furthermore, interference of HOTAIR could attenuate BC tumor growth in vivo.ConclusionIn short, this study demonstrated that HOTAIR promoted the proliferation, migration, invasion of BC through regulating the miR-601/ZEB1 axis, which provided a theoretical basis for the research on lncRNA-directed therapeutics in BC.

Alleviation of TGF-β1 induced tubular epithelial-mesenchymal transition via the δ-opioid receptor.

Renal fibrosis is a common pathological feature of progressive chronic kidney disease (CKD). It is indicated that transforming growth factor-β1 (TGF-β1) plays as a central mediator in renal fibrosis. The present study aimed to investigate the role of δ-opioid receptor (DOR) on renal fibrosis of the rat renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-β1 and to elucidate its underlying mechanism, as well as its involvement in signaling pathways. Cells were treated with TGF-β1 (10ng·mL-1 ), along with a specific DOR agonist (UFP-512) or naltrindole (a DOR antagonist). Cell viability and morphology, as well as cell migration, were measured after drug administration. Western blotting was employed to examine the extracellular matrix (ECM) protein Fibronectin, and the tubular epithelial-mesenchymal transition (EMT) markers (E-cadherin and α-smooth muscle actin (α-SMA)), signal transducer (p-Smad3), and EMT-regulatory gene (Snail). The expression level of phosphorylated Akt and p38 was also examined. Our results showed that TGF-β1 induced fibroblastic appearance and increased the expression of Fibronectin, α-SMA, P-Smad3, and Snail, while it decreased the expression of E-cadherin in NRK-52E cells. Moreover, TGF-β1 induced the activation of Akt and p38 MAPK signaling pathways. DOR activation was found to efficiently block morphological changes and cell migration, as long as the expression changes of Fibronectin, E-cadherin, α-SMA, P-Smad3, Snail, P-Akt, and P-p38 were induced by TGF-β1. These findings suggest that DOR may serve as an antifibrotic factor for renal proximal tubule cells by inhibiting the fibrosis process via TGF-β/Smad, Akt, and p38 MAPK signaling pathways.

MicroRNA-92b acts as an oncogene by targeting PTEN/AKT in NSCLC.

MicroRNAs can act as tumour suppressors or oncogenes by regulating cellular differentiation, proliferation and apoptosis, and the dysregulation of miRNA is involved in the occurrence and development of NSCLC. Here, we provided evidence that miR-92b as an oncogene in NSCLC by targeting PTEN/AKT. We found that miR-92b was up-regulated in human NSCLC tissues and cell lines. MiR-92b knockdown suppressed the NSCLC cells proliferation and migration in both in vivo and in vitro models. Conversely, miR-92b overexpression induced an aggressive phenotype. Moreover, miR-92b-mediated regulation of NSCLC cell proliferation and migration depended on binding to PTEN mRNA, which then led to the degradation of PTEN and activation of the downstream AKT signalling pathway. Overall, this study revealed the oncogenic roles of miR-92b in NSCLC by targeting PTEN/AKT, and provided novel insights for future treatments of NSCLC patients. SIGNIFICANCE OF THE STUDY: MiR-92b was up-regulated in human NSCLC tissues and cell lines. Our study demonstrated that miR-92b as an oncogene in NSCLC by targeting PTEN/AKT in both in vivo and in vitro models and provided novel insights for future treatments of NSCLC patients.