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The objective of this study was to investigate the potential mechanisms by which (+)-catechin alleviates neuropathic pain. Thirty-two male Sprague-Dawley rats were divided into four groups: the sham group, the chronic constriction injury (CCI)group, the CCI+ ibuprofen group, and the CCI+ (+)-catechin group. CCI surgery induces thermal hyperalgesia in rats and (+)-catechin ameliorated CCI-induced thermal hyperalgesia and repaired damaged sciatic nerve in rats. CCI decreased SOD levels in male rat spinal cord dorsal horn and promoted MDA production, induced oxidative stress by increasing NOX4 levels and decreasing antioxidant enzyme HO-1 levels, and also increased protein levels of TLR4, p-NF-κB, NLRP3 inflammasome components, and IL-1β. In contrast, (+)-catechin reversed the above results. In i vitro experiments, (+)-catechin reduced the generation of reactive oxygen species (ROS) in GMI-R1 cells after LPS stimulation and attenuated the co-expression of IBA-1 and NLRP3. It also showed significant inhibition of the NF-κB and NLRP3 inflammatory pathways and activation of the Nrf2-mediated antioxidant system. Overall, these findings suggest that (+)-catechin inhibits the activation of the NLRP3 inflammasome through the triggering of the Nrf2-induced antioxidant system, the inhibition of the TLR4/NF-κB pathway, and the production of ROS to alleviate CCI-induced neuropathic pain in male rats., (© 2024 Wiley Periodicals LLC.)

The aim of this study was to investigate the potential therapeutic applications of (+)-catechin in the treatment of neuropathic pain. In vivo study, 32 SD rats were randomly divided into four groups: sham group, chronic constriction injury (CCI) group, CCI + ibuprofen group and CCI+ (+)-catechin group. They were subjected to behavioural tests, ELISA, immunohistochemistry and Western blotting. The mechanisms involved were investigated using specific inhibitors in cell experiments. Results of in vivo experiments showed that (+)-catechin could reduce the cold sensitivity pain in a rat model of CCI; ELISA and immunohistochemistry results showed that (+)-catechin could decrease the levels of IL-8, IL-6, TNF-α, CCL2 and CCL5 in serum and the expression levels of nNOS, COX2, IL6, TNF-α, IBA-1 and CSF1R in DRG of CCI rats. Finally, western blot confirmed that (+)-catechin could diminish the levels of IL-34/CSF1R/JAK2/STAT3 signalling pathway in DRG of CCI rats. In vitro studies showed that (+)-catechin reduced IL-34 secretion in LPS-induced RSC96 cells. Meanwhile, (+)-catechin administration in LPS-induced Schwann cell-conditioned medium (L-CM) significantly inhibited the proliferation and migration of RAW264.7 cells; in addition, L-CM+(+)-catechin reduced the activation of the CSF1R/JAK2/STAT3 signalling pathway. (+)-Catechin attenuated the Schwann cell-macrophage cascade response in the DRG by modulating the IL34/CSFIR axis and inhibiting activation of the JAK2/STAT3 pathway, thereby attenuating CCI-induced neuropathic pain in rats., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms., Experimental Design: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models., Results: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models., Conclusions: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments., (©2024 The Authors; Published by the American Association for Cancer Research.)

Compelling evidences suggest that transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) can be therapeutically effective for central nervous system (CNS) injuries and neurodegenerative diseases. The therapeutic effect of BM-MSCs mainly attributes to their differentiation into neuron-like cells which replace injured and degenerative neurons. Importantly, the neurotrophic factors released from BM-MSCs can also rescue injured and degenerative neurons, which plays a biologically pivotal role in enhancing neuroregeneration and neurological functional recovery. Tetramethylpyrazine (TMP), the main bioactive ingredient extracted from the traditional Chinese medicinal herb Chuanxiong, has been reported to promote the neuronal differentiation of BM-MSCs. This study aimed to investigate whether TMP regulates the release of neurotrophic factors from BM-MSCs. We examined the effect of TMP on brain-derived neurotrophic factor (BDNF) released from BM-MSCs and elucidated the underlying molecular mechanism. Our results demonstrated that TMP at concentrations of lower than 200 μM increased the release of BDNF in a dose-dependent manner. Furthermore, the effect of TMP on increasing the release of BDNF from BM-MSCs was blocked by inhibiting the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT)/cAMP-response element binding protein (CREB) pathway. Therefore, we concluded that TMP could induce the release of BDNF from BM-MSCs through activation of the PI3K/AKT/CREB pathway, leading to the formation of neuroprotective and proneurogenic microenvironment. These findings suggest that TMP possesses novel therapeutic potential to promote neuroprotection and neurogenesis through improving the neurotrophic ability of BM-MSCs, which provides a promising nutritional prevention and treatment strategy for CNS injuries and neurodegenerative diseases via the transplantation of TMP-treated BM-MSCs., (© 2021 International Federation for Cell Biology.)

Myeloid differentiation factor 88 (MyD88) is a universal and essential adaptor protein required for the Toll-like receptors (TLRs) pathway activation in invertebrates as well as in vertebrates. Herein, we characterized a MyD88 (Pt-MyD88) cDNA sequence in the swimming crab (Portunus trituberculatus). The Pt-MyD88 ORF is predicted to encode 469 peptides with an N-terminal death domain and a typical C-terminal TIR domain. Real-Time quantitative PCR analysis showed that the Pt-MyD88 transcriptions were constitutively expressed in hemocytes, gill, intestine, heart and muscle in normal crab. The expressions of Pt-MyD88 would be down-regulated by V. alginolyticus or LPS challenge, and be up-regulated by WSSV infection in hemocytes. Intracellular localization showed Pt-MyD88 was distributed mainly in the cytoplasm when it was over-expressed in human cell HEK293T or in Drosophila Schneider 2 (S2). Functionally, over-expression of Pt-MyD88 could either activate the NF-κB in HEK293T cells or activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. In primary cultured hemocytes of swimming crab, after Pt-MyD88 was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), hyastatin3, crustin1 and crustin3 have been significantly inhibited, while the expression of other AMPs is normal compared to non-specific dsRNA treated cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many cytokines and chemokines that are important for hematopoiesis activate the PI3K signaling pathway. Because this pathway is frequently mutated and activated in cancer, PI3K inhibitors have been developed for the treatment of several malignancies, and are now being tested in the clinic in combination with chemotherapy. However, the role of PI3K in adult hematopoietic stem cells (HSCs), particularly during hematopoietic stress, is still unclear. We previously showed that the individual PI3K catalytic isoforms P110α or P110β have dispensable roles in HSC function, suggesting redundancy between PI3K isoforms in HSCs. We now demonstrate that simultaneous deletion of P110α and P110δ in double knockout (DKO) HSCs uncovers their redundant requirement in HSC cycling after 5-fluorouracil (5-FU) chemotherapy administration. In contrast, DKO HSCs are still able to exit quiescence in response to other stress stimuli, such as LPS. We found that DKO HSCs and progenitors have impaired sensing of inflammatory signals ex vivo, and that levels of IL1-β and MIG are higher in the bone marrow after LPS than after 5-FU administration. Furthermore, exogenous in vivo administration of IL1-β can induce cell cycle entry of DKO HSCs. Our findings have important clinical implications for the use of PI3K inhibitors in combination with chemotherapy.

BRCA1 is an established breast and ovarian tumor suppressor gene that encodes multiple protein products whose individual contributions to human cancer suppression are poorly understood. BRCA1-IRIS (also known as "IRIS"), an alternatively spliced BRCA1 product and a chromatin-bound replication and transcription regulator, is overexpressed in various primary human cancers, including breast cancer, lung cancer, acute myeloid leukemia, and certain other carcinomas. Its naturally occurring overexpression can promote the metastasis of patient-derived xenograft (PDX) cells and other human cancer cells in mouse models. The IRIS-driven metastatic mechanism results from IRIS-dependent suppression of phosphatase and tensin homolog ( PTEN ) transcription, which in turn perturbs the PI3K/AKT/GSK-3β pathway leading to prolyl hydroxylase-independent HIF-1α stabilization and activation in a normoxic environment. Thus, despite the tumor-suppressing genetic origin of IRIS, its properties more closely resemble those of an oncoprotein that, when spontaneously overexpressed, can, paradoxically, drive human tumor progression., Competing Interests: Conflict of interest statement: D.M.L. is a grantee of and consultant to the Novartis Institute of Biomedical Research. W.G.K. owns equity in and consults for Lilly. A.L.K. was, during the course of some of this work, a grantee of and consultant to the Novartis Institute of Biomedical Research., (Copyright © 2018 the Author(s). Published by PNAS.)

The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins., Competing Interests: Conflict of interest statement: B.B.L. and J.J.Z. are coinventors on patent application PCT/US2017/039001. All other authors declare no conflict of interest.

To explore the effects of IL-7/IL-7R on the RANKL-mediated osteoclast differentiation in vitro and OVX-induced bone loss in vivo. BMMs and RAW264.7 were transfected with IL-7, IL-7R siRNA, c-Fos siRNA, and c-jun siRNA and later stimulated by RANKL. TRAP and toluidine blue staining were used to observe osteoclast formation and bone resorption, respectively. HE and TRAP staining were used to detect trabecular bone microstructure and osteoclasts of mice, respectively. qRT-PCR and Western blot analysis were used to examine expression. IL-7 unregulated the expression of CTSK, NFATc1, MMP9, and the phosphorylation of p38 and Akt by activating the c-Fos/c-Jun pathway, which increased osteoclast numbers and bone resorption in RANKL-stimulated macrophages. While IL-7R siRNA and c-Fos siRNA decreased the expression, as well as and the phosphorylation of p38 and Akt.IL-7 decreased the BMD and OPG expression in OVX-induced mice and increased the TRAP positive cells, the mRNA expression of c-fos, c-jun, and RANKL, which was contradictory to IL-7R siRNA, and c-Fos siRNA. Furthermore, IL-7R siRNA and c-Fos siRNA caused thicker trabeculae, increased trabecular number, and decreased osteolysis in OVX mice. IL-7/IL-7R can promote RANKL-mediated osteoclast formation and bone resorption by activating the c-Fos/c-Jun pathway, as well as inducing bone loss in OVX mice., (© 2018 Wiley Periodicals, Inc.)

It was aimed at exploring the potential pharmacological effects of alkaloids in Sophora alopecuroides by means of network pharmacology in this study. The main alkaloids in S. alopecuroides were collected for analysis of drug properties, prediction of potential targets and screening of signaling pathways. DAVID analysis tool combined with KEGG database was used to annotate and analyze the signaling pathway. The alkaloids-targets-signaling pathways network was built through Cytoscape software. Results showed that 17 alkaloids in S. alopecuroides involved 49 targets (170 times in all) and 22 important signaling pathways. Three nodes in model of network pharmacology were cross-linked, and the metabolic pathways were coordinated and regulated by each other. It indicated that alkaloids in S. alopecuroides may have therapeutic effect on diseases of cancer, metabolic disorder, endocrine system, digestive system, nervous system and so on., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Frutescones H-R (1-11), new sesqui- or monoterpene-based meroterpenoids, were isolated from the aerial parts of Baeckea frutescens. Their structures and absolute configurations were established by means of spectroscopic analyses (HRESIMS, 1D and 2D NMR, and ECD), as well as single-crystal X-ray crystallography of 1, (-)-7, and 9. The anti-inflammatory activities of all isolates were evaluated by measuring their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophages, and the structure-activity relationships of 1-11 are also discussed. Compound 8 exhibited anti-inflammatory activity with an IC 50 value of 0.36 μM, which might be related to the regulation of the NF-κB signaling pathway via the suppression of p65 nuclear translocation and the consequent decrease of IL-6 and TNF-α.

The p110β isoform of PI3K is preferentially activated in many tumors deficient in the phosphatase and tensin homolog (PTEN). However, the mechanism(s) linking PTEN loss to p110β activation remain(s) mysterious. Here, we identify CRKL as a member of the class of PI3Kβ-interacting proteins. Silencing CRKL expression in PTEN-null human cancer cells leads to a decrease in p110β-dependent PI3K signaling and cell proliferation. In contrast, CRKL depletion does not impair p110α-mediated signaling. Further study showed that CRKL binds to tyrosine-phosphorylated p130Cas in PTEN-null cancer cells. Since Src family kinases are known both to be regulated by PTEN and to phosphorylate and activate p130Cas, we tested and found that Src inhibition cooperated with p110β inhibition to suppress the growth of PTEN-null cells. These data suggest both a potential mechanism linking PTEN loss to p110β activation and the possible benefit of dual inhibition of Src and PI3K for PTEN-null tumors., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the effect of antisense non-coding RNA in the INK4 locus (ANRIL) on invasion and metastasis of thyroid cancer (TC)., Results: ANRIL expression was significantly up-regulated in TC tissues and cells (P < 0.001), and ANRIL expression was significantly different regarding histological grade and LNM (both P < 0.01). The siRNA-mediated ANRIL silencing inhibits proliferation, invasion, and metastasis of TPC-1 and SW579 cells, and lung metastasis, which can be reversed by TGF-β1 siRNA. The mRNA levels of p15INK4b, p14ARF and p16INK4a in TPC-1 and SW579 cells increased significantly after silencing ANRIL (all P < 0.001), and TGF-β1 siRNA could reverse the ANRIL siRNA induced increase of p15INK4b; expressions of TGF-β1 and p-Smad2/3 were increased after silencing ANRIL (both P < 0.05)., Materials and Methods: TC and adjacent normal tissues were collected from 105 TC patients. LncRNA ANRIL expressions were detected by qRT-PCR. The siRNA ANRIL and siRNA TGF-β1 were constructed for TPC-1 and SW579 cell line transfection: si-ANRIL group, si-TGF-β1 group, si-ANRIL + si-TGF-β1 group, negative control group and blank group. Effects of ANRIL silencing on proliferation, invasion and metastasis of TC cells was detected by MTT assay, Transwell assay and tail vein injection of nude mice in vitro and in vivo. TGF-β1 and p-Smad2/3 expressions in TGF-β/Smad signaling pathway were detected by western blot., Conclusions: ANRIL may reduce p15INK4B expression through inhibiting TGF-β/Smad signaling pathway, promoting invasion and metastasis of TC cells, and the silencing of ANRIL inhibits the invasion and metastasis of TPC-1 cells., Competing Interests: We declare that we have no conflicts of interest.

Adenosine triphosphate-binding cassette transporter A1 (ABCA1) is a crucial cholesterol transporter and plays a central role in the high density lipoproteins (HDL) cholesterol metabolism and lipid clearance from the foam cell. Lipoxin A4 (LXA4) is an endogenous lipid mediator that requires cell-cell interaction or cell-platelet interaction for its synthesis. The roles of LXA4 on inflammatory responses are well described, while its effects on mediating ABCA1 and underlying mechanisms remain unclear. In this study, we showed that LXA4 significantly increases expression of ABCA1 and LXRα in a dose-dependent manner in THP-1 macrophage-derived foam cells. Cellular cholesterol content was decreased while cholesterol efflux was increased by LXA4 treatment. However, after short interfering RNA of LXRα, the effects of LXA4 on ABCA1 expression and cholesterol metabolism were significantly abolished. These results provide evidence that LXA4 increases ABCA1 expression and promotes cholesterol efflux through LXRα pathway in THP-1 macrophage-derived foam cells.

Purpose: To investigate the regulatory mechanism of miR-218 in human hepatocellular carcinoma (HCC)., Methods: qPCR was used to compare the expression levels miR-218 among six hepatocellular carcinoma cell lines and normal liver tissues. After transfecting MHCC97L cells with either miR-218 mimics or miR-218 inhibitor, western blotting was used to examine the expressing patterns of cyclinD1, p21, and PTEN/AKT/PI3K signaling pathway-related proteins. MTT and colony forming assay was used to assess the capability of cell proliferation. Bioinformatic method was applied to predict the binding of miR-218 on HoxA10, and western blotting was used to examine the modulatory effect of miR-218 AND HoxA10 on PTEN/AKT/PI3K pathway in HCC., Results: The expression levels of miR-218 were frequently lower in HCC cell lines than in normal liver tissues. Over-expression of miR-218 in HCC cells significantly decreased cell proliferation whereas inhibiting miR-218 promoted cancer cell proliferation. Western blotting analysis demonstrated that tumorigenesis related protein cyclin D1 and p21, as well as PTEN/AKT/PI3K signaling pathways were actively modulated by miR-218 in HCC cells. The expression of endogenous HoxA10 was also down-regulated by miR-218 over-expression, and silencing HoxA10 directly activated PTEN in HCC cells., Conclusion: Modulation of miR-218 actively affected HCC cancer cell development. The regulatory mechanism of miR-218 in HCC cells was acting through PTEN/AKT/PI3K pathway and possibly associated with HoxA10.

Glucagon-like peptides (GLP-1/GLP-2) are coproduced and highlighted as key modulators to improve glucose homeostasis and insulin sensitivity after bariatric surgery. However, it is unknown if CNS GLP-2 plays any physiological role in the control of glucose homeostasis and insulin sensitivity. We show that mice lacking GLP-2 receptor (GLP-2R) in POMC neurons display glucose intolerance and hepatic insulin resistance. GLP-2R activation in POMC neurons is required for GLP-2 to enhance insulin-mediated suppression of hepatic glucose production (HGP) and gluconeogenesis. GLP-2 directly modulates excitability of POMC neurons in GLP-2R- and PI3K-dependent manners. GLP-2 initiates GLP-2R-p85α interaction and facilitates PI3K-Akt-dependent FoxO1 nuclear exclusion in POMC neurons. Central GLP-2 suppresses basal HGP and enhances insulin sensitivity, which are abolished in POMC-p110α KO mice. Thus, CNS GLP-2 plays a key physiological role in the control of HGP through activating PI3K-dependent modulation of membrane excitability and nuclear transcription of POMC neurons in the brain., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To explore the effects of reduced glutathione (GSH) on the proliferation of hepatic stellate cells and the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway., Methods: The rat HSC-T6 cell line was activated by lipopolysaccharide (LPS; 0.1 mug/ml) and incubated with various concentrations of GSH (0, 1, 2, 5 and 10 mmol/L) for 24 h. Cell proliferation was evaluated by the MTT colorimetric assay. Collagen IV and hyaluronic acid (HA) contents were measured by chemiluminescence immunoassay of cell supernatants. Nrf2 and HO-1 protein expression was observed by immunohistochemistry. Nrf2 and HO-1 mRNA expression was assessed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). HO-1 activity was analyzed by spectrophotometer., Results: GSH treatment inhibited HSC-T6 proliferation and decreased the secretion of HA and collagen IV (P less than 0.05); GSH treatment of HSC-T6 cells also led to increased expression of Nrf2 and HO-1, and increased activity of HO-1 (P less than 0.05)., Conclusion: GSH can inhibit the proliferation of hepatic stellate cells in vitro and reduce secretion of hyaluronic acid and collagen IV. The underlying mechanism in HSC-T6 cells may involve regulation of the Nrf2/HO-1 signaling pathway.

Evidence suggests that the role played by the adipocyte-derived hormone leptin in female reproductive physiology is mediated in part by neurons located within the ventral premammillary nucleus (PMV). Leptin activates PMV neurons; however, the intracellular signaling pathway and channel(s) involved remain undefined. Notably, leptin's excitatory and inhibitory effects within hypothalamic and brainstem nuclei share the intracellular signaling cascade phosphoinositide 3 kinase (PI3K). Therefore, we assessed whether PI3K signaling is required for the acute effect of leptin to alter cellular activity of PMV neurons that express leptin receptors (LepR PMV neurons). Leptin caused a rapid depolarization in the majority of LepR PMV neurons in patch-clamp recordings of hypothalamic slices, while a subset of LepR PMV neurons were hyperpolarized in response to leptin. Data were obtained from both male and female mice and results demonstrate that the acute effect of leptin on LepR PMV neurons was identical for both sexes. Pharmacological inhibition of PI3K prevented the acute leptin-induced change in neuronal activity of LepR PMV neurons, indicating a PI3K-dependent mechanism of leptin action. Similarly, mice with genetically disrupted PI3K signaling in LepR PMV neurons failed to alter cellular activity in response to leptin. Moreover, the leptin-induced depolarization was dependent on a putative TRPC channel. In contrast, the leptin-induced-hyperpolarization required the activation of a putative Katp channel. Collectively, these results suggest that PI3K signaling in LepR PMV neurons is essential for leptin-induced alteration in cellular activity, and these data may suggest a cellular correlate in which leptin contributes to the initiation of reproductive development.

PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy, making phosphatidylinositol 3-kinase (PI3K) a target for cancer therapy. Despite the promise of targeted therapy, resistance often develops, leading to treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CA(H1047R). Notably, most PIK3CA(H1047R)-driven mammary tumors recurred after PIK3CA(H1047R) inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of Met or Myc (also known as c-Met and c-Myc, respectively). Whereas Met amplification led to tumor survival dependent on activation of endogenous PI3K, tumors with Myc amplification became independent of the PI3K pathway. Functional analyses showed that Myc contributed to oncogene independence and resistance to PI3K inhibition. Notably, PIK3CA mutations and c-MYC elevation co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.

Phosphatidyl inositol 3-kinase (PI3K) signaling in the hypothalamus has been implicated in the regulation of energy homeostasis, but the critical brain sites where this intracellular signal integrates various metabolic cues to regulate food intake and energy expenditure are unknown. Here, we show that mice with reduced PI3K activity in the ventromedial hypothalamic nucleus (VMH) are more sensitive to high-fat diet-induced obesity due to reduced energy expenditure. In addition, inhibition of PI3K in the VMH impaired the ability to alter energy expenditure in response to acute high-fat diet feeding and food deprivation. Furthermore, the acute anorexigenic effects induced by exogenous leptin were blunted in the mutant mice. Collectively, our results indicate that PI3K activity in VMH neurons plays a physiologically relevant role in the regulation of energy expenditure., (Copyright 2010 Elsevier Inc. All rights reserved.)

Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85alpha alone induced resistance to diet-induced obesity. In contrast, deletion of the p110alpha catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.

Aim: Insulin-like growth factor-1 (IGF-1) is an important hypertrophic and cell cycle progression factor for a number of cell types. It has been proven that IGF-1 is involved in the regulation of thyroid proliferation and cell cycle progression; however, the exact mechanism of this regulation has not been fully elucidated. In the present study, we investigated the effect of IGF-1 on the expression of cyclin D1, an important cell cycle regulatory protein, and a signaling pathway involved in IGF-1's effect on cyclinD1 expression in FRTL thyroid cells., Methods: FRTL thyroid cells were treated with IGF-1 or vector control for 24 h. As appropriate to individual experiments, a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and/or a nuclear factor-kappaB (NF-kappaB) inhibitor, BAY11-7082, were added 1 h prior to IGF-1 treatment. Western blotting was used to detect cyclin D1 protein expression. Immunofluorescence was performed to analyze the expression of IkappaBalpha, an NF-kappaB inhibitory protein. Cell cycle analysis was performed by fluorescence activated cell sorting (FACS)., Results: IGF-1 increased the cyclin D1 expression in thyroid cells. This increase was blocked by pretreatment with LY294002 or BAY11-7082. Further studies showed that IGF-1 specifically induced NF-kappaB activity. Treatment with IGF-1 could accelerate cell cycle progression from G(0)/G(1) to S phase, whereas this progression was inhibited by the presence of LY294002 or BAY11-7082., Conclusion: In summary, the results of the present study show that in FRTL cells, IGF-1 promotes cell cycle progression via an upregulation of cyclin D1 expression, at least partially through the PI3K/NF-kappaB signaling pathway.

Transforming growth factor beta (TGF-beta) signaling facilitates metastasis in advanced malignancy. While a number of protein-encoding genes are known to be involved in this process, information on the role of microRNAs (miRNAs) in TGF-beta-induced cell migration and invasion is still limited. By hybridizing a 515-miRNA oligonucleotide-based microarray library, a total of 28 miRNAs were found to be significantly deregulated in TGF-beta-treated normal murine mammary gland (NMuMG) epithelial cells but not Smad4 knockdown NMuMG cells. Among upregulated miRNAs, miR-155 was the most significantly elevated miRNA. TGF-beta induces miR-155 expression and promoter activity through Smad4. The knockdown of miR-155 suppressed TGF-beta-induced epithelial-mesenchymal transition (EMT) and tight junction dissolution, as well as cell migration and invasion. Further, the ectopic expression of miR-155 reduced RhoA protein and disrupted tight junction formation. Reintroducing RhoA cDNA without the 3' untranslated region largely reversed the phenotype induced by miR-155 and TGF-beta. In addition, elevated levels of miR-155 were frequently detected in invasive breast cancer tissues. These data suggest that miR-155 may play an important role in TGF-beta-induced EMT and cell migration and invasion by targeting RhoA and indicate that it is a potential therapeutic target for breast cancer intervention.

Middle T antigen (MT) of polyomavirus is known to play an important role in virus-mediated cellular transformation. While MT has been extensively examined in spontaneously immortalized rodent fibroblasts, its interactions with cells of other types and species are less well understood. We have undertaken a cross-species and cross-cell-type comparison of MT-induced transformation in cells with genetically defined backgrounds. We tested the transforming abilities of a panel of MT mutants, Y250F, Y315F, and Y322F, that are selectively mutated in the binding sites for the principal effectors of MT--Src homology 2 domain-containing transforming protein, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-gamma, respectively--in fibroblasts and epithelial cells of murine or human origin. We found that the Y315F mutation disabled the ability of MT to induce transformation in all cell types and species tested. While Y315F also failed to activate the PI3K pathway in these cells, genetic evidence has indicated Y315 may make other contributions to transformation. To confirm the role of PI3K, the PIK3CA gene, encoding p110alpha, the prime effector of PI3K signaling downstream from activated growth factor receptors, was genetically ablated. This abolished the transforming activity of MT, demonstrating the essential role for this PI3K isoform in MT-mediated transformation. The Y250F mutant was able to transform the human, but not the murine, cells that were examined. Interestingly, this mutant fully activates the PI3K pathway in human cells but activated PI3K signaling poorly in the murine cells used in the study. This again points to the importance of PI3K activation for transformation and suggests that the mechanism by which MT activates the PI3K pathway differs in different species.

Growth factor signaling is mediated through Class IA phosphatidylinositol 3-kinases (PI3Ks). Among this class of enzymes, only p110alpha, encoded by the PIK3CA gene, has been found to be mutant in human cancers. To determine the specific functions of p110alpha, we generated mice carrying a conditionally targeted allele of the PIK3CA gene. Here, we report that PIK3CA-knockout mouse embryonic fibroblasts are deficient in cellular signaling in response to various growth factors, unable to differentiate into adipocytes, and resistant to oncogenic transformation induced by a variety of oncogenic receptor tyrosine kinases, indicating a fundamental role for p110alpha in these biological processes.