PI3K Signaling Pathway Research Articles

EZH2 inhibition confers PIK3CA-driven lung tumors enhanced sensitivity to PI3K inhibition.

Members of the PI3K signaling pathway, especially PIK3CA, the gene encoding the catalytic subunit of the PI3K complex, are highly mutated and amplified in various cancer types, including non-small cell lung cancer. Although PI3K inhibitors have been used in clinics for follicular lymphoma and chronic lymphocytic leukemia, no agents targeting PI3K aberrations in lung cancer have been approved by the FDA so far. In this study, we observed that PIK3CA-E545K, the most common mutation in lung cancer, harbored a modest induction of stem-like properties in lung epithelial cells, and drove development of adenocarcinoma autochthonously when paired with p53 loss in a murine mouse model. We also found that PIK3CA-mutant of amplified lung cancer cells were sensitive to EZH2 inhibition. EZH2 inhibition synergized with PI3K inhibition in human cancer cells in vitro and worked together efficiently in vivo. Mechanistically, EZH2 inhibition cooperated with PI3K inhibition to produce a more potent suppression of phospho-AKT downstream of PI3K. This study suggests a promising combination therapy to combat lung cancers with PIK3CA mutation or amplification. Both copanlisib, the PI3K inhibitor, and tazemetostat, the EZH2 inhibitor, are FDA-approved, which should enhance the clinical translation of this work.

Molecular structure, expression, and the emerging role of Siglec-15 in skeletal biology and cancer.

Siglec-15, a Siglec family member and type-1 transmembrane protein, is expressed mainly in human macrophages and dendritic cells. It is comprised of a lysine-containing transmembrane domain, two extracellular immunoglobulin (Ig)-like domains and a short cytoplasmic domain. Siglec-15 is highly conserved in vertebrates and acts as an immunoreceptor. It exerts diverse functions on osteoclast physiology as well as the tumor microenvironment. Siglec-15 interacts with adapter protein DAP12 - Syk signaling pathway to regulate the RANKL/RANK-mediated PI3K, AKT, and ERK signaling pathways during osteoclast formation in vitro. Consistently, the lack of the Siglec-15 gene in mice leads to impaired osteoclast activity and osteopetrosis in vivo. In addition, Siglec-15 is expressed by tumor-associated macrophages (TAMs) and regulates the tumor microenvironment by activating the SYK/MAPK signaling pathway. Interestingly, Siglec-15 shares sequence homology to programmed death-ligand 1 (PD-L1) and has a potential immune-regulatory role in cancer immunology. Thus, Siglec-15 might also represent an alternative target for the treatment of cancers that do not respond to anti-PD-L1/PD-1 immunotherapy. Understanding the role of Siglec-15 in osteoclastogenesis and the tumor microenvironment will help us to develop new treatments for bone disorders and cancer.

Fluroxypyr-1-methylheptyl ester causes apoptosis of bovine mammary gland epithelial cells by regulating PI3K and MAPK signaling pathways and endoplasmic reticulum stress

Fluroxypyr-1-methylheptyl ester (FPMH) is an auxin herbicide that is widely applied to crops and pastures to block growth of post-emergence weeds. Several studies have reported the toxicity of FPMH in aquatic vertebrates. However, the adverse impacts of FPMH on mammals, including domestic animals, have not been reported. The purpose of our current study is to assess the impact of FPMH on the bovine mammary system and milk production. To evaluate the toxicity of FPMH on the mammary glands of lactating cows, the bovine mammary gland epithelial cell line, MAC-T, was exposed to various concentrations (0, 5, 7.5, 10, 15, and 20 μM) of FPMH for 24 h, and then various assessments were performed. The results showed that FPMH dose-dependently reduced MAC-T cell viability following exposure to FPMH and induced mitochondrial depolarization and apoptosis. FPMH also modulated signaling through the PI3K and MAPK pathways. In addition, the expression levels of proteins related to endoplasmic reticulum (ER) stress were upregulated, indicating induction of ER stress, and calcium homeostasis was disrupted following FPMH treatment. In conclusion, our investigation suggests that FPMH may be toxic to the bovine mammary system and may decrease dairy production.

Abstract P197: An anti-HER3 antibody, HMBD-001, that uniquely binds to and blocks the HER3 heterodimerization interface, shows superior tumor growth inhibition in biomarker-defined preclinical cancer models including NRG1-fusion driven cancers

Abstract HER3 activation, through NRG1 ligand-dependent and independent heterodimerization with HER2 or EGFR, can drive tumor growth and survival via potent PI3K pathway signaling. HER3 activation has emerged as an important mechanism for both tumor progression and acquired resistance to standard of care therapies in multiple indications. With an effective biomarker strategy to select for patients with HER3 driven cancer, potent inhibition of HER3-driven heterodimerization has the potential to confer profound clinical impact. HER3 targeting approaches to date have not shown expected clinical efficacy. Suboptimal HER3 inhibition is one possible explanation; to prevent downstream PI3K signalling, it is critical to fully block both ligand-dependent and independent HER3 activation. Further, despite the clinical evaluation of several anti-HER3 antibodies, only limited progress has been made to establish predictive biomarkers of response to HER3 inhibition. The most thoroughly studied biomarkers predicting response have been increased expression and genomic rearrangements of the HER3 ligand, NRG1. For example, oncogenic NRG1-fusions have been identified as tumor drivers in up to 0.2% of all solid cancers. Few biomarkers predicting lack of response have been reported to date. HMBD-001, is an unique anti-HER3 antibody rationally developed to bind the dimerization interface of HER3 in order to block all HER3 heterodimerization. In contrast to other anti-HER3 antibodies that predominantly bind to the NRG1 binding domain, we observed potent and superior in vitro and in vivo tumor growth inhibition for HMBD-001 in multiple cancer models, including NRG1-fusion driven models. We show that HMBD-001 binds to HER3 with high affinity even in the presence of high concentrations of NRG1, in contrast to other antibodies in which NRG1 competes for the HER3 ligand binding site, thereby decreasing affinity and likely potency. Notably, HMBD-001 treatment of an NRG1-fusion ovarian PDX model showed superior tumor growth inhibition compared with anti-HER3 and anti-HER2/HER3 bispecific antibodies targeting the NRG1 binding domain of HER3. To develop a robust biomarker signature for patient selection beyond NRG1-fusions, we interrogated the genomic and transcriptomic data of all preclinical models for which HMBD-001 efficacy data was available, including in vitro and in vivo models representing multiple HER3 associated cancer sub-types. We identified a novel gene signature that was robustly predictive of HMBD-001 response across a wide range of cancers, including loss of function and gain of function mutations in specific downstream mediators of the MAPK and PI3K signaling pathways. In conclusion, the superior potency of HMBD-001 in preclinical models predicts more complete inhibition of HER3 and better responses in HER3 driven cancers that can be identified using a novel gene signature biomarker. Clinical trials of HMBD-001 in HER3 driven cancers, including those with NRG1-fusions, are expected to commence in 2021. Citation Format: Dipti Thakkar, Shalini Paliwal, Shreya Kar, Namita Gandhi, Konrad Paszkiewicz, Piers Ingram, Jerome Boyd-Kirkup. An anti-HER3 antibody, HMBD-001, that uniquely binds to and blocks the HER3 heterodimerization interface, shows superior tumor growth inhibition in biomarker-defined preclinical cancer models including NRG1-fusion driven cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P197.

Abstract P123: Antitumor activity of tipifarnib and PI3K pathway inhibitors in HRAS-associated HNSCC

Abstract HRAS-MAPK and PI3K-AKT-mTOR are important oncogenic pathways in head and neck squamous cell carcinoma (HNSCC) and other squamous cell carcinomas (SCCs). HRAS is mutated in ~5% and overexpressed in approximately 30% of HNSCC patients, raising the possibility that some HRAS wild-type (WT) HNSCCs may also display a degree of dependence on HRAS. RAS proteins undergo several post-translational modifications, including the addition of a farnesyl isoprenoid moiety by the enzyme farnesyltransferase (FT), which facilitate their attachment to membranes. This dependence prompted the development of selective inhibitors (FTIs) for the treatment of RAS-driven cancers. Tipifarnib is a potent and selective FTI that has demonstrated antitumor activity in recurrent/metastatic HNSCC carrying HRAS mutations. Based on these data, a pivotal study (NCT03719690) evaluating the efficacy of tipifarnib in HRAS mutant HNSCC (AIM-HN) is currently ongoing. PIK3CA (the catalytic subunit of PI3K), another prominent driver in HNSCC, is commonly activated either by gain of function mutations or gene amplification with some overlap between the two subsets. Multiple reports indicate that HRAS and PIK3CA pathways cooperate and crosstalk in driving tumor progression in SCCs and resistance to inhibitors of respective pathways. In this study, we explored whether combined inhibition of HRAS farnesylation (by tipifarnib) and inhibition of PI3K pathway signaling (with inhibitors of PI3K-a, AKT or mTORC1/2) would be more effective in CDX and PDX models of HRAS-associated SCCs relative to the monotherapy approaches. In a panel of HNSCC cell lines harboring HRAS and/or PIK3CA alterations (mutation, overexpression or amplification), tipifarnib reduced cell growth and, in combination with PI3K-a inhibitor alpelisib, induced cytotoxicity. Consistent with in vitro findings, robust inhibition of tumor growth was observed in majority of animals treated with the combination of tipifarnib and alpelisib. Similar activity was noted with the AKT inhibitor uprosertib and the mTORC1/2 inhibitor sapanisertib. Regressions with the FTI-PI3K-a inhibitor doublet were observed both in tumors that were HRAS mutant or HRAS overexpressed with or without mutation or amplification of the PIK3CA gene or carried the latter without alterations in HRAS, suggesting that concomitant blockade of both targets may have surprisingly broad and potent anti-tumor activity in HNSCC. In dose-scheduling experiments in PDX models, simultaneous blockade of both targets was superior to split intermittent dosing of the two drugs, underlining the cooperativity of the two pathways in these models. Mechanistically, tipifarnib repressed the compensatory MAPK pathway activation induced by alpelisib at the level of phosphorylated ribosomal S6 protein. Impact of tipifarnib and alpelisib combination on additional MAPK and AKT signaling mediators will be reported. Citation Format: Francis Burrows, Shivani Malik, Stacia Chan, Asako Mccloskey, Zhiyong Wang, Mara Gilard, Silvio Gutkind. Antitumor activity of tipifarnib and PI3K pathway inhibitors in HRAS-associated HNSCC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P123.

Feruloylated arabinoxylan from wheat bran inhibited M1-macrophage activation and enhanced M2-macrophage polarization

The effects of feruloylated arabinoxylan (AX) on typically activated inflammatory macrophages (M1) and alternatively anti-inflammatory macrophages (M2) and its possible mechanisms were investigated. The results revealed that feruloylated AX was composed of 37.63% arabinose and 52.23% xylose, with a weight-average molecular weight of 1.1374 × 104 Da, and bound ferulic acid content of 10.84 mg/g. Besides, feruloylated AX (50–1000 μg/mL) markedly downregulated the mRNA expressions of NO, IL-1β, TNF-α, IL-6, and IL-23a, and reduced the phosphorylation levels of p38, ERK, and JNK in M1. In contrast, the mRNA expressions of Arg-1, Mrc-1, and CCL22 were significantly upregulated by feruloylated AX (50–1000 μg/mL), and the phosphorylation level of AKT was significantly increased in M2. Overall, our results indicated that feruloylated AX could have an inhibitory or a promoting effect on already activated macrophages, and MAPK or PI3K signaling pathways might be involved in this regulation.

Structural effects of morpholine replacement in ZSTK474 on Class I PI3K isoform inhibition: Development of novel MEK/PI3K bifunctional inhibitors

Established roles for PI3K and MAPK signaling pathways in tumorigenesis has prompted extensive research towards the discovery of small-molecule inhibitors as cancer therapeutics. However, significant compensatory regulation exists between these two signaling cascades, leading to redundancy among survival pathways. Consequently, initial clinical trials aimed at either PI3K or MEK inhibition alone have proven ineffective and highlight the need for development of targeted and innovative therapeutic combination strategies. We designed a series of PI3K inhibitor derivatives wherein a single morpholine group of the PI3K inhibitor ZSTK474 was substituted with a variety of 2-aminoethyl functional groups. Analogs with pendant hydroxyl or methoxy groups maintained low nanomolar inhibition towards PI3Kα, PI3Kγ, and PI3Kδ isoforms in contrast to those with pendant amino groups which were significantly less inhibitory. Synthesis of prototype PI3K/MEK bifunctional inhibitors (6r, 6s) was guided by the structure-activity data, where a MEK-targeting inhibitor was tethered directly via a short PEG linker to the triazine core of the PI3K inhibitor analogs. These compounds (6r, 6s) displayed nanomolar inhibition towards PI3Kα, δ, and MEK (IC50 ∼105–350 nM), and low micromolar inhibition for PI3Kβ and PI3Kγ (IC50 ∼1.5–3.9 μM) in enzymatic inhibition assays. Cell viability assays demonstrated superior anti-proliferative activity for 6s over 6r in three tumor-derived cell lines (A375, D54, SET-2), which correlated with inhibition of downstream AKT and ERK1/2 phosphorylation. Compounds 6r and 6s also demonstrated in vivo tolerability with therapeutic efficacy through reduction of kinase activation and amelioration of disease phenotypes in the JAK2V617F mutant myelofibrosis mouse cancer model. Taken together, these results support further structure optimization of 6r and 6s as promising leads for combination therapy in human cancer as a new class of PI3K/MEK bifunctional inhibitors.

Different HCV Exposure Drives Specific miRNA Profile in PBMCs of HIV Patients.

Micro RNAs (miRNAs) are essential players in HIV and HCV infections, as both viruses modulate cellular miRNAs and interact with the miRNA-mediated host response. We aim to analyze the miRNA profile of HIV patients with different exposure to HCV to explore specific signatures in the miRNA profile of PBMCs for each type of infection. We massively sequenced small RNAs of PBMCs from 117 HIV+ infected patients: 45 HIV+ patients chronically infected with HCV (HIV/HCV+), 36 HIV+ that spontaneously clarified HCV after acute infection (HIV/HCV-) and 36 HIV+ patients without previous HCV infection (HIV). Thirty-two healthy patients were used as healthy controls (HC). Differential expression analysis showed significantly differentially expressed (SDE) miRNAs in HIV/HCV+ (n = 153), HIV/HCV- (n = 169) and HIV (n = 153) patients. We found putative dysregulated pathways, such as infectious-related and PI3K signaling pathways, common in all contrasts. Specifically, putatively targeted genes involved in antifolate resistance (HIV/HV+), cancer-related pathways (HIV/HCV-) and HIF-signaling (HIV) were identified, among others. Our findings revealed that HCV strongly influences the expression profile of PBMCs from HIV patients through the disruption of its miRNome. Thus, different HCV exposure can be identified by specific miRNA signatures in PBMCs.

Development of Notch1 Positive T-Lineage Lymphomas or Splenic Marginal Zone Lymphomas with Pan-Hematopoietic or Pro-B Cell Specific Deletion of Trp53 with Distinct Differentially Dysregulated Pathways

Deletion of the tumor suppressor gene TP53 (Trp53 in mice) has been associated with the development of numerous human malignancies. TP53 acts as a central coordinator of the DNA damage response. In mice, pan-Trp53 deletion leads predominantly to the development of T-cell lymphomas, followed by B-cell lymphomas, sarcomas and teratomas. In order to dissect the role of Trp53 in the hematopoietic system, we created two different loss of function mouse models: Pan-hematopoietic Trp53 deletion using Vav1-Cre based deletion; and a B-cell-specific deletion was created using CD19-Cre.Vav1-p53CKO mice developed hematolymphoid malignancies with 100% penetrance by 12 months. Most malignancies observed were CD3e+ T-lineage lymphomas involving the thymus or spleen (37/45). Beyond 200 days, these mice predominantly developed mixed myeloid malignancies. The shift away from T-lineage malignancies in older mice may reflect aging-related decline of pre-malignant lymphoid progenitors and skewing to myeloid progenitors.Flow cytometric characterization of the T-lineage lymphomas identified a mix of tumors, including double-negative (CD4-CD8-), double-positive (CD4+CD8+), or single positive (CD4/CD8). In pre-malignant mice, Vav1-p53CKO thymocytes showed accelerated maturation with most of the cells in the DN4 stage, suggesting a bypass of the p53-dependent DN3 β-selection checkpoint. All T-lineage lymphomas showed overexpression of surface Notch1 as well as overexpression of Notch1 targets Hes1 and p21 at the transcript level. Consistent with prior data, normal murine thymocyte subsets showed high levels of expression of Notch1 target genes at the DN3 stage of development, which appears dysregulated in these T-lineage lymphomas. This Notch1 activation was found to be multifactorial with increased Mdm2 and decreased Numb levels seen in tumors. Overall, we demonstrate Notch1 activation and subsequent acceleration through the T-cell developmental stages in this model of pan-hematopoietic Trp53 deletion.The B-cell specific Trp53 knockout mice (CD19-P53CKO) (n=54) were followed up for up to 2 years. The majority (47/54) developed splenomegaly in an age-dependent manner. Histologic examination showed marginal zone expansion (6/54), frank low-grade marginal zone lymphoma (16/54) or diffuse splenic lymphoma (25/54). The disease was confined to the spleen in the case of lower-grade histology while higher grades correlated with liver and kidney involvement. Flow cytometric analysis of tumors showed B220+ CD19+ IgM+ cells. Interestingly, these tumors demonstrated low levels of Notch2 expression, which normally is highly expressed in marginal zone B-cells.In order to characterize pathogenesis, we sorted follicular and marginal zone B-cells from floxed P53 and pre-malignant CD19-P53CKO mice. RNA was isolated from all these fractions and the spleens of 5 CD19-P53KO mice with diffuse lymphoma and subjected to RNA-Seq. A comparison of the floxed p53 with the CD19-P53CKO fractions (follicular and marginal zone) revealed a highly similar transcriptome. On the other hand, p53-deficient lymphomas showed >10,000 genes significantly differentially expressed demonstrating the unique transcriptome which developed during malignant transformation.Pathway analysis of these genes using Gene Set Enrichment Analysis (GSEA) identified enrichment of PI3K, Rap1 and MAPK signaling pathways, which are associated with cellular proliferation. Overexpression of the PI3K pathway genes Ccne1, Sgk1, Mapk13 and Pik3cb were validated by qPCR in 10 independent tumor samples when compared to the splenic marginal zone fractions. In the B-cell lineage, Trp53 deficiency leads to the dysregulation of multiple genes involved in key cellular signaling pathways, including the PI3K/MAPK pathway.In summary, pan hematopoietic deletion of Trp53 led to T-lineage lymphoma in young mice and myeloid tumors in older mice; with activation of Notch1 signaling in the former. B-cell specific deletion of Trp53 led to splenic marginal zone and diffuse B-cell lymphoma with transcriptional dysregulation of key signaling molecules. Hence, tumorigenesis by Trp53 deletion is tightly linked to lineage and appears to dysregulate key signaling pathways that are operant in those lineages, potentially identifying novel strategies for therapeutic interventions in P53 dependent human hematolymphoid malignancies. DisclosuresNo relevant conflicts of interest to declare.

INVESTIGATION OF SALIVARY miR-9, miR-34a ve miR-196a LEVELS IN FANCONI ANEMIA AND ORAL SQUAMOUS CELL CARCINOMA PATIENTS

Fanconi anemia (FA) is a rare bone marrow deficiency syndrome due to the DNA repair gene mutations, and Oral Squamous Cell Carcinoma (OSCC) is seen more frequently in FA patients than in the general population. The dysregulation of PI3K and Wnt signaling has been implicated in OSCC pathogenesis and abnormal expressions of miRNAs (a class of non-coding small regulatory RNAs) associated with these signaling pathways has been reported in OSHK patients. Salivary miRNAs are valuable biomarker candidates for OSCC development and prognosis. In this study, salivary levels of miR-9, miR-34a and miR-196a miRNAs related to PI3K and Wnt signaling pathways were examined in OSCC and FA patients and compared with the healthy control group. Saliva samples were collected from 89 subjects including 25 OSCC patients, 24 FA patients and 40 healthy controls. Total RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research) due to the kit instructions. cDNA was generated with miRCURY LNA miRNA PCR Assay (Qiagen, Hilden, Germany) and Quantitative real-time PCR was performed with miRCURY LNA SYBR Green PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For the normalization of the expression levels of each miRNA, the mean expression U6 SnRNA was used as reference. The ΔΔCt value and the normalized miR-9, miR-34a and miR-196a salivary levels were calculated with Livak Method. Our results showed that miR-9 and miR-34a levels in OSCC patients were significantly lower compared to healthy control groups (p= 0,01 and p= 0,012), and there was no significant difference in miR-196a levels (p> 0,05). In FA patients, miR-9 and miR-34 levels were lower than in control groups, likewise the OSCC patients (p =0,017 and p =0,014). There was no significant difference between miR-9, miR-34a, and miR-196a levels of FA patients and OSCC patients (p >0.05). According to our results, low levels of miR-9 and miR-34a in saliva are biomarker candidates that may be important for OSCC development. In FA patients, close follow-up of the levels of miR-9 and miR-34 would be appropriate considering OSCC development. Further studies are needed to confirm the potential of miR-9 and miR-34a as biomarkers for OSCC.

Exploring the selective mechanism of inhibitors towards different subtypes of class I PI3K

The activation of the PI3K signaling pathway is closely related to the formation of various cancers. However, four subtypes of PI3K have a high degree of sequence homology, which is challenging to develop isomeric selective inhibitors. In this work, MD simulations and binding affinity calculations are combined to elucidate the interaction mechanism of different inhibitors towards PI3K subtypes. Key residues, hydrophobic interactions, and hydrogen bonds forming between the receptor and inhibitor are pointed out, which is critical to the understanding of the binding selectivity. This work can provide a theoretical reference for the design of more promising selective inhibitors.

Revelation of Pivotal Genes Pertinent to Alzheimer's Pathogenesis: A Methodical Evaluation of 32 GEO Datasets.

Alzheimer’s disease (AD), a dreadful neurodegenerative disorder that affects cognitive and behavioral function in geriatric populations, is characterized by the presence of amyloid deposits and neurofibrillary tangles in brain regions. The International D World Alzheimer Report 2018 noted a global prevalence of 50 million AD cases and forecasted a threefold rise to 139 million by 2050. Although there exist numerous genetic association studies pertinent to AD in different ethnicities, critical genetic factors and signaling pathways underlying its pathogenesis remain ambiguous. This study was aimed to analyze the genetic data retrieved from 32 Gene Expression Omnibus datasets belonging to diverse ethnic cohorts in order to identify overlapping differentially expressed genes (DEGs). Stringent selection criteria were framed to shortlist appropriate datasets based on false discovery rate (FDR) p-value and log FC, and relevant details of upregulated and downregulated DEGs were retrieved. Among the 32 datasets, only six satisfied the selection criteria. The GEO2R tool was employed to retrieve significant DEGs. Nine common DEGs, i.e., SLC5A3, BDNF, SST, SERPINA3, RTN3, RGS4, NPTX, ENC1 and CRYM were found in more than 60% of the selected datasets. These DEGs were later subjected to protein–protein interaction analysis with 18 AD-specific literature-derived genes. Among the nine common DEGs, BDNF, SST, SERPINA3, RTN3 and RGS4 exhibited significant interactions with crucial proteins including BACE1, GRIN2B, APP, APOE, COMT, PSEN1, INS, NEP and MAPT. Functional enrichment analysis revealed involvement of these genes in trans-synaptic signaling, chemical transmission, PI3K pathway signaling, receptor–ligand activity and G protein signaling. These processes are interlinked with AD pathways.

Bombyxin II Regulates Glucose Absorption and Glycogen Synthesis through the PI3K Signaling Pathway in HepG2 Cells.

Bombyxin, as an insulin-like insect hormone, was discovered in the silkmoth Bombyx mori. It can regulate the metabolism of trehalose and glycogen in Bombyx mori, but whether it has glucose absorption and glycogen synthesis effect on mammalian cells was not clear. BombyxinII (BbxII) and mutant BbxII (mBbxII) genes were cloned into pcDNA3.1(+) vector, respectively; then, gene vectors were transfected into 293FT cells using Lipofectamine 2000. Levels of mRNA and protein expression of BbxII and mBbxII were detected by PCR and Western blot in 293FT cells, respectively. Glucose consumption and glycogenesis were determined by glucose oxidase-peroxidase (GOD-POD) and periodic acid-Schiff (PAS) staining in HepG2 cells; the PI3K signaling pathway was inhibited with wortmannin S1952 in HepG2 cells. Result showed that BbxII and mBbxII genes were being successfully expressed in 293FT cells, respectively. The expression protein of BbxII gene is 10kd pre-bombyxinII, and yet, the expression protein of mBbxII gene is 4kd mature bombyxinII. Only the 4kd bombyxinII showed increased glucose uptake and glycogenesis in HepG2 cells, and the ability of increasing glucose uptake was equal to the human insulin (10 nM). PI3K-wortmannin S1952 inhibitor can decrease the glycogen synthesis induced by bombyxin II protein in HepG2 cells. In conclusion, mature bombyxin II may adjust glucose absorption and glycogen synthesis in HepG2 cells through the PI3K signaling pathway.

Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation

Variants of the oncogenic EML4-ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4-ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4-ALK V3 but not V1 protein re-localises to microtubules, an effect recapitulated in a catalytically inactive EML4-ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild-type and catalytically inactive EML4-ALK V3 proteins, but not a Lys-Glu salt bridge mutant. We propose that EML4-ALK foci formation occurs as a result of transient association of stable EML4-ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4-ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.

Histological and Immunohistochemical Assessment of Ovarian Tissue and Endometrium in Patients with Diminished Ovarian Reserve

<h3>Study Objective</h3> To analyze the histological and immunohistochemical features of ovarian tissue and endometrium in women with diminished ovarian reserve and to investigate the correlation between these parameters and the effectiveness of infertility treatment <h3>Design</h3> Prospective study, Level II. <h3>Setting</h3> National Medical Research Center. <h3>Patients or Participants</h3> 70 patients with diminished ovarian reserve who underwent drug-free in vitro activation using a modified surgical technique. Two bilateral ovarian cortical tissue samples and one endometrial sample were obtained from each patient during the surgery. 40 patients out of 70 underwent ovarian stimulation after at least a one-month postoperative period. <h3>Interventions</h3> Bilateral ovarian cortex biopsy via laparoscopy, ovarian tissue fragmentation in aliquots of 1 mm², and hysteroscopy with endometrial biopsy were performed in all patients, followed by histological and immunohistochemical analysis (expression of proteins of the Hippo signaling pathway (YAP, MST-1, LATS-1) and PTEN (inhibitor of PI3K signaling pathway). The Human Protein Atlas datasets were used as a comparison group. <h3>Measurements and Main Results</h3> We revealed an impaired functioning of intracellular signaling pathways (primarily YAP- and PTEN-dependent) both in ovarian tissue and endometrial samples. Moreover, the expression of the proteins targeted in the study was significantly reduced in patients who responded to stimulation (n=31). Histological analysis of tissue samples of patients with diminished ovarian reserve indicated profound changes in various structures of the ovaries: surface epithelium, vascular component, stroma of ovarian cortex. In 25% of patients, chronic endometritis was also determined. <h3>Conclusion</h3> Specific histological and immunohistochemical features of ovarian tissue and endometrium associated with diminished ovarian reserve and the outcome of ovarian stimulation were shown.

KLK6 mediates stemness and metabolism of gastric carcinoma cells via the PI3K/AKT/mTOR signaling pathway.

Gastric cancer is a common tumor of the digestive system, which can occur in any part of the stomach. Kallikrein 6 (KLK6) is a trypsin-like serine protease and has been found to be involved in extracellular matrix remodeling, tumor invasion and nervous system plasticity. Our previous study reported that KLK6 suppressed HGC-27 gastric cancer cell growth by inhibiting epithelial-mesenchymal transition; however, the mechanism of action underlying the effect of KLK6 still remains unclear. The aim of the present study was to investigate the effect and the underlying mechanism of KLK6 on stem cell-like properties and metabolism in gastric carcinoma cells. The HGC-27 cell line was transfected with KLK6 overexpression (OV-KLK6) and interference (short hairpin-KLK6) vectors, then the transfection efficiency was confirmed using western blot analysis and reverse transcription-quantitative PCR. The percentage of CD133+ and CD44+ cells was detected using flow cytometry, while the protein expression levels of the stem-associated genes, Nanog, Oct-4, SOX2 and Notch1, the metabolic markers, hexokinase (HK)1, HK2, GLUT1, and the proteins within the PI3K signaling pathway, phosphorylated (p)-PI3K, p-AKT and p-mTOR, were determined using western blot analysis. Biochemical kits were used to measure ATP production, lactic acid content and glucose uptake. A tumorigenicity assay was performed with nude mice to detect gastric tumor volume, and the protein expression level of Oct-4, Nanog, HK1, HK2 and GLUT1, and the mRNA expression level of KLK6 was also determined in gastric tumor tissues of mice. Compared with that in the control group, KLK6 protein and mRNA expression levels were significantly decreased in the four sh-RNA groups (P<0.05). Among them, sh-RNA-3 induced the lowest KLK6 expression and was used to silence KLK6 in subsequent experiments. Compared with that in the control and negative control groups, the percentage of CD133+ and CD44+ cells, the protein expression level of Oct-4, Nanog, HK1, HK2, GLUT1, p-PI3K, p-AKT and p-mTOR, and ATP content, lactic acid production, glucose uptake and gastric tumor volume were significantly decreased by sh-KLK6 (P<0.05), whereas KLK6 overexpression induced the opposite effect (P<0.05). In conclusion, KLK6 modulated stemness properties and cell metabolic profile in gastric carcinoma cells and the mechanism may be associated with the PI3K/AKT/mTOR signaling pathway.

Material Basis and Mechanism of Chansu Injection for COVID-19 Treatment Based on Network Pharmacology and Molecular Docking Technology.

Purpose The clinical efficacy of Chansu injection for COVID-19 treatment has been confirmed. Its mechanism of action remains unclear. We used network pharmacology and molecular docking technology to explore the potential material basis and mechanism of action of Chansu injection for COVID-19. Methods The main components of Chansu injection were determined using HPLC. The PharmMapper, SwissTargetPrediction, SEA, and TCMID databases were used to screen for the active ingredients and therapeutic targets of Chansu injection, while the OMIM and GeneCards Suite databases were used to search for COVID-19-related targets. The STRING database was used for protein-protein interaction (PPI) network construction and topological analysis, while DAVID was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the core targets. The main active compounds of Chansu injection were docked with 3CL protease, ACE2, RdRp, and spike protein. Results The three Chansu injection compounds were identified using HPLC. A total of 236 drug-related targets and 16,611 disease-related targets were identified, and 77 common targets were determined through mapping. The PPI mapping results revealed that 16 core targets were obtained through topological analysis and screening. Furthermore, GO and KEGG pathway enrichment analyses revealed that the PI3K and JAK-STAT signaling pathways are the major pathways. The molecular docking results suggest that the three Chansu injection components have high binding energies to the S protein. Conclusions The potential mechanism of Chansu injection for COVID-19 involves multiple targets and pathways, thereby providing a scientific basis for its clinical application and further research.

RTK-Dependent Inducible Degradation of Mutant PI3Kα Drives GDC-0077 (Inavolisib) Efficacy.

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. SIGNIFICANCE: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors.See related commentary by Vanhaesebroeck et al., p. 20.This article is highlighted in the In This Issue feature, p. 1.

Macrophage migration inhibitory factor (MIF) inhibitor 4-IPP downregulates stemness phenotype and mesenchymal trans-differentiation after irradiation in glioblastoma multiforme.

Radiation therapy is among the most essential treatment methods for glioblastoma multiforme (GBM). Radio-resistance and cancer stem cell properties can cause therapeutic resistance, cancer heterogeneity, and poor prognoses in association with GBM. Furthermore, the GBM subtype transition from proneural to the most malignant mesenchymal subtype after radiation therapy also accounts for high resistance to conventional treatments. Here, we demonstrate that the inhibition of macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (DDT) by 4-iodo-6-phenylpyrimidine (4-IPP), a dual inhibitor targeting MIF and DDT, downregulates stemness phenotype, intracellular signaling cascades, mesenchymal trans-differentiation, and induces apoptosis in proneural glioma stem cells (GSCs). In an analysis of The Cancer Genome Atlas, high MIF and DDT expression were associated with poor prognosis. GSC growth was effectively inhibited by 4-IPP in a time- and dose-dependent manner, and 4-IPP combined with radiation therapy led to significantly reduced proliferation compared with radiation therapy alone. The expression of stemness factors, such as Olig2 and SOX2, and the expression of pAKT, indicating PI3K signaling pathway activation, were decreased in association with both 4-IPP monotherapy and combination treatment. The expression of mesenchymal markers, TGM2 and NF-κB, and expression of pERK (indicating MAPK signaling pathway activation) increased in association with radiation therapy alone but not with 4-IPP monotherapy and combination therapy. In addition, the combination of 4-IPP and radiation therapy significantly induced apoptosis compared to the monotherapy of 4-IPP or radiation. In vivo results demonstrated a significant tumor-suppressing effect of 4-IPP when combined with radiation therapy. Collectively, our results showed that the targeted inhibition of MIF and DDT has the potential to strengthen current clinical strategies by enhancing the anticancer effects of radiation therapy.

Bradykinin Protects Human Endothelial Progenitor Cells from High-Glucose-Induced Senescence through B2 Receptor-Mediated Activation of the Akt/eNOS Signalling Pathway.

Background Circulating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown. Methods Cord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated β-galactosidase (SA-β-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot. Results B2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK. Conclusion BK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.