Triclosan induces earlier puberty onset in female mice via interfering with L-type calcium channels and activating Pik3cd.

Triclosan (TCS) is a broad-spectrum antibacterial chemical widely presents in people's daily lives. Epidemiological studies have revealed that TCS exposure may affect female puberty development. However, the developmental toxicity after low-dose TCS continuous exposure remains to be confirmed. In our study, 8-week-old ICR female mice were continuously exposed to TCS (30, 300, 3000 µg/kg/day) or vehicle (corn oil) from 2 weeks before mating to postnatal day 21 (PND 21) of F1 female mice, while F1 female mice were treated with TCS intragastric administration from PND 22 until PND 56. Vaginal opening (VO) observation, hypothalamic-pituitary-ovarian (HPO) axis related hormones and genes detection, and ovarian transcriptome analysis were carried out to investigate the effects of TCS exposure on puberty onset. Meanwhile, human granulosa-like tumor cell lines (KGN cells) were exposed to TCS to further explore the biological mechanism of the ovary in vitro. The results showed that long-term exposure to low-dose TCS led to approximately a 3-day earlier puberty onset in F1 female mice. Moreover, TCS up-regulated the secretion of estradiol (E2) and the expression of ovarian steroidogenesis genes. Notably, ovarian transcriptomes analysis as well as bidirectional validation in KGN cells suggested that L-type calcium channels and Pik3cd were involved in TCS-induced up-regulation of ovarian-related hormones and genes. In conclusion, our study demonstrated that TCS interfered with L-type calcium channels and activated Pik3cd to up-regulate the expression of ovarian steroidogenesis and related genes, thereby inducing the earlier puberty onset in F1 female mice.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Cell-Penetrating Peptide-Modified Graphene Oxide Nanoparticles Loaded with Rictor siRNA for the Treatment of Triple-Negative Breast Cancer.

INTRODUCTION: Breast cancer is a malignant tumor that seriously threatens women's life and health. METHODS: In this study, we proposed to use graphene nanoparticles loaded with siRNA that can silence Rictor molecules essential for the mammalian target of rapamycin (mTOR) complex 2 (mTORC2) complex to enhance gene delivery to tumor cells through modification of cell-penetrating peptide (CPP) for the treatment of breast cancer. RESULTS: Remarkably, we successfully synthesized graphene oxide (GO)/polyethyleneimine (PEI)/polyethylene glycol (PEG)/CPP/small interfering RNA (siRNA) system, and the results were observed by atomic force microscopy (AFM) and ultraviolet visible (UV-Vis) absorption spectra. The optimum mass ratio of siRNA to GO-PEI-PEG-CPP was 1:0.5. We screened out Rictor siRNA-2 from 9 candidates, which presented the highest inhibition rate, and this siRNA was selected for the subsequent experiments. We validated that Rictor siRNA-2 significantly reduced the Rictor expression in triple negative breast cancer (TNBC) cells. Confocal fluorescence microscope and flow cytometry analysis showed that GO-PEI-PEG-CPP/siRNA was able to be effectively uptake by TNBC cells. GO-PEI-PEG-CPP/siRNA improved the effect of siRNA on the inhibition of TNBC cell viability and the induction of TNBC cell apoptosis. The expression of Rictor and the phosphorylation of Akt and p70s6k were inhibited by GO-PEI-PEG-CPP/siRNA. Tumorigenicity analysis in nude mice showed that GO-PEI-PEG-CPP/siRNA significantly repressed the tumor growth of TNBC cells in vivo. The levels of ki-67 were repressed by GO-PEI-PEG-CPP/siRNA, and the apoptosis was induced by GO-PEI-PEG-CPP/siRNA in the system. DISCUSSION: Therefore, we concluded that CPP-modified GO nanoparticles loaded with Rictor siRNA significantly repressed TNBC progression by the inhibition of PI3K/Akt/mTOR signaling. Our finding provides a promising therapeutic strategy for the treatment of TNBC.

Novel Therapies for Metastatic Triple-Negative Breast Cancer: Spotlight on Immunotherapy and Antibody-Drug Conjugates.

Background: Triple-negative breast cancer (TNBC) is a biologically heterogeneous disease that is often associated with worse outcomes compared with other subtypes such as hormone receptor-positive tumors and HER2-positive tumors. While chemotherapy remains the mainstay of standard therapy for metastatic TNBC (mTNBC), several novel treatments have been developed over the past few years. In this review article, we review the major developments in the management of patients with mTNBC. Summary: The combination of chemotherapy and immunotherapy is a potential therapeutic option for PD-L1-positive mTNBC, as the FDA recently approved atezolizumab (Tecentriq) and pembrolizumab (Keytruda) in combination with chemotherapy. Also, 2 targeted therapies-olaparib (Lynparza) and talazoparib (Talzenna)-are FDA approved for the management of mTNBC with germline BRCA mutations, and sacituzumab govitecan, an anti-Trop2 antibody-drug conjugate (ADC), was recently approved for previously treated mTNBC. A number of promising therapies are on the horizon, including AKT inhibitors for PI3K-altered TNBC as well as other ADCs. Key Message: The successful clinical development of immunotherapies, PARP inhibitors, and ADCs for the management of mTNBC has improved the survival outcome of patients. Over the coming years, the therapeutic developments in precision medicine will likely change the mTNBC landscape, and might make the current definition of TNBC as breast cancer that is estrogen receptor negative, progesterone receptor negative, and HER2 negative obsolete.

CD44/HA signaling mediates acquired resistance to a PI3Kα inhibitor.

Most luminal breast carcinomas (BrCas) bearing PIK3CA mutations initially respond to phosphoinositide-3-kinase (PI3K)-α inhibitors, but many eventually become resistant. The underlying mechanisms of this resistance remain obscure. In this work, we showed that a CD44high state due to aberrant isoform splicing was acquired from adaptive resistance to a PI3Kα inhibitor (BLY719) in luminal BrCas. Notably, the expression of CD44 was positively correlated with estrogen receptor (ER) activity in PIK3CA-mutant breast cancers, and ER-dependent transcription upon PI3Kα pathway inhibition was in turn mediated by CD44. Furthermore, the interaction of CD44 with the ligand hyaluronan (HA) initiated the Src-ERK signaling cascade, which subsequently maintained AKT and mTOR activity in the presence of a PI3Kα inhibitor. Activation of this pathway was prevented by disruption of the CD44/HA interaction, which in turn restored sensitivity to BLY719. Our results revealed that an ER-CD44-HA signaling circuit that mediates robust compensatory activation of the Src-ERK signaling cascade may contribute to the development of acquired resistance to PI3Kα inhibitors. This study provides new insight into the mechanism of adaptive resistance to PI3Kα inhibition therapy.

Antagonists of the serotonin receptor 5A target human breast tumor initiating cells.

BACKGROUND: Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR). METHODS: We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line. RESULTS: We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551. CONCLUSIONS: Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.

Ex vivo blockade of PI3K gamma or delta signaling enhances the antitumor potency of adoptively transferred CD8+ T cells.

Adoptive T cell transfer therapy induces objective responses in patients with advanced malignancies. Despite these results, some individuals do not respond due to the generation of terminally differentiated T cells during the expansion protocol. As the gamma and delta catalytic subunits in the PI3K pathway are abundant in leukocytes and involved in cell activation, we posited that blocking both subunits ex vivo with the inhibitor IPI-145 would prevent their differentiation, thereby increasing antitumor activity in vivo. However, IPI-145 treatment generated a product with reduced antitumor activity. Instead, T cells inhibited of PI3Kγ (IPI-549) or PI3Kδ (CAL-101 or TGR-1202) alone were more potent in vivo. While T cells coinhibited of PI3Kγ and PI3Kδ were less differentiated, they were functionally impaired, indicated by reduced production of effector cytokines after antigenic re-encounter and decreased persistence in vivo. Human CAR T cells expanded with either a PI3Kγ or PI3Kδ inhibitor possessed a central memory phenotype compared to vehicle cohorts. We also found that PI3Kδ-inhibited CARs lysed human tumors in vitro more effectively than PI3Kγ-expanded or traditionally expanded CAR T cells. Our data imply that sole blockade of PI3Kγ or PI3Kδ generates T cells with remarkable antitumor properties, a discovery that has substantial clinical implications.

A high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells.

Elevated glucose consumption is fundamental to cancer, but selectively targeting this pathway is challenging. We develop a high-throughput assay for measuring glucose consumption and use it to screen non-small-cell lung cancer cell lines against bioactive small molecules. We identify Milciclib that blocks glucose consumption in H460 and H1975, but not in HCC827 or A549 cells, by decreasing SLC2A1 (GLUT1) mRNA and protein levels and by inhibiting glucose transport. Milciclib blocks glucose consumption by targeting cyclin-dependent kinase 7 (CDK7) similar to other CDK7 inhibitors including THZ1 and LDC4297. Enhanced PIK3CA signaling leads to CDK7 phosphorylation, which promotes RNA Polymerase II phosphorylation and transcription. Milciclib, THZ1, and LDC4297 lead to a reduction in RNA Polymerase II phosphorylation on the SLC2A1 promoter. These data indicate that our high-throughput assay can identify compounds that regulate glucose consumption and that CDK7 is a key regulator of glucose consumption in cells with an activated PI3K pathway.

A mutation in PIK3CD gene causing pediatric systemic lupus erythematosus: A case report.

RATIONALE: Gain of function (GOF) mutations in PIK3CD gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, cytomegalovirus and/or epstein-Barr virus (EBV) viremia, and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. PATIENT CONCERNS: We report a patient who was diagnosed with systemic lupus erythematosus (SLE) at a young age and was recently found to carry heterozygous mutations in PIK3CD. The patient not only presented with recurrent sinopulmonary infections, CD4 lymphopenia, lymphadenopathy, EBV viremia, and elevated serum IgM, but also met classification criteria of SLE based on persistent proteinuria and hematuria, leukopenia and anemia, low level of serum complement, and positive autoantibody for antinuclear antibodies. DIAGNOSES: Activated PI3Kδ syndrome. INTERVENTIONS: Oral prednisolone and hydroxychloroquine combined with mycophenolate mofetil was given to the patient. He was currently receiving intravenous immunoglobulin per month in association with hydroxychloroquine, low-dose prednisolone, and mycophenolate mofetil. OUTCOMES: At present, the level of complement restored to normal, hematuria and proteinuria disappeared, and liver function returned to normal. LESSONS: SLE may be a novel phenotype of GOF mutation in PI3CKD gene (GOF PIK3CD).

Herpesviruses in the Activated Phosphatidylinositol-3-Kinase-δ Syndrome.

The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is important for multiple stages of herpesvirus replication including virus entry, replication, latency, and reactivation. Recently, patients with gain-of-function mutations in the p110δ-catalytic subunit of PI3K or in the p85-regulatory subunit of PI3K have been reported. These patients have constitutively active PI3K with hyperactivation of Akt. They present with lymphoproliferation and often have infections, particularly recurrent respiratory infections and/or severe virus infections. The most frequent virus infections are due to Epstein-Barr virus (EBV) and cytomegalovirus (CMV); patients often present with persistent EBV and/or CMV viremia, EBV lymphoproliferative disease, or CMV lymphadenitis. No patients have been reported with CMV pneumonia, colitis, or retinitis. Other herpesvirus infections have included herpes simplex pneumonia, recurrent zoster, and varicella after vaccination with the varicella vaccine. Additional viral infections have included adenovirus viremia, severe warts, and extensive Molluscum contagiosum virus infection. The increased susceptibility to virus infections in these patients is likely due to a reduced number of long-lived memory CD8 T cells and an increased number of terminally differentiated effector CD8 T cells.