bims-pideca 2019-12-22 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2019–12–22
seventeen papers selected by
Ralitsa Radostinova Madsen, University College London

Efficacy of PI3K inhibitors in advanced breast cancer.
Lab review: Molecular dissection of the signal transduction pathways associated with PTEN deletion-induced optic nerve regeneration.
Overview of the relevance of PI3K pathway in HR-positive breast cancer.
Sustained activation of autophagy suppresses adipocyte maturation via a lipolysis-dependent mechanism.
Individual Cells Can Resolve Variations in Stimulus Intensity along the IGF-PI3K-AKT Signaling Axis.
Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling.
Biomarkers of response and resistance to PI3K inhibitors in estrogen receptor-positive breast cancer patients and combination therapies involving PI3K inhibitors.
Capivasertib Plus Paclitaxel Versus Placebo Plus Paclitaxel As First-Line Therapy for Metastatic Triple-Negative Breast Cancer: The PAKT Trial.
Combination of KRAS gene silencing and PI3K inhibition for ovarian cancer treatment.
First-In-Human Phase I Study Of A Dual mTOR Kinase And DNA-PK Inhibitor (CC-115) In Advanced Malignancy.
Fluorescent biosensors for multiplexed imaging of phosphoinositide dynamics.
Synergistic activity of mTORC1/2 kinase and MEK inhibitors suppresses pediatric low-grade glioma tumorigenicity and vascularity.
Direct physical interaction of active Ras with mSIN1 regulates mTORC2 signaling.
Somatic Mutation of PIK3CA (H1047R) Is a Common Driver Mutation Hotspot in Canine Mammary Tumors as Well as Human Breast Cancers.
The PI3k/Akt pathway is associated with angiogenesis, oxidative stress and survival of mesenchymal stem cells in pathophysiologic condition in ischemia.
Wortmannin targeting phosphatidylinositol 3-kinase suppresses angiogenic factors in shear-stressed endothelial cells.
Stearoyl-CoA desaturase and tumorigenesis.

Ann Oncol. 2019 Dec 01. 30(Supplement_10): x12-x20

Efficacy of PI3K inhibitors in advanced breast cancer.

B Verret, J Cortes, T Bachelot, F Andre, M Arnedos.

  The phosphoinositide 3 (PI3)-kinase/Akt signaling pathway has always been a focus of interest in breast cancer due to its role in cell growth, cell proliferation, cell migration and deregulated apoptosis. Its activation has been linked to endocrine resistance and worse prognosis in certain subgroups of breast cancer. In addition, deregulation of the PI3K/Akt pathway including PIK3CA activating mutation is frequently present in breast cancer. Multiple efforts have been carried out to target this pathway, initially with pan-PI3K inhibitors with some hint of activity but hampered by their limiting side-effects. A recent large randomized trial in patients with endocrine-resistant PIK3CA-mutant hormone receptor (HR)-positive tumors led to the approval of the first PI3K inhibitor, alpelisib, in combination with fulvestrant. The specificity of alpelisib against the p110α catalytic isoform provided additional efficacy and a better toxicity profile. In this review, we summarize the main research with PI3K inhibitors in breast cancer and we provide some insight of potential future combinations of this treatment in breast cancer patients.

Keywords:  PI3K inhibitors; breast cancer; targeted therapy

DOI:  https://doi.org/10.1093/annonc/mdz381
Restor Neurol Neurosci. 2019 ;37(6): 545-552

Lab review: Molecular dissection of the signal transduction pathways associated with PTEN deletion-induced optic nerve regeneration.

Haoliang Huang, Simran Kaur, Yang Hu.

   BACKGROUND: Permanent loss of vital functions after central nervous system (CNS) injury occurs in part because axons in the adult mammalian CNS do not regenerate after injury. PTEN was identified as a prominent intrinsic inhibitor of CNS axon regeneration about 10 years ago. The PTEN negatively regulated PI3K-AKT-mTOR pathway, which has been intensively explored in diverse models of axon injury and diseases and its mechanism for axon regeneration is becoming clearer.
OBJECTIVE: It is timely to summarize current knowledge about the PTEN/AKT/mTOR pathway and discuss future directions of translational regenerative research for neural injury and neurodegenerative diseases.
METHODS: Using mouse optic nerve crush as an in vivo retinal ganglion cell axon injury model, we have conducted an extensive molecular dissection of the PI3K-AKT-mTORC1/mTORC2 pathway to illuminate the cross-regulating mechanisms in axon regeneration.
RESULTS: AKT is the nodal point that coordinates both positive (PI3K-PDK1-pAKT-T308) and negative (PI3K-mTORC2-pAKT-S473) signals to regulate adult CNS axon regeneration through two parallel pathways, activating mTORC1 and inhibiting GSK3β. However, mTORC1/S6K1-mediated feedback inhibition after PTEN deletion prevents potent AKT activation.
CONCLUSIONS: A key permissive signal from an unidentified AKT-independent pathway is required for stimulating the neuron-intrinsic growth machinery. Future studies into this complex neuron-intrinsic balancing mechanism involving necessary and permissive signals for axon regeneration is likely to lead to safe and effective regenerative strategies for CNS repair.

Keywords:  AKT; Axon regeneration; Optic Nerve; PTEN; RGC; mTOR

DOI:  https://doi.org/10.3233/RNN-190949
Ann Oncol. 2019 Dec 01. 30(Supplement_10): x3-x11

Overview of the relevance of PI3K pathway in HR-positive breast cancer.

N Vasan, E Toska, M Scaltriti.

  One of the hallmarks of hormone receptor (HR)-positive breast cancer is its dependence on the phosphatidylinositol-3-kinase (PI3K) pathway. Here, we review the epidemiologic, functional, and pharmacologic interactions between oncogenic PI3K and the estrogen receptor (ER). We discuss the epidemiology of PI3K pathway alterations, mechanisms of resistance to PI3K inhibitors, and the current mechanistic landscape of crosstalk between PI3K and ER, which provide the rationale for dual ER and PI3K inhibition and is now a standard of care in the treatment of ER+ PIK3CA-mutant metastatic breast cancer. We outline newer studies in this field that delineate the clinically relevant overlaps between PI3K and parallel signaling pathways, insulin signaling, and ER epigenetic modifiers. We also identify several caveats with the current data and propose new strategies to overcome these bottlenecks.

Keywords:   PIK3CA ; AKT; PI3K inhibitors; PI3K pathway; breast cancer; estrogen receptor

DOI:  https://doi.org/10.1093/annonc/mdz281
Autophagy. 2019 Dec 18. 1-15

Sustained activation of autophagy suppresses adipocyte maturation via a lipolysis-dependent mechanism.

Xing Zhang, Dandan Wu, Chunqing Wang, Yan Luo, Xiaofeng Ding, Xin Yang, Floyd Silva, Sara Arenas, John Michael Weaver, Michael Mandell, Vojo Deretic, Meilian Liu.

  Dysregulation of macroautophagy/autophagy is implicated in obesity and insulin resistance. However, it remains poorly defined how autophagy regulates adipocyte development. Using adipose-specific rptor/raptor knockout (KO), atg7 KO and atg7 rptor double-KO mice, we show that inhibiting MTORC1 by RPTOR deficiency led to autophagic sequestration of lipid droplets, formation of LD-containing lysosomes, and elevation of basal and isoproterenol-induced lipolysis in vivo and in primary adipocytes. Despite normal differentiation at an early phase, progressive degradation and shrinkage of cellular LDs and downregulation of adipogenic markers PPARG and PLIN1 occurred in terminal differentiation of rptor KO adipocytes, which was rescued by inhibiting lipolysis or lysosome. In contrast, inactivating autophagy by depletion of ATG7 protected adipocytes against RPTOR deficiency-induced formation of LD-containing lysosomes, LD degradation, and downregulation of adipogenic markers in vitro. Ultimately, atg7 rptor double-KO mice displayed decreased lipolysis, restored adipose tissue development, and upregulated thermogenic gene expression in brown and inguinal adipose tissue compared to RPTOR-deficient mice in vivo. Collectively, our study demonstrates that autophagy plays an important role in regulating adipocyte maturation via a lipophagy and lipolysis-dependent mechanism.Abbreviations: ATG7: autophagy related 7; BAT: brown adipose tissue; CEBPB/C/EBPβ: CCAAT enhancer binding protein beta; DGAT1: diacylglycerol O-acyltransferase 1; eWAT: epididymal white adipose tissue; iWAT: inguinal white adipose tissue; KO: knockout; LD: lipid droplet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; PLIN1: perepilin 1; PNPLA2/ATGL: patatin-like phospholipase domain containing 2; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; RPTOR: regulatory associated protein of MTOR complex1; TG: triglyceride; ULK1: unc-51 like kinase 1; UCP1: uncoupling protein 1; WAT: white adipose tissue.

Keywords:  Adipocyte maturation; MTORC1; autophagy; lipolysis; lysosome

DOI:  https://doi.org/10.1080/15548627.2019.1703355
Cell Syst. 2019 Dec 18. pii: S2405-4712(19)30391-6. [Epub ahead of print]9(6): 580-588.e4

Individual Cells Can Resolve Variations in Stimulus Intensity along the IGF-PI3K-AKT Signaling Axis.

Sean M Gross, Mark A Dane, Elmar Bucher, Laura M Heiser.

  Cells sense and respond to signals in their local environment by activating signaling cascades that lead to phenotypic changes. Differences in these signals can be discriminated at the population level; however, single cells have been thought to be limited in their capacity to distinguish ligand doses due to signaling noise. We describe here the rational development of a genetically encoded FoxO1 sensor, which serves as a down-stream readout of insulin growth factor-phosphatidylinositol 3-kinase IGF-PI3K-AKT signaling pathway activity. With this reporter, we tracked individual cell responses to multiple IGF-I doses, pathway inhibitors, and repeated treatments. We observed that individual cells can discriminate multiple IGF-I doses, and these responses are sustained over time, are reproducible at the single-cell level, and display cell-to-cell heterogeneity. These studies imply that cell-to-cell variation in signaling responses is biologically meaningful and support the endeavor to elucidate mechanisms of cell signaling at the level of the individual cell.

Keywords:  AKT; FoxO1; IGF-I; fluorescent reporters; information theory; live-cell imaging; signaling dynamics; signaling pathways; single cell

DOI:  https://doi.org/10.1016/j.cels.2019.11.005
Cell Chem Biol. 2019 Dec 10. pii: S2451-9456(19)30396-4. [Epub ahead of print]

Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling.

Inchul You, Emily C Erickson, Katherine A Donovan, Nicholas A Eleuteri, Eric S Fischer, Nathanael S Gray, Alex Toker.

  The PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over half of tumors exhibiting aberrant AKT activation. Although potent small-molecule AKT inhibitors have entered clinical trials, robust and durable therapeutic responses have not been observed. As an alternative strategy to target AKT, we report the development of INY-03-041, a pan-AKT degrader consisting of the ATP-competitive AKT inhibitor GDC-0068 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon (CRBN). INY-03-041 induced potent degradation of all three AKT isoforms and displayed enhanced anti-proliferative effects relative to GDC-0068. Notably, INY-03-041 promoted sustained AKT degradation and inhibition of downstream signaling effects for up to 96 h, even after compound washout. Our findings suggest that AKT degradation may confer prolonged pharmacological effects compared with inhibition, and highlight the potential advantages of AKT-targeted degradation.

Keywords:  AKT; PI 3-kinase; PROTAC; degrader; oncogene; protein kinase

DOI:  https://doi.org/10.1016/j.chembiol.2019.11.014
Ann Oncol. 2019 Dec 01. 30(Supplement_10): x27-x42

Biomarkers of response and resistance to PI3K inhibitors in estrogen receptor-positive breast cancer patients and combination therapies involving PI3K inhibitors.

M Brandão, R Caparica, D Eiger, E de Azambuja.

  In this review, we discuss biomarkers of response and resistance to PI3K inhibitors (PI3Ki) in estrogen receptor-positive breast cancer, both in the early and advanced settings. We analyse data regarding PIK3CA mutations, PI3K pathway activation, PTEN expression loss, Akt signalling, insulin levels, 18FFDG-PET/CT imaging, FGFR1/2 amplification, KRAS and TP53 mutations. Most of the discussed data comprise retrospective and exploratory studies, hence many results are not conclusive. Therefore, among all of these biomarkers, only PIK3CA mutations have proved to have a predictive value for treatment with the α-selective PI3Ki alpelisib (SOLAR-1 trial) and the β-sparing PI3Ki taselisib (SANDPIPER trial) in the advanced setting. Since the accuracy of current individual biomarkers is not optimal, a composite biomarker, including DNA, RNA and protein expression data, to more precisely assess the PI3K/AKT/mTOR pathway activation status, may arise as a promising approach. Finally, we describe the rational for new combination therapies involving PI3Ki and anti-HER2 agents, chemotherapy, CDK4/6 inhibitors, mTOR inhibitors or new endocrine treatments and discuss the ongoing trials in this field.

Keywords:  PI3K inhibitors; PIK3CA; breast neoplasms; gene sequencing; predictive biomarkers

DOI:  https://doi.org/10.1093/annonc/mdz280
J Clin Oncol. 2019 Dec 16. JCO1900368

Capivasertib Plus Paclitaxel Versus Placebo Plus Paclitaxel As First-Line Therapy for Metastatic Triple-Negative Breast Cancer: The PAKT Trial.

Peter Schmid, Jacinta Abraham, Stephen Chan, Duncan Wheatley, Adrian Murray Brunt, Gia Nemsadze, Richard D Baird, Yeon Hee Park, Peter S Hall, Timothy Perren, Robert C Stein, László Mangel, Jean-Marc Ferrero, Melissa Phillips, John Conibear, Javier Cortes, Andrew Foxley, Elza C de Bruin, Robert McEwen, Daniel Stetson, Brian Dougherty, Shah-Jalal Sarker, Aaron Prendergast, Max McLaughlin-Callan, Matthew Burgess, Cheryl Lawrence, Hayley Cartwright, Kelly Mousa, Nicholas C Turner.

PURPOSE: The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently activated in triple-negative breast cancer (TNBC). The AKT inhibitor capivasertib has shown preclinical activity in TNBC models, and drug sensitivity has been associated with activation of PI3K or AKT and/or deletions of PTEN. The PAKT trial was designed to evaluate the safety and efficacy of adding capivasertib to paclitaxel as first-line therapy for TNBC.
PATIENTS AND METHODS: This double-blind, placebo-controlled, randomized phase II trial recruited women with untreated metastatic TNBC. A total of 140 patients were randomly assigned (1:1) to paclitaxel 90 mg/m2 (days 1, 8, 15) with either capivasertib (400 mg twice daily) or placebo (days 2-5, 9-12, 16-19) every 28 days until disease progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). Secondary end points included overall survival (OS), PFS and OS in the subgroup with PIK3CA/AKT1/PTEN alterations, tumor response, and safety.
RESULTS: Median PFS was 5.9 months with capivasertib plus paclitaxel and 4.2 months with placebo plus paclitaxel (hazard ratio [HR], 0.74; 95% CI, 0.50 to 1.08; 1-sided P = .06 [predefined significance level, 1-sided P = .10]). Median OS was 19.1 months with capivasertib plus paclitaxel and 12.6 months with placebo plus paclitaxel (HR, 0.61; 95% CI, 0.37 to 0.99; 2-sided P = .04). In patients with PIK3CA/AKT1/PTEN-altered tumors (n = 28), median PFS was 9.3 months with capivasertib plus paclitaxel and 3.7 months with placebo plus paclitaxel (HR, 0.30; 95% CI, 0.11 to 0.79; 2-sided P = .01). The most common grade ≥ 3 adverse events in those treated with capivasertib plus paclitaxel versus placebo plus paclitaxel, respectively, were diarrhea (13% v 1%), infection (4% v 1%), neutropenia (3% v 3%), rash (4% v 0%), and fatigue (4% v 0%).
CONCLUSION: Addition of the AKT inhibitor capivasertib to first-line paclitaxel therapy for TNBC resulted in significantly longer PFS and OS. Benefits were more pronounced in patients with PIK3CA/AKT1/PTEN-altered tumors. Capivasertib warrants further investigation for treatment of TNBC.

DOI: https://doi.org/10.1200/JCO.19.00368
J Control Release. 2019 Dec 12. pii: S0168-3659(19)30738-2. [Epub ahead of print]

Combination of KRAS gene silencing and PI3K inhibition for ovarian cancer treatment.

Min Ju Kim, So Jin Lee, Ju Hee Ryu, Sun Hwa Kim, Ick Chan Kwon, Thomas M Roberts.

  The phosphoinositide 3-kinase (PI3K) and RAS signaling pathways are frequently co-activated and altered during oncogenesis. Owing to their regulatory cross-talk, the early attempts of targeting only one pathway have mostly ended up promoting the development of drug resistance. Here, we propose using small interfering RNA (siRNA) therapeutics to directly target the undruggable KRAS (siKRAS) in combination with the pan-PI3K inhibitor GDC-0941 (GDC) to simultaneously block both PI3K and RAS signaling, thereby exerting synergistic anti-tumor effects on ovarian cancers with PTEN deficiency and KRASG12D mutation. For successful delivery of siKRAS, tGC/psi-nanoparticle formulation of polymerized siRNA and thiol-modified glycol chitosan nanoparticle-was used for KRAS specific inhibition in vitro and in vivo. GDC or siKRAS monotherapy each impede downstream signaling, leading to some delay in cell proliferation and migration. When combined, however, they engender much higher inhibition of PI3K signaling and stimulation of apoptosis in an ovarian allograft model compared to monotherapies. Our results show the feasibility of developing new combination strategies for the management of multiple oncogenic mutations activating PI3K and RAS signaling.

Keywords:  Combination therapy; KRAS; Ovarian Cancer; PI3K; siRNA

DOI:  https://doi.org/10.1016/j.jconrel.2019.12.019
Cancer Manag Res. 2019 ;11 10463-10476

First-In-Human Phase I Study Of A Dual mTOR Kinase And DNA-PK Inhibitor (CC-115) In Advanced Malignancy.

Pamela Munster, Monica Mita, Amit Mahipal, John Nemunaitis, Christophe Massard, Tom Mikkelsen, Cristina Cruz, Luis Paz-Ares, Manuel Hidalgo, Dana Rathkopf, George Blumenschein, David C Smith, Barbara Eichhorst, Tim Cloughesy, Ellen H Filvaroff, Shaoyi Li, Heather Raymon, Hans de Haan, Kristen Hege, Johanna C Bendell.

   Purpose: This first-in-human Phase I study investigated the safety, pharmacokinetics (PK), pharmacodynamic profile, and preliminary efficacy of CC-115, a dual inhibitor of mammalian target of rapamycin (mTOR) kinase and DNA-dependent protein kinase.
Patients and Methods: Patients with advanced solid or hematologic malignancies were enrolled in dose-finding and cohort expansion phases. In dose-finding, once-daily or twice-daily (BID) ascending oral doses of CC-115 (range: 0.5-40 mg/day) in 28-day continuous cycles identified the maximum-tolerated dose for cohort expansion in 5 specified tumor types. Twelve additional patients with mixed solid tumors participated in a bioavailability substudy.
Results: Forty-four patients were enrolled in the dose-finding cohort. Dose-limiting toxicity included thrombocytopenia, stomatitis, hyperglycemia, asthenia/fatigue, and increased transaminases. CC-115 10 mg BID was selected for cohort expansion (n=74) in which fatigue, nausea, and decreased appetite were the most frequent toxicities. Dose-proportional PK was found. CC-115 distributed to glioblastoma tissue (mean tumor/plasma concentration ratio: 0.713). Total exposure of CC-115 was similar under fasting and fed conditions. A patient with endometrial carcinoma remained in complete remission >4 years. Partial response (PR; n=2) and stable disease (SD; n=4) were reported in the bioavailability substudy; SD was reached in 53%, 22%, 21%, and 64% of patients with head and neck squamous cell carcinoma, Ewing sarcoma, glioblastoma multiforme, and castration-resistant prostate cancer, respectively. Chronic lymphocytic leukemia/small lymphocytic lymphoma showed 38% PR and 25% SD.
Conclusion: CC-115 was well-tolerated, with toxicities consistent with mTOR inhibitors. Together with biomarker inhibition and preliminary efficacy, oral CC-115 10 mg BID is a promising novel anticancer treatment.
Clinical trial registration: NCT01353625.

Keywords:  CC-115; DNA-PK inhibitor; Phase I study; mTOR inhibitor; mTORC1/mTORC2

DOI:  https://doi.org/10.2147/CMAR.S208720
ACS Chem Biol. 2019 Dec 19.

Fluorescent biosensors for multiplexed imaging of phosphoinositide dynamics.

Fabian Hertel, Simin Li, Mingyuan Chen, Lutz Pott, Sohum Mehta, Jin Zhang.

Phosphoinositides constitute a critical family of lipids that regulate numerous cellular processes. Phosphatidylinositol 4,5-bisphosphate (PIP2) is arguably the most important plasma membrane phosphoinositide and is involved in regulating diverse processes. It is also the precursor of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is critical for growth factor signaling, as well as membrane polarization and dynamics. Studying these lipids remains challenging due to their compartmentalized activities and location-dependent signaling profiles. Here we introduce several new genetically encoded fluorescent biosensors that enable real-time monitoring of PIP2 levels in live cells, including FRET-based and dimerization-dependent fluorescent protein (ddFP)-based biosensors that enable real-time monitoring of PIP2 levels in live cells. In addition, we developed a red fluorescent biosensor for 3' phosphoinositides that can be co-imaged with the green PIP2 indicator. Simultaneous visualization of dynamics of PIP2 and 3' phosphoinositides in the same cell shows that plasma membrane PIP3 formation upon EGF stimulation is coupled to a decrease in the local pool of PIP2.

DOI: https://doi.org/10.1021/acschembio.9b00691
Neuro Oncol. 2019 Dec 16. pii: noz230. [Epub ahead of print]

Synergistic activity of mTORC1/2 kinase and MEK inhibitors suppresses pediatric low-grade glioma tumorigenicity and vascularity.

Antje Arnold, Ming Yuan, Antionette Price, Lauren Harris, Charles G Eberhart, Eric H Raabe.

   BACKGROUND: Pediatric low-grade glioma (pLGG) is the most common childhood brain tumor. Many patients with unresectable or recurrent/refractory tumors have significant life-long disability. The majority of pLGG have mutations increasing the activity of the RAS/MAP kinase pathway. Activation of mTOR is also a hallmark of pLGG. We therefore hypothesized that the dual TORC1/2 kinase inhibitor TAK228 would synergize with the MEK inhibitor trametinib in pLGG.
METHODS: We tested TAK228 and trametinib in patient-derived pLGG cell lines harboring drivers of pLGG including BRAFV600E and NF1 loss. We measured cell proliferation, pathway inhibition, cell death, and senescence. Synergy was analyzed via MTS assay using the Chou-Talalay method. In vivo, we tested for overall survival, pathway inhibition, and performed immunohistochemistry for proliferation and vascularization. We performed scratch assay and measured angiogenesis protein activation in human umbilical vein endothelial cells (HUVECs).
RESULTS: TAK228 synergized with trametinib in pLGG at clinically relevant doses in all tested cell lines, suppressing proliferation, inducing apoptosis, and causing senescence in a cell-line-dependent manner. Combination treatment increased median survival by 70% and reduced tumor volume compared to mono-treatment and control cohorts. Vascularization of tumors decreased as measured by CD31 and CD34. Combination treatment blocked activation of FAK and SRC in HUVEC cells and reduced HUVEC migration compared with each drug alone.
CONCLUSION: The combination of TAK228 and trametinib synergized to suppress the growth of pLGG. These agents synergized to reduce tumor vascularity and endothelial cell growth and migration by blocking activation of FAK and SRC.

Keywords:  INK 128; MLN0128; Sapanisertib; angiogenesis; trametinib

DOI:  https://doi.org/10.1093/neuonc/noz230
BMC Cancer. 2019 Dec 19. 19(1): 1236

Direct physical interaction of active Ras with mSIN1 regulates mTORC2 signaling.

Mehraj-U-Din Lone, Javed Miyan, Mohammad Asif, Showkat A Malik, Parul Dubey, Varsha Singh, Kavita Singh, Kalyan Mitra, Deepali Pandey, Wahajul Haq, Himanshi Amita, Prince Kumar Singh, Wieland Kiess, Franziska Kaessner, Antje Garten, Smrati Bhadauria.

   BACKGROUND: The mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking.
METHODS: Protein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion.
RESULTS: Here, we present experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. We report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling.
CONCLUSIONS: Our study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2.

Keywords:  Cancer; Mammalian target of rapamycin (mTOR); Ras; Signaling; Superoxide anion

DOI:  https://doi.org/10.1186/s12885-019-6422-6
Cancers (Basel). 2019 Dec 12. pii: E2006. [Epub ahead of print]11(12):

Somatic Mutation of PIK3CA (H1047R) Is a Common Driver Mutation Hotspot in Canine Mammary Tumors as Well as Human Breast Cancers.

Kang-Hoon Lee, Hyeon-Ji Hwang, Hyun Ji Noh, Tae-Jin Shin, Je-Yoel Cho.

  Breast cancer is one of the most frequently diagnosed cancers in both women and female dogs. Genome-wide association studies in human breast cancer (HBC) have identified hundreds of genetic variations and somatic driver mutations. However, only a handful of variants have been studied for rare HBC and their associations remain inconclusive. Spontaneous canine mammary tumor (CMT) is a great model for HBC, with clinical similarity. We thus performed whole-exome sequencing in 20 pairs of CMT and normal tissues in dogs. We newly found that PIK3CA was the most frequently mutated gene in CMT (45%). Furthermore, canine PIK3CA A3140G (H1047R), at what is known as the mutational hotspot of HBC, is also a hotspot in CMT. Targeted sequencing confirmed that 29% of CMTs had the same PIK3CA A3140G mutation. Integration of the transcriptome suggests that the PIK3CA (H1047R) induced cell metabolism and cell cycle via an increase of PCK2 and a decrease of CDKN1B but had no effect on cell apoptosis. We identified additional significantly mutated genes, including SCRN1 and CLHC1, which have not been reported in HBC. Our study recapitulated some known HBC-associated genes and human cancer signatures in CMT, and identified novel genes that may be relevant to HBC. This study may allow us to better understand both HBC and CMT and lend new insights into the development of biomarkers.

Keywords:  CMT; WES; breast cancer; dog; exome-seq; mammary gland tumor; transcriptome

DOI:  https://doi.org/10.3390/cancers11122006
Physiol Res. 2019 Nov 30. 68(Suppl 2): S131-S138

The PI3k/Akt pathway is associated with angiogenesis, oxidative stress and survival of mesenchymal stem cells in pathophysiologic condition in ischemia.

A Samakova, A Gazova, N Sabova, S Valaskova, M Jurikova, J Kyselovic.

Ischemic diseases are characterized by reduced blood supply to a tissue or an organ due to obstruction of blood vessels. The most serious and most common ischemic diseases include ischemic heart disease, ischemic stroke, and critical limb ischemia. Revascularization is the first choice of therapy, but the cell therapy is being introduced as a possible way of treatment for no-option patients. One of the possibilities of cell therapy is the use of mesenchymal stem cells (MSCs). MSCs are easily isolated from bone marrow and can be defined as non-hematopoietic multipotent adult stem cells population with a defined capacity for self-renewal and differentiation into cell types of all three germ layers depending on their origin. Since 1974, when Friedenstein and coworkers (Friedenstein et al. 1974) first time isolated and characterized MSCs, MSC-based therapy has been shown to be safe and effective. Nevertheless, many scientists and clinical researchers want to improve the success of MSCs in regenerative therapy. The secret of successful cell therapy may lie, along with the homing, in secretion of biologically active molecules including cytokines, growth factors, and chemokines known as MSCs secretome. One of the intracellular signalling mechanism includes the activity of phosphatidylinositol-3-kinase (phosphoinositide 3-kinase) (PI3K) - protein kinase B (serine-threonine protein kinase Akt) (Akt) pathway. This PI3K/Akt pathway plays key roles in many cell types in regulating cell proliferation, differentiation, apoptosis, and migration. Pre-conditioning of MSCs could improve efficacy of signalling mechanism.
J Cell Physiol. 2019 Dec 19.

Wortmannin targeting phosphatidylinositol 3-kinase suppresses angiogenic factors in shear-stressed endothelial cells.

Anderson M Gomes, Thais S Pinto, Célio J da Costa Fernandes, Rodrigo A da Silva, Willian F Zambuzzi.

  Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.

Keywords:  PI3K; VEGF; angiogenesis; eNOS; methylation; shear stress; wortmannin

DOI:  https://doi.org/10.1002/jcp.29412
Chem Biol Interact. 2019 Dec 12. pii: S0009-2797(19)31346-8. [Epub ahead of print]316 108917

Stearoyl-CoA desaturase and tumorigenesis.

Kohtaro Kikuchi, Hidekazu Tsukamoto.

  Stearoyl-CoA desaturase (SCD) generates monounsaturated fatty acids (MUFAs) which contribute to cell growth, survival, differentiation, metabolic regulation and signal transduction. Overexpression of SCD is evident and implicated in metabolic diseases such as diabetes and non-alcoholic fatty liver disease. SCD also stimulates canonical Wnt pathway and YAP activation in support of stemness and tumorigenesis. SCD facilitates metabolic reprogramming in cancer which is mediated, at least in part, by regulation of AKT, AMPK, and NF-kB via MUFAs. Our research has revealed the novel positive loop to amplify Wnt signaling through stabilization of LRP5/6 in both hepatic stellate cells and liver tumor-initiating stem cell-like cells. As such, this loop is pivotal in promoting liver fibrosis and liver tumor development. This review summarizes the mechanisms of SCD-mediated tumor promotion described by recent studies and discusses the future prospect for SCD-mediated signaling crosstalk as a potential therapeutic target for cancer.

Keywords:  Hepatic stellate cells; Wnt; YAP; β-catenin

DOI:  https://doi.org/10.1016/j.cbi.2019.108917