bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2019‒08‒11
twenty-six papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute


  1. Mol Oncol. 2019 Aug 08.
    Ruicci KM, Plantinga P, Pinto N, Khan MI, Stecho W, Dhaliwal SS, Yoo J, Fung K, MacNeil D, Mymryk JS, Barrett JW, Howlett CJ, Nichols AC.
      PI3-kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signalling, leading to cell survival and proliferation. Phosphorylation of Akt Thr308 by PI3K-PDK1 and Akt Ser473 by mTOR complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K-activated Akt signalling is well-established in HNSCC, the significance of mTORC2-driven Akt signalling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2-specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy. This article is protected by copyright. All rights reserved.
    Keywords:  PI3-kinase; RICTOR; head and neck cancer; mTORC2; targeted therapy
    DOI:  https://doi.org/10.1002/1878-0261.12558
  2. Sci Signal. 2019 Aug 06. pii: eaav9150. [Epub ahead of print]12(593):
    Mayer AE, Löffler MC, Loza Valdés AE, Schmitz W, El-Merahbi R, Viera JT, Erk M, Zhang T, Braun U, Heikenwalder M, Leitges M, Schulze A, Sumara G.
      Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity.
    DOI:  https://doi.org/10.1126/scisignal.aav9150
  3. Nat Immunol. 2019 Aug 05.
    Charbonnier LM, Cui Y, Stephen-Victor E, Harb H, Lopez D, Bleesing JJ, Garcia-Lloret MI, Chen K, Ozen A, Carmeliet P, Li MO, Pellegrini M, Chatila TA.
      Regulatory T cells (Treg cells) deficient in the transcription factor Foxp3 lack suppressor function and manifest an effector T (Teff) cell-like phenotype. We demonstrate that Foxp3 deficiency dysregulates metabolic checkpoint kinase mammalian target of rapamycin (mTOR) complex 2 (mTORC2) signaling and gives rise to augmented aerobic glycolysis and oxidative phosphorylation. Specific deletion of the mTORC2 adaptor gene Rictor in Foxp3-deficient Treg cells ameliorated disease in a Foxo1 transcription factor-dependent manner. Rictor deficiency re-established a subset of Treg cell genetic circuits and suppressed the Teff cell-like glycolytic and respiratory programs, which contributed to immune dysregulation. Treatment of Treg cells from patients with FOXP3 deficiency with mTOR inhibitors similarly antagonized their Teff cell-like program and restored suppressive function. Thus, regulatory function can be re-established in Foxp3-deficient Treg cells by targeting their metabolic pathways, providing opportunities to restore tolerance in Treg cell disorders.
    DOI:  https://doi.org/10.1038/s41590-019-0442-x
  4. Breast Cancer Res. 2019 Aug 07. 21(1): 90
    Sobral-Leite M, Salomon I, Opdam M, Kruger DT, Beelen KJ, van der Noort V, van Vlierberghe RLP, Blok EJ, Giardiello D, Sanders J, Van de Vijver K, Horlings HM, Kuppen PJK, Linn SC, Schmidt MK, Kok M.
      INTRODUCTION: The presence of tumor-infiltrating lymphocytes (TILs) is correlated with good prognosis and outcome after (immuno)therapy in triple-negative and HER2-positive breast cancer. However, the role of TILs in luminal breast cancer is less clear. Emerging evidence has now demonstrated that genetic aberrations in malignant cells influence the immune landscape of tumors. Phosphatidylinositol 3-kinase (PI3K) is the most common altered pathway in ER-positive breast cancer. It is unknown whether changes in the PI3K pathway result in a different composition of the breast tumor microenvironment. Here we present the retrospective analysis of a prospective randomized trial in ER-positive breast cancer on the prognostic and predictive value of specific tumor-associated lymphocytes in the context of PI3K alterations.METHODS: We included 563 ER-positive tumors from a multicenter trial for stage I to III postmenopausal breast cancer patients, who were randomized to tamoxifen or no adjuvant therapy. The amount of CD8-, CD4-, and FOXP3-positive cells was evaluated by immunohistochemistry and quantified by imaging-analysis software. We analyzed the associations between PIK3CA hotspot mutations, PTEN expression, phosphorylated proteins of the PI3K and MAPK pathway (p-AKT, p-ERK1/2, p-4EBP1, p-p70S6K), and recurrence-free interval after adjuvant tamoxifen or no adjuvant treatment.
    RESULTS: CD8-positive lymphocytes were significantly more abundant in PIK3CA-mutated tumors (OR = 1.65; 95% CI 1.03-2.68). While CD4 and FOXP3 were not significantly associated with prognosis, patients with tumors classified as CD8-high had increased risk of recurrence (HR = 1.98; 95% CI 1.14-3.41; multivariable model including PIK3CA status, treatment arm, and other standard clinicopathological variables). Lymphocytes were more often present in tumors with increased PI3K downstream phosphorylation. This was most pronounced for FOXP3-positive cells.
    CONCLUSION: These exploratory analyses of a prospective trial in luminal breast cancer suggest high CD8 infiltration is associated with unfavorable outcome and that PI3K pathway alterations might be associated with the composition of the tumor microenvironment.
    Keywords:  Luminal breast cancer; PI3K pathway; PIK3CA mutations; Tumor-infiltrating lymphocytes
    DOI:  https://doi.org/10.1186/s13058-019-1176-2
  5. Cancer Res. 2019 Aug 08. pii: canres.0708.2019. [Epub ahead of print]
    Martínez-Carreres L, Puyal J, Leal-Esteban LC, Orpinell M, Castillo-Armengol J, Giralt A, Dergai O, Moret C, Barquissau V, Nasrallah A, Pabois A, Zhang L, Romero P, Lopez-Mejia IC, Fajas L.
      Cyclin-dependent kinase 4 (CDK4) is well-known for its role in regulating the cell cycle, however, its role in cancer metabolism, especially mTOR signaling, is undefined. In this study, we established a connection between CDK4 and lysosomes, an emerging metabolic organelle crucial for mTORC1 activation. On the one hand, CDK4 phosphorylated the tumor suppressor FLCN, regulating mTORC1 recruitment to the lysosomal surface in response to amino acids. On the other hand, CDK4 directly regulated lysosomal function and was essential for lysosomal degradation, ultimately regulating mTORC1 activity. Pharmacological inhibition or genetic inactivation of CDK4, other than retaining FLCN at the lysosomal surface, led to the accumulation of undigested material inside lysosomes, which impaired the autophagic flux and induced cancer cell senescence in vitro and in xenograft models. Importantly, the use of CDK4 inhibitors in therapy is known to cause senescence but not cell death. To overcome this phenomenon and based on our findings, we increased the autophagic flux in cancer cells by using an AMPK activator in combination with a CDK4 inhibitor. The cotreatment induced autophagy (AMPK activation), and impaired lysosomal function (CDK4 inhibition), resulting in cell death and tumor regression. Altogether, we uncover a previously unknown role for CDK4 in lysosomal biology and propose a novel therapeutic strategy to target cancer cells.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-0708
  6. Nat Commun. 2019 Aug 09. 10(1): 3622
    Xu ZX, Tan JW, Xu H, Hill CJ, Ostrovskaya O, Martemyanov KA, Xu B.
      Caspase-2 is the most evolutionarily conserved member in the caspase family of proteases and is constitutively expressed in most cell types including neurons; however, its physiological function remains largely unknown. Here we report that caspase-2 plays a critical role in synaptic plasticity and cognitive flexibility. We found that caspase-2 deficiency led to deficits in dendritic spine pruning, internalization of AMPA receptors and long-term depression. Our results indicate that caspase-2 degrades Rictor, a key mTOR complex 2 (mTORC2) component, to inhibit Akt activation, which leads to enhancement of the GSK3β activity and thereby long-term depression. Furthermore, we found that mice lacking caspase-2 displayed elevated levels of anxiety, impairment in reversal water maze learning, and little memory loss over time. These results not only uncover a caspase-2-mTORC2-Akt-GSK3β signaling pathway, but also suggest that caspase-2 is important for memory erasing and normal behaviors by regulating synaptic number and transmission.
    DOI:  https://doi.org/10.1038/s41467-019-11575-1
  7. Breast Cancer Res Treat. 2019 Aug 06.
    He J, McLaughlin RP, van der Noord V, Foekens JA, Martens JWM, van Westen G, Zhang Y, van de Water B.
      PURPOSE: Owing to its genetic heterogeneity and acquired resistance, triple-negative breast cancer (TNBC) is not responsive to single-targeted therapy, causing disproportional cancer-related death worldwide. Combined targeted therapy strategies to block interactive oncogenic signaling networks are being explored for effective treatment of the refractory TNBC subtype.METHODS: A broad kinase inhibitor screen was applied to profile the proliferative responses of TNBC cells, revealing resistance of TNBC cells to inhibition of the mammalian target of rapamycin (mTOR). A systematic drug combination screen was subsequently performed to identify that AEE788, an inhibitor targeting multiple receptor tyrosine kinases (RTKs) EGFR/HER2 and VEGFR, synergizes with selective mTOR inhibitor rapamycin as well as its analogs (rapalogs) temsirolimus and everolimus to inhibit TNBC cell proliferation.
    RESULTS: The combination treatment with AEE788 and rapalog effectively inhibits phosphorylation of mTOR and 4EBP1, relieves mTOR inhibition-mediated upregulation of cyclin D1, and maintains suppression of AKT and ERK signaling, thereby sensitizing TNBC cells to the rapalogs. siRNA validation of cheminformatics-based predicted AEE788 targets has further revealed the mTOR interactive RPS6K members (RPS6KA3, RPS6KA6, RPS6KB1, and RPS6KL1) as synthetic lethal targets for rapalog combination treatment.
    CONCLUSIONS: mTOR signaling is highly activated in TNBC tumors. As single rapalog treatment is insufficient to block mTOR signaling in rapalog-resistant TNBC cells, our results thus provide a potential multi-kinase inhibitor combinatorial strategy to overcome mTOR-targeted therapy resistance in TNBC cells.
    Keywords:  Drug resistance; Multi-kinase inhibitor; Polypharmacology; Triple-negative breast cancer (TNBC); mTOR-targeted therapy
    DOI:  https://doi.org/10.1007/s10549-019-05380-z
  8. Oncologist. 2019 Aug 08. pii: theoncologist.2019-0297. [Epub ahead of print]
    Bardia A, Gounder M, Rodon J, Janku F, Lolkema MP, Stephenson JJ, Bedard PL, Schuler M, Sessa C, LoRusso P, Thomas M, Maacke H, Evans H, Sun Y, Tan DSW.
      BACKGROUND: This multicenter, open-label, phase Ib study investigated the safety and efficacy of binimetinib (MEK inhibitor) in combination with buparlisib (phosphatidylinositol 3-kinase [PI3K] inhibitor) in patients with advanced solid tumors with RAS/RAF alterations.MATERIALS AND METHODS: Eighty-nine patients were enrolled in the study. Eligible patients had advanced solid tumors with disease progression after standard therapy and/or for which no standard therapy existed. Evaluable disease was mandatory, per RECIST version 1.1 and Eastern Cooperative Oncology Group performance status 0-2. Binimetinib and buparlisib combinations were explored in patients with KRAS-, NRAS-, or BRAF-mutant advanced solid tumors until the maximum tolerated dose and recommended phase II dose (RP2D) were defined. The expansion phase comprised patients with epidermal growth factor receptor (EGFR)-mutant, advanced non-small cell lung cancer, after progression on an EGFR inhibitor; advanced RAS- or BRAF-mutant ovarian cancer; or advanced non-small cell lung cancer with KRAS mutation.
    RESULTS: At data cutoff, 32/89 patients discontinued treatment because of adverse events. RP2D for continuous dosing was buparlisib 80 mg once daily/binimetinib 45 mg twice daily. The toxicity profile of the combination resulted in a lower dose intensity than anticipated. Six (12.0%) patients with RAS/BRAF-mutant ovarian cancer achieved a partial response. Pharmacokinetics of binimetinib were not altered by buparlisib. Pharmacodynamic analyses revealed downregulation of pERK and pS6 in tumor biopsies.
    CONCLUSION: Although dual inhibition of MEK and the PI3K pathways showed promising activity in RAS/BRAF ovarian cancer, continuous dosing resulted in intolerable toxicities beyond the dose-limiting toxicity monitoring period. Alternative schedules such as pulsatile dosing may be advantageous when combining therapies.
    IMPLICATIONS FOR PRACTICE: Because dysregulation of the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) pathways are both frequently involved in resistance to current targeted therapies, dual inhibition of both pathways may be required to overcome resistance mechanisms to single-agent tyrosine kinase inhibitors or to treat cancers with driver mutations that cannot be directly targeted. A study investigating the safety and efficacy of combination binimetinib (MEK inhibitor) and buparlisib (PI3K inhibitor) in patients harboring alterations in the RAS/RAF pathway was conducted. The results may inform the design of future combination therapy trials in patients with tumors harboring mutations in the PI3K and MAPK pathways.
    Keywords:  Binimetinib; Buparlisib; Ovarian cancer; Phase Ib; RAS/RAF
    DOI:  https://doi.org/10.1634/theoncologist.2019-0297
  9. Nat Commun. 2019 Aug 07. 10(1): 3554
    Wilson MR, Reske JJ, Holladay J, Wilber GE, Rhodes M, Koeman J, Adams M, Johnson B, Su RW, Joshi NR, Patterson AL, Shen H, Leach RE, Teixeira JM, Fazleabas AT, Chandler RL.
      ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. Here we show that monoallelic loss of ARID1A in the mouse endometrial epithelium is sufficient for vaginal bleeding when combined with PI3K activation. Sorted mutant epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT). We further show that ARID1A is bound to promoters with open chromatin, but ARID1A loss leads to increased promoter chromatin accessibility and the expression of EMT genes. PI3K activation partially rescues the mesenchymal phenotypes driven by ARID1A loss through antagonism of ARID1A target gene expression, resulting in partial EMT and invasion. We propose that ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates, and that coexistent ARID1A and PI3K mutations promote epithelial transdifferentiation and collective invasion. Broadly, our findings support a role for collective epithelial invasion in the spread of abnormal endometrial tissue.
    DOI:  https://doi.org/10.1038/s41467-019-11403-6
  10. Commun Biol. 2019 ;2 292
    Yamamoto M, Abe C, Wakinaga S, Sakane K, Yumiketa Y, Taguchi Y, Matsumura T, Ishikawa K, Fujimoto J, Semba K, Miyauchi M, Akiyama T, Inoue JI.
      Receptor activator of nuclear factor (NF)-κB (RANK) signaling promotes pregnancy-dependent epithelial cell differentiation and expansion for mammary gland development, which requires NF-κB pathway-dependent Cyclin D1 induction and inhibitor of DNA binding 2 (Id2) pathway-dependent anti-apoptotic gene induction. However, the roles of tumor necrosis factor receptor-associated factor 6 (TRAF6) remain unclear despite its requirement in RANK signaling. Here we show that TRAF6 is crucial for both mammary stem cell maintenance and pregnancy-induced epithelial cell expansion. TRAF6 deficiency impairs phosphoinositide 3-kinase (PI3K)/AKT and canonical NF-κB pathways, whereas noncanonical NF-κB signaling remains functional. Therefore, we propose that TRAF6 promotes cell proliferation by activating PI3K/AKT signaling to induce retinoblastoma phosphorylation in concert with noncanonical NF-κB pathway-dependent Cyclin D1 induction. Furthermore, TRAF6 inhibits apoptosis by activating canonical NF-κB signaling to induce anti-apoptotic genes with the Id2 pathway. Therefore, proper orchestration of TRAF6-dependent and -independent RANK signals likely establishes mammary gland formation.
    Keywords:  Cell growth; Cell proliferation; Organogenesis
    DOI:  https://doi.org/10.1038/s42003-019-0547-7
  11. Adv Biol Regul. 2019 Jul 30. pii: S2212-4926(19)30058-2. [Epub ahead of print] 100637
    Clarke BP, Logeman BL, Hale AT, Luka Z, York JD.
      Inositide lipid (PIP) and soluble (IP) signaling pathways produce essential cellular codes conserved in eukaryotes. In many cases, deconvoluting metabolic and functional aspects of individual pathways are confounded by promiscuity and multiplicity of PIP and IP kinases and phosphatases. We report a molecular genetic approach that reconstitutes eukaryotic inositide lipid and soluble pathways in a prokaryotic cell which inherently lack inositide kinases and phosphatases in their genome. By expressing synthetic cassettes of eukaryotic genes, we have reconstructed the heterologous formation of a range of inositide lipids, including PI(3)P, PI(4,5)P2 and PIP3. In addition, we report the reconstruction of lipid-dependent production of inositol hexakisphosphate (IP6). Our synthetic system is scalable, reduces confounding metabolic issues, for example it is devoid of inositide phosphatases and orthologous kinases, and enables accurate characterization gene product enzymatic activity and substrate selectivity. This genetically engineered tool is designed to help interpret metabolic pathways and may facilitate in vivo testing of regulators and small molecule inhibitors. In summary, heterologous expression of inositide pathways in bacteria provide a malleable experimental platform for aiding signaling biologists and offers new insights into metabolism of these essential pathways.
    Keywords:  Cell signaling; Inositol phosphate; Phosphatidylinositol; Synthetic biology
    DOI:  https://doi.org/10.1016/j.jbior.2019.100637
  12. JCI Insight. 2019 Aug 08. pii: 124945. [Epub ahead of print]4(15):
    Wang Y, Zhou H, Palyha O, Mu J.
      Type 2 diabetes mellitus (T2DM), also known as adult-onset diabetes, is characterized by ineffective insulin action due to insulin resistance in key metabolic tissues. Insulin receptor (IR) plays an important role in insulin signal transduction, defect of which has been considered the fundamental cause of T2DM. IR content reduction in diabetes is one key contributor to the defective insulin signaling and diabetes progression. Rescuing IR levels by transgenic complementation has not been considered as a treatment option because it is limited by uncontrollable expression level, tissue selectivity, or developmental defects. In the current study, we demonstrated that single-dose adeno-associated virus (AAV) vector delivered expression of human IR (hIR) in the liver of inducible IR-knockout mice and significantly improved the diabetic phenotype caused by IR deletion during adulthood. Such an approach was also applied, for the first time to our knowledge, to treating ob/ob mice, a model of severe T2DM attributed to superfluous calorie intake and insulin resistance. Interestingly, similar treatment with AAV-hIR had no obvious effect in healthy animals, indicative of low hypoglycemic risk as a consequence of potential excessive insulin action. The results described here support restoration of IR expression as a safe and effective T2DM therapeutic with a long-lasting profile.
    Keywords:  Diabetes; Gene therapy; Insulin signaling; Metabolism; Therapeutics
    DOI:  https://doi.org/10.1172/jci.insight.124945
  13. Exp Neurol. 2019 Aug 01. pii: S0014-4886(19)30178-5. [Epub ahead of print] 113029
    LaSarge CL, Pun RYK, Gu Z, Santos VR, Danzer SC.
      Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway is associated with epilepsy, autism and brain growth abnormalities in humans. mTOR hyperactivation often results from developmental somatic mutations, producing genetic lesions and associated dysfunction in relatively restricted populations of neurons. Disrupted brain regions, such as those observed in focal cortical dysplasia, can contain a mix of normal and mutant cells. Mutant cells exhibit robust anatomical and physiological changes. Less clear, however, is whether adjacent, initially normal cells are affected by the presence of abnormal cells. To explore this question, we used a conditional, inducible mouse model approach to delete the mTOR negative regulator phosphatase and tensin homolog (PTEN) from <1% to >30% of hippocampal dentate granule cells. We then examined the morphology of PTEN-expressing granule cells located in the same dentate gyri as the knockout (KO) cells. Despite the development of spontaneous seizures in higher KO animals, and disease worsening with increasing age, the morphology and physiology of PTEN-expressing cells was only modestly affected. PTEN-expressing cells had smaller somas than cells from control animals, but other parameters were largely unchanged. These findings contrast with the behavior of PTEN KO cells, which show increasing dendritic extent with greater KO cell load. Together, the findings indicate that genetically normal neurons can exhibit relatively stable morphology and intrinsic physiology in the presence of nearby pathological neurons and systemic disease.
    Keywords:  Dentate gate; Dentate granule cell; Neurolucida; PTEN; Soma area; Somatic hypertrophy; mTOR
    DOI:  https://doi.org/10.1016/j.expneurol.2019.113029
  14. Cancers (Basel). 2019 Aug 07. pii: E1126. [Epub ahead of print]11(8):
    Bhateja P, Cherian M, Majumder S, Ramaswamy B.
      The hedgehog (Hh) pathway plays a key role in embryonic development and stem cell programs. Deregulation of the Hh pathway is a key driver of basal cell carcinoma, and therapeutic targeting led to approval of Hh inhibitor, vismodegib, in the management of this cancer. The Hh pathway is implicated in other malignancies including hormone receptor (HR+) positive and triple negative breast cancer (TNBC). Hh signaling, which is activated in human mammary stem cells, results in activation of glioma-associated oncogene (GLI) transcription factors. High GLI1 expression correlates with worse outcomes in breast cancer. Non-canonical GLI1 activation is one mechanism by which estrogen exposure promotes breast cancer stem cell proliferation and epithelial-mesenchymal transition. Tamoxifen resistant cell lines show aberrant activation of Hh signaling, and knockdown of Hh pathway inhibited growth of tamoxifen resistant cells. As in other cancers Hh signaling is activated by the PI3K/AKT pathway in these endocrine resistant cell lines. Hh pathway activation has also been reported to mediate chemotherapy resistance in TNBC via various mechanisms including paracrine signaling to tumor micro-environment and selective proliferation of cancer stem cells. Co-activation of Hh and Wnt signaling pathways is a poor prognostic marker in TNBC. Early phase clinical trials are evaluating the combination of smoothened (SMO) inhibitors and chemotherapy in TNBC. In addition to SMO inhibitors like vismodegib and sonidegib, which are in clinical use for basal cell carcinoma, GLI1 inhibitors like GANT58 and GANT61 are in preclinical drug development and might be an effective mechanism to overcome drug resistance in breast cancer. Gene signatures predictive of Hh pathway activation could enrich for patients likely to respond to these agents.
    Keywords:  GLI1; breast cancer; hedgehog
    DOI:  https://doi.org/10.3390/cancers11081126
  15. Nat Commun. 2019 Aug 07. 10(1): 3545
    Ramadori G, Ljubicic S, Ricci S, Mikropoulou D, Brenachot X, Veyrat-Durebex C, Aras E, Ioris RM, Altirriba J, Malle E, Foell D, Vogl T, Coppari R.
      Tens of millions suffer from insulin deficiency (ID); a defect leading to severe metabolic imbalance and death. The only means for management of ID is insulin therapy; yet, this approach is sub-optimal and causes life-threatening hypoglycemia. Hence, ID represents a great medical and societal challenge. Here we report that S100A9, also known as Calgranulin B or Myeloid-Related Protein 14 (MRP14), is a leptin-induced circulating cue exerting beneficial anti-diabetic action. In murine models of ID, enhanced expression of S100A9 alone (i.e. without administered insulin and/or leptin) slightly improves hyperglycemia, and normalizes key metabolic defects (e.g. hyperketonemia, hypertriglyceridemia, and increased hepatic fatty acid oxidation; FAO), and extends lifespan by at least a factor of two. Mechanistically, we report that Toll-Like Receptor 4 (TLR4) is required, at least in part, for the metabolic-improving and pro-survival effects of S100A9. Thus, our data identify the S100A9/TLR4 axis as a putative target for ID care.
    DOI:  https://doi.org/10.1038/s41467-019-11498-x
  16. Aging Cell. 2019 Aug 06. e13013
    Guo Y, Ayers JL, Carter KT, Wang T, Maden SK, Edmonds D, Newcomb P P, Li C, Ulrich C, Yu M, Grady WM.
      The risk of colorectal cancer (CRC) varies between people, and the cellular mechanisms mediating the differences in risk are largely unknown. Senescence has been implicated as a causative cellular mechanism for many diseases, including cancer, and may affect the risk for CRC. Senescent fibroblasts that accumulate in tissues secondary to aging and oxidative stress have been shown to promote cancer formation via a senescence-associated secretory phenotype (SASP). In this study, we assessed the role of senescence and the SASP in CRC formation. Using primary human colon tissue, we found an accumulation of senescent fibroblasts in normal tissues from individuals with advanced adenomas or carcinomas in comparison with individuals with no polyps or CRC. In in vitro and ex vivo model systems, we induced senescence using oxidative stress in colon fibroblasts and demonstrated that the senescent fibroblasts secrete GDF15 as an essential SASP factor that promotes cell proliferation, migration, and invasion in colon adenoma and CRC cell lines as well as primary colon organoids via the MAPK and PI3K signaling pathways. In addition, we observed increased mRNA expression of GDF15 in primary normal colon tissue from people at increased risk for CRC in comparison with average risk individuals. These findings implicate the importance of a senescence-associated tissue microenvironment and the secretory factor GDF15 in promoting CRC formation.
    Keywords:  GDF15; colon organoids; colorectal cancer; microenvironment; senescence
    DOI:  https://doi.org/10.1111/acel.13013
  17. PLoS Biol. 2019 Aug 05. 17(8): e3000399
    Johnson KE, Howard G, Mo W, Strasser MK, Lima EABF, Huang S, Brock A.
      Most models of cancer cell population expansion assume exponential growth kinetics at low cell densities, with deviations to account for observed slowing of growth rate only at higher densities due to limited resources such as space and nutrients. However, recent preclinical and clinical observations of tumor initiation or recurrence indicate the presence of tumor growth kinetics in which growth rates scale positively with cell numbers. These observations are analogous to the cooperative behavior of species in an ecosystem described by the ecological principle of the Allee effect. In preclinical and clinical models, however, tumor growth data are limited by the lower limit of detection (i.e., a measurable lesion) and confounding variables, such as tumor microenvironment, and immune responses may cause and mask deviations from exponential growth models. In this work, we present alternative growth models to investigate the presence of an Allee effect in cancer cells seeded at low cell densities in a controlled in vitro setting. We propose a stochastic modeling framework to disentangle expected deviations due to small population size stochastic effects from cooperative growth and use the moment approach for stochastic parameter estimation to calibrate the observed growth trajectories. We validate the framework on simulated data and apply this approach to longitudinal cell proliferation data of BT-474 luminal B breast cancer cells. We find that cell population growth kinetics are best described by a model structure that considers the Allee effect, in that the birth rate of tumor cells increases with cell number in the regime of small population size. This indicates a potentially critical role of cooperative behavior among tumor cells at low cell densities with relevance to early stage growth patterns of emerging and relapsed tumors.
    DOI:  https://doi.org/10.1371/journal.pbio.3000399
  18. Biochem Soc Trans. 2019 Aug 05. pii: BST20190158. [Epub ahead of print]
    Ebner M, Koch PA, Haucke V.
      Lysosomes are the main degradative compartments of mammalian cells and serve as platforms for cellular nutrient signaling and sterol transport. The diverse functions of lysosomes and their adaptation to extracellular and intracellular cues are tightly linked to the spatiotemporally controlled synthesis, turnover and interconversion of lysosomal phosphoinositides, minor phospholipids that define membrane identity and couple membrane dynamics to cell signaling. How precisely lysosomal phosphoinositides act and which effector proteins within the lysosome membrane or at the lysosomal surface recognize them is only now beginning to emerge. Importantly, mutations in phosphoinositide metabolizing enzyme cause lysosomal dysfunction and are associated with numerous diseases ranging from neurodegeneration to cancer. Here, we discuss the phosphoinositides and phosphoinositide metabolizing enzymes implicated in lysosome function and homeostasis and outline perspectives for future research.
    Keywords:  autophagy; lysosomes; membrane contact sites; nutrient signaling; phosphosinositides
    DOI:  https://doi.org/10.1042/BST20190158
  19. Nat Commun. 2019 Aug 08. 10(1): 3558
    Kunkel J, Luo X, Capaldi AP.
      The growth rate of a yeast cell is controlled by the target of rapamycin kinase complex I (TORC1) and cAMP-dependent protein kinase (PKA) pathways. To determine how TORC1 and PKA cooperate to regulate cell growth, we performed temporal analysis of gene expression in yeast switched from a non-fermentable substrate, to glucose, in the presence and absence of TORC1 and PKA inhibitors. Quantitative analysis of these data reveals that PKA drives the expression of key cell growth genes during transitions into, and out of, the rapid growth state in glucose, while TORC1 is important for the steady-state expression of the same genes. This circuit design may enable yeast to set an exact growth rate based on the abundance of internal metabolites such as amino acids, via TORC1, but also adapt rapidly to changes in external nutrients, such as glucose, via PKA.
    DOI:  https://doi.org/10.1038/s41467-019-11540-y
  20. Reproduction. 2019 Aug 01. pii: REP-19-0201.R2. [Epub ahead of print]
    Ford E, Beckett EL, Roman S, McLaughlin EA, Sutherland J.
      In women, the non-growing population of follicles that comprise the ovarian reserve is determined at birth and serves as the reservoir for future fertility. This reserve of dormant, primordial follicles and the mechanisms controlling their selective activation which constitute the committing step into folliculogenesis are essential for determining fertility outcomes in women. Much of the available data on the mechanisms responsible for primordial follicle activation focuses on a selection of key molecular pathways, studied primarily in animal models, with findings often not synonymous in humans. The excessive induction of primordial follicle activation may cause the development of premature ovarian insufficiency (POI), a condition characterised by menopause before age 40. POI affects 1-2% of all women and is accompanied by additional health risks. Therefore, it is critical to further our understanding of primordial follicle activation in order to diagnose, treat, and prevent premature infertility. Research in primordial follicle activation has focussed on connecting new molecules to already established key signalling pathways, such as phosphatidylinositol 3-Kinase (PI3K) and mammalian target of rapamycin (mTOR). Additionally, other aspects of the ovarian environment, such as the function of the extracellular matrix, in contributing to primordial follicle activation have gained traction. Clinical applications are examining replication of this extracellular environment through the construction of biological matrices mimicking the three-dimensional ovary, to support follicular growth through to ovulation. This review outlines the importance of the events leading to the establishment of the ovarian reserve and highlights the fundamental factors known to influence primordial follicle activation in humans presenting new horizons for female infertility treatment.
    DOI:  https://doi.org/10.1530/REP-19-0201
  21. J Endocrinol. 2019 Aug 01. pii: JOE-19-0067.R1. [Epub ahead of print]
    Bradley EA, Premilovac D, Betik AC, Hu D, Attrill E, Richards S, Rattigan S, Keske M.
      Insulin stimulates glucose disposal in skeletal muscle in part by increasing microvascular blood flow and this effect is blunted during insulin resistance. We aimed to determine whether metformin treatment improves insulin-mediated glucose disposal and vascular insulin responsiveness in skeletal muscle of insulin resistant rats. Sprague Dawley rats were fed a normal (ND) or high-fat (HFD) diet for four weeks. A separate HFD group was given metformin in drinking water (HFD+MF, 150 mg/kg/day) during the final two weeks. After the intervention, overnight-fasted (food and metformin removed) anaesthetised rats underwent a 2-hr euglycemic-hyperinsulinemic clamp (10 mU/min/kg) or saline infusion. Femoral artery blood flow, hindleg muscle microvascular blood flow, muscle glucose disposal and muscle signalling (Ser473-Akt and Thr172-AMPK phosphorylation) were measured. HFD rats had elevated body weight, epididymal fat pad weight, fasting plasma insulin and free fatty acid levels when compared to ND. HFD-fed animals displayed whole body and skeletal muscle insulin resistance and blunting of insulin-stimulated femoral artery blood flow, muscle microvascular blood flow and skeletal muscle insulin-stimulated Ser473-Akt phosphorylation. Metformin treatment of HFD rats reduced fasting insulin and non-esterified fatty acid concentrations and lowered body weight and adiposity. During hyperinsulinemic-euglycemic clamp, metformin-treated animals showed improved vascular responsiveness to insulin, improved insulin-stimulated muscle Ser473-Akt phosphorylation but only partially restored (60%) muscle glucose uptake. This occurred without any detectable levels of metformin in plasma or change in muscle Thr172-AMPK phosphorylation. We conclude that two-week metformin treatment is effective at improving vascular and metabolic insulin responsiveness in muscle of HFD-induced insulin resistant rats.
    DOI:  https://doi.org/10.1530/JOE-19-0067
  22. Cell Rep. 2019 Aug 06. pii: S2211-1247(19)30905-2. [Epub ahead of print]28(6): 1439-1446.e5
    Suzawa M, Muhammad NM, Joseph BS, Bland ML.
      Chronic enteropathogen infection in early childhood reduces circulating insulin-like growth factor 1 (IGF1) levels and restricts growth. Pathogen-derived molecules activate host Toll-like receptors to initiate the immune response, but whether this pathway contributes to growth inhibition is unclear. In Drosophila, activation of Toll receptors in larval fat body suppresses whole-animal growth. Here, using a transcriptomic approach, we identify Drosophila insulin-like peptide 6 (Dilp6), a fat-body-derived IGF1 ortholog, as a selective target of Toll signaling induced by infection or genetic activation of the pathway. Using a tagged allele that we generated to measure endogenous Dilp6, we find a marked reduction in circulating hormone levels. Restoring Dilp6 expression in fat body rescues growth in animals with active Toll signaling. Our results establish that Toll signaling reduces growth by inducing hormone insufficiency, implying a mechanistic link between innate immune signaling and endocrine regulation of growth.
    Keywords:  Dilp6; Drosophila; Toll; chronic infection; chronic inflammation; enteropathogen; fat body; growth; innate immunity; insulin signaling
    DOI:  https://doi.org/10.1016/j.celrep.2019.07.015
  23. Nat Rev Cancer. 2019 Aug 06.
    Urick ME, Bell DW.
      Endometrial cancer accounts for ~76,000 deaths among women each year worldwide. Disease mortality and the increasing number of new diagnoses make endometrial cancer an important consideration in women's health, particularly in industrialized countries, where the incidence of this tumour type is highest. Most endometrial cancers are carcinomas, with the remainder being sarcomas. Endometrial carcinomas can be classified into several histological subtypes, including endometrioid, serous and clear cell carcinomas. Histological subtyping is currently used routinely to guide prognosis and treatment decisions for endometrial cancer patients, while ongoing studies are evaluating the potential clinical utility of molecular subtyping. In this Review, we summarize the overarching molecular features of endometrial cancers and highlight recent studies assessing the potential clinical utility of specific molecular features for early detection, disease risk stratification and directing targeted therapies.
    DOI:  https://doi.org/10.1038/s41568-019-0177-x
  24. Pharmaceutics. 2019 Aug 02. pii: E384. [Epub ahead of print]11(8):
    Bonizzi A, Truffi M, Sevieri M, Allevi R, Sitia L, Ottria R, Sorrentino L, Sottani C, Negri S, Grignani E, Mazzucchelli S, Corsi F.
      Everolimus (Eve) is an FDA approved drug that inhibits mammalian target of rapamycin (mTOR). It is employed in breast cancer treatment even if its responsiveness is controversial. In an attempt to increase Eve effectiveness, we have developed a novel Eve nanoformulation exploiting H-ferritin nanocages (HEve) to improve its subcellular delivery. We took advantage of the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1). Breast cancer cells overexpressing TfR-1 were successfully recognized by H-Ferritin, displaying quick nanocage internalization. HEve has been tested and compared to Eve for in vitro efficacy in sensitive and resistant breast cancer cells. Nanoformulated Eve induced remarkable antiproliferative activity in vitro, making even resistant cell lines sensitive to Eve. Moreover, the antiproliferative activity of HEve is fully in accordance with cytotoxicity observed by cell death assay. Furthermore, the significant increase in anticancer efficacy displayed in HEve-treated samples is due to the improved drug accumulation, as demonstrated by UHPLC-MS/MS quantifications. Our findings suggest that optimizing Eve subcellular delivery, thanks to nanoformulation, determines its improved antitumor activity in a panel of Eve-sensitive or resistant breast cancer cell lines.
    Keywords:  Everolimus; H-ferritin; breast cancer; nanoparticles
    DOI:  https://doi.org/10.3390/pharmaceutics11080384
  25. Front Endocrinol (Lausanne). 2019 ;10 481
    Sarkar PL, Lee W, Williams ED, Lubik AA, Stylianou N, Shokoohmand A, Lehman ML, Hollier BG, Gunter JH, Nelson CC.
      Androgen deprivation therapy (ADT) is the standard treatment for advanced prostate cancer (PCa), yet many patients relapse with lethal metastatic disease. With this loss of androgens, increased cell plasticity has been observed as an adaptive response to ADT. This includes gain of invasive and migratory capabilities, which may contribute to PCa metastasis. Hyperinsulinemia, which develops as a side-effect of ADT, has been associated with increased tumor aggressiveness and faster treatment failure. We investigated the direct effects of insulin in PCa cells that may contribute to this progression. We measured cell migration and invasion induced by insulin using wound healing and transwell assays in a range of PCa cell lines of variable androgen dependency (LNCaP, 22RV1, DuCaP, and DU145 cell lines). To determine the molecular events driving insulin-induced invasion we used transcriptomics, quantitative real time-PCR, and immunoblotting in three PCa cell lines. Insulin increased invasiveness of PCa cells, upregulating Forkhead Box Protein C2 (FOXC2), and activating key PCa cell plasticity mechanisms including gene changes consistent with epithelial-to-mesenchymal transition (EMT) and a neuroendocrine phenotype. Additionally, analysis of publicly available clinical PCa tumor data showed metastatic prostate tumors demonstrate a positive correlation between insulin receptor expression and the EMT transcription factor FOXC2. The insulin receptor is not suitable to target clinically however, our data shows that actions of insulin in PCa cells may be suppressed by inhibiting downstream signaling molecules, PI3K and ERK1/2. This study identifies for the first time, a mechanism for insulin-driven cancer cell motility and supports the concept that targeting insulin signaling at the level of the PCa tumor may extend the therapeutic efficacy of ADT.
    Keywords:  FOXC2; androgen deprivation; epithelial to mesenchymal transition (EMT); hyperinsulinemia; invasion; prostate cancer
    DOI:  https://doi.org/10.3389/fendo.2019.00481
  26. Sci Rep. 2019 Aug 05. 9(1): 11316
    Lauzier A, Normandeau-Guimond J, Vaillancourt-Lavigueur V, Boivin V, Charbonneau M, Rivard N, Scott MS, Dubois CM, Jean S.
      Autophagy has both tumor-promoting and -suppressing effects in cancer, including colorectal cancer (CRC), with transformed cells often exhibiting high autophagic flux. In established tumors, autophagy inhibition can lead to opposite responses resulting in either tumor cell death or hyperproliferation. The functional mechanisms underlying these differences are poorly understood. The present study aimed to investigate the relationship between the autophagic capacities of CRC cells and their sensitivities to autophagy inhibition. All studied CRC cell lines showed high basal autophagic flux. However, only HCT116 and Caco-2/15 cells displayed regulated autophagic flux upon starvation. Knockdown of ATG5 (which disrupts autophagosome elongation) or RAB21 (which decreases autophagosome/lysosome fusion) had little effect on CRC cell proliferation in vitro. Nonetheless, inhibition of autophagy in vivo had a substantial cell line-dependent impact on tumor growth, with some cells displaying decreased (HCT116 and Caco-2/15) or increased (SW480 and LoVo) proliferation. RNA sequencing and Western blot analyses in hyperproliferative SW480 tumors revealed that the mTORC2 and AKT pathways were hyperactivated following autophagy impairment. Inhibition of either mTOR or AKT activities rescued the observed hyperproliferation in autophagy-inhibited SW480 and reduced tumor growth. These results highlight that autophagy inhibition can lead, in specific cellular contexts, to compensatory mechanisms promoting tumor growth.
    DOI:  https://doi.org/10.1038/s41598-019-47659-7