bims-pideca 2020-10-11 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2020–10–11
nineteen papers selected by
Ralitsa Radostinova Madsen, University College London

Parallel Single-Cell RNA-Seq and Genetic Recording Reveals Lineage Decisions in Developing Embryoid Bodies.
Ras, PI3K and mTORC2 - three's a crowd?
The Lipid Handling Capacity of Subcutaneous Fat Is Programmed by mTORC2 during Development.
Ubiquitous expression of Akt1 p.(E17K) results in vascular defects and embryonic lethality in mice.
Mutant SF3B1 promotes AKT and NF-kB driven mammary tumorigenesis.
Transient activation of mTORC1 signaling in skeletal muscle is independent of Akt1 regulation.
Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency.
CDK 4/6 Inhibition Overcomes Acquired and Inherent Resistance to PI3Kα Inhibition in Pre-Clinical Models of Head and Neck Squamous Cell Carcinoma.
Control of T lymphocyte fate decisions by PI3K signaling.
WAVE3 phosphorylation regulates the interplay between PI3K, TGF-β, and EGF signaling pathways in breast cancer.
Lipoblastoma phenotype contains a somatic PIK3CA mutation.
CD44/HA signaling mediates acquired resistance to a PI3Kα inhibitor.
Non-genetic mechanisms of therapeutic resistance in cancer.
Active turnover of DNA methylation during cell fate decisions.
The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest.
Sensitive detection of Cre-mediated recombination using droplet digital PCR reveals Tg(BGLAP-Cre) and Tg(DMP1-Cre) are active in multiple non-skeletal tissues.
Molecular stratification of endometrioid ovarian carcinoma predicts clinical outcome.
Body fatness and mTOR pathway activation of breast cancer in the Women's Circle of Health Study.
Estrogen receptor β regulates AKT activity through up-regulation of INPP4B and inhibits migration of prostate cancer cell line PC-3.

Cell Rep. 2020 Oct 06. pii: S2211-1247(20)31211-0. [Epub ahead of print]33(1): 108222

Parallel Single-Cell RNA-Seq and Genetic Recording Reveals Lineage Decisions in Developing Embryoid Bodies.

Ik Soo Kim, Jingyi Wu, Gilbert J Rahme, Sofia Battaglia, Atray Dixit, Elizabeth Gaskell, Huidong Chen, Luca Pinello, Bradley E Bernstein.

  Early developmental specification can be modeled by differentiating embryonic stem cells (ESCs) to embryoid bodies (EBs), a heterogeneous mixture of three germ layers. Here, we combine single-cell transcriptomics and genetic recording to characterize EB differentiation. We map transcriptional states along a time course and model cell fate trajectories and branchpoints as cells progress to distinct germ layers. To validate this inferential model, we propose an innovative inducible genetic recording technique that leverages recombination to generate cell-specific, timestamp barcodes in a narrow temporal window. We validate trajectory architecture and key branchpoints, including early specification of a primordial germ cell (PGC)-like lineage from preimplantation epiblast-like cells. We further identify a temporally defined role of DNA methylation in this PGC-epiblast decision. Our study provides a high-resolution lineage map for an organoid model of embryogenesis, insights into epigenetic determinants of fate specification, and a strategy for lineage mapping of rapid differentiation processes.

Keywords:  differentiation; embryogenesis; lineage tracing; single-cell RNA sequencing; stem cells

DOI:  https://doi.org/10.1016/j.celrep.2020.108222
J Cell Sci. 2020 Oct 08. pii: jcs234930. [Epub ahead of print]133(19):

Ras, PI3K and mTORC2 - three's a crowd?

Stephen F Smith, Shannon E Collins, Pascale G Charest.

  The Ras oncogene is notoriously difficult to target with specific therapeutics. Consequently, there is interest to better understand the Ras signaling pathways to identify potential targetable effectors. Recently, the mechanistic target of rapamycin complex 2 (mTORC2) was identified as an evolutionarily conserved Ras effector. mTORC2 regulates essential cellular processes, including metabolism, survival, growth, proliferation and migration. Moreover, increasing evidence implicate mTORC2 in oncogenesis. Little is known about the regulation of mTORC2 activity, but proposed mechanisms include a role for phosphatidylinositol (3,4,5)-trisphosphate - which is produced by class I phosphatidylinositol 3-kinases (PI3Ks), well-characterized Ras effectors. Therefore, the relationship between Ras, PI3K and mTORC2, in both normal physiology and cancer is unclear; moreover, seemingly conflicting observations have been reported. Here, we review the evidence on potential links between Ras, PI3K and mTORC2. Interestingly, data suggest that Ras and PI3K are both direct regulators of mTORC2 but that they act on distinct pools of mTORC2: Ras activates mTORC2 at the plasma membrane, whereas PI3K activates mTORC2 at intracellular compartments. Consequently, we propose a model to explain how Ras and PI3K can differentially regulate mTORC2, and highlight the diversity in the mechanisms of mTORC2 regulation, which appear to be determined by the stimulus, cell type, and the molecularly and spatially distinct mTORC2 pools.

Keywords:  Mechanistic target of rapamycin complex 2; Phosphatidylinositol 3-kinase; Ras GTPase

DOI:  https://doi.org/10.1242/jcs.234930
Cell Rep. 2020 Oct 06. pii: S2211-1247(20)31212-2. [Epub ahead of print]33(1): 108223

The Lipid Handling Capacity of Subcutaneous Fat Is Programmed by mTORC2 during Development.

Wen-Yu Hsiao, Su Myung Jung, Yuefeng Tang, John A Haley, Rui Li, Huawei Li, Camila Martinez Calejman, Joan Sanchez-Gurmaches, Chien-Min Hung, Amelia K Luciano, Victoria DeMambro, Kathryn E Wellen, Clifford J Rosen, Lihua Julie Zhu, David A Guertin.

  Overweight and obesity are associated with type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal, as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictor upregulate mature adipocyte markers but develop a striking lipid storage defect resulting in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARγ and ChREBP, including genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, which encodes a major mTORC2 substrate and insulin effector. Further exploring this pathway may uncover new strategies to improve insulin sensitivity.

Keywords:  AKT; ChREBP; PPAR-gamma; adipocyte; adipose tissue; lipid metabolism; mTORC2; obesity; type 2 diabetes

DOI:  https://doi.org/10.1016/j.celrep.2020.108223
Hum Mol Genet. 2020 Oct 08. pii: ddaa216. [Epub ahead of print]

Ubiquitous expression of Akt1 p.(E17K) results in vascular defects and embryonic lethality in mice.

Marjorie J Lindhurst, Wenling Li, Nathaniel Laughner, Jasmine J Shwetar, Hannah C Kondolf, Xuefei Ma, Yoh-Suke Mukouyama, Leslie G Biesecker.

Proteus syndrome is a progressive overgrowth disorder with vascular malformations caused by mosaic expression of the AKT1 c.49G > A, p.(E17K) activating variant which was predicted to cause lethality if expressed ubiquitously. To test that hypothesis, we used the ACTB-Cre gene to activate a conditional Akt1 p.(E17K) allele in the mouse. No offspring heterozygous for both Cre and the conditional allele (βA-Akt1WT/flx) were viable. Fewer than expected numbers of βA-Akt1WT/flx embryos were seen beginning at E11.5, but a few survived until E17.5. The phenotype ranged from mild to severe but generally βA-Akt1WT/flx embryos had fewer visible blood vessels and more hemorrhages than their wild type littermates suggestive of a vascular abnormality. Examination of E13.5 limb skin showed a primitive capillary network with increased branching complexity and abnormal patterning compared to wild type skin. By E15.5, wild type skin had undergone angiogenesis and formed a hierarchical network of remodeled vessels, whereas in βA-Akt1WT/flx embryos, the capillary network failed to remodel. Mural cell coverage of the blood vessels was also reduced in βA-Akt1WT/flx skin compared to that of wild type. Restricting expression of Akt1E17K to endothelial, cardiac or smooth muscle cells resulted in viable offspring and remodeled vasculature and did not recapitulate the βA-Akt1WT/flx phenotype. We conclude that ubiquitous expression of Akt1E17K suppresses remodeling and inhibits the formation of a normal skin vasculature. We postulate that this failure prevents proper circulation necessary to support the growing embryo and that it is the result of interactions of multiple cell types with increased AKT signaling.

DOI: https://doi.org/10.1093/hmg/ddaa216
J Clin Invest. 2020 Oct 08. pii: 138315. [Epub ahead of print]

Mutant SF3B1 promotes AKT and NF-kB driven mammary tumorigenesis.

Bo Liu, Zhaoqi Liu, Sisi Chen, Michelle Ki, Caroline Erickson, Jorge S Reis-Filho, Benjamin H Durham, Qing Chang, Elisa de Stanchina, Yiwei Sun, Raul Rabadan, Omar Abdel-Wahab, Sarat Chandarlapaty.

  Mutations in the core RNA splicing factor SF3B1 are prevalent in leukemias and uveal melanoma but hotspot SF3B1 mutations are also seen in epithelial malignancies such as breast cancer. Although hotspot mutations in SF3B1 alter hematopoietic differentiation, whether SF3B1 mutations contribute to epithelial cancer development and progression is unknown. Here, we identify that SF3B1 mutations in mammary epithelial and breast cancer cells induce a recurrent pattern of aberrant splicing leading to activation of AKT and NF-kB, enhanced cell migration, and accelerated tumorigenesis. Transcriptomic analysis of human cancer specimens, MMTV-cre Sf3b1K700E/WT mice, and isogenic mutant cell lines identified hundreds of aberrant 3' splice sites (3'ss) induced by mutant SF3B1. Consistently between mouse and human tumors, mutant SF3B1 promoted aberrant splicing (dependent on aberrant branchpoints as well as pyrimidines downstream of the cryptic 3'ss) and consequent suppression of PPP2R5A and MAP3K7, critical negative regulators of AKT and NF-kB. Coordinate activation of NF-kB and AKT signaling was observed in the knock-in models, leading to accelerated cell migration and tumor development in combination with mutant PIK3CA but also hypersensitizing cells to AKT kinase inhibitors. These data identify hotspot mutations in SF3B1 as an important contributor to breast tumorigenesis and reveal unique vulnerabilities in cancers harboring them.

Keywords:  Breast cancer; Oncology; RNA processing

DOI:  https://doi.org/10.1172/JCI138315
Physiol Rep. 2020 Oct;8(19): e14599

Transient activation of mTORC1 signaling in skeletal muscle is independent of Akt1 regulation.

Mitsunori Miyazaki, Nobuki Moriya, Tohru Takemasa.

  The regulation of cellular protein synthesis is a critical determinant of skeletal muscle growth and hypertrophy in response to an increased workload such as resistance exercise. The mechanistic target of rapamycin complex 1 (mTORC1) and its upstream protein kinase Akt1 have been implicated as a central signaling pathway that regulates protein synthesis in the skeletal muscle; however, the precise molecular regulation of mTORC1 activity is largely unknown. This study employed germline Akt1 knockout (KO) mice to examine whether upstream Akt1 regulation is necessary for the acute activation of mTORC1 signaling in the plantaris muscle following mechanical overload. The phosphorylation states of S6 kinase 1, ribosomal protein S6, and eukaryotic translation initiation factor 4E-binding protein 1 which show the functional activity of mTORC1 signaling, were significantly increased in the skeletal muscle of both wildtype and Akt1 KO mice following an acute bout (3 and 12 hr) of mechanical overload. Akt1 deficiency did not affect load-induced alteration of insulin-like growth factor-1 (IGF-1)/IGF receptor mRNA expression. Also, no effect of Akt1 deficiency was observed on the overload-induced increase in the gene expressions of pax7 and myogenic regulatory factor of myogenin. These observations show that the upstream IGF-1/Akt1 regulation is dispensable for the acute activation of mTORC1 signaling and regulation of satellite cells in response to mechanical overload.

Keywords:  Akt1; mechanistic target of rapamycin; protein synthesis; satellite cells

DOI:  https://doi.org/10.14814/phy2.14599
Nat Commun. 2020 10 05. 11(1): 4977

Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency.

Melanie Werner-Klein, Ana Grujovic, Christoph Irlbeck, Milan Obradović, Martin Hoffmann, Huiqin Koerkel-Qu, Xin Lu, Steffi Treitschke, Cäcilia Köstler, Catherine Botteron, Kathrin Weidele, Christian Werno, Bernhard Polzer, Stefan Kirsch, Miodrag Gužvić, Jens Warfsmann, Kamran Honarnejad, Zbigniew Czyz, Giancarlo Feliciello, Isabell Blochberger, Sandra Grunewald, Elisabeth Schneider, Gundula Haunschild, Nina Patwary, Severin Guetter, Sandra Huber, Brigitte Rack, Nadia Harbeck, Stefan Buchholz, Petra Rümmele, Norbert Heine, Stefan Rose-John, Christoph A Klein.

Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals.

DOI: https://doi.org/10.1038/s41467-020-18701-4
J Clin Med. 2020 Oct 07. pii: E3214. [Epub ahead of print]9(10):

CDK 4/6 Inhibition Overcomes Acquired and Inherent Resistance to PI3Kα Inhibition in Pre-Clinical Models of Head and Neck Squamous Cell Carcinoma.

Eric Remer, Mai Badarni, Elad Hikri, Avraham Dayan, Lirit Levi, Aron Popovtzer, Muhammed Iraqi, Angel Porgador, Ben-Zion Joshua, Gideon Bachar, Moshe Elkabets, Maurizio Scaltriti, Aviram Mizrachi.

  Activating alterations in PIK3CA, the gene coding for the catalytic subunit of phosphoinositide-3-kinase (PI3K), are prevalent in head and neck squamous cell carcinoma (HNSCC) and thought to be one of the main drivers of these tumors. However, early clinical trials on PI3K inhibitors (PI3Ki) have been disappointing due to the limited durability of the activity of these drugs. To investigate the resistance mechanisms to PI3Ki and attempt to overcome them, we conducted a molecular-based study using both HNSCC cell lines and patient-derived xenografts (PDXs). We sought to simulate and dissect the molecular pathways that come into play in PIK3CA-altered HNSCC treated with isoform-specific PI3Ki (BYL719, GDC0032). In vitro assays of cell viability and protein expression indicate that activation of the mTOR and cyclin D1 pathways is associated with resistance to PI3Ki. Specifically, in BYL719-resistant cells, BYL719 treatment did not induce pS6 and pRB inhibition as detected in BYL719-sensitive cells. By combining PI3Ki with either mammalian target of rapamycin complex 1 (mTORC1) or cyclin D1 kinase (CDK) 4/6 specific inhibitors (RAD001 and abemaciclib, respectively), we were able to overcome the acquired resistance. Furthermore, we found that PI3Ki and CDK 4/6 inhibitors have a synergistic anti-tumor effect when combined in human papillomavirus (HPV)-negative/PIK3CA-WT tumors. These findings provide a rationale for combining PI3Ki and CDK 4/6 inhibitors to enhance anti-tumor efficacy in HNSCC patients.

Keywords:  CDK 4/6; PI3K; PIK3CA; head and neck squamous cell carcinoma; human papillomavirus; mTOR

DOI:  https://doi.org/10.3390/jcm9103214
F1000Res. 2020 ;pii: F1000 Faculty Rev-1171. [Epub ahead of print]9

Control of T lymphocyte fate decisions by PI3K signaling.

Benjamin Murter, Lawrence P Kane.

  Virtually all aspects of T and B lymphocyte development, homeostasis, activation, and effector function are impacted by the interaction of their clonally distributed antigen receptors with antigens encountered in their respective environments. Antigen receptors mediate their effects by modulating intracellular signaling pathways that ultimately impinge on the cytoskeleton, bioenergetic pathways, transcription, and translation. Although these signaling pathways are rather well described at this point, especially those steps that are most receptor-proximal, how such pathways contribute to more quantitative aspects of lymphocyte function is still being elucidated. One of the signaling pathways that appears to be involved in this "tuning" process is controlled by the lipid kinase PI3K. Here we review recent key findings regarding both the triggering/enhancement of PI3K signals (via BCAP and ICOS) as well as their regulation (via PIK3IP1 and PHLPP) and how these signals integrate and determine cellular processes. Lymphocytes display tremendous functional plasticity, adjusting their metabolism and gene expression programs to specific conditions depending on their tissue of residence and the nature of the infectious threat to which they are responding. We give an overview of recent findings that have contributed to this model, with a focus on T cells, including what has been learned from patients with gain-of-function mutations in PI3K as well as lessons from cancer immunotherapy approaches.

Keywords:  Lymphocyte activation; lipid kinases; regulatory T cells; signalling

DOI:  https://doi.org/10.12688/f1000research.26928.1
Oncogenesis. 2020 Oct 05. 9(10): 87

WAVE3 phosphorylation regulates the interplay between PI3K, TGF-β, and EGF signaling pathways in breast cancer.

Wei Wang, Urna Kansakar, Vesna Markovic, Bingcheng Wang, Khalid Sossey-Alaoui.

Both TGF-β and the PI3K-AKT signaling pathways are known activators of various intracellular pathways that regulate critical cellular functions, including cancer cell survival and proliferation. The interplay between these two oncogenic pathways plays a major role in promoting the initiation, growth, and progression of tumors, including breast cancers. The molecular underpinning of the inter-relationship between these pathways is, however, not fully understood, as is the role of WAVE3 phosphorylation in the regulation of tumor growth and progression. WAVE3 has been established as a major driver of the invasion-metastasis cascade in breast cancer and other tumors of epithelial origin. WAVE3 phosphorylation downstream of PI3K was also shown to regulate cell migration. Here we show that, in addition to PI3K, WAVE3 tyrosine phosphorylation can also be achieved downstream of TGF-β and EGF and that WAVE3 tyrosine phosphorylation is required for its oncogenic activity. Our in vitro analyses found loss of WAVE3 phosphorylation to significantly inhibit cell migration, as well as tumorsphere growth and invasion. In mouse models for breast cancer, loss of WAVE3 phosphorylation inhibited tumor growth of two aggressive breast cancer cell lines of triple-negative subtype. More importantly, we found that WAVE3 phosphorylation is also required for the activation of PI3K, TGF-β, and EGF signaling and their respective downstream effectors. Therefore, our study identified a novel function for WAVE3 in the regulation of breast cancer development and progression through the modulation of a positive feedback loop between WAVE3 and PI3K-TGF-β-EGF signaling pathways.

DOI: https://doi.org/10.1038/s41389-020-00272-0
Pediatr Dermatol. 2020 Oct 09.

Lipoblastoma phenotype contains a somatic PIK3CA mutation.

Christopher L Sudduth, Dennis J Konczyk, Alyaa Al-Ibraheemi, Patrick J Smits, Arin K Greene.

  Lipoblastoma typically occurs in childhood and is associated with rearrangements of the PLAG1 gene. We present a patient with an isolated mass thought to be a lipoblastoma clinically, radiographically, and histologically. The lesion was diagnosed as a PIK3CA-adipose lesion after the tissue was negative for PLAG1 rearrangement and contained a somatic PIK3CA mutation (H1047R). Although PIK3CA variants are associated with PROS (PIK3CA-related overgrowth spectrum), this report illustrates a non-syndromic, lipoblastoma phenotype caused by a PIK3CA mutation.

Keywords:  PIK3CA; PROS; lipoblastoma; lipoma; overgrowth

DOI:  https://doi.org/10.1111/pde.14406
Cell Death Dis. 2020 Oct 06. 11(10): 831

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

Cuixia Yang, Yumeng Sheng, Xiaoxing Shi, Yiwen Liu, Yiqing He, Yan Du, Guoliang Zhang, Feng Gao.

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.

DOI: https://doi.org/10.1038/s41419-020-03037-0
Nat Rev Cancer. 2020 Oct 08.

Non-genetic mechanisms of therapeutic resistance in cancer.

Jean-Christophe Marine, Sarah-Jane Dawson, Mark A Dawson.

Therapeutic resistance continues to be an indominable foe in our ambition for curative cancer treatment. Recent insights into the molecular determinants of acquired treatment resistance in the clinical and experimental setting have challenged the widely held view of sequential genetic evolution as the primary cause of resistance and brought into sharp focus a range of non-genetic adaptive mechanisms. Notably, the genetic landscape of the tumour and the non-genetic mechanisms used to escape therapy are frequently linked. Remarkably, whereas some oncogenic mutations allow the cancer cells to rapidly adapt their transcriptional and/or metabolic programme to meet and survive the therapeutic pressure, other oncogenic drivers convey an inherent cellular plasticity to the cancer cell enabling lineage switching and/or the evasion of anticancer immunosurveillance. The prevalence and diverse array of non-genetic resistance mechanisms pose a new challenge to the field that requires innovative strategies to monitor and counteract these adaptive processes. In this Perspective we discuss the key principles of non-genetic therapy resistance in cancer. We provide a perspective on the emerging data from clinical studies and sophisticated cancer models that have studied various non-genetic resistance pathways and highlight promising therapeutic avenues that may be used to negate and/or counteract the non-genetic adaptive pathways.

DOI: https://doi.org/10.1038/s41568-020-00302-4
Nat Rev Genet. 2020 Oct 06.

Active turnover of DNA methylation during cell fate decisions.

Aled Parry, Steffen Rulands, Wolf Reik.

DNA methylation is a key layer of epigenetic regulation. The deposition of methylation marks relies on the catalytic activity of DNA methyltransferases (DNMTs), and their active removal relies on the activity of ten-eleven translocation (TET) enzymes. Paradoxically, in important biological contexts these antagonistic factors are co-expressed and target overlapping genomic regions. The ensuing cyclic biochemistry of cytosine modifications gives rise to a continuous, out-of-thermal equilibrium transition through different methylation states. But what is the purpose of this intriguing turnover of DNA methylation? Recent evidence demonstrates that methylation turnover is enriched at gene distal regulatory elements, including enhancers, and can give rise to large-scale oscillatory dynamics. We discuss this phenomenon and propose that DNA methylation turnover might facilitate key lineage decisions.

DOI: https://doi.org/10.1038/s41576-020-00287-8
Cell Rep. 2020 Oct 06. pii: S2211-1247(20)31219-5. [Epub ahead of print]33(1): 108230

The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest.

Mohamed Moustafa-Kamal, Thomas J Kucharski, Wissal El-Assaad, Yazan M Abbas, Valentina Gandin, Bhushan Nagar, Jerry Pelletier, Ivan Topisirovic, Jose G Teodoro.

  mTOR is a serine/threonine kinase and a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in cells arrested in mitosis. Expression of a non-phosphorylatable raptor mutant reactivates mTORC1 and significantly reduces cytotoxicity of the mitotic poison Taxol. This effect is mediated via degradation of PDCD4, a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A is able to enhance the effects of Taxol and restore sensitivity in Taxol-resistant cancer cells. These findings indicate that the mTORC1/S6K/PDCD4/eIF4A axis has a pivotal role in the death versus slippage decision during mitotic arrest and may be exploited clinically to treat tumors resistant to anti-mitotic agents.

Keywords:  PDCD4; S6K; Taxol; cell cycle; eIF4A; hippuristanol; mTORC1; mitosis; raptor

DOI:  https://doi.org/10.1016/j.celrep.2020.108230
Bone. 2020 Oct 05. pii: S8756-3282(20)30454-3. [Epub ahead of print] 115674

Sensitive detection of Cre-mediated recombination using droplet digital PCR reveals Tg(BGLAP-Cre) and Tg(DMP1-Cre) are active in multiple non-skeletal tissues.

Krishnakali Dasgupta, Samantha Lessard, Steven Hann, Megan A Fowler, Alexander G Robling, Matthew L Warman.

  In humans, somatic activating mutations in PIK3CA are associated with skeletal overgrowth. In order to determine if activated PI3K signaling in bone cells causes overgrowth, we used Tg(BGLAP-Cre) and Tg(DMP1-Cre) mouse strains to somatically activate a disease-causing conditional Pik3ca allele (Pik3caH1047R) in osteoblasts and osteocytes. We observed Tg(BGLAP-Cre);Pik3caH1047R/+ offspring were born at the expected Mendelian frequency. However, these mice developed cutaneous lymphatic malformations and died before 7 weeks of age. In contrast, Tg(DMP1-Cre);Pik3caH1047R/+ survived and had no cutaneous lymphatic malformations. Assuming that Cre-activity outside of the skeletal system accounted for the difference in phenotype between Tg(BGLAP-Cre);Pik3caH1047R/+ and Tg(DMP1-Cre);Pik3caH1047R/+ mice, we developed sensitive and specific droplet digital PCR (ddPCR) assays to search for and quantify rates of Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated recombination in non-skeletal tissues. We observed Tg(BGLAP-Cre)-mediated recombination in several tissues including skin, muscle, artery, and brain; two CNS locations, hippocampus and cerebellum, exhibited Cre-mediated recombination in >5% of cells. Tg(DMP1-Cre)-mediated recombination was also observed in muscle, artery, and brain. Although we cannot preclude differences in phenotype between mice with Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated PIK3CA activation being due their inducing Cre-recombination at different stages of osteoblast differentiation, differences in recombination at non-skeletal sites of are the more likely explanation. Since unanticipated sites of recombination can affect the interpretation of data from experiments involving conditional alleles, we recommend ddPCR as a good first step for assessing efficiency, leakiness, and off-targeting in experiments that employ Cre-mediated or Flp-mediated recombination.

Keywords:  BGLAP; Cre-recombination; Mouse; Osteoblasts; Osteocalcin; Osteocytes; PIK3CA

DOI:  https://doi.org/10.1016/j.bone.2020.115674
Nat Commun. 2020 10 05. 11(1): 4995

Molecular stratification of endometrioid ovarian carcinoma predicts clinical outcome.

Robert L Hollis, John P Thomson, Barbara Stanley, Michael Churchman, Alison M Meynert, Tzyvia Rye, Clare Bartos, Yasushi Iida, Ian Croy, Melanie Mackean, Fiona Nussey, Aikou Okamoto, Colin A Semple, Charlie Gourley, C Simon Herrington.

Endometrioid ovarian carcinoma (EnOC) demonstrates substantial clinical and molecular heterogeneity. Here, we report whole exome sequencing of 112 EnOC cases following rigorous pathological assessment. We detect a high frequency of mutation in CTNNB1 (43%), PIK3CA (43%), ARID1A (36%), PTEN (29%), KRAS (26%), TP53 (26%) and SOX8 (19%), a recurrently-mutated gene previously unreported in EnOC. POLE and mismatch repair protein-encoding genes were mutated at lower frequency (6%, 18%) with significant co-occurrence. A molecular taxonomy is constructed, identifying clinically distinct EnOC subtypes: cases with TP53 mutation demonstrate greater genomic complexity, are commonly FIGO stage III/IV at diagnosis (48%), are frequently incompletely debulked (44%) and demonstrate inferior survival; conversely, cases with CTNNB1 mutation, which is mutually exclusive with TP53 mutation, demonstrate low genomic complexity and excellent clinical outcome, and are predominantly stage I/II at diagnosis (89%) and completely resected (87%). Moreover, we identify the WNT, MAPK/RAS and PI3K pathways as good candidate targets for molecular therapeutics in EnOC.

DOI: https://doi.org/10.1038/s41467-020-18819-5
NPJ Breast Cancer. 2020 ;6 45

Body fatness and mTOR pathway activation of breast cancer in the Women's Circle of Health Study.

Ting-Yuan David Cheng, Angela R Omilian, Song Yao, Pamela V Sanchez, Latasia Z Polk, Weizhou Zhang, Susmita Datta, Wiam Bshara, Rochelle Payne Ondracek, Warren Davis, Song Liu, Chi-Chen Hong, Elisa V Bandera, Thaer Khoury, Christine B Ambrosone.

  Energy imbalance has an important role in breast cancer prognosis. Hyperactive mechanistic Target of Rapamycin (mTOR) pathway is associated with breast tumor growth, but the extent to which body fatness is associated with mTOR pathway activities in breast cancer is unclear. We performed immunostaining for mTOR, phosphorylated (p)-mTOR, p-AKT, and p-p70S6K in tumor tissue from 590 women (464 African Americans/Blacks and 126 Whites) with newly diagnosed invasive breast cancer in the Women's Circle of Health Study. Anthropometric measures were taken by study staff, and body composition was measured by bioelectrical impedance analysis. Linear regressions were used to estimate percent differences in protein expression between categories of body mass index (BMI), waist circumference, waist/hip ratio, fat mass, fat mass index, and percent body fat. We observed that BMI ≥ 35.0 vs. <25 kg/m2 was associated with 108.3% (95% CI = 16.9%-270.9%) and 101.8% (95% CI = 17.0%-248.8%) higher expression in p-mTOR and normalized p-mTOR, i.e., p-mTOR/mTOR, respectively. Quartiles 4 vs. 1 of waist/hip ratio was associated with 41.8% (95% CI = 5.81%-89.9%) higher mTOR expression. Similar associations were observed for the body fat measurements, particularly in patients with estrogen receptor-negative (ER-) tumors, but not in those with ER+ tumors, although the differences in associations were not significant. This tumor-based study found positive associations between body fatness and mTOR pathway activation, evident by a p-mTOR expression, in breast cancer. Our findings suggest that mTOR inhibition can be a treatment strategy to prevent the recurrence of these tumors in obese individuals.

Keywords:  Biomarkers; Cancer epidemiology; Risk factors

DOI:  https://doi.org/10.1038/s41523-020-00187-4
Proc Natl Acad Sci U S A. 2020 Oct 05. pii: 202007160. [Epub ahead of print]

Estrogen receptor β regulates AKT activity through up-regulation of INPP4B and inhibits migration of prostate cancer cell line PC-3.

Surendra Chaurasiya, Wanfu Wu, Anders M Strom, Margaret Warner, Jan-Åke Gustafsson.

  Loss of the tumor suppressor, PTEN, is one of the most common findings in prostate cancer (PCa). This loss leads to overactive Akt signaling, which is correlated with increased metastasis and androgen independence. However, another tumor suppressor, inositol-polyphosphate 4-phosphatase type II (INPP4B), can partially compensate for the loss of PTEN. INPP4B is up-regulated by androgens, and this suggests that androgen-deprivation therapy (ADT) would lead to hyperactivity of AKT. However, in the present study, we found that in PCa, samples from men treated with ADT, ERβ, and INPP4B expression were maintained in some samples. To investigate the role of ERβ1 in regulation of INPPB, we engineered the highly metastatic PCa cell line, PC3, to express ERβ1. In these cells, INPP4B was induced by ERβ ligands, and this induction was accompanied by inhibition of Akt activity and reduction in cell migration. These findings reveal that, in the absence of androgens, ERβ1 induces INPP4B to dampen AKT signaling. Since the endogenous ERβ ligand, 3β-Adiol, is lost upon long-term ADT, to obtain the beneficial effects of ERβ1 on AKT signaling, an ERβ agonist should be added along with ADT.

Keywords:  INPP4B; androgen deprivation therapy; estrogen receptor; prostate cancer

DOI:  https://doi.org/10.1073/pnas.2007160117