A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation.
Mutations in ARID1A rank amongst the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation. Genetic Wnt/B-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells.
View details for DOI 10.1158/2159-8290.CD-20-1109
View details for PubMedID 33451982
- Organoids as Oracles for Precision Medicine in Rectal Cancer CELL STEM CELL 2020; 26 (1): 4?6
- What's so special about lipid transport in the human placenta? JOURNAL OF PHYSIOLOGY-LONDON 2019; 597 (19): 4863?64
Endoscopic Submucosal Dissection is Associated with Less Pathologic Uncertainty than Endoscopic Mucosal Resection in Diagnosing and Staging Barrett's- Related Neoplasia.
Digestive endoscopy : official journal of the Japan Gastroenterological Endoscopy Society
BACKGROUND & AIMS: Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have demonstrated similar efficacy in removal of neoplastic esophageal lesions. However, significant controversy exists over the preferred resection technique. Our primary aim was to compare the pathologic specimens produced via EMR and ESD and secondarily gauge their effect on clinical decision making and patient outcomes.METHODS: Using a retrospective cohort study design, all esophageal Barrett's-associated neoplastic lesions resected by a single provider (SF) from 2012-2017 were reviewed. The pathology was re-reviewed by 2 blinded authors for diagnosis, margins, and. Adverse outcomes and recurrence rates were also collected.RESULTS: 31 EMR and 20 ESD cases were identified. Baseline demographics and lesion characteristics were similar. ESD produced more R0 resections and more en bloc resections compared to EMR. EMR produced more equivocal lateral (13/31, 41.9% vs 1/20, 5.0%) and vertical margins (13/31,41.9% vs. 0/20, 0%, both p<0.05). This led to an inability to reach a definitive diagnosis in 13/31 EMR vs 0/20 ESD pathology specimens (p=.003). Of the 13 EMR specimens with equivocal pathology, 11 were noted to have 'at least intramucosal adenocarcinoma'. 4/11 patients chose to undergo elective esophagectomy with final surgical pathology demonstrating ?T1a disease in 2, and ?T1b disease in 2.CONCLUSION: Compared to ESD, EMR was associated with greater pathologic uncertainty in Barrett's-associated neoplasia This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/den.13487
View details for PubMedID 31306525
Real-time microscopic assessment of fatty acid uptake kinetics in the human term placenta.
2018; 72-73: 1?9
The placenta employs an efficient and selective fatty acid transport system to supply lipids for fetal development. Disruptions in placental fatty acid transport lead to restricted fetal growth along with cardiovascular and neurologic deficits. Nevertheless, little is known about the molecular mechanisms involved in human placental fatty acid trafficking during the initial steps of uptake, or the importance of fatty acid chain length in determining uptake rates.We employed BODIPY fluorophore conjugated fatty acid analogues of three chain lengths, medium (BODIPY-C5), long (BODIPY-C12), and very-long (BODIPY-C16), to study fatty acid uptake in isolated human trophoblast and explants using confocal microscopy. The three BODIPY-labeled fatty acids were added to freshly isolated explants and tracked for up to 30?min. Fatty acid uptake kinetics were quantified in trophoblast (cytotrophoblast and syncytiotrophoblast together) and the fetal capillary lumen.Long- (BODIPY-C12) and Very long-chain (BODIPY-C16) fatty acids accumulated more rapidly in the trophoblast layer than did medium-chain (BODIPY-C5) whereas BODIPY-C5 accumulated more rapidly in the fetal capillary than did the longer chain length fatty acids. The long-chain fatty acids, BODIPY-C12 and BODIPY-C16, are esterified and stored in lipid droplets in the cytotrophoblast layer, but medium-chain fatty acid, BODIPY-C5, is not.Fatty acids accumulate in trophoblast and fetal capillaries inversely according to their chain length. BODIPY-C5 accumulates in the fetal capillary in concentrations far greater than in the trophoblast, suggesting that medium-chain length BODIPY-labeled fatty acids are capable of being transported against a concentration gradient.
View details for PubMedID 30501875
Cytotrophoblast, Not Syncytiotrophoblast, Dominates Glycolysis and Oxidative Phosphorylation in Human Term Placenta
The syncytiotrophoblast (SCT) at the maternal-fetal interface has been presumed to be the primary driver of placental metabolism, and the underlying progenitor cytotrophoblast cells (CTB) an insignificant contributor to placental metabolic activity. However, we now show that the metabolic rate of CTB is much greater than the SCT. The oxygen consumption and extracellular acidification rate, a measure of glycolysis, are both greater in CTB than in SCT in vitro (CTB: 96?±?16 vs SCT: 46?±?14?pmol O2?×?min-1?×?100?ng DNA-1, p?0.001) and (CTB: 43?±?6.7 vs SCT 1.4?±?1.0 ?mpH?×?min-1?×?100?ng DNA-1, p?0.0001). Mitochondrial activity, as determined by using the mitochondrial activity-dependent dye Mitotracker CM-H2TMRosa, is higher in CTB than in SCT in culture and living explants. These data cast doubt on the previous supposition that the metabolic rate of the placenta is dominated by the SCT contribution. Moreover, differentiation into SCT leads to metabolic suppression. The normal suppression of metabolic activity during CTB differentiation to SCT is prevented with a p38 MAPK signaling inhibitor and epidermal growth factor co-treatment. We conclude that the undifferentiated CTB, in contrast to the SCT, is highly metabolically active, has a high level of fuel flexibility, and contributes substantially to global metabolism in the late gestation human placenta.
View details for DOI 10.1038/srep42941
View details for Web of Science ID 000394768200001
View details for PubMedID 28230167
View details for PubMedCentralID PMC5322316
Metabolic reprogramming ensures cancer cell survival despite oncogenic signaling blockade.
Genes & development
2017; 31 (20): 2067?84
There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5 Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.
View details for PubMedID 29138276
View details for PubMedCentralID PMC5733498
Biological features of placental programming
2016; 48: S47-S53
The placenta is a key organ in programming the fetus for later disease. This review outlines nine of many structural and physiological features of the placenta which are associated with adult onset chronic disease. 1) Placental efficiency relates the placental mass to the fetal mass. Ratios at the extremes are related to cardiovascular disease risk later in life. 2) Placental shape predicts a large number of disease outcomes in adults but the regulators of placental shape are not known. 3) Non-human primate studies suggest that at about mid-gestation, the placenta becomes less plastic and less able to compensate for pathological stresses. 4) Recent studies suggest that lipids have an important role in regulating placental metabolism and thus the future health of offspring. 5) Placental inflammation affects nutrient transport to the fetus and programs for later disease. 6) Placental insufficiency leads to inadequate fetal growth and elevated risks for later life disease. 7) Maternal height, fat and muscle mass are important in combination with placental size and shape in predicting adult disease. 8) The placenta makes a host of hormones that influence fetal growth and are related to offspring disease. Unfortunately, our knowledge of placental growth and function lags far behind that of other organs. An investment in understanding placental growth and function will yield enormous benefits to human health because it is a key player in the origins of the most expensive and deadly chronic diseases that humans face.
View details for DOI 10.1016/j.placenta.2016.10.012
View details for Web of Science ID 000390630000010
View details for PubMedID 27817870
View details for PubMedCentralID PMC5278807
IFPA meeting 2015 workshop report IV: placenta and obesity; stem cells of the feto-maternal interface; placental immunobiology and infection
2016; 48: S17-S20
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At the 2015 IFPA annual meeting there were 12 themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology and collectively covered areas of obesity and the placenta, stem cells of the feto-maternal interface, and placental immunobiology and infection.
View details for DOI 10.1016/j.placenta.2016.08.001
View details for PubMedID 27506263
Real-Time Tracking of BODIPY-C12 Long-Chain Fatty Acid in Human Term Placenta Reveals Unique Lipid Dynamics in Cytotrophoblast Cells
2016; 11 (4)
While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C12, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism.
View details for DOI 10.1371/journal.pone.0153522
View details for Web of Science ID 000375211700018
View details for PubMedID 27124483
View details for PubMedCentralID PMC4849650
Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis.
2014; 155 (5): 1956-1969
The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.
View details for DOI 10.1210/en.2013-2081
View details for PubMedID 24684304
View details for PubMedCentralID PMC3990843
Effect of Substrate Stiffness on Early Mouse Embryo Development
2012; 7 (7)
It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68 ± 15% vs. 59 ± 18%), blastocyst (64 ± 9.1% vs. 50 ± 18%) and hatching blastocyst stages (54 ± 25% vs. 21 ± 16%) and an increase in the number of trophectodermal cell (TE,65 ± 13 vs. 49 ± 12 cells) compared to control embryos cultured in PD (mean ± S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.
View details for DOI 10.1371/journal.pone.0041717
View details for Web of Science ID 000307045600039
View details for PubMedID 22860009
View details for PubMedCentralID PMC3409240
Impaired Placental Nutrient Transport in Mice Generated by in Vitro Fertilization
2012; 153 (7): 3457-3467
More than 4.5 million children have been conceived by in vitro fertilization (IVF). Interestingly, singleton IVF offspring born at term have an increased incidence of low birth weight. The mechanism responsible for the lower birth weight is unknown, but alterations in placental function are possible. Hence, the goal of our study was to examine placental growth and function in mice generated in vivo or in vitro. To assess placental function, blastocysts were generated by IVF or produced by natural mating (control group); both IVF and control blastocysts were transferred to pseudopregnant recipients. Placental weights did not differ at embryonic d 15.5 (E15.5) but were increased at E18.5 in the IVF group (25.4%, P < 0.001) compared with control. Proliferation was increased in IVF placentae, whereas overall placental gross morphology and apoptosis were not affected. Both fetal weights (16.4% lower at E15.5 and 8.8% lower at E18.5, P < 0.05) and fetal to placental ratios were lower (P < 0.001) in the IVF compared with the control group at both time points, whereas birth weights did not differ. At E18.5, the mRNA for selected glucose, system A amino acid transporters, and imprinted genes were down-regulated in IVF placentae. GLUT3 protein level was decreased in the IVF group (P < 0.05). Importantly, intrajugular injections of (14)C-methyl-D-glucose or (14)C-MeAIB tracers (n = 6 litters per group) showed that placental transport of glucose and amino acids were 24.8% (not significant) and 58.1% (P < 0.05) lower in the IVF group. Fetal accumulation of glucose was not different, but amino acid accumulation was significantly (36 %) lower in IVF fetuses (P < 0.05). We conclude that IVF alters both fetal and placental growth and, importantly, decreases placental transport efficiency in mice conceived by IVF.
View details for DOI 10.1210/en.2011-1921
View details for Web of Science ID 000305781800051
View details for PubMedID 22562173
View details for PubMedCentralID PMC3380310
Mechanotransduction: a major regulator of homeostasis and development
WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE
2010; 2 (6): 625-639
In nearly all aspects of biology, forces are a relevant regulator of life's form and function. More recently, science has established that cells are exquisitely sensitive to forces of varying magnitudes and time scales, and they convert mechanical stimuli into a chemical response. This phenomenon, termed mechanotransduction, is an integral part of cellular physiology and has a profound impact on the development of the organism. Furthermore, malfunctioning mechanical properties or mechanotransduction often leads to pathology of the organism. In this review, we describe mechanotransduction and the theories underlying how forces may be sensed, from the molecular to organism scale. The influence of mechanotransduction on normal and abnormal development, such as stem cell differentiation and cancer, is also reviewed. Studies illustrate the diversity of mechanotransduction, and the major role it has on organism homeostasis. Cells employ a variety of mechanisms, which differ depending upon cell type and environment, to sense and respond to forces.
View details for DOI 10.1002/wsbm.79
View details for Web of Science ID 000283713500001
View details for PubMedID 20890961
Phosphorylation Facilitates the Integrin Binding of Filamin under Force
2009; 97 (12): 3095-3104
Filamins are actin binding proteins that contribute to cytoskeletal integrity and biochemical scaffolds during mechanochemical signal transductions. Structurally, human filamins are dimers composed of an actin-binding domain with 24 immunoglobulin (Ig)-like repeats. In this study, we focus on the recently solved high-resolution crystal structure of Ig-like repeats 19-21 of filamin-A (IgFLNa-R19-R21). IgFLNa-R19-21 is of marked importance because it contains the binding site for integrins and facilitates the dynamic ability of filamin-A to communicate with the extracellular environment. However, the structure of filamin-A shows an interesting domain arrangement where the integrin binding site on IgFLNa-R21 is hindered sterically by IgFLNa-R20. Thus, a number of hypotheses on the regulation of filamin-A exist. Using molecular dynamics simulations we evaluated the effects of two primary regulators of filamin-A, force and phosphorylation. We find that a tensile force of 40 pN is sufficient to initiate the partial removal of the autoinhibition on the integrin binding site of IgFLNa-R21. Force coupled to phosphorylation at Ser(2152), however, affords complete dissociation of autoinhibition with a decreased force requirement. Phosphorylation seems to decrease the threshold for removing the IgFLNa-R20 beta-strand inhibitor within 300 ps with 40 pN tensile force. Furthermore, the molecular dynamic trajectories illustrate phosphorylation of Ser(2152) without force is insufficient to remove autoinhibition. We believe the results of this study implicate filamin-A as a tunable mechanosensor, where its sensitivity can be modulated by the degree of phosphorylation.
View details for DOI 10.1016/j.bpj.2009.08.059
View details for Web of Science ID 000272765400006
View details for PubMedID 20006946
View details for PubMedCentralID PMC2793350
Quantitative analysis of epithelial morphogenesis in Drosophila oogenesis: New insights based on morphometric analysis and mechanical modeling
2009; 331 (2): 129-139
The process of epithelial morphogenesis is ubiquitous in animal development, but much remains to be learned about the mechanisms that shape epithelial tissues. The follicle cell (FC) epithelium encapsulating the growing germline of Drosophila is an excellent system to study fundamental elements of epithelial development. During stages 8 to 10 of oogenesis, the FC epithelium transitions between simple geometries-cuboidal, columnar and squamous-and redistributes cell populations in processes described as posterior migration, squamous cell flattening and main body cell columnarization. Here we have carried out a quantitative morphometric analysis of these poorly understood events in order to establish the parameters of and delimit the potential processes that regulate the transitions. Our results compel a striking revision of accepted views of these phenomena, by showing that posterior migration does not involve FC movements, that there is no role for columnar cell apical constriction in FC morphogenesis, and that squamous cell flattening may be a compliant response to germline growth. We utilize mechanical modeling involving finite element computational technologies to demonstrate that time-varying viscoelastic properties and growth are sufficient to account for the bulk of the FC morphogenetic changes.
View details for DOI 10.1016/j.ydbio.2009.04.028
View details for Web of Science ID 000267777900003
View details for PubMedID 19409378
View details for PubMedCentralID PMC3145632
Molecular mechanics of filamin's rod domain
2008; 94 (3): 1075-1083
Rearrangement of the actin cytoskeleton is integral to cell shape and function. Actin-binding proteins, e.g., filamin, can naturally contribute to the mechanics and function of the actin cytoskeleton. The molecular mechanical bases for filamin's function in actin cytoskeletal reorganization are examined here using molecular dynamics simulations. Simulations are performed by applying forces ranging from 25 pN to 125 pN for 2.5 ns to the rod domain of filamin. Applying small loads ( approximately 25 pN) to filamin's rod domain supplies sufficient energy to alter the conformation of the N-terminal regions of the rod. These forces break local hydrogen bond coordination often enough to allow side chains to find new coordination partners, in turn leading to drastic changes in the conformation of filamin, for example, increasing the hydrophobic character of the N-terminal rod region and, alternatively, activating the C-terminal region to become increasingly stiff. These changes in conformation can lead to changes in the affinity of filamin for its binding partners. Therefore, filamin can function to transduce mechanical signals as well as preserve topology of the actin cytoskeleton throughout the rod domain.
View details for DOI 10.1529/biophysj.107.118802
View details for Web of Science ID 000252243200033
View details for PubMedID 17921200
View details for PubMedCentralID PMC2186249