Publications by authors named "Geoffrey M Wahl"

66 Publications

Tuft Cells Inhibit Pancreatic Tumorigenesis in Mice by Producing Prostaglandin D.

Gastroenterology 2020 11 24;159(5):1866-1881.e8. Epub 2020 Jul 24.

Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California. Electronic address:

Background & Aims: Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis.

Methods: We performed studies with LSL-Kras;Ptf1a mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA.

Results: Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA.

Conclusions: In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.
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http://dx.doi.org/10.1053/j.gastro.2020.07.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680354PMC
November 2020

Stem Cell Determinant SOX9 Promotes Lineage Plasticity and Progression in Basal-like Breast Cancer.

Cell Rep 2020 06;31(10):107742

Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address:

Lineage plasticity is important for the development of basal-like breast cancer (BLBC), an aggressive cancer subtype. While BLBC is likely to originate from luminal progenitor cells, it acquires substantial basal cell features and contains a heterogenous collection of cells exhibiting basal, luminal, and hybrid phenotypes. Why luminal progenitors are prone to BLBC transformation and what drives luminal-to-basal reprogramming remain unclear. Here, we show that the transcription factor SOX9 acts as a determinant for estrogen-receptor-negative (ER) luminal stem/progenitor cells (LSPCs). SOX9 controls LSPC activity in part by activating both canonical and non-canonical nuclear factor κB (NF-κB) signaling. Inactivation of TP53 and RB via expression of SV40 TAg in a BLBC mouse tumor model leads to upregulation of SOX9, which drives luminal-to-basal reprogramming in vivo. Furthermore, SOX9 deletion inhibits the progression of ductal carcinoma in situ (DCIS)-like lesions to invasive carcinoma. These data show that ER LSPC determinant SOX9 acts as a lineage plasticity driver for BLBC progression.
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http://dx.doi.org/10.1016/j.celrep.2020.107742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658810PMC
June 2020

Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders.

Proc Natl Acad Sci U S A 2020 06 18;117(22):12121-12130. Epub 2020 May 18.

Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037;

HRAS, NRAS, and KRAS4A/KRAS4B comprise the RAS family of small GTPases that regulate signaling pathways controlling cell proliferation, differentiation, and survival. RAS pathway abnormalities cause developmental disorders and cancers. We found that KRAS4B colocalizes on the cell membrane with other RAS isoforms and a subset of prenylated small GTPase family members using a live-cell quantitative split luciferase complementation assay. RAS protein coclustering is mainly mediated by membrane association-facilitated interactions (MAFIs). Using the RAS-RBD (CRAF RAS binding domain) interaction as a model system, we showed that MAFI alone is not sufficient to induce RBD-mediated RAS inhibition. Surprisingly, we discovered that high-affinity membrane-targeted RAS binding proteins inhibit RAS activity and deplete RAS proteins through an autophagosome-lysosome-mediated degradation pathway. Our results provide a mechanism for regulating RAS activity and protein levels, a more detailed understanding of which should lead to therapeutic strategies for inhibiting and depleting oncogenic RAS proteins.
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http://dx.doi.org/10.1073/pnas.2000848117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275768PMC
June 2020

Cysteine depletion induces pancreatic tumor ferroptosis in mice.

Science 2020 04;368(6486):85-89

Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA.

Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system x is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system x subunit, , induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.
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http://dx.doi.org/10.1126/science.aaw9872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681911PMC
April 2020

Tuft Cell Formation Reflects Epithelial Plasticity in Pancreatic Injury: Implications for Modeling Human Pancreatitis.

Front Physiol 2020 14;11:88. Epub 2020 Feb 14.

Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA, United States.

Chronic pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma (PDA), is a serious, widespread medical condition characterized by inflammation, fibrosis, and acinar to ductal metaplasia (ADM). ADM is a cell type transdifferentiation event where pancreatic acinar cells become ductal-like under conditions of injury or oncogenic mutation. Here, we show that chronic pancreatitis and ADM in genetically wild type mice results in the formation of a significant population of chemosensory tuft cells. Transcriptomic analyses of pancreatitis tuft cells identify expression of inflammatory mediators, consistent with a role for tuft cells in injury progression and/or resolution. Though similar to tuft cell populations in other organs and disease systems, we identified a number of key differences that suggest context-specific tuft cell functions. We evaluated seven different mouse strains for tuft cell formation in response to chronic injury and identified significant heterogeneity reflecting varying proclivity for epithelial plasticity between strains. These results have interesting implications in the role of epithelial plasticity and heterogeneity in pancreatitis and highlight the importance of mouse strain selection when modeling human disease.
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http://dx.doi.org/10.3389/fphys.2020.00088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033634PMC
February 2020

Nup93 regulates breast tumor growth by modulating cell proliferation and actin cytoskeleton remodeling.

Life Sci Alliance 2020 01 20;3(1). Epub 2020 Jan 20.

Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA

Nucleoporin 93 (Nup93) expression inversely correlates with the survival of triple-negative breast cancer patients. However, our knowledge of Nup93 function in breast cancer besides its role as structural component of the nuclear pore complex is not understood. Combination of functional assays and genetic analyses suggested that chromatin interaction of Nup93 partially modulates the expression of genes associated with actin cytoskeleton remodeling and epithelial to mesenchymal transition, resulting in impaired invasion of triple-negative, claudin-low breast cancer cells. Nup93 depletion induced stress fiber formation associated with reduced cell migration/proliferation and impaired expression of mesenchymal-like genes. Silencing , a gene responsible for actin cytoskeleton remodeling and up-regulated upon Nup93 depletion, partially restored the invasive phenotype of cancer cells. Loss of Nup93 led to significant defects in tumor establishment/propagation in vivo, whereas patient samples revealed that high Nup93 and low LIMCH1 expression correlate with late tumor stage. Our approach identified Nup93 as contributor of triple-negative, claudin-low breast cancer cell invasion and paves the way to study the role of nuclear envelope proteins during breast cancer tumorigenesis.
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http://dx.doi.org/10.26508/lsa.201900623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971368PMC
January 2020

DIRAS3 (ARHI) Blocks RAS/MAPK Signaling by Binding Directly to RAS and Disrupting RAS Clusters.

Cell Rep 2019 12;29(11):3448-3459.e6

Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

Oncogenic RAS mutations drive cancers at many sites. Recent reports suggest that RAS dimerization, multimerization, and clustering correlate strongly with activation of RAS signaling. We have found that re-expression of DIRAS3, a RAS-related small GTPase tumor suppressor that is downregulated in multiple cancers, inhibits RAS/mitogen-activated protein kinase (MAPK) signaling by interacting directly with RAS-forming heteromers, disrupting RAS clustering, inhibiting Raf kinase activation, and inhibiting transformation and growth of cancer cells and xenografts. Disruption of K-RAS cluster formation requires the N terminus of DIRAS3 and interaction of both DIRAS3 and K-RAS with the plasma membrane. Interaction of DIRAS3 with both K-RAS and H-RAS suggests a strategy for inhibiting oncogenic RAS function.
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http://dx.doi.org/10.1016/j.celrep.2019.11.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948147PMC
December 2019

Single-Cell Chromatin Analysis of Mammary Gland Development Reveals Cell-State Transcriptional Regulators and Lineage Relationships.

Cell Rep 2019 10;29(2):495-510.e6

Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA. Electronic address:

Technological improvements enable single-cell epigenetic analyses of organ development. We reasoned that high-resolution single-cell chromatin accessibility mapping would provide needed insight into the epigenetic reprogramming and transcriptional regulators involved in normal mammary gland development. Here, we provide a single-cell resource of chromatin accessibility for murine mammary development from the peak of fetal mammary stem cell (fMaSC) functional activity in late embryogenesis to the differentiation of adult basal and luminal cells. We find that the chromatin landscape within individual cells predicts both gene accessibility and transcription factor activity. The ability of single-cell chromatin profiling to separate E18 fetal mammary cells into clusters exhibiting basal-like and luminal-like chromatin features is noteworthy. Such distinctions were not evident in analyses of droplet-based single-cell transcriptomic data. We present a web application as a scientific resource for facilitating future analyses of the gene regulatory networks involved in mammary development.
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http://dx.doi.org/10.1016/j.celrep.2019.08.089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887110PMC
October 2019

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Nature 2019 05 17;569(7754):131-135. Epub 2019 Apr 17.

Department of Surgery, Division of Surgical Oncology, University of California San Diego School of Medicine, La Jolla, CA, USA.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance. Furthermore, PSC activation occurs very early during PDAC tumorigenesis, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.
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http://dx.doi.org/10.1038/s41586-019-1130-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565370PMC
May 2019

Epigenetic and Transcriptomic Profiling of Mammary Gland Development and Tumor Models Disclose Regulators of Cell State Plasticity.

Cancer Cell 2018 09 30;34(3):466-482.e6. Epub 2018 Aug 30.

Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA. Electronic address:

Cell state reprogramming during tumor progression complicates accurate diagnosis, compromises therapeutic effectiveness, and fuels metastatic dissemination. We used chromatin accessibility assays and transcriptional profiling during mammary development as an agnostic approach to identify factors that mediate cancer cell state interconversions. We show that fetal and adult basal cells share epigenetic features consistent with multi-lineage differentiation potential. We find that DNA-binding motifs for SOX transcription factors are enriched in chromatin that is accessible in stem/progenitor cells and inaccessible in differentiated cells. In both mouse and human tumors, SOX10 expression correlates with stem/progenitor identity, dedifferentiation, and invasive characteristics. Strikingly, we demonstrate that SOX10 binds to genes that regulate neural crest cell identity, and that SOX10-positive tumor cells exhibit neural crest cell features.
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http://dx.doi.org/10.1016/j.ccell.2018.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152943PMC
September 2018

Single-Cell Transcriptomes Distinguish Stem Cell State Changes and Lineage Specification Programs in Early Mammary Gland Development.

Cell Rep 2018 08;24(6):1653-1666.e7

Huntsman Cancer Institute, Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA. Electronic address:

The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.
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http://dx.doi.org/10.1016/j.celrep.2018.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301014PMC
August 2018

Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy.

PLoS One 2017 6;12(12):e0189051. Epub 2017 Dec 6.

Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, United States of America.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely dense fibrotic stroma, which contributes to tumor growth, metastasis, and drug resistance. During tumorigenesis, quiescent pancreatic stellate cells (PSCs) are activated and become major contributors to fibrosis, by increasing growth factor signaling and extracellular matrix deposition. The p53 tumor suppressor is known to restrict tumor initiation and progression through cell autonomous mechanisms including apoptosis, cell cycle arrest, and senescence. There is growing evidence that stromal p53 also exerts anti-tumor activity by paracrine mechanisms, though a role for stromal p53 in PDAC has not yet been described. Here, we demonstrate that activation of stromal p53 exerts anti-tumor effects in PDAC. We show that primary cancer-associated PSCs (caPSCs) isolated from human PDAC express wild-type p53, which can be activated by the Mdm2 antagonist Nutlin-3a. Our work reveals that p53 acts as a major regulator of PSC activation and as a modulator of PDAC fibrosis. In vitro, p53 activation by Nutlin-3a induces profound transcriptional changes, which reprogram activated PSCs to quiescence. Using immunofluorescence and lipidomics, we have also found that p53 activation induces lipid droplet accumulation in both normal and tumor-associated fibroblasts, revealing a previously undescribed role for p53 in lipid storage. In vivo, treatment of tumor-bearing mice with the clinical form of Nutlin-3a induces stromal p53 activation, reverses caPSCs activation, and decreases fibrosis. All together our work uncovers new functions for stromal p53 in PDAC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189051PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5718507PMC
December 2017

Lgr5 is a marker for fetal mammary stem cells, but is not essential for stem cell activity or tumorigenesis.

NPJ Breast Cancer 2017 24;3:16. Epub 2017 Apr 24.

Salk Institute for Biological Studies, Gene Expression Laboratory, La Jolla, CA 92037 USA.

The search for the bipotent mammary stem cells that drive mammary development requires markers to enable their prospective isolation. There is general agreement that bipotent mouse mammary stem cells are abundant in late fetal development, but their existence in the adult is vigorously debated. Among markers useful for mammary stem cell identification, the Wnt co-receptor Lgr5 has been suggested by some to be both "necessary and sufficient" for bipotency and transplantation of adult mammary stem cell activity, though other studies disagree. Importantly, the relevance of Lgr5 to the bipotency of fetal mammary stem cells has not been studied. We show here that expression of a fluorescent protein driven by the endogenous Lgr5 promoter enables significant fetal mammary stem cell enrichment. We used lineage tracing to demonstrate embryonic cells expressing Lgr5 are bipotent, while their adult counterparts are myoepithelial restricted. Importantly, our data conclusively demonstrate that Lgr5 is dispensable for both fetal and adult mammary stem cell activity and for the development of mammary tumors.
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http://dx.doi.org/10.1038/s41523-017-0018-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460261PMC
April 2017

Cell state plasticity, stem cells, EMT, and the generation of intra-tumoral heterogeneity.

NPJ Breast Cancer 2017 19;3:14. Epub 2017 Apr 19.

Huntsman Cancer Institute, The University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112 USA.

Cellular heterogeneity in cancer represents a significant challenge. In order to develop effective and lasting therapies, it is essential to understand the source of this heterogeneity, and its role in tumor progression and therapy resistance. Here, we consider not only genetic and epigenetic mechanisms, but also inflammation and cell state reprogramming in creating tumor heterogeneity. We discuss similarities between normal mammary epithelial developmental states and various breast cancer molecular sub-types, and the cells that are thought to propagate them. We emphasize that while stem cell phenotypes and mesenchymal character have often been conflated, existing data suggest that the combination of intrinsic genetic and epigenetic changes, and microenvironmental influences generate multiple types of tumor propagating cells distinguishable by their positions along a continuum of epithelial to mesenchymal, stem to differentiated and embryonic to mature cell states. Consequently, in addition to the prospect of stem cell-directed tumor therapies, there is a need to understand interrelationships between stem cell, epithelial-mesenchymal, and tumor-associated reprogramming events to develop new therapies that mitigate cell state plasticity and minimize the evolution of tumor heterogeneity.
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http://dx.doi.org/10.1038/s41523-017-0012-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460241PMC
April 2017

Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.

Nature 2017 03 8;543(7643):122-125. Epub 2017 Feb 8.

Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California 92093, USA.

Human cells have twenty-three pairs of chromosomes. In cancer, however, genes can be amplified in chromosomes or in circular extrachromosomal DNA (ecDNA), although the frequency and functional importance of ecDNA are not understood. We performed whole-genome sequencing, structural modelling and cytogenetic analyses of 17 different cancer types, including analysis of the structure and function of chromosomes during metaphase of 2,572 dividing cells, and developed a software package called ECdetect to conduct unbiased, integrated ecDNA detection and analysis. Here we show that ecDNA was found in nearly half of human cancers; its frequency varied by tumour type, but it was almost never found in normal cells. Driver oncogenes were amplified most commonly in ecDNA, thereby increasing transcript level. Mathematical modelling predicted that ecDNA amplification would increase oncogene copy number and intratumoural heterogeneity more effectively than chromosomal amplification. We validated these predictions by quantitative analyses of cancer samples. The results presented here suggest that ecDNA contributes to accelerated evolution in cancer.
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http://dx.doi.org/10.1038/nature21356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5334176PMC
March 2017

Establishment of human iPSC-based models for the study and targeting of glioma initiating cells.

Nat Commun 2016 Feb 22;7:10743. Epub 2016 Feb 22.

Gene Expression Laboratory Belmonte, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis.
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http://dx.doi.org/10.1038/ncomms10743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764898PMC
February 2016

Sox10 Regulates Stem/Progenitor and Mesenchymal Cell States in Mammary Epithelial Cells.

Cell Rep 2015 Sep 10;12(12):2035-48. Epub 2015 Sep 10.

Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA. Electronic address:

To discover mechanisms that mediate plasticity in mammary cells, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor Sox10. Here, we show that Sox10 is specifically expressed in mammary cells exhibiting the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence whereas its overexpression increases stem/progenitor activity. Intriguingly, we also show that Sox10 overexpression causes mammary cells to undergo a mesenchymal transition. Consistent with these findings, Sox10 is preferentially expressed in stem- and mesenchymal-like breast cancers. These results demonstrate a signaling mechanism through which stem and mesenchymal states are acquired in mammary cells and suggest therapeutic avenues in breast cancers for which targeted therapies are currently unavailable.
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http://dx.doi.org/10.1016/j.celrep.2015.08.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591253PMC
September 2015

The Trp53 delta proline (Trp53ΔP) mouse exhibits increased genome instability and susceptibility to radiation-induced, but not spontaneous, tumor development.

Mol Carcinog 2016 09 27;55(9):1387-96. Epub 2015 Aug 27.

Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California.

The tumor suppressor TP53 can initiate a plethora of anti-proliferative effects to maintain genomic integrity under conditions of genotoxic stress. The N-terminal proline-rich domain (PRD) of TP53 is important in the regulation of TP53 activity and stability. A common polymorphism at codon 72 in this region has been associated with altered cancer risk in humans. The Trp53ΔP mouse, which carries a germline homozygous deletion of a region of the PRD, does not develop spontaneous tumors in a mixed 129/Sv and C57BL/6 genetic background, but is highly susceptible to a broad range of tumor types following total body exposure to 4 Gy gamma (γ) radiation. This contrasts with the tumor spectrum in Trp53 null (-/-) mice, which mainly develop thymic lymphomas and osteosarcomas. Analysis of genomic instability in tissues and cells from Trp53ΔP mice demonstrated elevated basal levels of aneuploidy, but this is not sufficient to drive spontaneous tumorigenesis, which requires an additional DNA damage stimulus. Levels of genomic instability did not increase significantly in Trp53ΔP mice following irradiation exposure, suggesting that other radiation effects including tissue inflammation, altered metabolism or autophagy, may play an important role. The Trp53ΔP mouse is a novel model to dissect the mechanisms of tumor development induced by radiation exposure. © 2015 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/mc.22377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891300PMC
September 2016

A Critical Examination of the "Bad Luck" Explanation of Cancer Risk.

Cancer Prev Res (Phila) 2015 Sep 29;8(9):762-4. Epub 2015 Jun 29.

Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado.

Tomasetti and Vogelstein (1) argue that lifetime cancer risk for particular tissues is mostly determined by the total number of stem cell (SC) divisions within the tissue, whereby most cancers arise due to "bad luck"—mutations occurring during DNA replication. We argue that the poorly substantiated estimations of SC division parameters and assumptions that oversimplify somatic evolution prevent such a conclusion from being drawn.
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http://dx.doi.org/10.1158/1940-6207.CAPR-15-0229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849411PMC
September 2015

A versatile platform to analyze low-affinity and transient protein-protein interactions in living cells in real time.

Cell Rep 2014 Dec 20;9(5):1946-1958. Epub 2014 Nov 20.

Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.
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http://dx.doi.org/10.1016/j.celrep.2014.10.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4269221PMC
December 2014

Vitamin D receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy.

Cell 2014 Sep;159(1):80-93

Gene Expression Laboratory, Salk Institute, La Jolla, CA 92037, USA; Howard Hughes Medical Institute, Salk Institute, La Jolla, CA 92037, USA. Electronic address:

The poor clinical outcome in pancreatic ductal adenocarcinoma (PDA) is attributed to intrinsic chemoresistance and a growth-permissive tumor microenvironment. Conversion of quiescent to activated pancreatic stellate cells (PSCs) drives the severe stromal reaction that characterizes PDA. Here, we reveal that the vitamin D receptor (VDR) is expressed in stroma from human pancreatic tumors and that treatment with the VDR ligand calcipotriol markedly reduced markers of inflammation and fibrosis in pancreatitis and human tumor stroma. We show that VDR acts as a master transcriptional regulator of PSCs to reprise the quiescent state, resulting in induced stromal remodeling, increased intratumoral gemcitabine, reduced tumor volume, and a 57% increase in survival compared to chemotherapy alone. This work describes a molecular strategy through which transcriptional reprogramming of tumor stroma enables chemotherapeutic response and suggests vitamin D priming as an adjunct in PDA therapy. PAPERFLICK:
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http://dx.doi.org/10.1016/j.cell.2014.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177038PMC
September 2014

CRIPTO/GRP78 signaling maintains fetal and adult mammary stem cells ex vivo.

Stem Cell Reports 2014 Apr 3;2(4):427-39. Epub 2014 Apr 3.

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers.
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http://dx.doi.org/10.1016/j.stemcr.2014.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986630PMC
April 2014

Transcriptomic classification of genetically engineered mouse models of breast cancer identifies human subtype counterparts.

Genome Biol 2013 Nov 12;14(11):R125. Epub 2013 Nov 12.

Background: Human breast cancer is a heterogeneous disease consisting of multiple molecular subtypes. Genetically engineered mouse models are a useful resource for studying mammary cancers in vivo under genetically controlled and immune competent conditions. Identifying murine models with conserved human tumor features will facilitate etiology determinations, highlight the effects of mutations on pathway activation, and should improve preclinical drug testing.

Results: Transcriptomic profiles of 27 murine models of mammary carcinoma and normal mammary tissue were determined using gene expression microarrays. Hierarchical clustering analysis identified 17 distinct murine subtypes. Cross-species analyses using three independent human breast cancer datasets identified eight murine classes that resemble specific human breast cancer subtypes. Multiple models were associated with human basal-like tumors including TgC3(1)-Tag, TgWAP-Myc and Trp53-/-. Interestingly, the TgWAPCre-Etv6 model mimicked the HER2-enriched subtype, a group of human tumors without a murine counterpart in previous comparative studies. Gene signature analysis identified hundreds of commonly expressed pathway signatures between linked mouse and human subtypes, highlighting potentially common genetic drivers of tumorigenesis.

Conclusions: This study of murine models of breast carcinoma encompasses the largest comprehensive genomic dataset to date to identify human-to-mouse disease subtype counterparts. Our approach illustrates the value of comparisons between species to identify murine models that faithfully mimic the human condition and indicates that multiple genetically engineered mouse models are needed to represent the diversity of human breast cancers. The reported trans-species associations should guide model selection during preclinical study design to ensure appropriate representatives of human disease subtypes are used.
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http://dx.doi.org/10.1186/gb-2013-14-11-r125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053990PMC
November 2013

AMP-activated protein kinase induces p53 by phosphorylating MDMX and inhibiting its activity.

Mol Cell Biol 2014 Jan 4;34(2):148-57. Epub 2013 Nov 4.

Department of Biochemistry & Molecular Biology, Tulane University School of Medicine and Tulane Cancer Center, New Orleans, Louisiana, USA.

AMP-activated protein kinase (AMPK) has been shown to activate p53 in response to metabolic stress. However, the underlying mechanisms remain unclear. Here we show that metabolic stresses induce AMPK-mediated phosphorylation of human MDMX on Ser342 in vitro and in cells, leading to enhanced association between MDMX and 14-3-3. This markedly inhibits p53 ubiquitylation and significantly stabilizes and activates p53. By striking contrast, no phosphorylation of MDM2 by AMPK was noted. AMPK-mediated MDMX phosphorylation, MDMX-14-3-3 binding, and p53 activation were drastically reduced in mouse embryo fibroblasts harboring endogenous MDMX with S341A (mouse homologue of human serine 342), S367A, and S402A (mouse homologue of human serine 403) mutations. Moreover, deficiency of AMPK prevented MDMX-14-3-3 interaction and p53 activation. The activation of p53 through AMPK-mediated MDMX phosphorylation and inactivation was further confirmed by using cell and animal model systems with two AMPK activators, metformin and salicylate (the active form of aspirin). Together, the results unveil a mechanism by which metabolic stresses activate AMPK, which, in turn, phosphorylates and inactivates MDMX, resulting in p53 stabilization and activation.
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http://dx.doi.org/10.1128/MCB.00670-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911293PMC
January 2014

Receptor tyrosine kinase-like orphan receptor 2 (Ror2) expression creates a poised state of Wnt signaling in renal cancer.

J Biol Chem 2013 Sep 26;288(36):26301-26310. Epub 2013 Jul 26.

Lineberger Comprehensive Cancer Center,; Departments of Medicine and Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and. Electronic address:

Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.
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http://dx.doi.org/10.1074/jbc.M113.466086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764835PMC
September 2013

Stem cells and the developing mammary gland.

J Mammary Gland Biol Neoplasia 2013 Jun 27;18(2):209-19. Epub 2013 Apr 27.

Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada.

The mammary gland undergoes dynamic changes throughout life. In the mouse, these begin with initial morphogenesis of the gland in the mid-gestation embryo followed by hormonally regulated changes during puberty and later in adulthood. The adult mammary gland contains a hierarchy of cell types with varying potentials for self-maintenance and differentiation. These include cells able to produce complete, functional mammary glands in vivo and that contain daughter cells with the same remarkable regenerative potential, as well as cells with more limited clonogenic activity in vitro. Here we review how applying in vitro and in vivo methods for quantifying these cells in adult mammary tissue to fetal mammary cells has enabled the first cells fulfilling the functional criteria of transplantable, isolated mammary stem cells to be identified a few days before birth. Thereafter, the number of these cells increases rapidly. Populations containing these fetal stem cells display growth and gene expression programs that differ from their adult counterparts but share signatures characteristic of certain types of breast cancer. Such observations reinforce growing evidence of important differences between tissue-specific fetal and adult cells with stem cell properties and emphasize the merits of investigating their molecular basis.
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http://dx.doi.org/10.1007/s10911-013-9284-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161372PMC
June 2013

Paracrine Wnt signaling both promotes and inhibits human breast tumor growth.

Proc Natl Acad Sci U S A 2013 Apr 4;110(17):6991-6. Epub 2013 Apr 4.

Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.
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http://dx.doi.org/10.1073/pnas.1303671110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3637696PMC
April 2013

Use of a molecular genetic platform technology to produce human Wnt proteins reveals distinct local and distal signaling abilities.

PLoS One 2013 13;8(3):e58395. Epub 2013 Mar 13.

The Salk Institute for Biological Studies, La Jolla, California, USA.

Functional and mechanistic studies of Wnt signaling have been severely hindered by the inaccessibility of bioactive proteins. To overcome this long-standing barrier, we engineered and characterized a panel of Chinese hamster ovary (CHO) cell lines with inducible transgenes encoding tagged and un-tagged human WNT1, WNT3A, WNT5A, WNT7A, WNT11, WNT16 or the soluble Wnt antagonist Fzd8CRD, all integrated into an identical genomic locus. Using a quantitative real-time bioluminescence assay, we show that cells expressing WNT1, 3A and 7A stimulate Wnt/beta-catenin reporter activity, while the other WNT expressing cell lines interfere with this activation. Additionally, in contrast to WNT3A, WNT1 only exhibits activity when cell-associated, and thus only signals to neighboring cells. The reporter assay also revealed a rapid decline of Wnt activity at 37°C, indicating that Wnt activity is highly labile. These engineered cell lines will reduce the cost of making and purifying Wnt proteins and serve as a continuous, reliable and regulatable source of Wnts to research laboratories around the world.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058395PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596392PMC
September 2013

MDM2, MDMX and p53 in oncogenesis and cancer therapy.

Nat Rev Cancer 2013 Feb 10;13(2):83-96. Epub 2013 Jan 10.

Center for Genomic Science of [email protected], Fondazione Istituto Italiano di Tecnologia, Via Adamello 16, 20139 Milan, Italy.

The MDM2 and MDMX (also known as HDMX and MDM4) proteins are deregulated in many human cancers and exert their oncogenic activity predominantly by inhibiting the p53 tumour suppressor. However, the MDM proteins modulate and respond to many other signalling networks in which they are embedded. Recent mechanistic studies and animal models have demonstrated how functional interactions in these networks are crucial for maintaining normal tissue homeostasis, and for determining responses to oncogenic and therapeutic challenges. This Review highlights the progress made and pitfalls encountered as the field continues to search for MDM-targeted antitumour agents.
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http://dx.doi.org/10.1038/nrc3430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161369PMC
February 2013

What a difference a phosphate makes: life or death decided by a single amino acid in MDM2.

Cancer Cell 2012 May;21(5):595-596

Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92130, USA. Electronic address:

In this issue of Cancer Cell, Gannon and colleagues create genetically engineered mice to test the role phosphorylation plays in the modification of one serine long thought to play a critical role in controlling the activity of MDM2, one of p53's main negative regulators.
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http://dx.doi.org/10.1016/j.ccr.2012.04.033DOI Listing
May 2012
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