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1.
Nature ; 528(7581): 267-71, 2015 Dec 10.
Article in English | MEDLINE | ID: mdl-26633636

ABSTRACT

Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.


Subject(s)
Breast Neoplasms/physiopathology , Carcinoma, Ductal, Breast/physiopathology , Cell Transformation, Neoplastic , Mammary Glands, Human/physiopathology , Animals , Breast Neoplasms/genetics , Carcinoma, Ductal, Breast/genetics , Cell Lineage/genetics , Cells, Cultured , DNA Barcoding, Taxonomic , Female , Gene Expression Profiling , Heterografts , Humans , Lentivirus/genetics , Mammary Glands, Human/cytology , Mice , Mice, Inbred Strains , Mice, SCID , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Time Factors , Transduction, Genetic , ras Proteins/genetics
2.
Nat Commun ; 5: 5871, 2014 Dec 23.
Article in English | MEDLINE | ID: mdl-25532760

ABSTRACT

Genomic and phenotypic analyses indicate extensive intra- as well as intertumoral heterogeneity in primary human malignant cell populations despite their clonal origin. Cellular DNA barcoding offers a powerful and unbiased alternative to track the number and size of multiple subclones within a single human tumour xenograft and their response to continued in vivo passaging. Using this approach we find clone-initiating cell frequencies that vary from ~1/10 to ~1/10,000 cells transplanted for two human breast cancer cell lines and breast cancer xenografts derived from three different patients. For the cell lines, these frequencies are negatively affected in transplants of more than 20,000 cells. Serial transplants reveal five clonal growth patterns (unchanging, expanding, diminishing, fluctuating or of delayed onset), whose predominance is highly variable both between and within original samples. This study thus demonstrates the high growth potential and diverse growth properties of xenografted human breast cancer cells.


Subject(s)
Breast Neoplasms/genetics , Cell Proliferation , Animals , Breast Neoplasms/physiopathology , Cell Line, Tumor , Clone Cells , DNA Barcoding, Taxonomic , Female , Humans , Kinetics , Mice , Neoplasm Transplantation , Tumor Cells, Cultured
3.
PLoS One ; 8(6): e64164, 2013.
Article in English | MEDLINE | ID: mdl-23750206

ABSTRACT

Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer's disease (LOAD) risk and plasma amyloid ß (Aß) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aß, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5'UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02-0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Aspartic Acid Endopeptidases/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Polymorphism, Single Nucleotide , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Aspartic Acid Endopeptidases/genetics , Cell Line, Tumor , Gene Expression Regulation , Gene Knockdown Techniques , Genetic Loci/genetics , Genetic Predisposition to Disease/genetics , Humans , Intracellular Space/metabolism , Membrane Proteins/deficiency , Mice , Nerve Tissue Proteins/deficiency , Protein Binding
4.
Nat Cell Biol ; 13(12): 1395-405, 2011 Nov 20.
Article in English | MEDLINE | ID: mdl-22101514

ABSTRACT

How the proto-oncogene c-Myc balances the processes of stem-cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc's transcriptional roles at the epidermal differentiation complex, a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovol-1) and displace (Cebpa, Mxi1 and Sin3a) specific sets of differentiation-specific transcriptional regulators to epidermal differentiation complex genes. We found that Sin3a causes deacetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3a, genomic recruitment of c-Myc to the epidermal differentiation complex is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3a reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis through a negative feedback loop.


Subject(s)
Epidermis/physiology , Feedback, Physiological/physiology , Homeostasis/genetics , Keratinocytes/physiology , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Repressor Proteins/physiology , Transcription, Genetic/physiology , Animals , Epidermal Cells , Female , Keratinocytes/cytology , Kruppel-Like Factor 4 , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Primary Cell Culture , Proto-Oncogene Proteins c-myc/genetics , Repressor Proteins/genetics , Sin3 Histone Deacetylase and Corepressor Complex
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