Lysine-specific demethylase 1 (LSD1) is an important histone demethylase that mediates epithelial to mesenchymal transition (EMT). The E239K mutation of LSD1 was identified in a luminal breast cancer patient from the COSMIC Breast Cancer dataset. To investigate the functional effects of the E239K mutation of LSD1, a stable LSD1 knockdown MCF7 cell line was generated. Rescue with WT LSD1, but not E239K mutated LSD1, suppressed the invasion and migration of the LSD1 knockdown cells, indicating that the E239K mutation abolished the suppressive effects of LSD1 on the invasion and migration of MCF7 cells. Further analysis showed that the E239K mutation abolished LSD1-mediated invasion and migration of MCF7 cells through downregulation of estrogen receptor α (ERα). Most importantly, the E239K mutation disrupted the interaction between LSD1 and GATA3, which reduced the enrichment of LSD1 at the promoter region of the ERα gene; the reduced enrichment of LSD1 at the promoter region of the ERα gene caused enhanced histone H3K9 methylation, which subsequently suppressed the transcription of the ERα gene. In summary, the E239K mutation abolishes the suppressive function of LSD1 on migration and invasion of breast cancer cells by disrupting the interaction between LSD1 and GATA3.
Ovarian and uterine cancers are the most prevalent types of gynecological malignancies originating from mesothelial and/or Müllerian-derived epithelial cells. Recent genomic studies have identified common mutations in them that affect signaling pathways such as p53, PTEN/PI3K, RAS, and WNT pathways. However, how these mutations and their corresponding deregulated pathways affect gynecological cancer development from their cells-of-origin remains largely elusive. To address this, we performed the intrabursal injection of Cre-expressing adenovirus under the control of Krt8 promoter (Ad-K8-Cre) to mice carrying combinations of various conditional alleles for cancer genes. We found that Ad-K8-Cre specifically targeted mesothelial cells, including ovarian surface epithelial (OSE) cells (mainly the LGR5+ subset of OSE cells) and mesothelial cells lining the fallopian tube (FT) serosa; the injected Ad-K8-Cre also targeted Müllerian-derived epithelial cells, including FT epithelial cells and uterine endometrial epithelial cells. The loss of p53 may preferentially affect Müllerian-derived epithelial cells, leading to the development of uterine and ovarian malignancies, whereas PTEN-loss may preferentially affect mesothelial cells, leading to the development of ovarian endometrioid malignancies (upon KRAS-activation or APC-loss) or adenoma on the FT surface (upon DICER-loss). Overall, our data suggest that different Krt8+ mesothelial and epithelial cell types in the female reproductive system may have different sensitivities toward oncogenic mutations and, as a result, oncogenic events may dominantly determine the locations and types of the gynecological malignancies developed from them.
LSD1 (KDM1A), a histone demethylase, plays important roles in breast cancer. The breast cancer patients with LSD1 mutation show significantly worse outcomes compared to those without LSD1 mutation. The R251Q mutation of LSD1 increases the invasion and migration of luminal breast cancer cells. Furthermore, the R251Q mutation of LSD1 alters the expression of genes that modulates the epithelial to mesenchymal transition. Additionally, the R251Q mutation impairs the H3K4me2 demethylation activity of LSD1 by abolishing the interaction between LSD1 and CoREST, which leads to the increased expression of TRIM37, a histone H2A ubiquitin ligase that regulates the expression of E-cadherin. Collectively, our results suggest that the R251Q mutation abolishes the tumor suppressive effects of LSD1 on luminal breast cancer cells by disrupting the formation of functional LSD1/CoREST/HDAC complexes.
BRCA1 promotes error-free, homologous recombination-mediated repair (HRR) of DNA double-stranded breaks (DSBs). When excessive and uncontrolled, BRCA1 HRR activity promotes illegitimate recombination and genome disorder. We and others have observed that the BRCA1-associated protein RAP80 recruits BRCA1 to postdamage nuclear foci, and these chromatin structures then restrict the amplitude of BRCA1-driven HRR. What remains unclear is how this process is regulated. Here we report that both BRCA1 poly-ADP ribosylation (PARsylation) and the presence of BRCA1-bound RAP80 are critical for the normal interaction of BRCA1 with some of its partners (e.g., CtIP and BACH1) that are also known components of the aforementioned focal structures. Surprisingly, the simultaneous loss of RAP80 and failure therein of BRCA1 PARsylation results in the dysregulated accumulation in these foci of BRCA1 complexes. This in turn is associated with the intracellular development of a state of hyper-recombination and gross chromosomal disorder. Thus, physiological RAP80-BRCA1 complex formation and BRCA1 PARsylation contribute to the kinetics by which BRCA1 HRR-sustaining complexes normally concentrate in nuclear foci. These events likely contribute to aneuploidy suppression.
Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.
LSD1 (KDM1A) is a histone demethylase that plays both oncogenic and tumor suppressor roles in breast cancer. However, the exact contexts under which it plays these opposite functions remain largely elusive. By characterizing its role in luminal breast epithelial cells, here we show that inhibition of LSD1 by both genetic and pharmacological approaches increases their invasion and migration, whereas its inhibition by genetic approach, but not by pharmacological approach, impairs their proliferation/survival. Induced loss of LSD1 in luminal cells in a mouse model of luminal breast cancer, MMTV-PyMT, leads to a profound increase in lung metastasis. Mechanistically, LSD1 interacts with GATA3, a key luminal-specific transcription factor (TF), and their common target genes are highly related to breast cancer. LSD1 positively regulates GATA3 expression. It also represses expression of TRIM37, a breast epithelial oncogene encoding a histone H2A ubiquitin ligase, and ELF5, a key TF gene for luminal progenitors and alveolar luminal cells. LSD1-loss also leads to reduced expression of several cell-cell adhesion genes (e.g., CDH1, VCL, CTNNA1), possibly via TRIM37-upregulation and subsequently TRIM37-mediated repression. Collectively, our data suggest LSD1 largely plays a tumor suppressor role in luminal breast cancer and the oncogenic program associated with LSD1-inhibition may be suppressed via TRIM37-inhibition.
Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.
Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.
Thomas U Ahearn, Sam Peisch, Andreas Pettersson, Ericka M Ebot, Cindy Ke Zhou, Rebecca E Graff, Jennifer A Sinnott, Ladan Fazli, Gregory L Judson, Tarek A Bismar, Jennifer R Rider, Travis Gerke, June M Chan, Michelangelo Fiorentino, Richard Flavin, Howard D Sesso, Stephen Finn, Edward L Giovannucci, Martin Gleave, Massimo Loda, Zhe Li, Michael Pollak, and Lorelei A Mucci. 2018. “Expression of IGF/insulin receptor in prostate cancer tissue and progression to lethal disease.” Carcinogenesis, 39, 12, Pp. 1431-1437.Abstract
Circulating insulin-like growth factor-1 (IGF-1) is consistently associated with prostate cancer risk. IGF-1 binds to IGF-1 receptor (IGF1R) and insulin receptor (IR), activating cancer hallmark pathways. Experimental evidence suggests that TMPRSS2:ERG may interact with IGF/insulin signaling to influence progression. We investigated IGF1R and IR expression and its association with lethal prostate cancer among 769 men. Protein expression of IGF1R, IR and ERG (i.e. a surrogate of ERG fusion genes) were assayed by immunohistochemistry. Cox models estimated hazard ratios (HR) and 95% confidence intervals (CI) adjusted for clinical characteristics. Among patients, 29% had strong tumor IGF1R expression and 10% had strong IR expression. During a mean follow-up of 13.2 years through 2012, 80 men (11%) developed lethal disease. Tumors with strong IGF1R or IR expression showed increased cell proliferation, decreased apoptosis and a higher prevalence of ERG. In multivariable models, strong IGF1R was associated with a borderline increased risk of lethal prostate cancer (HR 1.7; 95% CI 0.9-3.1). The association appeared greater in ERG-positive tumors (HR 2.8; 95% CI 0.9-8.4) than in ERG-negative tumors (HR 1.3; 95% CI 0.6-3.0, p-heterogeneity 0.08). There was no association between IR and lethal prostate cancer (HR 0.8; 95% CI 0.4-1.9). These results suggest that tumor IGF1R expression may play a role in prostate cancer progression to a lethal phenotype and that ERG-positive tumors may be more sensitive to IGF signaling. These data may improve our understanding of IGF signaling in prostate cancer and suggest therapeutic options for disease subtypes.
A recent paradigm shift in ovarian cancer research is the finding that many ovarian cancers may originate from fallopian tube epithelial (FTE) cells. As tissue stem and progenitor cells often serve as cells of origin of cancer, a better understanding of FTE stem/progenitor cells and how they become transformed is essential for early detection and prevention of ovarian cancer. To facilitate study of FTE stem/progenitor cells in model systems, we established an organoid culture system for mouse FTE cells. We find that EPCAM+ mouse FTE cells can be stably cultured long-term under a minimal condition of activated EGF signaling and suppressed TGFbeta signaling. We show that both Notch and Wnt signaling are required for growth of FTE cells in organoids, and further activation of Wnt signaling supports their maturation toward the ciliated cell lineage. Lastly, by analyzing the frequency of organoid-forming cells in different portions of the fallopian tube (FT), we find that the distal portion of the FT, which includes the fimbria, is enriched with organoid-forming FTE stem cells.
TMPRSS2/ERG is the most common type of gene fusions found in human prostate cancer. There are two important features of TMPRSS2/ERG fusions. One is that these gene fusions lead to ectopic expression of ERG, an ETS family transcription factor, in prostate epithelial cells from the 5' control region of an androgen/estrogen dual-responsive gene, TMPRSS2; the other is that ~60% of these fusions are generated via intrachromosomal deletion of the interstitial region between TMPRSS2 and ERG. To recapitulate these important aspects of TMPRSS2/ERG fusions, we generated several TMPRSS2/ERG knockin mouse models based on the endogenous Tmprss2 locus. We found that TMPRSS2/ERG represents an early event in prostate tumorigenesis, by sensitizing prostate cells for cooperation with other oncogenic events, such as PTEN-deficiency. We also found that the interstitial region between TMPRSS2 and ERG harbors at least one prostate tumor suppressor, ETS2, whose loss contributes to prostate cancer progression. In this protocol, we describe how these knockin mouse models can be utilized to study roles of TMPRSS2/ERG fusions in prostate cancer development both in vivo and in vitro.
The exact identity of castrate-resistant (CR) cells and their relation to CR prostate cancer (CRPC) is unresolved. We use single-cell gene profiling to analyze the molecular heterogeneity in basal and luminal compartments. Within the luminal compartment, we identify a subset of cells intrinsically resistant to castration with a bi-lineage gene expression pattern. We discover LY6D as a marker of CR prostate progenitors with multipotent differentiation and enriched organoid-forming capacity. Lineage tracing further reveals that LY6D+ CR luminal cells can produce LY6D- luminal cells. In contrast, in luminal cells lacking PTEN, LY6D+ cells predominantly give rise to LY6D+ tumor cells, contributing to high-grade PIN lesions. Gene expression analyses in patients' biopsies indicate that LY6D expression correlates with early disease progression, including progression to CRPC. Our studies thus identify a subpopulation of luminal progenitors characterized by LY6D expression and intrinsic castration resistance. LY6D may serve as a prognostic maker for advanced prostate cancer.
Most breast cancers may have a luminal origin. TP53 is one of the most frequently mutated genes in breast cancers. However, how p53 deficiency contributes to breast tumorigenesis from luminal cells remains elusive. Here we report that induced p53 loss in Krt8+ mammary luminal cells leads to their clonal expansion without directly affecting their luminal identity. All induced mice develop mammary tumours with 9qA1 (Yap1) and/or 6qA2 (Met) amplification(s). These tumours exhibit a mammary stem cell (MaSC)-like expression signature and most closely resemble claudin-low breast cancer. Thus, although p53 does not directly control the luminal fate, its loss facilitates acquisition of MaSC-like properties by luminal cells and predisposes them to development of mammary tumours with loss of luminal identity. Our data also suggest that claudin-low breast cancer can develop from luminal cells, possibly via a basal-like intermediate state, although further study using a different luminal promoter is needed to fully support this conclusion.
ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Ergkd ) in which Erg expression can be conditionally restored by Cre recombinase. Ergkd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Ergkd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin- Sca-1+ c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Ergkd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785.
Prostate cancer (PCa) is one of the most common cancers and among the leading causes of cancer deaths for men in industrialized countries. It has long been recognized that the prostate is an androgen-dependent organ and PCa is an androgen-dependent disease. Androgen action is mediated by the androgen receptor (AR). Androgen deprivation therapy (ADT) is the standard treatment for metastatic PCa. However, almost all advanced PCa cases progress to castration-resistant prostate cancer (CRPC) after a period of ADT. A variety of mechanisms of progression from androgen-dependent PCa to CRPC under ADT have been postulated, but it remains largely unclear as to when and how castration resistance arises within prostate tumors. In addition, AR signaling may be modulated by extracellular factors among which are the cysteine-rich glycoproteins WNTs. The WNTs are capable of signaling through several pathways, the best-characterized being the canonical WNT/β-catenin/TCF-mediated canonical pathway. Recent studies from sequencing PCa genomes revealed that CRPC cells frequently harbor mutations in major components of the WNT/β-catenin pathway. Moreover, the finding of an interaction between β-catenin and AR suggests a possible mechanism of cross talk between WNT and androgen/AR signaling pathways. In this review, we discuss the current knowledge of both AR and WNT pathways in prostate development and tumorigenesis, and their interaction during development of CRPC. We also review the possible therapeutic application of drugs that target both AR and WNT/β-catenin pathways. Finally, we extend our review of AR and WNT signaling to the mammary gland system and breast cancer. We highlight that the role of AR signaling and its interaction with WNT signaling in these two hormone-related cancer types are highly context-dependent.
Recent high-throughput studies revealed recurrent RUNX1 mutations in breast cancer, specifically in oestrogen receptor-positive (ER(+)) tumours. However, mechanisms underlying the implied RUNX1-mediated tumour suppression remain elusive. Here, by depleting mammary epithelial cells of RUNX1 in vivo and in vitro, we demonstrate combinatorial regulation of AXIN1 by RUNX1 and oestrogen. RUNX1 and ER occupy adjacent elements in AXIN1's second intron, and RUNX1 antagonizes oestrogen-mediated AXIN1 suppression. Accordingly, RNA-seq and immunohistochemical analyses demonstrate an ER-dependent correlation between RUNX1 and AXIN1 in tumour biopsies. RUNX1 loss in ER(+) mammary epithelial cells increases β-catenin, deregulates mitosis and stimulates cell proliferation and expression of stem cell markers. However, it does not stimulate LEF/TCF, c-Myc or CCND1, and it does not accelerate G1/S cell cycle phase transition. Finally, RUNX1 loss-mediated deregulation of β-catenin and mitosis is ameliorated by AXIN1 stabilization in vitro, highlighting AXIN1 as a potential target for the management of ER(+) breast cancer.
Gene fusions involving ETS family transcription factors (mainly TMPRSS2-ERG and TMPRSS2-ETV1 fusions) have been found in ~50% of human prostate cancer cases. Although expression of TMPRSS2-ERG or TMPRSS2-ETV1 fusion alone is insufficient to initiate prostate tumorigenesis, they appear to sensitize prostate epithelial cells for cooperation with additional oncogenic mutations to drive frank prostate adenocarcinoma. To search for such ETS-cooperating oncogenic events, we focused on a well-studied prostate tumor suppressor NKX3.1, as loss of NKX3.1 is another common genetic alteration in human prostate cancer. Previous studies have shown that deletions at 8p21 (harboring NKX3.1) and 21q22 (resulting in TMPRSS2-ERG fusion) were both present in a subtype of prostate cancer cases, and that ERG can lead to epigenetic silencing of NKX3.1 in prostate cancer cells, whereas NKX3.1 can in turn negatively regulate TMPRSS2-ERG fusion expression via suppression of the TMPRSS2 promoter activity. We recently generated knockin mouse models for TMPRSS2-ERG and TMPRSS2-ETV1 fusions, utilizing the endogenous Tmprss2 promoter. We crossed these knockin models to an Nkx3.1 knockout mouse model. In Tmprss2-ERG;Nkx3.1+/- (or -/-) male mice, although we observed a slight but significant upregulation of Tmprss2-ERG fusion expression upon Nkx3.1 loss, we did not detect any significant cooperation between these two genetic events to enhance prostate tumorigenesis in vivo. Furthermore, retrospective analysis of a previously published human prostate cancer dataset revealed that within ERG-overexpressing prostate cancer cases, NKX3.1 loss or deletion did not predict biochemical relapse after radical prostatectomy. Collectively, these data suggest that although TMPRSS2-ERG fusion and loss of NKX3.1 are among the most common mutational events found in prostate cancer, and although each of them can sensitize prostate epithelial cells for cooperating with other oncogenic events, these two events themselves do not appear to cooperate at a significant level in vivo to enhance prostate tumorigenesis.
It has been shown that the mammary luminal lineage could be maintained by luminal stem cells or long-lived progenitors, but their identity and role in breast cancer remain largely elusive. By lineage analysis using Wap-Cre mice, we found that, in nulliparous females, mammary epithelial cells (MECs) genetically marked by Wap-Cre represented a subpopulation of CD61+ luminal progenitors independent of ovarian hormones for their maintenance. Using a pulse-chase lineage-tracing approach based on Wap-Cre adenovirus (Ad-Wap-Cre), we found that Ad-Wap-Cre-marked nulliparous MECs were enriched with CD61+ alveolar progenitors (APs) that gave rise to CD61- alveolar luminal cells during pregnancy/lactation and could maintain themselves long term. When transformed by different oncogenes, they could serve as cells of origin of heterogeneous mammary tumors. Thus, our study revealed a type of long-lived AP within the luminal lineage that may serve as the cellular origin of multiple breast cancer subtypes.