Esther Baena

ETV1 directs androgen metabolism and confers aggressive prostate cancer in targeted mice and patients.

Distinguishing aggressive from indolent disease and developing effective therapy for advanced disease are the major challenges in prostate cancer research. Chromosomal rearrangements involving ETS transcription factors, such as ERG and ETV1, occur frequently in prostate cancer. How they contribute to tumorigenesis and whether they play similar or distinct in vivo roles remain elusive. Here we show that in mice with ERG or ETV1 targeted to the endogenous Tmprss2 locus, either factor cooperated with loss of a single copy of Pten, leading to localized cancer, but only ETV1 appeared to support development of invasive adenocarcinoma under the background of full Pten loss. Mechanistic studies demonstrated that ERG and ETV1 control a common transcriptional network but largely in an opposing fashion. In particular, while ERG negatively regulates the androgen receptor (AR) transcriptional program, ETV1 cooperates with AR signaling by favoring activation of the AR transcriptional program. Furthermore, we found that ETV1 expression, but not that of ERG, promotes autonomous testosterone production. Last, we confirmed the association of an ETV1 expression signature with aggressive disease and poorer outcome in patient data. The distinct biology of ETV1-associated prostate cancer suggests that this disease class may require new therapies directed to underlying programs controlled by ETV1.

c-Myc-deficient B lymphocytes are resistant to spontaneous and induced cell death

AbstractC-myc gene is a member of the myc family of proto-oncogenes involved in proliferation, differentiation, and apoptosis. Overexpression of c-myc in fibroblasts causes apoptosis under low serum conditions in a process that requires the interaction of CD95 and CD95L on the surface. We have previously reported an in vivo conditional model to inactivate the c-myc gene in B lymphocytes. Here, we show that c-Myc-deficient primary B lymphocytes are resistant to different apoptotic stimuli. Nonactivated c-Myc-deficient B cells are resistant to spontaneous cell death. Upon activation, c-Myc-deficient B lymphocytes express normal surface levels of activation markers, and show resistance to staurosporine and CD95-induced cell death.

B Lymphocyte Commitment Program Is Driven by the Proto-Oncogene c-myc

c-Myc, a member of the Myc family of transcription factors, is involved in numerous biological functions including the regulation of cell proliferation, differentiation, and apoptosis in various cell types. Of all of its functions, the role of c-Myc in cell differentiation is one of the least understood. We addressed the role of c-Myc in B lymphocyte differentiation. We found that c-Myc is essential from early stages of B lymphocyte differentiation in vivo and regulates this process by providing B cell identity via direct transcriptional regulation of the ebf-1 gene. Our data show that c-Myc influences early B lymphocyte differentiation by promoting activation of B cell identity genes, thus linking this transcription factor to the EBF-1/Pax-5 pathway.

Cell death during lymphocyte development and activation

The development and homeostasis of the immune system requires an exquisite balance between cell proliferation and cell death. In this review, we discuss several in vivo and in vitro models that have been developed to help understand the importance of apoptosis during B and T cell development and activation.

An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4–/– mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.

Identification and evaluation of clinically significant prostate cancer: a step towards personalized diagnosis

Purpose of review Prostate cancer (PCa) diagnostics are evolving rapidly. The quest to differentiate ‘clinically significant’ from ‘clinically insignificant’ disease has gathered momentum, leading to substantial change in traditional diagnostic approaches. Herein, we review the relevant information on currently available biomarkers and assess their ability to help physicians and patients in making a shared and personalized decision based on their individual risk of harbouring clinically significant disease. Recent findings Serum, urine, tissue and imaging biomarkers have been evaluated to improve the identification of clinically significant disease, and this international effort has yielded promising, but not always consistent results. Changes in MRI technology have realized a quantum change, and this facility is now becoming more widely incorporated into diagnostic and disease risk-stratification protocols. However, standardization and further validation is required. Summary Acceptance and widespread adoption of serum, urine and genetic markers is awaited, but novel and promising techniques alone and in combination have emerged. With validation and further focus, these may be adopted more widely.

WDR5 modulates cell motility and morphology and controls nuclear changes induced by a 3D environment

Significance Cells require nuclear deformation to squeeze through tissue matrices. We have discovered that WD repeat domain 5 (WDR5; an epigenetic modulator of H3K4 methylation) is fundamental for cell polarity and migration in vitro and in vivo, independent of transcription. We have uncovered that the interactions between cells and the surrounding 3D confined conditions induce the upregulation of H3K4me3. Moreover, 3D environments control the deformability and the mechanical properties of the nucleus. We have identified that loss of WDR5 abrogates the H3K4 methylation and the nuclear changes induced by 3D conditions. Mechanistically, we found that myosin light chain kinase and myosin function were required for WDR5-mediated H3K4 methylation in 3D matrices. Our findings uncover functions of the epigenetic machinery when cells move through constricted conditions. Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability, and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting nongenomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5 and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking, and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared with cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn up-regulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a nongenomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity, and cell migratory capacity.

Abstract LB-275: Single cell analysis identified LY6D as a novel marker of castration-resistant multipotent luminal progenitors

The cellular origin of castration-resistant prostate cancer (CRPC) remains largely unsolved. We compared single-cell expression profiles of prostate cells sorted from hormone-naive and castrated mice. Our single-cell analysis reveals that the prostate luminal lineage is highly heterogeneous. A subset of prostate cells in the luminal lineage characterized by high expression levels of luminal lineage markers including cytokeratin 8 (Krt8) and androgen receptor (Ar) genes, as well as prostate stem/progenitor marker genes (e.g., Trop2/Tacstd2, Sca1/Ly6a), and intermediate levels of basal genes (e.g. Trp63, Krt5), is enriched upon castration. We validated LY6D as a novel marker of castration-resistant (CR) multipotent luminal progenitor. Organoid culture revealed that LY6D+ prostate cells, including LY6D+ prostate luminal cells, were enriched with multipotent organoid-forming cells under androgen-deprivation. Lineage-tracing using Krt8-CreER mice revealed that in wild-type mice, LY6D+ CR luminal cells appeared to produce LY6D- luminal cells upon regeneration; however, when under the Pten-loss background, LY6D+ Pten-null luminal cells gave rise to LY6D+ luminal cancer cells. Furthermore, upon induced loss of Pten, prostate cancers originating from Krt8+ CR luminal cells were LY6D+ high-grade and were more advanced than those originating from hormone-naive luminal cells. Lastly, in prostate cancer patients, LY6D amplification/upregulation correlates with advanced disease. Overall, our results uncover the luminal lineage heterogeneity and identify LY6D as a novel tumor-initiating cell marker of CRPC. Citation Format: Douglas E. Linn, Joao Barros-Silva, Guoji Guo, Michael Brown, Garry Ashton, Noel W. Clarke, Guo-Cheng Yuan, Stuart H. Orkin, Esther Baena, Zhe Li. Single cell analysis identified LY6D as a novel marker of castration-resistant multipotent luminal progenitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-275.

Abstract B157: Understanding the role of transcription factor ETV1 in metabolic reprogramming of prostate cells as a route to novel therapeutics.

ETV1 is the second most frequently rearranged ETS transcription factor gene in human prostate cancer. It is widely believed that ectopically expressed ETV1 (due to gene fusions) plays a similar role in prostate tumorigenesis to that of ERG. However, further analysis of clinical data revealed that high levels of ETV1 expression are often found in more advanced prostate cancers, whereas ERG overexpression is mainly associated with localized prostate cancer and elevated androgen receptor signaling. High expression of ETV1, rather than ERG, in the prostate has been associated with disease recurrence in some patients. Interestingly, many metabolic pathways often associated with cancer cells are enriched in the prostates of mice engineered to carry a Tmprss2-ETV1 knockin allele and ETV1-expressing human prostate cancer samples. To determine the dominant metabolic programs that accompany ETV1 expression, we have performed metabolic profiling, and integrated the data with human prostate cells and mouse transcriptome data. Our analyses have identified ETV1 as a regulator of lipid metabolism, glycolysis and glutamine metabolism. Of note, ETV1 expression reprograms the steroid biosynthesis pathway, leading to an increase in testosterone production at least in part through 17-β hydroxysteorid dehydrogenases activation. Indeed, one of the main causes of prostate cancer recurrence is local androgen production due to metabolic changes in prostate cancer cells. This novel finding may explain, at least in part, how ETV1 expression promotes castration-resistant disease, opening a new path to the development of novel therapies directed to progressive and lethal prostate cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B157. Citation Format: Esther Baena, Shahan Mamoor, Zhe Li, Stuart H. Orkin. Understanding the role of transcription factor ETV1 in metabolic reprogramming of prostate cells as a route to novel therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B157.

Abstract 2965: ERG and ETV1 regulate a common transcriptional network in prostate cells but in an opposite fashion

Chromosomal rearrangements involving ERG or ETV1 have been reported in >50% prostate cancer cases. It is presently unknown how ERG and ETV1 function leads to prostate tumorigenesis, and whether the factors play similar roles in this process. To address this, we focused on identification of their direct chromosomal targets relevant for prostate cancer initiation and progression. We also generated Tmprss2-ERG and Tmprss2-ETV1 knockin mouse models to interrogate the in vivo roles of these two most prevalent gene fusions. To investigate the transcriptional program regulated by ERG and ETV1, we performed siRNA silencing experiments in prostate cancer cells and overexpression studies in normal prostate immortalized cells. With the aid of a biotin-tagging approach, we also identified chromatin sites occupied by ERG and ETV1 in prostate cells. Cross-referencing ETV1 and ERG gene promoter binding to global expression profiling data permitted characterization of ERG and ETV1 -regulated programs in prostate cells. These combined transcriptional studies revealed that a common transcriptional network regulated by ERG and ETV1 but in an opposite fashion. Indeed, ETV1 enhanced the androgen receptor (AR) pathway, favoring tumor growth, while ERG showed a negative effect over the same pathway. Consistent with this, in the mouse models, ERG and ETV1 also exhibited differential regulation of common pathways: exogenous expression of ERG from the endogenous Tmprss2 promoter was much lower than that of ETV1, possibly due to a negative regulation of AR target genes (including Tmprss2) by ERG; however, transgenic overexpression of a mutated version of AR was able to break this negative regulation and significantly upregulated Tmprss2-ERG expression in vivo. In contrast, Tmprss2-ETV1 expression alone was able to upregulate expression of AR targets in vivo. Moreover, although ectopic expression of either ERG or ETV1 from the endogenous Tmprss2 locus alone in mouse prostate was insufficient to initiate prostate tumorigenesis in vivo, Tmprss2-ETV1 cooperated with Pten loss to enhance the prostate cancer phenotype, whereas Tmprss2-ERG failed to significantly affect the prostate phenotype induced by Pten-loss. Finally, analysis of patient data supported a differential outcome based on the presence of ERG or ETV1 fusions. In summary, we conclude that ERG and ETV1 play different roles in prostate tumorigenesis, and accordingly prostate cancers with ERG or ETV1 fusions should be considered different molecular subtypes. These findings have important implications for the development of targeted therapy for ERG- and ETV1-associated prostate cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2965. doi:1538-7445.AM2012-2965