Splicing factor ESRP1 controls ER-positive breast cancer by altering metabolic pathways.

The epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) control the epithelial-to-mesenchymal transition (EMT) splicing program in cancer. However, their role in breast cancer recurrence is unclear. In this study, we report that high levels of ESRP1, but not ESRP2, are associated with poor prognosis in estrogen receptor positive (ER+) breast tumors. Knockdown of ESRP1 in endocrine-resistant breast cancer models decreases growth significantly and alters the EMT splicing signature, which we confirm using TCGA SpliceSeq data of ER+ BRCA tumors. However, these changes are not accompanied by the development of a mesenchymal phenotype or a change in key EMT-transcription factors. In tamoxifen-resistant cells, knockdown of ESRP1 affects lipid metabolism and oxidoreductase processes, resulting in the decreased expression of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), and phosphoglycerate dehydrogenase (PHGDH) at both the mRNA and protein levels. Furthermore, ESRP1 knockdown increases the basal respiration and spare respiration capacity. This study reports a novel role for ESRP1 that could form the basis for the prevention of tamoxifen resistance in ER+ breast cancer.

Expression levels of SF3B3 correlate with prognosis and endocrine resistance in estrogen receptor-positive breast cancer.

De novo or acquired resistance to endocrine therapy limits its utility in a significant number of estrogen receptor-positive (ER-positive) breast cancers. It is crucial to identify novel targets for therapeutic intervention and improve the success of endocrine therapies. Splicing factor 3b, subunit 1 (SF3B1) mutations are described in luminal breast cancer albeit in low frequency. In this study, we evaluated the role of SF3B1 and SF3B3, critical parts of the SF3b splicing complex, in ER-positive endocrine resistance. To ascertain the role of SF3B1/SF3B3 in endocrine resistance, their expression levels were evaluated in ER-positive/endocrine-resistant cell lines (MCF-7/LCC2 and MCF-7/LCC9) using a real-time quantitative reverse transcription PCR (qRT-PCR). To further determine their clinical relevance, expression analysis was performed in a cohort of 60 paraffin-embedded ER-positive, node-negative breast carcinomas with low, intermediate, and high Oncotype DX recurrence scores. Expression levels of SF3B1 and SF3B3 and their prognostic value were validated in large cohorts using publicly available gene expression data sets including The Cancer Genome Atlas. SF3B1 and SF3B3 levels were significantly increased in ERα-positive cells with acquired tamoxifen (MCF-7/LCC2; both P<0.0002) and fulvestrant/tamoxifen resistance (MCF-7/LCC9; P=0.008 for SF3B1 and P=0.0006 for SF3B3). Expression levels of both MCF-7/LCC2 and MCF-7/LCC9 were not affected by additional treatments with E2 and/or tamoxifen. Furthermore, qRT-PCR analysis confirmed that SF3B3 expression is significantly upregulated in Oncotype DX high-risk groups when compared with low risk (P=0.019). Similarly, in publicly available breast cancer gene expression data sets, overexpression of SF3B3, but not SF3B1, was significantly correlated with overall survival. Furthermore, the correlation was significant in ER-positive, but not in ER-negative tumors.This is the first study to document the role of SF3B3 in endocrine resistance and prognosis in ER-positive breast cancer. Potential strategies for therapeutic targeting of the splicing mechanism(s) need to be evaluated.

Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23.

BACKGROUND: The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. In this study, we analyzed the role of Awd in controlling Notch signaling during development. RESULTS: To study the awd gene function we used genetic mosaic approaches to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. Our results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments we show that Notch receptor accumulates in early endosomes in awd mutant follicle cells. A trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation. CONCLUSIONS: In this report we demonstrate that awd is essential for Notch signaling via its endocytic role. In addition, we identify the endocytic step at which Awd function is required for Notch signaling and we obtain evidence indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.

Developmental function of Nm23/awd: a mediator of endocytosis.

The metastasis suppressor gene Nm23 is highly conserved from yeast to human, implicating a critical developmental function. Studies in cultured mammalian cells have identified several potential functions, but many have not been directly verified in vivo. Here, we summarize the studies on the Drosophila homolog of the Nm23 gene, named a bnormal w ing d iscs (awd), which shares 78% amino acid identity with the human Nm23-H1 and H2 isoforms. These studies confirmed that awd gene encodes a nucleoside diphosphate kinase, and provided strong evidence of a role for awd in regulating cell differentiation and motility via regulation of growth factor receptor signaling. The latter function is mainly mediated by control of endocytosis. This review provides a historical account of the discovery and subsequent analyses of the awd gene. We will also discuss the possible molecular function of the Awd protein that underlies the endocytic function.

Drosophila von Hippel-Lindau tumor suppressor gene function in epithelial tubule morphogenesis.

Mutations in the human von Hippel-Lindau (VHL) gene are the cause of VHL disease that displays multiple benign and malignant tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function against the alpha subunit of hypoxia-inducible factor (HIF-alpha). However, many HIF-independent functions of VHL have been identified. Recent evidence also indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes, although it is still unclear how many of these noncanonical functions relate to the pathophysiological processes because of a lack of tractable genetic systems. Here, we report the first genomic mutant phenotype of Drosophila melanogaster VHL (dVHL) in the epithelial tubule network, the trachea, and show that dVHL regulates branch migration and lumen formation via its endocytic function. The endocytic function regulates the surface level of the chemotactic signaling receptor Breathless and promotes clearing of the lumen matrix during maturation of the tracheal tubes. Importantly, the regulatory function in tubular morphogenesis is conserved in the mammalian system, as conditional knockout of Vhl in mouse kidney also resulted in similar cell motility and lumen phenotypes.

The Drosophila metastasis suppressor gene Nm23 homolog, awd, regulates epithelial integrity during oogenesis.

The expression levels of the metastasis suppressor gene Nm23 have been shown to correlate positively or inversely with prognosis in different cancer cohorts. This indicates that Nm23 may be needed at different expression levels and may function differently in various tissues. Here we report a novel epithelial function of the Drosophila melanogaster homolog of human Nm23, abnormal wing discs (awd). We show a dynamic expression pattern of the Awd protein during morphogenesis of the Drosophila follicle cells during oogenesis. Loss-of-function awd mutant cells result in the accumulation and spreading of adherens junction components, such as Drosophila E-cadherin, beta-catenin/Armadillo, and alpha-spectrin, and the disruption of epithelial integrity, including breaking up of the epithelial sheet and piling up of follicle cells. In contrast, overexpression of awd diminishes adherens junction components and induces a mesenchymal-cell-like cell shape change. The gain-of-function phenotype is consistent with a potential oncogenic function of this metastasis suppressor gene. Interestingly, we demonstrate that the epithelial function of awd is mediated by Rab5 and show that the Rab5 expression level is downregulated in awd mutant cells. Therefore, awd modulates the level and localization of adherens junction components via endocytosis. This is the first demonstration of an in vivo function of Nm23 family genes in regulating epithelial morphogenesis.

Awd, the homolog of metastasis suppressor gene Nm23, regulates Drosophila epithelial cell invasion.

Border cell migration during Drosophila melanogaster oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, or Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. Here we show that the metastasis suppressor gene homolog Nm23/awd is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominant-negative dynamin. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion.