The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer.

RNA-binding proteins (RBPs) are increasingly identified as post-transcriptional drivers of cancer progression. The RBP LARP1 is an mRNA stability regulator, and elevated expression of the protein in hepatocellular and lung cancers is correlated with adverse prognosis. LARP1 associates with an mRNA interactome that is enriched for oncogenic transcripts. Here we explore the role of LARP1 in epithelial ovarian cancer, a disease characterized by the rapid acquisition of resistance to chemotherapy through the induction of pro-survival signalling. We show, using ovarian cell lines and xenografts, that LARP1 is required for cancer cell survival and chemotherapy resistance. LARP1 promotes tumour formation in vivo and maintains cancer stem cell-like populations. Using transcriptomic analysis following LARP1 knockdown, cross-referenced against the LARP1 interactome, we identify BCL2 and BIK as LARP1 mRNA targets. We demonstrate that, through an interaction with the 3' untranslated regions (3' UTRs) of BCL2 and BIK, LARP1 stabilizes BCL2 but destabilizes BIK with the net effect of resisting apoptosis. Together, our data indicate that by differentially regulating the stability of a selection of mRNAs, LARP1 promotes ovarian cancer progression and chemotherapy resistance.

Activation of SGK1 in Endometrial Epithelial Cells in Response to PI3K/AKT Inhibition Impairs Embryo Implantation.

BACKGROUND: Serum & Glucocorticoid Regulated Kinase 1 (SGK1) plays a fundamental role in ion and solute transport processes in epithelia. In the endometrium, down-regulation of SGK1 during the window of receptivity facilitates embryo implantation whereas expression of a constitutively active mutant in the murine uterus blocks implantation. METHODS/RESULTS: Here, we report that treatment of endometrial epithelial cells with specific inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT activity pathway results in reciprocal activation of SGK1. Flushing of the uterine lumen of mice with a cell permeable, substrate competitive phosphatidylinositol analogue that inhibits AKT activation (AKT inhibitor III) resulted in Sgk1 phosphorylation, down-regulation of the E3 ubiquitin-protein ligase Nedd4-2, and increased expression of epithelial Na+ channels (ENaC). Furthermore, exposure of the uterine lumen to AKT inhibitor III prior to embryo transfer induced a spectrum of early pregnancy defects, ranging from implantation failure to aberrant spacing of implantation sites. CONCLUSION: Taken together, our data indicate that the balanced activities of two related serine/threonine kinases, AKT and SGK1, critically govern the implantation process.

The transcriptional co-factor RIP140 regulates mammary gland development by promoting the generation of key mitogenic signals.

Nuclear receptor interacting protein (Nrip1), also known as RIP140, is a co-regulator for nuclear receptors that plays an essential role in ovulation by regulating the expression of the epidermal growth factor-like family of growth factors. Although several studies indicate a role for RIP140 in breast cancer, its role in the development of the mammary gland is unclear. By using RIP140-null and RIP140 transgenic mice, we demonstrate that RIP140 is an essential factor for normal mammary gland development and that it functions by mediating oestrogen signalling. RIP140-null mice exhibit minimal ductal elongation with no side-branching, whereas RIP140-overexpressing mice show increased cell proliferation and ductal branching with age. Tissue recombination experiments demonstrate that RIP140 expression is required in both the mammary epithelial and stromal compartments for ductal elongation during puberty and that loss of RIP140 leads to a catastrophic loss of the mammary epithelium, whereas RIP140 overexpression augments the mammary basal cell population and shifts the progenitor/differentiated cell balance within the luminal cell compartment towards the progenitors. For the first time, we present a genome-wide global view of oestrogen receptor-α (ERα) binding events in the developing mammary gland, which unravels 881 ERα binding sites. Unbiased evaluation of several ERα binding sites for RIP140 co-occupancy reveals selectivity and demonstrates that RIP140 acts as a co-regulator with ERα to regulate directly the expression of amphiregulin (Areg), the progesterone receptor (Pgr) and signal transducer and activator of transcription 5a (Stat5a), factors that influence key mitogenic pathways that regulate normal mammary gland development.

The transcriptional corepressor RIP140 regulates oxidative metabolism in skeletal muscle.

Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.

667C>T and 1298A>C polymorphisms of MTHFR do not predict response to methotrexate in patients with gestational trophoblastic neoplasia.

OBJECTIVE: Approximately one third of patients treated with methotrexate for gestational trophoblastic neoplasia (GTN) following a molar pregnancy are reported to develop resistance to methotrexate and need to change to different chemotherapeutic agents. Previous studies, in other clinical settings, have suggested that polymorphisms in key folate metabolising enzymes such as 5,10-methylenetetrahydrofolate reductase (MTHFR) influence both toxicity and efficacy of methotrexate. Our objective was to investigate the impact of two common functional MTHFR polymorphisms, 677C>T and 1298A>C, on the efficacy of methotrexate in women treated for GTN following a molar pregnancy. METHODS: DNA from 121 women treated with methotrexate for GTN was genotyped for the 677C>T and 1298A>C polymorphisms using TaqMan SNP Genotyping Assays. In 64 cases these polymorphisms were also genotyped in the antecedent molar pregnancy, using DNA extracted from archival blocks of tissue. Response to methotrexate was evaluated with reference to serial human chorionic gonadotrophin (hCG) levels in patient serum. RESULTS: No significant association was found between the genotype of the patient, or presence of the variant allele, and clinical response to methotrexate therapy for either the 677C>T or the 1298A>C SNP. No significant association was found between the genotypes of the molar tissue and response to methotrexate. In molar tissue there was a significant reduction in the expected number with the 677TT genotype suggesting the 677C>T SNP may identify a subgroup of molar pregnancies less likely to progress to GTN. CONCLUSION: Neither the genotype for the 677C>T SNP or the 1298A>C SNP in MTHFR predict the therapeutic outcomes of women treated with single agent methotrexate for GTN.

Author Correction: Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SREBP1 drives Keratin-80-dependent cytoskeletal changes and invasive behavior in endocrine-resistant ERα breast cancer.

Approximately 30% of ERα breast cancer patients relapse with metastatic disease following adjuvant endocrine therapies. The connection between acquisition of drug resistance and invasive potential is poorly understood. In this study, we demonstrate that the type II keratin topological associating domain undergoes epigenetic reprogramming in aromatase inhibitors (AI)-resistant cells, leading to Keratin-80 (KRT80) upregulation. KRT80 expression is driven by de novo enhancer activation by sterol regulatory element-binding protein 1 (SREBP1). KRT80 upregulation directly promotes cytoskeletal rearrangements at the leading edge, increased focal adhesion and cellular stiffening, collectively promoting cancer cell invasion. Shearwave elasticity imaging performed on prospectively recruited patients confirms KRT80 levels correlate with stiffer tumors. Immunohistochemistry showed increased KRT80-positive cells at relapse and, using several clinical endpoints, KRT80 expression associates with poor survival. Collectively, our data uncover an unpredicted and potentially targetable direct link between epigenetic and cytoskeletal reprogramming promoting cell invasion in response to chronic AI treatment.

Author Correction: SREBP1 drives keratin-80-dependent cytoskeletal changes and invasive behavior in endocrine-resistant ERα breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1.

Aberrant transforming growth factor-ß (TGF-ß) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-ß signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-ß signaling activity and that stromal cell-conditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-ß-induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment.

LEFTY2 inhibits endometrial receptivity by downregulating Orai1 expression and store-operated Ca2+ entry.

Early embryo development and endometrial differentiation are initially independent processes, and synchronization, imposed by a limited window of implantation, is critical for reproductive success. A putative negative regulator of endometrial receptivity is LEFTY2, a member of the transforming growth factor (TGF)-ß family. LEFTY2 is highly expressed in decidualizing human endometrial stromal cells (HESCs) during the late luteal phase of the menstrual cycle, coinciding with the closure of the window of implantation. Here, we show that flushing of the uterine lumen in mice with recombinant LEFTY2 inhibits the expression of key receptivity genes, including Cox2, Bmp2, and Wnt4, and blocks embryo implantation. In Ishikawa cells, a human endometrial epithelial cell line, LEFTY2 downregulated the expression of calcium release-activated calcium channel protein 1, encoded by ORAI1, and inhibited store-operated Ca2+ entry (SOCE). Furthermore, LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, as well as YM-58483, inhibited, whereas the Ca2+ ionophore, ionomycin, strongly upregulated COX2, BMP2 and WNT4 expression in decidualizing HESCs. These findings suggest that LEFTY2 closes the implantation window, at least in part, by downregulating Orai1, which in turn limits SOCE and antagonizes expression of Ca2+-sensitive receptivity genes. KEY MESSAGES: •Endometrial receptivity is negatively regulated by LEFTY2. •LEFTY2 inhibits the expression of key murine receptivity genes, including Cox2, Bmp2 and Wnt4, and blocks embryo implantation. •LEFTY2 downregulates the expression of Orai1 and inhibits SOCE. •LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, and YM-58483 inhibit COX2, BMP2, and WNT4 expression in endometrial cells. •Targeting LEFTY2 and Orai1 may represent a novel approach for treating unexplained infertility.

Biomarker Assessment of HR Deficiency, Tumor BRCA1/2 Mutations, and CCNE1 Copy Number in Ovarian Cancer: Associations with Clinical Outcome Following Platinum Monotherapy.

The current study evaluated three biomarkers [homologous recombination deficiency (HRD), tumor BRCA1/2 (tBRCA) mutations, and CCNE1 copy-number variation (CNV)] in ovarian tumors from patients enrolled on the SCOTROC4 clinical trial for associations with outcome following carboplatin monotherapy. Ovarian tumors (n = 250), with high-grade serous (HGSOC) subgroup analysis (n = 179) were classified as HRD positive (HRD score ≥42 or tBRCA mutation) and as CCNE1 amplification positive (CCNE1 CNV score >2.4). Seventy-four (30%) tumors were HRD positive, including 34 (14%) with tBRCA mutations. Forty-seven (19%) were CCNE1 amplification positive, all of which were tBRCA wild-type. HRD and tBRCA, but not CCNE1 amplification, were significantly associated with CA125 complete response in the entire cohort (HRD, P = 0.00015; tBRCA P = 0.0096), and the HGSOC subgroup (HRD, P = 0.0016; tBRCA P = 0.032). HRD and lack of CCNE1 amplification were associated with improved progression-free survival (PFS) and overall survival (OS) in the full cohort and HGSOC subgroup (HRD, P = 0.00021; CCNE1 status P = 0.038). HRD remained significant for OS and PFS after adjusting for clinical factors, while CCNE1 status only remained significant for PFS. Patients with HRD-positive tumors had greater PFS and OS benefit from platinum dose intensification than HRD-negative tumors (P = 0.049 and P = 0.035, respectively). An alternative exploratory HRD score threshold (≥33 or tBRCA mutation) was also significantly associated with both PFS and OS in the HGSOC subset.Implications: HRD, tumor BRCA1/2 mutations, and absence of CCNE1 amplification are associated with improved survival of ovarian cancer patients treated with platinum monotherapy and HRD-positive patients may benefit from platinum dose intensification. Mol Cancer Res; 16(7); 1103-11. ©2018 AACR.

The nuclear cofactor receptor interacting protein-140 (RIP140) regulates the expression of genes involved in Aß generation.

The receptor interacting protein-140 (RIP140) is a cofactor for several nuclear receptors and has been involved in the regulation of metabolic and inflammatory genes. We hypothesize that RIP140 may also affect Aß generation because it modulates the activity of transcription factors previously implicated in amyloid precursor protein (APP) processing, such as peroxisome proliferator-activated receptor-γ (PPARγ). We found that the levels of RIP140 are reduced in Alzheimer's disease (AD) postmortem brains compared with healthy controls. In addition, in situ hybridization experiments revealed that RIP140 expression is enriched in the same brain areas involved in AD pathology, such as cortex and hippocampus. Furthermore, we provide evidence using cell lines and genetically modified mice that RIP140 is able to modulate the transcription of certain genes involved in AD pathology, such as ß-APP cleaving enzyme (BACE1) and GSK3. Consequently, we found that RIP140 overexpression reduced the generation of Aß in a neuroblastoma cell line by decreasing the transcription of ß-APP cleaving enzyme via a PPARγ-dependent mechanism. The results of this study therefore provide molecular insights into common signaling pathways linking metabolic disease with AD.

Expression of CDK7, Cyclin H, and MAT1 Is Elevated in Breast Cancer and Is Prognostic in Estrogen Receptor-Positive Breast Cancer.

PURPOSE: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. EXPERIMENTAL DESIGN: mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. RESULTS: We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. CONCLUSIONS: Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. Clin Cancer Res; 22(23); 5929-38. ©2016 AACR.

Multiple signaling defects in the absence of RIP140 impair both cumulus expansion and follicle rupture.

The nuclear receptor corepressor RIP140 is essential in the ovary for ovulation, but is not required for follicle growth and luteinization. To identify genes that may be subject to regulation by RIP140 or play a role in ovulation, we compared ovarian gene expression profiles in untreated immature wild-type and RIP140 null mice and after treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin. Many genes involved in signaling, extracellular matrix formation, cell-cell attachment, and adhesion were aberrantly regulated in the absence of RIP140, varying according to the hormone status of the mice. Notable among these was the reduced expression of a number of genes that encode components of signaling pathways and matrix proteins required for cumulus expansion, a key remodeling process necessary for ovulation. Histological analysis confirmed that cumulus expansion in RIP140 null mice is reduced, oocyte detachment from the mural cell wall is impaired, and follicles fail to rupture in response to LH. Although the expression of many genes involved in cumulus cell expansion was reduced, there was a subset of genes involved in extracellular matrix formation and cell-cell interactions that was up-regulated and may interfere with ovarian tissue remodeling. We propose that widespread gene dysregulation in ovarian tissues in the absence of RIP140 leads to the anovulatory phenotype. This helps to define an important role for RIP140 in the regulation of multiple processes leading to ovulation.

Role of the RIP140 corepressor in ovulation and adipose biology.

RIP140 is a ligand-dependent corepressor for most, if not all, nuclear receptors. It is expressed widely in many different tissues, but the phenotype of mice devoid of RIP140 indicates that it plays a crucial role in the ovary and in adipose biology. Ovarian expression of RIP140 is cell-type-specific during follicular development and it is essential for oocyte release during ovulation, but not for luteinization of mature ovarian follicles. In adipose tissue, RIP140 is essential for normal fat accumulation and RIP140-null mice show decreased lipid storage even on a high-fat diet, with upregulation of mitochondrial uncoupling protein (UCP1) in some fat depots. Thus RIP140 plays a crucial role in female fertility and in energy homeostasis, and could be a target for infertility treatment, new contraceptive strategies or prevention of obesity.

The thyroid hormone receptor-associated protein TRAP220 is required at distinct embryonic stages in placental, cardiac, and hepatic development.

Recent work indicates that thyroid hormone receptor-associated protein 220 (TRAP220), a subunit of the multiprotein TRAP coactivator complex, is essential for embryonic survival. We have generated TRAP220 conditional null mice that are hypomorphic and express the gene at reduced levels. In contrast to TRAP220 null mice, which die at embryonic d 11.5 (E11.5), hypomorphic mice survive until E13.5. The reduced expression in hypomorphs results in hepatic necrosis, defects in hematopoiesis, and hypoplasia of the ventricular myocardium, similar to that observed in TRAP220 null embryos at an earlier stage. The embryonic lethality of null embryos at E11.5 is due to placental insufficiency. Tetraploid aggregation assays partially rescues embryonic development until E13.5, when embryonic loss occurs due to hepatic necrosis coupled with poor myocardial development as observed in hypomorphs. These findings demonstrate that, for normal placental function, there is an absolute requirement for TRAP220 in extraembryonic tissues at E11.5, with an additional requirement in embryonic tissues for hepatic and cardiovascular development thereafter.

DMXL2 drives epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation.

The acquisition of endocrine therapy resistance in estrogen receptor α (ERα) breast cancer patients represents a major clinical problem. Notch signalling has been extensively linked to breast cancer especially in patients who fail to respond to endocrine therapy. Following activation, Notch intracellular domain is released and enters the nucleus where activates transcription of target genes. The numerous steps that cascade after activation of the receptor complicate using Notch as biomarker. Hence, this warrants the development of reliable indicators of Notch activity. DMXL2 is a novel regulator of Notch signalling not yet investigated in breast cancer. Here, we demonstrate that DMXL2 is overexpressed in a subset of endocrine therapy resistant breast cancer cell lines where it promotes epithelial to mesenchymal transition through hyper-activation of Notch signalling via V-ATPase dependent acidification. Following DMXL2 depletion or treatment with Bafilomycin A1, both EMT targets and Notch signalling pathway significantly decrease. We show for the first time that DMXL2 protein levels are significantly increased in ERα positive breast cancer patients that progress after endocrine therapy. Finally, we demonstrate that DMXL2 is a transmembrane protein with a potential extra-cellular domain. These findings identify DMXL2 as a novel, functional biomarker for ERα positive breast cancer.

Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion.

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients.

The pioneer factor PBX1 is a novel driver of metastatic progression in ERα-positive breast cancer.

Over 30% of ERα breast cancer patients develop relapses and progress to metastatic disease despite treatment with endocrine therapies. The pioneer factor PBX1 translates epigenetic cues and mediates estrogen induced ERα binding. Here we demonstrate that PBX1 plays a central role in regulating the ERα transcriptional response to epidermal growth factor (EGF) signaling. PBX1 regulates a subset of EGF-ERα genes highly expressed in aggressive breast tumours. Retrospective stratification of luminal patients using PBX1 protein levels in primary cancer further demonstrates that elevated PBX1 protein levels correlate with earlier metastatic progression. In agreement, PBX1 protein levels are significantly upregulated during metastatic progression in ERα-positive breast cancer patients. Finally we reveal that PBX1 upregulation in aggressive tumours is partly mediated by genomic amplification of the PBX1 locus. Correspondingly, ERα-positive breast cancer patients carrying PBX1 amplification are characterized by poor survival. Notably, we demonstrate that PBX1 amplification can be identified in tumor derived-circulating free DNA of ERα-positive metastatic patients. Metastatic patients with PBX1 amplification are also characterized by shorter relapse-free survival. Our data identifies PBX1 amplification as a functional hallmark of aggressive ERα-positive breast cancers. Mechanistically, PBX1 amplification impinges on several critical pathways associated with aggressive ERα-positive breast cancer.

Complex formation and function of estrogen receptor α in transcription requires RIP140.

RIP140 is a transcriptional coregulator involved in energy homeostasis, ovulation, and mammary gland development. Although conclusive evidence is lacking, reports have implicated a role for RIP140 in breast cancer. Here, we explored the mechanistic role of RIP140 in breast cancer and its involvement in estrogen receptor α (ERα) transcriptional regulation of gene expression. Using ChIP-seq analysis, we demonstrate that RIP140 shares more than 80% of its binding sites with ERα, colocalizing with its interaction partners FOXA1, GATA3, p300, CBP, and p160 family members at H3K4me1-demarcated enhancer regions. RIP140 is required for ERα-complex formation, ERα-mediated gene expression, and ERα-dependent breast cancer cell proliferation. Genes affected following RIP140 silencing could be used to stratify tamoxifen-treated breast cancer cohorts, based on clinical outcome. Importantly, this gene signature was only effective in endocrine-treated conditions. Cumulatively, our data suggest that RIP140 plays an important role in ERα-mediated transcriptional regulation in breast cancer and response to tamoxifen treatment.