Lisa A. Hurley

Nonclassical estrogen receptor α signaling mediates negative feedback in the female mouse reproductive axis

Ovarian estrogen exerts both positive and negative feedback control over luteinizing hormone (LH) secretion during the ovulatory cycle. Estrogen receptor (ER) α but not ERβ knockout mice lack estrogen feedback. Thus, estrogen feedback appears to be primarily mediated by ERα. However, it is now recognized that, in addition to binding to estrogen response elements (EREs) in DNA to alter target gene transcription, ERα signals through ERE-independent or nonclassical pathways, and the relative contributions of these pathways in conveying estrogen feedback remain unknown. Previously we created a knockin mouse model expressing a mutant form of ERα (AA) with ablated ERE-dependent but intact ERE-independent activity. Breeding this allele onto the ERα-null (−/−) background, we examine the ability of ERE-independent ERα signaling pathways to convey estrogen feedback regulation of the female hypothalamic–pituitary axis in vivo. ERα−/AA exhibited 69.9% lower serum LH levels compared with ERα−/− mice. Additionally, like wild type, ERα−/AA mice exhibited elevated LH after ovariectomy (OVX). Furthermore, the post-OVX rise in serum LH was significantly suppressed by estrogen treatment in OVX ERα−/AA mice. However, unlike wild type, both ERα−/AA and ERα−/− mice failed to exhibit estrous cyclicity, spontaneous ovulation, or an afternoon LH surge response to estrogen. These results indicate that ERE-independent ERα signaling is sufficient to convey a major portion of estrogens negative feedback actions, whereas positive feedback and spontaneous ovulatory cyclicity require ERE-dependent ERα signaling.

Sox3 Is Required for Gonadal Function, but Not Sex Determination, in Males and Females

ABSTRACT Sox3 is expressed in developing gonads and in the brain. Evolutionary evidence suggests that the X-chromosomal Sox3 gene may be the ancestral precursor of Sry, a sex-determining gene, and Sox3 has been proposed to play a role in sex determination. However, patients with mutations in SOX3 exhibit normal gonadal determination but are mentally retarded and have short stature secondary to growth hormone (GH) deficiency. We used Cre-LoxP targeted mutagenesis to delete Sox3 from mice. Null mice of both sexes had no overt behavioral deficits and exhibited normal GH gene expression. Low body weight was observed for some mice; overgrowth and misalignment of the front teeth was observed consistently. Female Sox3 null mice (−/−) developed ovaries but had excess follicular atresia, ovulation of defective oocytes, and severely reduced fertility. Pituitary (luteinizing hormone and follicle-stimulating hormone) and uterine functions were normal in females. Hemizygous male null mice (−/Y) developed testes but were hypogonadal. Testis weight was reduced by 42%, and there was extensive Sertoli cell vacuolization, loss of germ cells, reduced sperm counts, and disruption of the seminiferous tubules. We conclude that Sox3 is not required for gonadal determination but is important for normal oocyte development and male testis differentiation and gametogenesis.

miR-182 integrates apoptosis, growth, and differentiation programs in glioblastoma

Glioblastoma multiforme (GBM) is a lethal, therapy-resistant brain cancer consisting of numerous tumor cell subpopulations, including stem-like glioma-initiating cells (GICs), which contribute to tumor recurrence following initial response to therapy. Here, we identified miR-182 as a regulator of apoptosis, growth, and differentiation programs whose expression level is correlated with GBM patient survival. Repression of Bcl2-like12 (Bcl2L12), c-Met, and hypoxia-inducible factor 2α (HIF2A) is of central importance to miR-182 anti-tumor activity, as it results in enhanced therapy susceptibility, decreased GIC sphere size, expansion, and stemness in vitro. To evaluate the tumor-suppressive function of miR-182 in vivo, we synthesized miR-182-based spherical nucleic acids (182-SNAs); i.e., gold nanoparticles covalently functionalized with mature miR-182 duplexes. Intravenously administered 182-SNAs penetrated the blood-brain/blood-tumor barriers (BBB/BTB) in orthotopic GBM xenografts and selectively disseminated throughout extravascular glioma parenchyma, causing reduced tumor burden and increased animal survival. Our results indicate that harnessing the anti-tumor activities of miR-182 via safe and robust delivery of 182-SNAs represents a novel strategy for therapeutic intervention in GBM.

Estrogen Receptor α Signaling Pathways Differentially Regulate Gonadotropin Subunit Gene Expression and Serum Follicle-Stimulating Hormone in the Female Mouse

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

Estrogen Actions in the Male Reproductive System Involve Estrogen Response Element-Independent Pathways

The estrogen receptor-alpha (ERalpha) acts through multiple pathways, including estrogen response element (ERE)-dependent (classical) and ERE-independent (nonclassical) mechanisms. We previously created a mouse model harboring a two-amino-acid mutation of the DNA-binding domain (E207A, G208A) that precludes direct binding of ERalpha to an ERE. After crossing heterozygous mutant mice with an ERalpha knockout (ERKO) line, it was possible to assess the degree of physiological rescue by the isolated ERalpha nonclassical allele (-/AA; AA) when compared with ERKO mice (-/-) and to wild type (+/+; WT). In male ERKO mice up to 8 months of age, testosterone levels were high, although LH levels were similar to WT. Testosterone was normal in the AA mice, indicating that the AA allele rescues the enhanced testosterone biosynthesis in ERKO mice. Male ERKO mice exhibited distention of the seminiferous tubules as early as 2-3 months of age as a consequence of decreased water resorption in the efferent ducts. By 3-4 months of age, ERKO mice had impaired spermatogenesis in approximately 40% of their tubules, and sperm counts and motility declined in association with the histological changes. In the AA mice, histological defects were greatly reduced or absent, and sperm counts and motility were rescued. Levels of aquaporins 1 and 9, which contribute to water uptake in the efferent ducts, were reduced in ERKO mice and partially or fully rescued in AA mice, whereas another water transporter, sodium-hydrogen exchanger-3, was decreased in both ERKO and AA mice. We conclude that non-ERE-dependent estrogen pathways are sufficient to rescue the defective spermatogenesis observed in ERKO mice and play a prominent role in ERalpha action in the testis, including pathways that regulate water resorption and androgen biosynthesis.

Bcl2L13 is a ceramide synthase inhibitor in glioblastoma

Significance Molecular mechanisms of therapy (apoptosis) resistance in cancer are poorly understood. Here, we have identified Bcl2-like 13 (Bcl2L13) as a ceramide synthase inhibitor that is overexpressed in glioblastoma (GBM) and other malignancies. Bcl2L13 inhibits therapy-induced apoptosis and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) and blocks CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors, thereby providing a molecular explanation for the low levels of proapoptotic ceramide species in high-grade gliomas, which are associated with poor survival. To our knowledge, this work provides the first evidence of direct regulation of CerS activity by a Bcl-2 family member and establishes the Bcl2L13–CerS axis as a target for therapeutic intervention. Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13–CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.

ENU mutagenesis in mice identifies candidate genes for hypogonadism

Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.

Dual bioluminescence and near-infrared fluorescence monitoring to evaluate spherical nucleic acid nanoconjugate activity in vivo

Significance Small interfering (si) and micro (mi)RNA-carrying nanomaterials emerged as a new class of anticancer therapeutics. To enable quantification of intratumoral gene expression in response to RNAi nanoconjugate treatment in vivo, we developed a dual reporter glioblastoma xenograft model using cell lines that stably coexpress optical reporters for luciferase and a near-infrared fluorescent protein (iRFP670). We generated orthotopic glioblastoma multiforme tumors expressing an iRFP670-O6-methylguanine-DNA-methyltransferase (MGMT) fusion protein for real-time assessment of MGMT-targeting spherical nucleic acids (i.e., gold-based nanoconjugates functionalized with siRNA oligonucleotides targeted to MGMT). We demonstrate that dual noninvasive bioluminescence and fluorescence imaging can determine intratumoral protein expression in response to systemic spherical nucleic acid treatment and represents an in vivo testing platform to facilitate preclinical investigations of nanoscale gene silencing therapeutics. RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood–brain barrier-/blood–tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.

A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism

Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2DG/DG mice compared to Rxfp2+/DG and Rxfp2+/+ mice. Testicular histology of the Rxfp2DG/DG mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2−) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2DG/−) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2+/− littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7–9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.

Internalized CD44s splice isoform attenuates EGFR degradation by targeting Rab7A

Significance Abnormal EGFR signaling is frequently observed in glioblastoma multiforme (GBM). However, the clinical benefit of EGFR inhibitors has been limited. Here we show that the cell surface molecule CD44s splice isoform acts as a signaling modulator that attenuates EGFR degradation and sustains EGFR’s downstream AKT signaling. CD44s internalizes in cells and inhibits Rab7A-mediated EGFR trafficking for degradation, resulting in sustained EGFR protein levels. CD44 depletion combined with EGFR inhibitor results in a robust and synergistic GBM cancer cell killing. Because CD44s-mediated inhibition on Rab7A also affects other receptor tyrosine kinases (RTKs), inhibiting CD44s may be an exciting approach for perturbation of multiple RTKs in GBMs. CD44 has been postulated as a cell surface coreceptor for augmenting receptor tyrosine kinase (RTK) signaling. However, how exactly CD44 triggers RTK-dependent signaling remained largely unclear. Here we report an unexpected mechanism by which the CD44s splice isoform is internalized into endosomes to attenuate EGFR degradation. We identify a CD44s-interacting small GTPase, Rab7A, and show that CD44s inhibits Rab7A-mediated EGFR trafficking to lysosomes and subsequent degradation. Importantly, CD44s levels correlate with EGFR signature and predict poor prognosis in glioblastomas. Because Rab7A facilitates trafficking of many RTKs to lysosomes, our findings identify CD44s as a Rab7A regulator to attenuate RTK degradation.