Hai-Yun Yen

Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest.

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2α, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.

Cell-Nonautonomous Induction of Ovarian and Uterine Serous Cystadenomas in Mice Lacking a Functional Brca1 in Ovarian Granulosa Cells

Women with germline mutations in BRCA1 have a 40% risk of developing ovarian cancer by age 70 and are also predisposed to cancers of the fallopian tubes. Given that ovulatory activity is a strong risk factor for sporadic ovarian cancer, we hypothesized that reduced BRCA1 expression might predispose to gynecological cancers indirectly, by influencing ovarian granulosa cells. These cells secrete sex steroids that control the ovulatory cycle and influence the growth of ovarian epithelial tumors. Granulosa cells also secrete mullerian inhibiting substance (MIS), a hormone that inhibits both the formation of female reproductive organs in male embryos and the proliferation of ovarian epithelial tumor cells. We tested this hypothesis by using the Cre-lox system to inactivate the Brca1 gene in mouse ovarian granulosa cells. A truncated form of the Fsh receptor promoter served as the Cre driver. Here, we show that indeed, inactivation of the Brca1 gene in granulosa cells led to the development of cystic tumors in the ovaries and uterine horns. These tumors carried normal Brca1 alleles, supporting the view that Brca1 may influence tumor development indirectly, possibly through an effector secreted by granulosa cells.

Jagged1 functions downstream of Twist1 in the specification of the coronal suture and the formation of a boundary between osteogenic and non-osteogenic cells

The Notch pathway is crucial for a wide variety of developmental processes including the formation of tissue boundaries. That it may function in calvarial suture development and figure in the pathophysiology of craniosynostosis was suggested by the demonstration that heterozygous loss of function of JAGGED1 in humans can cause Alagille syndrome, which has craniosynostosis as a feature. We used conditional gene targeting to examine the role of Jagged1 in the development of the skull vault. We demonstrate that Jagged1 is expressed in a layer of mesoderm-derived sutural cells that lie along the osteogenic-non-osteogenic boundary. We show that inactivation of Jagged1 in the mesodermal compartment of the coronal suture, but not in the neural crest compartment, results in craniosynostosis. Mesodermal inactivation of Jagged1 also results in changes in the identity of sutural cells prior to overt osteogenic differentiation, as well as defects in the boundary between osteogenic and non-osteogenic compartments at the coronal suture. These changes, surprisingly, are associated with increased expression of Notch2 and the Notch effector, Hes1, in the sutural mesenchyme. They are also associated with an increase in nuclear β-catenin. In Twist1 mutants, Jagged1 expression in the suture is reduced substantially, suggesting an epistatic relationship between Twist1 and Jagged1. Consistent with such a relationship, Twist1-Jagged1 double heterozygotes exhibit a substantial increase in the severity of craniosynostosis over individual heterozygotes. Our results thus suggest that Jagged1 is an effector of Twist1 in coronal suture development.

Changes in the Mouse Estrus Cycle in Response to Brca1 Inactivation Suggest a Potential Link between Risk Factors for Familial and Sporadic Ovarian Cancer

Menstrual cycle activity is the most important risk factor for sporadic serous ovarian carcinoma, whereas a germ-line mutation in BRCA1 is the most important risk factor for the familial form. Given the rarity of BRCA1 mutations in sporadic ovarian cancers, we hypothesized that BRCA1 influences the menstrual cycle in a way that mimics the factors underlying sporadic ovarian cancer predisposition, making BRCA1 mutations redundant in such cancers. We compared the length of each phase of the estrus cycle (equivalent to the human menstrual cycle) and of circulating levels of estradiol in control mice and in mice carrying a Brca1 mutation in their ovarian granulosa cells, two thirds of which develop ovarian or uterine epithelial tumors. We also compared the length of the different phases of the cycle in mutants that subsequently developed tumors with those in mutants that remained tumor-free. Mutant mice as well as oophorectomized wild-type mice harboring mutant ovarian grafts showed a relative increase in the average length of the proestrus phase of the estrus cycle, which corresponds to the estrogen-dominated follicular phase of the human menstrual cycle. Total circulating levels of estradiol were also increased in mutant mice injected with pregnant mare serum gonadotropins. The relative increase in proestrus length was highest in mutant mice that subsequently developed reproductive epithelial tumors. We conclude that loss of a functional Brca1 increases murine ovarian epithelial tumor predisposition by increasing estrogen stimulation in the absence of progesterone, recapitulating conditions associated with sporadic ovarian cancer predisposition in humans.

Alterations in Brca1 expression in mouse ovarian granulosa cells have short-term and long-term consequences on estrogen-responsive organs.

Incessant menstrual cycle activity, uninterrupted by either pregnancy or oral contraceptive use, is the most important risk factor for sporadic ovarian cancer. Menstrual cycle progression is partly controlled by steroid hormones such as estrogens and others that are secreted by ovarian granulosa cells. We showed earlier that mice carrying a homozygous granulosa cell specific knock out of Brca1, the homolog of BRCA1that is associated with familial ovarian cancer predisposition in humans, develop benign epithelial tumors in their reproductive tract. These tumors are driven, at least in part, by a prolongation of the proestrus phase of the estrus cycle (equivalent to the follicular phase of the menstrual cycle) in Brca1 mutant mice, resulting in prolonged unopposed estrogen stimulation. Mutant mice synchronized in proestrus also showed increased circulating estradiol levels, but the possibility that this changealso plays a role in tumor predisposition was not investigated. We sought to determine whether these changes in hormonal stimulation result in measurable changes in tissues targeted by estrogen outside the ovary. Here we show that mice carrying a Brca1 mutation in ovarian granulosa cells show increased endometrial proliferation during proestrus, implying that the effects of Brca1 inactivation on estrogen stimulation have short-term consequences, at least on this target organ. We further show that mutant mice develop increased femoral trabecular thickness and femoral length, which are well-known consequences of chronic estrogen stimulation. Estrogen biosynthesis by granulosa cells was increased not only in mice carrying a homozygous Brca1 mutation, but also in heterozygous mutants mimicking the mutational status in granulosa cells of human BRCA1 mutation carriers. The results suggestthat humangermline BRCA1 mutations, although associated with increased cancer risk, may also have beneficial consequences, such as increased bone strength, that may have contributed to the maintenance of mutated BRCA1 alleles in the human gene pool.Incessant menstrual cycle activity, uninterrupted by either pregnancy or oral contraceptive use, is the most important risk factor for sporadic ovarian cancer. Menstrual cycle progression is partly controlled by steroid hormones such as estrogens and others that are secreted by the ovarian granulosa cells. We showed earlier that mice carrying a homozygous granulosa cell-specific knockout of Brca1, the homolog of BRCA1 that is associated with familial ovarian cancer predisposition in humans, develop benign epithelial tumors in their reproductive tract. These tumors are driven, at least in part, by a prolongation of the proestrus phase of the estrus cycle (equivalent to the follicular phase of the menstrual cycle) in Brca1 mutant mice, resulting in prolonged unopposed estrogen stimulation. Mutant mice synchronized in proestrus also showed increased circulating estradiol levels, but the possibility that this change also has a role in tumor predisposition was not investigated. We sought to determine whether these changes in hormonal stimulation result in measurable changes in tissues targeted by estrogen outside the ovary. Here we show that mice carrying a Brca1 mutation in their ovarian granulosa cells show increased endometrial proliferation during proestrus, implying that the effects of Brca1 inactivation on estrogen stimulation have short-term consequences, at least on this target organ. We further show that mutant mice develop increased femoral trabecular thickness and femoral length, which are well-known consequences of chronic estrogen stimulation. Estrogen biosynthesis by granulosa cells was increased not only in mice carrying a homozygous Brca1 mutation, but also in heterozygous mutants mimicking the mutational status in granulosa cells of human BRCA1 mutation carriers. The results suggest that human germline BRCA1 mutations, although associated with increased cancer risk, may also have beneficial consequences, such as increased bone strength, that may have contributed to the maintenance of mutated BRCA1 alleles in the human gene pool.

Targeting the glucose-regulated protein-78 abrogates Pten-null driven AKT activation and endometrioid tumorigenesis

Rates of the most common gynecologic cancer, endometrioid adenocarcinoma (EAC), continue to rise, mirroring the global epidemic of obesity, a well-known EAC risk factor. Thus, identifying novel molecular targets to prevent and/or mitigate EAC is imperative. The prevalent Type 1 EAC commonly harbors loss of the tumor suppressor, Pten, leading to AKT activation. The major endoplasmic reticulum (ER) chaperone, GRP78, is a potent pro-survival protein to maintain ER homeostasis, and as a cell surface protein, is known to regulate the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. To determine whether targeting GRP78 could suppress EAC development, we created a conditional knockout mouse model using progesterone receptor-Cre-recombinase to achieve Pten and Grp78 (cPtenf/fGrp78f/f) deletion in the endometrial epithelium. Mice with a single Pten (cPtenf/f) deletion developed well-differentiated EAC by 4 weeks. In contrast, no cPtenf/fGrp78f/f mice developed EAC, even after more than 8 months of observation. Histologic examination of uteri from cPtenf/fGrp78f/f mice also revealed no complex atypical hyperplasia, a well-established EAC precursor. These histologic observations among the cPtenf/fGrp78f/f murine uteri also corresponded to abrogation of AKT activation within the endometrium. We further observed that GRP78 co-localized with activated AKT on the surface of EAC, thus providing an opportunity for therapeutic targeting. Consistent with previous findings that cell surface GRP78 is an upstream regulator of PI3K/AKT signaling, we show here that in vivo short-term systemic treatment with a highly specific monoclonal antibody against GRP78 suppressed AKT activation and increased apoptosis in the cPtenf/f tumors. Collectively, these findings present GRP78-targeting therapy as an efficacious therapeutic option for EAC.

A Mouse Model That Reproduces the Developmental Pathways and Site Specificity of the Cancers Associated With the Human BRCA1 Mutation Carrier State.

Predisposition to breast and extrauterine Müllerian carcinomas in BRCA1 mutation carriers is due to a combination of cell-autonomous consequences of BRCA1 inactivation on cell cycle homeostasis superimposed on cell-nonautonomous hormonal factors magnified by the effects of BRCA1 mutations on hormonal changes associated with the menstrual cycle. We used the Müllerian inhibiting substance type 2 receptor (Mis2r) promoter and a truncated form of the Follicle stimulating hormone receptor (Fshr) promoter to introduce conditional knockouts of Brca1 and p53 not only in mouse mammary and Müllerian epithelia, but also in organs that control the estrous cycle. Sixty percent of the double mutant mice developed invasive Müllerian and mammary carcinomas. Mice carrying heterozygous mutations in Brca1 and p53 also developed invasive tumors, albeit at a lesser (30%) rate, in which the wild type alleles were no longer present due to loss of heterozygosity. While mice carrying heterozygous mutations in both genes developed mammary tumors, none of the mice carrying only a heterozygous p53 mutation developed such tumors (P < 0.0001), attesting to a role for Brca1 mutations in tumor development. This mouse model is attractive to investigate cell-nonautonomous mechanisms associated with cancer predisposition in BRCA1 mutation carriers and to investigate the merit of chemo-preventive drugs targeting such mechanisms.

Requirement for Jagged1-Notch2 signaling in patterning the bones of the mouse and human middle ear

Whereas Jagged1-Notch2 signaling is known to pattern the sensorineural components of the inner ear, its role in middle ear development has been less clear. We previously reported a role for Jagged-Notch signaling in shaping skeletal elements derived from the first two pharyngeal arches of zebrafish. Here we show a conserved requirement for Jagged1-Notch2 signaling in patterning the stapes and incus middle ear bones derived from the equivalent pharyngeal arches of mammals. Mice lacking Jagged1 or Notch2 in neural crest-derived cells (NCCs) of the pharyngeal arches display a malformed stapes. Heterozygous Jagged1 knockout mice, a model for Alagille Syndrome (AGS), also display stapes and incus defects. We find that Jagged1-Notch2 signaling functions early to pattern the stapes cartilage template, with stapes malformations correlating with hearing loss across all frequencies. We observe similar stapes defects and hearing loss in one patient with heterozygous JAGGED1 loss, and a diversity of conductive and sensorineural hearing loss in nearly half of AGS patients, many of which carry JAGGED1 mutations. Our findings reveal deep conservation of Jagged1-Notch2 signaling in patterning the pharyngeal arches from fish to mouse to man, despite the very different functions of their skeletal derivatives in jaw support and sound transduction.

Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent

We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type Brca1 to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional Brca1 gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that Brca1 -/- female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by Brca1-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers.

Abstract 4356: EphB4 as a novel therapeutic target for head-neck SCC.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Receptor tyrosine kinase EphB4 and its cognate trans-membrane ligand EphrinB2 regulate venous-artery determination and are critically required for fusion of vein and arterial compartments, as well as vessel maturation in development and in pathological angiogenesis. Aberrant induction of EphB4 in certain tumors and pro-tumor cell survival function independent of its role in tumor angiogenesis have been reported. We have studied the expression and function of EphB4 in head neck squamous cell cancer. Methods: EphB4 expression in 78 freshly frozen head/neck squamous cell carcinoma tumor samples were studied by immunostaining, western blot analysis, gene copy number, and correlated with stage. Head/neck tumor cell lines also were studied for expression and significance of EphB4 expression on survival by knock down studies using EphB4 lentiviral shRNA. Inhibition of EphB4 activation using decoy soluble EphB4 receptor fused in frame at the C-terminus with human albumin (sEphB4-HSA) was evaluated for in vivo studies of human tumor xenografts. Results: EphB4 was expressed in all human tumor tissues, and the levels correlated directly with stage. High EphB4 also predicted shortened survival. EphB4 protein levels varied in tumor cell lines, and loss of cell viability with EphB4 shRNA knock down was highest in cell lines with highest EphB4 levels (range 25-78%). Tumor xenografts in sEphB4-HSA treated mice were reduced over 80% compared to the control PBS treatment. Tissue analysis revealed reduction in tumor vessel density, reduced recruitment of pericytes, and decreased perfusion of the tumor vessels. sEphB4-HSA treatment also decreased tumor tissue PI3K activity manifested by reduced phosphorylated AKT and S6 level. Tumor cell death by apoptosis was widespread consistent with combined anti-angiogenic activity and direct tumor cell toxicity. Conclusion: EphB4 receptor tyrosine kinase is highly expressed in head/neck squamous cell cancer. EphB4 provides survival signal to the tumor cells directly. Decoy EphB4 inhibitor sEphB4-HSA is highly potent in in vivo tumor xenograft models. sEphB4-HSA is thus a candidate for investigation in human trials and the clinical trials are underway. Citation Format: Ren Liu, Hai-yun Yen, Rizwan Masood, Uttam Sinha, Anthony El-Khoueiry, Stephen Liu, Barbara Gitlitz, Parkash S. Gill. EphB4 as a novel therapeutic target for head-neck SCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4356. doi:10.1158/1538-7445.AM2013-4356