Aberrant Morphogenesis Research Articles

Prevotella Denticola Dacryocystitis and Abscess in a Child with Goldenhar Syndrome

Dacryocystitis is the inflammation of the nasolacrimal sac and is due to obstruction of the nasolacrimal system or lacrimal stagnation, leading to favorable environments for infection. Any disruptions to the nasolacrimal pathway can increase the risk of dacryocystitis. Here is a unique case of Prevotella denticola dacryocystitis that progressed into cellulitis and abscess formation in a 7-year-old female with a medical history of Goldenhar syndrome, recurrent dacryocystitis, and periodontal disease. The patient presented with standard dacryocystitis symptoms—tenderness to palpation, erythema, and edema to the medial canthus. The patient was treated empirically with vancomycin, ceftriaxone, and clindamycin. She was discharged home on clindamycin with symptomatic improvement and plans for dacryocystorhinostomy. Goldenhar syndrome is a rare congenital craniofacial malformation disorder due to aberrant morphogenesis in structures derived from the first and second branchial arches. P. denticola is an anaerobic gram-negative bacillus significant to the oral microbiome. There is currently limited research suggesting the relationship between Goldenhar syndrome and dacryocystitis complicated by Prevotella denticola. Based on the patient’s medical history, we predict that her complex presentation was secondary to the dissemination of Prevotella from her oral cavity. This case report emphasizes the importance of gram-negative anaerobic coverage in complicated dacryocystitis in patients with nasolacrimal defects.

In toto light sheet fluorescence microscopy live imaging datasets of Ceratitis capitata embryonic development

The Mediterranean fruit fly (medfly), Ceratitis capitata, is an important model organism in biology and agricultural research with high economic relevance. However, information about its embryonic development is still sparse. We share nine long-term live imaging datasets acquired with light sheet fluorescence microscopy (484.5 h total recording time, 373 995 images, 256 Gb) with the scientific community. Six datasets show the embryonic development in toto for about 60 hours at 30 minutes intervals along four directions in three spatial dimensions, covering approximately 97% of the entire embryonic development period. Three datasets focus on germ cell formation and head involution. All imaged embryos hatched morphologically intact. Based on these data, we suggest a two-level staging system that functions as a morphogenetic framework for upcoming studies on medfly. Our data supports research on wild-type or aberrant morphogenesis, quantitative analyses, comparative approaches to insect development as well as studies related to pest control. Further, they can be used to test advanced image processing approaches or to train machine learning algorithms and/or neuronal networks.

Deciphering the mutation spectrum in south Indian children with congenital anomalies of the kidney and urinary tract

BackgroundCongenital anomalies of the kidney and urinary tract (CAKUT) cover a spectrum of structural malformations that result from aberrant morphogenesis of kidney and urinary tract. It is the most prevalent cause of kidney failure in children. Hence, it is important from a clinical perspective to unravel the molecular etiology of kidney and urinary tract malformations. Causal variants in genes that direct various stages of development of kidney and urinary tract in fetal life have been identified in 5–20% of CAKUT patients from Western countries. Recent advances in next generation sequencing technology and decreasing cost offer the opportunity to characterize the genetic profile of CAKUT in Indian population and facilitate integration of genetic diagnostics in care of children with CAKUT.MethodsCustomized targeted panel sequencing was performed to identify pathogenic variants in 31 genes known to cause human CAKUT in 69 south Indian children with CAKUT. The NGS data was filtered using standardized pipeline and the variants were classified using ACMG criteria. Genotype and phenotype correlations were performed.ResultsThe cohort consisted of children mostly with posterior urethral valve (PUV) (39.1%), vesico-ureteric reflux (VUR) (33.3%) and multi-cystic dysplastic kidney (MCDK) (7.2%). No pathogenic or likely pathogenic variants were identified in the study. Most of our variants (n = 39, 60%) were variants of unknown significance with 25.6% (10/39) of them were identified as potentially damaging but were novel variants.ConclusionsThe present study did not identify any disease-causing monogenic variants in the cohort. The absence of genetic cause may be due to limitations of panel-based testing and also due to higher proportion of children with abnormalities in lower urinary tract than hypodysplasia of kidneys. Clinical, larger targeted panel or whole exome sequencing may be a better method to characterize the genetic profile of Indians patients with CAKUT.

Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration.

Rab-GTPases and associated effectors mediate cargo transport through the endomembrane system of eukaryotic cells, regulating key processes such as membrane turnover, signal transduction, protein recycling and degradation. Using developmental transcriptome data, we identified Rabgef1 (encoding the protein RabGEF1 or Rabex-5) as the only gene associated with Rab GTPases that exhibited strong concordance with retinal photoreceptor differentiation. Loss of Rabgef1 in mice (Rabgef1-/-) resulted in defects specifically of photoreceptor morphology and almost complete loss of both rod and cone function as early as eye opening; however, aberrant outer segment formation could only partly account for visual function deficits. RabGEF1 protein in retinal photoreceptors interacts with Rabaptin-5, and RabGEF1 absence leads to reduction of early endosomes consistent with studies in other mammalian cells and tissues. Electron microscopy analyses reveal abnormal accumulation of macromolecular aggregates in autophagosome-like vacuoles and enhanced immunostaining for LC3A/B and p62 in Rabgef1-/- photoreceptors, consistent with compromised autophagy. Transcriptome analysis of the developing Rabgef1-/- retina reveals altered expression of 2469 genes related to multiple pathways including phototransduction, mitochondria, oxidative stress and endocytosis, suggesting an early trajectory of photoreceptor cell death. Our results implicate an essential role of the RabGEF1-modulated endocytic and autophagic pathways in photoreceptor differentiation and homeostasis. We propose that RabGEF1 and associated components are potential candidates for syndromic traits that include a retinopathy phenotype.

Yap and its subcellular localization have distinct compartment-specific roles in the developing lung.

Although the Hippo-yes-associated protein (Yap) pathway has been implicated in lung development, the specific roles for Yap and its nucleocytoplasmic shuttling in the developing airway and alveolar compartments remain elusive. Moreover, conflicting results from expression studies and differences in the lung phenotypes of Yap and Hippo kinase null mutants caused controversy over the dynamics and significance of Yap subcellular localization in the developing lung. Here, we show that the aberrant morphogenesis of Yap-deficient lungs results from the disruption of developmental events specifically in distal epithelial progenitors. We also show that activation of nuclear Yap is enough to fulfill the Yap requirements to rescue abnormalities in these lungs. Remarkably, we found that Yap nucleocytoplasmic shuttling is largely dispensable in epithelial progenitors for both branching morphogenesis and sacculation. However, if maintained transcriptionally active in airways, nuclear Yap profoundly alters proximal-distal identity and halts epithelial differentiation. Taken together, these observations provide novel insights into the crucial importance of Hippo-Yap signaling in the lung prenatally.

3D Mammary Epithelial Cell Models: A Goldmine of DCIS Biomarkers and Morphogenetic Mechanisms.

Breast ductal carcinoma in situ (DCIS) has been typically recognized by pathologists on the basis of aberrant mammary duct morphology. Thus, there are increasing efforts to detect DCIS biomarkers and druggable targets. In this study we focused on the molecular mechanism involving Annexin A8 (ANXA8), a Ca2+ and phospholipid binding protein, which is regulated by all-trans Retinoic Acid (RA), and it is highly expressed in breast DCIS tissue samples relative to atypical ductal hyperplasia, and normal breast tissue. Using a panel of human mammary epithelial HME1 cell lines that share a common protein signature, and develop in vitro three dimensional (3D) “DCIS-like” amorphous structures, we identified by bioinformatics analysis protein-miRNA pairs, potentially involved in mammary morphogenetic mechanisms, including the ANXA8 mechanism. HME1 cells with genetic mutations hampering the physiological RA regulation of the RA receptor alpha (RARA) transcriptional function, but retain the RARA function controlling the PI3KCA-AKT signaling, develop 3D “DCIS-like” amorphous structures with upregulated ANXA8. Consistently, ectopic ANXA8 expression, by affecting the RARA transcriptional function, induced HME1 DCIS-like amorphous acini expressing phosphorylated AKT (P-AKT). Apparently, a RA-RARA-ANXA8 feedback loop fosters a vicious circle of aberrant morphogenesis. Interestingly, a few miRNAs regulated by RA are predicted to target ANXA8 mRNA. These miRNAs are candidate components of the RA-RARA-ANXA8 mechanism, and their deregulation might induce DCIS initiation.

Neurog3-dependent pancreas dysgenesis causes ectopic pancreas in Hes1 mutant mice.

Mutations in Hes1, a target gene of the Notch signalling pathway, lead to ectopic pancreas by a poorly described mechanism. Here, we use genetic inactivation of Hes1 combined with lineage tracing and live imaging to reveal an endodermal requirement for Hes1, and show that ectopic pancreas tissue is derived from the dorsal pancreas primordium. RNA-seq analysis of sorted E10.5 Hes1+/+ and Hes1-/- Pdx1-GFP+ cells suggested that upregulation of endocrine lineage genes in Hes1-/- embryos was the major defect and, accordingly, early pancreas morphogenesis was normalized, and the ectopic pancreas phenotype suppressed, in Hes1-/-Neurog3-/- embryos. In Mib1 mutants, we found a near total depletion of dorsal progenitors, which was replaced by an anterior Gcg+ extension. Together, our results demonstrate that aberrant morphogenesis is the cause of ectopic pancreas and that a part of the endocrine differentiation program is mechanistically involved in the dysgenesis. Our results suggest that the ratio of endocrine lineage to progenitor cells is important for morphogenesis and that a strong endocrinogenic phenotype without complete progenitor depletion, as seen in Hes1 mutants, provokes an extreme dysgenesis that causes ectopic pancreas.

AhR Deletion Promotes Aberrant Morphogenesis and Synaptic Activity of Adult-Generated Granule Neurons and Impairs Hippocampus-Dependent Memory

Newborn granule cells are continuously produced in the subgranular zone of dentate gyrus throughout life. Once these cells mature, they integrate into pre-existing circuits modulating hippocampus-dependent memory. Subsequently, mechanisms controlling generation and maturation of newborn cells are essential for proper hippocampal function. Therefore, we have studied the role of aryl hydrocarbon receptor (AhR), a ligand-activated bHLH-PAS transcription factor, in hippocampus-dependent memory and granule neuronal morphology and function using genetic loss-of-function approaches based on constitutive and inducible-nestin AhR-/- mice. The results presented here show that the impaired hippocampus-dependent memory in AhR absence is not due to its effects on neurogenesis but to aberrant dendritic arborization and an increased spine density, albeit with a lower number of mature mushrooms spines in newborn granule cells, a finding that is associated with an immature electrophysiological phenotype. Together, our data strongly suggest that AhR plays a pivotal role in the regulation of hippocampal function, by controlling hippocampal granule neuron morphology and synaptic maturation.

Stomatal Complex Development and F-Actin Organization in Maize Leaf Epidermis Depend on Cellulose Synthesis.

Cellulose microfibrils reinforce the cell wall for morphogenesis in plants. Herein, we provide evidence on a series of defects regarding stomatal complex development and F-actin organization in Zea mays leaf epidermis, due to inhibition of cellulose synthesis. Formative cell divisions of stomatal complex ontogenesis were delayed or inhibited, resulting in lack of subsidiary cells and frequently in unicellular stomata, with an atypical stomatal pore. Guard cells failed to acquire a dumbbell shape, becoming rounded, while subsidiary cells, whenever present, exhibited aberrant morphogenesis. F-actin organization was also affected, since the stomatal complex-specific arrays were scarcely observed. At late developmental stages, the overall F-actin network was diminished in all epidermal cells, although thick actin bundles persisted. Taken together, stomatal complex development strongly depends on cell wall mechanical properties. Moreover, F-actin organization exhibits a tight relationship with the cell wall.

Constitutive metanephric mesenchyme-specific expression of interferon-gamma causes renal dysplasia by regulating Sall1 expression.

Transplacental viral and parasitic infections have been shown to initiate an innate response in the mammalian embryo by increasing the expression of pro-inflammatory cytokines such as interferon-gamma (Ifng). However, the developmental consequences of an activated innate immunity and, in particular, the effects of induction of Ifng expression independent of infection have been largely overlooked. Here, we demonstrate in vivo that the conditional overexpression of Ifng in metanephric mesenchymal (MM) progenitors results in renal agenesis or hypoplasia. Cell death was observed in and around the MM region of E10.5–11.5 mutants where Ifng was constitutively expressed during early kidney development and resulted in a retardation of branching morphogenesis. Furthermore, isolated mutant or normal Ifng-treated metanephroi replicated this phenotype in culture, demonstrating the inherent nature of the aberrant morphogenesis. The expression of renal progenitor marker Sall1 was significantly decreased in the MM of mutant kidneys, suggesting that a reduction in Sall1 may be the cause of cell death in the MM during early kidney development and that, in turn, retards UB branching in the mutants. Therefore, the aberrant induction of Ifng expression, as part of an innate immune response, may contribute to renal agenesis or hypoplasia during early metanephric development by regulating the MM progenitor population.

Morphogenetic defects underlie Superior Coloboma, a newly identified closure disorder of the dorsal eye.

The eye primordium arises as a lateral outgrowth of the forebrain, with a transient fissure on the inferior side of the optic cup providing an entry point for developing blood vessels. Incomplete closure of the inferior ocular fissure results in coloboma, a disease characterized by gaps in the inferior eye and recognized as a significant cause of pediatric blindness. Here, we identify eight patients with defects in tissues of the superior eye, a congenital disorder that we term superior coloboma. The embryonic origin of superior coloboma could not be explained by conventional models of eye development, leading us to reanalyze morphogenesis of the dorsal eye. Our studies revealed the presence of the superior ocular sulcus (SOS), a transient division of the dorsal eye conserved across fish, chick, and mouse. Exome sequencing of superior coloboma patients identified rare variants in a Bone Morphogenetic Protein (Bmp) receptor (BMPR1A) and T-box transcription factor (TBX2). Consistent with this, we find sulcus closure defects in zebrafish lacking Bmp signaling or Tbx2b. In addition, loss of dorsal ocular Bmp is rescued by concomitant suppression of the ventral-specific Hedgehog pathway, arguing that sulcus closure is dependent on dorsal-ventral eye patterning cues. The superior ocular sulcus acts as a conduit for blood vessels, with altered sulcus closure resulting in inappropriate connections between the hyaloid and superficial vascular systems. Together, our findings explain the existence of superior coloboma, a congenital ocular anomaly resulting from aberrant morphogenesis of a developmental structure.

Tracing anti-cancer and cancer-promoting actions of all-trans retinoic acid in breast cancer to a RARα epigenetic mechanism of mammary epithelial cell fate.

A hallmark of cancer cells is the ability to evade the growth inhibitory/pro-apoptotic action of physiological all-trans retinoic acid (RA) signal, the bioactive derivative of Vitamin A. However, as we and others reported, RA can also promote cancer cell growth and invasion. Here we show that anticancer and cancer-promoting RA actions in breast cancer have roots in a mechanism of mammary epithelial cell morphogenesis that involves both transcriptional (epigenetic) and non-transcriptional RARα (RARA) functions. We found that the mammary epithelial cell-context specific degree of functionality of the RARA transcriptional (epigenetic) component of this mechanism, by tuning the effects of the non-transcriptional RARA component, determines different cell fate decisions during mammary morphogenesis. Indeed, factors that hamper the RARA epigenetic function make physiological RA drive aberrant morphogenesis via non-transcriptional RARA, thus leading to cell transformation. Remarkably, also the cell context-specific degree of functionality of the RARA epigenetic component retained by breast cancer cells is critical to determine cell fate decisions in response to physiological as well as supraphysiological RA variation. Overall this study supports the proof of principle that the epigenetic functional plasticity of the mammary epithelial cell RARA mechanism, which is essential for normal morphogenetic processes, is necessary to deter breast cancer onset/progression consequent to the insidious action of physiological RA.

Plant development regulated by cytokinin sinks.

Morphogenetic signals control the patterning of multicellular organisms. Cytokinins are mobile signals that are perceived by subsets of plant cells. We found that the responses to cytokinin signaling during Arabidopsis development are constrained by the transporter PURINE PERMEASE 14 (PUP14). In our experiments, the expression of PUP14 was inversely correlated to the cytokinin signaling readout. Loss of PUP14 function allowed ectopic cytokinin signaling accompanied by aberrant morphogenesis in embryos, roots, and the shoot apical meristem. PUP14 protein localized to the plasma membrane and imported bioactive cytokinins, thus depleting apoplastic cytokinin pools and inhibiting perception by plasma membrane-localized cytokinin sensors to create a sink for active ligands. We propose that the spatiotemporal cytokinin sink patterns established by PUP14 determine the cytokinin signaling landscape that shapes the morphogenesis of land plants.

Cardiac troponin T is necessary for normal development in the embryonic chick heart.

The heart is the first functioning organ to develop during embryogenesis. The formation of the heart is a tightly regulated and complex process, and alterations to its development can result in congenital heart defects. Mutations in sarcomeric proteins, such as alpha myosin heavy chain and cardiac alpha actin, have now been associated with congenital heart defects in humans, often with atrial septal defects. However, cardiac troponin T (cTNT encoded by gene TNNT2) has not. Using gene‐specific antisense oligonucleotides, we have investigated the role of cTNT in chick cardiogenesis. TNNT2 is expressed throughout heart development and in the postnatal heart. TNNT2‐morpholino treatment resulted in abnormal atrial septal growth and a reduction in the number of trabeculae in the developing primitive ventricular chamber. External analysis revealed the development of diverticula from the ventricular myocardial wall which showed no evidence of fibrosis and still retained a myocardial phenotype. Sarcomeric assembly appeared normal in these treated hearts. In humans, congenital ventricular diverticulum is a rare condition, which has not yet been genetically associated. However, abnormal haemodynamics is known to cause structural defects in the heart. Further, structural defects, including atrial septal defects and congenital diverticula, have previously been associated with conduction anomalies. Therefore, to provide mechanistic insights into the effect that cTNT knockdown has on the developing heart, quantitative PCR was performed to determine the expression of the shear stress responsive gene NOS3 and the conduction gene TBX3. Both genes were differentially expressed compared to controls. Therefore, a reduction in cTNT in the developing heart results in abnormal atrial septal formation and aberrant ventricular morphogenesis. We hypothesize that alterations to the haemodynamics, indicated by differential NOS3 expression, causes these abnormalities in growth in cTNT knockdown hearts. In addition, the muscular diverticula reported here suggest a novel role for mutations of structural sarcomeric proteins in the pathogenesis of congenital cardiac diverticula. From these studies, we suggest TNNT2 is a gene worthy of screening for those with a congenital heart defect, particularly atrial septal defects and ventricular diverticula.

Retinoic Acid Induces Myogenic Tongue Abnormalities in Fetal Mice

RETINOIC ACID INDUCES MYOGENIC TONGUE ABNORMALITIES IN FETAL MICE Wei Cong, Bo Liu, Ru Wang, Jing Xiao, Department of Oral Pathology, College of Stomatology, Dalian Medical University, Dalian, China; Department of Stomatology, the First Affiliated Hospital, Dalian Medical University, Dalian, China Objective: To study the aberrant morphogenesis and underlying mechanism of RA induced tongue malformation in fetal mice. Study Design: Exogenous RA was used to induce tongue malformation. The histological changes of tongue were characterized by HE staining and transmission electron microscopy. Myogenic differentiation was measured by Immunohistochemical assays and qRT-PCR of Myf5 and MyoD. The signaling genes were evaluated by qRT-PCR. Results: In +RA fetal mice, the tongues failed to descend and flatten while maintaining a higher position at E14.5. Subcellular changes of genioglossus showed the myofibrils failed to form normal structures of sarcomeres, and muscle fibers were disorderly arranged. The levels of Myf5 and MyoD were reduced. The downregulation of Wnt5a was positively correlated with Camk2d and inversely correlated with Tbx1, Ror2, and PKCd. Conclusions: The Wnt5a/CaMKII pathway may be involved in hypoplasia and disorder of tongue with decreased myogenic differentiation induced by RA.

TGFβ1a regulates zebrafish posterior lateral line formation via Smad5 mediated pathway.

The zebrafish sensory posterior lateral line (pLL) has become an attractive model for studying collective cell migration and cell morphogenesis. Recent studies have indicated that chemokine, Wnt/β-catenin, Fgf, and Delta-Notch signaling pathways participate in regulating pLL development. However, it remains unclear whether TGFβ signaling pathway is involved in pLL development. Here we report a critical role of TGFβ1 in regulating morphogenesis of the pLL primordium (pLLP). The tgfβ1a gene is abundantly expressed in the lateral line primordium. Knockdown or knockout of tgfβ1a leads to a reduction of neuromast number, an increase of inter-neuromast distance, and a reduced number of hair cells. The aberrant morphogenesis in embryos depleted of tgfβ1a correlates with the reduced expression of atoh1a, deltaA, and n-cadherin/cdh2, which are known important regulators of the pLLP morphogenesis. Like tgfβ1a depletion, knockdown of smad5 that expresses in the pLLP, affects pLLP development whereas overexpression of a constitutive active Smad5 isoform rescues the defects in embryos depleted of tgfβ1a, indicating that Smad5 mediates tgfβ1a function in pLLP development. Therefore, TGFβ/Smad5 signaling plays an important role in the zebrafish lateral line formation.

Abstract 2501: Proteomics signature of lack of retinoic acid signal surveillance at early breast cancer stages

Abstract Endogenous retinoic acid (RA), the bioactive derivative of Vitamin A, is necessary for breast acinar morphogenesis. One of the hallmarks of early stage breast cancer, such as ductal carcinoma in situ (DCIS), is the inability to respond to RA morphogenetic action, which results in the uncontrolled growth of the mammary duct luminal cells. By imaging endogenous RA signaling during normal and aberrant breast acinar development in three dimensional (3D) culture, we found that the inability to form the acinar lumen can be traced to lack of activation of the RA receptor alpha (RARA) genomic function by endogenous RA variation. We further found that aberrant acinar morphogenesis, regardless of the causing factors, is marked by a distinct protein signature, which includes the upregulation of Annexin A8 (ANXA8). Forced expression of ANXA8 in breast epithelial cells was sufficient to disrupt 3D acinar architecture. Moreover, by screening DCIS tissue microarrays (TMAs), we found that ANXA8 expression correlates with tumor grade and stage. Thus, further development of this proteomics signature indicative of lack RA signal surveillance is expected to assist in the early detection of initial breast cancer stages. This work was funded by R01 CA127614, the Friends for an Earlier Breast Cancer Test Foundation, and the Terri Brodeur Breast Cancer Foundation. Citation Format: Stefano Rossetti, Wiam Bshara, Nicoletta Sacchi. Proteomics signature of lack of retinoic acid signal surveillance at early breast cancer stages. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2501. doi:10.1158/1538-7445.AM2014-2501

Cytomegalovirus miRNAs Target Secretory Pathway Genes to Facilitate Formation of the Virion Assembly Compartment and Reduce Cytokine Secretion

Herpesviruses, including human cytomegalovirus (HCMV), encode multiple microRNAs (miRNA) whose targets are just being uncovered. Moreover, miRNA function during the virus life cycle is relatively unknown. We find that HCMV miRs UL112-1, US5-1, and US5-2 target multiple components of the host secretory pathway, including VAMP3, RAB5C, RAB11A, SNAP23, and CDC42. A HCMV miR UL112-1, US5-1, and US5-2 triple mutant displayed aberrant morphogenesis of the virion assembly compartment (VAC), increased secretion of noninfectious particles, and increased IL-6 release from infected cells. Ectopic expression of miRs UL112-1, US5-1, and US5-2 or siRNAs directed against RAB5C, RAB11A, SNAP23, and CDC42 caused the loss of Golgi stacks with reorganization into structures that resemble the VAC and a decrease in cytokine release. These observations indicate that multiple HCMV miRNAs coordinately regulate reorganization of the secretory pathway to control cytokine secretion and facilitate formation of the VAC for efficient infectious virus production.

Abstract 2497: Upregulation of annexin A8 is a common molecular phenotype of aberrant breast acinar morphogenesis induced by different genetic factors.

Abstract It is estimated that up to 90% of women have benign breast lesions, some of which are pre-malignant. In situ lesions, particularly the ones that involve the ducts (ductal carcinoma in situ, DCIS), have the potential to progress into invasive malignant lesions and become life threatening. These breast premalignant states are identified because of specific morphological tissue abnormalities. Three-dimensional (3D) cultures of human mammary epithelial cells closely recapitulate breast acinar morphogenesis. While untransformed mammary epithelial cells form hollow acini, lined by polarized, non-proliferating cells, breast cancer cells form acini filled with non-polarized, actively proliferating cells, similar to DCIS. By using a proteomic approach in a panel of morphologically aberrant acini derived from human breast epithelial cells, we found that the disruption of acinar morphogenesis by different genetic factors generates a common proteomic signature. Among the proteins that we found consistently upregulated, we identified Annexin A8 (ANXA8), a member of the annexin family of calcium and membrane-binding proteins with a wide range of cellular functions, including cell division, apoptosis, and cell growth regulation. We tested ANXA8 expression in tissue samples from patients with atypical ductal hyperplasia (ADH), and tissue microarrays of DCIS, with or without, history of invasive breast cancer. Interestingly, this analysis detected ANXA8 increased expression in atypical proliferating ducts relative to non-proliferative ducts, making ANXA8 a potential marker of breast pre-malignancies. Acknowledgments: this study was funded by grants of the Susan Komen Foundation, the Breast Cancer Coalition of Rochester, the Friends for an Earlier Test of Breast Cancer Foundation. Citation Format: Stefano Rossetti, Francesca Corlazzoli, Nicoletta Sacchi. Upregulation of annexin A8 is a common molecular phenotype of aberrant breast acinar morphogenesis induced by different genetic factors. [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 2497. doi:10.1158/1538-7445.AM2013-2497 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3568: Induction of aberrant breast acinar morphogenesis by deregulation of an estrogen receptor alpha circadian circuit.

Abstract Altered estrogen receptor alpha (ERA) signaling and altered circadian rhythms are both features of breast cancer. ERA mRNA oscillates in a circadian fashion in ERA-positive breast epithelial cells, but not in ERA-positive breast cancer cells (Rossetti et al., 2012a). Interestingly, we identified a circuit consisting of ERA and an estrogen-regulated loop consisting of PER2 and BMAL1, two circadian clock genes, in entrained ERA-positive breast epithelial cells. This estrogen-regulated circuit is apparently necessary for breast epithelial acinar morphogenesis, and its disruption by ERA knock down negatively affects both the estrogen-sustained circadian PER2-BMAL1mechanism and the formation of acini in three-dimensional (3D) culture on basement membrane. Conversely, knock down of either PER2 or BMAL1, by hampering the PER2-BMAL1 loop of the circadian clock, negatively affects both ERA circadian oscillations and 3D breast acinar morphogenesis (Rossetti et al., 2012b). This is the first evidence that a circuit comprising ERA and key circadian clock components is implicated in the acinar developmental processes of breast epithelial cells. Acknowledgments: this work was supported by the Department of Defense (DOD) Concept Award W81XWH0610657 (NS), the Breast Cancer Coalition of Rochester (NS), and the Friends for an Earlier Test for Breast Cancer Foundation (NS). Rossetti, S, Esposito, J, Corlazzoli, F, Gregorski, A, and Sacchi, N. (2012a). Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes. Cell Cycle 11, 350-360. Rossetti, S, Corlazzoli, F, Gregorski, A, Azmi, NH, and Sacchi, N. (2012b). Identification of an estrogen-regulated circadian mechanism necessary for breast acinar morphogenesis. Cell Cycle 11, 3691-3700. Citation Format: Stefano Rossetti, Francesca Corlazzoli, Alex Gregorski, Nicoletta Sacchi. Induction of aberrant breast acinar morphogenesis by deregulation of an estrogen receptor alpha circadian circuit. [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 3568. doi:10.1158/1538-7445.AM2013-3568