Acinar Formation Research Articles

Clinical significance of histopathologic and ultrastructural pathologic examination in etiological diagnosis of infantile cholestatic diseases

To study the features of histopathologic and ultrastructural pathologic changes of liver biopsy in patients with infantile cholestatic disease, and to investigate its diagnostic significance combining with the clinical data. Thirty-six children diagnosed as infantile cholestatic disease and received liver biopsy in Chongqing Medical University Children's Hospital from Jun 2007 to Oct 2008 were enrolled and the pathologic and ultrastructural pathologic changes of liver were analyzed. Morphologic changes under light microscope in liver tissues included hepatocyte swelling, hepatocyte denaturation, hepatocyte necrosis, multinucleated giant cell formation, bile duct proliferation, fiber tissues proliferation and inflammatory cells infiltration in liver lobules and portal regions. The characteristics of cholestasis including intralobular cholestasis, acinus formation, feather-like cytoplasmic filaments and bile stasis in bile canaliculi were observed. The morphologic changes of biliary atresia were observed in 7 cases whose image investigations showed no obstruction of biliary tract. Nuclear changes, resolution of cytoplasm, inflammatory cell infiltration, collagen fiber proliferation and increased number of lysosomes were observed under electromicroscope. Two cases of glycogen storage disease, 1 case of Niemann-Pick disease and 1 case of lipid storage disease with unknown cause were confirmed by the combination of histological changes and clinical manifestations. Common pathologic changes of liver tissues existed under light microscope or electroscope. The diagnosis of hereditary metabolic disorders could be made increasingly by application of these two technologies in clinical practice. It is difficult to diagnose biliary atresia in early childhood by image investigations and the pathological changes of liver tissues are helpful.

A sub-cellular viscoelastic model for cell population mechanics.

Understanding the biomechanical properties and the effect of biomechanical force on epithelial cells is key to understanding how epithelial cells form uniquely shaped structures in two or three-dimensional space. Nevertheless, with the limitations and challenges posed by biological experiments at this scale, it becomes advantageous to use mathematical and ‘in silico’ (computational) models as an alternate solution. This paper introduces a single-cell-based model representing the cross section of a typical tissue. Each cell in this model is an individual unit containing several sub-cellular elements, such as the elastic plasma membrane, enclosed viscoelastic elements that play the role of cytoskeleton, and the viscoelastic elements of the cell nucleus. The cell membrane is divided into segments where each segment (or point) incorporates the cell's interaction and communication with other cells and its environment. The model is capable of simulating how cells cooperate and contribute to the overall structure and function of a particular tissue; it mimics many aspects of cellular behavior such as cell growth, division, apoptosis and polarization. The model allows for investigation of the biomechanical properties of cells, cell-cell interactions, effect of environment on cellular clusters, and how individual cells work together and contribute to the structure and function of a particular tissue. To evaluate the current approach in modeling different topologies of growing tissues in distinct biochemical conditions of the surrounding media, we model several key cellular phenomena, namely monolayer cell culture, effects of adhesion intensity, growth of epithelial cell through interaction with extra-cellular matrix (ECM), effects of a gap in the ECM, tensegrity and tissue morphogenesis and formation of hollow epithelial acini. The proposed computational model enables one to isolate the effects of biomechanical properties of individual cells and the communication between cells and their microenvironment while simultaneously allowing for the formation of clusters or sheets of cells that act together as one complex tissue.

Human 21T breast epithelial cell lines mimic breast cancer progression in vivo and in vitro and show stage-specific gene expression patterns

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.

PTEN and p53 cross-regulation induced by soy isoflavone genistein promotes mammary epithelial cell cycle arrest and lobuloalveolar differentiation

The tumor suppressors phosphatase and tensin homologue deleted on chromosome ten (PTEN) and p53 are closely related to the pathogenesis of breast cancer, yet pathway-specific mechanisms underlying their participation in mediating the protective actions of dietary bioactive components on breast cancer risk are poorly understood. We recently showed that dietary exposure to the soy isoflavone genistein (GEN) induced PTEN expression in mammary epithelial cells in vivo and in vitro, consistent with the breast cancer preventive effects of soy food consumption. Here, we evaluated PTEN and p53 functional interactions in the nuclear compartment of mammary epithelial cells as a mechanism for mammary tumor protection by GEN. Using the non-tumorigenic human mammary epithelial cells MCF10-A, we demonstrate that GEN increased PTEN expression and nuclear localization. We show that increased nuclear PTEN levels initiated an autoregulatory loop involving PTEN-dependent increases in p53 nuclear localization, PTEN-p53 physical association, PTEN-p53 co-recruitment to the PTEN promoter region and p53 transactivation of PTEN promoter activity. The PTEN-p53 cross talk induced by GEN resulted in increased cell cycle arrest; decreased pro-proliferative cyclin D1 and pleiotrophin gene expression and the early formation of mammary acini, indicative of GEN promotion of lobuloalveolar differentiation. Our findings provide support to GEN-induced PTEN as both a target and regulator of p53 action and offer a mechanistic basis for PTEN pathway activation to underlie the antitumor properties of dietary factors, with important implications for reducing breast cancer risk.

Unregulated ARF6 Activation in Epithelial Cysts Generates Hyperactive Signaling Endosomes and Disrupts Morphogenesis

Tumor development in glandular tissues is associated with structural alterations in the hollow ducts and spherical structures that comprise such tissues. We describe a signaling axis involving sustained activation of the GTP-binding protein, ARF6, that provokes dramatic changes in the organization of epithelial cysts, reminiscent of tumorigenic glandular phenotypes. In reconstituted basement membrane cultures of renal epithelial cysts, enhanced ARF6 activation induces the formation of cell-filled glandular structures with multiple lumens and disassembled cadherin-based cell-cell contacts. All of these alterations are accompanied by growth factor receptor internalization into signaling endosomes and reversed by blocking ARF6 activation or receptor endocytosis. Receptor localization in signaling endosomes results in hyperactive extracellular signal-regulated kinase signaling leading to Bcl-2 stabilization and aberrant cysts. Similarly, formation of hyperproliferative and disorganized mammary acini induced by chronic stimulation of colony-stimulating factor 1 receptor is coupled to endogenous ARF6 activation and constitutive receptor internalization and is reversed by ARF6 inhibition. These findings identify a previously unrecognized link between ARF6-regulated receptor internalization and events that drive dramatic alterations in cyst morphogenesis providing new mechanistic insight into the molecular processes that can promote epithelial glandular disruption.

Krüppel-like Factor 4 Inhibits Epithelial-to-Mesenchymal Transition through Regulation of E-cadherin Gene Expression

The Krüppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. Although KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition. Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing, while chromatin immunoprecipitation identified endogenous KLF4 binding to the GC-rich/E-box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent epithelial-to-mesenchymal transition in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.

Transforming growth factor-beta co-receptor endoglin suppresses breast cancer invasion and metastasis

Transforming growth factor-beta (TGFβ) signaling in cancer has been implicated in both growth suppression in early lesions as well as enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine the signaling output in individual tumors are still largely unknown. In endothelial cells, TGFβ signaling is modulated by the TGFβ co-receptor endoglin (CD105). Here we demonstrate that endoglin is differentially expressed in invasive breast cancers and breast cancer cell lines, and is subject to epigenetic silencing by gene methylation. Downregulation of endoglin expression in nontumorigenic MCF10A cells leads to the formation of abnormal acini in 3D culture but does not promote cell migration or result in cell transformation. In contrast, in the presence of an activated oncogene, loss of endoglin in MCF10A cells leads to enhanced migration and invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in the endoglin-negative MDA-MB-231 cell line blocks TGFβ-enhanced cell motility and invasion and reduces the ability of cells to successfully colonize the lung parenchyma in an in vivo metastasis model. Unlike endothelial cells, endoglin does not does not modulate canonical TGFβ signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, lack of endoglin expression in clinical samples significantly correlates with ENG gene methylation and poor clinical outcome. Together these data identify endoglin as a key component suppressing the invasive activities of breast cancer cells.

Abstract 5056: Signaling pathways critical for insulin receptor substrate (IRS)-mediated disruption of MCF-10A acini formation

Abstract Insulin receptor substrate (IRS)-1 and IRS-2 are important adaptor proteins in insulin-like growth factor-I receptor (IGF-IR) signaling. IRS-1 and IRS-2 are expressed during mammary gland development and also in human breast tumors. In order to study the role of IRS-1 and IRS-2 in breast tumorigenesis, we overexpressed both IRS proteins in immortalized, non-transformed human mammary epithelial MCF-10A cells. It has been shown that MCF-10A cells form uniform, empty-lumen acini in three dimensional (3D) culture. We previously showed that overexpression of either IRS-1 or IRS-2 in MCF-10A cells disrupted the acini in 3D culture and resulted in extensive proliferation and disrupted apical-basal polarity. In both IRS-1 and IRS-2 overexpressing MCF-10A cells, an increase in tyrosine phosphorylation and association with p85 of both IRS proteins indicated that IRS-mediated signaling was activated. siRNA knockdown of the overexpressed IRS proteins reversed the disruption of MCF-10A acini, confirming that this is indeed an IRS-related event. In order to identify the critical effectors in IRS-mediated disruption of MCF-10A polarity and growth, we used small molecule inhibitors to screen multiple kinase signaling pathways upstream and downstream of the IRS proteins. Inhibition of Rac or atypical PKC (aPKC) didn't affect MCF-10A cell acini formation, however it abolished the IRS-mediated disruption of MCF-10A acini formation. Consistent with this, knockdown of Rac and aPKC by siRNA suppressed IRS-mediated disruption in acini formation. Rac belongs to the family of GTPase that regulates cell motility. aPKC is a key component of the PAR polarity complex, and has previously been shown to be involved in IRS-signaling in adipocytes. While the signaling mechanism of Rac and aPKC in the IRSs-induced disruption of acini is still unknown, we found that another component in the PAR polarity complex Par3 may associate with IRS proteins and leads to dissociation of the polarity complex. Taken together, overexpression of IRS-1 and IRS-2 disrupts the MCF-10A acini in 3D culture in part through Rac and aPKC pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5056.

Abstract 463: Hypoxia leads to impaired differentiation and sustained global histone acetylation in immortalized mammary epithelial cells cultured as acini

Abstract We study the effect of hypoxia on mammary epithelial cell differentiation at a non-malignant stage as a putative step in early malignant progression. Due to inadequate vasculature solid tumors often contain regions with low oxygen levels (hypoxia). Hypoxia is associated with increased metastatic potential and worse patient outcome. The hypoxic response is in large driven by the two transcription factors hypoxia inducible factor (HIF) 1 and HIF 2. We have reported that hypoxia leads to a less differentiated phenotype in breast cancer and in neuroblastoma, and in the clinic low tumor cell differentiation is related to poor prognosis in these diseases. To investigate the effect of hypoxia on mammary epithelial cell differentiation we employed the model of acinar morphogenesis. In this model immortalized mammary epithelial cells in three-dimensional (3D) culture on a laminin-rich matrix differentiate and form acini-like structures with evacuated lumen surrounded by polarized cells. We have previously seen that hypoxia (1% oxygen) impairs the process of acinar morphogenesis. The hypoxic cells failed to obtain polarity and markers of differentiation were affected at expression level as well as cellular localization. Addressing the mechanism behind the hypoxia-impaired differentiation, we find that hypoxia impairs the global histone deacetylation that is associated with changes in cell morphology and differentiation of the mammary epithelial cells. Three days post-seeding, during the proliferation stage, both normoxic and hypoxic cells stained positive for acetylated histone 4 (AcH4). When the normoxic cells go through morphological changes and acinar formation the acetylation of histone 4 is reduced whereas it is maintained in the hypoxic cell-clusters 10 days after seeding. Transfer of acini from normoxia to hypoxia at this time-point leads to an increase in AcH4. Thus, the hypoxic condition prevents global deacetylation, which is generally associated with high level of transcription and an undifferentiated proliferative cell state, rendering the cells an open chromatin state and hindering acinar formation. These data indicate that hypoxia promotes loss of differentiation also at a non-malignant stage. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 463.

Abstract 312: P190-B RhoGAP overexpression disrupts mitosis, alters cell polarity, and increases adhesion and invasion in mammary epithelial cells

Abstract Breast cancer causes approximately 40,000 deaths each year in the United States alone. Collective epithelial invasion is an important process in tumor progression, and it also occurs in the normal mammary gland (MG) through specialized structures called terminal end buds (TEBs) that proliferate and migrate to drive ductal outgrowth during MG development. P190-B RhoGAP is highly expressed in TEBs, suggesting it may regulate the invasive process of MG development. Indeed, tetracycline-inducible p190-B overexpression causes perturbations in TEB morphology, stromal composition, and MG branching morphogenesis. In the current study, we analyzed the gene expression patterns of MECs isolated from p190-B overexpressing mice to determine how p190-B regulates epithelial morphogenesis as well as stromal-epithelial interactions. We investigated the expression patterns of p190-B and investigated the effects of overexpression in MCF-7 breast cancer cells. We further investigated the effects of p190-B overexpression on primary MECs in a three-dimensional (3D) culture MEC morphogenesis assay in reconstituted basement membrane (rBM) and in adhesion assays. Microarray results demonstrated that a large number of genes involved in regulating mitotic cell division and the PI3K pathway of proliferation/survival signaling are altered in p190-B overexpressing primary MECs. Real-time PCR confirmed a number of genes are significantly up- or down-regulated in these pathways. Consistent with these findings, p190-B localizes to the mitotic spindle and kinetochores during metaphase of mitosis and to the midbody at cytokinesis. Furthermore, overexpression of p190-B caused abnormal centrosome formation, multipolar spindles, failed cytokinesis, and multinucleation in MCF-7 cells. In addition, primary MECs overexpressing p190-B demonstrated increased adhesion to laminin and type IV collagen, components of the basement membrane, and primary MECs overexpressing p190-B cultured in rBM showed increased disrupted acinar formation, loss of cell polarity, and invasion. P190-B overexpression alters cell polarity and mitosis and regulates adhesive and invasive properties in MECs. These phenotypes may be critical for TEB structure and function during MG branching morphogenesis. We propose that p190-B overexpression may contribute to tumorigenesis by disrupting mitosis and promoting invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 312.

Peroxisome Proliferator-Activated Receptor-γ Inhibits Transformed Growth of Non-Small Cell Lung Cancer Cells through Selective Suppression of Snail

Work from our laboratory and others has demonstrated that activation of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) inhibits transformed growth of non-small cell lung cancer (NSCLC) cell lines in vitro and in vivo. We have demonstrated that activation of PPARγ promotes epithelial differentiation of NSCLC by increasing expression of E-cadherin, as well as inhibiting expression of COX-2 and nuclear factor-κB. The Snail family of transcription factors, which includes Snail (Snail1), Slug (Snail2), and ZEB1, is an important regulator of epithelial-mesenchymal transition, as well as cell survival. The goal of this study was to determine whether the biological responses to rosiglitazone, a member of the thiazolidinedione family of PPARγ activators, are mediated through the regulation of Snail family members. Our results indicate that, in two independent NSCLC cell lines, rosiglitazone specifically decreased expression of Snail, with no significant effect on either Slug or ZEB1. Suppression of Snail using short hairpin RNA silencing mimicked the effects of PPARγ activation, in inhibiting anchorage-independent growth, promoting acinar formation in three-dimensional culture, and inhibiting invasiveness. This was associated with the increased expression of E-cadherin and decreased expression of COX-2 and matrix metaloproteinases. Conversely, overexpression of Snail blocked the biological responses to rosiglitazone, increasing anchorage-independent growth, invasiveness, and promoting epithelial-mesenchymal transition. The suppression of Snail expression by rosiglitazone seemed to be independent of GSK-3 signaling but was rather mediated through suppression of extracellular signal-regulated kinase activity. These findings suggest that selective regulation of Snail may be critical in mediating the antitumorigenic effects of PPARγ activators.

Derepression of HMGA2 via Removal of ZBRK1/BRCA1/CtIP Complex Enhances Mammary Tumorigenesis

The high mobility group AT-hook 2 (HMGA2), a DNA architectural protein, is highly regulated during development and plays an important role in tumorigenesis. Indeed, HMGA2 was overexpressed in many different kinds of tumors. However, the mechanisms regulating HMGA2 expression remain elusive. Using microarray analysis, we found that HMGA2, along with a dozen of other genes, was co-repressed by ZBRK1, BRCA1, and CtIP. BRCA1 exerts its transcriptional repression activity through interaction with the transcriptional repressor ZBRK1 in the central domain, and with CtIP in the C-terminal BRCT domain. Here, we show that ZBRK1, BRCA1, and CtIP form a repression complex that coordinately regulates HMGA2 expression via a ZBRK1 recognition site in the HMGA2 promoter. Depletion of any of the proteins in this complex via adenoviral RNA interference in MCF10A mammary epithelial cells activates HMGA2 expression, resulting in increased colony formation in soft agar. Similarly, depletion of ZBRK1, or ectopic overexpression of HMGA2, in MCF10A cells induces abnormal acinar size with increased cell number and inhibits normal acinar formation. Consistently, many BRCA1-deficient mouse breast tumors express higher levels of HMGA2 than BRCA1-proficient tumors. These results suggest that activation of HMGA2 gene expression through derepression of the ZBRK1/BRCA1/CtIP complex is a significant step in accelerating breast tumorigenesis.

Tükürük Bezinde Pleomorfik Adenoma Tanısında Hangi Sitolojik Özellikler Önemli?

Objective: The aim of this present study is to evaluate which cytologic features of fine needle aspiration cytology (FNAC) are important in the diagnosis of pleomorphic adenoma (PA) in salivary gland. Material and Methods: May-Grunwald-Giemsa stained smears, and hematoxylin and eosin stained paraffin sections of 60 FNAC of parotid (n= 55), submandibular (n= 3) glands and palate (n= 2) were reviewed. Sixty patients, who have both biopsies and FNACs were included in the study. We analyzed eight cytological and three architectural features in a series of 32 FNACs from PA which were correctly diagnosed cytologically (Group A) and compared them with 14 FNACs from non-PA lesions and tumors which were correctly diagnosed cytologically (Group B) and with 14 FNACs from salivary gland tumors or lesions in which, cytological/histological diagnosis were different (Group C). The specimens were evaluated according to the presence or absence of the cytologic features; plasmocytoid appearance and abundant cytoplasm (PAAC), chondromyxoid matrix (CMM), intermingled CMM and tumor cell, spindle cell, oncocytic cell, squamoid cell, bipolar/multipolar or reniform nucleus, and cellular arrangement; acinus formation, large loose clusters with irregular edges, and tumor groups with arborising vascular pattern. Results: The cell which was PAAC, CMM and a mixture of epithelial cells with CMM were selected as the three most predictive parameters of differentiating of non-PA lesions and tumors from PA. Conclusion: In the 60 patients who were suspected of having PA, a correct diagnosis was made by a combination of the above features. The sensitivity of this procedure for PA was 100%, and the specificity was 92%.

Induction of PTEN-p53 Crosstalk in Mammary Epithelial Cells: A Novel Mechanism of Breast Cancer Prevention by the Dietary Factor Genistein.

Abstract Consumption of soy foods either at an early age or for lifetime has been associated with reduced risk for developing breast cancer in humans and in animal models. However, this association continues to be controversial and the precise mechanisms for protection remain elusive. Among the soy products, the isoflavone genistein (GEN) has been widely suggested to confer mammary tumor protection. Previously we demonstrated the increased expression of tumor suppressors PTEN and p53 in mammary epithelial cells (MECs) isolated from young adult female rats fed dietary soy protein isolate (SPI) or casein (CAS) supplemented with GEN, when compared to MECs from rats fed the control (CAS) diet. Since NMU-administered rats fed SPI had reduced tumor incidence and increased tumor latency than those fed CAS, PTEN and p53 likely mediate the observed tumor resistance with SPI in vivo. We hypothesized that GEN induction of PTEN and p53 in MECs results in the formation of a PTEN/p53 functional complex to negatively regulate breast cancer development. Here, we used the human non-tumorigenic, ER-negative mammary epithelial cell line, MCF-10A, as an in vitro system to mechanistically dissect ER-independent actions of GEN involving PTEN and p53. GEN (40 nM, 2μM) augmented PTEN and p53 expression in treated relative to control cells. GEN also induced nuclear co-localization and physical association of PTEN and p53. To test a functional consequence of GEN-induced PTEN/p53 cross-talk on mammary epithelial phenotype, we analyzed GEN effects on cell cycle progression and acini formation in 3D cultures. Our results showed attenuated cell proliferation and lower cyclin D1 and pleiotrophin transcript levels in GEN-treated cells, which were abrogated by small interfering RNA to PTEN, indicating PTEN-dependence. Using FACS analysis, we showed that GEN induced cell cycle arrest at G0-G1 phase. Treatment with GEN promoted early acini formation of MECs grown in Matrigel, which temporally coincided with PTEN-dependent suppression of p21 and p27 transcript levels. Further analyses of GEN effects on MECs demonstrated induction by GEN of PTEN promoter-luc reporter activity as measured by dual-luciferase assay. Interestingly, treatment with siRNA to either PTEN or p53 reduced basal and GEN-induced PTEN promoter activity. Given that p53 binds to the PTEN promoter, our results suggest a feed-forward cycle in which dietary factor (GEN) induction of nuclear PTEN leads to PTEN promotion of its own signaling. By maintaining a stable pool of nuclear p53 to boost its transcription, PTEN ensures its continuous expression in MECs to favor cell differentiation. These data elucidate a novel mechanism by which dietary factors with PTEN-inducing activity may attenuate breast cancer risk and development. Funding by USDA-CRIS 6251-5100002-06S and the Department of Defense Breast Cancer Program (0810548). Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3149.

Abstract IA-13: Mechanisms regulating anchorage independent survival

Abstract One of the earliest recognized features of tumor cells in culture is their ability to proliferate and survive without attachment to extracellular matrix. This ‘anchorage independence’ of tumor cells distinguishes their behavior from normal cells and is believed to be important to allow tumor cells to proliferate outside of their natural niches during early stages of tumor formation as well as during invasion and metastasis. My presentation will focus on findings from our laboratory on the mechanisms responsible for death of cells deprived of matrix and how oncogenes and other factors promote anchorage independence. We have found that three distinct death processes — apoptosis, metabolic impairment, and a novel non-apoptotic mechanism, referred to as entosis, involving invasion of one cell within another and lysosomal degradation — contribute to death of cells deprived of their appropriate matrix. The major topic of my presentation will be new unpublished findings on metabolic pathways that are regulated by matrix attachment. We have found that detachment of mammary epithelial cells from ECM causes an ATP deficiency due to loss of glucose transport. Overexpression of ErbB2 rescues the ATP deficiency by restoring glucose uptake through PI(3)K activation. Interestingly, we found that the ATP deficiency could also be rescued by antioxidant treatment without rescue of glucose uptake. This restoration was found to be dependent on stimulation of fatty acid oxidation (FAO), which is inhibited by detachment-induced reactive oxygen species (ROS). The significance of these findings was supported by studies in 3D acinar cultures and anchorage-independent colony formation. These results reveal both the importance of matrix attachment in regulating metabolic activity and an unanticipated mechanism for cell survival in altered matrix environments through antioxidant restoration of ATP generation. Citation Information: Cancer Res 2009;69(23 Suppl):IA-13.

Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis

The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.

Interaction of E-cadherin and PTEN Regulates Morphogenesis and Growth Arrest in Human Mammary Epithelial Cells

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a dual-function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two nonmalignant human mammary epithelial cell lines that form polarized, growth-arrested structures (acini) when cultured in three-dimensional laminin-rich extracellular matrix gels (lrECM). As acini begin to form, PTEN accumulates both in the cytoplasm and at cell-cell contacts where it colocalizes with the E-cadherin/beta-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in Skbr-3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in three-dimensional lrECM, indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus seems to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.

Novel egg white based 3D cell culture system for breast cancer research.

Abstract Abstract #4060 Introduction: Over the last few years, much attention has been paid to the development and utilization of three-dimensional (3D) cell culture to replace classical two-dimensional (2D) monolayer cell culture systems grown as a flat layer on plastic. Not completely surprisingly, there are tremendous differences between cells in 2D and 3D culture. However, currently available 3D cell culture media are expensive for regular use by the majority of research laboratories and thus large scale use of 3D cell culture system is presently cost prohibited. In our search for a reliable and economically viable replacement for the expensive commercial 3D media, we hypothesized that avian egg white could be a potential alternative.
 Methods: Egg white-based chamber slide: Separated egg white from the chicken eggs and coated each wells of an eight well culture chamber with 80μl of egg white by careful heating at 600C on a heating block. After 30-60 minutes, the egg white becomes a semi-solid that adheres to the bottom of the well. The unstuck egg white is washed out by adding 500µl of growth medium and removing it slowly using a Pasteur pipette.
 Cell culture and analysis: To each well 2x103 immortalized human breast epithelial cells (MCF10A), were added in 0.5ml of appropriate culture medium. We have analyzed the growth curve of the acini, lumen formation, apoptosis and cell proliferation at different days post-culture. Apico/basal polarization of the acini was analyzed using appropriate antibodies. We have also cultured IGFR1-transformed MCF10A cells, different established human cell lines (MCF7, HEK293, HeLa, LNCaP, and Saos-2), and MMTV-PyMT transformed mouse mammary epithelial cells in their appropriate growth media. For comparative studies, we used a well-established reconstituted basement membrane matrix preparation (BM).
 Results: Our analysis shows that this simple avian egg white based system supports growth of cells in 3D, with significantly decreased cost. Specifically, the growth of MCF10A in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apico/basal polarization comparable to what has been described using established 3D culture media. There was no significant difference in MCF10A proliferation and acini size between egg white and BM. We have also observed similar morphology for different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells between egg white and BM.
 Conclusion: Our data convincingly argue that egg white can be used as a suitable alternative model for 3D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3D cell culture on a regular basis, and thus result in a dramatic increase of utilization of the 3D cell culture in research. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4060.

Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis

Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis

TEAD1 and c-Cbl are novel prostate basal cell markers that correlate with poor clinical outcome in prostate cancer.

Prostate cancer is the most frequently diagnosed male cancer, and its clinical outcome is difficult to predict. The disease may involve the inappropriate expression of genes that normally control the proliferation of epithelial cells in the basal layer and their differentiation into luminal cells. Our aim was to identify novel basal cell markers and assess their prognostic and functional significance in prostate cancer. RNA from basal and luminal cells isolated from benign tissue by immunoguided laser-capture microdissection was subjected to expression profiling. We identified 112 and 267 genes defining basal and luminal populations, respectively. The transcription factor TEAD1 and the ubiquitin ligase c-Cbl were identified as novel basal cell markers. Knockdown of either marker using siRNA in prostate cell lines led to decreased cell growth in PC3 and disrupted acinar formation in a 3D culture system of RWPE1. Analyses of prostate cancer tissue microarray staining established that increased protein levels of either marker were associated with decreased patient survival independent of other clinicopathological metrics. These data are consistent with basal features impacting on the development and clinical course of prostate cancers.