Acinar Formation Research Articles

Effect of ionizing radiation on acinar morphogenesis of human prostatic epithelial cells under three-dimensional culture conditions

Homeostasis is maintained by the interplay of multiple factors that directly or indirectly regulate cell proliferation and cell death. Complex multiple interactions between cells and the extracellular matrix occur during acinar morphogenesis and changes in these might indicate carcinogenesis of cells from a normal to a malignant, invasive phenotype. In this study, the human prostatic epithelial cell line RWPE-1 was cultured under three-dimensional (3-D) culture conditions, and the effect of ionizing radiation on acinar morphogenesis and its association with autophagy were discussed. The results illustrated that formation of specific spheroid (acinar) structures was detectable under 3-D culture conditions. Radiation induced the disruption of acini in different cell models using either gene overexpression (Akt) or gene knock-down (Beclin 1 and ATG7). Introduction of Akt not only accelerated the growth of cells (i.e., caused the cells to manifest elongating and microspike-like structures that are obviously different from structures seen in wild-type RWPE-1 cells under two-dimensional conditions), but also changed their morphological characteristics under 3-D culture conditions. Knock-down of autophagy-related genes (Beclin 1 and ATG7) increased the radiosensitivity of cells under 3-D culture conditions, and cells died of non-apoptotic death after radiation. The results suggested that ionizing radiation may change the cell phenotype and the formation of acini. Additionally even the autophagy mechanism may play a role in these processes.

Myoepithelial hamartoma as a solitary mass in the pancreatic parenchyma: the first case report

Solid hamartoma of the pancreas is very rare, and only 3 cases have been reported thus far. A patient underwent pancreaticoduodenectomy due to a mass in the head of the pancreas which was suspected to be a borderline malignant tumor, but the histologic diagnosis turned out to be myoepithelial hamartoma (MEH) or adenomyoma. It was characterized by benign duct and glandular structures surrounded by proliferating smooth muscle, and acinus formation was not observed. Immunohistochemical stain for smooth muscle actin (SMA) was positive in spindle cells, and CD34 was negative, differentiating it from the three previously reported cases of solid hamartoma of the pancreas. MEH is an entity that is on the same spectrum as heterotopic pancreas. MEH is rare and has usually been reported in the gastrointestinal tract. To the best of our knowledge, MEH has never been reported in the pancreas. Therefore we report the world's first documented case of MEH of the pancreas.

Atypical Neurofibroma and Osteosclerotic Metastasis

35-year-old male presented with multiple swellings in left leg, headache, weakness of limbs for 4 months, and blurring of vision for the last 15 days. On examination, he was pale, cachexic with generalized lymphadenopathy and lower motor neuron type weakness of limbs sparing right upper limb. Blood investigations showed anemia with high alkaline phosphatase. Chest radiograph revealed osteosclerotic metastatic lesion in humerus. Biopsy of leg lesion revealed atypical neurofibroma. Computed tomography (CT) of thorax revealed osteoblastic metastasis. Bone marrow aspiration showed cells with round to oval nuclei, fine granular chromatin with large central prominent nucleoli and eosinophilic cytoplasm with acini formation. Magnetic resonance imaging (MRI) of brain and spinal cord defined metastatic leptomeningeal deposits. Cerebrospinal fluid (CSF) cytology was positive for malignant cells. Gastroscopy showed an ulceroinfiltrative growth from stomach which on histopathology revealed diffuse adenocarcinoma. Palliative treatment was given with intrathecal methotrexate and systemic corticosteroid with chemotherapy. Patient's symptom improved drastically, but we lost him to followup.

P5-07-05: Hypoxic Mammary Epithelial Cells in Three-Dimensional Culture Develop a Cancer-Like Phenotype.

Abstract Due to inadequate vascularization and tumor cell proliferation solid tumors are hypoxic compared to corresponding normal tissues. Tumor hypoxia has been associated with increased metastatic potential and worse patient outcome. The hypoxic response is mainly driven by the hypoxia inducible transcription factors; HIF −1α and HIF-2α. We have reported that hypoxia leads to a less differentiated phenotype in breast cancer and neuroblastoma, and in the clinic low tumor cell differentiation is related to poor prognosis in these diseases. We have shown that high levels of HIF-2α protein correlate to distant metastasis and poor survival in invasive breast cancer. The aim of the present study was to investigate the effect of hypoxia on mammary epithelial cell differentiation and epithelial organization. We employed the well-characterized model of acinar morphogenesis on extra cellular matrix substrate. In this model mammary epithelial cells that are cultured in three-dimensional culture on a laminin-rich matrix differentiate and form acini-like structures with evacuated lumen surrounded by polarized palisade cells. We found that hypoxia (1% oxygen) impaired the process of acinar morphogenesis and the hypoxic cells failed to obtain polarity and that markers of differentiation were affected at expression levels as well as cellular localization. The hypoxic cells remained proliferative whereas normoxic cells lost Ki-67 expression within a week. We found that hypoxia impaired the global histone deacetylation that has been shown by others to take place in the process of acini formation in mammary epithelial cells. Three days post-seeding, during the proliferation stage, both normoxic and hypoxic cells stained positive for acetylated histone 4. When the normoxic cells went through morphological changes and acinar formation, the acetylation of histone 4 was reduced whereas it was maintained in the hypoxic cell-clusters at day 10 post-seeding. Thus, the hypoxic conditions maintained the epithelial cells in an acetylated state, which is generally associated with an open chromatin structure, high level of transcription and an undifferentiated stage. These data indicate that hypoxia promotes loss of differentiation also at a non-malignant stage and render the cells a cancer-like phenotype. We are currently extending this study in primary cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-07-05.

P2-02-08: Int6 Regulates Both Proteasomal Degradation and Translation Initiation and Is Critical for Proper Formation of Acini by Human Mammary Epithelium.

Abstract The mammalian INT6/EIF3E gene has been implicated in breast tumor formation, but its functional activities remain poorly defined. Consistent with the fact that Int6 is frequently down-regulated in human breast tumors, we found that repressing INT6 expression induced transformed properties in normal human mammary epithelial cells (MCF10A) in both 2D and 3D cultures. Int6 contains a PCI domain, found in several components of both the proteasome and the translation initiation factor eIF3, suggesting that it can functionally interact with both. Indeed, our data show that Int6 associates with proteasomes in human cells, and that INT6 knockdown prevents proper assembly of active proteasomes. In addition, we show that down-regulation of INT6 reduces translation initiation (both polysome formation and global protein synthesis). However, Int6 regulates translation selectively, reducing translation from cap-dependent and Bcl2- IRES reporters, but stimulating translation from the CVB3 IRES. These data collectively suggest that Int6 controls both protein synthesis and protein degradation, and is thus capable of fine-tuning protein levels. Abnormal levels of regulatory proteins caused by Int6 abnormalities may thus be responsible for disrupting normal morphogenesis of mammary epithelial cells and causing transformed phenotypes. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-08.

TRIM29 Functions as a Tumor Suppressor in Nontumorigenic Breast Cells and Invasive ER+ Breast Cancer

Tripartite motif-containing 29 (TRIM29) is a member of the TRIM protein family that has been implicated in hematologic and solid tumor cancers. We found that TRIM29 functions as a tumor suppressor in both the nontumorigenic MCF10A [estrogen receptor (ER)-/TRIM29+] breast cell line and the invasive MCF7 (ER+/TRIM29-) breast cell line. Silencing TRIM29 in MCF10A cells resulted in preneoplastic changes that included loss of polarity in three-dimensional culture, increased proliferation, anchorage-independent growth, and increased migration and invasion. Conversely, the introduction of TRIM29 into MCF7 cells caused reversion to a less aggressive phenotype by antagonizing the growth effect of 17β-estradiol. The interaction between TRIM29 and ER signaling in MCF7 cells was supported by a reduction in ERE binding in the presence of TRIM29 and suppression of ER-dependent gene expression of TFF1, FOS, and GREB1. By microarray analyses, we showed that younger women (<55 years of age) with early-stage, ER+ breast cancer who were given no adjuvant systemic therapy had a significantly lower risk of relapse when their tumor had high TRIM29 expression (P = 0.02). This effect was not observed in older women (>55 years of age) and thus may be due to menopause and loss of circulating estrogens. Our results suggest that loss of TRIM29 expression in normal breast luminal cells can contribute to malignant transformation and lead to progression of ER+ breast cancer in premenopausal women.

Inhibitory Activity of YKL-40 in Mammary Epithelial Cell Differentiation and Polarization Induced by Lactogenic Hormones: A Role in Mammary Tissue Involution

We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells.

The Rho GTPase Cdc42 is required for primary mammary epithelial cell morphogenesis in vitro

The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast cancer, and several studies have described mechanisms by which it may promote tumor formation and progression. However, little is known about the role of Cdc42 during normal mammary epithelial cell (MEC) morphogenesis. Here we aimed to define the precise role for Cdc42 during primary mammary acinus formation in vitro. For these studies, MECs were isolated from Cdc42fl/fl conditional knockout mice, transduced with Adeno-cre-GFP virus to delete Cdc42 or Adeno-GFP control virus, and effects on morphogenesis were investigated using a three-dimensional (3D) culture assay. Interestingly, markedly fewer mammary acini developed in Cdc42 deficient cultures, and the acini that formed were significantly smaller and disorganized. Cellular proliferation and survival were reduced in the Cdc42 deficient acini. However, control and knockout MECs cultured as monolayers displayed similar cell cycle profiles, suggesting that Cdc42 is important for MEC proliferation in the context of 3D polarity. Overexpression of cyclin D1, which promotes cell cycle progression downstream of Cdc42, failed to rescue the defect in acinus size. Furthermore, lumen formation and apical-basal polarity were disrupted, and mitotic spindle orientation and Cdc42/aPKC polarity complex defects likely contributed to these phenotypes. Studies using dominant negative Cdc42 and siRNA to knockdown Cdc42 in MDCK and Caco-2 cell lines undergoing cystogenesis in 3D cultures revealed critical roles for Cdc42 in spindle orientation, polarity, and lumen formation. Our studies, using complete knockout in primary epithelial cells, demonstrate that Cdc42 is not only an important regulator of polarity and lumen formation; it is also essential for proliferation and survival, which are key cellular processes that drive MEC morphogenesis in vitro and in vivo.

Metastatic mixed acinar‐neuroendocrine carcinoma of the pancreas to the liver: A cytopathology case report with review of the literature

A case of metastatic mixed acinar-neuroendocrine carcinoma (MANEC) of the pancreas to the liver is reported. A diagnostic percutaneous US-guided FNA and core biopsy of a liver nodule was performed. The FNA smears were cellular and showed neoplastic cells in clusters with acinar formation, isolated single cells, and scattered naked nuclei. The cytoplasm was finely granular. The nuclei were relatively uniform, some with speckled chromatin and prominent nucleoli. The immunohistochemistry performed on the cell block showed strong positivity for cytokeratin AE1/AE3, chromogranin, and synaptophysin. Furthermore, the tumor cells were weakly positive for α1-antichymotrypsin. The Ki-67 mitotic index was up to 50%. Based on the morphology and supporting immunohistochemical stains, the final cytopathologic diagnosis rendered was "Positive for malignant cells. Carcinoma with mixed acinar and endocrine features." To our knowledge, this is the first report of a metastatic MANEC to the liver diagnosed based on cytology with confirmatory histology. The difficulties in the cytopathologic diagnosis and differential diagnosis of MANEC are discussed in this article.

The lipid kinase PI4KIIIβ and the eEF1A2 oncogene co-operate to disrupt three-dimensional in vitro acinar morphogenesis

The study of in vitro morphogenesis is fundamental to understanding neoplasia since the dysregulation of morphogenic pathways that create multi-cellular organisms is a common hallmark of oncogenesis. The in vitro culture of human breast epithelial cells on reconstituted basement membranes recapitulate some features of in vivo breast development, including the formation of a three-dimensional structure termed an acinus. Importantly, the capacity to disrupt in vitro acinar morphogenesis is a common property of human breast oncogenes. In this report, we find that phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar devlopment. Furthermore, expression of an oncogenic PI4KIIIβ activator, eEF1A2 (eukaryotic elongation factor 1 alpha 2), phenocopies the PI4KIIIβ multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters the localization of PI(4)P and PI(4,5)P 2 within acini, indicating the importance of these lipids in acinar development. Our work shows that PI4KIIIβ, eEF1A2 and PI(4)P likely play an important role in mammary neoplasia and acinar development.

Use of gene expression signature to predict response to combination chemotherapy in breast cancer clinical subgroups.

e21071 Background: Accurate stratification of patients prior to treatment with combination chemotherapy would allow non-responders to receive an alternative treatment in a timely manner and potentially improve clinical outcomes. We describe a 22-gene signature that accurately predicts response to antimitotic combination chemotherapy for breast cancer. This signature was selected in a well characterized system that models a key step disrupted in tumorigenesis: the formation of spatially accurate mammary ductal units by breast epithelial cells. Hence the 22 genes represent a biological process that is independent of any specific patient set or predefined clinical classification. Methods: To determine whether genes with differential expression during human mammary acinar morphogenesis predict response to combination chemotherapy in breast cancer, we have analyzed results from two published microarray datasets (Fournier, et al., 2006; Popovici et al., 2010). Expression levels of the majority of genes that were coordinately down regulated during acini formation were significantly associated with response to combination chemotherapy treatment. A 22-gene signature representing the down regulated genes was evaluated independently in each of three breast cancer clinical subgroups, ER-positive (n=146), HER2-positive (n=41), and triple negative (n=90) using two methods of analysis, hierarchical clustering and logistic regression. Results: Hierarchical cluster analysis results showed that the 22 genes accurately stratified patients in each of the three subgroups by response (Fisher’s Exact p<0.05). Logistic regression with 3-fold cross validation demonstrated that different models accurately predicted response in these subgroups (AUC ≥0.7). We have observed that the 22-gene signature is broadly effective across independent patient clinical subgroups in its ability to stratify patients according to chemotherapy response in breast cancer. Conclusions: The 22-gene signature has the potential to provide patients, early in the care process, with accurate and personalized information to predict response to combination chemotherapy.

Mutant p53 Disrupts MCF-10A Cell Polarity in Three-dimensional Culture via Epithelial-to-mesenchymal Transitions

Mutant p53 is not only deficient in tumor suppression but also acquires additional activity, called gain of function. Mutant p53 gain of function is recapitulated in knock-in mice that carry one null allele and one mutant allele of the p53 gene. These knock-in mice develop aggressive tumors compared with p53-null mice. Recently, we and others showed that tumor cells carrying a mutant p53 are addicted to the mutant for cell survival and resistance to DNA damage. To further define mutant p53 gain of function, we used the MCF-10A three-dimensional model of mammary morphogenesis. MCF-10A cells in three-dimensional culture undergo a series of morphological changes and form polarized and growth-arrested spheroids with hollow lumen, which resembles normal glandular architectures in vivo. Here, we found that endogenous wild-type p53 in MCF-10A cells was not required for acinus formation, but knockdown of endogenous wild-type p53 (p53-KD) led to partial clearance of cells in the lumen due to decreased apoptosis. Consistent with this, p53-KD altered expression patterns of the cell adhesion molecule E-cadherin, the cytoskeletal marker β-catenin, and the extracellular matrix protein laminin V. We also found that ectopic expression of the mutant G245S led to a phenotype similar to p53-KD, whereas a combination of ectopic expression of siRNA-resistant G245S with p53-KD led to a less cleared lumen. In contrast, ectopic expression of mutant R248W, R175H, and R273H disrupted normal acinus architectures with filled lumen and led to formation of irregular and multiacinus structures regardless of p53-KD. In addition, these mutants altered normal expression patterns and/or levels of E-cadherin, β-catenin, laminin V, and tight junction marker ZO-1. Furthermore, epithelial-to-mesenchymal transitions (EMT) markers, Snail, Slug, and Twist, were highly induced by mutant p53 and/or p53-KD. Together, we postulate that EMT represents a mutant p53 gain of function and mutant p53 alters cell polarity via EMT.

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.

Abstract 2061: Impaired acinar morphogenesis and cancer-like phenotype in hypoxic mammary epithelial cells

Abstract Due to inadequate vascularization and tumor cell proliferation solid tumors are hypoxic compared to corresponding normal tissues. Tumor hypoxia has been associated with increased metastatic potential and worse patient outcome. The hypoxic response is mainly driven by the hypoxia inducible transcription factors; HIF -1α and HIF-2α. We have reported that hypoxia leads to a less differentiated phenotype in breast cancer and neuroblastoma, and in the clinic low tumor cell differentiation is related to poor prognosis in these diseases. We have shown that high levels of HIF-2α protein correlate to distant metastasis and poor survival in invasive breast cancer. The aim of the present study was to investigate the effect of hypoxia on mammary epithelial cell differentiation and epithelial organization. We employed the well-characterized model of acinar morphogenesis on extra cellular matrix substrate. In this model mammary epithelial cells MCF-10A that are cultured in three-dimensional culture on a laminin-rich matrix differentiate and form acini-like structures with evacuated lumen surrounded by polarized palisade cells. We found that hypoxia (1% oxygen) impaired the process of acinar morphogenesis and the hypoxic cells failed to obtain polarity and that markers of differentiation were affected at expression levels as well as cellular localization. The hypoxic cells remained proliferative whereas normoxic cells lost Ki-67 expression within a week. Addressing the mechanism behind the loss of differentiation in hypoxia, we found that hypoxia impaired the global histone deacetylation that has been shown by others to take place in the process of acini formation in mammary epithelial cells. Three days post-seeding, during the proliferation stage, both normoxic and hypoxic cells stained positive for acetylated histone 4. When the normoxic cells went through morphological changes and acinar formation the acetylation of histone 4 was reduced whereas it was maintained in the hypoxic cell-clusters at day10 post-seeding. Thus, the hypoxic conditions prevented global deacetylation, which is generally associated with high level of transcription and an undifferentiated proliferative cell stage, rendering the cells an open chromatin and hindering acinar formation. Transcription factors of the basic helix-loop-helix family are instrumental in mammary epithelial differentiation. The Inhibitor of Differentiation, ID, proteins are modulators of basic helix-loop-helix transcription factor activity and are reportedly hypoxia-induced. We therefore investigated their expression in the hypoxic cells and found increased expression of ID1, known to inhibit mammary epithelial differentiation and promote proliferation. These data indicate that hypoxia promotes loss of differentiation also at a non-malignant stage and render the cells a cancer-like phenotype. We are currently extending this study in primary cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2061. doi:10.1158/1538-7445.AM2011-2061

Abstract 5093: The molecular genetics of radiogenic breast cancer

Abstract The aim of this study is to identify the underlying molecular genetic mechanisms of radiation induced breast carcinogenesis. Studies have shown that women exposed to ionising radiation at a young age are more at risk of developing breast cancer than older women exposed to the same level of radiation. Higher levels of estrogen are present in young women and estrogen has a known transforming effect on breast epithelial cells. One hypothesis suggests that radiation and estrogen synergise to drive breast epithelial cell transformation. We have developed an in vitro model of radiation-induced breast epithelial cell transformation in order to investigate genetic alterations associated with breast cell transformation. The immortalised, non-transformed breast epithelial cell line MCF-10A was exposed to fractionated doses of X-rays in the presence or absence of exogenous estrogen. We have shown that radiation treated cells display evidence of transformation, including loss of contact inhibition, increased cell invasion, disrupted acini formation and tumor formation in immunocompromised Rag2−/− γ−/− mice. Analysis of radiation treated cells by SNP array karyotyping identified focal gene deletion (EP300 and OCT-1) and amplification (c-MYC) which may be linked to radiation-induced breast cell transformation. Gene deletion and amplification was confirmed by Fluorescent In Situ Hybridization analysis and alterations in expression have been confirmed by western analysis. EP300 is a transcriptional co-activator and a putative tumor suppressor. Somatic mutations in EP300 have been found in solid tumours and the gene is located in a region of chromosome 22 affected by loss of heterozygosity in numerous cancers. Oct-1 encodes an octamer binding transcription factor that has been associated with regulating DNA damage response through interactions with BRCA1 and GADD45. c-MYC is a known proto-oncogene that encodes a transcription factor involved in cell proliferation, cell cycle regulation and apoptosis. Analysis of our in vitro model of radiation-induced breast epithelial cell transformation has identified genetic alterations that have established roles in mediating cellular response to DNA damage and which might be key events in the development of radiogenic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2011-5093

C-Jun N-terminal kinase activity supports multiple phases of 3D-mammary epithelial acinus formation

Primary murine mammary epithelial cells cultured on a laminin-rich-extracellular matrix (ECM) require c-Jun N-terminal kinase (JNK) activity for acinus formation. Inhibition of JNK (using SP600125) or small interfering RNA-mediated knockdown of JNK1 blocked acinus formation, impaired cell polarisation and lumen clearance and allowed sustained extracellular signal-regulated kinase (ERK) phosphorylation, cell proliferation, adhesion-independent cell survival and expression of epithelial-mesenchymal transition markers. ERK inhibition abolished the effects of JNK blockade. Interestingly, inhibition of JNK from the time of cell seeding blocked cell polarisation and lumen clearance; later inhibition (≥ 6 h) only affected lumen clearance. ERK inhibition effectively protected cell polarisation but less so, lumen clearance. SP600125-treatment similarly affected acinus formation by the 'normal' human mammary epithelial MCF10A cell line. Expression of dominant-negative JNK1 in MCF10A cells also undermined acinus formation, generating large 'multi-acinar spheres' whose formation is probably driven by excessive luminal cell proliferation and cell survival. As JNK activity must be suppressed from the time of cell seeding to block cell polarisation, we studied the behaviour of MCF10A cells immediately after seeding in laminin rich matrix: we detected engagement of cells with the matrix, early polarisation, movement of cells into clusters and 'epithelial-cell- like' behaviour of clustered cells. Inhibition of JNK activity or expression of dominant-negative JNK1 allowed cell engagement to the matrix, but blocked cell polarisation and all subsequent 'behaviours'. While integrin activation occurred, tyrosine-phosphorylation of paxillin, Fak and Src was significantly damped by JNK inhibition. These results emphasise the multi-phase dependency of the organisation of mammary cells in 3D on JNK activity and suggest a 'permissive' support of ECM-integrin 'outside-in' signalling and a 'damping' of growth-factor ERK signalling as its two key cell physiological effects.

KLF8 promotes human breast cancer cell invasion and metastasis by transcriptional activation of MMP9

Epithelial to mesenchymal transition (EMT) and extracellular matrix degradation are critical for the initiation and progression of tumor invasion. We have recently identified Krüppel-like factor 8 (KLF8) as a critical inducer of EMT and invasion. KLF8 induces EMT primarily by repressing E-cadherin transcription. However, how KLF8 promotes invasion is unknown. Here we report a novel KLF8-to-MMP9 signaling that promotes human breast cancer invasion. To identify the potential KLF8 regulation of MMPs in breast cancer, we established two inducible cell lines that allow either KLF8 overexpression in MCF-10A or knockdown in MDA-MB-231 cells. KLF8 overexpression induced a strong increase in MMP9 expression and activity as determined by quantitative real-time PCR and zymography. This induction was well correlated with the MMP inhibitor-sensitive Matrigel invasion. Conversely, KLF8 knockdown caused the opposite changes that could be partially prevented by MMP9 overexpression. Promoter-reporter assays and chromatin and oligonucleotide precipitations determined that KLF8 directly bound and activated the human MMP9 gene promoter. Three-dimensional (3D) glandular culture showed that KLF8 expression disrupted the normal acinus formation which could be prevented by the MMP inhibitor, whereas KLF8 knockdown corrected the abnormal 3D architecture which could be protected by MMP9 overexpression. KLF8 knockdown promoted MDA-MB-231 cell aggregation in suspension culture which could be prevented by MMP9 overexpression. KLF8 knockdown inhibited the lung metastasis of MDA-MB-231 cells in nude mice. Immunohistochemical staining strongly correlated the co-expression of KLF8 and MMP9 with the patient tumor invasion, metastasis and poor survival. Taken together, this work identified the KLF8 activation of MMP9 as a novel and critical signaling mechanism underlying human breast cancer invasion and metastasis.

Endoglin expression in breast tumor cells suppresses invasion and metastasis and correlates with improved clinical outcome

Tumor growth factor-β (TGF-β) signaling in cancer has been implicated in growth suppression of early lesions and enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine this signaling output in individual tumors are still largely unknown. In endothelial cells, TGF-β signaling is modulated by the TGF-β co-receptor endoglin (CD105). Here we demonstrate that endoglin is expressed in a subset of invasive breast cancers and cell lines and is subject to epigenetic silencing by gene methylation. Endoglin downregulation in non-tumorigenic MCF10A breast cells leads to the formation of abnormal acini in 3D culture, but does not promote cell migration or transformation. In contrast, in the presence of activated ErbB2, endoglin downregulation in MCF10A cells leads to enhanced invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in MDA-MB-231 cells blocks TGF-β-enhanced cell motility and invasion and reduces lung colonization in an in vivo metastasis model. Unlike endothelial cells, endoglin does not modulate Smad-mediated TGF-β signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, in a large cohort of invasive breast cancers, lack of endoglin expression in the tumor cell compartment correlates with ENG gene methylation and poor clinical outcome.

PKCζ regulates cell polarisation and proliferation restriction during mammary acinus formation

Mammary epithelial cells organize in three dimensions and generate acini when supported on laminin-rich extracellular matrix. Acinus formation begins with the apicobasal polarisation of the outer cells of the assembly and the withdrawal of these cells from the cell cycle. Internal cells then clear out to form a hollow lumen. Here, we show that PKCζ is phosphorylated (at T410) and activated in the early stages of acinus formation in both primary cells and MCF10A cells, and during mammary tree maturation in vivo. Phospho-PKCζ colocalised with tight junction components and bound to the Par polarising complex in developing acini. To further investigate the importance of PKCζ phosphorylation in this context, acinus formation was studied in MCF10A cells overexpressing non-phosphorylatable (T410A) or 'constitutively phosphorylated' (T410E) PKCζ. In both cell types, acinus-associated cell polarisation and lumen clearance were compromised, emphasising the importance of regulated phosphorylation of PKCζ at T410 for successful acinus formation. PKCζ can be activated in a phosphorylation (at T410)-dependent and a phosphorylation-independent manner. Cells overexpressing a complete kinase-deficient PKCζ (K281W) displayed a cell polarising deficit, but also generated large 'multi-acinar' structures with associated early lumenal cell hyperproliferation. Therefore our data shows, for the first time, that two separable PKCζ activities (one phosphorylation-dependent, the other not) are required to support the cell polarisation and proliferation restriction that underpins successful acinus formation. Paralleling these contributions, we found that low levels of PKCζ mRNA expression are associated with more 'poorly differentiated' tumours and a poor outcome in a cohort of 295 breast cancer patients.

PKCζ regulates cell polarisation and proliferation restriction during mammary acinus formation

Mammary epithelial cells organize in three dimensions and generate acini when supported on laminin-rich extracellular matrix. Acinus formation begins with the apicobasal polarisation of the outer cells of the assembly and the withdrawal of these cells from the cell cycle. Internal cells then clear out to form a hollow lumen. Here, we show that PKCζ is phosphorylated (at T410) and activated in the early stages of acinus formation in both primary cells and MCF10A cells, and during mammary tree maturation in vivo. Phospho-PKCζ colocalised with tight junction components and bound to the Par polarising complex in developing acini. To further investigate the importance of PKCζ phosphorylation in this context, acinus formation was studied in MCF10A cells overexpressing non-phosphorylatable (T410A) or 'constitutively phosphorylated' (T410E) PKCζ. In both cell types, acinus-associated cell polarisation and lumen clearance were compromised, emphasising the importance of regulated phosphorylation of PKCζ at T410 for successful acinus formation. PKCζ can be activated in a phosphorylation (at T410)-dependent and a phosphorylation-independent manner. Cells overexpressing a complete kinase-deficient PKCζ (K281W) displayed a cell polarising deficit, but also generated large 'multi-acinar' structures with associated early lumenal cell hyperproliferation. Therefore our data shows, for the first time, that two separable PKCζ activities (one phosphorylation-dependent, the other not) are required to support the cell polarisation and proliferation restriction that underpins successful acinus formation. Paralleling these contributions, we found that low levels of PKCζ mRNA expression are associated with more 'poorly differentiated' tumours and a poor outcome in a cohort of 295 breast cancer patients.