Multicellular Architecture of Malignant Breast Epithelia Influences Mechanics

Cell–matrix and cell–cell mechanosensing are important in many cellular processes, particularly for epithelial cells. A crucial question, which remains unexplored, is how the mechanical microenvironment is altered as a result of changes to multicellular tissue structure during cancer progression. In this study, we investigated the influence of the multicellular tissue architecture on mechanical properties of the epithelial component of the mammary acinus. Using creep compression tests on multicellular breast epithelial structures, we found that pre-malignant acini with no lumen (MCF10AT) were significantly stiffer than normal hollow acini (MCF10A) by 60%. This difference depended on structural changes in the pre-malignant acini, as neither single cells nor normal multicellular acini tested before lumen formation exhibited these differences. To understand these differences, we simulated the deformation of the acini with different multicellular architectures and calculated their mechanical properties; our results suggest that lumen filling alone can explain the experimentally observed stiffness increase. We also simulated a single contracting cell in different multicellular architectures and found that lumen filling led to a 20% increase in the “perceived stiffness” of a single contracting cell independent of any changes to matrix mechanics. Our results suggest that lumen filling in carcinogenesis alters the mechanical microenvironment in multicellular epithelial structures, a phenotype that may cause downstream disruptions to mechanosensing.     

Combined Inhibition of ErbB1/2 and Notch Receptors Effectively Targets Breast Ductal Carcinoma In Situ (DCIS) Stem/Progenitor Cell Activity Regardless of ErbB2 Status

Pathways involved in DCIS stem and progenitor signalling are poorly understood yet are critical to understand DCIS biology and to develop new therapies. Notch and ErbB1/2 receptor signalling cross talk has been demonstrated in invasive breast cancer, but their role in DCIS stem and progenitor cells has not been investigated. We have utilised 2 DCIS cell lines, MCF10DCIS.com (ErbB2-normal) and SUM225 (ErbB2-overexpressing) and 7 human primary DCIS samples were cultured in 3D matrigel and as mammospheres in the presence, absence or combination of the Notch inhibitor, DAPT, and ErbB1/2 inhibitors, lapatinib or gefitinib. Western blotting was applied to assess downstream signalling. In this study we demonstrate that DAPT reduced acini size and mammosphere formation in MCF10DCIS.com whereas there was no effect in SUM225. Lapatinb reduced acini size and mammosphere formation in SUM225, whereas mammosphere formation and Notch1 activity were increased in MCF10DCIS.com. Combined DAPT/lapatinib treatment was more effective at reducing acini size in both DCIS cell lines. Mammosphere formation in cell lines and human primary DCIS was reduced further by DAPT/lapatinib or DAPT/gefitinib regardless of ErbB2 receptor status. Our pre-clinical human models of DCIS demonstrate that Notch and ErbB1/2 both play a role in DCIS acini growth and stem cell activity. We report for the first time that cross talk between the two pathways in DCIS occurs regardless of ErbB2 receptor status and inhibition of Notch and ErbB1/2 was more efficacious than either alone. These data provide further understanding of DCIS biology and suggest treatment strategies combining Notch and ErbB1/2 inhibitors should be investigated regardless of ErbB2 receptor status.     

Targeting CD44-STAT3 Signaling by Gemini Vitamin D Analog Leads to Inhibition of Invasion in Basal-Like Breast Cancer

Background

CD44, a transmembrane glycoprotein, is a major receptor for extracellular proteins involved in invasion and metastasis of human cancers. We have previously demonstrated that the novel Gemini vitamin D analog BXL0124 [1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluro-cholecalciferol] repressed CD44 expression in MCF10DCIS.com basal-like human breast cancer cells and inhibited MCF10DCIS xenograft tumor growth. In the present study, we investigated potential factors downstream of CD44 and the biological role of CD44 repression by BXL0124 in MCF10DCIS cells.

Methods and Findings

The treatment with Gemini vitamin D BXL0124 decreased CD44 protein level, suppressed STAT3 signaling, and inhibited invasion and proliferation of MCF10DCIS cells. The interaction between CD44 and STAT3 was determined by co-immunoprecipitation. CD44 forms a complex with STAT3 and Janus kinase 2 (JAK2) to activate STAT3 signaling, which was inhibited by BXL0124 in MCF10DCIS cells. The role of CD44 in STAT3 signaling and invasion of MCF10DCIS cells was further determined by the knockdown of CD44 using small hairpin RNA in vitro and in vivo. MCF10DCIS cell invasion was markedly decreased by the knockdown of CD44 in vitro. The knockdown of CD44 also significantly decreased mRNA expression levels of invasion markers, matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), in MCF10DCIS cells. In MCF10DCIS xenograft tumors, CD44 knockdown decreased tumor size and weight as well as invasion markers.

Conclusions

The present study identifies STAT3 as an important signaling molecule interacting with CD44 and demonstrates the essential role of CD44-STAT3 signaling in breast cancer invasion. It also suggests that repression of CD44-STAT3 signaling is a key molecular mechanism in the inhibition of breast cancer invasion by the Gemini vitamin D analog BXL0124.     

Nek2A contributes to tumorigenic growth and possibly functions as potential therapeutic target for human breast cancer

Nek2A (NIMA-related kinases 2A) has been known as an important centrosome regulatory factor. The aim of this study was to investigate the expression of Nek2A and the role it played in different stages of breast cancer. We detected the expression of Nek2A in both mRNA and protein levels in MCF10 cell lines including MCF-10A, MCF-10DCIS.com, MCF-10CA1a and in human breast samples which contained normal breast tissue (NBT), breast ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). Our study revealed that the mRNA and protein expression of Nek2A were significantly up-regulated in MCF-10DCIS.com and MCF-10CA1a cell lines as well as in human primary breast cancer tissue (DCIS and IDC). Our study also presented a correlation between Nek2A mRNA expression and some clinic pathological factors. We found that Nek2A mRNA expression was associated with molecular subtypes, ER, PR and Ki-67 immunoreactivity (P<0.05) in DCIS and associated with histological grade, lymph node metastasis, molecular subtypes, c-erbB-2, and Ki-67 expression (P<0.05) in IDC. In addition, we observed that ectopic expression of Nek2A in "normal" immortalized MCF-10A breast epithelial cell resulted in increased Nek2A which lead to abnormal centrosomes. Furthermore, knockdown of Nek2A in MCF-10DCIS.com could remarkably inhibit cell proliferation and induce cell cycle arrest in MCF-10DCIS.com cell line. These data suggested that Nek2A might bear a close relationship with development and progression of breast carcinoma, and highlighted its role as a novel potential biomarker for diagnosis and a possible therapeutic target for human breast cancer especially for DCIS.     

Scanning electron microscopy of 7,12-dimethylbenz(a)-anthracene-induced mammary carcinoma in the female Sprague-Dawley rat

The surface morphology of normal mammary glands and mammary carcinomas was examined under the scanning electron microscope after digestion of connective tissue and the basal lamina with collagenase, hyaluronidase and hydrochloric acid (HCl). Two types of cells were clearly identified in the acini of normal glands; granular epithelial cells and stellate myoepithelial cells. Spindle-shaped myoepithelial cells lying longitudinally along the mammary ducts were also recognized. 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas consisted of irregular masses of cells which had polypoid or columnar processes with rounded heads; the masses appeared to be composed of a single type of rhomboid cell. The tumors lacked the stellate or spindle-shaped myoepithelial cells found in normal acini and ducts.     

Expression of Putative Stem Cell Marker,Hepatocyte Nuclear Factor 4 Alpha,in Mammary Gland of Water Buffalo

Buffaloes account for more than 56% of total milk production in India. Cyclic remodeling of mammary glands of human, mice, cow, sheep, and goat is determined by mammary stem cells. It is logical to assume that buffalo mammary gland will have mammary stem/progenitor cells. Thus far, no report exists on identification of buffalo mammary stem cells. Hepatocyte nuclear factor 4 alpha (HNF4A) is a candidate marker for hepatic progenitor cells and has recently been suggested as a marker of bovine mammary stem/progenitor cells. We hypothesized that (1 Pasha TN, Hayat Z. Present situation and future perspective of buffalo production in Asia. J Anim Plant Sci 2012; 22(3 supple.):250–256. [Google Scholar]) HNF4A identifies putative buffalo mammary stem/progenitor cells and (2 NDDB. National Dairy Development Board. 2015. http://www.nddb.org/English/Statistics/Pages/Milk-Production.aspx. Accessed May 10, 2015. [Google Scholar]) the number of HNF4A-positive cells increases during mastitis. Sixteen buffalo mammary samples were collected from a local slaughterhouse. Hematoxylin and eosin staining were performed on 5-micron thick sections and on the basis of gross examination and histomorphology of the mammary glands, physiological stages of the animals were estimated as non-lactating (n = 4), mastitis (n = 9), and prepubertal (n = 3). In total, 24048 cells were counted (5–10 microscopic fields/animal; n = 16 animals) of which, 40% cells were mammary epithelial cells (MEC) and 60% cells were the stromal cells. The percentage of MEC in non-lactating animals was higher compared to mastitic animals (47.3% vs. 37.3%), which was likely due to loss of MEC in mastitis. HNF4A staining was observed in nuclei of MEC of ducts, alveoli, and stromal cells. Basal location and low frequency of HNF4A-positive MEC (ranges from 0.4–4.5%) were consistent with stem cell characteristics. Preliminary study showed coexpression of HNF4A with MSI1 (a mammary stem cell marker in sheep), suggesting HNF4A was likely to be a putative mammary stem/progenitor cell marker in buffalo. HNF4A-positive MEC (basal and luminal; light and dark stained) tended to be higher in non-lactating than the mastitic animals (8.73 ± 1.71% vs. 4.29 ± 1.19%; P = 0.07). The first hypothesis that HNF4A identify putative mammary stem/progenitor cells was confirmed but the second hypothesis that the number of mammary stem/progenitor cells decreases during mastitis was unsupported. This is the first report outlining the expression of HNF4A and identification of putative mammary stem/progenitor cells in buffalo mammary gland.     

Cell–Cell Adhesion Molecule CEACAM1 is Expressed in Normal Breast and Milk and Associates with β1 Integrin in a 3D Model of Morphogenesis

CEA cell adhesion molecule-1 (CEACAM1) is a cell-cell adhesion molecule that, paradoxically, is expressed in an apical location in normal breast epithelium. Strong lumenal membrane staining is observed in 100% of normal glands (11/11), low in atypical hyperplasia (2/6), high in cribiform ductal carcinoma in situ (DCIS) (8/8), but low in other types of DCIS (2/15). Although most invasive ductal carcinomas express CEACAM1 (21/26), the staining pattern tends to be weak and cytoplasmic in tumours with minimal lumena formation (grades 2-3), while there is membrane staining in well-differentiated tumours (grade 1). The 'normal' breast epithelial line MCF10F forms acini with lumena in Matrigel with apical membrane expression of CEACAM1. MCF7 cells that do not express CEACAM1 and fail to form lumena in Matrigel, revert to a lumen forming phenotype when transfected with the CEACAM1-4S but not the -4L isoform. CEACAM1 directly associates with and down-regulates the expression of beta1-integrin. Immuno-electron microscopy reveals numerous vesicles coated with CEACAM1 within the lumena, and as predicted by this finding, CEACAM1 is found in the lipid fraction of breast milk. Thus, CEACAM1 is a critical molecule in mammary morphogenesis and may play a role in the absorption of the lipid vesicles of milk in the infant intestinal tract.     

Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: Implications for breast cancer and DCIS therapy with autophagy inhibitors

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating “synthetic” cancer associated fibroblasts (CAFs). More specifically, these “synthetic” CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti-cancer agents, to therapeutically restore the expression of stromal Cav-1 in cancer associated fibroblasts. We discuss this possibility, in light of the launch of a new clinical trial that uses chloroquine to treat DCIS patients: PINC (Preventing Invasive Breast Neoplasia with Cholorquine) [See http://clinicaltrials.gov/show/NCT01023477].     

Lapatinib inhibits stem/progenitor proliferation in preclinical in vitro models of ductal carcinoma in situ (DCIS)

Breast-conserving surgery for ductal carcinoma in situ (DCIS) is often combined with irradiation, reducing recurrence rates to 20% within 10 years; however, there is no change in overall survival. Evidence in the invasive breast indicates that breast cancer stem cells (CSCs) are radiotherapy-resistant and are capable of re-initiating a tumor recurrence; hence, targeting CSCs in high risk DCIS patient may improve survival. HER2 is overexpressed in 20% of DCIS and is known to be highly active in breast CSCs; we therefore investigated the effect of Lapatinib on DCIS CSC activity using 2 in vitro culture systems. Two DCIS cell lines DCIS.com (HER2 normal) and SUM225 (HER2 overexpressed) as well as DCIS cells from patient samples (n = 18) were cultured as mammospheres to assess CSC activity and in differentiated 3D-matrigel culture to determine effects within the non-CSCs. Mammosphere formation was reduced regardless of HER2 status, although this was more marked within the HER2-positive samples. When grown as differentiated DCIS acini in 3D-matrigel culture, Lapatinib only reduced acini size in the HER2-positive samples via decreased proliferation. Further investigation revealed lapatinib did not reduce self-renewal activity in the CSC population, but their proliferation was decreased regardless of HER2 status. In conclusion we show Lapatinib can reduce DCIS CSC activity, suggesting that the use of Lapatinib in high-risk DCIS patients has the potential to reduce recurrence and the progression of DCIS to invasive disease.     

Low CD10 mRNA Expression Identifies High-Risk Ductal Carcinoma In Situ (DCIS)

Purpose

Optimal management of breast ductal carcinoma in situ (DCIS) is controversial, and many patients are still overtreated. The local death of myoepithelial cells (MECs) is believed to be a pre-requisite to tumor invasion. We thus hypothesized that loss of CD10 expression, a MEC surface peptidase, would signify basement membrane disruption and confer increased risk of relapse in DCIS. The aim of our study was to retrospectively evaluate the prognostic value of CD10 in DCIS.

Experimental Design

CD10 expression was evaluated by quantitative RT-PCR and immunohistochemistry using paraffin-embedded samples of normal breast tissue (n = 11); of morphologically normal ducts associated with DCIS (n = 10); and of DCIS without an invasive component (n = 154).

Results

CD10 immunostaining was only observed in MECs in normal tissue and in DCIS. Normal tissue showed high mRNA expression levels of CD10, whereas DCIS showed a variable range. After a median follow-up of 6 years, DCIS with CD10 expression below the levels observed in normal tissue (71%) demonstrated a higher risk of local relapse (HR = 1.88; [95CI:1.30–2.70], p = 0.001) in univariate analysis. No relapse was observed in patients expressing high CD10 mRNA levels (29%) similar to the ones observed in normal tissue. In multivariate analysis including known prognostic factors, low CD10 mRNA expression remained significant (HR = 2.25; [95%CI:1.24–4.09], p = 0.008), as did the recently revised Van Nuys Prognostic Index (VNPI) score (HR = 2.03; [95%CI:1.23–3.35], p = 0.006).

Conclusion

The decrease of CD10 expression in MECs is associated with a higher risk of relapse in DCIS; this knowledge has the potential to improve DCIS management.     

Hydrocortisone and progesterone regulation of the proliferation,morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum-free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1–10 μg/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01–1 μg/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 μg/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross-talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells. © 1995 Wiley-Liss, Inc.     

From Single Cells to Tissues: Interactions between the Matrix and Human Breast Cells in Real Time

Background

Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma - epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.

Results

The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.

Conclusions

Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.     

Mammary epithelial cell polarity is regulated differentially by p73 isoforms via epithelial-to-mesenchymal transition

p73 is expressed as TA and ΔN isoforms, both of which are implicated in tumor suppression and/or promotion. To address how p73 possesses these opposing functions, we developed three-dimensional culture of MCF10A cells, which undergo cell morphogenesis to form polarized spheroids with hollow lumen similar to normal mammary acini in vivo. Here, we showed that upon knockdown of p73, particularly TAp73 but not ΔNp73, MCF10A cells formed irregular and near-normal acini without hollow lumen in three-dimensional culture. We also found that upon knockdown of p73 or TAp73, but not ΔNp73, MCF10A cells underwent epithelial-to-mesenchymal transition (EMT) via down-regulation of E-cadherin coupled with up-regulation of β-catenin and laminin V. In addition, we found that Snail-1, Slug, and Twist, all of which are known to act as EMT inducers by repressing E-cadherin expression, were increased markedly upon knockdown of p73 and TAp73 but little if any by ΔNp73. Furthermore, we showed that knockdown of p73 or TAp73 in MCF10A cells led to a marked increase in cell proliferation and migration. Together, our data suggest that TAp73 is necessary for maintaining normal cell polarity by suppressing EMT.     

Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells

Summary Stromal-epithelial interactions play a profound role in regulating normal and tumor development in the mammary gland. The molecular details of these events, however, are incompletely understood. A novel serum-free transwell coculture system was developed to study the natural paracrine interactions between mammary epithelial cells (MEC) and mammary fibroblasts (MFC) isolated from normal rats during puberty. The MEC were cultured within a reconstituted basement membrane (RBM) in transwell inserts with or without MFC in the lower well. The presence of MFC stimulated epithelial cell growth, induced alveolar morphogenesis, and enhanced casein accumulation, a marker of the functional differentiation of MEC, but did not induced ductal morphogenesis. Potent mitogenic, morphogenic, and lactogenic effects were observed after 1 wk in serum-free medium, fibroblast survival was enhanced significantly when the MFC were cultured within the RBM. Taken together, this in vitro model effectively reconstitutes a physiologically relevant three-dimensional microenvironment for MEC and MFC, and seems ideal for studying the locally derived factors that regulate the developmental fate of the epithelial and fibroblast compartments of the mammary gland.     

Exogenous thyroid hormone affects myoepithelium and proliferation in the developing rat parotid gland

Abstract

In the mature rat parotid gland, myoepithelial cells (MEC) invest intercalated ducts, but not acini. During postnatal development, however, these cells differentiate around both intercalated ducts and acini, then translocate to only intercalated ducts during weaning. Previously, we found that thyroxine (T₄) accelerates translocation of cells with small secretory granules from acini into intercalated ducts and the number of apoptotic cells increased tremendously with high doses. We present here additional analysis of the effects of T₄ on developing rat parotid gland, namely, the distribution of MEC and the proliferation of parenchymal cells. Beginning at age four days, pups were given daily subcutaneous injections of low, medium, and high doses of T₄ or vehicle or no injection. At ages 4, 7, 10, and 15 days, glands were excised and processed for light microscopy. Sections were double-immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and actin, and counterstained with hematoxylin. Proliferative activity was assessed via PCNA histochemistry and MEC were identified using actin histochemistry. MEC in the T₄ groups invested mostly acini at 15 days in vehicle/normal glands and mostly intercalated ducts after 10 days in the T₄ groups. The proliferative activity of acinar cells and MEC in vehicle/normal glands declined progressively with age and T₄ increased the rate of this decline in the MEC in a dose-dependent manner. We conclude that T₄ accelerates the translocation of MEC from acini to intercalated ducts and that an important mechanism is the more rapid decline in the proliferative activity of MEC than in acinar cells in the T₄ groups. Some of the decline in the proliferative activity of all cells in the high and medium dose T₄ groups after seven days may have been due to dose-related thyroxine toxicity.     

HuR Is Necessary for Mammary Epithelial Cell Proliferation and Polarity at Least in Part via ΔNp63

HuR, a RNA binding protein, is known to function as a tumor maintenance gene in breast cancer and associated with tumor growth and poor prognosis. However, the cellular function of this protein remains largely unknown in normal mammary epithelial cells. Here, we showed that in immortalized MCF10A mammary epithelial cells, HuR knockdown inhibits cell proliferation and enhances premature senescence. We also showed that in three-dimensional culture, MCF10A cells with HuR knockdown form abnormal acini with filled lumen and an aberrant expression pattern of the extracellular matrix protein laminin V. In addition, we showed that HuR knockdown increases ΔNp63, but decreases wild-type p53, expression in MCF10A cells. Moreover, we showed that ΔNp63 knockdown partially rescues the proliferative defect induced by HuR knockdown in MCF10A cells. Consistent with this, we identified two U-rich elements in the 3′-untranslated region of p63 mRNA, to which HuR specifically binds. Finally, we showed that HuR knockdown enhances ΔNp63 mRNA translation but has no effect on p63 mRNA turnover. Together, our data suggest that HuR maintains cell proliferation and polarity of mammary epithelial cells at least in part via ΔNp63.     

PUMA Cooperates with p21 to Regulate Mammary Epithelial Morphogenesis and Epithelial-To-Mesenchymal Transition

Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini. Previously, we showed that knockdown of p53 or p73 leads to aberrant mammary acinus formation accompanied with decreased expression of p53 family targets PUMA and p21, suggesting that PUMA, an inducer of apoptosis, and p21, an inducer of cell cycle arrest, directly regulate mammary morphogenesis. To address this, we generated multiple MCF10A cell lines in which PUMA, p21, or both were stably knocked down. We found that morphogenesis of MCF10A cells was altered modestly by knockdown of either PUMA or p21 alone but markedly by knockdown of both PUMA and p21. Moreover, we found that knockdown of PUMA and p21 leads to loss of E-cadherin expression along with increased expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, we found that knockdown of ΔNp73, which antagonizes the ability of wide-type p53 and TA isoform of p73 to regulate PUMA and p21, mitigates the abnormal morphogenesis and EMT induced by knockdown of PUMA or p21. Together, our data suggest that PUMA cooperates with p21 to regulate normal acinus formation and EMT.     

On the Role of the Microenvironment in Mammary Gland Development and Cancer

Knowledge of the specific microenvironmental cues involved at the earliest stages of breast cancer development is currently limited.Breast cancer can be viewed as a disease of defective development, wherein the processes that guide growth and morphogenesis of the mammary gland are inappropriately activated during tumor proliferation and invasion. Research over the last couple of decades, reviewed by Polyak and Kalluri (2011), has defined some of the key microenvironmental signals that underlie both tissue development and disease progression. Meticulous investigation of animal models has revealed how processes controlling mammary gland development during puberty, pregnancy, lactation, and involution become activated in cancer. For example, some of the same stromally produced matrix metalloproteinases (MMPs) that facilitate outgrowth and branching morphogenesis as the glandular epithelium grows into the fat pad during puberty are also involved in the penetration of the basement membrane by the developing cancer (Wiseman and Werb 2002). In parallel, development of physiologically relevant 3D culture systems has enabled identification of specific biochemical and biophysical signals, required for maintenance of normal tissue structure, that become dysregulated as tumors grow. For example, recent studies have found that increasing the stiffness and collagen composition of the extracellular matrix can cause normal mammary epithelial structures to acquire invasive characteristics (Egeblad et al. 2010).Although models for studying the impact of the microenvironment on mammary tissue behavior have become increasingly sophisticated, a significant impediment in elucidating the most important changes in breast cancer development is a limited understanding of the specific microenvironmental cues involved at the earliest stages of disease development. The most commonly hypothesized model of breast cancer development posits an evolution through incremental steps of accumulating cellular abnormalities from normal epithelium through proliferative disease without atypia (PDWA), atypical hyperplasia, ductal carcinoma in situ (DCIS), and then invasive breast cancer (Santen and Mansel 2005). This model is supported by epidemiologic studies that show a stepwise increase in relative risk (RR) of subsequent development of invasive breast cancer from PDWA (RR = 2) to atypical hyperplasia (RR = 4) to DCIS (RR = 10) (Arpino et al. 2005). What are the critical factors that influence whether a premalignant lesion will develop into invasive cancer? Although seminal work by Polyak and coworkers (Hu et al. 2005), as well as other researchers, has identified some of the specific characteristics associated with subsequent disease progression for patients with DCIS, such lesions have already accumulated a broad array of genetic and structural abnormalities. Investigations of yet earlier stages of disease may help us to identify which alterations are the key drivers of progression to malignancy. This information could lead to entirely novel approaches targeting these processes, toward the ultimate goal of prevention of breast cancer formation.