A molecular landscape of quiescence and proliferation highlights the role of Pten in mammary gland acinogenesis.

Cell context is key for cell state. Using physiologically relevant models of laminin-rich extracellular matrix (lrECM) induction of mammary epithelial cell quiescence and differentiation, we provide a landscape of the key molecules for the proliferation-quiescence decision, identifying multiple layers of regulation at the mRNA and protein levels. Quiescence occurred despite activity of Fak (also known as PTK2), Src and phosphoinositide 3-kinases (PI3Ks), suggesting the existence of a disconnecting node between upstream and downstream proliferative signalling. Pten, a lipid and protein phosphatase, fulfils this role, because its inhibition increased proliferation and restored signalling via the Akt, mTORC1, mTORC2 and mitogen-activated protein kinase (MAPK) pathways. Pten and laminin levels were positively correlated in developing murine mammary epithelia, and Pten localized apicolaterally in luminal cells in ducts and near the nascent lumen in terminal end buds. Consistently, in three-dimensional acinogenesis models, Pten was required for triggering and sustaining quiescence, polarity and architecture. The multilayered regulatory circuitry that we uncovered provides an explanation for the robustness of quiescence within a growth-suppressive microenvironment, which could nonetheless be disrupted by perturbations in master regulators such as Pten.

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM), a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM) displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB) cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

The role of cellular quiescence in cancer - beyond a quiet passenger.

Quiescence, the ability to temporarily halt proliferation, is a conserved process that initially allowed survival of unicellular organisms during inhospitable times and later contributed to the rise of multicellular organisms, becoming key for cell differentiation, size control and tissue homeostasis. In this Review, we explore the concept of cancer as a disease that involves abnormal regulation of cellular quiescence at every step, from malignant transformation to metastatic outgrowth. Indeed, disrupted quiescence regulation can be linked to each of the so-called 'hallmarks of cancer'. As we argue here, quiescence induction contributes to immune evasion and resistance against cell death. In contrast, loss of quiescence underlies sustained proliferative signalling, evasion of growth suppressors, pro-tumorigenic inflammation, angiogenesis and genomic instability. Finally, both acquisition and loss of quiescence are involved in replicative immortality, metastasis and deregulated cellular energetics. We believe that a viewpoint that considers quiescence abnormalities that occur during oncogenesis might change the way we ask fundamental questions and the experimental approaches we take, potentially contributing to novel discoveries that might help to alter the course of cancer therapy.

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling.

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.

Viral infection and smell loss: The case of COVID-19.

Olfactory disorders have been increasingly reported in individuals infected with SARS-CoV-2, the virus causing the coronavirus disease 2019 (COVID-19). Losing the sense of smell has a strong impact on the quality of life, since it may lead to malnutrition, weight loss, food poisoning, depression, and exposure to dangerous chemicals. Individuals who suffer from anosmia (inability to smell) also cannot sense the flavor of food, which is a combination of taste and smell. Interestingly, infected individuals have reported sudden loss of smell with no congested nose, as is frequently observed in common colds or other upper respiratory tract infections. These observations suggest that SARS-CoV-2 infection leads to olfactory loss through a distinct mechanism, which is still unclear. This article provides an overview of olfactory loss and the recent findings relating to COVID-19. Possible mechanisms of SARS-CoV-2-induced olfactory loss are also discussed.

Mitochondrial morphology regulates organellar Ca2+ uptake and changes cellular Ca2+ homeostasis.

Changes in mitochondrial size and shape have been implicated in several physiologic processes, but their role in mitochondrial Ca2+ uptake regulation and overall cellular Ca2+ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics toward increased fusion through expression of a dominant negative (DN) form of the fission protein [dynamin-related protein 1 (DRP1)] markedly increased both mitochondrial Ca2+ retention capacity and Ca2+ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion-promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin (MFN)-2 strongly reduced the mitochondrial Ca2+ uptake speed and capacity in these cells. These changes were not dependent on modifications in mitochondrial calcium uniporter expression, inner membrane potentials, or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as indicated by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium-replenishing process of store-operated calcium entry was impaired in MFN2 knockdown cells, whereas DRP1-DN-promoted fusion resulted in faster cytosolic Ca2+ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca2+ uptake, which impacts cellular Ca2+ homeostasis.-Kowaltowski, A. J., Menezes-Filho, S. L., Assali, E. A., Gonçalves, I. G., Cabral-Costa, J. V., Abreu, P., Miller, N., Nolasco, P., Laurindo, F. R. M., Bruni-Cardoso, A., Shirihai, O. Mitochondrial morphology regulates organellar Ca2+ uptake and changes cellular Ca2+ homeostasis.

Heparanase-1 activity and the early postnatal prostate development.

Ventral prostate (VP) morphogenesis starts during embryonic development and continues for the first three postnatal weeks. Heparan sulfate (HS) affects paracrine signaling. Heparanase-1 (HPSE) is the only enzyme capable of cleaving HS. HPSE releases the HS bioactive fragment and mobilizes growth factors. Little is known, however, about HS turnover and HPSE function during VP morphogenesis. In this study, we measured HSPG expression and analyzed the expression and distribution of HPSE in the rat VP. HPSE was predominantly expressed by the VP epithelium. The VP was treated with heparin in ex vivo cultures to interfere with HS and resulted in delayed epithelial growth. Hpse knockdown using siRNA delayed epithelial growth in the first postnatal week ex vivo, which was similar to treating with the lower concentration of heparin. Hpse silencing was related to changes in HS chain length (as determined by size-exclusion chromatography, up-regulation of Mmp9, and down-regulation of Mmp2 expression). It also down-modulated ERK1/2 phosphorylation, suggesting a reduction in signaling, likely due to decreased HS cleavage and growth factor bioavailability. Our results showed that HPSE played a role in early epithelial growth during the first week of VP postnatal development. Developmental Dynamics 248:211-220, 2019. © 2019 Wiley Periodicals, Inc.

The role of SDF1 in prostate epithelial morphogenesis.

Androgens induce rat prostate induction from the urogenital sinus epithelium at embryonic day 17.5. Subsequent morphogenesis, including epithelial cord growth, branching, and canalization, results from concerted paracrine interactions with the stroma. A significant number of paracrine factors bind heparan sulfate (HS). We hypothesized that interfering with overall sulfation could disrupt the signaling mediated by HS-binding factors and that the undersulfated environment would allow investigation of individual exogenous morphogens. First, we investigated whether acinar morphogenesis involved HS-proteoglycan expression and found that syndecans 1 and 3 were upregulated in RWPE1 cells in the transition from two- to three-dimensional (3D) Matrigel, capable of promoting spheroid formation. We then investigated whether sodium chlorate, a general sulfation inhibitor, interfered with spheroid formation by RWPE1 cells and acinar morphogenesis in ex vivo ventral prostate (VP) organ culture. As expected, treatment with sodium chlorate inhibited spheroid formation by RWPE1 cells in 3D culture. Chlorate also inhibited ex vivo VP epithelial branching and canalization, resulting in long branchless epithelial structures. We then investigated whether the HS-binding factors, FGF10, TGFß1, and SDF1, could reverse the effect of sodium chlorate. Although no effect was seen in the FGF10- and TGFß1-treated samples, SDF1 promoted epithelial canalization in the low sulfated environment, highlighting its specific role in lumen formation. Altogether, the results show that sodium chlorate perturbed prostate morphogenesis and allowed investigation of factors involved in branching and/or canalization, implicating SDF1 signaling in epithelial canalization.

Castration-induced prostate epithelial cell apoptosis results from targeted oxidative stress attack of M1142 -macrophages.

Prostate development and function are regulated by androgens. Epithelial cell apoptosis in response to androgen deprivation is caspase-9-dependent and peaks at Day 3 after castration. However, isolated epithelial cells survive in the absence of androgens. Znf142 showed an on-off expression pattern in intraepithelial CD68-positive macrophages, with the on-phase at Day 3 after castration. Rats treated with gadolinium chloride to deplete macrophages showed a significant drop in apoptosis, suggesting a causal relationship between macrophages and epithelial cell apoptosis. Intraepithelial M1-polarization was also limited to Day 3, and the inducible nitric oxide synthase (iNOS) knockout mice showed significantly less apoptosis than wild-type controls. The epithelial cells showed focal DNA double-strand breaks (DSB), 8-oxoguanine, and protein tyrosine-nitrosylation, fingerprints of exposure to peroxinitrite. Cultured epithelial cells induced M1-polarization and showed focal DSB and underwent apoptosis. The same phenomena were reproduced in LNCaP cells cocultured with Raw 264.7 macrophages. In conclusion, the M1 142 -macrophage (named after Znf142) attack causes activation of the intrinsic apoptosis pathway in epithelial cells after castration.

Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture.

The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.

Macrophage roles in the clearance of apoptotic cells and control of inflammation in the prostate gland after castration.

BACKGROUND: Androgen deprivation results in massive apoptosis in the prostate gland. Macrophages are actively engaged in phagocytosing epithelial cell corpses. However, it is unknown whether microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis (LAP) is involved and contribute to prevent inflammation. METHODS: Flow cytometry, RT-PCR and immunohistochemistry were used to characterize the macrophage subpopulation residing in the epithelial layer of the rat ventral prostate (VP) after castration. Stereology was employed to determine variations in the number of ED1 and ED2. Mice were treated with either chloroquine or L-asparagine to block autophagy. RESULTS: M1 (iNOS-positive) and M2 macrophages (MRC1+ and ARG1+) were not found in the epithelium at day 5 after castration. The percentage of CD68+ (ED1) and CD163+ (ED2) phenotypes increased after castration but only CD68+ cells were present in the epithelium. RT-PCR showed increased content of the autophagy markers Bcl1 and LC3 after castration. In addition, immunohistochemistry showed the presence of LC3+ and ATG5+ cells in the epithelium. Double immunohistochemistry showed these cells to be CD68+ /LC3+ , compatible with the LAP phenotype. LC3+ cells accumulate significantly after castration. Chloroquine and L-asparagine administration caused inflammation of the glands at day 5 after castration. CONCLUSIONS: CD68+ macrophages phagocytose apoptotic cell corpses and activate the LAP pathway, thereby contributing to the preservation of a non-inflammed microenvironment. Marked inflammation was detected when autophagy blockers were administered to castrated animals.

Methylene blue photodynamic therapy induces selective and massive cell death in human breast cancer cells.

BACKGROUND: Breast cancer is the main cause of mortality among women. The disease presents high recurrence mainly due to incomplete efficacy of primary treatment in killing all cancer cells. Photodynamic therapy (PDT), an approach that causes tissue destruction by visible light in the presence of a photosensitizer (Ps) and oxygen, appears as a promising alternative therapy that could be used adjunct to chemotherapy and surgery for curing cancer. However, the efficacy of PDT to treat breast tumours as well as the molecular mechanisms that lead to cell death remain unclear. METHODS: In this study, we assessed the cell-killing potential of PDT using methylene blue (MB-PDT) in three breast epithelial cell lines that represent non-malignant conditions and different molecular subtypes of breast tumours. Cells were incubated in the absence or presence of MB and irradiated or not at 640 nm with 4.5 J/cm2. We used a combination of imaging and biochemistry approaches to assess the involvement of classical autophagic and apoptotic pathways in mediating the cell-deletion induced by MB-PDT. The role of these pathways was investigated using specific inhibitors, activators and gene silencing. RESULTS: We observed that MB-PDT differentially induces massive cell death of tumour cells. Non-malignant cells were significantly more resistant to the therapy compared to malignant cells. Morphological and biochemical analysis of dying cells pointed to alternative mechanisms rather than classical apoptosis. MB-PDT-induced autophagy modulated cell viability depending on the cell model used. However, impairment of one of these pathways did not prevent the fatal destination of MB-PDT treated cells. Additionally, when using a physiological 3D culture model that recapitulates relevant features of normal and tumorous breast tissue morphology, we found that MB-PDT differential action in killing tumour cells was even higher than what was detected in 2D cultures. CONCLUSIONS: Finally, our observations underscore the potential of MB-PDT as a highly efficient strategy which could use as a powerful adjunct therapy to surgery of breast tumours, and possibly other types of tumours, to safely increase the eradication rate of microscopic residual disease and thus minimizing the chance of both local and metastatic recurrence.

Coherent angular motion in the establishment of multicellular architecture of glandular tissues.

Glandular tissues form ducts (tubes) and acini (spheres) in multicellular organisms. This process is best demonstrated in the organization of the ductal tree of the mammary gland and in 3D models of morphogenesis in culture. Here, we asked a fundamental question: How do single adult epithelial cells generate polarized acini when placed in a surrogate basement membrane 3D gel? Using human breast epithelial cells from either reduction mammoplasty or nonmalignant breast cell lines, we observed a unique cellular movement where single cells undergo multiple rotations and then maintain it cohesively as they divide to assemble into acini. This coherent angular motion (CAMo) was observed in both primary cells and breast cell lines. If CAMo was disrupted, the final geometry was not a sphere. The malignant counterparts of the human breast cell lines in 3D were randomly motile, did not display CAMo, and did not form spheres. Upon "phenotypic reversion" of malignant cells, both CAMo and spherical architecture were restored. We show that cell-cell adhesion and tissue polarity are essential for the formation of acini and link the functional relevance of CAMo to the establishment of spherical architecture rather than to multicellular aggregation or growth. We propose that CAMo is an integral step in the formation of the tissue architecture and that its disruption is involved in malignant transformation.

Cardioprotective mechanism of S-nitroso-N-acetylcysteine via S-nitrosated betadrenoceptor-2 in the LDLr-/- mice.

Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with ß2-AR signaling in mediating this protection. Ventricular superoxide (O2⁻) and hydrogen peroxide (H2O2) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O2⁻ and H2O2 production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H2O2 and O2⁻ production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in ß2-AR expression associated with coupling change to Gi; ß2-ARs-S-nitrosation (ß2-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with ß2-ARs overexpression and ß2-AR-SNO via an anti-apoptotic pathway.

A practical and robust method to evaluate metabolic fluxes in primary pancreatic islets.

OBJECTIVE: Evaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables, is expensive and poorly reproducible. Our aim was to establish a practical method to assess primary islet metabolic fluxes using standard commercial consumables. METHODS: Pancreatic islets were isolated from mice/rats, dispersed with trypsin, and adhered to pre-coated standard Seahorse or Resipher microplates. Oxygen consumption was evaluated using a Seahorse Extracellular Flux Analyzer or a Resipher Real-time Cell Analyzer. RESULTS: We provide a detailed protocol with all steps to optimize islet isolation with high yield and functionality. Our method requires a few islets per replicate; both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators and glucose. The technique was validated by other functional assays, which show these cells present conserved calcium influx and insulin secretion in response to glucose. We also show that our dispersed islets maintain robust basal respiration levels, in addition to maintaining up to 89% viability after five days in dispersed cultures. Furthermore, OCRs can be measured in Seahorse analyzers and in other plate respirometry systems, using standard materials. CONCLUSIONS: Overall, we established a practical and robust method to assess islet metabolic fluxes and oxidative phosphorylation, a valuable tool to uncover basic ß-cell metabolic mechanisms as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols.

Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis.

Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.

Extracellular matrix stiffness regulates degradation of MST2 via SCF ßTrCP.

The Hippo pathway plays central roles in relaying mechanical signals during development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we found that chemical inhibition of proteasomal proteolysis resulted in increased levels of MST2 in human breast epithelial cells. MST2 binds SCFßTrCP E3 ubiquitin ligase and silencing ßTrCP resulted in MST2 accumulation. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that ßTrCP binds MST2 via a non-canonical degradation motif. Additionally, stiffer extracellular matrix, as well as hyperactivation of integrins resulted in enhanced MST2 degradation mediated by integrin-linked kinase (ILK) and actomyosin stress fibers. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and underscores how alterations in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.

Prognostic value of integrin αV expression and localization pattern in invasive breast carcinomas.

Invasion of surrounding stroma is an early event in breast cancer metastatic progression, and involves loss of cell polarity, loss of myoepithelial layer, epithelial-mesenchymal transition (EMT) and remodeling of the extracellular matrix (ECM). Integrins are transmembrane receptors responsible for cell-ECM binding, which triggers signals that regulate many aspects of cell behavior and fate. Changes in the expression, localization and pairing of integrins contribute for abnormal responses found in transformed epithelia. We analyzed 345 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of integrin αV and correlation with clinical parameters. Patients with lower levels of integrin αV staining showed reduced cancer specific survival. A subset of cases presented a peripheral staining of integrin αV surrounding tumor cell clusters, possibly matching the remaining myoepithelial layer. Indeed, the majority of ductal carcinoma in situ (DCIS) components found in the TMA presented integrin αV at their periphery, whereas this pattern was mostly lost in invasive components, even in the same sample. The lack of peripheral integrin αV correlated with decreased cancer specific survival. In addition, we observed that the presence of integrin αV in the stroma was an indicative of poor survival and metastatic disease. Consistently, by interrogating publicly available datasets we found that, although patients with higher mRNA levels of integrin αV had increased risk of developing metastasis, high co-expression of integrin αV and a myoepithelial cell marker (MYH11) mRNA levels correlated with better clinical outcomes. Finally, a 3D cell culture model of non-malignant and malignant cells reproduced the integrin αV pattern seen in patient samples. Taken together, our data indicate that both the expression levels of integrin αV and its tissue localization in primary tumors have prognostic value, and thus, could be used to help predict patients at higher risk of developing metastasis.

MMP-2 contributes to the development of the mouse ventral prostate by impacting epithelial growth and morphogenesis.

Epithelial growth, branching, and canalization are important morphogenetic events of the rodent ventral prostate (VP) that take place during the first postnatal week. In this study, we evaluated the effect of knocking out MMP-2 (MMP-2(-/-)), by examining developmental and structural aspects of the VP in MMP-2(-/-) mice. Neonate (day 6) MMP-2(-/-) mice showed fewer epithelial tips, a lower epithelial cell proliferation rate, and also reticulin fiber accumulation. The VP of adult MMP-2(-/-) mice showed lower relative weight, smaller epithelial and smooth-muscle cell volume, and a larger amount of thicker reticulin fibers. No differences in cell proliferation or apoptotic index were noted between adult MMP-2(-/-) and wild-type mice. MMP-9 was found in the adult MMP-2(-/-), but not in the wild-type. In conclusion, MMP-2 function is essential for the epithelial morphogenesis of the mouse VP, and expression of MMP-9 is not sufficient for acquisition of the normal adult histology.

MMP-2 regulates rat ventral prostate development in vitro.

We have hypothesized that epithelial growth, branching, and canalization in the rodent ventral prostate (VP) would require matrix remodeling, and hence matrix metalloproteinase (MMP) activity. Therefore, the aim of this study was to evaluate the impact of blocking MMP-2, using whole organ culture. siRNA was employed to inhibit MMP-2 expression, and this was compared to GM6001's (a broad-spectrum MMP inhibitor) inhibition of general MMPs. These blocks impaired VP morphogenesis. MMP-2 silencing reduced organ size, epithelial area, and the number of tips, as well as caused a dilation of the distal parts of the epithelium. Histology, 3-D reconstruction, biochemistry, and second harmonic generation (SHG) revealed that MMP-2 silencing affected VP architecture by interfering in epithelial cell proliferation, lumen formation, and cellular organization of both epithelium and stroma, besides intense accumulation of collagen fibers. These data suggest that MMP-2 plays important roles in prostate growth, being directly involved with epithelial morphogenesis.