Sprouty genes regulate activated fibroblasts in mammary epithelial development and breast cancer.

Stromal fibroblasts are a major stem cell niche component essential for organ formation and cancer development. Fibroblast heterogeneity, as revealed by recent advances in single-cell techniques, has raised important questions about the origin, differentiation, and function of fibroblast subtypes. In this study, we show in mammary stromal fibroblasts that loss of the receptor tyrosine kinase (RTK) negative feedback regulators encoded by Spry1, Spry2, and Spry4 causes upregulation of signaling in multiple RTK pathways and increased extracellular matrix remodeling, resulting in accelerated epithelial branching. Single-cell transcriptomic analysis demonstrated that increased production of FGF10 due to Sprouty (Spry) loss results from expansion of a functionally distinct subgroup of fibroblasts with the most potent branching-promoting ability. Compared to their three independent lineage precursors, fibroblasts in this subgroup are "activated," as they are located immediately adjacent to the epithelium that is actively undergoing branching and invasion. Spry genes are downregulated, and activated fibroblasts are expanded, in all three of the major human breast cancer subtypes. Together, our data highlight the regulation of a functional subtype of mammary fibroblasts by Spry genes and their essential role in epithelial morphogenesis and cancer development.

Proteomic and functional analyses of the periodic membrane skeleton in neurons.

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of potential candidate MPS-interacting proteins that span diverse functional categories. We examined representative proteins in several of these categories using super-resolution imaging, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, and observed periodic distributions characteristic of the MPS along the neurites for ~20 proteins. Genetic perturbations of the MPS and its interacting proteins further suggested functional roles of the MPS in axon-axon and axon-dendrite interactions and in axon diameter regulation, and implicated the involvement of MPS interactions with cell adhesion molecules and non-muscle myosin in these roles. These results provide insights into the interactome of the MPS and suggest previously unknown functions of the MPS in neurons.

LGL1 binds to Integrin ß1 and inhibits downstream signaling to promote epithelial branching in the mammary gland.

Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin ß1 and inhibits its downstream signaling, and Integrin ß1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin ß1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.

Occludin is a target of Src kinase and promotes lipid secretion by binding to BTN1a1 and XOR.

Lipid droplets (LDs) have increasingly been recognized as an essential organelle for eukaryotes. Although the biochemistry of lipid synthesis and degradation is well characterized, the regulation of LD dynamics, including its formation, maintenance, and secretion, is poorly understood. Here, we report that mice lacking Occludin (Ocln) show defective lipid metabolism. We show that LDs were larger than normal along its biogenesis and secretion pathway in Ocln null mammary cells. This defect in LD size control did not result from abnormal lipid synthesis or degradation; rather, it was because of secretion failure during the lactation stage. We found that OCLN was located on the LD membrane and was bound to essential regulators of lipid secretion, including BTN1a1 and XOR, in a C-terminus-dependent manner. Finally, OCLN was a phosphorylation target of Src kinase, whose loss causes lactation failure. Together, we demonstrate that Ocln is a downstream target of Src kinase and promotes LD secretion by binding to BTN1a1 and XOR.

Born to Run? Diverse Modes of Epithelial Migration.

Bundled with various kinds of adhesion molecules and anchored to the basement membrane, the epithelium has historically been considered as an immotile tissue and, to migrate, it first needs to undergo epithelial-mesenchymal transition (EMT). Since its initial description more than half a century ago, the EMT process has fascinated generations of developmental biologists and, more recently, cancer biologists as it is believed to be essential for not only embryonic development, organ formation, but cancer metastasis. However, recent progress shows that epithelium is much more motile than previously realized. Here, we examine the emerging themes in epithelial collective migration and how this has impacted our understanding of EMT.

3D in vitro culture system to study collective migration in mammary organoid epithelium.

We recently established an in vitro culture system in which mammary gland organoid undergoes directional migration in response to an FGF10 concentration gradient. Here, we describe a step-by-step protocol for preparing organoids, the setup of the 3D culture system, and the image acquisition approach. The technical difficulties in conducting the 3D migration assay are choosing epithelial organoids of appropriate sizes and manually paring organoids and beads pre-soaked in FGF10 within a desirable distance (∼100 µm). For complete details on the use and execution of this protocol, please refer to Lu et al. (2020).

Asymmetric Stratification-Induced Polarity Loss and Coordinated Individual Cell Movements Drive Directional Migration of Vertebrate Epithelium.

Collective migration is essential for development, wound repair, and cancer metastasis. For most collective systems, "leader cells" determine both the direction and the power of the migration. It has remained unclear, however, how the highly polarized vertebrate epithelium migrates directionally during branching morphogenesis. We show here that, unlike in other systems, front-rear polarity of the mammary epithelium is set up by preferential cell proliferation in the front in response to the FGF10 gradient. This leads to frontal stratification, loss of apicobasal polarity, and leader cell formation. Leader cells are a dynamic population and move faster and more directionally toward the FGF10 signal than do follower cells, partly because of their intraepithelial protrusions toward the signal. Together, our data show that directional migration of the mammary epithelium is a unique multistep process and that, despite sharing remarkable cellular and molecular similarities, vertebrate and invertebrate epithelial branching are fundamentally distinct processes.

Occludin protects secretory cells from ER stress by facilitating SNARE-dependent apical protein exocytosis.

Tight junctions (TJs) are fundamental features of both epithelium and endothelium and are indispensable for vertebrate organ formation and homeostasis. However, mice lacking Occludin (Ocln) develop relatively normally to term. Here we show that Ocln is essential for mammary gland physiology, as mutant mice fail to produce milk. Surprisingly, Ocln null mammary glands showed intact TJ function and normal epithelial morphogenesis, cell differentiation, and tissue polarity, suggesting that Ocln is not required for these processes. Using single-cell transcriptomics, we identified milk-producing cells (MPCs) and found they were progressively more prone to endoplasmic reticulum (ER) stress as protein production increased exponentially during late pregnancy and lactation. Importantly, Ocln loss in MPCs resulted in greatly heightened ER stress; this in turn led to increased apoptosis and acute shutdown of protein expression, ultimately leading to lactation failure in the mutant mice. We show that the increased ER stress was caused by a secretory failure of milk proteins in Ocln null cells. Consistent with an essential role in protein secretion, Occludin was seen to reside on secretory vesicles and to be bound to SNARE proteins. Taken together, our results demonstrate that Ocln protects MPCs from ER stress by facilitating SNARE-dependent protein secretion and raise the possibility that other TJ components may participate in functions similar to Ocln.

Mammary stem cells, where art thou?

Tremendous progress has been made in the field of stem cell biology. This is in part due to the emergence of various vertebrate organs, including the mammary gland, as an amenable model system for adult stem cell studies and remarkable technical advances in single cell technology and modern genetic lineage tracing. In the current review, we summarize the recent progress in mammary gland stem cell biology at both the adult and embryonic stages. We discuss current challenges and controversies, and potentially new and exciting directions for future research. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cell Differentiation and Reversion Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.

Human erythrocyte band 3 is a host receptor for Plasmodium falciparum glutamic acid-rich protein.

Malaria remains a major global threat to human health and economic development. Microvascular lesions caused by Plasmodium falciparum-infected human erythrocytes/red blood cells are hallmarks of severe pathogenesis contributing to high mortality, particularly in children from sub-Saharan Africa. In this study, we used a phage display complementary DNA library screening strategy to identify P falciparum glutamic acid-rich protein (PfGARP) as a secreted ligand that recognizes an ectodomain of human erythrocyte anion-exchanger, band 3/AE1, as a host receptor. Domain mapping of PfGARP revealed distinct nonoverlapping repeats encoding the immune response epitopes and core erythrocyte-binding activity. Synthetic peptides derived from the erythrocyte-binding repeats of PfGARP induced erythrocyte aggregation reminiscent of the rosetting phenomenon. Using peptides derived from the immunogenic repeats, a quantitative immunoassay was developed to detect a selective immune response against PfGARP in human plasma samples obtained from patients in rural Mali, suggesting the feasibility of PfGARP as a potential biomarker of disease progression. Collectively, our results suggest that PfGARP may play a functional role in enhancing the adhesive properties of human erythrocytes by engaging band 3 as a host receptor. We propose that immunological and pharmacological inhibition of PfGARP may unveil new therapeutic options for mitigating lesions in cerebral and pregnancy-associated malaria.

Calpain-1 regulates platelet function in a humanized mouse model of sickle cell disease.

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5'-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, and partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa (αIIbß3) integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD.

Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia.

Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex.

Damage-induced BRCA1 phosphorylation by Chk2 contributes to the timing of end resection.

The BRCA1 tumor suppressor plays an important role in homologous recombination (HR)-mediated DNA double-strand-break (DSB) repair. BRCA1 is phosphorylated by Chk2 kinase upon γ-irradiation, but the role of Chk2 phosphorylation is not understood. Here, we report that abrogation of Chk2 phosphorylation on BRCA1 delays end resection and the dispersion of BRCA1 from DSBs but does not affect the assembly of Mre11/Rad50/NBS1 (MRN) and CtIP at DSBs. Moreover, we show that BRCA1 is ubiquitinated by SCF(Skp2) and that abrogation of Chk2 phosphorylation impairs its ubiquitination. Our study suggests that BRCA1 is more than a scaffold protein to assemble HR repair proteins at DSBs, but that Chk2 phosphorylation of BRCA1 also serves as a built-in clock for HR repair of DSBs. BRCA1 is known to inhibit Mre11 nuclease activity. SCF(Skp2) activity appears at late G1 and peaks at S/G2, and is known to ubiquitinate phosphodegron motifs. The removal of BRCA1 from DSBs by SCF(Skp2)-mediated degradation terminates BRCA1-mediated inhibition of Mre11 nuclease activity, allowing for end resection and restricting the initiation of HR to the S/G2 phases of the cell cycle.

The F-box protein FBXO44 mediates BRCA1 ubiquitination and degradation.

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF(FBXO44)) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF(FBXO44) reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF(FBXO44) is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF(FBXO44)-mediated BRCA1 degradation might contribute to sporadic breast tumor development.

Attenuated Salmonella typhimurium carrying shRNA-expressing vectors elicit RNA interference in murine bladder tumors.

AIM: To examine whether attenuated Salmonella typhimurium (S typhimurium) could be used as an anti-cancer agent or a tumor-targeting vehicle for delivering shRNA-expressing pDNA into cancer cells in a mouse tumor model. METHODS: Mouse bladder transitional cancer cell line (BTT-T739) expressing GFP was used, in which the GFP expression level served as an indicator of RNA interference (RNAi). BTT-T739-GFP tumor-bearing mice (4-6 weeks) were treated with S typhimurium carrying plasmids encoding shRNA against gfp or scrambled shRNA. The mRNA and protein expression levels of GFP were assessed 5 d after the bacteria administration, and the antitumor effects of S typhimurium were evaluated. RESULTS: In BTT-T739-GFP tumor-bearing mice, S typhimurium (1×10(9) cfu, po) preferentially accumulated within tumors for as long as 40 d, and formed a tumor-to-normal tissue ratio that exceeded 1000/1. S typhimurium carrying plasmids encoding shRNA against gfp inhibited the expression of GFP in tumor cells by 73.4%. Orally delivered S typhimurium significantly delayed tumor growth and prolonged the survival of tumor-bearing mice. CONCLUSION: This study demonstrates that attenuated S typhimurium can be used for both delivering shRNA-expressing vectors into tumor cells and eliciting RNAi, thus exerting anti-tumor activity, which may represent a new strategy for the treatment of solid tumors.

BRCA1/BARD1 complex interacts with steroidogenic factor 1--A potential mechanism for regulation of aromatase expression by BRCA1.

Germline mutations in BRCA1 predispose women to early onset of breast and ovarian cancers. Findings from previous studies support the notion that the tissue- and gender-specific tumor suppression function of BRCA1 is associated with its role in negative regulation of aromatase expression, the rate-limiting step in estrogen biosynthesis. The molecular mechanism of BRCA1 in regulating aromatase promoter activity remains to be elucidated. In this study, we demonstrate that, in an ovarian granulosa cell line KGN, steroidogenic factor 1 (SF-1) is required for aromatase PII promoter basal activity as well as the elevated aromatase expression mediated by BRCA1 knockdown. Furthermore, BRCA1 in KGN cells exists mainly as a heterodimer with BARD1. We provide evidence that the BRCA1/BARD1 complex interacts with SF-1 both in vivo and in vitro. However, the intrinsic ubiquitin E3 ligase activity of BRCA1/BARD1 does not appear to contribute to ubiquitynation of SF-1. We propose that the interaction between SF-1 and BRCA1/BARD1 may recruit BRCA1/BARD1 complex to the aromatase PII promoter for BRCA1/BARD1-mediate transcriptional repression.

A Role of CREB in BRCA1 Constitutive Promoter Activity and Aromatase Basal Expression.

Aromatase is the rate-limiting enzyme in estrogen biosynthesis and a key target in breast cancer treatment. Its ovary-specific promoter, PII, is induced in response to protein kinase A (PKA) activation. It has been proposed that breast cancer susceptibility gene 1, BRCA1, is involved in negative regulation of aromatase PII activity. Surprisingly, inhibition of PKA pathway by inhibitor H89 elevates basal aromatase expression while abolishes cAMP-mediated aromatase induction in an ovarian granulosa cell line, KGN. In this report, we decipher the mechanism by which the PKA pathway negatively regulates aromatase basal expression. We show that PKA pathway plays a positive role in the expression of BRCA1. H89 effectively reduces endogenous BRCA1 mRNA levels as well as reporter gene expression from a BRCA1 promoter. Mutation of a cAMP-responsive element (CRE) in the BRCA1 promoter reduces BRCA1 expression. Chromatin immunoprecipitation (ChIP) shows that CRE-binding protein, CREB, binds to the BRCA1 promoter. Furthermore, knockdown of CREB in KGN cells leads to decreased BRCA1 level as well as elevated basal aromatase mRNA expression. These data demonstrate that both the CRE site in the BRCA1 promoter and CREB are required for BRCA1 constitutive expression. Our study suggests that PKA pathway exerts its negative impact on basal aromatase expression indirectly by contributing to the constitutive expression of BRCA1.

Tumor suppressor BRCA1 inhibits a breast cancer-associated promoter of the aromatase gene (CYP19) in human adipose stromal cells.

Adipose tissue provides an important extragonadal source of estrogen. Obesity-associated elevation of estrogen production increases risk of breast cancer in postmenopausal women. Aromatase (CYP19), which converts androgen to estrogen, is a key enzyme in estrogen biosynthesis. In normal adipose tissue, transcription of the aromatase gene is initiated from a relatively weak adipose-specific promoter (I.4). However, in breast cancer, a switch of promoter utilization from I.4 to a strong ovary-specific promoter, PII, leads to increased aromatase expression and, hence, elevated estrogen production. Here, we report an intriguing relationship between the breast cancer susceptibility gene BRCA1 and aromatase expression in human adipose stromal cells (ASCs). Upon stimulation by phorbol ester or dexamethasone, increased aromatase expression in ASCs was accompanied by significant reduction of the BRCA1 level. In addition, adipogenesis-induced aromatase expression was also inversely correlated with BRCA1 abundance. Downregulation of BRCA1 expression in response to various stimuli was through distinct transcription or posttranscription mechanisms. Importantly, siRNA-mediated knockdown of BRCA1 led to specific activation of the breast cancer-associated PII promoter. Therefore, in addition to its well-characterized activities in breast epithelial cells, a role of BRCA1 in modulation of estrogen biosynthesis in ASCs may also contribute to its tissue-specific tumor suppressor function.

Ubiquitination and proteasome-mediated degradation of BRCA1 and BARD1 during steroidogenesis in human ovarian granulosa cells.

Germ-line mutations in BRCA1 predispose women to early-onset, familial breast and ovarian cancers. However, BRCA1 expression is not restricted to breast and ovarian epithelial cells. For example, ovarian BRCA1 expression is enriched in ovarian granulosa cells, which are responsible for ovarian estrogen production in premenopausal women. Furthermore, recent tissue culture and animal studies suggest a functional role of BRCA1 in ovarian granulosa cells. Although levels of BRCA1 are known to fluctuate significantly during folliculogenesis and steroidogenesis, the mechanism by which BRCA1 expression is regulated in granulosa cells remains to be elucidated. Here we show that the ubiquitin-proteasome degradation pathway plays a significant role in the coordinated protein stability of BRCA1 and its partner BARD1 in ovarian granulosa cells. Our work identifies the amino-terminal RING domain-containing region of BRCA1 as the degron sequence that is both necessary and sufficient for polyubiquitination and proteasome-mediated protein degradation. Interestingly, mutations in the RING domain that abolish the ubiquitin E3 ligase activity of BRCA1 do not affect its own ubiquitination or degradation in ovarian granulosa cells. The proteasome-mediated degradation of BRCA1 and BARD1 also occurs during the cAMP-dependent steroidogenic process. Thus, the dynamic changes of BRCA1/BARD1 protein stability in ovarian granulosa cells provide an excellent paradigm for investigating the regulation of this protein complex under physiological conditions.