Structural and Molecular Mechanisms of Cytokine-Mediated Endocrine Resistance in Human Breast Cancer Cells.

Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKß establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKß signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance.

Cancer cell extravasation requires iplectin-mediated delivery of MT1-MMP at invadopodia.

BACKGROUND: Invadopodia facilitate cancer cell extravasation, but the molecular mechanism whereby invadopodia-specific proteases such as MT1-MMP are called to invadopodia is unclear. METHODS: Mass spectrometry and immunoprecipitation were used to identify interactors of MT1-MMP in metastatic breast cancer cells. After identification, siRNA and small molecule inhibitors were used to assess the effect these interactors had on cellular invasiveness. The chicken embryo chorioallantoic membrane (CAM) model was used to assess extravasation and invadopodia formation in vivo. RESULTS: In metastatic breast cancer cells, MT1-MMP was found to associate with plectin, a cytolinker and scaffolding protein. Complex formation between plectin and MT1-MMP launches invadopodia formation, a subtype we termed iplectin (i = invadopodial). iPlectin delivers MT1-MMP to invadopodia and is indispensable for regulating cell surface levels of the enzyme. Genetic depletion of plectin with siRNA reduced invadopodia formation and cell invasion in vitro. In vivo extravasation efficiency assays and intravital imaging revealed iplectin to be a key contributor to invadopodia ultrastructure and essential for extravasation. Pharmacologic inhibition of plectin using the small molecule Plecstatin-1 (PST-1) abrogated MT1-MMP delivery to invadopodia and extravasation efficiency. CONCLUSIONS: Anti-metastasis therapeutic approaches that target invadopodia are possible by disrupting interactions between MT1-MMP and iplectin. CLINICAL TRIAL REGISTRATION NUMBER: NCT04608357.

Nanoscale flow cytometry-based quantification of blood-based extracellular vesicle biomarkers distinguishes MCI and Alzheimer's disease.

INTRODUCTION: Accurate testing for Alzheimer's disease (AD) represents a crucial step for therapeutic advancement. Currently, tests are expensive and require invasive sampling or radiation exposure. METHODS: We developed a nanoscale flow cytometry (nFC)-based assay of extracellular vesicles (EVs) to screen biomarkers in plasma from mild cognitive impairment (MCI), AD, or controls. RESULTS: Circulating amyloid beta (Aß), tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, p-tauS235, ubiquitin, and lysosomal-associated membrane protein 1-positive EVs distinguished AD samples. p-tau181, p-tau217, p-tauS235, and ubiquitin-positive EVs distinguished MCI samples. The most sensitive marker for AD distinction was p-tau231, with an area under the receiver operating characteristic curve (AUC) of 0.96 (sensitivity 0.95/specificity 1.0) improving to an AUC of 0.989 when combined with p-tauS235. DISCUSSION: This nFC-based assay accurately distinguishes MCI and AD plasma without EV isolation, offering a rapid approach requiring minute sample volumes. Incorporating nFC-based measurements in larger populations and comparison to "gold standard" biomarkers is an exciting next step for developing AD diagnostic tools. HIGHLIGHTS: Extracellular vesicles represent promising biomarkers of Alzheimer's disease (AD) that can be measured in the peripheral circulation. This study demonstrates the utility of nanoscale flow cytometry for the measurement of circulating extracellular vesicles (EVs) in AD blood samples. Multiple markers including amyloid beta, tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, and p-tauS235 accurately distinguished AD samples from healthy controls. Future studies should expand blood and cerebrospinal fluid-based EV biomarker development using nanoflow cytometry approaches.

Native Mass Spectrometry Quantitation of α2-3-Linked N-Acetylneuraminic Acid Content of Prostate-Specific Antigen: An Accurate Liquid Biopsy for Clinically Significant Prostate Cancer.

Application of the prostate-specific antigen (PSA) test, which measures PSA levels in blood, is standard in prostate cancer (PCa) screening. However, because PSA levels may be elevated for reasons other than PCa, it leads to high rates of misdiagnosis and overtreatment. Recently, alteration in the N-glycan sialylation of PSA, specifically increased levels of α2-3-linked N-acetylneuraminic acid (α2-3-Neu5Ac or α2-3-sialic acid), was identified as a potential biomarker for clinically significant PCa. Here, we introduce a robust top-down native mass spectrometry (MS) approach, performed using a combination of α2-3-Neu5Ac-specific and nonspecific neuraminidases and employing center-of-mass monitoring (CoMMon), for quantifying the levels of α2-3-Neu5Ac as a fraction of total N-linked Neu5Ac present on PSA extracted from blood serum. To illustrate the potential of the assay for clinical diagnosis and disease staging of PCa, the percentages of α2-3-Neu5Ac on PSA (%α23PSA) in the serum of low-grade (International Society of Urological Pathology Grade Group/GG1), intermediate-grade (GG2), and high-grade (GG3,4,5) PCa individuals were measured. We observed a high sensitivity (85.5%) and specificity (84.6%) for discrimination of GG1 from clinically significant GG2-5 patients when using a %α23PSA test cut-off of 28.0%. Our results establish that the %α23PSA in blood serum PSA, which can be precisely measured in a non-invasive manner with our dual neuraminidase native MS/CoMMon assay, can discriminate between clinically significant PCa (GG2-5) and low-grade PCa (GG1). Such discrimination has not been previously achieved and represents an important clinical need. This assay could greatly improve the standard PSA test and serve as a valuable PCa diagnostic tool.

TBX3 promotes progression of pre-invasive breast cancer cells by inducing EMT and directly up-regulating SLUG.

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial-mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Nanoscale flow cytometry to distinguish subpopulations of prostate extracellular vesicles in patient plasma.

OBJECTIVE: To determine if prostate-derived extracellular vesicles (EVs) present in patient plasma samples are of exocytotic origin (exosomes) or released by the cell membrane (microparticles/microvesicles). Both malignant and normal prostate cells release two types of EVs into the circulation, exosomes, and microparticles/microvesicles which differ in size, origin, and mode of release. Determining what proportion of prostate-derived EVs are of exosomal versus microparticle/microvesicle EV subtype is of potential diagnostic significance. MATERIALS AND METHODS: Multi-parametric analytical platforms such as nanoscale flow cytometry (nFC) were used to analyze prostate derived extracellular vesicles. Plasmas from prostate cancer (PCa) patient plasmas representing benign prostatic hyperplasia (BPH), low grade prostate cancer (Gleason Score 3 + 3) and high grade prostate cancer (Gleason Score ≥4 + 4) were analyzed for various exosome markers (CD9, CD63, CD81) and a prostate specific tissue marker (prostate specific membrane antigen/PSMA). RESULTS: By using nanoscale flow cytometry, we determine that prostate derived EVs are primarily of cell membrane origin, microparticles/microvesicles, and not all PSMA expressing EVs co-express exosomal markers such as CD9, CD63, and CD81. CD9 was the most abundant exosomal marker on prostate derived EVs (12-19%). There was no trend observed in terms of more PSMA + CD9 or PSMA + CD63 co-expressing EVs versus increasing grade of prostate cancer. CONCLUSION: The majority of prostate derived EVs present in plasmas are from the cell membrane as evidenced by their size and most importantly, lack of co-expression of exosomal markers such as CD9/CD63/CD81. In fact, CD81 was not present on any prostate derived EVs in patient plasmas whereas CD9 was present on a minority of prostate derived EVs. The addition of an exosomal marker for detection of prostate-derived EVs does not provide greater clarity in distinguishing EVs released by the prostate.

RSPO3 is a prognostic biomarker and mediator of invasiveness in prostate cancer.

BACKGROUND: While prostate cancer can often manifest as an indolent disease, the development of locally-advanced or metastatic disease can cause significant morbidity or mortality. Elucidation of molecular mechanisms contributing to disease progression is crucial for more accurate prognostication and effective treatments. R-Spondin 3 (RSPO3) is a protein previously implicated in the progression of colorectal and lung cancers. However, a role for RSPO3 in prostate cancer prognosis and behaviour has not been explored. METHODS: We compare the relative levels of RSPO3 expression between normal prostate tissue and prostate cancer in two independent patient cohorts (Taylor and GSE70768-Cambridge). We also examine the association of biochemical relapse with RSPO3 levels in these cohorts. For elucidation of the biological effect of RSPO3, we use siRNA technology to reduce the levels of RSPO3 in established prostate cancer cell lines, and perform in vitro proliferation, invasion, western blotting for EMT markers and clonogenic survival assays for radiation resistance. Furthermore, we show consequences of RSPO3 knockdown in an established chick chorioallantoic membrane (CAM) assay model of metastasis. RESULTS: RSPO3 levels are lower in prostate cancer than normal prostate, with a tendency for further loss in metastatic disease. Patients with lower RSPO3 expression have lower rates of biochemical relapse-free survival. SiRNA-mediated loss of RSPO3 results in no change to clonogenic survival and a lower proliferative rate, but increased invasiveness in vitro with induction of epithelial-mesenchymal transition (EMT) markers. Consistent with these results, lower RSPO3 expression translates to greater metastatic capacity in the CAM assay. Together, our preclinical findings identify a role of RSPO3 downregulation in prostate cancer invasiveness, and provide a potential explanation for how RSPO3 functions as a positive prognostic marker in prostate cancer.

The role of extracellular vesicles in cancer microenvironment and metastasis: myths and challenges.

The concept of vesicles or cell debris released by cancer cells to promote metastasis is not new, but the mechanisms used to currently ascribe their impact in metastasis are of intense debate. A significant increase in reports describing the role of cancer-derived EVs in cancer metastasis has been followed by a growing amount of uncertainty behind these claims. This review will delve into the role of EVs in promoting cancer metastasis by relying on a balanced perspective that looks at challenges faced previously by extracellular vesicle biologists, current technical limitations in the field, and overlooked physiologic mechanisms that may play a confounding role. This review will also discuss how certain experimental approaches are misleading which ultimately lead to overly optimistic mechanisms that have minimally contributed to the pathophysiology of metastasis.

The cytoplasmic domain of MT1-MMP is dispensable for migration augmentation but necessary to mediate viability of MCF-7 breast cancer cells.

Membrane-type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease that regulates ECM degradation, proMMP-2 activation, and varied cellular processes including migration and viability. MT1-MMP is believed to be a central mediator of tumourigenesis whose role is dictated by its functionally distinct protein domains. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain, exemplifying diverse regulatory functions. To further our understanding of the multifunctional contributions of MT1-MMP to cellular processes, we overexpressed cytoplasmic domain altered constructs in MCF-7 breast cancer cells and analyzed migration and viability in 2D culture conditions, morphology in 3D Matrigel culture, and tumorigenic ability in vivo. We found that the cytoplasmic domain was not needed for MT1-MMP mediated migration promotion, but was necessary to maintain viability during serum depravation in 2D culture. Similarly, during 3D Matrigel culture the cytoplasmic domain of MT1-MMP was not needed to initiate a protrusive phenotype, but was necessary to prevent colony blebbing when cells were serum deprived. We also tested in vivo tumorigenic potential to show that cells expressing cytoplasmic domain altered constructs demonstrated a reduced ability to vascularize tumours. These results suggest that the cytoplasmic domain regulates MT1-MMP function in a manner required for cell survival, but is dispensable for cell migration.

Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.

BACKGROUND: The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. METHODS: We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. RESULTS: To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. CONCLUSIONS: While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.

Targeting of CCBE1 by miR-330-3p in human breast cancer promotes metastasis.

BACKGROUND: MicroRNAs (miRs) are involved in the regulation of many processes that contribute to malignancy, including cell proliferation, radiation resistance, invasion and metastasis. The role of miR-330-3p, an miR upregulated in breast cancer, remains unclear. METHODS: We examine the association of miR-330-3p with distant relapse-free survival in the Oxford cohort of breast cancer patients. We also study miR-330-3p function using in vitro invasion and ex ovo metastasis assays. Using in vitro luciferase assays, we validate a novel target gene for miR-330-3p, Collagen And Calcium Binding EGF Domains 1 (CCBE1). We assess functional consequences of CCBE1 loss by using siRNA-mediated knockdown followed by in vitro invasion assays. Lastly, we examine the expression profile of CCBE1 in breast carcinomas in the Curtis and TCGA Breast Cancer data sets using Oncomine Platform as well as distant relapse-free and overall survival of patients in the Helsinki University breast cancer data set according to CCBE1 expression status. RESULTS: miR-330-3p is enriched in breast cancer, and higher levels of miR-330-3p expression are associated with lower distant relapse-free survival in a cohort of breast cancer patients. Consistent with these observations, overexpression of miR-330-3p in breast cancer cell lines results in greater invasiveness in vitro, and miR-330-3p-overexpressing cells also metastasise more aggressively ex ovo. We identify CCBE1 as a direct target of miR-330-3p, and show that knockdown of CCBE1 results in a greater invasive capacity. Accordingly, in breast cancer patients CCBE1 is frequently downregulated, and its loss is associated with reduced distant relapse-free and overall survival. CONCLUSIONS: We show for the first time that miR-330-3p targets CCBE1 to promote invasion and metastasis. miR-330-3p and CCBE1 may represent promising biomarkers in breast cancer.

Cellular heterogeneity profiling by hyaluronan probes reveals an invasive but slow-growing breast tumor subset.

Tumor heterogeneity confounds cancer diagnosis and the outcome of therapy, necessitating analysis of tumor cell subsets within the tumor mass. Elevated expression of hyaluronan (HA) and HA receptors, receptor for HA-mediated motility (RHAMM)/HA-mediated motility receptor and cluster designation 44 (CD44), in breast tumors correlates with poor outcome. We hypothesized that a probe for detecting HA-HA receptor interactions may reveal breast cancer (BCa) cell heterogeneity relevant to tumor progression. A fluorescent HA (F-HA) probe containing a mixture of polymer sizes typical of tumor microenvironments (10-480 kDa), multiplexed profiling, and flow cytometry were used to monitor HA binding to BCa cell lines of different molecular subtypes. Formulae were developed to quantify binding heterogeneity and to measure invasion in vivo. Two subsets exhibiting differential binding (HA(-/low) vs. HA(high)) were isolated and characterized for morphology, growth, and invasion in culture and as xenografts in vivo. F-HA-binding amounts and degree of heterogeneity varied with BCa subtype, were highest in the malignant basal-like cell lines, and decreased upon reversion to a nonmalignant phenotype. Binding amounts correlated with CD44 and RHAMM displayed but binding heterogeneity appeared to arise from a differential ability of HA receptor-positive subpopulations to interact with F-HA. HA(high) subpopulations exhibited significantly higher local invasion and lung micrometastases but, unexpectedly, lower proliferation than either unsorted parental cells or the HA(-/low) subpopulation. Querying F-HA binding to aggressive tumor cells reveals a previously undetected form of heterogeneity that predicts invasive/metastatic behavior and that may aid both early identification of cancer patients susceptible to metastasis, and detection/therapy of invasive BCa subpopulations.

Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells.

BACKGROUND: Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease implicated in metastatic progression ostensibly due to its ability to degrade extracellular matrix (ECM) components and allow migration of cells through the basement membrane. Despite in vitro studies demonstrating this principle, this knowledge has not translated into the use of MMP inhibitors (MMPi) as effective cancer therapeutics, or been corroborated by evidence of in vivo ECM degradation mediated by MT1-MMP, suggesting that our understanding of the role of MT1-MMP in cancer progression is incomplete. METHODS: MCF-7 and MDA-MB 231 breast cancer cell lines were created that stably overexpress different levels of MT1-MMP. Using 2D culture, we analyzed proMMP-2 activation (gelatin zymography), ECM degradation (fluorescent gelatin), ERK signaling (immunoblot), cell migration (transwell/scratch closure/time-lapse imaging), and viability (colorimetric substrate) to assess how different MT1-MMP levels affect these cellular parameters. We also utilized Matrigel 3D cell culture and avian embryos to examine how different levels of MT1-MMP expression affect morphological changes in 3D culture, and tumourigenecity and extravasation efficiency in vivo. RESULTS: In 2D culture, breast cancer cells expressing high levels of MT1-MMP were capable of widespread ECM degradation and TIMP-2-mediated proMMP-2 activation, but were not the most migratory. Instead, cells expressing low levels of MT1-MMP were the most migratory, and demonstrated increased viability and ERK activation. In 3D culture, MCF-7 breast cancer cells expressing low levels of MT1-MMP demonstrated an invasive protrusive phenotype, whereas cells expressing high levels of MT1-MMP demonstrated loss of colony structure and cell fragment release. Similarly, in vivo analysis demonstrated increased tumourigenecity and metastatic capability for cells expressing low levels of MT1-MMP, whereas cells expressing high levels were devoid of these qualities despite the production of functional MT1-MMP protein. CONCLUSIONS: This study demonstrates that excessive ECM degradation mediated by high levels of MT1-MMP is not associated with cell migration and tumourigenesis, while low levels of MT1-MMP promote invasion and vascularization in vivo.

Probiotic Lactobacillus rhamnosus Reduces Organophosphate Pesticide Absorption and Toxicity to Drosophila melanogaster.

Organophosphate pesticides used in agriculture can pose health risks to humans and wildlife. We hypothesized that dietary supplementation with Lactobacillus, a genus of commensal bacteria, would reduce absorption and toxicity of consumed organophosphate pesticides (parathion and chlorpyrifos [CP]). Several Lactobacillus species were screened for toleration of 100 ppm of CP or parathion in MRS broth based on 24-h growth curves. Certain Lactobacillus strains were unable to reach stationary-phase culture maxima and displayed an abnormal culture morphology in response to pesticide. Further characterization of commonly used, pesticide-tolerant and pesticide-susceptible, probiotic Lactobacillus rhamnosus strain GG (LGG) and L. rhamnosus strain GR-1 (LGR-1), respectively, revealed that both strains could significantly sequester organophosphate pesticides from solution after 24-h coincubations. This effect was independent of metabolic activity, as L. rhamnosus GG did not hydrolyze CP and no difference in organophosphate sequestration was observed between live and heat-killed strains. Furthermore, LGR-1 and LGG reduced the absorption of 100 µM parathion or CP in a Caco-2 Transwell model of the small intestine epithelium. To determine the effect of sequestration on acute toxicity, newly eclosed Drosophila melanogaster flies were exposed to food containing 10 µM CP with or without supplementation with live LGG. Supplementation with LGG simultaneously, but not with administration of CP 3 days prior (prophylactically), mitigated CP-induced mortality. In summary, the results suggest that L. rhamnosus may be useful for reducing toxic organophosphate pesticide exposure via passive binding. These findings could be transferable to clinical and livestock applications due to affordability and practical ability to supplement products with food-grade bacteria. IMPORTANCE: The consequences of environmental pesticide pollution due to widespread usage in agriculture and soil leaching are becoming a major societal concern. Although the long-term effects of low-dose pesticide exposure for humans and wildlife remain largely unknown, logic suggests that these chemicals are not aligned with ecosystem health. This observation is most strongly supported by the agricultural losses associated with honeybee population declines, known as colony collapse disorder, in which pesticide usage is a likely trigger. Lactobacilli are bacteria used as beneficial microorganisms in fermented foods and have shown potentials to sequester and degrade environmental toxins. This study demonstrated that commonly used probiotic strains of lactobacilli could sequester, but not metabolize, organophosphate pesticides (parathion and chlorpyrifos). This Lactobacillus-mediated sequestration was associated with decreased intestinal absorption and insect toxicity in appropriate models. These findings hold promise for supplementing human, livestock, or apiary foods with probiotic microorganisms to reduce organophosphate pesticide exposure.

The transcriptional regulator TBX3 promotes progression from non-invasive to invasive breast cancer.

BACKGROUND: TBX3 is a T-box transcription factor repressor that is elevated in metastatic breast cancer and is believed to promote malignancy of tumor cells, possibly by promoting cell survival and epithelial-mesenchymal transition. METHODS: The relative expression of TBX3 was assessed in the 21T cell lines which were derived from an individual patient and represent three distinct stages of breast cancer progression: 21PT, atypical ductal hyperplasia; 21NT, ductal carcinoma in situ; and 21MT-1, invasive mammary carcinoma. Two different isoforms of TBX3 (TBX3iso1 and TBX3iso2) were overexpressed to evaluate cell survival/colony forming ability, growth, and invasion in the ductal carcinoma in situ-like 21NT cell line using an in vitro Matrigel model of cancer progression. In addition, TBX3 expression was knocked down to evaluate the effects of downregulating TBX3 on the invasive mammary carcinoma-like 21MT-1 cell line. Finally, PCR array profiling was used to assess alterations in gene expression due to TBX3 overexpression in the 21NT cells. RESULTS: TBX3 is abundant in the invasive 21MT-1 cell line, while being minimally expressed in the non-invasive 21NT and 21PT cell lines. Overexpression of either TBX3iso1 or TBX3iso2 in 21NT cells resulted in increased cell survival/colony forming ability, growth vs. apoptosis and invasion in Matrigel. In contrast, short hairpin RNA-mediated knockdown of TBX3 in the 21MT-1 cells resulted in smaller colonies, with a more regular, less dispersed (less infiltrative) morphology. Array profiling of the 21NT TBX3 iso1 and iso2 transfectants showed that there are common alterations in expression of several genes involved in signal transduction, cell cycle control/cell survival, epithelial-mesenchymal transition and invasiveness. CONCLUSIONS: Overall, these results indicate that TBX3 (isoform 1 or 2) expression can promote progression in a model of early breast cancer by altering cell properties involved in cell survival/colony formation and invasiveness, as well as key regulatory and EMT/invasiveness-related gene expressions.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential.

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel-Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease.

E-cadherin interacts with EGFR resulting in hyper-activation of ERK in multiple models of breast cancer.

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success.

Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Re-examining the size/charge paradigm: differing in vivo characteristics of size- and charge-matched mesoporous silica nanoparticles.

The combination of nanoparticle (NP) size, charge, and surface chemistry (e.g., extent of modification with polyethylene glycol (PEG)) is accepted as a key determinant of NP/cellular interactions. However, the influence of spatial arrangement and accessibility of the charged molecules on the NP surface vis-à-vis the average surface charge (zeta (ζ) potential) is incompletely understood. Here we demonstrate that two types of mesoporous silica nanoparticles (MSNP) that are matched in terms of primary and hydrodynamic particle size, shape, pore structure, colloidal stability, and ζ potential, but differ in surface chemistry, viz. the spatial arrangement and relative exposure of surface amines, have profoundly different interactions with cells and tissues when evaluated in vitro and in vivo. While both particles are ∼50 nm in diameter, PEGylated, and positively charged (ζ = +40 mV), PEG-PEI (MSNPs modified with exposed polyamines), but not PEG-NMe3(+) (MSNP modified with distributed, obstructed amines) rapidly bind serum proteins, diverse cells types in vitro, and endothelial and white blood cells in vivo (ex ovo chick embryo model). This finding helps elucidate the relative role of surface exposure of charged molecules vs ζ potential in otherwise physicochemically matched MSNP and highlights protein corona neutrality as an important design consideration when synthesizing cationic NPs for biological applications.

Histological Characterization of Class I HLA Molecules in Whole Umbilical Cord Tissue Towards an Inexhaustible Graft Alternative for Reconstructive Surgery.

BACKGROUND: Limited graft availability is a constant clinical concern. Hence, the umbilical cord (UC) is an attractive alternative to autologous grafts. The UC is an inexhaustible tissue source, and its removal is harmless and part of standard of care after the birth of the baby. Minimal information exists regarding the immunological profile of a whole UC when it is considered to be used as a tissue graft. We aimed to characterize the localization and levels of class I human leukocyte antigens (HLAs) to understand the allogenicity of the UC. Additionally, HLA-E and HLA-G are putative immunosuppressive antigens that are abundant in placenta, but their profiles in UC whole tissue are unclear. HYPOTHESIS: The UC as a whole expresses a relatively low but ubiquitous level of HLA-ABC and significant levels of HLA-G and HLA-E. METHODS: Healthy patients with no known pregnancy-related complications were approached for informed consent. UCs at term and between 12 and 19 weeks were collected to compare HLA profiles by gestational age. Formalin-fixed paraffin-embedded tissues were sectioned to 5 µm and immunohistochemically stained with a pan-HLA-ABC, two HLA-G-specific, or an HLA-E-specific antibody. RESULTS: HLA-ABC was consistently found present in UCs. HLA-ABC was most concentrated in the UC vessel walls and amniotic epithelium but more dispersed in the Wharton's Jelly. HLA-E had a similar localization pattern to HLA-ABC in whole UC tissues at both gestational ages, but its protein level was lower. HLA-G localization and intensity were poor in all UC tissues analyzed, but additional analyses by Western immunoblot and mass spectrometry revealed a low level of HLA-G in the UC. CONCLUSION: The UC may address limitations of graft availability. Rather than the presence of HLA-G, the immunosuppressive properties of the UC are more likely due to the abundance of HLA-E and the interaction known to occur between HLA-E and HLA-ABC. The co-localization of HLA-E and HLA-ABC suggests that HLA-E is likely presenting HLA-ABC leader peptides to immune cells, which is known to have a primarily inhibitory effect.