PLK1 Regulates MicroRNA Biogenesis through Drosha Phosphorylation.

Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3ß, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S300 and/or S302. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S300 and S302 as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.

Direct targeting of FOXP3 in Tregs with AZD8701, a novel antisense oligonucleotide to relieve immunosuppression in cancer.

BACKGROUND: The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered, unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man. METHODS: We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons. RESULTS: AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs, reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO, which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs, reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo, strongly modulated Treg effector molecules (eg, ICOS, CTLA-4, CD25 and 4-1BB), and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy. CONCLUSIONS: Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669).

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing.

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods.

VEGF pathway inhibition potentiates PARP inhibitor efficacy in ovarian cancer independent of BRCA status.

Poly ADP-ribose polymerase inhibitors (PARPi) have transformed ovarian cancer (OC) treatment, primarily for tumours deficient in homologous recombination repair. Combining VEGF-signalling inhibitors with PARPi has enhanced clinical benefit in OC. To study drivers of efficacy when combining PARP inhibition and VEGF-signalling, a cohort of patient-derived ovarian cancer xenografts (OC-PDXs), representative of the molecular characteristics and drug sensitivity of patient tumours, were treated with the PARPi olaparib and the VEGFR inhibitor cediranib at clinically relevant doses. The combination showed broad anti-tumour activity, reducing growth of all OC-PDXs, regardless of the homologous recombination repair (HRR) mutational status, with greater additive combination benefit in tumours poorly sensitive to platinum and olaparib. In orthotopic models, the combined treatment reduced tumour dissemination in the peritoneal cavity and prolonged survival. Enhanced combination benefit was independent of tumour cell expression of receptor tyrosine kinases targeted by cediranib, and not associated with change in expression of genes associated with DNA repair machinery. However, the combination of cediranib with olaparib was effective in reducing tumour vasculature in all the OC-PDXs. Collectively our data suggest that olaparib and cediranib act through complementary mechanisms affecting tumour cells and tumour microenvironment, respectively. This detailed analysis of the combined effect of VEGF-signalling and PARP inhibitors in OC-PDXs suggest that despite broad activity, there is no dominant common mechanistic inter-dependency driving therapeutic benefit.

Macrophage Activation Status Rather than Repolarization Is Associated with Enhanced Checkpoint Activity in Combination with PI3Kγ Inhibition.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

Implementation of an antibiotic stewardship quality improvement initiative in a community hospital for infants born at ≥35 weeks.

We present our experience in the implementation of an antibiotic stewardship quality improvement initiative directed toward infants born at ≥35 weeks using as a primary tool the Kaiser Permanente early onset sepsis calculator (KP-EOS-C) at the Baylor Scott & White Medical Center - Frisco. After the approval and support of the medical staff and administration, we proceeded to launch an extensive educational program for all women's services nursing staff on how to utilize this calculator to communicate results to the pediatricians on staff. After implementation, we saw a 54% reduction in the number of infants undergoing sepsis workup evaluations and a 51% reduction in the number of infants receiving antibiotics (P < 0.001). We conclude that the implementation of this type of initiative may be feasible and worthwhile in other similar community hospitals, provided there is buy-in by physicians and administration as well as an extensive educational program to the hospital medical and nursing staff.

Longitudinal immune characterization of syngeneic tumor models to enable model selection for immune oncology drug discovery.

BACKGROUND: The ability to modulate immune-inhibitory pathways using checkpoint blockade antibodies such as αPD-1, αPD-L1, and αCTLA-4 represents a significant breakthrough in cancer therapy in recent years. This has driven interest in identifying small-molecule-immunotherapy combinations to increase the proportion of responses. Murine syngeneic models, which have a functional immune system, represent an essential tool for pre-clinical evaluation of new immunotherapies. However, immune response varies widely between models and the translational relevance of each model is not fully understood, making selection of an appropriate pre-clinical model for drug target validation challenging. METHODS: Using flow cytometry, O-link protein analysis, RT-PCR, and RNAseq we have characterized kinetic changes in immune-cell populations over the course of tumor development in commonly used syngeneic models. RESULTS: This longitudinal profiling of syngeneic models enables pharmacodynamic time point selection within each model, dependent on the immune population of interest. Additionally, we have characterized the changes in immune populations in each of these models after treatment with the combination of α-PD-L1 and α-CTLA-4 antibodies, enabling benchmarking to known immune modulating treatments within each model. CONCLUSIONS: Taken together, this dataset will provide a framework for characterization and enable the selection of the optimal models for immunotherapy combinations and generate potential biomarkers for clinical evaluation in identifying responders and non-responders to immunotherapy combinations.

Early Recognition and Treatment of Acute Disseminated Encephalomyelitis in Pediatrics: A Case Series.

OBJECTIVE: Our aim is to emphasize the varied presentation of acute disseminated encephalomyelitis (ADEM) to help health care professionals improve recognition of the disease in a timely manner, thereby allowing for the selection of an appropriate treatment regimen. Therefore, this may avoid neurocognitive consequences and the ultimate fatality of the patient. PATIENTS AND METHODS: This is a retrospective case series involving 7 cases of children presenting to the Pediatric Emergency Department of Hackensack University Medical Center who were ultimately diagnosed with ADEM. RESULTS: In many of the cases, a preceding viral-like illness with nonspecific symptomatology made it difficult to accurately establish an initial diagnosis. Ultimately, the neurologic symptoms spontaneously resolved or improved with administration of high-dose steroids. CONCLUSIONS: Children presenting to the emergency department with nonspecific symptoms associated with any neurological deficits should undergo further investigation using magnetic resonance imaging and lumbar puncture to rule out rare yet possibly fatal diseases such as ADEM.

PI3Kα/δ inhibition promotes anti-tumor immunity through direct enhancement of effector CD8+ T-cell activity.

PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835, a dual PI3Kα/δ inhibitor, specifically on the tumor immune microenvironment using syngeneic models. Continuous suppression of PI3Kα/δ was not required for anti-tumor activity, as tumor growth inhibition was potentiated by an intermittent dosing/schedule in vivo. Moreover, PI3Kα/δ inhibition delivered strong single agent anti-tumor activity, which was associated with dynamic suppression of T-regs, improved CD8+ T-cell activation and memory in mouse syngeneic tumor models. Strikingly, AZD8835 promoted robust CD8+ T-cell activation dissociated from its effect on T-regs. This was associated with enhancing effector cell viability/function. Together these data reveal novel mechanisms by which PI3Kα/δ inhibitors interact with the immune system and validate the clinical compound AZD8835 as a novel immunoncology drug, independent of effects on tumor cells. These data support further clinical investigation of PI3K pathway inhibitors as immuno-oncology agents.

Combination of dual mTORC1/2 inhibition and immune-checkpoint blockade potentiates anti-tumour immunity.

mTOR inhibition can promote or inhibit immune responses in a context dependent manner, but whether this will represent a net benefit or be contraindicated in the context of immunooncology therapies is less understood. Here, we report that the mTORC1/2 dual kinase inhibitor vistusertib (AZD2014) potentiates anti-tumour immunity in combination with anti-CTLA-4 (αCTLA-4), αPD-1 or αPD-L1 immune checkpoint blockade. Combination of vistusertib and immune checkpoint blocking antibodies led to tumour growth inhibition and improved survival of MC-38 or CT-26 pre-clinical syngeneic tumour models, whereas monotherapies were less effective. Underlying these combinatorial effects, vistusertib/immune checkpoint combinations reduced the occurrence of exhausted phenotype tumour infiltrating lymphocytes (TILs), whilst increasing frequencies of activated Th1 polarized T-cells in tumours. Vistusertib alone was shown to promote a Th1 polarizing proinflammatory cytokine profile by innate primary immune cells. Moreover, vistusertib directly enhanced activation of effector T-cell and survival, an effect that was critically dependent on inhibitor dose. Therefore, these data highlight direct, tumour-relevant immune potentiating benefits of mTOR inhibition that complement immune checkpoint blockade. Together, these data provide a clear rationale to investigate such combinations in the clinic.

Functional Roles for Exosomal MicroRNAs in the Tumour Microenvironment.

Extracellular microRNAs are released from cells both passively and actively. The presence of these microRNAs in the tumour microenvironment (TME) can significantly impact on the plasticity of cancer cells leading to the promotion of metastatic and angiogenic processes. These extracellular microRNAs can act not only on other cancer cells, but also cells present in the TME, such as immune cells, endothelial cells, fibroblasts, and others acting to subvert the host immune system and drive tumour progression. In this review we highlight the current understanding of both the mechanisms by which microRNAs are released from tumour cells and the downstream functional effects that extracellular microRNAs have on recipient cells.

Inhibition of oxidative phosphorylation suppresses the development of osimertinib resistance in a preclinical model of EGFR-driven lung adenocarcinoma.

Metabolic plasticity is an emerging hallmark of cancer, and increased glycolysis is often observed in transformed cells. Small molecule inhibitors that target driver oncogenes can potentially inhibit the glycolytic pathway. Osimertinib (AZD9291) is a novel EGFR tyrosine kinase inhibitor (TKI) that is potent and selective for sensitising (EGFRm) and T790M resistance mutations. Clinical studies have shown osimertinib to be efficacious in patients with EGFRm/ T790M advanced NSCLC who have progressed after EGFR-TKI treatment. However experience with targeted therapies suggests that acquired resistance may emerge. Thus there is a need to characterize resistance mechanisms and to devise ways to prevent, delay or overcome osimertinib resistance. We show here that osimertinib suppresses glycolysis in parental EGFR-mutant lung adenocarcinoma lines, but has not in osimertinib-resistant cell lines. Critically, we show osimertinib treatment induces a strict dependence on mitochondrial oxidative phosphorylation (OxPhos), as OxPhos inhibitors significantly delay the long-term development of osimertinib resistance in osimertinib-sensitive lines. Accordingly, growth conditions which promote a less glycolytic phenotype confer a degree of osimertinib resistance. Our data support a model in which the combination of osimertinib and OxPhos inhibitors can delay or prevent resistance in osimertinib-naïve tumour cells, and represents a novel strategy that warrants further pre-clinical investigation.

Circulating MicroRNAs as Biomarkers and Mediators of Cell-Cell Communication in Cancer.

The realization of personalized medicine for cancer will rely not only on the development of new therapies, but on biomarkers that direct these therapies to the right patient. MicroRNA expression profiles in the primary tumor have been shown to differ between cancer patients and healthy individuals, suggesting they might make useful biomarkers. However, examination of microRNA expression in the primary tumor requires an invasive biopsy procedure. More recently, microRNAs have been shown to be released from the primary tumor into the circulation where they can be utilized as non-invasive biomarkers to diagnose patients, predict prognosis, or indicate therapeutic response. This review provides an overview of the current use of circulating microRNAs as biomarkers as well as recent findings on their role in regulating cell signaling interactions in the tumor microenvironment.

Contraception or eugenics? Sterilization and "mental retardation" in the 1970s and 1980s.

Nonconsensual sterilization is usually seen as the by-product of a classist and racist society; disability is ignored. This article examines the 1973 sterilization of two young black girls from Alabama and other precedent-setting court cases involving the sterilization of "mentally retarded" white women to make disability more central to the historical analysis of sterilization. It analyzes the concept of mental retardation and the appeal of a surgical solution to birth control, assesses judicial deliberations over the "right to choose" contraceptive sterilization when the capacity to consent is in doubt, and reflects on the shadow of eugenics that hung over the sterilization debate in the 1970s and 1980s.

Fibroblast growth factor receptor splice variants are stable markers of oncogenic transforming growth factor ß1 signaling in metastatic breast cancers.

INTRODUCTION: Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) facilitate breast cancer (BC) metastasis; however, stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, with the hope of targeting unique aspects of metastatic tumor outgrowth, we sought to identify molecular markers that could identify tumor cells that had completed the EMT:MET cycle. METHODS: An in vivo reporter system for epithelial cadherin (E-cad) expression was used to quantify its regulation in metastatic BC cells during primary and metastatic tumor growth. Exogenous addition of transforming growth factor ß1 (TGF-ß1) was used to induce EMT in an in situ model of BC. Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-ß1, thus completing one full EMT:MET cycle. Changes in fibroblast growth factor receptor type 1 (FGFR1) isoform expression were validated using PCR analyses of patient-derived tumor tissues versus matched normal tissues. FGFR1 gene expression was manipulated using short hairpin RNA depletion and cDNA rescue. Preclinical pharmacological inhibition of FGFR kinase was employed using the orally available compound BGJ-398. RESULTS: Metastatic BC cells undergo spontaneous downregulation of E-cad during primary tumor growth, and its expression subsequently returns following initiation of metastatic outgrowth. Exogenous exposure to TGF-ß1 was sufficient to drive the metastasis of an otherwise in situ model of BC and was similarly associated with a depletion and return of E-cad expression during metastatic progression. BC cells treated and withdrawn from TGF-ß stably upregulate a truncated FGFR1-ß splice variant that lacks the outermost extracellular immunoglobulin domain. Identification of this FGFR1 splice variant was verified in metastatic human BC cell lines and patient-derived tumor samples. Expression of FGFR1-ß was also dominant in a model of metastatic outgrowth where depletion of FGFR1 and pharmacologic inhibition of FGFR kinase activity both inhibited pulmonary tumor outgrowth. Highlighting the dichotomous nature of FGFR splice variants and recombinant expression of full-length FGFR1-α also blocked pulmonary tumor outgrowth. CONCLUSION: The results of our study strongly suggest that FGFR1-ß is required for the pulmonary outgrowth of metastatic BC. Moreover, FGFR1 isoform expression can be used as a predictive biomarker for therapeutic application of its kinase inhibitors.

Therapeutic Opportunities for Targeting microRNAs in Cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that can function as either powerful tumor promoters or suppressors in numerous types of cancer. The ability of miRs to target multiple genes and biological signaling pathways has created intense interest in their potential clinical utility as predictive and diagnostic biomarkers, and as innovative therapeutic agents. Recently, accumulating preclinical studies have illustrated the feasibility of slowing tumor progression by either overexpressing tumor suppressive miRNAs, or by neutralizing the activities of oncogenic miRNAs in cell- and animal-based models of cancer. Here we highlight prominent miRNAs that may represent potential therapeutic targets in human malignancies, as well as review current technologies available for inactivating or restoring miRNA activity in clinical settings.

Upregulated WAVE3 expression is essential for TGF-ß-mediated EMT and metastasis of triple-negative breast cancer cells.

Breast cancer is the second leading cause of cancer death in women in the United States. Metastasis accounts for the death of ~90 % of these patients, yet the mechanisms underlying this event remain poorly defined. WAVE3 belongs to the WASP/WAVE family of actin-binding proteins that play essential roles in regulating cell morphology, actin polymerization, cytoskeleton remodeling, cell motility, and invasion. Accordingly, we demonstrated previously that WAVE3 promotes the acquisition of invasive and metastatic phenotypes by human breast cancers. Herein, we show that transforming growth factor-ß (TGF-ß) selectively and robustly induced the expression of WAVE3 in metastatic breast cancer cells, but not in their nonmetastatic counterparts. Moreover, the induction of WAVE3 expression in human and mouse triple-negative breast cancer cells (TNBCs) by TGF-ß likely reflects its coupling to microRNA expression via a Smad2- and ß3 integrin-dependent mechanism. We further demonstrate the requirement for WAVE3 expression in mediating the initiation of epithelial-mesenchymal transition (EMT) programs stimulated by TGF-ß. Indeed, stable depletion of WAVE3 expression in human TNBC cells prevented TGF-ß from inducing EMT programs and from stimulating the proliferation, migration, and the formation of lamellipodia in metastatic TNBC cells. Lastly, we observed WAVE3 deficiency to abrogate the outgrowth of TNBC cell organoids in 3-dimensional organotypic cultures as well as to decrease the growth and metastasis of 4T1 tumors produced in syngeneic Balb/C mice. Indeed, WAVE3 deficiency significantly reduced the presence of sarcomatoid morphologies indicative of EMT phenotypes in pulmonary TNBC tumors as compared to those detected in their parental counterparts. Collectively, these findings indicate the necessity for WAVE3 expression and activity during EMT programs stimulated by TGF-ß; they also suggest that measures capable of inactivating WAVE3 may play a role in alleviating metastasis stimulated by TGF-ß.

TGF-ß upregulates miR-181a expression to promote breast cancer metastasis.

Late-stage breast cancer metastasis is driven by dysregulated TGF-ß signaling, but the underlying molecular mechanisms have not been fully elucidated. We attempted to recapitulate tumor and metastatic microenvironments via the use of biomechanically compliant or rigid 3D organotypic cultures and combined them with global microRNA (miR) profiling analyses to identify miRs that were upregulated in metastatic breast cancer cells by TGF-ß. Here we establish miR-181a as a TGF-ß-regulated "metastamir" that enhanced the metastatic potential of breast cancers by promoting epithelial-mesenchymal transition, migratory, and invasive phenotypes. Mechanistically, inactivation of miR-181a elevated the expression of the proapoptotic molecule Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a expression was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression was dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and was highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings strongly implicate miR-181a as a predictive biomarker for breast cancer metastasis and patient survival, and consequently, as a potential therapeutic target in metastatic breast cancer.

Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer.

Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-ß (TGF-ß) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-ß. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated ß1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating ß1 integrin expression necessary for metastatic outgrowth.