Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression.

Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5 h to ∼1.5 h. Loss of K2 inhibits focal adhesion turnover and ß1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.

The WAVE3/ß-catenin oncogenic signaling regulates chemoresistance in triple negative breast cancer.

BACKGROUND: Metastatic breast cancer is responsible for the death of the majority of breast cancer patients. In fact, metastatic BC is the 2nd leading cause of cancer-related deaths in women in the USA and worldwide. Triple negative breast cancer (TNBC), which lacks expression of hormone receptors (ER-α and PR) and ErbB2/HER2, is especially lethal due to its highly metastatic behavior, propensity to recur rapidly, and for its resistance to standard of care therapies, through mechanisms that remain incompletely understood. WAVE3 has been established as a promoter of TNBC development and metastatic progression. In this study, we investigated the molecular mechanisms whereby WAVE3 promotes therapy-resistance and cancer stemness in TNBC, through the regulation of ß-catenin stabilization. METHODS: The Cancer Genome Atlas dataset was used to assess the expression of WAVE3 and ß-catenin in breast cancer tumors. Kaplan-Meier Plotter analysis was used to correlate expression of WAVE3 and ß-catenin with breast cancer patients' survival probability. MTT assay was used to quantify cell survival. CRISPR/Cas9-mediated gene editing, 2D and 3D tumorsphere growth and invasion assays, Immunofluorescence, Western blotting, Semi-quantitative and real-time quantitative PCR analyses were applied to study the WAVE3/ß-catenin oncogenic signaling in TNBC. Tumor xenograft assays were used to study the role of WAVE3 in mediating chemotherapy resistance of TNBC tumors. RESULTS: Genetic inactivation of WAVE3 in combination of chemotherapy resulted in inhibition of 2D growth and 3D tumorsphere formation and invasion of TNBC cells in vitro, as well as tumor growth and metastasis in vivo. In addition, while re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 did not. Further studies revealed that dual blocking of WAVE3 expression or phosphorylation in combination with chemotherapy treatment inhibited the activity and expression and stabilization of ß-catenin. Most importantly, the combination of WAVE3-deficiency or WAVE3-phospho-deficiency and chemotherapy suppressed the oncogenic behavior of chemoresistant TNBC cells, both in vitro and in vivo. CONCLUSION: We identified a novel WAVE3/ß-catenin oncogenic signaling axis that modulates chemoresistance of TNBC. This study suggests that a targeted therapeutic strategy against WAVE3 could be effective for the treatment of chemoresistant TNBC tumors.

Neighborhood and racial influences on triple negative breast cancer: evidence from Northeast Ohio.

PURPOSE: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) with higher recurrence rates and poorer prognoses and most prevalent among non-Hispanic Black women. Studies of multiple health conditions and care processes suggest that neighborhood socioeconomic position is a key driver of health disparities. We examined roles of patients' neighborhood-level characteristics and race on prevalence, stage at diagnosis, and mortality among patients diagnosed with BC at a large safety-net healthcare system in Northeast Ohio. METHODS: We used tumor registry to identify BC cases from 2007 to 2020 and electronic health records and American Community Survey for individual- and area-level factors. We performed multivariable regression analyses to estimate associations between neighborhood-level characteristics, measured by the Area Deprivation Index (ADI), race and comparative TNBC prevalence, stage at diagnosis, and total mortality. RESULTS: TNBC was more common among non-Hispanic Black (53.7%) vs. non-Hispanic white patients (46.4%). Race and ADI were individually significant predictors of TNBC prevalence, stage at diagnosis, and total mortality. Race remained significantly associated with TNBC subtype, adjusting for covariates. Accounting for TNBC status, a more disadvantaged neighborhood was significantly associated with a worse stage at diagnosis and higher death rates. CONCLUSION: Our findings suggest that both neighborhood socioeconomic position and race are strongly associated with TNBC vs. other BC subtypes. The burden of TNBC appears to be highest among Black women in the most socioeconomically disadvantaged neighborhoods. Our study suggests a complex interplay of social conditions and biological disease characteristics contributing to racial disparities in BC outcomes.

Inducible Nitric Oxide Synthase Embedded in Alginate/Polyethyleneimine Hydrogel as a New Platform to Explore NO-Driven Modulation of Biological Function.

Nitric oxide (NO), a small free radical molecule, turned out to be pervasive in biology and was shown to have a substantial influence on a range of biological activities, including cell growth and apoptosis. This molecule is involved in signaling and affects a number of physiologic functions. In recent decades, several processes related to cancer, such as angiogenesis, programmed cell death, infiltration, cell cycle progression, and metastasis, have been linked with nitric oxide. In addition, other parallel work showed that NO also has the potential to operate as an anti-cancer agent. As a result, it has gained attention in cancer-related therapeutics. The nitric oxide synthase enzyme family (NOS) is required for the biosynthesis of nitric oxide. It is becoming increasingly popular to develop NO-releasing materials as strong tumoricidal therapies that can deliver sustained high concentrations of nitric oxide to tumor sites. In this paper, we developed NO-releasing materials based on sodium alginate hydrogel. In this regard, alginate hydrogel discs were modified by adsorbing layers of polyethyleneimine and iNOS-oxygenase. These NO-releasing hydrogel discs were prepared using the layer-by-layer film building technique. The iNOS-oxygenase is adsorbed on the positively charged polyethyleneimine (PEI) matrix layer, which was formed on a negatively charged sodium alginate hydrogel. We show that nitric oxide is produced by enzymes contained within the hydrogel material when it is exposed to a solution containing all the components necessary for the NOS reaction. The electrostatic chemical adsorption of the layer-by-layer process was confirmed by FTIR measurements as well as scanning electron microscopy. We then tested the biocompatibility of the resulting modified sodium alginate hydrogel discs. We showed that this NOS-PEI-modified hydrogel is overall compatible with cell growth. We characterized the NOS/hydrogel films and examined their functional features in terms of NO release profiles. However, during the first 24 h of activity, these films show an increase in NO release flux, followed by a gradual drop and then a period of stable NO release. These findings show the inherent potential of using this system as a platform for NO-driven modulation of biological functions, including carcinogenesis.

The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma.

Inhibition of the protein neddylation process by the small-molecule inhibitor MLN4924 has been recently indicated as a promising direction for cancer treatment. However, the knowledge of all biological consequences of MLN4924 for cancer cells is still incomplete. Here, we report that MLN4924 inhibits tumor necrosis factor-alpha (TNF-α)-induced matrix metalloproteinase 9 (MMP9)-driven cell migration. Using real-time polymerase chain reaction (PCR) and gelatin zymography, we found that MLN4924 inhibited expression and activity of MMP9 at the messenger RNA (mRNA) and protein levels in both resting cells and cells stimulated with TNF-α, and this inhibition was closely related to impaired cell migration. We also revealed that MLN4924, similar to TNF-α, induced phosphorylation of inhibitor of nuclear factor kappa B-alpha (IκB-α). However, contrary to TNF-α, MLN4924 did not induce IκB-α degradation in treated cells. In coimmunoprecipitation experiments, nuclear IκB-α which formed complexes with nuclear factor kappa B p65 subunit (NFκB/p65) was found to be highly phosphorylated at Ser32 in the cells treated with MLN4924, but not in the cells treated with TNF-α alone. Moreover, in the presence of MLN4924, nuclear NFκB/p65 complexes were found to be enriched in c-Jun and cyclin dependent kinase inhibitor 1 A (CDKN1A/p21) proteins. In these cells, NFκB/p65 was unable to bind to the MMP9 gene promoter, which was confirmed by the chromatin immunoprecipitation (ChIP) assay. Taken together, our findings identified MLN4924 as a suppressor of TNF-α-induced MMP9-driven cell migration in esophageal squamous cell carcinoma (ESCC), likely acting by affecting the nuclear ubiquitin-proteasome system that governs NFκB/p65 complex formation and its DNA binding activity in regard to the MMP9 promoter, suggesting that inhibition of neddylation might be a new therapeutic strategy to prevent invasion/metastasis in ESCC patients.

Surfing the big WAVE: Insights into the role of WAVE3 as a driving force in cancer progression and metastasis.

WAVE3 belongs to the WASP/WAVE family of actin cytoskeleton remodeling proteins. These proteins are known to be involved in several biological functions ranging from controlling cell shape and movement, to being closely associated with pathological conditions such as cancer progression and metastasis. Last decade has seen an explosion in the literature reporting significant scientific advances on the molecular mechanisms whereby the WASP/WAVE proteins are regulated both in normal physiological as well as pathological conditions. The purpose of this review is to present the major findings pertaining to how WAVE3 has become a critical player in the regulation of signaling pathways involved in cancer progression and metastasis. The review will conclude with suggesting options for the potential use of WAVE3 as a therapeutic target to prevent the progression of cancer to the lethal stage that is the metastatic disease.

Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced ß1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of ß1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between ß1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.

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.

Role of WAVE3 as an of actin binding protein in the pathology of triple negative breast cancer.

Breast cancer, a prevalent global health concern, has sparked extensive research efforts, particularly focusing on triple negative breast cancer (TNBC), a subtype lacking estrogen receptor (ER), progesterone receptor, and epidermal growth factor receptor. TNBC's aggressive nature and resistance to hormone-based therapies heightens the risk of tumor progression and recurrence. Actin-binding proteins, specifically WAVE3 from the Wiskott-Aldrich syndrome protein (WASP) family, have emerged as major drivers in understanding TNBC biology. This review delves into the intricate molecular makeup of TNBC, shedding light on actin's fundamental role in cellular processes. Actin, a structural element in the cytoskeleton, regulates various cellular pathways essential for homeostasis. Its dynamic nature enables functions such as cell migration, motility, intracellular transport, cell division, and signal transduction. Actin-binding proteins, including WAVE3, play pivotal roles in these processes. WAVE3, a member of the WASP family, remains the focus of this review due to its potential involvement in TNBC progression. While actin-binding proteins are studied for their roles in healthy cellular cycles, their significance in TNBC remains underexplored. This review aims to discuss WAVE3's impact on TNBC, exploring its molecular makeup, functions, and significance in tumor progression. The intricate structure of WAVE3, featuring elements like the verprolin-cofilin-acidic domain and regulatory elements, plays a crucial role in regulating actin dynamics. Dysregulation of WAVE3 in TNBC has been linked to enhanced cell migration, invasion, extracellular matrix remodeling, epithelial-mesenchymal transition, tumor proliferation, and therapeutic resistance. Understanding the role of actin-binding proteins in cancer biology has potential clinical implications, making them potential prognostic biomarkers and promising therapeutic targets. The review emphasizes the need for further research into actin-binding proteins' clinical applications, diagnostic value, and therapeutic interventions. In conclusion, this comprehensive review explores the complex interplay between actin and actin-binding proteins, with special emphasis on WAVE3, in the context of TNBC. By unraveling the molecular intricacies, structural characteristics, and functional significance, the review paves the way for future research directions, clinical applications, and potential therapeutic strategies in the challenging landscape of TNBC.

Advances in 3D Culture Models to Study Exosomes in Triple-Negative Breast Cancer.

Breast cancer, a leading cause of cancer-related deaths globally, exhibits distinct subtypes with varying pathological, genetic, and clinical characteristics. Despite advancements in breast cancer treatments, its histological and molecular heterogeneity pose a significant clinical challenge. Triple-negative breast cancer (TNBC), a highly aggressive subtype lacking targeted therapeutics, adds to the complexity of breast cancer treatment. Recent years have witnessed the development of advanced 3D culture technologies, such as organoids and spheroids, providing more representative models of healthy human tissue and various malignancies. These structures, resembling organs in structure and function, are generated from stem cells or organ-specific progenitor cells via self-organizing processes. Notably, 3D culture systems bridge the gap between 2D cultures and in vivo studies, offering a more accurate representation of in vivo tumors' characteristics. Exosomes, small nano-sized molecules secreted by breast cancer and stromal/cancer-associated fibroblast cells, have garnered significant attention. They play a crucial role in cell-to-cell communication, influencing tumor progression, invasion, and metastasis. The 3D culture environment enhances exosome efficiency compared to traditional 2D cultures, impacting the transfer of specific cargoes and therapeutic effects. Furthermore, 3D exosomes have shown promise in improving therapeutic outcomes, acting as potential vehicles for cancer treatment administration. Studies have demonstrated their role in pro-angiogenesis and their innate therapeutic potential in mimicking cellular therapies without side effects. The 3D exosome model holds potential for addressing challenges associated with drug resistance, offering insights into the mechanisms underlying multidrug resistance and serving as a platform for drug screening. This review seeks to emphasize the crucial role of 3D culture systems in studying breast cancer, especially in understanding the involvement of exosomes in cancer pathology.

Kindlin-2 Regulates the Oncogenic Activities of Integrins and TGF-ß In Triple Negative Breast Cancer Progression and Metastasis.

Background: Kindlin-2, an adaptor protein, is dysregulated in various human cancers, including triple negative breast cancer (TNBC), where it drives tumor progression and metastasis by influencing several cancer hallmarks. One well-established role of Kindlin-2 involves the regulation of integrin signaling, achieved by directly binding to the cytoplasmic tail of the integrin ß subunit. In this study, we present novel insights into Kindlin-2's involvement in stabilizing the ß1-Integrin:TGF-ß type 1 receptor (TßRI) complexes, acting as a physical bridge that links ß1-Integrin to TßRI. The loss of Kindlin-2 results in the degradation of this protein complex, leading to the inhibition of downstream oncogenic pathways. Methods: Our methodology encompassed a diverse range of in vitro assays, including CRISPR/Cas9 gene editing, cell migration, 3D tumorsphere formation and invasion, solid binding, co-immunoprecipitation, cell adhesion and spreading assays, as well as western blot and flow cytometry analyses, utilizing MDA-MB-231 and 4T1 TNBC cell lines. Additionally, preclinical in vivo mouse models of TNBC tumor progression and metastasis were employed to substantiate our findings. Results: The investigation revealed that the direct interaction between Kindlin-2 and ß1-Integrin is mediated through the C-terminal F3 domain of Kindlin-2, while the interaction between Kindlin-2 and TßRI is facilitated through the F2 domain of Kindlin-2. Disruption of this bridge, achieved via CRISPR/Cas9-mediated knockout of Kindlin-2, led to the degradation of ß1-Integrin and TßRI, resulting in the inhibition of oncogenic pathways downstream of both proteins, subsequently hindering tumor growth and metastasis. Treatment of Kindlin-2-deficient cells with the proteasome inhibitor MG-132 restored the expression of both ß1-Integrin and TßRI. Furthermore, the rescue of Kindlin-2 expression reinstated their oncogenic activities both in vitro and in vivo. Conclusions: This study identifies a novel function of Kindlin-2 in stabilizing the ß1-Integrin:TßR1 complexes and regulating their downstream oncogenic signaling. The translational implications of these findings are substantial, potentially unveiling new therapeutically targeted pathways crucial for the treatment of TNBC tumors.

Role of Kindlin 2 in prostate cancer.

Kindlin-2 is a cytoskeletal adapter protein that is present in many different cell types. By virtue of its interaction with multiple binding partners, Kindlin-2 intercalates into numerous signaling pathways and cytoskeletal nodes. A specific interaction of Kindlin-2 that is of paramount importance in many cellular responses is its direct binding to the cytoplasmic tails of integrins, an interaction that controls many of the adhesive, migratory and signaling responses mediated by members of the integrin family of cell-surface heterodimers. Kindlin-2 is highly expressed in many cancers and is particularly prominent in prostate cancer cells. CRISPR/cas9 was used as a primary approach to knockout expression of Kindlin-2 in both androgen-independent and dependent prostate cancer cell lines, and the effects of Kindlin-2 suppression on oncogenic properties of these prostate cancer cell lines was examined. Adhesion to extracellular matrix proteins was markedly blunted, consistent with the control of integrin function by Kindlin-2. Migration across matrices was also affected. Anchorage independent growth was markedly suppressed. These observations indicate that Kindlin-2 regulates hallmark features of prostate cancer cells. In androgen expressing cells, testosterone-stimulated adhesion was Kindlin-2-dependent. Furthermore, tumor growth of a prostate cancer cell line lacking Kindlin-2 and implanted into the prostate gland of immunocompromised mice was markedly blunted and was associated with suppression of angiogenesis in the developing tumor. These results establish a key role of Kindlin-2 in prostate cancer progression and suggest that Kindlin-2 represents an interesting therapeutic target for treatment of prostate cancer.

Expression of the αVß3 integrin affects prostate cancer sEV cargo and density and promotes sEV pro-tumorigenic activity in vivo through a GPI-anchored receptor, NgR2.

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVß3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVß3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVß3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVß3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVß3 and an αVß3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVß3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVß3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVß3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVß3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.

miRNA-548c: a specific signature in circulating PBMCs from dilated cardiomyopathy patients.

High fidelity genome-wide expression analysis has strengthened the idea that microRNA (miRNA) signatures in peripheral blood mononuclear cells (PBMCs) can be potentially used to predict the pathology when anatomical samples are inaccessible like the heart. PBMCs from 48 non-failing controls and 44 patients with relatively stable chronic heart failure (ejection fraction of ≤ 40%) associated with dilated cardiomyopathy (DCM) were used for miRNA analysis. Genome-wide miRNA-microarray on PBMCs from chronic heart failure patients identified miRNA signature uniquely characterized by the downregulation of miRNA-548 family members. We have also independently validated downregulation of miRNA-548 family members (miRNA-548c & 548i) using real time-PCR in a large cohort of independent patient samples. Independent in silico Ingenuity Pathway Analysis (IPA) of miRNA-548 targets shows unique enrichment of signaling molecules and pathways associated with cardiovascular disease and hypertrophy. Consistent with specificity of miRNA changes with pathology, PBMCs from breast cancer patients showed no alterations in miRNA-548c expression compared to healthy controls. These studies suggest that miRNA-548 family signature in PBMCs can therefore be used to detect early heart failure. Our studies show that cognate networking of predicted miRNA-548 targets in heart failure can be used as a powerful ancillary tool to predict the ongoing pathology.

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.

Alginate-based hydrogel platform embedding silver nanoparticles and cisplatin: characterization of the synergistic effect on a breast cancer cell line.

Introduction: Breast cancer is a significant cause of mortality in women globally, and current treatment approaches face challenges due to side effects and drug resistance. Nanotechnology offers promising solutions by enabling targeted drug delivery and minimizing toxicity to normal tissues. Methods: In this study, we developed a composite platform called (Alg-AgNPs-CisPt), consisting of silver nanoparticles coated with an alginate hydrogel embedding cisplatin. We examined the effectiveness of this nanocomplex in induce synergistic cytotoxic effects on breast cancer cells. Results and Discussion: Characterization using various analytical techniques confirmed the composition of the nanocomplex and the distribution of its components. Cytotoxicity assays and apoptosis analysis demonstrated that the nanocomplex exhibited greater efficacy against breast cancer cells compared to AgNPs or cisplatin as standalone treatments. Moreover, the nanocomplex was found to enhance intracellular reactive oxygen species levels, further validating its efficacy. The synergistic action of the nanocomplex constituents offers potential advantages in reducing side effects associated with higher doses of cisplatin as a standalone treatment. Overall, this study highlights the potential of the (Alg-AgNPs-CisPt) nanocomplex as a promising platform embedding components with synergistic action against breast cancer cells.

The WAVE2/miR-29/Integrin-ß1 Oncogenic Signaling Axis Promotes Tumor Growth and Metastasis in Triple-negative Breast Cancer.

Breast cancer is the most frequently diagnosed malignancy in women and the major cause of death because of its invasion, metastasis, and resistance to therapies capabilities. The most aggressive subtype of breast cancer is triple-negative breast cancer (TNBC) due to invasive and metastatic properties along with early age of diagnosis and poor prognosis. TNBC tumors do not express estrogen, progesterone, and HER2 receptors, which limits their treatment with targeted therapies. Cancer invasiveness and metastasis are known to be promoted by increased cell motility and upregulation of the WAVE proteins. While the contribution of WAVE2 to cancer progression is well documented, the WAVE2-mediated regulation of TNBC oncogenic properties is still under investigated, as does the molecular mechanisms by which WAVE2 regulates such oncogenic pathways. In this study, we show that WAVE2 plays a significant role in TNBC development, progression, and metastasis, through the regulation of miR-29 expression, which in turn targets Integrin-ß1 (ITGB1) and its downstream oncogenic activities. Conversely, we found WAVE2 expression to be regulated by miR-29 in a negative regulatory feedback loop. Reexpression of exogenous WAVE2 in the WAVE2-deficient TNBC cells resulted in reactivation of ITGB1 expression and activity, further confirming the specificity of WAVE2 in regulating Integrin-ß1. Together, our data identify a novel WAVE2/miR-29/ITGB1 signaling axis, which is essential for the regulation of the invasion-metastasis cascade in TNBC. Our findings offer new therapeutic strategies for the treatment of TNBC by targeting WAVE2 and/or its downstream effectors. Significance: Identification of a novel WAVE2/miR-29/ITGB1 signaling axis may provide new insights on how WAVE2 regulates the invasion-metastasis cascade of TNBC tumors through the modulation of ITGB1 and miR-29.

Time-resolved live-cell spectroscopy reveals EphA2 multimeric assembly.

Ephrin type-A receptor 2 (EphA2) is a receptor tyrosine kinase that initiates both ligand-dependent tumor-suppressive and ligand-independent oncogenic signaling. We used time-resolved, live-cell fluorescence spectroscopy to show that the ligand-free EphA2 assembles into multimers driven by two types of intermolecular interactions in the ectodomain. The first type entails extended symmetric interactions required for ligand-induced receptor clustering and tumor-suppressive signaling that inhibits activity of the oncogenic extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) protein kinases and suppresses cell migration. The second type is an asymmetric interaction between the amino terminus and the membrane proximal domain of the neighboring receptors, which supports oncogenic signaling and promotes migration in vitro and tumor invasiveness in vivo. Our results identify the molecular interactions that drive the formation of the EphA2 multimeric signaling clusters and reveal the pivotal role of EphA2 assembly in dictating its opposing functions in oncogenesis.

miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer.

BACKGROUND: microRNAs have been established as powerful regulators of gene expression in normal physiological as well as in pathological conditions, including cancer progression and metastasis. Recent studies have demonstrated a key role of miR-31 in the progression and metastasis of breast cancer. Downregulation of miR-31 enhances several steps of the invasion-metastasis cascade in breast cancer, i.e., local invasion, extravasation and survival in the circulation system, and metastatic colonization of distant sites. miR-31 exerts its metastasis-suppressor activity by targeting a cohort of pro-metastatic genes, including RhoA and WAVE3. The molecular mechanisms that lead to the loss of miR-31 and the activation of its pro-metastatic target genes during these specific steps of the invasion-metastasis cascade are however unknown. RESULTS: In the present report, we identify promoter hypermethylation as one of the major mechanisms for silencing miR-31 in breast cancer, and in the triple-negative breast cancer (TNBC) cell lines of basal subtype, in particular. miR-31 maps to the intronic sequence of a novel long non-coding (lnc)RNA, LOC554202 and the regulation of its transcriptional activity is under control of LOC554202. Both miR-31 and the host gene LOC554202 are down-regulated in the TNBC cell lines of basal subtype and over-expressed in the luminal counterparts. Treatment of the TNBC cell lines with either a de-methylating agent alone or in combination with a de-acetylating agent resulted in a significant increase of both miR-31 and its host gene, suggesting an epigenetic mechanism for the silencing of these two genes by promoter hypermethylation. Finally, both methylation-specific PCR and sequencing of bisulfite-converted DNA demonstrated that the LOC554202 promoter-associated CpG island is heavily methylated in the TNBC cell lines and hypomethylated in the luminal subtypes. CONCLUSION: Loss of miR-31 expression in TNBC cell lines is attributed to hypermethylation of its promoter-associated CpG island. Together, our results provide the initial evidence for a mechanism by which miR-31, an important determinant of the invasion metastasis cascade, is regulated in breast cancer.

Lgi1 null mutant mice exhibit myoclonic seizures and CA1 neuronal hyperexcitability.

LGI1 in humans is responsible for a predisposition to autosomal dominant partial epilepsy with auditory features (ADPEAF). However, mechanisms of how LGI1 mutations cause epilepsy remain unclear. We have used a mouse chromosome engineering strategy to create a null mutation for the gene ortholog encoding LGI1. The Lgi1 null mutant mice show no gross overall developmental abnormalities from routine histopathological analysis. After 12-18 days of age, the homozygous mutant mice all exhibit myoclonic seizures accompanied by rapid jumping and running and die shortly thereafter. The heterozygous mutant mice do not develop seizures. Electrophysiological analysis demonstrates an enhanced excitatory synaptic transmission by increasing the release of the excitatory neurotransmitter glutamate, suggesting a basis for the seizure phenotype. This mouse model, therefore, provides novel insights into the mechanism behind ADPEAF and offers a new opportunity to study the mechanism behind the role of LGI1 in susceptibility to myoclonic seizures.