Metastatic Breast Cancer Cells Research Articles

3D Culture Modeling of Metastatic Breast Cancer Cells in Additive Manufactured Scaffolds.

Cancer biology research is increasingly moving toward innovative in vitro 3D culture models, as conventional and current 2D cell cultures fail to resemble in vivo cancer biology. In the current study, porous 3D scaffolds, designed with two different porosities along with 2D tissue culture polystyrene (TCP) plates were used with a model breast cancer human cell line. The 3D engineered system was evaluated for the optimal seeding method (dynamic versus static), adhesion, and proliferation rate of MDA-MB-231 breast cancer cells. The expression profiles of proliferation-, stemness-, and dormancy-associated cancer markers, namely, ki67, lamin A/C, SOX2, Oct3/4, stanniocalcin 1 (STC1), and stanniocalcin 2 (STC2), were evaluated in the 3D cultured cells and compared to the respective profiles of the cells cultured in the conventional 2D TCP. Our data suggested that static seeding was the optimal seeding method with porosity-dependent efficiency. Moreover, cells cultured in 3D scaffolds displayed a more dormant phenotype in comparison to 2D, which was manifested by the lower proliferation rate, reduced ki67 expression, increased lamin A/C expression, and overexpression of STCs. The possible relationship between the cell affinity to different extracellular matrix (ECM) proteins and the RANK expression levels was also addressed after deriving collagen type I (COL-I) and fibronectin (FN) MDA-MB-231 filial cell lines with enhanced capacity to attach to the respective ECM proteins. The new derivatives exhibited a more mesenchymal like phenotype and higher RANK levels in relation to the parental cells, suggesting a relationship between ECM cell affinity and RANK expression. Therefore, the present 3D cell culture model shows that cancer cells on printed scaffolds can work as better representatives in cancer biology and drug screening related studies.

HER3 expression in metastatic colorectal cancer: Defining the clinicomolecular profile of an emerging target.

3588 Background: The success of tailored systemic therapies in treating distinct molecular subsets of patients (e.g., deficient mismatch repair, BRAF mutant, HER2 amplified) has spurred further exploration of novel targetable subsets within the heterogeneous landscape of metastatic colorectal cancer (mCRC). Human epidermal growth factor receptor 3 [HER3 (ErbB3)], a member of the HER (ErbB) receptor tyrosine kinase family, plays an important role in tumorigenesis and metastases and has emerged as a promising therapeutic target in a diverse array of cancers. For example, patritumab deruxtecan (U3-1402; HER3-DXd) is a HER3-directed antibody drug conjugate that has demonstrated clinically meaningful antitumor activity and acceptable safety profiles in metastatic breast cancer and EGFR-mutated non-small cell lung cancer. There is limited data, however, on the clinicopathological characterization of HER3 expression in mCRC. Methods: Tissue samples (surgical-metastatectomy) (N = 115) were obtained from a clinical cohort of patients (N = 99) with histologically proven mCRC and liver metastases who underwent liver resection with/without perioperative systemic chemotherapy. HER3 expression was analyzed on whole-mount preparations by immunohistochemistry (IHC). Staining was performed and visualized using the HER3 (D22C5) XP Rabbit-mAb (Cell Signaling Technology). Patients were categorized based on membranous intensity score as follows: Low with IHC 0 (absence of staining or staining in < 10% of tumor cells), 1+ (faint/barely perceptible staining in ≥10% of tumor cells) or 2+ (weak to moderate staining in ≥10% of tumor cells), or High with IHC 3+ (strong staining in ≥10% of tumor cells). Clinicomolecular and treatment data, including gender, tumor sidedness, mutational status (RAS or BRAF), and prior chemotherapy were collected by review of patient electronic medical records. Chi-squared (or Fisher’s exact) test were used to determine associations between groups. Overall survival (OS) was calculated using Kaplan-Meier method and compared using log-rank tests. Results: Among 99 analyzed patients, 98 were evaluable for HER3 expression. Of these 25.5%, 26.5%, 40.8% and 7.2% showed HER3 IHC scores of 3+, 2+, 1+ and 0, respectively. No significant association was seen with HER3 expression and clinicopathological variables, mutational status, or prior treatment. Among patients with 2 samples analyzed from the same liver surgery, there was a moderate level of heterogeneity with concordance of 78.5% (kappa 0.43). Patients with high HER3 expression had poorer OS (5-year OS: 52%; median: 90.2 months) compared to low HER3 expression (5-year OS: 85%; median: not reached). Conclusions: In this large cohort of mCRC, HER3 expression was observed in 92.8% of patients and across diverse clinical and molecular features, supporting HER3 as a promising targetable biomarker in a large subset of mCRC.

Comparing extracellular vesicles and cell membranes as biocompatible coatings for gold nanorods: Implications for targeted theranostics

Extracellular vesicles (EVs) and cell membrane nanoghosts are excellent coatings for nanomaterials, providing enhanced delivery in the target sites and evasion of the immune system. These cell-derived coatings allow the exploration of the delivery properties of the nanoparticles without stimulation of the immune system. Despite the advances reported on the use of EVs and cell-membrane coatings for nanomedicine applications, there are no standards to compare the benefits and main differences between these technologies. Here we investigated macrophage-derived EVs and cell membranes-coated gold nanorods and compared both systems in terms of target delivery in cancer and stromal cells. Our results reveal a higher tendency of EV-coated nanorods to interact with macrophages yet both EV and cell membrane-coated nanorods were internalized in the metastatic breast cancer cells. The main differences between these nanoparticles are related to the presence or absence of CD47 in the coating material, not usually addressed in EVs characterization. Our findings highlight important delivery differences exhibited by EVs- or cell membranes- coated nanorods which understanding may be important to the design and development of theragnostic nanomaterials using these coatings for target delivery.

Increased Ammonium Toxicity in Response to Exogenous Glutamine in Metastatic Breast Cancer Cells.

Several cancers, including breast cancers, show dependence on glutamine metabolism. The purpose of the present study was to determine the mechanistic basis and impact of differential glutamine metabolism in nonmetastatic and metastatic murine mammary cancer cells. Universally labeled 13C5-glutamine metabolic tracing, qRT-PCR, measures of reductive–oxidative balance, and exogenous ammonium chloride treatment were used to assess glutamine reprogramming. Results show that 4 mM media concentration of glutamine, compared with 2 mM, reduced viability only in metastatic cells, and that this decrease in viability was accompanied by increased incorporation of glutamine-derived carbon into the tricarboxylic acid (TCA) cycle. While increased glutamine metabolism in metastatic cells occurred in tandem with a decrease in the reduced/oxidized glutathione ratio, treatment with the antioxidant molecule N-acetylcysteine did not rescue cell viability. However, the viability of metastatic cells was more sensitive to ammonium chloride treatment compared with nonmetastatic cells, suggesting a role of metabolic reprogramming in averting nitrogen cytotoxicity in nonmetastatic cells. Overall, these results demonstrate the ability of nonmetastatic cancer cells to reprogram glutamine metabolism and that this ability may be lost in metastatic cells.

Bisphenol A exposure induces metastatic aggression in low metastatic MCF-7 cells via PGC-1α mediated mitochondrial biogenesis and epithelial-mesenchymal plasticity

AimsThe frequency of estrogen receptor alpha (ERα)-positive breast cancers and their metastatic progression is prevalent in females globally. Aberrant interaction of estrogen-like endocrine-disrupting chemicals (EDCs) is highly implicated in breast carcinogenesis. Studies have shown that single or acute exposures of weak EDCs such as bisphenol A (BPA) may not have a substantial pro-carcinogenic/metastatic effect. However, repeated exposure to EDCs is expected to strongly induce carcinogenic/metastatic progression, which remains to be studied. Main methodsLow metastatic ERα-positive human breast cancer cells (MCF-7) were exposed to nanomolar doses of BPA every 24 h (up to 200 days) to study the effect of repeated exposure on metastatic potential. Following the designated treatment of BPA, markers of epithelial-mesenchymal transition (EMT), migration and invasion, mitochondrial biogenesis, ATP levels, and peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) knockdown assays were performed. Key findingsA repeated exposure of low dose BPA induced stable epithelial-mesenchymal plasticity in MCF-7 cells to augment migration and invasion in the ERα-dependent pathway. Repeated exposures of BPA increased the levels of several mesenchymal markers such as N-cadherin, vimentin, cluster of differentiation 44 (CD44), slug, and alpha-smooth muscle actin (α-SMA), whereas reduced the level of E-cadherin drastically. BPA-induced mitochondrial biogenesis favored metastatic aggression by fulfilling bioenergetics demand via PGC-1α/NRF1/ERRα signaling. Knockdown of PGC-1α resulted in suppressing both mitochondrial biogenesis and EMT in BPA exposed MCF-7 cells. SignificanceRepeated exposures of low dose BPA may induce metastatic aggression in ERα-positive breast cancer cells via PGC-1α-mediated mitochondrial biogenesis and epithelial-mesenchymal plasticity.

Transcriptional Repression by FoxM1 Suppresses Tumor Differentiation and Promotes Metastasis of Breast Cancer.

This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

Effects of Melatonin and Doxorubicin on Primary Tumor And Metastasis in Breast Cancer Model.

Melatonin exerts oncostatic effects on breast cancer via immunomodulation and antioxidation. Doxorubicin is an effective chemotherapeutic agent, but parallel studies also provide ample evidence of an off-target effect of Doxorubicin in breast cancer patients. Combinatorial use of doxorubicin and melatonin has not been comprehensively analyzed in breast cancer models. We hypothesized that the anti-oxidative, anti-proliferative and anti-inflammatory effects of melatonin could ameliorate the off-target effects of doxorubicin in breast cancer patients and enhance the anti-tumoral effects of doxorubicin. The goal of the study is to test this hypothesis in cancer cell lines and xenografted mice. The effects of Melatonin and doxorubicin on the cell viability were evaluated in 4T1-Brain Metastatic Tumor (4TBM). Furthermore, the effects of melatonin and doxorubicin on the primary tumors and systemic metastasis were evaluated in the xenografted mice. Lung and liver tissues were removed and metastasis analyses were performed. The levels of p65, phospho-STAT3, CD11b+, GR1+, Ki67, and cleaved caspase-3 proteins were determined with immunohistochemistry and western blot analysis. We examined the effects of melatonin and Melatonin+Doxorubicin combination therapy on 4TBM cells. Our results showed that doxorubicin inhibited the proliferation of metastatic breast cancer cells while melatonin did not affect cells. Tumor growth and metastasis were markedly suppressed in melatonin alone and in combination with doxorubicin. The expression of CD11b+ and GR1+ proteins, which are indicators of myeloid-derived suppressor cells (MDSCs), were noted to be reduced in both primary tumor and metastatic tissues in melatonin and doxorubicin groups. The combination of melatonin with doxorubicin reduced primary tumor growth and distant metastasis. Based on these results, melatonin is a promising candidate for combinatory use with conventional chemotherapeutics for breast cancer treatment.

Production and optimization of a vasostatin-30 and vasoinhibin fusion protein that inhibits tumor angiogenesis and dissemination of breast cancer cells in a zebrafish model

Angiogenesis plays a key role in tumor growth and metastasis. Current antiangiogenic drugs have shown poor clinical benefit making the development of new antiangiogenic agents a clinical need. In this work, we optimized the production of a recombinant fusion protein derived from vasostatin-30 (VS30) and vasoinhibin (Vi), named VS_VI, using Escherichia coli and the central composite experimental design. The bioactivity of soluble VS_VI was compared to VS30, Vi, and angiostatin using in vitro cell cultures and the zebrafish model. Results showed that 21.3 and 17.5 mg/L of total VS_VI and soluble VS_VI, respectively, were attained at pH 7.0 and 28.5 °C. In vitro studies showed that purified soluble VS_VI decreased by 2-fold the VEGF-induced HUVEC proliferation compared to VS30 and Vi, showing no effect on 3T3 NIH cell proliferation at 10 nM. In vivo using zebrafish, VS_VI significantly reduced intratumoral angiogenesis at 1 mg/kg compared to a control, VS30, Vi, and angiostatin. VS_VI decreased metastatic dissemination of MDA-MB-468 and MDA-MB-231 cells at 1 mg/kg. Meanwhile, vasoinhibin and angiostatin at 1 mg/kg and vasostatin-30 at 10 mg/kg decreased the dissemination of T47D cells. Results showed VS_VI fusion protein impairs tumor angiogenesis and early dissemination of metastatic breast cancer cells, making it a promising agent in breast cancer treatment.

Activation of Host-NLRP3 Inflammasome in Myeloid Cells Dictates Response to Anti-PD-1 Therapy in Metastatic Breast Cancers.

Tumor-associated inflammation leads to dysregulated cytokine production that promotes tumor immune evasion and anti-tumor immunity dysfunction. In advanced stage breast cancer, the proinflammatory cytokine IL-1β is overexpressed due to large proportions of activated myeloid cells in the tumor microenvironment (TME). Here, we demonstrate the role of the host nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing 3 (NLRP3) inflammasome in metastatic breast cancer. In vitro, we show that stimulation of THP-1 cells with conditioned media collected from MDA-MB-468 cells induced NLRP3 activation and increased Pdcd1l1 expression. In vivo, mice deficient in NLRP3 orthotopically implanted with metastatic breast cancer cell line (E0771) showed significant reduction in tumor growth (p < 0.05) and increased survival (p < 0.01). Inhibition of NLRP3 with the small molecule OLT1177® reduced expression of Pdcd1l1 (p < 0.001), Casp1 (p < 0.01) and Il1b (p < 0.01) in primary tumors. Furthermore, tumor-bearing mice receiving OLT1177® showed reduced infiltration of myeloid-derived suppressor cells (MDSCs) (p < 0.001) and increased CD8+ T cells (p < 0.05) and NK cells (p < 0.05) in the TME. NLRP3 inhibition in addition to anti-PD-1 treatment significantly reduced tumor growth from the monotherapies (p < 0.05). These data define NLRP3 activation as a key driver of immune suppression in metastatic breast cancers. Furthermore, this study suggests NLRP3 as a valid target to increase efficacy of immunotherapy with checkpoint inhibitor in metastatic breast cancers.

OGT/CDK5/ACSS2 Axis Regulates Breast Cancer Brain Metastasis Growth

Onset of brain metastases (BMs) in breast cancer patients is considered an end‐stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Thus, there is an urgent need to develop novel treatment strategies. Metastatic breast cancer cells colonizing the brain encounter adverse ‘nutritional environment’, as the levels of key metabolic fuels such as glucose are much lower, in part because they are avidly consumed by neurons. To overcome this challenge, brain‐metastatic cancer cells rely on acetate as a fuel source and convert it into acetyl‐CoA via upregulation of acetyl‐CoA synthetase 2 (ACSS2) enzyme. We have previously shown that brain tumors increase the nutrient sensing post‐translational modification O‐GlcNAcylation and O‐GlcNAc transferase (OGT) levels to regulate acetate metabolism into acetyl‐CoA via increased phosphorylation of ACSS2 on Ser‐267 in a cyclin dependent kinase 5 (CDK5)‐dependent manner, increasing brain tumor growth. Here, we show that human breast cancer cells selected to metastasize to the brain contain increased levels of O‐GlcNAc, OGT and ACSS2‐Ser267 phosphorylation compared to parental cells and show that human breast cancer brain metastatic patient samples contain elevated ACSS2‐Ser‐267 levels. Moreover, overexpression of OGT or ACSS2‐S267D phospho‐mimetic mutant confer a growth advantage on brain metastatic breast cancer cells in an in vivo intracranial xenograft model, may be due to an increase in lipogenic proteins. Additionally, we show that pharmacologically targeting CDK5 and ACSS2 with small molecule inhibitors reduces tumor growth in a novel orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling in transducing nutritional state to regulate acetate metabolism in breast cancer BM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastasis.

Increased Fatty Acid Synthesis and Catabolism Supports Metastatic Breast Cancer Cell Migration

Lipid accumulation is positively associated with breast cancer aggressiveness and poorer clinical outcomes. Despite this observation, mechanisms underlying the increase and role of stored lipid in breast cancer progression remain incompletely understood. The accumulation of triacylglycerol (TAG) is dependent on the balance of uptake, synthesis, and utilization of fatty acids (FAs). In the current study, we utilized non‐metastatic MCF10A‐ras and metastatic MCF10CA1a human breast cancer cells to determine differences in TAG storage and catabolism for sustaining migration—a critical step in the metastatic cascade. Our results demonstrate that MCF10CA1a cells have 90% more TAG than MCF10A‐ras (TAG Assay and Transmission Electron Microscopy), although no significant difference in 14C‐palmitate uptake. MCF10CA1a cells have greater FA synthesis as shown by greater incorporation of 13C‐acetate (60%), 13C‐glucose (65%), and 13C‐glutamine (50%) into palmitate compared to the MCF10A‐ras cells (LC‐MS/MS), as well as higher protein levels of FA synthase (FASN). Additionally, MCF10CA1a cells display greater flux of uniformly‐labeled 13C‐glucose and 13C‐glutamine to the FA synthesis precursor, citrate, and lower intracellular citrate pool size compared to MCF10A‐ras. In addition to increased FA anabolism, MCF10CA1a cells rely on FA oxidation (FAO) for cellular migration compared to MCF10A‐ras cells, as determined by inhibiting the rate‐limiting enzyme of FAO, carnitine palmitoyltransferase 1, with etomoxir (transwell and wound‐healing assays). Pretreatment with the FASN inhibitor TVB‐3166 (3 d) significantly reduced TAG levels and subsequent migration by 67% compared to vehicle‐treated cells. Similarly, pretreatment with inhibitors of the TAG‐synthesizing enzymes diacylglycerol O‐acyltransferase 1 and 2 reduced TAG accumulation and subsequent migration (75%) of MCF10CA1a cells compared to vehicle. Together, the data suggest that FA synthesis may contribute to TAG storage necessary to sustain breast cancer migration. To test this hypothesis, we inhibited the initial enzyme of the TAG lipolysis pathway, adipose triacylglycerol lipase (ATGL; inhibitor ATGListatin), and measured migration. Inhibition of ATGL alone reduced MCF10CA1a cell migration (20% decrease compared to vehicle), and addition of the FAO inhibitor (etomoxir) provided no further effect. Our study indicates that metastatic MCF10CA1a cells accumulate FAs by increasing de novo lipogenesis, store them as TAG, and that catabolism of these stores drives FAO‐dependent migration.

Effect of Knock‐out of the a4 Isoform of Vacuolar H+‐ATPase following CRISPR/Cas9 on Activation of Cathepsins in MDA‐MB231 Breast Cancer Cell

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases creates a low pH in a localized environment which promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells cleave extracellular matrix proteins and activate other proteases, facilitating tumor cell invasion. Our lab is investigating a connection between the isoforms of the a subunit for V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but not cathepsin L. However, although there was a trend, differences in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data supports the conclusion that there is a significant difference in the effects of V‐ATPase inhibition on secreted cathepsins. Inhibiting V‐ATPases does not appear to affect the activity of secreted cathepsin L, however it reduces the activity of secreted cathepsins B and D. To further investigate the role of V‐ATPases in the activation of cathepsins, we are using CRISPR/Cas9 to knockout the genes of the four V‐ATPase “a” subunit isoforms, a1, a2, a3 and a4. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. The a1 and a2 isoforms are found in intracellular compartments. Thus far we have made the most progress with knocking out the a4 isoform. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsins B and D while not effecting activation of cathepsin L.

The apoptotic effect of ozone therapy on mitochondrial activity of highly metastatic breast cancer cell line MDA-MB-231 using in vitro approaches

Ozone (O3) gas is the triatomic state of oxygen and it is used as a disinfection agent due to its strong oxidizing effect, since its discovery in the mid-nineteenth century. Ozone therapy is also an alternative therapeutic approach for some diseases like circulatory disorders, AIDS, asthma, cardiovascular diseases, and certain types of cancer by increasing the oxygen levels in the blood by external addition of ozone to the body. In this study, the therapeutic potential of ozone therapy was examined by inhibiting the growth of breast cancer cells in a dose-dependent procedure. Ozone concentrations varying from 5 to 20 ​μg/ml were applied to the MDA-MB-231, human breast adenocarcinoma and HUVEC, human umbilical vein endothelium, cell lines, and MDA cells demonstrated an increased rate of death while its migration potential decreases. RT-PCR analysis showed mRNA expression levels of pro-apoptotic genes demonstrated higher folds in MDA cells after 10 ​μg/ml treatment. In the same context, Annexin V/PI and cell cycle analysis also concluded that ozone therapy causes apoptotic cell death on breast tumor cells. The use of ozone therapy for cancer treatment requires further and extensive research. However, this research has shown that ozone therapy is a promising source for cancer treatment in a way by inhibiting the proliferation of breast tumor cells.

A Therapeutic Sheep in Metastatic Wolf’s Clothing: Trojan Horse Approach for Cancer Brain Metastases Treatment

HighlightsDOX-PLGA@CM employs the whole set of membrane molecules of a brain-homing metastatic breast cancer cell optimized through a natural selection process. Thus, the hetero and multivalent effects of these molecules greatly facilitate the nanoparticle crossing the blood-brain barrier.Attributed to the homotypic effect of the nanocarrier, DOX-PLGA@CM shows stronger anticancer efficacy than free DOX for its parenteral cells.DOX-PLGA@CM effectively reaches the metastatic tumor lesions in the brain, and slows down the progression of brain metastatic breast cancer.

Learning the rules of collective cell migration using deep attention networks.

Collective, coordinated cellular motions underpin key processes in all multicellular organisms, yet it has been difficult to simultaneously express the 'rules' behind these motions in clear, interpretable forms that effectively capture high-dimensional cell-cell interaction dynamics in a manner that is intuitive to the researcher. Here we apply deep attention networks to analyze several canonical living tissues systems and present the underlying collective migration rules for each tissue type using only cell migration trajectory data. We use these networks to learn the behaviors of key tissue types with distinct collective behaviors-epithelial, endothelial, and metastatic breast cancer cells-and show how the results complement traditional biophysical approaches. In particular, we present attention maps indicating the relative influence of neighboring cells to the learned turning decisions of a 'focal cell'-the primary cell of interest in a collective setting. Colloquially, we refer to this learned relative influence as 'attention', as it serves as a proxy for the physical parameters modifying the focal cell's future motion as a function of each neighbor cell. These attention networks reveal distinct patterns of influence and attention unique to each model tissue. Endothelial cells exhibit tightly focused attention on their immediate forward-most neighbors, while cells in more expansile epithelial tissues are more broadly influenced by neighbors in a relatively large forward sector. Attention maps of ensembles of more mesenchymal, metastatic cells reveal completely symmetric attention patterns, indicating the lack of any particular coordination or direction of interest. Moreover, we show how attention networks are capable of detecting and learning how these rules change based on biophysical context, such as location within the tissue and cellular crowding. That these results require only cellular trajectories and no modeling assumptions highlights the potential of attention networks for providing further biological insights into complex cellular systems.

Does Oncocalyxone A (oncoA) have intrinsic fluorescence?

BackgroundOncocalyxone A (oncoA) is a quinone extracted from the Cordia oncocalyx plant. This compound has pharmacological properties, such as anti-inflammatory, analgesic, and cytotoxic activities, among others. OncoA presents a similar chemical structure to doxorubicin, a drug used in cancer treatment, which possesses an intrinsic fluorescence explored in various studies, including those using doxorubicin-loaded nanoparticles. Thus, due to the chemical structural similarity, the question arose whether oncoA could also show autofluorescence. Therefore, this study proposed to characterize the absorption and emission spectral profiles of oncoA and analyze if this compound could be used as a fluorescent probe. MethodsFor this, fucoidan-coated polyisobutylcyanoacrylate (PIBCA) nanoparticles containing oncoA were prepared, and an uptake study was performed using a human metastatic breast cancer cell line (MDA-MB-231 cells). ResultsOncoA presented a maximum emission wavelength in the blue region, near 430 nm, at 350 nm excitation, compatible with standard microscope optics. Fluorescence microscopy analyses showed that oncoA-loaded PIBCA nanoparticles were internalized by MDA-MB-231 cells under incubation times as shorter as 15 min. ConclusionAccording to these findings, oncoA-encapsulated nanoparticles are promising fluorescent probes and could be useful for cellular uptake studies.

Transcription factor Runx2 changes the expression of some matricellular proteins in metastatic breast cancer cells.

Runx2 is one of the runt-related genes that are overexpressed in human cancers and contribute to metastasis. The cancer cell metastasis requires modifications of the extracellular matrix (ECM) and reduction in ECM-cell interaction. This process is performed by various enzymes and proteins secreted by cancer and surrounding cells. This study aimed to investigate the effect of the Runx2 transcription factor on the expression of matricellular proteins such as HPA1, LOX, SPARC, and OPN, which have important roles in ECM modification and ECM-cell interaction in human breast cancer. Also, the changes in their associated oncogenic pathways including Akt, Erk, FAK activities, and c-jun protein expression were investigated. Runx2 knockdown model was created using runx2 siRNA in MDA-MB-231 human metastatic breast cancer cells. The changes in the mRNA and protein expressions of ECM proteins were shown by the qPCR and Western blotting, respectively. The results showed that there was a decrease in both mRNA and protein expressions of HPA1, SPARC, and LOX, whereas there was no change in those of OPN. Phosphorylated Akt, Erk, FAK levels, and protein expression of c-jun, however, decreased in the cells. Our results revealed that Runx2 affected matricellular protein expression, which is important for metastasis and invasion of breast cancer. Hence, we have concluded that runx2 appears to be efficient for regulating breast cancer metastasis through an expression of matricellular proteins.

Cooperative interaction between ER\u03b1 and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

The epithelial to mesenchymal transition (EMT) has been proposed to contribute to the metastatic spread of breast cancer cells. EMT-promoting transcription factors determine a continuum of different EMT states. In contrast, estrogen receptor α (ERα) helps to maintain the epithelial phenotype of breast cancer cells and its expression is crucial for effective endocrine therapies. Determining whether and how EMT-associated transcription factors such as ZEB1 modulate ERα signaling during early stages of EMT could promote the discovery of therapeutic approaches to suppress metastasis. Here we show that, shortly after induction of EMT and while cells are still epithelial, ZEB1 modulates ERα-mediated transcription induced by estrogen or cAMP signaling in breast cancer cells. Based on these findings and our ex vivo and xenograft results, we suggest that the functional interaction between ZEB1 and ERα may alter the tissue tropism of metastatic breast cancer cells towards bone.

Clinically relevant CHK1 inhibitors abrogate wild-type and Y537S mutant ER\u03b1 expression and proliferation in luminal primary and metastatic breast cancer cells

BackgroundChallenges exist in the clinical treatment of luminal estrogen receptor α (ERα)-positive breast cancers (BCs) both to prevent resistance to endocrine therapy (ET) and to treat ET-resistant metastatic BCs (MBC). Therefore, we evaluated if kinases could be new targets for the treatment of luminal primary and MBCs.Methods ~ 170 kinase inhibitors were applied to MCF-7 cells either with adaptative or genetic resistance to ET drugs and both ERα levels and cell proliferation were measured. Robust-Z-score calculation identified AZD7762 (CHK1/CHK2 inhibitor) as a positive hit. Subsequently, Kaplan–Meier analyses of CHK1 and CHK2 impact on ERα-positive BC patients relapse-free-survival (RFS), bioinformatic evaluations of CHK1 and CHK2 expression and activation status as a function of ERα activation status as well as drug sensitivity studies in ERα-positive BC cell lines, validation of the impact of the ATR:CHK1 and ATM:CHK2 pathways on the control of ERα stability and BC cell proliferation via inhibitor- and siRNA-based approaches, identification of the molecular mechanism required for inhibitor-dependent ERα degradation in BC and the impact of CHK1 and CHK2 inhibition on the 17β-estradiol (E2):ERα signaling, synergy proliferation studies between ET-drugs and clinically relevant CHK1 inhibitors in different luminal BC cell lines, were performed.ResultsA reduced CHK1 expression correlates with a longer RFS in women with ERα-positive BCs. Interestingly, women carrying luminal A BC display an extended RFS when expressing low CHK1 levels. Accordingly, CHK1 and ERα activations are correlated in ERα-positive BC cell lines, and the ATR:CHK1 pathway controls ERα stability and cell proliferation in luminal A BC cells. Mechanistically, the generation of DNA replication stress rather than DNA damage induced by ATR:CHK1 pathway inhibition is a prerequisite for ERα degradation. Furthermore, CHK1 inhibition interferes with E2:ERα signaling to cell proliferation, and drugs approved for clinical treatment of primary and MBC (4OH-tamoxifen and the CDK4/CDK6 inhibitors abemaciclib and palbociclib) exert synergic effects with the CHK1 inhibitors in clinical trials for the treatment of solid tumors (AZD7762, MK8776, prexasertib) in preventing the proliferation of cells modeling primary and MBC.ConclusionsCHK1 could be considered as an appealing novel pharmacological target for the treatment of luminal primary and MBCs.

Design, Synthesis, and Molecular Docking Studies of Curcumin Hybrid Conjugates as Potential Therapeutics for Breast Cancer.

Curcumin (CUR) has received great attention over the past two decades due to its anticancer, anti-inflammatory, and antioxidant properties. Similarly, Dichloroacetate (DCA), an pyruvate dehydrogenase kinase 1 (PKD1) inhibitor, has gained huge attention as a potential anticancer drug. However, the clinical utility of these two agents is very limited because of the poor bioavailability and unsolicited side effects, respectively. We have synthesized fusion conjugates of CUR and DCA with an amino acids linker to overcome these limitations by utilizing the molecular hybridization approach. The molecular docking studies showed the potential targets of Curcumin-Modified Conjugates (CMCs) in breast cancer cells. We synthesized six hybrid conjugates named CMC1-6. These six CMC conjugates do not show any significant toxicity in a human normal immortalized mammary epithelial cell line (MCF10A) in vitro and C57BL/6 mice in vivo. However, treatment with CMC1 and CMC2 significantly reduced the growth and clonogenic survival by colony-formation assays in several human breast cancer cells (BC). Treatment by oral gavage of a transgenic mouse BC and metastatic BC tumor-bearing mice with CMC2 significantly reduced tumor growth and metastasis. Overall, our study provides strong evidence that CUR and DCA conjugates have a significant anticancer properties at a sub-micromolar concentration and overcome the clinical limitation of using CUR and DCA as potential anticancer drugs.