Aggressive Cancer Cells Research Articles

Acidosis overrides molecular heterogeneity to shape therapeutically targetable metabolic phenotypes in colon cancers.

Colorectal cancer (CRC) represents a prototypical example of a cancer type for which inter- and intra-tumor heterogeneities remain major challenges for the clinical management of patients. Besides genotype-mediated phenotypic alterations, tumor microenvironment (TME) conditions are increasingly recognized to promote intrinsic diversity and phenotypic plasticity and sustain disease progression. In particular, acidosis is a common hallmark of solid tumors, including CRC, and it is known to induce aggressive cancer cell phenotypes. In this study, we report that long-term adaptation to acidic pH conditions is associated with common metabolic alterations, including a glycolysis-to-respiration switch and a higher reliance on the activity of phosphoglycerate dehydrogenase (PHGDH), in CRC cells initially displaying molecularly heterogeneous backgrounds. Pharmacological inhibition of PHGDH activity or mitochondrial respiration induces greater growth-inhibitory effects in acidosis-exposed CRC cells in 2D and 3D culture conditions, and in patient-derived CRC organoids. These data pave the way for drugs targeting the acidic tumor compartment as a "one-size-fits-all" therapeutic approach to delay CRC progression.

FXYD6 is transcriptionally activated by KLF10 to suppress the aggressiveness of gastric cancer cells.

Despite improvements in therapeutic approaches, the mortality rate of gastric cancer (GC) remains unacceptably high. Evidence suggests that FXYD domain containing ion transport regulator 6 (FXYD6) is downregulated in GC. However, its exact function and the molecular mechanism in GC are still unclear. FXYD6 expression in different cell lines was estimated using RT-qPCR. Western blotting was employed for protein expression detection. Cell counting kit-8 assay, colony formation assay, and flow cytometry were implemented to assess GC cell viability, proliferation, and apoptosis, respectively. Bioinformatics analysis as well as chromatin immunoprecipitation and luciferase reporter assays were utilized for verifying FXYD6 interaction with the transcription factor Krüppel-like factor 10 (KLF10). The results showed thatFXYD6 displayed a decreased level in GC cell lines. Impaired proliferative ability and enhanced apoptotic capacity were observed in GC cells overexpressing FXYD6. KLF10 expression is positively correlated with FXYD6 expression in GC samples. KLF10 binds to the FXYD6 promoter to enhance its transcription. FXYD6 depletion counteracted KLF10 upregulation-triggered reduction in GC cell proliferation and elevation in apoptosis. In conclusion,KLF10 activates FXYD6 transcription, thereby impeding GC cell proliferation and promoting cell apoptosis.

Synergistic activity of Pitstop-2 and 1,6-hexanediol in aggressive human lung cancer cells

Metastatic cancer cells undergo metabolic reprogramming, which involves changes in the metabolic fluxes, including endocytosis, nucleocytoplasmic transport, and mitochondrial metabolism, to satisfy their massive demands for energy, cell division, and proliferation compared to normal cells. We have previously demonstrated the ability of two different types of compounds to interfere with linchpins of metabolic reprogramming, Pitstop-2 and 1,6-hexanediol (1,6-HD). 1,6-HD disrupts glycolysis enzymes and mitochondrial function, enhancing reactive oxygen species production and reducing cellular ATP levels, while Pitstop-2 impedes clathrin-mediated endocytosis and small GTPases activity. Besides, both compounds interfere with the integrity of nuclear pore complexes, the gatekeepers for all nucleocytoplasmic transport. Herein, we investigate the possible synergistic effects of both compounds on lowly, highly metastatic, and erlotinib-resistant non-small cell lung cancer. We observe a synergistic cytotoxic effect on erlotinib-resistant cells. Moreover, motility assays show that the compounds combination significantly impedes the motility of all cells. Drug safety and tolerability assessments were validated using the in vivo model organism Caenorhabditis elegans, where fairly high doses showed negligible impact on survival, development, or behavioral parameters. Our findings propose that the 1,6-HD and Pitstop-2 combination may usher in the design of potent strategies for treating advanced lung cancer.

OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

Embryonic development and tumor genesis share numerous similarities, with OCT4 standing out as a pivotal transcription factor in embryonic development. Expression of OCT4 is associated with poor prognosis of lung adenocarcinoma. VEGF-correlated chemokine-1 (VCC-1), also known as C-X-C motif chemokine ligand 17 (CXCL17), has been suggested to play a role in promoting tumor angiogenesis and metastasis. In the present study, we show a positive correlation between OCT4 expression levels and tumor metastatic potential, where an increase in OCT4 expression parallels an upregulation of VCC-1 in lung cancer. This relationship was substantiated through DNA microarray analysis and further confirmed by tissue staining of clinical lung cancer samples, demonstrating a positive correlation between OCT4 and VCC-1 expression. In A549 and H1299 human lung cancer cells, modulations in OCT4 expression directly influenced VCC-1 levels, as evidenced by the reporter assay of the VCC-1 promoter, indicating the regulatory role of OCT4 in transactivating VCC-1 expression. Furthermore, enhanced VCC-1 expression in H1299 cells promoted transforming growth factor-β (TGF-β) secretion, contributing to lung cancer cell aggressiveness. Additionally, VCC-1 secretion by H1299 cells could attract THP-1 macrophages, further implicating its role in tumor progression. NOD/SCID mice inoculated with VCC-1-knockdown A549 lung cancer cells exhibited significantly smaller tumors than those inoculated with control cells. On the basis of these findings, we highlight the importance of the OCT4-VCC-1 axis in lung cancer progression. Our findings also provide therapeutic targets for lung cancer.

3D Cell Culture Models as a Platform for Studying Tumor Progression, Testing Treatment Responses, and Discovering Biomarkers.

In this chapter, we present a detailed protocol for establishing a three-dimensional (3D) multicellular tumor spheroids (MCTSs) model to simulate the tumor microenvironment (ME) associated with metabolic dysfunction-associated steatotic liver disease (MASLD) for the study of hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cell aggressiveness, growth, and metastasis potential. The MASLD microenvironment (MASLD-ME) is recreated by embedding hepatic stellate cells in a collagen I matrix within a Boyden chamber system. The metabolic medium mimics MASLD conditions, enriched with high glucose, fructose, insulin, and fatty acids, to simulate metabolic stresses associated with the disease.In the protocol, cancer cells are loaded in the upper compartment to analyze their migration toward the MASLD-ME, thereby facilitating studies on cancer cell invasiveness and metastatic capacity. This method offers an adaptable, reproducible model to research disease progression and investigate therapeutic interventions, contributing to preclinical research on MASLD-related liver cancer pathophysiology and potential drug responses.

Valosin-containing protein (VCP), a component of tumor-derived extracellular vesicles, impairs the barrier integrity of brain microvascular endothelial cells.

Metastases are the leading cause of cancer-related deaths, and their origin is not fully elucidated. Recently, studies have shown that extracellular vesicles (EVs), particularly small extracellular vesicles (sEV), can disrupt the homeostasis of organs, promoting the development of a secondary tumor. However, the role of sEV in brain endothelium and their association with metastasis related to breast cancer is unknown. Thus, this study aimed to investigate sEV-triggered changes in the phosphorylation state of proteins on the surface of brain endothelial cells, as they form the first barrier in contact with circulating tumor cells and EVs, and once identified, to modulate its interactors and effects from this through different functional assays. We used the most aggressive breast cancer cell line, MDA-MB-231, and its brain-seeking variant, MDA-MB-231-br. From these cells, small and large extracellular vesicles were harvested to treat hCMEC/D3 cells, an immortalized cell line from the human brain microvasculature. Higher levels of phosphorylation of VEGFR1 and VEGFR2 were found in hCMEC/D3 cells treated with MDA-MB-231-br sEV. By computational analysis, the Valosin-Containing Protein (VCP) was predicted to be an important sEV cargo affecting the VEGFR2 intracellular trafficking, validated by western blotting analysis. Then, VCP was modulated by cell transfection or chemical inhibition in hCMEC/D3 cells and assessed in different functional in vitro assays evidencing a significant effect on the functionality of these cells. Thus, this study demonstrates that the VCP-containing sEVs induce modifications at different phosphor sites of VEGFR2 and effectively modulate the state of brain microvascular endothelial cells.

Silencing of Epidermal Growth Factor-like Domain 8 Promotes Proliferation and Cancer Aggressiveness in Human Ovarian Cancer Cells by Activating ERK/MAPK Signaling Cascades.

Ovarian cancer (OC) is the second most common female reproductive cancer and the most lethal gynecological malignancy worldwide. Most human OCs are characterized by high rates of drug resistance and metastasis, leading to poor prognosis. Improving the outcomes of patients with relapsed and treatment-resistant OC remains a challenge. This study aimed to investigate the role of epidermal growth factor-like domain 8 (EGFL8) in human OC by examining the effects of siRNA-mediated EGFL8 knockdown on cancer progression. EGFL8 knockdown in human OC cells promoted aggressive traits associated with cancer progression, including enhanced proliferation, colony formation, migration, invasion, chemoresistance, and reduced apoptosis. Additionally, knockdown upregulated the expression of epithelial-mesenchymal transition (EMT) markers (Snail, Twist1, Zeb1, Zeb2, and vimentin) and cancer stem cell biomarkers (Oct4, Sox2, Nanog, KLF4, and ALDH1A1), and increased the expression of matrix metallopeptidases (MMP-2 and MMP-9), drug resistance genes (MDR1 and MRP1), and Notch1. Low EGFL8 expression also correlated with poor prognosis in human OC. Overall, this study provides crucial evidence that EGFL8 inhibits the proliferation and cancer aggressiveness of human OC cells by suppressing ERK/MAPK signaling. Therefore, EGFL8 may serve as a valuable prognostic biomarker and a potential target for developing novel human OC therapies.

Raman Imaging of Targeted Drug Delivery with DNA-Based Nano-Optical Devices.

Raman spectroscopy (RS) has emerged as a novel optical imaging modality by identifying molecular species through their bond vibrations, offering high specificity and sensitivity in molecule detection. However, its application in intracellular molecular probing has been limited due to challenges in combining vibrational tags with functional probes. DNA nanostructures, known for their high programmability, have been instrumental in fields like biomedicine and nanofabrication. So far, their ability to customize Raman signals remains largely untapped. In this study, a new class of Raman active DNA origami-based hybrid nanodevice (ND) for targeted cancer cell drug delivery and imaging is engineered. The ND is specifically engineered for metastatic prostate cancer treatment, featuring a legumain enzyme-responsive sequence for the controlled release of the chemotherapeutic agent doxorubicin. Integrating RS with precise targeting, the ND enables imaging of aggressive cancer cells and efficient drug delivery with minimal off-target effects. The developed device offers stimuli-responsive behavior, enhanced stability, exceptional tunability, and potent targeting abilities, positioning it as a highly promising strategy for advancing precision cancer imaging and therapy.

Lack of canonical activities of connexins in highly aggressive human prostate cancer cells

Connexins (Cxs) have the ability to form channels that allow the exchange of ions/metabolites between adjacent cells (gap junction channels, GJC) or between the intra- and extra-cellular compartments (hemichannels, HC). Cxs were initially classified as tumor suppressors. However, more recently, it has been shown that Cxs exert anti- and pro-tumorigenic effects depending on the cell and tissue context. In prostate cancer (PCa), the expression and functionality of Cxs remain highly controversial. Here, we analyzed the expression pattern of Cx26, Cx32, Cx37, Cx40, Cx43 and Cx45 in PCa cell lines with increasing levels of tumor aggressiveness (LNCaP < LNCaP-C4-2 < Du-145 < PC-3). In addition, GJ and HC activities were evaluated in the PCa cell lines using dye coupling and dye uptake assays, respectively. Lastly, the cellular localization of Cx26, Cx32, and Cx43 was analyzed in LNCaP and PC-3 cell lines using immunofluorescence analyses. Our results showed a positive association between the mRNA levels of Cx26, Cx37 and Cx45 and the degree of aggressiveness of PCa cells, a negative association in the case of Cx32 and Cx43, and no clear pattern for Cx40. At the protein level, a negative relationship between the expression of Cx26, Cx32 and Cx43 and the degree of aggressiveness of PCa cell lines was observed. No significant differences were observed for the expression of Cx37, Cx40, and Cx45 in PCa cell lines. At the functional level, only LNCaP cells showed moderate GJ activity and LNCaP and LNCaP-C4-2 cells showed HC activity. Immunofluorescence analyses confirmed that the majority of Cx26, Cx32, and Cx43 expression was localized in the cytoplasm of both LNCaP and PC3 cell lines. This data indicated that GJ and HC activities were moderately detected only in the less aggressive PCa cells, which suggest that Cxs expression in highly aggressive PCa cells could be associated to channel-independent roles.

Toll-Like receptor 3-mediated interferon-β production is suppressed by oncostatin m and a broader epithelial-mesenchymal transition program

BackgroundPatients with Triple Negative Breast Cancer (TNBC) currently lack targeted therapies, and consequently face higher mortality rates when compared to patients with other breast cancer subtypes. The tumor microenvironment (TME) cytokine Oncostatin M (OSM) reprograms TNBC cells to a more stem-like/mesenchymal state, conferring aggressive cancer cell properties such as enhanced migration and invasion, increased tumor-initiating capacity, and intrinsic resistance to the current standards of care. In contrast to OSM, Interferon-β (IFN-β) promotes a more differentiated, epithelial cell phenotype in addition to its role as an activator of anti-tumor immunity. Importantly, OSM suppresses the production of IFN-β, although the mechanism of IFN-β suppression has not yet been elucidated.MethodsIFN-β production and downstream autocrine signaling were assessed via quantitative real-time PCR (qRT-PCR) and Western blotting in TNBC cells following exposure to OSM. RNA-sequencing (RNA-seq) was used to assess an IFN-β metagene signature, and to assess the expression of innate immune sensors, which are upstream activators of IFN-β. Cell migration was assessed using an in vitro chemotaxis assay. Additionally, TNBC cells were exposed to TGF-β1, Snail, and Zeb1, and IFN-β production and downstream autocrine signaling were assessed via RNA-seq, qRT-PCR, and Western blotting.ResultsHere, we identify the repression of Toll-like Receptor 3 (TLR3), an innate immune sensor, as the key molecular event linking OSM signaling and the repression of IFN-β transcription, production, and autocrine IFN signaling. Moreover, we demonstrate that additional epithelial-mesenchymal transition-inducing factors, such as TGF-β1, Snail, and Zeb1, similarly suppress TLR3-mediated IFN-β production and signaling.ConclusionsOur findings provide a novel insight into the regulation of TLR3 and IFN-β production in TNBC cells, which are known indicators of treatment responses to DNA-damaging therapies. Furthermore, strategies to stimulate TLR3 in order to increase IFN-β within the TME may be ineffective in stem-like/mesenchymal cells, as TLR3 is strongly repressed. Rather, we propose that therapies targeting OSM or OSM receptor would reverse the stem-like/mesenchymal program and restore TLR3-mediated IFN-β production within the TME, facilitating improved responses to current therapies.

Elongated Particles Show a Preferential Uptake in Invasive Cancer Cells.

Mechanically driven cellular preference for drug carriers can enhance selectivity in cancer therapy, underscoring the importance of understanding the physical aspects of particle uptake. In this study, it was hypothesized that elongated particles might be preferentially taken up by deformable, aggressive cancer cells compared to normal cells. Two film-stretching methods were tested for 0.8-2.4 μm polystyrene (PS) particles: one based on solubility in organic solvents and the other on heat-induced softening. The heat-induced method produced more homogenous particle batches, with a standard deviation in the particle aspect ratio of 0.42 compared to 0.91 in the solvent-based method. The ability of cells to engulf elongated PS particles versus spherical particles was assessed in two subsets of human melanoma A375 cells. In the more aggressive cancer cell subset (A375+), uptake of elongated PS particles increased by 10% compared to spherical particles. In contrast, the less aggressive subset (A375-) showed a 25% decrease in uptake of elongated particles. This resulted in an uptake ratio between A375+ and A375- that was 1.5 times higher for elongated PS particles than for spherical ones. To further demonstrate relevance to drug delivery, elongated paclitaxel-loaded biodegradable, slow-releasing poly(lactic-co-glycolic) acid (PLGA) particles were synthesized. No significant difference in cytotoxic effect was observed between A375+ and A375- cells treated with spherical drug-loaded particles. However, treatment with ellipsoidal particles led to a significantly enhanced cytotoxic effect in aggressive cells compared to less aggressive cells. These findings present promising directions for tailored cancer drug delivery and demonstrate the importance of particle physical properties in cellular uptake and drug delivery mechanisms.

FMNL3 Promotes Migration and Invasion of Breast Cancer Cells via Inhibiting Rad23B-Induced Ubiquitination of Twist1.

Breast cancer is a heterogeneous malignant tumor, and its high metastasis rate depends on the abnormal activation of cell dynamics. Formin-like protein 3 (FMNL3) plays an important role in the formation of various cytoskeletons that participate in cell movement. The objective of this study was to explore the function of FMNL3 in breast cancer progression and endeavor to reveal the molecular mechanism of this phenomenon. We found that FMNL3 was abnormally highly expressed in aggressive breast cancer cells and tissues, and it significantly inhibited E-cadherin expression. FMNL3 could specifically interact with Twist1 rather than other epithelial-mesenchymal transition transcription factors (EMT-TFs). We also found that FMNL3 enhanced the repressive effect of Twist1 on CDH1 transcription in breast cancer cells. Further mechanism studies showed that FMNL3 suppressed the ubiquitin degradation of Twist1 by inhibiting the interaction between Twist1 and Rad23B, the ubiquitin transfer protein of Twist1. In vitro functional experiments, it was confirmed that FMNL3 promoted the migration and invasion of breast cancer cells by regulating Twist1. Furthermore, Twist1 could directly bind to the fmnl3 promoter to facilitate FMNL3 transcription. To conclude, this study indicated that FMNL3 acted as a pro-metastasis factor in breast cancer by promoting Twist1 stability to suppress CDH1 transcription.

Downregulated Regucalcin Expression Induces a Cancer-like Phenotype in Non-Neoplastic Prostate Cells and Augments the Aggressiveness of Prostate Cancer Cells: Interplay with the G Protein-Coupled Oestrogen Receptor?

Regucalcin (RGN) is a calcium-binding protein and an oestrogen target gene, which has been shown to play essential roles beyond calcium homeostasis. Decreased RGN expression was identified in several cancers, including prostate cancer (PCa). However, it is unknown if the loss of RGN is a cause or a consequence of malignancy. Also, it needs confirmation if RGN oestrogenic regulation occurs through the G-protein-coupled oestrogen receptor (GPER). This study investigates how RGN knockdown affects prostate cell fate and metabolism and highlights the GPER/RGN interplay in PCa. Bioinformatic analysis assessed the relationship between RGN expression levels and patients' outcomes. RGN knockdown (siRNA) was performed in non-neoplastic prostate and castration-resistant PCa. Wild-type and RGN knockdown PCa cells were treated with the GPER agonist G1. Viability (MTT), proliferation (Ki-67 immunocytochemistry), apoptosis (caspase-3-like activity) and migration (Transwell assays) were evaluated. Spectrophotometric analysis was used to determine glucose consumption, lactate production and lactate dehydrogenase activity. Lipid content was assessed using the Oil Red assay. Bioinformatic analysis showed that the loss of RGN correlates with the development of metastatic PCa and poor survival outcomes. RGN knockdown induced a cancer-like phenotype in PNT1A cells, indicated by increased cell viability and proliferation and reduced apoptosis. In DU145 PCa cells, RGN knockdown augmented migration and enhanced the glycolytic profile, which indicates increased aggressiveness, in line with patients' data. GPER activation modulated RGN expression in PCa cells and RGN knockdown in DU145 cells influenced GPER actions, which highlighted an interplay between these molecular players with relevance for their potential use as biomarkers or therapeutic targets.

Abstract B005: Exploring breast cancer heterogeneity in the context of metastatic tumour microenvironments and therapy resistance

Abstract Breast tumors comprise several sub-populations of cells, so-called “clones”, with specific cellular and molecular characteristics. Some cancer clones can be more ‘aggressive’ than others in particular tumor microenvironments (TMEs), contributing to metastatic progression and drug resistance. Understanding the specific genes/pathways deregulated in these aggressive cancer cells is essential for developing new tailored treatment strategies. To accomplish this, we optimized the use of a lentiviral-based barcoding technique (Berthelet, J., et.al., Sci Adv 2021, Serrano, A., et.al., Nat Rev Cancer 2022) where cancer cells from cell lines and patient-derived xenografts are tagged with optical barcodes and transplanted into the mammary fat pads of immunodeficient mice. Upon resection of tumors and collection of metastatic organs at the experimental endpoint, we analyzed clonal fate and fitness using flow cytometry and imaging. The subsequent molecular characterization of individual clones was determined by bulk and single-cell RNA sequencing. We also leverage the use of these barcoded models to investigate the mechanisms of innate and acquired resistance to Sacituzumab Govitecan (SG), a drug recently approved for the treatment of patients with advanced breast cancer. Our studies revealed unique gene signatures and pathways associated with preferential organ tropism (in the lungs, liver and brain TMEs) and SG resistance. This comprehensive analysis, currently validated using autopsy samples and patient databases, will support the design of new predictive markers and targeted therapies for metastatic breast cancer. Citation Format: Sreeja Reddy Gadipally, Jean Berthelet, Samuel C. Lee, Dharmesh D. Bhuva, Michael Roach, Farrah El-Saafin, Caroline Bell, Yunjian Wu, David Baloyan, Christphe Ginestier, Charafe Jauffret Emmanuelle, Melissa Davis, Robin L. Anderson, Luciano Martelotto, Belinda Yeo, Delphine Merino. Exploring breast cancer heterogeneity in the context of metastatic tumour microenvironments and therapy resistance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B005.

Drug-loaded nanomaterial kills aggressive breast cancer cells

Drug-loaded nanomaterial kills aggressive breast cancer cells

Molecular Mechanism for Malignant Progression of Gastric Cancer Within the Tumor Microenvironment.

Gastric cancer (GC) is one of the most common cancers worldwide. Most patients are diagnosed at the progressive stage of GC, and progress in the development of effective anti-GC drugs has been insufficient. The tumor microenvironment (TME) regulates various functions of tumor cells, and interactions between the cellular and molecular components of the TME-e.g., inflammatory cells, fibroblasts, vasculature cells, and innate and adaptive immune cells-promote the aggressiveness of cancer cells and dissemination to distant organs. This review summarizes the roles of various TME cells and molecules in regulating the malignant progression and metastasis of GC. We also address the important roles of signaling pathways in mediating the interaction between cancer cells and the different components of the GC TME. Finally, we discuss the implications of these molecular mechanisms for developing novel and effective therapies targeting molecular and cellular components of the GC TME to control the malignant progression of GC.

Serum from Hypertensive Patients Induces Cancer-Supporting Phenotype of Vascular Endothelium In Vitro.

Large-scale epidemiological studies have established a bidirectional association between hypertension and cancer. However, the underlying mechanisms explaining this connection remain unclear. In our study, we investigated whether serum from patients with hypertension (HT) could enhance the aggressiveness of cancer cells in vitro through alterations in endothelial cell phenotype. Experiments were performed using EAhy926 endothelial cells and ovarian (SKOV-3), colorectal (SW480), pancreatic (PSN-1), breast (MCF-7), and lung (A549) cancer cell lines. This study showed that conditioned medium (CM) produced by EAhy926 cells, when exposed to serum from patients with untreated hypertension (HT-CM), promotes the proliferation, migration, and invasion of every cancer cell line tested. In addition, endothelial cells subjected to HT serum promote the adhesion of all cancer cell types except PSN-1. An intensified transendothelial invasion of cancer cells was accompanied by decreased expression of junctional proteins (connexin 43, E-cadherin, occluding, desmoglein) in HT serum-treated endothelial cells. Quantitative analysis of the secretome of endothelial cells subjected to HT serum showed that they secrete increased amounts of CCL2, CXCL1, CXCL8, bFGF, HGF, IL-6, PAI-1, and TGF-β1. Moreover, cancer cells exposed to HT-CM display increased mRNA expression for several pro-cancerogenic agents, including CXCL8, tPA, and VEGF. Our report shows that hypertension may potentiate cancer cell aggressiveness by modulating endothelial cell phenotype. Further tests with antihypertensive drugs are required to assess whether effective treatment of hypertension can mitigate its cancer-promoting potential.

Single-cell chemoproteomics identifies metastatic activity signatures in breast cancer.

Protein activity state, rather than protein or mRNA abundance, is a biologically regulated and relevant input to many processes in signaling, differentiation, development, and diseases such as cancer. While there are numerous methods to detect and quantify mRNA and protein abundance in biological samples, there are no general approaches to detect and quantify endogenous protein activity with single-cell resolution. Here, we report the development of a chemoproteomic platform, single-cell activity-dependent proximity ligation, which uses automated, microfluidics-based single-cell capture and nanoliter volume manipulations to convert the interactions of family-wide chemical activity probes with native protein targets into multiplexed, amplifiable oligonucleotide barcodes. We demonstrate accurate, reproducible, and multiplexed quantitation of a six-enzyme (Ag-6) panel with known ties to cancer cell aggressiveness directly in single cells. We further identified increased Ag-6 enzyme activity across breast cancer cell lines of increasing metastatic potential, as well as in primary patient-derived tumor cells and organoids from patients with breast cancer.

The effects of an aggressive breast tumor on thrombosis after antithrombin downregulation in a hypercoagulable mouse model

BackgroundDespite improvements in therapy, breast cancer still contributes to high mortality rates. Survival of these patients becomes progressively worse upon diagnosis with cancer-associated thrombosis (CAT). Unfortunately, the mechanism causing CAT has remained unclear. ObjectiveSet up an acute and non-invasive hypercoagulable mouse model with an aggressive breast cancer and study the mechanism of cancer-associated thrombosis. MethodsMice were grafted with the aggressive breast cancer cell line MDA-MB-231 or sham-treated. Subsequently, an acute imbalance in coagulation was introduced by injecting a synthetic small interfering (si) RNA targeting hepatic Serpinc1 to knockdown antithrombin – a condition known to predispose to cause a hypercoagulant state in vivo. ResultsSilencing Serpinc1 with siRNA decreased plasma antithrombin levels. siRNA treatment had no short-term effects on tumor characteristics, but increased distant metastasis within the timeframe of this study. The systemic pro-inflammatory status, with elevated platelet counts and fibrinogen levels in tumor-bearing mice, was also not affected by antithrombin silencing. While elevated fibrin deposition in the liver upon Serpinc1 targeting was not significantly affected by the presence of breast cancer, knockdown of antithrombin did significantly increase intratumoral fibrin deposition and inflammation. Surprisingly, in the presence of an aggressive tumor, a protective outcome with less clinical features coinciding with venous thrombosis were observed in mice with antithrombin knockdown. ConclusionWe conclude that the presence of a breast tumor protects hypercoagulant mice from severe consumption of coagulation factors after lowering hepatic antithrombin levels, possibly due to elevated platelet counts. However, the consequences on cancer-associated thrombosis remained inconclusive.

Protective Effects of Nettle Tea on SKOV-3 Ovarian Cancer Cells Through ROS Production, Apoptosis Induction, and Motility Inhibition Without Altering Autophagy.

Urtica dioica L. (UD), also known as the stinging nettle, has long been used in traditional medicine for its wide range of health benefits. The current study focuses on the effect of nettle tea on the growth and proliferation of one of the most aggressive ovarian adenocarcinoma cell line, SKOV-3 cells. To examine this, cytotoxicity, cell cycle analysis, and ROS assays were performed, along with Annexin V/PI dual staining, cell death ELISA, Western blot analysis, and motility assays. The results showed that a UD aqueous extract (UDAE) can inhibit the growth and proliferation of SKOV-3 cells in a dose- and time-dependent manner by promoting cellular fragmentation. This was accompanied by an increase in two apoptotic hallmarks, the flipping of phosphatidylserine to the outer membrane leaflet and DNA fragmentation as revealed by cell death ELISA. This aqueous extract showed a pro-oxidant activity while also activating the extrinsic caspase-dependent apoptotic pathway with no alteration in autophagy markers. Furthermore, the extract showed promising inhibitory effect on the migratory capacities of aggressive ovarian cancer cells, in vitro.