Breast Cancer Proliferation Research Articles

Notch3 regulates Mybl2 via HeyL to limit proliferation and tumor initiation in breast cancer

Notch signaling is a conserved signaling pathway that participates in many aspects of mammary gland development and homeostasis, and has extensively been associated with breast tumorigenesis. Here, to unravel the as yet debated role of Notch3 in breast cancer development, we investigated its expression in human breast cancer samples and effects of its loss in mice. Notch3 expression was very weak in breast cancer cells and was associated with good patient prognosis. Interestingly, its expression was very strong in stromal cells of these patients, though this had no prognostic value. Mechanistically, we demonstrated that Notch3 prevents tumor initiation via HeyL-mediated inhibition of Mybl2, an important regulator of cell cycle. In the mammary glands of Notch3-deficient mice, we observed accelerated tumor initiation and proliferation in a MMTV-Neu model. Notch3-null tumors were enriched in Mybl2 mRNA signature and protein expression. Hence, our study reinforces the anti-tumoral role of Notch3 in breast tumorigenesis.

The novel role of etoposide in inhibiting the migration and proliferation of small cell lung cancer and breast cancer via targeting Daam1

ObjectivesDaam1 (Dishevelled-associated activator of morphogenesis 1) is a Wnt/PCP signaling protein that engages in cytoskeleton reorganization and is abnormally activated in certain tumors. Daam1 is closely related to cancer metastasis, which is expected to become a target for cancer treatment. However, the natural small molecules targeting Daam1 have not been identified. Materials and methodsWe screened several natural small molecules that may bind to Daam1 by Sybyl molecular simulation docking technique. As a first-line drug for the treatment of small cell lung cancer, etoposide was chosen for further investigation. Next, we used Micro Scale Thermophoresis (MST) to verify the interaction of etoposide and Daam1. Small cell lung cancer H446 cells and breast cancer MCF-7 cells were treated with etoposide and subjected to Western blotting to measure the Daam1 expression. The effect of etoposide on cell proliferation was determined by CCK-8 assay in vitro and by a tumor-bearing mouse model in vivo. Wound healing assay and Boyden chamber assay were used to evaluate the role of etoposide in the migration and invasion ability of tumor cells. The effect of etoposide on the microfilament assembly was visualized by immunofluorescence staining with phalloidine. Finally, the possible mechanism of down-regulation of Daam1 expression after etoposide-induced small cell lung cancer cells was detected by a half-life experiment and immunofluorescence staining with lysosomal marker LAMP1. ResultsSybyl molecular modeling docking technique was performed to screen a natural chemical library for molecules that bound to the FH2 domain of Daam1 and found etoposide was virtually interacted with Daam1. MST validated etoposide directly bound to the FH2 domain of Daam1. Etoposide significantly down-regulated the expression of Daam1 in small cell lung cancer H446 cells and breast cancer MCF-7 cells. Moreover, 270 μmol/L etoposide largely inhibited the proliferation, migration, and invasion of H446 cells and MCF-7 cells. Immunofluorescence staining experiments revealed that etoposide induced the disassembly of microfilaments in H446 cells and MCF-7 cells, which were rescued by Daam1 overexpression. In nude mice transplanted with H446 cells, 5, 10, 20 mg/kg etoposide (drug/weight) injected via tail vein largely retarded the proliferation of subcutaneous tumors. Etoposide induced Daam1 to shorten its half-life and enter the lysosome degradation pathway, and eventually leading to the downregulation of Daam1 expression. ConclusionsEtoposide is a novel natural small molecule targeting Daam1. Etoposide inhibits the proliferation, migration and invasion of small cell lung cancer cells and breast cancer cells, and also suppresses tumor proliferation of small cell lung cancer in vivo.

Circ_0001667 accelerates breast cancer proliferation and angiogenesis through regulating CXCL10 expression by sponging miR-6838-5p.

An increasing number of circular RNAs (circRNAs) have been shown to play an important role in the tumorigenesis of breast cancer. The aim of this study was to investigate the expression and function of circ_0001667 and its potential molecular mechanisms in breast cancer. The expression levels of circ_0001667, miR-6838-5p and CXC chemokine ligand 10 (CXCL10) in breast cancer tissues and cells were detected by quantitative real-time PCR. Cell counting kit-8 assay, EdU assay, flow cytometry, colony formation and tube formation assays were used to detect cell proliferation and angiogenesis. The binding relationship between miR-6838-5p and circ_0001667 or CXCL10 was predicted using the starBase3.0 database and verified by dual-luciferase reporter gene assay, RIP and RNA pulldown. Animal experiments were performed to assess the function of circ_0001667 knockdown on breast cancer tumor growth. Circ_0001667 was highly expressed in breast cancer tissues and cells, and its knockdown inhibited proliferation and angiogenesis of breast cancer cells. Circ_0001667 sponged miR-6838-5p, and inhibition of miR-6838-5p reversed the inhibitory effect of silencing circ_0001667 on proliferation and angiogenesis of breast cancer cells. MiR-6838-5p targeted CXCL10, and overexpression of CXCL10 reverses the effect of miR-6838-5p overexpression on breast cancer cell proliferation and angiogenesis. In addition, circ_0001667 interference also reduced breast cancer tumor growth in vivo. Circ_0001667 is involved in breast cancer cell proliferation and angiogenesis through regulation of the miR-6838-5p/CXCL10 axis.

Elevated expression of myosin VI contributes to breast cancer progression via MAPK/ERK signaling pathway

Breast cancer (BC) is one of the most common malignancies occurring in women worldwide, and its incidence is increasing each year. Accumulating evidence indicated that Myosin VI (MYO6) functions as a gene associated with tumor progression in several cancers. However, the potential role of MYO6 and its underlying mechanisms in the development and progression of BC remains unknown. Herein, we examined the expression levels of MYO6 in BC cells and tissues by western blot and immunohistochemistry. Loss- and gain-of-function investigations in vitro were performed to determine the biological functions of MYO6. And in vivo effects of MYO6 on tumorigenesis were investigated in nude mice. Our findings showed that the expression of MYO6 was up-regulated in breast cancer, and its high expression was correlated with poor prognosis. Further investigation exhibited that silencing the expression of MYO6 significantly inhibited cell proliferation, migration and invasion, whereas overexpression of MYO6 enhanced these abilities in vitro. Also, reduced expression of MYO6 significantly retarded the tumor growth in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) revealed that MYO6 was involved in mitogen-activated protein kinase (MAPK) pathway. Moreover, we proved that MYO6 enhanced BC proliferation, migration and invasion via increasing the expression of phosphorylated ERK1/2. Taken together, our findings highlight the role of MYO6 in promoting BC cell progression through MAPK/ERK pathway, suggesting it may be a new potential therapeutic and prognostic target for BC patients.

Overexpression of POU3F2 promotes radioresistance in triple-negative breast cancer via Akt pathway activation.

POU3F2 is associated with malignant behaviors and poor prognosis in cancer. However, the function and mechanism of POU3F2 in breast cancer remain to be elucidated. Our study aimed to explore the role of POU3F2 in triple-negative breast cancer and radiotherapy. POU3F2 expression was examined by RT-PCR and Western blot. The proliferation of cancer cells was measured by MTT assay. Migration of cancer cells was determined by Transwell assay and wound healing assay. To determine which protein interacts with POU3F2, Co-IP was performed. Survival analysis was performed based on the online database GEPIA. DNA damage after radiation was examined by Comet Assay. Radiosensitivity was evaluated with clonogenic survival assays. A tumor xenograft model was established with MDA-MB-231 breast cancer cells in BALB/c nude mice to explore the effect of POU3F2 in vivo. We found that the expression of POU3F2 was significantly elevated in breast cancer cells, especially in TNBC, and higher POU3F2 expression was related to poor prognosis of patients with breast cancer. Functional assays revealed that POU3F2 promoted proliferation, migration, and invasion of triple-negative breast cancer (TNBC) cells in vitro and in vivo. In addition, the knockdown of POU3F2 decreased the radioresistance of TNBC cells in vitro. Furthermore, POU3F2 could enhance the activation of the Akt pathway by interacting with ARNT2, thereby promoting proliferation and radioresistance in TNBC cells. Our results provide evidence that high expression of POU3F2 promotes radioresistance in triple-negative breast cancer via Akt pathway activation by interacting with ARNT2.

MiR‑221 and miR‑222 regulate cell cycle progression and affect chemosensitivity in breast cancer by targeting ANXA3.

Breast malignancy remains one of the most common causes of cancer-associated mortalities among women. MicroRNA (miR)-221 and miR-222 are homologous miRs and have a substantial impact on cancer progression. In the present study, the regulatory mechanisms of miR-221/222 and its target annexin A3 (ANXA3) in breast cancer cells were investigated. Breast tissue samples were collected to evaluate the expression patterns of miR-221/222 levels in breast cancer cell lines and cancer tissues according to clinical characteristics. The levels of miR-221/222 were increased or decreased in cancer cell lines compared with normal breast cell lines according to cell line subtype. Subsequently, the changes in the progression and invasion of breast cancer cells were investigated using cell proliferation, invasion assay, gap closure and colony formation assays. Western blotting of cell cycle proteins and flow cytometry were performed to evaluate the possible pathway of miR-221/222 and ANXA3 axis. Chemosensitivity tests were performed to explore the suitability of the miR-221/222 and ANXA3 axis as a therapeutic target in breast cancer. The expression levels of miR-221/222 were associated with aggressive characteristics of breast cancer subtypes. Cell transfection assay demonstrated the regulation of breast cancer proliferation and invasiveness by miR-221/222. MiR-221/222 directly targeted the 3'-untranslated region of ANXA3 and suppressed the expression of ANXA3 at the mRNA and protein levels. In addition, miR-221/222 negatively regulated cell proliferation and the cell cycle pathway in breast cancer cells by targeting ANXA3. In combination with adriamycin, downregulation of ANXA3 may sensitize adriamycin-induced cell death to induction of persistent G2/M and G0/G1 arrest. Decreased expression of ANXA3 through increased expression of miR-221/222 reduced breast cancer progression and increased the effectiveness of the chemotherapy drug. The present results indicated the miR-221/222 and ANXA3 axis to be a possible novel therapeutic target for the treatment of breast cancer.

Isopimaric acid, an ion channel regulator, regulates calcium and oxidative phosphorylation pathways to inhibit breast cancer proliferation and metastasis

Breast cancer is the globally most common malignant tumor and the biggest threat to women. Even though the diagnosis and treatment of breast cancer are progressing continually, a large number of breast cancer patients eventually develop a metastatic tumor, especially triple-negative breast cancer (TNBC). Recently, metal ion homeostasis and ion signaling pathway have become important targets for cancer therapy. In this study, We analyzed the effects and mechanisms of isopimaric acid (IPA), an ion channel regulator, on the proliferation and metastasis of breast cancer cells (4 T1, MDA-MB-231and MCF-7) by cell functional assay, flow cytometry, western blot, proteomics and other techniques in vitro and in vivo. Results found that IPA significantly inhibited the proliferation and metastasis of breast cancer cells (especially 4 T1). Further studies on the anti-tumor mechanism of IPA suggested that IPA might affect EMT and Wnt signaling pathways by targeting mitochondria oxidative phosphorylation and Ca2+ signaling pathways, and then inducing breast cancer cell cycle arrest and apoptosis. Our research reveals the therapeutic value of IPA in breast cancer and provides a theoretical basis for the new treatment of breast cancer.

Chalcone-3 Inhibits the Proliferation of Human Breast Cancer MDA-MB-231 Cell Line.

Chalcone-3 has been shown to be cytotoxic and selective against luminal subtype breast cancer cell lines, which are suspected to occur through the mechanism of epidermal growth factor receptors (EGFR) inhibition. However, the cytotoxic effect has never been tested on cell strains from patients with triple negative breast cancer (TNBC), where EGFR expression is known to increase. This study aimed to identify the role of chalcone-3 in one of the downstream targets of EGFR as an antiproliferative agent. Chalcone-3 was examined for its effect on proliferation in human breast cancer MDA-MB-231 cell lines. The percentage of proliferation inhibition was analyzed using methyl-thiazol tetrazolium assay. Flow cytometry was used to analyze the population of cell cycle distribution and the expression of cyclin-D1 and pEGFR. Chalcone-3 inhibited the proliferation of MDA-MB-231 cells in a dose and time-dependent manner with an IC50 value of 17.98±6.36 µg/mL by inducing cell cycle arrest at the G2/M phase. Flow cytometry assays showed that chalcone-3 significantly reduced the expression of pEGFR and cyclin-D1, contributing to cell cycle arrest. Chalcone-3 might have potential as an anti-proliferative drug to treat TNBC.

Echinacoside hampers malignant progression of breast cancer MCF-7 cells by modulating AKR1B10/ERK signal transduction

This study analyzes the impact of echinacoside(ECH) in the proliferation, metastasis and adriamycin(ADR) resistance of breast cancer(BC) MCF-7 cells via the modulation of aldo-keto reductase family 1 member 10(AKR1B10)/extracellular signal-regulated kinase(ERK) pathway. The chemical structure of ECH was firstly confirmed. MCF-7 cells were treated with different concentration(0, 10, 20, 40 μg·mL~(-1)) of ECH for 48 h. Western blot was used to analyze expression of AKR1B10/ERK pathway-associated proteins and cell counting kit-8(CCK-8) assay to determine cell viability. MCF-7 cells were collected and classified into control group, ECH group, ECH + Ov-NC group, and ECH + Ov-AKR1B10 group. Then Western blot was employed to analyze the expression of AKR1B10/ERK pathway-associated proteins. CCK-8 and 5-ethynyl-2'-deoxyuridine(EdU) assay were used to examine cell proliferation. Cell migration was appraised with scratch assay, Transwell assay, and Western blot. Eventually, MCF-7 cells were treated with ADR for 48 h to induce ADR resistance. Cell viability was tested by CCK-8 assay and cell apoptosis was estimated based on terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) assay and Western blot. Based on Protein Data Bank(PDB) and molecular docking, the binding affinity of ECH to AKR1B10 was assessed. Various doses of ECH decreased the expression of AKR1B10/ERK pathway-associated proteins in a dose-dependent manner and declined cell viability compared with the control group. Compared with the control group, 40 μg·mL~(-1) ECH blocked the AKR1B10/ERK pathway in MCF-7 cells and inhibited the proliferation, metastasis and ADR resistance of the cells. Compared with the ECH + Ov-NC group, ECH + Ov-AKR1B10 group showed the recovery of some biological behaviors of MCF-7 cells. ECH also targeted AKR1B10. ECH can inhibit the proliferation, metastasis, and ADR resistance of BC cells by blocking AKR1B10/ERK pathway.

The Evolution of Ki-67 and Breast Carcinoma: Past Observations, Present Directions, and Future Considerations.

The 1983 discovery of a mouse monoclonal antibody-the Ki-67 antibody-that recognized a nuclear antigen present only in proliferating cells represented a seminal discovery for the pathologic assessment of cellular proliferation in breast cancer and other solid tumors. Cellular proliferation is a central determinant of prognosis and response to cytotoxic chemotherapy in patients with breast cancer, and since the discovery of the Ki-67 antibody, Ki-67 has evolved as an important biomarker with both prognostic and predictive potential in breast cancer. Although there is universal recognition among the international guideline recommendations of the value of Ki-67 in breast cancer, recommendations for the actual use of Ki-67 assays in the prognostic and predictive evaluation of breast cancer remain mixed, primarily due to the lack of assay standardization and inconsistent inter-observer and inter-laboratory reproducibility. The treatment of high-risk ER-positive/human epidermal growth factor receptor-2 (HER2) negative breast cancer with the recently FDA-approved drug abemaciclib relies on a quantitative assessment of Ki-67 expression in the treatment decision algorithm. This further reinforces the urgent need for standardization of Ki-67 antibody selection and staining interpretation, which will hopefully lead to multidisciplinary consensus on the use of Ki-67 as a prognostic and predictive marker in breast cancer. The goals of this review are to highlight the historical evolution of Ki-67 in breast cancer, summarize the present literature on Ki-67 in breast cancer, and discuss the evolving literature on the use of Ki-67 as a companion diagnostic biomarker in breast cancer, with consideration for the necessary changes required across pathology practices to help increase the reliability and widespread adoption of Ki-67 as a prognostic and predictive marker for breast cancer in clinical practice.

Tetrahydrocurcumin regulates the tumor immune microenvironment to inhibit breast cancer proliferation and metastasis via the CYP1A1/NF-κB signaling pathway

The NF-κB signaling pathway is overactivated in tumor cells, and the activation of the NF-κB signaling pathway releases a large number of inflammatory factors, which enhance tumor immunosuppression and promote tumor metastasis. The cytochrome P450 (CYP450) system consists of important metabolic enzymes present in different tissues and progressive tumors, which may lead to changes in the pharmacological action of drugs in inflammatory diseases such as tumors. In this study, the anticancer effect of tetrahydrocurcumin (THC), an active metabolite of curcumin, on breast cancer cells and the underlying mechanism were investigated. Result showed that THC selectively inhibited proliferation and triggered apoptosis in breast cancer cells in a concentration- and time-dependent manner. Moreover, THC-induced cell apoptosis via a mitochondria-mediated pathway, as indicated by the upregulated ratio of Bax/Bcl-2 and reactive oxygen species (ROS) induction. In addition, THC could affect the CYP450 enzyme metabolic pathway and inhibit the expression of CYP1A1 and activation of the NF-κB pathway, thereby inhibiting the migration and invasion of breast cancer cells. Furthermore, after overexpression of CYP1A1, the inhibitory effects of THC on the proliferation, metastasis, and induction of apoptosis in breast cancer cells were weakened. The knockdown of CYP1A1 significantly enhanced the inhibitory effect of THC on the proliferation, metastasis, and apoptosis induction of breast cancer cells. Notably, THC exhibited a significant tumor growth inhibition and anti-pulmonary metastasis effect in a tumor mouse model of MCF-7 and 4T1 cells by regulating the tumor immunosuppressive microenvironment. Collectively, these results showed that TH could effectively trigger apoptosis and inhibit the migration of breast cancer cells via the CYP1A1/NF-κB signaling pathway, indicating that THC serves as a potential candidate drug for the treatment of breast cancer.

Deregulation of miR-375 Inhibits HOXA5 and Promotes Migration, Invasion, and Cell Proliferation in Breast Cancer.

Breast cancer (BC) is a highly aggressive tumour and one of the women's leading causes of cancer-related deaths in worldwide. MiR-375 overexpressed in BC cells, and its biological relevance is largely unknown. Here in, we explored the function of miR-375 in BC. MicroRNA-375 targets were predicted by online target prediction tools and found that HOXA5 is one of the potential targets. MTT assay was employed to assess the effect of miR-375 on cell proliferation, where migration and invasion transwell assays were applied to detect cell migratory and invasive ability. Besides, relative expression of miR-375 and HOXA5 was measured in BC and HEK-293 cells, and its downstream gene target expressions were evaluated by qRT-PCR and western blot. In this study, we found that miR-375 expression was higher in BC cell lines than in the HEK-293 cell line, whereas HOXA5 expression was significantly lower. Our study showed that exogenous inhibition of miR-375 promoted HOXA5 expression; on the contrary, miR-375 mimics down-regulated HOXA5 expression level. Knockdown of miR-375 expression in BC cells reduces cell proliferation, migration, and invasion by inverse correlation expression of HOXA5. Our findings associated that miR-375 accelerated apoptosis evasion, proliferation, migration, and invasion by targeting HOXA5. In addition, nucleolin interferes in miR-375 biogenesis while silencing of nucleolin significantly reduced miR-375 expression and increased HOXA5 expression in BC. Thus, miR-375/HOXA5 axis may represent a potential therapeutic target for BC treatment.

M6A reader HNRNPA2B1 destabilization of ATG4B regulates autophagic activity, proliferation and olaparib sensitivity in breast cancer

N6-methyladenosine RNA (m6A) is the most extensive epigenetic modification in mRNA and influences tumor progression. However, the role of m6A regulators and specific mechanisms in breast cancer still need further study. Here, we investigated the significance of the m6A reader HNRNPA2B1 and explored its influence on autophagy and drug sensitivity in breast cancer. HNRNPA2B1 was selected by bioinformatics analysis, and its high expression level was identified in breast cancer tissues and cell lines. HNRNPA2B1 was related to poor prognosis. Downregulation of HNRNPA2B1 reduced proliferation, enhanced autophagic flux, and partially reversed de novo resistance to olaparib in breast cancer. ATG4B was determined by RIP and MeRIP assays as a downstream gene of HNRNPA2B1, by which recognized the m6A site in the 3′UTR. Overexpression of ATG4B rescued the malignancy driven by HNRNPA2B1 in breast cancer cells and increased the olaparib sensitivity. Our study revealed that the m6A reader HNRNPA2B1 mediated proliferation and autophagy in breast cancer cell lines by facilitating ATG4B mRNA decay and targeting HNRNPA2B1/m6A/ATG4B might enhance the olaparib sensitivity of breast cancer cells.

Synthesis and Characterization of New Mixed-Ligand Complexes; Density Functional Theory, Hirshfeld, and In Silico Assays Strengthen the Bioactivity Performed In Vitro.

N'-Acetyl-2-cyanoacetohydrazide (H2L1) and 2-cyano-N-(6-ethoxybenzo thiazol-2-yl) acetamide (HL2) ligands were used to synthesize [Cr(OAc)(H2L1)(HL2)]·2(OAc) and [Mn(H2L1)(HL2)]·Cl2·2H2O as mixed ligand complexes. All new compounds were analyzed by analytical, spectral, and computational techniques to elucidate their chemical formulae. The bidentate nature was suggested for each coordinating ligand via ON donors. The electronic transitions recorded are attributing to 4A2g(F) → 4T2g(F)(υ2) and 4A2g(F) → 4T1g(F)(υ3) types in the octahedral Cr(III) complex, while 6A1 → 4T2(G) and 6A1 → 4T1(G) transitions are attributing to the tetrahedral Mn(II) complex. These complexes were optimized by the density functional theory method to verify the bonding mode which was suggested via N(3), O(8), N(9), and N(10) donors from the mixed-ligands. Hirshfeld crystal models were demonstrated for the two ligands to indicate the distance between the functional groups within the two ligands and supporting the exclusion of self-interaction in between. Finally, the biological activity of the two mixed ligand complexes was tested by in silico ways as well as in vitro ways for confirmation. Three advanced programs were applied to measure the magnitude of biological efficiency of the two complexes toward kinase enzyme (3nzs) and breast cancer proliferation (3hy3). All in silico data suggest the superiority of the Mn(II) complex. Moreover, the in vitro assays for the two complexes that measure their antioxidant and cytotoxic activity support the distinguished activity of the Mn(II) complex.

Discovery of IHMT-337 as a potent irreversible EZH2 inhibitor targeting CDK4 transcription for malignancies

Enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of PRC2 complex, plays an important role in tumor development and progression through its catalytic and noncatalytic activities. Overexpression or gain-of-function mutations of EZH2 have been significantly associated with tumor cell proliferation of triple-negative breast cancer (TNBC) and diffuse large B-cell lymphoma (DLBCL). As a result, it has gained interest as a potential therapeutic target. The currently available EZH2 inhibitors, such as EPZ6438 and GSK126, are of benefit for clinical using or reached clinical trials. However, certain cancers are resistant to these enzymatic inhibitors due to its noncatalytic or transcriptional activity through modulating nonhistone proteins. Thus, it may be more effective to synergistically degrade EZH2 in addition to enzymatic inhibition. Here, through a rational design and chemical screening, we discovered a new irreversible EZH2 inhibitor, IHMT-337, which covalently bounds to and degrades EZH2 via the E3 ligase CHIP-mediated ubiquitination pathway. Moreover, we revealed that IHMT-337 affects cell cycle progression in TNBC cells through targeting transcriptional regulating of CDK4, a novel PRC2 complex- and enzymatic activity-independent function of EZH2. More significantly, our compound inhibits both DLBCL and TNBC cell proliferation in different preclinical models in vitro and in vivo. Taken together, our findings demonstrate that in addition to enzymatic inhibition, destroying of EZH2 by IHMT-337 could be a promising therapeutic strategy for TNBC and other malignancies that are independent of EZH2 enzymatic activity.

Thioalbamide inhibits FoF1-ATPase in breast cancer cells and reduces tumor proliferation and invasiveness in breast cancer in vivo models

ObjectiveThioalbamide is a ribosomally synthesized and post-translationally modified peptide (RiPP) belonging to the family of thioamitides, a rare class of microbial specialized metabolites with unusual post-translational modifications and promising biological activities. Recent studies have demonstrated the ability of thioalbamide to exert highly selective cytotoxic effects on tumor cells by affecting their energy metabolism, thus causing abnormal ROS production and triggering apoptosis. This study is aimed to investigate the molecular mechanisms underlying the antitumor activity of thioalbamide in order to identify its exact molecular target. MethodsWild type MCF-7 and MDA-MB-231 breast cancer cell lines as well as cancer cells deprived of mitochondrial DNA (ρ0 cells) were employed in order to assess thioalbamide effects on tumor bioenergetics. In this regard, metabolic profile was evaluated by a Seahorse XFe96 analyzer, and the activity of the enzyme complexes involved in oxidative phosphorylation was quantified by spectrophotometric assays. Thioalbamide effects on tumor invasiveness were assessed by gelatin zymography experiments and invasion assays. In vivo experiments were carried out on breast cancer xenograft and “experimental metastasis” mouse models. ResultsExperiments carried out on ρ0 breast cancer cells, together with Seahorse analysis and the application of spectrophotometric enzymatic assays, highlighted the ability of thioalbamide to affect the mitochondrial respiration process, and allowed to propose the FoF1-ATPase complex as its main molecular target in breast cancer cells. Additionally, thioalbamide-mediated OXPHOS inhibition was shown, for the first time, to reduce tumor invasiveness by inhibiting metalloproteinase-9 secretion. Furthermore, this study has confirmed the antitumor potential of thioalbamide in two different in vivo models. In particular, experiments on MCF-7 and MDA-MB-231 xenograft mouse models have confirmed in vivo its high anti-proliferative and pro-apoptotic activity, while experiments on MDA-MB-231 ″experimental metastasis” mouse models have highlighted its ability to inhibit breast cancer cell invasiveness. ConclusionsOverall, our results shed more light on the molecular mechanisms underlying the pharmacological potential of thioamidated peptides, thus reducing the gap that separates this rare class of microbial metabolites from clinical studies, which could validate them as effective tools for cancer treatment.

Abstract B008: The role of the androgen receptor in reciprocal activation of breast cancer metastases and the lung epithelium during metastatic outgrowth

Abstract The lung is one of the most common sites of breast cancer (BC) metastasis. A major gap in our ability to effectively treat BC patients with established lung metastases lies in our lack of knowledge regarding how the lung microenvironment is remodeled during metastasis and how this contributes to disease progression. Our data suggest that the lung epithelium, in particular lung type II alveolar epithelial (AT2) cells, contribute to metastatic outgrowth. We hypothesize that BC lung micrometastases activate surrounding lung epithelial cells which, in turn, support the survival and outgrowth of BC metastases within the lung. The overall goal of these studies is to identify factors secreted by resident lung cells that could be used as metastasis-specific therapeutic targets/agents. We utilized immunocompetent mouse metastasis models to study metastatic progression within the lung. Multispectral fluorescent imagining of metastases demonstrated that a wound repair-related phenotype, characterized by chronic inflammation, develops within the lung microenvironment during metastatic outgrowth. We observed an increase in the number and activation of AT2 cells surrounding metastases as they grow. To gain mechanistic insight into how growing metastases may impact AT2 cells, we conducted single-cell RNA-sequencing (scRNAseq) on mouse lungs with high vs. low metastatic burden. This investigation showed that metastatic outgrowth significantly changes AT2 gene expression resulting in a modified secretome. Cell culture experiments, using multiple AT2 cell models, indicated that no contact co-culture with TNBC also caused significant changes in AT2 gene expression. Upstream regulator analysis of these datasets predicted that the androgen receptor (AR) may contribute to AT2 activation during metastatic outgrowth. Compared to non-adjacent tumor-free MMTV-PyMT lung tissue, there was an increased percentage of AR+ cells in the lung adjacent to metastases, which was further associated with large metastases only. Co-immunofluorescence experiments demonstrated that these lung-specific AR+ cells were primarily AT2 cells and fibroblasts, while in human lungs the majority of AR+ cells were AT2 cells and macrophages. Interestingly, co-culturing of TNBC and AT2 cells enhanced BC proliferation, suggesting that following activation, AT2 secreted factors may reciprocally impact metastatic growth. In conclusion, these data indicate that a tumor supportive microenvironment develops as lung metastases grow and suggests that by the time patients present with established/detectable lung metastases, the lung microenvironment has already undergone fundamental alterations that promote metastatic progression. Future studies will elucidate how activated lung cells support metastatic progression and could lead to the development or repurposing of therapeutic strategies to prevent destructive metastatic outgrowth. Citation Format: Jessica L. Christenson, Nicole S. Spoelstra, G. Devon Trahan, Kathleen I. O'Neill, Michelle M. Williams, Jennifer K. Richer. The role of the androgen receptor in reciprocal activation of breast cancer metastases and the lung epithelium during metastatic outgrowth [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B008.

Abstract B003: Hypoxia-induced secretion of fucosylated PVR/CD155 from brain met-associated fibroblasts drives breast cancer invasive capacity by altering cell-cell contacts & focal adhesion

Abstract Background: Brain metastasis (BM) develops in ~10-30% of breast cancer (BC) patients; triple negative breast cancer (TNBC), the most aggressive subtype of BC, exhibits the highest incidence of BM. Treatment options for BCBM are extremely limited due to the poor biological understanding. Hence, there is an urgent need to elucidate molecular mechanisms driving BCBM. We aim to study BCBM pathogenesis by dissecting the role of tumorigenic fucosylated proteins secreted by BM-associated fibroblasts (bmCAFs). Methods: We assessed global fucosylation (post-translational protein modification by L-fucose) patterns by fucose-binding lectin pulldown and immunoblot (IB) analysis. Conditioned media (CM) derived from CAFs that were depleted or not of fucosylated proteins were used to treat BC cells to assess motility and invasion. Fuco-proteomics and phosphoproteomic profiling identified bmCAF-secreted fucosylated (sf) proteins in bmCAF-derived CM and associated global signaling changes induced in BC cells, respectively. qRT-PCR was performed to analyze FUT11 levels under normoxia and hypoxia. Immunofluorescence and IB analyses of BC cells treated ± with bmCAF-derived CM was performed to validate PVR downstream signaling changes. Stereotactic intracranial implantation of BC cells alone or with ctrl/PVR knocked-down- bmCAFs was used for the BCBM mouse model. Results: We discovered that fucosylated proteins secreted uniquely by bmCAFs but not by normal-breast fibroblasts (NBF) or primary tumor-CAFs (tCAF), potently drives BC proliferation and invasiveness. Fucosylated proteomic profiling of the bmCAF secretome identified a soluble Polio Virus Receptor (PVR) isoform as uniquely upregulated, fucosylated, and secreted by bmCAFs. Of the 13 fucosyltransferases (FUTs), we found that bmCAFs upregulate FUT11, a key hypoxia-related gene. FUT11 and secreted fucosylated PVR (sfPVR) are significantly increased in bmCAFs exposed to hypoxia, consistent with the hypoxic environment of the brain. PVR can exist as transmembrane and secreted isoforms. Whereas transmembrane PVR is known to contribute to poor prognosis in a number of cancers by facilitating tumorigenic cell:matrix interactions and attenuating anti-tumor immunity, roles of secreted fucosylated PVR (sfPVR) in cancer are unknown. Our phosphoproteomics analyses of BC cells identified cellular adhesion/cytoskeletal and EPHA2 signaling as significantly modulated by bmCAF sfPVR to drive BC cell motility and invasiveness. Indeed, PVR knockdown abrogates the ability of bmCAFs to enhance BC growth/spread in the brain in a mouse model. Conclusion: Our data demonstrate that pathological hypoxia drives FUT11-mediated fucosylation and secretion of PVR by bmCAFs, which potently drives BC cells motility and invasiveness, representing a mechanism by which bmCAFs can promote BCBM. We expect our ongoing and further studies to advance our understanding of how sfPVR from bmCAFs promotes BCBM and to establish a basis for therapeutic targeting of PVR and/or use of fucosylated PVR and its signaling effectors as biomarkers. Citation Format: Emma Adhikari, Qian Liu, Viktoriya Marusyk, Victoria Lzumi, John M. Koomen, Andriy Marusyk, Eric Lau. Hypoxia-induced secretion of fucosylated PVR/CD155 from brain met-associated fibroblasts drives breast cancer invasive capacity by altering cell-cell contacts & focal adhesion [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B003.

Anti-colorectal cancer of Ardisia gigantifolia Stapf. and targets prediction via network pharmacology and molecular docking study

BackgroundArdisia gigantifolia Stapf. (AGS), a Chinese folk medicine widely grows in the south of China and several studies reported that AGS could inhibit the proliferation of breast cancer, liver cancer, and bladder cancer cell lines. However, little is known about its anti-colorectal cancer (CRC) efficiency.MethodsIn the present study, a combination of MTT assay, network pharmacological analysis, bioinformatics, molecular docking, and molecular dynamics simulation study was used to investigate the active ingredients, and targets of AGS against CRC, as well as the potential mechanism.ResultsMTT assay showed that three kinds of fractions from AGS, including the n-butanol extract (NBAGS), ethyl acetate fraction (EAAGS), and petroleum ether fraction (PEAGS) significantly inhibited the proliferation of CRC cells, with the IC50 values of 197.24, 264.85, 15.45 µg/mL on HCT116 cells, and 523.6, 323.59, 150.31 µg/mL on SW620 cells, respectively. Eleven active ingredients, including, 11-O-galloylbergenin, 11-O-protocatechuoylbergenin, 11-O-syringylbergenin, ardisiacrispin B, bergenin, epicatechin-3-gallate, gallic acid, quercetin, stigmasterol, stigmasterol-3-o-β-D-glucopyranoside were identified. A total of 173 targets related to the bioactive components and 21,572 targets related to CRC were picked out through database searching. Based on the crossover targets of AGS and CRC, a protein-protein interaction network was built up by the String database, from which it was concluded that the core targets would be SRC, MAPK1, ESR1, HSP90AA1, MAPK8. Besides, GO analysis showed that the numbers of biological process, cellular component, and molecular function of AGS against CRC were 1079, 44, and 132, respectively, and KEGG pathway enrichment indicated that 96 signaling pathways in all would probably be involved in AGS against CRC, among which MAPK signaling pathway, lipid, and atherosclerosis, proteoglycans in cancer, prostate cancer, adherens junction would probably be the major pathways. The docking study verified that AGS had multiple ingredients and multiple targets against CRC. Molecular dynamics (MD) simulation analysis showed that the binding would be stable via forming hydrogen bonds.ConclusionOur study showed that AGS had good anti-CRC potency with the characteristics of multi-ingredients, -targets, and -signaling pathways.

The N6-methyladenosine METTL3 regulates tumorigenesis and glycolysis by mediating m6A methylation of the tumor suppressor LATS1 in breast cancer

BackgroundPosttranscriptional modification of tumor-associated factors plays a pivotal role in breast cancer progression. However, the underlying mechanism remains unknown. M6A modifications in cancer cells are dynamic and reversible and have been found to impact tumor initiation and progression through various mechanisms. In this study, we explored the regulatory mechanism of breast cancer cell proliferation and metabolism through m6A methylation in the Hippo pathway. MethodsA combination of MeRIP-seq, RNA-seq and metabolomics-seq was utilized to reveal a map of m6A modifications in breast cancer tissues and cells. We conducted RNA pull-down assays, RIP-qPCR, MeRIP-qPCR, and RNA stability analysis to identify the relationship between m6A proteins and LATS1 in m6A regulation in breast cancer cells. The expression and biological functions of m6A proteins were confirmed in breast cancer cells in vitro and in vivo. Furthermore, we investigated the phosphorylation levels and localization of YAP/TAZ to reveal that the activity of the Hippo pathway was affected by m6A regulation of LATS1 in breast cancer cells. ResultsWe demonstrated that m6A regulation plays an important role in proliferation and glycolytic metabolism in breast cancer through the Hippo pathway factor, LATS1. METTL3 was identified as the m6A writer, with YTHDF2 as the reader protein of LATS1 mRNA, which plays a positive role in promoting both tumorigenesis and glycolysis in breast cancer. High levels of m6A modification were induced by METTL3 in LATS1 mRNA. YTHDF2 identified m6A sites in LATS1 mRNA and reduced its stability. Knockout of the protein expression of METTL3 or YTHDF2 increased the expression of LATS1 mRNA and suppressed breast cancer tumorigenesis by activating YAP/TAZ in the Hippo pathway.ConclusionsIn summary, we discovered that the METTL3-LATS1-YTHDF2 pathway plays an important role in the progression of breast cancer by activating YAP/TAZ in the Hippo pathway.