The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization.

Mitophagy is a vital process that controls mitochondria quality, dysregulation of which can promote cancer. Oncoprotein mucin 1 (MUC1) targets mitochondria to attenuate drug-induced apoptosis. However, little is known about whether and how MUC1 contributes to mitochondrial homeostasis in cancer cells. We identified a novel role of MUC1 in promoting mitophagy. Increased mitophagy is coupled with the translocation of MUC1 to mitochondria, where MUC1 interacts with and induces degradation of ATPase family AAA domain-containing 3A (ATAD3A), resulting in protection of PTEN-induced kinase 1 (Pink1) from ATAD3A-mediated cleavage. Interestingly, MUC1-induced mitophagy is associated with increased oncogenicity of cancer cells. Similarly, inhibition of mitophagy significantly suppresses MUC1-induced cancer cell activity in vitro and in vivo. Consistently, MUC1 and ATAD3A protein levels present an inverse relationship in tumor tissues of breast cancer patients. Our data validate that MUC1/ATAD3A/Pink1 axis-mediated mitophagy constitutes a novel mechanism for maintaining the malignancy of cancer cells, providing a novel therapeutic approach for MUC1-positive cancers.

MUC1 triggers lineage plasticity of Her2 positive mammary tumors.

Aberrant overexpression of mucin 1 (MUC1) and human epidermal growth factor receptor 2 (HER2) are often observed in breast cancer. However, the role of concomitant MUC1/HER2 in the development of breast cancer has not been fully illustrated. Following analysis of public microarray datasets that revealed a correlation between double MUC1 and HER2 positivity and a worse clinical outcome, we generated a mouse model overexpressing both Her2 and MUC1 cytoplasmic domain (MUC1-CD) to investigate their interaction in mammary carcinogenesis. Coexpression of Her2 and MUC1-CD conferred a growth advantage and promoted the development of spontaneous mammary tumors. Genomic analysis revealed that enforced expression of MUC1-CD and Her2 induces mammary tumor lineage plasticity, which is supported by gene reprogramming and mammary stem cell enrichment. Through gain- and loss-of-function strategies, we show that coexpression of Her2 and MUC1-CD is associated with downregulation of tricarboxylic acid (TCA) cycle genes in tumors. Importantly, the reduction in TCA cycle genes induced by MUC1-CD was found to be significantly connected to poor prognosis in HER2+ breast cancer patients. In addition, MUC1 augments the Her2 signaling pathway by inducing Her2/Egfr dimerization. These findings collectively demonstrate the vital role of MUC1-CD/Her2 collaboration in shaping the mammary tumor landscape and highlight the prognostic and therapeutic implications of MUC1 in patients with HER2+ breast cancer.

Glass-activated regeneration of volumetric muscle loss.

Volumetric muscle loss (VML) resulting from injuries to skeletal muscles has profound consequences in healthcare. Current VML treatment based on the use of soft materials including biopolymers and decellularized extracellular matrix (dECM) is challenging due to their incapability of stimulating the formation of satellite cells (SCs), muscle stem cells, which are required for muscle regeneration. Additional stem cells and/or growth factors have to be incorporated in these constructs for improved efficacy. Here we report an approach by using bioactive glasses capable of regenerating VML without growth factors or stem cells. One silicate and two borate compositions with different degradation rates (2.4% for silicate 45S5; 5.3% and 30.4% for borate 8A3B and 13-93B3, respectively, in simulated body fluid (SBF) at 37 °C for 30 days) were used for this study. Our in vitro models demonstrate the ability of ions released from bioactive glasses in promoting angiogenesis and stimulating cells to secrete critical muscle-related growth factors. We further show the activation of SCs and the regeneration of skeletal muscles in a rat VML model. Considering these promising results, this work reveals a potentially simple and safe approach to regenerating skeletal muscle defects. STATEMENT OF SIGNIFICANCE: (1) This is the first report on an inorganic material used in skeletal muscle regeneration through in vitro and in vivo models. (2) Bioactive glass is found to activate the production of satellite cells (SCs), muscle stem cells, without the incorporation of extra stem cells or growth factors. (3) The work represents a simple, safe, low-cost yet efficient means for healing muscle defects.

Erlotinib overcomes paclitaxel-resistant cancer stem cells by blocking the EGFR-CREB/GRß-IL-6 axis in MUC1-positive cervical cancer.

Cancer stem cells (CSCs) are often enriched after chemotherapy and contribute to tumor relapse. While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are widely used for the treatment of diverse types of cancer, whether EGFR-TKIs are effective against chemoresistant CSCs in cervical cancer is largely unknown. Here, we reveal that EGFR correlates with reduced disease-free survival in cervical cancer patients with chemotherapy. Erlotinib, an EGFR-TKI, effectively impedes CSCs enrichment in paclitaxel-resistant cells through inhibiting IL-6. In this context, MUC1 induces CSCs enrichment in paclitaxel-resistant cells via activation of EGFR, which directly enhances IL-6 transcription through cAMP response element-binding protein (CREB) and glucocorticoid receptor ß (GRß). Treatment with erlotinib sensitizes CSCs to paclitaxel therapy both in vitro and in vivo. More importantly, positive correlations between the expressions of MUC1, EGFR, and IL-6 were found in 20 cervical cancer patients after chemotherapy. Mining TCGA data sets also uncovered the expressions of MUC1-EGFR-IL-6 correlates with poor disease-free survival in chemo-treated cervical cancer patients. Collectively, our work has demonstrated that the MUC1-EGFR-CREB/GRß axis stimulates IL-6 expression to induce CSCs enrichment and importantly, this effect can be abrogated by erlotinib, uncovering a novel strategy to treat paclitaxel-resistant cervical cancer.

MUC1 induces M2 type macrophage influx during postpartum mammary gland involution and triggers breast cancer.

The microenvironment of postpartum mammary gland involution (PMI) has been linked to the increased risk of breast cancer and poor outcome of patients. Nevertheless the mechanism underlying regulates the microenvironment remains largely unknown. MUC1, which is abnormally overexpressed in most breast cancer, is physiologically expressed in PMI. Using MUC1 cytoplasm domain (MUC1-CD) transgenic mice, we reveal that the overexpression of MUC1-CD in mammary epithelial cells increases M2 type macrophage infiltration in PMI. By sustain activating p50, MUC1 upregulates M2 macrophage chemo-attractants and the anti-apoptotic protein Bcl-xL. Because of the tumor promotional microenvironments and reduced apoptosis, MUC1-CD delays PMI process and results in atypical phenotype in multiparous mice mammary. This finding is further supported by the positive association between the expression of MUC1 and p50 in Luminal A and Luminal B subtypes through analyzing breast cancer databases. Taken together, our study demonstrates that MUC1-CD plays an important role in regulating microenvironment of PMI and promoting postpartum mammary tumorigenicity, providing novel prevention and treatment strategies against postpartum breast cancer.

Chemical genomic analysis of GPR35 signaling.

GPR35, a family A orphan G protein-coupled receptor, has been implicated in inflammatory, neurological, and cardiovascular diseases. However, not much is known about the signaling and functions of GPR35. We performed a label-free kinome short hairpin RNA screen and identified a putative signaling network of GPR35 in HT-29 cells, some of which was validated using gene expression, biochemical and cellular assays. The results showed that GPR35 induced hypoxia-inducible factor 1α, and was involved in synaptic transmission, sensory perception, the immune system, and morphogenetic processes. Collectively, our data suggest that GPR35 may play an important role in response to hypoxic stress and be a potential target for the treatment of inflammatory, cardiovascular, and neurological disorders.

Label-Free Cell Phenotypic Identification of D-Luciferin as an Agonist for GPR35.

D-Luciferin (also known as beetle or firefly luciferin) is one of the most widely used bioluminescent reporters for monitoring in vitro or in vivo luciferase activity. The identification of several natural phenols and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as agonists for GPR35, an orphan G protein-coupled receptor, had motivated us to examine the pharmacological activity of D-Luciferin, given that it also contains phenol and carboxylic acid moieties. Here, we describe label-free cell phenotypic assays that ascertain D-Luciferin as a partial agonist for GPR35. The agonistic activity of D-Luciferin at the GPR35 shall evoke careful interpretation of biological data when D-Luciferin or its analogues are used as probes.

Spatial colocalization and functional link of purinosomes with mitochondria.

Purine biosynthetic enzymes organize into dynamic cellular bodies called purinosomes. Little is known about the spatiotemporal control of these structures. Using super-resolution microscopy, we demonstrated that purinosomes colocalized with mitochondria, and these results were supported by isolation of purinosome enzymes with mitochondria. Moreover, the number of purinosome-containing cells responded to dysregulation of mitochondrial function and metabolism. To explore the role of intracellular signaling, we performed a kinome screen using a label-free assay and found that mechanistic target of rapamycin (mTOR) influenced purinosome assembly. mTOR inhibition reduced purinosome-mitochondria colocalization and suppressed purinosome formation stimulated by mitochondria dysregulation. Collectively, our data suggest an mTOR-mediated link between purinosomes and mitochondria, and a general means by which mTOR regulates nucleotide metabolism by spatiotemporal control over protein association.

Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops.

Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

In vivo selection for spine-derived highly metastatic lung cancer cells is associated with increased migration, inflammation and decreased adhesion.

We developed a murine spine metastasis model by screening five metastatic non-small cell lung cancer cell lines (PC-9, A549, NCI-H1299, NCI-H460, H2030). A549 cells displayed the highest tendency towards spine metastases. After three rounds of selection in vivo, we isolated a clone named A549L6, which induced spine metastasis in 80% of injected mice. The parameters of the A549L6 cell spinal metastatic mouse models were consistent with clinical spine metastasis features. All the spinal metastatic mice developed symptoms of nerve compression after 40 days. A549L6 cells had increased migration, invasiveness and decreased adhesion compared to the original A549L0 cells. In contrast, there was no significant differences in cell proliferation, apoptosis and sensitivity to chemotherapeutic agents such as cisplatin. Comparative transcriptomic analysis and real-time PCR analysis showed that expression of signaling molecules regulating several tumor properties including migration (MYL9), metastasis (CEACAM6, VEGFC, CX3CL1, CST1, CCL5, S100A9, IGF1, NOTCH3), adhesion (FN1, CEACAM1) and inflammation (TRAF2, NFκB2 and RelB) were altered in A549L6 cells. We suggest that migration, adhesion and inflammation related genes contribute to spine metastatic capacity.

Inhibition of non-small cell lung cancer (NSCLC) growth by a novel small molecular inhibitor of EGFR.

The epidermal growth factor receptor (EGFR) is a therapeutic target (oncotarget) in NSCLC. Using in vitro EGFR kinase activity system, we identified a novel small molecule, WB-308, as an inhibitor of EGFR. WB-308 decreased NSCLC cell proliferation and colony formation, by causing G2/M arrest and apoptosis. Furthermore, WB-308 inhibited the engraft tumor growths in two animal models in vivo (lung orthotopic transplantation model and patient-derived engraft mouse model). WB-308 impaired the phosphorylation of EGFR, AKT, and ERK1/2 protein. WB-308 was less cytotoxic than Gefitinib. Our study suggests that WB-308 is a novel EGFR-TKI and may be considered to substitute for Gefitinib in clinical therapy for NSCLC.

Oridonin inhibits tumor growth and metastasis through anti-angiogenesis by blocking the Notch signaling.

While significant progress has been made in understanding the anti-inflammatory and anti-proliferative effects of the natural diterpenoid component Oridonin on tumor cells, little is known about its effect on tumor angiogenesis or metastasis and on the underlying molecular mechanisms. In this study, Oridonin significantly suppressed human umbilical vascular endothelial cells (HUVECs) proliferation, migration, and apillary-like structure formation in vitro. Using aortic ring assay and mouse corneal angiogenesis model, we found that Oridonin inhibited angiogenesis ex vivo and in vivo. In our animal experiments, Oridonin impeded tumor growth and metastasis. Immunohistochemistry analysis further revealed that the expression of CD31 and vWF protein in xenografts was remarkably decreased by the Oridonin. Furthermore, Oridonin reinforced endothelial cell-cell junction and impaired breast cancer cell transendothelial migration. Mechanistically, Oridonin not only down-regulated Jagged2 expression and Notch1 activity but also decreased the expression of their target genes. In conclusion, our results demonstrated an original role of Oridonin in inhibiting tumor angiogenesis and propose a mechanism. This study also provides new evidence supporting the central role of Notch in tumor angiogenesis and suggests that Oridonin could be a potential drug candidate for angiogenesis related diseases.

Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel.

Current technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

The Three Catecholics Benserazide, Catechol and Pyrogallol are GPR35 Agonists.

Nearly 1% of all clinically used drugs are catecholics, a family of catechol-containing compounds. Using label-free dynamic mass redistribution and Tango ß-arrestin translocation assays, we show that several catecholics, including benserazide, catechol, 3-methoxycatechol, pyrogallol, (+)-taxifolin and fenoldopam, display agonistic activity against GPR35.

Label-free cell phenotypic assessment of the biased agonism and efficacy of agonists at the endogenous muscarinic M3 receptors.

INTRODUCTION: Efficacy describes the property of a ligand that enables the receptor to change its behavior towards the host cell, while biased agonism defines the ability of a ligand to differentially activate some of the vectorial pathways over others mediated through the receptor. However, little is known about the molecular basis defining the efficacy of ligands at G protein-coupled receptors. Here we characterize the biased agonism and cell phenotypic efficacy of seven agonists at the endogenous muscarinic M3 receptors in six different cell lines including HT-29, PC-3, HeLa, SF268, CCRF-CEM and HCT-15 cells. METHODS: Quantitative real-time PCR and multiple label-free whole cell dynamic mass redistribution (DMR) assays were used to determine the functional muscarinic receptors in each cell line. DMR pathway deconvolution assay was used to determine the pathway biased activity of the muscarinic agonists. Operational agonism model was used to quantify the pathway bias, while macro-kinetic data reported in literature was used to analyze the biochemical mechanism of action of these agonists. RESULTS: Quantitative real-time PCR and ligand pharmacology studies showed that all the native cell lines endogenously express functional M3 receptors. Furthermore, different agonists triggered distinct DMR signals in a specific cell line as well as in different cell lines. DMR pathway deconvolution using known G protein modulators revealed that the M3 receptor in all the six cell lines signals through multiple G protein-mediated pathways, and certain agonists display biased agonism in a cell line-dependent manner. The whole cell efficacy and potency of these agonists were found to be sensitive to the assay time as well as the cell background. Correlation analysis suggested that the whole cell efficacy of agonists is correlated well with their macro-dissociation rate constants. DISCUSSION: This study implicates that the endogenous M3 receptors are coupled to multiple pathways, and the muscarinic agonists can display distinct biased agonism and whole cell phenotypic efficacy.

A label-free optical biosensor with microfluidics identifies an intracellular signalling wave mediated through the ß(2)-adrenergic receptor.

The canonical model of G protein-coupled receptor (GPCR) signalling states that it is solely initiated at the cell surface. In recent years, a handful of evidence has started emerging from high-resolution molecular assays that the internalized receptors can mediate the third wave of signalling, besides G protein- and ß-arrestin-mediated signalling both initiating at the cell surface. However, little is known about the functional consequences of distinct waves of GPCR signalling, in particular, at the whole cell system level. We here report the development of label-free biosensor antagonist reverse assays and their use to differentiate the signalling waves of an endogenous ß2-adrenergic receptor (ß2-AR) in A431 cells. Results showed that the persistent agonist treatment activated the ß2-ARs, leading to a long-term sustained dynamic mass redistribution (DMR) signal, a whole cell phenotypic response. Under the persistent treatment scheme in microplates, a panel of known ß-blockers all dose-dependently and completely reversed the DMR signal of epinephrine at a relatively low dose (10 nM), except for sotalol which partially reversed the DMR. Under the perfusion conditions with microfluidics, the subsequent perfusion with sotalol only reversed the DMR induced by epinephrine or isoproterenol at 10 nM, but not at 10 µM. Furthermore, the degree of the DMR reversion by sotalol was found to be in an opposite relation with the duration of the initial agonist treatment. Together, these results suggest that the hydrophilic antagonist sotalol is constrained outside the cells throughout the assays, and the early signalling wave initiated at the cell surface dominates the DMR induced by epinephrine or isoproterenol at relatively low doses, while a secondary and late signalling wave is initiated once the receptors are internalized and contributes partially to the long-term sustainability of the DMR of epinephrine or isoproterenol at high doses.

Probing biochemical mechanisms of action of muscarinic M3 receptor antagonists with label-free whole cell assays.

Binding kinetics of drugs is increasingly recognized to be important for their in vivo efficacy and safety profiles. However, little is known about the effect of drug binding kinetics on receptor signaling in native cells. Here we used label-free whole cell dynamic mass redistribution (DMR) assays under persistent and duration-controlled stimulation conditions to investigate the influence of the binding kinetics of four antagonists on the signaling of endogenous muscarinic M3 receptor in native HT-29 cells. Results showed that DMR assays under different conditions differentiated the biochemical mechanisms of action of distinct M3 antagonists. When co-stimulated with acetylcholine, tiotropium, a relatively slow binding antagonist, was found to selectively block the late signaling of the receptor, suggesting that acetylcholine attains its binding equilibrium faster than tiotropium does, thereby still being able to initiate its rapid response until the antagonist draws up and fully blocks the signaling. Furthermore, DMR assays under microfluidics allowed estimation of the residence times of these antagonists acting at the receptor in native cells, which were found to be the determining factor for the blockage efficiency of M3 receptor signaling under duration-controlled conditions. This study demonstrates that DMR assays can be used to elucidate the functional consequence of kinetics-driven antagonist occupancy in native cells.

Cucurbitacin E inhibits breast tumor metastasis by suppressing cell migration and invasion.

Tumor metastasis is the main cause of cancer-related deaths of patients. Breast cancer is highly malignant with considerable metastatic potential, which urges the necessity for developing novel potential drug candidate to prevent tumor metastasis. Here, we report our finding with Cucurbitacin E (CuE, α-elaterin), a tetracyclic triterpenes compound isolated from Cucurbitaceae. The potency of CuE on breast cancer metastasis inhibition was assessed in vivo and in vitro. In our animal experiments, intraperitoneal administrations of CuE significantly inhibited breast tumor metastasis to the lung without affecting apoptosis or proliferation of inoculated 4T1 and MDA-MB-231 breast cancer cells. Treatment of metastatic breast tumor cells with CuE markedly blocked tumor cell migration and invasion in vitro. Subsequent studies showed that CuE impaired Arp2/3-dependent actin polymerization and suppressed Src/FAK/Rac1/MMP involved pathway. Overall, our data demonstrate that CuE blocks breast cancer metastasis by suppressing tumor cell migration and invasion. We provide first evidence of a novel role for CuE as a potential candidate for treating breast cancer metastasis.

Thieno[3,2-b]thiophene-2-carboxylic acid derivatives as GPR35 agonists.

The optimization of a series of thieno[3,2-b]thiophene-2-carboxylic acid derivatives for agonist activity against the GPR35 is reported. Compounds were optimized to achieve ß-arrestin-biased agonism for developing probe molecules that may be useful for elucidating the biology and physiology of GPR35. Compound 13 was identified to the most potent GPR35 agonist, and compounds 30 and 36 exhibited the highest efficacy to cause ß-arrestin translocation.