Hua-Jung Li’s research while affiliated with National Chung Hsing University and other places

Metastasis is the leading cause of mortality in breast cancer, with lung metastasis being particularly detrimental. Identification of the processes determining metastatic organotropism could enable the development of approaches to prevent and treat breast cancer metastasis. Here, we found that lung-tropic and non-lung-tropic breast cancer cells differ in their response to sialic acids, affecting the sialylation of surface proteins. Lung-tropic cells showed higher levels of ST6GAL1, while non-lung-tropic cells had more ST3GAL1. ST6GAL1-mediated α-2,6-sialylation, unlike ST3GAL1-mediated α-2,3-sialylation, increased lung metastasis by promoting cancer cell migration through pulmonary endothelial layers and reducing junction protein levels. α-2,6-sialylated PECAM-1 on breast cancer cells facilitated extravasation through the pulmonary endothelium, a critical step in lung metastasis. Knockdown of ST6GAL1 or PECAM-1 significantly reduced lung metastasis. Human pulmonary endothelium displayed high PECAM-1 levels. Through transhomophilic interaction with pulmonary PECAM-1, α-2,6-sialylated PECAM-1 on ST6GAL1-positive cancer cells increased pulmonary extravasation in a diapedesis-like, cell-autonomous manner. Additionally, lung-tropic cells and their exosomes increased the permeability of pulmonary endothelial cells, promoting metastasis in a non-cell-autonomous manner. Analysis of human breast cancer samples showed a correlation between elevated ST6GAL1/PECAM-1 expression and lung metastasis. These results suggest that targeting ST6GAL1-mediated α-2,6-sialylation could be a potential therapeutic strategy to prevent lung metastasis in breast cancer patients.

Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.

Mesenchymal stem cells (MSCs) have been used in clinical studies to treat neurological diseases and damage. However, implanted MSCs do not achieve their regenerative effects by differentiating into and replacing neural cells. Instead, MSC secretome components mediate the regenerative effects of MSCs. MSC-derived extracellular vesicles (EVs)/exosomes carry cargo responsible for rescuing brain damage. We previously showed that EP4 antagonist-induced MSC EVs/exosomes have enhanced regenerative potential to rescue hippocampal damage, compared with EVs/exosomes from untreated MSCs. Here we show that EP4 antagonist-induced MSC EVs/exosomes promote neurosphere formation in vitro and increase neurogenesis and neuritogenesis in damaged hippocampi; basal MSC EVs/exosomes do not contribute to these regenerative effects. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNP) levels in EP4 antagonist-induced MSC EVs/exosomes are 20-fold higher than CNP levels in basal MSC EVs/exosomes. Decreasing elevated exosomal CNP levels in EP4 antagonist-induced MSC EVs/exosomes reduced the efficacy of these EVs/exosomes in promoting β3-tubulin polymerization and in converting toxic 2′,3′-cAMP into neuroprotective adenosine. CNP-depleted EP4 antagonist-induced MSC EVs/exosomes lost the ability to promote neurogenesis and neuritogenesis in damaged hippocampi. Systemic administration of EV/exosomes from EP4-antagonist derived MSC EVs/exosomes repaired cognition, learning, and memory deficiencies in mice caused by hippocampal damage. In contrast, CNP-depleted EP4 antagonist-induced MSC EVs/exosomes failed to repair this damage. Exosomal CNP contributes to the ability of EP4 antagonist-elicited MSC EVs/exosomes to promote neurogenesis and neuritogenesis in damaged hippocampi and recovery of cognition, memory, and learning. This experimental approach should be generally applicable to identifying the role of EV/exosomal components in eliciting a variety of biological responses. Significance statement Mesenchymal stem cells (MSCs) have therapeutic effects for neurological diseases and damage; however, their therapeutic effects are mediated by components of the MSC secretome. Compared with basal MSC EVs/exosomes, EP4 antagonist-induced MSC EVs/exosomes have superior regenerative ability to rescue damaged brain functions. Elevated CNP levels are required for the enhanced ability of EP4-induced MSC EVs/exosomes to promote β3-tubulin polymerization, decrease toxic 2′,3′-cAMP, produce neuroprotective adenosine, induce neurogenesis, and elicit neuritogenesis in damaged hippocampi. At a functional level, elevated EV/exosome CNP levels are required to rescue cognition and learning deficiencies caused by this damage. These data suggest that CNP modulation is a potential target for treating brain damage and neural degeneration diseases. Moreover, these results suggest a generalized approach to identifying causal roles for EV/exosome cargo components in a variety of regenerative applications.

Adult brains have limited regenerative capacity. Consequently, both brain damage and neurodegenerative diseases often cause functional impairment for patients. Mesenchymal stem cells (MSCs), one type of adult stem cells, can be isolated from various adult tissues. MSCs have been used in clinical trials to treat human diseases and the therapeutic potentials of the MSC‐derived secretome and extracellular vesicles (EVs) have been under investigation. We found that blocking the prostaglandin E2/prostaglandin E2 receptor 4 (PGE2/EP4) signaling pathway in MSCs with EP4 antagonists increased EV release and promoted the sorting of specific proteins, including anti‐inflammatory cytokines and factors that modify astrocyte function, blood–brain barrier integrity, and microglial migration into the damaged hippocampus, into the EVs. Systemic administration of EP4 antagonist‐elicited MSC EVs repaired deficiencies of cognition, learning and memory, inhibited reactive astrogliosis, attenuated extensive inflammation, reduced microglial infiltration into the damaged hippocampus, and increased blood–brain barrier integrity when administered to mice following hippocampal damage. Stem Cells Translational Medicine 2019 Brain damage is associated with reactive astrogliosis, extensive inflammation, broken blood–brain barrier, and microglial infiltration into the damaged area. Prostaglandin E2 receptor 4 antagonist‐elicited mesenchymal stem cell extracellular vesicles, which are enriched in IL‐2, IL‐10, vascular endothelial growth factor‐a, and BDNF, suppress reactive astrogliosis and inflammation and allow the recovery of CNS neurons, blood–brain barrier, and behavioral function.

Cells expressing mesenchymal/basal phenotypes in tumors have been associated with stem cell properties. Cancer stem cells (CSCs) are often resistant to conventional chemotherapy. We explored overcoming mesenchymal CSC resistance to chemotherapeutic agents. Our goal was to reduce CSC numbers in vivo, in conjunction with chemotherapy, to reduce tumor burden. Analysis of clinical samples demonstrated that COX‐2/PGE2/EP4 signaling is elevated in basal‐like and chemoresistant breast carcinoma and is correlated with survival and relapse of breast cancer. EP4 antagonism elicts a striking shift of breast cancer cells from a mesenchymal/CSC state to a more epithelial non‐CSC state. The transition was mediated by EP4 antagonist‐induced extracellular vesicles [(EVs)/exosomes] which removed CSC markers, mesenchymal markers, integrins, and drug efflux transporters from the CSCs. In addition, EP4 antagonism‐induced CSC EVs/exosomes can convert tumor epithelial/non‐CSCs to mesenchymal/CSCs able to give rise to tumors and to promote tumor cell dissemination. Because of its ability to induce a CSC‐to‐non‐CSC transition, EP4 antagonist treatment in vivo reduced the numbers of CSCs within tumors and increased tumor chemosensitivity. EP4 antagonist treatment enhances tumor response to chemotherapy by reducing the numbers of chemotherapy‐resistant CSCs available to repopulate the tumor. EP4 antagonism can collaborate with conventional chemotherapy to reduce tumor burden. This article is protected by copyright. All rights reserved.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

Prostaglandin E2 (PGE2)-initiated signaling contributes to stem cell homeostasis and regeneration. However, it is unclear how PGE2 signaling controls cell stemness. This study identifies a previously unknown mechanism by which PGE2/prostaglandin E receptor 4 (EP4) signaling regulates multiple signaling pathways (e.g., PI3K/Akt signaling, TGFβ signaling, Wnt signaling, EGFR signaling) which maintain the basal mammary stem cell phenotype. A shift of basal mammary epithelial stem cells (MaSCs) from a mesenchymal/stem cell state to a non-basal-MaSC state occurs in response to prostaglandin E receptor 4 (EP4) antagonism. EP4 antagonists elicit release of signaling components, by controlling their trafficking into extracellular vesicles/exosomes in a lipid raft/caveolae-dependent manner. Consequently, EP4 antagonism indirectly inactivates, through induced extracellular vesicle/exosome release, pathways required for mammary epithelial stem cell homeostasis, e.g. canonical/noncanonical Wnt, TGFβ and PI3K/Akt pathways. EP4 antagonism causes signaling receptors and signaling components to shift from non-lipid raft fractions to lipid raft fractions, and to then be released in EP4 antagonist-induced extracellular vesicles/exosomes, resulting in the loss of the stem cell state by mammary epithelial stem cells. In contrast, luminal mammary epithelial cells can acquire basal stem cell properties following ingestion of EP4 antagonist-induced stem cell extracellular vesicles/exosomes, and can then form mammary glands. These findings demonstrate that PGE2/EP4 signaling controls homeostasis of mammary epithelial stem cells through regulating extracellular vesicle/exosome release. Reprogramming of mammary epithelial cells can result from EP4-mediated stem cell property transfer by extracellular vesicles/exosomes containing caveolae-associated proteins, between mammary basal and luminal epithelial cells. This article is protected by copyright. All rights reserved.

Unlabelled: Mesenchymal cells of the tumor-associated stroma are critical determinants of carcinoma cell behavior. We focus here on interactions of carcinoma cells with mesenchymal stem cells (MSC), which are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells. We find that carcinoma cell-derived interleukin-1 (IL-1) induces prostaglandin E(2) (PGE(2)) secretion by MSCs. The resulting PGE(2) operates in an autocrine manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines by the MSCs. The PGE(2) and cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of β-catenin signaling and formation of cancer stem cells. These observations indicate that MSCs and derived cell types create a cancer stem cell niche to enable tumor progression via release of PGE(2) and cytokines. Significance: Although PGE2 has been implicated time and again in fostering tumorigenesis, its effects on carcinoma cells that contribute specifically to tumor formation are poorly understood. Here we show that tumor cells are able to elicit a strong induction of the COX-2/microsomal prostaglandin-E synthase-1 (mPGES-1)/PGE(2) axis in MSCs recruited to the tumor-associated stroma by releasing IL-1, which in turn elicits a mesenchymal/stem cell–like phenotype in the carcinoma cells.