SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A

SET is known as a potent PP2A inhibitor, however, its oncogenic role including its tumorigenic potential and involvement in the development of chemoresistance in non-small cell lung cancer (NSCLC) has not yet been fully discussed. In present study, we investigated the oncogenic role of SET by SET-knockdown and showed that SET silencing impaired cell growth rate, colony formation and tumor sphere formation in A549 cells. Notably, silencing SET enhanced the pro-apoptotic effects of paclitaxel, while ectopic expression of SET diminished the sensitivity of NSCLC cells to paclitaxel. Since the SET protein was shown to affect chemosensitivity, we next examined whether combining a novel SET antagonist, EMQA, sensitized NSCLC cells to paclitaxel. Both the in vitro and in vivo experiments suggested that EMQA and paclitaxel combination treatment was synergistic. Importantly, we found that downregulating p-Akt by inhibiting SET-mediated protein phosphatase 2A (PP2A) inactivation determined the pro-apoptotic effects of EMQA and paclitaxel combination treatment. To dissect the critical site for EMQA functioning, we generated several truncated SET proteins. By analysis of the effects of EMQA on the binding affinities of different truncated SET proteins to PP2A-catalytic subunits, we revealed that the 227–277 amino-acid sequence is critical for EMQA-induced SET inhibition. Our findings demonstrate the critical role of SET in NSCLC, particularly in the development of chemoresistance. The synergistic effects of paclitaxel and the SET antagonist shown in current study encourage further validation of the clinical potential of this combination.     

Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathway

Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1-4). In this study, we investigated the role of EP receptors in PGE2-induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM-10 microM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2-induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2'5'-dideoxyadenosine, at concentrations that inhibited PGE2-induced cAMP, significantly blocked PGE2-induced VEGF secretion in PC-3 cells. We conclude that PGE2-induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways.     

Epstein‐Barr virus‐encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan‐2 and synaptotagmin‐like‐4 in nasopharyngeal carcinoma cells

Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell‐to‐cell communication. The Epstein‐Barr virus (EBV)‐encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan‐2 (SDC2) and synaptotagmin‐like‐4 (SYTL4) through nuclear factor (NF)‐κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF‐κB signaling to promote EV secretion, and further enhance cancer progression of NPC.     

Targeting cancer stem cells via integrin β4

Integrins mediate cell-cell interactions and communication with the extracellular matrix (ECM). These transmembrane protein receptors allow binding between a cell and its surroundings, initiating a breadth of intracellular signaling resulting in proliferation, differentiation, survival, or migration. Such responses have made integrins an attractive target for cancer therapy. Self-renewing and highly tumorigenic cancer stem cells (CSCs) are most resistant to traditional radiation treatment and chemotherapy, and therefore may contribute directly to the metastasis and relapse of the disease. In both the 4T1 mouse metastatic mammary tumor model and SCC7 head and neck squamous cell carcinoma model, integrin β4 (ITGB4) was expressed on ALDH^high 4T1 and SCC7 CSCs. Using two immunological approaches, we targeted ITGB4 through 1) ITGB4 protein-pulsed dendritic cell (ITGB4-DC) vaccination or 2) via anti-CD3/anit-ITGB4 bispecific antibody (ITGB4 BiAb)-armed T cell adoptive transfer. These two therapies reduced ITGB4-expressing CSCs and inhibited local tumor growth and lung metastasis through ITGB4 specific cellular and humoral immune responses. Additionally, the combination of anti-PD-L1 immunotherapy with our two ITGB4-targeted approaches significantly improved treatment efficacy. We also found increased concentrations of serum IFN-γ and IL-6 in the 4T1 and SCC7 models which may help define future directions of this ITGB4-targeted study. Together, these results emphasize ITGB4 as a practical CSC immunological target with possible therapeutic benefits across tumor types with high ITGB4 expression.     

口腔鳞状细胞癌组织前列腺素E2受体EP3的表达及临床意义

目的 前列腺素E2 (prostaglandin E2,PGE2)可以通过促进癌细胞的增殖、抑制癌细胞的凋亡、促进癌细胞的转移等方式促进口腔鳞癌的发生发展,但关于其具体作用机制罕见报道.因此,本研究旨在检测口腔鳞状细胞癌组织和相应癌旁组织中花生四烯酸(arachidonic acid,AA)及前列腺素E2受体3亚型(E-prostanoid 3,EP3)的表达.方法 选取南方医科大学南方医院2013-05-01-2015-05-01行手术治疗的口腔鳞癌患者40例,将口腔鳞状细胞癌组织作为观察组(40例),相应癌旁组织作为对照组(40例).ELISA方法检测AA的表达,Real-time PCR方法检测EP3受体mRNA的表达,免疫组织化学染色方法检测EP3受体蛋白的表达.结果 与癌旁组织相比,AA在口腔鳞状细胞癌组织中的表达明显升高.40例口腔鳞癌组织中AA的平均质量分数为(477.4±39.84) pg/g,40例癌旁组织中AA的平均质量分数为(38.4±4.92) pg/g,差异有统计学意义,t=3.146,P=0.001.与癌旁组织相比,鳞癌组织中EP3受体mRNA的表达明显降低.40例鳞癌组织EP3受体mRNA的相对平均表达量为0.43±0.15,差异有统计学意义,t=2.365,P=0.011.与癌旁组织相比,鳞癌组织中EP3受体蛋白的表达明显降低,20例癌旁组织呈阳性表达,而18例鳞癌组织呈弱阳性表达,差异有统计学意义,T=-114,P=0.016.结论 口腔鳞状细胞癌组织中,AA表达量的显著上调,EP3受体表达量的显著下调,可能在口腔鳞状细胞癌的发生发展过程中发挥了重要作用.     

Exosome‐mediated regulation of tumor immunology

Exosomes are representative extracellular vesicles (EV) derived from multivesicular endosomes (MVE) and have been described as new particles in the communication of neighborhood and/or distant cells by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and nucleotides including micro (mi) RNAs. Exosomes from immune cells and tumor cells act in part as a regulator in tumor immunology. CD8⁺ T cells that show potent cytotoxic activity against tumor cells reside as an inactive naïve form in the T‐cell zone of secondary lymphoid organs. Once receiving tumor‐specific antigenic stimulation by dendritic cells (DC), CD8⁺ T cells are activated and differentiated into effector CTL. Subsequently, CTL circulate systemically, infiltrate into tumor lesions through the stromal neovasculature where mesenchymal stromal cells, for example, mesenchymal stem cells (MSC) and cancer‐associated fibroblasts (CAF), abundantly exist, destroy mesenchymal tumor stroma in an exosome‐mediated way, go into tumor parenchyma, and attack tumor cells by specific interaction. DC‐derived and regulatory T (Treg) cell‐derived exosomes, respectively, promote and inhibit CTL generation in this setting. In this review, we describe the roles of exosomes from immune cells and tumor cells on the regulation of tumor progression.     

E‐cadherin‐Fc chimera protein matrix enhances cancer stem‐like properties and induces mesenchymal features in colon cancer cells

Cancer stem cells (CSC) are a subpopulation of tumor cells with properties of high tumorigenicity and drug resistance, which lead to recurrence and poor prognosis. Although a better understanding of CSC is essential for developing cancer therapies, scarcity of the CSC population has hindered such analyses. The aim of the present study was to elucidate whether the E‐cadherin‐Fc chimera protein (E‐cad‐Fc) enhances cancer stem‐like properties because studies show that soluble E‐cadherin stimulates human epithelial growth factor receptor (EGFR) and downstream signaling pathways that are reported to play a crucial role in CSC. For this purpose, we used ornithine decarboxylase (ODC)‐degron–transduced (Degron(+)) KM12SM cells as a CSC model that retains relatively low CSC properties. Compared to cultures without E‐cad‐Fc treatment, we found that E‐cad‐Fc treatment further suppressed proteasome activity and largely enhanced cancer stem‐like properties of ODC‐degron–transduced KM12SM cells. These results include increased expression of stem cell markers Lgr5, Bmi‐1, SOX9, CD44, and CD44v9, aldehyde dehydrogenase (ALDH), and enhancement of robust spheroid formation, and chemoresistance to 5‐fluorouracil (5‐FU) and oxaliplatin (L‐OHP). These effects could be attributed to activation of the EGFR pathway as identified by extensive phosphorylation of EGFR, ERK, PI3K, AKT, and mTOR. In SW480 cells, E‐cad‐Fc matrix induced some CSC markers such as CD44v9 and ALDH. We also found that E‐cad‐Fc matrix showed high efficiency of inducing mesenchymal changes in colon cancer cells. Our data suggest that the E‐cad‐Fc matrix may enhance CSC properties such as enhancement of chemoresistance and sphere formation.     

PGE2-mediated upregulation of iNOS in murine breast cancer cells through the activation of EP4 receptors

We report here that endogenous prostaglandin E(2) (PGE(2)) resulting from cyclooxygenase (COX)-2 expression in a highly metastatic murine breast cancer cell line C3L5 upregulates IFN-gamma + LPS-induced nitric oxide (NO) synthase (iNOS) expression and NO production. This action of PGE(2) is mediated through the EP(4) receptor in a cAMP-dependent manner. Both nonselective and selective COX-2 inhibitors suppressed IFN-gamma + LPS-induced NO production, which was largely restored by exogenous PGE(2) or EP(4) receptor agonist PGE(1) alcohol. EP(4) antagonist AH-23848B inhibited NO production with a concomitant downregulation of iNOS mRNA in IFN-gamma + LPS-stimulated cells. cAMP dependence of NO production by cells under inducible conditions was demonstrated by the use of known modulators of intracellular cAMP. Since both COX-2 and iNOS are implicated in breast cancer progression, our findings of EP(4) receptor-mediated upregulation of iNOS in COX-2-expressing breast cancer cells suggest that blocking COX-2 and/or EP(4) may provide a simple therapeutic modality in this tumor model.     

Prostaglandin E2 receptor EP4 as the common target on cancer cells and macrophages to abolish angiogenesis,lymphangiogenesis, metastasis,and stem‐like cell functions

We previously established that COX‐2 overexpression promotes breast cancer progression and metastasis. As long‐term use of COX‐2 inhibitors (COX‐2i) can promote thrombo‐embolic events, we tested an alternative target, prostaglandin E2 receptor EP4 subtype (EP4), downstream of COX‐2. Here we used the highly metastatic syngeneic murine C3L5 breast cancer model to test the role of EP4‐expressing macrophages in vascular endothelial growth factor (VEGF)‐C/D production, angiogenesis, and lymphangiogenesis in situ, the role of EP4 in stem‐like cell (SLC) functions of tumor cells, and therapeutic effects of an EP4 antagonist RQ‐15986 (EP4A). C3L5 cells expressed all EP receptors, produced VEGF‐C/D, and showed high clonogenic tumorsphere forming ability in vitro, functions inhibited with COX‐2i or EP4A. Treating murine macrophage RAW 264.7 cell line with COX‐2i celecoxib and EP4A significantly reduced VEGF‐A/C/D production in vitro, measured with quantitative PCR and Western blots. Orthotopic implants of C3L5 cells in C3H/HeJ mice showed rapid tumor growth, angiogenesis, lymphangiogenesis (CD31/LYVE‐1 and CD31/PROX1 immunostaining), and metastasis to lymph nodes and lungs. Tumors revealed high incidence of EP4‐expressing, VEGF‐C/D producing macrophages identified with dual immunostaining of F4/80 and EP4 or VEGF‐C/D. Celecoxib or EP4A therapy at non‐toxic doses abrogated tumor growth, lymphangiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF‐A/C/D and phosphorylated Akt/ERK proteins, VEGF‐C/D positive macrophage infiltration, and proliferative/apoptotic cell ratios. Knocking down COX‐2 or EP4 in C3L5 cells or treating cells in vitro with celecoxib or EP4A and treating tumor‐bearing mice in vivo with the same drug reduced SLC properties of tumor cells including preferential co‐expression of COX‐2 and SLC markers ALDH1A, CD44, OCT‐3/4, β‐catenin, and SOX‐2. Thus, EP4 is an excellent therapeutic target to block stem‐like properties, angiogenesis, and lymphangiogenesis induced by VEGF‐A/C/D secreted by cancer cells and tumor infiltrating macrophages.     

Necrosis in DU145 prostate cancer spheroids induces COX‐2/mPGES‐1‐derived PGE2 to promote tumor growth and to inhibit T cell activation

Cyclooxygenase (COX)‐2‐derived prostaglandin E2 (PGE₂) supports the growth of a spectrum of cancers. The potential benefit of COX‐2‐inhibiting non‐steroidal anti‐inflammatory drugs (NSAIDs) for cancer treatment is however limited by their well‐known cardiovascular side‐effects. Therefore, targeting microsomal PGE synthase 1 (mPGES‐1), the downstream enzyme in the COX‐2‐dependent pathway of PGE₂ production might be attractive, although conflicting data regarding a potential tumor‐supporting function of mPGES‐1 were reported. We determined the impact of mPGES‐1 in human DU145 prostate cancer cell growth. Surprisingly, knockdown of mPGES‐1 did not alter growth of DU145 monolayer cells, but efficiently inhibited the growth of DU145 multicellular tumor spheroids (MCTS). Opposed to MCTS, monolayer cells did not secrete PGE₂ due to a lack of COX‐2 expression, which was induced during spheroid formation. Pharmacological inhibition of COX‐2 and mPGES‐1 supported the crucial role of PGE₂ for growth of MCTS. The functionality of spheroid‐derived PGE₂ was demonstrated by its ability to inhibit cytotoxic T cell activation. When investigating mechanisms of spheroid‐induced COX‐2 induction, we observed that among microenvironmental factors neither glucose deprivation, hypoxia nor tumor cell apoptosis enhanced COX‐2 expression. Interestingly, interfering with apoptosis in spheroids triggered a shift towards necrosis, thus augmenting COX‐2 expression. We went on to demonstrate that necrotic cells induced COX‐2 mRNA expression and PGE₂ secretion from live tumor cells. In conclusion, necrosis‐dependent COX‐2 upregulation in MCTS promoted PGE₂‐dependent tumor growth and inhibited activated cytotoxic T cells. Hence, blocking mPGES‐1 as a therapeutic option may be considered for COX‐2/mPGES‐1‐positive solid cancers.     

PD‐1 blockade enhances the antitumor efficacy of GM‐CSF surface‐modified bladder cancer stem cells vaccine

Eliminating cancer stem cells (CSCs) is a key issue in eradicating tumor. The streptavidin–granulocyte‐macrophage‐colony stimulating factor (SA–GM‐CSF) surface‐modified bladder CSCs vaccine previously developed using our protein–anchor technology could effectively induce specific immune response for eliminating CSCs. However, program death receptor‐1 (PD‐1)/program death ligand 1 (PD‐L1) signaling in tumor microenvironment results in tumor‐adaptive immune resistance. Although the CSCs vaccine could increase the number of CD8⁺T cells, a part of these CD8⁺T cells expressed PD‐1. Moreover, the CSCs vaccine upregulated the PD‐L1 expression of tumor cells, resulting in immune resistance. Adding PD‐1 blockade to the CSCs vaccine therapy increased the population of CD4⁺, CD8⁺ and CD8⁺IFN‐γ⁺ but not CD4⁺ Foxp3⁺T cells and induced the highest production of IFN‐γ. PD‐1 blockade could effectively enhance the functions of tumor‐specific T lymphocytes generated by the CSCs vaccine. This combination therapy improved the cure rate among mice and effectively protected the mice against a second CSCs cell challenge, but not a RM‐1 cell challenge. These results indicate that PD‐1 blockade combined with the GM‐CSF‐modified CSCs vaccine effectively induced a strong and specific antitumor immune response against bladder cancer.     

Cell‐based selection provides novel molecular probes for cancer stem cells

Cancer stem cells (CSC) represent a malignant subpopulation of cells in hierarchically organized tumors. They constitute a subpopulation of malignant cells within a tumor mass and possess the ability to self‐renew giving rise to heterogeneous tumor cell populations with a complex set of differentiated tumor cells. CSC may be the cause of metastasis and therapeutic refractory disease. Because few markers exist to identify and isolate pure CSC, we used cell‐based Systematic Evolution of Ligands by EXponential enrichment (cell‐SELEX) to create DNA aptamers that can identify novel molecular targets on the surfaces of live CSC. Out of 22 putative DNA sequences, 3 bound to ～90% and 5 bound to ～15% of DU145 prostate cancer cells. The 15% of cells that were positive for the second panel of aptamers expressed high levels of E‐cadherin and CD44, had high aldehyde dehydrogenase 1 activity, grew as spheroids under nonadherent culture conditions, and initiated tumors in immune‐compromised mice. The discovery of the molecular targets of these aptamers could reveal novel CSC biomarkers.     

Regulation of nonsmall‐cell lung cancer stem cell like cells by neurotransmitters and opioid peptides

Nonsmall‐cell lung cancer (NSCLC) is the leading type of lung cancer and has a poor prognosis. We have shown that chronic stress promoted NSCLC xenografts in mice via stress neurotransmitter‐activated cAMP signaling downstream of beta‐adrenergic receptors and incidental beta‐blocker therapy was reported to improve clinical outcomes in NSCLC patients. These findings suggest that psychological stress promotes NSCLC whereas pharmacologically or psychologically induced decreases in cAMP may inhibit NSCLC. Cancer stem cells are thought to drive the development, progression and resistance to therapy of NSCLC. However, their potential regulation by stress neurotransmitters has not been investigated. In the current study, epinephrine increased the number of cancer stem cell like cells (CSCs) from three NSCLC cell lines in spheroid formation assays while enhancing intracellular cAMP and the stem cell markers sonic hedgehog (SHH), aldehyde dehydrogenase‐1 (ALDH‐1) and Gli1, effects reversed by GABA or dynorphin B via Gα_i‐mediated inhibition of cAMP formation. The growth of NSCLC xenografts in a mouse model of stress reduction was significantly reduced as compared with mice maintained under standard conditions. Stress reduction reduced serum levels of corticosterone, norepinephrine and epinephrine while the inhibitory neurotransmitter γ‐aminobutyric acid (GABA) and opioid peptides increased. Stress reduction significantly reduced cAMP, VEGF, p‐ERK, p‐AKT, p‐CREB, p‐SRc, SHH, ALDH‐1 and Gli1 in xenograft tissues whereas cleaved caspase‐3 and p53 were induced. We conclude that stress neurotransmitters activate CSCs in NSCLC via multiple cAMP‐mediated pathways and that pharmacologically or psychologically induced decreases in cAMP signaling may improve clinical outcomes in NSCLC patients.