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Supplementary Figure S4. The prognostic significance of miR-663 and CXCR4 in astrocytic glioma.

Supplementary tables from S1-S6.

Purpose: To identify the miRNA regulators of C–X–C motif chemokine receptor 4 (CXCR4) and the underlying mechanism as well as the therapeutic and prognostic values in human glioblastoma (GBM).Experimental Design: miRNA profile analyses and bioinformatics predictions were used to identify the mediators of CXCR4, which were confirmed by luciferase reporter assay, Western blot assay and immunohistochemistry. The effects of miR-663 on CXCR4-mediated GBM malignancy were investigated by gain-of-function experiments. Orthotopic xenografts derived from constitutive or induced miR-663–expressing GBM cells were used to determine the antitumor effects of miR-663 and CXCR4-specific antagonist AMD3100. Bivariate correlation analyses were used to examine the correlation of miR-663 and CXCR4 levels in glioma. The prognostic values of miR-663 and CXCR4 were examined in 281 cases of astrocytic glioma from our hospital and 476 cases of GBM from The Cancer Genome Atlas database using the multivariate Cox regression analysis and Kaplan–Meier analysis.Results: miR-663 negatively regulated CXCR4 expression by targeting its coding sequence in GBM and compromised the proliferative and invasive capacities of GBM cells induced by CXCR4 overexpression. Constitutive or induced miR-663 overexpression combined with CXCR4 antagonist AMD3100 suppressed orthotopic GBM growth and prolonged tumor-bearing mice survival. Clinically, miR-663 and CXCR4 were inversely correlated in GBM and composed a valuable biomarker set in predicting the outcomes of GBM patients.Conclusions: miR-663 negatively regulated CXCR4 to inhibit its oncogenic effect. Combination of miR-663 and CXCR4 can serve as a valuable prognostic biomarker set as well as molecular targets for therapeutic intervention of GBM. Clin Cancer Res; 21(17); 4004–13. ©2015 AACR.

PDF file - 455K, Supplementary Fig. S1. The prognostic values of miR-663 in GBM patients stratified by IDH1/ATRX status.

Purpose: To determine the prognostic significance of miR-663 in glioblastoma, its effect in tumor progression, and the underlying mechanism.Experimental Design: Specimens from 256 cases of patients with glioma, including 239 patients with follow-up information, were used to analyze the association between miR-663 and patients' prognosis by Kaplan–Meier and multivariate Cox regression analyses. The effects of miR-663 on glioblastoma cell proliferation and invasion were examined both in vitro and in vivo. Bioinformatics prediction and signal network analysis were applied to identify the putative targets of miR-663, which were further verified by luciferase reporter assay, rescue experiments as well as the immunohistochemistry (IHC) and Western blotting examination of downstream effectors. Quantitative reverse transcriptase PCR (qRT-PCR) and IHC were applied to investigate the clinical association between miR-663 and its target in human glioblastoma specimens.Results: miR-663 was inversely correlated with glioma grades but positively correlated with patients' survival. Furthermore, two distinct subgroups of patients with glioblastoma with different prognoses were identified on the basis of miR-663 expression in our specimens and that from The Cancer Genome Atlas (TCGA) database. Overexpression of miR-663 significantly suppressed the proliferation and invasion of glioblastoma cells in vitro and in vivo. Mechanistically, we discovered PIK3CD as a direct target of miR-663 and found that phosphorylated AKT and three key downstream effectors of PIK3CD, i.e., CCND1, MMP2, and MMP7, were downregulated by miR-663 overexpression. Moreover, PIK3CD was inversely correlated with miR-663 in glioblastoma specimens and predicted poor prognosis of patients with glioblastoma.Conclusion: miR-663 is a novel prognostic biomarker and a potential therapeutic candidate for glioblastoma. Clin Cancer Res; 20(7); 1803–13. ©2014 AACR.

PDF file - 747K, Supplementary Fig. S2. The effects of miR-663 on GBM cells.

PDF file - 1654K, Supplementary Fig. S4. The suppressive effects of miR-663 on PIK3CD and its downstream proliferation and invasion-related targets.

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Supplementary Tables S1-S2 from Endogenous Axon Guiding Chemorepulsant Semaphorin-3F Inhibits the Growth and Metastasis of Colorectal Carcinoma

Supplementary Figure legends. Supplementary Figure legends for Fig. S1-S4.

Purpose: To elucidate the role of Semaphorin-3F (SEMA3F), originally described as an axon guiding chemorepulsant implicated in nerve development, in the progression of colorectal carcinoma.Experimental Design: SEMA3F and its receptor NRP2 were examined in 72 cases of human colorectal carcinoma specimens and cell lines LoVo, SW480, and SW620 with immunohistochemistry and Western blotting. SEMA3F mRNA expression in the frozen tissue specimens and cell lines was examined with quantitative reverse transcriptase-PCR. Confocal laser scanning microscopy was used for detection of cellular localization of the proteins by immunofluorescent staining. MTT assay, flow cytometry, cell adhesion and migration, and xenografts were used to evaluate biological significance of SEMA3F.Results: SEMA3F was significantly reduced in colorectal carcinoma tissues and cell lines. Overexpression of SEMA3F resulted in reduced proliferation, adhesion to fibronectin, and migratory capability as well as reduced S-phase population and integrin αvβ3 expression of SW480 colon cancer cells. In addition, SEMA3F-overexpressing cells exhibited diminished tumorigenesis when transplanted orthotopically in nude mice and reduced liver metastases. Moreover, transfection of siRNA targeting SEMA3F in colon cancer cells increased their tumorigenicity in vivo.Conclusions: Endogenous SEMA3F acts as a suppressor of the growth and metastasis of human colorectal cancer cells. Clin Cancer Res; 17(9); 2702–11. ©2011 AACR.

PDF file - 546K, Supplementary Fig. S3. Identification of PIK3CD as a target by miR-663.

Differential expression of the ligands for NK cell inhibitory and stimulatory receptor by BCSCs or non-BCSCs.

Supplementary Tables 1-8 PDF file 178K, Supplementary Table 1. The expression of IGF-1R, �-catenin and POU5F1 in 200 cases of lung adenocarcinoma; Supplementary Table 2. Top 10 gene ontology classification of genes related to the biological process of LACSLCs; Supplementary Table 3. Top 10 gene ontology classification of genes related to the cellular components of LACSLCs; Supplementary Table 4. Top 10 gene ontology classification of genes involved in the molecular functions of LACSLCs; Supplementary Table 5. Expression profile of up-regulated genes identified by cDNA array and qRT-PCR in A549 LACSLCs; Supplementary Table 6. Expression profile of down-regulated genes identified by cDNA array and qRT-PCR in A549 LACSLCs; Supplemental Table 7. Antibodies used for immunostaining analysis; Supplemental Table 8. Sequences of the primers used for qRT-PCR

Characterization of stem-like properties of ALDH+ cells isolated from primary breast cancer specimens.

Breast cancer stem-like cells (BCSC) are crucial for metastasis but the underlying mechanisms remain elusive. Here, we report that tumor-infiltrating natural killer (NK) cells failed to limit metastasis and were not associated with improved therapeutic outcome of BCSC-rich breast cancer. Primary BCSCs were resistant to cytotoxicity mediated by autologous/allogeneic NK cells due to reduced expression of MICA and MICB, two ligands for the stimulatory NK cell receptor NKG2D. Furthermore, the downregulation of MICA/MICB in BCSCs was mediated by aberrantly expressed oncogenic miR20a, which promoted the resistance of BCSC to NK cell cytotoxicity and resultant lung metastasis. The breast cancer cell differentiation–inducing agent, all-trans retinoic acid, restored the miR20a–MICA/MICB axis and sensitized BCSC to NK cell–mediated killing, thereby reducing immune escape–associated BCSC metastasis. Together, our findings reveal a novel mechanism for immune escape of human BCSC and identify the miR20a–MICA/MICB signaling axis as a therapeutic target to limit metastatic breast cancer. Cancer Res; 74(20); 5746–57. ©2014 AACR.

Activation and cytotoxicity of NK cells by interaction with BCSCs or non-BCSCs.

Table S1. The levels of ALDH1+ cells and CD56+ cells and the clinical parameters of breast cancers. Table S2. Univariate analysis of potential prognostic factors for the ALDH1low and ALDH1high subgroups of breast cancers. Table S3. Multivariate analysis of potential prognostic factors for the ALDH1low and ALDH1high subgroups of breast cancers. Table S4. Patient characteristics of primary breast cancers for culture. Table S5. Antibodies used in the study. Table S6. Primers used for cloning coding sequence of MICA and MICB. Table S7. Primers sequences that were used in the qRT-PCR. Table S8. Small interference RNA sequences for knockdown of MICA and MICB expression. Table S9. Primers used for cloning of MICA-3’UTR and MICB-3’UTR. Table S10. Nucleotide sequences of MICA-3’UTR and MICB-3’UTR with mutant binding sites for miRNA-20a.

The effect of ATRA on the sensitivity to NK cell killing, survival, and lung metastasis of BCSCs

Cancer stem-like cells (CSLC) are crucial in tumor initiation and progression; however, the underlying mechanism for the self-renewal of cancer cells remains undefined. In the study, immunohistochemical analysis of specimens freshly excised from patients with lung adenocarcinoma showed that high expression of insulin-like growth factor I receptor (IGF-IR) in lung adenocarcinoma cells was positively correlated with the expressions of cancer stem cell markers CD133 and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). IGF-IR activation enhanced POU class 5 homeobox 1 (POU5F1) expression on human lung adenocarcinoma stem-like cells (LACSLC) through PI3K/AKT/GSK3β/β-catenin cascade. POU5F1 could form a novel complex with β-catenin and SOX2 to bind Nanog promoter for transcription to maintain self-renewal of LACSLCs, which was dependent on the functional IGF-IR. Genetic and pharmacologic inhibition of IGF-IR abrogated LACSLC capabilities for self-renewal and tumorigenicity in vitro. In an in vivo xenograft tumor model, knockdown of either IGF-IR or POU5F1 impeded tumorigenic potentials of LACSLCs. By analyzing pathologic specimens excised from 200 patients with lung adenocarcinoma, we found that colocalization of highly expressed IGF-IR with β-catenin and POU5F1 predicted poor prognosis. Taken together, we show that IGF-IR—mediated POU5F1 expression to form a complex with β-catenin and SOX2 is crucial for the self-renewal and oncogenic potentials of LACSLCs, and the integrative clinical detection of the expressions of IGF-IR, β-catenin, and POU5F1 is indicatory for predicting prognosis in the patients of lung adenocarcinoma. Cancer Res; 73(10); 3181–9. ©2013 AACR.

Knockdown of MICA or MICB in non-BCSCs.

Supplementary Table 9 PDF file 167K, Supplementary Table 9. Clinicopathological features and IHC staining index of ALDH1A1, IGF-1R, �-catenin and POU5F1 in 200 patients with LAC

Supplementary Figures 1-8 PDF file 1850K, Supplementary Figure 1. LACSLCs derived from A549 and H358 cell lines; Supplementary Figure 2. Sphere LACSLCs derived from A549 cell line expressed high levels of CSLCs transcription factors including SOX2 and POU5F1 with low expression of differentiated markers CK8/CK18;Supplementary Figure 3. Immunofluorescence staining of CD133 and ALDH1A1;Supplementary Figure 4. Correlation of IGF-1R and ALDH1A1 with poor DSS in LAC patients;Supplementary Figure 5. Insulin signaling/IGF pathway is highly correlated with expression genes in LACSLCs;Supplementary Figure 6. Disruption of IGF-1R signaling pathway inhibits the self-renewal and the tumorigenic potential of human LACSLCs;Supplementary Figure 7. MEK inhibitor PD98059 has no effect on POU5F1; Expression of IGF-1, �-catenin and POU5F1 in fresh LAC specimens and A549 LACSLCs

Suppression of MICA/B by overexpressing miR-20a in BCSCs.

Supplementary Text including Supplementary Methods and Supplementary Figure Legends

Current conventional treatment strategies for glioblastoma (GBM) have limited efficacy due to the rapid development of resistance to temozolomide (TMZ). It is particularly urgent to develop novel therapeutic strategies that can overcome TMZ resistance and provide patients with better prognoses. Here, a TMZ-resistant GBM cell strain and a mouse model of TMZ resistance are established as valuable tools to explore novel therapeutic strategies against TMZ resistance. Experimentally, p38MAPK inhibitor reduces the accumulation of F4/80+/CD11b+ macrophages/microglia in glioma and prolongs the survivals of glioma-bearing mice. Glioma-associated macrophages/microglia have a significanct expression of PD-L1. p38MAPK inhibitor in combination with PD-L1 antibody can effectively prolongs the survivals of TMZ-resistant GBM-bearing hosts, and differentially reduces the accumulation of circulating monocytes-derived tumor-associated macrophages and PD-L1 abundances of resident glioma-associated microglia. This combination therapy could be a treatment option for patients at the recurrence or chronic TMZ maintenance stages. A clinical study to confirm the safety and effectiveness of this combination therapy is warranted.

Aberrant activation of Wnt/β-catenin signaling by dysregulated post-translational protein modifications, especially ubiquitination is causally linked to cancer development and progression. Although Lys48-linked ubiquitination is known to regulate Wnt/β-catenin signaling, it remains largely obscure how other types of ubiquitination, such as linear ubiquitination governs its signaling activity. The expression and regulatory mechanism of linear ubiquitin chain assembly complex (LUBAC) on Wnt/β-catenin signaling was examined by immunoprecipitation, western blot and immunohistochemical staining. The ubiquitination status of β-catenin was detected by ubiquitination assay. The impacts of SHARPIN, a core component of LUBAC on malignant behaviors of gastric cancer cells were determined by various functional assays in vitro and in vivo. Unlike a canonical role in promoting linear ubiquitination, SHARPIN specifically interacts with β-catenin to maintain its protein stability. Mechanistically, SHARPIN competes with the E3 ubiquitin ligase β-Trcp1 for β-catenin binding, thereby decreasing β-catenin ubiquitination levels to abolish its proteasomal degradation. Importantly, SHARPIN is required for invasiveness and malignant growth of gastric cancer cells in vitro and in vivo, a function that is largely dependent on its binding partner β-catenin. In line with these findings, elevated expression of SHARPIN in gastric cancer tissues is associated with disease malignancy and correlates with β-catenin expression levels. Our findings reveal a novel molecular link connecting linear ubiquitination machinery and Wnt/β-catenin signaling via SHARPIN-mediated stabilization of β-catenin. Targeting the linear ubiquitination-independent function of SHARPIN could be exploited to inhibit the hyperactive β-catenin signaling in a subset of human gastric cancers.

Tumor-associated macrophages (TAMs) constitute a large population of glioblastoma and facilitate tumor growth and invasion of tumor cells, but the underlying mechanism remains undefined. In this study, we demonstrate that chemokine (C-C motif) ligand 8 (CCL8) is highly expressed by TAMs and contributes to pseudopodia formation by GBM cells. The presence of CCL8 in the glioma microenvironment promotes progression of tumor cells. Moreover, CCL8 induces invasion and stem-like traits of GBM cells, and CCR1 and CCR5 are the main receptors that mediate CCL8-induced biological behavior. Finally, CCL8 dramatically activates ERK1/2 phosphorylation in GBM cells, and blocking TAM-secreted CCL8 by neutralized antibody significantly decreases invasion of glioma cells. Taken together, our data reveal that CCL8 is a TAM-associated factor to mediate invasion and stemness of GBM, and targeting CCL8 may provide an insight strategy for GBM treatment.

Immunotherapies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand (PD-L1) axis have been emerging as a promising therapeutic strategy to treat lung cancer. PD-1 is preferentially expressed by activated T lymphocytes; but whether/how its expression by tumor-associated macrophages (TAMs) in lung adenocarcinoma remains elusive. Herein, we investigate the frequency of PD-1 expression on TAMs in mouse allografts by flow cytometry analysis and evaluate the spatial distribution and clinicopathological significance of PD-1+ TAMs in 213 cases of human lung adenocarcinoma specimens by immunohistochemical staining. We find the expression of PD-1 by both mouse and human TAMs. Mouse PD-1+ TAMs possess unique transcriptional profile as compared to PD-1- TAMs. Furthermore, PD-1 is preferentially expressed by CD163+ TAMs in the tumor stroma than those in the tumor islets of lung adenocarcinoma. Stromal PD-1+ TAM infiltration is an independent predictor of reduced survival as determined by univariate (P < .001) and multivariate (P = .023) analysis. Moreover, patients with high stromal PD-1+ TAMs but low tumor cell PD-L1 expression have the shortest survival (P = .0001). Our study demonstrates that PD-1+ TAMs have unique gene expression characteristics and PD-1+ TAMs in the tumor stroma is a potential prognostic factor in lung adenocarcinoma, suggesting that a better understanding of PD-1+ TAMs will be beneficial for immunotherapy of lung adenocarcinoma patients.

Autophagy is a highly conserved process that is vital for tumor progression and treatment response. Although autophagy is proposed to maintain the stemness phenotype in adult diffuse glioma, the molecular basis of the link between autophagy and stemness is poorly understood, which makes it impossible to effectively screen for the population that will benefit from autophagy-targeted treatment. Here, ATG9B as essential for self-renewal capacity and tumor-propagation potential is identified. Notably, ASCL2 transcriptionally regulates the expression of ATG9B to maintain stemness properties. The ASCL2-ATG9B axis is an independent prognostic biomarker and indicator of autophagic activity. Furthermore, the highly effective blood-brain barrier (BBB)-permeable autophagy inhibitor ROC-325, which can significantly inhibit the progression of ASCL2-ATG9B axis

Plexin-domain containing 2 (PLXDC2) has been reported as an oncoprotein in several human malignancies. However, its expression and roles in gastric cancer remain largely unclear. In this study, we found that PLXDC2 was highly expressed in gastric cancer tissues, and the expression levels were positively correlated with clinicopathological features, but negatively with the patients’ outcome. Cox regression analysis identified PLXDC2 as an independent prognostic indicator for the patients. Knockdown of PLXDC2 markedly suppressed the in vitro invasion and in vivo metastasis of gastric cancer cells, while overexpression of PLXDC2 resulted in opposite effects. Mechanistically, PLXDC2 enhanced the level of phosphorylated Cortactin (p-Cortactin) by physically interacting with protein tyrosine phosphatase 1B (PTP1B), an important dephosphorylase, to prevent its dephosphorylating of p-Cortactin, thereby promoting the formation of invadopodia. Collectively, our results indicate that PLXDC2 contributes to the invasion and metastasis of gastric cancer by inhibiting PTP1B to facilitate the invadopodium formation, and may serve as a potential prognostic biomarker and a therapeutic target for this disease.

Glioblastoma is a lethal primary brain tumor with abundant immune-suppressive glioblastoma-associated macrophage (GAM) infiltration. Skewing immune suppressive GAMs towards an immune-activating phenotype represents a promising immunotherapeutic strategy against glioblastoma. Herein, we reported that genetic deletion of miRNA-processing enzyme Dicer in macrophages inhibited the growth of GL261 murine glioblastoma xenografts and prolonged survival of tumor-bearing mice. Single cell RNA sequencing (scRNA-seq) of the tumor-infiltrating immune cells revealed that Dicer deletion in macrophages reduced the proportion of cell-cycling GAM cluster and reprogramed the remaining GAMs towards a proinflammatory activation state (enhanced phagocytotic and IFN-producing signature). Dicer-deficient GAMs showed reduced level of cyclin-dependent kinases (CDK1 and CDK2) and increased expression of CDK inhibitor p27 Kip1, thus manifesting impaired proliferation. Dicer knockout enhanced phagocytotic activity of GAMs to eliminate GL261 tumor cells. Increased proinflammatory GAM clusters in macrophage Dicer-deficient mice actively interacted with tumor-infiltrating T cells and NK cells through TNF paracrine signaling to create a pro-inflammatory immune microenvironment for tumor cell elimination. Our work identifies the role of Dicer deletion in macrophages in generating an immune-activating microenvironment, which could be further developed as a potential immunotherapeutic strategy against glioblastoma.

Aims The aim of this study was to investigate the tumor microenvironment immune types (TMIT) based on tumor cell programmed cell death ligand 1 (PD‐L1) expression and tumor‐infiltrating lymphocytes (TILs) distribution and whether distinct TMIT subtypes (TMIT I, PD‐L1high/TILhigh; TMIT II, PD‐L1low/TILlow; TMIT III, PD‐L1high/TILlow; and TMIT IV, PD‐L1low/TILhigh) differentially affect clinical outcomes of patients with lung adenocarcinoma (LAC) and squamous cell carcinoma (SCC). Methods and results Immunohistochemistry (IHC) was applied to evaluate the expression of PD‐L1 and the spatial distribution of programmed cell death 1 (PD‐1) and CD8 TILs on the surgically resected specimens from 205 cases of LAC and 149 cases of SCC. PD‐1 and CD8 TILs were more frequently distributed in SCC than those in LAC, regardless of their infiltrating in the tumor islets or stroma. The density of TILs was a poor prognostic factor in LAC but a favorable one in SCC. PD‐L1 levels and its clinical prognostic significance differed in LAC vs SCC. LAC patients with TMIT III and SCC patients with TMIT I had the longest survival, respectively (P = .0197 and .0049). Moreover, TMIT stratification based on tumor cell PD‐L1 expression and stromal CD8+ TILs could be considered as an independent prognostic factor of SCC patients' survival as determined by both univariate and multivariate analysis. Conclusion Our study indicates that different type of TMIT provides its specific microenvironment with diverse impact on survival of LAC and SCC patients and highlights the importance of the integrative assessment of PD‐L1 status and TILs' spatial distribution to predict patients' prognosis., Our study indicates that different type of TMIT provides its specific microenvironment with diverse impact on survival of lung adenocarcinoma and squamous cell carcinoma patients and highlights the importance of the integrative assessment of programmed cell death ligand 1 status and tumor‐infiltrating lymphocytes' spatial distribution to predict patients' prognosis.

BackgroundThe existing cell surface markers used for sorting glioma stem cells (GSCs) have obvious limitations, such as vulnerability to the enzymatic digestion and time-consuming labeling procedure. Reduced nicotinamide adenine dinucleotide (NADH) as a cellular metabolite with property of autofluorescence has the potential to be used as a new biomarker for sorting GSCs.MethodsA method for sorting GSCs was established according to the properties of the autofluorescence of NADH. Then, the NADHhighand NADHlowsubpopulations were sorted. The stem-like properties of the subpopulations were evaluated by qRT-PCR, western blot analyses, limiting dilution assay, cell viability assay, bioluminescence imaging, and immunofluorescence analysis in vitro and in vivo. The relationship between CD133+/CD15+cells and NADHhighsubpopulation was also assessed.ResultsNADHhighcells expressed higher stem-related genes, formed more tumor spheres, and harbored stronger pluripotency in vitro and higher tumorigenicity in vivo, compared to NADHlowsubpopulation. NADHhighglioma cells had the similar stemness with CD133+or CD15+GSCs, but the three subpopulations less overlaid each other. Also, NADHhighglioma cells were more invasive and more resistant to chemotherapeutic drug temozolomide (TMZ) than NADHlowcells. In addition, the autofluorescence of NADH might be an appropriate marker to sort cancer stem cells (CSCs) in other cancer types, such as breast and colon cancer.ConclusionOur findings demonstrate that intracellular autofluorescence of NADH is a non-labeling, sensitive maker for isolating GSCs, even for other CSCs.

Aberrant expression of neuropilin and tolloid-like 2 (NETO2) has been observed during the progression of some human carcinomas. However, the expression pattern and clinical relevance of NETO2 in gastric cancer (GC) remain to be elucidated. In this study, we found that NETO2 expression was higher in GC tissues compared with paired non-cancerous tissues. Moreover, the expression of NETO2 was positively correlated with clinical stage, invasion depth, lymph node metastasis, and tumor size, but inversely correlated with overall and disease-free survival rates. Cox regression analysis identified NETO2 as an independent prognostic indicator for GC patients. Overexpression of NETO2 facilitated migration and invasion of GC cells in vitro and metastasis in vivo in association with induction of epithelial-mesenchymal transition. Conversely, knockdown of NETO2 had the opposite effects. Mechanistically, silencing NETO2 reduced the phosphorylation of PI3K, AKT, and NF-κB p65 as well as the expression of Snail, whereas NETO2 overexpression achieved the opposite results. Furthermore, we identified TNFRSF12A as a mediator for NETO2 to activate PI3K/AKT/NF-κB/Snail axis. Collectively, our results demonstrate that NETO2 promotes invasion and metastasis of GC cells and represents a novel prognostic indicator as well as a potential therapeutic target in GC.

Epithelial-mesenchymal transition (EMT) plays a critical role in initiating tumor invasion and metastasis of colorectal cancer (CRC), although the underlying mechanisms remain to be clarified. Herein, we demonstrate that the active form of Rac family small GTPase 1 (RAC1-GTP) is overexpressed in CRCs and promotes the EMT-mediated invasion of CRC cells through activation of the signal transducers and activators of transcription 3 (STAT3) pathway. Increased expression of RAC1-GTP in CRC tissues was positively correlated with the TNM stages of CRCs and indicated poor prognosis of CRC patients. Targeting RAC1-GTP activity by its specific inhibitor NSC23766 markedly suppressed the migration and invasion of CRC cells. Mechanistically, RAC1-GTP directly interacted with STAT3 to promote STAT3 phosphorylation, thus promoted EMT of CRC cells. Enforced expression of constitutively activated STAT3 (STAT3-C) abrogated the suppressive effect of RAC1-GTP disruption on the migration and invasion of CRC cells. Importantly, NSC23766 disrupted EMT in CRC cells and significantly diminished growth of CRC xenografts. Taken together, our data indicate that RAC1-GTP is an important player in EMT-mediated tumor invasion and a potential therapeutic target for CRCs.

Capillary morphogenesis protein 2 (CMG2) was originally identified through its participation in capillary morphogenesis, and subsequently identified as the second receptor for anthrax toxin (ANTXR2). Although tumor-associated functions of CMG2 have also been reported, the clinical significance and functional mechanism of CMG2 in glioma remain to be elucidated. We assessed the clinicopathological relevance of CMG2 in a cohort of 48 glioma patients as well as through public glioma databases, and explored the function of CMG2 using glioblastoma (GBM) models in vitro and in vivo. CMG2 overexpression was associated with increased tumor grade and poor patient survival. CMG2 promoted G2/M-phase transition during the cell cycle of GBM cells in vitro and contributed to tumor growth in vivo. We also observed that CMG2 is implicated in the activation of extracellular signal-regulated kinases (ERKs), epithelial-mesenchymal transition (EMT), migration, and invasion in GBM cells. Transcriptomic analysis of GBM cells with or without CMG2 overexpression indicated that a panel of oncogenic signaling pathways was altered with CMG2 upregulation, implying that CMG2 might orchestrate these signaling pathways to regulate the growth of GBM cells. Yes-associated protein 1 (YAP1) activity was enhanced by CMG2 overexpression but suppressed with CMG2 deficiency. Since YAP1 is critically implicated in GBM, the oncogenic roles of CMG2 in GBM cells might thus be mediated, at least partially, by YAP1. Altogether, CMG2 functioned as an oncogene in glioma cells and is a potential prognostic biomarker or therapeutic target for the clinical treatment of glioma. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A growing body of evidence shows that the development and progression of gastric cancer (GC) is mainly associated to the presence of gastric cancer stem-like cells (GCSLCs). However, it is unclear how GCSLC population is maintained. This study aimed to explore the role of capillary morphogenesis gene 2 (CMG2) in GCSLC maintenance and the relevance to GC progression. We found that CMG2 was highly expressed in GC tissues and the expression levels were associated with the invasion depth and lymph node metastasis of GC, and inversely correlated with the survival of GC patients. Sorted CMG2High GC cells preferentially clustered in CD44High stem-like cell population, which expressed high levels of stemness-related genes with increased capabilities of self-renewal and tumorigenicity. Depletion of CMG2 gene resulted in reduction of GCSLC population with attenuated stemness and decrease of invasive and metastatic capabilities with subdued epithelial–mesenchymal transition phenotype in GC cells. Mechanistically, CMG2 interacted with LRP6 in GCSLCs to activate a Wnt/β-catenin pathway. Thus, our results demonstrate that CMG2 promotes GC progression by maintaining GCSLCs and can serve as a new prognostic indicator and a target for human GC therapy.

Glioblastoma (GBM) is a fatal tumor and comprises heterogeneous cells in which a subpopulation with stem cell-like properties is included. Cancer cells with stem cell-like properties account for tumor initiation, drug resistance and recurrence. To identify and characterize specific factors in regulating stem-like traits is critical for GBM therapeutic. Here, we showed that Stanniocalcin-1 (STC1), a secretory glycoprotein, functions as a novel stimulator for stem-like traits of GBM cells. We found STC1 was prominently expressed in glioma spheres which are mainly comprised of glioma stem-like cells. The stem-like traits of GBM cells, as determined by the expression of stem cell markers, tumor-sphere formation efficiency and colony-forming ability, were enhanced by STC1 overexpression and inhibited by STC1 knockdown. Furthermore, introduction of STC1 enhanced tumorigenesis in vivo while knockdown of STC1 showed reverse effect. Finally, we demonstrated that STC1 interacted with the extracellular domain of NOTCH1 to activate NOTCH1-SOX2 signaling pathway, by which STC1 augmented the stem-like traits of GBM cells. Taken together, our data herein indicate that STC1 is a novel non-canonical NOTCH ligand and acts as a crucial regulator of stemness in GBM.

The 66 kDa estrogen receptor alpha (ERα66) is the main molecular target for endocrine therapy such as tamoxifen treatment. However, many patients develop resistance with unclear mechanisms. In a large cohort study of breast cancer patients who underwent surgery followed by tamoxifen treatment, we demonstrate that ERα36, a variant of ERα66, correlates with poor prognosis. Mechanistically, tamoxifen directly binds and activates ERα36 to enhance the stemness and metastasis of breast cancer cells via transcriptional stimulation of aldehyde dehydrogenase 1A1 (ALDH1A1). Consistently, the tamoxifen-induced stemness and metastasis can be attenuated by either ALDH1 inhibitors or a specific ERα36 antibody. Thus, tamoxifen acts as an agonist on ERα36 in breast cancer cells, which accounts for hormone therapy resistance and metastasis of breast cancer. Our study not only reveals ERα36 as a stratifying marker for endocrine therapy but also provides a promising therapeutic avenue for tamoxifen-resistant breast cancer.