Cancer-associated fibroblast-derived PAI-1 promotes lymphatic metastasis via the induction of EndoMT in lymphatic endothelial cells.

BACKGROUND: Endothelial-mesenchymal transition (EndoMT) is an emerging adaptive process that modulates lymphatic endothelial function to drive aberrant lymphatic vascularization in the tumour microenvironment (TME); however, the molecular determinants that govern the functional role of EndoMT remain unclear. Here, we show that cancer-associated fibroblast (CAF)-derived PAI-1 promoted the EndoMT of lymphatic endothelial cells (LECs) in cervical squamous cell carcinoma (CSCC). METHODS: Immunofluorescent staining of α-SMA, LYVE-1 and DAPI were examined in primary tumour samples obtained from 57 CSCC patients. Assessment of cytokines secreted by CAFs and normal fibroblasts (NFs) was performed using human cytokine antibody arrays. The phenotype of EndoMT in lymphatic endothelial cells (LECs), gene expression levels, protein secretion and activity of signaling pathways were measured by real-time RT-PCR, ELISA or western blotting. The function of lymphatic endothelial monolayers was examined by transwell, tube formation assay, transendothelial migration assay in vitro. Lymphatic metastasis was measured using popliteal lymph node metastasis model. Furthermore, association between PAI-1 expression and EndoMT in CSCC was analyzed by immunohistochemistry. The Cancer Genome Atlas (TCGA) databases was used to assess the association of PAI-1 with survival rate in CSCC. RESULTS: CAF-derived PAI-1 promoted the EndoMT of LECs in CSCC. LECs undergoing EndoMT could initiate tumour neolymphangiogenesis that facilitated cancer cell intravasation/extravasation, which in turn promoted lymphatic metastasis in CSCC. Mechanistically, PAI-1 activated the AKT/ERK1/2 pathways by directly interacting with low-density lipoprotein receptor-related protein (LRP1), thereby leading to elevated EndoMT activity in LECs. Blockade of PAI-1 or inhibition of LRP1/AKT/ERK1/2 abrogated EndoMT and consequently attenuated CAF-induced tumour neolymphangiogenesis. Furthermore, clinical data revealed that increased PAI-1 levels positively correlated with EndoMT activity and poor prognosis in CSCC patients. CONCLUSION: Our data indicate that CAF-derived PAI-1 acts as an important neolymphangiogenesis-initiating molecular during CSCC progression through modulating the EndoMT of LECs, resulting in promotion of metastasis ability in primary site. PAI-1 could serve as an effective prognostic biomarker and therapeutic target for CSCC metastasis.

Tumor-secreted exosomal Wnt2B activates fibroblasts to promote cervical cancer progression.

The activation of stromal fibroblasts into cancer-associated fibroblasts (CAFs) has been suggested to promote primary tumor growth and progression; however, the mechanisms underlying the crosstalk between tumors and fibroblasts that drives stromal heterogeneity remain unknown. Here, we show that high Wnt2B levels were positively correlated with the number of CAFs in cervical cancer (CC). More importantly, Wnt2B was characteristically enriched in CC cell-secreted exosomes and transferred into fibroblasts to promote fibroblast activation via Wnt/ß-catenin signaling, and inhibiting exosomal release or the Wnt/ß-catenin signaling pathway diminished the activation induced by exosomal Wnt2B. Moreover, circulating exosomal Wnt2B also promoted CAF conversion in vitro and its expression was significantly higher in CC patients. In conclusion, our findings indicate that CC cell-derived Wnt2B can induce the activation of fibroblasts into CAFs, mainly via exosome-dependent secretion, thus providing directions for the development of diagnostic and therapeutic targets for CC progression.

A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner.

Lymphatic remodelling in the hypoxic tumour microenvironment (TME) is critically involved in the metastasis of cervical squamous cell carcinoma (CSCC); however, its underlying mechanisms remain unclear. Here, we uncovered a novel lymphatic pattern in the hypoxic TME, wherein lymphatic vessels (LVs) are encapsulated by tumour-associated macrophages (TAMs) to form an interconnected network. We describe these aggregates as LVEM (LVs encapsulated by TAMs) considering their advantageous metastatic capacity and active involvement in early lymph node metastasis (LNM). Mechanistic investigations revealed that interleukin-10 (IL-10) derived from hypoxic TAMs adjacent to LVs was a prerequisite for lymphangiogenesis and LVEM formation through its induction of Sp1 upregulation in lymphatic endothelial cells (LECs). Interestingly, Sp1high LECs promoted the transactivation of C-C motif chemokine ligand 1 (CCL1) to facilitate TAM and tumour cell recruitment, thereby forming a positive feedback loop to strengthen the LVEM formation. Knockdown of Sp1 or blockage of CCL1 abrogated LVEM and consequently attenuated LNM. Notably, CSCCnon-LNM is largely devoid of hypoxic TAMs and the resultant LVEM, which might explain its metastatic delay. These findings identify a novel and efficient metastasis-promoting lymphatic pattern in the hypoxic TME, which might provide new targets for anti-metastasis therapy and prognostic assessment.

Periostin+ cancer-associated fibroblasts promote lymph node metastasis by impairing the lymphatic endothelial barriers in cervical squamous cell carcinoma.

Lymph node metastasis (LNM), a critical prognostic determinant in cancer patients, is critically influenced by the presence of numerous heterogeneous cancer-associated fibroblasts (CAFs) in the tumor microenvironment. However, the phenotypes and characteristics of the various pro-metastatic CAF subsets in cervical squamous cell carcinoma (CSCC) remain unknown. Here, we describe a CAF subpopulation with elevated periostin expression (periostin+ CAFs), located in the primary tumor sites and metastatic lymph nodes, that positively correlated with LNM and poor survival in CSCC patients. Mechanistically, periostin+ CAFs impaired lymphatic endothelial barriers by activating the integrin-FAK/Src-VE-cadherin signaling pathway in lymphatic endothelial cells and consequently enhanced metastatic dissemination. In contrast, inhibition of the FAK/Src signaling pathway alleviated periostin-induced lymphatic endothelial barrier dysfunction and its related effects. Notably, periostin- CAFs were incapable of impairing endothelial barrier integrity, which may explain the occurrence of CAF-enriched cases without LNM. In conclusion, we identified a specific periostin+ CAF subset that promotes LNM in CSCC, mainly by impairing the lymphatic endothelial barriers, thus providing the basis for potential stromal fibroblast-targeted interventions that block CAF-dependent metastasis.

Low expression of transferrin receptor 2 predict poor prognosis in gastric cancer patients.

This study was to detect the expression level of transferrin receptor 2 (TfR2) in gastric cancer and to analyze its value in predicting the prognosis of gastric cancer patients. Real-time PCR (RT-qPCR) was applied to detect the mRNA expression of TfR2 in gastric cancer tissues and paired adjacent nontumorous tissues. Immunohistochemistry (IHC) and western blotting were used to determine the protein expression of TfR2 in gastric cancer, and the relationship with the prognosis of gastric cancer patients was analyzed. The results were: (1) The mRNA and protein levels of TfR2 in gastric cancer tissues were remarkably lower than those in adjacent nontumorous gastric tissue (P < .05). (2) Clinicopathological analysis showed that the expression level of TfR2 was significantly correlated with TNM stage of patients (P = .047). (3) The results of univariate survival analysis showed that histological grade, T stage, N stage, M stage, Lauren's classification, and TfR2 expression level influenced the overall survival of gastric cancer patients. Multivariate survival analysis showed that low TfR2 expression (HR = 1.671, 95%CI: 1.006-2.774, P = .047), poor differentiation (HR = 2.123, 95%CI: 1.188-3.795, P = .011), and M1 stage (HR = 8.541, 95%CI: 3.416-21.353, P < .001) were independent risk factors affecting the overall survival of gastric cancer patients. In conclusion, TfR2 exhibited low expression in gastric cancer tissue, and is an independent prognostic factor of gastric cancer patients.

Correction to: miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells.

An amendment to this paper has been published and can be accessed via the original article.

miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells.

BACKGROUND: miRNA expression profiles in ectopic endometrium (EC) serving as pathophysiologic genetic fingerprints contribute to determining endometriosis progression; however, the underlying molecular mechanisms remain unknown. METHODS: miRNA microarray analysis was used to determine the expression profiling of EC fresh tissues. qRT-PCR was performed to screen miR-205-5p expression in EC tissues. The roles of miR-205-5p and its candidate target gene, angiopoietin-2 (ANGPT2), in endometriosis progression were confirmed on the basis of both in vitro and in vivo systems. miR-205-5p and ANGPT2 expression were measured by in situ hybridization and immunochemistry, and their clinical significance was statistically analysed. RESULTS: miR-205-5p was screened as a novel suppressor of endometriosis through primary ectopic endometrial stromal cell migration, invasion, and apoptosis assay in vitro, along with endometrial-like xenograft growth and apoptosis in vivo. In addition, ANGPT2 was identified as a direct target of miR-205-5p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of ANGPT2 could respectively rescue and simulate the effects induced by miR-205-5p. Importantly, the miR-205-5p-ANGPT2 axis was found to activate the ERK/AKT pathway in endometriosis. Finally, miR-205-5p and ANGPT2 expression were closely correlated with the endometriosis severity. CONCLUSION: The newly identified miR-205-5p-ANGPT2-AKT/ERK axis illustrates the molecular mechanism of endometriosis progression and may represent a novel diagnostic biomarker and therapeutic target for disease treatment.

Hypoxia-induced ZEB1 promotes cervical cancer progression via CCL8-dependent tumour-associated macrophage recruitment.

The accumulation of tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is associated with malignant progression in cancer. However, the mechanisms by which the hypoxic TME facilitates TAM infiltration are not fully understood. This study showed that high ZEB1 expression in hypoxic cervical cancer cell islets was positively correlated with CD163+ TAM accumulation. ZEB1 in hypoxic cancer cells promoted the migration of TAMs in vitro and altered the expression of multiple chemokines, especially CCL8. Mechanistically, hypoxia-induced ZEB1 activated the transcription of CCL8, which attracted macrophages via the CCR2-NF-κB pathway. Furthermore, ZEB1 and CCL8 were independent prognostic factors in cervical cancer patients based on The Cancer Genome Atlas (TCGA) data analysis. In conclusion, hypoxia-induced ZEB1 exerts unexpected functions in cancer progression by fostering a prometastatic environment through increased CCL8 secretion and TAM recruitment; thus, ZEB1 may serve as a candidate biomarker of tumour progression and provide a potential target for disrupting hypoxia-mediated TME remodelling.

Cancer-derived exosomal miR-221-3p promotes angiogenesis by targeting THBS2 in cervical squamous cell carcinoma.

AIMS: Recently, cancer-derived exosomes were shown to have pro-metastasis function in cancer, but the mechanism remains unclear. Angiogenesis is essential for tumor progression and is a great promising therapeutic target for advanced cervical cancer. Here, we investigated the role of cervical cancer cell-secreted exosomal miR-221-3p in tumor angiogenesis. METHODS AND RESULTS: miR-221-3p was found to be closely correlated with microvascular density in cervical squamous cell carcinoma (CSCC) by evaluating the microvascular density with immunohistochemistry and miR-221-3p expression with in situ hybridization in clinical specimens. Using the groups of CSCC cell lines (SiHa and C33A) with miR-221-3p overexpression and silencing, the CSCC exosomes were characterized by electron microscopy, western blotting, and fluorescence microscopy. The enrichment of miR-221-3p in CSCC exosomes and its transfer into human umbilical vein endothelial cells (HUVECs) were confirmed by qRT-PCR. CSCC exosomal miR-221-3p promoted angiogenesis in vitro in Matrigel tube formation assay, spheroid sprouting assay, migration assay, and wound healing assay. Then, exosome intratumoral injection indicated that CSCC exosomal miR-221-3p promoted tumor growth in vivo. Thrombospondin-2 (THBS2) was bioinformatically predicted to be a direct target of miR-221-3p, and this was verified by using the in vitro and in vivo experiments described above. Additionally, overexpression of THBS2 in HUVECs rescued the angiogenic function of miR-221-3p. CONCLUSIONS: Our results suggest that CSCC exosomes transport miR-221-3p from cancer cells to vessel endothelial cells and promote angiogenesis by downregulating THBS2. Therefore, CSCC-derived exosomal miR-221-3p could be a possible novel diagnostic biomarker and therapeutic target for CSCC progression.

Cervical squamous cell carcinoma-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis by targeting VASH1.

Cancer-secreted exosomal miRNAs are emerging mediators of cancer-stromal cross-talk in the tumor environment. Our previous miRNAs array of cervical squamous cell carcinoma (CSCC) clinical specimens identified upregulation of miR-221-3p. Here, we show that miR-221-3p is closely correlated with peritumoral lymphangiogenesis and lymph node (LN) metastasis in CSCC. More importantly, miR-221-3p is characteristically enriched in and transferred by CSCC-secreted exosomes into human lymphatic endothelial cells (HLECs) to promote HLECs migration and tube formation in vitro, and facilitate lymphangiogenesis and LN metastasis in vivo according to both gain-of-function and loss-of-function experiments. Furthermore, we identify vasohibin-1 (VASH1) as a novel direct target of miR-221-3p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of VASH1 could respectively rescue and simulate the effects induced by exosomal miR-221-3p. Importantly, the miR-221-3p-VASH1 axis activates the ERK/AKT pathway in HLECs independent of VEGF-C. Finally, circulating exosomal miR-221-3p levels also have biological function in promoting HLECs sprouting in vitro and are closely associated with tumor miR-221-3p expression, lymphatic VASH1 expression, lymphangiogenesis, and LN metastasis in CSCC patients. In conclusion, CSCC-secreted exosomal miR-221-3p transfers into HLECs to promote lymphangiogenesis and lymphatic metastasis via downregulation of VASH1 and may represent a novel diagnostic biomarker and therapeutic target for metastatic CSCC patients in early stages.

The role of the hypoxia-Nrp-1 axis in the activation of M2-like tumor-associated macrophages in the tumor microenvironment of cervical cancer.

To explore the mechanisms through which hypoxic tumor microenvironment (TME) modulates the transition of tumor-associated macrophages (TAMs). The migration ability of RAW264.7 macrophages was determined by transwell assay. Flow cytometric, western blot and immunofluorescence analyses of CD206 further validated the M2 polarization of macrophages. Immunofluorescence, western blot and qRT-PCR were performed to detect the expression of neuropilin-1 (Nrp-1) and carbonic anhydrase IX (CAIX). An intermittent hypobaric hypoxia (IH) animal model was established to evaluate the role of hypoxia in activating M2-like TAMs in vivo. We also used immunohistochemistry to analyze the association between CAIX, CD163+ macrophages and Nrp-1 in a series of 72 human cervical cancer specimens. We found that the hypoxic cervical TME educated the recruited macrophages to transform into the M2 phenotype. Nrp-1 expression was significantly increased in hypoxia-primed cervical cancer cells. Blocking Nrp-1 expression prevented hypoxic cells from recruiting and polarizing macrophages towards the M2 phenotype. Hypoxia exposure significantly increased the expression of Nrp-1 as well as the infiltration of macrophages in vivo. Consistently, immunochemical staining in serial tissue sections of cervical cancer revealed upregulated levels of Nrp-1 in CAIX-positive hypoxic regions along with a concurrent significant elevation of M2 macrophages. Nrp-1 and M2-like TAMs were related to the malignant properties of cervical cancer, such as the FIGO stage and lymph node metastasis. Nrp-1 plays critical roles in hypoxic TME-induced activation and pro-tumoral effects of TAMs in cervical cancer. Interfering with Nrp-1 may be a potential therapeutic strategy in treating cervical cancer.

MicroRNA-221-3p, a TWIST2 target, promotes cervical cancer metastasis by directly targeting THBS2.

MicroRNAs have implicated in the relapse and metastasis of cervical cancer, which is the leading cause of cervical cancer-related mortality. However, the underlying molecular mechanisms need further elucidation. Our present study revealed that miR-221-3p is transcriptionally promoted in metastatic cervical cancer tissues compared with non-metastatic cervical cancer tissues. Forced overexpression of miR-221-3p facilitated EMT and promoted cell migration and invasion in vitro and lymphatic metastasis in vivo. Twist homolog 2 (TWIST2) was found to be a key transcription factor binding to the promoter of miR-221-3p. Inhibitors of miR-221-3p drastically reduced the induction of EMT and decreased cell migration and invasion mediated by TWIST2. By combined computational and experimental approaches, THBS2 was recognized to be an important downstream target gene of miR-221-3p. In cervical cancer tissues, especially with lymphatic metastasis, miR-221-3p and TWIST2 were increased and THBS2 was decreased, suggesting that TWIST2 induces miR-221-3p expression and consequently suppresses its direct target THBS2 in lymphatic metastasis CC. Our findings uncover a mechanistic role for miR-221-3p in lymph node metastasis, suggesting that miR-221-3p is upregulated by the transcription factor TWIST2 and downregulates its target THBS2, which may potentially promote lymph node metastasis in cervical cancer.

[Oxidized low-density lipoprotein modulates differentiation of murine memory CD8+ T cell subpopulations].

OBJECTIVE: To investigate effect of oxidized low-density lipoprotein (ox-LDL) on memory CD8+ T cell subpopulation differentiation in mice with autoimmune diabetes. METHODS: Cultured splenic CD8+ T cells from pre-diabetic NOD mice isolated with magnetic beads were treated with 30 µg/mL ox-LDL and 10 U/mL interleukin-2 (IL-2) for 24 h and the control cells were treated with IL-2 only. Flow cytometry was used to determine the percentage of splenic CD8+IFN-γ+ T cells, expressions of CD8, CD44 and CD62L on the T cells, and the activation of T cell factor-1 (TCF-1) and STAT-3. The CD127+ memory T cells were purified and transplanted into the pre-diabetic NOD mice via the tail vein, and the blood glucose was recorded weekly and survival time of the mice was monitored. RESULTS: Treatment with ox-LDL significantly reduced islet ß cell-specific cytotoxic CD8+T cells as compared with the control group [(0.7∓0.03)% vs (2.7∓0.14)%, P<0.01]. The percentage of effector memory CD8+T cells (Tem) in the total memory CD8+T cells was reduced [(10.3∓0.71)% vs (30.3∓1.36)%, P<0.01] and that of stem cell-like memory T cells was significantly increased [(72.3∓3.8)% vs (55.1∓2.61)%, P<0.05] following ox-LDL treatment, which also resulted in significantly decreased activation of TCF-1 [(14.5∓0.82)% vs (34.2∓1.23)%, P<0.01] and pSTAT-3 [(3.3∓0.12)% vs (22.1∓1.1)%, P<0.01]. Transplantation of ox-LDL-treated memory T cells in pre-diabetic NOD mice obviously inhibited the increase of blood glucose and prolonged the survival time of the mice (P<0.05). CONCLUSION: Ox-LDL decreases the activation of transcriptional factors TCF-1 and phosphorylation of STAT-3, inhibits the formation of effector memory CD8+ T cells with long-term cytotoxicity, but promote the generation of stem cell-like memory CD8+ T cells, which result in suppression of islet ß cell-specific effector cytotoxic CD8+ T cell differentiation to lessen autoimmune injury to the islet ß cells.

Relationship between UGT1A1*6/*28 polymorphisms and severe toxicities in Chinese patients with pancreatic or biliary tract cancer treated with irinotecan-containing regimens.

PURPOSE: The aim of this retrospective study was to investigate the relationship between UGT1A1 polymorphisms and toxicities in Chinese patients with pancreatic or biliary tract cancer receiving irinotecan-containing regimens as the second- or third-line chemotherapy. PATIENTS AND METHODS: A total of 36 patients with unresectable pancreatic cancer and 12 patients with unresectable biliary tract cancer were included. Approximately 33 patients were treated with FOLFIRI regimen, a chemotherapy regimen, where FOL stands for folinic acid, F for fluorouracil, and IRI for irinotecan (irinotecan 180 mg/m(2) at day 1, CF 200 mg/m(2) at day 1-2, 5-FU 400 mg/m(2) at day 1-2, followed by continuous infusion of 5-FU 600 mg/m(2) for 22 hours at day 1-2, every 2 weeks). The other 15 patients were treated with irinotecan monotherapy (180 mg/m(2), every 2 weeks). UGT1A1*6/*28 polymorphisms were detected by direct sequencing. RESULTS: The frequencies of GG, GA, AA genotypes for UGT1A1*6 were 70.8% (n=34), 25.0% (n=12), and 4.2% (n=2), respectively. And those of TA6/TA6, TA6/TA7, TA7/TA7 for UGT1A1*28 were 79.2% (n=38), 18.8% (n=9), and 2.0% (n=1), respectively. A total of 22 patients (45.8%) had grade III-IV neutropenia, and six patients (12.5%) experienced grade III-IV diarrhea. The incidence of grade III-IV neutropenia in patients with UGT1A1*6 GA or AA genotype was 71.4%, which was significantly higher than that with GG genotype (35.3%, P=0.022). No relationship was found between grade III-IV neutropenia and UGT1A1*28 polymorphism. The statistical analysis between grade III-IV diarrhea and UGT1A1*6/*28 polymorphisms was not conducted in view of the limited number of patients. CONCLUSION: In Chinese patients with pancreatic or biliary tract cancer administered irinotecan-containing regimens, those with UGT1A1*6 variant may have a high risk of severe neutropenia.

UGT1A1 gene polymorphisms and the toxicities of FOLFIRI in Chinese Han patients with gastrointestinal cancer.

BACKGROUND: Neutropenia and diarrhea are the common dose-limiting toxicities of irinotecan, and uridine diphosphate glucuronosyltransferases (UGTs) gene polymorphisms are considered to be one of the predictive markers of irinotecan related toxicities. This study aims to investigate the association between UGT1A1 gene polymorphisms and irinotecan related toxicities in Chinese Han gastrointestinal cancer patients receiving FOLFIRI regimen chemotherapy. METHODS: A total of 94 gastrointestinal cancer patients with metastasis (72 colon and rectum, 20 stomach, 1 pancreas and 1 duodenum), were enrolled from 2007 to 2010 in Shanghai Ruijin Hospital. All patients received standard dosage of FOLFIRI regimen (irinotecan 180mg/m2 d1, CF 200mg/m2 d1-d2, 5-FU 400mg/m2 d1-d2, followed by continuous infusion of 5-FU 600mg/m2 for 22h d1-d2, every 2 weeks). UGT1A1 gene polymorphisms (*60 -3279T > G, *93 -3156G > A, *28 -53TA6 > TA7, *6 211G > A) were detected by direct sequencing of DNA extracted from peripheral blood. The incidence of neutropenia, diarrhea, and time to severe toxicity were recorded. The relationship between UGT1A1 gene polymorphisms and toxicities was analyzed. RESULTS: The frequencies of UGT1A1*60 (-3279 G/G), UGT1A1*93 (-3156 A/A), UGT1A1*28 (-53 7/7) and UGT1A1*6 (211 A/A) homozygote were 6.4% (6/94), 1.1% (1/94), 1.1% (1/94) and 2.1% (2/94), respectively. The incidences of grade 3/4 neutropenia and diarrhea were 53.2% (50/94) and 12.8% (12/94), respectively. Comparing those with wild type, patients with UGT1A1*28 or *93 allele had significantly high risk of grade 3/4 diarrhea (6/7, 7/7 vs. 6/6: 27.8% vs. 9.2%, OR=3.791, P=0.034; G/A, A/A vs. G/G: 29.4% vs. 9.1%, OR=4.167, P=0.023). No relationship was found between UGT1A1 allele polymorphism and grade 3/4 neutropenia. The baseline serum total bilirubin was elevated in UGT1A1*28, *93 allele carriers and *60 homozygote, but with no relationship with severe toxicities. CONCLUSION: In Chinese Han gastrointestinal cancer patients receiving standard FOLFIRI regimen, UGT1A1*28 or *93 allele carriers may have high risk of severe diarrhea.