Hypoxia-induced ZEB1 promotes cervical cancer immune evasion by strengthening the CD47-SIRPα axis.

BACKGROUND: The dynamic interaction between cancer cells and tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is an active barrier to the effector arm of the antitumour immune response. Cancer-secreted exosomes are emerging mediators of this cancer-stromal cross-talk in the TME; however, the mechanisms underlying this interaction remain unclear. METHODS: Exosomes were isolated with ExoQuick exosome precipitation solution. The polarizing effect of TAMs was evaluated by flow cytometry, western blot analysis, immunofluorescence staining and in vitro phagocytosis assays. Clinical cervical cancer specimens and an in vivo xenograft model were also employed. RESULTS: Our previous study showed that hypoxia increased the expression of ZEB1 in cervical squamous cell carcinoma (CSCC) cells, which resulted in increased infiltration of TAMs. Here, we found that hypoxia-induced ZEB1 expression is closely correlated with CD47-SIRPα axis activity in CSCC, which enables cancer cells to evade phagocytosis by macrophages and promotes tumour progression. ZEB1 was found to directly activate the transcription of the CD47 gene in hypoxic CSCC cells. We further showed that endogenous ZEB1 was characteristically enriched in hypoxic CSCC cell-derived exosomes and transferred into macrophages via these exosomes to promote SIRPα+ TAM polarization. Intriguingly, exosomal ZEB1 retained transcriptional activity and reprogrammed SIRPα+ TAMs via activation of the STAT3 signalling pathway in vitro and in vivo. STAT3 inhibition reduced the polarizing effect induced by exosomal ZEB1. Knockdown of ZEB1 increased the phagocytosis of CSCC cells by macrophages via decreasing CD47 and SIRPα expression. CONCLUSIONS: Our results suggest that hypoxia-induced ZEB1 promotes immune evasion in CSCC by strengthening the CD47-SIRPα axis. ZEB1-targeted therapy in combination with CD47-SIRPα checkpoint immunotherapy may improve the outcomes of CSCC patients in part by disinhibiting innate immunity.

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.

Cancer-Associated Fibroblast Heterogeneity: A Factor That Cannot Be Ignored in Immune Microenvironment Remodeling.

Cancer-associated fibroblasts (CAFs) are important, highly heterogeneous components of the tumor extracellular matrix that have different origins and express a diverse set of biomarkers. Different subtypes of CAFs participate in the immune regulation of the tumor microenvironment (TME). In addition to their role in supporting stromal cells, CAFs have multiple immunosuppressive functions, via membrane and secretory patterns, against anti-tumor immunity. The inhibition of CAFs function and anti-TME therapy targeting CAFs provides new adjuvant means for immunotherapy. In this review, we outline the emerging understanding of CAFs with a particular emphasis on their origin and heterogeneity, different mechanisms of their regulation, as well as their direct or indirect effect on immune cells that leads to immunosuppression.

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.

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.

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.

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.

[Proteasome inhibitors sensitize ovarian cancer cells to cisplatin].

OBJECTIVE: To explore the sensitivity and the molecular mechanism of cisplatin-resistance ovarian cancer cell line C13 to proteasome inhibitors and the combination with cisplatin. METHODS: After different treatments, methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability, annexin-V/propidium iodide (PI) apoptosis detection kit was used to determine the apoptosis rate of different groups, western blot assay was introduced to evaluate the expression levels of Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein (cFLIPs), and the activity of caspase-8 was examined. RESULTS: MTT assay shown that the cell viability ratios of combination group at serial time points from 12, 24, 36, 48, 60, 72 hours were (56.0 ± 8.4) %, (44.7 ± 7.3) %, (33.7 ± 11.2) %, (27.6 ± 8.0) %, (27.6 ± 7.6) % and (28.1 ± 2.4) %, which were much lower than those of cisplatin group (P < 0.05). After treated for 24 hours, apoptosis rates of cisplatin group, bortezomib group and combination group were (16.7 ± 1.7) %, (23.4 ± 2.1) % and (26.9 ± 1.6) %, respectively. The rate of combination group was much higher than that of non-treated group and that of cisplatin group or bortezomib group (P < 0.05). Western blot assay showed the changes of expression levels of cFLIPs, which were down-regulated seriously after cisplatin, bortezomib or combination treatment [(43.2 ± 2.3) % vs (75.7 ± 3.0) % vs (67.9 ± 2.1) %, P < 0.05]. The caspase-8 activity of combination group was (5.6 ± 1.6) folds than that of non-treated group, which was higher than those of other two groups [(2.3 ± 1.0) and (4.2 ± 0.9) folds, P < 0.05]. CONCLUSIONS: The tumor cell lethal effect of cisplatin could be increase significantly by the combination application of proteasome inhibitors, bortezomib. And the cFLIPs/caspase-8 signaling pathway may be play an important role in the molecular mechanism of the combination treatment.

[Research advance on role of Coxsackie and adenovirus receptor (CAR) in tumor progression].

Coxsackie and adenovirus receptor (CAR) is originally identified as the cellular receptor of 2-and 5-type adenoviruses. Many researches have suggested that CAR can affect the growth, adhesive ability and cytoskeleton of tumor cells, and has complicated functions in metastasis and invasion of tumors. Moreover, the expression of CAR has close relationship with tumor prognosis and cytoreduction mediated by adenoviruses. CAR has become a new hotspot in the research on mechanism of tumor progression and gene therapy. Our review focuses on the structure and function of CAR and its role in mediating occurrence and progression of tumor.

[Proteasome inhibitors sensitize ovarian cancer cells to paclitaxel induced apoptosis].

OBJECTIVE: To explore the sensitivity of ovarian cancer cell line SKOV3 to paclitaxel, proteasome inhibitors, bortezomib, and their combination. METHODS: The methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability after treatment. The annexin V-propidium iodide apoptosis detection kit was used to determine the apoptosis rate of different groups. Western blot assay was used to evaluate the expression levels of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK-3beta). RESULTS: In MTT assay, the cell viability ratios of the combination group at serial time points from 12, 24, 36, 48 and 72 hours were (65.2 +/- 5.8)%, (58.3 +/- 14.4)%, (35.3 +/- 5.0)%, (19.2 +/- 1.5)%, and (11.4 +/- 2.5)%, which were significantly lower than those of the paclitaxel group (P < 0.05). After drug treatments, apoptosis rates of paclitaxel group, bortezomib group and the combination group were (14.7 +/- 0.5)%, (15.1 +/- 0.8)% and (20.5 +/- 0.7)% respectively. The rate of the combination group was significantly higher than that of non-treated group and paclitaxel group (P < 0.05). Western blot assay showed the changes in expression levels of phosphorylated AKT and GSK-3beta, which were decreased significantly after paclitaxel and bortezomib combination treatment [(3.2 +/- 0.8)%, (19.3 +/- 0.4)%; P < 0.05]. CONCLUSIONS: The lethal effect of paclitaxel on tumor cells could be increased significantly by its combination with proteasome inhibitors, bortezomib. The AKT/GSK-3beta signaling pathway plays an important role in the molecular mechanism of the combination treatment.