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.

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.

Clinical Significance of CD163+ and CD68+ Tumor-associated Macrophages in High-risk HPV-related Cervical Cancer.

Objective. To explore the influence of M2-polarized tumor-associated macrophages (TAMs) on high-risk human papillomavirus (hr-HPV)-related cervical carcinogenesis and metastasis. Methods. CD68+ and CD163+ macrophages were examined immunohistochemically in a series of 130 samples, including 26 cases of normal cervical tissues, 59 cases of cervical intraepithelial neoplasia (CIN), and 45 cases of squamous cell carcinoma (SCC), and the results were statistically analyzed. The macrophage count was corrected for the epithelial and stromal compartments respectively. Clinical data were also obtained. Results. High counts of CD68+ and CD163+ macrophages were associated with hr-HPV infection (both p < 0.05) and positively correlated with cervical carcinogenesis (Spearman's rho = 0.478, p = 0.000; Spearman's rho = 0.676, p =0.000, respectively). The immunostaining pattern of CD163 exhibited clearer background than that of CD68. CD163+ macrophages showed a more obviously increasing migration into the epithelium along with the progression of CIN to invasive cancer. Notably, a high index of CD163+ macrophages was significantly associated with higher FIGO stages (p = 0.009) and lymph node metastasis (p = 0.012), but a similar finding was not found for CD68+ macrophages (p = 0.067, p = 0.079, respectively). Conclusions. Our study supported a critical role of TAMs as a prospective predictor for hr-HPV-related cervical carcinogenesis. CD163, as a promising TAMs marker, is superior to CD68 for predicting the malignant transformation and metastatic potential of cervical cancer.