Elevated expression of ECT2 as a diagnostic marker and prognostic indicator in endometrial cancer.

OBJECTIVES: The study aims to investigate genes associated with endometrial cancer (EC) progression to identify new biomarkers for early detection. METHODS: Differentially expressed genes (DEGs), Series test of cluster (STC) and protein-protein interaction analyses identified hub genes in EC. Clinical samples were utilized to examine the expression pattern of ECT2, assess its prognostic value, and evaluate its diagnostic potential. RESULTS: Upregulated DEGs were significantly enriched in cancer-related processes and pathways. Validations across databases identified ASPM, ATAD2, BUB1B, ECT2, KIF14, NUF2, NCAPG, and SPAG5 as potential hub genes, with ECT2 exhibiting the highest diagnostic efficacy. The expression levels of ECT2 varied significantly across different clinical stages, pathological grades, and metastasis statuses in UCEC. Furthermore, ECT2 mRNA was upregulated in the p53abn group, indicating a poorer prognosis, and downregulated in the MMRd and NSMP groups, suggesting a moderate prognosis. In clinical samples, ECT2 expression increased from normal endometria and endometrial hyperplasia without atypia (EH) to atypical endometrial hyperplasia (AH) and EC, effectively distinguishing between benign and malignant endometria. High ECT2 expression was associated with an unfavourable prognosis. CONCLUSIONS: ECT2 expression significantly rises in AH and EC, showing high accuracy in distinguishing between benign and malignant endometria. ECT2 emerges as a promising biomarker for diagnosing endometrial neoplasia and as a prognostic indicator in EC.

Tumor-to-stroma cd8+ t cells ratio combined with cancer-associated fibroblasts: an innovative approach to predicting lymph node metastases of cervical cancer.

PURPOSE: Precise identification of lymph node metastases is vital for the management of cervical cancer. However, the existing diagnostic methods for lymph node metastases have certain drawbacks. In this study, we aim to explore the expression of cancer-associated fibroblasts (CAFs) and tumor-to-stroma CD8+ T cells ratio (CD8+ T cells T:S ratio) and its association with lymph node metastases of cervical cancer. METHODS: Hundred and ten cervical cancer tissues and 39 biopsy tissues from patients were investigated immunocytochemically for the expression of CAFs and CD8+ T cells. The statistical correlation analysis was carried out using the SPSS system. RESULTS: A strong and statistically significant negative correlation (r= - 0.690; P < 0.001) was observed between CAF density and CD8+ T cells T:S ratio. Not only were CAFs density and CD8+ T cells T:S ratio correlated with lymph node metastases respectively (P < 0.001), but the combination of them also significantly correlated with lymph node metastases (P < 0.001). Then, we constructed the combined diagnosis model (Logit (P) = - 4.446 + 0.300 × CAFs + 0.752 × CD8+ T cells T:S Ratio) of cervical cancer lymph node metastases. ROC curves analysis showed that the ROC curves areas for CAFs, CD8+ T cells T:S ratio, and a combination of both are 0.879, 0.747, and 0.951. Then, the prediction model was verified by biopsy specimens and consistent results were obtained. CONCLUSIONS: The combination of CAF density and CD8+ T cells T:S ratio has a significant predictive value for lymph node metastases in patients with cervical cancer.

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.

Adjuvant chemotherapy versus adjuvant concurrent chemoradiotherapy after radical surgery for early-stage cervical cancer: a randomized, non-inferiority, multicenter trial.

We conducted a prospective study to assess the non-inferiority of adjuvant chemotherapy alone versus adjuvant concurrent chemoradiotherapy (CCRT) as an alternative strategy for patients with early-stage (FIGO 2009 stage IB-IIA) cervical cancer having risk factors after surgery. The condition was assessed in terms of prognosis, adverse effects, and quality of life. This randomized trial involved nine centers across China. Eligible patients were randomized to receive adjuvant chemotherapy or CCRT after surgery. The primary end-point was progression-free survival (PFS). From December 2012 to December 2014, 337 patients were subjected to randomization. Final analysis included 329 patients, including 165 in the adjuvant chemotherapy group and 164 in the adjuvant CCRT group. The median follow-up was 72.1 months. The three-year PFS rates were both 91.9%, and the five-year OS was 90.6% versus 90.0% in adjuvant chemotherapy and CCRT groups, respectively. No significant differences were observed in the PFS or OS between groups. The adjusted HR for PFS was 0.854 (95% confidence interval 0.415-1.757; P = 0.667) favoring adjuvant chemotherapy, excluding the predefined non-inferiority boundary of 1.9. The chemotherapy group showed a tendency toward good quality of life. In comparison with post-operative adjuvant CCRT, adjuvant chemotherapy treatment showed non-inferior efficacy in patients with early-stage cervical cancer having pathological risk factors. Adjuvant chemotherapy alone is a favorable alternative post-operative treatment.

Comprehensive Analysis of Copy Number Variation, Nucleotide Mutation, and Transcription Level of PPAR Pathway-Related Genes in Endometrial Cancer.

Endometrial cancer is a common malignant tumor in gynecology, and the prognosis of advanced patients is dismal. Recently, many studies on the peroxisome proliferator-activated receptor pathway have elucidated its crucial involvement in endometrial cancer. Copy number variation (CNA) and nucleotide mutations often occur in tumor tissues, leading to abnormal protein expression and changes in protein structure. We analyzed the exon sequencing data of endometrial cancer patients in the TCGA database and found that somatic changes in PPAR pathway-related genes (PPAR-related-gene) often occur in UCEC patients. Patients with CNA or mutation changes in the exon region of the PPAR-related-gene usually have different prognostic outcomes. Furthermore, we found that the mRNA transcription and protein translation levels of PPAR-related-gene in UCEC are significantly different from that of adjacent tissues/normal uterus. The transcription level of some PPAR-related-gene (DBI, CPT1A, CYP27A1, and ME1) is significantly linked to the prognosis of UCEC patients. We further constructed a prognostic predicting tool called PPAR Risk score, a prognostic prediction tool that is a strong independent risk factor for the overall survival rate of UCEC patients. Comparing to the typical TNM classification system, this tool has higher prediction accuracy. We created a nomogram by combining PPAR Risk score with clinical characteristics of patients in order to increase prediction accuracy and promote clinical use. In summary, our study demonstrated that PPAR-related-gene in UCEC had significant alterations in CNA, nucleotide mutations, and mRNA transcription levels. These findings can provide a fresh perspective for postoperative survival prediction and individualized therapy of UCEC patients.

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.

Erratum: Loss of E-cadherin promotes prostate cancer metastasis via upregulation of metastasis-associated gene 1 expression.

[This corrects the article DOI: 10.3892/ol.2012.934.].

Treatment and clinical outcomes of cervical cancer during pregnancy.

BACKGROUND: This study aims to investigate clinicopathological factors associated with survival rate and treatment of patients with cervical cancer during pregnancy (CCP). METHODS: A total of 92 patients diagnosed CCP were retrospectively reviewed. One patient was from Nanfang Hospital of Southern Medical University, 5 patients were from Tongji Hospital, and 86 patients were from case reports in the PubMed database from 1961 to 2019. Patients and tumor characteristics were evaluated. Kaplan-Meier and Cox regression methods were used to analyze the 5-year disease-specific survival (DSS). RESULTS: Most patients (73 cases) were stage I according to the 2018 International Federation of Gynecology and Obstetrics (FIGO) standards. Twelve patients (13.04%) terminated pregnancy once diagnosed. These patients were diagnosed at the mean gestational age (GA) of 11±3 weeks, during early pregnancy. For the rest of the patients (80 cases) who continued pregnancy, the mean GA was 35±2 weeks at delivery. There was a significant difference in survival whether the treatment was performed once diagnosed or not. The 5-year DSS was 75% in adenocarcinoma (AC), 68.5% in squamous cell carcinoma (SCC), and 43.7% in the rare subtype. Among the 38 patients who underwent neoadjuvant chemotherapy (NACT), one patient suffered from spontaneous abortion, and one baby experienced acute myeloid leukemia (AML) ex-FAB (French-American-British)-M7 subtype and received bone marrow transplantation. Other delivered newborns showed no abnormality or malformation. Cox multi-factorial analysis demonstrated that tumor size (2 cm) was an independent overall survival predictor for CCP patients (P<0.05). CONCLUSIONS: Tumor size was an independent prognostic factor of survival in CCP patients. Pregnancy has adverse effects on the prognosis of cervical cancer. Personalized treatment is a priority for CCP patients.

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.

Clinicopathological Aspects of Small Cell Neuroendocrine Carcinoma of the Uterine Cervix: a Multicenter Retrospective Study and Meta-Analysis.

BACKGROUND/AIMS: To evaluate the clinicopathologic aspects of small cell neuroendocrine carcinoma of the uterine cervix (SCNEC). METHODS: A retrospective review of 40 patients with SCNEC in 3 hospitals from 2009 to 2015 was conducted to assess the survival rates and examine the associations between clinicopathological variables and overall survival (OS). A meta-analysis of 22 studies containing 1901 patients was also conducted to further confirm the results. RESULTS: In the clinical group of 40 patients, the 5-year OS rate was 20%. Advanced International Federation of Gynecology and Obstetrics (FIGO) stage and radiation therapy (RT) were associated with poor survival. However, radical surgery was associated with prolonged survival. In the meta-analysis of 1901 patients, the 2-year disease-free survival (DFS) rate, 5-year DFS rate, 2-year OS rate, 3-year OS rate and 5-year OS rate of SCNEC were 48%, 35%, 62%, 35%, and 35% respectively. Advanced FIGO stage, larger tumor size, lymph node metastasis (LNM) (+), lymphovascular space involvement (LVSI) (+), parametrial involvement (PI) (+), depth of stromal invasion (DSI) > 2/3, and RT were associated with poor survival. However, a chemotherapy regimen similar to that for small cell lung cancer was associated with prolonged survival. CONCLUSION: Advanced FIGO stage, larger tumor size, LNM (+), LVSI (+), DSI > 2/3, PI (+), and RT were independent predictors of poor prognosis of SCNEC. Radical surgery combined with a chemotherapy regimen similar to that of small cell lung cancer may be a potential therapeutic approach for SCNEC.

Preoperative SCC-Ag and thrombocytosis as predictive markers for pelvic lymphatic metastasis of squamous cervical cancer in early FIGO stage.

Objectives: To explore the clinical significance of squamous cell carcinoma antigen (SCC-Ag) and thrombocytosis to predict pelvic lymphatic metastasis (PLM) of squamous cervical cancer (SCC) in International Federation of Gynecology and Obstetrics (FIGO) stages IA-IIA. Methods: A retrospective clinicopathologic review of 782 patients of a primary cohort in three Chinese hospitals from 2010 to 2015, and 407 patients of a validation cohort in another institution from 2015 to 2017. A receiver operating characteristic curve was used to determine the optimal SCC-Ag threshold to predict PLM in the groups. Univariate and multivariate logistic analyses for PLM were performed to assess differences in outcome. Results: In the primary and validation cohort, 15.6% (122/782) and 25.3% (103/407) patients were classified into the thrombocytosis group (platelet count >300 × 109/L), respectively. Optimal cutoff values of SCC-Ag for predicting PLM of the thrombocytosis group and the normal group were 3.26 ng/mL (AUC 0.754; sensitivity 73.08%; specificity 72.92%; P = 0.000) and 4.58 ng/mL (AUC 0.706; sensitivity 53.26%; specificity 83.98%; P = 0.000), respectively, in the primary cohort, and 1.55 ng/mL (AUC 0.705; sensitivity 79.31%; specificity 55.41%; P = 0.000) and 1.75 ng/mL (AUC 0.655; sensitivity 69.57%; specificity 64.26%; P = 0.000), respectively, in the validation cohort. In multivariate logistic analysis, preoperative SCC-Ag over 3.26 ng/mL and lymphovascular space involvement were the significant predictors of PLM for SCC in FIGO stages IA-IIA. Conclusions: Preoperative SCC-Ag alone or combined with thrombocytosis might be used as predictive markers for PLM before initial treatment in early stage SCC.

The novel fusion protein sTRAIL-TMTP1 exhibits a targeted inhibition of primary tumors and metastases.

UNLABELLED: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells and enhance therapy in vivo. However, concerns regarding its considerable liver toxicity limit its use in humans as a cancer therapy. Tumor molecular targeted peptide 1 (TMTP1) has previously been reported by our laboratory to target primary tumors and metastatic foci. Here, we report a novel recombinant fusion protein, sTRAIL-TMTP1, which not only induced apoptosis in cancer cells in vitro but also inhibited tumor growth and metastases in vivo. Moreover, sTRAIL-TMTP1 impacted tumor angiogenesis. The biodistribution results demonstrated that sTRAIL-TMTP1 accumulation peaked 6 h after injection and persisted for 24 h in the tumor, while faint sTRAIL-TMTP1 accumulation was detected in normal organs in tumor-bearing mice. Thus, combining sTRAIL with TMTP1 resulted in high anti-tumor activity and low toxicity. In conclusion, the novel fusion protein sTRAIL-TMTP1 was successfully developed in our laboratory and was observed to induce apoptosis both in primary and metastatic cancer, which may lead to the development of novel, targeted anticancer agents. KEY MESSAGE: Construction, purification, and characterization of the novel recombinant fusion protein, sTRAIL-TMTP1. sTRAIL-TMTP1 not only induce apoptosis in cancer cells but inhibit tumor growth and metastasis. sTRAIL-TMTP1 showed an impact on caspase activity and tumor angiogenesis. sTRAIL-TMTP1's accumulate in tumor with little accumulation in normal organs.

DT390-triTMTP1, a novel fusion protein of diphtheria toxin with tandem repeat TMTP1 peptide, preferentially targets metastatic tumors.

Peptide-based therapies have emerged as one of the most promising therapeutics strategy in cancer-targeted therapy. Using our laboratory newly identified peptide TMTP1 and diphtheria toxin, we developed a new fusion protein that showed remarkable ability to target highly metastatic tumors. Fusion protein toxins were generated by fusing the first 390 amino acids of diphtheria toxin [truncated diphtheria toxin (DT390)] to different repeats of peptide TMTP1 (DT390-TMTP1, DT390-biTMTP1, and DT390-triTMTP1). Efficacies of the recombinant fusion proteins on tumor growth and metastasis were evaluated in vitro and in vivo. DT390-triTMTP1 showed the most powerful toxicity against cancer, which led to tumor growth retardation or regression and prolonged survival of human prostate cancer PC-3M-1E8 subcutaneously bearing or gastric cancer MKN-45 orthotopic nude mice. Increased TUNEL and caspase-3 staining and reduced ki67 staining in tumor cells suggested that the anticancer effects of DT390-triTMTP1 were through selectively inducing apoptosis and inhibiting proliferation of cancer cells. In a murine model of human orthotopic gastric carcinoma, DT390-biTMTP1 significantly inhibited metastases to liver and spleen, while DT390-triTMTP1 not only totally suppressed metastasis but also reduced primary tumors by 66.6%. In the biodistribution test, DT390-triTMTP1 was observed to home to tumor tissue rapidly and lasted over 48 h, with only a transient appearance in liver and kidney immediately after injection. Thus, our present study provided a novel recombinant fusion protein DT390-triTMTP1 with preferential targeting and high cytotoxicity, which may be a promising strategy for the targeted therapy of cancer metastasis.

Akt mediates metastasis-associated gene 1 (MTA1) regulating the expression of E-cadherin and promoting the invasiveness of prostate cancer cells.

Human metastasis-associated gene 1 (MTA1) is highly associated with the metastasis of prostate cancer; however, the molecular functions of MTA1 that facilitate metastasis remain unclear. In this study, we demonstrate that the silencing of MTA1 by siRNA treatment results in the upregulation of E-cadherin expression by the phosphorylation of AKT (p-AKT) and decreases the invasiveness of prostate cancer cells. We show that MTA1 is expressed in over 90% of prostate cancer tissues, especially metastatic prostate cancer tissue, comparing to non-expression in normal prostate tissue. RT-PCR analysis and Western blot assay showed that MTA1 expression is significantly higher in highly metastatic prostate cancer PC-3M-1E8 cells (1E8) than in poorly metastatic prostate cancer PC-3M-2B4 cells (2B4). Silencing MTA1 expression by siRNA treatment in 1E8 cells increased the cellular malignant characters, including the cellular adhesive ability, decreased the cellular invasive ability and changed the polarity of cellular cytoskeleton. 1E8 cells over-expressing MTA1 had a reduced expression of E-cadherin, while 1E8 cells treated with MTA1 siRNA had a higher expression of E-cadherin. The expression of phosphorylated AKT (p-AKT) or the inhibition of p-AKT by wortmannin treatment (100 nM) significantly altered the function of MTA1 in the regulation of E-cadherin expression. Alterations in E-cadherin expression changed the role of p-AKT in cellular malignant characters. All of these results demonstrate that MTA1 plays an important role in controlling the malignant transformation of prostate cancer cells through the p-AKT/E-cadherin pathway. This study also provides a new mechanistic role for MTA1 in the regulation of prostate cancer metastasis.

Loss of E-cadherin promotes prostate cancer metastasis via upregulation of metastasis-associated gene 1 expression.

E-cadherin is a key cell-to-cell adhesion molecule associated with the invasion and metastasis of tumor cells; however, the molecular mechanisms are not entirely understood. In this study, we investigated whether downregulation of E-cadherin by E-cadherin-specific small intefering RNA (siRNA) was able to promote malignant phenotypes of prostate cancer cells through upregulating the metastasis-associated gene 1 (MTA1) in vitro. The expression levels of E-cadherin in human paired prostate adenocarcinoma cell lines, PC-3M-2B4 (2B4) and PC-3M-1E8 (1E8), were investigated using western blot analysis. The alteration of malignant phenotypes associated with decreasing E-cadherin expression were assessed in 2B4 cells using wound-healing assays, solid-phase adhesion assays, invasion assays and cytoskeletal staining. The expression of E-cadherin and MTA1 in normal, localized and metastatic prostate cancer cells was analyzed using immunohistochemistry. Downregulation of E-cadherin using an RNA interference approach led to the upregulation of MTA1 expression, decreased tumor cell adhesion ability as well as enhanced cell mobility, invasion and cellular polarity compared with the controls (P<0.05). E-cadherin regulated MTA1 in a time-dependent manner. The correlation between E-cadherin and MTA1 was inversed in the prostate cancer group (P<0.05; r(s)=-0.434). The data suggest that E-cadherin plays an important role in prostate cancer metastasis, which is likely to be due to the regulation of MTA1 expression. E-cadherin may combine with MTA1 and alter the malignant phenotype of prostate cancer cells. A combined testing strategy for detecting MTA1 and E-cadherin may be sufficient for selecting high-risk patients with metastasis. Therefore, E-cadherin and MTA1 may be potential powerful factors for the treatment of various types of cancer.