Correction: Systemic Delivery of MicroRNA-101 Potently Inhibits Hepatocellular Carcinoma In Vivo by Repressing Multiple Targets.

[This corrects the article DOI: 10.1371/journal.pgen.1004873.].

Microvascular fractal dimension predicts prognosis and response to chemotherapy in glioblastoma: an automatic image analysis study.

The microvascular profile has been included in the WHO glioma grading criteria. Nevertheless, microvessels in gliomas of the same WHO grade, e.g., WHO IV glioblastoma (GBM), exhibit heterogeneous and polymorphic morphology, whose possible clinical significance remains to be determined. In this study, we employed a fractal geometry-derived parameter, microvascular fractal dimension (mvFD), to quantify microvessel complexity and developed a home-made macro in Image J software to automatically determine mvFD from the microvessel-stained immunohistochemical images of GBM. We found that mvFD effectively quantified the morphological complexity of GBM microvasculature. Furthermore, high mvFD favored the survival of GBM patients as an independent prognostic indicator and predicted a better response to chemotherapy of GBM patients. When investigating the underlying relations between mvFD and tumor growth by deploying Ki67/mvFD as an index for microvasculature-normalized tumor proliferation, we discovered an inverse correlation between mvFD and Ki67/mvFD. Furthermore, mvFD inversely correlated with the expressions of a glycolytic marker, LDHA, which indicated poor prognosis of GBM patients. Conclusively, we developed an automatic approach for mvFD measurement, and demonstrated that mvFD could predict the prognosis and response to chemotherapy of GBM patients.

High-mobility group box 1 released by autophagic cancer-associated fibroblasts maintains the stemness of luminal breast cancer cells.

Cancer stem cells/cancer-initiating cells (CICs) and their microenvironmental niche play a vital role in malignant tumour recurrence and metastasis. Cancer-associated fibroblasts (CAFs) are major components of the niche of breast cancer-initiating cells (BCICs), and their interactions may profoundly affect breast cancer progression. Autophagy has been considered to be a critical process for CIC maintenance, but whether it is involved in the cross-talk between CAFs and CICs to affect tumourigenesis and pathological significance has not been determined. In this study, we found that the presence of CAFs containing high levels of microtubule-associated protein 1 light chain 3 (LC3II), a marker of autophagosomes, was associated with more aggressive luminal human breast cancer. CAFs in human luminal breast cancer tissues with high autophagy activity enriched BCICs with increased tumourigenicity. Mechanistically, autophagic CAFs released high-mobility group box 1 (HMGB1), which activated its receptor, Toll-like receptor (TLR) 4, expressed by luminal breast cancer cells, to enhance their stemness and tumourigenicity. Furthermore, immunohistochemistry of 180 luminal breast cancers revealed that high LC3II/TLR4 levels predicted an increased relapse rate and a poorer prognosis. Our findings demonstrate that autophagic CAFs play a critical role in promoting the progression of luminal breast cancer through an HMGB1-TLR4 axis, and that both autophagy in CAFs and TLR4 on breast cancer cells constitute potential therapeutic targets. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Systemic delivery of microRNA-101 potently inhibits hepatocellular carcinoma in vivo by repressing multiple targets.

Targeted therapy based on adjustment of microRNA (miRNA)s activity takes great promise due to the ability of these small RNAs to modulate cellular behavior. However, the efficacy of miR-101 replacement therapy to hepatocellular carcinoma (HCC) remains unclear. In the current study, we first observed that plasma levels of miR-101 were significantly lower in distant metastatic HCC patients than in HCCs without distant metastasis, and down-regulation of plasma miR-101 predicted a worse disease-free survival (DFS, P<0.05). In an animal model of HCC, we demonstrated that systemic delivery of lentivirus-mediated miR-101 abrogated HCC growth in the liver, intrahepatic metastasis and distant metastasis to the lung and to the mediastinum, resulting in a dramatic suppression of HCC development and metastasis in mice without toxicity and extending life expectancy. Furthermore, enforced overexpression of miR-101 in HCC cells not only decreased EZH2, COX2 and STMN1, but also directly down-regulated a novel target ROCK2, inhibited Rho/Rac GTPase activation, and blocked HCC cells epithelial-mesenchymal transition (EMT) and angiogenesis, inducing a strong abrogation of HCC tumorigenesis and aggressiveness both in vitro and in vivo. These results provide proof-of-concept support for systemic delivery of lentivirus-mediated miR-101 as a powerful anti-HCC therapeutic modality by repressing multiple molecular targets.

Ubiquitin specific peptidase 5 mediates Histidine-rich protein Hpn induced cell apoptosis in hepatocellular carcinoma through P14-P53 signaling.

Hpn is a small histidine-rich cytoplasmic protein from Helicobacter pylori and has been recognized as a high-risk factor for several cancers including gastric cancer, colorectal cancer, and MALT lymphoma. However, the relationship between Hpn and cancers remains elusive. In this study, we discovered that Hpn protein effectively suppressed cell growth and induced apoptosis in hepatocellular carcinoma (HCC). A two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics was performed to find the molecular targets of Hpn in HCC cells. It was identified that twelve proteins were differentially expressed, with USP5 being one of the most significantly downregulated protein. The P14ARF -P53 signaling was activated by USP5 knockdown in HCC cells. Furthermore, USP5 overexpression significantly rescued the suppressive effect of Hpn on the viability of HCC cells. In conclusion, our study suggests that Hpn plays apoptosis-inducing roles through suppressing USP5 expression and activating the P14ARF -P53 signaling. Therefore, Hpn may be a potential candidate for developing novel anti-HCC drugs.

MiR-34a modulates ErbB2 in breast cancer.

Breast cancer is the second highest cause of carcinoma-related death caused by distant metastasis in women. Estrogen receptor (ER), human epidermal growth factor receptor 2, (HER2) and progesterone receptor (PR) are three classified makers of breast cancer, which are defined as ER+, HER2+, and the most serious ER-PR-HER2- (triple-negative). It is well known that ErbB2 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2) plays an important part in breast cancer. However, the molecular mechanisms underlying ErbB2 action needs to be well studied. In this report, we discovered that the decreased expression levels of miR-34a were inversely correlated with the increased ErbB2 levels in breast cancer. A luciferase reporter assay was done to understand the potential correlation between ErbB2 and miR-34a. Over-expression of miR-34a reduces ErbB2 expression and suppresses breast cancer cell invasion and growth in vitro. What's more, reduced expression of ErbB2 inhibits breast Cancer cell proliferation and re-expression of ErbB2 reversed miR-34a-dependent tumor suppression. Meanwhile, miR-34a levels were correlated inversely with breast cancer malignancy. Our study demonstrates that miR-34a, like ErbB2, might be a diagnostic target in breast cancer.

Vastatin, an Endogenous Antiangiogenesis Polypeptide That Is Lost in Hepatocellular Carcinoma, Effectively Inhibits Tumor Metastasis.

Hepatocellular carcinoma (HCC) is a hypervascular cancer without effective treatment. Here we report that polypeptide of NC1 domain of type VIII collagen (Vastatin) is an endogenous polypeptide expressed in normal liver tissue but lost in the liver of most HCC patients (73.1%). Its expression level is negatively associated with tumor size (P = 0.035) and metastasis (P = 0.016) in HCC patients. To evaluate its potential use as a therapeutic, we constructed a recombinant adeno-associated virus carrying Vastatin (rAAV-Vastatin) to treat HCC in an orthotopic Buffalo rat model. rAAV-Vastatin treatment significantly prolonged the median survival, inhibited tumor growth, and completely prevented metastasis in HCC-bearing rats by decreasing microvessel density and increasing tumor necrosis. No detectable toxicity in nontumor-bearing mice was observed. To investigate its molecular mechanisms, we performed DNA microarray, western blotting assays, and bioinformatic analysis to determine its effect on global gene expression patterns and signal transduction pathways. Our results indicated that rAAV-Vastatin significantly reduced the expressions of Pck1, JAG2, and c-Fos, thus inhibiting the cellular metabolism, Notch and AP-1 signaling pathways, respectively. Hence, we demonstrated for the first time that Vastatin is a novel, safe, and effective antiangiogenic therapeutic and a potential biomarker for HCC.

Comparison of Iron-Binding Ability Between Thr70-NapA and Ser70-NapA of Helicobacter pylori.

BACKGROUND: The neutrophil-activating protein (NapA) of Helicobacter pylori (H. pylori), with DNA-binding and iron seizing properties, is a fundamental virulence factor involved in H. pylori-related diseases. Compared with Ser70-NapA strain, Thr70-NapA strain is more intimately correlated with iron-deficiency anemia. METHODS: To investigate whether two types of proteins differ in iron-binding ability, mutated Thr70-NapA and Ser70-NapA strains were established. Isothermal titration calorimetry (ITC) method was conducted to measure the binding between the NapA protein and Fe(2+) . The structural changes of NapA protein were also tested during iron interaction by fast protein liquid chromatography (FPLC) and circular dichroism (CD) methods. DNA-binding assay was performed for evaluate the affinity of both mutated and wild types of NapA with DNA. RESULTS: Mutated Thr70-NapA had higher iron-binding ability than wild Ser70-NapA. The structural stability of Thr70-NapA was disrupted and became more active along with the rising concentration of Fe(2+) , whereas no similar association was observed between Ser70-NapA and Fe(2+) level. When the iron/protein molar ratio ranged from 10 to 20, both Ser70-NapA and Thr70-NapA displayed weaker DNA-binding ability. CONCLUSIONS: Thr70-NapA has much stronger ability to sequester ferrous ion compared with Ser70-NapA in H. pylori. In addition, the DNA-binding property of NapA is dependent upon the Fe(2+) concentration.

Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer.

BACKGROUND: Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. OBJECTIVE: To identify recurrent somatic mutations with prognostic significance in patients with CRC. METHOD: Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. RESULTS: Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6-14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. CONCLUSIONS: The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging.

Hotair mediates hepatocarcinogenesis through suppressing miRNA-218 expression and activating P14 and P16 signaling.

BACKGROUND & AIMS: Long non-coding RNA Hotair has been considered as a pro-oncogene in multiple cancers. Although there is emerging evidence that reveals its biological function and the association with clinical prognosis, the precise mechanism remains largely elusive. METHODS: We investigated the function and mechanism of Hotair in hepatocellular carcinoma (HCC) cell models and a xenograft mouse model. The regulatory network between miR-218 and Hotair was elucidated by RNA immunoprecipitation and luciferase reporter assays. Finally, the correlation between Hotair, miR-218 and the target gene Bmi-1 were evaluated in 52 paired HCC specimens. RESULTS: In this study, we reported that Hotair negatively regulated miR-218 expression in HCC, which might be mediated through an EZH2-targeting-miR-218-2 promoter regulatory axis. Further investigation revealed that Hotair knockdown dramatically inhibited cell viability and induced G1-phase arrest in vitro and suppressed tumorigenicity in vivo by promoting miR-218 expression. Oncogene Bmi-1 was shown to be a functional target of miR-218, and the main downstream targets signaling, P16(Ink4a) and P14(ARF), were activated in Hotair-suppressed tumorigenesis. In primary human HCC specimens, Hotair and Bmi-1 were concordantly upregulated whereas miR-218 was downregulated in these tissues. Furthermore, Hotair was inversely associated with miR-218 expression and positively correlated with Bmi-1 expression in these clinical tissues. CONCLUSION: Hotair silence activates P16(Ink4a) and P14(ARF) signaling by enhancing miR-218 expression and suppressing Bmi-1 expression, resulting in the suppression of tumorigenesis in HCC.

MiR-218-targeting-Bmi-1 mediates the suppressive effect of 1,6,7-trihydroxyxanthone on liver cancer cells.

Traditional Chinese medicine is recently emerged as anti-cancer therapy or adjuvant with reduced side-effects and improved quality of life. In the present study, an active ingredient, 1,6,7-trihydroxyxanthone (THA), derived from Goodyera oblongifolia was found to strongly suppress cell growth and induce apoptosis in liver cancer cells. MicroRNAs are a group of small non-coding RNAs that regulate gene expression at post-transcriptional levels. Our results demonstrated that miR-218 was up-regulated and oncogene Bmi-1 was down-regulated by THA treatment. Further investigation showed that THA-induced-miR-218 up-regulation could lead to activation of tumor suppressor P16(Ink4a) and P14(ARF), the main down-stream targets of Bmi-1. In conclusion, THA might be a potential anti-cancer drug candidate, at least in part, through the activation of miR-218 and suppression of Bmi-1 expression.

The interaction between human enteroviruses and type I IFN signaling pathway.

Human enteroviruses (HEV), very common and important human pathogens, cause infections in diverse ways. Recently, the large epidemic of HFMD caused by HEV infection became a growing threat to public health in China. As the first line of immune response, the type I interferon (IFN-α/ß) pathway plays an essential role in antiviral infection, particularly in limiting both the early and late stages of infection. Because of co-evolution with the host, the viruses have evolved multiple strategies to evade or subvert the host immunity to ensure their survival. In this paper, we systematically reviewed and summarized the interaction between HEV infections and host type I IFN responses. We firstly described the recent findings of HEV recognition and IFN induction, specifically on host pattern-recognition receptors (PRRs) in HEV infection. Then we discussed the antiviral effect of IFN in HEV infection. Finally, we timely summarized the mechanisms of HEV to circumvent the IFN responses. Clarification of the complexity in this battle may provide us new strategies for prevention and antiviral treatment.

Overexpression of NKX6.1 is closely associated with progressive features and predicts unfavorable prognosis in human primary hepatocellular carcinoma.

The homeobox gene NKX6.1 was recently identified in cervical tumors. This study was designed to explore the clinical and prognostic significance of NKX6.1 further in patients with primary hepatocellular carcinoma (HCC). The expression levels of NKX6.1 were examined using real-time PCR, Western blotting, and immunohistochemistry in HCC cell lines and HCC tissues. The invasion capability of cell lines following silencing or overexpression of NKX6.1 was investigated by Transwell assay. Cells proliferation was tested by MTT assays. Epithelial-mesenchymal transition (EMT) marker expression levels were detected in relation to NKX6.1 expression. Correlation between NKX6.1 immunohistochemical staining, clinicopathologic parameters, and follow-up data of HCC patients was analyzed statistically. NKX6.1 expression was higher in HCC tissues compared to the adjacent noncancerous tissue. NKX6.1 overexpression was significantly correlated with tumor size, tumor differentiation, clinical stage, metastasis, and relapse. Kaplan-Meier analysis revealed that NKX6.1 overexpression was related to unfavorable 5-year disease-free survival and overall survival. Importantly, multivariate analysis indicated that NKX6.1 overexpression was an independent unfavorable marker for overall survival. Moreover, a significant relationship was observed between NKX6.1 and EMT marker expression levels, and NKX6.1 knockdown inhibited cell invasion, and overexpression of NKX6.1 promotes cell proliferation in vitro. NKX6.1 is upregulated in HCC and is a reliable prognostic marker for patients with HCC.

Low expression of BARX2 in human primary hepatocellular carcinoma correlates with metastasis and predicts poor prognosis.

AIM: The homeobox gene Barx2 was recently identified as a regulator of ovarian and breast cancer; however, the expression level of BARX2 and its significance in hepatocellular carcinoma (HCC) remain unknown. METHODS: Protein and mRNA expression levels of Barx2 were examined using Western blotting and real-time PCR respectively, in paired HCC tissue and matched adjacent non-cancerous tissue from 12 patients. The expression levels of epithelial-mesenchymal transition (EMT) markers were also detected in relation to BARX2 expression. Lastly, immunohistochemistry for BARX2 was also performed on a tissue microarray containing 231 HCC tissue samples. RESULTS: We observed that BARX2 expression was lower in HCC tissues compared to matching adjacent non-cancerous tissue. The low expression level of BARX2 was significantly correlated with metrics of tumor size, tumor differentiation, clinical stage, metastasis and relapse. Furthermore, the patients with low BARX2 expression had adverse survival outcomes. Importantly, multivariate Cox regression analysis revealed that low BARX2 expression was an independent marker for lower overall survival (P = 0.007). Moreover, a significant negative relationship was observed between the expression of BARX2 and markers of EMT. CONCLUSION: These findings provide evidence that the low expression level of BARX2 in HCC is significantly correlated with tumor metastasis, and that BARX2 may be an independent prognostic biomarker for patients with HCC.

Staphylococcal enterotoxin C2 promotes osteogenesis and suppresses osteoclastogenesis of human mesenchymal stem cells.

As a super-antigen, staphylococcal enterotoxin C2 (SEC2) stimulates the release of massive inflammatory cytokines such as interferon-gamma (IFN-γ), interleukin-1 (IL-1) and interleukin-2 (IL-2) which are documented to implicate osteoblast differentiation. In the present study, SEC2 was found to significantly improve the osteoblast differentiation by up-regulating BMP2 and Runx2/Cbfa1 expression. Interferon (IFN)-inducible gene IFI16, a co-activator of Runx2/Cbfa1, was also activated by SEC2 in the osteoblast differentiation. In addition, exogenous introduction of SEC2 stimulated OPG expression and suppressed RANKL, suggesting suppression of osteoclastogenesis in hMSCs. Therefore, our results displayed that SEC2 plays an important role in the commitment of MSC to the osteoblast and it might be a potential new therapeutic candidate for bone regeneration.

Suppression of tumorigenicity by microRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme.

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to identify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.

1,3,5-Trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone directly targets heat shock protein 27 in hepatocellular carcinoma.

We previously showed that the small molecule 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP) induces apoptosis in hepatocellular carcinoma (HCC) by suppressing Hsp27 expression, although the mechanism is not fully understood. To investigate the functional association between TDP and Hsp27 protein in HCC, recombinant Hsp27 protein was incubated with TDP at room temperature, and assayed by mass spectrum (MS) and natural electrophoresis. TDP effectively stimulated Hsp27 to form aggregates ex vitro, leading to suppression of its chaperone activity. The aggregates were degraded by the ubiquitin-proteasome (UPS) pathway. TDP directly interacted with Asp17 and Phe55 in chain C of Hsp27 on the basis of bioinformatic prediction. In conclusion, Hsp27 is a direct target of TDP in its anti-cancer activity, which provides strong support for a clinical application.

MicroRNA-218 inhibits melanogenesis by directly suppressing microphthalmia-associated transcription factor expression.

The microphthalmia-associated transcription factor (MITF) is a pivotal regulator of melanogenic enzymes for melanogenesis, and its expression is modulated by many transcriptional factors at the transcriptional level or post-transcriptional level through microRNAs (miRNAs). Although several miRNAs modulate melanogenic activities, there is no evidence of their direct action on MITF expression. Out of eight miRNAs targeting the 3'-UTR of Mitf predicted by bioinformatic programs, our results show miR-218 to be a novel candidate for direct action on MITF expression. Ectopic miR-218 dramatically reduced MITF expression, suppressed tyrosinase activity, and induced depigmentation in murine immortalized melan-a melanocytes. MiR-218 also suppressed melanogenesis in human pigmented skin organotypic culture (OTC) through the repression of MITF. An inverse correlation between MITF and miR-218 expression was found in human primary skin melanocytes and melanoma cell lines. Taken together, our findings demonstrate a novel mechanism involving miR-218 in the regulation of the MITF pigmentary process and its potential application for skin whitening therapy.

The telomere/telomerase binding factor PinX1 is a new target to improve the radiotherapy effect of oesophageal squamous cell carcinomas.

Chemoradiotherapy (CRT) is a standard treatment for oesophageal squamous cell carcinoma (ESCC) in its advanced stages. The telomerase/telomere interacting protein PinX1 contributes to telomere maintenance, tumourigenicity, and influences the DNA damage agent-induced apoptotic response in telomerase-positive cancer cells. However, the clinical and biological significance of PinX1 in human ESCCs remains unclear. We examined the expression dynamics of PinX1 by immunohistochemistry in a learning cohort (n = 98) and a validation cohort (n = 59) of ESCC patients treated with definite chemoradiotherapy (CRT). A series of in vivo and in vitro assays were performed to elucidate the effect of PinX1 on ESCC cells' CRT response and underlying mechanisms. Knockdown of PinX1 did not affect ESCC cells' chemosensitivities to 5-fluorouracil and cisplatin, but substantially increased ESCC cells' therapeutic efficacy of radiation both in vitro and in vivo. Ectopic overexpression of PinX1 dramatically enhanced ESCC cells' resistance to radiotherapy. Furthermore, we demonstrated that PinX1 resistance to radiotherapy (RT) was attributed to PinX1 maintaining telomere stability, reducing ESCC cell death by RT-induced mitosis catastrophe (MC). High expression of Pinx1 correlated positively with ESCC's resistance to CRT, and was a strong and independent predictor for short disease-specific survival (DSS) of ESCC patients. Our data suggest that PinX1 could serve as a novel predictor for a CRT response to ESCC patients, and the pathway of PinX1-mediated telomere stability might represent a new target to improve the RT effect of ESCC.

De-oncogenic HPV E6/E7 vaccine gets enhanced antigenicity and promotes tumoricidal synergy with cisplatin.

In order to develop more effective therapeutic vaccines against cancers with high-risk human papillomavirus (HPV) infection, it is crucial to enhance the immunogenicity, eliminate the oncogenicity of oncoproteins, and take a combination of E7- and E6-containing vaccines. It has been shown recently that PE(ΔIII)-E7-KDEL3 (E7), a fusion protein containing the HPV16 oncoprotein E7 and the translocation domain of Pseudomonas aeruginosa exotoxin A, is effective against TC-1 tumor cells inoculated in mice, therefore, we engineered PE(ΔIII)-E6-CRL-KDEL3 (E6), the de-oncogenic versions of the E7 and E6 fusion proteins [i.e. PE(ΔIII)-E7(d)-KDEL3, E7(d), and PE(ΔIII)-E6(d)-CRL-KDEL3, E6(d)] and tested the immunoefficacies of these fusion proteins as mono- and bivalent vaccines. Results indicated that the E7(d) get higher immunogenicity than its wild type and the E6 fusion proteins augmented the immunogenicity and antitumor effects of their E7 counterparts. Furthermore, the bivalent vaccine system E7(d) plus E6(d), in the presence of cisplatin, showed the best tumoristatic and tumoricidal effects against established tumors in vivo. Therefore, it can be concluded that this novel therapeutic vaccine system, upon further optimization, may shed new light on clinical management of HPV-related carcinomas.