[Roles of Myeloid-derived Suppressor Cells in Breast Cancer Metastasis].

Breast cancer patients with bone,liver and lung metastases tend to have a poor prognosis.According to Paget's "seed and soil" theory,metastatic cancer cell "seeds" must fall on congenial target organ "soil".Studies have shown that myeloid-derived suppressor cells(MDSCs)can be recruited at the site of breast cancer metastasis in advance and play a role in the metastasis of breast cancer cells.This paper reviews the biological characteristics of MDSCs,the roles of MDSCs in peripheral circulation,prometastatic niche,and metastatic site during breast cancer metastasis,as well as the research progress of MDSCs-targeted treatment of breast cancer metastasis.

Identification of a multidimensional transcriptome prognostic signature for lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is one of the leading contributors to cancer-related deaths worldwide. The objective of the current study is to identify a multidimensional transcriptome prognostic signature by combining protein-coding gene (PCG) with long non-coding RNA (lncRNA) for patients with LUAD. METHODS: We obtained LUAD PCG and lncRNA expression profile data from three datasets in the Gene Expression Omnibus database and conducted survival analyzes for these individuals. RESULTS: We established a predictive model comprising the three PCGs (NHLRC2, PLIN5, GNAI3), and one lncRNA (AC087521.1). This model segregated patients with LUAD into low- and high-risk groups based on significant differences in survival in the training dataset (GSE31210, n = 226, log-rank test P < .001). Risk stratification of the model was subsequently validated in other two test datasets (GSE37745, n = 106, log-rank test P < .001; GSE30219, n = 85, log-rank test P = .006). Time-dependent receiver operating characteristic (timeROC) curve analysis demonstrated that the model correlated strongly with disease progression and outperformed pathological stage in terms of prognostic ability. Cox proportional hazards regression analysis revealed that the signature could serve as an independent predictor of clinical outcomes in patients with LUAD. CONCLUSIONS: We describe a novel multidimensional transcriptome signature that can predict survival probabilities in patients with LUAD.

Candida albicans-induced acute lung injury through activating several inflammatory signaling pathways in mice.

Candida albicans infection-induced acute lung injury is one of the most prevalent diseases in immunosuppressive individual. Nevertheless, the mechanism by which Candida albicans induced acute lung injury remains unclear. The present study investigated the mechanism by which Candida albicans induced acute lung injury in mice. Mice were randomly divided into four groups and intratracheally injected with 60 µl Candida albicans (106 CFU) or normal saline. Half of the mice were sacrificed at 6 h after Candida albicans. The rest of the mice for survival test were observed until 7 d after Candida albicans. As expected, immunosuppression aggravated Candida albicans-induced acute lung injury and death in mice. Additionally, Candida albicans infection elevated mRNA levels of pro-inflammatory and chemokines in lungs and the levels of IL-6, IL-1ß and IL-17 in serum. Further study showed that Candida albicans promoted nuclear translocation of NF-κB p50 and p65 subunits in pulmonary epithelial cells and interstitial cells. Candida albicans induced pulmonary p38, ERK1/2 and Akt phosphorylation in normal and immunosuppressive mice. Moreover, Candida albicans infection activated pulmonary STAT3 signaling in normal and immunosuppressive mice. Overall, these results suggest that Candida albicans induced acute lung injury and death may be through activating several inflammatory signaling pathways.

B7­H3 promotes malignant progression of muscle­invasive bladder cancer.

The objective of the present study was to investigate the expression of B7 homologue 3 (B7­H3) in muscle­invasive bladder cancer (MIBC) tissues, evaluate its correlation with patient clinicopathological characteristics, and to explore the effect of B7­H3 on MIBC cells. B7­H3 expression levels in tumor tissues from 115 patients undergoing radical cystectomy for MIBC were detected by immunohistochemical staining, followed by analysis of the association with clinicopathological characteristics and survival. A B7­H3­silenced cell line was established by RNA interference (RNAi). Alterations in cell proliferation, cell cycle, migration and invasion were analyzed in vitro. The proteins associated with cancer cell behavior were detected by western blot analysis. In addition, we utilized a xenograft tumor assay in nude mice to test the inhibitory effect of B7­H3 shRNA on MIBC in vivo. The results revealed that, among the 115 patients, the B7­H3 expression level was significantly associated with an increased incidence of distant metastasis (P=0.014) and vascular invasion (P=0.031), whereas it was not statistically associated with sex, age, pathologic grade, tumor stage, recurrence and lymphatic metastasis. Overall survival (OS) and progression­free survival (PFS) were significantly worse for patients with high B7­H3 expression (P<0.001 and P<0.001, respectively) among the 115 MIBC patients. Suppression of B7­H3 significantly inhibited the proliferation, caused G2 phase arrest, as well as declined migration and invasion abilities in vitro. The protein expression of Ki67, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP2) and MMP9 were decreased in the T24/B7­H3 shRNA group compared with the control (P<0.05, respectively). Finally, we were able to inhibit tumor development by decreasing B7­H3 expression in vivo. In conclusion, a high expression level of B7­H3 in MIBC tissues is associated with a poor clinicopathological status and poor prognosis, and promotes the development of MIBC in vitro and in vivo. Thus, B7­H3 may be a potential novel biomarker for the poor prognosis of MIBC patients.

A network meta-analysis of the short-term efficacy of five chemotherapy regimens based on cisplatin and fluorouracil for esophagogastric junctional adenocarcinoma.

The primary purpose of this study was to explore the short-term efficacy of different cisplatin and fluorouracil-based chemotherapy regimens in the treatment of patients with esophagogastric junctional adenocarcinoma (EGJA) using a network meta-analysis (NMA). Randomized controlled trials (RCTs) related to chemotherapy regimens based on cisplatin and fluorouracil for EGJA were included from the PubMed, EMBASE and Cochrane Library electronic databases (from inception to June 2016). Direct and indirect evidence were combined to calculate the pooled odds ratio (OR) and its 95% confidence interval (95% CI) as well as to draw the surface under the cumulative ranking (SUCRA) curves. This NMA finally enrolled ten eligible RCTs with the following five regimens: cisplatin plus fluorouracil (cisplatin+fluorouracil), cisplatin+fluorouracil-based chemotherapy (cisplatin+fluorouracil+docetaxel/epirubicin/irinotecan), fluorouracil-based chemotherapy (fluorouracil+docetaxel/doxorubicin/methotrexate/irinotecan), cisplatin-based chemotherapy (cisplatin+docetaxel/epirubicin/irinotecan/capecitabine/s-1) and other drug-based chemotherapy (docetaxel/irinotecan/capecitabine). These results revealed that compared with a cisplatin+ fluorouracil-based chemotherapy regimen, the fluorouracil-based chemotherapy regimen had a lower overall response rate (ORR) and partial response (PR) for EGJA patients (ORR: OR=0.43, 95% CI=0.22-0.86; PR: OR=0.46, 95% CI=0.23-0.91). Cluster analyses suggested that the cisplatin+fluorouracil-based chemotherapy regimen had the best short-term efficacy for EGJA in terms of the complete response (CR), PR, ORR, stable disease (SD) and progression disease (PD). Our results indicated that cisplatin+fluorouracil-based chemotherapy regimens may have the best short-term efficacy in the treatment of EGJA.

A case of systemic amyloidosis beginning with purpura.

Primary systemic amyloidosis is a relatively rare disease, caused when abnormal extracellular deposition of fibrillary protein builds up in a variety of target organs, such as heart, kidneys, lungs liver, and so forth. The symptoms of the disease are usually vague, while many kinds of auxiliary or laboratory examinations especially pathologic biopsy can provide a clue for the diagnosis. Here we described a case who had purpura-like lesions in the initial stage, followed by progressive malfunctions in the kidneys, the heart, the lungs, as well as the liver. The final diagnosis was primary systemic amyloidosis determined by skin pathologic biopsy. And the disease led to a fatal outcome within three months after the diagnosis.

Dkk-1 secreted by mesenchymal stem cells inhibits growth of breast cancer cells via depression of Wnt signalling.

We determined the molecular mechanism of inhibitory effect of human mesenchymal stem cells (hMSCs) on the growth of human MCF-7 breast cancer cells. Our finding showed that beta-catenin was down-regulated in MCF-7 cells by conditioned media from Z3 hMSCs, and the expression level of dickkopf-1 (Dkk-1) was higher in Z3 cells than that in MCF-7 cells. Neutralization of Dkk-1 and small interference RNA targeting Dkk-1 mRNA in Z3 cells attenuated the inhibitory effect of Z3 cells on MCF-7 cells. Overexpression of Dkk-1 in Z3 cells enhanced the inhibition. Therefore, Dkk-1 secreted by Z3 cells involves the inhibition via the Wnt pathway.

Efficient regulation of gene expression using self-contained fiber-modified adenovirus vectors containing the tet-off system.

Previously, we developed single adenovirus (Ad) vectors that contained the gene of interest in the E1 deletion region and the transactivator gene for the tetracycline-controllable expression system in the E3 deletion region. In the present study, we improved the Ad vector-mediated tetracycline-controllable expression system by the fiber modification of Ad. We developed fiber-modified Ad vectors containing the tet-off system, which are effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Ad vectors containing the tet-off system with an Arg-Gly-Asp (RGD) peptide in the HI loop of the fiber knob or the Ad type 35 fiber greatly improved transduction efficiency (more than 1-2-log orders) into the cells lacking CAR expression but expressing alphav integrin or CD46, respectively. They exhibited vastly higher regulation of gene expression by doxycycline. The combination of fiber-modified Ad vectors and the tetracycline-controllable expression system should offer a powerful tool for gene therapy and gene transfer experiment.

Approaches to improving the kinetics of adenovirus-delivered genes and gene products.

Adenovirus (Ad) vectors have been expected to play a great role in gene therapy because of their extremely high transduction efficiency and wide tropism. However, due to the intrinsic deficiency of their immunogenic toxicities, Ad vectors are rapidly cleared from the host, transgene expression is transient, and readministration of the same serotype Ad vectors is problematic. As a result, Ad vectors are continually undergoing refinement to realize their potential for gene therapy application. Even after 1999, when a patient fatally succumbed to the toxicity associated with Ad vector administration at a University of Pennsylvania (U.S.) experimental clinic, enthusiasm of gene therapists for Ad vectors has not waned. With great efforts from various research groups, significant advances have been achieved through comprehensive approaches to improving the kinetics of Ad vector-delivered genes and gene products.

Adenovirus vector-mediated doxycycline-inducible RNA interference.

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.

Tight positive regulation of transgene expression by a single adenovirus vector containing the rtTA and tTS expression cassettes in separate genome regions.

We previously developed single adenovirus (Ad) vectors that contained the components for a tetracycline-regulatable gene-expression system in the E1 and E3 deletion regions, and showed that the Ad vectors containing the tet-on system exhibit a much inferior regulation of transgene expression than those containing the tet-off system. In many cases, the tet-on system may be preferable because of its positive regulation of transgene expression. To this end, in the present study, by introducing the latest generation reverse tetracycline-responsive transcriptional activator (rtTA2s-M2 or rtTA2s-S2) and the tetracycline-controlled transcriptional silencer (tTS) into the original tet-on system, we constructed various modified Ad-mediated tet-on systems. Among them, the novel single Ad vector, which contained three heterologous gene-expression cassettes of the gene of interest, rtTA2s-S2, and tTS in the E1 deletion region, the E3 deletion region, and the region between E4 and 3'ITR, respectively, displayed vastly improved doxycycline-inducible gene expression in terms of low basal expression, high induced expression, and high responsiveness to doxycycline both in vitro and in vivo. These results also suggest that the low responsiveness to doxycycline may explain why the original tet-on system in the context of the Ad vector is not effective in vivo. This is the first report describing the cloning of three heterologous gene-expression cassettes into three separate regions of the E1/E3-deleted Ad genome. This improved Ad-mediated tet-on system might be useful for gene therapy and greatly facilitate the analyses of gene function.

Woodchuck hepatitis virus post-transcriptional regulation element enhances transgene expression from adenovirus vectors.

In studies of both gene therapy and gene function, transgene expression can be modulated at both the transcriptional and post-transcriptional levels. In a previous study, we optimized the transcriptional regulatory elements for adenovirus (Ad) vectors to mediate efficient transgene expression, including promoter, enhancer, intron, and poly(A) sequence. In the present study, we systematically investigated the ability of the Woodchuck hepatitis virus post-transcriptional regulation element (WPRE) to enhance the expression of the luciferase gene, as a model gene, in the context of Ad vectors. We found that the WPRE in the sense orientation cloned between the luciferase gene and the poly(A) sequence stimulated 2- to 7-fold more luciferase expression in vitro and 2- to 50-fold more in the liver, kidney and lung of mouse than occurred without the use of the WPRE. The most efficient Ad vector in this study, which contained the improved CMV promoter (the conventional CMV promoter with the intron A) and the WPRE, showed more than 700-fold luciferase expression in mouse liver than did the Ad vector containing the conventional CMV promoter but no WPRE. These results indicate that inclusion of the WPRE, combined with the optimization of transcriptional regulatory elements in Ad vectors, will permit a given therapeutic goal to be achieved with substantially fewer viral particles. This information would be helpful for the construction of adenovirus vectors for studies regarding both gene therapy and gene function.

Regulated gene expression from adenovirus vectors: a systematic comparison of various inducible systems.

Positively and tightly regulated gene expression is essential for gene function and gene therapy research. The currently-used inducible gene expression systems include tetracycline (Tet-on and T-REx), ecdysone, antiprogestin and dimerizer-based systems. Adenovirus (Ad) vectors play an important role in gene function and gene therapy research for their various advantages over other vector systems. Previously, we reported the inferiority of the Tet-on system as an inducible gene expression system in the context of Ad vectors in comparison with the Tet-off system. In this study, to identify an optimal system for regulated gene expression from Ad vectors, we made a rigorous direct comparison of these five inducible gene expression systems in three cell lines using the luciferase reporter gene. The highest sensitivity to the respective inducer was that of the dimerizer system, followed by the antiprogestin system. The lowest basal expression and the highest induction factor were both characteristic of the dimerizer system. Furthermore, the dimerizer and T-REx systems exhibited much higher induced expression levels than the other three systems. The elucidation of the characteristic features of each system should provide important information for widespread and feasible application of these systems. Overall, these results suggest the most appropriate inducible gene expression system in the context of Ad vectors to be the dimerizer system.

Strength evaluation of transcriptional regulatory elements for transgene expression by adenovirus vector.

In studies of both gene function and gene therapy, transgene expression may be assisted considerably through the use of transcriptional regulatory elements with high activity. In this study, we evaluated the strength of various transcriptional regulatory elements both in vitro (six types of cell line) and in vivo (mouse heart, lung, kidney, spleen, and liver) by adenovirus-mediated gene transfer. In the case of the promoter/enhancer (P/E), the activity of CMV P/E (from the human cytomegalovirus immediate-early 1 gene) and hybrid CA P/E (composed of the CMV enhancer and chicken beta-actin promoter) were investigated, both of which are known to be strong and widely used. While hybrid CA P/E showed a higher transgene expression activity than CMV P/E, the addition of the intron A sequence (the largest intron of CMV) to CMV P/E increased the activity of CMV P/E to the same or higher level than that of hybrid CA P/E. Concerning the polyadenylation signal (P(A)) sequence, one from the bovine growth hormone (BGH) gene was about two times more efficient than that from the Simian virus 40 (SV40) late gene, both in vitro and in vivo. In the context of the CMV P/E containing the intron A sequence, a further increase in transgene expression was obtained by the addition of a SV40 enhancer downstream from the P(A) sequence. The combination of the SV40 P(A) and a SV40 enhancer showed almost comparable activity to BGH P(A). This information would be helpful for the construction of adenovirus vectors for studies regarding both gene function and gene therapy.