Dynamic m6A mRNA Methylation Reveals the Role of METTL3/14-m6A-MNK2-ERK Signaling Axis in Skeletal Muscle Differentiation and Regeneration.

N6-methyladenosine (m6A) RNA methylation has emerged as an important factor in various biological processes by regulating gene expression. However, the dynamic profile, function and underlying molecular mechanism of m6A modification during skeletal myogenesis remain elusive. Here, we report that members of the m6A core methyltransferase complex, METTL3 and METTL14, are downregulated during skeletal muscle development. Overexpression of either METTL3 or METTL14 dramatically blocks myotubes formation. Correspondingly, knockdown of METTL3 or METTL14 accelerates the differentiation of skeletal muscle cells. Genome-wide transcriptome analysis suggests ERK/MAPK is the downstream signaling pathway that is regulated to the greatest extent by METTL3/METTL14. Indeed, METTL3/METTL14 expression facilitates ERK/MAPK signaling. Via MeRIP-seq, we found that MNK2, a critical regulator of ERK/MAPK signaling, is m6A modified and is a direct target of METTL3/METTL14. We further revealed that YTHDF1 is a potential reader of m6A on MNK2, regulating MNK2 protein levels without affecting mRNA levels. Furthermore, we discovered that METTL3/14-MNK2 axis was up-regulated notably after acute skeletal muscle injury. Collectively, our studies revealed that the m6A writers METTL3/METTL14 and the m6A reader YTHDF1 orchestrate MNK2 expression posttranscriptionally and thus control ERK signaling, which is required for the maintenance of muscle myogenesis and may contribute to regeneration.

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. RESULTS: We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3'-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. CONCLUSIONS: Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study.

LncRNA DSCAM-AS1 interacts with YBX1 to promote cancer progression by forming a positive feedback loop that activates FOXA1 transcription network.

Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.

Adrenomedullin inhibits tumor metastasis and is associated with good prognosis in triple-negative breast cancer patients.

BACKGROUND: Cancer metastasis is the major reason for cancer-related deaths, but the mechanism of cancer metastasis still unclear. Adrenomedullin (ADM), a peptide hormone, functions as a local paracrine and autocrine mediator with multiple biological activities, such as angiogenesis, cell proliferation, and anti-inflammation. However, the expression and potential function of ADM in triple-negative breast cancer (TNBC) remain unclear. METHODS: Real-time polymerase chain reaction and western blotting were performed to examine the expression of ADM in TNBC tissues and cell lines. A total of 458 TNBC tissue samples and adjacent nontumor tissue samples were detected by immunochemistry to determine the correlation between ADM expression and clinicopathological characteristics. We determined the role and mechanistic pathways of ADM in tumor metastasis in cell lines. RESULTS: Our data showed that ADM expression was noticeably decreased in TNBC samples and cell lines. Low expression levels correlate with an increased risk of recurrence and metastasis. Furthermore, low ADM expression was associated with poor prognosis and was an independent marker for TNBC. In vitro, ADM may decrease cancer cell invasion, which is likely the result of its effect on the cancer cell epithelial-mesenchymal transition. CONCLUSIONS: Our findings suggest that ADM is a valuable biomarker for TNBC prognosis and an anti-metastasis candidate therapeutic target in triple-negative breast cancer.

EuRBPDB: a comprehensive resource for annotation, functional and oncological investigation of eukaryotic RNA binding proteins (RBPs).

RNA binding proteins (RBPs) are a large protein family that plays important roles at almost all levels of gene regulation through interacting with RNAs, and contributes to numerous biological processes. However, the complete list of eukaryotic RBPs including human is still unavailable. Here, we systematically identified RBPs in 162 eukaryotic species based on both computational analysis of RNA binding domains (RBDs) and large-scale RNA binding proteomic data, and established a comprehensive eukaryotic RBP database, EuRBPDB (http://EuRBPDB.syshospital.org). We identified a total of 311 571 RBPs with RBDs (corresponding to 6368 ortholog groups) and 3,651 non-canonical RBPs without known RBDs. EuRBPDB provides detailed annotations for each RBP, including basic information and functional annotation. Moreover, we systematically investigated RBPs in the context of cancer biology based on published literatures, PPI-network and large-scale omics data. To facilitate the exploration of the clinical relevance of RBPs, we additionally designed a cancer web interface to systematically and interactively display the biological features of RBPs in various types of cancers. EuRBPDB has a user-friendly web interface with browse and search functions, as well as data downloading function. We expect that EuRBPDB will be a widely-used resource and platform for both the communities of RNA biology and cancer biology.

miR-665 expression predicts poor survival and promotes tumor metastasis by targeting NR4A3 in breast cancer.

Cancer metastasis is the main cause of death in breast cancer (BC) patients. Therefore, prediction and treatment of metastasis is critical for enhancing the survival of BC patients. In this study, we aimed to identify biomarkers that can predict metastasis of BC and elucidate the underlying mechanism of the functional involvement of such markers in metastasis. miRNA expression profile was analyzed using a custom microarray system in 422 BC tissues. The relationship between the upregulated miR-665, metastasis and survival of BC was analyzed and verified in another set of 161 BC samples. The biological function of miR-665 in BC carcinogenesis was explored with in vitro and in vivo methods. The target gene of miR-665 and its signaling cascade were also analyzed. There are 399 differentially expressed miRNAs between BC and noncancerous tissues, of which miR-665 is the most upregulated miRNA in the BC tissues compared with non-tumor breast tissues (P < 0.001). The expression of miR-665 predicts metastasis and poor survival in 422 BC patients, which is verified in another 161 BC patients and 2323 BC cases from online databases. Ectopic miR-665 expression promotes epithelial-mesenchymal transition (EMT), proliferation, migration and invasion of BC cells, and increases tumor growth and metastasis of BC in mice. Bioinformatics, luciferase assay and other methods showed that nuclear receptor subfamily 4 group A member 3 (NR4A3) is a target of miR-665 in BC. Mechanistically, we demonstrated that miR-665 promotes EMT, invasion and metastasis of BC via inhibiting NR4A3 to activate MAPK/ERK kinase (MEK) signaling pathway. Our study demonstrates that miR-665 upregulation is associated with metastasis and poor survival in BC patients, and mechanistically, miR-665 enhances progression of BC via NR4A3/MEK signaling pathway. This study provides a new potential prognostic biomarker and therapeutic target for BC patients.

A group of tissue-specific microRNAs contribute to the silencing of CUX1 in different cell lineages during development.

Cut-like homeobox 1 (CUX1) is a highly conserved homeoprotein that functions as a transcriptional repressor of genes specifying terminal differentiation. We previously showed that liver-specific microRNA-122 (miR-122) regulates the timing of liver development by silencing CUX1 post-transcriptionally. Since the CUX1 protein is expressed in a subset of embryonic tissues, we hypothesized that it is regulated by specific microRNAs (miRNAs) in each cell type during development. Using a large-scale screening method, we identified ten tissue-specific miRNAs from different cell lineages that directly targeted CUX1. An analysis of the interaction between heart-specific microRNA-208a (miR-208a) and CUX1 in the hearts of developing mouse embryos and in P19CL6 cells undergoing cardiac differentiation indicated that CUX1 is regulated by miR-208a during heart development and cardiomyocyte differentiation. Functional analysis of miR-208a in P19CL6 cells using lentiviral-mediated over-expression showed that it regulates the transition between cellular proliferation and differentiation. These results suggest that these tissue-specific miRNAs might play a common role in timing the progression of terminal differentiation of different cell lineages, possibly by silencing the differentiation repressor CUX1.

Genome-wide study of ER-regulated lncRNAs shows AP000439.3 may function as a key regulator of cell cycle in breast cancer.

Estrogen receptor (ER) plays important roles in cell growth, development and tumorigenesis. Although ER-regulated genes have been extensively investigated, little is known about roles of ER-regulated lncRNAs in breast cancer. Here, we conducted genome-wide study of ER-regulated lncRNAs by using RNA-seq, ChIP-seq and TCGA data. A total of identified 114 ER-regulated lncRNAs were identified, many of them were overexpressed in ER+ breast cancer and co-expressed with some key regulators. Silencing one of most prominent lncRNA, AP000439.3, resulted in inhibition of cell cycle progression and proliferation. Further study revealed AP000439.3 can regulate expression of CCND1 through enhancing estrogen receptor induction of CCND1. This finding revealed lncRNAs may serve as important effectors of ER in regulation of gene expression and cell phenotype in breast cancer.

Deep sequencing reveals a global reprogramming of lncRNA transcriptome during EMT.

Several studies have shown that long non-coding RNAs (lncRNAs) may play an essential role in Epithelial-Mesenchymal Transition (EMT), which is an important step in tumor metastasis; however, little is known about the global change of lncRNA transcriptome during EMT. To investigate how lncRNA transcriptome alterations contribute to EMT progression regulation, we deep-sequenced the whole-transcriptome of MCF10A as the cells underwent TGF-ß-induced EMT. RESULTS: Deep-sequencing results showed that the long RNA transcriptome of MCF10A had undergone global changes as early as 8h after treatment with TGF-ß. The expression of 3403 known and novel lncRNAs, and 570 known and novel circRNAs were altered during EMT. To identify the key lncRNA-regulator, we constructed the co-expression network and found all junction nodes in the network are lncRNAs. One junction node, RP6-65G23.5, was further verified as a key regulator of EMT. Intriguingly, we identified 216 clusters containing lncRNAs which were located in "gene desert" regions. The expressions of all lncRNAs in these clusters changed concurrently during EMT, strongly suggesting that these clusters might play important roles in EMT. Our study reveals a global reprogramming of lncRNAs transcriptome during EMT and provides clues for the future study of the molecular mechanism of EMT.

Cyclin D1 expression predicts postoperative distant metastasis and survival in resectable esophageal squamous cell carcinoma.

PURPOSE: We aim to identify esophageal squamous cell carcinoma patients with increased risk of postoperative metastases. RESULTS: A high level of cyclin D1 expression, together with poor tumor cell differentiation and advanced tumor stages, increased risk of postoperative metastasis and decreased distant metastasis-free survival in ESCC in both cohorts. A high level of cyclin D1 expression also decreased overall survival in the training cohort (p < 0.01) but not in the validation cohort (p = 0.415). However, when the two cohorts of patients were pooled to obtain a larger case number, a high level of cyclin D1 expression was again demonstrated as an independent predictor that decreased overall survival (p < 0.01). METHODS: We used data from two institutions to establish training (n = 319) and validation (n = 164) cohorts. Tissue microarrays were generated for immunohistochemical evaluation. The correlation among cyclin D1 expression, clinicopathologic variables, postoperative distant metastases, overall survival, and distant metastasis-free survival were analyzed. Multivariate analyses were used to test the independent factors impacting postoperative distant metastases and survival. The outcomes generated from the training cohort were then tested using the validation cohort and pooled dataset. CONCLUSIONS: High level of cyclin D1 expression increased distant metastasis, decreased overall survival and distant metastasis-free survival in resectable ESCC. Using a combination of cyclin D1 expression, tumor cell differentiation grade, and tumor stages, identifying patients with increased risk of postoperative metastases becomes possible.

A new prognostic histopathologic classification of nasopharyngeal carcinoma.

BACKGROUND: The current World Health Organization (WHO) classification of nasopharyngeal carcinoma (NPC) conveys little prognostic information. This study aimed to propose an NPC histopathologic classification that can potentially be used to predict prognosis and treatment response. METHODS: We initially developed a histopathologic classification based on the morphologic traits and cell differentiation of tumors of 2716 NPC patients who were identified at Sun Yat-sen University Cancer Center (SYSUCC) (training cohort). Then, the proposed classification was applied to 1702 patients (retrospective validation cohort) from hospitals outside SYSUCC and 1613 patients (prospective validation cohort) from SYSUCC. The efficacy of radiochemotherapy and radiotherapy modalities was compared between the proposed subtypes. We used Cox proportional hazards models to estimate hazard ratios (HRs) with 95% confidence intervals (CI) for overall survival (OS). RESULTS: The 5-year OS rates for all NPC patients who were diagnosed with epithelial carcinoma (EC; 3708 patients), mixed sarcomatoid-epithelial carcinoma (MSEC; 1247 patients), sarcomatoid carcinoma (SC; 823 patients), and squamous cell carcinoma (SCC; 253 patients) were 79.4%, 70.5%, 59.6%, and 42.6%, respectively (P < 0.001). In multivariate models, patients with MSEC had a shorter OS than patients with EC (HR = 1.44, 95% CI = 1.27-1.62), SC (HR = 2.00, 95% CI = 1.76-2.28), or SCC (HR = 4.23, 95% CI = 3.34-5.38). Radiochemotherapy significantly improved survival compared with radiotherapy alone for patients with EC (HR = 0.67, 95% CI = 0.56-0.80), MSEC (HR = 0.58, 95% CI = 0.49-0.75), and possibly for those with SCC (HR = 0.63; 95% CI = 0.40-0.98), but not for patients with SC (HR = 0.97, 95% CI = 0.74-1.28). CONCLUSIONS: The proposed classification offers more information for the prediction of NPC prognosis compared with the WHO classification and might be a valuable tool to guide treatment decisions for subtypes that are associated with a poor prognosis.

Stomatin-like protein 2 is overexpressed in epithelial ovarian cancer and predicts poor patient survival.

BACKGROUND: Stomatin-like protein 2 (SLP-2, also known as STOML2) is a stomatin homologue of uncertain function. SLP-2 overexpression has been suggested to be associated with cancer progression, resulting in adverse clinical outcomes in patients. Our study aim to investigate SLP-2 expression in epithelial ovarian cancer cells and its correlation with patient survival. METHODS: SLP-2 mRNA and protein expression levels were analysed in five epithelial ovarian cancer cell lines and normal ovarian epithelial cells using real-time PCR and western blotting analysis. SLP-2 expression was investigated in eight matched-pair samples of epithelial ovarian cancer and adjacent noncancerous tissues from the same patients. Using immunohistochemistry, we examined the protein expression of paraffin-embedded specimens from 140 patients with epithelial ovarian cancer, 20 cases with borderline ovarian tumours, 20 cases with benign ovarian tumours, and 20 cases with normal ovarian tissues. Statistical analyses were applied to evaluate the clinicopathological significance of SLP-2 expression. RESULTS: SLP-2 mRNA and protein expression levels were significantly up-regulated in epithelial ovarian cancer cell lines and cancer tissues compared with normal ovarian epithelial cells and adjacent noncancerous ovarian tissues. Immunohistochemistry analysis revealed that the relative overexpression of SLP-2 was detected in 73.6 % (103/140) of the epithelial ovarian cancer specimens, 45.0 % (9/20) of the borderline ovarian specimens, 30.0 % (6/20) of the benign ovarian specimens and none of the normal ovarian specimens. SLP-2 protein expression in epithelial ovarian cancer was significantly correlated with the tumour stage (P < 0.001). Epithelial ovarian cancer patients with higher SLP-2 protein expression levels had shorter progress free survival and overall survival times compared to patients with lower SLP-2 protein expression levels. Multivariate analyses showed that SLP-2 expression levels were an independent prognostic factor for survival in epithelial ovarian cancer patients. CONCLUSIONS: SLP-2 mRNA and proteins were overexpressed in epithelial ovarian cancer tissues. SLP-2 protein overexpression was associated with advanced stage disease. Patients with higher SLP-2 protein expression had shorter progress free survival and poor overall survival times. Thus, SLP-2 protein expression was an independent prognostic factor for patients with epithelial ovarian cancer.

Vascular invasion as an independent predictor of poor prognosis in nonmetastatic gastric cancer after curative resection.

The prognostic significance of vascular invasion (VI) in nonmetastatic gastric cancer (GC) remains a matter of controversy. The purpose of this study was to assess the impact of VI on survival in this group of GC patients. We enrolled 361 GC patients without metastasis who underwent curative gastrectomy between 1996 and 2009 in Sun Yat-sen University Cancer Center. A retrospective analysis of the clinicopathological data was performed, focusing on the impact of VI detected by routine H&E staining on disease-free survival (DFS) and cancer-specific survival (CSS). The presence of VI was detected in 13.9% of our cohort. The VI status was significantly correlated with the tumor size, infiltration depth, and TNM stage (P < 0.05). Patients with VI showed significantly lower DFS and CSS compared with patients without VI (P < 0.0001 for both). The subgroup analysis indicated that the presence of VI was a negative predictor of DFS in all TNM stages and a predictor of lower CSS only in stage I (P < 0.05 for all). A multivariate Cox proportional analysis identified VI as an independent predictor of CSS (P = 0.022). The presence of VI is a risk factor for recurrence and an independent predictor of poor survival in nonmetastatic GC after curative resection. The VI status should be considered to stratify with this group of GC patients for adjuvant treatment and more effective follow-up protocol.

Vascular Endothelial Growth Factor is a Useful Predictor of Postoperative Distant Metastasis and Survival Prognosis in Esophageal Squamous Cell Carcinoma.

BACKGROUND: The correlation between vascular endothelial growth factor (VEGF) and prognosis for patients with esophageal squamous cell carcinoma (ESCC) is controversial. This study investigated the correlation of VEGF expression with distant metastases and prognosis in resectable ESCC to improve the identification of patients with increased risk of postoperative metastases. METHODS: Data from two centers were used to establish a training cohort (n = 319) and a validation cohort (n = 164). Tissue microarrays were generated for immunohistochemical evaluation. The correlations among VEGF expression, clinicopathologic variables, and prognosis were analyzed. The outcomes generated from the training cohort then were tested using the validation cohort. Multivariate analyses were used to test the independent factors that had an impact on postoperative distant metastases, overall survival (OS), and distant metastasis-free survival (DMFS). RESULTS: Tumor stages, tumor cell grade, and VEGF expression were prognostic factors independent of ESCC outcome. The data indicated that high levels of VEGF expression were correlated with a high risk of postoperative distant metastases (p = 0.013) in the training cohort. This result was confirmed by the validation cohort (p < 0.01) and logistic regression analyses. A high level of VEGF expression also was correlated with poor DMFS (p = 0.011) and OS (p = 0.033) in the training cohort, which also was confirmed by the validation cohort and Cox regression analyses. CONCLUSIONS: Expression of VEGF is a predictor of distant metastasis, OS, and DMFS in resectable ESCC patients. Using a combination of VEGF expression, tumor stages, and tumor cell grade, identification of patients with increased risk of postoperative metastases may become possible.

Afatinib enhances the efficacy of conventional chemotherapeutic agents by eradicating cancer stem-like cells.

Cancer stem cells (CSC) have garnered significant attention as a therapeutic focus, based on evidence that they may represent an etiologic root of treatment-resistant cells. Indeed, expression of the multidrug resistance protein ATP-binding cassette subfamily G member 2 (ABCG2) confers chemoresistance to CSCs, where it serves as a potential biomarker and therapeutic target. Here, we show that afatinib, a small-molecule inhibitor of the tyrosine kinases EGFR, HER2, and HER4, preferentially eliminated side population cells with CSC character, in both cell lines and patient-derived leukemia cells, by decreasing ABCG2 expression. In these cells, afatinib also acted in parallel to suppress self-renewal capacity and tumorigenicity. Combining afatinib with the DNA-damaging drug topotecan enhanced the antitumor effect of topotecan in vitro and in vivo. Mechanistic investigations suggested that ABCG2 suppression by afatinib did not proceed by proteolysis through the ubiquitin-dependent proteosome, lysosome, or calpain. Instead, we found that afatinib increased DNA methyltransferase activity, thereby leading to methylation of the ABCG2 promoter and to a decrease in ABCG2 message level. Taken together, our results advocate the use of afatinib in combination with conventional chemotherapeutic drugs to improve efficacy by improving CSC eradication.

Application of endobronchial ultrasound-guided transbronchial needle aspiration in the management of mediastinal and hilar lymphadenopathy without intrapulmonary mass: experience from the largest cancer center of southern China.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is useful for lung cancer diagnosis and lymph node staging. The purpose of this study was to investigate EBUS-TBNA for managing mediastinal and hilar lymphadenopathies without intrapulmonary masses. We retrospectively reviewed our EBUS-TBNA database that was obtained between August 2010 and October 2012. Mediastinal and hilar lymphadenopathies of unknown origin and in the absence of known pulmonary malignancies were included. Final diagnoses were determined by EBUS-TBNA, surgery, and/or clinical follow-up for at least 6 months. Sensitivity, specificity, accuracy, and positive and negative predictive values were determined using standard statistical methods. We identified 128 patients with mediastinal and hilar lymphadenopathies and without intrapulmonary masses. EBUS-TBNA was successfully performed to obtain samples from 161 lymph nodes and mediastinal masses. EBUS-TBNA was diagnostic for 119 of 128 patients (93.0%) for all disease categories. The sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy of EBUS-TBNA were 89.8, 100, 100, 81.6, and 93.0%, respectively. The procedures were uneventful and there were no severe complications. EBUS-TBNA is a safe, minimally invasive approach for diagnosing mediastinal and hilar lymphadenopathies without intrapulmonary masses. It obviates the need for more invasive procedures for tissue sampling of the mediastinum and hilum.

Overexpression of cystatin SN positively affects survival of patients with surgically resected esophageal squamous cell carcinoma.

BACKGROUND: Cystatin SN is a secreted protein and a cysteine proteinase inhibitor. It has been considered to be a tumor marker for gastrointestinal tract cancer in several functional researches. However, the clinicopathological and prognostic significance of Cystatin SN expression in esophageal squamous cell carcinoma (ESCC) has not been elucidated. METHODS: In our study, the expression of Cystatin SN was detected in 209 surgically resected ESCC tissues and 170 peritumoral normal esophageal mucosae by immunohistochemistry. The prognostic significance of Cystatin SN expression was analysed with Kaplan-Meier plots and the Cox proportional hazards regression models. RESULTS: The results showed that the immunostaining of Cystatin SN in ESCC tissues was less intense than that in the normal control tissue (P < 0.001). Compared with patients with low tumoral Cystatin SN expression, ESCC patients with tumors high-expression Cystatin SN exhibited increased disease-free survival (DFS) and overall survival (OS) (P < 0.001 and P < 0.001, respectively). Furthermore, the expression level of Cystatin SN could further stratify the ESCC patients by survival (DFS and OS) in the stage II subgroup (P < 0.001 and P < 0.001, respectively). Multivariate analyses showed that Cystatin SN expression, N status and differentiation were independent and significant predictors of survival. CONCLUSIONS: We concluded that ESCC patients whose tumors express high levels of Cystatin SN have favourable survival compared with those patients with low Cystatin SN expression. Tumoral Cystatin SN expression may be an independent predictor of survival for patients with resectable ESCCs.

Prognostic analysis of early-stage squamous cell carcinoma of the vulva.

AIM: The aim of this study was to analyze prognostic factors of early-stage squamous cell carcinoma of the vulva. METHODS: A retrospective analysis was conducted on 35 patients who were treated for early-stage squamous cell carcinoma of the vulva at Sun Yat-sen University Cancer Center from January 1980 to December 2005. The Statistical Package for Social Science (SPSS) was used to compare the different strategies of operation and to analyze the prognostic factors. RESULTS: Thirty-five patients had early-stage squamous cell carcinoma of the vulva. Of these cases, 26 were well differentiated, seven were moderately differentiated, and two were poorly differentiated. The five-year survival rate was 77.1%. Five cases were in FIGO stage 1a and 30 cases were in stage 1b; median survival times were 182.3 months and 152.5 months, and the five-year survival rates were 100% and 81.5% (P >0.05), respectively. The five-year survival of the patients who underwent local excision; radical vulvectomy and en bloc resection of inguinofemoral lymphadenectomy; orradical vulvectomyen bloc resection of inguinofemoral lymphadenectomy, and pelvic lymph nodes was 50%, 81.8%, and 83.9%, respectively. For these cases, 74.3% of the tumors were medial while 25.7% were lateral, and the five-year survival rates of patients according to tumor location were 87.0% and 64.8% (P <0.05), respectively. The inguinal lymph node not increased and active were 16 cases (45.7%), and increased, active and hard were 17 cases (48.6%), and syncretic were two cases (5.7%), five-year survival rates were 73.3%, 92.9% and 50% (P <0.05), respectively. Of these cases, 74.3% of the tumors were cauliflower-like and 25.7% were nodular; five-year survival rates by tumor type were 91.3% and 66.7% (P <0.05), respectively. CONCLUSIONS: For patients with early-stage squamous cell carcinoma of the vulva, surgical operation is the primary, yet the best, treatment. The related prognostic factors were tumor location (lateral/medial), stage, gross morphology, and clinical state of the inguinal lymph node.

Decreased expression of light chain 3 (LC3) increased the risk of distant metastasis in triple-negative breast cancer.

The aim of this study was to evaluate the prognostic value of light chain 3 (LC3) expression in triple-negative breast cancer (TNBC) and describe the association of LC3 expression with the occurrence of metastasis. LC3 expression in tissue microarray was evaluated by immunohistochemistry in 163 patients with TNBC. The prognostic value of LC3 expression was assessed by a Cox regression model adjusted for clinical characteristics. Low LC3 expression in TNBC was observed in 56 (34.4 %) of 163 TNBC. Low LC3 expression significantly correlated with a higher risk of distant metastasis, rather than locoregional relapse. The 10-year distant metastases-free survival for LC3-negative and LC3-positive patients was 57.2 and 95.1 %, respectively (p < 0.0001). Accordingly, a significant correlation was found between LC3 expression and disease-free survival (DFS) and overall survival (OS). Multivariate analysis indicated that LC3 negative was a significant independent prognostic factor of DFS (p = 0.019), but not for OS (p = 0.545) in all patients. Our results suggested that expression of LC3 in TNBC was associated with higher distant metastases. This finding could open new avenues for the development of novel therapy strategies to TNBC.

Saracatinib (AZD0530) is a potent modulator of ABCB1-mediated multidrug resistance in vitro and in vivo.

Saracatinib, a highly selective, dual Src/Abl kinase inhibitor, is currently in a Phase II clinical trial for the treatment of ovarian cancer. In our study, we investigated the effect of saracatinib on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters in vitro and in vivo. Our results showed that saracatinib significantly enhanced the cytotoxicity of ABCB1 substrate drugs in ABCB1 overexpressing HeLa/v200, MCF-7/adr and HEK293/ABCB1 cells, an effect that was stronger than that of gefitinib, whereas it had no effect on the cytotoxicity of the substrates in ABCC1 overexpressing HL-60/adr cells and its parental sensitive cells. Additionally, saracatinib significantly increased the doxorubicin (Dox) and Rho 123 accumulation in HeLa/v200 and MCF-7/adr cells, whereas it had no effect on HeLa and MCF-7 cells. Furthermore, saracatinib stimulated the ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner. In addition, the homology modeling predicted the binding conformation of saracatinib within the large hydrophobic drug-binding cavity of human ABCB1. However, neither the expression level of ABCB1 nor the phosphorylation level of Akt was altered at the reversal concentrations of saracatinib. Importantly, saracatinib significantly enhanced the effect of paclitaxel against ABCB1-overexpressing HeLa/v200 cancer cell xenografts in nude mice. In conclusion, saracatinib reverses ABCB1-mediated MDR in vitro and in vivo by directly inhibiting ABCB1 transport function, without altering ABCB1 expression or AKT phosphorylation. These findings may be helpful to attenuate the effect of MDR by combining saracatinib with other chemotherapeutic drugs in the clinic.