SUMMARY Cancer-secreted miRNAs are emerging mediators of cancer–host crosstalk. Here we show that miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is a potent regulator of migration through targeting the tight junction protein ZO-1. In endothelial monolayers, exosome-mediated transfer of cancer-secreted miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-105 in non-metastatic cancer cells induces metastasis and vascular permeability in distant organs, whereas inhibition of miR-105 in highly metastatic tumors alleviates these effects. MiR-105 can be detected in the circulation at the pre-metastatic stage, and its levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer.

BackgroundMicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.MethodsThe pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.ResultsMore than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.ConclusionsOur study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.

Fibroblast growth factor (FGF) receptor (FGFR) substrate 2 (FRS2) is an adaptor protein that plays a critical role in FGFR signaling. FRS2 is located on chromosome 12q13-15 that is frequently amplified in liposarcomas. The significance of FRS2 and FGFR signaling in high-grade liposarcomas is unknown. Herein, we first comparatively examined the amplification and expression of FRS2 with CDK4 and MDM2 in dedifferentiated liposarcoma (DDLS) and undifferentiated high-grade pleomorphic sarcoma (UHGPS). Amplification and expression of the three genes were identified in 90% to 100% (9-11 of 11) of DDLS, whereas that of FRS2, CDK4, and MDM2 were observed in 55% (41 of 75), 48% (36 of 75), and 44% (33/75) of clinically diagnosed UHGPS, suggesting that these "UHGPS" may represent DDLS despite lacking histologic evidence of lipoblasts. Immunohistochemical analysis of phosphorylated FRS2 protein indicated that the FGFR/FRS2 signaling axis was generally activated in about 75% of FRS2-positive high-grade liposarcomas. Moreover, we found that FRS2 and FGFRs proteins are highly expressed and functional in three high-grade liposarcoma cell lines: FU-DDLS-1, LiSa-2, and SW872. Importantly, the FGFR selective inhibitor NVP-BGJ-398 significantly inhibited the growth of FU-DDLS-1 and LiSa-2 cells with a concomitant suppression of FGFR signal transduction. Attenuation of FRS2 protein in FU-DDLS-1 and LiSa-2 cell lines decreased the phosphorylated extracellular signal-regulated kinase 1/2 and AKT and repressed cell proliferation. These findings indicate that analysis of FRS2 in combination with CDK4 and MDM2 will more accurately characterize pathologic features of high-grade liposarcomas. Activated FGFR/FRS2 signaling may play a functional role in the development of high-grade liposarcomas, therefore, serve as a potential therapeutic target.

Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here we report that berbamine (BBM) and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca2+/calmodulin-dependent protein kinase II (CAMKII). Furthermore, BBM inhibited the in vivo tumorigenicity of liver cancer cells in NOD/SCID mice, and down-regulated the self-renewal abilities of liver cancer initiating cells. Chemical inhibition or short hairpin RNAs-mediated knockdown of CAMKII recapitulated the effects of BBM, while overexpression of CAMKII promoted cancer cell proliferation and increased the resistance of liver cancer cells to BBM treatments. Western blot analyses of human liver cancer specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with the paired peri-tumor tissues, which supports a role of CAMKII in promoting human liver cancer progression and the potential clinical use of BBM for liver cancer therapies. Our data suggests that BBM and its derivatives are promising agents to suppress liver cancer growth by targeting CAMKII.

BackgroundTriple negative breast cancer (TNBC) has higher rates of recurrence and distant metastasis, and poorer outcome as compared to non-TNBC. Aberrant activation of WNT signaling has been detected in TNBC, which might be important for triggering oncogenic conversion of breast epithelial cell. Therefore, we directed our focus on identifying the WNT ligand and its underlying mechanism in TNBC cells.MethodsWe performed large-scale analysis of public microarray data to screen the WNT ligands and the clinical significance of the responsible ligand in TNBC. WNT5B was identified and its overexpression in TNBC was confirmed by immunohistochemistry staining, Western blot and ELISA. ShRNA was used to knockdown WNT5B expression (shWNT5B). Cellular functional alteration with shWNT5B treatment was determined by using wound healing assay, mammosphere assay; while cell cycle and apoptosis were examined by flowcytometry. Mitochondrial morphology was photographed by electron microscope. Biological change of mitochondria was detected by RT-PCR and oxygen consumption assay. Activation of WNT pathway and its downstream targets were evaluated by liciferase assay, immunohistochemistry staining and immunoblot analysis. Statistical methods used in the experiments besides microarray analysis was two-tailed t-test.ResultsWNT5B was elevated both in the tumor and the patients’ serum. Suppression of WNT5B remarkably impaired cell growth, migration and mammosphere formation. Additionally, G0/G1 cell cycle arrest and caspase-independent apoptosis was observed. Study of the possible mechanism indicated that these effects occurred through suppression of mitochondrial biogenesis, as evidenced by reduced mitochondrial DNA (MtDNA) and compromised oxidative phosphorylation (OXPHOS). In Vivo and in vitro data uncovered that WNT5B modulated mitochondrial physiology was mediated by MCL1, which was regulated by WNT/β-catenin responsive gene, Myc. Clinic data analysis revealed that both WNT5B and MCL1 are associated with enhanced metastasis and decreased disease-free survival.ConclusionsAll our findings suggested that WNT5B/MCL1 cascade is critical for TNBC and understanding its regulatory apparatus provided valuable insight into the pathogenesis of the tumor development and the guidance for targeting therapeutics.

Ribonucleotide reductase subunit RRM2B (p53R2) has been reported to suppress invasion and metastasis in colorectal cancer (CRC). Here we report that high levels of RRM2B expression is correlated with markedly better survival in CRC patients. In a fluorescence-labeled orthotopic mouse xenograft model, we confirmed that overexpression of RRM2B in non-metastatic CRC cells prevented lung and/or liver metastasis, relative to control cells that did metastasize. Clinical outcome studies were conducted on a training set with 103 CRCs and a validation set with 220 CRCs. All participants underwent surgery with periodic follow-up to determine survivability. A newly developed specific RRM2B antibody was employed to perform immunohistochemistry (IHC) for determining RRM2B expression levels on tissue arrays. In the training set, the Kaplan-Meier and multivariate COX analysis revealed that RRM2B is associated with better survival of CRCs, especially in stage IV patients (Hazard ratio, HR=0.40; 95% CI 0.18–0.86, p=0.016). In the validation set, RRM2B was negatively related to tumor invasion (odds ratio, OR=0.45, 95% CI 0.19–0.99, p=0.040) and lymph node involvement (OR=0.48, 95% CI 0.25–0.92, p=0.026). Further, elevated expression of RRM2B was associated with better prognosis in this set as determined by multivariate analyses (HR=0.48, 95% CI 0.26–0.91, p=0.030). Further investigations revealed that RRM2B was correlated with better survival of CRCs with advanced stage III–IV tumors rather than earlier stage I–II tumors. Taken together, our findings establish that RRM2B suppresses invasiveness of cancer cells and that its expression is associated with a better survival prognosis for CRC patients.

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PurposeThe development of resistance against anticancer drugs has been a persistent clinical problem for the treatment of locally advanced malignancies in the head and neck mucosal derived squamous cell carcinoma (HNSCC). Recent evidence indicates that the DNA translesion synthesis (TLS) polymerase η (Pol η; hRad30a gene) reduces the effectiveness of gemcitabine/cisplatin. The goal of this study is to examine the relationship between the expression level of Pol η and the observed resistance against these chemotherapeutic agents in HNSCC, which is currently unknown. MethodsSixty-four mucosal derived squamous cell carcinomas of head and neck (HNSCC) from 1989 and 2007 at the City of Hope National Medical Center (Duarte, CA) were retrospectively analyzed. Pretreatment samples were immunostained with anti-Pol η antibody and the correlation between the expression level of Pol η and clinical outcomes were evaluated. Forty-nine cases treated with platinum (n=40) or gemcitabine (n=9) based chemotherapy were further examined for Pol η expression level for comparison with patient response to chemotherapy. ResultsThe expression of Pol η was elevated in 67% of the head and neck tumor samples. Pol η expression level was significantly higher in grade 1 to grade 2 tumors (well to moderately differentiated). The overall benefit rate (complete response+ partial response) in patients treated with platinum and gemcitabine based chemotherapy was 79.5%, where low Pol η level was significantly associated with high complete response rate (p=0.03), although not associated with overall survival. Furthermore, no significant correlation was observed between Pol η expression level with gender, age, tobacco/alcohol history, tumor stage and metastatic status.ConclusionsOur data suggest that Pol η expression may be a useful prediction marker for the effectiveness of platinum or gemcitabine based therapy for HNSCC.