A novel long non-coding RNA MIR4500HG003 promotes tumor metastasis through miR-483-3p-MMP9 axis in triple-negative breast cancer.

Breast cancer (BC) is the most common cancer and the leading cause of cancer-related deaths in women worldwide. The 5-year survival rate is over 90% in BC patients, but once BC cells metastasis into distal organs, it is dramatically decreasing to less than 30%. Especially, triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. Understanding the underline mechanisms of TNBC metastasis is a critical issue. Non-coding RNAs, including of lncRNAs and microRNAs, are non-protein-coding transcripts and have been reported as important regulators in TNBC metastasis. However, the underline mechanisms for non-coding RNAs regulating TNBC metastasis remain largely unclear. Here, we found that lncRNA MIR4500HG003 was highly expressed in highly metastatic MDA-MB-231 TNBC cells and overexpression of MIR4500HG003 enhanced metastasis ability in vitro and in vivo and promoted MMP9 expression. Furthermore, we found MIR4500HG003 physically interacted with miR-483-3p and reporter assay showed miR-483-3p attenuated MMP9 expression. Importantly, endogenous high expressions of MIR4500HG003 were correlated with tumor recurrence in TNBC patients with tumor metastasis. Taken together, our findings suggested that MIR4500HG003 promotes metastasis of TNBC through miR-483-3p-MMP9 signaling axis and may be used as potential prognostic marker for TNBC patients.

Advance Care Planning Affects End-of-Life Treatment Preferences Among Patients With Heart Failure: A Randomized Controlled Trial.

This study explored the effects of advance care planning interventions on end-of-life treatment decisions among patients with heart failure. The study design was a randomized controlled trial. An intervention involving a motivational video, a cartoon version educational brochure, and a guided discussion was implemented. A total of 82 hospitalized patients with heart failure were recruited. Half of the participants received the intervention, and the other half received routine care. The Life Support Preferences Questionnaire was the primary measurement instrument. Before the advance care planning intervention, a significant difference between the experimental and control groups was observed in the cardiopulmonary resuscitation score but not the total, antibiotics, surgery, and artificial nutrition and hydration scores. In the experimental group but not in the control group, significant differences were observed between pretest and posttest total, antibiotics, cardiopulmonary resuscitation, surgery, and artificial nutrition and hydration scores. Significant differences in mean score changes were observed in total and each treatment score between the experimental and control groups. The advance care planning intervention led participants to select fewer medical treatments. This intervention may be suitable for societies where people are unfamiliar with advance care planning and may feel uncomfortable discussing death.

ICAM2 initiates trans-blood-CSF barrier migration and stemness properties in leptomeningeal metastasis of triple-negative breast cancer.

Leptomeningeal metastasis (LM) occurs when tumor cells spread to the leptomeningeal space surrounding the brain and the spinal cord, thereby causing poor clinical outcomes. The triple-negative breast cancer (TNBC) has been associated with symptoms of LM and mechanism remained unclear. Through proteomic analysis, we identified high expression of ICAM2 in leptomeningeal metastatic TNBC cells, which promoted the colonization of the spinal cord and resulted in poor survival in vivo. Two-way demonstration indicated that high levels of ICAM2 promoted blood-cerebrospinal fluid barrier (BCB) adhesion, trans-BCB migration, and stemness abilities and determined the specificity of LM in vivo. Furthermore, pull-down and antibody neutralizing assay revealed that ICAM2 determined the specificity of LM through interactions with ICAM1 in the choroid plexus epithelial cells. Therefore, neutralizing ICAM2 can attenuate the progression of LM and prolong survival in vivo. The results suggested that targeting ICAM2 is a potential therapeutic strategy for LM in TNBC.

Carboxyl-terminal modulator protein facilitates tumor metastasis in triple-negative breast cancer.

Currently, the survival rate for breast cancer is more than 90%, but once the cancer cells metastasize to distal organs, the survival rate is dramatically reduced, to less than 30%. Triple-negative breast cancer accounts for 15-20% of all breast cancers. Triple-negative breast cancer (TNBC) is associated with poor prognostic and diagnostic outcomes due to the limiting therapeutic strategies, relative to non-TNBC breast cancers. Therefore, the development of targeted therapy for TNBC metastasis remains an urgent issue. In this study, high Carboxyl-terminal modulator protein (CTMP) is significantly associated with recurrence and disease-free survival rate in TNBC patients. Overexpression of CTMP promotes migration and invasion abilities in BT549 cells. Down-regulating of CTMP expression inhibits migration and invasion abilities in MDA-MB-231 cells. In vivo inoculation of high-CTMP cells enhances distant metastasis in mice. The metastasis incidence rate is decreased in mice injected with CTMP-downregulating MDA-MB-231 cells. Gene expression microarray analysis indicates the Akt-dependent pathway is significantly enhanced in CTMP overexpressing cells compared to the parental cells. Blocking Akt activation via Akt inhibitor treatment or co-expression of the dominant-negative form of Akt proteins successfully abolishes the CTMP mediating invasion in TNBC cells. Our findings suggest that CTMP is a potential diagnostic marker for recurrence and poor disease-free survival in TNBC patients. CTMP promotes TNBC metastasis via the Akt-activation-dependent pathway.

MiR-211 determines brain metastasis specificity through SOX11/NGN2 axis in triple-negative breast cancer.

Brian metastasis, which is diagnosed in 30% of triple-negative breast cancer (TNBC) patients with metastasis, causes poor survival outcomes. Growing evidence has characterized miRNAs involving in breast cancer brain metastasis; however, currently, there is a lack of prognostic plasma-based indicator for brain metastasis. In this study, high level of miR-211 can act as brain metastatic prognostic marker in vivo. High miR-211 drives early and specific brain colonization through enhancing trans-blood-brain barrier (BBB) migration, BBB adherence, and stemness properties of tumor cells and causes poor survival in vivo. SOX11 and NGN2 are the downstream targets of miR-211 and negatively regulate miR-211-mediated TNBC brain metastasis in vitro and in vivo. Most importantly, high miR-211 is correlated with poor survival and brain metastasis in TNBC patients. Our findings suggest that miR-211 may be used as an indicator for TNBC brain metastasis.

Downregulation of STK4 promotes colon cancer invasion/migration through blocking ß-catenin degradation.

Mammalian STE20-like kinase 1 (MST1/STK4/KRS2) encodes a serine/threonine kinase that is the mammalian homolog of Drosophila Hippo. STK4 plays an important role in controlling cell growth, apoptosis, and organ size. STK4 has been studied in many cancers with previous studies indicating an involvement in colon cancer lymph node metastasis and highlighting its potential as a diagnostic marker for colon cancer. However, the role of STK4 defect in promoting colon cancer progression is still understudied. Here, we found that STK4 was significantly downregulated in colon cancer and was associated with distal metastasis and poor survival. Furthermore, STK4 knockdown enhanced sphere formation and metastasis in vitro and promoted tumor development in vivo. We found that STK4 colocalized with ß-catenin and directly phosphorylated ß-catenin resulting in its degradation via the ubiquitin-mediated pathway. This may suggest that STK4 knockdown causes ß-catenin phosphorylation failure and subsequently ß-catenin accumulation, consequently leading to anchorage-independent growth and metastasis in colon cancer. Our results support that STK4 may act as a potential candidate for the assessment of ß-catenin-mediated colon cancer prognosis.

High-CLDN4 ESCC cells harbor stem-like properties and indicate for poor concurrent chemoradiation therapy response in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the major type of esophageal cancer in Asia and demonstrates poor survival rates following a therapeutic regimen. METHODS: Cancer stem cells (CSCs) are responsible for tumor initiation, progression, and treatment failure in cancers. Therefore, identification and characterization of CSCs may help to improve clinical outcomes for ESCC patients. Tumor sphere formation assay are performed to isolate cancer stem-like ESCC cells. QRT-PCR, tumor initiation, metastasis, CCRT treatment are used to evaluate ESCC cells' stemness properties in vitro and in vivo. RESULTS: The authors' data demonstrates that cancer stem-like ESCC cells harbored stemness characteristics including self-renewal, differentiation, and transdifferentiation, and possess tumor initiation, metastasis, and treatment inefficiency properties. For the identification of useful biomarkers of cancer stem-like ESCC cells, the authors further identified that CLDN4 was upregulated in cancer stem-like ESCC cells when compared with bulk cancer cells. High-CLDN4 cells harbored stemness and cisplatin/concurrent chemoradiation therapy (CCRT) resistance properties and a high level of CLDN4 was correlated with poor prognosis and poor CCRT response in ESCC patients. Importantly, thiamine tetrahydrofurfuryl disulfide (TTFD) decreased CLDN4 and attenuated stemness in ESCC cells, and TTFD combined with CCRT improved CCRT response in vivo. CONCLUSIONS: CLDN4 was suggested as prognostic and a CCRT response indicator for ESCC patients. TTFD combined with CCRT has potential to improve ESCC patient's clinical outcomes in the future.

A novel long non-coding RNA linc-ZNF469-3 promotes lung metastasis through miR-574-5p-ZEB1 axis in triple negative breast cancer.

Triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. The major sites for TNBC metastasis include the lungs, brain, liver, and bone. Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nucleotides and have been reported as important regulators in BC metastasis. However, the underlying mechanisms for lncRNAs regulating TNBC metastasis are not fully understood. Here we found that linc-ZNF469-3 was highly expressed in lung-metastatic LM2-4175 TNBC cells and overexpression of linc-ZNF469-3 enhanced invasion ability and stemness properties in vitro and lung metastasis in vivo. Furthermore, we found linc-ZNF469-3 physically interacted with miR-574-5p and overexpression of miR-574-5p attenuated ZEB1 expression. Importantly, endogenous high expressions of linc-ZNF469-3 and ZEB1 were correlated with tumor recurrence in TNBC patients with lung metastasis. Taken together, our findings suggested that linc-ZNF469-3 promotes lung metastasis of TNBC through miR-574-5p-ZEB1 signaling axis and may be used as potential prognostic marker for TNBC patients.

CTMP, a predictive biomarker for trastuzumab resistance in HER2-enriched breast cancer patient.

Trastuzumab is regarded as the primary therapy for patients with HER2-enriched breast cancer, but the pathological complete response for advanced cases is less than 30%. The underlying mechanism of trastuzumab resistance remains unclear and there are currently no conclusive biomarkers for patient response to trastuzumab. Identifying predictive biomarkers for trastuzumab response may allow treatments to be individually tailored and optimized multi-target therapies may be developed. CTMP activates AKT signaling in breast cancer and over-activation of AKT has been reported to contribute to trastuzumab resistance. In this study, we examined samples from 369 patients to investigate the correlation between CTMP expression level and patient outcome. Elevated CTMP expression was correlated with adverse outcomes in HER2-enriched patients including overall and disease-free survival as well as trastuzumab resistance. Ectopic expression of varying levels of CTMP in SkBR3 cells dose-dependently attenuated trastuzumab-mediated growth inhibition through AKT activation. In addition, inhibition of AKT signaling by AKT inhibitor IV and Rapamycin reversed CTMP-mediated trastuzumab resistance. In clinical samples, the high expression of CTMP was showed in trastuzumab non-responders and positively correlated with AKT activity. Taken together, we demonstrated that CTMP promotes AKT activation resulting in trastuzumab resistance in patients with HER2-enriched breast cancer. High CTMP expression not only predicted poor prognosis, but may also predict resistance to trastuzumab in HER2-enriched patients. Therefore, CTMP expression may be considered as a prognostic biomarker in HER2-enriched breast cancer and high expression may indicate a utility for AKT-inhibition in these patients.

Huntingtin-Interacting Protein-1 Is an Early-Stage Prognostic Biomarker of Lung Adenocarcinoma and Suppresses Metastasis via Akt-mediated Epithelial-Mesenchymal Transition.

RATIONALE: Non-small cell lung cancer (NSCLC) carries a poor survival rate mainly because of metastasis. However, the molecular mechanisms that govern NSCLC metastasis have not been described. Because huntingtin-interacting protein-1 (HIP1) is known to play a role in tumorigenesis, we tested the involvement of HIP1 in NSCLC progression and metastasis. OBJECTIVES: HIP1 expression was measured in human NSCLC tumors, and correlation with survival outcome was evaluated. Furthermore, we investigated the ability of HIP1 to suppress metastasis. The molecular mechanism by which HIP1 contributes to suppress metastasis was investigated. METHODS: We used tissue arrays containing samples from 121 patients with NSCLC to analyze HIP1 expression by immunohistochemistry. To investigate the role of HIP1 expression on metastasis, we evaluated cellular mobility, migration, and invasion using lung adenocarcinoma (AdCA) cells with modified HIP1 expression levels. The human disease mouse models with the same cells were applied to evaluate the HIP1 suppressing metastasis and its mechanism in vivo. MEASUREMENTS AND MAIN RESULTS: HIP1 expression in AdCA progression was found to be an early-stage prognostic biomarker, with low expression correlated to poor prognosis. We also found HIP1 to be a metastatic suppressor in AdCA. HIP1 significantly repressed the mobility of lung cancer cells in vitro and in vivo and regulated the epithelial-mesenchymal transition by repressing AKT/glycogen synthase kinase-3ß/ß-catenin signaling. CONCLUSIONS: HIP1 serves as an early-stage prognostic biomarker and a metastatic suppressor. Reduced expression during AdCA progression can relieve HIP1 suppression of Akt-mediated epithelial-mesenchymal transition and thereby lead to development of late metastases and poor prognosis.

Pin1 positively affects tumorigenesis of esophageal squamous cell carcinoma and correlates with poor survival of patients.

BACKGROUND: Pin1 promotes oncogenesis by regulating multiple oncogenic signaling. In this study, we investigated the involvement of Pin1 in tumor progression and in the prognosis of human esophageal squamous cell carcinoma (ESCC). RESULTS: We observed that proliferation, clonogenicity and tumorigenesis of CE81T cells were inhibited by Pin1 knockdown. We next analyzed Pin1 expression in clinical ESCC specimens. When compared to the corresponding non-tumor part, Pin1 protein and mRNA levels in tumor part were higher in 84% and 62% patients, respectively. By immunohistochemistry, we identified that high Pin1 expression was associated with higher primary tumor stage (p = 0.035), higher overall cancer stage (p = 0.047) and poor overall survival (p < 0.001). Furthermore, the association between expression of Pin1 and levels of ß-catenin and cyclin D in cell line and clinical specimens was evaluated. ß-catenin and cyclin D1 were decreased in CE81T cells with Pin1 knockdown. Cyclin D1 level correlated with Pin1 expression in clinical ESCC specimens. CONCLUSIONS: Pin1 upregulation was associated with advanced stage and poor prognosis of ESCC. Pin1 knockdown inhibited aggressiveness of ESCC cells. ß-catenin and cyclin D1 were positively regulated by Pin1. These results indicated that targeting Pin1 pathway could represent a potential modality for treating ESCC.

High expression of CHRNA1 is associated with reduced survival in early stage lung adenocarcinoma after complete resection.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths around the world, and a high recurrence rate after complete resection is an important issue reducing the cure rate and survival of patients with early stage NSCLC. Several pathologic biomarkers are associated with recurrence in early stage lung cancer after complete resection. METHODS: We evaluated the expression and prognostic value of the α1 subunit of the nicotinic acetylcholine receptor (CHRNA1) as well as other pathologic features of tumor tissues resected from patients with stage I adenocarcinoma of the lung. RESULTS: A high ratio (173/185) of CHRNA1 expression (93.5 %) was found in stage I lung adenocarcinoma. In the multivariate survival analysis, tumor necrosis, angiolymphatic invasion, perineural invasion, and CHRNA1 expression were independent poor prognostic factors for both recurrence-free and overall survival (OS). Patients expressing CHRNA1 had worse median recurrence-free survival (60.6 vs. 77.9 months, P = 0.03) and OS (65.1 vs. 77.9 months, P = 0.04) compared with CHRNA1-negative patients. CONCLUSIONS: CHRNA1 expression could be directly tested from the tumor after complete resection. In early stage NSCLC, it could be a useful prognostic factor for recurrence and survival.

Loss of RUNX3 increases osteopontin expression and promotes cell migration in gastric cancer.

Loss of RUNX3 expression is frequently observed in gastric cancer and is highly associated with lymph node metastasis and poor prognosis. However, the underlying molecular mechanisms of gastric cancer remain unknown. In this study, we found that the protein levels of RUNX3 and osteopontin (OPN) are inversely correlated in gastric cancer clinical specimens and cell lines. Furthermore, similar inverse trends between RUNX3 and OPN messenger RNA (mRNA) expression were demonstrated in six out of seven normal-tumor-paired gastric cancer clinical specimens. In addition, low RUNX3 and high OPN expression were associated with poor prognosis in gastric cancer patients. Ectopic expression of green fluorescent protein-RUNX3 reduced OPN protein and mRNA expression in the AGS and SCM-1 gastric cancer cell lines. In contrast, knockdown of RUNX3 in GES-1, a normal gastric epithelial cell line, increased OPN expression. Although three RUNX3-binding sequences have been identified in the OPN promoter region, direct binding of RUNX3 to the specific binding site, -142 to -137bp, was demonstrated by chromatin immunoprecipitation assay. The binding of RUNX3 to the OPN promoter significantly decreased OPN promoter activity. The knockdown of OPN or overexpression of RUNX3 inhibited cell migration in AGS and SCM-1 cells; however, the coexpression of RUNX3 and OPN reversed the RUNX3-reduced migration ability in AGS and SCM-1 cells. In contrast, the knockdown of both RUNX3 and OPN inhibited RUNX3-knockdown-induced migration of GES-1 cells. Together, our data demonstrated that RUNX3 is a transcriptional repressor of OPN and that loss of RUNX3 upregulates OPN, which promotes migration in gastric cancer cells.

Protein phosphorylation profiling using an in situ proximity ligation assay: phosphorylation of AURKA-elicited EGFR-Thr654 and EGFR-Ser1046 in lung cancer cells.

The epidermal growth factor receptor (EGFR), which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations.

Analysis of protein-protein interactions in cross-talk pathways reveals CRKL protein as a novel prognostic marker in hepatocellular carcinoma.

Deciphering the network of signaling pathways in cancer via protein-protein interactions (PPIs) at the cellular level is a promising approach but remains incomplete. We used an in situ proximity ligation assay to identify and quantify 67 endogenous PPIs among 21 interlinked pathways in two hepatocellular carcinoma (HCC) cells, Huh7 (minimally migratory cells) and Mahlavu (highly migratory cells). We then applied a differential network biology analysis and determined that the novel interaction, CRKL-FLT1, has a high centrality ranking, and the expression of this interaction is strongly correlated with the migratory ability of HCC and other cancer cell lines. Knockdown of CRKL and FLT1 in HCC cells leads to a decrease in cell migration via ERK signaling and the epithelial-mesenchymal transition process. Our immunohistochemical analysis shows high expression levels of the CRKL and CRKL-FLT1 pair that strongly correlate with reduced disease-free and overall survival in HCC patient samples, and a multivariate analysis further established CRKL and the CRKL-FLT1 as novel prognosis markers. This study demonstrated that functional exploration of a disease network with interlinked pathways via PPIs can be used to discover novel biomarkers.

Overexpression of T-LAK cell-originated protein kinase predicts poor prognosis in patients with stage I lung adenocarcinoma.

Tumor recurrence is the most common cause of disease failure after surgical resection in early-stage lung adenocarcinoma. Identification of clinically relevant prognostic markers could help to predict patients with high risk of disease recurrence. A meta-analysis of available lung adenocarcinoma microarray datasets revealed that T-LAK cell-originated protein kinase (TOPK), a serine/threonine protein kinase, is overexpressed in lung cancer. Using stable cell lines with overexpression or knockdown of TOPK, we have shown that TOPK can promote cell migration, invasion, and clonogenic activity in lung cancer cells, suggesting its crucial role in lung tumorigenesis. To evaluate the prognostic value of TOPK expression in resected stage I lung adenocarcinoma, a retrospective analysis of 203 patients diagnosed with pathological stage I lung adenocarcinoma was carried out to examine the expression of TOPK by immunohistochemistry (IHC). The prognostic significance of TOPK overexpression was examined. Overexpression of TOPK (IHC score >3) was detected in 67.0% of patients, and these patients were more frequently characterized with disease recurrence and angiolymphatic invasion. Using multivariate analysis, patient age (>65 years old; P = 0.002) and TOPK overexpression (IHC score >3; P < 0.001) significantly predicted a shortened overall survival. Moreover, TOPK overexpression (IHC score >3; P = 0.005) also significantly predicted a reduced time to recurrence in the patients. Our results indicate that overexpression of TOPK could predetermine the metastatic capability of tumors and could serve as a significant prognostic predictor of shortened overall survival and time to recurrence.