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Nitric oxide (NO), a free radical, plays a critical role in a wide range of physiological and pathological processes. Due to its pleiotropic function, it has been widely investigated in various types of cancers and is strongly associated with cancer development. Mounting pieces of evidence show that NO regulates various cancer-related events, which mainly depends on phosphorylating the key proteins in several signaling pathways. However, phosphorylation of proteins modulated by NO signaling pathway may lead to different effects in different types of cancer, which is complex and remains unclear. Therefore, in this review, we focus on the effect of protein phosphorylation modulated by NO signaling pathway in different types of cancers including breast cancer, lung cancer, prostate cancer, colon cancer, gastric cancer, pancreatic cancer, ovarian cancer, and neuroblastoma. Phosphorylation of key proteins, including p38 MAPK, ERK, PI3K, STAT3, and p53, modified by NO in various signaling pathways affects different cancer-related processes including cell apoptosis, proliferation, angiogenesis, metastasis, and several cancer therapies. Our review links the NO signaling pathway to protein phosphorylation in cancer development and provides new insight into potential targets and cancer therapy.

Deregulation of E3 ubiquitin ligases is intimately implicated in breast cancer pathogenesis and progression, but the underlying mechanisms still remain elusive. Here we report that RING finger protein 144A (RNF144A), a poorly characterized member of the RING-in-between-RING family of E3 ubiquitin ligases, functions as a tumor suppressor in breast cancer. RNF144A was downregulated in a subset of primary breast tumors and restoration of RNF144A suppressed breast cancer cell proliferation, colony formation, migration, invasion in vitro, tumor growth, and lung metastasis in vivo. In contrast, knockdown of RNF144A promoted malignant phenotypes of breast cancer cells. Quantitative proteomics and biochemical analysis revealed that RNF144A interacted with and targeted heat-shock protein family A member 2 (HSPA2), a putative oncoprotein that is frequently upregulated in human cancer and promotes tumor growth and progression, for ubiquitination and degradation. Notably, the ligase activity-defective mutants of RNF144A impaired its ability to induce ubiquitination and degradation of HSPA2, and to suppress breast cancer cell proliferation, migration, and invasion as compared with its wild-type counterpart. Moreover, RNF144A-mediated suppression of breast cancer cell proliferation, migration, and invasion was rescued by ectopic HSPA2 expression. Clinically, low RNF144A and high HSPA2 expression in breast cancer patients was correlated with aggressive clinicopathological characteristics and decreased overall and disease-free survival. Collectively, these findings reveal a previously unappreciated role for RNF144A in suppression of breast cancer growth and metastasis, and identify RNF144A as the first, to our knowledge, E3 ubiquitin ligase for HSPA2 in human cancer.

Simple Summary There is clinical evidence that ulcerated and inflammatory cell-infiltrated oral cancer is frequently associated with early metastases. Our results from genomic screening in patients with metastatic oral cancer identified specific changes in genes that regulate macrophage chemotaxis and drive tumor progression. This opens up potential therapeutic opportunities toward personalized medicine tailored to manage patients with advanced disease. Abstract Invasive oral squamous cell carcinoma (OSCC) is often ulcerated and heavily infiltrated by pro-inflammatory cells. We conducted a genome-wide profiling of tissues from OSCC patients (early versus advanced stages) with 10 years follow-up. Co-amplification and co-overexpression of TWIST1, a transcriptional activator of epithelial-mesenchymal-transition (EMT), and colony-stimulating factor-1 (CSF1), a major chemotactic agent for tumor-associated macrophages (TAMs), were observed in metastatic OSCC cases. The overexpression of these markers strongly predicted poor patient survival (log-rank test, p = 0.0035 and p = 0.0219). Protein analysis confirmed the enhanced expression of TWIST1 and CSF1 in metastatic tissues. In preclinical models using OSCC cell lines, macrophages, and an in vivo matrigel plug assay, we demonstrated that TWIST1 gene overexpression induces the activation of CSF1 while TWIST1 gene silencing down-regulates CSF1 preventing OSCC invasion. Furthermore, excessive macrophage activation and polarization was observed in co-culture system involving OSCC cells overexpressing TWIST1. In summary, this study provides insight into the cooperation between TWIST1 transcription factor and CSF1 to promote OSCC invasiveness and opens up the potential therapeutic utility of currently developed antibodies and small molecules targeting cancer-associated macrophages.

Uveal melanoma (UVM) is an adult intraocular malignancy which is the most frequent and has a high tendency for metastasis. This study aims to develop significant differential gene subnetwork between primary and metastatic UVM to identify potential prognostic biomarkers. Differentially expressed genes (DEGs) among three chip datasets were downloaded from Gene Expression Omnibus and identified according to standardization annotation information. Genetic enrichment analyses were utilized to describe biologic functions. The protein-protein interaction network of DEGs was developed and the module analysis was constructed by STRING and Cytoscape. Kaplan-Meier method of the integrated expression score was applied to analyze survival outcomes. Functional annotation was assessed to perform GO and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. In addition, ClueGO and gene set enrichment analysis were analyzed to detect underlying significant genes and involved signaling pathways. A total of 103 DEGs with function enrichment were recognized and might be considered as candidate prognostic biomarkers between primary and metastatic UVM. Furthermore, Kaplan-Meier method suggested that SCD5, SPTBN1, FABP5, SQLE, PTPLA (HACD1), and CDC25B were independent prognostic factors in UVM. Functional annotations indicated that the most involved significant pathways including interferon-gamma response, IL-6 JAK STAT3 signaling, TNFA signaling via NFKB and inflammatory response. Significant DEGs between primary and metastatic UVM tissue were identified and might have involved in the metastasis of UVM. SCD5, SPTBN1, FABP5, SQLE, PTPLA (HACD1), and CDC25B transcription levels were of high prognostic value, which might assist us to understand the underlying carcinogenesis or advancement of UVM better.

Objective. To investigate the expression of kinesin family member 20A (KIF20A) in bladder cancer, the effect of KIF20A on the proliferation and metastasis of bladder cancer cells, and the effect of KIF20A expression on the prognosis of bladder cancer patients.Methods. Bladder cancer tissue and its adjacent tissues were collected from tumour patients. The mRNA and protein expression levels of KIF20A in the tissue samples were detected by qRT-PCR and western blot. Immunohistochemical (IHC) staining was used to identify the expression and distribution of KIF20A proteins in the tissue samples. The relationship between the KIF20A expression and the clinical pathology of bladder cancer was analysed. The effect of the differential expression of KIF20A on the prognosis of patients with bladder cancer was analysed by the TCGA database. The plasmid was transfected into the bladder cell lines T24 and 5637 to construct two stable cell lines with knocked down KIF20A. The effect of KIF20A expression on the proliferation and invasion of T24 and 5637 bladder cells was explored in vitro using the abovementioned stable cell lines. The effect of the KIF20A expression on the proliferation of bladder cancer cells was evaluated by a mouse xenograft model.Results. The expression of KIF20A was significantly higher in the bladder cancer tissues than in the adjacent control tissues. The expression of KIF20A was significantly associated with the degree of pathological differentiation of bladder cancer. Patients with a higher expression of KIF20A had a higher tumour grade and a more advanced stage. The mean survival of patients with a high KIF20A expression was significantly lower than the mean survival of patients with a low KIF20A expression. The in vitro experiments demonstrated that the knockdown of KIF20A significantly inhibited T24 and 5637 cell proliferation and invasion. The in vivo experiments showed that the knockdown of KIF20A significantly inhibited the proliferation of the bladder tumours.Conclusion. KIF20A promotes the proliferation and metastasis of bladder cancer cells. Bladder cancer patients with a high KIF20A expression have a worse tumour differentiation and a poor prognosis. KIF20A may become an independent factor that affects the prognosis of bladder cancer patients and a therapeutic target for bladder cancer.

Multidrug resistance (MDR) remains a major clinical obstacle in the treatment of gastric cancer (GC) since it causes tumor recurrence and metastasis. The transcription factor activator protein-2α (AP-2α) has been implicated in drug-resistance in breast cancer; however, its effects on MDR of gastric cancer are far from understood. In this study, we aimed to explore the effects of AP-2α on the MDR in gastric cancer cells selected by vincristine (VCR). Decreased AP-2α levels were markedly detected by RT-PCR and Western blot in gastric cancer cell lines (BGC-823, SGC-7901, AGS, MKN-45) compared with that in the gastric epithelial cell line (GES-1). Furthermore, we found that the expression of AP-2α in SGC7901/VCR or SGC7901/adriamycin (ADR) cells was lower than in SGC7901 cells. Thus, a vector overexpressing AP-2α was constructed and used to perform AP-2α gain-of-function studies in SGC7901/VCR cells. The decreased IC50 values of the anti-cancer drugs in sensitive and resistant cells after transfect with pcDNA3.1/AP-2α were determined in SGC7901/VCR cells by MTT assay. Moreover, flow cytometry analysis indicated that overexpressed AP-2α induced cell cycle arrest in the G0/G1 phase and promoted cell apoptosis of VCR-selected SGC7901/VCR cells. RT-PCR and Western blot demonstrated that overexpressed AP-2α can significantly induce the down-regulation of Notch1, Hes-1, P-gp and MRP1 in SGC7901/VCR cells. Similar effects can be observed when Numb (Notch inhibitor) was introduced. In addition, the intracellular ADR accumulation was markedly detected in AP-2α overexpressed or Numb cells. In conclusion, our results indicate that AP-2α can reverse the MDR of gastric cancer cells, which may be realized by inhibiting the Notch signaling pathway.

Patients with metastatic rhabdomyosarcoma (RMS) have a poor prognosis. The detection of contaminating RMS cells in the bone marrow (BM) is important in clinical staging and risk assessment. The cytological examination of the BM remains the gold standard for the diagnosis of RMS, but has a limited sensitivity. In the present study, 32 BM and two cerebrospinal fluid (CSF) samples from 11 patients with suspected metastasis were analyzed by flow cytometry (FCM) with ganglioside D2 (GD2) conjugated with fluorescein isothiocyanate, cluster of differentiation (CD)90-phycoerythrin, CD45-peridinin chlorophyll protein and CD56-allophycocyanin monoclonal antibody cocktail in parallel to morphological examination at diagnosis or during treatment. Five samples (14.7%) were positive for RMS onup morphological examination. By FCM, 16 samples (47.1%) were positive for RMS. A significant difference was identified between the two methods. The four-color FCM assay successfully detected RMS cells in BM samples to a level of 0.01% (1 per 104 cells). RMS cells demonstrated a phenotype with CD56+/CD90+/CD45−/GD2− expression, which is different from the CD56+/CD90+/CD45−/GD2+ expression phenotype in neuroblastoma cells. The follow-up of four patients by FCM demonstrated that two patients became minimal residual disease-negative following two and four cycles of chemotherapy, respectively, and survived. The other two cases remained FCM-positive despite receiving four courses of chemotherapy and consequently succumbed to progressive disease. In addition, FCM analysis of the CSF samples from one patient confirmed a diagnosis of CSF metastasis with RMS. In conclusion, FCM may have a role not only in staging and monitoring the effects of therapy, but also in providing diagnostic confirmation of CSF metastasis with RMS.

BACKGROUND Many studies have investigated the prognostic role of E-cadherin in patients with NSCLC; however, the result still remains inconclusive. An up-to data system review and meta-analysis was necessary to give a comprehensive evaluation of prognostic role of E-cadherin in NSCLC. METHODS Eligible studies were searched in Pubmed, Embase and Web of Science databases. The inclusion criteria were studies that assessed the relationship between E-cadherin expression detected by immunohistochemistry (IHC) and the prognosis or clinicopathological features in patients with NSCLC. Subgroup analysis according to race, percentage of reduced/negative E-cadherin expression, histological type, and sample size were also conducted. Odds ratio (OR) or hazard ratio (HR) with 95% confidence interval (CI) were calculated to examine the risk or hazard association. RESULTS A total of 29 studies including 4010 patients were qualified for analysis. The analysis suggested that downregulated E-cadherin expression was significant associated with unfavorable overall survival (OS) and disease-free survival/progression-free survival (DFS/PFS) in patients with NSCLC. Subgroup analysis by race, percentage of reduced/negative E-cadherin expression, sample size also found the significant association in OS. When only the stage I NSCLC were considered, downregulated E-cadherin expression still had an unfavorable impact on OS. Additionally, downregulated E-cadherin expression was significantly associated with differentiation grade, lymphnode metastasis, vascular invasion, and TNM stage. CONCLUSION Downregulated E-cadherin expression detected by IHC seems to correlate with tumour progression and could serve as an important prognostic factor in patients with NSCLC.

We assessed the MSKCC nomogram performance in predicting SLN metastases in a Chinese breast cancer population. A new model (the SCH nomogram) was developed with clinically relevant variables and possible advantages. Data were collected from 1,545 patients who had a successful SLN biopsy between March 2005 and November 2011. We validated the MSKCC nomogram in the modeling and validation group. Clinical and pathologic features of SLN biopsy in modeling group of 1,000 patients were assessed with multivariable logistic regression to predict the presence of SLN metastasis in breast cancer. The SCH nomogram was created from the logistic regression model and subsequently applied to 545 consecutive SLN biopsies. By multivariate analysis, age, tumor size, tumor location, tumor type, and lymphovascular invasion were identified as independent predictors of SLN metastasis. The SCH nomogram was then developed using the five variables. The new model was accurate and discriminating (with an AUC of 0.7649 in the modeling group) compared to the MSKCC nomogram (with an AUC of 0.7105 in the modeling group). The area under the ROC curve for the SCH nomogram in the validation population is 0.7587. The actual probability trends for the various deciles were comparable to the predicted probabilities. The false-negative rates of the SCH nomogram were 1.67, 3.54, and 8.20 % for the predicted probability cut-off points of 5, 10, and 15 %, respectively. Compared with the MSKCC nomogram, the SCH nomogram has a better AUC with fewer variables and has lower false-negative rates for the low-probability subgroups. The SCH nomogram could serve as a more acceptable clinical tool in preoperative discussions with patients, especially very-low-risk patients. When applied to these patients, the SCH nomogram could be used to safely avoid a SLN procedure. The nomogram should be validated in various patient populations to demonstrate its reproducibility.

Accurate intraoperative diagnosis of sentinel lymph node (SLN) metastasis reduces the need for additional surgery in patients with involved nodes. The present study evaluates the clinical value of multiple cross-sectional touch imprint cytology (TIC) as an intraoperative assessment for the diagnosis of SLN metastasis.This study consisted of 366 patients with surgically harvested SLNs that were sliced along their long axis at 2.0-3.0-mm intervals and 122 patients with SLNs that were sliced along their short axis at 1.5-mm intervals using a cutting apparatus designed by our group. The first group of patients was enrolled in this study between February 2005 and February 2008, while the second group was enrolled between March 2008 and January 2009. Serial sectioning of the SLNs at 100-microm intervals with hematoxylin-eosin (HE) staining was used as the gold standard for pathological diagnosis.Multiple cross-sectional TIC has a sensitivity, specificity, and overall accuracy rate of 92.0, 99.0, and 97.5%, respectively, on a per-patient basis, and it is superior to the standard imprint preparation protocol. Furthermore, the multiple cross-sectional TIC technique developed in this study was observed to detect more accurately macrometastases on a per-patient basis in comparison to the typical protocol (P = 0.023). Of the patients included in this study, 97.7% had a positive SLN within their first three harvested SLNs.Multiple cross-sectional TIC is superior to the standard protocol, especially due to its ability to locate macrometastasis. Limiting intraoperative TIC to the first three harvested SLNs in the diagnosis of SLN metastasis may make this diagnostic procedure significantly cheaper and easier for pathologists to perform.

Background and Objectives Phyllodes tumors (PTs) are uncommon biphasic breast tumors that usually occur in adult females. They are composed of a benign epithelial component and a cellular, spindle cell stroma forming a leaf-like structure. No one morphologic finding is reliable in predicting the clinical behavior of the tumor. The purpose of this study was to explore the clinicopathologic factors associated with outcome and metastasis. Methods We retrospectively reviewed the records of 172 patients seen at Mackay Memorial Hospital from January 1985 to December 2003. Clinical data analyzed included age, presenting symptoms and signs, tumor size, location, type of surgery, time to recurrence, and metastasis. The clinicopathologic factors associated with outcome and metastasis were analyzed statistically using the chi-square test with Yate correction. Results The mean follow-up was 71 months (range 7–237). The mean age was 37 years (range 11–73). The majority of tumors were found in the upper outer quadrant (46.0%), with an equal propensity to occur in either breast (48.8% vs. 50.0%). The pathologic diagnoses included 131 benign, 12 borderline, and 29 malignant lesions. Nineteen patients (11%) had a recurrence and three (1.7%) had metastases. The initial diagnosis of all 19 recurrent tumors were benign. Age, surgical approach, mitotic activity, and surgical margin were significantly correlated with recurrence (P = 0.029, 0.020, 0.048, and 0.00018, respectively). Stromal cellularity, stromal overgrowth, stromal atypia, mitotic activity, tumor margin, and heterologous stromal elements were significantly correlated with metastases (P = 0.032, 0.00008, 0.000002, 0.004, 0.005, and 0.046, respectively). Mammography and breast echo were not reliable for differentiating PTs from fibroadenomas (6.9% vs. 37.9% and 3.3% vs. 45%, respectively). Frozen section was of limited value (41.6%). The role of adjuvant radiotherapy and chemotherapy remains to be defined. Local excision, wide excision, or mastectomy with negative surgical margins yielded high local control rates (88.7%, 88.2%, and 100%, respectively), but local excision was associated with a relatively high percentage of positive surgical margins (18.3%). A total of 42 modified radical mastectomies were performed. The reasons for these procedures included a diagnosis of malignancy on frozen section or because the tumors were so large, they were assumed to be carcinomas. No axillary lymph node metastases were found. Fifteen patients in our series had tumors with infiltrating tumor margin, severe stromal overgrowth, atypia, and cellularity. All three patients with metastases (3/15) were in this group. Presence of metastases was significantly correlated with this group (P = 0.0000038). Conclusions Wide excision with a clear margin may be the preferable initial therapy, even for malignant PTs. Routine axillary lymph node dissection is not recommended. Patients have tumors with infiltrating tumor margin, severe stromal overgrowth, atypia, and cellularity are at high risk for metastases. J. Surg. Oncol. 2005;91:185–194. © 2005 Wiley-Liss, Inc.

Background: Liver metastasis is a common and often fatal occurrence in cancer patients. A better understanding of the biology of liver metastasis is essential to improve survival statistics for these patients. We have previously shown that metastatic lung and colon carcinoma cells that invade the liver initiate a rapid host inflammatory response that entails increased production of cytokines TNF-α and IL-1 and results in increased tumor adhesion, transmigration and metastasis. TNF-α is a pleiotropic cytokine that can also induce tumor cell apoptosis. We hypothesized that the ability of tumor cells to form liver metastases in the face of increased local TNF-α levels is due to an acquired resistance to the tumoricidal effects of TNF-α. The objective of the present study was therefore to identify molecular mechanism(s) that regulate the tumor cell response and sensitivity to TNF-α. Methodology: We used a Lewis lung carcinoma-based tumor model consisting of poorly metastatic subline M-27 cells in which ectopic expression of the type 1 insulin-like growth factor receptor (IGF-IR) markedly increased the liver-metastasizing potential of the cells. TNF-α-mediated signalling in these (M-27IGFIR) and wild-type (M-27) cells were analyzed by a combination of Western blotting, electrophoretic mobility shift assays, cytokine profiling, immunofluorescence labelling and flow cytometry. Results: We found that IGF-I receptor overexpression in the tumor cells altered TNF-α-mediated signalling by accelerating TNF-α-induced IκBα phosphorylation and degradation and enhancing nuclear translocation of NFκB. This resulted in increased IL-6 production in M-27IGFIR, but not M-27 cells in response to TNF-α. IL-6 expression could be further augmented by co-stimulation with IGF-I, an effect that was mediated via PI3-K signalling. Furthermore, the level of activated STAT-3 in M-27IGFIR cells was significantly increased, suggesting that IL-6 could activate STAT-3 in these cells through an autocrine mechanism. Finally, when apoptosis induction by TNF-α in these cells was compared using antibodies to cleaved caspase 3, a significant reduction in the proportion of apoptotic cells was seen in TNF-α-treated M-27IGFIR as compared to M-27 cells. Conclusions: Taken together, the results suggest that overexpression of IGF-IR altered TNF-α signalling in the tumor cells, shifting the response towards increased IL-6 production and autocrine STAT-3 activation, thereby providing the cells with a survival advantage in the presence of TNF-α. Supported by Canadian Institute for Health Research grant MOP- 81201 (PB) and a McGill University Health Center Research Institute fellowship (SL). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 554.