Malignant Phenotype Of Cancer Cells Research Articles

The cancer glycome: Carbohydrates as mediators of metastasis

Glycosylation is a frequent post-translational modification which results in the addition of carbohydrate determinants, “glycans”, to cell surface proteins and lipids. These glycan structures form the “glycome” and play an integral role in cell–cell and cell–matrix interactions through modulation of adhesion and cell trafficking.Glycosylation is increasingly recognized as a modulator of the malignant phenotype of cancer cells, where the interaction between cells and the tumor micro-environment is altered to facilitate processes such as drug resistance and metastasis. Changes in glycosylation of cell surface adhesion molecules such as selectin ligands, integrins and mucins have been implicated in the pathogenesis of several solid and hematological malignancies, often with prognostic implications. In this review we focus on the functional significance of alterations in cancer cell glycosylation, in terms of cell adhesion, trafficking and the metastatic cascade and provide insights into the prognostic and therapeutic implications of recent findings in this fast-evolving niche.

Mechanisms of hypoxia-mediated immune escape in cancer.

An important aspect of malignant progression is the acquired ability of tumor cells to avoid recognition and destruction by the immune system (immune escape). Clinical cancer progression is also associated with the development of tumor hypoxia, which is mechanistically linked to the acquisition of malignant phenotypes in cancer cells. Despite the well-established role of hypoxia in tumor cell invasion and metastasis, and resistance to therapy, relatively few studies have examined the contribution of hypoxia to cancer immune escape. Accumulating evidence reveals that hypoxia can impair anticancer immunity by altering the function of innate and adaptive immune cells and/or by increasing the intrinsic resistance of tumor cells to the cytolytic activity of immune effectors. Here, we discuss certain aspects of the contribution of hypoxia to tumor immune escape and provide evidence for a novel role of cyclic guanosine monophosphate (cGMP) signaling in the regulation of hypoxia-induced immune escape. Thus, we propose that activation of cGMP signaling in cancer cells may have important immunotherapeutic applications.

Abstract 1111: Hypoxia inducible factor regulation of galectin3 expression in neuroblastoma cells:Implications in controlling malignant phenotype of neuroblastoma cells

Abstract Neuroblastoma (NB) is an extra cranial tumor arising from neural crest progenitor cells. Effective angiogenesis, or new blood vessel formation, is critical for NB tumor growth and progression. Elevated levels of galectin-3 (Gal-3), an angiogenic cytokine is extensively correlated with the malignant behavior and poor prognosis of many cancers. Our studies have shown that Gal-3 expression is significantly up-regulated in aggressive NB cells, and Gal-3 promotes angiogenesis and migration of microglial cells. We have also previously reported that Gal-3 expression is up regulated under low oxygen/hypoxic conditions. The specific mechanisms of Gal-3 induction in NB are not understood. Hypoxic microenvironment drives malignant phenotypes of cancer cells through the transcription factor hypoxia-inducible factor (HIF1). Because NB cells reside in a low oxygen environment, we determined if the hypoxic status modulates Gal-3 expression. In this study, we utilized RT-PCR, western blot analysis, immunofluorescence staining, RNA interference and biochemical approaches. The hypoxia-mimetic, cobalt chloride (CoCl2)-treated neuroblastoma Neuro-2a cells showed enhanced levels of HIF 1 protein. In correlation with the increased HIF1 levels, Gal-3 expression was increased at both RNA and protein levels. Silencing of HIF1 expression by small hairpin interfering RNA in Neuro-2a cells blocked hypoxia induced Gal-3 expression indicating that HIF1 regulates Gal-3 expression in NB cells. Down regulation of Gal-3 resulted in decreased levels of the angiogenic factors IL-6 and urokinase plasminogen activator in association with decreased proliferation, migration, and angiogenesis. Thus our data suggest that HIF1alpha plays a role in the regulation of Gal-3 expression and malignant phenotype of NB cells. Note: This abstract was not presented at the meeting. Citation Format: Umadevi V. Wesley, Esat Resad, Robert J. Dempsey. Hypoxia inducible factor regulation of galectin3 expression in neuroblastoma cells:Implications in controlling malignant phenotype of neuroblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1111. doi:10.1158/1538-7445.AM2014-1111

STAT3 in Cancer-Friend or Foe?

The roles and significance of STAT3 in cancer biology have been extensively studied for more than a decade. Mounting evidence has shown that constitutive activation of STAT3 is a frequent biochemical aberrancy in cancer cells, and this abnormality directly contributes to tumorigenesis and shapes many malignant phenotypes in cancer cells. Nevertheless, results from more recent experimental and clinicopathologic studies have suggested that STAT3 also can exert tumor suppressor effects under specific conditions. Importantly, some of these studies have demonstrated that STAT3 can function either as an oncoprotein or a tumor suppressor in the same cell type, depending on the specific genetic background or presence/absence of specific coexisting biochemical defects. Thus, in the context of cancer biology, STAT3 can be a friend or foe. In the first half of this review, we will highlight the “evil” features of STAT3 by summarizing its oncogenic functions and mechanisms. The differences between the canonical and non-canonical pathway will be highlighted. In the second half, we will summarize the evidence supporting that STAT3 can function as a tumor suppressor. To explain how STAT3 may mediate its tumor suppressor effects, we will discuss several possible mechanisms, one of which is linked to the role of STAT3β, one of the two STAT3 splicing isoforms. Taken together, it is clear that the roles of STAT3 in cancer are multi-faceted and far more complicated than one appreciated previously. The new knowledge has provided us with new approaches and strategies when we evaluate STAT3 as a prognostic biomarker or therapeutic target.

Cell type-specific reciprocal regulation of HIF1A gene expression is dependent on 5′- and 3′-UTRs

In the present study, we demonstrated the reciprocal regulation of hypoxia-inducible factor 1 alpha (HIF1A) gene expression via untranslated region-(UTR) dependent mechanisms. A 151 nucleotide sequence found in the HIF1A 5′-UTR is sufficient for significant translational up-regulation. On the other hand, the 3′-UTR of HIF1A has been implicated in mRNA degradation. In the non-metastatic breast cancer cell line MCF7, the 3′-UTR-dependent down-regulatory machinery predominates over the 5′-UTR-dependent up-regulation of HIF1A. However, 5′-UTR-dependent up-regulation is dominant among metastatic cell lines (MDA-MB453, U87MG). It is therefore likely that the predominance of 5′-UTR-dependent translational enhancement of HIF1A is critical for the malignant phenotype of cancer cells. PTBP-1, but not HuR, is a candidate RNA binding protein for the translational control of HIF1A.

Role of 3’-untranslated region translational control in cancer development, diagnostics and treatment

The messenger RNA 3'-untranslated region (3'UTR) plays an important role in regulation of gene expression on the posttranscriptional level. The 3'UTR controls gene expression via orchestrated interaction between the structural components of mRNAs (cis-element) and the specific trans-acting factors (RNA binding proteins and non-coding RNAs). The crosstalk of these factors is based on the binding sequences and/or direct protein-protein interaction, or just functional interaction. Much new evidence that has accumulated supports the idea that several RNA binding factors can bind to common mRNA targets: to the non-overlapping binding sites or to common sites in a competitive fashion. Various factors capable of binding to the same RNA can cooperate or be antagonistic in their actions. The outcome of the collective function of all factors bound to the same mRNA 3'UTR depends on many circumstances, such as their expression levels, affinity to the binding sites, and localization in the cell, which can be controlled by various physiological conditions. Moreover, the functional and/or physical interactions of the factors binding to 3'UTR can change the character of their actions. These interactions vary during the cell cycle and in response to changing physiological conditions. Abnormal functioning of the factors can lead to disease. In this review we will discuss how alterations of these factors or their interaction can affect cancer development and promote or enhance the malignant phenotype of cancer cells. Understanding these alterations and their impact on 3'UTR-directed posttranscriptional gene regulation will uncover promising new targets for therapeutic intervention and diagnostics. We will also discuss emerging new tools in cancer diagnostics and therapy based on 3'UTR binding factors and approaches to improve them.

Hyperglycemia, a neglected factor during cancer progression.

Recent evidence from large cohort studies suggests that there exists a higher cancer incidence in people with type 2 diabetes (DM2). However, to date, the potential reasons for this association remain unclear. Hyperglycemia, the most important feature of diabetes, may be responsible for the excess glucose supply for these glucose-hungry cells, and it contributes to apoptosis resistance, oncogenesis, and tumor cell resistance to chemotherapy. Considering associations between diabetes and malignancies, the effect of hyperglycemia on cancer progression in cancer patients with abnormal blood glucose should not be neglected. In this paper, we describe the role that hyperglycemia plays in cancer progression and treatment and illustrate that hyperglycemia may contribute to a more malignant phenotype of cancer cells and lead to drug resistance. Therefore, controlling hyperglycemia may have important therapeutic implications in cancer patients.

Micro-scaffold array chip for upgrading cell-based high-throughput drug testing to 3D using benchtop equipment

Cell-based high throughput drug screening accelerates the pace of drug discovery which is routinely operated on planar high-density multi-well plates with sophisticated robotic liquid-dispensing systems for cell seeding and drug administration. Considerable efforts have been made to upgrade in vitro cellular models from 2D to a more biomimetic 3D configuration. For instance, in anti-cancer drug screening, tumor spheroids are increasingly applied as a gold-standard 3D model exhibiting cellular behaviors and drug responses distinguishable from the 2D counterpart. However, translation of spheroids to high throughput drug screening is challenging since pre-formation of spheroids and subsequent translocation to multi-well plates for drug testing are usually uncontrollable and time/reagent consuming and cell loss is inevitable during medium exchange for drug testing. Here we present an off-the-shelf micro-scaffold array chip which enables high throughput 3D cell culture, drug administration and quantitative in situ assays entirely on the same chip. The sponge-like micro-scaffolds functioned both as absorbents to realize parallel auto-loading of cells or drugs and as barriers to prevent cell loss during medium exchange via centrifugation. Rapid manual loading of cell suspensions or drugs into the 96 isolated micro-scaffolds on the chip was achieved in the timescale of several seconds, meanwhile with total medium consumption reduced to the order of microliters. Proof of concept demonstration of drug cytotoxicity testing was performed on multiple cancer cells using common benchtop equipment, making it accessible to most biomedical labs with basic cell culture setups. Higher cellular drug resistance was constantly obtained with this platform compared to the planar cultures, which was partially attributed to the malignant phenotype of cancer cells yielded by enhanced cell-matrix interactions in the micro-scaffolds. Interestingly, the high drug resistance of 3D cultured cells in the micro-scaffold was shown to be density-independent in contrast to the density-dependent drug response for 2D cultured cells, indicating intrinsic differences between the two culture models. This platform is expected to facilitate upgrade of the current cell-based high throughput drug testing to the 3D level and be widely applicable across various disciplines.

Metastasis-associated gene 1 promotes invasion and migration potential of laryngeal squamous cell carcinoma cells

Overexpression of the metastasis-associated gene 1 (MTA1) has previously been found to be associated with progression of various cancer types to the metastasis stage. The function of MTA1 in laryngeal squamous cell carcinoma (LSCC) remains unclear. To explore the significance of MTA1 in the invasion and migration processes in LSCC, gene transfection and RNA interference (RNAi) were performed to study the biological function of MTA1 in the LSCC cell line, HEP-2. Results showed that MTA1 promoted the invasion, adhesion and migration behavior of LSCC cells. RNAi against MTA1 significantly decreased the malignant phenotypes of cancer cells. MTA1 may be important in the process of LSCC invasion and metastasis.

Mechanical properties of cancer cytoskeleton depend on actin filaments to microtubules content: Investigating different grades of colon cancer cell lines

Biomechanical properties of cancer cells have been proposed as promising biomarkers to investigate cancer progression. Cytoskeletal reorganization alters these characteristics in different grades of cancer cells. In the present study based on the micropipette aspiration method, whole body evaluation for two different colon cancer cells was performed to determine viscoelastic parameters of the cells. A finite element model was developed for verification of experiments and predicting some behaviors of cells. Western blot analysis and fluorescence intensity for actin microfilaments and microtubules were performed to measure cell content of the proteins. It was illustrated that the proportion of microtubules and actin microfilaments is different in grade I and grade IV colon cancer cells in a manner that microtubules attain an effectual role in progressive reorganization of cytoskeleton in transition from nonaggressive to malignant phenotypes in cancer cells. Furthermore, it was concluded that larger instantaneous Young's modulus value for high grade cells is related to the existence of extensively build-up actin networks at the cell cortex. Based on the cell mechanics results, a simple parameter is suggested for sorting different grades of colon cancer cells.

The Clinical Significance Of Activated p-AKT Expression In Peripheral T-Cell Lymphomas

BackgroundThe oncogenic phosphatidylinositol-3-kinase/serine-threonine kinase (PI3K/AKT) pathway mediates diverse prosurvival signals and promotes the malignant phenotype of cancer cells through multiple downstream pathways. We aimed to define the prognostic role and clinical significance of p-AKT expression in peripheral T-cell lymphomas (PTCLs). MethodsWe evaluated the p-Akt expression in patients with PTCLs using tissue microarray(TMA) technology. The intensity of p-AKT staining was scored as 0, 1, 2, and 3 and the proportion of p-AKT positive cells was scored from 0% to 100%. p-AKT expression score was expressed as arbitrary units (AUs), calculated by multiplying the intensity score by the proportion score. ResultsA total of 63 PTCL patients were analyzed (PTCL not otherwise specified [PTCL-NOS, n=16], angioimmunoblastic T-cell lymphoma [AITL, n=19], and anaplastic large cell lymphoma [ALCL, n=11] and natural killer T-cell lymphoma [NKTCL, n=17]). The upper limit of the third quartile (Q3) of the AU values was 120. High p-AKT group (AU>Q3) included a higher proportion of NKTCL (41.7%) and ALCL (33.3%) subtypes, while low p-AKT group (AU≤Q3) included higher proportion of AITL (37.3%) and PTCL-NOS (25.0%) subtypes. High p-AKT group showed substantially poorer overall survival (OS) (median OS, 2.3 months vs. 25.2 months, P< 0.001) compared with the low p-AKT group. Multivariate analysis showed that high p-AKT group retained its significance as an independent prognostic factor for poor OS (hazard ratio [HR] 6.5; 95% confidence interval [CI], 2.7 – 15.9; P< 0.001) and poor PFS (HR 4.7, 95% CI; 2.1 – 10.6, P< 0.001).< 0.001) compared with the low p-AKT group. ConclusionPTCLs having high p-AKT expression showed a significantly worse survival than patients with low p-AKT expression. Thus, more effective treatment approaches are needed for this subset of patients with PTCLs, and we suggest inhibition of PI3K/AKT pathway may be a promising therapeutic strategy in PTCLs. Disclosures:No relevant conflicts of interest to declare.

RIP1 potentiates BPDE-induced transformation in human bronchial epithelial cells through catalase-mediated suppression of excessive reactive oxygen species

Cell survival signaling is important for the malignant phenotypes of cancer cells. Although the role of receptor-interacting protein 1 (RIP1) in cell survival signaling is well documented, whether RIP1 is directly involved in cancer development has never been studied. In this report, we found that RIP1 expression is substantially increased in human non-small cell lung cancer and mouse lung tumor tissues. RIP1 expression was remarkably increased in cigarette smoke-exposed mouse lung. In human bronchial epithelial cells (HBECs), RIP1 was significantly induced by cigarette smoke extract or benzo[a]pyrene diol epoxide (BPDE), the active form of the tobacco-specific carcinogen benzo(a)pyrene. In RIP1 knockdown HBECs, BPDE-induced cytotoxicity was significantly increased, which was associated with induction of cellular reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs), including c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38. Scavenging ROS suppressed BPDE-induced MAPK activation and inhibiting ROS or MAPKs substantially blocked BPDE-induced cytotoxicity, suggesting ROS-mediated MAPK activation is involved in BPDE-induced cell death. The ROS-reducing enzyme catalase is destabilized in an ERK- and JNK-dependent manner in RIP1 knockdown HBECs and application of catalase effectively blocked BPDE-induced ROS accumulation and cytotoxicity. Importantly, BPDE-induced transformation of HBECs was significantly reduced when RIP1 expression was suppressed. Altogether, these results strongly suggest an oncogenic role for RIP1, which promotes malignant transformation through protecting DNA-damaged cells against carcinogen-induced cytotoxicity associated with excessive ROS production.

Abstract 3110: CBF regulates cell senescence and autophagy through sphingolipid metabolism.

Abstract Core Binding Factor (CBF) is a heterodimeric transcription factor comprised of one of three RUNX DNA binding proteins (Runx1, Runx2, or Runx3) and a non-DNA binding component termed CBFβ. CBFβ is a dedicated and required component of CBF. CBF is commonly altered by chromosomal translocations leading to acute myeloid and lymphocytic leukemias. More recently, altered CBF activity was associated with the malignant phenotype of various solid tumors. Recent studies placed RUNX factors in direct transcriptional control of several sphingolipid pathway enzymes as well as autophagy in murine fibroblast and myofibrils. However, CBF regulation of sphingolipid metabolism in cancer cells has not been investigated. Purpose: To study CBF function in solid tumors. Hypothesis: Previous work in the laboratory lead to a hypothesis that suggested a requirement for CBF in the malignant phenotype of prostate and ovarian cancers. Method and Results: To determine if CBF contributed to the malignant phenotype of cancer cells, we used lentiviral delivery of CBFβ-specific shRNA to decrease CBFβ expression. Cells knocked down for CBFβ expression exhibited a growth defect that was not associated with large perturbances in the cell cycle or with increased apoptosis. Since both sphingolipid metabolism and autophagy are central to survival pathways, these processes were examined in the CBFβ knockdown cell lines. Prostate (PPC1 and PC-3) and ovarian (SKOV-3) cancer cell lines that were infected with the CBFβ-specific shRNA but not those cells infected with a non-target-shRNA displayed a significant increase in autophagic markers, including LC3B-II, suggesting that autophagy was upregulated in CBFβ knocked-down cells. Treatment of CBFβ knockdown cells with the autophagy inhibitors chloroquine and 3-methyladenosine modestly increased cell survival, suggesting that cell death was associated with increased autophagy. Real time PCR analysis revealed that Ugcg, an enzyme critical for sphingolipid metabolism, was decreased in SKOV-3 cells after CBFβ knockdown. These data lead to a hypothesis that loss of CBFβ increases cell death and autophagy by, in part, perturbing sphinglolipid metabolism and, thus, increasing ceramide pools. Since sphingolipid metabolism is linked to senescence the process was examined using senescence-associated B-galactosidase activity, which was found to be increased in CBFβ knockdown cells. Treatment of cells knocked down for CBFβ with Fumonsin B1 (FB1), which decreases ceramide production, improved cell survival and decreased autophagy. Further, direct lipodomic analysis of the CBFβ knockdown cells identified significant increases in the levels of ceramide. Conclusion: Together, these data suggest that CBF regulates senescence and autophagy via control of the sphingolipid pathway. These studies further suggest that CBF is a putative therapeutic target in prostate and ovarian cancers. Citation Format: Adam H. Greer, Thomas Yong, Katie Fennell, Shari Meyers, Nathan Davis. CBF regulates cell senescence and autophagy through sphingolipid metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3110. doi:10.1158/1538-7445.AM2013-3110

Abstract 2910: Suppressing excessive reactive oxygen species accumulation by RIP1 potentiates BPDE-induced transformation of human bronchial epithelial cells.

Abstract Cell survival signaling is important for the malignant phenotypes of cancer cells. Although the role of receptor-interacting protein 1 (RIP1) in cell survival signaling is well documented, whether RIP1 is directly involved in cancer development has never been studied. In this report, we found that RIP1 expression is substantially increased in human non-small cell lung cancer (NSCLC) tissues and cell lines. RIP1 expression in human bronchial epithelial cells (HBECs) was significantly induced by cigarette smoke extract (CSE) or benzo[a]pyrene diol epoxide (BPDE), the active form of the tobacco-specific carcinogen benzo (a) pyrene (BaP). In RIP1 knockdown HBECs, BPDE-induced cytotoxicity was significantly increased, which was associated with induction of cellular reactive oxygen species (ROS), mainly hydrogen peroxide (H2O2), and activation of mitogen-activated protein kinases (MAPKs) including JNK, ERK and p38. Scavenging ROS suppressed BPDE-induced MAPK activation and inhibiting ROS or MAPKs substantially blocked BPDE-induced cytotoxicity, suggesting ROS-mediated MAPK activation is involved in BPDE-induced cell death. The H2O2-reducing enzyme catalase is destabilized in an ERK- and JNK-dependent manner in RIP1 knockdown HBECs and application of catalase effectively blocked BPDE-induced H2O2 accumulation and cytotoxicity. Importantly, BPDE-induced transformation of HBECs was significantly reduced when RIP1 expression was suppressed. Altogether, these results strongly suggest an oncogenic role for RIP1, which promotes malignant transformation through protecting DNA-damaged cells against carcinogen-induced cytotoxicity associated with excessive ROS production. Citation Format: Qiong Wang, Wenshu Chen, Xiuling Xu, Bilan Li, Weiyang He, Mabel T. Padilla. Suppressing excessive reactive oxygen species accumulation by RIP1 potentiates BPDE-induced transformation of human bronchial epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2910. doi:10.1158/1538-7445.AM2013-2910

NPM‐ALK up‐regulates iNOS expression through a STAT3/microRNA‐26a‐dependent mechanism

NPM-ALK chimeric oncogene is aberrantly expressed in an aggressive subset of T-cell lymphomas that frequently occurs in children and young adults. The mechanisms underlying the oncogenic effects of NPM-ALK are not completely elucidated. Inducible nitric oxide synthase (iNOS) promotes the survival and maintains the malignant phenotype of cancer cells by generating NO, a highly active free radical. We tested the hypothesis that iNOS is deregulated in NPM-ALK(+) T-cell lymphoma and promotes the survival of this lymphoma. In line with this possibility, an iNOS inhibitor and NO scavenger decreased the viability, adhesion, and migration of NPM-ALK(+) T-cell lymphoma cells, and an NO donor reversed these effects. Moreover, the NO donor salvaged the viability of lymphoma cells treated with ALK inhibitors. In further support of an important role of iNOS, we found iNOS protein to be highly expressed in NPM-ALK(+) T-cell lymphoma cell lines and in 79% of primary tumours but not in human T lymphocytes. Although expression of iNOS mRNA was identified in NPM-ALK(+) T-cell lymphoma cell lines and tumours, iNOS mRNA was remarkably elevated in T lymphocytes, suggesting post-transcriptional regulation. Consistently, we found that miR-26a contains potential binding sites and interacts with the 3'-UTR of iNOS. In addition, miR-26a was significantly decreased in NPM-ALK(+) T-cell lymphoma cell lines and tumours compared with T lymphocytes and reactive lymph nodes. Restoration of miR-26a in lymphoma cells abrogated iNOS protein expression and decreased NO production and cell viability, adhesion, and migration. Importantly, the effects of miR-26a were substantially attenuated when the NO donor was simultaneously used to treat lymphoma cells. Our investigation of the mechanisms underlying the decrease in miR-26a in this lymphoma revealed novel evidence that STAT3, a major downstream substrate of NPM-ALK tyrosine kinase activity, suppresses MIR26A1 gene expression.

Overexpression and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 6 in colorectal cancer

BackgroundCarcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits anoikis and affects the malignant phenotype of cancer cells. In this study, we analyzed CEACAM6 as a gene that is highly upregulated in colon cancer tissues, and examined the assertion that CEACAM6 might be a suitable candidate tumor marker for the diagnosis of colon cancer. MethodsCEACAM6 gene expression in human colon tissues was performed by tissue microarray and analyzed using RT-PCR (each of normal and tumor tissue, n=40) and immunohistochemical and clinicopathological (colon cancer patients, n=143) analyses. ResultsCEACAM6 transcriptional and translational levels were significantly upregulated in human tumor tissues compared to non-tumor regions, and clinicopathological analysis revealed a significant correlation between CEACAM6 protein expression and Dukes' stage (p<0.001). High expression levels of CEACAM6 were significantly associated with lower overall survival (p<0.001) and shorter recurrence-free survival (p<0.001). We demonstrated that knockdown of CEACAM6 with CEACAM6-specific small interfering RNA in colorectal cancer cells attenuated invasivity (35%); conversely, the overexpression of CEACAM6 increased invasiveness. ConclusionsCEACAM6 is significantly upregulated in colon cancer tissues and is closely associated with poor prognosis, indicating that CEACAM6 might be used as a tumor biomarker and a potential therapeutic target for colon cancer.

Regulation of Human β-1,4-Galactosyltransferase V Gene Expression in Cancer Cells

β-1,4-Galactosyltransferase (β-1,4-GalT) V - whose human and mouse genes were cloned by us - has been suggested to be involved in the biosyntheses of N-glycans, O-glycans, and lactosylceramide by in vitro studies. Our recent study showed that β-1,4-GalT V-knockout mice are embryonic lethal, suggesting the importance of the glycans synthesized by β-1,4-GalT V for embryonic development. A subsequent study showed that murine β-1,4-GalT V is involved in the biosynthesis of lactosylceramide. It is well known that the glycosylation of cell surface glycoproteins and glycolipids changes dramatically upon the malignant transformation of cells. We found that among six β-1,4-GalTs the gene expression of only β-1,4-GalT V increases upon malignant transformation. The expression of the β-1,4-GalT V gene has been shown to be regulated by transcription factors Sp1 and Ets-1 in cancer cells. Both transcription factors regulate the gene expression levels of not only glycosyltransferases, but also key molecules involved in tumor growth, invasion and metastasis. Therefore, the abnormal glycosylation and malignant phenotypes of cancer cells are considered to be suppressed by regulating the expression levels of the transcription factor genes. This review gives a summary account of the gene discovery, in vivo function, and transcriptional mechanism of β-1,4-GalT V. Also, a perspective on applications of the manipulation of transcription factor genes to cancer therapy will be discussed.

Biomarkers for the targeted therapies of non-small cell lung cancer

Biomarkers for the targeted therapies of non-small cell lung cancer Antonio Rossi1, Domenico Galetta21Division of Medical Oncology, S.G. Moscati Hospital, Avellino, Italy; 2Department of Medical Oncology, Giovanni Paolo II Oncology Institute, Bari, ItalyAbstract: Targeted therapy includes new biologic agents specifically designed to selectively target molecular pathways responsible for, or that substantially drive, the malignant phenotype of cancer cells. A lot of new biologic agents have been introduced in clinical development for the treatment of advanced non-small cell lung cancer (NSCLC), but unfortunately negative results were more frequent than successes. Two pathways have been deeply studied and have led to the development of corresponding biomarkers for defining the most appropriate therapeutic approach in advanced NSCLC patients. The epidermal growth factor receptor (EGFR) pathway is targeted by tyrosine kinase inhibitors, gefitinib and erlotinib, and monoclonal antibody, cetuximab. EGFR mutation status, EGFR gene copy number determined by fluorescent in situ hybridization, and EGFR protein expression determined by immunohistochemistry have been evaluated as potential markers for clinical decision making regarding anti-EGFR therapy. Among these, EGFR mutation status resulted in the most important predictive/prognostic factor for EGFR-tyrosine kinase inhibitor therapy. EGFR protein expression seems to be important for cetuximab plus chemotherapy treatment, but further data are needed to define its role in this setting. In the last few years, the anaplastic lymphoma kinase (ALK) gene fusion is becoming an important biomarker in defining the specific NSCLC subtype to target with the corresponding inhibitor, crizotinib. To date, considering the EGFR activating mutations and the ALK gene fusion, generally mutually exclusive, and the availability of the correspondent inhibitors, 20% to 50% of advanced NSCLC could now be treated in Western and Eastern countries, respectively, with a targeted therapy.Keywords: biomarkers, EGFR, ALK, NSCLC, TKI, crizotinib

Expression of ezrin correlates with malignant phenotype of lung cancer, and in vitro knockdown of ezrin reverses the aggressive biological behavior of lung cancer cells

Ezrin, one of the ezrin-radixin-moesin proteins, is involved in the formation of cell membrane processes such as lamellipodia and filopodia and acts as a membrane-cytoskeleton linker. Its aberrant expression correlates with development and progression of several human cancers. However, the expression of ezrin and its role in lung cancer are currently unknown. In this study, we performed ezrin small interfering RNA transfection in two lung cancer cell lines and examined the effects on malignant phenotypes in cancer cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and chamber transwell assays. Ezrin knockdown significantly reduced the proliferation, migration, and invasion of lung cancer cells in vitro. To address the possible mechanisms, we evaluated the expression of adhesion molecules E-cadherin and β-catenin by Western blot and reverse transcriptase-polymerase chain reaction analyses. The results demonstrated that downregulation of ezrin reduced β-catenin and increased E-cadherin at the protein level but had no effects on their mRNA levels, suggesting posttranscriptional regulation of these two adhesion molecules. Immunofluorescence assays revealed that ezrin knockdown restored membranous expression of E-cadherin and decreased cytoplasmic β-catenin in lung cancer cells. In addition, ezrin expression was immunohistochemically evaluated on 135 normal and 183 lung cancer tissues. The expression of ezrin was significantly higher in cancer samples than paired autologous normal lung tissues. In normal bronchial epithelium, ezrin was mainly localized on the apical membrane, while in lung cancers and metastatic foci, ezrin was primarily distributed in cytoplasm. Among lung cancer tissues, expression of ezrin was higher in the invasive front of primary lesions and the highest in lymphatic metastasis. Statistical analysis demonstrated that ezrin expression correlated significantly with lymphatic metastasis and advanced TNM stage. Our data suggest that ezrin may play a crucial role in governing the biological behavior of lung cancer.

The mouse Mageb18 gene encodes a ubiquitously expressed type I MAGE protein and regulates cell proliferation and apoptosis in melanoma B16-F0 cells

Although many cancer vaccines have been developed against typeI MAGE (melanoma antigen) genes owing to their shared tumour-specific expression properties, studies about their expression and functions are relatively limited. In the present study, we first identify a non-testis-specific typeI MAGE gene, Mageb18 (melanoma antigen family B 18). Mouse Mageb18 is also expressed in digestion- and immune-related tissues as well as testis, and its expression in testis is age-dependent. Mageb18 is expressed in many mouse-derived cell lines, and DNA demethylation and histone acetylation mediate the reactivation of Mageb18 in Mageb18-negtive H22 and C6 cells. We also show that mouse Mageb18 encodes a 46kDa protein which is predominantly localized in the cytoplasm. In testis, the endogenous MAGEB18 protein is mainly expressed in proliferative spermatogonia and primary and secondary spermatocytes, but less so in spermatids. Finally, we demonstrate that knockdown of MAGEB18 inhibits the growth of B16-F0 cells and induces apoptosis, which correlates with increased levels of TP53 (tumour protein 53), p21, Bax and caspase 3. The results of the present study thus uncover an important phenomenon that the expression of certain typeI MAGE genes, at least for Mageb18, is non-testis-specific. Although they can regulate various malignant phenotypes of cancer cells, it is necessary to study further their expression pattern in normal tissues before using them to develop more effective and safer cancer vaccines.