CUL4A Ubiquitin Ligase Is an Independent Predictor of Overall Survival in Pancreatic Adenocarcinoma.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with dismal prognosis. Genomic instability due to defects in cell-cycle regulation/mitosis or deficient DNA-damage repair is a major driver of PDAC progression with clinical relevance. Deregulation of licensing of DNA replication leads to DNA damage and genomic instability, predisposing cells to malignant transformation. While overexpression of DNA replication-licensing factors has been reported in several human cancer types, their role in PDAC remains largely unknown. We aimed here to examine the expression and prognostic significance of the DNA replication-licensing factors chromatin licensing and DNA replication factor 1 (CDT1), cell-division cycle 6 (CDC6), minichromosome maintenance complex component 7 (MCM7) and also of the ubiquitin ligase regulator of CDT1, cullin 4A (CUL4A), in PDAC. MATERIALS AND METHODS: Expression levels of CUL4, CDT1, CDC6 and MCM7 were evaluated by immunohistochemistry in 76 formalin-fixed paraffin-embedded specimens of PDAC patients in relation to DNA-damage response marker H2AX, clinicopathological parameters and survival. We also conducted bioinformatics analysis of data from online available databases to corroborate our findings. RESULTS: CUL4A and DNA replication-licensing factors were overexpressed in patients with PDAC and expression of CDT1 positively correlated with H2AX. Expression of CUL4A and CDT1 positively correlated with lymph node metastasis. Importantly, elevated CUL4A expression was associated with reduced overall survival and was an independent indicator of poor prognosis on multivariate analysis. CONCLUSION: Our findings implicate CUL4A, CDT1, CDC6 and MCM7 in PDAC progression and identify CUL4A as an independent prognostic factor for this disease.

Ras suppressor-1 (RSU1) exerts a tumor suppressive role with prognostic significance in lung adenocarcinoma.

Ras suppressor-1 (RSU1), originally described as a suppressor of Ras oncogenic transformation, localizes to focal adhesions interacting with the ILK-PINCH-PARVIN (IPP) complex that exerts a well-established oncogenic role in cancer. However, RSU1 implication in lung cancer is currently unknown. Our study aims to address the role of RSU1 in lung adenocarcinoma (LUADC). We here show that RSU1 protein expression by immunohistochemistry is downregulated in LUADC human tissue samples and represents a significant prognostic indicator. In silico analysis of gene chip and RNA seq data validated our findings. Depletion of RSU1 by siRNA in lung cancer cells promotes anchorage-independent cell growth, cell motility and epithelial to mesenchymal transition (EMT). Silencing of RSU1 also alters IPP complex expression in lung cancer cells. The p29 RSU1 truncated isoform is detected in lung cancer cells, and its expression is downregulated upon RSU1 silencing, whereas it is overexpressed upon ILK overexpression. These findings suggest that RSU1 exerts a tumor suppressive role with prognostic significance in LUADC.

Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage.

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.

Glucagon-like Peptide-1 Receptor in the Human Hypothalamus Is Associated with Body Mass Index and Colocalizes with the Anorexigenic Neuropeptide Nucleobindin-2/Nesfatin-1.

Data on animals emphasize the importance of the neuronal glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) for feeding suppression, although it is unclear whether astrocytes participate in the transduction of anorectic GLP-1R-dependent signals. In humans, the brain circuitry underlying these effects remains insufficiently investigated. The present study aimed to explore GLP-1R protein expression in the human hypothalamus and its correlation with body mass index (BMI). Sections of hypothalamus from 28 autopsy cases, 11 with normal weight (BMI < 25 kg/m2) and 17 with non-normal weight (BMI ≥ 25 kg/m2), were examined using immunohistochemistry and double immunofluorescence labeling. Prominent GLP-1R immunoexpression was detected in neurons of several hypothalamic nuclei, including paraventricular, supraoptic, and infundibular nuclei; the lateral hypothalamic area (LH); and basal forebrain nuclei. Interestingly, in the LH, GLP-1R was significantly decreased in individuals with BMI ≥ 25 kg/m2 compared with their normal weight counterparts (p = 0.03). Furthermore, GLP-1R was negatively correlated (τb = −0.347, p = 0.024) with BMI levels only in the LH. GLP-1R extensively colocalized with the anorexigenic and antiobesogenic neuropeptide nucleobindin-2/nesfatin-1 but not with the astrocytic marker glial fibrillary acidic protein. These data suggest a potential role for GLP-1R in the regulation of energy balance in the human hypothalamus. In the LH, an appetite- and reward-related brain region, reduced GLP-1R immunoexpression may contribute to the dysregulation of homeostatic and/or hedonic feeding behavior. Possible effects of NUCB2/nesfatin-1 on central GLP-1R signaling require further investigation.

The Role of Hippo Signaling Pathway and ILK in the Pathophysiology of Human Hypertrophic Scars and Keloids: An Immunohistochemical Investigation.

BACKGROUND: Keloids and hypertrophic scars are characterized by abnormal fibroblast activation and proliferation. While their molecular pathogenesis remains unclear, myofibroblasts have been associated with their development. Hippo pathway effectors YAP/TAZ promote cell proliferation and matrix stiffening. Integrin-linked kinase (ILK), a central component of focal adhesions that mediates cell-matrix interactions, has been linked to tissue repair and fibrosis. The aim of this study was to investigate the expression of key Hippo pathway molecules and ILK in hypertrophic scars and keloids. METHODS: YAP/TAZ, TEAD4, ILK and a-SMA expression were evaluated by immunohistochemistry in keloids (n = 55), hypertrophic scars (n = 38) and normal skin (n = 14). RESULTS: The expression of YAP/TAZ, TEAD4, ILK and a-SMA was higher in fibroblasts of keloids compared to hypertrophic scars while negative in normal skin. There was a significant positive correlation between the expression of ILK and Hippo pathway effectors. CONCLUSIONS: Our results suggest that the deregulation of Hippo signaling and ILK are implicated in keloid and hypertrophic scar formation.

Focal adhesion proteins in hepatocellular carcinoma: RSU1 a novel tumour suppressor with prognostic significance.

AIM: Hepatocellular carcinoma (HCC) is a common cause a cancer-related death. Focal adhesions (FAs) represent multiprotein complexes at integrin-mediated cell-extracellular matrix adhesion sites that orchestrate vital cellular functions. The heterotrimeric ILK-PINCH-PARVB (IPP) complex, RSU1, a PINCH binding protein and CTEN, a member of the tensin family of proteins exert a critical role in FAs, where they regulate important cancer related functions such as cell adhesion, migration, proliferation and survival. Previous studies implicate these FA proteins in liver pathophysiology but their detailed role in human HCC is not fully understood. Here in we investigated expression and function of IPP, RSU1 and CTEN in human HCC. METHODS: The expression of focal adhesion proteins was studied in human HCC by immunohistochemistry in relation to clinicopathological parameters, previous studied genomic instability markers and patient's survival. Effects on cell proliferation and FA proteins expression upon ILK inhibition and RSU1 silencing were also investigated in HCC in vitro. RESULTS: IPP complex and CTEN proteins are overexpressed while RSU1 expression is decreased in human HCC. CTEN expression correlates with reduced patients' survival while RSU1 represents an independent favorable prognostic indicator in human HCC. Nuclear ILK expression correlates with markers of genomic instability. Pharmacological targeting of ILK suppresses, while RSU1 silencing promotes cell growth of HCC cells in vitro, while in both experimental conditions expression and/or localization of focal adhesion proteins is deregulated. CONCLUSION: Our results suggest that FA signaling is implicated in hepatocellular carcinogenesis with prognostic significance. RSU1 seems to exert tumor suppressive functions in HCC and represents a novel favorable prognostic indicator.

Intrinsic neural stem cell properties define brain hypersensitivity to genotoxic stress.

Impaired replication has been previously linked to growth retardation and microcephaly; however, why the brain is critically affected compared with other organs remains elusive. Here, we report the differential response between early neural progenitors (neuroepithelial cells [NECs]) and fate-committed neural progenitors (NPs) to replication licensing defects. Our results show that, while NPs can tolerate altered expression of licensing factors, NECs undergo excessive replication stress, identified by impaired replication, increased DNA damage, and defective cell-cycle progression, leading eventually to NEC attrition and microcephaly. NECs that possess a short G1 phase license and activate more origins than NPs, by acquiring higher levels of DNA-bound MCMs. In vivo G1 shortening in NPs induces DNA damage upon impaired licensing, suggesting that G1 length correlates with replication stress hypersensitivity. Our findings propose that NECs possess distinct cell-cycle characteristics to ensure fast proliferation, although these inherent features render them susceptible to genotoxic stress.

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications.

In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.

Liquid biopsy genotyping by a simple lateral flow strip assay with visual detection.

Liquid biopsy, as a minimally invasive method that allows real-time monitoring of the tumor genome, represents a competing approach for cancer diagnosis, prognosis and therapy decision making. Liquid biopsy in cancer patients mainly includes analysis of circulating tumor cells (CTC) and cell-free circulating tumor DNA (ctDNA). ctDNA is the tumor-derived fraction of the cell-free DNA present in the blood. ctDNA is detected based on cancer-specific genomic aberrations (mainly mutations) and represents a challenging analyte due to high fragmentation and low concentration. Several methodologies have been developed for ctDNA analysis in cancer patients but many of these technologies are too time-intensive, complicated and expensive for implementation in diagnostic testing. Herein, we developed a novel lateral flow strip assay for mutational analysis of ctDNA in blood samples and visual detection that is based on gold nanoparticles as reporters. As a model, common single-point mutations of the KRAS gene, related to colorectal cancer (CRC), have been selected for method development. The proposed DNA biosensor has been successfully applied for the detection of three KRAS mutations (KRAS G12D/A/V), along with the wild-type KRAS gene in synthetic DNA targets, cancer cell lines and cfDNA from blood samples of healthy individuals and CRC patients. The main advantages of the proposed lateral flow assay are simplicity, rapid analysis time (∼10 min) and visual detection without the requirement of special instrumentation. The assay is also cost-effective with high detectability, specificity and reproducibility and has the potential to be used as a portable and universal device. In conclusion, the proposed assay offers a rapid diagnostic strip test for visual genotyping, as an alternative approach for liquid biopsy applications.

Integrin-Linked-Kinase Overexpression Is Implicated in Mechanisms of Genomic Instability in Human Colorectal Cancer.

BACKGROUND: Genomic instability is a hallmark of cancer cells contributing to tumor development and progression. Integrin-linked kinase (ILK) is a focal adhesion protein with well-established role in carcinogenesis. We have previously shown that ILK overexpression is critically implicated in human colorectal cancer (CRC) progression. In light of the recent findings that ILK regulates centrosomes and mitotic spindle formation, we aimed to determine its implication in mechanisms of genomic instability in human CRC. METHODS: Association of ILK expression with markers of genomic instability (micronuclei formation, nucleus size, and intensity) was investigated in diploid human colon cancer cells HCT116 upon ectopic ILK overexpression, by immunofluorescence and in human CRC samples by Feulgen staining. We also evaluated the role of ILK in mitotic spindle formation, by immunofluorescence, in HCT116 cells upon inhibition and overexpression of ILK. Finally, we evaluated association of ILK overexpression with markers of DNA damage (p-H2AX, p-ATM/ATR) in human CRC tissue samples by immunohistochemistry and in ILK-overexpressing cells by immunofluorescence. RESULTS: We showed that ILK overexpression is associated with genomic instability markers in human colon cancer cells and tissues samples. Aberrant mitotic spindles were observed in cells treated with specific ILK inhibitor (QLT0267), while ILK-overexpressing cells failed to undergo nocodazole-induced mitotic arrest. ILK overexpression was also associated with markers of DNA damage in HCT116 cells and human CRC tissue samples. CONCLUSIONS: The above findings indicate that overexpression of ILK is implicated in mechanisms of genomic instability in CRC suggesting a novel role of this protein in cancer.

Nucleobindin 2/nesfatin-1 expression and colocalisation with neuropeptide Y and cocaine- and amphetamine-regulated transcript in the human brainstem.

Feeding is a complex behaviour entailing elaborate interactions between forebrain, hypothalamic and brainstem neuronal circuits via multiple orexigenic and anorexigenic neuropeptides. Nucleobindin-2 (NUCB2)/nesfatin-1 is a negative regulator of food intake and body weight with a widespread distribution in rodent brainstem nuclei. However, its localisation pattern in the human brainstem is unknown. The present study aimed to explore NUCB2/nesfatin-1 immunoexpression in human brainstem nuclei and its possible correlation with body weight. Sections of human brainstem from 20 autopsy cases (13 males, seven females; eight normal weight, six overweight, six obese) were examined using immunohistochemistry and double immunofluorescence labelling. Strong immunoreactivity for NUCB2/nesfatin-1 was displayed in various brainstem areas, including the locus coeruleus, medial and lateral parabrachial nuclei, pontine nuclei, raphe nuclei, nucleus of the solitary tract, dorsal motor nucleus of vagus (10N), area postrema, hypoglossal nucleus, reticular formation, inferior olive, cuneate nucleus, and spinal trigeminal nucleus. NUCB2/nesfatin-1 was shown to extensively colocalise with neuropeptide Y and cocaine- and amphetamine-regulated transcript in the locus coeruleus, dorsal raphe nucleus and solitary tract. Interestingly, in the examined cases, NUCB2/nesfatin-1 protein expression was lower in obese than normal weight subjects in the solitary tract (P = 0.020). The findings of the present study provide neuroanatomical support for a role for NUCB2/nesfatin-1 in feeding behaviour and energy balance. The widespread distribution of NUCB2/nesfatin-1 in the human brainstem nuclei may be indicative of its pleiotropic effects on autonomic, neuroendocrine and behavioural processes. In the solitary tract, a key integrator of energy status, altered neurochemistry may contribute to obesity. Further research is necessary to decipher human brainstem energy homeostasis circuitry, which, despite its importance, remains inadequately characterised.

Integrin-linked kinase (ILK) regulates KRAS, IPP complex and Ras suppressor-1 (RSU1) promoting lung adenocarcinoma progression and poor survival.

Integrin-linked kinase (ILK) forms a heterotrimeric protein complex with PINCH and PARVIN (IPP) in Focal Adhesions (FAs) that acts as a signaling platform between the cell and its microenvironment regulating important cancer-related functions. We aimed to elucidate the role of ILK in lung adenocarcinoma (LUADC) focusing on a possible link with KRAS oncogene. We used immunohistochemistry on human tissue samples and KRAS-driven LUADC in mice, analysis of large scale publicly available RNA sequencing data, ILK overexpression and pharmacological inhibition as well as knockdown of KRAS in lung cancer cells. ILK, PINCH1 and PARVB (IPP) proteins are overexpressed in human LUADC and KRAS-driven LUADC in mice representing poor prognostic indicators. Genes implicated in ILK signaling are significantly enriched in KRAS-driven LUADC. Silencing of KRAS, as well as, overexpression and pharmacological inhibition of ILK in lung cancer cells provide evidence of a two-way association between ILK and KRAS. Upregulation of PINCH, PARVB and Ras suppressor-1 (RSU1) expression was demonstrated in ILK overexpressing lung cancer cells in addition to a significant positive correlation between these factors in tissue samples, while KRAS silencing downregulates IPP and RSU1. Pharmacological inhibition of ILK in KRAS mutant lung cancer cells suppresses cell growth, migration, EMT and increases sensitivity to platinum-based chemotherapy. ILK promotes an aggressive lung cancer phenotype with prognostic and therapeutic value through functions that involve KRAS, IPP complex and RSU1, rendering ILK a promising biomarker and therapeutic target in lung adenocarcinoma.

Expression and prognostic significance of YAP, TAZ, TEAD4 and p73 in human laryngeal cancer.

OBJECTIVES: The Hippo signaling pathway plays a critical role in organ size control and tissue homeostasis and its perturbation is associated with tumorigenesis. YAP (Yes associated protein) and TAZ (transcriptional co-activator with PDZ- binding motif) are the major nuclear effectors of the Hippo pathway interacting with TEADs (TEA domain) and p73 transcriptional factors to regulate gene expression. Altered expression of the above proteins promotes tumor initiation, progression and metastasis in a variety of cancer types. This study addresses their expression and prognostic significance in human laryngeal carcinoma. METHODS: Protein expression of YAP, TAZ, TEAD4 and p73 was examined by immunohistochemistry in 121 human laryngeal squamous cell carcinomas. Correlations with clinicopathological data and survival were evaluated. RESULTS: All proteins were overexpressed in human laryngeal carcinomas compared to non-neoplastic adjacent epithelium. High expression of YAP, TAZ, TEAD4 and p73 correlated significantly with high grade, advanced stage, supraglottic location of tumor, nodal metastases and recurrence. Furthermore, high expression of all proteins was significantly associated with poor overall and disease- free survival. p73 expression proved to be an independent predictive factor of survival and YAP expression proved to be an independent predictive factor of disease recurrence. CONCLUSIONS: Deregulation of the expression of the Hippo pathway proteins is implicated in human laryngeal carcinogenesis and YAP and p73 have prognostic significance in the outcome of the disease.

Nucleobindin-2/Nesfatin-1 in the Human Hypothalamus Is Reduced in Obese Subjects and Colocalizes with Oxytocin, Vasopressin, Melanin-Concentrating Hormone, and Cocaine- and Amphetamine-Regulated Transcript.

BACKGROUND/AIMS: Nesfatin-1, processed from nucleobindin-2 (NUCB2), is a potent anorexigenic peptide being expressed in rodent hypothalamic nuclei and involved in the regulation of feeding behavior and body weight in animals. The present study aimed to investigate NUCB2/nesfatin-1 protein expression in the human hypothalamus as well as its correlation with body weight. METHODS: Sections of hypothalamus and adjacent cholinergic basal forebrain nuclei, including the nucleus basalis of Meynert (NBM) and the diagonal band of Broca (DBB), from 25 autopsy cases (17 males, 8 females; 8 lean, 9 overweight, 8 obese) were examined using immunohistochemistry and double immunofluorescence labeling. RESULTS: Prominent NUCB2/nesfatin-1 immunoexpression was detected in supraoptic, paraventricular, and infundibular nuclei, lateral hypothalamic area (LHA)/perifornical region, and NBM/DBB. NUCB2/nesfatin-1 was found to extensively colocalize with (a) oxytocin and vasopressin in paraventricular and supraoptic nuclei, (b) melanin-concentrating hormone in the LHA, and (c) cocaine- and amphetamine-regulated transcript in infundibular and paraventricular nuclei and LHA. Interestingly, in the LHA, NUCB2/nesfatin-1 protein expression was significantly decreased in obese, compared with lean (p < 0.01) and overweight (p < 0.05) subjects. CONCLUSIONS: The findings of the present study are suggestive of a potential role for NUCB2/nesfatin-1 as an integral regulator of food intake and energy homeostasis in the human hypothalamus. In the LHA, an appetite- and reward-related brain area, reduced NUCB2/nesfatin-1 immunoexpression may contribute to dysregulation of homeostatic and/or hedonic feeding behavior and obesity. NUCB2/nesfatin-1 localization in NBM/DBB might imply its participation in the neuronal circuitry controlling cognitive influences on food intake and give impetus towards unraveling additional biological actions of NUCB2/nesfatin-1 in human neuronal networks.

Geminin ablation in vivo enhances tumorigenesis through increased genomic instability.

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham-treated controls in urethane-induced lung and azoxymethane/dextran sodium sulfate-induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non-monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

High PINCH1 Expression in Human Laryngeal Carcinoma Associates with Poor Prognosis.

Focal adhesion signaling to actin cytoskeleton is critically implicated in cell migration and cancer invasion and metastasis. Actin-binding proteins cofilin and N-WASP regulate actin filament turnover, and focal adhesion proteins parvins and PINCH mediate integrin signaling to the actin cytoskeleton. Altered expression of these proteins has been implicated in human cancer. This study addresses their expression and prognostic significance in human laryngeal carcinoma. Protein expressions of cofilin, N-WASP, α-parvin, ß-parvin, and PINCH1 were examined by immunohistochemistry in 72 human laryngeal squamous cell carcinomas. Correlations with clinicopathological data and survival were evaluated. All proteins examined were overexpressed in human laryngeal carcinomas compared to adjacent nonneoplastic epithelium. High expression of PINCH1 was associated significantly with high grade, lymph node-positive, and advanced stage disease. Moreover, high PINCH1 expression significantly associated with poor overall and disease-free survival and high cytoplasmic PINCH1 expression was shown by multivariate analysis to independently predict poor overall survival. In conclusion, we provide novel evidence that focal adhesion signaling to actin cytoskeleton is implicated in human laryngeal carcinogenesis and PINCH1 has prognostic significance in the disease.

Expression of EMT inducers integrin-linked kinase (ILK) and ZEB1 in phyllodes breast tumors is associated with aggressive phenotype.

Phyllodes tumors (PTs) of the breast constitute an uncommon group of mammary fibroepithelial lesions with ambiguous biologic behavior. Recent evidence suggests that epithelial mesenchymal transition (EMT), a driving force of cancer progression is implicated in PTs pathogenesis. Integrin-linked kinase (ILK), a focal adhesion kinase, has been implicated in cancer and EMT and represents a novel cancer therapeutic target. In this study, we aimed to investigate ILK and EMT markers expression in phyllodes breast tumors in relation to tumor grade. Expression of ILK and EMT markers E-cadherin, ß-catenin, Ν-cadherin, vimentin, Snail, ZEB1 and Twist was evaluated by immunohistochemistry in paraffin-embedded tissue sections from 96 human phyllodes breast tumors (48 benign, 27 borderline, 21 malignant). Cytoplasmic and nuclear immunopositivity of ILK were observed in both the epithelial and the stromal component of phyllodes breast tumors and were significantly higher with increasing tumor grade. An EMT-related expression profile consisting of decreased membranous and increased nuclear/cytoplasmic immunoreactivity of E-cadherin and ß-catenin and increased expression of N-cadherin, vimentin, Snail, ZEB1 and Twist was observed in tumor epithelial and stromal component and was significantly associated with malignant phyllodes breast tumor histopathology. Interestingly, there was a significant correlation of ILK expression with all of the EMT markers examined. Our results suggest that EMT significantly contributes to phyllodes tumor pathogenesis and originally implicate ILK and ZEB1 in phyllodes tumors malignant phenotype.

ILK Expression in Colorectal Cancer Is Associated with EMT, Cancer Stem Cell Markers and Chemoresistance.

BACKGROUND/AIM: Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critically implicated in cancer metastasis and chemoresistance. Herein, we investigated integrin-linked kinase (ILK)'s role in human colon cancer (CRC) progression and chemoresistance in relation to EMT and CSC markers. PATIENTS AND METHODS: Expression of ILK, EMT and CSC markers were evaluated by immunohistochemistry in 149 CRC samples. We also generated colon cancer cells resistant to 5-FU and oxaliplatin and studied the effect of ILK inhibition on drug response by MTT assay and on EMT and CSC markers' expression. RESULTS: ILK expression in human CRC correlates with EMT and CSC markers and is associated with metastasis and chemoresistance. ILK inhibition increases sensitivity of resistant cells to 5-FU and oxaliplatin and reduces the levels of EMT and CSC markers in 5-FU resistant cells. CONCLUSION: ILK overexpression in human CRC associates with EMT and CSC traits, contributing to tumor progression and chemoresistance.

LIMK/cofilin pathway and Slingshot are implicated in human colorectal cancer progression and chemoresistance.

Cofilin phospho-regulation is important for actin filament turnover and is implicated in cancer. Phosphorylation of cofilin is mediated by LIM kinases (LIMKs) and dephosphorylation by Slingshot phosphatases (SSH). LIMKs and SSH promote cancer cell invasion and metastasis and represent novel anti-cancer targets. However, little is known regarding LIMK/cofilin and SSH in human colorectal cancer (CRC). In this study, we aimed to address their expression and significance in human CRC. We evaluated expression of non-phosphorylated (active) and phosphorylated cofilin, LIMK1, LIMK2, and SSH1 by immunohistochemistry in 143 human CRC samples in relation to clinicopathologic parameters, response of metastatic disease to chemotherapy, and epithelial-mesenchymal transition (EMT) markers ß-catenin, E-cadherin, and ZEB. We show that active cofilin, LIMK1, LIMK2, and SSH1 are overexpressed in human CRC and are associated with tumor progression parameters. SSH1 is an independent predictor of lymph node metastasis by multivariate analysis. LIMK1 and SSH1 expression is also higher in non-responders to chemotherapy, and SSH1 is shown by multivariate analysis to independently predict response of metastatic disease to chemotherapy. Active cofilin, LIMK1, LIMK2, and SSH1 also correlated with the EMT markers examined. In addition, immunofluorescence analysis showed increased expression of active cofilin, LIMK1, LIMK2, and SSH1 in HT29 colon cancer cells resistant to 5-fluorouracil compared to parental HT29 cells. Our results suggest that F-actin regulators LIMK/cofilin pathway and SSH1 are associated with CRC progression and chemoresistance representing promising tumor biomarkers and therapeutic targets in CRC.

Focal Adhesion Proteins α- and ß-Parvin are Overexpressed in Human Colorectal Cancer and Correlate with Tumor Progression.

This study aims to address the role of focal adhesion proteins α- and ß-parvin in human colorectal carcinoma (CRC). Expression of α- and ß-parvin was examined by immunohistochemistry and real-time RT-PCR in a series of human CRC. Parvins were overexpressed in CRC and their expression correlated significantly with tumor invasion, lymph node metastasis, and disease stage. A significant positive correlation of parvins protein expression with overexpression of integrin-linked kinase, p-AKT, and nuclear ß-catenin, as well as with downregulation of E-cadherin was also observed. In conclusion, overexpression of α- and ß-parvin seems to be implicated in human colorectal cancer progression.