Increased expression of importin-ß, exportin-5 and nuclear transportable proteins in Alzheimer's disease aids anatomic pathologists in its diagnosis.

Understanding the metabolic profile of neurons with the hyperphosphorylated tau protein characteristic of Alzheimer's disease is essential to unraveling new potential therapies and diagnostics for the surgical pathologist. We stratified 75 brain tissues from Alzheimer's disease into hyperphosphorylated tau positive or negative and did co-expression analyses and qRTPCR for importin-ß and exportin-5 plus several bcl2 family members and compared the data to controls, Down's dementia and Parkinson's disease. There was a significant increase in the expression of importin-ß and exportin-5 in Alzheimer's disease relative to the three other categories (each p value<0.0001) where each protein co-localized with hyperphosphorylated tau. Both apoptotic and anti-apoptotic proteins were each significantly increased in Alzheimer's disease relative to the three other groups. Neurons with hyperphosphorylated tau in Alzheimer's disease have the profile of metabolically active cells including increased exportin-5 and importin-ß mRNA and proteins which indicates that immunohistochemistry testing of these proteins may aid the surgical pathologist in making a definitive diagnosis.

Expression of CRM1 and CDK5 shows high prognostic accuracy for gastric cancer.

AIM: To evaluate the predictive value of the expression of chromosomal maintenance (CRM)1 and cyclin-dependent kinase (CDK)5 in gastric cancer (GC) patients after gastrectomy. METHODS: A total of 240 GC patients who received standard gastrectomy were enrolled in the study. The expression level of CRM1 and CDK5 was detected by immunohistochemistry. The correlations between CRM1 and CDK5 expression and clinicopathological factors were explored. Univariate and multivariate survival analyses were used to identify prognostic factors for GC. Receiver operating characteristic analysis was used to compare the accuracy of the prediction of clinical outcome by the parameters. RESULTS: The expression of CRM1 was significantly related to size of primary tumor (P = 0.005), Borrmann type (P = 0.006), degree of differentiation (P = 0.004), depth of invasion (P = 0.008), lymph node metastasis (P = 0.013), TNM stage (P = 0.002) and distant metastasis (P = 0.015). The expression of CDK5 was significantly related to sex (P = 0.048) and Lauren's classification (P = 0.011). Multivariate Cox regression analysis identified that CRM1 and CDK5 co-expression status was an independent prognostic factor for overall survival (OS) of patients with GC. Integration of CRM1 and CDK5 expression could provide additional prognostic value for OS compared with CRM1 or CDK5 expression alone (P = 0.001). CONCLUSION: CRM1 and CDK5 co-expression was an independent prognostic factors for GC. Combined CRM1 and CDK5 expression could provide a prognostic model for OS of GC.

Both high expression of nucleophosmin/B23 and CRM1 predicts poorer prognosis in human gastric cancer.

Nucleophosmin/B23 and CRM1 are molecular markers which play an important role in tumorigenesis and tumor progression in gastric cancer (GC). However, the association between the two remains unclear. This study evaluated the expression and the correlation of B23 and CRM1 in GC. B23 and CRM1 expression in GC and adjacent noncancerous tissues (ANCT) of gastrectomy specimens from 131 GC patients was measured by immunohistochemistry. Positive expression rates of B23 and CRM1 were significantly higher in GC tissues than in ANCT. The high expression rates of B23 and CRM1 were significantly higher in patients with more advanced tumor stages and distant metastasis (all p < 0.05). Only high expression of CRM1was correlated with positive Her2 status (p = 0.01). B23 expression was positively correlated with CRM1expression in GC tissues (p = 0.038). Univariate analysis showed that TNM stage (p = 0.0001), metastasis (p = 0.027), B23 (p = 0.0111), and CRM1 expression (p = 0.0019) were significant risk factors affecting overall survival. Both high expression of B23 and CRM1 in GC patients suggests poor prognosis, co-expression of the two (p = 0.043) even worse. Cox multivariate analysis showed that positive B23 (p = 0.0231) and CRM1 (p = 0.0048) expression were both independent prognostic factors that negatively correlated with survival. We revealed the co-expression of B23 or CRM1 in GC. The expression levels of B23 or CRM1 were closely related to poor prognosis in GC, and both B23 or CRM1 were independent risk factor.

Overexpression of CRM1: A Characteristic Feature in a Transformed Phenotype of Lung Carcinogenesis and a Molecular Target for Lung Cancer Adjuvant Therapy.

Our previous study showed that chromosome region maintenance 1 (CRM1), a nuclear export receptor for various cancer-associated "cargo" proteins, was important in regulating lung carcinogenesis in response to a tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The objectives of this study are to comprehensively evaluate the significance of CRM1 in lung cancer development and investigate the therapeutic potential of targeting CRM1 for lung cancer treatment using both in vitro and in vivo models. We showed that CRM1 was overexpressed not only in lung tumor tissues from both lung cancer patients and mice treated with NNK but also in NNK-transformed BEAS-2B human bronchial epithelial cells. Furthermore, stable overexpression of CRM1 in BEAS-2B cells by plasmid vector transfection led to malignant cellular transformation. Moreover, a decreased CRM1 expression level in A549 cells by short hairpin siRNA transfection led to a decreased tumorigenic activity both in vitro and in nude mice, suggesting the potential to target CRM1 for lung cancer treatment. Indeed, we showed that the cytotoxic effects of cisplatin on A549 cells with CRM1 down-regulated by short hairpin siRNA were significantly increased, compared with A549 cells, and the cytotoxic effects of cisplatin became further enhanced when the drug was used in combination with leptomycin B, a CRM1 inhibitor, in both in vitro and in vivo models. Cancer target genes were significantly involved in these processes. These data suggest that CRM1 plays an important role in lung carcinogenesis and provides a novel target for lung cancer adjuvant therapy.

Involvement of chromosome region maintenance 1 (CRM1) in the formation and progression of esophageal squamous cell carcinoma.

Chromosome region maintenance 1 (CRM1) has been related to several malignancies. The predictive value of CRM1 in the malignance and prognosis of esophageal squamous cell carcinoma (ESCC), however, is not clear yet. In this study, we displayed that CRM1 expression was up-regulated in ESCC using immunohistochemistry and Western blot. Statistical analysis demonstrated that patients with high CRM1 levels indicated shorter survival period. We further found that silencing CRM1 caused apoptosis in ESCC cell lines. Moreover, knockdown of CRM1 disturbed the expression of tumor suppressor proteins and inhibited NF-κB activity in ESCC cell lines, especially if the cell line was treated with 5-fluorouracil. In consequence, our results for the first time indicated that CRM1 was dysregulated in ESCC, and suppression of CRM1 expression which resulted in inhibiting of NF-κB signaling might be developed into a new strategy in ESCC therapy.

Expression of proteins involved in epigenetic regulation in human cutaneous melanoma and peritumoral skin.

Epigenetic processes play a critical role in melanoma development. However, little is known about proteins responsible for epigenetic transformations in melanoma cells. The processes in the peritumoral skin within the excision margin are almost unstudied. We studied the changes in expression of 112 proteins involved in epigenetic regulation of gene expression in the human cutaneous melanoma and its peritumoral zone using "The Proteomic Antibody Microarrays" (GRAA2, Sigma-Aldrich). Dimethylated histone H3 at lysines 4 and 9 as well as proteins involved in the regulation of transcription (histone deacetylases HDAC-1 and HDAC-11, DNA methyl-binding protein Kaiso), cell cycle control (protein kinases Aurora-В and PKR, chromosome protein CENP-E , and phosphorylated and acetylated histone H3), DNA repair (phosphorylated histone H2AX), and nuclear protein import (importins α3 and α5/7) were over-expressed in the melanoma tissue as compared with normal skin. At the same time, HDAC-10 and proliferating cell nuclear antigen PCNA were downregulated. In the peritumoral skin, at the excision margin (1-2 cm from the melanoma edge), we observed similar changes in expression of these proteins and, additionally, over-expression of arginine methyltransferases PRMT5 and NAD-dependent histone deacetylase SIR2. Histone methyltransferase G9a and metastasis-associated protein 2 were downregulated. Therefore, epigenetic regulation that requires histone modifications and expression of some regulatory proteins is of importance for melanoma development and propagation. The observed changes in the peritumoral skin may indicate the epigenetic pre-tuning in this zone possibly involved in malignant transformation. These results can be potentially useful for melanoma diagnostics and targeted therapy.

An intracellular targeted antibody detects EGFR as an independent prognostic factor in ovarian carcinomas.

BACKGROUND: In ovarian cancer, the reported rate of EGFR expression varies between 4-70% depending on assessment method and data on patient outcome are conflicting. METHODS: In this study we investigated EGFR expression and its prognostic value in a cohort of 121 invasive ovarian carcinomas, using a novel antibody against the intracellular domain of the receptor. We further evaluated an association between EGFR, the nuclear transporter CRM1 as well as COX-2. Furthermore, we evaluated EGFR expression in ten ovarian cancer cell lines and incubated cancer cells with Leptomycin B, a CRM1 specific inhibitor. RESULTS: We observed a membranous and cytoplasmic EGFR expression in 36.4% and 64% of ovarian carcinomas, respectively. Membranous EGFR was an independent prognostic factor for poor overall survival in ovarian cancer patients (HR 2.7, CI 1.1-6.4, p = 0.02) which was also found in the serous subtype (HR 4.6, CI 1.6-13.4, p = 0.004). We further observed a significant association of EGFR with COX-2 and nuclear CRM1 expression (chi-square test for trends, p = 0.006 and p = 0.013, respectively). In addition, combined membranous EGFR/COX-2 expression was significantly related to unfavorable overall survival (HR 7.2, CI 2.3-22.1, p = 0.001).In cell culture, we observed a suppression of EGFR protein levels after exposure to Leptomycin B in OVCAR-3 and SKOV-3 cells. CONCLUSIONS: Our results suggest that the EGFR/COX-2/CRM1 interaction might be involved in progression of ovarian cancer and patient prognosis. Hence, it is an interesting anti-cancer target for a combination therapy. Further studies will also be needed to investigate whether EGFR is also predictive for benefit from EGFR targeted therapies.

JAB1 expression is associated with inverse expression of p27(kip1) in hepatocellular carcinoma.

BACKGROUND/AIMS: Recent studies have shown that overexpression of c-jun activation domain binding protein 1 (JAB1) and reduced expression of p27(kip1) are associated with advanced tumor stage and poor prognosis in several human cancers. Here, We investigated the functional role and correlation of JAB1 and p27(kip1) in hepatocellular carcinoma (HCC). METHODOLOGY: Immunohistochemical study for JAB1, p27(kip1) was performed on 76 cases of HCC and adjacent nontumorous tissues. 6 Fresh specimens of HCC and the adjacent liver tissue were collected for Western blot analysis. The influence of As2O3 on HCC SMMC-7721 cells, was detected by flow cytometry and Hochest staining. The expression and subcellular localization of p27(kip1) and JAB1 were investigated by Western blot and immunofluorescence. RESULTS: The expression of JAB1 was higher but p27(kip1) was lower in HCC than that in adjacent liver tissue. As2O3 treatment inhibited the growth of SMMC-7721 cells. In As2O3-treated cells, p27(kip1) expression was increased while JAB1 was decreased. The location of p27(kip1) and JAB1 were transferred from cytoplasm to nucleus. CONCLUSIONS: In HCC, JAB1 was inversely correlated with p27(kip1). As2O3 attenuated the expression of JAB1, disturbed the location and expression of p27(kip1), which may participate in regulating the growth of human hepatoma cells.

Expression of the nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor in human ovarian cancer.

BACKGROUND: The human nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 (CRM1) mediates the nuclear export of proteins and messenger RNAs and, thus, is an important regulator of subcellular distribution of key molecules. Whereas cell-biologic studies have suggested a fundamental role for CRM1 in the regulation of mitosis, the expression of this protein in human tumor tissue has not been investigated to date. METHODS: In this study, the expression of CRM1 was analyzed in a cohort of 88 ovarian tumors and 12 ovarian cell lines for the first time to the authors' knowledge. RESULTS: Immunohistochemistry revealed increased nuclear (52.7%) and cytoplasmic (56.8%) expression of CRM1 in 74 carcinomas compared with the expression revealed in borderline tumors and benign lesions. Similarly, CRM1 expression was increased in ovarian cancer cell lines compared with human ovarian surface epithelial cells. Cytoplasmic CRM1 expression was related significantly to advanced tumor stage (P= .043), poorly differentiated carcinomas (P= .011), and higher mitotic rate (P= .008). Nuclear CRM1 was associated significantly with cyclooxygenase-2 (COX-2) expression (P= .002) and poor overall survival (P= .01). Because it was demonstrated previously that blocking of CRM1 by leptomycin B (LMB) contributes to the inhibition of nuclear export, the authors used a set of mechanistic assays to study the effects of CRM1 inhibition in cancer cells. Treatment of OVCAR-3 cells with LMB revealed a significant reduction of cell proliferation and increased apoptosis as well as suppressed interleukin-1beta-induced COX-2 expression. CONCLUSIONS: The current results indicated that CRM1 is expressed in a subpopulation of ovarian carcinomas with aggressive behavior and is related to poor patient outcome. A correlation also was demonstrated between CRM1 and COX-2 expression in ovarian cancer tissue. Furthermore, the treatment of ovarian cancer cells with LMB revealed a reduction in COX-2 expression. Therefore, the authors suggest that CRM1 may be an interesting biomarker for the assessment of patient prognosis and a molecular target for anticancer treatment.

Dynamic intracellular survivin in oral squamous cell carcinoma: underlying molecular mechanism and potential as an early prognostic marker.

Survivin functions as an apoptosis inhibitor and a regulator of cell division in many tumours. The intracellular localization of survivin in tumours has been suggested as a prognostic marker. However, current reports are inconsistent and the underlying molecular mechanisms are not understood. The present study has examined the localization and prognostic value of nuclear and cytoplasmic survivin in the pre-therapeutic biopsies from 71 oral and oropharyngeal squamous carcinoma (OSCC) patients. Statistical analysis indicated that preferential nuclear versus cytoplasmic survivin correlated with favourable versus unfavourable disease outcome. Uni- and multi-variate analysis showed that in contrast to total survivin expression, the difference between nuclear and cytoplasmic survivin was a strong predictor for relapse-free survival (p=0.0003). As a potential underlying molecular mechanism, it is shown in OSCC cell lines that predominantly cytoplasmic survivin mediates protection against chemo- and radio-therapy-induced apoptosis. Importantly, the cytoplasmic localization of survivin is regulated by its nuclear export signal (NES), and export-deficient nuclear survivin is not cytoprotective. This study suggests that the difference between cytoplasmic and nuclear survivin is an indicator for survivin activity in tumour cells. Thus, this difference may serve as a predictive marker of outcome in OSCC patients undergoing multi-modality therapy. The pharmacogenetic interference with survivin's cytoplasmic localization is also to be pursued as a potential therapeutic strategy.

Intramolecular masking of nuclear localization signals: analysis of importin binding using a novel AlphaScreen-based method.

Active nuclear import of proteins requires the recognition of a nuclear localization sequence (NLS) by members of the importin (IMP) family of proteins. We have developed a modified AlphaScreen-based assay able to estimate the solution binding affinities of such interactions using biotinylated IMPs and His6-tagged NLS-containing proteins. We describe this assay in detail as well as its application in documenting the phenomenon of intramolecular masking of NLSs using recombinant green fluorescent protein (GFP) fusion proteins containing sequences from the SV40 large tumor T antigen (T-ag). We also use it to examine, for the first time, IMP binding to the cancer cell-specific proapoptotic factor viral protein 3 (VP3) from the chicken anemia virus (CAV). High-affinity binding of the IMPalpha/beta heterodimer to the T-ag NLS was observed when the GFP tag was fused to its N terminus but not to its C terminus. Effects of flanking residues were also observed in GFP-T-ag fusion derivatives containing the Thr128 NLS-inactivating mutation, whereby the absence of flanking sequences N terminal to the T-ag NLS appeared to decrease the specificity of the mutation in terms of oblating IMPalpha/beta binding. IMPbeta, but not IMPalpha or the IMPalpha/beta heterodimer, was found to bind to CAV VP3 with high affinity. Interestingly, GFP-VP3(74-121) bound to IMPbeta with threefold higher affinity than the full-length protein, GFP-VP3(1-121), implying that the NLS is masked to a significant extent in the context of full-length protein. This may represent a regulatory mechanism to control nuclear import in a tumor cell-specific fashion.

The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase.