Heparanase is overexpressed in lung cancer and correlates inversely with patient survival

BACKGROUND: Heparanase is an endo-beta-D-glucuronidase that is capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, yielding HS fragments of still appreciable size (approximately 5-7 kDa). Heparanase activity has been detected frequently in several cell types and tissues. Heparanase activity correlates with the metastatic potential of tumor-derived cells, a correlation that has been attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix barrier. METHODS: In this study, the authors examined heparanase expression in 114 patients with lung cancer by means of immunohistochemistry and correlated clinical-pathologic data with heparanase immunostaining and cellular localization. RESULTS: Heparanase was overexpressed in 75% of the study patients. Heparanase expression was correlated with lung cancer lymph node status and metastasis classification (P = .04 and P = .01, respectively) and was correlated inversely with patient survival (P = .007). It is noteworthy that this adverse effect depended largely on the cellular localization of heparanase. Thus, whereas cytoplasmic staining of heparanase is associated with a poor prognosis, nuclear heparanase predicts a favorable outcome for patients with lung cancer. CONCLUSIONS: The current findings suggest that heparanase expression and cellular localization are decisive for lung cancer patients' prognosis, most likely because of heparanase-mediated tumor cell dissemination by blood and lymph vessels.     

Human Rad17 is phosphorylated upon DNA damage and also overexpressed in primary non-small cell lung cancer tissues

The spRAD17 gene is an essential component of the DNA damage and replication checkpoints in the fission yeast Schizosaccharomyces pombe. Cloning of the human homologue of spRAD17, hRAD17, indicated that it exhibits structural similarity with the replication accessory protein family, which include subunits of the Replication factor C complex. We have analyzed the phosphorylation status of hRad17 in response to DNA damaging agents. Our results showed that phosphorylation of hRad17 occurred immediately after UV and ionizing radiation treatment and reached peak level at approximately 3 h, suggesting that hRad17 may be a component of the DNA damage checkpoint. When primary tumor samples were analyzed, we observed that the majority (74%) of non-small cell lung carcinoma samples exhibited a significantly higher level of hRad17 expression compared with matched normal tissue controls. In contrast, hRad17 protein levels in a panel of primary colon carcinoma samples did not show an elevated level of expression compared with normal colon tissues. This observation suggests that the function of the hRAD17 gene may be involved in lung cancer development and may serve as a potential tumor marker.     

A BRCA1 deficient-like signature is enriched in breast cancer brain metastases and predicts DNA damage-induced poly (ADP-ribose) polymerase inhibitor sensitivity

Introduction

There is an unmet clinical need for biomarkers to identify breast cancer patients at an increased risk of developing brain metastases. The objective is to identify gene signatures and biological pathways associated with human epidermal growth factor receptor 2-positive (HER2+) brain metastasis.

Methods

We combined laser capture microdissection and gene expression microarrays to analyze malignant epithelium from HER2+ breast cancer brain metastases with that from HER2+ nonmetastatic primary tumors. Differential gene expression was performed including gene set enrichment analysis (GSEA) using publicly available breast cancer gene expression data sets.

Results

In a cohort of HER2+ breast cancer brain metastases, we identified a gene expression signature that anti-correlates with overexpression of BRCA1. Sequence analysis of the HER2+ brain metastases revealed no pathogenic mutations of BRCA1, and therefore the aforementioned signature was designated BRCA1 Deficient-Like (BD-L). Evaluation of an independent cohort of breast cancer metastases demonstrated that BD-L values are significantly higher in brain metastases as compared to other metastatic sites. Although the BD-L signature is present in all subtypes of breast cancer, it is significantly higher in BRCA1 mutant primary tumors as compared with sporadic breast tumors. Additionally, BD-L signature values are significantly higher in HER2-/ER- primary tumors as compared with HER2+/ER + and HER2-/ER + tumors. The BD-L signature correlates with breast cancer cell line pharmacologic response to a combination of poly (ADP-ribose) polymerase (PARP) inhibitor and temozolomide, and the signature outperformed four published gene signatures of BRCA1/2 deficiency.

Conclusions

A BD-L signature is enriched in HER2+ breast cancer brain metastases without pathogenic BRCA1 mutations. Unexpectedly, elevated BD-L values are found in a subset of primary tumors across all breast cancer subtypes. Evaluation of pharmacological sensitivity in breast cancer cell lines representing all breast cancer subtypes suggests the BD-L signature may serve as a biomarker to identify sporadic breast cancer patients who might benefit from a therapeutic combination of PARP inhibitor and temozolomide and may be indicative of a dysfunctional BRCA1-associated pathway.     

Cyclooxygenase-2 is overexpressed in human cervical cancer.

Multiple lines of evidence suggest that cyclooxygenase-2 (COX-2) is an important target for preventing epithelial malignancies. Little is known, however, about the expression of COX-2 in gynecological malignancies. By immunoblot analysis, COX-2 was detected in 12 of 13 cases of cervical cancer but was undetectable in normal cervical tissue. Immunohistochemistry revealed COX-2 in malignant epithelial cells. COX-2 was also expressed in cervical intraepithelial neoplasia. The mechanism by which COX-2 is up-regulated in cervical cancer is unknown. Because the epidermal growth factor (EGF) receptor is commonly overexpressed in cervical cancer, we investigated whether EGF could induce COX-2 in cultured human cervical carcinoma cells. Treatment with EGF markedly induced COX-2 protein, COX-2 mRNA, and stimulated COX-2 promoter activity. The induction of COX-2 by EGF was suppressed by inhibitors of tyrosine kinase activity, phosphatidylinositol 3-kinase, mitogen-activated protein kinase kinase, and p38 mitogen-activated protein kinase. Moreover, overexpressing dominant-negative forms of extracellular signal-regulated kinase 1, c-Jun NH2-terminal kinase, p38, and c-Jun blocked EGF-mediated induction of COX-2 promoter activity. Taken together, these findings suggest that deregulation of the EGF receptor signaling pathway may lead to enhanced COX-2 expression in cervical cancer.     

TIMELESS is overexpressed in lung cancer and its expression correlates with poor patient survival

TIMELESS (TIM) is a mammalian homolog of a Drosophila circadian rhythm gene, but its circadian properties in mammals have yet to be determined. TIM appears to be essential for replication protection and genomic stability. Recently, the involvement of TIM in human malignancies has been reported; therefore, we investigated the role of TIM in lung cancer. Microarray expression analysis of lung cancer cell lines showed that TIM expression was elevated 3.7‐fold (P < 0.001) in non‐small cell lung cancer cell lines (n = 116) compared to normal lung controls (n = 59). In addition, small cell lung cancer cell lines (n = 29) expressed TIM at levels 2.2‐fold (P < 0.001) higher than non‐small cell lung cancer. Western blot analysis of 22 lung cancer cell lines revealed that all of them expressed TIM protein and that 20 cell lines (91%) expressed TIM protein at higher levels than a normal control line. Remarkably, immunohistochemistry of 30 surgically resected lung cancer specimens showed that all lung cancer specimens but no matched normal lung tissues were positive for TIM expression. Moreover, immunohistochemistry of surgically resected specimens from 88 consecutive patients showed that high TIM protein levels correlated with poor overall survival (P = 0.013). Mutation analysis for TIM in 23 lung cancer cell lines revealed no mutation. TIM knockdown suppressed proliferation and clonogenic growth, and induced apoptosis in H157 and H460 cells. Taken together, our findings suggest that TIM could be useful as a diagnostic and prognostic marker for lung cancer and targeting it would be of high therapeutic value for this disease.     

HMGA2在肺癌细胞系中的表达及与转移的相关性

目的:检测在肺癌细胞系中HMGA2的表达及分布情况并分析其与肺癌细胞转移能力间的关系。方法:采用Western blot、RT-PCR方法检测肺癌细胞系A549、BE1、LH7、95-D、95-C中HMGA2蛋白及mRNA的表达并和人支气管上皮细胞系HBE进行对比,结果由计算机图像系统采集并分析。结果:Western blot检测HMGA2蛋白在肺癌细胞系中过表达,而在人支气管上皮细胞系弱表达。而Western blot和RT-PCR结果均显示高转移能力的BE1和95-D肺癌细胞系的表达强于低转移能力的LH7和95-C肺癌细胞系（P<0.05）。结论:HMGA2表达在肺癌细胞中显著高于正常上皮细胞,其表达水平与细胞的转移能力正相关。HMGA2的过表达可能促进肺癌的转移。     

Much of spontaneous mutagenesis in Escherichia coli is due to error-prone DNA repair: implications for spontaneous carcinogenesis

The role of DNA repair genes (uvrA, uvrB, uvrD, recA, recB,lexA, and umuC) in spontaneous mutagenesis was examined in Escherichiacoli. The spontaneous mutation rate per bacterium per cell division(µ) was determined for the reversion of UAA (his-4 andtrpE65), UAG (lacZ53), and frameshift (trpE9777) mutations,and for the occurrence of forward mutations to valine resistance.Rich growth medium enhanced µ in a wild-type strain butnot in a uvrB5 strain. In minimal growth medium, the uvrA anduvrB strains had the largest µ (1.9–6.2-fold greaterthan that for isogenic wild-type strains, depending on the mutationassay). The uvrB strains carrying lexA, recA, umuC, or boththe uvrD and rec B mutations (in combination), i.e., mutationsthat inhibit error-prone DNA repair, had the lowest µvalues ({small tilde}10-fold less than the uvrB strain). TherecA and lexA mutations also reduced µ (by {small tilde}2-fold) in uvr⁺ strains. The genetic control of the error pronerepair-dependent sector of spontaneous mutagenesis was shownto be qualitatively similar to the genetic control for u.v.radiation mutagenesis. The umuC mutation, which drasticallyreduced spontaneous mutagenesis, had no effect on genetic recombination.It is proposed that the low level of spontaneous mutagenesisobserved in the recA, lexA, umuC, and the uvrD recB strainsis due to errors made during DNA replication, while the enhancedlevel of spontaneous mutagenesis observed in the wild type,and especially in the uvrA and uvrB strains, is due to excisablelesions that are produced in the DNA by normal metabolic reactions,and that such unexcised lesions induce mutations via error-proneDNA repair. These results are discussed in terms of their relevanceto spontaneous carcinogenesis.     

Mitogen-activated protein kinase phosphatase-1 is overexpressed in non-small cell lung cancer and is an independent predictor of outcome in patients.

PURPOSE: An increase in the activity of the mitogen-activated protein kinases (MAPKs) has been correlated with a more malignant phenotype in several tumor models in vitro and in vivo. A key regulatory mechanism of the MAPKs [extracellular signal-regulated kinase (ERK); c-jun NH(2)-terminal kinase (JNK); and p38] is the dual specificity phosphatase CL100, also called MAPK phosphatase-1 (MKP-1). This study was designed to examine the involvement of CL100/MKP-1 and stress-related MAPKs in lung cancer. EXPERIMENTAL DESIGN: We assessed the expression of CL100/MKP-1 and the activation of the MAPKs in a panel of 18 human cell lines [1 primary normal bronchial epithelium, 8 non-small cell lung cancer (NSCLC), 7 small cell lung cancer (SCLC), and 2 carcinoids] and in 108 NSCLC surgical specimens. RESULTS: In the cell lines, CL100/MKP-1 expression was substantially higher in NSCLC than in SCLC. P-ERK, P-JNK, and P-p38 were activated in SCLC and NSCLC, but the degree of their activation was variable. Immunohistochemistry in NSCLC resection specimens showed high levels of CL100/MKP-1 and activation of the three MAPK compared with normal lung. In univariate analysis, no relationship was found among CL100/MKP-1 expression and P-ERK, P-JNK, or P-p38. Interestingly, high CL100/MKP-1 expression levels independently predicted improved survival in multivariate analysis. JNK activation associated with T(1-2) and early stage, whereas ERK activation correlated with late stages and higher T and N. Neither JNK nor ERK activation were independent prognostic factors when studied for patient survival. CONCLUSIONS: Our data indicate the relevance of MAPKs and CL100/MKP-1 in lung cancer and point at CL100/MKP-1 as a potential positive prognostic factor in NSCLC. Finally, our study supports the search of new molecular targets for lung cancer therapy within the MAPK signaling pathway.     

DNA damage-induced signalling in ataxia-telangiectasia and related syndromes.

ATM, the protein mutated in the human genetic disorder ataxia-telangiectasia, functions by responding to radiation damage to DNA, primarily DNA double strand breaks (dsb), to reduce the risk of genome instability, cancer and neurodegeneration. ATM is rapidly activated as an existing protein to phosphorylate a number of downstream proteins that are involved in DNA repair and cell cycle checkpoint activation. While the exact mechanism of activation of ATM has not been determined, it is now evident that it relies heavily on the Mre11 complex (Mre11/Rad50/Nbs1) and a series of post-translational events for this activation. The Mre11 complex acts as a sensor for the break, recruits ATM to this site where it is autophosphorylated and then is capable of phosphorylating substrates that participate in DNA repair and cell cycle control. A greater understanding of how ATM is activated and functions through different signalling pathways is paramount to devising therapeutic strategies for the treatment of A-T patients. This knowledge can also be used to advantage in sensitizing cells to radiation and ultimately deriving novel therapeutic approaches for the treatment of cancer.     

MicroRNA-224 is implicated in lung cancer pathogenesis through targeting caspase-3 and caspase-7

We recently reported that miR-224 was significantly up-regulated in non-small cell lung cancer (NSCLC) tissues, in particular in resected NSCLC metastasis. We further demonstrated that miR-224 functions as an oncogene in NSCLC by directly targeting TNFAIP1 and SMAD4. However, the biological functions of miR-224 in NSCLC are controversial and underlying mechanisms of miR-224 in the progression and metastasis of lung cancer remain to be further explored. Here we report that caspase3 (CASP3) and caspase7 (CASP7) are previously unidentified targets of miR-224 in NSCLC, and that miR-224 promotes lung cancer cells proliferation and migration in part by directly targeting CASP7 and down-regulating its expression. In addition, miR-224 attenuated TNF-α induced apoptosis by direct targeting of CASP3 resulting in reduction of cleaved PARP1 expression in lung cancer cells. Furthermore, the expression of miR-224 negatively correlates with the expression of CASP7 and CASP3 in tissue samples from patients with lung cancer. Finally, we found that activated NF-κB signaling is involved in the regulation of miR-224 expression in lung cancer. Our study provides new insight in understanding of oncogenic role of miR-224 in the lung cancer pathogenesis and suggests that NF-κB/miR-224/CASP3, 7 pathway could be a putative therapeutic target in lung cancer.     

Multidrug resistance-associated protein (MRP1) is overexpressed in DNA aneuploid carcinomatous cells in non-small cell lung cancer (NSCLC)

Resistance to chemotherapy is intrinsically present in most nonsmall-cell lung carcinomas (NSCLC). No parameter has yet been determined to predict the response to chemotherapy. However, MRP1 (multidrug resistance-associated protein) is suspected to play an important role in resistance to treatment. The genetic basis for this resistance is not clearly understood, but it could result from chromosome reassortments catalyzed by aneuploidy. The aim of this study was to investigate MRP1 expression concurrently to DNA ploidy analysis in order to evaluate the link between MRP1 expression and chromosome 16 (MRP1 gene location) aberrations in NSCLC before treatment. Eighty-four surgical tumor specimens, 18 selected samples containing more than 80% of carcinomatous cells and 11 samples from normal bronchial epithelium were studied. Samples were stained by MRP1 FITC indirect staining and propidium iodide and analyzed by Flow Cytometry. Fifty tumors contained at least 1 DNA-aneuploid clone and the percentage of MRP1-positive cells was higher in DNA-aneuploid cells (p = 0.0003). All tumors expressed MRP1, but the level of expression was 3-fold higher in DNA-aneuploid cells than in DNA-diploid cells (normal bronchial cells as well as carcinomatous cells) (p < 0.0001). FISH analysis of 24 tumor imprints using a chromosome 16 alpha-satellite centromere probe demonstrated significantly more frequent gain of chromosome 16 in DNA-aneuploid tumors. These results suggest that MRP1 overexpression in NSCLC could be a consequence of chromosome 16 reassortments catalyzed by aneuploidy and that DNA-aneuploid tumors could require different treatment modalities from those applied to DNA-diploid tumors.     

Fatty acid binding protein 6 is overexpressed in colorectal cancer.

PURPOSE: Fatty acid binding protein 6 (FABP6) is a cancer-related protein that acts as an intracellular transporter of bile acid in the ileal epithelium. Because bile acids are implicated in the carcinogenesis of colorectal cancer, we evaluated FABP6 expression in colorectal cancer. EXPERIMENTAL DESIGN: The expression of FABP6 mRNA was evaluated in 78 paired samples of cancer/normal tissue representing colorectal cancer cases, plus 16 adenomas, and 16 metastatic lymph nodes. An immunohistochemical study was conducted with paraffin sections. In vitro transfection was done to determine FABP6's biological roles. RESULTS: The expression of FABP6 mRNA was significantly higher in cancer (75 of 78, 96.2%) than in normal tissue (P<0.001). The expression of mRNA was increased in cancer compared with adenoma, but was dramatically decreased in node metastases. Tumors with high FABP6 expression were smaller in size (P<0.01), more often in the left colon (P<0.05), and had shallower invasion into the bowel wall (P<0.05) compared with those with low expression. There was no significant difference between high- and low-expression tumors regarding clinicopathologic variables such as histologic type, lymph node, or liver metastasis, Dukes' classification, and prognosis. Immunohistochemical study revealed that FABP6 expression was primarily observed in cancer cells. In vitro transfection revealed that transfectants showed weaker invasiveness (P<0.05), more dominant proliferation (P<0.001), and less apoptosis than mock cells. CONCLUSIONS: The expression of FABP6 was higher in primary colorectal cancers and adenomas than in normal epithelium, but was dramatically decreased in lymph node metastases, suggesting that FABP6 may play an important role in early carcinogenesis.     

Prostate stem cell antigen is overexpressed in prostate cancer metastases.

PURPOSE: Prostate stem cell antigen (PSCA) is expressed by a majority of prostate cancers and is a promising therapeutic target. PSCA protein and mRNA expression was examined in prostate cancer bone, lymph node, and visceral metastases to assess the potential of PSCA as an immunotherapeutic target in advanced prostate cancer. EXPERIMENTAL DESIGN: Immunohistochemical analysis of PSCA protein expression and quantitative mRNA expression analysis of PSCA was done on clinical specimens of prostate cancer bone, lymph node, and visceral metastases. PSCA protein and mRNA expression levels were quantified and compared between available matched pairs of bone and lymph node or visceral metastases. RESULTS: Bone metastases stained with higher intensity of PSCA compared with lymph node or liver metastases in seven of eight (87.5%) matched pairs (P = 0.035). PSCA mRNA expression was equal or greater than that of LAPC-9, a PSCA expressing xenograft, in 12 of 24 (50%) cases of prostate cancer metastases and was significantly correlated with PSCA protein expression (sigma = 0.84, P = 0.0019). Overall, PSCA protein expression was detected in 41 of 47 (87.2%), four of six (66.7%), and two of three (66.7%) cases of bone, lymph node, and liver metastases, respectively. Mean PSCA staining intensity was significantly higher in prostate cancer bone metastases compared with lymph node metastases (2.0 +/- 0.02 versus 0.83 +/- 0.31, P = 0.014). CONCLUSIONS: Prostate cancer metastases express PSCA. However, greater PSCA staining intensity and level of PSCA mRNA expression was associated with bone metastases compared with lymph node metastases. This study suggests that PSCA is a promising tumor marker and potential therapeutic target for patients with metastatic prostate cancer.