MMP-10 is overexpressed, proteolytically active, and a potential target for therapeutic intervention in human lung carcinomas

Matrix metalloproteinase (MMP)-mediated degradation of the extracellular matrix is a major factor for tumor development and expansion. This study analysed MMP-10 protein expression and activity in human lung tumors of various grade, stage, and type to address the relationship between MMP-10 and tumor characteristics and to evaluate MMP-10 as a therapeutic target in non small cell lung carcinoma (NSCLC). Unlike the majority of MMPs, MMP-10 was located in the tumor mass as opposed to tumor stroma. MMP-10 protein was observed at low levels in normal human lung tissues and at significantly higher levels in all types of NSCLC. No correlation was observed between MMP-10 protein expression and tumor type, stage, or lymph node invasion. To discriminate between active and inactive forms of MMP-10 in samples of human NSCLC, we have developed an ex vivo fluorescent assay. Measurable MMP-10 activity was detected in 42 of 50 specimens of lung cancer and only 2 of 10 specimens of histologically normal lung tissue. No relationship was observed between MMP-10 activity levels and clinicopathologic characteristics. Our results suggest that MMP-10 is expressed and active at high levels in human NSCLC compared to normal lung tissues, and, as such, is a potential target for the development of novel therapeutics for lung cancer treatment.     

UbcH10 overexpression in human lung carcinomas and its correlation with EGFR and p53 mutational status

IntroductionUbcH10 codes for the cancer related E2 Ubiquitin Conjugating Enzyme, an enzymatic molecule with a key role in the ubiquitin–proteasome pathway. Current studies have suggested a critical role of UbcH10 in a variety of malignancies, including human thyroid, breast, ovarian and colorectal carcinomas. The aim of this study has been to extend the analysis of UbcH10 expression to lung cancer. This neoplasia represents one of the leading cause of cancer mortality worldwide, and new tools for an accurate diagnosis/prognosis are needed.MethodsThe expression levels of UbcH10 were analysed in human non-small cell lung carcinoma (NSCLC) by quantitative RT-PCR and tissue microarray immunohistochemistry, and these values were correlated with the clinicopathological features of the patients affected by NSCLC.ResultsOur results demonstrate that UbcH10 is overexpressed in NSCLC compared to the normal lung tissue. Moreover, UbcH10 expression is significantly higher in squamous cell and large cell carcinomas than in adenocarcinomas, and directly and inversely correlated with the mutational status of p53 and EGFR, respectively. The suppression of UbcH10 expression by RNAi resulted in a drastic reduction of proliferation and migration abilities of lung carcinoma cell lines.ConclusionThese results, taken together, indicate that UbcH10 overexpression has a critical role in lung carcinogenesis, and the evaluation of UbcH10 expression levels may be a new tool for the characterisation of NSCLC.     

Overexpression of UbcH10 alternates the cell cycle profile and accelerate the tumor proliferation in colon cancer

Background  

UbcH10 participates in proper metaphase to anaphase transition, and abrogation of UbcH10 results in the premature separation of sister chromatids. To assess the potential role of UbcH10 in colon cancer progression, we analyzed the clinicopathological relevance of UbcH10 in colon cancer.     

UbcH10 expression may be a useful tool in the prognosis of ovarian carcinomas

The UbcH10 gene codes for a protein that belongs to the ubiquitin-conjugating enzyme family. Previous studies of our group suggest UbcH10 expression as a valid indicator of the proliferative and aggressive status of thyroid carcinomas. Therefore, to better understand the process of ovarian carcinogenesis, and to look for possible tools to be used as prognostic markers in these neoplasias, we decided to extend the analysis of the UbcH10 expression to the ovarian neoplastic disease. We found that the UbcH10 gene was upregulated in some ovarian carcinoma cell lines analysed. Then, immunohistochemical studies demonstrate that UbcH10 expression significantly correlates with the tumor grade and the undifferentiated histotype of the ovarian carcinomas. Furthermore, a significant relationship between UbcH10 expression and overall survival was observed. Finally, the block of UbcH10 protein synthesis by RNA interference inhibited the growth of ovarian carcinoma cell lines, suggesting a role of UbcH10 overexpression in ovarian carcinogenesis. Therefore, all these data taken together suggest the possibility to use UbcH10 detection as a marker for the diagnosis and prognosis of these neoplastic diseases and open the perspective of a therapy of some ovarian carcinomas based on the suppression of the UbcH10 synthesis and/or function.     

Overexpression and amplification of STK15 in human gliomas

The serine/threonine kinase 15 (STK15) at chromosome 20q13.2 is frequently shown to be amplified and overexpressed in several human cancers. STK15 has been reported to act as a cell cycle regulator and its overexpression induces centrosome amplification and aneuploidy. Recently we showed that STK15 even plays a role in human malignant brain tumours and we described an amplification of the gene in 31% of the investigated gliomas. In this study we scrutinized the correlation of increased STK15 on DNA and mRNA levels in gliomas of different histological grades. Southern blotting confirmed the amplification frequency of the STK15 gene, which had been previously detected by comparative PCR. In total, DNA gains were found in 26% of the investigated gliomas. Interestingly, we detected overexpression of STK15 mRNA in 60% of the analyzed brain tumours. The elevated expression does not strongly correlate with gains on DNA level, but all cases with an amplification of the STK15 gene display overexpression. Gains of the STK15 gene seem to occur irrespectively of the histological grades of the tumours, so that STK15 probably is not a progression associated factor. Amplification and overexpression of the kinase rather represent a primary alteration in human gliomas, which could play an important role as an early event in all glioma subtypes.     

Overexpression of the c-MET/HGF receptor gene in human thyroid carcinomas.

The receptor for Hepatocyte Growth Factor is a transmembrane tyrosine kinase encoded by the c-MET oncogene. We have previously shown that the Met protein is expressed in several human epithelial tissues. The receptor is barely detectable, however, in normal thyroids and in specimens from patients affected by non-neoplastic thyroid diseases. Now we report that the expression of the Met/HGF receptor is increased a hundred fold in 22 out of 41 human carcinomas derived from the thyroid follicular epithelium. A comprehensive analysis of 15 cases showed that the overexpressing carcinomas belong to histotype variants correlated with negative prognosis and in all but one case there were evidences of locally advanced disease and/or distant metastases. The 11 benign adenomas and the 5 medullary carcinomas tested were negative. Western blot analysis with monoclonal antibodies directed against either the intracellular or the extracellular receptor domains failed to reveal major structural alterations. Southern blot analysis also demonstrated that the c-MET gene was not amplified nor rearranged. These data suggest a role for the overexpression of c-MET oncogene in the pathogenesis and progression of thyroid tumors derived from the follicular epithelium.     

Overexpression of gastrin and c-met protein involved in human gastric carcinomas and intestinal metaplasia

Many studies have investigated the expression of c-met and c-erbB2 protein in human gastric adenocarcinomas, but the expression of gastrin protein in human gastric cancer and the relationship between gastrin and c-met are unknown. We have constructed a tissue microarray containing 408 formalin-fixed and paraffin-embedded human tissue blocks, including tissues containing intestinal metaplasia (IM, n=72) and primary tumors (n=232), as well as normal gastric mucosa (n=104) from patients with gastric cancer. Immunohistochemistry (IHC) was used for detecting gastrin, c-met and c-erbB2 proteins. Gastrin was detected in 13.5% (7/52) and c-met in 15.3% (11/72) of IM cases. In gastric carcinomas, 48.4% (103/213) of cases expressed gastrin, 68.8% (148/215) expressed c-met, and 5.5% (11/200) expressed c-erbB2. Gastrin and c-met protein expression were significantly higher in gastric tumor tissue than in IM (P<0.0001). Overexpression of c-erbB2 protein was detected in gastric carcinomas but not in normal gastric mucosa (P<0.05). Expression of gastrin and c-met protein was associated (P<0.01), but no significant difference was found on the changes of gastrin, c-met and c-erbB2 expression in gastric cancer with tumor stage, grade of differentiation or tumor type. These results indicate that gastrin and c-met play a role in the early process during malignant transformation of the gastric mucosa.     

Preliminary exploration of potential molecular therapeutic targets in recurrent and metastatic parathyroid carcinomas

Parathyroid carcinoma (PC) is a rare endocrine malignancy. Surgical resection is curative for local lesions, while effective therapies are lacking for recurrent or metastatic PCs. To study whether targeted therapies could be applied in recurrent or metastatic PCs, potential therapeutic targets were identified with next-generation sequencing (NGS). DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) sections from 19 recurrent or metastatic PC samples. A panel of 560 genes was sequenced with NGS to identify genomic alterations at an average sequencing depth of 581×. In total, 190 genomic alterations were identified. Nine PC samples (47%) harbored at least one potentially actionable genomic alteration including in the after genes: ROS1 (5/19; 26%), PTEN (3/19; 16%), TSC1 (2/19; 11%), PIK3CA (1/19; 5%), AKT1 (1/19; 5%), MTOR (1/19; 5%), ERBB2 (1/19; 5%), NTRK1 (1/19; 5%), IDH1 (1/19; 5%) and FGFR3 (1/19; 5%). CDC73 mutations were detected in 9/19 (47%) PC samples. Additional recurrent genomic alterations were identified in MSH2 (15/19; 79%), AR (9/19; 47%), BCR (8/19; 42%), SLC45A3 (6/19; 32%), MAGI1 (5/19; 26%), ZNF521 (4/19; 21%), KMT2C (4/19; 21%) and NOTCH4 (4/19; 21%). Our study identified a relatively high frequency of potentially actionable genomic alterations in PC patients in a Chinese population for the first time. A series of recurrent mutant genes was detected as well. Our study contributes to both the selection of novel targeted therapies for PC and further molecular understanding of this refractory malignancy.     

Overexpression, amplification, and androgen regulation of TPD52 in prostate cancer

Gains in the long arm of chromosome 8 (8q) are believed to be associated with poor outcome and the development of hormone-refractory prostate cancer. Based on a meta-analysis of gene expression microarray data from multiple prostate cancer studies (D. R. Rhodes et al., Cancer Res 2002;62:4427-33), a candidate oncogene, Tumor Protein D52 (TPD52), was identified in the 8q21 amplicon. TPD52 is a coiled-coil motif-bearing protein, potentially involved in vesicle trafficking. Both mRNA and protein levels of TPD52 were highly elevated in prostate cancer tissues. Array comparative genomic hybridization and amplification analysis using single nucleotide polymorphism arrays demonstrated increased DNA copy number in the region encompassing TPD52. Fluorescence in situ hybridization on tissue microarrays confirmed TPD52 amplification in prostate cancer epithelia. Furthermore, our studies suggest that TPD52 protein levels may be regulated by androgens, consistent with the presence of androgen response elements in the upstream promoter of TPD52. In summary, these findings suggest that dysregulation of TPD52 by genomic amplification and androgen induction may play a role in prostate cancer progression.     

Critical and diverse involvement of Akt/mammalian target of rapamycin signaling in human lung carcinomas

BACKGROUND: Aberrant signaling cascades emanating from epidermal growth factor receptor (EGFR) are involved in the complex network of oncogenic signaling in lung carcinomas. One representative cascade is the phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin (mTOR) pathway. METHODS: The authors investigated the involvement of mTOR in the pathobiologic profiles of 150 specimens of lung carcinoma by immunohistochemistry and immunoblotting in correlation with the upstream and downstream proteins Akt and p70S6‐kinase (S6K), respectively. RESULTS: Immunohistochemistry revealed Akt activation in 44% of tumors and mTOR expression in 68.7% of tumors, and the preponderance of activation was observed in adenocarcinoma (AC) (100%). Phosphorylated mTOR (p‐mTOR) was observed in 53.3% of tumors and had the highest frequency in AC (89.7%). In AC, the frequency of p‐mTOR staining was higher in the well differentiated subtype, in particular, in the acinar structure. However, little correlation was observed between the activation of mTOR and Akt, except in the 5 AC specimens that harbored an EGFR gene mutation, which exhibited constitutive activation of both Akt and mTOR. Conversely, in squamous cell carcinomas, mTOR activation was associated with a significantly higher frequency of lymph node metastasis. CONCLUSIONS: The results of this study suggested the dual functions of mTOR. First, mTOR may function not only in the proliferation of tumor cells as an effector molecule downstream of EGFR but also possibly in the morphogenesis of AC. Second, the activation of mTOR may play a key role in metastasis in squamous cell carcinoma. Overall, the current results demonstrated the potential for the application of rapamycin, an mTOR inhibitor, as an additional novel component of chemotherapy for a defined subset of patients with lung carcinoma. Cancer 2009. © 2008 American Cancer Society.     

Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer.     

The prognostic significance of amplification and overexpression of c-met and c-erb B-2 in human gastric carcinomas

BACKGROUND: The c-met and the c-erb B-2 protooncogenes belong to a family of tyrosine kinase growth factor receptors. Abnormalities of these oncogenes and protein products have been reported in several cancers. The authors investigated the correlation between clinical factors and amplification or overexpression of the c-met and/or c-erb B-2 gene in Japanese patients with gastric carcinoma patients, with a focus on prognostic significance. METHODS: Amplification and overexpression of c-met and c-erb B-2 were investigated retrospectively in 128 gastric carcinoma patients by using immunohistochemistry and Southern blot hybridization. Survival analysis was performed with the Kaplan-Meier test, and the log rank test was used for statistical analysis. RESULTS: Overexpression of c-met and c-erb B-2 was observed in 46.1% and 16.4% of gastric carcinoma cases, respectively. Gene amplification of c-met and c-erb B-2 was detected in 10.2% and 11.7% of gastric carcinoma cases, respectively. Amplification and overexpression of c-met were correlated significantly with depth of tumor invasion and lymph node metastasis, whereas amplification and overexpression of c-erb B-2 were correlated significantly with histologic type. The survival rate of patients with amplification and/or overexpression of c-met or c-erb B-2 was significantly poorer than that of patients with no amplification or overexpression. Multivariate analysis revealed that c-met overexpression and lymph node metastasis were independent prognostic factors. CONCLUSIONS: These data suggest that overexpression and/or gene amplification of c-met and c-erb B-2 may be prognostic factors in gastric carcinoma.     

Prevalence of human papillomavirus DNA sequences in verrucous carcinoma of the lip: genomic and therapeutic approaches

We examined the presence of human papillomavirus (HPV) DNA sequences in tissues of verrucous carcinoma (VC) of the lip. Detection and typing of HPV DNA was performed using polymerase chain reaction with sequence analysis and restriction fragment length polymorphism analysis. All tissues of VC contained HPV DNA and one of recurrent case was infected with four different HPV DNAs including high risk types. Seven different HPV types were detected in VC, of which (accession no. in EMBL/GeneBank/DDBJ databases) has been described as a partial sequences from an unknown HPV type. Sequence analysis showed that HPVX is related to HPV-20 (74.8% sequence homology). These results indicate that various mucosal and cutaneous HPVs of high risk types are associated with the one of pathogenesis of VC of the lip. In addition, the relationship between VC of the lip and HPV infection is discussed, as is therapy against VC.     

Comparison of chromosomal and array-based comparative genomic hybridization for the detection of genomic imbalances in primary prostate carcinomas

Background  

In order to gain new insights into the molecular mechanisms involved in prostate cancer, we performed array-based comparative genomic hybridization (aCGH) on a series of 46 primary prostate carcinomas using a 1 Mbp whole-genome coverage platform. As chromosomal comparative genomic hybridization (cCGH) data was available for these samples, we compared the sensitivity and overall concordance of the two methodologies, and used the combined information to infer the best of three different aCGH scoring approaches.     

Frequent gains and losses of specific chromosome segments in human ovarian carcinomas shown by comparative genomic hybridization

Total genomic DNA obtained from 24 ovarian carcinomas was examined for genomic imbalances by comparative genomic hybridization (CGH). A varying number of gains and losses (1 up to 31) of specific chromosomal segments was detected per tumor. Chromosomal segments which were most often present in increased copy numbers were (in decreasing order): 1q21, 8q24, 8q23, 3q26, 12p12-p13, 20q, 7q31, and 7q33-qter. Loss of material was found most frequently at 16q12, 13q13-q14, Xq, 8p21-p22, 5q13-q14, and 5q21. All these chromosomal segments involved in gains and losses may carry gene loci playing a more or less causal role in the process of ovarian malignancies. Based on these findings CGH can be regarded as a valuable tool for rapid screening of genomic imbalances in human tumors.     

Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target

Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.