Runx2, a bone-specific transcriptional regulator, is abnormally expressed in highly metastatic prostate cancer cells. Here, we identified the functional activities of Runx2 in facilitating tumor ...growth and osteolysis. Our studies show that negligible Runx2 is found in normal prostate epithelial and non-metastatic LNCaP prostate cancer cells. In the intra-tibial metastasis model, high Runx2 levels are associated with development of large tumors, increased expression of metastasis-related genes (MMP9, MMP13, VEGF, Osteopontin) and secreted bone-resorbing factors (PTHrP, IL8) promoting osteolytic disease. Runx2 siRNA treatment of PC3 cells decreased cell migration and invasion through Matrigel in vitro, and in vivo shRunx2 expression in PC3 cells blocked their ability to survive in the bone microenvironment. Mechanisms of Runx2 function were identified in co-culture studies showing that PC3 cells promote osteoclastogenesis and inhibit osteoblast activity. The clinical significance of these findings is supported by human tissue microarray studies of prostate tumors at stages of cancer progression, in which Runx2 is expressed in both adenocarcinomas and metastatic tumors. Together these findings indicate that Runx2 is a key regulator of events associated with prostate cancer metastatic bone disease.
Pancreatic cancer is one of the most aggressive and lethal types of cancer, and more effective therapeutic agents are urgently needed. Overexpressed cell surface antigens are ideal targets for ...therapy with monoclonal antibody (mAb)-based drugs, but none have been approved for the treatment of pancreatic cancer. Here, we report development of two novel mouse mAbs, KU42.33C and KU43.13A, against the human pancreatic cancer cell line BxPC-3. Using ELISA, flow cytometry, competitive assay and immunoprecipitation followed by mass spectrometry, we discovered that these two mAbs target two distinct epitopes on the external domain of CD109 that are overexpressed by varying amounts in human pancreatic cancer cell lines. Treatment with these two naked antibodies alone did not affect tumour cell growth or migration
. Of the two mAbs, only KU42.33C was useful in determining the expression of CD109 in tumour cells by Western blot and immunohistochemistry. Interestingly, immunohistochemistry of human pancreatic carcinoma tissue arrays with mAb KU42.33C showed that 94% of the 65 human pancreatic adenocarcinoma cases were CD109 positive, with no expression in normal pancreatic tissues. Our results suggest that these two novel mAbs are excellent tools for determining the expression level of CD109 in the tumour specimens and sera of patients with a wide range of cancers, in particular pancreatic cancer, and for investigating its diagnostic, prognostic and predictive value. Further research is warranted and should aim to unravel the therapeutic potential of the humanised forms or conjugated versions of such antibodies in patients whose tumours overexpress CD109 antigen.
Toll-like receptors (TLRs) have raised an extraordinary interest in cancer research due to their role in tumor progression. By activating the production of several biological factors, TLRs drive an ...inflammatory response and activate the adaptive immune system. The aim of this study was to investigate the expression and clinical relevance of TLR3, TLR4, and TLR9 in gastric cancer. For this purpose, an immunohistochemical study on cancer specimens from 106 patients with gastric cancer was performed using tissue arrays and specific antibodies against TLR3, TLR4, and TLR9. The results indicate that gastric carcinomas samples show high expression of TLR3, TLR4, and TLR9 by cancer cells. The expression of TLR3 by cancer cells was significantly associated with a poor overall survival in patients with resectable tumors. Moreover, in patients with resectable tumors and lymph node invasion, a high TLR3 expression defines a population with even worse prognosis. Therefore, TLR3 may have clinical interest as indicator of tumor aggressiveness and as a prognostic indicator in gastric cancer.
Purpose: Inflammatory breast cancer (IBC) is associated with very poor prognosis. The aims of this study are ( a ) to prospectively identify differential gene expression patterns associated with IBC ...and ( b ) to confirm these pathways using tissue arrays.
Experimental Design: For gene expression analysis, IBC ( n = 14) was clinically defined as rapid-onset cancer associated with erythema and skin changes, whereas non-IBC patients ( n = 20) had stage III breast cancers, and cDNA analysis was carried out using the Affymetrix (Santa Clara, CA) HG-U133A microarrays.
Tissue arrays were constructed from paraffin-embedded material, and the molecular phenotype of 75 IBC was compared with results
from >2,000 non-IBC.
Results: Gene expression analyses indicated that IBC has higher expression of genes associated with increased metabolic rate, lipid
signaling, and cell turnover relative to non-IBC tumors. Consistent with the expression analysis, IBC had statistically higher
Ki-67 (93% versus 11%; P < 0.001). BAX expression, reflecting increased apoptosis and cell turnover, was significantly uniformly higher in almost
all IBC (98% versus 66%; P < 0.05), whereas the expression of Bcl-2 was not significantly different. IBC tumors were more likely to be steroid hormone
receptor negative (estrogen receptor, 49% versus 30%; P = 0.002; progesterone receptor, 68% versus 42%; P = 0.001). The expression of tyrosine kinases was not significantly different. E-cadherin was found to be expressed in 87%
of IBC, whereas the expression p53 was not significantly different.
Conclusion: This study is one of the largest molecular analyses of IBC. Both IBC and non-IBC are genetically heterogeneous with consistent
differences in the molecular phenotype of IBC.
Amplification of the 11q13 locus is commonly observed in a number of human cancers including both breast and ovarian cancer.
Cyclin D1 and
EMS1 have been implicated as candidate oncogenes involved in ...the emergence of amplification at this locus. Detailed analysis of the 11q13 amplicon in breast cancer led to the discovery of four regions of amplification suggesting the involvement of other genes. Here, we investigate the role of EMSY, a recently described BRCA2 interacting protein, as a key element of the 11q13 amplicon in ovarian cancer.
EMSY maps to 11q13.5 and is amplified in 13% of breast and 17% of ovarian carcinomas.
EMSY amplification was assessed by fluorescent in-situ hybridization (FISH) in 674 ovarian cancers in a tissue microarray and correlated with histopathological subtype and tumor grade. A detailed map of the 11q13 amplicon in 51 cases of ovarian cancer was obtained using cDNA-array-based comparative genomic hybridization (aCGH). To further characterize the role of
EMSY within this amplicon, we evaluated both the amplification profiles and RNA expression levels of
EMSY and two other genes from the 11q13 amplicon in an additional series of 22 ovarian carcinomas.
EMSY amplification was seen in 52/285 (18%) high grade papillary serous carcinomas, 4/27 (15%) high grade endometrioid carcinomas, 3/38 (8%) clear cell carcinomas, and 3/10 (30%) undifferentiated carcinomas. aCGH mapping of 11q13 in ovarian cancer showed that
EMSY localized to the region with the highest frequency of copy number gain.
Cyclin D1 and
EMS1 showed a lower frequency of copy number gain. A highly significant correlation between
EMSY gene amplification and RNA expression was also observed (
P = 0.0001). This was a stronger correlation than for other genes at 11q13 including
Cyclin D1 and
PAK1.
These findings support the role of
EMSY as a key oncogene within the 11q13 amplicon in ovarian cancer.
Array-CGH represents a comprehensive tool to discover genomic disease alterations that could potentially be applied to body fluids. In this report, we aimed at applying array-CGH to urinary samples ...to characterize bladder cancer.
Urinary DNA from bladder cancer patients and controls were hybridized on 44K oligonucleotide arrays. Validation analyses of identified regions and candidates included fluorescent in situ hybridization (FISH) and immunohistochemistry in an independent set of bladder tumors spotted on custom-made tissue arrays (n = 181).
Quality control of array-CGH provided high reproducibility in dilution experiments and when comparing reference pools. The most frequent genomic alterations (minimal recurrent regions) among bladder cancer urinary specimens included gains at 1q and 5p, and losses at 10p and 11p. Supervised hierarchical clustering identified the gain at 1q23.3-q24.1 significantly correlated to stage (p = 0.011), and grade (p = 0.002). The amplification and overexpression of Prefoldin (PFND2), a selected candidate mapping to 1q23.3-q24.1, correlated to increasing stage and tumor grade by means of custom-designed and optimized FISH (p = 0.013 and p = 0.023, respectively), and immunohistochemistry (p ≤0.0005 and p = 0.011, respectively), in an independent set of bladder tumors included in tissue arrays. Moreover, PFND2 overexpression was significantly associated with poor disease-specific survival (p ≤0.0005). PFND2 was amplified and overexpressed in bladder tumors belonging to patients providing urinary specimens where 1q23.3q24.1 amplification was detected by array-CGH.
Genomic profiles of urinary DNA mirrowed bladder tumors. Molecular profiling of urinary DNA using array-CGH contributed to further characterize genomic alterations involved in bladder cancer progression. PFND2 was identified as a tumor stratification and clinical outcome prognostic biomarker for bladder cancer patients.
Pancreatic ductal adenocarcinoma is characterized by a paucity of neoplastic cells embedded in a densely desmoplastic stroma. Therefore, laser capture microdissection was performed to obtain ...homogeneous populations of normal and neoplastic ductal cells. These were subjected to a comparative study of gene expression utilizing human cDNA arrays. A variety of dysregulated genes were identified, comprising cell cycle and growth regulators, invasion regulators, signalling and developmental molecules. In addition to genes already found to be overexpressed in pancreatic cancer, such as TIMP1, MMP7, CD59, rhoC and NDKA, we present evidence to implicate genes which have not previously been reported in this tumour type. These include the overexpressed genes ABL2, Notch4 and SOD1, as well as XRCC1, a DNA repair gene whose transcript was found downregulated. Quantitative real-time RT-PCR (QRT-PCR) was employed to confirm differential expression of ABL2, Notch4 and SOD1 and immunohistochemical analysis was used to verify decreased protein expression of XRCC1 using a custom-built pancreatic tissue array. Combining microarray-derived gene expression profiles of pure pancreatic cell populations, QRT-PCR and pancreas-specific tissue arrays therefore proved to be highly informative in elucidating the molecular pathology of this highly malignant tumour type.
Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial–mesenchymal transition in development and tumor metastasis. Due to the lack of ...human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development.
Summary We assessed differences in the patterns of expression of matrix metalloproteases and their inhibitors (tissue inhibitors of metalloproteases) in ductal carcinoma in situ alone and admixed ...with invasive ductal carcinomas (n = 40), as well as in pure invasive ductal carcinomas (n = 40), immunohistochemically and using tissue arrays. The invasive ductal carcinoma components showed higher expression of matrix metalloprotease–9 and –13 than did the admixed ductal carcinoma in situ, whereas stromal fibroblasts of the invasive components showed higher expression of matrix metalloprotease–2, –7, –9, –13, and –14 and tissue inhibitor of metalloprotease–1 and –3 than did fibroblasts around the neoplastic ducts of the admixed ductal carcinoma in situ. Expression of matrix metalloprotease–14 and tissue inhibitor of metalloprotease–3 was significantly higher in the mononuclear inflammatory cells of the invasive components. By contrast, matrix metalloprotease–1 expression was significantly higher in stromal cells of the ductal carcinoma in situ admixed with invasive ductal carcinoma. The pure invasive ductal carcinomas had significantly higher expression of matrix metalloprotease–1, –9, –11, and –14 and tissue inhibitor of metalloprotease–1 and –3 than the invasive ductal carcinomas admixed with ductal carcinoma in situ. Our findings indicate a significant association of matrix metalloprotease expression by the periductal stromal cells of the ductal carcinoma in situ component of mixed tumors and the occurrence of distant metastasis. Our data suggest that the molecular matrix metalloprotease/tissue inhibitor of metalloprotease profile can contribute to better characterization of early breast carcinomas.
Semiquantitative immunohistochemical assessment of estrogen receptor (ER) is used to predict the likelihood of response to antiestrogen therapy in breast carcinoma. If semiquantitative ...immunohistochemical analysis leads to therapeutic decisions, the importance of standardization and quality control increases. ER assessment reproducibility was studied among 172 laboratories using tissue microarray slides with 20 tissue spots negative and 10 tissue spots expressing ER at low, medium, or high levels. More than 80% of the laboratories demonstrated ER positivity in the medium- and high-expressing tissue spots, but only about 43% succeeded with tissue spots with low expression. Poor interlaboratory agreement was based on insufficient retrieval efficacy as shown by additional tests using autoclave pretreatment. The immunohistochemical scores used to quantify therapeutic target molecules remain inconclusive as long as progress toward standardized immunohistochemical procedures and evaluation is not achieved. Tissue microarray technology has proved its suitability for large-scale immunohistochemical trials, giving rise to new dimensions in control assessment.
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