Expression of metastasis suppressor gene product, nm23 protein, is not inversely correlated with the tumour progression in human malignant melanomas

An inverse correlation between the nm23 RNA level and tumour progression of melanocytes has been reported. To elucidate whether the expression of nm23 gene product in malignant melanoma is also inversely correlated with metastatic potential, conventional prognostic parameters or the tumour suppressor protein p53, immunohistochemical studies using a monoclonal antibody against nm23-H1 protein were performed on 138 benign and malignant melanocytic tumours. The expression of nm23 protein was compared with that of p53 protein and conventional clinicopathological prognostic factors. The nm23 protein level in benign melanocytes and metastatic melanoma cells was also studied by Western blot analysis. No significant difference regarding the protein was observed between naevi and melanomas, either at histological or protein levels. The expression correlated with local recurrence within 1 year after surgery, level of invasion and tumour thickness, but no parallels were observed between the nm23 and p53 proteins, suggesting that gene is regulated by independent mechanisms, although located on the same chromosome. There was no inverse correlation between the nm23 protein and melanoma metastasis which suggested that the nm23 protein does not appear to be lost during melanoma metastasis.     

Overexpression of UQCRC2 is correlated with tumor progression and poor prognosis in colorectal cancer

Ubiquinol-cytochrome c reductase complex core protein 2 (UQCRC2) is an important subunit of mitochondrial respiratory complex III. However, its role in tumorigenesis and tumor progression remains unknown, especially with regards to colorectal cancer (CRC). In this research, we measured the expression of UQCRC2 protein by immunohistochemistry assay in 89 paired paraffin-embedded tumor tissues and corresponding adjacent normal tissues from patients with colorectal adenocarcinoma and investigated possible correlations of UQCRC2 expression with clinicopathological parameters and prognosis. We found that UQCRC2 was significantly upregulated in CRC tissues compared with adjacent normal tissues, and immunohistochemical UQCRC2 status was correlated to the depth of invasion (T), lymph node metastasis (N), advanced TNM stage. Multivariate analysis indicated that UQCRC2 remained an independent prognostic factor for poorer overall survival. Furthermore, we determined the role of UQCRC2-knockdown in CRC cells (RKO and HCT116) using lentivirus-mediated small hairpin RNAs (shRNAs). The effects of UQCRC2 knockdown on CRC cells (RKO and HCT116) proliferation were analyzed by cell proliferation and colony formation assay, and cell cycle and apoptosis were assessed by flow cytometry. We found that silencing UQCRC2 suppressed cell proliferation and colony formation in RKO and HCT116 cells, led to a cell cycle arrest and induced cell apoptosis in vitro. These results provided novel insights into the potential role of UQCRC2 in the tumorigenesis and progression of CRC, and revealed that UQCRC2 may serve as a new prognostic and therapeutic target in CRC.     

MYC gene amplification is often acquired in lethal distant breast cancer metastases of unamplified primary tumors

In breast cancer, amplification of MYC is consistently observed in aggressive forms of disease and correlates with poor prognosis and distant metastases. However, to date, a systematic analysis of MYC amplification in metastatic breast cancers has not been reported. Specifically, whether the MYC amplification status may change in metastases in comparison to the corresponding primary breast tumor, and potential variability among different metastases within the same patient have also not been assessed. We generated single patient tissue microarrays consisting of both primary breast carcinomas and multiple matched systemic metastases from 15 patients through our previously described rapid autopsy program. In total, the 15 tissue microarrays contained 145 primary tumor spots and 778 spots derived from 180 different metastases. In addition, two separate tissue microarrays were constructed composed of 10 matched primary breast cancers and corresponding solitary metastases sampled not at autopsy but rather in routine surgical resections. These two tissue microarrays totaled 50 primary tumor spots and 86 metastatic tumor spots. For each case, hormone receptor status, HER2/neu, EGFR and CK5/6 expression were assessed, and the cases were characterized as luminal, basal-like or HER2 based on published criteria. Both fluorescence in situ hybridization and immunohistochemistry for MYC was performed on all cases. Of the 25 cases, 24 were evaluable. While 4 of 24 primary tumors (16%) demonstrated MYC amplification, an additional 6 (25% of total evaluable cases) acquired MYC amplification in their systemic metastases. Of note, there was remarkably little heterogeneity in MYC copy number among different metastases from the same patient. MYC immunoreactivity was increased in metastases relative to matched primaries in the surgical cohort, although there was no perfect correlation with MYC amplification. In conclusion, amplification of MYC is a frequent event in breast cancer, but occurs more frequently as a diffuse, acquired event in metastatic disease than in the corresponding primary. These observations underscore the importance of MYC in breast cancer progression/metastasis, as well as its relevance as a potential therapeutic target in otherwise incurable metastatic disease.     

Loss of raf-1 kinase inhibitor protein expression is associated with tumor progression and metastasis in colorectal cancer

Raf-1 kinase inhibitor protein (RKIP) is known as a critical down-regulator of the mitogen-activated protein kinase signaling pathway and a potential molecular determinant of malignant metastasis. The aim of this study was to determine the prognostic significance of RKIP expression in colorectal cancer (CRC).Immunohistochemical staining for RKIP was performed on a tissue microarray comprising 1,197 mismatch repair (MMR)-proficient and 141 MMRdeficient CRCs. The association of RKIP with clinicopathologic features was analyzed. Loss of cytoplasmic RKIP was associated with distant metastasis (P = .038), higher N stage (P = .032), vascular invasion (P = .01), and worse survival (P = .001) in the MMR-proficient group. In MMR-deficient CRCs, loss of cytoplasmic RKIP was associated with distant metastasis (P = .043) and independently predicted worse survival (P = .004). Methylation analysis of 28 cases showed that loss of RKIP expression is unlikely to be due to promoter methylation.Loss of RKIP expression is a marker of tumor progression and distant metastasis in MMR-proficient and MMR-deficient CRCs.     

Downregulation of GLTSCR2 expression is correlated with breast cancer progression

Glioma tumor-suppressor candidate region gene2 (GLTSCR2) is a recently identified nucleolus-localized protein participating in the regulation of cell cycle progression and apoptosis. Down-regulation of GLTSCR2 in several types of cancers and increased transforming activity in GLTSCR2-downregulated cancer cells indicated its tumor suppressive potential. The aim of this study was to evaluate GLTSCR2 expression in breast cancer and to investigate the question of whether reduced expression of GLTSCR2 may have any pathological significance in breast cancer development or progression. In this study, we performed quantitative RT-PCR and immunohistochemistry to evaluate the expression of GLTSCR2 and relevance with clinicopathological factors in the invasive ductal carcinoma (IDC). GLTSCR2 expression was reduced in 48% of IDC (n = 426) by a semi-quantitative scoring system using tissue microarray. GLTSCR2 mRNA was significantly reduced by 0.16 fold in 15 out of 17 (88%) cases of IDC. Reduction of GLTSCR2 was significantly correlated with increased histological grade (p < 0.005), increased tumor size (p < 0.001), axillary lymph node involvement (p < 0.001) and decreased disease free survival (p < 0.025). In addition, we show that upregulation of GLTSCR2 decreases the invasive potential of breast cancer cells. Taken together, our data suggest that GLTCR2 may play a role in the tumorigenesis, progression and biological behavior in breast cancer.     

CD10 inhibits cell motility but expression is associated with advanced stage disease in colorectal cancer

Introduction

CD10 is a cell membrane-bound endopeptidase which is expressed in normal small bowel but not in normal colon. It is aberrantly expressed in a small proportion of colorectal cancers (CRC) and this has been associated with liver metastasis and poor prognosis. We sought to investigate the mechanism of CD10 activity and its association with clinicopathological features.

Identification of secretory gelsolin as a plasma biomarker associated with distant organ metastasis of colorectal cancer

Colorectal cancer (CRC) is one of the most common cancers worldwide. More than half of all CRC patients will develop metastases, which represents the major cause of death for CRC patients. CRC metastases confined in other organs are potentially resectable, and patients who receive curative resections appear to have better outcomes. Thus, the early detection of metastasis in CRC patients could improve their survival rate after curative surgery. Here, we report the use of Cy-dye labeling combined with multi-dimensional fractionation and mass spectrometry as a proteomics-based approach for identifying CRC metastasis-associated biomarker(s) in plasma samples collected from three CRC patients upon diagnosis of their primary and metastatic tumors. Among the eight identified proteins, we used Western blot analysis and an in-house-developed ELISA to validate the increased plasma levels of one, secretory (plasma) gelsolin, in >80% of CRC patients with distal metastases in a larger sample cohort (32 patients). We also found a significant increase of secretory gelsolin in plasma samples of stage IV versus stages I-III CRC patients before treatment. Furthermore, immunohistochemistry showed that secretory gelsolin was highly overexpressed in CRC tissue specimens compared to adjacent normal tissues, and a cell model study showed that secretory gelsolin may help regulate CRC cell migration.     

MDM2 expression is associated with progress of disease and WAF1 expression in resected lung cancer

Although MDM2, p21/WAF1, and p53 are considered as regulating each other based on in vitro studies, the relation in human lung cancer is not fully understood. The expressions of these proteins were examined immunohistochemically in 112 resected non-small cell lung cancer specimens and the correlation between them were analyzed. MDM2 was expressed in 45% of all lung cancers. In advanced stage, MDM2-positive cases were observed more frequently than in early stage, showing significant difference. No significant difference was observed in the prognosis of the patients regardless of the expression of any protein. Although no correlation was observed between MDM2 expression and p53 expression, or between p21/WAF1 expression and p53 expression, MDM2 expression was strongly related with p21/WAF1 expression. Therefore, MDM2 expression may relate to the progress of the stage of lung cancer, and MDM2 expression and p21/WAF1 expression may be associated not through the p53-related pathway.     

Inactivation of the PTEN gene protein product is associated with the invasiveness and metastasis, but not angiogenesis, of breast cancer

PTEN is a novel tumor-suppressor gene located on chromosomal band 10q23. Loss of PTEN function has been implicated in the progression of several types of cancer, but the correlation between loss of PTEN expression and advanced carcinomas is not well established. The capacity for angiogenesis of a tumor is known to play a very important role in growth and metastasis, and there have been reports that PTEN relates to angiogenesis. In the present study, formalin-fixed and paraffin embedded tissues from 101 patients with breast carcinomas, including 88 cases of invasive ductal carcinomas and 13 cases of ductal carcinoma in situ (DCIS), were evaluated by immunohistochemical methods for the expression of PTEN and vascular endothelial growth factor (VEGF), as well as microvessel density (MVD). The results were compared with the clinicopathologic parameters. There was no loss of PTEN expression in any of the cases of DCIS, but 28 (32%) of the 88 invasive cases did not express PTEN. Loss of PTEN expression was associated with lymph node metastasis ( P  = 0.03), but did not correlate with tumor size, tumor grade, MVD or recurrence. VEGF expression significantly correlated with lymph node metastasis in invasive ductal carcinoma ( P  = 0.01). There was no correlation between the expression of PTEN and that of VEGF ( P  = 0.63). The present study suggests that loss of PTEN expression is common and correlates with tumor progression and lymph node metastasis in breast carcinoma. The relationship between loss of PTEN and progression of breast cancer may not be explained by modulation of angiogenesis.     

KRAS gene mutation in colorectal cancer is correlated with increased proliferation and spontaneous apoptosis

KRAS mutation occurs in 30% to 50% of colorectal cancers (CRCs) and has been suggested to be associated with proliferation and decreased apoptosis. In this study, we analyzed KRAS in 198 CRCs and compared the clinicopathologic variables between KRAS-mutated and wild-type CRCs. Also, a subset of 90 and 66 CRCs from this cohort underwent microsatellite instability testing and histomorphologic evaluation, and the frequency of microsatellite instability-high (MSI-H) and histomorphologic variables were compared between KRAS-mutated and wild-type CRCs. Clinicopathologic features (age, sex, and tumor site, depth, size, grade, and metastasis) were not different between KRAS-mutated and wild-type CRCs. Compared with wild-type KRAS CRCs, KRAS-mutated CRCs had a lower frequency of MSI-H (15% vs 42%; P = .015), a higher chance of having brisk mitosis (77% vs 43%, P = .022) and apoptosis (77% vs 28%; P = .00012), and a greater mean of mitotic figures (P = .0002) and apoptotic cells (P = .0008). KRAS mutation was associated with higher tumor cell turnover.     

MDM2 gene amplification correlates with ring chromosomes in soft tissue tumors

The human homolog of the murine double minute type 2 gene (MDM2) has been cloned and mapped to 12q 13-14. The gene presumably functions as a cellular regulator and mediator of TP53 function. Amplification of the MDM2 gene has recently been observed in soft tissue sarcoma and in osteosarcoma. We studied MDM2 amplification in a series of 94 mesenchymal tumors and found 3-20-fold amplification in 20 tumors: in 10 of 49 malignant fibrous histiocytomas (MFH), in 1 of 2 pleomorphic liposarcomas, in 6 of 7 atypical lipomas, and in 3 of 12 typical lipomas. Normal hybridization patterns were detected in all 16 myxoid liposarcomas, in all 3 leiomyosarcomas, and in all 5 leiomyomas studied. The MDM2 amplification correlated with the presence of marker ring chromosomes; of the 10 MFH with MDM2 amplification, 5 had ring chromosomes, compared to 4 of 39 without MDM2 amplification, and all 9 lipomas with MDM2 amplification had ring chromosomes, in 5 of the tumors as the sole karyotypic anomaly. The correlation between ring chromosomes and MDM2 gene amplification indicates that the marker rings of MFH and of atypical lipoma often harbor genetic material derived from chromosome 12. Genes Chrom Cancer 9:26 1-265 (1994). © 1994 Wiley-Liss, Inc.     

Down‐regulated expression of SATB2 is associated with metastasis and poor prognosis in colorectal cancer

To identify novel biomarkers of metastasis of colorectal cancer (CRC), we developed an orthotopic implantation model of murine CRC and selected in vivo M5, a subclone of the SW480 CRC cell line with enhanced potential for metastasis to the liver. We compared the differences in the gene expression profiles between M5 and SW480 cells using gene expression profiling. We found that expression of special AT‐rich sequence‐binding protein 2 (SATB2) was down‐regulated in M5 cells. Immunohistochemical analysis of 146 colorectal tumour samples showed that underexpression of SATB2 was strongly correlated with poor prognosis, tumour invasion, lymph node metastasis, distant metastasis, and Dukes' classification for CRC. Univariate and multivariate survival analyses further showed that SATB2 expression was a potential favourable prognostic factor for CRC. These results demonstrated not only that SATB2 is a potential novel prognostic factor for CRC, but also that selection of a highly metastatic clone of SW480 in vivo coupled with gene expression profiling is a powerful approach to identifying prognostic markers for CRC. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Acinar morphology in colorectal cancer is associated with survival but is not an independent prognostic variable

AIMS: Microacinar morphology has been reported as a stage-independent prognostic variable for colorectal cancer. We have undertaken a retrospective analysis to test this observation in patients with colonic as well as rectal adenocarcinoma, and to assess interobserver agreement for identifying microacinar morphology. METHODS AND RESULTS: One hundred and eighty patients with colorectal cancer, 144 colonic and 36 rectal, were assigned to either macroacinar (144) or microacinar (36) groups. There was excellent correlation between two observers examining the colorectal cancers independently (kappa statistic, kappa = 0.87). Patients with microacinar tumours had a significantly reduced median survival compared with macroacinar lesions (46 and 87 months, respectively, P = 0.022) and this morphology was significantly associated with higher Dukes' stage (P = 0.007). Microacinar morphology lacked statistical significance when examined in a multivariate analysis with other prognostic variables, both when colonic and rectal tumours were examined either separately or as a combined colorectal group, but small numbers of rectal tumours in this study make it difficult to draw useful conclusions about microacinar morphology in rectal adenocarcinoma. CONCLUSIONS: Microacinar morphology was strongly associated with poor prognosis when examined in isolation but was not found to be an independent factor when examined with other prognostic variables.     

Hyaluronan synthase 2 overexpression is correlated with the tumorigenesis and metastasis of human breast cancer

Extracellular matrix (ECM) is closely correlated with the malignant behavior of breast cancer cells. Hyaluronan (HA) is one of the main components of ECM, and actively regulates cell adhesion, migration and proliferation by interacting with specific cell surface receptors such as CD44 and RHAMM. HA synthase 2 (HAS2) catalyzes the sysnthesis of HA, but its role in breast tumorigenesis remains unclear. This study assessed the roles of HAS2 in malignant behavior of human breast cancer and sought to provide mechanistic insights into the biological and pivotal roles of HAS2. We observed HAS2 was overexpressed in breast cancer cell lines and invasive duct cancer tissues, compared with the nonmalignant breast cell lines and normal breast tissues. In addition, a high level of HAS2 expression was statistically correlated with lymph node metastasis. Functional assays showed that knockdown of HAS2 expression inhibited breast tumor cell proliferation in vivo and in vitro, through the induction of apoptosis or cell cycle arrest. Further studies showed that the HA were elevated in breast cancer, and HAS2 could upregulate HA expression. In conclusion, HAS2-HA system influences the biological characteristics of human breast cancer cells, and HAS2 may be a potential prognostic marker and therapeutic target in breast cancer.     

VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis

Angiogenesis is essential for tumour growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF-B, a new VEGF family member that binds to the tyrosine kinase receptor flt-1, has been identified. Although the importance of VEGF has been shown in many human tumour types, the contribution of VEGF-B to tumour neovascularization is unknown in any tumour type. This study therefore measured the mRNA level of VEGF-B and its receptor flt-1 by ribonuclease protection assay and the pattern of VEGF-B expression by immunohistochemistry in 13 normal breast samples and 68 invasive breast cancers. Flt-1 expression was significantly higher in tumours than in normal breast (p=0.02) but no significant difference was seen in VEGF-B between normal and neoplastic breast (p=0.3). There was a significant association between VEGF-B and node status (p=0.02) and the number of involved nodes (p=0.01), but not with age (p=0.7), size (p=0.6), oestrogen receptor (ER) (p=0.2), grade (p=0.5) or vascular invasion (p=0.16). No significant relationship was present between VEGF-B and flt-1 (p=0.2) or tumour vascularity (p=0.4). VEGF-B was expressed mostly in the cytoplasm of tumour cells, although occasional stromal components including fibroblasts and endothelial cells were also positive. No difference in VEGF-B expression was observed adjacent to regions of necrosis, in keeping with this VEGF family member not being hypoxically regulated. These findings suggest that VEGF-B may contribute to tumour progression by a non-angiogenic mechanism, possibly by increasing plasminogen activators and hence metastasis, as has been described in vitro. Measurement of VEGF-B together with other angiogenic factors may identify a poor prognostic patient group, which may benefit from anti-VEGF receptor therapy targeted to flt-1 (VEGFR1) as well as kdr (VEGFR2).