A switch from stromal to tumor cell expression of stromelysin-1 mRNA associated with the conversion of squamous to spindle carcinomas during mouse skin tumor progression

We previously reported that the expression of stromelysin-1 (ST-1), a matrix-degrading metalloproteinase, correlates with tumor progression in the mouse skin model of carcinogenesis. Using in situ hybridization techniques, we confirmed in this study the expression of ST-1 mRNA in mouse skin keratinocytes treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and also observed dramatic expression of ST-1 message in underlying fibroblastic cells. Benign tumors formed by an initiation/promotion protocol expressed low levels of ST-1 mRNA, which was localized exclusively to stromal tissue surrounding the tumor cells. Squamous cell carcinomas, produced either by chemical carcinogenesis or by injection of cultured cells derived from chemically initiated squamous cell tumors, expressed high levels of ST-1 mRNA, which was also localized to adjacent stromal tissues. In contrast, aggressive, highly metastatic spindle cell tumors expressed ST-1 mRNA in the tumor cells as well as in normal, adjacent stroma. These results suggest that the change from ST-1 expression in surrounding stromal cells to its expression in the tumor cells themselves is associated with the conversion of squamous to spindle carcinomas and may play a causal role in the ability of these cells to invade and metastasize. © 1994 Wiley-Liss, Inc.     

H-ras mutation is an additional event of sarcomatous transformation in aerodigestive spindle cell carcinoma

The conversion from a carcinomatous component to a sarcomatous one in spindle cell carcinoma (SPCC) of the upper aerodigestive tract is thought to occur via a series of molecular alterations; however the detailed mechanism is still unknown. We examined mutations at the H-ras and p53 genes in 16 SPCCs of upper aerodigestive tracts using PCR-RFLP, PCR-SSCP and direct sequencing analysis. The two distinct components, sarcomatous and carcinomatous components in SPCC, were analyzed independently. p53 mutations were detected in both components of SPCC (50.0%, 8/16), and those in the sarcomatous component were completely in accordance with those in the carcinomatous one. In contrast, H-ras mutations were detected only in the sarcomatous component (12.5%, 2/16), and not in the carcinomatous one (0%, 0/16). There was a statistically significant difference in prognosis between the patients with the H-ras mutation (n=2) and those without (n=14); the former had poorer prognosis (P=0.0049). Our results seem to suggest that the H-ras mutation is a relatively uncommon event in SPCC; however, the presence of H-ras mutations may be associated with a more malignant potential in SPCC, while actually occurring during the sarcomatous change itself.     

Mechanism of H-ras oncogene activation in mouse squamous carcinoma induced by an alkylating agent

A mouse skin squamous cell carcinoma induced by topical application of the direct-acting alkylating agent beta-propiolactone contains an activated H-ras oncogene with an A----T transversion at the second nucleotide of codon 61. The mutation was detected in NIH3T3 transfectant and original tumor DNA by an XbaI restriction enzyme polymorphism and confirmed by oligonucleotide "mismatch" hybridization. The mutation was not seen in the liver of the same animal. The activated oncogene also exhibited several restriction enzyme polymorphisms in transfectant DNA due to a reciprocal translocation 3' to the coding region of the gene, which occurred during transfection. The activating mutation was found in only 1 of 6 beta-propiolactone induced mouse skin tumors examined, the only tumor with a transforming H-ras oncogene. This is a much lower frequency of activation than that previously reported for the same tumor type induced by polycyclic aromatic hydrocarbons. The A----T transversion mutation is consistent with a potentially direct mutagenic effect of a specific beta-propiolactone-DNA adduct.     

Sirt7在宫颈鳞状细胞癌组织中的表达及意义

目的:作为七个Sirtuin(SIRT,又称抗衰老酶)家族成员(Sirt1-Sirt7)之一,Sirt7已被发现在很多肿瘤中异常表达,但其在宫颈癌中的表达仍不清楚.本研究中重点探索Sirt7在宫颈鳞状细胞癌中的表达以及其在宫颈癌发生、发展及转移方面的临床意义.方法:本研究共纳入手术切除宫颈癌组织石蜡标本113例,采用免疫组化(SP)法检测宫颈癌标本39例,宫颈上皮内瘤变标本62例和正常宫颈组织12 例中Sirt7的表达情况,并分析Sirt7与宫颈癌临床病理之间的关系.结果:Sirt7在正常宫颈、宫颈鳞状上皮内瘤变(低度)、宫颈鳞状上皮内瘤变(高度)及宫颈鳞癌组织中的阳性表达率依次上升,分别为 8.3%,13.1%,41.7% 及69.2%(P<0.05).另外,Sirt7的表达情况与宫颈鳞癌的组织分化程度相关(P<0.05),具有统计学意义,而与临床分期、淋巴结转移及患者年龄无关(P>0.05).结论:Sirt7可能在宫颈鳞癌的恶性进展中发挥作用,有可能是宫颈鳞癌治疗的一个潜在靶点.     

Frequent somatic imbalance of marker alleles for chromosome 1 in human primary breast carcinoma.

Loss of heterozygosity at particular chromosomal loci in the tumor cell, as evidenced by restriction fragment length polymorphism analysis, has been taken as a hallmark of the presence of tumor suppressor genes. Recent studies of breast carcinoma have suggested that such genes might be located on the short as well as on the long arm of chromosome 1. We report here that comparison of constitutional and tumor genotypes of 84 breast tumors at 7 polymorphic chromosome 1 loci indicates a frequent imbalance of alleles on both 1p (12 of 61 informative patients) and 1q (37 of 71 informative patients). In about one-half of these cases, however, this imbalance was consistent with a gain in copy number of one allele in tumor DNA relative to normal DNA, rather than loss of the other. In 10 tumors we performed chromosome 1 enumeration in the interphase nucleus using in situ hybridization with a probe detecting the heterochromatin region at 1q12. These experiments confirmed the supernumerary presence of region 1q12 in those tumors showing an allelic copy number gain of 1q. We suggest that there are several genes on chromosome 1 serving as targets for these changes, some of them associated with breast cancer development through their deletion and others through an increase in copy number.     

Metastasis is an early event in mouse mammary carcinomas and is associated with cells bearing stem cell markers

Introduction  

It is still uncertain whether metastasis is predominantly an early or late event in tumor progression. The detection of early metastases and cells responsible for the dissemination may therefore have significant clinical implications.     

Allelic imbalance on the long arm of chromosome 5 in oral squamous cell carcinoma

To obtain a more detailed estimate of chromosome 5 loci involvement in oral squamous cell carcinoma (SCC), thirty-two oral SCCs were examined for loss of heterozygosity (LOH) on the long arm of chromosome 5 (5q) by PCR-LOH assay using thirteen microsatellite markers. LOH was observed in 16 (51.6%) of 31 informative cases. The incidence or the number of regions showing LOH was significantly higher in moderately and poorly differentiated tumors than in the well differentiated ones. Among the loci tested, D5S178 exhibited LOH in 11 (39.3%) of 28 cases, suggesting this to be the site, at 5q, of a novel candidate tumor suppressor gene. These data indicate that the incidence of LOH at chromosome 5q is high and is associated with oral tumor differentiation.     

N-Nitrosocimetidine as an initiator of murine skin tumors with associated H-ras oncogene activation

N-Nitrosocimetidine (NCM) is a derivative of the drug cimetidine,a methylguanidine derivative used in the treatment of pepticulcer, and is known to be inactive as a complete mouse skincarcinogen, even when given in repeated high doses for a longperiod. In the crurrent experiment, NCM was tested for its abilityto initiate skin tumors on Sencar mice. It was applied at dosesof 1 or 0.3 mg, 5 times/week for 6 weeks, followed by the tumorpromoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 1 µg,2 times/week for 50 weeks. Controls received acetone. The higherNCM dose had significant effects on TPA-promotable tumors, resultingin shortened time to first tumor, increased incidence of alltumors (2-fold) and of mafignant tumors (4-fold), and greatertumor growth rate (2-fold), compared with the acetone/TPA-treatedmice. The mice given the lower NCM dose did not exhibit increasedtumor incidence, but their tumors had a significantly. highergrowth rate (3-fold) than those of the TPA controls. NCM withoutTPA treatment caused no tumors. Thus, NCM is a definitive, thoughweak initiator of TPA-promotable tumors. Nine tumors from theNCM-treatment groups were adyzed for activated oncogenes bythe NIH 3T3 cell transfection assay. Five were positive andfour of these were found by selective oligonucleotide hybridizationanalysis to have an A to T transversion in the second podtionof codon 61 of the H-ras oncogene. One of two tumors from theacetone/TPA group also contained transforming DNA and demonstratedthis mutation. None of the tumors had a G-A transition mutationat the second position of codon 12 of this oncogene. Tumor initiationby NCM may then be associated with the same oncogene mutationreported for mouse skin tumors initiated by other types of carcinogens,although occurrence of the mutated oncogene in TPA controlsprecludes a definitive conclusion.     

Lymph node metastasis is associated with allelic loss on chromosome 13q12-13 in esophageal squamous cell carcinoma.

Allelotype analysis of whole chromosomes showed that loss of heterozygosity (LOH) on 13q was exclusively associated with lymph node metastasis and poor prognosis in esophageal squamous cell carcinoma (ESC). To identify a locus responsible for lymph node metastasis, we performed fine deletion mapping on 13q by analyzing 60 ESCs with 18 polymorphic markers. Allelic loss was observed with at least one marker in 34 tumors (56.7%), and lymph node metastasis was significantly correlated with LOH (P = 0.0053). We found frequent loss at D13S260 (43.7%), D13S171 (38.6%), and D13S267 (43.6%) on 13q12-13. Among these markers, LOH at D13S171 showed a significant correlation with lymph node metastasis (P = 0.0441). Because these markers flank the BRCA2 gene, we intensively examined a mutation of the gene through all coding exons and exon-intron junctions by PCR-single-strand conformational polymorphism analysis under two different assays. We found only seven nucleotide substitutions as normal polymorphic changes; tumor-specific mutations were not detected, and loss of expression was not observed, indicating that the BRCA2 gene might not be a target of allelic loss in this region. Relatively frequent LOH was also found at the RB1 locus (34.7%), but a significant correlation with lymph node metastasis was not observed (P = 0.7430). Protein expression of RB1 was examined in 31 ESC cell lines, and loss of expression was infrequent (6.5%), indicating that inactivation of the RB1 gene might not be responsible for lymph node metastasis. Taken together, allelic loss at 13q12-13 of the primary ESC was closely associated with lymph node metastasis, and unidentified tumor suppressor gene(s) in this region might be involved.     

Allelic loss on chromosome 1 is associated with tumor progression of cervical carcinoma.

BACKGROUND: Alterations in chromosome 1 are common in human malignancies. The frequency of loss of heterozygosity (LOH) on chromosome 1 in cervical carcinoma and its clinical significance are not clearly understood. METHODS: LOH on chromosome 1 was studied in 100 cervical carcinomas by the polymerase chain reaction (PCR) using 29 highly polymorphic microsatellite markers spaced approximately 10 centimorgans apart. Loci with high frequencies of LOH were identified and the findings were correlated with clinicopathologic characteristics. RESULTS: LOH on chromosome 1 at 1 or more loci was detected in 93% of tumors. The frequencies of LOH at locus D1S2829 (1p31), D1S2663 (1p36.3), and D1S2725 (1q25) exceeded 30%, and 12 other loci exhibited frequencies of LOH of 20-30%. Advanced stage tumors had a significantly higher percentage of informative microsatellite markers with LOH than early stage tumors. Of the 29 microsatellite markers studied, 4 loci had a significantly higher frequency of LOH in Stage III and IV tumors than in earlier stage tumors. CONCLUSIONS: Frequent aberrations on chromosome 1 in cervical carcinoma suggest that inactivation of tumor suppressor genes is important in cervical tumorigenesis. Higher frequencies of LOH in Stage III and IV tumors suggest that chromosome 1 changes are late events in cervical carcinoma. The findings of this study are consistent with earlier reports that suggest that tumor suppressor genes are present at 1p36.3 and 1p31. To the authors' knowledge, the high frequency of LOH mapped to 1q25 has not been reported previously. Its significance awaits further clarification.     

The Mcs7 quantitative trait locus is associated with an increased susceptibility to mammary cancer in congenic rats and an allele-specific imbalance

Identification of high-penetrance breast cancer genes such as Brca1 has been accomplished by analysing familial cases. However, these genes occur at low frequency and do not account for the majority of genetic risk. Identification of low-penetrance alleles that occur commonly in populations may benefit from unbiased genome-wide screening. One such approach uses linkage studies in rodent models to identify homologous human candidates. The Wistar Kyoto (WKy) rat is resistant to mammary carcinomas induced with 7,12-dimethybenz[a]anthracene (DMBA), whereas the Wistar Furth (WF) strain is susceptible. Previous genome-wide linkage studies in crosses of these strains identified three WKy resistance quantitative trait loci, Mcs5, Mcs6 and Mcs8, and one predicted to increase susceptibility, Mcs7. The Mcs7 region on rat chromosome 10 (RNO10) is orthologous to human 17q, a common site of genetic aberrations in breast cancer. Here, we establish the independent phenotype conferred by Mcs7 using congenic rats carrying the WKy Mcs7 locus on a WF background. Tumor multiplicity was significantly higher ( approximately 50%) in DMBA-treated congenics homozygous and heterozygous for the WKy allele at the Mcs7 locus, compared to controls. We also investigated allelic imbalance (AI) in mammary carcinomas from (WKy x WF)F1 rats and Mcs7 heterozygous congenics. Of the four known WKy Mcs loci tested, only Mcs7 displayed AI. The pattern of AI in carcinomas from both F1 and Mcs7 congenic rats was similar, suggesting a WF allelic loss. Together, these data suggest that one or more breast cancer-related genes are located within the dominantly acting WKy allele at the Mcs7 locus.     

Frequent loss of heterozygosity of the long arm of chromosome 7 is closely associated with progression of human gastric carcinomas

Loss of heterozygosity (LOH) on the long arm of chromosome 7 was examined using 5 polymorphic marker probes on 98 gastric carcinomas to elucidate a novel locus for development and progression of the tumors. Twenty-six (32%) of 82 informative cases showed LOH on 7q on at least one locus of 5 loci. Among 5 loci, LOH at D7S95 locus was most frequent, the incidence being 53% in well-differentiated gastric carcinomas and 33% in poorly differentiated and scirrhous gastric carcinomas respectively. At 3 loci, c-met, D7S63 and D7S22, the incidence of LOH was about 30% and 10% in well-differentiated and poorly differentiated gastric carcinoma cases respectively. In contrast, LOH at D7S64 was not detected in any gastric-carcinoma cases. Deletion mapping of 7q revealed that D7S95 locus was the essential region of LOH. Eight (62%) of 13 cases with LOH at D7S95 locus belonged to the most advanced stage grouping. Furthermore, 6 (75%) of 8 cases with abdominal dissemination showed LOH at D7S95. Therefore, cases with LOH at D7S95 showed significantly worse prognosis than the cases without the LOH in the stage-III and stage-IV groups. These findings overall suggest that D7S95 locus on 7q may contain a candidate suppressor gene for the progression of gastric carcinoma.     

GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development

Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse‐free survival, hazard ratio = 2.37, 95% confidence interval, 1.27–4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42–5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap‐cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland‐specific GANP‐deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP‐heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance.     

SAGE profiling of UV-induced mouse skin squamous cell carcinomas, comparison with acute UV irradiation effects

Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV-induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH-1 mice epidermis with that of UV-induced squamous cell carcinomas (SCCs) from SKH-1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase-14 (reduced 8.5-fold in SCCs); cathepsins D and S (reduced 3-fold and increased 11.3-fold, respectively, in SCCs); decorin, glutathione S-transferase omega-1, hypoxia-inducible factor 1 alpha, insulin-like growth factor binding protein-7, and matrix metalloproteinase-13 (increased 18-, 12-, 12-, 18.3-, and 11-folds, respectively, in SCCs). Chemokine (C-C motif), ligand 27 (CCL27), which was found downregulated 12.7-fold in SCCs by SAGE, was also observed to be strongly downregulated 6-24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV-irradiated versus sham-treated SKH-1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV-treated skin and SCCs included keratins 6 and 16, small proline-rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.     

DNA amplification on chromosome 7q in squamous cell carcinoma of the tongue

Squamous cell carcinoma of the head and neck exhibit a highly variable picture of chromosomal aberrations. In the present study the clearly defined anatomical region of the tongue was analyzed for potentially specific patterns of chromosomal alterations. Fresh tumor samples from 18 patients afflicted by squamous cell carcinoma of the tongue constituted the clinical basis of the present investigation. The tumor samples were analyzed on the basis of comparative genomic hybridization (CGH), a molecular cytogenetic FISH-approach. Gains in DNA copy numbers were detected as the predominant imbalance on chromosomes 7q (9/18), 3q (48/18), 16p (7/18) and 20q (7/18). The regions of minimal overlap on these chromosomes were mapped to 7q11.2q11.3 and 3q26. A conspicuous finding was the frequent detection of amplifications in the 7q11 region. Gains in the 7q region have been rarely reported in CGH studies of tumors derived from different regions of the head and neck. Amplifications on 7q could thus be specifically linked with the tongue region and could correlate with specific clinical factors of this tumor entity.     

Fascin over-expression is associated with aggressiveness of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is associated with a high potential of tumor recurrence and metastasis, leading to poor prognosis. Cell motility is an important factor in the progression and metastasis of cancers. Recently, Fascin has been linked to tumor progression by induction of cell motility. However, the precise roles of Fascin in OSCC have not been elucidated clearly. The aim of this study was to analyze the roles of Fascin in OSCC progression using OSCC clinical samples. We demonstrated that Fascin over-expression was found in OSCC clinical samples and its expression was significantly associated with nodal metastasis (p=0.027), tumor recurrence (p<0.001) and poor patients' overall survival (p=0.013). Consistently, Fascin proteins were detected in all OSCC cell lines with the expression level corresponding to the invasion ability. To specifically investigate the mechanism of Fascin in OSCC, we examined the E-cadherin expression in the same set of OSCC specimens. Fascin was negatively correlated with E-cadherin expression (p=0.018, r=-0.513). In conclusion, our findings suggested that Fascin over-expression might enhance OSCC aggressiveness, possibly by interacting with E-cadherin expression.     

Expression of chemokine receptor CCR7 is associated with cervical lymph node metastasis of oral squamous cell carcinoma

Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. The present study was designed to examine the expression of chemokine receptor CCR7 in oral squamous cell carcinoma (OSCC), and to investigate the possible role of CCR7/CCL21 interaction in neck lymph node metastasis of OSCC. By using immunohistochemistry, RT-PCR and Western Blot, expression of CCR7 was examined in 85 cases of oral squamous cell carcinoma, and Tca8113 and ACC cell lines. CCL21-mediated cell migration was assayed in Matrigel-coated chemotaxis chamber. In vitro adhesion assay was shown for banding of tumor cell lines to submandibular lymph nodes with or without anti-CCR7 antibody treatment. Immunohistochemical staining showed 65.9% (56/85) of positive CCR7 expression in OSCC tissues. CCR7 expression was significantly higher in patients with lymph node metastasis compared with those without lymph node metastasis (P=0.015) and was also associated with tumor size (P=0.014), and clinical stage (P=0.009). RT-PCR and Western Blot also confirmed positive CCR7 expression in oral squamous cell carcinoma and Tca8113 cell line, and negative CCR7 expression in normal oral mucosa and ACC cell line. CCL21 stimulation increased the ability of CCR7-positive Tca8113 cells passing through the Matrigel membrane. CCR7-positive Tca8113 cells also showed stronger adhesion to lymph nodes, which could be partly blocked by anti-CCR7 antibody incubation. These results indicated that the chemotactic CCR7/CCL21 interaction may be a possible mechanism for induction of directional lymph node metastasis by oral squamous cell carcinoma.     

Understanding genetic progression of squamous cell carcinoma to spindle cell carcinoma in a mouse model of head and neck cancer

Reports have suggested that spindle cell carcinoma of the head and neck occurs following radiation therapy of incompletely resected SCC, representing anaplastic progression of the primary tumor. Examination of differences between spindle cell carcinoma and SCC may provide important information about anaplastic progression, clinical behavior, and response to therapy. We created a mouse model that developed spindle cell carcinoma. Spindle cell carcinoma was characterized by marked downregulation of epithelial differentiation markers and cell adhesion genes. Expression of growth factors and receptors important for epithelial proliferation was inhibited while those which regulate fibroblast and mesenchymal cell proliferation were increased. By far the largest class of upregulated genes in spindle cell carcinomas was chemokine receptors and ligands which are involved in tumor cell invasion and metastasis. These changes in gene expression clearly show loss of epithelial characteristics, acquisition of mesenchymal phenotypes, and increased propensity for invasion and metastasis by spindle cell carcinomas.