The histogenesis of mammary tumours induced in the rat by chemical carcinogens

126 Wistar female rats were given carcinogenic compounds, 2-acetylaminofluorene (2-AAF), 20-methylcholanthrene (MC) and 7,12-dimethylbenz(a)anthracene (DMBA), for induction of breast tumors to determine the site of origin and biological behavior of mammary carcinoma in the rat. 18 virgin rats served as control material of a corresponding age to the tumor-bearing rats. A dissecting microscope was used to identify early neoplastic lesions in the breast glands for histological study. 17 weeks after carcinogen treatment, breast gland tumors, especially adenocarcinomas became evident by palpation. Mammary carcinoma was found to be multifocal in origin, arising almost exclusively in ducts, ductules, and end-buds without the intervention of any recognizable lesion preceding the appearance of tumor cells. Fibroadenomas and adenomas occurred in rats with generally well-developed breast glands, while mammary carcinomas occurred in younger rats with less developed breast glands. For successful tumor induction, the important factors to consider are the architectural state of the mammary gland, the action of hormonal factors, genetically-determined susceptibility, and the carcinogenic agent and its dosage. Mast cells did not play a significant role in tumor formation, nor were significant myoepithelial cell contributions found in the early carcinomas and fibroadenomas.     

The insulin receptor content is increased in breast cancers initiated by three different oncogenes in transgenic mice

The Insulin Receptor (IR) is a potential oncogenefor mammary epithelial cells since its content isincreased in most human breast cancer specimens, andboth ligand-dependent malignant transformation and ligand-dependent enhanced growthoccurs in cultured breast cells overexpressing the IR.To better understand whether the IR plays arole in mammary carcinogenesis which is independent ofother initiation factors, we measured IR content intransgenic mouse models of breast cancer induced by3 known oncogenes (Wnt-1, Neu, and Ret). Insulinreceptor content was measured by a specific radioimmunoassay.In normal mammary gland tissues IR content was14.6 ± 1.4 ng/mg of protein (mean ±SEM, n=6). In the 3 cancersIR content was elevated (Neu=36.1 ±4.6, n=8, p < 0.002; Wnt-1= 38.3 ± 2.6, n=13, p< 0.001; and Ret=53.6 ± 7.1,n=7, p < 0.001). These dataindicate that IR overexpression, in addition to beinga potential oncogene, is increased in mouse tumorsinitiated by other oncogenes, and therefore may alsoplay a supportive role in the growth ofbreast cancers.     

SCC-S2 is overexpressed in colon cancers and regulates cell proliferation

SCC-S2 was recently reported to be overexpressed in various cancers and associated with the malignant behavior of cancer cells. However, the expression of SCC-S2 and its biological roles in colon cancers have not been reported. The aim of this study is to investigate the expression and clinical significance of SCC-S2 in colon cancers and explore its biological function in colon cancer cells. We analyzed the expression pattern of SCC-S2 in 92 colon cancer tissues by immunohistochemistry and found that SCC-S2 was overexpressed in 45 (48.9?%) of 92 colon cancer specimens. There was a significant association between SCC-S2 overexpression and TNM stage (p?=?0.0387), lymph node metastasis (p?=?0.0219), and proliferation index (p?=?0.0279). siRNA knockdown of SCC-S2 expression in CACO2 and HCT116 cells decrease cell proliferation, colony formation, and soft agar colony formation ability. Furthermore, western blot analysis showed that SCC-S2 depletion decreased cyclin D1 and phospho-Rb levels. In conclusion, we demonstrated that SCC-S2 is overexpressed in colon cancers and contributes to malignant cell growth by cyclin D1 and phospho-Rb modulation, which makes SCC-S2 a candidate therapeutic target in colon cancer.     

The DNA2 nuclease/helicase is an estrogen-dependent gene mutated in breast and ovarian cancers

Genomic instability, a hallmark of cancer, is commonly caused by failures in the DNA damage response. Here we conducted a bioinformatical screen to reveal DNA damage response genes that are upregulated by estrogen and highly mutated in breast and ovarian cancers. This screen identified 53 estrogen-dependent cancer genes, some of which are novel. Notably, the screen retrieved 9 DNA helicases as well as 5 nucleases. DNA2, which functions as both a helicase and a nuclease and plays a role in DNA repair and replication, was retrieved in the screen. Mutations in DNA2, found in estrogen-dependent cancers, are clustered in the helicase and nuclease domains, suggesting activity impairment. Indeed, we show that mutations found in ovarian cancers impair DNA2 activity. Depletion of DNA2 in cells reduces their tumorogenicity in mice. In human, high expression of DNA2 correlates with poor survival of estrogen receptor-positive patients but not of estrogen receptor-negative patients. We also demonstrate that depletion of DNA2 in cells reduces proliferation, while addition of estrogen restores proliferation. These findings suggest that cells responding to estrogen will proliferate despite being impaired in DNA2 activity, potentially promoting genomic instability and triggering cancer development.     

Insig2 is overexpressed in pancreatic cancer and its expression is induced by hypoxia

A hypoxic microenvironment is a characteristic feature of pancreatic cancer, and induces the expressions of various genes involved in malignant behaviors. Insulin‐induced gene 2 (Insig2) has recently been shown to be correlated with cellular invasion in colon cancer. However, there have been no reports regarding its expression in pancreatic cancer. In this study, we evaluated Insig2 mRNA expression and the biological function of Insig2 in pancreatic cancer. We measured Insig2 mRNA expression in cultured pancreatic cancer cell lines and invasive ductal carcinoma (IDC) cells, normal pancreatic epithelial cells, and pancreatic intraepithelial neoplasia cells obtained by laser‐capture microdissection. We also investigated the effects of Insig2‐targeting siRNAs on the cell proliferation and cell invasion of pancreatic cancer cell lines. All pancreatic cancer cell lines expressed Insig2 mRNA. The PANC‐1 and MIA PaCa‐2 pancreatic cancer cell lines showed >2‐fold higher Insig2 mRNA expression levels under hypoxic conditions (1% O₂) than under normoxic conditions (21% O₂). Cell proliferation was significantly decreased in SUIT‐2 cells and cell invasion was significantly decreased in SUIT‐2, Capan‐2, and CFPAC‐1 cells after transfection of the Insig2‐targeting siRNAs. In analyses of microdissected cells, cells from IDC tissues expressed significantly higher levels of Insig2 mRNA than normal pancreatic cells (P < 0.001) and pancreatic intraepithelial neoplasia cells (P = 0.082). In analyses of IDC cells, the levels of Insig2 mRNA expression were significantly higher in late‐stage patients than in early‐stage patients. The present data suggest that Insig2 is associated with the malignant potential of pancreatic cancer under hypoxic conditions. (Cancer Sci 2011; 102: 1137–1143)     

Overexpression of human H-ras transgene is responsible for tumors induced by chemical carcinogens in mice

Level of human prototype H-ras transgene expression in tumors induced by chemical carcinogens (N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea) was analyzed in human H-ras transgenic mice (CB6F1-TgrasH2 Jic mice). All forestomach tumors examined revealed about 2-fold overexpression of the human H-ras transgene with or without point mutation at codon 12 or codon 61. However, endogenous mouse H- and K-ras genes exhibited neither point mutation nor overexpression. These results suggested that increased levels of ras gene products in the cell played an important role in facilitating chemical carcinogenesis in transgenic mice.     

Transformation of human breast epithelial cells by chemical carcinogens

The present study was carried out to determine whether humanbreast epithelial cells (HBEC) are transformed by chemicalsthat have been proven to be carcinogenic in other model systems.A spontaneously immortalized human breast epithelial cell line,MCF-10F, was treated with dimethylbenz[a]anthracene (DMBA),benzo[a]pyrene (B[a]P), N-methyl-N-nitrosoguanidine (MNNG) orN-methyl-N-nitrosourea (NMU) for 24 h. MCF-7 and T24 malignantcell lines were used as positive controls. All the carcinogensinduced alterations in both cell morphology and pattern of growth,increased growth rate and anchorage-independent growth in agar—methocel,which became evident by the 8th to 10th passages post-treatment,at     

Detection of private and common tumor-associated antigens in murine sarcomas induced by different chemical carcinogens.

Two fibrosarcomas of similar histological type, induced in C3Hf mice by either methylcholanthrene or 3,4-benz(a)pyrene, were shown to have individually unique tumor-rejection antigens in classical transplantation-type experiments. By contrast, sera of autochthonous mice, which resisted only transplants of the immunizing sarcoma, were found to contain complement-dependent cytotoxic antibodies, specific for both sarcomas, in vitro. The existence of individually unique as well as common tumor-associated antigens in chemically-induced murine sarcomas is suggested. The private "tumor transplantation-type" antigens elicited tumor rejection responses in vivo. The common tumor-associated antigens, although immunogenic in autochthonous hosts, inducing the production of tumor-specific antibodies, failed to induce transplantation cross-resistance in vivo. This study supports the contention that, in carcinogen-induced murine tumors, and perhaps in human neoplasms as well the evaluation of humoral (and cell-mediated) immune responses in vitro may not reflect tumor rejection-type immune responses in vivo.     

CD44 is overexpressed in basal-like breast cancers but is not a driver of 11p13 amplification

Overexpression and alternative splicing of CD44 have been implicated in tumour progression. Here we describe the identification of a high level amplification of human 11p13, encompassing the CD44 gene, in primary breast cancers and cell lines and test whether CD44 acts as the driver of this amplicon. aCGH analysis revealed 11p13 amplification in 3% (3/100) of primary breast carcinomas and in two cell lines. The minimal region of amplification was 34.38–37.62 Mb. Amplification was confirmed by dual-colour FISH in these cell lines and further validated by CISH in an independent tumour cohort. CD44 expression in primary breast cancers was significantly associated with features of basal-like breast cancer. Detection of CD44 expression in breast cancer cell lines confirmed moderate to high expression in basal-like cell lines and minimal expression in luminal cell lines. In both, primary breast cancers and cell lines, 11p13 amplification was associated with high levels of CD44 mRNA expression. CD44 alternative splicing was detected in four of nine cell lines and in tumour samples, irrespective of the amplification status. RNAi mediated knock down of CD44 failed to reveal an increased dependence on CD44 expression for proliferation or survival in amplified cell lines. Given that expression of CD44 is not an absolute requirement for the survival of cells harbouring CD44 gene amplification, CD44 is unlikely to be a driver of the 11p13 amplicon.     

Identification and validation of an ERBB2 gene expression signature in breast cancers

ERBB2 is a transmembrane tyrosine kinase receptor encoded by a gene located in chromosome region 17q12. Overexpression of ERBB2, generally by way of gene amplification, plays a role in mammary oncogenesis. This alteration can be overcome by use of the humanized monoclonal antibody trastuzumab (Herceptin). Accurate determination of ERBB2 status is required for appropriate use of this targeted therapy and is currently analysed by immunohistochemistry (IHC) on tissue sections and/or fluorescence in situ hybridisation (FISH) on interphase chromosomes. We have studied the gene expression profiles of a series of 213 breast tumours and 16 breast cancer cell lines with known ERBB2 status, using Ipsogen's DiscoveryChip microarrays with approximately 9000 cDNAs. We have identified 36 genes and expressed sequence tags that were differentially expressed in tumours and in cell lines with and without ERBB2 protein overexpression. This ERBB2-specific gene expression signature (GES) contained 29 overexpressed genes including the ERBB2 gene itself, five genes located in its immediate vicinity on 17q12, non-17q genes such as GATA4 and eight downregulated genes including oestrogen receptor alpha (ER). Some correlations were validated at the protein level using IHC on tissue microarrays. The GES was able to distinguish ERBB2-negative and -positive cancer samples, as well as FISH-negative and FISH-positive ERBB2 2+ IHC samples.     

5-Azacytidine potentiates initiation induced by carcinogens in rat liver

To test the validity of the hypothesis that hypomethylationof DNA plays an important role in the initiation of carcinogenicprocess, 5-azacytidine (5-AzC) (10 mg/kg), an inhibitor of DNAmethylation, was given to rats during the phase of repair synthesisinduced by the three carcinogens, benzo[a]-pyrene (200 mg/kg),N-methyl-N-nltrosourea (60 mg/kg) and 1,2-dimethylhydrazine(1,2-DMH) (100 mg/kg). The initiated hepatocytes in the liverwere assayed as the -glutamyltransferase (-GT) positive fociformed following a 2-week selection regimen consisting of dietary0.02% 2-acetylaminofluorene coupled with a necrogenic dose ofCCl₄. The results obtained indicate that with all three carcinogens,administration of 5-AzC during repair synthesis increased theincidence of initiated hepatocytes, for example 10–20foci/cm² in 5-AzC and carcinogen-treated rats compared with3–5 foci/cm² in rats treated with carcinogen only. Administrationof [³H]-5-azadeoxycytidine during the repair synthesis inducedby 1,2-DMH further showed that 0.019 mol% of cytosine residuesin DNA were substituted by the analogue, indicating that incorporationof 5-AzC occurs during repair synthesis. In the absence of thecarcinogen, 5-AzC given after a two thirds partial hepatectomy,when its incorporation should be maximum, failed to induce any-GT positive foci. The results suggest that hypomethylationof DNA per se may not be sufficient for initiation. Perhapstwo events might be necessary for initiation, the first causedby the carcinogen and a second involving hypomethylation ofDNA.     

Enzyme deviation patterns in primary rat hepatomas induced by sequential administration of two chemically different carcinogens

Enzyme deviation patterns were examined in primary rat hepatomas induced by short-term sequential administration of two chemical carcinogens from among 2-fluorenylacetamide (FAA), diethylnitrosamine (DENA), and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) or by FAA or 3'-Me-DAB followed by phenobarbital as a promoter. The purpose was to discern how the patterns are influenced by different administration schedules of carcinogens and which of the two carcinogens in the sequence affects the pattern more. Biochemical differentiation of hyperplastic hepatic nodules and hepatomas was determined by simultaneous assays of activities and isozyme composition of glucose-adenosine triphosphate phosphotransferase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and gamma-glutamyltransferase with consideration of histological classification of nodules and tumors. Poorly differentiated hepatomas were predominantly induced by 3'-Me-DAB followed by FAA or DENA except for hepatomas induced by 3'-Me-DAB followed by phenobarbital, which were mainly well and moderately differentiated; well and moderately differentiated hepatomas were predominantly induced by FAA followed by 3'-Me-DAB or phenobarbital. The degree of enzyme deviation of the hepatomas induced by DENA as the first carcinogen was intermediate between those of hepatomas induced by FAA or 3'-Me-DAB, although the degree tended to increase with increased dose or term of DENA. These results indicate that deviations of some enzymes, such as pyruvate kinase and fructose-1,6-bisphosphatase, as well as histological differentiation of the primary hepatomas are more strongly influenced by the first carcinogen than by the second under our administration schedules and that the degree of enzyme deviation shown by hepatomas produced by a particular carcinogen treatment regimen principally related to the potential of that regimen to induce the more anaplastic tumors.     

Chromosome bands induced in human and Syrian hamster cells by chemical carcinogens

After 24 hours of treatment of human peripheral leucocyte cultures or Syrian hamster embryonic secondary cultures with some known chemical carcinogens at concentrations that produce transformation in vitro of hamster cells, the chromosomes of both types of cells exhibited typical G banding. Chromosome bands did not result with urethane, a compound which does not produce cytotoxicity or cause transformation by direct exposure of cells. Non-carcinogenic chemicals which do not inhibit cell multiplication also failed to produce bands. Cytogenetic analysis following removal of the carcinogens from the cultures indicated non-banded uniformly stained chromosomes. It is concluded that metaphases with chromosome bands are more likely to be the result of nonspecific toxicity rather than being related to the carcinogenic properties of the chemicals.     

c-fos gene expression in rat liver is induced by phenobarbital

In the present study we investigated c-fos expression in rat livers, that was initiated with the three arylamines, 2-acetylaminofluorene, 2-acetylaminophenanthrene and trans-4-acetylaminostilbene. The tumor promoter phenobarbital was applied chronically for 26, 52 and 100 weeks. Gene expression, determined by the mRNA level, and FOS protein were increased after 52 weeks of treatment in arylamine initiated as well as in phenobarbital only treated animals. Expression of c-fos seems to be a phenobarbital induced effect that is independent of additional initiator treatments. This finding was supported by immunohistochemical studies demonstrating increased FOS levels to be localized around the central vein. The results indicate that phenobarbital, a widely used tumor promoter, induces c-fos expression. In addition, we demonstrated enhanced FOS in GST-P-positive foci and in tumors.