Candidate serous cancer precursors in fallopian tube epithelium of BRCA1/2 mutation carriers.

Occult invasive and intraepithelial carcinomas have been identified in the tubal fimbria of BRCA mutation carriers undergoing prophylactic surgery, and recently described lesions overexpressing p53 in the distal tubes of mutation carriers, and non-carriers, have been proposed as histological precursors of high-grade serous carcinoma. The aim of this study was to confirm these findings in a larger, independent case set, to further characterize the cancer precursor lesions, and to determine their frequency in BRCA mutation-positive (n=176) and control groups (n=64). For the purposes of this study, we excluded cases without documentation of a germline mutation of BRCA1/2, and without histological examination of the entire tube, and cases with a diagnosis of invasive carcinoma. Controls included salpingectomies from women undergoing surgery for reasons other than ovarian malignancy. Diagnostic categories were assigned based on combined histological review and immunostaining results. Histological abnormalities were identified in 23% of the BRCA group and in 25% of the control group, and included localized p53 overexpression in 20% of the BRCA group and 25% of the control group. Tubal intramucosal carcinoma was identified in 8% of the BRCA cases and in 3% of the control group. Four cases of intraepithelial carcinoma (21%) did not overexpress p53. There was no significant difference in the median age, frequency of histological abnormalities, p53 signatures, or tubal intraepithelial carcinoma between the BRCA mutation-positive and control groups. This large, blinded review of tubes from BRCA mutation carriers confirms previous reports of putative cancer precursors in distal tubal mucosa, and that p53 signatures occur with similar frequency in women at low and high genetic risk of tubal/ovarian carcinoma. Tubal intraepithelial carcinoma, which, like invasive serous cancer, usually but not always overexpresses p53 protein, is more frequent in BRCA mutation carriers.

Evidence for a latent precursor (p53 signature) that may precede serous endometrial intraepithelial carcinoma.

Both serous intraepithelial carcinoma and endometrial glandular dysplasia are associated with uterine serous carcinoma. Recently a candidate serous cancer precursor containing p53 mutations (p53 signature) was described in the fallopian tube. We analyzed normal and neoplastic endometrium for a similar entity. In total 10 endometrial polyps involved by intraepithelial and/or invasive carcinoma and 137 benign polyps were studied. All were stained for p53 and MIB-1. A subset of p53 signatures and carcinomas were analyzed for gamma-H2AX and p53 mutations. p53 signatures were identified in 7 of 10 cases intraepithelial carcinoma and were multicentric in 2. In one case, the signature was in continuity with intraepithelial carcinoma. Of 137 benign polyps (4%), 6 contained p53 signatures. The MIB-1 fraction in most signatures was less than 5%, and ranged from 50 to 90% in carcinomas. DNA damage (gamma-H2AX) was demonstrated in both p53 signatures and adjacent carcinomas but not in benign polyps. Shared identical p53 mutations were found in paired signatures and carcinomas in two of three cases analyzed, including one case with multiple signatures. In one, a coexistent invasive serous cancer was not found to contain a p53 mutation. In a third, a p53 signature and an invasive cancer harbored two different p53 mutations. This is the first description of p53 signatures adjacent to carcinoma, suggesting a role for this entity in the genesis of serous malignancy. The significance of p53 signatures in benign conditions (polyps) remains to be determined. The role of the p53 signature in early serous neoplasia is discussed.

Anomalous expression of the HLA-DR alpha and beta chains in ovarian and other cancers.

Tumor formation in immunocompetent hosts is believed to be dependent on the ability of tumor cells to evade the immune system, as suggested by the alterations of expression of the major histocompatibility complex (MHC) and related molecules in a number of cancers. Our previous serial analysis of gene expression (SAGE) study revealed that HLA-DRA (encoding the alpha chain of HLA-DR) is one of the most highly overexpressed genes in ovarian cancer. This finding was unanticipated, as overexpression of MHC molecules would be expected to increase tumor immunogenicity, therefore compromising tumor growth. We have now examined the expression of HLA-DR alpha chain in ovarian and a variety of other cancers using tissue arrays and found it overexpressed in a majority of the cancer tissues investigated. In contrast, the HLA-DR beta chain, which together with the alpha chain forms the functional HLA-DR complex, was not frequently found expressed in cancer, resulting to a lack of mature HLA-DR in these tissues. Interestingly, HLADRA and HLADRB transcripts were both found expressed in many other cancer types, including ovarian cancer, suggesting that the downregulation of HLADR beta chain is a post-transcriptional or post-translational mechanism. In addition, we observed high levels of the invariant chain (Ii/CD74) expression in both the cytoplasm and plasma membrane of ovarian tumor cells, possibly contributing to the lack of mature HLA-DR protein expression. Interestingly, we found that IFN-gamma could induce mature HLA-DR at the surface of normal ovarian cells, while this ability was reduced in tumor cells. Together, these data suggest that, while ovarian tumors overexpress HLA-DR alpha, perhaps as a result of inflammatory events in the tumor microenvironment, the tumor cells may have compensatory mechanisms to reduce the production of functional MHC class II molecules, thus reducing immunogenicity and favoring tumor growth. In addition, because of its ubiquitous expression in ovarian and other cancers, HLA-DR alpha may represent a novel biomarker for malignancy.

Role of the RNA-binding protein HuR in colon carcinogenesis.

Immunohistochemical analysis of paired tumor and normal tissue specimens revealed that the expression and cytoplasmic abundance of the RNA-binding protein HuR increased with malignancy, particularly in colon carcinomas. Interventions to modulate HuR expression in human RKO colon cancer cells altered gene expression profiles and identified beta-catenin mRNA as a novel HuR target. Subcutaneous injection of HuR-overexpressing RKO cells into nude mice produced significantly larger tumors than those arising from control populations; conversely, RKO cells expressing reduced HuR through small interference RNA- or antisense HuR-based approaches developed significantly more slowly. We propose that HuR-regulated target mRNA expression contributes to colon cancer growth. Our results suggest a pivotal function for HuR in colon carcinogenesis.

Tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer but not in ovarian cystadenomas.

PURPOSE: Claudin proteins represent a large family of integral membrane proteins crucial for tight junction (TJ) formation and function. Claudins have been shown to be up-regulated in various cancers and have been suggested as possible biomarkers and targets for cancer therapy. Because claudin-3 and claudin-4 have been proposed to be expressed in epithelial ovarian cancer, we have performed a detailed analysis of CLDN3 and CLDN4 expression in a panel of ovarian tumors of various subtypes and cell lines. We also investigated whether high expression of claudin-3 and claudin-4 was associated with TJ function in ovarian cancer cells. EXPERIMENTAL DESIGN: RNA was obtained from a panel of 39 microdissected epithelial ovarian tumors of various histological subtypes for real-time reverse transcription-PCR analysis. In addition, a total of 70 cases of ovarian carcinomas, ovarian cysts, and normal ovarian epithelium from a tissue array were analyzed by immunohistochemistry. Finally, a panel of cell lines was used for Western analysis of claudin expression and TJ permeability studies. RESULTS: Although expressed at low levels in some normal human tissues, including the ovary, CLDN3 and CLDN4 are highly up-regulated in epithelial ovarian cancers of all subtypes. Immunohistochemical analyses using our ovarian tissue array confirmed the high level of expression of claudin-3 and claudin-4 in the majority of ovarian carcinomas, including many tumors exhibiting cytoplasmic staining. Ovarian cystadenoma did not frequently overexpress these proteins, suggesting that the expression of these proteins is associated with malignancy. In ovarian cancer cell lines, claudin-3 and claudin-4 expression was not associated with functional TJs as measured by transepithelial electrical resistance. CONCLUSIONS: These results show that CLDN3 and CLDN4 are frequently up-regulated in ovarian tumors and cell lines and may represent novel markers for this disease. Overexpression of these genes in ovarian cancer also suggests interesting scenarios for the involvement of TJ in tumorigenesis. A better knowledge of the mechanisms underlying ovarian tumorigenesis will likely result in the development of novel approaches for the diagnosis and therapy of this deadly disease.

Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells.

The mechanisms of drug resistance in cancer are poorly understood. Serial analysis of gene expression (SAGE) profiling of cisplatin-resistant and sensitive cells revealed many differentially expressed genes. Remarkably, many ECM genes were elevated in cisplatin-resistant cells. COL6A3 was one of the most highly upregulated genes, and cultivation of cisplatin-sensitive cells in the presence of collagen VI protein promoted resistance in vitro. Staining of ovarian tumors with collagen VI antibodies confirmed collagen VI expression in vivo and suggested reorganization of the extracellular matrix in the vicinity of the tumor. Furthermore, the presence of collagen VI correlated with tumor grade, an ovarian cancer prognostic factor. These results suggest that tumor cells may directly remodel their microenvironment to increase their survival in the presence of chemotherapeutic drugs.

Immunohistochemical expression and prognostic significance of FAS and GLUT1 in bladder carcinoma.

BACKGROUND: Fatty Acid Synthase (FAS) and Human Erythrocyte Glucose Transporter 1 (GLUT1) are new markers involved in the biological activities of cancer cells. FAS is a multifunctional enzyme that synthesizes palmitate from acetyl-CoA and malonyl-CoA. GLUT1 is a transmembrane protein normally expressed in perineurium and erythrocytes. FAS and GLUT1 expression have been recently described in many aggressive tumors. We explored the immunohistochemical expression of FAS and GLUT1 in bladder carcinomas to reveal statistical associations with clinico-pathological features and recurrence. MATERIALS AND METHODS: Thirty-one node- and distant metastasis-negative transitional cell carcinomas from patients with a five-year follow-up were evaluated for FAS and GLUT1 expression. RESULTS: Univariate analysis showed that low-grade, pTa stage and FAS-negative expression were associated with indolent tumors. Multivariate analysis showed that FAS expression (p = 0.006) and pT1-2 stage tumors (p = 0.001) were independent predictors of recurrence. CONCLUSION: Endogenous fatty acids are an exploitable storage of energy for aggressive human bladder carcinomas. Glucose uptake is not required by bladder tumors.

Regulation of fatty acid synthase expression in breast cancer by sterol regulatory element binding protein-1c.

Activation of fatty acid synthase (FAS) expression and fatty acid synthesis is a common event in human breast cancer. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate genes involved in lipid metabolism, including FAS. SREBP-1c expression is induced in liver and adipose tissue by insulin and by fasting/refeeding and is critical for nutritional regulation of lipogenic gene expression. In contrast, upregulation of fatty acid metabolism during in vitro transformation of human mammary epithelial cells and in breast cancer cells was driven by increased MAP kinase and PI 3-kinase signaling, which increased SREBP-1 levels. SREBP-1a was more abundant than SREBP-1c in many proliferative tissues and cultured cells and was thus a candidate to regulate lipogenesis for support of membrane synthesis during cell growth. We now show that SREBP-1c and FAS mRNA were both increased by H-ras transformation of MCF-10a breast epithelial cells and were both reduced by exposure of MCF-7 breast cancer cells to the MAP kinase inhibitor, PD98059, or the PI 3-kinase inhibitor, wortmannin, while SREBP-1a and SREBP-2 showed less variation. Similarly, the mRNA levels for FAS and SREBP-1c in a panel of primary human breast cancer samples showed much greater increases than did those for SREBP-1a and SREBP-2 and were significantly correlated with each other, suggesting coordinate regulation of SREBP-1c and FAS in clinical breast cancer. We conclude that regulation of FAS expression in breast cancer is achieved through modulation of SREBP-1c, similar to the regulation in liver and adipose tissue, although the upstream regulation of liopgenesis differs in these tissues.

Activation of fatty acid synthesis during neoplastic transformation: role of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

Activation of fatty acid synthase (FAS) expression and fatty acid synthesis is a common event in tumor cells from a variety of human cancers and is closely linked to malignant transformation and to tumor virulence in population studies of human cancer. We now show that, in contrast to nutritional regulation of lipogenesis in liver or adipose tissue, changes in fatty acid metabolism during in vitro transformation of the human mammary epithelial cell line MCF-10a are driven by increases in epidermal growth factor signaling, acting in major part through the mitogen-activated protein (MAP) kinase and phosphatidylinositol (PI) 3-kinase signaling cascades. H-ras transformation of MCF-10a cells resulted in upregulation of MAP kinase and PI 3-kinase signals, upregulation of sterol regulatory element binding protein 1 (SREBP-1) transcription factor levels, and upregulation of FAS expression and FA synthesis. Deletion of the major SREBP binding site from the FAS promoter abrogated transcription in transformed MCF-10a cells. Inhibitors of MAP and PI 3-kinases downregulated SREBP-1 levels and decreased transcription from the FAS promoter, reducing FAS expression and fatty acid synthesis in transformed MCF-10a cells and in MCF-7 and HCT116 carcinoma cells. H-ras transformation sensitized MCF-10a cells to the FAS inhibitors cerulenin and C-75. These results confirm an important role for SREBP-1 in neoplastic lipogenesis, and provide a likely basis for the linkage of upregulated fatty acid metabolism with neoplastic transformation and with tumor virulence, since MAP and PI 3-kinase signaling contributes to both.