Distribution of adenosine triphosphatase in infiltrating ductal carcinoma and non-neoplastic breast.

The histochemical reaction for adenosine triphosphatase (ATPase) has previously been used to differentiate myoepithelial from epithelial cells in the breast and to investigate the possible contribution of myoepithelial cells to mammary carcinoma. Discrepancies in published reports prompted this study of ATPase in non-neoplastic breast and infiltrating ductal carcinoma. ATPase was localized mainly on myoepithelial cells of normal breast and was identified with significant frequency on epithelial cells in hyperplastic ducts. Infiltrating ductal carcinomas usually displayed a variable reactivity. In one instance, malignant cells demonstrating mucin production were found to be ATPase-positive. An infiltrating ductal carcinoma of the papillary type with apocrine features was also strongly ATPase-reactive. It is concluded that ATPase is not an exclusive marker of myoepithelial cells and, therefore, data resulting from the use of this enzyme to study the role of the myoepithelium in mammary carcinoma must be interpreted with caution.     

Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.

Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested.     

Transient receptor potential melastatin-related 7 channel is overexpressed in human pancreatic ductal adenocarcinomas and regulates human pancreatic cancer cell migration

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancer with a tendency to invade surrounding healthy tissues, leading to a largely incurable disease. Despite many advances in modern medicine, there is still a lack of early biomarkers as well as efficient therapeutical strategies. The melastatin-related transient receptor potential 7 channel (TRPM7) is a nonselective cation channel that is involved in maintaining Ca(2+) and Mg(2+) homeostasis. It has been recently reported to regulate cell differentiation, proliferation and migration. However, the role of TRPM7 in PDAC progression is far to be understood. In our study, we show that TRPM7 is 13-fold overexpressed in cancer tissues compared to the healthy ones. Furthermore, TRPM7 staining is stronger in tumors with high grade, suggesting a correlation between TRPM7 expression and PDAC progression. Importantly, TRPM7 expression is inversely related to patient survival. In BxPC-3 cell line, dialyzing the cytoplasm during the patch-clamp whole-cell recording with a 0-Mg(2+) solution activated a nonselective current with a strong outward rectification. This cation current is inhibited by intracellular Mg(2+) and by TRPM7 silencing. The downregulation of TRPM7 by small interference RNA dramatically inhibited intracellular Mg(2+) fluorescence and cell migration without affecting cell proliferation, suggesting that TRPM7 contributes to Mg(2+) entry and cell migration. Moreover, external Mg(2+) following TRPM7 silencing fully restored the cell migration. In summary, our results indicate that TRPM7 is involved in the BxPC-3 cell migration via a Mg(2+)-dependent mechanism and may be a potential biomarker of poor prognosis of PDAC.     

COP1, the negative regulator of p53, is overexpressed in breast and ovarian adenocarcinomas

The tumor suppressor protein p53 plays a central role in protecting normal cells from undergoing transformation. Thus, it is fitting that cancer cells selectively dampen the p53 response to gain a selective growth advantage. In fact, the p53 gene is the most commonly mutated tumor suppressor gene in human cancers, and if the gene is not mutated, then other components of the p53 pathways are skewed to dampen the p53 response to stress. We recently identified COP1 as a novel and critical negative regulator of p53. COP1 is a RING finger-containing protein that targets p53 for degradation to the proteasome and is necessary for p53 turnover in normal and cancer cells. However, the association between COP1 and cancer remains to be determined. We performed expression analysis of COP1 in ovarian and breast cancer tissue microarrays. COP1 is significantly overexpressed in 81% (25 of 32) of breast and 44% (76 of 171) of ovarian adenocarcinoma as assessed by in situ hybridization and immunohistochemistry. Overexpression of COP1 correlated with a striking decrease in steady state p53 protein levels and attenuation of the downstream target gene, p21, in cancers that retain a wild-type p53 gene status. Overall, these results suggest that overexpression of COP1 contributes to the accelerated degradation of p53 protein in cancers and attenuates the tumor suppressor function of p53.     

Dickkopf‐1 is overexpressed in human pancreatic ductal adenocarcinoma cells and is involved in invasive growth

The protein products of the Dickkopf (DKK) genes are antagonists of Wnt glycoproteins, which participate in tumor development and progression by binding to frizzled receptors. In this study, the expression of DKK‐1 was analyzed in a panel of 43 human cultured carcinoma cell lines. DKK‐1 expression was consistently and significantly upregulated in pancreatic carcinoma cell lines. Low level of DKK‐3 expression was also seen. In contrast, the expression of DKK‐2 and ‐4 was not detectable in most pancreatic carcinoma cell lines. The overexpression of DKK‐1 was confirmed in surgically resected human pancreatic cancer tissues, in which the mRNA level was evaluated in paired samples from cancerous and noncancerous pancreatic tissues. In ductal adenocarcinomas (23 cases), DKK‐1 mRNA levels were significantly upregulated compared to corresponding noncancerous tissues in a statistically significant level. To test the biological role of DKK‐1 in pancreatic carcinoma cells, we performed a knockdown of DKK‐1 in SUIT‐2 human pancreatic adenocarcinoma cell line and S2‐CP8, its metastatic subline, using a retroviral short hairpin RNA expression vector. DKK‐1 knockdown resulted in reduced migratory activity of SUIT‐2 in vitro. The in vitro growth rate and Matrigel invasion were also suppressed by DKK‐1 knockdown in S2‐CP8 cells. Collectively, the evidence suggests that, despite of its presumed antagonistic role in Wnt signaling, DKK‐1 may have a role in the aggressiveness of pancreatic carcinoma cells and could, therefore, serve as a novel biomarker of pancreatic cancer.     

Centrosomal kinase AIK1 is overexpressed in invasive ductal carcinoma of the breast

A centrosomal serine/threonine kinase, AIK1(3)/breast tumor amplified kinase/aurora2, which was recently identified as an oncogene, shows high amino acid identity with chromosome segregation kinases, fly Aurora, and yeast Ipl1. Immunohistochemical analyses of invasive ductal adenocarcinomas of the breast revealed that overexpression of AIK1 was observed in 94% of the cases, irrespective of the histopathological type, whereas the protein was not detected in normal ductal and lobular cells. Benign breast lesions including fibrocystic disease and fibroadenoma (epithelial components) displayed weakly detectable AIK1 expression in part of the lesions. This is the first immunohistochemical report of AIK1 expression in primary human breast carcinomas. Although the physiological function(s) of AIK1 kinase during cell division remains to be determined, the markedly high positivity of AIK1 staining in the cancer lesions suggested a possible involvement of its overexpression in the tumorigenesis of some of breast cancer cells.     

Expression of the "stem cell marker" CD133 in pancreas and pancreatic ductal adenocarcinomas

Background  

It has been suggested that a small population of cells with unique self-renewal properties and malignant potential exists in solid tumors. Such "cancer stem cells" have been isolated by flow cytometry, followed by xenograft studies of their tumor-initiating properties. A frequently used sorting marker in these experiments is the cell surface protein CD133 (prominin-1). The aim of this work was to examine the distribution of CD133 in pancreatic exocrine cancer.     

Lectin agglutinability of non-neoplastic and neoplastic human lymphoid cells in vitro.

Agglutination by two lectins, Concanavalin A (Con A) and Ricinus communis agglutinin (RCA), has been investigated in a human lymphoid cell system. The main conclusions of this study are: (1) no systematic correlation exists between the neoplastic state and sensitivity to Con A or RCA; (2) cells of neoplastic lines vary unsystematically in their surface properties as evaluated by Con A agglutination, with the possible exception that presence of Epstein-Barr virus (EBV) is associated with a high degree of agglutination and (3) cells of diploid lymphoblastoid lines and phytohemagglutinin (PHA)-stimulated lymphoctes agglutinate similarly and significantly better than unstimulated T- or B-lymphocytes. The relatively simple Con A agglutination assay can be used as an adjunct in classification of human lymphoid cell lines.     

Induction and chemotherapeutic response of two transplantable ductal adenocarcinomas of the pancreas in C57BL/6 mice

Following implant of cotton thread-carrying 3-methyl-cholanthrene into the pancreas tissue of 90 C57BL/6 and 60 BALB/c mice, 13 developed ductal adenocarcinomas. Two of these tumors, both of C57BL/6 origin (Panc 02 and 03), were established in serial s.c. transplant. Panc 02 was treated with 37 different anticancer drugs representing all of the chemical and functional classes of clinically useful anticancer agents including alkylating agents, antimetabolites, agents that bind to or cause scission of DNA, and others that inhibit mitosis or inhibit protein synthesis. When drug treatment was started within 3 to 4 days after tumor implant, Panc 02 showed only limited response to treatment with two nitrosoureas, [N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N- nitrosourea, monohydrochloride and N-(2-chloroethyl)-N'-(2,6-dioxo-3-piperdinyl)-N-nitrosourea)], and N-phosphonacetyl-L-aspartate. Drug response of Panc 03 was determined only with Adriamycin, 5-fluorouracil, cyclophosphamide, cis-(SP-4-2)-diamminedichloroplatinum, or N,N'-bis(2-chloroethyl)-N-nitrosourea. When drug treatment was started 3 days after tumor implant, high cure rates were obtained with Adriamycin treatment, and limited therapeutic responses were seen to treatment with cis-diamminedichloroplatinum or cyclophosphamide. A comparison of the biological characteristics and drug responsiveness of Panc 02 and Panc 03 with those of a number of other transplantable tumors of mice is reported.     

EP300—A miRNA‐regulated metastasis suppressor gene in ductal adenocarcinomas of the pancreas

Genetic and epigenetic alterations during development of pancreatic ductal adenocarcinomas (PDACs) are well known. This study investigates genetic and epigenetic data together with tumor biology to find specific alterations responsible for metastasis formation. Using 16 human PDAC cell lines in a murine orthotopic PDAC model, local infiltration and metastatic spread were assessed by standardized dissemination scores. The cell lines were further classified into 3 hierarchical groups according to their metastatic potential. Their mRNA and microRNA (miRNA) expression was profiled via mRNA‐microarray as well as Taqman Low Density Array, and validated by single quantitative RT‐PCR and Western blotting. In the highly metastatic group, a significant induction of EP300 targeting miRNAs miR‐194 (fold change: 26.88), miR‐200b (fold change: 61.65), miR‐200c (fold change: 19.44) and miR‐429 (fold change: 21.67) (p < 0.05) was detected. Corresponding to this, decreased expression of EP300 mRNA (p < 0.0001) and protein (p < 0.05) were detected in the highly metastatic PDAC cell lines with liver metastases compared to the nonmetastatic or marginally metastatic cell lines, while no correlation with local tumor growth was found. In conclusion, epigenetic alterations with upregulated EP300 targeting miRNAs miR‐194, miR‐200b, miR‐200c and miR‐429 are related to reduced EP300 mRNA and protein in PDAC. These results demonstrate that miRNAs might be able to modulate the expression of metastasis‐specific suppressor genes and metastatic behavior in PDAC, suggesting diagnostic and therapeutic opportunities for EP300 and its targeting miRNAs in PDAC.     

CML28 is a broadly immunogenic antigen,which is overexpressed in tumor cells

Donor lymphocyte infusion (DLI) reliably induces durable remission in 75-80% of patients with relapsed chronic myelogenous leukemia (CML) after allogeneic hematopoietic stem cell transplantation. To identify immunological targets of the graft-versus-leukemia response (GVL) after DLI, we used CML post-DLI responder sera to screen a CML cDNA expression library. One of the antigens identified in this screen is a M(r) 28,000 protein, termed CML28. CML28 is identical to hRrp46p, a component of the human exosome, a multiprotein complex involved in the 3' processing of RNA. Components of the human exosome include known autoantigens, such as PMScl-100, an autoantibody target in patients with polymyositis, scleroderma, or polymyositis-scleroderma overlap syndrome. Recombinant CML28-GST fusion protein was purified, and used in Western blot and ELISA to demonstrate the development of a high-titer CML28-specific IgG antibody response in a patient with relapsed CML who responded to DLI. Northern blotting demonstrated that CML28 is highly expressed in a variety of hematopoietic and epithelial tumor cell lines, but not in normal hematopoietic tissues or other normal tissue, with the exception of testis. Purified recombinant CML28 was used to generate a CML28-specific murine monoclonal antibody. Western blotting with CML28 monoclonal antibody against whole-cell lysates derived from blood and marrow of normal donors and patients with leukemia revealed high expression of this antigen in tumor but not in normal samples. Because CML28 was highly expressed in epithelial tumor cell lines, anti-CML28 responses were also examined in patients with solid tumors. By ELISA, we found specific serological responses in 10-33% of patients with lung cancer, melanoma, and prostate cancer. Our studies suggest that immunogenicity of CML28 is likely because of overexpression of this antigen in tumor cells. Moreover, given its expression and immunogenicity in a wide variety of malignancies, CML28 merits additional evaluation as a target for antigen-specific immunotherapy.     

Progesterone, glucocorticoid, but not estrogen receptor mRNA is altered in breast cancer stroma

Our laboratory has previously found that anti-mitogenic nuclear receptor mRNA is elevated in late stage tumours and this study was performed to scrutinize the possibility of cancer-stroma crosstalk using hormone signaling in these tissues. RNA levels in stromal tissue were examined for the estrogen alpha, estrogen beta, androgen, progesterone and glucocorticoid nuclear receptors by a semi-quantitative PCR. Significant differences in expression between the cancer stroma and control tissue were seen, analyzing for both cancer grade and estrogen receptor status. Stroma and control tissue were significantly different for the progesterone and glucocorticoid nuclear receptors (p=5.908 x 10(-7) and 2.761 x 10(-5), respectively). Glucocorticoid receptor also showed a significant increase to mRNA levels in the stroma of estrogen receptor negative tumours (p=5.85 x 10(-5)). By contrast, the estrogen receptors alpha and beta, those most closely associated with breast tissue growth, showed no significant change in mRNA (p=0.372 and 0.655, respectively). Androgen receptor mRNA also remained unaffected (p=0.174).     

Solid, infiltrating variety of papillary cystic neoplasm of the pancreas

Four tumors with histologic, histochemical, and ultrastructural characteristics of papillary cystic neoplasm of the pancreas but lacking the capsule commonly observed in papillary cystic neoplasm and infiltrating interiorly into the pancreatic parenchyma were seen in two men and two women who were 39 to 51 years old. These tumors contrasted with typical encapsulated papillary cystic neoplasms that show extrapancreatic growth and that are detected as a palpable abdominal mass in young women. These four tumors, although unpalpable, were all detected by nodular images of calcification in abdominal plain radiography. The tumors had a mean diameter of 3.1 cm (range, 1.7 to 4.5 cm), were confined within the pancreatic parenchyma, and showed central fibrosis accompanied by dystrophic changes such as calcification and ossification. A small necrotic cyst contiguous to the fibrous focus also was observed in the tumor in three patients. Tumor cells similar to those of a papillary cystic neoplasm had infiltrated among the pancreatic parenchyma in solid and pseudopapillary patterns, and pleomorphic atypism was observed in some parts of the tumor in three patients. These tumors were tentatively designated solid, infiltrating variety of papillary cystic neoplasms to differentiate them from ordinary encapsulated papillary cystic neoplasms. Although no signs of recurrence have been noticed after surgical treatment in these patients, the infiltrating growth pattern and the presence of pleomorphic atypism suggest higher malignancy than the ordinary papillary cystic neoplasm. The contrasting growth pattern and possible malignancy may warrant establishment of these tumors as a papillary cystic neoplasm subclass.     

Promoter hypermethylation of the Chfr gene in neoplastic and non-neoplastic gastric epithelia

While chromosomal instability is a common feature of human solid tumours, no abnormalities in genes involved in the mitotic checkpoint have been identified. However, recently, Chfr (checkpoint with forkhead associated and ring finger), a mitotic stress checkpoint gene, has been reported to be inactivated due to promoter hypermethylation in several types of human malignancy. To clarify whether Chfr promoter hypermethylation is involved in gastric carcinogenesis, we investigated the promoter methylation status of the Chfr gene in gastric cancer cell lines and primary gastric cancers. Non-neoplastic gastric epithelia from cancer-bearing and noncancer-bearing stomachs were also examined for Chfr promoter hypermethylation to study its cancer specificity. Two of 10 gastric cancer cell lines (20%) showed Chfr promoter hypermethylation with resultant loss of expression, which could be restored by 5-aza-2' deoxycytidine treatment. Chfr promoter hypermethylation was present in 35% (25 of 71) of primary tumours and occurred at similar frequencies in early and advanced stages. As for non-neoplastic gastric epithelia, 1% (one of 91) from noncancer-bearing and 5% (four of 71) from cancer-bearing stomachs exhibited Chfr promoter hypermethylation. Thus, Chfr promoter hypermethylation is mostly cancer specific and frequently leads to chromosome instability in gastric cancer.     

Cisplatin, 5-Fluorouracil, and leucovorin in the therapy of adenocarcinomas of the pancreas

Pancreatic cancer is resistant to most chemotherapeutic regimens. Based upon evidence of synergy between 5-fluorouracil (5-FU) and cisplatin (CDDP), and of enhanced 5-FU activity in the presence of leucovorin in other neoplasms, a phase II study of CDDP, 5-FU, and leucovorin was conducted to determine the efficacy of this regimen in patients with unresectable adenocarcinomas of the pancreas. Nineteen patients were enrolled, and all were evaluable for toxicity and response. One complete and two partial responses were observed (15.8%). Adverse effects were reversible and tolerable. This regimen has limited activity against pancreatic cancer, and cannot be recommended as standard therapy in this disease.     

p53 accumulation confers prognostic information in resectable adenocarcinomas with ductal but not with intestinal differentiation in the pancreatic head

The aim of the study was to examine the relation between p53 protein accumulation, clinicopathological variables and prognosis in resectable adenocarcinomas of the pancreatic head. The clinical records and tissue specimens of 82 consecutive patients resected for adenocarcinomas located in the head of the pancreas were reviewed retrospectively. Formalin-fixed and paraffin-embedded specimens from each tumour were stained with the monoclonal antibody DO7, and the nuclear p53 positivity within each tumour was assessed. Histopathological reclassification showed that 60 tumours exhibited ductal differentiation and 22 tumours intestinal differentiation. Twenty-five percent (15/60) of the ductal tumours and 50% (11/22) of the intestinal tumours were positive for p53 accumulation. p53 immunoreactivity was significantly correlated to a worse prognosis in the tumours of ductal differentiation, with median survival 0.76 years for p53 positive and 1.44 years for p53 negative patients. The p53 positivity of tumours with intestinal differentiation showed no such correlation. No correlation was found between p53 accumulation and other known prognostic factors in either the ductal or the intestinal type of tumours. Our results indicate that the tumour biology of ductal adenocarcinomas differs significantly from that of adenocarcinomas of the intestinal type located in the pancreatic head, and that p53 accumulation confers a worse prognosis only of ductal tumours. Subclassification of these tumours based on type of differentiation is therefore suggested since periampullary tumours include ductally as well as intestinally differentiated adenocarcinomas.