Frequent DYRK2 gene amplification in micropapillary element of lung adenocarcinoma - an implication in progression in EGFR-mutated lung adenocarcinoma.

The present study aimed to discern the molecular alterations involved in the progression of EGFR-mutated lung adenocarcinoma (LADC). We previously demonstrated that the micropapillary (mPAP) element is the most important histological factor for assessing malignant grades in LADCs. Therefore, mPAP and other elements were separately collected from three cases of EGFR-mutated LADC using laser capture microdissection and subjected to a comprehensive mRNA expression analysis. We focused on DYRK2 in this study because its level showed a substantial increase in EGFR-mutated LADCs with mPAP. We also immunohistochemically examined 130 tumors for the expression of DYRK2. The results confirmed a strong expression of DYRK2 in EGFR-mutated LADC with mPAP. Fluorescent in situ hybridization (FISH) analyses targeting the DYRK2 locus revealed frequent gene amplification in EGFR-mutated LADC, specifically occurring in the high-grade components, like mPAP. In summary, the results of this study suggest that DYRK2 overexpression through gene amplification is one of the molecular mechanisms responsible for promoting the progression of EGFR-mutated LADC.

Kallikrein family proteases KLK6 and KLK7 are potential early detection and diagnostic biomarkers for serous and papillary serous ovarian cancer subtypes.

BACKGROUND: Early detection of ovarian cancer remains a challenge due to widespread metastases and a lack of biomarkers for early-stage disease. This study was conducted to identify relevant biomarkers for both laparoscopic and serum diagnostics in ovarian cancer. METHODS: Bioinformatics analysis and expression screening in ovarian cancer cell lines were employed. Selected biomarkers were further validated in bio-specimens of diverse cancer types and ovarian cancer subtypes. For non-invasive detection, biomarker proteins were evaluated in serum samples from ovarian cancer patients. RESULTS: Two kallikrein (KLK) serine protease family members (KLK6 and KLK7) were found to be significantly overexpressed relative to normal controls in most of the ovarian cancer cell lines examined. Overexpression of KLK6 and KLK7 mRNA was specific to ovarian cancer, in particular to serous and papillary serous subtypes. In situ hybridization and histopathology further confirmed significantly elevated levels of KLK6 and KLK7 mRNA and proteins in tissue epithelium and a lack of expression in neighboring stroma. Lastly, KLK6 and KLK7 protein levels were significantly elevated in serum samples from serous and papillary serous subtypes in the early stages of ovarian cancer, and therefore could potentially decrease the high "false negative" rates found in the same patients with the common ovarian cancer biomarkers human epididymis protein 4 (HE4) and cancer antigen 125 (CA-125). CONCLUSION: KLK6 and KLK7 mRNA and protein overexpression is directly associated with early-stage ovarian tumors and can be measured in patient tissue and serum samples. Assays based on KLK6 and KLK7 expression may provide specific and sensitive information for early detection of ovarian cancer.

ERK phosphorylation is not increased in papillary thyroid carcinomas with BRAF(V600E) mutation compared to that of corresponding normal thyroid tissues.

BACKGROUND: An association between a BRAF(V600E) mutation and upregulation of mitogen-activated protein kinase (MAPK) pathways in human papillary thyroid carcinoma (PTC) tissues has not been demonstrated well outside of in vitro studies. The aims of this study were to evaluate the activation status of extracellular signal-regulated kinase 1/2 (ERK1/2) in human PTCs with BRAF(V600E) mutations compared to that of corresponding normal thyroid tissue and to determine the expressions of Raf kinase inhibitor protein (RKIP) and MAPK phosphatase 3 (MKP-3), possible regulators of ERK1/2 activation. METHODS: We analyzed the presence of BRAF(V600E) mutation and the expressions of BRAF, total ERK, p-ERK, RKIP, and MKP-3 in 33 PTCs and corresponding normal thyroid gland tissues using western blot analysis. RESULTS: BRAF(V600E) mutation was found in 28 (84.8%) of 33 PTCs, 96.4% (27/28) of which showed decreased p-ERK activity, while 75% (21/28) showed increased MKP-3 expression. There were significant differences in p-ERK and MKP-3 expressions between BRAF(V600E) (+) PTCs and normal thyroid glands (p < 0.001). There were no differences in expressions of BRAF, total ERK, and RKIP between PTCs and normal thyroid tissue, irrespective of the presence of BRAF(V600E) mutation. CONCLUSIONS: In human BRAF(V600E) (+) PTCs, ERK phosphorylation is decreased compared to normal thyroid glands and the observed decrease in ERK1/2 MAPK phosphorylation in BRAF(V600E) (+) PTCs may be associated with increased MKP-3 activity.

Correlation between chemosensitivity to anticancer drugs and telomerase reverse transcriptase mRNA expression in gastric cancer.

BACKGROUND: The determination of sensitive chemotherapy drugs for gastric cancer (GC) is one of the greatest challenges of adjuvant therapy. Here we evaluated the chemosensitivity of GC to anticancer drugs and the telomerase reverse transcriptase (hTERT) mRNA expression, and investigated the relationship of them. METHODS: The GC cells which were collected from 68 patients with primary GC were primary cultured. The chemosensitivity of GC cells to anticancer drugs was evaluated successfully using the MTT assay for 60 cases of GC cells, and the hTERT mRNA expression was examined in 60 cases of GC tissues and corresponding normal gastric mucosa and 6 cases of chronic superficial gastritis mucosa by in situ hybridization. RESULTS: Taxol, cisplatin and 5-fluorouracil were in general more effective than adriamycin and mitomycin for GC cells, and the chemosensitivity to anticancer drugs was associated with tumor histological types and a worse tumor grade. Compared to normal gastric mucosa tissues, hTERT mRNA expression was significantly increased in GC (P<0.05), which was related with a worse differentiation and drug-resistance to 5-fluorouracil or adriamycin in GC. CONCLUSIONS: These data demonstrate for the first time that examinations of hTERT mRNA expression as an important factor could be used to select the chemotherapeutic drugs for GC patients. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1793217009875483.

Indoleamine 2,3-dioxygenase affects the aggressiveness of intraductal papillary mucinous neoplasms through Foxp3+CD4+CD25+ T cells in peripheral blood.

OBJECTIVE: Intraductal papillary mucinous neoplasms (IPMNs) have a high malignant potential. We previously reported that peripheral Foxp3(+)CD4(+)CD25(+) T-cell (Foxp3(+) Treg) populations significantly increase with IPMN pathological aggressiveness. Dendritic cell-mediated induction of active Tregs from naive CD4(+) T cells requires indoleamine 2,3-dioxygenase (IDO). Here, we evaluated whether an IDO-Foxp3(+) Treg interaction plays a role in IPMN pathological aggressiveness. METHODS: We evaluated peripheral blood samples and resected specimens from 12 patients with IPMN. We analyzed Foxp3(+)CD4(+)CD25(+) T cells in peripheral blood by fluorescence-activated cell sorting, evaluated the resected specimens by anti-IDO antibody staining, and compared them with the patients' clinicopathological factors. RESULTS: The pathological aggressiveness of IPMN was significantly associated with the number of peripheral Foxp3(+) Tregs (P < 0.05) and IDO-positive cells per high-power field (HPF) (P < 0.01). There was a significant correlation between the numbers of peripheral Foxp3(+) Tregs and IDO-positive cells/HPF (r = 0.625, P < 0.01). Patients with 7 or more IDO-positive cells/HPF had a significantly higher recurrence rate than those with less than 7 IDO-positive cells/HPF (P < 0.01, log-rank test). CONCLUSIONS: Peripheral Foxp3(+) Tregs accurately reflect the aggressiveness of IPMNs. An increase in Foxp3(+) Tregs can be induced by local IDO-positive cells in IPMN.

Histologic and cytomorphologic features of ALK-rearranged lung adenocarcinomas.

Chromosomal rearrangements leading to constitutive activation of anaplastic lymphoma receptor tyrosine kinase (ALK) define a category of lung adenocarcinomas that may be amenable to targeted therapy with the ALK inhibitor crizotinib. Defining distinctive features of ALK-rearranged (ALK+) lung adenocarcinomas may help identify cases that merit molecular testing. However, data describing the morphologic features of ALK+ lung adenocarcinomas are conflicting and are primarily based on analysis of resected primary lung tumors. It is unclear whether the findings from prior studies are applicable to metastatic lung tumors or to small biopsy/cytology specimens. To address these issues, we examined resection, excision, small biopsy, and cytology cell block specimens from 104 ALK+ and 215 ALK- lung adenocarcinomas from primary and metastatic sites. All cases were evaluated for ALK rearrangements by fluorescence in situ hybridization. The predominant histologic subtypes and distinctive cytomorphologic features were assessed in each case. Primary ALK+ lung adenocarcinomas showed a significant association with solid, micropapillary, and papillary-predominant histologic patterns and tumor cells with a signet ring or hepatoid cytomorphology. Among metastatic lung tumors and small biopsy/cytology specimens, the only distinguishing morphologic feature of ALK+ tumors was the presence of signet ring cells. Based on these results, we developed a morphology-based scoring system for predicting ALK rearrangements in lung adenocarcinomas. The scoring system predicted ALK rearrangements in a new cohort of 78 lung adenocarcinomas (29 ALK+ and 49 ALK-) with a sensitivity of 88% and a specificity of 45%. In conclusion, ALK+ lung adenocarcinomas have distinctive morphologic features, with signet ring cells showing a significant association with ALK rearrangements irrespective of tumor site (primary vs metastatic) or specimen type. However, morphologic screening alone will not detect a minority of ALK+ lung adenocarcinomas, and the routine use of ancillary studies may be warranted to identify all patients who may benefit from crizotinib treatment.

Global tyrosine kinome profiling of human thyroid tumors identifies Src as a promising target for invasive cancers.

BACKGROUND: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using a bead-based, high-throughput system. METHODS: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells. RESULTS: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p<0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation. CONCLUSION: Global kinome analysis enables the discovery of novel targets for thyroid cancer therapy. Further investigation of Src targeted therapy for advanced thyroid cancer is warranted.

PARP-1 activity in normal and cancerous human endometrium and its relationship with quantity of abasic sites (AP).

OBJECTIVES: Poly (ADP-ribose) polymerase (PARP-1) is involved in the processes of DNA repair contributing to the maintenance of genomic stability. Recent data suggest that polymerase is involved in the development of endometrial adenocarcinomas and more advanced tumors displaying lowest enzyme protein expression. Data on PARP-1 activity regarding carcinogenesis in human endometrium are scarce. That was the reason why the authors of the present work wished to investigate the enzyme activity in human uterine hormone-dependent cancer and to compare the results with those obtained for normal endometrial tissue. The next aim was to check whether enzyme activity in normal and cancerous endometrium depends on the number of AP sites, which are widely known as oxidative stress DNA damage markers and PARP-1 activity stimulators. MATERIAL AND METHODS: Universal Colorimetric PARP Assay Kit was used to estimate the enzyme activity in units/ mg protein. Apurinic sites/105 base pairs (bp) were measured by Oxidative DNA Damage Kit Quantitative. Results were calculated for 47 endometrial samples and 15 uterine adenocarcinomas specimens. Finally the PARP-1 activity was analyzed for histological and some clinical features of neoplasms. RESULTS AND CONCLUSIONS: 1. no differences in PARP-1 activity were found in non-cancerous types of human endometrium; 2. mean enzyme activity was lower in sporadic endometrial cancers than in noncancerous endometrial specimens (2.89 +/- 0.55 vs 6.39 +/- 0.06; p < 0.005); 3. mean PARP-1 activity in lower grade neoplasms was higher than in G3 tumors and was lower in adenocarcinomas displaying deep uterine wall infiltration; 4. there was no relationship between PARP-1 activity and AP level.

Modulation of matrix metalloproteinase activity in human thyroid cancer cell lines using demethylating agents and histone deacetylase inhibitors.

BACKGROUND: The purpose of this study was to investigate the effects of treating human thyroid cancer cell lines with demethylating agents and histone deacetylase (HDAC) inhibitors to see if they would downregulate expression and activity of the matrix metalloproteinases (MMP)-2 and MMP-9, resulting in inhibition of growth and invasion. METHODS: A total of 1 papillary cancer cell line (TPC-1) and 3 follicular thyroid cancer cell lines (FTC-133, FTC-236, and FTC-238) were treated with the demethylating agent 5-azacytidine (5-AZC) and the HDAC inhibitors trichostatin A (TSA) and valproic acid (VA). The activity of MMP proteins was determined using gelatin zymography, and commercially available assays were used to quantify growth inhibition and thyroid cancer cell invasion. RESULTS: Treatment with TSA and VA resulted in decreased protein activity of MMP-2 and MMP-9 in all cell lines in a dose-dependent manner after 48 hours of treatment compared with untreated controls. In addition, 5-, TSA, and VA caused inhibition of growth in the range of 25-80% for all cell lines at 24, 48, and 72 hours. VA and TSA significantly decreased cell invasion in the FTC-133 and TPC-1 cell lines. CONCLUSION: The HDAC inhibitors TSA and VA decreased the protein activity of MMP-2 and MMP-9 and, in combination with the demethylating agent 5-AZC, inhibited cellular growth in human papillary and follicular thyroid cancer cell lines. These results elucidate our understanding of the pathways affected by the demethylating agents and HDAC inhibitors, and provide further evidence that MMPs are a potentially useful target for molecular therapies in patients with aggressive or refractory thyroid cancers.

Caspase-3 activity in papillary thyroid carcinomas.

AIM: The aim of this work was to assess caspase-3 activity in the tissue of papillary thyroid carcinomas of patients and analyze the peculiarities of changes in this activity depending upon a number of pathomorphological and clinical features of tumoral process. METHODS: Caspase-3 activity was determined by spectrophotometry with regard to acetyl-asp-glu-val-asp-paranitroanilide. RESULTS: At initial stages of tumor development, in the absence of metastases to lymph nodes, blood and lymphatic vessel invasion by tumor cells, extrathyroid spreading of tumor, sclerotic and fibrous changes in tumor stroma, and in the presence of tumor capsule, caspase-3 activity in papillary carcinoma tissue was higher compared to unchanged thyroid tissue of normofollicular structure. In case of a more aggressive behaviour of tumor, enzyme activity in carcinoma tissue did not differ significantly or (in case of extrathyroid spreading of tumor) was decreased compared to that in extratumoral tissue. In combination, this was expressed by a progressive decrease in caspase-3 activity in tumor tissue with increasing T category. Сaspase 3 activity was found to be much higher in the tissue of papillary carcinomas of follicular-papillary structure and lower in the tissue of tumors of mixed structure with solid areas, compared to that in the tissue of papillary carcinomas of typical papillary structure. CONCLUSIONS: The data obtainеd in assessing caspase-3 activity suggest that the intensity of spontaneous apoptosis of human papillary thyroid carcinoma cells depends upon the stage and aggressiveness of tumoral process.

[The mitogen-activated protein kinase (MAPK) signaling pathway in papillary thyroid cancer. From the molecular bases to clinical practice]. / Vía de señalización dependiente de la proteincinasa de activación mitogénica en el carcinoma papilar de tiroides. De las bases moleculares a la práctica clínica.

In recent years, significant progress has been made in elucidating the genetic bases promoting tumorigenesis in various human neoplasms. Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway is a major event in the carcinogenesis of papillary thyroid carcinoma (PTC), the most prevalent endocrine malignancy. Affected elements include RET/PTC rearrangements and point mutations of the Ras and BRAF genes. Mutations in these genes are found in over 70% of PTC. Chromosomal RET rearrangements, called RET/PTC, result in constitutive ligand-independent activation of RET kinase, which was the first genetic anomaly detected in PTC and is found in 5-70% of tumoral samples. Although less frequent, the activation of other tyrosine kinase receptors, such as NTRK1, c-Met or EGFR, has also been reported in PTC. The BRAF mutation represents the most common genetic alteration found in PTC. More than 90% of BRAF mutations lead to a change of a valine to a glutamic acid at position 600 (V600E). Finally, Ras is the least affected molecule in the pathway. A relationship between clinical behavior and these genetic alterations has been proposed. Thus, the BRAF mutation is associated with a more aggressive PTC phenotype and is correlated with poorer outcomes. However, no clear association has been found between RET/PTC and clinical features. The discovery of these alterations opens the way to new therapeutic strategies, especially to treat those patients in whom conventional therapy is not effective. Several new drugs are being tested, such as small molecule tyrosine kinase inhibitors. Some of these recently developed agents have begun to be used with promising results.

[Clinical significance of functional variants of matrix metalloproteinase genes in thyroid cancer].

Under analysis were results of genetic investigations of 105 patients with thyroid cancer (TC) treated from 2004 through 2007 in the St. Petersburg City Center of surgery and oncology of the endocrine system organs. The patients' age was from 17 to 80 years (mean age 51.6 +/- 1.9 years). The influence of functional variants of matrix metalloproteinase genes in TC was determined. A reliable correlation was noted between certain gene markers and TC patients' age. An association was revealed between matrix metalloproteinase-3 5A (more active allele) and size of the tumor. A reliably decreased frequency of hyperactive genotype MMP-1 2G was detected in a group of women with metastatic forms of papillary cancer as compared with patients without metastases. It was shown that MMP genes could be a substantial factor of slowing down the rate of malignant growth and invasive properties of cancer of the thyroid gland.

Usefulness of human telomerase reverse transcriptase in pancreatic juice as a biomarker of pancreatic malignancy.

OBJECTIVES: Human telomerase reverse transcriptase (hTERT), one of the subunits of telomerase, is a promising diagnostic marker for pancreatic cancer. In the present study, we did a large-scale analysis of 115 preoperative pancreatic juice specimens to evaluate the feasibility of detection of hTERT expression by immunohistochemistry for preoperative diagnosis of pancreatic malignancy. METHODS: The expression of hTERT was examined by immunohistochemistry in preoperative pancreatic juice samples. RESULTS: In pancreatic juice samples, hTERT expression was detectable in 84% of pancreatic ductal adenocarcinomas (PDACs), whereas 62% of PDACs were positive by cytology. In intraductal papillary mucinous neoplasms (IPMNs), hTERT expression was detectable in 88% of malignant IPMNs, whereas only 22% were positive by cytology. The sensitivity, specificity, and overall accuracy of hTERT expression for differentiation between carcinoma and other benign diseases were 85.1%, 82.1%, and 84.3%, respectively, whereas the same values for cytologic accuracy were 47.1%, 89.3%, and 57.4%, respectively. When the results of cytology and hTERT expression were combined, the sensitivity and overall accuracy increased to 92.0% and 87.8%, respectively. CONCLUSIONS: Our results suggested that the assessment of hTERT expression in preoperative pancreatic juice increased the sensitivity and accuracy of diagnosis of PDACs and malignant IPMNs without using special techniques.

In situ telomerase activity in pancreatic juice may discriminate pancreatic cancer from other pancreatic diseases.

OBJECTIVES: Detection of telomerase activity in pancreatic juice samples by telomeric repeat amplification protocol (TRAP) has been shown to be a useful diagnostic test for pancreatic cancer. However, some investigations suggested that the activity was not specific to pancreatic juice from pancreatic cancer. In the present study using an in situ TRAP assay, we investigated the specificity of telomerase activity in pancreatic juice for pancreatic cancer. METHODS: Pancreatic juice were obtained endoscopically at endoscopic retrograde cholangiopancreatography from 10 patients with pancreatic cancer, 4 with intraductal papillary neoplasm, and 3 with normal pancreas. In situ telomerase activity in pancreatic juice samples was examined by in situ TRAP assay and was compared with the results of cytological examination. RESULTS: Of 10 samples from pancreatic cancer, high telomerase activity in situ was detected in 9 and cancer cells in 7. Of 4 samples from intraductal papillary neoplasm, low telomerase activity in situ and mild atypical cells were detected in 2. Telomerase activity in situ was detected in none of 3 samples from normal pancreas. CONCLUSIONS: Detection of in situ telomerase activity in pancreatic juice might help discriminate pancreatic cancer from other pancreatic diseases.

Chemopreventive effects of a selective cyclooxygenase-2 inhibitor (etodolac) on chemically induced intraductal papillary carcinoma of the pancreas in hamsters.

The present study was designed to determine whether etodolac, a selective cyclooxygenase-2 inhibitor, prevents chemically induced intraductal papillary carcinoma (IPC) in the main pancreatic duct of hamsters. Hamsters were subjected to cholecystoduodenostomy with dissection of the distal end of the common duct. Four weeks after surgery, the surviving hamsters received subcutaneous injections of N-nitrosobis(2-oxopropyl)amine four times at a dose of 10 mg/kg body wt, every 2 weeks. The animals were divided into three groups according to the simultaneous oral intake of a standard pelleted diet containing etodolac at 0% (group CE, n = 30), 0.01% (group ET, n = 21) and 0.04% (group ET4, n = 25), respectively. Hamsters were killed for pathological examination at 36 weeks after the operation. The incidence of induced pancreatic carcinoma was 93, 81 and 72% in groups CE, ET and ET4, respectively. The pancreatic carcinomas were histologically classified into four types, i.e. tubular, papillary, cyst adenocarcinoma and IPC. The incidence of IPC and the number of IPCs per animal were significantly lower in groups ET4 (36% and 0.48) and ET (48% and 0.62) when compared with group CE (67% and 1.30). The proliferating cell nuclear antigen labeling indices in the non-cancerous epithelial cells of the main pancreatic duct were 2.8 and 6.8% in groups ET4 and ET, respectively, and were significantly lower than that in group CE (10.8%). In conclusion, etodolac inhibited N-nitrosobis(2-oxopropyl)amine-induced IPC in hamsters. Suppression of epithelial cell proliferation of the main pancreatic duct was considered as a possible mechanism of cancer prevention in this hamster model.

Expression of cyclooxygenase-2 and inducible nitric oxide synthase in ovarian surface epithelial carcinomas: is there any correlation with angiogenesis or clinicopathologic parameters?

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) products have been implicated in the regulation of immune system, tumor cell apoptosis, and angiogenesis in many human tumors. In this study, we investigated the expression of COX-2 and iNOS in ovarian carcinomas by immunohistochemistry and correlated the results with other prognostic parameters. Specimens from 100 ovarian carcinomas were studied by immunohistochemistry for COX-2 and iNOS expression, and angiogenesis microvessel density (MVD) was evaluated by CD34-stained microvessels. High COX-2 expression was observed in 85% of carcinomas. No correlation was found between COX-2 expression and clinicopathologic variables. Patients with high COX-2-expressed tumors had shorter overall survival, but it is not statistically significant. Expression of iNOS in serous and low-grade carcinomas was significantly higher than that in nonserous and high-grade carcinomas (P < 0.05). There was a positive correlation between COX-2 and iNOS expression (P= 0.009). No correlation of COX-2 and iNOS expression with MVD was found. Expression of iNOS showed no effect on survival of the patients. We found that iNOS expression might act in the first steps of carcinogenesis, whereas COX-2 expression was seen in more advanced tumors. Shorter overall survival of patients with high COX-2 expression might indicate new targets for therapy.

Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1beta in human papillary thyroid cancer cells.

We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2-7) and lower (e.g. BHP 14-9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2-7 and BHP 14-9 cells. In BHP 2-7 cells, promoter activity was dependent on an upstream sequence. In BHP 14-9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1beta (HNF-1beta) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1beta was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1beta-positive BHP 2-7 cells, but not in the HNF-1beta-negative BHP 14-9 cells. HNF-1beta bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1beta-specific antibody. Cotransfection of a HNF-1beta expression plasmid increased NNMT promoter activity significantly in both HNF-1beta-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1beta in BHP 14-9 cells increased endogenous NNMT protein levels. In summary, HNF-1beta functions as a transcription activator for NNMT gene expression in some papillary thyroid cancer cells.

Increased expression of matrix metalloproteinase-7 in invasive early gastric cancer.

BACKGROUND: Matrix metalloproteinase-7 (MMP-7), a proteolytic enzyme, is suspected to play an important role in the progression of various cancers. To clarify the clinical importance of MMP-7, we retrospectively analyzed MMP-7 expression in gastric epithelial tumors. METHODS: We tested 201 lesions (from 172 patients) of surgically or endoscopically resected gastric epithelial tumors (gastric cancer, 158 lesions; gastric adenoma, 32 lesions; hyperplastic polyp, 11 lesions). MMP-7 expression was immunohistochemically examined. Sections with immunostaining signals in more than 30% of tumor cells were judged to show positive expression. RESULTS: MMP-7 was expressed in 33.3% (67/201) of all lesions. MMP-7-positive tumors were significantly more frequent in diffuse-type adenocarcinomas (62.2%; 28/45) compared with intestinal-type lesions (31.9%; 36/113; P < 0.001). Cancers invading the submucosa or deeper (60.5%; 46/76) were showed positivity significantly more frequently than mucosal cancers (22.0%; 18/82; P < 0.001). MMP-7-positive lesions increased with the progression of gastric epithelial tumors, including adenomas, mucosal cancers, and cancers invading the submucosal layer or deeper (P < 0.001). MMP-7 expression occurred significantly more often in lymphatic invasion-positive cancers (65.1%; 41/63) than in lymphatic invasion-negative cancers (24.2%; 23/95; P < 0.001). CONCLUSIONS: The MMP-7-positive rate increased with the progression of gastric epithelial tumors, such as adenoma, mucosal cancer, and cancer invading the submucosal layer or deeper. MMP-7 was significantly associated with aggressive pathological phenotypes of cancer. The detection of the MMP-7 protein may be useful in pretherapeutic diagnosis.

Malignant myoepithelial cells are associated with the differentiated papillary structure and metastatic ability of a syngeneic murine mammary adenocarcinoma model.

BACKGROUND: The normal duct and lobular system of the mammary gland is lined with luminal and myoepithelial cell types. Although evidence suggests that myoepithelial cells might suppress tumor growth, invasion and angiogenesis, their role remains a major enigma in breast cancer biology and few models are currently available for exploring their influence. Several years ago a spontaneous transplantable mammary adenocarcinoma (M38) arose in our BALB/c colony; it contains a malignant myoepithelial cell component and is able to metastasize to draining lymph nodes and lung. METHODS: To characterize this tumor further, primary M38 cultures were established. The low-passage LM38-LP subline contained two main cell components up to the 30th subculture, whereas the higher passage LM38-HP subline was mainly composed of small spindle-shaped cells. In addition, a large spindle cell clone (LM38-D2) was established by dilutional cloning of the low-passage MM38-LP cells. These cell lines were studied by immunocytochemistry, electron microscopy and ploidy, and syngeneic mice were inoculated subcutaneously and intravenously with the different cell lines, either singly or combined to establish their tumorigenic and metastatic capacity. RESULTS: The two subpopulations of LM38-LP cultures were characterized as luminal and myoepithelium-like cells, whereas LM38-HP was mainly composed of small, spindle-shaped epithelial cells and LM38-D2 contained only large myoepithelial cells. All of them were tumorigenic when inoculated into syngeneic mice, but only LM38-LP cultures containing both conserved luminal and myoepithelial malignant cells developed aggressive papillary adenocarcinomas that spread to lung and regional lymph nodes. CONCLUSION: The differentiated histopathology and metastatic ability of the spontaneous transplantable M38 murine mammary tumor is associated with the presence and/or interaction of both luminal and myoepithelial tumor cell types.