Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter.

Key molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) are well described. However, the mechanisms through which clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we used an integrative genomics approach to examine CRC progression. We used laser capture microdissection to isolate colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas and metastases, and used gene expression profiling to identify pathways that were differentially expressed between the different cell types. We identified a number of potentially important transcriptional changes in developmental and oncogenic pathways, and noted a marked upregulation of EREG in primary and metastatic cancer cells. We confirmed this pattern of gene expression by in situ hybridization and observed staining consistent with autocrine expression in the tumor cells. Upregulation of EREG during the adenoma-carcinoma transition was associated with demethylation of two key sites within its promoter, and this was accompanied by an increase in the levels of epidermal growth factor receptor (EGFR) phosphorylation, as assessed by reverse-phase protein analysis. In CRC cell lines, we demonstrated that EREG demethylation led to its transcriptional upregulation, higher levels of EGFR phosphorylation, and sensitization to EGFR inhibitors. Low levels of EREG methylation in patients who received cetuximab as part of a phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers, including those of the head and neck, lung and bladder. Therefore, we propose that upregulation of EREG expression through promoter demethylation might be an important means of activating the EGFR pathway during the genesis of CRC and potentially other cancers.

Activation of Akt signaling in prostate induces a TGFß-mediated restraint on cancer progression and metastasis.

Mutations in the PTEN tumor suppressor gene are found in a high proportion of human prostate cancers, and in mice, Pten deletion induces high-grade prostate intraepithelial neoplasia (HGPIN). However, progression from HGPIN to invasive cancer occurs slowly, suggesting that tumorigenesis is subject to restraint. We show that Pten deletion, or constitutive activation of the downstream kinase AKT, activates the transforming growth factor (TGF)ß pathway in prostate epithelial cells. TGFß signaling is known to have a tumor suppressive role in many cancer types, and reduced expression of TGFß receptors correlates with advanced human prostate cancer. We demonstrate that in combination either with loss of Pten or expression of constitutively active AKT1, inactivation of TGFß signaling by deletion of the TGFß type II receptor gene relieves a restraint on tumorigenesis. This results in rapid progession to lethal prostate cancer, including metastasis to lymph node and lung. In prostate epithelium, inactivation of TGFß signaling alone is insufficient to initiate tumorigenesis, but greatly accelerates cancer progression. The activation of TGFß signaling by Pten loss or AKT activation suggests that the same signaling events that have key roles in tumor initiation also induce the activity of a pathway that restrains disease progression.

The role of SPARC in the TRAMP model of prostate carcinogenesis and progression.

SPARC (Secreted Protein Acidic and Rich in Cysteine), is a matricellular glycoprotein that is produced by tumor and/or neighboring stroma. In human prostate cancer, SPARC immunoreactivity is highest in metastatic lesions but distinct contributions of tumoral and stromal SPARC to tumorigenesis and progression are unclear. To determine the role of SPARC in primary prostate tumorigenesis, we crossed SPARC-null (SP(-/-)) with TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. TRAMP(+)/SP(-/-) mice exhibited accelerated cancer development and progression. Compared to their TRAMP(+)/SP(-/-) counterparts, TRAMP(+)/SP(+/+) tumors had fewer proliferating cells, and decreased cyclins A and D1 with increased p21(Cip) and p27(Kip). Similar effects on proliferation and cell-cycle regulators were observed in human prostate cancer cell lines, transiently transfected with pSPARC. TRAMP(+)/SP(-/-) tumors exhibited decreased stromal collagen, enhanced matrix metalloproteinase activity and increased vascular endothelial growth factor, proinflammatory cytokines. To determine the contribution of stromal SPARC, we evaluated subcutaneous tumor growth of TRAMP cell lines in syngeneic SP(+/+) and SP(-/-) mice. Enhanced growth, decreased stromal collagen and increased proteolysis were noted in SP(-/-) mice. Our findings demonstrate that both tumor and stromal SPARC are limiting for primary prostate tumorigenesis and progression, through effects on the cell cycle and the creation of a less favorable tumor microenvironment.

Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate cancer.

The gene for E3 ubiquitin ligase WWP1 is located at 8q21, a region frequently amplified in human cancers, including prostate cancer. Recent studies have shown that WWP1 negatively regulates the TGFbeta tumor suppressor pathway by inactivating its molecular components, including Smad2, Smad4 and TbetaR1. These findings suggest an oncogenic role of WWP1 in carcinogenesis, but direct supporting evidence has been lacking. In this study, we examined WWP1 for gene dosage, mRNA expression, mutation and functions in a number of human prostate cancer samples. We found that the WWP1 gene had copy number gain in 15 of 34 (44%) xenografts and cell lines from prostate cancer and 15 of 49 (31%) clinical prostate cancer samples. Consistently, WWP1 was overexpressed in 60% of xenografts and cell lines from prostate cancer. Mutation of WWP1 occurred infrequently in prostate cancer. Functionally, WWP1 overexpression promoted colony formation in the 22Rv1 prostate cancer cell line. In PC-3 prostate cancer cells, WWP1 knockdown significantly suppressed cell proliferation and enhanced TGFbeta-mediated growth inhibition. These findings suggest that WWP1 is an oncogene that undergoes genomic amplification at 8q21 in human prostate cancer, and WWP1 overexpression is a common mechanism involved in the inactivation of TGFbeta function in human cancer.

Neuromedin U is regulated by the metastasis suppressor RhoGDI2 and is a novel promoter of tumor formation, lung metastasis and cancer cachexia.

Most deaths from urinary bladder cancer are owing to metastatic disease. A reduction in Rho GDP Dissociation Inhibitor 2 (RhoGDI2) protein has been associated with increased risk of metastasis in patients with locally advanced bladder cancer, whereas in animal models, RhoGDI2 reconstitution in cells without expression results in lung metastasis suppression. Recently, we noted an inverse correlation between tumor RhoGDI2 and Neuromedin U (NMU) expression, suggesting that NMU might be a target of the lung metastasis suppressor effect of RhoGDI2. Here we evaluated whether NMU is regulated by RhoGDI2 and is functionally important in tumor progression. We used small interfering RNA knockdown of endogenous RhoGDI2 in poorly tumorigenic and non-metastatic human bladder cancer T24 cells and observed increased NMU RNA expression. Although NMU overexpression did not increase the monolayer growth of T24 or related T24T poorly metastatic human bladder cancer cells, it did augment anchorage-independent growth for the latter. Overexpression of NMU in T24 and T24T cells significantly promoted tumor formation of both cell lines in nude mice, but did not alter the growth rate of established tumors. Furthermore, NMU-overexpressing xenografts were associated with lower animal body weight than control tumors, indicating a possible role of NMU in cancer cachexia. NMU overexpression in T24T cells significantly enhanced their lung metastatic ability. Bioluminescent in vivo imaging revealed that lung metastases in T24T grew faster than the same tumors in the subcutaneous microenvironment. In conclusion, NMU is a RhoGDI2-regulated gene that appears important for tumorigenicity, lung metastasis and cancer cachexia, and thus a promising therapeutic target in cancer.

A novel method for gene expression mapping of metastatic competence in human bladder cancer.

Expression profiling by DNA microarray analysis has provided insights into molecular alterations that underpin cancer progression and metastasis. Although differential expression of microarray-defined probes can be related to numerical or structural chromosomal alterations, it is unclear if such changes are also clustered in distinct chromosomes or genomic regions and whether chromosomal alterations always reflect changes in gene expression. Here we apply the dChip algorithm and a novel technique to test the hypothesis that expression changes occurring as a function of tumor progression and metastasis are nonrandomly distributed. Expression profiling of a human xenograft model of lung metastasis phenotype indicates that chromosomes 2, 11, and 20 contain higher percentages of differentially expressed genes (P < .05). Furthermore, we show that a number of differentially expressed probes mapped to chromosome 17q, defining the existence of an expression "hot spot" corresponding to an area of gain determined by comparative genomic hybridization (CGH). Interestingly, other areas of gains detected by CGH were not associated with expression hot spots. In summary, we show that gene expression changes during bladder cancer lung metastasis occur nonrandomly in specific chromosomes and intrachromosomal locations.

The serine/threonine protein kinase, p90 ribosomal S6 kinase, is an important regulator of prostate cancer cell proliferation.

An increase in the activity of mitogen-activated protein kinase (MAPK) has been correlated with the progression of prostate cancer to advanced disease in humans. The serine/threonine protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of MAPK but its role in prostate cancer has not previously been examined. Increasing RSK isoform 2 (RSK2) levels in the human prostate cancer line, LNCaP, enhanced prostate-specific antigen (PSA) expression, an important diagnostic marker for prostate cancer, whereas inhibiting RSK activity using a RSK-specific inhibitor, 3Ac-SL0101, decreased PSA expression. The RSK2 regulation of PSA expression occurred via a mechanism involving both RSK2 kinase activity and its ability to associate with the coactivator, p300. RNA interference of the androgen receptor (AR) showed that the AR was important in the RSK2-mediated increase in PSA expression. RSK levels are higher in approximately 50% of human prostate cancers compared with normal prostate tissue, which suggests that increased RSK levels may participate in the rise in PSA expression that occurs in prostate cancer. Furthermore, 3Ac-SL0101 inhibited proliferation of the LNCaP line and the androgen-independent human prostate cancer line, PC-3. These results suggest that proliferation of some prostate cancer cells is dependent on RSK activity and support the hypothesis that RSK may be an important chemotherapeutic target for prostate cancer.

Reduced expression of metastasis suppressor RhoGDI2 is associated with decreased survival for patients with bladder cancer.

PURPOSE: RhoGDI2 was recently shown to be a metastasis suppressor gene in models of bladder cancer. We sought to further understand its importance in human cancer by determining the level of its expression and the distribution of its encoded protein in normal human tissues and cell lines and to evaluate whether its protein expression is a determinant of human bladder cancer progression. EXPERIMENTAL DESIGN: RhoGDI2 mRNA and protein expression was evaluated in cell lines and human tissues using Affymetrix and tissue microarrays, respectively. Tissue microarrays represented most human normal adult tissues and material from 51 patients that had undergone radical cystectomy for bladder cancer. In these 51 patients, the chi(2) test was used to test for associations between RhoGDI2 and stage, grade of urothelial carcinoma, histological type, and disease-specific survival status. Cox proportional hazards regression analyses were used to estimate the effect of RhoGDI2 expression level on time to development of metastatic disease and disease-specific survival time, adjusting for grade, stage, and histological type. RESULTS: In normal tissues, there was strong RhoGDI2 protein expression in WBCs, endothelial cells, and transitional epithelium. RhoGDI2 mRNA expression was inversely related to the invasive and metastatic phenotype in human bladder cancer cell lines. In patients with bladder cancer, univariate analysis indicated that reduced tumor RhoGDI2 protein expression was associated with a lower actuarial 5-year disease-free and disease-specific survival (P = 0.01). In addition, patients with tumors that had low or absent RhoGDI2 had a shorter time to disease-specific death (P

Retrospective study of Chlamydia trachomatis using the polymerase chain reaction on archival Papanicolaou-stained cytologic smears.

OBJECTIVE: To detect chlamydial DNA on archived Papanicolaou-stained (Pap) smears using the polymerase chain reaction (PCR) technique. STUDY DESIGN: A PCR assay was designed to identify chlamydial DNA using consensus sequences unique to the genus Chlamydia in the 16S rRNA gene. This assay produced a 109 base pair product containing a single Pvu II restriction site. One hundred cervicovaginal Pap smears from a teen clinic population were processed for DNA isolation and PCR. Amplifiable DNA was isolated from 93 of the 100 cases as determined by a human growth hormone gene. These specimens were subjected to chlamydial PCR. RESULTS: PCR analysis of the 93 samples yielded 6 that were positive for the chlamydial 16S rRNA sequence. The six positive chlamydial amplicons were purified and subjected to Pvu II restriction enzyme analysis to validate their identity. The analysis confirmed the identity of the products, as a single Pvu II restriction site resulted in 41 base pair and 68 base pair products, as predicted. CONCLUSION: PCR testing for Chlamydia trachomatis can be performed on DNA isolated from archival Pap smears. Using this methodology, 6.5% of young women in our teen clinic population were positive for chlamydial DNA.

Loss of heterozygosity at 13q14 and 13q21 in high grade, high stage prostate cancer.

BACKGROUND: Loss of heterozygosity (LOH) at chromosome 13q has been frequently detected in prostate cancer, and three regions (i.e., 13q14, 13q21, and 13q33) may harbor tumor suppressor genes important in this neoplasm. In this study, we examined the frequency of 13q LOH in advanced prostate cancers, in order to determine the clinicopathologic relevance of 13q LOH. METHODS: LOH was determined by analyzing microsatellite markers in 41 cases of microdissected predominantly high grade prostate cancer tissues and their matched nonneoplastic cells. The results were compared with those generated previously for lower grade, asymptomatic cancers. RESULTS: The frequencies of LOH at 13q14, 13q21, and 13q33 were 62% (21/34), 57% (20/35), and 34% (11/32), respectively. In comparison to previous results, LOH at 13q14 and 13q21 but not 13q33 was more frequent in prostate cancers that produced local clinical symptoms (bladder outlet obstruction) than those that did not (P < 0.05). LOH at 13q14 was also significantly more frequent in high grade and high stage cancers than those that were lower grade and lower stage (P < 0.05). CONCLUSIONS: Although the target genes on 13q have not been identified in carcinomas of the prostate, LOH at 13q14 in particular is associated with clinically significant prostate cancers. Further fine mapping of these loci may lead to identification of tumor suppressor genes that are deleted in aggressive carcinomas of the prostate.

An 800-kb region of deletion at 13q14 in human prostate and other carcinomas.

Deletions of regions at 13q14 have been detected by various genetic approaches in human cancers including prostate cancer. Several studies have defined one region of loss of heterozygosity (LOH) at 13q14 that seems to reside in a DNA segment of 7.1 cM between genetic markers D13S263 and D13S153. To define the smallest region of overlap (SRO) for deletion at 13q14, we first applied tissue microdissection and multiplex PCR to detect homozygous deletion and/or hemizygous deletion at 13q14 in 134 prostate cancer specimens from 114 patients. We detected deletions at markers D13S1227, D13S1272, and A005O48 in 13 (10%) of these tumor specimens. Of the 13 tumors with deletions, 12 were either poorly differentiated primary tumors or metastases of prostate cancer. To fine-map the deletion region, we then constructed a high-resolution YAC/BAC/STS/EST physical map based on experimental and database analyses. Several markers encompassing the deletion region were analyzed for homozygous deletion and/or hemizygous deletion in 61 cell lines/xenografts derived from human cancers of the prostate, breast, ovary, endometrium, cervix, and bladder, and a region of deletion was defined by duplex PCR assay between markers A005X38 and WI-7773. We also analyzed LOH at 13q14 in the 61 cell lines/xenografts using the homozygosity mapping of deletion approach and 26 microsatellite markers. We found 24 (39%) of the cell lines/xenografts to show LOH at 13q14 and defined a region of LOH by markers M1 and M5. Combination of homozygous or hemizygous deletion and LOH results defined the SRO for deletion to be an 800-kb DNA interval between A005X38 and M5. There are six known genes located in or close to the SRO for deletion. This region of deletion is at least 2 Mb centromeric to the RB1 tumor-suppressor gene and the leukemia-associated genes 1 and 2, each of which is located at 13q14. These data suggest that the 800-kb DNA segment with deletion contains a gene whose deletion may be important for the development of prostate and other cancers. This study also provides a framework for the fine-mapping, cloning, and identification of a novel tumor-suppressor gene at 13q14.

Inactivation of the E-cadherin gene in sporadic diffuse-type gastric cancer.

Loss of the cell adhesion molecule E-cadherin has been observed in a variety of human carcinomas, and germline E-cadherin mutations have been found in several familial cases of diffuse gastric cancer. We sought to determine the prevalence and nature of E-cadherin alterations in "sporadic" gastric carcinomas. We performed comprehensive sequencing of the coding region, loss of heterozygosity (LOH) analysis, and immunohistochemical protein expression determination on 40 sporadic gastric adenocarcinomas. In total, 7 of 25 diffuse-type cancers harbored genetic alterations in the E-cadherin gene. Novel mutations predicted to significantly compromise protein function were found within 4 of these cancers, 2 of which harbored alterations resulting in biallelic inactivation of the gene product. Three diffuse cancers failed to amplify Exon 8 of E-cadherin, suggesting the presence of a homozygous abnormality. Notably, one germline E-cadherin mutation was also identified within these "sporadic" diffuse cancers. Significant gene mutations were not found in the 14 intestinal-type or histologically mixed cancer. Immunohistochemistry revealed aberrant or negative protein expression in seven diffuse-type tumors, four of which correlated with the genetic alterations. Both diffuse and intestinal-type tumors exhibited low rates of LOH, suggesting that allelic loss at the locus is not a common mechanism for E-cadherin inactivation during gastric tumorigenesis. Our observations suggest that inactivation of the E-cadherin gene occurs only in a subset of diffuse-type gastric cancers, as the majority of cases did not contain genetic alterations or identifiable protein abnormalities. Germline E-cadherin alterations, although rare, may underlie some diffuse gastric cancer cases that have important biologic and practical implications

Molecular classification of human carcinomas by use of gene expression signatures.

Classification of human tumors according to their primary anatomical site of origin is fundamental for the optimal treatment of patients with cancer. Here we describe the use of large-scale RNA profiling and supervised machine learning algorithms to construct a first-generation molecular classification scheme for carcinomas of the prostate, breast, lung, ovary, colorectum, kidney, liver, pancreas, bladder/ureter, and gastroesophagus, which collectively account for approximately 70% of all cancer-related deaths in the United States. The classification scheme was based on identifying gene subsets whose expression typifies each cancer class, and we quantified the extent to which these genes are characteristic of a specific tumor type by accurately and confidently predicting the anatomical site of tumor origin for 90% of 175 carcinomas, including 9 of 12 metastatic lesions. The predictor gene subsets include those whose expression is typical of specific types of normal epithelial differentiation, as well as other genes whose expression is elevated in cancer. This study demonstrates the feasibility of predicting the tissue origin of a carcinoma in the context of multiple cancer classes.

Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer.

Detection, treatment, and prediction of outcome for men with prostate cancer increasingly depend on a molecular understanding of tumor development and behavior. We characterized primary prostate cancer by monitoring expression levels of more than 8900 genes in normal and malignant tissues. Patterns of gene expression across tissues revealed a precise distinction between normal and tumor samples, and revealed a striking group of about 400 genes that were overexpressed in tumor tissues. We ranked these genes according to their differential expression in normal and cancer tissues by selecting for highly and specifically overexpressed genes in the majority of cancers with correspondingly low or absent expression in normal tissues. Several such genes were identified that act within a variety of biochemical pathways and encode secreted molecules with diagnostic potential, such as the secreted macrophage inhibitory cytokine, MIC-1. Other genes, such as fatty acid synthase, encode enzymes known as drug targets in other contexts, which suggests new therapeutic approaches.

Novel DNA copy number losses in chromosome 12q12--q13 in adenoid cystic carcinoma.

In order to find common genetic abnormalities that may identify loci of genes involved in the development of adenoid cystic carcinoma (ACC), we investigated DNA copy number changes in 24 of these tumors by comparative genomic hybridization (CGH). Our results indicate that unlike many carcinomas, ACCs have relatively few changes in DNA copy number overall. Twenty tumors had DNA copy number changes, which were mostly restricted to a few chromosomal arms. A frequent novel finding was the loss of DNA copy number in chromosome 12q (eight tumors, 33%) with the minimal common overlapping region at 12q12--q13. Deletion in this region has not been reported to be frequent in other types of cancer analyzed by CGH. In addition, deletions in 6q23-qter and 13q21--q22 and gains of chromosome 19 were observed in 25% to 38% of ACCs. Deletion of 19q, previously reported in a small series of ACC, was not identified in the current group of carcinomas. The current CGH results for chromosomes 12 and 19 were confirmed by microsatellite allelotyping. These results indicate that DNA copy number losses in 12q may be important in the oncogenesis of ACC and suggest that the 12q12--q13 region may harbor a new tumor-suppressor gene.

Defining a common region of deletion at 13q21 in human cancers.

Previous molecular genetic analyses identified a region of deletion at 13q21 in a variety of human cancers, suggesting the existence of a tumor suppressor gene(s) at this locus. In our earlier study on prostate cancer, the region of deletion was confined to a 3.1 cM interval between D13S152 and D13S162. At present, however, no known gene located in this interval has been firmly implicated in cancer, and the region remains too large for gene identification. To fine-map the area of interest, we established a contig of bacterial artificial chromosome (BAC) clones, narrowed the region of deletion by loss of heterozygosity (LOH) and homozygosity-mapping-of-deletion (HOMOD) analyses in different types of cancers, and tested a candidate gene from the region for mutation and alteration of expression in prostate cancers. The contig consisted of 75 overlapping BAC clones. In addition to the generation of 47 new sequence-tagged-site (STS) markers from the ends of BAC inserts, 76 known STS and expressed sequence tag markers were mapped to the contig (25 kb per marker on average). The minimal region of deletion was further defined to be about 700 kb between markers D13S791 and D13S166 by LOH analysis of 42 cases of prostate cancer, and by HOMOD analysis of eight prostate cancer cell lines/xenografts and 49 cell lines from cancers of the breast, ovary, endometrium, and cervix, using 18 microsatellite markers encompassing the deletion region. A gene that is homologous to the WT1 tumor suppressor gene, AP-2rep (KLF12), was mapped in this region and was analyzed for its expression and genetic mutation. In addition to low levels of expression in both normal and neoplastic cells of the prostate, this gene did not have any mutations in a group of aggressive prostate cancers and cell lines/xenografts, as assessed by the methods of polymerase chain reaction-single strand conformational polymorphism analysis and direct sequencing. These studies suggest that a 700 kb interval at 13q21 harbors a tumor suppressor gene(s) that seems to be involved in multiple types of cancer, and that the AP-2rep gene is unlikely to be an important tumor suppressor gene in prostate cancer. The BAC contig and high-resolution physical map of the defined region of deletion should facilitate the cloning of a tumor suppressor gene(s) at 13q21.

Osteocalcin is incompletely spliced in non-osseous tissues.

Osteocalcin (OC) is known to be a bone tissue-specific protein, expression of which is believed to be controlled by the OC promoter. In this communication, we provided evidence to demonstrate that tissue-specific expression of OC was also regulated at the RNA splicing level. We identified incompletely spliced variants of human OC mRNA, which retain one or more introns during RNA splicing, existing dominantly in non-osseous organs. Northern blot analysis identified two OC RNA transcripts expressed in normal human tissues, but the expression level of the transcripts varied between the tissues. Most non-osseous tissues expressed transcripts with higher molecular weight, prominent in ovary, kidney, pancreas, spleen, thymus, prostate, and testis, than the expected size of OC mRNA as seen in bone marrow. RT-PCR analysis identified up to six OC transcripts in most tissues tested except bone marrow. Sequence analysis showed that four of five RNA variants contained intron 1 in common and the dominant one contained all three introns. MG63, an osteoblastic osteosarcoma cell, expressed only the completely-spliced form of OC, whereas incompletely spliced RNA was dominant in most prostate tumor cells. Combined study of in situ hybridization and immunohistochemistry revealed that OC RNA was highly expressed in prostate tumor epithelial cells while only very low levels of protein were detected, which confirms that there are OC RNA variants in non-osseous tissues. In conclusion, we demonstrated that OC mRNA is also expressed in several non-osseous tissues. However, only bone preferentially underwent the complete splicing event of all three introns. The function of other splicing variants of OC mRNA needs to be further investigated.

cDNA and genomic cloning of lacritin, a novel secretion enhancing factor from the human lacrimal gland.

Multiple extracellular factors are hypothesized to promote the differentiation of unstimulated and/or stimulated secretory pathways in exocrine secretory cells, but the identity of differentiation factors, particularly those organ-specific, remain largely unknown. Here, we report on the identification of a novel secreted glycoprotein, lacritin, that enhances exocrine secretion in overnight cultures of lacrimal acinar cells which otherwise display loss of secretory function. Lacritin mRNA and protein are highly expressed in human lacrimal gland, moderately in major and minor salivary glands and slightly in thyroid. No lacritin message or protein is detected elsewhere among more than 50 human tissues examined. Lacritin displays partial similarity to the glycosaminoglycan-binding region of brain-specific neuroglycan C (32 % identity over 102 amino acid residues) and to the possibly mucin-like amino globular region of fibulin-2 (30 % identity over 81 amino acid residues), and localizes primarily to secretory granules and secretory fluid. The lacritin gene consists of five exons, displays no alternative splicing and maps to 12q13. Recombinant lacritin augments unstimulated but not stimulated acinar cell secretion, promotes ductal cell proliferation, and stimulates signaling through tyrosine phosphorylation and release of calcium. It binds collagen IV, laminin-1, entactin/nidogen-1, fibronectin and vitronectin, but not collagen I, heparin or EGF. As an autocrine/paracrine enhancer of the lacrimal constitutive secretory pathway, ductal cell mitogen and stimulator of corneal epithelial cells, lacritin may play a key role in the function of the lacrimal gland-corneal axis.

Expression profiling of gastric adenocarcinoma using cDNA array.

To investigate the expression profile of gastric adenocarcinoma, cDNA array experiments were performed using Atlas Human Cancer 1.2 K Array (Clontech Laboratories, Palo Alto, CA) on nine xenografted and two primary gastric cancer samples. The expression of the tumor samples was compared to that of two normal gastric epithelial tissues. The expression pattern of the primary tumors was similar to that of xenografted tumors. The up-regulated genes had expression ratios ranging from 2.5 to 16, whereas the down-regulated genes had a range from -2.5 to -16. No variation in gene expression was detected in the analysis of the xenografted tumors versus the primary tumors, indicating that the xenografts represented the primary tumors well. Thirty-eight genes showed altered gene expression in 5 or more samples (>45%). Thirty-one genes were up-regulated and seven genes were down-regulated. The most abundantly up-regulated genes (ratio >5) included genes such as S100A4, CDK4, MMP14 and beta catenin. The GIF was markedly down-regulated (ratio < -10). To confirm our findings, six genes (three up- and three down-regulated) were selected for semi-quantitative RT-PCR analysis. The RT-PCR results were consistent with the array findings. Our approach revealed that several genes are abnormally expressed in gastric cancer and found that genes known to interact in several common molecular pathway(s) were consistently altered.