An in-vitro model of scirrhous carcinoma of the stomach using stomach fibroblasts derived from gastric cancer patients

BACKGROUND/AIMS: The mechanisms of the particular stromal changes that occur upon cancer invasion by scirrhous carcinoma of the stomach, in particular, the relationships among cancer cells, stomach fibroblasts and collagen, a major constituent of the stroma of the invasive tumor, have yet to be clarified. METHODOLOGY: Three different human fibroblast cell lines (TIG-101, MF-2, MKF-1) and a cancer cell line derived from scirrhous carcinoma of the stomach (KATO III) were cultured three-dimensionally in collagen gels to investigate collagen gel contraction by these cells as a model of scirrhous carcinoma of the stomach. RESULTS: The gels contracted and gradually decreased in size in all of the fibroblast (TIG-101, MF-2 and MKF-1) cultures, but not in the KATO III culture, and the extent of gel contraction was not uniform among the fibroblast cell lines. The extent of gel contraction when fibroblasts derived from stomach (MF-2, MKF-1) were co-cultured with KATO III cells in collagen gel was almost similar to that of fibroblasts alone. Moreover, microscopic examination following Masson's trichrome staining revealed condensation and remodeling of collagen fibrils only around the fibroblast cells. CONCLUSIONS: The extent of collagen gel contraction by fibroblasts may depend on their in vivo origin. This property appears to be characteristic of fibroblasts, but not of malignant epithelial cells, under this culture system. Furthermore, the results of the present study demonstrate that stomach fibroblasts may play an important role in the stromal changes associated with scirrhous gastric cancer.     

Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon

BACKGROUND AND AIMS: The recent findings of bone morphogenetic protein (BMP) receptor Ia mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer suggest a role for BMPs in the colonic epithelium and colon cancer. We investigated the role of BMP2 in the colon. METHODS: We assessed BMP receptor expression in cell lines using the reverse-transcribed polymerase chain reaction and immunoblotting. We investigated the effect of BMP2 on cell lines using the MTT assay and by immunoblotting for markers of differentiation, proliferation, and apoptosis. We assessed the expression of BMP2, its receptors, and signal transduction elements in mouse and human colon tissue using immunohistochemistry. We also investigated the effect of the BMP antagonist noggin in vivo in mice by assessing colon tissue with immunohistochemistry and immunoblotting. Finally, we investigated the expression of BMP2 in microadenomas from familial adenomatous polyposis patients. RESULTS: BMP receptors (BMPR) Ia, BMPR Ib, and BMPR II are all expressed in colonic epithelial cell lines. BMP2 inhibits colonic epithelial cell growth in vitro, promoting apoptosis and differentiation and inhibiting proliferation. BMP2, BMPRIa, BMPRIb, BMPRII, phosphorylated Smad1, and Smad4 are expressed predominantly in mature colonocytes at the epithelial surface in normal adult human and mouse colon. Noggin inhibits apoptosis and proliferation in mouse colonic epithelium in vivo. BMP2 expression is lost in the microadenomas of familial adenomatous polyposis patients. CONCLUSIONS: These data suggest that BMP2 acts as a tumor suppressor promoting apoptosis in mature colonic epithelial cells.     

Upregulation of mucosal soluble fas ligand and interferon-gamma may be involved in ulcerogenesis in patients with Helicobacter pylori-positive gastric ulcer

BACKGROUND: Excessive upregulation of gastric epithelial cell apoptosis is speculated to be associated with ulcerogenesis in Helicobacter pylori-positive peptic ulcer disease. H. pylori may have an ulcerogenic effect through induction of gastric epithelial cell apoptosis mediated by infiltrating T cells and their soluble products. METHODS: The contents of soluble Fas ligand (sFasL) and interferon-gamma (IFN-gamma) in organ cultures and the degree of apoptosis and the expression of apoptosis-related proteins in the gastric epithelium were examined using the mucosal tissues obtained from the antrum and the ulcer site in patients with H. pylori-positive gastric ulcer (GU). The molecular mechanisms of gastric epithelial cell apoptosis induced by sFasL and IFN-gamma were analyzed using epithelial cell lines, MKN 45 and KATO III. RESULTS: The mucosal tissues of the ulcer site had substantially higher contents of sFasL and IFN-gamma in organ cultures regardless of its healing stage in association with increased numbers of apoptotic cells and enhanced expression of proapoptotic proteins Bak and Bax in the surface foveolar epithelium as compared with the antral tissues in patients with H. pylori-positive GU. The addition of sFasL caused increases in cytotoxic cell death and caspase-3 activation in MKN 45 and KATO III cells in which IFN-gamma treated cells had more prominent effects than untreated cells. The expression of Bak in MKN 45 cells increased when they were treated with IFN-gamma. CONCLUSIONS: Upregulation of mucosal sFasL and IFN-gamma may be involved in ulcerogenesis in patients with H. pylori-positive GU through induction of gastric epithelial cell apoptosis.     

Epithelial-stromal interactions in the mouse and human mammary gland in vivo

This review deals with the development and hormonal responses of mouse and human mammary glands. A major focus of the review is the role of mesenchymal-epithelial interactions in embryonic mammary development and the role of stromal-epithelial interactions in mammary gland biology. Finally, we present a new model for studying growth, differentiation and hormonal response in human breast epithelium grown in vivo in nude mouse hosts. This new model involves the construction of tissue recombinants composed of human or mouse mammary fibroblasts plus human breast epithelium in polymerized collagen gels. In the model, mouse mammary fibroblasts and human breast fibroblasts appear to support the normal differentiation and growth of human breast epithelium equally. This observation raises the possibility of using mouse mammary fibroblasts from various mutant mice to assess the role of specific paracrine-acting gene products in human mammary gland biology and carcinogenesis.     

Targeting the stromal androgen receptor in primary prostate tumors at earlier stages

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates. The differential proliferation rates, yet with similarly expanded intermediate cell populations, indicated that the prostate stromal AR might play a more dominant role than the epithelial AR to promote primary tumor proliferation at an early stage of tumor. Tissue recombination of human prostate stromal cell lines (WPMY1-v or WPMY1-ARsi) with human prostate cancer epithelial cell lines (PC3-v or PC3-AR9) further demonstrated that the AR might function as a suppressor in epithelial cells and a proliferator in stromal cells in the primary prostate tumors. The dual roles of the AR in prostate epithelium and stroma may require us to reevaluate the target and timing of androgen-deprivation therapy for prostate cancer patients and may suggest a need to develop new drugs to selectively target stromal AR in the primary prostate tumors at earlier stages.     

Mucosal in Vitro Permeability in the Intestinal Tract of the Pig,the Rat,and Man: Species- and Region-Related Differences

Background: Excessive upregulation of gastric epithelial cell apoptosis is speculated to be associated with ulcerogenesis in Helicobacter pylori -positive peptic ulcer disease. H. pylori may have an ulcerogenic effect through induction of gastric epithelial cell apoptosis mediated by infiltrating T cells and their soluble products. Methods: The contents of soluble Fas ligand (sFasL) and interferon- &;#110 (IFN- &;#110 ) in organ cultures and the degree of apoptosis and the expression of apoptosis-related proteins in the gastric epithelium were examined using the mucosal tissues obtained from the antrum and the ulcer site in patients with H. pylori -positive gastric ulcer (GU). The molecular mechanisms of gastric epithelial cell apoptosis induced by sFasL and IFN- &;#110 were analyzed using epithelial cell lines, MKN 45 and KATO III. Results: The mucosal tissues of the ulcer site had substantially higher contents of sFasL and IFN- &;#110 in organ cultures regardless of its healing stage in association with increased numbers of apoptotic cells and enhanced expression of proapoptotic proteins Bak and Bax in the surface foveolar epithelium as compared with the antral tissues in patients with H. pylori -positive GU. The addition of sFasL caused increases in cytotoxic cell death and caspase-3 activation in MKN 45 and KATO III cells in which IFN- &;#110 -treated cells had more prominent effects than untreated cells. The expression of Bak in MKN 45 cells increased when they were treated with IFN- &;#110. Conclusions: Upregulation of mucosal sFasL and IFN- &;#110 may be involved in ulcerogenesis in patients with H. pylori- positive GU through induction of gastric epithelial cell apoptosis.     

Human prolactin receptors are insensitive to mouse prolactin: implications for xenotransplant modeling of human breast cancer in mice

Experimental testing of growth, metastatic progression and drug responsiveness of human breast cancer in vivo is performed in immunodeficient mice. Drug candidates need to show promise against human breast cancer in mice before being allowed into clinical trials. Breast cancer growth is under endocrine control by ovarian steroids and the pituitary peptide hormone prolactin. While it is recognized that the most relevant biologic effects of prolactin are achieved with prolactin from the matching species, the biologic efficacy of mouse prolactin for human prolactin receptors has not been recorded. Thus, it is unclear whether the mouse endocrine environment adequately reflects the hormonal environment in breast cancer patients with regard to prolactin. We now show both recombinant and natural pituitary-derived mouse prolactin to be a poor agonist for human prolactin receptors. Mouse prolactin failed to induce human prolactin receptor-mediated biologic responses of cell clustering, proliferation, gene induction and signal transduction, including activation of Stat5, Stat3, Erk1/2 and Akt pathways. Consistent data were derived from human breast cancer lines T-47D, MCF-7 and ZR-75.1, as well as human prolactin receptor-transfected COS-7 and 32D cells. Failure of mouse prolactin to activate human prolactin receptors uncovers a key deficiency of the mouse endocrine environment for human xenotransplant studies. Since most human breast cancers express prolactin receptors, human breast cancer transferred into mice is unnaturally selected for growth in the absence of circulating prolactin. The new insight raises concerns about the validity of analyzing biology and drug responsiveness of human breast cancer in existing mouse xenotransplant models.     

STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell-cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.     

Primers on Molecular Pathways — Notch

Notch, initially discovered and well characterized in Drosophila due to the notches apparent in the wing blades of mutant strains, and its signaling pathway play a key role in cell-cell communication, which involves gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life, including timely cell lineage specification of both the endocrine and exocrine pancreas. This pathway serves as an excellent model signaling cascade for the regulation of the transition from normal ductal epithelium to metaplasia to cancer. In human pancreatic cancer, the change in epithelial differentiation programming is an early hallmark. Several of these changes, including activation of the Notch pathway, which are observed in pathological scenarios, are also seen during normal embryonic development of the pancreas. Notch receptors, ligands as well as downstream targets have been identified to be upregulated in preneoplastic lesions to invasive pancreatic cancers in humans and mice, suggesting that Notch signaling may be an early event leading to accumulation of undifferentiated precursor cells in pancreatic cancers.     

Effects of oestrogen on gene expression in epithelium and stroma of normal human breast tissue

Oestrogen (E) is essential for normal and cancer development in the breast, while anti-oestrogens have been shown to reduce the risk of the disease. However, little is known about the effect of E on gene expression in the normal human breast, particularly when the epithelium and stroma are intact. Previous expression profiles of the response to E have been performed on tumour cell lines, in the absence of stroma. We investigated gene expression in normal human breast tissue transplanted into 9-10-week-old female athymic nude (Balb/c nu/nu) mice. After 2 weeks, when epithelial proliferation is minimal, one-third of the mice were treated with 17beta-oestradiol (E2) to give human luteal-phase levels in the mouse, which we have previously shown to induce maximal epithelial cell proliferation. RNA was isolated from treated and untreated mice, labelled and hybridized to Affymetrix HG-U133A (human) GeneChips. Gene expression levels were generated using BioConductor implementations of the RMA and MAS5 algorithms. E2 treatment was found to represent the largest source of variation in gene expression and cross-species hybridization of mouse RNA from xenograft samples was demonstrated to be negligible. Known E2-responsive genes (such as TFF1 and AREG), and genes thought to be involved in breast cancer metastasis (including mammoglobin, KRT19 and AGR2), were upregulated in response to E treatment. Genes known to be co-expressed with E receptor alpha in breast cancer cell lines and tumours were both upregulated (XBP-1 and GREB1) and downregulated (RARRES1 and GATA3). In addition, genes that are normally expressed in the myoepithelium and extracellular matrix that maintain the tissue microenvironment were also differentially expressed. This suggests that the response to oestrogen in normal breast is highly dependent upon epithelial-stromal/myoepithelial interactions which maintain the tissue microenvironment during epithelial cell proliferation.     

Mammary stroma modulates hormonal responsiveness of mammary epithelium in vivo in the mouse

Several lines of evidence indicate that there may be important stromal influences on epithelial behavior in a number of adult organs. The present studies were undertaken to examine the contribution of mammary gland stroma to mammary epithelial cell hormonal responsiveness in vivo. To accomplish this, mouse mammary epithelium from a developmental state that is not responsive to estrogen (E)-regulation of progesterone receptors (3-week-old) was surgically recombined with mammary stroma from a developmental state that is E-responsive (10-week-old) and vice versa. The recombinants were then tested in vivo for the ability of E to regulate epithelial progesterone receptors. The results demonstrate that when immature nonresponsive epithelium is transplanted to mature stroma of E-responsive mice, the epithelium prematurely acquires E responsiveness. When the converse experiment was carried out, mature E-responsive epithelium retained its responsiveness when transplanted to stroma of immature nonresponsive mice. The contribution of the host systemic milieu to changes observed upon transplantation was also assessed. To do this, mature stroma was transplanted to nonresponsive mice before transplantation with nonresponsive epithelium. Under these conditions, the mature stroma was still able to promote responsiveness. Analysis of mammary gland morphology of the surgical recombinants revealed no significant differences in epithelial structures or organization that could account for the observed differences in hormone responsiveness. From these results we conclude that the local environment of mammary stroma has the capacity to influence mammary epithelial cell behavior and can modulate epithelial hormonal responsiveness in vivo. The mechanisms underlying stromal influences on epithelial behavior remain to be elucidated.     

AKT activation promotes metastasis in a mouse model of follicular thyroid carcinoma

The phosphatidylinositol 3-kinase/AKT pathway is crucial to many cell functions, and its dysregulation in tumors is a common finding. The molecular basis of follicular thyroid cancer metastasis is not well understood but may also be influenced by AKT activation. We previously created a knockin mutant mouse that expresses a mutant thyroid hormone receptor-beta gene (TRbetaPV mouse) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. In this study, we investigated whether our mouse model exhibits similar AKT activation as human follicular thyroid cancer. Western blot analysis on thyroids from both wild-type and TRbeta(PV/PV) mice revealed elevation of activated AKT in TRbeta(PV/PV) mice. Immunohistochemistry and confocal microscopy reveal activated AKT in both the thyroid and metastatic lesions of TRbeta(PV/PV) mice. Whereas all three AKT isoforms were overexpressed in primary tumors from TRbeta(PV/PV) mice in the cytoplasm of thyroid cancer cells, only AKT1 was also found in the nucleus, matching the localization of activated AKT in a pattern similar to human follicular thyroid cancer. In the metastases, all AKT isoforms correlated with phosphorylated AKT nuclear localization. We created primary thyroid cell lines derived from TRbeta(PV/PV) mice and found reduction of phosphorylated AKT levels or AKT downstream targets diminishes cell motility. Activated AKT is common to both human and mouse follicular thyroid cancer and is correlated with increased cell motility in vitro and metastasis in vivo. Thus, TRbeta(PV/PV) mice could be used to further dissect the detailed pathways underlying the progression and metastasis of follicular thyroid carcinoma.     

Models of ovarian cancer--are we there yet?

Ovarian cancer is the most lethal of all gynecological cancers and arises most commonly from the surface epithelium. Successful clinical management of patients with epithelial ovarian cancer is limited by the lack of a reliable and specific method for early detection, and the frequent recurrence of chemoresistant disease. Experimental models are of crucial importance not only to understand the biological and genetic factors that influence the phenotypic characteristics of the disease but also to utilize as a basis for developing rational intervention strategies. Ovarian cancer cell lines derived from ascites or primary ovarian tumors have been used extensively and can be very effective for studying the processes controlling growth regulation and chemosensitivity or evaluating novel therapeutics, both in vitro and in xenograft models. While our limited knowledge of the initiating events of ovarian cancer has restricted the development of models in which the early pathogenic events can be studied, recent advances in the ability to manipulate gene expression in ovarian surface epithelial cells in vitro and in vivo have begun to provide insights into the molecular changes that may contribute to the development of ovarian cancer. This review highlights the strengths and weaknesses of some of the current models of ovarian cancer, with special consideration of the recent progress in modeling ovarian cancer using genetically engineered mice.     

Transforming growth factor-beta 1 and its receptors in human lung cancer and mouse lung carcinogenesis

The transforming growth factor-betas (TGF-beta s) are multifunctional proteins that inhibit the proliferation of many epithelial cells through a set of cell protein receptors that includes the TGF-beta type I (RI) and type II (RII) receptors. Loss of growth inhibition by TGF-beta is thought to contribute to the development of many types of tumors. In the present study, we have examined expression of the proteins and mRNAs for TGF-beta 1, TGF-beta RI, and TGF-beta RII in normal human lung, well-characterized non-small cell lung cancer (NSCLC) cell lines, and primary NSCLC specimens. Immunohistochemical staining for TGF-beta 1, TGF-beta RI, and TGF-beta RII using specific antibodies in normal human lung showed expression of the 3 proteins in the epithelium of bronchi and bronchioles as well as in alveoli. Differential expression of TGF-beta RI and TGF-beta RII proteins was detected in 5 NSCLC cell lines using Western blot analysis, with reduced levels in 3 cell lines. A panel of 45 formalin-fixed and paraffin-embedded NSCLC specimens showed positive immunostaining for TGF-beta 1, TGF-beta RI, and TGF-beta RII, with reduced TGF-beta RII in poorly differentiated adenocarcinomas and squamous cell carcinomas and some moderately differentiated adenocarcinomas. In situ hybridization studies conducted with specific riboprobes for TGF-beta 1, TGF-beta RI, and TGF-beta RII showed corresponding localization of expression of the mRNAs in the specimens that showed positive immunostaining for the proteins. To investigate the roles of TGF-beta 1, TGF-beta RI, and TGF-beta RII in chemically induced mouse lung tumorigenesis, we examined the expression of their proteins and mRNAs in 2 mouse model systems. Whereas expression of the proteins and mRNAs for TGF-beta 1 and TGF-beta RI was comparable in lung adenomas and bronchioles of A/J mice treated with benzo(alpha)pyrene, decreased immunostaining and hybridization for TGF-beta RII protein and mRNA was detected in 50% of lung adenomas in these mice. Interestingly, expression of TGF-beta 1 and the TGF-beta receptor proteins was similar to that of bronchioles in C57B1/6 mice and their littermates heterozygous for deletion of the TGF-beta 1 gene treated with diethylnitrosamine. These data show that reduced levels of expression of TGF-beta RII occur in some, but not all, human and mouse lung tumors. This suggests that different mechanisms of action, some of which may involve the TGF-beta signaling pathway, may contribute to the progression of lung tumorigenesis.     

A small composite probasin promoter confers high levels of prostate-specific gene expression through regulation by androgens and glucocorticoids in vitro and in vivo

Transient transfection studies have shown that the probasin (PB) promoter confers androgen selectivity over other steroid hormones, and transgenic animal studies have demonstrated that the PB promoter will target androgen, but not glucocorticoid, regulation in a prostate-specific manner. Previous PB promoters either targeted low levels of transgene expression or became too large to be conveniently used. The goal was to design a PB promoter that would be small, yet target high levels of prostate-specific transgene expression. Thus, a composite probasin promoter (ARR2PB) coupled to the bacterial chloramphenicol acetyltransferase reporter (ARR2PBCAT) was generated and tested in prostatic and nonprostatic cell lines and in a transgenic mouse model. In PC-3, LNCaP, and DU145 prostate cancer cell lines, the ARR2PB promoter gave basal expression and was induced in response to androgen and glucocorticoid treatment after cotransfection with the respective steroid receptor. Basal expression of ARR2PBCAT in the nonprostatic COS-1, MCF-7, ZR-75-1, and PANC-1 cell lines was very low; however, CAT activity could be induced in response to androgens and glucocorticoids when cells were cotransfected with either the AR or GR. In contrast to the transfection studies, ARR2PBCAT transgene expression remained highly specific for prostatic epithelium in transgenic mice. CAT activity decreased after castration, and could be induced by androgens and, in addition, glucocorticoids. This demonstrates that the necessary sequences required to target prostate-specific epithelial expression are contained within the composite ARR2PB minimal promoter, and that high transgene expression can now be regulated by both androgens and glucocorticoids. The ARR2PB promoter represents a novel glucocorticoid inducible promoter that can be used for the generation of transgenic mouse models and in viral gene therapy vectors for the treatment of prostate cancer in humans.