Antibodies blocking adhesion and matrix binding domains of laminin-332 inhibit tumor growth and metastasis in vivo.

Laminin-332 (LN-332), which is essential for epithelial cell adhesion and migration, is up-regulated in most invasive carcinomas. Association between LN-332 and carcinoma cell integrins and stroma collagen is thought to be important for tumor growth and metastasis. Here, we show that function blocking LN-332 antibodies interfering with cellular adhesion and migration in vitro evoke apoptotic pathways. The antibodies also target epithelial tumors in vivo. Antibodies against the cell binding domain of the alpha3 chain of LN-332 inhibited tumor growth by up to 68%, and antibodies against the matrix binding domains of the beta3 and gamma2 chains significantly decreased lung metastases. The LN-332 antibodies appear to induce tumor cell anoikis and subsequent programmed cell death and reduce migration by interfering with tumor cell matrix interactions.

Targeting a tumor-specific laminin domain critical for human carcinogenesis.

Laminin-332 is critical for squamous cell carcinoma (SCC) tumorigenesis, but targeting it for cancer therapy has been unachievable due to key role of laminin-332 in promoting tissue integrity. Here, we show that a portion of laminin-332, termed G45, which is proteolytically removed and absent in normal tissues, is prominently expressed in most human SCC tumors and plays an important role in human SCC tumorigenesis. Primary human keratinocytes lacking G45 (DeltaG45) showed alterations of basal receptor organization, impaired matrix deposition, and increased migration. After SCC transformation, the absence of G45 domain in DeltaG45 cells was associated with deficient extracellular signal-regulated kinase and phosphotidylinositol 3-kinase (PI3K) pathway activation, impaired invasion, deficient metalloproteinase activity, and absent tumorgenicity in vivo. Expression of G45 or activated PI3K subunit in DeltaG45 cells reversed these abnormalities. G45 antibody treatment induced SCC tumor apoptosis, decreased SCC tumor proliferation, and markedly impaired human SCC tumorigenesis in vivo without affecting normal tissue adhesion. These results show a remarkable selectivity of expression and function for laminin-332 G45 in human SCC tumorigenesis and implicate it as a specific target for anticancer therapy.

Mature human thymocytes migrate on laminin-5 with activation of metalloproteinase-14 and cleavage of CD44.

We have previously shown that laminin-5 is expressed in the human thymic medulla, in which mature thymocytes are located. We now report that laminin-5 promotes migration of mature medullary thymocytes, whereas it has no effect on cortical immature thymocytes. Migration was inhibited by blocking mAbs directed against laminin-5 integrin receptors and by inhibitors of metalloproteinases. Interactions of thymocytes with laminin-5 induced a strong up-regulation of active metalloproteinase-14. However, we found that thymocytes did not cleave the laminin-5 gamma(2) chain, suggesting that they do not use the same pathway as epithelial cells to migrate on laminin-5. Interactions of thymocytes with laminin-5 also induced the release of a soluble fragment of CD44 cell surface molecule. Moreover, CD44-rich supernatants induced thymocyte migration in contrast with supernatants depleted in CD44 by immunoadsorption. CD44 cleavage was recently reported to be due to metalloproteinase-14 activation and led to increased migration in cancer cells. Thus, in this study, we show that laminin-5 promotes human mature thymocyte migration in vitro via a multimolecular mechanism involving laminin-5 integrin receptors, metalloproteinase-14 and CD44. These data suggest that, in vivo, laminin-5 may function in the migration of mature thymocytes within the medulla and be part of the thymic emigration process.

Production, purification, and functional analysis of recombinant human and mouse 17beta-hydroxysteroid dehydrogenase type 7.

17beta-Hydroxysteroid dehydrogenases (17HSDs) have a central role in the regulation of the biological activity of sex steroid hormones. There is increasing evidence that in addition to their importance in gonads, these hormones also have substantial metabolic roles in a variety of peripheral tissues. In the present study, the cDNA of human 17HSD type 7 was cloned. In silico, the gene corresponding to the cDNA was localized on chromosome 1q23, close to the locus of hereditary prostate cancer 1 (HPC1) (1q24-25) and primary open-angle glaucoma (GLC1A) (1q23-25). Further, a pseudogene was found on chromosome 1q44, close to the locus of predisposing for early-onset prostate cancer (PCaP) (1q42.2-43). Both human (h17HSD7) and mouse 17HSD type 7 (m17HSD7) were for the first time produced as recombinant proteins and purified for functional analyses. Further, kinetic parameters and specific activities were described. h17HSD7 converted estrone (E1) to a more potent estrogen, estradiol (E2), and dihydrotestosterone (DHT), a potent androgen, to an estrogenic metabolite 5alpha-androstane-3beta, 17beta-diol (3betaA-diol) equally, thereby catalyzing the reduction of the keto group in either 17- or 3-position of the substrate. Minor 3betaHSD-like activity towards progesterone (P) and 20-hydroxyprogesterone (20-OH-P), leading to the inactivation of P by h17HSD7, was also detected. m17HSD7 efficiently catalyzed the reaction from E1 to E2 and moderately converted DHT to an inactive metabolite 5alpha-androstane-3alpha,17beta-diol (3alphaA-diol) and to an even lesser degree 3betaA-diol. The mouse enzyme did not metabolize P or 20-OH-P. The expression of 17HSD type 7 was observed widely in human tissues, most distinctly in adrenal gland, liver, lung, and thymus. Based on the enzymatic characteristics and tissue distribution, we conclude that h17HSD7 might be an intracrine regulator of steroid metabolism, fortifying the estrogenic milieu in peripheral tissues.

Mammalian tolloid metalloproteinase, and not matrix metalloprotease 2 or membrane type 1 metalloprotease, processes laminin-5 in keratinocytes and skin.

Laminin-5, a major adhesive ligand for epithelial cells, undergoes processing of its gamma2 and alpha3 chains. This study investigated the mechanism of laminin-5 processing by keratinocytes. BI-1 (BMP-1 isoenzyme inhibitor-1), a selective inhibitor of a small group of astacin-like metalloproteinases, which includes bone morphogenetic protein 1 (BMP-1), mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mammalian Tolloid-like 2 (mTLL-2), inhibited the processing of laminin-5 gamma2 and alpha3 chains in keratinocyte cultures in a dose-dependent manner. In a proteinase survey, all BMP-1 isoenzymes processed human laminin-5 gamma2 and alpha3 chains to 105- and 165-kDa fragments, respectively. In contrast, MT1-MMP and MMP-2 did not cleave the gamma2 chain of human laminin-5 but processed the rat laminin gamma2 chain to an 80-kDa fragment. An immunoblot and quantitative PCR survey of the BMP-1 isoenzymes revealed expression of mTLD in primary keratinocyte cultures but little or no expression of BMP-1, mTLL-1, or mTLL-2. mTLD was shown to cleave the gamma2 chain at the same site as the previously identified BMP-1 cleavage site. In addition, mTLD/BMP-1 null mice were shown to have deficient laminin-5 processing. Together, these data identify laminin-5 as a substrate for mTLD, suggesting a role for laminin-5 processing by mTLD in the skin.

Downregulation of estrogen-metabolizing 17 beta-hydroxysteroid dehydrogenase type 2 expression correlates inversely with Ki67 proliferation marker in colon-cancer development.

The 17HSDs are a group of isozymes that catalyze the interconversion between high-activity 17 beta-hydroxysteroids and low-activity 17-ketosteroids. In the present study, we characterized the expression of 17HSD types 1 and 2 in normal and malignant gastrointestinal tissues and cells. Using the colon as a model for cancer of the gastrointestinal tract, expression of the 17HSD enzymes in cancer development was studied and correlated with proliferation and differentiation markers as assessed by Ki67 and mucin staining, respectively. In normal colon and small intestine, 17HSD type 2 mRNA was expressed in the surface epithelial cells and, to a lesser extent, in the cryptal epithelial cells. In colon-cancer specimens, 17HSD type 2 expression was downregulated both in the tissues and in the cell lines and correlated inversely with the proliferation marker. No expression for the 17HSD type 1 enzyme was observed in normal or cancerous gastrointestinal tract tissues. In line with the expression studies, 17HSD activity measurements with colon cells showed that only the oxidative conversion of E2 to E1 was present, and Northern blot analysis showed the signal only for 17HSD type 2. Localization of the ERs alpha and beta, assessed by immunohistochemistry and in situ hybridization, showed the presence of ER beta in the lamina propria of the colon. Our study shows that 17HSD type 2 expression is associated with the functional integrity of the gastrointestinal tract. The decrease in expression of the type 2 enzyme may increase estrogen influence in colon cancer.