Citations

Abstract Metastatic invasion is the primary cause of breast cancer mortality, and adhesion receptors, such as CD44, are believed to be critical in this process. Historically, primary breast tumor epithelium has been investigated in isolation from other tissue components, leading to the common interpretation that CD44 and its primary ligand, hyaluronan, promote invasion. Here, we provide in vivo evidence showing CD44 antagonism to breast cancer metastasis. In a mouse model of spontaneously metastasizing breast cancer (MMTV-PyV mT), we found that loss of CD44 promotes metastasis to the lung. Localization studies, in combination with a novel hyaluronan synthase-GFP transgenic mouse, show a restricted pattern of expression for CD44 and hyaluronan. Whereas CD44 is expressed in tumor epithelium, hyaluronan synthase expression is restricted to stromal-associated cells. This distinct CD44 and hyaluronan pattern of distribution suggests a role for epithelial-stromal interaction in CD44 function. To define the relevance of this spatial regulation, we developed an in vitro invasion assay to emulate invasion into the extracellular matrix. Invasion of CD44-positive tumor cells was inhibited in hyaluronan-containing matrices, whereas blocking CD44-hyaluronan association increased invasion. Collectively, these data show that during breast cancer progression, hyaluronan-CD44 dynamics occurring through epithelial-stromal interactions are protective against metastasis.

Abstract Targeting tumor-associated vascular endothelium by replication-competent viral vectors is a promising strategy for cancer gene therapy. Here we describe the development of a viral vector based on the Edmonston vaccine strain of measles virus targeted to integrin αvβ3, which is expressed abundantly on activated but not quiescent vascular endothelium. We displayed a disintegrin, M28L echistatin that binds with a high affinity to integrin αvβ3 on the COOH terminus of the viral attachment (H) protein and rescued the replication-competent recombinant virus by reverse genetics. The new targeted virus was named measles virus echistatin vector (MV-ERV). Its native binding to CD46 was purposefully retained to allow virus infection of tumor cells expressing this receptor. MV-ERV correctly displayed echistatin on the outer surface of its envelope and produced interesting ring formation phenomena due to cell detachment upon infection of susceptible Vero cells in vitro. MV-ERV grew to 106 plaque-forming units/mL, slightly lower than the parental Edmonston strain of measles virus (MV-Edm), but it selectively infected Chinese hamster ovary cells expressing integrin αvβ3. It also selectively infected both bovine and human endothelial cells on matrigels and unlike MV-Edm, MV-ERV infected newly formed blood vessels in chorioallantoic membrane assays. In animal models, MV-ERV but not the control MV-Edm caused the regression of s.c. xenografts of resistant multiple myeloma tumors (MM1) in severe combined immunodeficient mice. The tumors were either completely eradicated or their growth was significantly retarded. The specificity, potency, and feasibility of MV-ERV infection clearly show the potential use of MV-ERV in gene therapy for targeting tumor-associated vasculature for the treatment of solid tumors.

AbstractCarney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a+/− mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at ∼6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP–responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation.

AbstractWe have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8+ T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8+ T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy.

Abstract Prostate cancer is a leading and increasingly prevalent cause of cancer death in men. Whereas family history of disease is one of the strongest prostate cancer risk factors and suggests a hereditary component, the predisposing genetic factors remain unknown. We first showed that KLF6 is a tumor suppressor somatically inactivated in prostate cancer and since then, its functional loss has been further established in prostate cancer cell lines and other human cancers. Wild-type KLF6, but not patient-derived mutants, suppresses cell growth through p53-independent transactivation of p21. Here we show that a germline KLF6 single nucleotide polymorphism, confirmed in a tri-institutional study of 3,411 men, is significantly associated with an increased relative risk of prostate cancer in men, regardless of family history of disease. This prostate cancer–associated allele generates a novel functional SRp40 DNA binding site and increases transcription of three alternatively spliced KLF6 isoforms. The KLF6 variant proteins KLF6-SV1 and KLF6-SV2 are mislocalized to the cytoplasm, antagonize wtKLF6 function, leading to decreased p21 expression and increased cell growth, and are up-regulated in tumor versus normal prostatic tissue. Thus, these results are the first to identify a novel mechanism of self-encoded tumor suppressor gene inactivation and link a relatively common single nucleotide polymorphism to both regulation of alternative splicing and an increased risk in a major human cancer.

Abstract Galectin-3 (Gal-3), a β-galactoside-binding protein is expressed in a specific cell–type manner in pituitary tumors. Here we questioned the mechanism of Gal-3 expression in pituitary tumors, by using methylation-specific PCR and DNA sequence analyses to analyze the methylation status of the promoter region of the LGALS3 gene. DNA analysis of a human pituitary tumor, breast carcinoma cell lines, and thyroid carcinoma cell lines showed that in cells expressing Gal-3 protein, the LGALS3 gene was unmethylated, whereas in Gal-3 null cells, the promoter of the LGALS3 gene was methylated. Treatment of cells with 30 μmol/L 5-aza-2′-deoxycytidine induced Gal-3 mRNA and protein expression. Among pituitary tumors, 30% (7/23), mainly in follicle-stimulating hormone/luteinizing hormone–producing (38%) and null cell (57%) adenomas, the promoter of the LGALS3 was found to be methylated and silenced, although prolactin- and adrenocorticotropic hormone–producing tumors, which were unmethylated, expressed the Gal-3 protein. These results show for the first time that Gal-3 expression is regulated in part by promoter methylation in pituitary as well as in other tumors. Because it is functionally involved in cancer progression and metastasis, Gal-3 may serve as a possible therapeutic target in the treatment of pituitary tumors.

Abstract Glycosphingolipids are known to play roles in integrin-mediated cell adhesion and migration; however, the mechanisms by which glycosphingolipids affect integrins are unknown. Here, we show that addition of the glycosphingolipid, C8-lactosylceramide (C8-LacCer), or free cholesterol to human fibroblasts at 10°C causes the formation of glycosphingolipid-enriched plasma membrane domains as shown by visualizing a fluorescent glycosphingolipid probe, BODIPY-LacCer, incorporated into the plasma membrane of living cells. Addition of C8-LacCer or cholesterol to cells initiated the clustering of β1-integrins within these glycosphingolipid-enriched domains and the activation of the β1-integrins as assessed using a HUTS antibody that only binds activated integrin. On warming to 37°C, β1-integrins were rapidly internalized via caveolar endocytosis in cells treated with C8-LacCer or cholesterol, whereas little β1-integrin was endocytosed in untreated fibroblasts. Incubation of cells with C8-LacCer or cholesterol followed by warm-up caused src activation, a reorganization of the actin cytoskeleton, translocation of RhoA GTPase away from the plasma membrane as visualized using total internal reflection fluorescence microscopy, and transient cell detachment. These studies show that LacCer can regulate integrin function both by modulating integrin clustering in microdomains and by regulating integrin endocytosis via caveolae. Our findings suggest the possibility that aberrant levels of glycosphingolipids found in cancer cells may influence cell attachment events by direct effects on integrin clustering and internalization.

Abstract It is well known that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) acts synergistically with the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (ADC) to reactivate DNA methylation-silenced genes. Moreover, in several studies, TSA was capable of inducing DNA demethylation even in the absence of ADC. Here we describe a mechanism by which HDAC inhibitors affect DNA methylation through their regulation on DNMT3B, a methyltransferase responsible for de novo DNA methylation. Using quantitative real-time PCR and Western blot analysis, we show that TSA down-regulates DNMT3B mRNA and protein expression in human endometrial cancer cells. This decrease in DNMT3B mRNA results in a significant reduction in de novo methylation activities. Further experiments indicated that TSA decreases DNMT3B mRNA stability and reduces its half-life from ∼4 to 2.5 hours. We established that protein synthesis is required for posttranscriptional regulation, suggesting the involvement of an RNase and/or key mRNA stabilization factor(s) controlling the DNMT3B mRNA stability. Therefore, TSA may not only modify histone acetylation, but also potentially alter DNA methylation. Since the HDAC inhibitors are frequently used in epigenetic studies and are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.

Abstract Prostate cancer is the most frequent non–skin cancer in men. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor has been shown to play an important role. Androgen receptor is expressed in both early and late-stage prostate cancer. Also, androgen-regulated pathways are thought to be active as evidenced by elevated levels of prostate-specific antigen (PSA). In addition, several androgen receptor coactivators and cytokines are involved in prostate cancer progression. In this regard, we have shown previously that the coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), a cytokine involved in late-stage prostate cancer. In this study, we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronically with IL-6. We found that p300 but not CREB-binding protein induced activation of PSA in these cells and that the histone acetyltransferase activity of p300 was critical. This effect was independent of the presence of androgens or antiandrogens. Moreover, we found markedly reduced levels of androgen receptor in these cells and p300 transfection did not affect those levels, suggesting that the p300 effect on PSA could be bypassing the androgen receptor. Transfection with exogenous androgen receptor showed minimal response of PSA to androgens but higher response to p300. We found similar effects of p300 on the androgen response element III, which mediates the androgen receptor–dependent activation of PSA. Finally, we showed that p300 alone regulates expression of the endogenous PSA gene in the IL-6–treated cells. These findings reveal a new insight in the progression of prostate cancer, suggesting that coactivators, such as p300, play more important roles in late-stage prostate cancer, and could regulate androgen-dependent genes in the absence or with very low levels of androgen receptor.

Abstract Recent studies using glycogen synthase kinase-3β (GSK-3β)–deficient mouse embryonic fibroblasts suggest that GSK-3β positively regulates nuclear factor κB (NFκB)–mediated gene transcription. Because NFκB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3β in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3β and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3β by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3β influences NFκB-mediated gene transcription at a point distal to the Iκ kinase complex, as only ectopic expression of the NFκB subunits p65/p50, but not an Iκ kinase β constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3β inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3β in cancer cell survival and proliferation and suggest GSK-3β as a potential therapeutic target in the treatment of pancreatic cancer.