Showing papers on "Tumor progression published in 1998"

Alterations in dna methylation : a fundamental aspect of neoplasia

Abstract: Neoplastic cells simultaneously harbor widespread genomic hypomethylation, more regional areas of hypermethylation, and increased DNA-methyltransferase (DNA-MTase) activity. Each component of this "methylation imbalance" may fundamentally contribute to tumor progression. The precise role of the hypomethylation is unclear, but this change may well be involved in the widespread chromosomal alterations in tumor cells. A main target of the regional hypermethylation are normally unmethylated CpG islands located in gene promoter regions. This hypermethylation correlates with transcriptional repression that can serve as an alternative to coding region mutations for inactivation of tumor suppressor genes, including p16, p15, VHL, and E-cad. Each gene can be partially reactivated by demethylation, and the selective advantage for loss of gene function is identical to that seen for loss by classic mutations. How abnormal methylation, in general, and hypermethylation, in particular, evolve during tumorigenesis are just beginning to be defined. Normally, unmethylated CpG islands appear protected from dense methylation affecting immediate flanking regions. In neoplastic cells, this protection is lost, possibly by chronic exposure to increased DNA-MTase activity and/or disruption of local protective mechanisms. Hypermethylation of some genes appears to occur only after onset of neoplastic evolution, whereas others, including the estrogen receptor, become hypermethylated in normal cells during aging. This latter change may predispose to neoplasia because tumors frequently are hypermethylated for these same genes. A model is proposed wherein tumor progression results from episodic clonal expansion of heterogeneous cell populations driven by continuous interaction between these methylation abnormalities and classic genetic changes.

Modulation of Apoptosis and Bcl-2 Expression by Prostaglandin E2 in Human Colon Cancer Cells

Abstract: Previously, we have shown that forced expression of prostaglandin endoperoxide synthase-2 [also called cyclooxygenase (COX) 2] leads to inhibition of programmed cell death in intestinal epithelial cells. More recently, we have demonstrated that growth of human colonic cancer xenografts is inhibited by treatment with a highly selective COX-2 inhibitor in tumors that express COX-2 (HCA-7) but not in those that lack COX-2 expression (HCT-116). To explore the biochemical mechanisms involved in these effects, we have evaluated the role of COX-2-derived eicosanoid products on programmed cell death in human colon cancer cells. Here we report that PGE2 treatment of human colon cancer cells leads to increased clonogenicity of HCA-7, but not HCT-116 cells. Treatment with a highly selective COX-2 inhibitor (SC-58125) decreases colony formation in monolayer culture and this growth inhibition was reversed by treatment with PGE2. Additionally, PGE2 inhibits programmed cell death caused by SC-58125 and induces Bcl-2 expression, but did not affect Bcl-x or Bax expression in human colon cancer (HCA-7) cells. Therefore, decreased cell death caused by PGE2 would enhance the tumorigenic potential of intestinal epithelial cells. Thus, these results may help to explain a component of the mechanism by which COX inhibitors prevent colorectal cancer in humans.

Mutational Analysis of the APC/β-Catenin/Tcf Pathway in Colorectal Cancer

Abstract: Mutation of the adenomatous polyposis coli ( APC ) tumor suppressor gene initiates the majority of colorectal (CR) cancers. One consequence of this inactivation is constitutive activation of β-catenin/Tcf-mediated transcription. To further explore the role of the APC/β-catenin/Tcf pathway in CR tumorigenesis, we searched for mutations in genes implicated in this pathway in CR tumors lacking APC mutations. No mutations of the γ-catenin ( CTNNG1 ), GSK-3α ( GSK3A ), or GSK-3β ( GSK3B ) genes were detected. In contrast, mutations in the NH 2 -terminal regulatory domain of β-catenin ( CTNNB1 ) were found in 13 of 27 (48%) CR tumors lacking APC mutations. Mutations in the β-catenin regulatory domain and APC were observed to be mutually exclusive, consistent with their equivalent effects on β-catenin stability and Tcf transactivation. In addition, we found that CTNNB1 mutations can occur in the early, adenomatous stage of CR neoplasia, as has been observed previously with APC mutations. These results suggest that CTNNB1 mutations can uniquely substitute for APC mutations in CR tumors and that β-catenin signaling plays a critical role in CR tumorigenesis.

Reduced angiogenesis and tumor progression in gelatinase A-deficient mice.

Abstract: Matrix proteolysis is thought to play a crucial role in several stages of tumor progression, including angiogenesis, and the invasion and metastasis of tumor cells. We investigated the specific role of gelatinase A (matrix metalloproteinase 2) on these events using gelatinase A-deficient mice. In these mice, tumor-induced angiogenesis was suppressed according to dorsal air sac assay. When B16-BL6 melanoma cells or Lewis lung carcinoma cells were implanted intradermally, the tumor volumes at 3 weeks after implantation in the gelatinase A-deficient mice decreased by 39% for B16-BL6 melanoma and by 24% for Lewis lung carcinoma (P < 0.03 for each tumor). The number of lung colonies of i.v. injections fell by 54% for B16-BL6 melanoma and 77% for Lewis lung carcinoma (P < 0.014 and P < 0.0015, respectively). These results indicated that host-derived gelatinase A plays an important role in angiogenesis and tumor progression, suggesting the usefulness of gelatinase A inhibitors for anticancer chemotherapy.

Induction of antigen-specific T cell anergy: An early event in the course of tumor progression

Abstract: The priming of tumor-antigen-specific T cells is critical for the initiation of successful anti-tumor immune responses, yet the fate of such cells during tumor progression is unknown. Naive CD4+ T cells specific for an antigen expressed by tumor cells were transferred into tumor-bearing mice. Transient clonal expansion occurred early after transfer, accompanied by phenotypic changes associated with antigen recognition. Nevertheless, these cells had a diminished response to peptide antigen in vitro and were unable to be primed in vivo. The development of antigen-specific T cell anergy is an early event in the tumor-bearing host, and it suggests that tolerance to tumor antigens may impose a significant barrier to therapeutic vaccination.

Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization

Abstract: Acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression. High levels of components of the plasminogen activation system, including urokinase, but paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI1), have been correlated with a poor prognosis for some cancers. We report here that deficient PAI1 expression in host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When this PAI1 deficiency was circumvented by intravenous injection of a replication-defective adenoviral vector expressing human PAI1, invasion and associated angiogenesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis.

Role of nitric oxide in tumor progression: Lessons from experimental tumors

Abstract: Nitric oxide (NO), a potent biological mediator, plays a key role in physiological as well as pathological processes, including inflammation and cancer. The role of NO in tumor biology remains incompletely understood. While a few reports indicate that the presence of NO in tumor cells or their microenvironment is detrimental to tumor cell survival and consequently their metastatic ability, a large body of clinical and experimental data suggest a promoting role of NO in tumor progression and metastasis. We suggest that tumor cells capable of very high levels of NO production die in vivo, and those producing or exposed to lower levels of NO, or capable of resisting NO-mediated injury undergo a clonal selection because of their survival advantage; they also utilize certain NO-mediated mechanisms for promotion of growth, invasion and metastasis. The possible mechanism(s) are: (a) a stimulatory effect on tumor cell invasiveness, (b) a promotion of tumor angiogenesis and blood flow in the tumor neovasculature, and (c) a suppression of host anti-tumor defense. In this review, we discuss these mechanisms on the basis of data derived from experimental models, in particular, a mouse mammary tumor model in which the expression of eNOS by tumor cells is positively correlated with invasive and metastatic abilities. Tumor-derived NO was shown to promote tumor cell invasiveness and angiogenesis. The invasion-stimulating effects of NO were due to an upregulation of matrix metalloproteases and a downregulation of their natural inhibitors. Treatment of tumor-bearing mice with NO-blocking agents reduced the growth and vascularity of primary tumors and their spontaneous metastases. We propose that selected NO-blocking drugs may be useful in treating certain human cancers either as single agents or as a part of combination therapies.

Frequent Nitric Oxide Synthase-2 Expression in Human Colon Adenomas: Implication for Tumor Angiogenesis and Colon Cancer Progression

Abstract: An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P

Tumor cell growth inhibition by caveolin re-expression in human breast cancer cells

Abstract: Cancer development is a multistage process that results from the step-wise acquisition of somatic alterations in diverse genes. Recent studies indicate that caveolin-1 expression correlates with the level of oncogenic transformation in NIH3T3 cells, suggesting that caveolin in caveolae may regulate normal cell proliferation. In order to better understand potential functions of caveolin-1 in cancer development, we have studied expression levels of caveolin-1 in human breast cancer cells, and have found that caveolin expression is significantly reduced in human breast cancer cells compared with their normal mammary epithelial counterparts. When the caveolin cDNA linked to the CMV promoter is transfected into human mammary cancer cells having no detectable endogenous caveolin, overexpression of caveolin-1 resulted in substantial growth inhibition, as seen by the 50% decrease in growth rate and by approximately 15-fold reduction in colony formation in soft agar. In addition, characterization of caveolin-1 expression during cell cycle progression indicates that expression of alpha-caveolin-1 is regulated during cell cycle. Furthermore p53-deficient cells showed a loss in caveolin expression. In summary, the overall expression patterns, its ability to inhibit tumor growth in culture, its regulation during the cell cycle, and the loss of expression in p53-deficient cells all are consistent with an important growth regulating function for caveolin-1 in normal human mammary cells, that needs to be repressed in oncogenic transformation and tumor cell growth.

Orthotopic models are necessary to predict therapy of transplantable tumors in mice

Abstract: Rapid evaluation of new cytotoxic agents and biological response modifiers for therapy of cancer and elucidation of their mechanisms of action require the use of relevant animal models. It is well established that the faithful reproduction of the tumor microenvironment that allows the emergence of subpopulations of tumor cells with the biological and metastatic properties observed in clinical cancer occurs with orthotopic tumor models (transplantable and transgenic). This review summarizes the evidence that phenotypic properties of metastatic cells are governed by the expression of genes that are regulated by interaction with the relevant organ environment. While ectopic models of cancer allow rapid screening of new compounds and transgenic models afford opportunities to study early cellular and molecular events in tumor progression and metastasis, orthotopic transplantation of tumor cells remains an affordable, reproducible and reliable methodology for the study of organ-specific determinants of the biology and therapy of cancer.

Thrombin receptor overexpression in malignant and physiological invasion processes

Abstract: Although the involvement of soluble and matrix-immobilized proteases in tumor cell invasion and metastasis is well recognized, the role of proteolytically activated cell surface receptors has not been elucidated. We report here that thrombin receptor, a member of the protease-activated receptor family, is preferentially expressed in highly metastatic human breast carcinoma cell lines and breast carcinoma biopsy specimens. Introduction of thrombin receptor antisense cDNA considerably inhibited the invasion of metastatic breast carcinoma cells in culture through a reconstituted basement membrane. During placental implantation of the human embryo, thrombin receptor is transiently expressed in the invading cytotrophoblasts. These results emphasize the involvement of thrombin receptor in cell invasion associated with tumor progression and normal embryonic development.

Role of Nitric Oxide in Angiogenesis and Tumor Progression in Head and Neck Cancer

Abstract: Background: Angiogenesis (formation of new blood vessels) is associated with tumor growth and metastasis in patients with solid tumors, including those of the head and neck. Nitric oxide (NO) production may contribute to these processes. We assessed the role of the NO pathway in angiogenesis and tumor progression in patients with head and neck cancer. Methods: Biochemical assays were used to measure NO synthase (NOS) activity and cyclic guanosine monophosphate (cGMP) levels in specimens of tumor and normal mucosa obtained from 27 patients. Microvessels in tumor specimens were identified by CD-31-specific immunohistochemical staining. Associations between microvessel densities, levels of NOS, and cGMP were examined by use of two-sided statistical tests. Tumor specimens and human squamous carcinoma A-431 cells were grown as explants on the corneas of rabbits, and the effect of the NOS inhibitor N v -nitro-L-arginine-methyl ester (L-NAME) was tested. Results: Levels of total NOS, inducible NOS, and cGMP were higher in tumor specimens than in specimens of normal mucosa (all P<.0001). Tumor specimens from patients with lymph node metastases presented a higher total NOS activity (P = .005) and were markedly more vascularized than tumor specimens from patients with no lymph node involvement (P = .0002). Microvessel density at the tumor edge was an independent predictor of metastasis for this series of patients (odds ratio = 1.19; 95% confidence interval = 1.07‐2.89; P = .04). A-431 cells and tumor specimens exhibiting high levels of NOS activity induced angiogenesis in the rabbit cornea assay; when NO production was blocked, tumor angiogenesis and growth were repressed. Conclusions: The NO pathway appears to play a key role in tumor angiogenesis and spread in patients with head and neck cancer. [J Natl Cancer Inst 1998;90:587‐96]

Reduction of Established Spontaneous Mammary Carcinoma Metastases following Immunotherapy with Major Histocompatibility Complex Class II and B7.1 Cell-based Tumor Vaccines

Abstract: For many cancer patients, removal of primary tumor is curative; however, if metastatic lesions exist and are not responsive to treatment, survival is limited. Although immunotherapy is actively being tested in animal models against primary tumors and experimental metastases (i.v. induced), very few studies have examined immunotherapy of spontaneous, established metastatic disease. The shortage of such studies can be attributed to the paucity of adequate animal models and to the concern that multiple metastatic lesions may be more resistant to immunotherapy than a localized primary tumor. Here, we use the BALB/c-derived mouse mammary carcinoma, 4T1, and show that this tumor very closely models human breast cancer in its immunogenicity, metastatic properties, and growth characteristics. Therapy studies demonstrate that treatment of mice with established primary and metastatic disease with MHC class II and B7.1-transfected tumor cells reduces or eliminates established spontaneous metastases but has no impact on primary tumor growth. These studies indicate that cell-based vaccines targeting the activation of CD4+ and CD8+ T cells may be effective agents for the treatment of malignancies, such as breast cancer, where the primary tumor is curable by conventional methods, but metastatic lesions remain refractile to current treatment modalities.

β-Catenin Mutations in Human Prostate Cancer

Abstract: Beta-catenin plays essential roles in both intercellular adhesion and signal transduction. As a signaling molecule, beta-catenin supplies an activating domain to the T-cell factor/lymphoid enhancer-binding factor family of DNA-binding proteins and activates gene transcription. Posttranslational stabilization of beta-catenin, leading to elevated protein levels and constitutive gene activation, has been proposed as an important step in oncogenesis. Stabilization of beta-catenin can occur through mutation to highly conserved amino acids encoded in exon 3 of the beta-catenin gene (CTNNB1). To determine whether this pathway of malignant transformation is important in prostate cancer, we analyzed 104 prostate cancer tissue specimens, 4 prostate cancer cell lines, and 3 prostate tumor xenografts for activating mutations in exon 3 of CTNNB1. Mutations were detected in 5 of the 104 prostate cancer tissue samples. Four of the five mutations involved serine or threonine residues implicated in the degradation of beta-catenin. A fifth tumor had a mutation at codon 32, changing a highly conserved aspartic acid to a tyrosine. Mutational analysis of multiple regions from several tumor samples showed that the beta-catenin mutations were present focally and therefore may occur during tumor progression.

Elevated expression of caveolin is associated with prostate and breast cancer.

Abstract: To identify genes associated with prostate cancer progression, we developed a strategy involving the use of differential display-PCR with a panel of genetically matched primary tumor- and metastasis-derived mouse prostate cancer cell lines. We isolated a cDNA fragment with homology to the mouse caveolin-1 gene. Northern blotting with this fragment revealed increased caveolin expression in metastasis-derived cell lines relative to primary tumor-derived cell lines. Western blotting with a polyclonal caveolin antibody confirmed increased caveolin protein in metastasis-derived mouse cell lines and expression in three of four human prostate cancer cell lines. Immunohistochemical analysis of a human prostate cancer cell line demonstrated a prominent granular pattern of caveolin accumulation. Subsequent analysis of mouse and human prostate specimens revealed minimal caveolin expression in normal epithelium with abundant staining of smooth muscle and endothelium. The frequency of caveolin-positive cells was increased in prostate cancer with markedly increased accumulation of caveolin and a granular staining pattern in lymph node metastatic deposits. In human breast cancer specimens, increased caveolin staining was detected in intraductal and infiltrating ductal carcinoma as well as nodal disease. Caveolin therefore appears to be associated with human prostate cancer progression and is also present in primary and metastatic human breast cancer.

Multinuclearity and Increased Ploidy Caused by Overexpression of the Aurora- and Ipl1-like Midbody-associated Protein Mitotic Kinase in Human Cancer Cells

Abstract: Aurora- and Ipl1-like midbody-associated protein (AIM-1) is a serine/threonine kinase that is structurally related to Drosophila aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. A kinase-negative form of AIM-1 inhibits the formation of cleavage furrow without affecting nuclear division, indicating that the gene controls entry into cytokinesis during M phase in mammalian cells. A human gene that encodes the protein AIM-1 was overexpressed in colorectal and other tumor cell lines. The regulation of AIM-1 expression was cell cycle dependent in normal and tumor cells, and the maximum accumulation was observed at G 2 -M. Exogenous overexpression of wild-type AIM-1 produced multinuclearity in human cells, suggesting that the excess amount of AIM-1 had a dominant-negative effect on the overexpressing cells. In long-term culture of AIM-1-overexpressing cells, multiple nuclei in a cell were occasionally fused, and then an increased ploidy and aneuploidy were induced. Thus, the overexpression of AIM-1 in colorectal tumor cell lines is thought to have a causal relationship with multinuclearity and increased ploidy. Cytokinesis error caused by AIM-1 overexpression is a major factor in the predisposition of tumor cells to the perturbation of chromosomal integrity that is commonly observed in human neoplasia. Thus, defects of pathways essential for mitotic regulation are important during human cancer development.

Tumor Cell-associated Hyaluronan as an Unfavorable Prognostic Factor in Colorectal Cancer

Abstract: Hyaluronan (HA) is a linear high molecular weight extracellular polysaccharide. It is thought to be involved in mitosis and the enhancement of wound healing, tumor invasion, and metastasis. Because its clinical relevance in cancer has not been explored, we scored HA in colorectal adenocarcinoma and studied its relationship with patient survival. A specific probe prepared from cartilage proteoglycan aggregates was used to stain paraffin-embedded tumor samples from 202 colorectal adenocarcinoma patients treated in Kuopio University Hospital and followed up for a mean of 14 years. The hypothesis that the percentage of HA-positive carcinoma cells (HA%) and HA intensity in cancer cells correlated with survival was tested with the log-rank test, hazard ratios, and their confidence intervals. Ninety-three % of tumors had at least a proportion of carcinoma cells positive for HA. HA intensity in tumor epithelium was stronger in Dukes' stages C and D tumors and in high-grade tumors. The cancer-related survival rate was lower among patients with strong HA intensity in tumor epithelium (P < 0.001) and high HA% (P < 0.001). Recurrence-free survival was also shorter in patients with an intense signal for HA (P = 0.001) and high HA% in tumor epithelium (P = 0.04). HA intensity in tumor epithelium independently predicted survival and recurrence-free survival (Cox's analysis). We conclude that a high proportion of HA-positive cancer cells and high intensity of the HA-signal predicts a poor survival rate. The abnormal expression of HA in the neoplastic colon epithelial cells is suggested to provide a distinct advantage for invasive growth and metastasis.

Transforming growth factor β1 is associated with angiogenesis, metastasis, and poor clinical outcome in prostate cancer

Abstract: BACKGROUND Prostate tumors express high levels of transforming growth factor-β1 (TGF-β1) and seem to acquire resistance to its antiproliferative effects with tumor progression. Moreover, TGF-β1 could be involved in tumor-promoting processes such as angiogenesis, cell migration, and immunosuppression. METHODS Immunoreactivity for TGF-β1 and its receptors type I and type II (TGFβ-RI and TGFβ-RII), tumor vascular count, and cell proliferation were studied in 73 cases of prostate cancer, diagnosed between 1975–1983 and followed with surveillance. RESULTS Patients with tumor overproduction of TGF-β1 had shorter median cancer-specific survival than patients with normal TGF-β1 immunoreactivity (5.0 vs. 10 years, P = 0.006). Furthermore, increased TGF-β1 staining was associated with tumor grade, high vascular counts, and metastasis (P = 0.02, 0.02, and 0.01, respectively). Patients with loss of tumor TGFβ-RII expression in combination with TGF-β1 overproduction showed particularly short survival (2.6 vs. 10 years, P = 0.0000), when compared to patients with normal immunoreactivity. CONCLUSIONS Overproduction of TGF-β1 and loss of TGFβ-RII expression are associated with poor clinical outcome in prostate cancer, and TGF-β1 may promote tumor progression by stimulating angiogenesis and metastasis. Prostate 37:19–29, 1998. © 1998 Wiley-Liss, Inc.

Genetic Alterations in Hormone-Refractory Recurrent Prostate Carcinomas

Abstract: To study the genetic basis of tumor progression, we have screened 37 hormone-refractory prostate carcinomas for genetic changes by comparative genomic hybridization (CGH). All recurrent tumors showed genetic aberrations, with a mean total number of changes per tumor of 11.4 (range, 3 to 23). The most common genetic aberrations were losses of 8p (72.5%), 13q (50%), 1p (50%), 22 (45%), 19 (45%), 10q (42.5%), and 16q (42.5%) and gains of 8q (72.5%), 7q (40%), Xq (32.5%), and 18q (32.5%). The CGH results were further validated with fluorescence in situ hybridization (FISH) using probes for pericentromeric regions of chromosomes 7, 8, and 18 as well as probes for caveolin (7q31), c-myc (8q24), and bcl-2 (18q21.3). In addition, the samples had previously been analyzed for androgen receptor gene copy number. CGH and FISH results were concordant in 78% of cases. Seventeen of twenty-two tumors showed an increased copy number of c-myc by FISH. However, only 5 of 17 (29%) of the cases showed high-level (more than threefold) amplification. Both CGH and FISH findings suggested that in most of the cases 8q gain involves the whole q-arm of the chromosome. Four of seventeen (24%) cases showed increased copy number of bcl-2 by FISH; however, no high-level amplifications were found. To evaluate the clonal relationship of the primary and recurrent tumors, six primary-recurrent tumor pairs from the same patients were studied by CGH. In three of six cases (50%), the recurrent tumor had more than one-half of the aberrations found in the corresponding primary tumor, indicating a close clonal relationship. In the rest of the cases, such a linear clonal relationship was less evident. Altogether, these results suggest that recurrent prostate carcinomas are genetically unstable. The resulting heterogeneity may well underlie the poor responsiveness of hormone-refractory tumors to treatment.

RET/PTC oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes.

Abstract: Malignant tumors of the thyroid gland vary considerably in aggressiveness, ranging from a well-differentiated, clinically indolent, to an undifferentiated, often lethal phenotype. Undifferentiated (anaplastic) thyroid tumors are supposed to be derived, through a process of progression, from previously differentiated neoplasms. A common genetic alteration in thyroid tumors is the rearrangement of the tyrosine kinase-encoding RET proto-oncogene, leading to the generation of chimeric RET/PTC oncogenes. To define the characteristics of the thyroid tumor subset with RET rearrangements, we have investigated its activation by a combined immunohistochemistry and reverse transcription-PCR approach in a series of 316 well-characterized thyroid tumors representative of the main diagnostic groups. RET activation was detected in 81 of 201 (40.3%) papillary carcinomas. It correlated with tumors exhibiting the "classic" morphological features of papillary cancer or with the microcarcinoma subtype (P = 0.017). RET activation in papillary carcinoma was not associated with clinical markers (such as large tumor size, extrathyroidal extension, or metastases) of increased morbidity. Follicular-type neoplasms (61 adenomas and 22 carcinomas), as well as the aggressive poorly differentiated (15 cases) or undifferentiated (anaplastic) carcinomas (17 cases), were negative. This study demonstrates that all thyroid carcinomas harboring activating RET rearrangements exhibit a well-differentiated phenotype, that of papillary carcinoma, and indicates that the subset of RET/PTC-positive papillary carcinomas do not progress to more aggressive, less differentiated tumor phenotypes.

Elevated and Absent pRb Expression Is Associated with Bladder Cancer Progression and Has Cooperative Effects with p53

Abstract: Rb protein (pRb) expression was evaluated in 185 cases of transitional cell carcinoma of the bladder from patients that underwent radical cystectomy Tumors were stratified into three categories based on the percentage of nuclei expressing pRb: (a) 0, 0% of tumor cells showing nuclear reactivity; (b) 1+, 1-50% of tumor cells showing nuclear reactivity; and (c) 2+, >50% of tumor cells showing nuclear reactivity Cases with undetectable (pRb 0) and high (pRb 2+) pRb reactivity had identical rates of recurrence These cases had significantly higher recurrence (P = 00001) and lower survival rates (P = 00002) compared to cases with moderate (pRb 1+) pRb reactivity, indicating that high levels of pRb expression may reflect a dysfunctional (altered) Rb pathway The tumors were also examined for alterations in p53 expression; patients with tumors altered in both p53 and pRb had significantly increased rates of recurrence (P < 00001) and survival (P < 00001) compared to patients with no alterations in either p53 or pRb; patients with alterations in only one of these proteins had intermediate rates of recurrence and survival These results suggest that: (a) bladder cancers with high pRb expression do not show the tumor suppressor effects of the protein; and (b) alteration in both p53 and pRb may act in cooperative or synergistic ways to promote tumor progression

p53 and vascular endothelial growth factor regulate tumor growth of NOS2-expressing human carcinoma cells

Abstract: The finding of frequent nitric oxide synthase expression in human cancers indicates that nitric oxide has a pathophysiological role in carcinogenesis. To determine the role of nitric oxide in tumor progression, we generated human carcinoma cell lines that produced nitric oxide constitutively. Cancer cells expressing inducible nitric oxide synthase that had wild-type p53 had reduced tumor growth in athymic nude mice, whereas those with mutated p53 had accelerated tumor growth associated with increased vascular endothelial growth factor expression and neovascularization. Our data indicate that tumor-associated nitric oxide production may promote cancer progression by providing a selective growth advantage to tumor cells with mutant p53, and that inhibitors of inducible nitric oxide synthase may have therapeutic activity in these tumors.

Effect of p21WAF1/CIP1 Expression on Tumor Progression in Bladder Cancer

Abstract: Background: Altered expression of p53 protein is an important predictor of progression in bladder cancer. The action of p53 on cell cycle regulation is mediated, in part, through expression of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 (p21). Loss of p21 expression may, therefore, contribute to tumor progression. We sought to determine the relationship between p21 expression in bladder cancer and disease progression. Methods: Tumor specimens were obtained from 242 patients who underwent cystectomy for bladder cancer. Median follow-up was 8.5 years (range, 0.1‐11.8 years). Nuclear p21 status was determined by immunohistochemistry and was then analyzed in relationship to the probability of tumor recurrence, overall survival, and tumor p53 status. Reported P values are two-sided. Results: Nuclear p21 expression was detected in the tumors of 156 (64%) of the 242 patients. Patients with p21-positive tumors had a decreased probability of tumor recurrence (P<.00001) and an increased probability of overall survival (P<.00001) in comparison with patients with p21-negative tumors. In a multivariable analysis, p21 expression was an independent predictor of tumor recurrence (P = .0017) and of survival (P = .006) when assessed with tumor grade, tumor stage, lymph node status, and p53 status. p21 expression was associated with p53 status (P<.001); 56% of tumors with p53 alterations showed loss of p21 expression, whereas 79% of tumors expressing wildtype p53 were p21 positive. Patients with p53-altered/p21negative tumors demonstrated a higher rate of recurrence and worse survival compared with those with p53-altered/ p21-positive tumors (P<.0001). Patients with p53-altered/ p21-positive tumors demonstrated a similar rate of recurrence and survival as those with p53-wild type tumors. Conclusion: Loss of p21 expression is a statistically significant and independent predictor of bladder cancer progression. Maintenance of p21 expression appears to abrogate the deleterious effects of p53 alterations on bladder cancer progression. [J Natl Cancer Inst 1998;90:1072‐9]

A Dominant Negative Mutant of the Insulin-like Growth Factor-I Receptor Inhibits the Adhesion, Invasion, and Metastasis of Breast Cancer

Abstract: The 5-year survival rate for women with metastatic breast cancer is only 25-30%; thus, the need to improve treatment is apparent. Overexpression of insulin-like growth factor-I receptor (IGF-IR) correlates with poor prognosis and local recurrence. In this study, we addressed whether functional impairment of IGF-IR affects adhesion, invasion, and metastasis of breast cancer. Impairment of IGF-IR function was achieved by transfecting a dominant negative form of the receptor, termed 486stop, into MDA-MB-435 metastatic breast cancer cells. The protein product of 486stop is secreted extracellularly, resulting in a bystander effect. Cellular adhesion to laminin and collagen was inhibited 94 and 88%, respectively. Furthermore, 486stop inhibited insulin-like growth factor-I-stimulated invasion through collagen IV by 75%. The dominant negative receptor was secreted, as evidenced by the observation that MDA-MB-435 and MDA-MB-231 cells were prevented from binding to laminin by 90% when treated with conditioned medium (CM) from 486stop-transfected cells. CM also inhibited the invasion of MDA-MB-231 cells across collagen IV by 80%. Finally, CM made MDA-MB-231 cells 30% more sensitive to Taxol-induced cell death. Growth in soft agar was suppressed by 486stop, but growth in monolayer was unaffected. When injected into the mammary fat pad, 486stop did not significantly suppress growth of the primary tumor, but metastasis to the lungs, livers, lymph nodes, and lymph vessels was significantly decreased compared to the vector control. In conclusion, inhibition of IGF-IR resulted in suppression of adhesion, invasion, and metastasis, providing a mechanistic rationale for targeting IGF-IR in the treatment of metastatic breast cancer.

Vascular integrin alpha(v)beta3: a new prognostic indicator in breast cancer.

Abstract: Blood vessel density is a prognostic indicator of multiple tumor types. Recently, it has been established that tumor-associated blood vessels express elevated levels of integrin alpha(v)beta3. In fact, there is evidence that integrin alpha(v)beta3 identifies the most proliferative endothelial cells within human breast carcinomas. Therefore, we evaluated breast cancer tissue in terms of both blood vessel density and alpha(v)beta3 expression. We found that the antibody LM609 to integrin alpha(v)beta3 preferentially stains the blood vessels of small caliber. Furthermore, comparative studies between LM609 and anti-CD31 antibodies on normal breast indicate that very low and weak expression of integrin alpha(v)beta3 was found on vessels within normal tissue, whereas CD31 antigen was expressed in almost all vasculature. Indeed, expression of integrin alpha(v)beta3 was significantly higher in tumors of patients with metastasis than in those without metastasis. In a series of 197 consecutive patients with invasive breast cancer and long follow-up, vascular expression of integrin alpha(v)beta3 in tumor vascular "hot spots" was found to be the most significant prognostic factor predictive of relapse-free survival in both node-negative and node-positive patients. These findings support the contention that angiogenesis plays a critical role in breast cancer progression and suggest that integrin alpha(v)beta3 is an endothelial cell marker with significant prognostic value and potential usefulness as a target for specific antiangiogenic therapy.

Tumor growth inhibition induced in a murine model of human Burkitt's lymphoma by a proteasome inhibitor

Abstract: Cell growth and viability are dependent on the function of the multicatalytic proteinase complex (proteasome), a multisubunit particle that affects progression through the mitotic cycle by degradation of cyclins. Exposure of rodent fibroblasts and human lymphoblasts in culture to benzyloxycarbonyl-leucyl-leucyl-phenylalaninal (Z-LLF-CHO), a cell-permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the proteasome, resulted in the induction of apoptosis in a rapid, dose-dependent fashion. Fibroblasts transformed with ras and myc, lymphoblasts transformed by c-myc alone, and a Burkitt's lymphoma (BL) cell line that overexpresses c-Myc were up to 40-fold more susceptible to apoptosis than were either primary rodent fibroblasts or immortalized nontransformed human lymphoblasts, respectively. To determine whether such preferential apoptosis could impact upon tumor growth in vivo, toxicological studies were performed in mice with severe combined immunodeficiency and showed that mice tolerated single interscapular doses of Z-LLF-CHO without unacceptable toxicity. Severe combined immunodeficient mice bearing s.c. BL tumors in the flank were treated interscapularly with Z-LLF-CHO or a comparable dose of the peptidyl alcohol (Z-LLF-OH), which does not induce proteasome inhibition or apoptosis. Single doses of Z-LLF-CHO induced statistically significant (P < 0.0001) early tumor regression and a significant (P < 0.0001) delay in tumor progression. Analysis of tumor specimens revealed increased apoptosis in BL tumors from mice treated with Z-LLF-CHO. These results, showing a 42% tumor growth delay, indicate that proteasome inhibitors have the potential of curbing the growth of a c-myc-related tumor.

Altered Estrogen Receptor α and β Messenger RNA Expression during Human Breast Tumorigenesis

Abstract: Using a multiplex reverse transcription-PCR assay, we compared the relative expression of estrogen receptor (ER) a and ER-AŸiiiKN A between adjacent samples of normal breast tissue and matched primary breast tumors obtained from 18 different patients. Within this cohort, 7 tumors were ER negative, and 11 tumors were ER positive, as determined by the ligand binding assay. No differences in the ratio of ER-a:ER-AŸ expression were observed in the ER-negative cohort. However, in the ER-positive cohort, a significantly (P < 0.02) higher ER-o:ER-AŸ ratio was observed in the tumor compared with that of the normal tissue component. Our data revealed that the increase in the ER-a:ER-AŸ ratio was due primarily to a significant (/" < 0.05) increase in ER-a mRNA expression in conjunction with a lower ER-AŸmRNA expression in the tumor compared with that of the normal compartment in some, but not all, ER-positive cases. These results suggest that the role of ER-a- and ER-AŸ-driven pathways and/or their interaction change during breast tumorigenesis.

Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.

Abstract: An in vitro carcinogenesis model of human skin keratinocytes has been developed based on the spontaneously immortalized keratinocyte cell line HaCaT. Immortalization, the initial stage in human carcinogenesis in vitro, was induced by ultraviolet-type mutations in the p53 gene followed by further genetic alterations leading to the loss of senescence genes, in particular on chromosome 3p. Despite multiple genetic changes, the HaCaT cell line sustained its genomic balance up to high passage levels and maintained a non-tumorigenic phenotype. Tumorigenic transformation was induced by ras oncogene transfection but also by culture stress and elevated temperature, resulting in benign and malignant tumorigenic clones. Malignant conversion was associated with the loss of a copy of chromosome 15, leading to a decrease in thrombospondin-1 (TSP-1) expression. Heat-induced malignant conversion was associated with a gain of material on chromosome 11, including the cyclin D1 gene. The microenvironment plays a major role in tumorigenic transformation and the control of malignant cells. Overexpression of platelet-derived growth factor in HaCaT cells caused mesenchyme activation and formation of benign tumors. Halting tumor angiogenesis completely prevented invasion of malignant cells and induced a benign tumor phenotype. Transfer of a normal chromosome 15 or TSP-1 transfection into a skin carcinoma line resulted in tumor suppression due to TSP-1-blocked tumor vascularization. Because of the reduced TSP-1 expression, blood vessels infiltrated the tumor, and it expanded. Progression to more aggressive tumor phenotypes required the in vivo environment and was caused by selection of a subpopulation and further genetic modifications. The improved autonomous growth of these cells was associated with new expression of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which acted in an autocrine manner to stimulate proliferation and migration. With this in vitro skin carcinogenesis model we were able to demonstrate multiple stages in the transformation process that were associated with different genetic and phenotypic characteristics. In addition, we documented that modulation of the tumor stroma plays an important and decisive role in tumor development and progression. From this we hypothesize that the growth restraints of the microenvironment are increasingly lost with advancing stages of carcinogenesis but can be restored by modulation of the tumor stroma.