Loss of tumorigenicity of human breast cancer cells engineered to produce IL-2, IL-4 or GM-CSF in nude mice.

Human breast cancer cells (OCUB-M), retrovirally transduced with granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2) or IL-4 gene were examined for their antitumor activities in nude mice. Although cell proliferation rates in vitro of these cytokine-producing cells were not significantly different from that of wild-type cells, nude mice that were subcutaneously inoculated with cytokine-producing cells did not develop tumors in contrast to mice that were injected with wild-type cells. Injection of GM-CSF-producing cells into the vicinity of growing wild-type tumors retarded subsequent growth of wild-type tumors. Histological examination of tumors which received GM-CSF-producing cells revealed marked infiltration of mononuclear cells around the tumors. Irradiation of cytokine-producing cells diminished their proliferation capacity but production of cytokine(s) was retained. Therefore, inoculation of irradiated cytokine producer cells into growing tumors can be used as a therapeutic maneuver for breast cancer.     

Stat1 negatively regulates angiogenesis, tumorigenicity and metastasis of tumor cells

Stat1 is deficient or inactive in many types of human tumors whereas some tumors have activated Stat1. Whether Stat1 affects tumor growth and metastasis is unclear. In the present study, we used Stat1 knockout tumor cells to determine (1) whether Stat1 can regulate angiogenesis, growth, and metastasis of tumor cells; and (2) whether Stat1 is required for the inhibitory effect of IFN-beta on the expression of angiogenic factor bFGF. Highly tumorigenic and metastatic RAD-105 tumor cells derived from a fibrosarcoma of a Stat1 knockout mouse were reconstituted with a Stat1 expression vector. The reconstitution of Stat1 suppressed the tumorigenicity and metastasis of RAD-105 cells in nude mice which correlated with a decreased microvessel density and decreased expression of proangiogenic molecules bFGF, MMP-2, and MMP-9 in vivo. Moreover, noncytotoxic concentrations of IFN-beta significantly inhibited the in vitro expression of bFGF in the Stat1-reconstituted cells but not in the Stat1-deficient cells, which was consistent with decreased bFGF expression of Stat1-reconstituted tumors in vivo. Therefore, Stat1 is essential for IFN-mediated inhibition of bFGF production, suggesting that tumor-intrinsic Stat1 is an important mediator for antiangiogenic signals, such as IFN. Collectively, these data demonstrate that Stat1 expressed by tumor cells is a negative regulator of tumor angiogenesis and, hence, tumor growth and metastasis.     

A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell invasion and tumorigenicity

Androgen deprivation therapy causes a paradoxical elevation of matrix metalloproteinases (MMPs) including MMP-9 resulting in aggressive tumor phenotype in many patients with prostate cancer. In this study, we have evaluated a novel antisense phosphorodiamidate Morpholino oligomer (PMO) targeted against MMP-9 in models of angiogenesis and in human prostate xenograft in athymic mice. The treatment of androgen-independent DU145 human prostate cells with a 21-mer MMP-9 antisense PMO caused a dose-dependent inhibition of cell proliferation compared to scrambled or MMP-2 antisense PMO at similar concentrations. This was associated with decreases in MMP-9 expression, gelatinolytic activity and increased stability of the insulin-like growth factor-binding protein (IGFBP-3), a proapoptotic factor and MMP-9 substrate. In vitro invasion assays revealed a 40-60% inhibition of DU145 cell invasion in the presence of 25 microM MMP-9 antisense PMO. A significant decrease in endothelial cell migration and vascularization was observed in the Matrigel plug assay in mice when treated intraperitoneally with 300 microg/day MMP-9 antisense for 21 days. In the highly vascular DU145 tumor xenografts, MMP-9 inhibition caused decreased tumor growth with regression in 50% of the animals. Histological analysis revealed increased apoptosis and fibrous tissue deposits in the MMP-9 antisense-treated tumors compared to the scrambled and saline controls. No apparent toxicity or mortality was associated with the MMP-9 PMO treatment. In summary, the MMP-9 antisense PMO inhibited in vitro prostate cancer cell proliferation, invasion and in vivo angiogenesis. These data establish the feasibility of developing a site-directed, nontoxic antisense therapeutic agent for inhibiting local invasion and metastasis.     

EphA6 promotes angiogenesis and prostate cancer metastasis and is associated with human prostate cancer progression

Metastasis is the primary cause of prostate cancer (CaP)-related death. We investigate the molecular, pathologic and clinical outcome associations of EphA6 expression and CaP metastasis. The expression profiling of Eph receptors (Ephs) and their ephrin ligands was performed in parental and metastatic CaP cell lines. Among Ephs and ephrins, only EphA6 is consistently overexpressed in metastatic CaP cells. Metastatic potential of EphA6 is assessed by RNAi in a CaP spontaneous metastasis mouse model. EphA6 knock-down in human PC-3M cells causes decreased invasion in vitro and reduced lung and lymph node metastasis in vivo. In addition, knock-down of EphA6 decreases tube formation in vitro and angiogenesis in vivo. EphA6 mRNA expression is higher in 112 CaP tumor samples compared with benign tissues from 58 benign prostate hyperplasia patients. Positive correlation was identified between EphA6 expression and vascular invasion, neural invasion, PSA level, and TNM staging in CaP cases. Further, genome-wide gene expression analysis in EphA6 knock-down cells identified a panel of differentially regulated genes including PIK3IPA, AKT1, and EIF5A2, which could contribute to EphA6-regulated cancer progression. These findings identify EphA6 as a potentially novel metastasis gene which positively correlates with CaP progression. EphA6 may be a therapeutic target in metastatic CaP.     

Adenovirus-mediated interferon-beta gene therapy suppresses growth and metastasis of human prostate cancer in nude mice

The purpose of this study was to determine the effects of interferon-beta (IFN-beta) gene transfer on the growth of PC3MM2 human prostate cancer cells in nude mice. Intralesional delivery of an adenoviral vector encoding murine IFN-beta (AdIFN-beta), but not a vector encoding bacterial beta-galactosidase (AdLacZ), suppressed PC3MM2 tumors in a dose-dependent manner. At the highest dose (2x10(9) plaque-forming units, PFU), a single injection of AdIFN-beta (but not AdLacZ) suppressed orthotopic PC3MM2 tumors and development of metastasis by 80%, and eradicated the tumors in 20% of mice. Immunohistochemical staining showed that AdIFN-beta-treated tumors contained fewer microvessels, fewer proliferating cells, and more apoptotic cells than did the control tumors. Compared with controls, tumors injected with AdIFN-beta expressed higher levels of IFN-beta and inducible nitric oxide synthase (iNOS) and lower levels of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). In vitro analysis indicated that expression of bFGF and TGF-beta1 in PC3MM2 cells could be suppressed by the nitric oxide donor sodium nitroprusside. These data suggest that intratumoral delivery of the IFN-beta gene with adenoviral vectors could be an effective therapy for prostate cancer and that tumor suppression by AdIFN-beta correlated with up-regulation of iNOS and down-regulation of angiogenesis.     

Suppression of invasion and metastasis of prostate cancer cells by overexpression of NDRG2 gene

N-myc downstream regulated gene 2 (NDRG2) is involved in invasion and metastasis of cancer, furthermore it is frequently down-regulated in prostate cancer. Herein we evaluated the effect of NDRG2 overexpression on invasiveness and bone destruction in prostate cancer. The human prostate cancer cell line PC-3 and DU145 were infected with Ad-NDRG2 or Ad-LacZ. Overexpression of NDRG2 not only inhibited the growth of the cells, but also suppressed invasiveness of the cells in an in vitro assay. PC-3 cells infected with Ad-NDRG2 or Ad-LacZ were injected into the tibias of nude mice. Four weeks later, we found the mice injected with PC-3 cells overexpressing NDRG2 had smaller tumors and less bone destruction. These results demonstrate that NDRG2 overexpression can inhibit tumor growth and invasion, furthermore, it can decrease bone destruction caused by prostate cancer bone metastasis.     

SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells

Background

SULF2 is a 6-O-endosulfatase which removes 6-O sulfate residues from N-glucosamine present on heparan sulfate (HS). The sulfation pattern of HS influences signaling events mediated by heparan sulfate proteoglycans (HSPGs) located on cell surface, which are critical for the interactions with growth factors and their receptors. Alterations in SULF2 expression have been identified in the context of several cancer types but its function in cancer is still unclear where the precise molecular mechanism involved has not been fully deciphered. To further investigate SULF2 role in tumorigenesis, we overexpressed such gene in prostate cancer cell lines.

Methods

The normal prostate epithelial cell line RWPE-1 and the prostate cancer cells DU-145, and PC3 were transfected with SULF2-expressing plasmid pcDNA3.1/Myc-His(−)-Hsulf-2. Transfected cells were then submitted to viability, migration and colony formation assays.

Results

Transfection of DU-145 and PC3 prostate cancer cells with SULF2 resulted in increased viability, which did not occur with normal prostate cells. The effect was reverted by the knockdown of SULF2 using specific siRNAs. Furthermore, forced expression of SULF2 augmented cell migration and colony formation in both prostate cell lines. Detailed structural analysis of HS from cells overexpressing SULF2 showed a reduction of the trisulfated disaccharide UA(2S)-GlcNS(6S). There was an increase in epithelial-mesenchymal transition markers and an increase in WNT signaling pathway.

Conclusions

These results indicate that SULF2 have a pro-tumorigenic effect in DU-145 and PC3 cancer cells, suggesting an important role of this enzyme in prostatic cancer metastasis.     

Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer

Survivin plays an important role in cancer development. We aim to show here that suppression of survivin expression or function by antisense and dominant-negative (DN) mutant can inhibit gastric cancer carcinogenesis and angiogenesis in vivo. Plasmid constructs expressing survivin antisense and DN mutant replacing the cysteine residue at amino acid 84 with alanine (Cys84Ala) were prepared and introduced into BCG-823 and MKN-45 gastric cancer cells to establish stable transfectants. We showed that both antisense and DN mutant stable transfectants exhibited abnormal morphology, with decreased cell growth and increased rate of spontaneous apoptosis and mitotic catastrophe. Furthermore, in nude mice xenografts, these cells exhibited decreased de novo gastric tumor formation and reduced development of angiogenesis. Results from these studies strongly suggest that survivin is a promising target for gastric cancer treatment.     

Suppression of tumorigenicity, but not anchorage independence, of human cancer cells by new candidate tumor suppressor gene CapG

Previously, we isolated a series of cell lines from a human diploid fibroblast lineage as a model for multistep tumorigenesis in humans. After passaging a single LT-transfected fibroblast clone, differently progressed cell lines were obtained, including immortalized, anchorage-independent and tumorigenic cell lines. In the present paper, we analysed the gene expression profiles of these model cell lines, and observed that expression of the CapG protein was lost in the tumorigenic cell line. To examine the possibility that loss of CapG protein expression was required for tumorigenic progression, we transfected CapG cDNA into the tumorigenic cell line and tested for tumor-forming ability in nude mice. Results showed that ectopic expression of CapG suppressed tumorigenicity, but not growth in soft agar or liquid medium. We also found that certain cancer cell lines including stomach cancer, lung cancer and melanoma had also lost CapG expression. One such cancer cell line AZ521 also became non-tumorigenic after the introduction of CapG cDNA. Moreover, we showed that CapG expression was repressed in small-cell lung cancer tissues. Together, our findings indicated that CapG is a new tumor suppressor gene involved in the tumorigenic progression of certain cancers.     

AKT activation and response to interferon-beta in human cancer cells

Significant growth inhibition and induction of apoptosis by IFN-beta in cancer cells including colorectal cancer cells have been observed. We and others have previously reported the Stat 1 induction of TRAIL is a crucial step in the IFN-beta induced apoptosis pathway. However, when evaluating the sensitivity of a panel of colorectal cancer cell lines, we found no clear correlation between activation of the Jak/Stat signaling pathway and response to interferon. In the present study, we have evaluated the interaction of the PI3k/Akt pathway and IFN-beta induced apoptosis in human colorectal cancer cells. The results demonstrate a correlation between Akt activity, phosphorylation of Bad and resistance to interferon-induced apoptosis in these cells. The association of activation of Akt, phosphorylation of Bad and resistance to IFN-beta-induced apoptosis was further supported by the observation that disruption of the pathway in a more resistant cell line led to sensitization, and expression of an activated Akt in a more sensitive cell line led to increased resistance. Taken together, this data indicates that the PI3/Akt kinase pathway may be an important contributor to IFN-beta sensitivity and resistance in colorectal cancer cells. This data demonstrates a potential pathway by which cells may develop resistance to IFN, and further elucidation of this process may allow us to better target IFN therapy.     

Androgen-induced differentiation and tumorigenicity of human prostate epithelial cells

Androgen ablation is the primary treatment modality for patients with metastatic prostate cancer; however, the role of androgen receptor signaling in prostate cancer development remains enigmatic. Using a series of genetically defined immortalized and tumorigenic human prostate epithelial cells, we found that introduction of the androgen receptor induced differentiation of transformed prostate epithelial cells to a luminal phenotype reminiscent of organ-confined prostate cancer when placed in the prostate microenvironment. Moreover, androgen receptor expression converted previously androgen-independent, tumorigenic prostate epithelial cells into cells dependent on testosterone for tumor formation. These observations indicate that androgen receptor expression is oncogenic and addictive for the human prostate epithelium.     

Suppression of prostate cancer nodal and systemic metastasis by blockade of the lymphangiogenic axis

Lymph node involvement denotes a poor outcome for patients with prostate cancer. Our group, along with others, has shown that initial tumor cell dissemination to regional lymph nodes via lymphatics also promotes systemic metastasis in mouse models. The aim of this study was to investigate the efficacy of suppressive therapies targeting either the angiogenic or lymphangiogenic axis in inhibiting regional lymph node and systemic metastasis in subcutaneous and orthotopic prostate tumor xenografts. Both androgen-dependent and more aggressive androgen-independent prostate tumors were used in our investigations. Interestingly, we observed that the threshold for dissemination is lower in the vascular-rich prostatic microenvironment compared with subcutaneously grafted tumors. Both vascular endothelial growth factor-C (VEGF-C) ligand trap (sVEGFR-3) and antibody directed against VEGFR-3 (mF4-31C1) significantly reduced tumor lymphangiogenesis and metastasis to regional lymph nodes and distal vital organs without influencing tumor growth. Conversely, angiogenic blockade by short hairpin RNA against VEGF or anti-VEGFR-2 antibody (DC101) reduced tumor blood vessel density, significantly delayed tumor growth, and reduced systemic metastasis, although it was ineffective in reducing lymphangiogenesis or nodal metastasis. Collectively, these data clarify the utility of vascular therapeutics in prostate tumor growth and metastasis, particularly in the context of the prostate microenvironment. Our findings highlight the importance of lymphangiogenic therapies in the control of regional lymph node and systemic metastasis.     

Interleukin 8 expression regulates tumorigenicity and metastasis in human bladder cancer

Interleukin 8 (IL-8) is mitogenic and chemotactic for endothelial cells. Within a neoplasm, IL-8 is secreted by inflammatory and neoplastic cells. The highly tumorigenic and highly metastatic human transitional cell carcinoma (TCC) cell line 253J B-V overexpresses IL-8 relative to the nontumorigenic and nometastatic 253J-P cell line. To determine whether IL-8 expression regulates tumorigenicity and metastasis in human TCC, 253J B-V cells were transfected with the full-sequence antisense (AS) cDNA for IL-8, whereas 253J-P cells were transfected with the full-length IL-8 cDNA, and control cells for each were transfected with the neomycin resistance (Neo) gene. In vitro, sense-transfected 253J-P cells overexpressed IL-8-specific mRNA and protein, whereas both of these were markedly reduced in AS-IL-8-transfected 253J B-V cells relative to controls. Moreover, sense-transfected cells showed up-regulation in matrix metalloproteinase type 9 mRNA, collagenase activity, and increased invasiveness through Matrigel-coated filters, whereas these measures were lower in AS-transfected cells relative to controls. After implantation into the bladders of athymic nude mice, the sense-transfected 253J-P cells acquired increased tumorigenicity and metastasis, whereas the AS-transfected cells significantly inhibited tumorigenicity and metastases in the 253J B-V cell lines. This effect was accompanied by reduced IL-8 expression and microvessel density. These studies demonstrate that IL-8 expression enhances angiogenic activity through the induction of matrix metalloproteinase type 9 and subsequently regulates the tumorigenesis and production of spontaneous metastases of human TCC.     

Inhibition of human prostate cancer growth, osteolysis and angiogenesis in a bone metastasis model by a novel mechanism-based selective gelatinase inhibitor

Metastasis to the bone is a major clinical complication in patients with prostate cancer (PC). However, therapeutic options for treatment of PC bone metastasis are limited. Gelatinases are members of the matrix metalloproteinase (MMP) family and have been shown to play a key role in PC metastasis. Herein, we investigated the effect of SB-3CT, a covalent mechanism-based MMP inhibitor with high selectivity for gelatinases, in an experimental model of PC bone metastases. Intraperitoneal (i.p.) treatment with SB-3CT (50 mg/kg) inhibited intraosseous growth of human PC3 cells within the marrow of human fetal femur fragments previously implanted in SCID mice, as demonstrated by histomorphometry and Ki-67 immunohistochemistry. The anti-osteolytic effect of SB-3CT was confirmed by radiographic images. Treatment with SB-3CT also reduced intratumoral vascular density and bone degradation in the PC3 bone tumors. A direct inhibition of bone marrow endothelial cell invasion and tubule formation in Matrigel by SB-3CT in vitro was also demonstrated. The use of the highly selective gelatinase inhibitors holds the promise of effective intervention of metastases of PC to the bone.     

Stromal cells from human benign prostate hyperplasia produce a growth-inhibitory factor for LNCaP prostate cancer cells,identified as interleukin-6

To understand specific interactions between stromal cells and epithelial cells in benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma, we developed stromal-cell cultures from normal human prostate (PNX) and BPH (BH101), composed of fibroblasts and myofibroblasts. Their role in epithelial-cell growth was studied using the established cancer cell lines LNCaP, PC3 and DU 145 and an SV40 large T-immortalized normal epithelial-cell line, PNTIA, in double-diffusion co-culture chambers. PNTIA was stimulated by PNX (x 1.6) and more strongly by BH101 stromal cells (x 2.7). Conversely, LNCaP growth decreased by 50% in the presence of BH101 stromal cells (stromal/epithelial ratio: 10). A BH101-conditioned medium (CM), obtained in serum-free conditions, induced 90% inhibition of [³H]thymidine incorporation of the LNCaP androgen-sensitive cell line. Two other androgen-independent prostate cancer cell lines were either insensitive to BH101 CM (PC3) or slightly inhibited (40% for DU145). BH101 produced large amounts of IL-Iβ, IL-6 and IL-8. HPLC gel filtration enabled separation of an inhibitory fraction which contained IL-6. IL-6 was demonstrated to be responsible for the strong inhibitory effect since an IL-6-neutralizing antibody abolished this inhibition, which was reproduced by human recombinant IL-6. Recombinant IL-6 growth inhibition was observed only on LNCaP prostate cancer androgen-sensitive cells. © 1996 Wiley-Liss, Inc.     

Pim1 kinase is required to maintain tumorigenicity in MYC-expressing prostate cancer cells

PIM1 kinase and MYC are commonly co-expressed in human prostate cancer and synergize to induce rapidly progressing prostate cancer in mouse models. Deficiency of the Pim kinase genes is well tolerated in vivo, suggesting that PIM1 inhibition might offer an attractive therapeutic modality for prostate cancer, particularly for MYC-expressing tumors. Here we examine the molecular consequences of Pim1 and MYC overexpression in the prostate as well as the effects of depleting Pim1 in prostate carcinoma cells with high levels of MYC. Overexpression of Pim1 in the mouse prostate induces several pro-tumorigenic genetic programs including cell cycle genes and Myc-regulated genes before the induction of any discernible pathology. Pim1 depletion by RNA interference in mouse and human prostate cancer cells decreased cellular proliferation, survival, Erk signaling and tumorigenicity even when MYC levels were not significantly altered. These results indicate that PIM1 may be necessary to maintain tumorigenicity, and further support efforts aimed at developing PIM1 inhibitors for prostate cancer therapy.     

Suppression of telomerase, reexpression of KAI1, and abrogation of tumorigenicity by nerve growth factor in prostate cancer cell lines.

Nerve growth factor (NGF) is expressed in the prostate, where it appears to be involved in the control of epithelial cell growth and differentiation. NGF production is decreased in prostate tumors. However, the role of this neurotrophin in the control of proliferation and progression of prostate cancers is still a matter of investigation. Prostate adenocarcinomas are telomerase-positive tumors. Chronic exposure of DU145 and PC3 prostate tumor cell lines to NGF resulted in a dramatic down-regulation of telomerase activity. This effect was correlated in terms of concentrations and time with a remarkable down-regulation of cell proliferation both in vitro and in vivo but was not secondary to NGF-induced quiescence. No down-regulation of telomerase activity was, in fact, detectable during serum starvation-induced quiescence. LNCaP cells, which do not express NGF receptors, appear to be insensitive to the actions of NGF. DU145 and PC3 cells do not express the KAI1 metastasis suppressor gene, which is present in the prostate and is progressively lost during the progression of prostate cancers. Chronic NGF treatment strongly induced the reexpression of this gene in these cell lines, and this effect was correlated with the suppression of their invasive potential in vitro. The data presented here suggest that NGF reverts two metastatic prostate cancer cell lines to slowly proliferating, noninvasive phenotypes characterized by a very low telomerase activity and by the expression of the KAI1 metastasis suppressor gene.