Midkine, a heparin-binding growth factor, produced by the host enhances metastasis of Lewis lung carcinoma cells

Midkine is a heparin-binding growth factor and is expressed by a number of tumor cells, contributing to their growth both in vitro and in vivo. Spontaneous lung metastasis of Lewis lung carcinoma cells, which did not significantly express MK, was significantly less extensive in mice deficient in the midkine gene than in wild-type mice, when the tumor was subcutaneously grown above the thigh. Midkine strongly enhanced migration of Lewis lung carcinoma cells in vitro. Therefore, midkine is also a host factor enhancing tumor metastasis. Anti-midkine therapy for malignancy may act on midkine produced by both the tumor and host.     

VEGF反义RNA对人食管癌细胞生长转移的抑制作用

目的:探讨研究血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)反义RNA在抑制恶性肿瘤生长和转移及抗肿瘤血管生成治疗中的意义。方法:采用脂质体法将反义VEGFcDNA质粒转染入食管癌细胞TE1;MTT法检测细胞增殖情况;原位杂交和RTPCR技术检测VEGF的表达水平,FCM分析细胞周期,并对转染前后细胞进行裸鼠体内生长转移等生物学行为实验。结果:转染反义VEGFcDNA质粒的TE1细胞中VEGF表达水平降低,与对照组比较,裸鼠体内成瘤时间延长,肿瘤生长速度减慢,质量和体积差异有显著性意义(P<0.05)。结论:VEGF反义RNA能抑制食管癌TE1细胞VEGF表达,对裸鼠体内TE1细胞的生长有抑制作用,有望成为食管癌基因治疗的优选基因之一。     

Antisense phosphorothioate oligodeoxynucleotide down-regulation of the insulin-like growth factor I receptor in ovarian cancer cells

The insulin-like growth factors (IGF-I and IGF-II) play a key role in cellular proliferation and are involved in cellular transformation. The expression of the IGF-I receptor has been demonstrated in a variety of human tumor cell lines including ovarian cancer cells. Phosphorothioate antisense oligodeoxynucleotides (S-ODNs) were analyzed for their potential to suppress the IGF-I receptor in the NIH:OVCAR-3 ovarian cancer cell line. The application of the antisense S-ODN reduced potently the cell growth of unstimulated NIH:OVCAR-3 cells, whereas sense and mismatch S-ODNs were without any effect. This effect resembled that of the monoclonal antibody (MAb) αIR-3. In contrast to the antisense compound, this MAb only partially inhibited the IGF-I-induced proliferation of ovarian cancer cells. The concentration of the antisense S-ODN to exhibit an identical inhibition of cell proliferation was reduced to 50 nM when the oligonucleotides were delivered by the cationic lipid formulation lipofectin. The specificity of the antisense S-ODN action was confirmed by reduction of the receptor protein and of the receptor mRNA, as assayed by flow cytometry and by Northern blot hybridizations. Our data demonstrate the potency of antisense S-ODNs to target the IGF-I receptor message and show that antisense strategies against the IGF-I receptor may provide new strategies for the therapy of ovarian cancer. Int. J. Cancer 77:567–571, 1998. © 1998 Wiley-Liss, Inc.     

Down-regulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells.

Galectin-3 is an endogenous beta-galactoside-binding protein with specificity for type I and II ABH blood group epitopes and poly-N-acetyllactosamine glycan-containing cell surface glycoproteins and is the major nonintegrin cellular laminin-binding protein. Galectin-3 is expressed at an elevated level in a wide range of neoplasms, and expression was shown to be associated in some tumor cell systems with metastases. Here we determined the functional consequence of blocking galectin-3 expression in highly malignant human breast carcinoma MDA-MB-435 cells. Inhibition of galectin-3 expression led to reversion of the transformed phenotype as determined by altered morphology, loss of serum-independent growth, acquisition of growth inhibition properties by cell contact, and abrogation of anchorage-independent growth. The blockage of galectin-3 expression led to a significant suppression of tumor growth in nude mice. These results provide direct evidence that galectin-3 expression is necessary for the maintenance of the transformed and tumorigenic phenotype of MDA-MB-435 breast carcinoma cells.     

Interaction of androgen-induced autocrine heparin-binding growth factor with fibroblast growth factor receptor on androgen-dependent Shionogi carcinoma 115 cells

Stimulation of a Shionogi carcinoma 115-derived cultured cell line (SC-3) with androgen resulted in secretion of heparin-binding growth factor. In this study, we analyzed cell-surfaced receptors for growth factors. Binding data of growth factors on intact SC-3 cells revealed the presence of low and high affinity receptors for epidermal growth factor (EGF), insulin, and fibroblast growth factor (FGF). The dissociation constant values were 50 pM and 1.0 nM for EGF, 1.2 nM and 30 nM for insulin, and 34 pM and 7.5 nM for FGF. The numbers of maximal binding sites were 300 and 900/cell for EGF, 2,000 and 14,000/cell for insulin, and 13,000 and 810,000/cell for FGF. To examine the association of androgen-induced growth factor with one of these receptors, the conditioned medium prepared from androgen-stimulated SC-3 cells was fractionated through a heparin-Sepharose column. Growth factor activity adsorbed and eluted by 1 M NaCl from the column was comigrated with the activity inhibiting FGF-receptor association. In addition, basic 125I-FGF was cross-linked, using disuccinimidyl suberate, to the receptor with an apparent molecular weight of 130,000, whose labeling was inhibited when basic FGF, acidic FGF, or highly purified androgen-induced growth factor was present in excess. Furthermore, the highly purified growth factor-, basic FGF- or androgen-induced growth of SC-3 cells was significantly and similarly inhibited by anti-basic FGF antibody IgG. These results indicate that androgen-induced FGF-like factor acts as an autocrine growth factor via the FGF receptor in a process of SC-3 cell proliferation.     

Expression of heparin-binding epidermal growth factor-like growth factor in breast carcinoma

The expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated for 76 cases of breast carcinoma. HB-EGF was expressed in 71.8% of the carcinoma cases but only slightly in normal mammary glands. Interestingly, its expression was inversely related to biological aggressiveness of the breast carcinoma. These results suggest that HB-EGF may play a crucial role in the early stage of this carcinoma.     

Contextual effects of transforming growth factor beta on the tumorigenicity of human colon carcinoma cells.

Transforming growth factor betas (TGF-betas) are a growth factor family with negative autocrine growth functions for most epithelial cells including colon carcinoma cell lines. Both type I (RI) and type II (RII) transmembrane TGF-beta receptors have been shown to be indispensable for TGF-beta-mediated cell growth regulation. Previous studies using different model systems have shown that both overexpression of TGF-beta1 and transfection of antisense TGF-beta1 to reduce TGF-beta1 expression could lead to increased tumorigenicity. These results are seemingly contradictory and suggest that effects of TGF-beta modulation on malignant properties of cancer cells may be contextual. This study addresses this issue using human colon carcinoma cells (CBS and FET) to determine the effects of modulation of the various components of the TGF-beta system on in vitro and in vivo growth properties in two independent isogenic models of colon carcinoma. Cells were stably transfected with a tetracycline-repressible RII expression vector (CBS4-RII), a tetracycline-repressible expression vector containing a truncated RII cDNA lacking the serine/threonine kinase domain (CBS4-deltaRII and FET6-deltaRII), or with a vector containing the TGF-beta1 cDNA (CBS4-beta1S and FET-beta1S). Expression of the truncated RII reduced TGF-beta sensitivity, whereas overexpression of RII increased TGF-beta sensitivity. TGF-beta overexpression did not affect TGF-beta response. In vivo tumorigenicity assays revealed that CBS4-RII cells had lower tumorigenicity than control cells, whereas CBS4-deltaRII and CBS4-beta1S had higher tumorigenicity than controls. The CBS4 cells are poorly tumorigenic in athymic mice, and the wild-type FET6 cells are nontumorigenic. FET6-deltaRII cells formed rapidly growing tumors, and FET-beta1S cells also formed tumors. These data illustrate the paradoxical tumor-promoting and -suppressing effects of TGF-beta signaling activity in two isogenic model systems from human colon carcinomas, thus demonstrating that the effects of modulation of TGF-beta expression or TGF-beta signaling capability affects malignancy in a contextual manner.     

Expression of the TNF-alpha gene on mouse lung carcinoma cells suppresses spontaneous lung metastasis without affecting tumorigenicity

Forced expression of TNF-alpha in tumor cells has been shown to inhibit their tumor growth in vivo through a number of mechanism such as activation of an immune system and induction of an apoptotic process. We re-examined the anti-tumor effects caused by the TNF-alpha gene transfer using high-metastatic, murine lung carcinoma A11 cells. Expressed TNF-alpha molecules remained on cell surface and were not secreted into culture supernatants in vitro. Syngeneic immunocompetent mice developed tumors of TNF-alpha-expressed A11 cells and the growth of their subcutaneous tumors was not different from that of parent tumors. Spleen of the mice that developed TNF-alpha-expressed A11 tumors was significantly larger than that of the mice bearing parent tumors, but relative ratios of each cell population were not different. In contrast to subcutaneous tumors, the number of spontaneous lung foci metastasized from the subcutaneous TNF-alpha-expressed A11 tumors was markedly reduced compared with that from parent tumors. Expressed TNF-alpha on tumors is released by matrix metalloproteinases from surrounding tissues and anti-tumor effects by TNF-alpha can be influenced by local environmental conditions.     

Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line

The epidermal growth factor (EFG) family of receptors and their respective ligands play a major role in breast cancer progression and are the targets of new therapeutic approaches. Following immortalization with SV40 T antigen of normal human breast epithelial cells, a transformed variant cell line (NS2T2A1) was selected for its increased tumorigenicity in nude mice. This cell line was shown to have a higher expression of EGF receptors (EGFR) and amphiregulin (AR) when compared to their normal counterparts or less aggressive transformed cells. Dual staining of EGFR and AR was observed in 50-60% of NS2T2A1 cells, while 30-40% cells expressed AR only. To explore the potential tumorigenic role of AR, a 1.1 kb AR cDNA in an antisense orientation was transfected in NS2T2A1 cells. Three clones, selected by hygromycin B, expressed AR antisense RNA (AR AS1, AR AS2 and AR AS3 cell lines) in which AR protein expression was reduced (ranging from about 50 to < 5%). The anchorage-independent growth of AR AS cell lines was reduced to levels ranging from 32.4-6.8% relative to the control cell line transfected with the vector alone. The clones expressing AR antisense RNA showed a reversion of the malignant phenotype when injected in nude mice, since a significant reduction of tumor intake was observed coincident with a significant tumor mass reduction (> 96%). Moreover, intra-tumoral vascularization decreased significantly in tumors derived from AR AS cells (26.7, 70.7 and 50.4% of control). These in vitro and in vivo data reveal the oncogenic nature of AR in transformed breast epithelial cells and imply a role for AR in tumor angiogenesis.     

A soluble transforming growth factor beta type III receptor suppresses tumorigenicity and metastasis of human breast cancer MDA-MB-231 cells.

Transforming growth factor beta (TGF-beta) can promote late stage tumor progression in a number of model systems. In the present study, we have examined whether expression of a truncated soluble extracellular domain of TGF-beta type III receptor (sRIII) in human breast cancer MDA-MB-231 cells can antagonize the tumor-promoting activity of TGF-beta by sequestering active TGF-beta isoforms that are produced by the cancer cells. The secretion of sRIII reduced the amount of active TGF-beta1 and TGF-beta2 in the conditioned medium. This led to a significant reduction of the growth-inhibitory activity of the medium conditioned by sRIII-expressing cells on the growth of mink lung epithelial CCL64 cells in comparison with the medium conditioned by the control cells. The tumor incidence and growth rate of all of the three sRIII-expressing clones studied were significantly lower than those of the control cells in athymic nude mice. Four of five control cell-inoculated mice showed spontaneous metastasis in the lung, whereas none of the sRIII-expressing cell-inoculated mice had any lung metastasis. Thus, our results suggest that the sRIII may be used to antagonize the tumor-promoting activity of TGF-beta.     

Proteolytic activation of heparin-binding EGF-like growth factor by membrane-type matrix metalloproteinase-1 in ovarian carcinoma cells

Increased expression of heparin-binding EGF-like growth factor (HB-EGF) and membrane-type matrix metalloproteinase-1 (MT1-MMP) is frequently associated with various types of malignant tumor. HB EGF-like growth factor has been reported to promote the malignant progression of ovarian carcinoma. Based on this finding, inhibition of HB-EGF activity with CRM197 is now under phase I clinical evaluation. On the other hand, MT1-MMP expressed in ovarian carcinoma cells is thought to promote invasion and growth of tumor cells by degrading the extracellular matrix. However, we recently demonstrated that co-expression of MT1-MMP and HB-EGF in gastric carcinoma cells leads to cleavage of HB-EGF within its N-terminal heparin-binding region, converting it into a potent heparin-independent growth factor. In this study, we evaluated the importance of regulation of HB-EGF by MT1-MMP in clinical samples of ovarian carcinoma. We detected co-expression of HB-EGF and MT1-MMP in clear cell ovarian carcinoma tissues, particularly at the invasion front and in tumor cells that had disseminated into the ascites, whereas HB-EGF alone was expressed in non-invasive borderline ovarian tumor tissue. Furthermore, a soluble HB-EGF fragment that corresponds to that processed by MT1-MMP was detected in malignant ascites obtained from patients with metastatic ovarian carcinoma. Ovarian carcinoma cells that express MT1-MMP and HB-EGF exhibited enhanced cell growth in a 3D-collagen matrix and anchorage-independent growth in suspension. These results indicate that MT1-MMP co-expressed with HB-EGF in ovarian carcinoma cells potentiates the activity of HB-EGF to promote invasive tumor growth and spreading in vivo.     

Sp1 decoy transfected to carcinoma cells suppresses the expression of vascular endothelial growth factor, transforming growth factor beta1, and tissue factor and also cell growth and invasion activities

Vasculature development is thought to be an important aspect in the growth and metastasis of solid tumors. Among the angiogenic factors produced by tumor cells, vascular endothelial growth factor is considered to be the most potent and pathologically important. The synthesis of this growth factor has been shown to be modulated through Sp1 function following stimulation by tumor necrosis factor alpha (TNF-alpha). Oligodeoxynucleotides (ODNs) were synthesized with either the consensus sequence for Sp1 binding (Sp1 decoy ODNs) or a mutated form of this sequence (mt-Sp1 decoy ODNs). Using the hemagglutinating virus of Japan (HVJ)-liposome method, we transferred these ODNs into cultured cancer cells (A549 and U251 cells). The TNF-alpha-mediated expression of both VEGF and transforming growth factor beta1 and tissue factor (TF) by the cancer cells could be simultaneously suppressed to less than 30% by transfection of Sp1 decoy ODNs but not by mt-Sp1 decoy ODNs. In addition, in vitro invasiveness, synthesis of mRNA for urokinase-type plasminogen activator, and cell proliferation of both cell lines were also inhibited to 40% by the transfection of only Sp1 decoy ODNs. These results suggested that the Sp1 decoy strategy would be effective for regulating tumor growth by simultaneously reducing cancer cell (a) angiogenic growth factor expression, (b) proliferation, and (c) invasiveness.     

Expression of the wild-type insulin-like growth factor II receptor gene suppresses growth and causes death in colorectal carcinoma cells.

The insulin-like growth factor II receptor (IGFIIR) has been implicated as a tumor suppressor gene in human malignancy. Frequent mutation, loss of heterozygosity, and microsatellite instability (MSI) directly affecting the IGFIIR gene have been reported in several primary human tumor types. However, to our knowledge, dynamic functional evidence of a growth-suppressive role for IGFIIR has not yet been provided. We identified one MSI-positive colorectal carcinoma cell line, SW48, with monoallelic mutation in IGFIIR identical to that seen in primary colorectal carcinomas. A zinc-inducible construct containing the wild-type IGFIIR cDNA was stably transfected into SW48 cells. Growth rate and apoptosis were compared between zinc-treated, untreated, and untransfected cells. A twofold increase in IGFIIR protein expression was detected after zinc treatment in discrete clonal isolates of transfected SW48 cells. Moreover, zinc induction of exogenous wild-type IGFIIR expression reproducibly decreased growth rate and increased apoptosis. These data prove that wild-type IGFIIR functions as a growth suppressor gene in colorectal cancer cells and provide dynamic in vitro functional support for the hypothesis that IGFIIR is a human growth suppressor gene.     

Co-expression of heparin-binding EGF-like growth factor and related peptides in human gastric carcinoma

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of polypeptide growth factors, which includes EGF, transforming growth factor α (TGF-α), amphiregulin (AR) and betacellulin (BTC). To assess the potential role of HB-EGF in human gastric carcinomas, the expression of HB-EGF and EGF receptor (EGF-R) was examined in normal and cancerous gastric tissues and cultured gastric cancer cell lines. By Northern blot analysis, there was a 4.7-fold increase in HB-EGF mRNA levels in human gastric cancers compared with normal gastric tissues. There was a concomitant 3.9-fold increase in EGF-R mRNA levels in these cancers. Immunostaining revealed co-localization in 72% of the cancer cells of HB-EGF and EGF-R. AR and BTC moieties were not evident by Northern blot analysis. However, using PCR, both AR and BTC mRNA species were demonstrated in normal and cancerous gastric tissues. By Northern blot analysis, HB-EGF, TGF-α, AR, BTC and EGF-R mRNA moieties were co-expressed in KATO III and NCI-N87 gastric cancer cell lines. Furthermore, HB-EGF, EGF and TGF-α enhanced the growth of both cell lines in a dose-dependent manner. Our findings suggest that HB-EGF is relatively abundant in human gastric cancers and that co-expression of the EGF ligand family may lead to excessive activation of EGF-R in this disorder. © 1996 Wiley-Liss, Inc.     

Proliferation of Shionogi carcinoma 115 cells by glucocorticoid-induced autocrine heparin-binding growth factor(s) in serum-free medium

Shionogi carcinoma 115 (SC115) has been accepted for 20 years as an androgen-responsive mouse mammary tumor. Recently, the growth of the tumor was also found to be stimulated by pharmacological, but not physiological, doses of glucocorticoid. In a serum-free culture system [Ham's F-12:Eagle's minimal essential medium (1:1, v/v) containing 0.1% bovine serum albumin], we have established that 10(-8) M testosterone, or 10(-6) M dexamethasone significantly stimulates the growth of SC-3 cells (a cloned cell line from a SC115 tumor) via androgen and glucocorticoid receptors, respectively. Recently, we demonstrated that the testosterone-induced growth of SC-3 cells is mediated through autocrine fibroblast growth factor (FGF)-like peptide(s). In the present study, mechanisms of glucocorticoid-induced growth of SC-3 cells were investigated. Serum-free conditioned medium obtained from 10(-6) M dexamethasone-stimulated SC-3 cells was fractionated by heparin-Sepharose affinity chromatography; one sharp peak of growth-stimulatory activity for SC-3 cells, eluted at 1.3 M NaCl, was identified. When the peak fraction was added to serum-free medium, the shape of SC-3 cells changed from an epithelial to a fibroblast-like appearance, similar to that induced with testosterone or basic (b)FGF. Furthermore, the growth-stimulatory activity induced with the peak fraction as well as testosterone or bFGF was markedly inhibited by anti-bFGF antibody immunoglobulin G (75 to 90% inhibition was obtained), and the specific binding of 125I-bFGF on SC-3 cells was significantly inhibited by the peak fraction. These results suggest that the glucocorticoid-induced growth of SC-3 cells is also mediated through FGF-like peptide(s) in an autocrine mechanism, which is very similar to that induced by testosterone, if not identical.     

Hepatocyte growth factor activator inhibitor type 1 suppresses metastatic pulmonary colonization of pancreatic carcinoma cells

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a transmembrane protease inhibitor that regulates the activities of membrane-bound and extracellular serine proteases. HAI-1 has two Kunitz-type inhibitor domains with the N-terminal Kunitz domain (KD1) responsible for inhibiting known target proteases. Previously, we reported that knockdown of HAI-1 in the human pancreatic carcinoma cell line SUIT-2 resulted in epithelial to mesenchymal transition. To evaluate the role of HAI-1 in metastasis, we examined the metastatic capability of SUIT-2 cells that did or did not stably express HAI-1 short-hairpin RNA in an experimental pulmonary metastasis assay using nude mice. The extent of pulmonary metastasis was verified by histological examination and direct measurement of human cytokeratin 19 mRNA levels. One week after injecting SUIT-2 cells into mouse tail veins, apparent metastatic colonization was observed in 36% (4/11) of mice injected with HAI-1-knockdown SUIT-2, whereas none (0/11) of the control mice were positive for metastasis. After 2 weeks the metastasis positive ratios were 80% (4/5) and 40% (2/5), and after 4 weeks the ratios were 82% (9/11) and 45% (5/11) for HAI-1-knockdown and control SUIT-2 cells, respectively. Thus, loss of HAI-1 promoted pulmonary metastasis. Co-injection of recombinant KD1 abolished metastasis produced by HAI-1-knockdown SUIT-2 cells after 1 week. Moreover, recombinant KD1 restored E-cadherin levels in HAI-1 knockdown SUIT-2 cells and reduced their invasiveness in vitro. These data indicate that HAI-1 regulates pulmonary metastasis of SUIT-2, and KD1 may have therapeutic application for inhibiting metastatic cancer cell spreading.