Anti-tumor activity of motesanib in a medullary thyroid cancer model.

BACKGROUND: Medullary thyroid cancer (MTC) is frequently associated with mutations in the tyrosine kinase Ret and with increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Motesanib is an investigational, orally administered small molecule antagonist of VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR); Kit; and possibly Ret. AIM: The aim of this study was to investigate the effects of motesanib on wildtype and mutant Ret activity in vitro and on tumor xenograft growth in a mouse model of MTC. METHODS/RESULTS: In cellular phosphorylation assays, motesanib inhibited the activity of wild-type Ret (IC(50)=66 nM), while it had limited activity against mutant Ret C634W (IC(50)=1100 nM) or Ret M918T (IC(50)>2500 nM). In vivo, motesanib significantly inhibited the growth of TT tumor cell xenografts (expressing Ret C634W) and significantly reduced tumor blood vessel area and tumor cell proliferation, compared with control. Treatment with motesanib resulted in substantial inhibition of Ret tyrosine phosphorylation in TT xenografts and, at comparable doses, in equivalent inhibition of VEGFR2 phosphorylation in both TT xenografts and in mouse lung tissue. CONCLUSIONS: The results of this study demonstrate that motesanib inhibited thyroid tumor xenograft growth predominantly through inhibition of angiogenesis and possibly via a direct inhibition of VEGFR2 and Ret expressed on tumor cells. These data suggest that targeting angiogenesis pathways and specifically the VEGF pathway may represent a novel therapeutic approach in the treatment of MTC.

The coordinated functional expression of epidermal growth factor receptor and c-Met in colorectal carcinoma metastasis.

The process of metastasis is a highly selective, nonrandom process resulting in the clonal selection of a population of cells that is able to detach from the primary tumor, invade and survive in the circulation, arrest, extravasate, and ultimately survive and proliferate in the secondary organ-specific site. Tumor cell interactions with the microenvironment can profoundly influence the survival and proliferation of the cell at a secondary site. The epidermal growth factor receptor (EGFR) and the hepatocyte growth factor receptor (c-Met) are two such receptor tyrosine kinases (RTKs) that have been causally implicated in colorectal carcinoma (CRC) progression and metastasis. Activation of these RTKs can stimulate a number of specific pathways directly effecting tumor cell migration, survival and proliferation. The aberrant regulation of the RTKs is often noted in advanced CRC and its' liver metastases and can significantly effect the metastatic phenotype of tumor cells.

Treatment of human metastatic transitional cell carcinoma of the bladder in a murine model with the anti-vascular endothelial growth factor receptor monoclonal antibody DC101 and paclitaxel.

Vascular endothelial cell growth factor (VEGF) regulates angiogenesis and metastasis of bladder cancer (transitional cell carcinoma, TCC) through binding to VEGF receptor-2 (VEGFR-2). In this study, we evaluated whether the anti-VEGFR monoclonal antibody (Mab) DC101 in combination with paclitaxel inhibited tumorigenesis, angiogenesis, and metastasis of human TCC growing within the bladder of athymic nude mice. In vivo therapy with Mab DC101 and paclitaxel induced significant regression of bladder tumors compared with either agent alone. Median bladder weights were reduced from 601 mg in untreated controls, 422 mg in mice treated with paclitaxel alone (P < 0.005), 361 mg in mice treated with DC101 alone (P < 0.005), and 113 mg in mice that received combination therapy (P < 0.0005). Only one of nine mice developed spontaneous lymph node metastasis after combined treatment, compared with seven of seven untreated controls (P < 0.0005), six of eight after DC101 (P < 0.01), and five of eight mice after paclitaxel (P < 0.05). Combined treatment with both paclitaxel and DC101 inhibited tumor-induced neovascularity compared with all other groups (P < 0.005), without altering the expression of VEGF or flk1. Mab DC101 and paclitaxel combined enhanced apoptosis in the tumor and endothelial cells compared with other treatment (P < 0.005). These studies indicate that Mab DC101, which blocks VEGFR-2 function, has significant efficacy against human TCC, especially when combined with the chemotherapeutic agent paclitaxel. The antitumor effect was mediated by inhibition of angiogenesis and induction of both tumor cell and endothelial cell apoptosis.

Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma.

We determined whether down-regulation of the epidermal growth factor-receptor (EGF-R) signaling pathway by oral administration of a novel EGF-R tyrosine kinase inhibitor (PKI166) alone or in combination with gemcitabine (administered i.p.) can inhibit growth and metastasis of human pancreatic carcinoma cells implanted into the pancreas of nude mice. Therapy beginning 7 days after orthotopic injection of L3.6pl human pancreatic cancer cells reduced the volume of pancreatic tumors by 59% in mice treated with gemcitabine only, by 45% in those treated with PKI166 only, and by 85% in those given both drugs. The combination therapy also significantly inhibited lymph node and liver metastasis, which led to a significant increase in overall survival. EGF-R activation was significantly blocked by therapy with PKI166 and was associated with significant reduction in tumor cell production of VEGF and IL-8, which in turn correlated with a significant decrease in microvessel density and an increase in apoptotic endothelial cells. Collectively, our results demonstrate that oral administration of an EGF-R tyrosine kinase inhibitor decreased growth and metastasis of human pancreatic cancer growing orthotopically in nude mice and increased survival. The therapeutic effects were mediated in part by inhibition of tumor-induced angiogenesis attributable to a decrease in production of proangiogenic molecules by tumor cells and increased apoptosis of tumor-associated endothelial cells.

Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms.

Both epidermal growth factor receptor (EGF-R) signaling mechanisms and angiogenesis have been evaluated as independent targets for therapy of human pancreatic carcinoma, but a link between the two processes has been identified only recently. This study evaluated whether EGF-R blockade therapy with anti-EGF-R antibody C225 inhibits pancreatic carcinoma growth and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whether gemcitabine potentiates this effect. In vitro treatment of human pancreatic carcinoma L3.6pl cells with C225 inhibited EGF-R autophosphorylation, producing a maximum of 20% cytostasis. Treatment with C225 plus gemcitabine resulted in additive cytotoxic effects that increased with increasing gemcitabine concentrations. Dose-dependent decreases in expression of the angiogenic factors vascular endothelial growth factor and interleukin 8 (but not basic fibroblast growth factor) were observed in the C225-treated cells (mRNA and protein levels). In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with gemcitabine resulted in growth inhibition, tumor regression, and abrogation of metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm3, respectively. Gemcitabine treatment alone reduced median tumor volume from 538 to 152 mm3. Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and 20% of C225-treated animals. No macroscopically visible liver metastases were observed in the combination treatment group. As early as 11 days after C225 treatment, the median percentage of proliferating cell nuclear antigen-positive cells was substantially reduced compared with gemcitabine treatment alone (26% versus 73%, respectively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after 11 days treatment, production of vascular endothelial growth factor and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 days after C225 or combination treatments (but not gemcitabine alone) in direct correlation with the difference in percentage of apoptotic endothelial cells, as visualized by double immunofluorescence microscopy. These experiments indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6pl pancreatic carcinoma growing in nude mice which is mediated in part by inhibition of tumor-induced angiogenesis, leading to tumor cell apoptosis and regression. Furthermore, this effect is potentiated in combination with gemcitabine.

Interleukin 8 expression regulates tumorigenicity and metastases in androgen-independent prostate 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 metastatic PC-3M-LN4 cell line overexpresses IL-8 relative to the poorly metastatic PC-3P cell line. We evaluated whether IL-8 expression by human prostate cancer growing within the prostate of athymic nude mice regulates tumor angiogenesis, growth, and metastasis. PC-3P cells were transfected with the full-length sense IL-8 cDNA, whereas PC-3M-LN4 cells were transfected with the full-sequence antisense IL-8 cDNA. Control cells were transfected with the neomycin resistance gene (Neo). In vitro, sense-transfected PC-3P cells overexpressed IL-8-specific mRNA and protein, which resulted in up-regulation of matrix metalloproteinase 9 (MMP-9) mRNA, and collagenase activity, resulting in increased invasion through Matrigel. After antisense transfection of the PC-3M-LN4 cells, IL-8 and MMP-9 expression, collagenase activity, and invasion were markedly reduced relative to controls. After orthotopic implantation, the sense-transfected PC-3P cells were highly tumorigenic and metastatic, with significantly increased neovascularity and IL-8 expression compared with either PC-3P cells or controls. Antisense transfection significantly reduced the expression of IL-8 and MMP-9 and tumor-induced neovascularity, resulting in inhibition of tumorigenicity and metastasis. These results demonstrate that IL-8 expression regulates angiogenesis in prostate cancer, in part by induction of MMP-9 expression, and subsequently regulates the growth and metastasis of human prostate cancer.

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.

Paclitaxel enhances the effects of the anti-epidermal growth factor receptor monoclonal antibody ImClone C225 in mice with metastatic human bladder transitional cell carcinoma.

Previously we reported that when cells from the human transitional cell carcinoma cell line 253J B-V growing orthotopically within the bladder of athymic nude mice were treated with the anti-epidermal growth factor receptor monoclonal antibody C225, angiogenesis was inhibited, resulting in regression of the primary tumor and inhibition of metastasis. In this study, we evaluated whether paclitaxel enhanced this therapeutic effect of C225. In vitro, the proliferation of 253J B-V cells was inhibited more by the combination of C225 and paclitaxel than with either agent alone. In vivo therapy with C225 and paclitaxel resulted in significantly greater regression of tumors compared with either agent alone. Median bladder tumor weight was 85 mg (range, 69-133 mg) compared with 168 mg (range, 72-288 mg) after C225 alone (P < 0.05), and 273 mg (range, 83-563 mg) after paclitaxel alone (P < 0.005). The incidence of spontaneous lymph node metastasis was also reduced by the combination of C225 with paclitaxel, although this result did not significantly differ from results after the use of C225 alone. Treatment with paclitaxel and C225 down-regulated the expression of basic fibroblast growth factor, vascular endothelial cell growth factor, interleukin-8, and matrix metalloproteinase type 9 and inhibited tumor-induced neovascularity compared with untreated controls (P < 0.005). Moreover, the combination of C225 and paclitaxel enhanced apoptosis in tumor and endothelial cells compared with either agent alone (P < 0.005). These studies indicate that therapy with paclitaxel increases the ability of C225 to inhibit tumorigenicity and metastasis. This effect is mediated by inhibition of angiogenesis and induction of apoptosis.

Amplification of telomeric DNA directly correlates with metastatic potential of human and murine cancers of various histological origin.

Telomeres, repeated DNA sequences (T2AG3)n that guard the ends of chromosomes, serve as a checkpoint for cell-cycle progression and regulate cell senescence and apoptosis. Loss of the telomeric repeats promotes genomic instability, which is the hallmark of most cancer cells. Whether this loss differs among tumor cells with malignant potential is unknown and was the goal of this study. An all-human telomeric DNA probe was used to perform fluorescence in situ hybridization (FISH) and the telomeric signals in interphase nuclei were quantitated using a computer software package. Southern blot analysis was carried out to measure terminal restriction fragment length (TRFL) in multiple cancer cell lines, including nonmetastatic and metastatic human breast, lung, prostate, colon, brain, and renal carcinomas, as well as human and murine melanoma clones and somatic cell hybrids. The metastatic capability of all cell lines, clones and somatic cell hybrids was evaluated subsequent to orthotopic implantation into nude mice. FISH preparations with telomeric DNA probes showed that the mean percent telomeric area in the metastatic nuclei was significantly greater than their nonmetastatic counterparts and Southern blotting in selected samples confirmed our findings. These data suggest that amplification of telomeres is directly correlated with invasive and metastatic potential of murine or human tumor cells.

Growth hormone receptor expression and function in meningiomas: effect of a specific receptor antagonist.

OBJECT: This study was undertaken to explore the effects of growth hormone (GH) and the GH-stimulated peptide insulin-like growth factor-1 (IGF-1) on the growth rate of meningiomas. METHODS: Polymerase chain reaction and ribonuclease protection assays were used to demonstrate that GH receptor messenger RNA was present in all 14 meningioma specimens studied, regardless of tumor grade. Both wild type (GHRwt) and a previously described exon 3 deletion isoform (GHRd3) of the GH receptor were identified in individual tumor specimens. The importance of the GH receptor was assessed using a GH receptor antagonist (B2036). Blockade of the GH receptor with B2036 reduced serum-induced DNA synthesis, as measured by thymidine incorporation, by 8 to 33% (mean 20%) in primary meningioma cultures. Tumors that expressed the GHRwt and GHRd3 isoforms, or a combination of the two, were all responsive to antagonist treatment. The importance of IGF- in stimulating meningioma cell growth was also assessed. It was found that IGF-1 increased thymidine incorporation in primary meningioma cultures in a dose-dependent manner: 1 ng/ml, 5 ng/ml, and 10 ng/ml resulted in increases in thymidine incorporation of 21%, 43%, and 176%, respectively, over baseline values. CONCLUSIONS: In these studies the authors demonstrate that activation of the GH/IGF-1 axis significantly increases the growth rate of meningiomas. Blockade of the GH receptor on tumor cells inhibited tumor growth. If these findings are confirmed in animal studies, agents that downregulate the GH/IGF-1 axis might represent a potential adjuvant therapy in the management of patients with meningioma.

Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice.

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human transitional cell carcinoma (TCC) of the bladder. We have shown that therapy targeting EGFR inhibited the growth of human TCC established orthotopically in nude mice. The purpose of this study was to evaluate whether EGFR-directed therapy affects angiogenesis associated with the growth and metastasis of human TCC. We determined the cytostatic effect and the effect on production of angiogenic factors after in vitro treatment of the human TCC cell line 253J B-V with MAb C225, a chimerized monoclonal anti-EGFR antibody. The 253J B-V cells were implanted orthotopically into athymic nude mice, and established tumors (4 weeks) were treated with i.p. MAb C225. Expression of the angiogenic factors vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) was evaluated by immunohistochemistry and in situ mRNA hybridization analyses and correlated with microvessel density evaluated after immunohistochemical staining with anti-CD31. In vitro treatment with MAb C225 inhibited mRNA and protein production of VEGF, IL-8, and bFGF by 253J B-V cells in a dose-dependent manner. MAb C225 therapy of nude mice with established TCCs growing orthotopically resulted in inhibition of growth and metastasis compared with controls (P <0.0005). VEGF, IL-8, and bFGF expression was significantly lower in treated tumors than in controls. The down-regulation of these angiogenic factors preceded the involution of blood vessels. These studies indicate that therapy with anti-EGFR MAb C225 has a significant antitumor effect mediated, in part, by inhibition of angiogenesis.

The biology of melanoma brain metastasis.

Brain metastases are clinically diagnosed in the majority of patients with metastatic melanoma. The prognosis for patients with melanoma brain metastasis is poor with a median survival time of 6 months after diagnosis. Development of better therapies requires a better understanding of the biology of melanoma brain metastasis. The development of a relevant in vivo model offers this possibility. The intracarotid injection of different murine or human melanoma cells into syngeneic or nude mice produces metastases in different regions of the brain. This site-specific metastasis is not due to patterns of initial cell arrest, motility, or invasiveness, but rather to the ability of melanoma cells to proliferate in the brain parenchyma or the meninges. The blood-brain barrier is intact in metastases that are smaller than 0.25 mm in diameter. Although in larger metastases the blood-brain barrier is leaky, the lesions are resistant to many chemotherapeutic drugs. We have also analyzed the malignant behavior of several melanoma cell lines isolated from brain or visceral metastases of patients. The cells from brain metastases showed a slower growth rate and exhibited lower metastatic potential than cells from visceral metastases, indicating that brain metastases do not necessarily represent the end stage in the metastatic cascade. Rather, brain metastases are likely to originate from a unique subpopulation of cells within the primary neoplasm.

Preferential activation of the epidermal growth factor receptor in human colon carcinoma liver metastases in nude mice.

Increased epidermal growth factor receptor (EGF-R) gene expression and functional protein levels correlate with the metastatic potential of human colon carcinoma (HCC) cells in nude mice. The purpose of this study was to determine whether the production of liver metastases by HCC cells depends on the EGF-R activation status and whether different organ microenvironments influence this activation. Using two independent monoclonal antibodies specific for the activated (i.e., tyrosine-phosphorylated) EGF-R, increased immunoreactivity was observed in HCC cells growing as metastatic lesions in the livers of athymic nude mice. Staining was observed throughout these lesions, both peripherally and centrally. In contrast, little or no immunoreactivity for activated EGF-R was observed in primary tumors growing orthotopically in the cecum or ectopically in the subcutis of nude mice. Immunohistochemistry for total EGF-R levels (irrelevant of activation status) demonstrated similar levels of immunoreactivity in HCC tumors growing in the cecum, subcutis, or liver of nude mice, indicating that total EGF-R levels are not altered after growth in these different microenvironments. Controls included immunohistochemistry for total and activated EGF-R levels in HCC cells growing in vitro under serum-free or EGF-stimulated conditions and A431-epidermoid carcinoma growing in nude mice. Western blot analyses confirmed the specificity of the antibodies for the activated EGF-R. These results suggest that the production of liver metastasis by HCC cells depends in part on the response of tumor cells to organ-derived growth factors and hence the activation of specific cell surface tyrosine kinase receptors.

Epidermal growth factor receptor and insulin-like growth factor-I receptor expression and function in human soft-tissue sarcoma cells.

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) receptors are implicated in the development and progression of several malignancies including osteogenic and soft tissue sarcomas (STS). To determine a role for ligand-mediated receptor activation in sarcoma progression, the relative expression and function of EGF-R, IGF-I-R, and several other molecular determinants implicated in the progression of mesenchymal neoplasms were evaluated in human sarcoma cells established from surgical specimens of primary and metastatic tumors. mRNA blot analyses demonstrated the expression of c-Met, p53, and MDM2-specific transcripts. Western blot analyses confirmed the production of high levels of p53 protein; however, minimal levels of MDM2 and c-Met proteins were detected. Analysis of STS cells #23, #26, and #50 originating from an unclassified sarcoma lung metastasis, a malignant fibrous histiocytoma lung metastasis, and a dedifferentiated chondrosarcoma, respectively demonstrated high steady-state levels of EGF-R and IGF-I-R mRNA transcripts and protein correlating with receptor-specific tyrosine kinase activity and autophosphorylation in response to ligand. Treatment of these STS cells with EGF resulted in a >5 fold increase in DNA synthesis and mitogenesis compared with untreated controls. In contrast, treatment with IGF-I showed a variable STS growth response correlating with the origin of the tumor. These data support the involvement of EGF-R and IGF-I-R in the growth and metastasis of human soft tissue sarcoma and may offer new targets for therapeutic intervention in the management of this disease.

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

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.

Expression of epidermal growth factor receptor and human papillomavirus E6/E7 proteins in cervical carcinoma cells.

BACKGROUND: Epidermal growth factor receptor (EGF-R) proteins are highly expressed in many tumors, including those of the cervix. We have observed previously that the introduction of a transcription unit containing an antisense sequence for the E6/E7 genes of human papillomavirus (HPV) 18, along with a transcription unit containing a sense complementary DNA sequence for the wild-type retinoblastoma (Rb) gene, decreased the growth of human cervical carcinoma HeLa cells (HPV 18 positive) both in vitro and in vivo. To clarify the regulatory mechanisms by which this reduction in cell proliferation occurred, we studied the expression of EGF-R proteins in these cells. METHODS: Western blot and northern blot techniques were used to measure EGF-R expression, and a pulse-chase immunoprecipitation assay was used to measure the stability of EGF-R protein in HeLa cells and HeLa cells that had been transfected with the antisense E6/E7 or sense Rb sequences. Cell proliferation was measured by use of a tetrazolium-based colorimetric assay for numbers of viable cells. RESULTS: The introduction of sense Rb or antisense E6/E7 transcription units or a combination of these two transcription units into HeLa cells dramatically decreased the level of EGF-R proteins in these cells; EGF-R levels were not affected at the transcriptional level but at the post-transcriptional level. Addition of the anti-EGF-R-specific monoclonal antibody 225mAb to HeLa cells caused 53% (95% confidence interval = 44%-62%) growth inhibition. CONCLUSIONS: These results suggest that HeLa cervical carcinoma cells are dependent on EGF-R for proliferation and that changes in functional levels of the E6/E7 HPV proteins and endogenous Rb proteins may alter the growth rate of cervical cancer cell lines by reducing the stability of EGF-R at the post-transcriptional level.

Influence of the host microenvironment on the clonal selection of human colon carcinoma cells during primary tumor growth and metastasis.

The purpose of this study was to determine the subpopulation dynamics of human colon carcinoma (HCC) cells growing at orthotopic (cecum, liver) or ectopic (subcutis, kidney, spleen) sites in nude mice and to correlate any outgrowth of distinct clones with the differential expression of metastasis-related genes. Low metastatic KM12C HCC cells were genetically tagged with a retrovirus harboring the neomycin-resistance (Neo(R)) gene. Southern blot analyses demonstrated only minor resolution of the Neo(R) hybridization pattern in DNA isolated from primary tumors growing orthotopically or ectopically, suggesting a polyclonal outgrowth. In contrast, a major resolution of the Neo(R) hybridization pattern was observed in liver-specific metastases, demonstrating the outgrowth of single dominant clones. Expression of epidermal growth factor receptor (EGR-R) increased 20-60% in the liver metastases vs spleen tumors and the KM12C Neo(R) cells. Transforming growth factor alpha (TGF-alpha), amphiregulin (AR), and c-met showed only modest differences in mRNA expression. A 20-80% increase in type IV collagenase mRNA levels was also observed in all tumor specimens. Furthermore, expression of the multi-drug resistance gene PGY-1 and the carcinoembryonic antigen (CEA) gene were elevated in the liver metastases compared with the spleen tumors and cultured cells. Transcript levels of the angiogenic factors interleukin-8 and basic fibroblast growth factor did not correlate with clonal outgrowth. These data demonstrate a correlation between EGF-R, type IV collagenase, CEA, and PGY-1 gene expression and the production of liver metastases. Our results suggest that distinct HCC clones differentially expressing specific mRNA transcripts for metastasis-related genes are the forerunners of the experimental liver metastatic lesions.

Therapy of human transitional cell carcinoma of the bladder by oral administration of the epidermal growth factor receptor protein tyrosine kinase inhibitor 4,5-dianilinophthalimide.

Epidermal growth factor receptor (EGF-R), a transmembrane glycoprotein that mediates the mitogenic response of cells to epidermal growth factor, is highly expressed on malignant human bladder cancer cells. The 4,5-dianilinophthalimides represent a novel class of inhibitors of the EGF-R family of tyrosine kinase with selectivity at the enzymatic and cellular levels. Two compounds of this class, CGP 54211 and CGP 53353, inhibited tyrosine kinase activity of the EGF-R in five different human transitional cell carcinoma lines. The compounds also produced cytostasis in vitro. Highly metastatic human 253J B-V cells were implanted in the bladder wall of nude mice. The daily oral administration of CGP 54211 inhibited the level of EGF-R phosphorylation in this tumor; necrosis and inhibition of tumor growth paralleled this inhibition.