An antisense oligonucleotide targeting TGF-ß2 inhibits lung metastasis and induces CD86 expression in tumor-associated macrophages.

BACKGROUND: The transforming growth factor (TGF)-ß pathway is a well-described inducer of immunosuppression and can act as an oncogenic factor in advanced tumors. Several preclinical and clinical studies show that the TGF-ß pathway can be considered a promising molecular target for cancer therapy. The human genome has three TGF-ß isoforms and not much is known about the oncogenic response to each of the isoforms. Here, we studied the antitumor response to ISTH0047, a recently developed locked nucleic acid-modified antisense oligonucleotide targeting TGF-ß2. MATERIALS AND METHODS: We have studied the anticancer response to ISTH0047 using gymnotic delivery in tumor cell cultures and in in vivo preclinical orthotopic mouse models for primary tumors (breast and kidney tumors) and lung metastasis. RESULTS: We observed that ISTH0047 is able to significantly reduce TGF-ß2 mRNA and protein levels without altering the levels of TGF-ß1 and TGF-ß3. ISTH0047 prevented lung metastasis in syngeneic orthotopic renal cell carcinoma (RENCA) and breast cancer (4T1) tumor models. In addition, using an orthotopic xenograft model of a lung cancer cell line (CRL5807) that mainly expresses TGF-ß2, we observed that ISTH0047 had an important effect on the lung microenvironment inhibiting the growth of lung lesions. ISTH0047 treatment re-educated macrophages in the lung parenchyma to express the tumor-suppressive factor, CD86. CONCLUSION: Overall, our data point to TGF-ß2 as a therapeutic target and ISTH0047 as a novel anticancer drug to prevent lung metastasis by impacting on the tumor niche, in part, through the induction of CD86 in tumor-associated macrophages.

Reduced growth rate accompanied by aberrant epidermal growth factor signaling in drug resistant human breast cancer cells.

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.

Identification of epidermal growth factor receptor and c-erbB2 pathway inhibitors by correlation with gene expression patterns.

BACKGROUND: Growth factor receptor-signaling pathways are potentially important targets for anticancer therapy. The interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new agents that target and inhibit the activity of the epidermal growth factor (EGF) receptor and of c-erbB2 (also called HER2 or neu), by correlating EGF receptor, transforming growth factor (TGF)-alpha (a ligand for EGF receptor), and c-erbB2 messenger RNA (mRNA) expression levels with the results of cytotoxicity assays of the 49000 compounds in the National Cancer Institute (NCI) drug screen database. METHODS: The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-alpha, or c-erbB2 mRNA expression patterns. The putative EGF receptor-inhibiting compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity, and EGF-dependent growth. Putative ErbB2-inhibiting compounds were tested for effects on antibody-induced ErbB2 tyrosine kinase activity. RESULTS: EGF receptor mRNA and TGF-alpha mRNA levels were highest in cell lines derived from renal cancers, and c-erbB2 mRNA levels were highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs) were tested as inhibitors of the EGF receptor or c-erbB2 signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 microM. CONCLUSIONS: Novel EGF receptor or c-erbB2 pathway inhibitors can be identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels.

Altered gene expression in drug-resistant human breast cancer cells.

It is increasingly recognized that drug-resistant cells undergo transitions not directly linked to "classical" drug resistance. We examined the expression of growth factors, growth factor receptors, and the estrogen receptor in 17 drug-resistant and 2 revertant human breast cancer sublines to provide an understanding of the phenotypic changes that occur and how these changes could affect the biology of the cell. These sublines were derived from five parental human breast cancer cell lines (MCF-7, ZR75B, T47D, MDA-MB-231, and MDA-MB-453). The expression of estrogen receptor was absent or decreased in 6 of the 15 resistant MCF-7, ZR75B, and T47D sublines. Increases of as much as 49-fold compared to parental levels were observed in transforming growth factor alpha, epidermal growth factor receptor, c-erbB2, and/or c-erbB3 mRNA expression in 14 of the 17 resistant sublines. Altered amphiregulin and insulin-like growth factor-I receptor expression was observed in nine and four drug-resistant sublines, respectively. No major alterations were observed in epidermal growth factor and c-erbB4 expression. Few alterations were observed in two sublines derived from estrogen receptor-negative cells. Higher levels of phosphotyrosine residues were detected in a subset of the resistant sublines, indicating an increased tyrosine kinase activity in these cells. Interestingly, decreased growth rates were observed in all of the sublines, despite up-regulated growth factor-related gene expression. Taken together, these data suggest that loss of estrogen receptor, increased expression of growth factor pathway genes, and decreased growth rate regularly occur in drug-resistant breast cancer cells. Although we do not know whether the altered expression of growth factor pathway genes is linked as a cause or a consequence of the reduced growth rate, it is well established that decreased growth rate confers drug resistance. These phenotypic changes in drug-resistant human breast cancer cells could serve to initiate, support, or extend the drug resistance.

Brain-derived neurotrophic factor protects neuroblastoma cells from vinblastine toxicity.

Brain-derived neurotrophic factor (BDNF) and its receptors are necessary for the survival and development of many neuronal cells. Because BDNF and TrkB are expressed in many poor-prognosis neuroblastoma (NB) tumors, we evaluated the role of BDNF in affecting sensitivity to chemotherapeutic agents. We investigated the effects of activation of the BDNF-TrkB signal transduction pathway in two NB cell lines, 15N and SY5Y. 15N cells lack the high-affinity receptor p145TrkB and express BDNF; 15N cells were used along with 15N-TrkB cells, a subline transfected with a TrkB expression vector. In cytotoxicity assays, 15N-TrkB cells were consistently 1.4-2 fold more resistant to vinblastine than 15N cells. Drug accumulation assays showed a 50% reduction in[3H]vinblastine accumulation in 15N-TrkB cells compared with control 15N cells. Addition of 30 ng/ml BDNF resulted in a reduction to 46% of control in 15N cells and a reduction to 28% of control in 15N-TrkB cells. SY5Y cells were chosen as a second model because they lack both endogenous BDNF and TrkB expression. p145TrkB expression is induced by 1 nM retinoic acid. Vinblastine accumulation was not significantly affected by 1 nM retinoic acid in SY5Y cells. Addition of 30 ng/ml BDNF decreased [3H]vinblastine accumulation to 58% of control in SY5Y cells and decreased [3H]vinblastine accumulation to 62% of control in TrkB-expressing SY5Y cells. Although an increase in BDNF expression in seen in multidrug-resistant sublines of SY5Y and BE(2)-C NB cells, the protective effect of BDNF in vinblastine toxicity may be unrelated to mdr-1, because the activity of other agents transported by P-glycoprotein was not affected. There was no increase in mdr-1 expression in 1 nM RA SY5Y cells and 15N-TrkB cells, as assessed by Northern blot analysis. In addition to the effects of BDNF on vinblastine cytotoxicity and accumulation, there was an inhibition in the ability of vinblastine to depolymerize tubulin in BDNF-treated cells. Thus, BDNF and TrkB may partially rescue NB cells from vinblastine toxicity and thereby may contribute to a more chemoresistant phenotype.

Drug-resistant breast cancer cells frequently retain expression of a functional wild-type p53 protein.

Abnormalities in the p53 tumor suppressor gene have been shown to affect cellular processes related to cell cycle control and gene amplification. In this study we compare the status and function of wild-type p53 in MCF-7 breast cancer cells with sublines selected for resistance to chemotherapeutic agents having different mechanisms of action. Sublines that were resistant to melphalan, pyrazafurin, mitoxantrone, etoposide and PALA all retained expression of wild-type p53. Methotrexate-resistant MCF-7 cells were unusual heterozygotes that expressed a wild-type and dominant, in-frame p53 deletion mutant and the doxorubicin-resistant cells expressed only mutant p53. Analysis of the G1 checkpoint after treatment with ionizing radiation revealed that the pyrazafurin-, melphalan- and mitoxantrone-resistant cells arrested strongly in G1. The etoposide- and PALA-resistant cells had an intermediate G1 arrest phenotype and the methotrexate- and doxorubicin-resistant cells had a minimal G1 arrest phenotype. mRNA and protein analyses of downstream effector genes, including P21CIP1/Waf1, mdm2, Gadd 45 and the retinoblastoma protein, did not entirely differentiate sublines having a strong versus intermediate G1 arrest phenotype. Neither the p53 status nor the strength of the G1 arrest could be correlated with cell survival after ionizing radiation. When drug-sensitive MCF-7 cells were treated with the same chemotherapeutic agents, p53 and p21CIP1/Waf1 levels increased between 2- and 14-fold. Together these data suggest that other cellular factors likely play a role in overcoming the inhibitory effects of ionizing radiation on p53 in drug-resistant breast cancer cells.

Normal p53 status and function despite the development of drug resistance in human breast cancer cells.

Loss of or mutations in p53 protein have been shown to decrease both radio- and chemosensitivity. The present study assessed the p53 gene status, ability to arrest in G1 of the cell cycle, the functionality of the p53 transduction pathway, and apoptosis following treatment with radiation in a series of drug-resistant human breast cancer cells to determine whether p53 alterations occur during the development of drug resistance. We used 13 sublines derived from MCF-7, ZR75B, and T47D cells, which were resistant to doxorubicin, paclitaxel, vinblastine, cisplatin, etoposide, and amsacrine. Eleven of 12 drug-resistant sublines retained the parental p53 gene status, as determined by sequence analysis and functional yeast assay; only one subline was found to have acquired a mutation in the p53 gene. The MCF-7 TH subline was found to both acquire mutated p53 and to have major changes in p53 protein expression and function. In 12 other drug-resistant sublines, the G1 checkpoint was conserved or only slightly impaired. A normal accumulation of p53, p21Cip1/Waf1, and Mdm2 proteins and hypophosphorylation of Rb protein occurred in response to radiation with only small differences noted in the kinetics of p53 and p21Cip1/Waf1 induction. Increased susceptibility to apoptosis was found in the ZR75B drug-resistant sublines, whereas no evidence for apoptosis was observed in the ZR75B, MCF-7, and T47D parentals and the MCF-7 and T47D drug-resistant sublines. This effect could not be explained by alterations in bcl-2 or bax expression. Our results demonstrate that alterations in: (a) p53 gene status; (b) ability to arrest in G1; (c) induction of p53 protein and p53-dependent genes; and (d) decreased activation of apoptosis is not a requirement for the onset of drug resistance. The function of p53 appears to be dissociated from drug resistance in our model system.

Increased resistance to cytotoxic agents in ZR75B human breast cancer cells transfected with epidermal growth factor receptor.

Human breast cancer cells selected for multidrug resistance frequently overexpress ligands and receptors in the epidermal growth factor (EGF) receptor family. To determine whether this overexpression contributes to the drug resistant phenotype, EGF receptor transfected ZR75B human breast cancer cells were examined. Two EGF receptor overexpressing clones were evaluated: clone 11 with > 1 x 10(6) sites, and clone 13 with 310,000 receptor sites/cell. These were compared with clone 2-neo, which was transfected with the neomycin gene only and contained 43,000 receptor sites/cell. The EGF receptor overexpressing clones and the neo transfected control clone displayed comparable growth rates. Cytotoxicity analyses were performed with doxorubicin, vinblastine, cisplatin and 5-fluorouracil to determine the sensitivity of the clones to antineoplastic drugs. The EGF receptor overexpressing clones were found to be 1.5-5.6 times more resistant to the four drugs tested. This increase in the IC50 conferred a selective advantage when grown in the presence of 2, 3 and 6 ng/ml doxorubicin. Clone 13 cells overtook a mixed population which began with clone 2-neo comprising 95% of the cells. Clone 2-neo remained the dominant clone in the absence of drug. Finally, after long-term selection of the clones with 6 ng/ml doxorubicin, clone 2-neo became fourfold more resistant than the unselected clone 2-neo, a level which was comparable to that found in the EGF receptor overexpressing clones 11 and 13. No additional increase in resistance was observed for these clones, suggesting that clone 2-neo had developed additional resistance mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential signal transduction of epidermal-growth-factor receptors in hormone-dependent and hormone-independent human breast cancer cells.

In breast cancer, hormone dependency is inversely correlated with the number of surface epidermal-growth-factor (EGF) receptors on the tumor cells. In vitro, EGF stimulated only hormone-dependent immortalized human breast cancer cells to grow with an increased rate whereas hormone-independent cells were not affected by EGF. The number of EGF surface receptors is about 5-10-times smaller on hormone-dependent cells than on hormone-independent cells. Two cell lines representing the two cell types were used to demonstrate the signal-transduction capabilities of the EGF receptors. The two cell lines were the hormone-dependent MCF-7 cells and the hormone-independent MDA-MB-231 cells. Incubation at 37 degrees C for 15 min with 10(-8) M EGF increased the surface EGF-receptor density substantially on MCF-7 cells (50%) and reduced the number of these receptors on MDA-MB-231 cells to about 65% of the control. Both cell lines internalized a fluorescein-isothiocyanate-labeled EGF with similar kinetics. EGF triggered tyrosine phosphorylation of several targets in isolated MCF-7 cell membranes. One of these targets was shown by immunoprecipitation to be the EGF receptor. In MDA-MB-231 cell membranes, the EGF receptor was demonstrated to be the main target for tyrosine phosphorylation. The mRNA expression of the immediate early proto-oncogene c-fos was stimulated by EGF only in MCF-7 cells. In contrast, the mRNA of the EGF receptors was stimulated by EGF in both cell lines. These results demonstrate that, although EGF-binding sites are present on both cell lines, their signal-transduction capacity and activities are substantially different and resulted in a divergent response of the two cell types to EGF.

Increased epidermal growth factor receptor in an estrogen-responsive, adriamycin-resistant MCF-7 cell line.

We examined the expression of the estrogen and epidermal growth factor (EGF) receptors in a drug-resistant subline of MCF-7 cells in order to study potential alterations in hormone dependence or in the growth factor pathway that could be related to the development of drug resistance in human breast cancer. The drug-resistant subline was derived from MCF-7 cells by selection with Adriamycin in the presence of the P-glycoprotein antagonist, verapamil, to prevent acquisition of the classical multidrug resistance phenotype. The Adriamycin-resistant cells retain estrogen-binding, estrogen-responsive monolayer growth, and estrogen-dependent tumorigenesis. Estrogen-binding studies demonstrate 1.4 x 10(6) sites per cell with unaltered affinity when compared to parental MCF-7 cells, which have 2.7 x 10(5) sites per cell. An increase in expression of EGF receptor, eight to 12-fold, occurred early in the selection for drug resistance, and appears to be unrelated to verapamil exposure, since cells maintained in Adriamycin without verapamil also have increased EGF receptor expression. Partially drug-sensitive revertants carried a verapamil, but out of Adriamycin, demonstrate a decline in EGF receptor expression. We postulate that activation of growth factor pathways in drug-resistant cells may enhance mechanisms of drug resistance, or provide mitogenic stimuli for cells to recover after damage by drug exposure.

Inhibition of growth-factor-activated proliferation by anti-estrogens and effects on early gene expression of MCF-7 cells.

Recently, it was reported that the anti-estrogen tamoxifen not only inhibits estradiol-stimulated growth of MCF-7 cells but also significantly reduces the proliferation rate of cells stimulated by growth factors. We have confirmed this finding and also shown that the new anti-estrogen droloxifene inhibits the proliferation of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I)-stimulated MCF-7 cells. The growth-factor-induced proliferation was inhibited in a dose-dependent manner by the anti-estrogens in the complete absence of estrogen and FCS. Of the anti-estrogens, droloxifene was considerably more potent than tamoxifen. Because the expression of the proto-oncogenes c-fos and c-myc has been considered a key event in development of the mitogenic response, we examined the effects of anti-estrogens on c-myc and c-fos gene expression. We included in these investigations the steroidal anti-estrogen ICI 164,384 because this compound has no or very little estrogenic activity. The studies revealed that all 3 anti-estrogens transiently induced c-myc mRNA expression. However, the anti-estrogens inhibited estradiol-induced c-myc mRNA expression, although with different potencies. Pre-incubation of MCF-7 cells with droloxifene and tamoxifen resulted in elevated levels of growth-factor-induced c-myc mRNA expression. In contrast, the anti-estrogens did not induce c-fos mRNA or affect the expression of c-fos mRNA induced by growth factors. In conclusion, non-steroidal anti-estrogens inhibit growth-factor-stimulated proliferation of MCF-7 cells without inhibiting growth-factor-induced c-myc or c-fos mRNA expression.

c-fos, c-jun and c-myc expressions are not growth rate limiting for the human MCF-7 breast cancer cells.

Insulin-like growth factor I (IGF-I) was 3 times more potent in pagating MCF-7 cell proliferation than epidermal growth factor (EGF). IGF-I stimulated c-fos mRNA expression about 5 times less than EGF. Both growth factors were equipotent in inducing c-jun and c-myc mRNA expressions. The protein level of c-Myc correlated with the mRNA level. IGF-I and EGF stimulated the transcriptional activity dependent on the phorbol 12-myristate 13-acetate-responsive element (TRE) to the same extent, when measured by the chloramphenicol acetyl transferase activity of a transiently transfected multiple TRE construct. These results strongly indicate that the expression levels of the measured proto-oncogenes do not correlate with the increase of growth stimulation by IGF-I and EGF and are not growth rate limiting for the human MCF-7 breast cancer cells.

Pharmacologic and biologic properties of droloxifene, a new antiestrogen.

Pharmacologic investigations with droloxifene in vitro and in vivo revealed that droloxifene is a more efficient antiestrogen than tamoxifen. Droloxifene differs from tamoxifen in the following ways: it has a more than 10-fold higher binding affinity to the estrogen receptor; it shows lower estrogenic and higher antiestrogenic effects on rat uterus, indicating a higher therapeutic index; it more potently inhibits growth of various human ER-positive mammary carcinoma cell lines; short-term exposures with clinically relevant concentrations of droloxifene produce long-term growth inhibition of human ER-positive cancer cells and are more effective than continuous treatment with tamoxifen; it more effectively reduces S-phases and arrests ER-positive cells in G1-phase of the cell cycle; it antagonizes estrogen independent, growth factor stimulated proliferation of MCF-7 cells with higher efficiency; it blocks estrogen activated c-myc expression better than tamoxifen; it more effectively inhibits growth of various experimental tumors of animal (R 3230, DMBA) and human (T61) origin. Therefore, in all experimental systems, it was found that droloxifene is a more potent antiestrogen than tamoxifen.