Autocrine and paracrine growth regulation of human breast cancer

Summary We consider the hypothesis that estrogen control of hormone dependent breast cancer is mediated by autocrine and paracrine growth factors secreted by the breast cancer cells themselves. Though we show direct, unmediated effects of estrogen on specific cell functions, we also provide evidence that human breast cancer cells secrete a collection of growth factors (IGF-I, TGF, TGF, a PDGF-like competency factor, and at least one new epithelial colony stimulating factor). Some of these are estrogen-regulated in hormone dependent cells, and are constitutively increased in cells which acquire independence either spontaneously or byras transfection. Collectively, the secreted growth factors are capable of promoting tumor formation by MCF-7 cells in nude mice, though not to the same extent as estrogens. There would seem to be potential for clinical intervention in the autocrine and paracrine control of breast cancer cells, including some cells which are no longer dependent on estrogens.     

Pharmacokinetics, toxicity, and efficacy of ends-modified raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome.

Raf-1 protein serine threonine kinase plays an important role in cell survival and proliferation. Antisense inhibition of Raf-1 expression has been shown to enhance the cytotoxic effects of radiation and anticancer drugs. Here we have evaluated the toxicity, pharmacokinetics, and antitumor efficacy of a novel formulation of liposome-entrapped raf antisense oligodeoxyribonucleotide (LErafAON). The LErafAON preparation showed high liposome entrapment efficiency of rafAON (>85%) and stability at room temperature. In CD2F1 mice, administration of LErafAON produced no morbidity/mortality (5-35 mg/kg/dose, i.v., x12). Dose-related elevations in liver enzymes (alanine aminotransferase and aspartate aminotransferase) and histopathological changes in liver were noted in LErafAON and blank liposome groups. No morbidity/mortality and changes in clinical chemistry or histopathology were observed in New Zealand white rabbits (3.75 mg/kg/dose, i.v., x8; 6.5 mg/kg/dose, i.v., x6) or in cynomolgous monkeys (3.75 or 6.25 mg/kg/dose, i.v., x9). Transient decrease in total hemolytic complement activity (approximately 62-74%) and increases in C3a (approximately 3-fold) and Bb levels (approximately 5-12-fold) were observed in LErafAON and blank liposome groups of monkeys. A 30 mg/kg i.v. dose of LErafAON in human prostate tumor (PC-3)-bearing BALB/c athymic mice gave a terminal plasma half-life of 27 h, and intact rafAON could be detected in plasma and in normal and tumor tissues for up to at least 48 h. In monkeys, the terminal plasma half-life of 30.36 +/- 23.87 h was observed at an i.v. dose of 6.25 mg/kg. LErafAON (25 mg/kg/dose, i.v., x10) or ionizing radiation (3.8 Gy/day, x5) treatment of PC-3 tumor-bearing athymic mice led to tumor growth arrest, whereas a combination of LErafAON and ionizing radiation treatments resulted in tumor regression. LErafAON treatment caused inhibition of Raf-1 protein expression in normal and tumor tissues in these mice (>50%, versus controls). These data have formed a basis of the clinical Phase I studies of LErafAON for cancer treatment.     

Enhanced therapeutic effects of doxorubicin and paclitaxel in combination with liposome-entrapped ends-modified raf antisense oligonucleotide against human prostate, lung and breast tumor models

Raf-1 protein kinase plays an important role in cell growth, proliferation and cell survival. We have previously described the use of liposome-entrapped antisense raf oligonucleotide (LErafAON) to inhibit Raf-1 expression resulting in tumor growth inhibition and radiosensitization. The present study was undertaken to evaluate the chemosensitization effects of LErafAON in combination with doxorubicin or paclitaxel on a panel of human tumor xenografts. LErafAON (25.0 mg/kg i.v. x 10) displayed significant antitumor activity (P<0.05) when administered as a single agent in prostate (PC-3), lung (A549) and breast (MDA-MB 231) carcinoma models. Doxorubicin (1.0-4.0 mg/kg i.v. per week x 3) and paclitaxel (1.0-4.0 mg/kg i.v. on alternate days x 3) were administered as single agents at non-toxic doses that led to only minimal to moderate antitumor activity. However, a combination of LErafAON with doxorubicin or paclitaxel led to significantly enhanced antitumor activity in all the tumor types tested (PC-3, P<0.03; A549, P<0.035; MDA-MB 231, P<0.045) as compared with LErafAON alone or chemotherapeutic agents alone treated groups. This effect of chemosensitization appeared to be sequence-specific because a mismatch control oligonucleotide continued to show significant tumor growth. Additionally, no inhibition in Raf-1 expression in MDA-MB 231 tumor tissue was observed with mismatch oligonucleotide treatment. On the other hand, LErafAON treatment led to >75% inhibition of Raf-1 expression in tumor tissue. These preclinical observations support the use of LErafAON in combination with chemotherapeutic agents to improve the treatment of human cancers.     

Effect of v-rasH oncogene transfection on estrogen-independent tumorigenicity of estrogen-dependent human breast cancer cells

Spontaneous or therapeutically induced progression of hormone-dependent human breast cancer to a form not amenable to endocrine treatment has been frequently recorded in clinical settings. In an experimental model system, we have changed the estrogen-dependent tumorigenicity of a human breast cancer cell line, MCF-7, to an independent state by stably introducing a model oncogene, v-rasH, into this cell line by means of DNA transfection. We now show that the oncogene-transfected hormone-independent MCF-7 cells may secrete diffusible tumorigenic factors that not only support their own tumor growth in vivo, but are also humorally active in partially triggering the tumor growth of wild type previously nontumorigenic MCF-7 cells, even when the wild type cells are implanted at a distant anatomical site in the same animal. Estrogen-independent tumor formation by MCF-7 cells was also induced in 50% of animals given injection by continuous administration of conditioned media from MCF-7-ras cells. However, the wild type tumors had limited tumor growth. Tumors were verified as adenocarcinomas and by Southern blotting were shown to be derived from the cells injected. In an in vitro coculture assay, a 5- to 7-fold enhancement in anchorage-independent growth of MCF-7 cells was observed in the presence of MCF-7-ras feeder cell layer. These data suggest that v-rasH-induced estrogen-independent tumorigenicity of human breast cancer cells occurs by secretion of mitogens which may function in an endocrine manner.     

Cytokine regulation of tumor necrosis factor-alpha and -beta (lymphotoxin)-messenger RNA expression in human peripheral blood mononuclear cells.

The immune response at the molecular level is characterized by a carefully coordinated interplay of both cytokine production and receptor induction. The regulation of these molecules including the closely related tumor necrosis factors alpha (TNF) and beta (lymphotoxin, LT) is still incompletely understood. We have examined the effects of various cytokines on the expression of TNF and LT mRNA in human peripheral blood mononuclear cells (PBMC). Northern blot analysis with total cellular RNA from mixed populations of PBMC revealed that genes coding for TNF and LT were not spontaneously expressed. Treatment of PBMC with recombinant interleukin (IL)-2 resulted in a high level expression of TNF and LT mRNA. Whereas IL-1 beta was equally effective as IL-2 in inducing both TNF and LT mRNA, granulocyte-macrophage colony-stimulating factor selectively induced only TNF mRNA. Both TNF and LT mRNA were minimally induced by IL-1 alpha, IL-3, interferon (IFN)-alpha, or IFN-gamma. Similarly TNF alone had little effect on induction of TNF and LT mRNA. In conjunction with IL-2, cytokines such as IFN-alpha, IFN-gamma, or TNF did not interfere with IL-2 induction of TNF and LT mRNA. Interestingly, IL-4 in combination with IL-2 inhibited the IL-2-driven induction of TNF and LT mRNA. This inhibitory effect of IL-4 was also observed at the level of TNF and LT protein secretion. Furthermore, IL-4 was also inhibitory of IL-2-mediated induction of Tac mRNA in PBMC. These results extend the interrelationship of cytokine regulation of TNF and LT expression. In particular, they reveal the previously unrecognized function of IL-4 in antagonizing the IL-2 induction of TNF, LT, and Tac mRNA in PBMC.     

Relationship between DNA strand breaks and inhibition of poly(ADP-ribosylation): enhancement of carcinogen-induced transformation

Inhibition of poly(ADP-Rib) by benzamide (BA) or 3-aminobenzamide(3AB) for a limited period (i.e., when ADP-ribosylation is elevated)during and shortly following X-ray or MNNG-induced DNA damageof BALB/3T3 cells significantly (3- to 30-fold) enhanced transformationfrequency by these agents. Individual Type III foci isolatedfrom benzamide, X-ray, or X-ray plus benzamide treated cultureswere established and characterized for growth in soft agar andfor tumor induction in nude mice. DNA isolated from representativetransformed lines established as a result of BA, X-ray or X-rayand BA treatments was transfected onto NIH/3T3 cells. Transformationefficiencies ranging from 0.17 to 0.28 foci/ µg of DNAwere observed suggesting the possibility that dominant transforminggene(s) were responsible for the oncogenic phenotype of radiationand benzamide transformed DNA.     

The local effects of cachectin/tumor necrosis factor on wound healing.

Previous experimental studies have suggested that tumor necrosis factor (TNF) may have either a beneficial or a detrimental role in wound healing. Control and doxorubicin-treated (6 mg/kg, intravenously) rats underwent paired dorsal 5-cm linear wounds and had either vehicle or recombinant (r)TNF (0.5, 5, or 50 micrograms) applied locally to the wound. Paired wounds were harvested at 7 and 14 days after wounding and analyzed for wound-bursting strength (WBS) and activity of the gene for type 1 collagen and TNF. Doxorubicin treatment decreased WBS at 14 days but not at 7 days after wounding. Local application of 50 micrograms of rTNF decreased WBS in saline-treated rats and concentrations of 5 and 50 micrograms decreased WBS in doxorubicin-treated rats when measured 7 days after wounding. These effects dissipated when WBS was measured 14 days after wounding. Doxorubicin decreased wound collagen gene expression and local TNF treatment decreased wound collagen gene expression in saline-treated rats and further decreased it in doxorubicin-treated rats. The decrement in collagen gene expression induced by rTNF increased as the local dose of rTNF increased. The gene for TNF was not detectable in wounds from normal or doxorubicin-treated rats at 3, 7, 10, or 14 days after wounding. These data suggest that the gene for TNF is not expressed in wounds and that the local application of TNF is detrimental to wound healing as it decreases WBS and activity of the gene for collagen.     

Expression of mRNA for transforming growth factors-alpha and -beta and secretion of transforming growth factor-beta by renal cell carcinoma cell lines

Transforming growth factors (TGFs)-alpha and -beta are regulatory polypeptides that reversibly confer a transformed phenotype upon normal cultured fibroblasts. TGF-alpha is synthesized primarily by malignant cells and shares many properties with the tissue mitogen, epidermal growth factor (EGF). The expression of TGF-beta mRNA has been demonstrated in a variety of normal and malignant cell types, some of which secrete the mature protein in an inactive form. To investigate the role of TGFs in human renal cell carcinoma (RCC), we used two renal tumor-derived cell lines and one established RCC cell line for analysis of TGF-alpha and TGF-beta mRNA production and for evaluation of TGF-beta protein secretion. By northern blot hybridization, all three RCC cell lines expressed TGF-alpha and -beta mRNA. In addition, TGF-beta activity was found in the conditioned medium from these cells. The secreted TGF-beta protein, however, displayed biological activity only after activation by acid-treatment. These data demonstrate the constitutive expression of TGF-alpha and -beta mRNA by RCC cell lines and, also, the secretion by this tumor of endogenous TGF-beta protein in a latent form.     

Relationship between the expression of estrogen-regulated genes and estrogen-stimulated proliferation of MCF-7 mammary tumor cells

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.