Regulation of epidermal growth factor receptor levels by 1,25-dihydroxyvitamin D3 in human breast cancer cells

Specific, high affinity receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been demonstrated in human breast cancer cells. In addition, 1,25-(OH)2D3 has been shown to inhibit replication in some human breast cancer cell lines, although the mechanism(s) of this anti-tumor activity remain undefined. There is currently considerable interest in the role of autocrine growth factors in the control of breast cancer cell proliferation and the effects of steroid hormones on their production, receptor binding, and action. Since the epidermal growth factor (EGF) receptor mediates the effects of both EGF and the autocrine growth factor, alpha-transforming growth factor, we investigated the effect of 1,25-(OH)2D3 on EGF receptor levels in several human breast cancer cell lines. Preincubation of T-47D cells with 1,25-(OH)2D3 for 24 h resulted in a significant concentration-dependent decline in the specific binding of [125I]EGF. The effect was observed when EGF binding was assayed at either 0 or 37 degrees C, both before and after treatment with acid to remove receptor bound endogenous ligand. This indicated that the effect on [125I]-EGF binding was not due to effects of 1,25-(OH)2D3 on receptor internalization and degradation or receptor occupancy. The half-maximal inhibitory concentration of 1,25-(OH)2D3 was approximately 2 nM. The decrease in EGF binding was due to a decrease in receptor number from 2,900 sites/cell in control cultures to 2,330 and 1,730 sites/cell in cells treated for 24 h with 10(-8) and 10(-6) M 1,25-(OH)2D3, respectively. There was no change in the affinity of the receptor for EGF following treatment with 1,25-(OH)2D3 [Kd = 0.075 +/- 0.006 nM (+/- SEM) for control and Kd = 0.083 +/- 0.004 nM for treated cells]. Decreased EGF receptor levels were also achieved with a number of analogues of 1,25-(OH)2D3 in accordance with their affinities for the 1,25-(OH)2D3 receptor, i.e., potencies for decreasing EGF binding in T-47D cells were in the order: 1,25-(OH)2D3 greater than 1,24,25-trihydroxyvitamin D3 greater than 1,25,26-trihydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3 greater than or equal to 25-hydroxyvitamin D3. Specific, saturable EGF binding to MCF-7 cells was also reduced by 1,25-(OH)2D3 while binding to BT-20 and HBL-100 cells was unaffected by this treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of epidermal growth factor receptor by progestins and glucocorticoids in human breast cancer cell lines

Human breast cancer cells secrete a number of autocrine peptides which modulate their proliferation rates. The known effects of steroid hormones on breast cancer cell proliferation may be mediated in part by altering the production of these growth factors and/or their interactions with cellular receptor sites. Receptors for epidermal growth factor (EGF), which also bind the autocrine growth factor, alpha-transforming growth factor, are present on a number of breast cancer cell lines and it has previously been shown that T-47D and MCF-7 cells respond to progestins with an increase in the concentration of EGF receptors (EGF-R). In the present study we examined the effects of both progestins and glucocorticoids on EGF binding in 10 human breast cell lines. Five of these lines were progesterone receptor positive and all lines expressed the glucocorticoid receptor (GR). All cell lines were initially incubated for 24 hr with increasing concentrations of the synthetic progestin, medroxyprogesterone acetate (MPA), and the level of specifically bound EGF was determined. An increase in specific binding of EGF was confirmed in two PR-positive lines but, in addition, increases in EGF binding were observed in 4 PR-negative cell lines. In these last lines the synthetic glucocorticoid, dexamethasone, was a more potent inducer of EGF binding than MPA, a known glucocorticoid agonist, while the high-affinity PR ligand, ORG 2058, was without effect. Furthermore, MPA competed with dexamethasone for binding to GR in these cell lines, supporting the view that the induction of EGF binding by MPA in these cells was mediated via the GR. This conclusion was further supported by studies in which addition of the glucocorticoid and progestin antagonist, RU 486, inhibited the effect of ORG 2058 in two cell lines and completely abrogated the effect of dexamethasone in two other lines. Detailed binding studies revealed that the increase in EGF binding was accompanied by an increase in the concentration of EGF-R. This effect was observed when EGF binding was assayed at either 0 degree or 37 degrees C. Further studies demonstrated that the increases in EGF binding following ORG 2058 and dexamethasone treatment were accompanied by increases in EGF-R mRNA levels. Our data illustrate that the binding of EGF by some human breast cancer cells can be regulated by both progestins and glucocorticoids acting via their respective receptors and inducing increases in EGF-R mRNA levels.     

Induction of differentiation of human myeloid cell lines by tumor necrosis factor in cooperation with 1 alpha,25-dihydroxyvitamin D3

We analyzed the combined effect of tumor necrosis factor and 1 alpha,25-dihydroxyvitamin D3 on the differentiation of human myeloid cell lines HL-60, ML3, and U937. The two compounds synergize in inducing the morphological, phenotypic, enzymatic, and functional characteristics of cells of the monocytic lineage. Immune gamma-interferon synergizes with each compound to induce differentiation. However, recombinant tumor necrosis factor is much more effective than recombinant gamma-interferon in potentiating the effect of 1 alpha,25-dihydroxyvitamin D3 and, alone, is also more effective than recombinant gamma-interferon in inducing expression of the high-affinity Fc receptor on ML3 cells. The possible physiological or pathological relevance of the synergistic effect of tumor necrosis factor and 1 alpha,25-dihydroxyvitamin D3 on monocytic differentiation is discussed.     

Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines.

Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell lung cancer cell lines express the EGF receptor.     

Reduction of epidermal growth factor binding in human breast cancer cell lines by an alkyl-lysophospholipid

The effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), an alkyl lysophospholipid derivative, on the binding of epidermal growth factor (EGF) to human breast cancer cell lines (MCF-7, ZR-75-1, and BT-20), the human epidermoid cancer cell line (A431), and the rat fibroblast cell line (NIH3T3) were investigated. The addition of 10 micrograms/ml ET-18-OCH3 to the growth medium reduced the binding of EGF to hormone-dependent breast cancer cell lines (MCF-7 and ZR-75-1) and A431 but did not change that to the hormone-independent breast cancer cell line (BT-20). ET-18-OCH3 suppressed the EGF-binding prior to the onset of its inhibitory action on cell growth in MCF-7 and ZR-75-1. Scatchard plot analysis demonstrated that ET-18-OCH3 reduced the number of EGF receptor sites without affecting the affinity of EGF receptors in MCF-7 and ZR-75-1. Both EGF-binding and cell growth in NIH3T3 were not changed by treatment with 10 micrograms/ml ET-18-OCH3. These results suggest that ET-18-OCH3 inhibits the growth of hormone-dependent breast cancer cell lines (MCF-7 and ZR-75-1) by reducing the binding capacity of EGF receptors and consequently by disturbing the transfer of a variety of growth-promoting signals.     

Influence of tumor derived fibroblasts and 1,25-dihydroxyvitamin D3 on growth of breast cancer cell lines

Summary Fibroblasts are known to be present in variable amounts in human breast adenocarcinoma tissue. In order to investigate if they influence in some way the proliferation rate of the carcinoma cells, we developed a coculture model in which cells of well characterized breast epithelial cell lines were seeded and grown in microchamber slides along with fibroblasts derived from breast tumor biopsies. As representatives of hormone dependent and independent tumor cells, we used MCF-7 and BT-20 cell lines. A third line, NPM-21T, derived from non proliferating mastopathy cells immortalized by SV-40 T DNA transfection, was representative of non tumor epithelial cells. The proliferation rate of the adenocarcinoma and epithelial cells was assessed by measurement of the BrdU labeling index, the cells being identified by specific -actin immunostaining. It was found that the proliferation of the adenocarcinoma cells was significantly increased in the presence of fibroblasts, while that of immortalized cells was not. Moreover, 1,25(OH)₂D₃, which was known to be a negative regulator of carcinoma cell growth, was found to be able also to blunt the overgrowth in the presence of fibroblasts. The absence of response of NPM-21T cells to the presence of fibroblasts suggests that the tumor cells could be the origin of their own overgrowth, through an indirect mechanism mediated by the fibroblasts. The factors which are involved and the 1,25(OH)₂D₃ mechanism of action are not yet identified.Abbreviations 1,25(OH)₂D₃ 1,25-dihydroxycholecalciferol - BrdU 5-bromo-2'-deoxyuridine - LI labeling index - ER, PGR, VDR estrogen, progesterone, and 1,25(OH)₂D₃ receptors - EGF, EGF-R epidermal growth factor and epidermal growth factor receptor     

Modulating therapeutic effects of the c-Src inhibitor via oestrogen receptor and human epidermal growth factor receptor 2 in breast cancer cell lines

Purposec-Src is an important adapter protein with oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), which validates it as an attractive target for the treatment of breast cancer. A specific c-Src inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazolo[3,4-d]pyrinidine (PP2), was utilised to block c-Src activity to identify targeted vulnerabilities affected by ER and HER2 in a panel of breast cancer cell lines.MethodsER, growth factor receptors and signalling pathways were detected by Western-blot. The DNA content of the cells was determined by using a DNA fluorescence quantitation kit. Cell cycles were analysed by flow cytometry.ResultsThe antiproliferative effect of PP2 closely correlated with the inhibition of c-Src mediated extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt growth pathways. Inhibition of c-Src tyrosine kinase predominantly blocked ER negative breast cancer cell growth, particularly the triple (i.e. ER, progesteron receptor (PR), and HER2) negative cells. In contrast, ER negative Sk-Br-3 cells with highest HER2 phosphorylation were resistant to PP2, in which hyper-activated HER2 directly regulated growth pathways. However, blocking c-Src recovered ER expression and down-regulated HER2 which made Sk-Br-3 cells regain responsiveness to 4-hydroxytamoxifen. The majority of ER positive cells were not sensitive to PP2 regardless of wild-type or endocrine resistant cell lines.Conclusionsc-Src mediates the essential role of growth pathways in ER negative breast cancer cells. The ER positive and HER2 over-activation are two important predictive biomarkers for the resistance to a c-Src inhibitor. These data provided an important therapeutic rationale for patient selection in clinical trials with c-Src inhibitors in breast cancer.     

Inhibition by an alkyl-lysophospholipid of the uptake of epidermal growth factor in human breast cancer cell lines in relation to epidermal growth factor internalization

The effects of 1-O-octadecyl-2-O-methyl-sn-glycerol-3-phosphocholine (ET-18-OCH3), an alkyl-lysophospholipid derivative, on the binding and uptake of labeled epidermal growth factor (EGF) in hormone-dependent (MCF-7 and ZR-75-1) and hormone-independent (BT-20) breast cancer cell lines were investigated at 4 degrees C and 37 degrees C. The total (bound and intracellular) EGF associated with breast cancer cells tested were largely temperature dependent. By pretreatment of the cells with ET-18-OCH3 (10 micrograms/ml) for 12 h, the EGF uptake at 37 degrees C was greatly reduced in both MCF-7 and ZR-75-1 (ET-18-OCH3-susceptible) but not in BT-20 (ET-18-OCH3-resistant) cell lines. The ET-18-OCH3 pretreatment slightly decreased the EGF uptake at 4 degrees C in MCF-7 and ZR-75-1 and had little effect on that in BT-20. The EGF binding at 37 degrees C was unaffected by ET-18-OCH3 in MCF-7 and BT-20 and slightly decreased in ZR-75-1. The EGF binding at 4 degrees C was not changed by ET-18-OCH3 in all cell lines tested. These results suggest that labeled EGF is taken up by the cells in a temperature-dependent manner and ET-18-OCH3 may inhibit this internalization process only in ET-18-OCH3-sensitive human breast cancer cell lines. It is inferrable that the inhibition of the internalization process for EGF may be one of the modes of antitumoral action of ET-18-OCH3.     

Role of epidermal growth factor receptor in breast cancer

Decades of research in molecular oncology have brought about promising new therapies which are designed to target specific molecules which promote tumor growth and survival. The epidermal growth factor receptor (EGFR) is one of the first identified important targets of these novel antitumor agents. Approximately half of cases of triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) overexpress EGFR. Thus, EGFR inhibitors for treatment of breast cancer have been evaluated in several studies. However, results so far have been disappointing. One of the reasons for these unexpected results is the lack of biomarkers for predicting which patients are most likely to respond to EGFR inhibitors. Recent studies have shown that EGFR and its downstream pathway regulate epithelial-mesenchymal transition, migration, and tumor invasion and that high EGFR expression is an independent predictor of poor prognosis in IBC. Further, recent studies have shown that targeting EGFR enhances the chemosensitivity of TNBC cells by rewiring apoptotic signaling networks in TNBC. These studies indicate that EGFR-targeted therapy might have a promising role in TNBC and IBC. Further studies of the role of EGFR in TNBC and IBC are needed to better understand the best way to use EGFR-targeted therapy??e.g., as a chemosensitizer or to prevent metastases??to treat these aggressive diseases.     

Induction of anchorage-independent growth of JB6 mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3

1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3], a hormonally active form of vitamin D3, was shown previously to enhance chemically induced transformation of BALB 3T3 cells and Syrian hamster embryo cells. This report demonstrates that 1 alpha,25(OH)2D3, like phorbol ester tumor promoters, induces anchorage-independent growth of mouse JB6 epidermal cells. When plated on agar plates containing 1 alpha,25(OH)2D3 at concentrations higher than 0.05 ng/ml or 0.12 nM, JB6 cells formed colonies on the surface of agar plates dose dependently. This anchorage-independent growth was further confirmed by stimulation of DNA synthesis after liquefying the agar layer with NaI. A phorbol-ester resistant variant of JB6 cells was also resistant to 1 alpha,25(OH)2D3 in terms of induction of anchorage independency. Induction of anchorage-independent growth was specific for 1 alpha,25(OH)2D3: other derivatives of vitamin D3 also induced colony formation on agar plates but only at a higher concentration (500 ng/ml) and to much less extent than did 1 alpha,25(OH)2D3. JB6 cells were found to contain a receptor specific for 1 alpha,25(OH)2D3 with a Kd of 55.7 pM and Nmax of 102.5 fmol/mg protein, suggesting a receptor-mediated mechanism of the induction. The clone that was resistant to 1 alpha,25(OH)2D3 also contained the receptor. DNA-cellulose chromatography showed that a 1 alpha,25(OH)2D3-receptor complex interacted with DNA. In contrast to 1 alpha,25(OH)2D3, retinoic acid did not induce anchorage-independent growth of JB6 cells, but it inhibited the induction by 1 alpha,25(OH)2D3 when applied with it.     

Transforming growth factor alpha expression drives constitutive epidermal growth factor receptor pathway activation and sensitivity to gefitinib (Iressa) in human pancreatic cancer cell lines

The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-alpha (TGF-alpha)] expression (r(2) = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-alpha reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-alpha expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-alpha expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-alpha expression may therefore represent an effective means of identifying EGFR-responsive primary tumors.     

1alpha, 25-dihydroxyvitamin D3 suppresses interleukin-8-mediated prostate cancer cell angiogenesis

Angiogenesis is an essential step in initial tumor development and metastasis. Consequently, compounds that inhibit angiogenesis would be useful in treating cancer. A variety of antitumor effects mediated by 1alpha, 25-dihydroxyvitamin D3 (1,25-VD) have been reported, one of which is anti-angiogenesis; however, detailed mechanisms remain unclear. We have demonstrated that 1,25-VD inhibits prostate cancer (PCa) cell-induced human umbilical vein endothelial cell migration and tube formation, two critical steps involved in the angiogenesis. An angiogenesis factor, interleukin-8 (IL-8), secreted from PCa cell was suppressed by 1,25-VD at both mRNA and protein levels. Mechanistic dissection found that 1,25-VD inhibits NF-kappaB signal, one of the most important IL-8 upstream regulators. The 1,25-VD-mediated NF-kappaB signal reduction was shown to result from the blocking of nuclear translocation of p65, a subunit of the NF-kappaB complex, and was followed by attenuation of the NF-kappaB complex binding to DNA. The role of IL-8 in PCa progression was further examined by PCa tissue microarray analyses. We found that IL-8 expression was elevated during PCa progression, which suggests that IL-8 may play a role in tumor progression mediated through its stimulation on angiogenesis. These findings indicate that 1,25-VD could prevent PCa progression by interrupting IL-8 signaling, which is required in tumor angiogenesis, and thus applying vitamin D in PCa treatment may be beneficial for controlling disease progression.     

Different epidermal growth factor growth responses and receptor levels in human colon carcinoma cell lines

The growth response to epidermal growth factor (EGF) and the numbers and types of EGF receptors were studied in three human colon tumor cell lines from each of two groups of cell lines that differ markedly in their growth properties and extent of differentiation. Aggressively growing and poorly differentiated colon cells (group I) did not respond to EGF alone, while less aggressively growing and more differentiated cells (group III) responded with increased growth when EGF was added to their chemically defined, serum-free medium. The average number of EGF receptors (EGF-R) measured at the surface of group III cell lines by radioligand binding assays, was eight-fold higher than that measured for group I cell lines. These observations provide evidence for possible autocrine mechanisms that maintain available EGF-R levels in more differentiated group III colon tumor cells and down-regulate EGF-R levels in group I colon tumor cells.     

Epidermal growth factor receptor expression in human lung cancer cell lines

Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines were studied for epidermal growth factor (EGF) receptor expression. All NSCLC cell lines tested (eight of eight) had specific EGF binding sites, whereas only five of 11 SCLC cell lines bound EGF. NSCLC and SCLC cell lines expressed the same type of high affinity EGF binding sites with a Kd of 0.5 to 4.5 nM; however, NSCLC cells bound significantly more EGF than SCLC cell lines. The amount of binding sites in NSCLC cells ranged between 71 and 1,000 fmol/mg of protein and in SCLC cells, between 26 and 143 fmol/mg of protein. The two SCLC cell lines with EGF binding values within the range of NSCLC belonged to the variant subtype of SCLC. By means of an anti-erbB serum and indirect radioimmunoprecipitation, a strong Mr approximately 170,000 protein band could be detected in the NSCLC cell lines. This protein corresponds to the EGF receptor molecule. Its identity was proven by competition with excess erbB antigen for the antibody during the radioimmunoprecipitation. Furthermore, this Mr 170,000 protein exhibited protein kinase activity as evidenced by in vitro autophosphorylation. The radioactivity incorporated into the Mr 170,000 band in radioimmunoprecipitation and protein kinase assays was 10 to 100 times lower in these SCLC cell lines which were positive in the EGF binding assay compared to the NSCLC cell lines. We conclude that NSCLC in contrast to SCLC expresses high levels of EGF receptors which may be used to facilitate the differential diagnosis in some cases of lung cancer. These data suggest that EGF may play a role in growth and differentiation of NSCLC.     

Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts

Both permanent cultured cell lines and athymic mouse xenografts were established from two human glioblastomas. Biopsies from D-245 MG and D-270 MG contained amplified and rearranged epidermal growth factor receptor (EGFR) genes. Although the gene amplification and rearrangement seen originally was maintained in the xenografts, cultured cell lines established from these biopsies lost the amplified rearranged genes in vitro. Analysis of these cell lines and 11 additional permanent human glioma cell lines with normal EGFR gene copy number showed from 2.7 x 10(3) to 4.1 x 10(5) high affinity EGFRs/cell by radioreceptor assay. The RNase A protection assay showed minimal differences in the quantity of EGFR mRNA among the 13 glioma lines, while the D-245 MG and D-270 MG xenografts expressed approximately 10-20 times as much EGFR mRNA as the corresponding cell lines. Immunoprecipitation of EGFR from these lines, including D-245 MG and D-270 MG, demonstrated only the intact Mr 170,000 Da form, while truncated Mr 145,000 Da and 100,000 Da EGFR proteins were immunoprecipitated from the D-270 MG and D-245 MG xenografts, respectively. These studies demonstrate that gliomas with amplification of the EGFR gene are capable of establishing in culture but that the amplified rearranged genes are not maintained. Possible explanations are that the abnormal genes are lost during serial passage or that the cells with amplified rearranged genes only represent a minor subpopulation of cells, which are unable to grow in culture. In either case, these observations suggest that high expression and structural abnormalities of EGFR proteins generated by amplification and rearrangement of the EGFR gene provide a growth advantage for gliomas in vivo but not in vitro.     

Significance of epidermal growth factor receptor expression in breast cancer

Recent interest of many investigators is focused on epidermal growth factor receptor (EGFR) family, because of their potential role in the pathogenesis and progression of breast cancer. Paraffin tumor sections were collected retrospectively from 181 breast cancer patients diagnosed between 2002 and 2003. Immunohistochemical staining with ErbB-1, ErbB-2, ErbB-3, and ErbB-4 monoclonal antibodies was performed. The ErbB expression was correlated with the other clinicopathological variables. Overexpression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 was observed in 20.6, 18.2, 14.3, and 5.7% cases, respectively. Overexpression of ErbB-1 and ErbB-2 was associated with poor prognostic features and decreased 5-year disease-free survival. The patients with co-overexpression of ErbB-1 and ErbB-2 had a shorter DFS, although this difference was not statistically significant. ErbB-1 overexpression may indicate a subset of patients with a poor disease prognosis. Assays for ErbB-1 and ErbB-2 may be more useful than a single assay in predicting prognosis of a breast cancer patient.