A non-IGF binding mutant of IGFBP-3 modulates cell function in breast epithelial cells

We demonstrated previously that IGFBP-3 alone had no effect on cell death, but dramatically modulated apoptosis in Hs578T IGF non-responsive cells. We investigated whether a non-IGF binding mutant of IGFBP-3 retained its intrinsic actions in this cell line, prior to investigating its actions in IGF-responsive cells (MCF-7 and MCF-10A). In the Hs578T cells, the ceramide analogue, C2-induced apoptosis, non-glycosylated, glycosylated or mutant IGFBP-3 alone had no effect but on co-incubation with C2, all forms of IGFBP-3 markedly accentuated triggered apoptosis. In MCF-7 cells, IGFBP-3 was unable to modulate C2-induced death. In the MCF-10A cells, IGFBP-3 acted as a potent survival factor. IGFBP-3 also affected cell growth in the MCF-10A cells (inhibiting at low doses but increasing growth at higher concentrations). These actions of IGFBP-3 in the MCF-10A cells were independent of IGF-1. IGFBP-3 has differential IGF-independent effects on cell death and growth in normal breast and breast cancer cells.     

Oncogenic ras causes resistance to the growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3) in breast cancer cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation and promotes apoptosis in normal and malignant cells. In MCF-10A human mammary epithelial cells, 30 ng/ml human plasma-derived IGFBP-3 inhibited DNA synthesis to 70% of control. This inhibition appeared IGF-independent, since neither an IGF-receptor antibody nor IGFBP-6 inhibited DNA synthesis. Malignant transformation of MCF-10A cells by transfection with Ha-ras oncogene abolished the inhibitory effect of IGFBP-3, concomitant with an increase in IGFBP-3 secretion and cell association of approximately 60 and 300%, respectively. When mitogen-activated protein (MAP) kinase activation was partially inhibited using PD 98059, IGFBP-3 sensitivity in ras-transfected cells was restored, with a significant inhibitory effect at 10 ng/ml IGFBP-3. PD 98059 had no effect on IGFBP-3 secretion or cell association by ras-transfected or parent MCF-10A cells. Hs578T, a tumor-derived breast cancer cell line that expresses activated Ha-ras, similarly has a high level of secreted and cell-associated IGFBP-3. In the absence of PD 98059, DNA synthesis by Hs578T cells was reduced to 70% of control by 1000 ng/ml IGFBP-3. PD 98059 increased sensitivity to IGFBP-3, so that this level of inhibition was achieved with 100 ng/ml IGFBP-3. These results suggest that MAP kinase activation by oncogenic ras expression causes IGFBP-3 resistance, a possible factor in the dysregulation of breast cancer cell growth.     

Modeling human breast cancer metastasis in mice: maspin as a paradigm

Breast cancer is the most common cancer detected in women, accounting for nearly one out of every three cancers diagnosed in the United States. Most cancer patients do not die from the primary tumor but die due to metastasis. Therefore, the study of metastasis is of most importance both to the clinician and patient. In the past, animal models have been used in breast cancer research and mammary gland biology. Our group has also established several animal models to address the function of a novel tumor suppressor gene maspin in breast tumor progression. Maspin was initially isolated from normal mammary epithelial cells. Its expression was down regulated in breast tumors. To test the protective role of maspin overexpression in mammary tumor progression, we crossed maspin overexpression transgenic mice (WAP-maspin) with a strain of oncogenic WAP-SV40 T antigen mice. The bitransgenic mice had reduced tumor growth rate and metastasis. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. In a separate animal experiment, maspin overexpressing mammary tumor cells (TM40D) were implanted into the fat pad of syngeneic mice. TM40D tumor cells were very invasive and metastatic. However, both primary tumor growth and metastasis were significantly blocked in TM40D cells that overexpress maspin as a consequence of plasmid or retrovirus infection. These evidences demonstrate that maspin function to inhibit primary tumor growth as well as invasion and metastasis. Elucidating the molecular mechanism of maspin action will shed light on our understanding of breast cancer invasion and metastasis.     

Normal breast epithelial cells induce apoptosis of breast cancer cells via Fas signaling

Fas/Fas ligand (Fas L) death pathway is an important mediator of apoptosis. Deregulation of Fas pathway is reported to be involved in the immune escape of breast cancer and the resistance to anti-cancer drugs. In this study, we demonstrated that conditioned medium by normal breast epithelial cells (NBEC-CM) induced apoptosis of MCF-7 and T-47D Fas-sensitive cells but had no effect on MDA-MB-231 Fas-resistant cells. Inhibition of PI3 kinase or NF-kappaB by specific inhibitors or transient transfections restored the sensitivity of MDA-MB-231 cells to NBEC-induced apoptosis. Moreover, the constitutive activation of NF-kappaB was controlled by PI3 kinase because inhibition of PI3 kinase reduced NF-kappaB activity. Inducible activation of NF-kappaB rendered MCF-7 cells resistant to NBEC-CM- and Fas agonist antibody-triggered apoptosis. Therefore, constitutive or inducible activation of PI3 kinase and/or NF-kappaB in breast cancer cells rendered them resistant to NBEC-triggered apoptosis. In addition, Fas neutralizing antibody and dominant negative Fas abolished NBEC-triggered apoptosis. Western blot and confocal microscopy analysis showed an increase of membrane Fas/Fas L when cells were induced into apoptotis by NBEC-CM. Taken together, these data show that NBEC induced apoptosis in breast cancer cells via Fas signaling.     

Growth inhibition of breast epithelial cells by celecoxib is associated with upregulation of insulin-like growth factor binding protein-3 expression

Several experimental and epidemiological studies have suggested a role for the use of cyclooxygenase (COX)-2 inhibitors in the prevention of breast cancer. The relative lack of toxicity associated with these compounds favors their use as chemopreventive agents, but the underlying mechanism of their chemopreventive effect remains unclear. We have observed that the COX-2 inhibitor celecoxib inhibits growth and induces apoptosis in the immortalized breast epithelial cell line 184htert. Microarray gene expression analysis of 184htert cells treated with 50 microM celecoxib for 6h revealed the modulation of several genes of interest, including a significant induction of expression of the mRNA encoding insulin-like growth factor binding protein-3 (IGFBP-3). IGFBP-3 is a potent pro-apoptotic protein and growth inhibitor of breast cancer cells, which acts mainly by inhibiting the access of the mitogens IGF-I and IGF-II to their cell surface receptor, but also via IGF-independent effects. Quantitative real-time RT PCR demonstrated that 50 microM celecoxib induced a approximately 3-fold increase in expression of IGFBP-3 mRNA after 6h. Furthermore, ligand blot analysis revealed that celecoxib treatment was associated with the upregulation of IGFBP-3 at the protein level. IGFBP-3 (500 ng/ml) treatment of 184htert cells inhibited IGF-I and serum-induced proliferation, but had no effect on cell growth under serum-free conditions, indicating that IGF-independent effects of IGFBP-3 are not observed in this system. Our results suggest that celecoxib may decrease IGF-I-associated breast cancer risk by a mechanism involving induction of expression of IGFBP-3 and subsequent reduced proliferation of at-risk breast epithelial cells.     

IGFBP-3 Can Either Inhibit or Enhance EGF-mediated Growth of Breast Epithelial Cells Dependent upon the Presence of Fibronectin

Progression of breast cancer is associated with remodeling of the extracellular matrix, often involving a switch from estrogen dependence to a dependence on EGF receptor (EGFR)/HER-2 and is accompanied by increased expression of the main binding protein for insulin-like growth factors (IGFBP-3). We have examined the effects of IGFBP-3 on EGF responses of breast epithelial cells in the context of changes in the extracellular matrix. On plastic and laminin with MCF-10A normal breast epithelial cells, EGF and IGFBP-3 each increased cell growth and together produced a synergistic response, whereas with T47D breast cancer cells IGFBP-3 alone had no effect, but the ability of EGF to increase cell proliferation was markedly inhibited in the presence of IGFBP-3. In contrast on fibronectin with MCF-10A cells, IGFBP-3 alone inhibited cell growth and blocked EGF-induced proliferation. With the cancer cells, IGFBP-3 alone had no effect but enhanced the EGF-induced increase in cell growth. The insulin-like growth factor-independent effects of IGFBP-3 alone on cell proliferation were completely abrogated in the presence of an EGFR, tyrosine kinase inhibitor, Iressa. Although IGFBP-3 did not affect EGFR phosphorylation [Tyr¹⁰⁶⁸], it was found to modulate receptor internalization and was associated with activation of Rho and subsequent changes in MAPK phosphorylation. The levels of fibronectin and IGFBP-3 within breast tumors may determine their dependence on EGFR and their response to therapies targeting this receptor.     

IRF6 in development and disease: A mediator of quiescence and differentiation

Post utero development of the mammary gland is a complex developmental process characterized by states of rapid cell proliferation (branching morphogenesis) followed by functional differentiation (lactation) and the consequent apoptosis (involution) of the secretory mammary epithelial cell. This process is cyclical, such that involution returns the mammary gland to a near-virgin-like state capable of responding to morphogenic cues with each consecutive pregnancy. Importantly, many of the regulatory processes which oversee mammary gland development are corrupted or otherwise compromised during the development of breast cancer. For example, Interferon Regulatory Factor 6 (IRF6) is a novel protein with growth inhibitory properties that was initially identified in mammary epithelial cells through its interaction with maspin, a known tumor suppressor in normal breast tissue. Recent findings from our laboratory suggest that IRF6 functions synergistically with maspin to regulate mammary epithelial cell differentiation by acting on the cell cycle. This perspective focuses on the possible involvement of IRF6 in promoting differentiation by regulating exit from the cell cycle and entry into the G(0) phase of cellular quiescence, and how these new findings shed light on normal mammary gland development and the initiation and progression of breast cancer.     

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.     

Proapoptotic effects of antiestrogens, progestins and androgen in breast cancer cells

The promoting action of E2 in breast cancer cells has been, until now, mainly linked to its action on prolifieration. Because of the importance of an increase in apoptosis in breast cancer prevention, we have studied the possible effects of various antiestrogens, progestins and an androgen on its occurrence in three hormone-dependent breast cancer cell lines. The antiestrogens were, a triphenylethylene derivative, 4 hydroxytamoxifen(4OHTAM) and two steroidal antiestrogens, ICI182780 and RU58668. The progestins were Org2058, a pregnane derivative, tibolone (OrgOD14), a normethyltestosterone derivative and OrgOM38 (the Δ4 isomer of OrgOD14) and the androgen dihydrotestosterone (DHT). Apoptosis was studied in MCF-7, ZR75-1 and T47-D cells using morphological approaches and flow cytometry. The antiestrogens, the progestins and DHT were proapoptotic but to different potencies according to the cell line studied. Indeed, the ‘pure’ steroidal antiestrogens were more efficient than 4OHTam in increasing apoptosis. We have also studied the level of expression of some of the proteins involved in the regulation of apoptosis. Bcl-2 and bcx_L, two antiapoptotic members of the bcl-2 family proteins, were inhibited by the progestins and the antiestrogens. In contrast, the proapoptotic proteins, bax and bak seemed to be constitutively expressed. Thus, since the ratio of proapoptotic and antiapoptotic proteins determines apoptosis or cell survival, the hormone effects are operating by modulating the antiapoptotic regulators of the balance. These data demonstrate that antiestrogens, progestins, and androgens can promote apoptosis in breast cancer cells, an effect which could be of importance in the therapeutic prevention of breast cancer.     

Dose-dependent changes of the ratio of apoptosis to proliferation by norethisterone and medroxyprogesterone acetate in human breast epithelial cells.

OBJECTIVE: There is increasing evidence that adding progestogens to estrogen replacement therapy may do more harm than good; however, whether all progestogens act equally on breast cells is debatable. Apart from estrogens, mitogenic growth factors from stromal breast tissue are important in growth-regulation of breast cells, and may modify the response to progestogens. We investigated the effects of medroxyprogesterone acetate (MPA) as well as norethisterone (NET) in the presence of a growth factor mixture and/or estradiol in normal and cancerous human epithelial breast cells. METHODS: MCF10A cells (human epithelial, estrogen- and progesterone-receptor negative, normal breast cells), HCC1500 (human estrogen and progesterone receptor-positive primary breast cancer cells) and MCF-7 cells (human estrogen and progesterone receptor-positive metastatic breast cancer cell line) were used in the experiments. The cells were incubated with progestogens at concentrations of 10(-10) to 10(-6) M for 7 days and growth factors (GFs), estradiol (E2) alone and a combination of GFs + E2. Cell proliferation rate was measured by ATP assay. Apoptosis was measured by cell death assay. Ratios of cell death : proliferation were calculated from these results. RESULTS: In MCF10A cells growth factors elicited a decrease in the ratio of apoptosis to proliferation. This effect was further stimulated by the addition of MPA, whereas NET had no effect. In HCC cells growth factors and estradiol alone and in combination led to a reduction in the ratio. This effect could be partly reversed dose-dependently by the addition of MPA and NET, being more pronounced for MPA. Similar results were found for MCF-7 cells stimulated by estradiol. CONCLUSION: The results of our investigations demonstrate that there are differences between the two progestogens NET and MPA investigated with respect to their effects on normal and cancerous cells. By increasing the mitotic rate of normal epithelial cells, MPA may increase breast cancer risk in women when used in long-term treatment. In this respect NET reacts neutral. The mitosis of pre-existing cancerous cells may be partly inhibited by the addition of both progestogens. Thus, our results indicate that it is necessary to differentiate between normal and malignant breast cells concerning the assessment of progestogens as a risk factor for breast carcinogenesis.     

Evaluation of sphinganine and sphingosine as human breast cancer chemotherapeutic and chemopreventive agents

No comparative study of the effects of sphingolipid metabolites on proliferation and differentiation in normal human breast epithelial cells versus stem cells and tumorigenic cells has been reported. The purpose of this study was to evaluate the chemotherapeutic and chemopreventive potential of sphingoid bases (sphingosine and sphinganine) using a novel cell culture system of normal human breast epithelial cells (HBEC) developed from breast tissues of healthy women obtained during reduction mammoplasty (Type I HBEC with stem cell characteristics and Type II HBEC with basal epithelial cell phenotypes) and transformed tumorigenic Type I HBEC. The results show that sphinganine inhibited the growth and induced apoptosis of transformed tumorigenic Type I HBEC more potently than sphingosine (IC(50) for sphinganine 4 microM; sphingosine 6.4 microM). Both sphinganine and sphingosine at high concentrations (8-10 lM) arrested the cell cycle at G(2)/M. Sphinganine inhibited the growth and caused death of Type I HBEC more strongly than sphingosine. In comparison, Type II HBEC (normal differentiated cells) were less sensitive to the growth-inhibitory effects of sphingoid bases than Type I HBEC (stem cells) or transformed tumorigenic Type I HBEC, suggesting that sphingoid bases may serve as chemotherapeutic agents. At concentrations (0.05, 0.1, and 0.5 microM) that are below the growth-inhibitory range, sphingoid bases induced differentiation of Type I HBEC to Type II HBEC, as detected morphologically and via expression of a tumor suppressor protein, maspin, which is a marker of Type II HBEC. Thus, sphingoid bases may function as chemotherapeutic as well as chemopreventive agents by preferentially inhibiting cancer cells and eliminating stem cells from which most breast cancer cells arise.     

Insulin-like growth factor binding protein-related protein 1 inhibits proliferation of MCF-7 breast cancer cells via a senescence-like mechanism.

Elevated insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) mRNA in senescent human mammary epithelial cells suggested that the IGFBP-3 gene product may inhibit cell proliferation. To test this hypothesis, we used a retroviral vector to express IGFBP-rP1 cDNA in the IGFBP-rP1-deficient MCF-7 breast cancer cell line. Compared with control vector-transduced cells, cumulative cell numbers for IGFBP-rP1-transduced polyclonal or clonal cell cultures were reduced by 39 and 74%, respectively, after 1 week. Medium conditioned by IGFBP-rP1-producing cultures reduced cumulative cell numbers in parental MCF-7 cultures by 20% compared with medium from cultures of a control vector-transduced cell line. Nuclear fragmentation analysis and cell proliferation assays completed in the presence of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone excluded apoptosis as the responsible mechanism. The percentage of cells containing senescence-associated beta-galactosidase activity was doubled compared with control cell cultures. Flow cytometry analysis indicated that twice as many noncycling cells were present in the IGFBP-rP1-transduced MCF-7 cell cultures compared with controls. These findings indicate that IGFBP-rP1 is an inhibitor of MCF-7 breast cancer cell proliferation and may act via a cellular senescence-like mechanism.     

Quercetin regulates insulin like growth factor signaling and induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate cancer cells (PC-3)

Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRβ, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.