Analysis of cross-resistance of the selective estrogen receptor modulators arzoxifene (LY353381) and LY117018 in tamoxifen-stimulated breast cancer xenografts.

PURPOSE: Cross-resistance is the primary issue facing the evaluation of new antiestrogens to treat metastatic breast cancer because they may be tested, initially, in populations of patients that have failed long-term adjuvant tamoxifen (Tam) therapy. EXPERIMENTAL DESIGN: We have tested the benzothiophene derivatives, arzoxifene (Arzox; LY353381) and LY117018 in two models of Tam-stimulated tumor growth derived from either MCF-7 (M. M. Gottardis and V. C. Jordan, Cancer Res., 48: 5183-5187, 1988) or T47D (J. MacGregor Schafer et al., Clin. Cancer Res., 6: 4373-4380, 2000) breast cancer cells. RESULTS: Using the MCF-7:Tam model, we found that both Arzox and LY117018 (1.5 mg/day) resulted in tumor growth and, therefore, were partially cross-resistant with Tam. Next, using the T47D:17beta-estradiol (E(2)) model, we compared the antiestrogenic/antitumor properties of Arzox and LY117018 and determined that neither Arzox nor LY117018 caused T47D:E(2) tumor growth after 21 weeks. In addition, we determined that long-term treatment does not result in failure and subsequent development of transplantable Arzox- or LY117018-stimulated tumors. To establish whether Arzox and LY117018 are cross-resistant in T47D:Tam tumors, mice were treated with Arzox or LY117018 (1.5 mg/day), and, again, we found that neither resulted in the growth of transplantable tumors. Lastly, we showed that Arzox and LY117018 were only partially able to compete with postmenopausal E(2) (0.3 cm silastic capsule) in T47D:Tam tumors. However, when T47D:E(2) tumors were treated for 7 days instead of 5 days, both Arzox and LY117018 were more effective. CONCLUSIONS: Arzox is not cross-resistant with Tam in the T47D athymic mouse model but does exhibit cross-resistance in the MCF-7 model.     

Effects of a new clinically relevant antiestrogen (GW5638) related to tamoxifen on breast and endometrial cancer growth in vivo.

PURPOSE: Cross-resistance is an important issue for the evaluation of new antiestrogens to treat advanced breast cancer patients who have failed tamoxifen therapy. In addition, postmenopausal patients treated with long-term adjuvant tamoxifen show a 3-4-fold increase in the risk of developing endometrial cancer. Consequently, a new second line agent should be more antiestrogenic and less estrogen-like on the uterus, and be effective at controlling the growth of breast cancer after exposure to tamoxifen. The purpose was to evaluate the effects of the new tamoxifen analogue GW5638 on breast and endometrial cancer growth. EXPERIMENTAL DESIGN: Athymic mice were transplanted with an endometrial tumor model (ECC-1 E2) that is responsive to estrogen and has never been exposed to antiestrogen. In addition, we used three breast tumor models: a tamoxifen-na?ve tumor (T47D-E2) and two tamoxifen-stimulated tumors (MT2 TAM and MCF-7 TAM LT). The antiestrogen GW5638 (1.5 mg daily), tamoxifen (0.5 mg or 1.5 mg daily), and raloxifene (1.5 mg daily) were given p.o. The pure antiestrogen ICI182,780 (5 mg once a week) was given s.c. Western blots from MCF-7 TAM breast tumors were performed to demonstrate the regulation of estrogen receptor alpha expression by different ligands. RESULTS: Estradiol and GW5638 down-regulated the receptor compared with control. ICI182,780 completely degraded the receptor but tamoxifen had no effect. GW5638 did not promote tumor growth, and was effective in blocking the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve breast and endometrial tumors. However, raloxifene did not completely block the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve endometrial tumor after 14 weeks. GW5638 and ICI182,780 but not raloxifene were also effective in blocking the tamoxifen-stimulated breast tumor growth in athymic mice. CONCLUSIONS: GW5638 is more effective than raloxifene in blocking the effect of estrogen on tamoxifen-na?ve endometrial cancer. More importantly, GW5638, like the pure antiestrogen ICI182,780, is able to block the growth of breast cancer stimulated by tamoxifen differently from raloxifene. GW5638 down-regulates estrogen receptor but does not completely destroy the receptor. Therefore, based on our findings, GW5638 could be developed as a second line agent for advanced breast cancer patients and an important first line agent to evaluate as an adjuvant treatment or chemopreventive.     

Antitumor action of physiological estradiol on tamoxifen-stimulated breast tumors grown in athymic mice.

The estrogen receptor (ER)-positive MCF-7 breast cancer cell line can be transplanted into athymic mice and grown into tumors with estradiol (E2) support. Tamoxifen (TAM) blocks E2-stimulated tumor growth; however, continuous TAM treatment results in transplantable tumors within a year that will grow with either E2 or TAM (M. M. Gottardis and V. C. Jordan, Cancer Res., 48: 5183-5187, 1988). Although this model may represent the development of TAM resistance for the treatment of advanced breast cancer, no laboratory model exists to study the exposure of breast cancer to 5 years of adjuvant TAM therapy. We have addressed this issue and report the development and characterization of two tumor lines, MCF-7TAM and MT2, which have been serially transplanted into TAM-treated athymic mice for >5 years. The MCF-7TAM tumor rapidly regresses in response to E2 and then about 50% of tumors regrow in response to E2. Interestingly, tumor regression does not occur if TAM treatment is stopped, probably because E2 levels are too low in ovariectomized athymic mice. The development of the antitumor effect of E2 was documented for MT2 tumors over a 1-year period; TAM-stimulated tumor growth was retained, but E2 caused progressively less of a stimulatory effect. Most importantly, E2-stimulated tumors that regrew after initial tumor regression in both MCF-7TAM and MT2 lines were again responsive to TAM to block E2-stimulated growth. Unlike MCF-7 tumors, the MT2 tumor line contains a single point mutation, Asp351Tyr, in the ER, which was retained after the development of E2-stimulated regrowth. The mutation is associated with increased estrogen-like actions for the TAM-ER complex (A. S. Levenson et al., Br. J. Cancer, 77: 1812-1819, 1998), but we conclude that the mutant ER is not required for TAM resistance. On the basis of the new breast cancer models presented, we propose a cyclic sensitivity to TAM that may have important clinical implications: (a) it is possible that a woman's own estrogen may produce an antitumor effect on the presensitized micrometastatic disease after 5 years of TAM. Long-term antitumor action occurs because the drug is stopped, but resistance accumulates and tumors start to grow if adjuvant therapy is continued; and (b) although in the clinic TAM-resistant tumors respond to second-line therapies that cause estrogen withdrawal, e.g., pure antiestrogens or aromatase inhibitors, estrogen therapy may also be effective and return the tumor to TAM responsiveness. In this way, a hormone-responsive tumor may be controlled longer in the patient with advanced disease.     

Anti-proliferative and anti-estrogenic effects of ICI 164,384 and ICI 182,780 in 4-OH-tamoxifen-resistant human breast-cancer cells

The effects of the anti-estrogens 4-hydroxytamoxifen (OHTam), ICI 164,384 and ICI 182,780 were tested on the MCF-7/LCC2 breast-carcinoma cell line, which grows significantly in the presence of OHTam and serves as a model for studying anti-estrogen resistance of estrogen-receptor-positive breast cancer. Cell proliferation and cathepsin-D secretion were strongly inhibited by either ICI 182,780 or ICI 164,384 alone or ICI 164,384 in combination with 17-p-estradiol (E2) or OHTam. ICI 164,384 alone did not affect the cathepsin-D and pS2 mRNA levels, but antagonized the stimulatory effects of E2 or OHTam on these 2 mRNAs. OHTam was more effective than E2 in increasing cathepsin-D mRNA levels, supporting the idea that anti-estrogen-resistant breast cancer continues to over-express cathepsin-D. These data show that the steroidal anti-estrogens ICI 164,384 and ICI 182,780 retain their ability to inhibit cell proliferation and the estrogen-responsiveness of cathepsin-D and pS2 genes in the OHTam-resistant MCF-7/ LCC2 cell line. These pure anti-estrogens may thus be efficient second-line treatments of some Tamoxifen-resistant tumors.     

Expression of constitutively active Akt-3 in MCF-7 breast cancer cells reverses the estrogen and tamoxifen responsivity of these cells in vivo.

PURPOSE: Prior studies had suggested that Akt activity is elevated in a subset of breast cancers. In this study, to test the effect of active Akt-3 on estrogen receptor function, we have produced MCF-7 cells, which express active Akt-3 and examined the estrogen responsiveness of these cells in vivo and in vitro. Experimental Design: MCF-7 cells expressing active Akt-3 were studied for estradiol (E2) responsiveness in vitro by both using an estrogen receptor element reporter construct as well as looking at induction of endogenous genes. These cells were also studied in vivo after injection into nude, ovariectomized mice by following tumor growth rates in the presence or absence of E2, tamoxifen, or the pure antiestrogen, ICI 182,780 (fulvestrant). RESULTS: Akt-3-expressing cells were found to produce tumors in mice in the absence of E2 that were approximately equivalent in size to control cells in mice given E2. Moreover, the formation of tumors by the Akt-3 cells was greatly suppressed by E2, stimulated by tamoxifen, and unaffected by ICI 182,780. In the in vitro assays for gene induction by E2, the Akt-3-expressing cells exhibited similar E2 and tamoxifen responsiveness as the control cells. CONCLUSIONS: These results indicate that expression of active Akt-3 in MCF-7 cells results in E2-independent tumor growth. Moreover, the growth of these tumors is inhibited by E2 and enhanced by tamoxifen. Finally, these tumors are resistant to ICI 182,780. These findings suggest that the amount of active Akt present in breast cancers may be important in the relative efficacy of different treatments.     

Insulin receptor substrate 1 is a target for the pure antiestrogen ICI 182,780 in breast cancer cells

The pure antiestrogen ICI 182,780 inhibits insulin-like growth factor (IGF)-dependent proliferation in hormone-responsive breast cancer cells. However, the interactions of ICI 182,780 with IGF-I receptor (IGF-IR) intracellular signaling have not been characterized. Here, we studied the effects of ICI 182,780 on IGF-IR signal transduction in MCF-7 breast cancer cells and in MCF-7-derived clones overexpressing either the IGF-IR or its 2 major substrates, insulin receptor substrate 1 (IRS-1) or src/collagen homology proteins (SHC). ICI 182,780 blocked the basal and IGF-I-induced growth in all studied cells in a dose-dependent manner; however, the clones with the greatest IRS-1 overexpression were clearly least sensitive to the drug. Pursuing ICI 182,780 interaction with IRS-1, we found that the antiestrogen reduced IRS-1 expression and tyrosine phosphorylation in several cell lines in the presence or absence of IGF-I. Moreover, in IRS-1-overexpressing cells, ICI 182,780 decreased IRS-1/p85 and IRS-1/GRB2 binding. The effects of ICI 182,780 on IGF-IR protein expression were not significant; however, the drug suppressed IGF-I-induced (but not basal) IGF-IR tyrosine phosphorylation. The expression and tyrosine phosphorylation of SHC as well as SHC/GRB binding were not influenced by ICI 182,780. In summary, downregulation of IRS-1 may represent one of the mechanisms by which ICI 182,780 inhibits the growth of breast cancer cells. Thus, overexpression of IRS-1 in breast tumors could contribute to the development of antiestrogen resistance.     

Upregulation of PKC-delta contributes to antiestrogen resistance in mammary tumor cells

Acquired resistance to tamoxifen (Tam) in breast cancer patients is a serious therapeutic problem. We have previously reported that protein kinase C-delta (PKC-delta) plays a major role in estrogen (E2)-mediated cell proliferation. To determine if PKC-delta is one of the major alternate signaling pathways that supports cell growth in the presence of Tam, we determined the levels of PKC isoforms in four different models of antiestrogen-resistant cells. Three out of four antiestrogen resistance cell lines (Tam/MCF-7, ICI/MCF-7 and HER-2/MCF-7) expressed significantly high levels of both total and activated PKC-delta levels compared to sensitive cells. Estrogen receptor (ER) alpha content and function are maintained in all the antiestrogen-resistant cell lines. Overexpressing active PKC-delta in Tam-sensitive MCF-7 cells (PKC-delta/MCF-7) led to Tam resistance both in vitro and in vivo. Inhibition of PKC-delta by rottlerin (a relatively specific inhibitor of PKC-delta) or siRNA significantly inhibited estrogen- and Tam-induced growth in antiestrogen-resistant cells. PKC-delta levels are significantly higher in Tam-resistant tumors compared to Tam-sensitive tumors in xenograft model (P<0.05). Taken together, these data suggest that PKC-delta plays a major role in antiestrogen resistance in breast tumor cells and thus provides a new target for treatment.     

Rapid development of tamoxifen-stimulated mutant p53 breast tumors (T47D) in athymic mice.

MCF-7 cells are used routinely to study tamoxifen-stimulated drug resistance in vivo. However, unlike MCF-7 cells, T47D cells express mutant p53 protein and lose the estrogen receptor (ER) during long-term estrogen deprivation in vitro [Pink et al., Br. J. Cancer, 74: 1227-1236, 1996 (erratum, Br. J. Cancer, 75: 1557, 1997)]. As a result, T47D tumors may respond differently from MCF-7 tumors to long-term tamoxifen treatment. Ovariectomized athymic mice were given injections bilaterally with T47D cells (5 x 10(5)) into the mammary fat pads. A rapidly growing estradiol responsive tumor (T47D:E2) was established and 0.5 mg of tamoxifen given daily blocked estrogen-stimulated growth. In subsequent experiments, low doses of tamoxifen (0.17 mg or 0.5 mg) did not produce tamoxifen-stimulated tumors at 14 weeks, whereas high-dose tamoxifen (1.5 mg) consistently produced tamoxifen-stimulated tumors (T47D:Tam; 17 tumors/20 sites) at 8 weeks. In contrast, 1.5 mg of tamoxifen produced tamoxifen-stimulated MCF-7 tumors (MCF-7: Tam2) at a slower rate (20 weeks) and less consistently (14 tumors/26 sites). When the T47D:Tam tumor was passaged, it grew maximally with either 1.5 mg of tamoxifen or a 1-cm estradiol (premenopausal levels) capsule, and similar results were obtained with MCF-7:Tam2 tumors. Interestingly, when T47D:Tam tumors were treated with the 0.5 mg of tamoxifen, tumors grew only to 50% maximum. All of the tumors originating from MCF-7 and T47D cells expressed ER at similar levels; therefore, tamoxifen did not select for an ER-negative tumor. In conclusion, we have shown that tamoxifen-stimulated T47D p53 mutant tumors can be developed rapidly with high-dose therapy (1.5 mg daily). The results from this model provide new opportunities to investigate the rapid development of drug resistance to adjuvant tamoxifen in patients with mutant p53 breast tumors.     

Dietary genistein negates the inhibitory effect of tamoxifen on growth of estrogen-dependent human breast cancer (MCF-7) cells implanted in athymic mice

The use of dietary isoflavone supplements by postmenopausal women with breast cancer is increasing. We investigated interactions between the soy isoflavone, genistein, and an antiestrogen, tamoxifen (TAM), on the growth of estrogen (E)-dependent breast cancer (MCF-7) cells implanted in ovariectomized athymic mice. We hypothesized that weakly estrogenic genistein negate/overwhelm the inhibitory effect of TAM on the growth of E-dependent breast tumors. Six treatment groups were used: control (C); 0.25 mg estradiol (E2) implant (E); E2 implant + 2.5 mg TAM implant (2.5 TE); E2 implant + 2.5 mg TAM implant + 1000 ppm genistein (2.5 TEG); E2 implant + 5 mg TAM implant (5 TE), and E2 implant +5 mg TAM implant +1000 ppm genistein (5 TEG). Treatment with TAM (2.5 TE and 5 TE) suppressed E2-stimulated MCF-7 tumor growth in ovariectomized athymic mice. Dietary genistein negated/overwhelmed the inhibitory effect of TAM on MCF-7 tumor growth, lowered E2 level in plasma, and increased expression of E-responsive genes (e.g., pS2, PR, and cyclin D1). Therefore, caution is warranted for postmenopausal women consuming dietary genistein while on TAM therapy for E-responsive breast cancer.     

Dietary lariciresinol attenuates mammary tumor growth and reduces blood vessel density in human MCF-7 breast cancer xenografts and carcinogen-induced mammary tumors in rats

Lariciresinol is a dietary lignan that accounts for a significant portion of the total phytoestrogen intake from Western foods. Recent epidemiological studies suggest that high dietary intake of lignans and lariciresinol is associated with reduced breast cancer risk. However, no causal relationship between lariciresinol intake and breast cancer development has been established. In this study, we investigated for the first time the effects and possible mechanisms of action of lariciresinol on hormone responsive mammary cancer in vivo in dimethylbenz[a]anthracene induced mammary cancer in rats, and in human MCF-7 breast cancer xenografts in athymic mice. For tumor bearing rats, lariciresinol (3 or 15 mg/kg of body weight) or vehicle was administered p.o. daily for 9 weeks. For E2-maintained ovariectomized athymic mice bearing orthotopic MCF-7 tumors, control diet (AIN-93G) or lariciresinol containing diet (AIN-93G supplemented with 20 or 100 mg of lariciresinol/kg of diet) was administered for 5 weeks. In both models, lariciresinol administration inhibited the tumor growth and tumor angiogenesis. In MCF-7 cells, enterolactone significantly inhibited the E2-stimulated VEGF secretion. Moreover, in MCF-7 xenografts, lariciresinol administration enhanced tumor cell apoptosis and increased estrogen receptor beta expression. Lariciresinol and its further metabolites secoisolariciresinol, enterodiol and enterolactone were found in serum of both rats and athymic mice confirming a similar lignan metabolism pattern as in humans. These findings indicate conceivable importance of dietary lignan lariciresinol in inhibition of breast cancer development.     

In vitro regulation of vascular endothelial growth factor by estrogens and antiestrogens in estrogen-receptor positive breast cancer

BACKGROUND: The effects of antiestrogens on angiogenesis in breast cancer are not fully defined. In this study we investigated the in vitro effects of antiestrogens at different concentrations on vascular endothelial growth factor (VEGF) production in estrogen receptor (ER)-positive breast cancer cells. METHODS: The dose-dependent effects of 17beta-estradiol (E2), 4-hydroxytamoxifen (4OHT), and ICI182,780 were analyzed both with reference to growth rates and VEGF protein production using enzyme-linked immunosorbent assay (ELISA) in MCF-7 cells. RESULTS: E2 stimulated both the growth rates and VEGF production of MCF-7 cells in the same manner. Although 4OHT stimulated the growth rates as an agonistic effect in an estrogen-free media at levels ranging from 1 nM to 1 micro M, it did not stimulate VEGF expression at the same levels except for at 1 micro M. Although 4OHT had a weak agonistic effect on VEGF production at 1 micro M in an estrogen-free media, it significantly inhibited E2-stimulated VEGF production at the same level. A cytotoxic effect was observed with 10 micro M 4OHT that paradoxically caused a prominent increase in VEGF production. ICI182,780 had no significant effects on the growth rates or VEGF production in this cell line. CONCLUSIONS: These results support the hypothesis that tamoxifen could inhibit angiogenesis induced by estrogens in ER-positive breast cancer cells.     

Effects of the new selective estrogen receptor modulator LY353381.HCl (Arzoxifene) on human endometrial cancer growth in athymic mice.

PURPOSE: Arzoxifene (Arzox) is a novel benzothiophene analogue with selective estrogen receptor modulator activity similar to raloxifene. Arzox is being developed as a treatment for breast cancer and has a predominantly antiestrogenic effect on the rodent uterus. Our objectives were to verify whether the novel selective estrogen receptor modulator, Arzox, can be a good first-line agent and also be effective at controlling the growth of endometrial cancer after exposure to tamoxifen (Tam). EXPERIMENTAL DESIGN: We compared the effects of Tam and Arzox on the growth of estrogen responsive ECC-1 endometrial cancer cells in vitro, and we determined their antitumor effects on ECC-1 and EnCa101 endometrial carcinoma growth in athymic mice. RESULTS: We observed that estrogen receptor protein expression is down-regulated by Arzox to the same extent as raloxifene, whereas 4-hydroxytamoxifen, the active metabolite of Tam, does not affect estrogen receptor protein levels. Tam and Arzox inhibit the growth of Tam-na?ve ECC-1 tumors in athymic mice. However when Tam-stimulated or estrogen-stimulated (which had been treated with Tam previously) EnCa101 endometrial tumors were treated with Tam or Arzox, we observed a stimulatory effect of both compounds in these models. CONCLUSIONS: The results indicate that Arzox may be a good first-line agent, but it may be ineffective at controlling the growth of endometrial cancer after exposure to Tam. Our data suggest that Arzox stimulates endometrial tumor growth to at least the same extent as Tam, thereby suggesting a limited role as a second-line agent for the patient on Tam who develops occult endometrial cancer.     

Antiestrogenic glyceollins suppress human breast and ovarian carcinoma tumorigenesis.

PURPOSE: We have identified the phytoalexin compounds glyceollins I, II, and III, which exhibit marked antiestrogenic effects on estrogen receptor function and estrogen-dependent tumor growth in vivo. The purpose of this study was to investigate the interactions among the induced soy phytoalexins glyceollins I, II, and III on the growth of estrogen-dependent MCF-7 breast cancer and BG-1 ovarian cancer cells implanted in ovariectomized athymic mice. EXPERIMENTAL DESIGN: Four treatment groups for each cell line were used: vehicle control, 20 mg/kg/mouse/d glyceollin mixture injection, 0.72 mg estradiol (E2) implant, and E2 implant + 20 mg/kg/mouse/d glyceollin injection. RESULTS: Treatment with glyceollin suppressed E2-stimulated tumor growth of MCF-7 cells (-53.4%) and BG-1 cells (-73.1%) in ovariectomized athymic mice. These tumor-inhibiting effects corresponded with significantly lower E2-induced progesterone receptor expression in the tumors. In contrast to tamoxifen, the glyceollins had no estrogen-agonist effects on uterine morphology and partially antagonized the uterotropic effects of estrogen. CONCLUSIONS: These findings identify glyceollins as antiestrogenic agents that may be useful in the prevention or treatment of breast and ovarian carcinoma.     

Inhibition of the MAP kinase activity suppresses estrogen-induced breast tumor growth both in vitro and in vivo

Elevated expression of mitogen-activated protein kinase (Erk/MAPK) has been noted in a significant percentage of primary human breast cancers. To directly assess the importance of Erk/MAPK activation in estrogen (E2)-induced tumor progression, we blocked E2-signaling with MEK-inhibitor CI-1040 and/or tamoxifen (Tam). Our data show that both MEK-inhibitor CI-1040 and Tam blocked E2-induced MAPK phosphorylation and cell proliferation in MCF-7 breast cancer cells in vitro. However, in vivo studies show that anti-tumor efficacy of combining the CI-1040 and Tam was similar to single agent(s). Furthermore, sequential treatment with Tam followed by CI-1040 or CI-1040 followed by Tam did not significantly reduce E2-induced tumor growth. This suggests that the combination of CI-1040 and Tam may not be synergistic in inhibiting E2-induced tumor growth. However, these findings also indicate that MAPK plays a critical role in E2-induced tumor growth, and that this could be a potential therapeutic target to combat hormonally regulated growth in ER-positive tumors.     

Novel antitumor effect of estradiol in athymic mice injected with a T47D breast cancer cell line overexpressing protein kinase Calpha.

PURPOSE: Resistance to tamoxifen (TAM) represents a significant challenge to the management of breast cancer. We previously reported that the estrogen receptor (ER)-negative hormone-independent T47D:C42 cell line has both elevated protein kinase Calpha (PKCalpha) protein expression and basal activator protein-1 activity compared with the parental ER+ (hormone-dependent) T47D:A18 cell line. Stable transfection of PKCalpha to the T47D:A18 breast cancer cell line results in increased basal activator protein-1 activity, reduced ER function, increased proliferation rate, and hormone-independent growth (Tonetti et al., Br. J. Cancer, 83: 782-791, 2000). In this report, we further characterize the role of PKCalpha overexpression in vivo to elucidate a possible molecular mechanism of tamoxifen resistance. EXPERIMENTAL DESIGN: To determine whether the T47D:A18/PKCalpha cell line would produce hormone-independent tumors in athymic mice, we injected T47D:A18, T47D:A18/neo, or the T47D:A18/PKCalpha20 cell clones bilaterally into the mammary fat pads of athymic mice. Tumor growth was evaluated following treatment with estradiol (E2), TAM, and the pure antiestrogen, ICI 182,780. RESULTS: Mice receiving either T47D:A18 or T47D:A18/neo cells produced tumors that grew in response to E2 treatment, whereas the untreated control and TAM-treated groups showed no tumor growth. Interestingly, mice receiving the T47D:A18/PKCalpha20 clone produced tumors in both the control and TAM groups, whereas tumor growth was inhibited in mice treated with E2. PKCalpha was also overexpressed in an MCF-7 tumor model that also exhibited TAM-stimulated and E2-induced regression. CONCLUSIONS: These results suggest that overexpression of PKCalpha in breast tumors results in hormone-independent tumor growth that cannot be inhibited by TAM treatment. Furthermore, the finding that E2 has an antitumor effect on breast tumors overexpressing PKCalpha is a novel observation that may have important therapeutic implications.     

Treatment with the pure antiestrogen faslodex (ICI 182780) induces tumor necrosis factor receptor 1 (TNFR1) expression in MCF-7 breast cancer cells

Apoptosis induction by the pure antiestrogen faslodex, also known as ICI 182780 (ICI), is associated with an effective down-regulation of Bcl-2 expression in the human breast cancer cell line MCF-7. Recent observations point out that beside members of the Bcl-2 family also the TNFR1 signaling pathway may be involved in apoptosis induction by antiestrogens. In this report we have analyzed the expression of members of the TNFR1 signaling pathway during the apoptotic process induced by the pure antiestrogen faslodex and by tamoxifen (Tam) in MCF-7 breast cancer cells. Treatment with 10^–7M ICI or 10^–7M Tam leads to a time dependent increase of TNFR1 and TRADD steady-state mRNA levels in MCF-7 cells. In contrast, Bcl-2 expression was strongly decreased following administration of ICI but only weakly after administration of Tam. Western blot analysis and studies by the use of fluorescence microscopy and flow cytometry revealed a time dependent induction of TNFR1 protein and cell surface expression in MCF-7 cells in response to treatment with ICI. To investigate if TNFR1 is functionally involved in apoptosis induction by antiestrogens, we tested whether TNFR1 blocking antibodies can counteract the growth inhibitory action of Tam and ICI. Coincubation of MCF-7 cells with antiestrogens (ICI or Tam) and blocking TNFR1 antibodies lead to an increase in cell viability. Our results provide evidence for a cross talk between the TNFR1 signaling pathway and antiestrogens during the process of apoptosis induction in MCF-7 breast cancer cells. The superiority of the pure antiestrogen ICI to induce apoptosis in MCF-7 cells may result from its capability to modulate the induction of apoptosis via Bcl-2 as well as TNF-associated signal transduction pathways.     

Inhibition of estrogen-dependent tumorigenesis by the thyroid hormone receptor β in xenograft models

Association studies suggest that thyroid hormone receptor β (TRβ) could function as a tumor suppressor in breast cancer development, but unequivocal evidence is still lacking. To understand the role of TRβ in breast tumor development, we adopted the gain-of-function approach by stably expressing the THRB gene in a human breast cancer cell line, MCF-7 (MCF-7-TRβ). Parental MCF-7 cells express the estrogen receptor, but not TRs. MCF-7 cells, stably expressing only the selectable marker, the Neo gene, were also generated as control for comparison (MCF-7-Neo cells). Cell-based studies indicate that the estrogen (E2)-dependent growth of MCF-7 cells was inhibited by the expression of TRβ in the presence of the thyroid hormone (T3). In a xenograft mouse model, large tumors rapidly developed after inoculation of MCF-7-Neo cells in athymic mice. In contrast, markedly smaller tumors (98% smaller) were found when MCF-7-TRβ cells were inoculated in athymic mice, indicating that TRβ inhibited the E2-dependent tumor growth of MCF-7 cells. Further detailed molecular analysis showed that TRβ acted to activate apoptosis and decrease proliferation of tumor cells, resulting in inhibition of tumor growth. The TRβ-mediated inhibition of tumor growth was elucidated via down-regulation of the JAK-STAT-cyclin D pathways. This in vivo evidence shows that TRβ could act as a tumor suppressor in breast tumorigenesis. The present study provides new insights into the role of TR in breast cancer.