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Previous Article | Next Article 
Molecular and Cellular Biology, June 2005, p. 4591-4601, Vol. 25, No. 11
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.11.4591-4601.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
,
Kenna R. Mills,1,
Esther B. E. Becker,2
Danielle K. Lynch,1
Azad Bonni,2
Senthil K. Muthuswamy,1,
and
Joan S. Brugge1*
Department of Cell Biology,1 Department of Pathology, Harvard Medical School, Boston, Massachusetts 021152
Received 3 November 2004/ Returned for modification 2 December 2004/ Accepted 6 February 2005
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ABSTRACT |
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INTRODUCTION |
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We have investigated processes associated with lumen formation in the MCF-10A line of immortalized human mammary epithelial cells. When cultured on a reconstituted basement membrane derived from Engelbreth-Holm-Swarm tumor (Matrigel), immortalized MCF-10A mammary epithelial cells undergo a series of morphogenetic processes resulting in the formation of acinus-like structures containing a single layer of polarized cells surrounded by a hollow lumen (28). Formation of the luminal space in this model follows the development of apico-basal polarity and involves apoptosis and an autophagy-like process in the centrally localized cells (8). Cells that proliferate into the hollow cavity after lumen formation also undergo cell death, indicating that the luminal space is not compatible with cell survival. Oncogenes that induce filling of the luminal space appear to do so, in part, by blocking apoptosis (8).
The evidence that cells which proliferate into the luminal space undergo apoptosis raised the question whether filling of the luminal space by oncogenes that induce hyperproliferation requires antiapoptotic activities. Indeed, we found that filling of the luminal space in MCF-10A acini requires both constitutive stimulation of proliferation and antiapoptotic activities (8). For example, activation of homodimers of the oncogene ErbB2 (HER2/Neu) in preformed acinar structures leads to constitutive proliferation and filling of the luminal space (28), whereas acinar structures that express oncogenes, such as human papillomavirus type 16 (HPV E7) or cyclin D1, proliferate constitutively but contain hollow lumen (8). The ability of ErbB2 to induce luminal filling correlates with its ability to suppress activation of caspase 3 and apoptosis, whereas the proliferating cyclin D1 or HPV E7 expressing acinar cells are unable to promote survival. Moreover, only when HPV E7 or cyclin D1 acinar cells are supplied a prosurvival signal, by coexpressing the antiapoptotic protein Bcl-xL, can they block luminal apoptosis and fill the luminal space. The filled acinar structures induced by ErbB2 or the combination of cyclin D1 and Bcl-xL in this model resemble lesions associated with carcinoma in situ breast tumors. Indeed, amplification or overexpression of ErbB2 is detected in 60 to 85% of comedo-type carcinoma in situ tumors (42); thus, the effects of ErbB2 in this model may mimic events induced by ErbB2 in vivo.
To investigate the molecular mechanisms underlying luminal apoptosis during mammary acinar morphogenesis, we have examined the basis for Bcl-2 and Bcl-xL protection from luminal apoptosis. Pro-survival members of the Bcl-2 family inhibit apoptosis by heterodimerizing with the proapoptotic family members (4). Proapoptotic members fall into two categories; those containing BH domains 1 to 3 (e.g., Bax/Bak/Bok [Bax-like]) and those containing only a BH3 domain (e.g., Bad, Bid, Noxa, and Bim BH3-only]). The multidomain Bax-like members and the BH3-only members can both initiate apoptosis but differ in that the BH3-only members act as damage sensors and antagonists of prosurvival proteins (4), whereas Bax-like members contain innate cytotoxic function (40). The BH3-domain only proteins are well suited for setting thresholds for initiating apoptosis since they are regulated by divergent signaling cascades, and their expression is often tissue specific (17).
We demonstrate here that the ability of Bcl-xL to inhibit luminal apoptosis is dependent on binding to BH3-only proteins and identify the proapoptotic BH3-only protein Bim as a significant contributor to luminal apoptosis. Bim is upregulated during MCF-10A morphogenesis just prior to the induction of apoptosis, is required for early luminal apoptosis, and contributes to luminal clearance in acinar structures. Oncogenes that block luminal cell death inhibit Bim expression through stimulation of Mek/Erk kinase pathway. Thus, the regulation of a BH3-only member, Bim, plays a critical role in luminal apoptosis during acinar morphogenesis in this model system and may be an important target of oncogenes that disrupt glandular epithelial architecture.
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MATERIALS AND METHODS |
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Retrovirus vectors and MCF-10A cell lines. Vesicular stomatitis virus-pseudotyped retroviruses were produced as previously described (32). pMig, pMig-Bcl-xL, pMig-Bcl-xL (M1), and pMig-Bcl-xL (M8) were kindly provided by Stanley Korsmeyer. The construct pBabe-MEK2-DD was kindly provided by Sylvain Meloche. pBabe-vSRC-ER was a generous gift of Martin McMahon. Treatment of vSrc-ER cells with 4-hydroxytamoxifen (OHT) for 48 h caused a marked increase in tyrosine phosphorylation, and treatment with OHT for 48 h in preformed acini led to increased proliferation (data not shown). Vectors encoding the HPV E7 oncoprotein or the empty vector (pLXSN) were obtained as a gift from Denise Galloway and Peter Howley. Stable MCF-10A cells containing p75.B2, a chimeric ErbB2 receptor fused to the bivalent ligand FK-506 binding protein, and its activation via dimerization with the bivalent ligand AP1510 (ARIAD Pharmaceuticals) have been reported previously (28). Full-length BimEL cDNA was cloned by reverse transcription-PCR from RNA isolated from rat cerebellar granule neurons. BimEL cDNA was then subcloned into the polylinker of the mammalian expression vector pCDNA3 (Invitrogen) with an NH2-terminal hemagglutinin tag. Point mutations of Ser-65 were made by using the QuikChange site-directed mutagenesis kit (Stratagene). Similarly, the splicing donor site for BimL was mutated (T126 to G, without an amino acid change) to prevent splicing that generates both BimEL and BimL isoforms as previously been shown (34). Mutations were verified by sequencing. BimEL and BimEL-SA cDNAs were subsequently cloned into the retroviral vector pLPCX. For small interfering RNA (siRNA) experiments, methods for transfecting MCF-10A cells and sequence for control and Bim oligonucleotides have been previously described (32).
Morphogenesis assay. Assays were performed as previously described (28). Briefly, MCF-10A cells were resuspended in assay medium (DMEM/F12 supplemented with 2% donor horse serum, 10 µg of insulin/ml, 100 ng of cholera toxin/ml, 0.5 µg of hydrocortisone/ml, and 5 ng of EGF/ml). Eight-well RS glass slides (BD Falcon) were coated with 35 µl of Matrigel per well. Then, 5,000 cells were plated per well in assay medium containing a final concentration of 2% Matrigel. Assay medium containing 2% Matrigel was replaced every 4 days. For the stimulation of cells containing ErbB2 chimeras with AP1510, EGF was replaced with 1 µM AP1510 on day 8 or as indicated. For v-Src induction, acini were treated with 1 µM OHT (Sigma) or vehicle control (ethanol,1:2,000). For studies with chemical inhibitors, morphogenesis assays were performed as described until the medium was replaced with assay medium supplemented with dimethyl sulfoxide (1:1,000), UO126 (5 µM), SB202190 (5 µM), or LY294002 (25 µM) for 48 h as indicated.
Quantification of cell death. Assay media were removed from wells, and acini were washed once with phosphate-buffered saline (PBS). Structures were then incubated for 15 to 30 min at 37°C with 1 µM ethidium bromide (EtBr) in MCF10A growth medium. Cell death was quantified by counting the total number of acini in one well with at least two EtBr-positive cells. At least 100 structures were counted per well. All values are given as percentage of acinar structures exhibiting evidence of cell death in a total population at a given time point. For quantification of apoptosis in cells infected with BimEL or BimEL-SA we used a cell death detection enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics) according to the manufacturer's instructions. Error bars represent the standard error of the mean (SEM) of at least three independent experiments.
Protein extraction and Western analysis from MCF-10A acini. Acinar structures were washed briefly with 4°C PBS with protease inhibitors (phenylmethylsulfonyl fluoride [10 µg/ml], leupeptin [1 µg/ml], aprotinin [1 µg/ml], pepstatin [1 µg/ml]) and then treated for 15 min at 4°C with radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 20 mM Tris [pH 7.5], 0.1% sodium dodecyl sulfate, 1.0% sodium deoxycholate, 0.1.0% Triton X-100). Matrigel and acini were collected and pulled through a 27-gauge needle three to five times before being placed on ice for 15 min. Lysates were cleared by centrifugation at 16,000 x g for 20 min at 4°C, followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography.
Immunofluorescence and image acquisition. Structures were prepared as previously described (28). Briefly, acini were fixed in 4% formalin for 25 min at room temperature. Fixed structures were washed with PBS-glycine (130 mM NaCl, 7 mM Na2HPO4, 100 mM glycine) three times for 15 min each time. The structures were then blocked in IF buffer (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, 7.7 mM NaN3, 0.1% bovine serum albumin, 0.2% Triton X-100, 0.05% Tween 20) plus 10% goat serum for 1 to 2 h, followed by 20 blocking buffer [i.e., IF buffer containing 10% goat serum and 20 µg of goat anti-mouse F(ab')2/ml] for 40 min. Primary antibodies were diluted in 20 blocking buffer, followed by incubation overnight 4°C. Structures were washed three times in IF buffer for 15 min each. Anti-mouse or anti-rabbit secondary antibodies coupled with Alexa fluor dyes (Molecular Probes) were diluted in IF buffer containing 10% goat serum, followed by incubation for 60 min. After a wash with IF buffer as described above, structures were incubated with 5 µM Topro-3 Iodide (Molecular Probes) and 0.5 ng of DAPI (4',6'-diamidino-2-phenylindole; Sigma)/ml before being mounted with the antifade agent Prolong (Molecular Probes). Quantification of Bim staining in ErbB2 structures was performed at x40 magnification. Only fields including both multiacinar and normal structures were scored. The staining intensity for Bim in multiacinar structures was scored as less than, equal to, or greater than the normal structures visualized in the same field of view. A minimum of 100 multiacinar structures and 100 normal structures were counted per experiment, and each experiment was performed three independent times. Confocal analysis was performed by using the Nikon E800 Bio-Rad Laboratories confocal imaging system (Nikon Imaging Center at Harvard Medical School). Images were generated by using MetaMorph software, converted to Tiff format, and arranged by using Adobe Photoshop 7.0.
Histology. For Bim immunostaining, acinar structures were fixed and washed with PBS-glycine as described above. Prior to blocking, structures were incubated at 25°C for 15 min each time with 18 and 30% sucrose sequentially. Acini were then mixed with 250 µl of frozen section medium (Stephens Scientific). Samples were then frozen in a dry ice-methanol bath for 10 min and sectioned by using a cryostat. Next, 7 µM sections were prepared, placed on glass slides, and stored at –20°C until use. Immunofluorescence assays were then performed as described above beginning with the addition of in 10 IF blocking buffer. Because of the harsh treatment of cryosectioning, acinus structures were distorted in some sections.
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RESULTS |
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To determine whether Bim expression is required for luminal apoptosis during morphogenesis, we utilized RNA interference to inhibit expression of Bim. Transfection of cells with an siRNA that targeted Bim but not control oligonucleotides significantly reduced expression of Bim through day 15 (Fig. 3A). The expression of other BH3-only proteins such as Bid and Bad was not affected by the Bim siRNA transfection (data not shown). Luminal apoptosis was significantly reduced in cells transfected with the Bim siRNA oligonucleotide but not in control oligonucleotides, as measured by EtBr staining of acini. At day 8 and 10, only 15% of acini derived from cells transfected with Bim siRNA oligonucleotides contained centrally localized, EtBr-positive cells; in contrast, 50% of acini from control transfected cultures exhibited EtBr-positive cells in the luminal space (Fig. 3B). In corroboration with EtBr staining, we also found a dramatic decrease in staining for cleaved, activated caspse-3 in acini transfected with Bim siRNA oligonucleotides compared to control oligonucleotides (Fig. 3C). Day 10 structures transfected with Bim siRNA oligonucleotides contained 16% acini positive for caspase-3 staining compared to 48% acini transfected with control oligonucleotides (data not shown). This level of inhibition of luminal apoptosis was comparable to that seen with cells overexpressing Bcl-xL (Fig. 1B and C). We detected a similar reduction in luminal apoptosis by using an siRNA SMART pool (Dharmacon) targeting Bim (data not shown) and three vectors expressing short hairpin Bim-targeted sequences (T. Schmelzle, E. Lin, and J. S. Brugge, data not shown), supporting the Bim specificity of this siRNA. In addition to blocking apoptosis, Bim siRNA transfection delayed lumen formation since day 12 acini transfected with Bim siRNA oligonucleotides contained significantly greater number of acini with viable cells in the luminal space then control cells (62% filled acini [
2 cells in the lumen] in Bim siRNA-transfected cells compared to 38% in control acini) (Fig. 3D). The protection from luminal apoptosis in cells transfected with this Bim siRNA oligonucleotides was transient since we observed a complete loss of protection from cell death by day 15 (Fig. 3B). This reduced protection is likely due to loss of Bim siRNA oligonucleotides since the stable hairpin vectors provide constitutive protection from apoptosis. Taken together, these results implicate Bim in early luminal apoptosis and suggest that this proapoptotic protein contributes to luminal clearance.
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Studies in HEK293 cells indicated that phosphorylation of BimEL at this site reduces its apoptotic activity (23). To address whether loss of this phosphorylation site affects Bim's apoptotic activity in MCF-10A cells, we examined the induction of apoptosis in cells 48 h after infection with viruses encoding either wild-type Bim or BimSA. Indeed, we find that exogenous expression of the nonphosphorylatable BimEL-SA mutant induces a twofold increase in apoptosis compared to wild-type BimEL (as measured by DNA fragmentation cell death ELISA) (Fig. 4C) when expressed at equal levels in MCF-10A cells (data not shown). In corroboration, we also see increased caspase-8 cleavage in BimEL-SA-expressing cells compared to wild type (data not shown). These results support previous studies suggesting that Bim phosphorylation at S65 reduces its apoptotic activity. Based on these results, we hypothesize that outer cells in acini that are in direct contact with matrix may be protected from Bim's apoptotic function by reducing the apoptotic activity of Bim via phosphorylation of serine 69. However, reagents that allow us to prove S69 phosphorylation in situ are required to establish definitively whether S69 phosphorylation distinguishes Bim expressed in the inner and outer cells.
Oncogenic regulation of Bim during morphogenesis. We have previously shown that oncogenes differ in their ability to induce filling of luminal space. For example, whereas both activation of ErbB2 and expression of HPV E7 in MCF-10A cells allow escape from proliferative suppression and constitutive proliferation of acini, activation of the ErbB2 homodimers only leads to production of acini distinguished by a filled luminal space and a marked reduction in apoptosis (8) (Fig. 5C). In contrast, expression of the HPV E7 oncogene does not protect MCF-10A cells from apoptosis in the luminal space and therefore does not block lumen formation (Fig. 5A). We have subsequently found that activation of vSrc in preformed acini can also induce luminal filling. Stable MCF-10A cells expressing the vSrc oncogene fused to estrogen receptor was treated with OHT for 48 h to activate vSrc 8 days after plating cells in Matrigel. Induction of vSrc activation caused inhibition of luminal apoptosis as measured by EtBr staining (data not shown), staining with caspase-3, and filling of the luminal space (Fig. 5A). Treatment of normal acini with OHT during morphogenesis does not alter luminal apoptosis or filling (9), nor does it have any effect on Bim expression during morphogenesis (data not shown). Since Bim expression is critical for luminal apoptosis and lumen formation, we investigated whether the differential regulation of luminal apoptosis by distinct oncogenes is due, in part, to differences in their ability to regulate Bim during morphogenesis. Indeed, activation of v-Src during morphogenesis (day 7) reduced the level of Bim expression significantly (Fig. 5B). In contrast, cells expressing the HPV E7 oncogene did not inhibit Bim expression during morphogenesis (Fig. 5B).
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6, indicating that antibody accessibility is not altered in large ErbB2-induced structures (Fig. 5C, inset). Thus, two oncogenes, ErbB2 and vSrc, that inhibit luminal apoptosis and fill the luminal space block Bim expression, suggesting that Bim is an important target for oncogenes that are able to fill the luminal space. Acini expressing the HPV E7 oncogene contained elevated levels of Bim, and Bim was detected at earlier time points than control cells (Fig. 5B), suggesting that the failure of HPV E7-expressing cells to block luminal apoptosis may be due to its inability to inhibit Bim expression. To address this possibility, we used siRNA oligonucleotides to decrease Bim expression in HPV E7 cells. Cells expressing HPV E7 that were transfected with Bim siRNA oligonucleotides but not control oligonucleotides showed a marked reduction of apoptosis during early morphogenesis, as measured by EtBr staining (Fig. 5D). In addition, at a later stage of morphogenesis (day 12), HPV E7 cells transfected with Bim siRNA displayed a significant reduction in activated caspase-3 staining (Fig. 5D). Day 12 structures transfected with Bim siRNA contained a twofold reduction in the number of acini positive for caspase-3 staining (37% of acini) compared to cells transfected with control siRNA (82% acini) (data not shown). Thus, the failure of HPV E7 expressing cells to block Bim expression contributes to their inability to block luminal apoptosis and lumen formation.
Erk MAPK pathway regulates Bim expression and luminal filling during morphogenesis. ErbB2 can activate the phosphatidylinositol 3-OH kinase (PI 3-kinase), an activator of PKB/Akt kinase, and the Erk MAPK pathway (16), both of which are known pathways of cell survival. Bim expression can be regulated by either the Akt (11) or the Erk (41) pathway depending on the cell type. We and others have previously shown that in mammary epithelial cells the Erk pathway but not the Akt pathway can negatively regulate Bim levels during anoikis (26, 32). Indeed, we find that activation of ErbB2 in preformed acini leads to high levels of Erk activity compared to control acini (Fig. 6A). To test whether ErbB2 activation of Erk activity is involved in regulating Bim expression during morphogenesis, we examined the effects of blocking activation of Erk and PI 3-kinase. The ability of activated ErbB2 to inhibit Bim expression in large, filled acini was reversed when acini were treated for 48 h with an inhibitor of Mek (UO126) but not when acini were treated for 48 h with the PI 3-kinase inhibitor (LY294002) (Fig. 6B). UO126-treated cultures showed strong staining for Bim throughout the multi-acinar structures, and this inhibitor induced apoptosis in the ErbB2-expressing structures, as demonstrated by staining for activated caspase-3 (Fig. 6B). Similar findings were observed by using the Mek inhibitor PD98069 (data not shown). These results indicate that Bim expression is regulated by the Mek/Erk pathway in ErbB2-activated structures and that signals from Erk may be critical to prevent luminal apoptosis and lumen formation.
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DISCUSSION |
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These studies are the first to implicate a BH3-only protein in regulating luminal apoptosis and lumen formation of cyst-like epithelial structures. Although epithelial morphogenesis has not been extensively characterized in Bim-deficient mice, it is of interest that loss of Bcl-2 in mice leads to development and eventually death from polycystic kidney disease (29, 35), a disorder linked to alterations in epithelial tube size and morphogenesis (24). Intriguingly, the removal of a single bim allele in these bcl-2–/– mice was sufficient to eliminate polycystic kidney disease (3), suggesting that the interplay between proapototic and antiapoptotic Bcl-2 family members may regulate epithelial morphogenesis in vivo. In mammary epithelial cells, apoptosis has been detected in the presumptive luminal space of the terminal end bud (TEB) in vivo during early development of the mammary gland (19). Bcl-2 transgenic mice showed disruption of cellular organization of the TEB, including reduced levels of apoptosis in the TEBs.
Since we and others have recently shown that Bim expression is upregulated after detachment from matrix in multiple epithelial cells and contributes to anoikis in MCF-10A cells (26, 32) and that centrally localized acinar cells during morphogenesis are not in contact with basement membrane (8), we initially hypothesized that Bim might be induced specifically in the inner, matrix-deprived cells and thus contribute to the selective apoptosis in this population of cells. In contrast, we found that Bim is induced between days 6 and 8 in both outer and inner cells (Fig. 2B). Bim induction would sensitize the acinar cells to stress conditions, possibly those resulting from loss of matrix attachment and failure of the inner cells, to activate intracellular signaling proteins that protect cells from apoptosis. It is of interest that centrally localized cells are separated from matrix proteins and fail to activate Akt from day 5 onward during morphogenesis (8); however, cell death is not detected until the period when Bim is induced, suggesting that Bim induction may be a critical trigger for death of the inner cell population. The temporal regulation of apoptosis after polarization of the outer acinar cells in the MCF-10A model shares features of apoptosis associated with cavitation of embryoid bodies (EBs) during development (5, 6). Centrally localized cells in the ectodermal mass of the EBs undergo cell death after polarization of the outer, matrix-attached cells due to a signal transduced from surrounding endoderm cells when they undergo differentiation. Our results raise the question whether the centrally localized EB cells become sensitized to the absence of matrix attachment due to induction of a proapoptotic protein such as Bim. Studies of EBs lacking basement membrane due to loss of laminin-1 indicate that the EB cells are not dependent on matrix attachment for survival until they differentiate after induction by the endodermal cells (27). Together, these results suggest that cells may not undergo death when deprived of matrix until they develop into a matrix-dependent state, possibly by induction of proapoptotic proteins, like Bim, that sense matrix deprivation.
It is not clear how outer acinar cells are protected from Bim-induced cell death. We examined the possibility that Bim apoptotic function may be differentially regulated between matrix-attached cells and matrix-deprived inner cells. Recently, BimEL has been shown to be phosphorylated by the MAPK pathway, and this phosphorylation reduced BimEL apoptotic activity (22, 23). Indeed, we show that BimEL is phosphorylated at the major Erk phosphorylation site and that this phosphorylation is lost after loss of adhesion. A mutant form of BimEL that is unable to be phosphorylated at this site displayed higher apoptotic activity; thus, loss of matrix attachment may facilitate Bim's apoptotic function by preventing Erk phosphorylation of Bim. Extrapolating to the acinar cultures, we propose that outer-matrix attached acinar cells are protected from Bim's apoptotic effects during morphogenesis by maintaining Bim in a phosphorylated state, whereas inner cells, in the absence of matrix signals, become sensitized to Bim's highly apoptotic unphosphorylated form. Consistent with this model, we found that Bim was phosphorylated on serine 69 in acini derived from late-stage cultures that consisted exclusively of matrix-attached cells. Previous studies have indicated that phosphorylation of Bim at serine 69 targets Bim for proteosome-mediated degradation. Although this could be contributing to the protection from Bim expression, we consistently detect high levels of Bim under conditions in which there is detectable phosphorylation. Thus, it is possible that phosphorylation of Bim may also interfere with its apoptotic activity by other mechanisms. Further studies addressing the ability of pS69Bim to bind to pro- and antiapoptotic Bcl-2 family members will address this.
We have previously shown that the ability of oncogenes to induce luminal filling requires not only enhancement of proliferation but also inhibition of apoptosis (8). The data presented here suggest that oncogenes such as ErbB2 homodimers and v-Src may escape luminal apoptosis by inhibiting expression of Bim. The block of Bim expression, coupled with reinitiation of proliferation, may be sufficient to induce luminal filling in activated ErbB2 and v-Src structures. In contrast to ErbB2 expressing MCF-10A structures, acini expressing the oncogene HPV E7, which also induces uncontrolled proliferation but not protection from apoptosis, was unable to block Bim expression. Inhibition of Bim expression in HPV E7 acinar structures using Bim siRNA oligonucleotides reduced the level of apoptosis in these structures, suggesting that HPV E7's inability to block luminal apoptosis is due, in part, to its inability to inhibit Bim levels. This result is consistent with our previous finding that HPV E7 structures coexpressing Bcl-2 contained filled lumen and few apoptotic cells (8). The inability of HPV E7 to activate the Erk MAPK pathway (data not shown) is probably responsible for its failure to prevent Bim expression. These results suggest that Bim may function as a key molecule in keeping cells expressing proliferative oncogenes from progressing to more advanced tumor phenotypes, i.e., one in which the luminal space is infiltrated with tumor cells. Indeed, recent data suggest that Bim can also keep the proliferative oncogene c-Myc in check since inactivation of a single allele of bim accelerated Myc-induced tumor development in vivo (12). Thus, oncogenic inactivation of Bim, i.e., by ErbB2, may increase the probability of acquiring additional oncogenic mutations that may allow for complete disruption of epithelial acinus architecture and eventually lead to metastasis.
Bim expression has been found to be regulated at the transcriptional level by the Erk MAPK (32, 41) or the Akt kinase (10) pathway. We previously showed that activation of Erk, but not Akt, regulates Bim mRNA and protein expression in monolayer or detached cultures of MCF-10A cells (32). We present here several lines of evidence implicating the Erk pathway in regulation of Bim expression in 3D acinar structures: (i) ErbB2 homodimer induced inhibition of Bim expression in large filled acinar structures was reversed with treatment with Mek inhibitors and (ii) cells expressing an active form of Mek significantly inhibited Bim expression during morphogenesis. In contrast, the PI 3-kinase pathway is not involved in regulating Bim expression in acinar structures since Bim expression was not altered after inhibition of the PI 3-kinase pathway in ErbB2 activated structures. In addition, activation of Akt in MCF-10A acini does not cause decrease luminal apoptosis or inhibit lumen formation (9), a finding which is consistent with Akt not regulating Bim expression in MCF-10A cells. However, in ErbB2 expressing acini both the Erk pathway and the PI 3-kinase pathway contribute to cell survival as inhibitors to both pathways induces apoptosis (Fig. 6A), suggesting that ErbB2-mediated survival effects may involve multiple signaling pathways.
Although the Erk MAPK pathway has been implicated in branching morphogenesis during development of a number of epithelial tissues, including the mouse salivary gland (21) and developing mouse kidney (13), little is known about the role of the Erk MAPK pathway in normal mammary morphogenesis. Our studies suggest that Erk activation has profound effects on cell survival during epithelial morphogenesis in vitro and that oncogenes, such as ErbB2, that activate this pathway may provide dual oncogenic functions by enhancing proliferation and blocking apoptosis. Indeed, a high level of activated Erk has been found in breast tumors (33), and elevated Erk kinase activity has been associated with ErbB2/Neu expression (36) in breast tumors. In addition, Bissell and coworkers have shown that inhibition of the hyperactive Erk kinase pathway in 3D cultures of transformed human mammary epithelial cells induces conversion from disorganized, highly proliferative structures to ones that morphologically resemble normal acini (37, 38), thus supporting our data showing that expression of an overactive Erk kinase pathway can significantly distort acinar architecture.
The data described here provide important new insights into the molecular mechanisms underlying the generation of luminal space in acinus-like structures via regulation of the proapoptotic protein Bim. Furthermore, these data also identify one pathway whereby oncogenes may induce filling of the luminal space during early stages of tumorigenesis in vivo, i.e., by blocking Bim expression via the Mek/Erk pathway. ErbB2 is overexpressed in 46 to 80% of primary ductal carcinoma in situ lesions in the breast (42). It will be of interest to determine whether the expression of Bim or other BH3-only Bcl-2 family members is altered in these early tumor lesions and, if so, whether this correlates with activation of the Erk MAPK pathway.
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ACKNOWLEDGMENTS |
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This study was supported by grants from NIH-NCI, Aventis Pharmaceuticals, and American Cancer Society (to J.S.B.); by a Susan Komen Breast Cancer Postdoctoral Fellowship (to M.J.R.); and by an NSF predoctoral fellowship (to K.R.M.).
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FOOTNOTES |
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M.J.R. and K.R.M. contributed equally to this study.
Present address: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102.
Present address: Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 11274.
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