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Address correspondence to Mina J. Bissell, Life Science Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., Berkeley, CA 94720. Tel.: (510) 486-4368. Fax: (510) 486-5586. email: mjbissell{at}lbl.gov
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Abstract |
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Key Words: three-dimensional cultures; Akt; Rac1; tumor reversion; tissue polarity
F. Wang's present address is Department of Cellular and Molecular Pharmacology and the Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143.
Abbreviations used in this paper: 2D, two dimensional; 3D, three dimensional; EGFR, epidermal growth factor receptor; GSK 3ß, glycogen synthase kinase-3ß; lrBM, laminin-rich basement membrane; Myr-Akt, myristoylated Akt (constitutively active Akt); PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate.
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Introduction |
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During tumor progression, tissue polarity is lost and control of proliferation is compromised (Fish and Molitoris, 1994; Reichmann, 1994; Bissell and Radisky, 2001), and although these two phenomena have been suggested to be linked, previous investigations have not revealed the extent to which the increased cellular proliferation in tumors can directly produce tissue disorganization, and to what extent loss of polarity is an independent function of deregulated signaling pathways downstream of the oncogenic signal(s). To dissect the molecular mediators of these processes we have used an assay (Petersen et al., 1992) in which human mammary epithelial cells from the HMT-3522 tumor progression series are cultured in a physiologically relevant, three-dimensional (3D) laminin-rich basement membrane (lrBM). When cultured in 3D lrBM, the phenotypically normal, nonmalignant HMT-3522 S-1 (S-1) cells undergo growth arrest, produce an endogenous basement membrane, and form polarized acinus-like structures, very similar to primary cells from reduction mammoplasty. In contrast, the malignant HMT-3522 T4-2 (T4-2) cells continue to proliferate into apolar, amorphous structures, similar to structures formed by primary tumor cells in this assay (Petersen et al., 1992). In comparison to S-1 cells, expression levels of EGFR and ß1 integrin in T4-2 cells are greatly increased, and down-regulation of these signaling pathways in T4-2 cells grown in 3D lrBM can restore the formation of polarized acinus-like structures, resulting in a reversion similar to the normal phenotype of the S-1 cells (Weaver et al., 1997; Wang et al., 1998).
As PI3K is activated downstream of both EGFR and ß1 integrin (Chen and Guan, 1994; Lee and Juliano, 2000; Grant et al., 2002), we hypothesized that the phenotypic reversion affected by down-modulation of EGFR/ß1 integrin signaling in T4-2 cells was due to attenuation of PI3K activity. We showed previously that even highly malignant metastatic cancer cells, cultured in 3D lrBM, could be reverted to a normal phenotype by inhibition of PI3K, if treatment with PI3K inhibitors was performed in combination with appropriate manipulation of other signaling pathways (Wang et al., 2002a). Here we use inhibition of PI3K alone to dissect the signaling pathways that control proliferation and polarity in breast tumor cells. Our results reveal a new functional link between extracellular signaling mediators and tissue function that provides insight into processes that control the malignant phenotype if imbalanced. We also show that the PI3K and its lipid product, PIP3, are relocalized to the basal surface of the acini when the malignant cells are reverted in lrBM, a process that may play a role in integration of signaling pathways in reformation of polarity.
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Results |
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Phenotypic reversion of T4-2 cells by treatment with LY294002 results in cross-modulation of multiple signaling pathways
We had shown previously that inhibition of either EGFR or ß1-integrin results in phenotypic reversion of T4-2 cells associated with down-modulation of the total levels of both signaling molecules, and that this activity influences and is influenced by the MAPK signaling pathway (Wang et al., 1998). We now show that PI3K signaling is also an integral component of this cross-modulated signaling network. T4-2 cells treated with LY294002 show reduced levels of EGFR and ß1 integrin (Fig. 3 A). This effect depended upon 3D lrBM as it is not observed in cells cultured on two-dimensional (2D) plastic substrata (it should be noted that inhibition of PI3K activity, as measured by activation of downstream mediators Akt and GSK-3ß, was equally effective in cells on 2D or in 3D; Fig. 3 A). In addition, our results revealed that PTEN, the antagonist of PI3K that acts to dephosphorylate PIP3 and which becomes down-regulated in many carcinomas (Simpson and Parsons, 2001; Yamada and Araki, 2001), is also a component of the cross-modulated signaling network, as treatment of T4-2 cells with LY294002 resulted in an increase of PTEN to the level of the nonmalignant cells; this modulation, too, was seen only in cells cultured on 3D lrBM (Fig. 3 B). Taken together, these results demonstrate the existence of a retrodirectional control network that exists only when cells are cultured in a proper tissue context.
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Discussion |
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Normalization of signaling pathways in T4-2 cells in response to inhibition of PI3K is dependent upon culture in 3D lrBM, as T4-2 cells grown on 2D tissue culture plastic do not show the dramatic downmodulation of ß1 integrin and EGFR (Fig. 3 A), up-regulation of PTEN (Fig. 3 B), or the alterations in cellular morphology in response to treatment with inhibitors of PI3K (Figs. 2 and 3). Also, for T4-2 cells grown in 3D lrBM, the reduction in PI3K signaling is paralleled by a reorganization of signaling orientation, as both PI3K and its phospholipid product, PIP3, became repolarized to the basolateral surface of the reorganized T4-2 cell structures (Fig. 2). This basolateral distribution of PI3K and PIP3 might indeed reflect the localization of active cell surface receptors, e.g., integrins and receptor tyrosine kinases, many of which have particular functions when localized to the basal or basolateral surfaces (Playford et al., 1996; Weaver et al., 1997; Vermeer et al., 2003). Basolateral polarization of PIP3 has been suggested to be a critical determinant of differentiated tissue behavior in polarized MDCK cells grown as monolayers on filters (Watton and Downward, 1999) or as cysts in 3D collagen gels (Yu et al., 2003), and PIP3 becomes apolarly distributed in the plasma membrane during branching morphogenesis (Yu et al., 2003), a process believed to involve the transitory dedifferentiation to a migratory and invasive state that is highly reminiscent of the malignant phenotype. PI3K signaling polarization is also an essential component of chemotactic migration in neutrophils and Dictyostelium (Servant et al., 2000; Funamoto et al., 2002; Wang et al., 2002b), and the directionality of neuronal axon growth is controlled by spatially localized PI3K activity (Shi et al., 2003). Our observations in mammary epithelial cells do not reveal the extent to which the polarized distribution of PI3K and PIP3 causes, or is the consequence of, tissue polarity, but previous observations with these cells in 3D lrBM and with MDCK cysts have suggested that formation of cellcell contacts is an essential component of ECM-induced cell polarity (Weaver et al., 1997, 2002; Yeaman et al., 1999). If so, then formation of tight junctions at points of cellcell contact may provide boundaries for localization of PI3K and other signaling effectors such as integrins and growth factors that then provide the polarizing principle. These possibilities are under investigation.
We have found that the 3D presentation of lrBM is essential for coupling the expression levels and activity of EGFR and ß1 integrin in cultured mammary epithelial cells (Wang et al., 1998), and evidence in other systems also implicates reorganization of signaling pathways in cells cultured in 3D lrBM (Cukierman et al., 2001, 2002; Muthuswamy et al., 2001). We now show that components of the PI3K signaling pathway are involved in this cross-modulation process, as phenotypic reversion by inhibition of PI3K is associated with, and presumably, supported by, up-regulation of the PI3K antagonist, PTEN (Fig. 3). This also requires the establishment of organized structures in 3D lrBM, as treatment of T4-2 cells with PI3K inhibitors does not result in up-regulation of PTEN when cells are grown on 2D plastic substrata (Fig. 3 B). Given that the signaling reorganization associated with reversion of T4-2 cells is associated with global repolarization of signaling molecules, we suggest that directional orientation of signaling is an essential component of the cross-modulation process. In this regard, it is tempting to speculate that the 3D lrBMdirected basal localization of PI3K and PIP3 may explain why the cells in the outer layer of acini are more resistant to apoptosis than those not in contact with 3D lrBM (Debnath et al., 2002).
High expression of PI3K is commonly found in cancers and cancer cell lines (Vivanco and Sawyers, 2002; Wang et al., 2002a), and there is considerable evidence that the activity of this enzyme is a key component of the tumorigenic process. Cowden syndrome (an autosomal-dominant cancer predisposition syndrome caused by inherited mutations in PTEN) causes elevated risk of breast, thyroid, and skin tumors (Liaw et al., 1997); mice made heterozygous for expression of PTEN develop cancers at multiple sites (Di Cristofano et al., 1998), and transgenic mice deficient for PTEN expression in the mammary gland developed tumors at early stage (Li et al., 2002). Recent experiments using immortalized human mammary epithelial cells has shown that early passage cells require transfection of additional oncogenes along with PI3K (or Rac1/Akt) to become malignant, whereas late passage cells (which presumably accumulate more alterations) can be transformed with only PI3K (or Rac1/Akt) (Zhao et al., 2003); these results are also consistent with our model in which additional abnormalities must exist in addition to PI3K activation in order for HMT3522 mammary epithelial cells to become tumorigenic. Overexpression of constitutively active Akt in T lymphocytes, pancreatic cells, and the mammary gland increases cellular proliferation and promotes survival but does not induce cellular transformation or increase tumor incidence (Vivanco and Sawyers, 2002), suggesting that additional factors must be required. Rac1 isoforms are overexpressed in cancers of the breast and other organs, and increased activity of Rac1 or Rac3 has been found in breast carcinoma cell lines and Ras-transformed breast epithelial cells (Mira et al., 2000; Sahai and Marshall, 2002). Here, we have unified the roles of elevated Rac1 and Akt activities in a simple mechanistic framework: we have found that the high levels of PI3K in T4-2 cells contribute to the loss of polarity and increased proliferation through Akt and Rac1, and we find that these pathways and phenomena are functionally separable. We find that PI3K-Akt signaling is responsible for an appreciable increase in cell proliferation (Fig. 4), whereas the PI3K-Rac1 signaling is responsible for the loss of basal tissue polarity (Fig. 5), and that expression of both can completely prevent reversion by LY294002. These results show that overactive PI3K signaling activates these two effectors for separate but collaborative regulation of the distinct cellular behaviors of tumor tissues (Fig. 7). Significantly, our results also reveal that increased cell proliferation (in the absence of a polarity-disrupting signal) is not sufficient to result in loss of tissue organization (Fig. 4 E), a finding that may explain why Akt overexpression by itself is not sufficient to increase tumor incidence as well.
In conclusion, we have used the 3D lrBM assay to determine the role of PI3K signaling in the tumorigenic phenotype, signaling reorganization, and tissue polarity of mammary epithelial cells. Our discovery of asymmetric distribution of PI3K and PIP3 in these polarized acinus-like structures strongly implies that they might act as spatial determinants to regulate mammary epithelial polarity. Our elucidation of the events downstream of PI3K sheds light on the process by which increased proliferation and loss of tissue polarity act collaboratively to produce the malignant phenotype.
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Materials and methods |
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DNA constructs and gene transfection
A construct containing Rac1 L61HA (gift of Tung C. Chan, Thomas Jefferson University, Philadephia, PA) was digested with EcoRI and BamHI and cloned into the pLXSNNeo retroviral construct (CLONTECH Laboratories, Inc.). The myr-Akt pWZL retroviral construct myr4129 (Kohn et al., 1998) was provided by Richard Roth (Stanford University, Stanford, CA). Transfection of Phoenix packaging cells (gift of Garry P. Nolan, Stanford University) and production of retroviral stock were according to standard protocols. The HMT-3522 mammary epithelial cells were infected at 4050% confluence. Myr-Akt and Rac1 L61-double transfectants were produced by sequentially infecting cells with each construct. The stably expressing cells were selected in the presence of neomycin (500 µg/ml) or hygromycin B (50 µg/ml) and surviving clones were pooled.
Immunoblotting, immunoprecipitation, and indirect immunofluorescence
Immunoblotting and indirect immunofluorescence were performed as described previously (Weaver et al., 1997; Wang et al., 1998). For immunoprecipitation, cells were lysed in IP buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris/HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 2 mM NaF, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml E 64, and 1 mM Pefabloc) and centrifuged at 16,000 g at 4°C. Equal amounts of protein lysates were precleared by 50 µl of protein G plusconjugated agarose beads (Santa Cruz Biotechnology, Inc.) before the addition of 1 µg of primary antibody. Samples were incubated at 4°C with gentle rotation for 1 h. Subsequently, samples were incubated with 30 µl of protein G plusconjugated beads for 1 h at 4°C. The beads were washed three times with IP buffer before being heated with sample buffer at 95°C for 5 min and analyzed by SDS-PAGE and Western blotting.
For Rac1 activity assay, the cells from 10-d culture in 3D BM were lysed in GST-Fish buffer (10% glycerol, 50 mM Tris, pH 7.4, 100 mM NaCl, 1% NP-40, 2 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml E 64, and 1 mM Pefabloc) and centrifuged at 16,000 g at 4°C. Equal amounts of protein supernatants were incubated with recombinant GST-PAK-CD fusion protein (containing the Rac and Cdc42 binding region from human PAK1; Sander et al., 1998), bound to glutathione-coupled Sepharose beads (Amersham Biosciences) at 4°C for 30 min. The beads were washed with an excess of lysis buffer, eluted in sample buffer, and then analyzed by SDS-PAGE and Western blotting using antibody against Rac1.
For PIP3 immunofluorescence, the isolated colonies were fixed with 3.7% formaldehyde, washed with CSK buffer (10 mM Hepes, 138 mM KCl, 3 mM MgCl2, 1 mM EDTA), permeabilized by 0.1% Triton X-100 in CSK buffer, and blocked with 3% skim milk in blocking buffer (50 mM Tris/HCl, pH 7.5, 1 mM CaCl2). Primary antibody diluted 1:100 in blocking buffer was incubated with samples for 1 h in room temperature followed by FITC-conjugated secondary antibody.
Nuclei were counterstained with DAPI (Sigma-Aldrich). Control sections were stained with secondary antibodies only. The slides were sealed with Vectashield (Vector Laboratories). The images were collected with Zeiss 410 LSM confocal microscope (Zeiss Pluar 40x oil objective lenses; Carl Zeiss MicroImaging, Inc.) or RT SLIDER SPOT digital camera (SPOT RT v3.2 software; Diagnostic Instruments) attached to Zeiss Photomicroscope III (Zeiss Plan-Neofluar 40x oil objective lenses; Carl Zeiss MicroImaging, Inc.). Images for figures were colored and resized with Adobe Photoshop 7.0 software.
Anchorage-independent growth assays
For soft agar assay, 5,000 cells were plated in 1 ml of DME/F12 containing 0.3% agarose, overlaid with 1 ml of 1% agarose, and then exposed to treatment as indicated. Cultures were maintained for 15 d. Colonies from duplicated wells were measured and scored positive when the colony sizes exceeded a diameter of 50 µm.
For methyl cellulose anchorageindependent growth assay, 100,000 cells were seeded per 60-mm dishes in 5 ml of DME/F12 containing 1.5% methyl cellulose (Fisher Scientific) with inhibitor or vehicle only. Colonies were scored after 3 wk.
Online supplemental material
Supplemental figures show the following results; treatment of T4-2 cells with PI3K inhibitor LY294002 at 8-µM concentration when grown in 3D lrBM for 10 d does not affect cell viability or lead to increased cell death (Fig. S1 A); the same treatment decreases cell proliferation of T4-2 cells when cultured on 2D plastic (Fig. S1 B); expression of constitutively active Rac1 V12 in T4-2 cells transduced by adenovirus inhibits LY294002-induced repolarization of basal marker 6 integrin in 3D lrBM (Fig. S1 C); treatment of T4-2 cells with PI3K inhibitor does not change its expression levels (Fig. S2 A); overexpression of constitutively active Akt in T4-2 cells greatly increases anchorage-independent growth of T4-2 cells in soft agar (Fig. S2 B); and PIP3 staining intensity is significantly reduced when treating T4-2 cells with PI3K inhibitor (Fig. S2 C). Figs. S1 and S2 are available at http://www.jcb.org/cgi/content/full/jcb.200306090/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health (NIH; grant CA64786-08), the Department of Energy (OBER DEAC0376SF00098), and by an Innovator Award from the Department of Defense (DAMD17-02-1-0438) to M.J. Bissell. H. Liu is the recipient of a predoctoral fellowship from Breast Cancer Research Program of the Department of Defense. D.C. Radisky is the recipient of a postdoctoral fellowship from NIH and the American Cancer Society.
Submitted: 18 June 2003
Accepted: 5 January 2004
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