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Molecular Cancer Research 1:776-787 (2003)
© 2003 American Association for Cancer Research

Signaling and Regulation

Immortalized Mammary Epithelial Cells Overexpressing Protein Kinase C {gamma} Acquire a Malignant Phenotype and Become Tumorigenic in Vivo1

Esteban Mazzoni1,2, Alejandro Adam1, Elisa Bal de Kier Joffe1 and Julio A. Aguirre-Ghiso2,3

1 Department of Cell Biology, Research Area, Institute of Oncology "Angel H. Roffo," University of Buenos Aires, Argentina, and
2 Rochelle Belfer Chemotherapy Foundation Laboratory, Division of Hematology/Oncology, Department of Medicine, Mount Sinai School of Medicine, New York University, New York, NY

Requests for reprints: Julio A. Aguirre-Ghiso, Division of Hematology/Oncology, Department of Medicine, Mount Sinai School of Medicine, New York University (Box 1178), One Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-3194; Fax: (212) 996-5787. E-mail: julio.aguirre-ghiso{at}mssm.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
We have investigated the role of a classical isoform of protein kinase C (PKC{gamma}) in promoting immortalized mammary cell tumorigenesis in vivo and the contribution of proteases and adhesion molecules to this process. We hypothesized that overexpression of PKC{gamma} in immortalized mammary epithelial cells may initiate, by activating the mitogenic ERK pathway, early changes in proteases, adhesion molecules, and markers of an epithelium-to-mesenchyme transition that may contribute to in vivo tumorigenesis. Here we show that compared to vector-transfected cells, immortalized murine mammary epithelial cells (NMuMG) overexpressing PKC{gamma} have stronger activation of (~5-fold) ERK1/2 MAPKs, which results in a similar increase in cyclin D1. In addition, PKC{gamma}-expressing cells showed increased levels of vimentin, fibronectin (FN), ß1-integrins, enhanced adhesion to fibronectin, and its organization into fibrils. Concomitantly, PKC{gamma} induced a dramatic down-regulation of E-cadherin protein levels and its localization to cell-cell junctions. NMuMG cells expressing PKC{gamma} became resistant to death by anoikis and formed colonies in soft agar. This effect was dependent on ERK activation, because Mek1/2 inhibition with PD98059 abrogated anchorage-independent growth. Most importantly, unlike control NMuMG cells, PKC{gamma}-transfected cells inoculated s.c. into nude mice displayed tumorigenic and invasive capacity and were able to spontaneously metastasize. This behavior correlated with increased production of uPA and MMPs-9/-2 induced by PKC{gamma}. These results suggest that PKC{gamma} overexpression in immortalized mammary epithelial cells may generate, through an increase in ERK, signaling changes in the expression of genes associated with an epithelium-to-mesenchyme transition that may be sufficient to favor tumor growth in vivo.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
During its lifetime, the mammary gland undergoes cycles of growth and involution, requiring remodeling of the extracellular matrix (ECM) and proper tissue organization (1). Since the late 1970s, it has been known that the serine proteases uPA and plasmin (2) and more recently the family of metalloproteinases (3) are involved in the remodeling of the tissue during branching and involution of the mammary gland. In addition, it has been shown that a proper organization of the basal lamina and signaling through integrins in mammary gland cells is required to support normal tissue organization and function (4).

Growth factor receptor tyrosine kinases (RTKs) induce cell proliferation by activating, among other pathways, diacylglycerol (DAG) production and Ca2+ release through phospholipase C- and phospholipase D-dependent pathways, which in turn lead to the activation of different isoforms of protein kinase C (PKC; 5). There are 10 reported isoforms of PKC classified in classical, novel, and atypical according to their dependence on Ca2+ and DAG, DAG alone, or independence of these factors, respectively (5). In addition, the classical and novel isoforms of PKC are the best-characterized receptors for phorbol esters, which are well-known tumor promoters. PKC{delta} has been found elevated in mammary tumors (6) and forced overexpression of PKC{varepsilon} in the epidermis of transgenic mice leads to development of squamous cell carcinomas (7). PKC{alpha} overexpression correlates with the lack of estrogen receptor expression in mammary tumor cells, an indicator of poor prognosis (8, 9). In addition, PKC {alpha}, {delta}, {varepsilon}, and {zeta} isoforms are elevated during mammary gland involution (10), coincident with an increase in protease levels (2). PKC{gamma} expression was originally found in brain and adrenal glands (11). However, its distribution is not limited to those tissues and it was found in heart (12) and human pulmonary fibroblasts and epithelial cells (13, 14). When overexpressed, PKC{gamma} can transform NIH3T3 fibroblasts (15). Moreover, increased levels of PKC{gamma} and PKC{alpha} were found in ER-negative breast cancer cell lines (16). In this study, overexpression of PKC{alpha} in MCF7 cells caused an epithelium-to-mesenchyme transition (EMT), which was accompanied by PKC{gamma} overexpression (16), suggesting a functional role for this isoform in the EMT. The possibility that PKCs may be inducing the increase in proteases is supported by the fact that several PKC isoforms ({alpha}, ßII, {gamma}, {delta}, {varepsilon}, {zeta}, and {eta}) positively regulate both MMPs and uPA expression in fibroblasts and mammary tumor cells (for review, see Ref. 17). Moreover, it is well documented that increased protease activity correlates functionally with more aggressive mammary cancers (18). Thus, it is possible that the aberrantly activated PKC pathways facilitate the acquisition of a malignant invasive phenotype during tumor progression.

Targeting of Neu/ErbB2 and SV40 to the mammary gland of mice using transgenic technology induces benign lesions and, in some cases, malignant carcinomas (19). In addition, many of these oncogenes regulate the expression of both uPA and MMPs in vitro (17), although the role of these enzymes in the oncogene-targeted transgenic models is unclear. Proper tissue organization is lost during tumor progression due to absent E-cadherin-based cell-cell junctions and to alteration in tumor cell-ECM interactions through integrin receptors (20, 21). Integrin signaling has been shown to be required for the growth of different types of epithelial tumors and binding to FN matrix may be a prerequisite to initiate mitogenesis in culture or in vivo (20, 22). Thus, altered protease activity and matrix organization may be coincident or simultaneous events that contribute to mammary cancer progression.

Classical PKC isoforms can induce proteases overexpression (17) and mediate integrin signaling (23). Morse-Gaudio et al. (16) showed a correlation between increased expression of PKC{gamma} and expression of markers of mammary tumor malignancy on transformation with PKC{alpha}. However, whether PKC{gamma} overexpression has a functional role in the acquisition of malignant markers and properties in mammary epithelial cells has not been explored. We hypothesized that overexpression of PKC{gamma} in immortalized murine mammary gland cells (NMuMG) might induce changes in mitogenic signaling and tissue remodeling that may correlate with a malignant behavior. Here, we tested using a genetic approach whether PKC{gamma} overexpression in NMuMG cells activates Mek-ERKMAPK signaling, cyclin D1 expression, and the expression and/or function of adhesion molecules like ß1-integrin and fibronectin and proteases like uPA and MMPs. We further tested if these changes correlate with transformation, mammary tumor growth, local invasion, and metastasis development in vivo.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Normal Mammary Epithelial Cells Overexpressing PKC{gamma} Display Altered Morphology and Growth Properties in Culture
We set out to explore the functional link between overexpression of the classical DAG and Ca2+-dependent PKC{gamma} isoform, and acquisition of malignant properties in NMuMG-immortalized mammary epithelial cells. NMuMG cells do not express PKC{gamma}, allowing to study PKC{gamma}-induced signaling without interference of an endogenous counterpart. In contrast to PKC{gamma}, other isoforms of PKC (novel PKC{delta} and atypical PKC{zeta}) were not found to transform NMuMG cells as measured by agar colony formation assays.4 A viral vector encoding PKC{gamma} under the control of Moloney retrovirus LTR promoter or the same vector encoding only the G418 resistance gene was used to transduce NMuMG cells. G418-resistant colonies were pooled and two cell lines were obtained, one expressing PKC{gamma} (MG-PKC) and another only the empty vector (MG-Neo). Western blots from whole cell lysates showed expression of a band of Mr 80 in MG-PKC but not in MG-Neo cells (Fig. 1A ). Compared to MG-Neo cells, MG-PKC cells acquired a more fibroblast-like morphology and a larger size (data not shown).



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  		FIGURE 1. PKC{gamma} overexpression induces ERK activation and cyclin D1 expression in NMuMG cells. A. Expression of PKC{gamma} detected by Western blot in cell lysates of NMuMG cells transduced either with an empty vector or a viral vector encoding PKC{gamma}. The arrow indicates the MW corresponding to PKC{gamma}  or actin as indicated. B. ERK activation by PKC{gamma}. Whole lysates from MG-Neo or MG-PKC cells, incubated in the presence or absence of 10% fetal bovine serum (FBS) or 100 nM phorbol 12-myristate 13-acetate (PMA), were analyzed by Western blot using antibodies to phospho-ERK (top) and, after stripping off the membrane, to total ERK1/2 (bottom). The arrows indicate the position of phosphorylated and total ERK1/2. C. PKC{gamma} activation of ERK requires MEK signaling. Subconfluent MG-PKC monolayers were incubated in the absence of serum for 4 h. Cells were then treated with 50 µM PD98059 added to the culture medium. After the indicated times, cells were lysed and blotted as indicated in B. This experiment was repeated twice with identical results. D. Inhibition of PKC inhibits ERK activation. MG-Neo or MG-PKC cells were serum starved for 4 h, treated with or without PKC inhibitor calphostin C (200 nM) for the indicated times, lysed, and blotted as in B. Results shown are representative of two independent experiments. E. Cyclin D1 induction by PKC{gamma}. Serum-starved MG-Neo or MG-PKC cells cultured in the presence or absence of 200 nM calphostin C overnight were lysed and analyzed for cyclin D1 expression. Stripping and reblotting the membrane for actin expression served as loading control. In A, B, and E, figures are representative of at least three independent experiments.

 
We next tested whether the ERK pathway was activated, by analyzing the levels of phosphorylated (active) and total p44/p42 ERK1/2, respectively, by Western blot. We found that, compared to MG-Neo cells, PKC{gamma}-expressing cells had a ~4-fold increase in the basal level of active ERK, as determined by normalizing the phosphorylated ERK to total ERK signals which served as a loading control (Fig. 1B). This result suggests that PKC{gamma} signal is feeding into the Raf-Mek-ERK pathway constitutively. A brief treatment with 10% FBS or 100 nM PMA caused a ~7-fold increase in phosphorylated-ERK in MG-Neo cells but it caused a much lower (1.7- to 2.2-fold) activation of ERK over basal levels in MG-PKC cells, suggesting that PKC{gamma} expression causes close-to-maximal activation of the ERK pathway in these cells (Fig. 1B). Moreover, the response to PMA indicates that the PKC{gamma} appears to be responsive to ligand activation. Treatment with 10% FBS containing media or overexpression of PKC{gamma} did not induce a change in total ERK levels (Fig. 1, B and D). Transient transfection of NMuMG cells with a murine PKC{gamma} construct yielded a similar increase in ERK phosphorylation as that observed in cells stably expressing PKC{gamma} (data not shown). ERK phosphorylation in MG-PKC cells was sensitive to PD98059, suggesting that PKC-dependent activation of ERK was dependent on Raf-Mek activation (Fig. 1C). These results are in agreement with reports showing that activation of classical and novel PKC isoforms can lead to increased Raf-1 (24) and Mek-ERK signaling (23). PKC activation was responsible for ERK activation because calphostin C treatment of MG-PKC cells caused a reproducible ~40% decrease in ERK phosphorylation (Fig. 1D). However, this effect was reversible because the effect of calphostin C disappeared after 6 h (not shown). The PKC{gamma}-activated ERK signaling was transduced further downstream of the MAPK module as evidenced by a ~3-fold increase in cyclin D1 protein level in PKC{gamma} cells compared to MG-Neo control cells (Fig. 1E). The cyclin D1 increase was dependent on PKC{gamma} and ERK activation because calphostin C inhibited this increase (Fig. 1E). These data suggest that PKC{gamma}-mediated ERK activation is sufficient to activate both early gene response and the expression of cell cycle progression proteins as evidenced by an increase in the cyclin D1.

In studying growth properties in monolayers, we found that MG-PKC cells displayed a higher cell saturation density than control ones (Table 1). When grown at high confluence, only MG-PKC cells were able to grow beyond the limits of the monolayer and form overlays of cell aggregates, a behavior that correlates with loss of contact inhibition (Fig. 2A ). Although we expected an increased in vitro proliferation rate in PKC{gamma}-expressing cells with high ERK and cyclin D1 levels, these cells showed only a slight reduction in their population doubling times (Table 1), suggesting that the increase in basal ERK signaling does not provide an advantage for bidimensional growth.


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  		Table 1. Growth Characteristics of MG-Neo and MG-PKC Cells in Culture

Note: Results are representative of at least three independent experiments done in triplicate.

 


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  		FIGURE 2. PKC{gamma} expression induces anchorage-independent growth of NMuMG cells. A. Growth characteristics of MG-PKC and MG-Neo cells as monolayers. At high confluence, only MG-PKC cells were able to grow in multiple layers (arrows) while MG-Neo showed growth contact inhibition. Phase-contrast microscopy (x200). B. Colony formation in soft agar. Monocellular suspensions of both cell lines were embedded in 0.3% agar prepared in DMEM + insulin + 10% FBS, as described in "Materials and Methods." After 15–20 days, colonies with more than 20 cells were counted under an inverted microscope. For PKC or Mek inhibition, 20 µM H7 or 50 µM PD98059 were added every 3 days to culture medium. Data are representative of four independent assays (n = 8, P < 0.01). C. Growth in suspension (liquid medium). Monocellular suspensions of both cell lines were seeded in bacteriological petri dishes in growth medium. At 72 h, the number of cell aggregates (spheroids) per field was recorded. Experiment representative of four independent assays (10 fields/dish, n = 5, P < 0.01).

 
Effect of PKC{gamma} Expression on Anchorage-Independent Growth in Soft Agar and Liquid Medium
Although MG-PKC cells proliferated at relatively the same rate as MG-Neo cells in bidimensional culture conditions, we hypothesized that the stronger ERK activation in MG-PKC cells may be advantageous under more stringent growth conditions like growth in soft agar or liquid medium.

While MG-Neo cells formed very few colonies in agar, PKC{gamma} expression induced a 7- to 11-fold increase in colony formation (Fig. 2B). The ability of PKC{gamma}-expressing cells to grow in soft agar was reversed by the use of the Mek1/2 inhibitor PD98059 (50 µM) or the PKC inhibitor H7 (20 µM), indicating that active signaling through PKC and Raf-Mek-ERK pathway is required for growth in these conditions (Fig. 2B). When MG-Neo cells or MG-PKC cells were plated in bacterial culture plates, which do not support cell attachment, only MG-PKC cells survived and grew after a week. MG-Neo cells showed some attachment and spreading on bacterial plates 24–48 h after seeding (data not shown). However, a high number of dead cells was observed after 72 h and massive cell death occurred after a week (data not shown). In contrast, within the initial 48 h, MG-PKC cells organized in spheroids of 20–30 cells, which survived and grew over a period of 30 days (Fig. 2C and data not shown). Overall, these results suggest that MG-PKC, but not MG-Neo cells, are able to survive and proliferate under two different growth conditions that require anchorage independence.

PKC{gamma} Modulation of Fibronectin, ß1-Integrin, Vimentin, and E-Cadherin Expression and F-Actin Organization
It has been proposed that FN fibrillogenesis may provide a means to resist cell death by anoikis within agar colonies, providing a substratum for cell anchorage (25). Thus, we tested whether FN production and fibrillogenesis may associate with growth advantage in soft agar and liquid medium of MG-PKC cells. FN and ß1-integrin expression was tested by Western blot from whole cell lysates. We found that MG-PKC cells expressed ~2.5-fold more FN and ~4.5-fold more ß1-integrin compared to MG-Neo cells as determined by Western blot (Figs. 3A and 4A ). We next tested the adhesive capacity of MG-Neo or MG-PKC cells to FN, as a functional measure of integrin affinity/avidity. After 45 min, MG-PKC cells displayed ~3.1-fold increase in adhesion to immobilized FN (2 µg/well) compared to MG-Neo cells (Fig. 3B), indicating that, in addition to higher FN expression, the increase in ß1-integrin levels in MG-PKC cells may provide increased avidity for FN. Although higher ß1-integrin levels may explain the increased adhesion of MG-PKC cells to FN, a higher number of surface receptors does not directly imply that these integrins are in a high-affinity active conformation. Because one of the properties of active integrins is their ability to form FN-fibrils (26), we tested whether PKC{gamma} expression modulated this ability in NMuMG cells. MG-Neo and MG-PKC cells were allowed to grow for 72 h in 10% FBS-containing medium and, after fixation, they were stained with a monoclonal antibody specific for cellular-derived FN. Although some intracellular and intercellular FN staining was detected in MG-Neo cells, only MG-PKC cells were able to organize a rich ECM of FN fibrils which bridged the cells in a connecting network (Fig. 3C).



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  		FIGURE 3. PKC{gamma} expression in NMuMG cells modulates the expression and organization of fibronectin. A. Detection of FN expression by Western blot in whole cell lysates from MG-Neo and MG-PKC cells. PKC{gamma}-expressing cells had ~2.5-fold more FN than MG-Neo cells (experiment repeated three times). B. Determination of cell adhesion to immobilized FN after 45 min of incubation. MG-PKC cells displayed ~3.1-fold increase in adhesion to immobilized FN compared to MG-Neo (P < 0.01). Experiment representative of at least three independent assays. C. Immunohistochemical detection of extracellular FN. Exponentially growing MG-Neo or MG-PKC cells were stained for cellular-derived FN using a monoclonal antibody. Note that only some intracellular and extracellular staining for FN was found in MG-Neo cells, while MG-PKC cells were able to organize a rich extracellular matrix of FN fibrils which bridged the cells in a connecting network (arrows). Nuclei were counterstained with hematoxylin (top panel x400, bottom panel x1000).

 


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  		FIGURE 4. PKC{gamma} signaling induces an EMT. A. Detection of ß1-integrin and E-cadherin and PKC{gamma} expression by Western blot of whole cell lysates from MG-Neo and MG-PKC cells. B. Effect of PKC{gamma} expression on the organization of the actin cytoskeleton, ß1-integrin, and E-cadherin distribution and vimentin expression. Subconfluent monolayers were fixed and stained for F-actin using FITC-phalloidin (F-actin) or antibodies anti-ß1-integrin, E-cadherin or vimentin and TRITC- or HRP-conjugated secondary antibodies. MG-Neo cells mainly showed cortical F-actin, with short peripheral fibers and very few stress fibers (arrows). In contrast, MG-PKC cells exhibited large actin stress fibers and F-actin also organized in bundles that localized at cell protrusions. Note the elongated fibroblast-like morphology of MG-PKC cells accompanied by the long F-actin stress fibers (x1000). While MG-Neo cells presented only scarce, ß1-integrin-positive adhesion contacts that were distributed along the cell periphery, MG-PKC cells had a greatly enhanced number and size of these structures, which resemble large point contacts and were also predominantly observed with a basal distribution at the cell periphery (arrows) (x1000). Cells grown on coverslips were assayed for E-cadherin and vimentin expression by immunostaining. Note the almost complete absence of E-cadherin at the cell membrane or intracellular staining in PKC{gamma}-expressing cells and the significant cytoplasmic staining for vimentin in MG-PKC but not in MG-Neo cells. Nuclei were counterstained with Hoechst 33342 (fluorescence) or hematoxylin (cytochemistry; x400). Photographs are representative fields of three independent experiments.

 
To better understand the relationship between PKC{gamma} expression and FN fibrillogenesis, we investigated the F-actin cytoskeleton organization and ß1-integrin distribution. MG-Neo cells stained with FITC-phalloidin showed strong staining for F-actin that distributed mainly as cortical structures. Very few of these cells displayed stress fibers (Fig. 4B). In contrast, MG-PKC cells showed a dramatic change in cytoskeleton organization, with F-actin primarily organized in large stress fibers spanning the whole cell body and following the change in shape of these cells from cubical to fusiform morphology, characteristic of an EMT.

Integrins provide the link between FN matrix and the F-actin cytoskeleton and, because MG-PKC cells express higher levels of ß1-integrin, we tested whether their distribution was modulated by PKC{gamma} expression. MG-Neo cells showed occasionally a light staining for ß1 integrin at adhesion sites. In contrast, MG-PKC cells showed ß1-integrins organized into large adhesion contact sites in the basal plane of the cells (Fig. 4B). These did not resemble characteristic focal contacts because they did not display the typical arrowhead morphology, but rather larger point contacts, previously described in mammary tumor and neuronal cells (27, 28).

To determine whether PKC{gamma} overexpression induced other markers of an EMT, MG-Neo and MG-PKC cells were stained with an anti-vimentin antibody. As shown in Fig. 4B, unlike MG-Neo cells, MG-PKC cells displayed expression of vimentin. In support of the hypothesis that PKC{gamma} may be inducing an EMT, we found that MG-PKC cells had a dramatic down-regulation of E-cadherin protein as detected by Western blot and a reduction in E-cadherin-based cell-cell contacts, as determined by fluorescence microscopy (Fig. 4B). These results suggest that PKC{gamma} overexpression in NMuMG cells induces characteristic phenotypic changes associated with an EMT.

Production of uPA and MMPs by PKC{gamma}-Expressing Cells
We next tested whether PKC{gamma} overexpression induced a change in protease production by NMuMG cells. While MG-Neo cells secreted very low but measurable levels of plasminogen activator activity, MG-PKC cells produced ~6-fold more plasminogen activator activity as determined by radial caseinolysis (Fig. 5A ). This activity could be inhibited by 5-mM amiloride (a uPA catalytic inhibitor) and was not evident in plasminogen-free substrates (data not shown). In addition, zymographies for conditioned media (CM) from MG-Neo or MG-PKC cells revealed a single Mr ~48 caseinolytic activity band that was stronger in MG-PKC cells and corresponded to uPA molecular weight (data not shown). To assess the production of MMPs by MG-Neo or MG-PKC cells, CM of both cell lines were analyzed by gelatin zymography to detect secreted MMP activities. We found that, while MG-Neo cells secreted barely detectable levels of gelatinolytic activity at Mr 105 and 72 (corresponding to MMP-9 and MMP-2, respectively), MG-PKC cells displayed a strong increase in the secreted activity levels of these two MMPs (Fig. 5B). No evidence of gelatinolytic activity was detected in gels incubated in parallel in EDTA-containing incubation buffers, confirming that the observed activities corresponded to metalloproteinases (data not shown).



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  		FIGURE 5. Protease production is increased by PKC{gamma} expression. A. uPA activity in the conditioned media from MG-PKC and MG-Neo cells was quantified by radial caseinolysis. While MG-Neo cells secreted measurable but very low levels of uPA activity, MG-PKC cells secreted ~6-fold more enzyme activity (P < 0.01). B. Zymogram showing the MMP activities secreted to the conditioned media by MG-PKC and MG-Neo cells. While MG-Neo cells produced barely detectable levels of Mr 105 MMP-9 and Mr 72 MMP-2, MG-PKC cells displayed a strong increase in the levels of these two MMPs. Results shown in A and B are representative of at least four independent experiments.

 
NMuMG Cells Expressing PKC{gamma} Become Tumorigenic and Metastatic in Vivo
To test whether PKC{gamma} induced a tumorigenic phenotype in NMuMG cells, MG-Neo or MG-PKC cells (4 x 106/animal) were injected s.c. into the flank of athymic nude mice. In two independent experiments, after a latency period of ~45 days, all MG-PKC-injected animals (18 of 18) developed angiogenic, highly invasive undifferentiated carcinomas (Fig. 6, A and B ) that reached an average volume of ~340 mm3 2 weeks later and continued growing for at least 30 more days. These tumors were able to invade the underlying dermis and the s.c. muscle layer, which correlates with the increased protease activity displayed by MG-PKC cells. Tumor sections as well as MG-PKC cells in vitro showed a strong positive staining for cytokeratins (Fig. 6C), confirming their epithelial origin. In addition, MG-PKC tumors presented numerous mitosis and only scarce apoptotic images (data not shown). Moreover, we found metastatic nodules in lungs and/or lymph nodes in 8 of 10 MG-PKC cell-injected mice in which a complete necropsy was done. The cells in metastatic nodules largely resembled the cells in the original tumor (Fig. 6, D and E). In contrast, after a latency of 75 days, only 2 of 12 mice inoculated with MG-Neo cells developed small tumors. These results indicate that increased PKC{gamma} signaling in immortalized mammary gland cells is able to induce changes in the ERKMAPK signaling and in the expression of proteases and adhesion molecules that may favor the formation of highly undifferentiated, invasive, and metastatic carcinomas in vivo.



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  		FIGURE 6. In vivo tumorigenicity of MG-PKC cells in nude mice. MG-Neo (empty squares) or MG-PKC (empty diamonds) cells (4 x 106/animal) were injected s.c. into the flank of athymic nude mice. A. Growth curve of tumors measured with a caliper as indicated in "Materials and Methods." B. MG-PKC tumors were constituted by a mass of undifferentiated cells that invaded the dermis and the s.c. muscle layer, as shown by Masson trichromic staining (x100). C. The epithelial lineage was confirmed by immunohistochemical staining for cytokeratin (arrows) (x400). D. H&E staining of a lung (L) showing a metastatic (M) nodule (x400). E. Low-power magnification of a H&E staining of a lymph node (LN) invaded by metastatic (M) MG-PKC cells.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In the present work, we have searched for a functional link between overexpression of PKC{gamma} and the expression and function of molecules associated with an EMT and acquisition of a malignant phenotype in immortalized murine mammary cells. Although a correlation was established between high levels of PKC{gamma} and expression of markers of malignancy in breast cancer cell lines (16), a causal role for PKC{gamma} in these processes was unknown. Here we show that immortalized NMuMG cells overexpressing PKC{gamma} acquire malignant properties, which include changes in the expression of typical markers of an EMT observed in mammary epithelial tumors.

PKC{gamma} enhanced Mek-ERKMAPK signaling, which correlated with the induction of cyclin D1. Both ERK activation and cyclin D1 induction appear to partially depend on PKC activity because an inhibitor of PKC reduces, although not completely, these signals. It is possible that these and other still unknown changes promote NMuMG cells, which are spontaneously immortalized (29) to transform and acquire malignant behavior after PKC{gamma} transfection. PKC functions mainly as a tumor promoter and several of the classical isoforms including PKC{alpha} show this function in epithelial cells (5, 30), while others like ßII isoform show tumor suppressor or proapoptotic activity in colon cancer (30). Our results show that deregulated signaling through PKC{gamma} may induce, like PKC{alpha} classical isoform, the acquisition of malignant properties in this murine model. It remains to be determined if PKC{gamma} is overexpressed hyperactivated or mutated in human mammary tumors and if its transforming capacity in murine mammary cells is due to overlap of function with other PKC classical isoforms or if it is specific for this isoform.

We found that PKC{gamma} expression allows anchorage-independent growth and this effect was inhibited by PD98059 treatment, suggesting that increased mitogenic signaling through ERK may be a direct contributor to their proliferative phenotype. The mechanisms by which PKC{gamma} activates ERK and favors anchorage-independent growth are unclear. It is possible that PKC{gamma}-induced ERK activation is dependent on Shc phosphorylation induced by PKC (23) or on direct phosphorylation of Raf-1 by PKC (24) or as recently reported for the atypical isoform PKC{zeta}, through direct interaction with Mek, which results in ERK activation independently of Raf-1 (31). The ability to induce anchorage independence and protease production may depend on the regulation of downstream transcription factors (32). For example, v-Jun association with Fos promotes anchorage independence and uPA and MMPs production (32) and these transcription factors can be activated by classical and novel PKCs (33). It is possible, that PKC{gamma}-activated signaling may induce Jun-Fos association and activation of an anchorage-independent growth program. It is uncertain if Mek-ERK signaling is indispensable for PKC{gamma}-induced transformation and other pathways may be activated or inhibited by PKC{gamma} signaling to achieve a tumorigenic phenotype. We are currently exploring the role of signaling through p38 and Akt in PKC{gamma}-induced NMuMG transformation.

The changes observed in adhesion molecules' function and expression as well as the changes in F-actin organization and vimentin and E-cadherin expression deserve further discussion. MG-PKC cells showed increased ß1-integrin expression and these integrins seem to be in a high-affinity/avidity conformation, because adhesion of these cells to FN was enhanced and only PKC{gamma}-expressing cells were able to organize FN into fibrils. The mechanism responsible for ß1-integrins' activation in MG-PKC cells is still unclear. However, the increased numbers of stress fibers and ß1-receptors in these cells may increase the frequency of integrin clustering, favoring avidity and increasing integrin affinity (34). It is also possible that increased responsiveness of PKC{gamma}-expressing cells to the initial interaction between ß1-integrins and FN may cause F-actin cytoskeleton reorganization into stress fibers promoting integrin clustering, enhanced integrin affinity, and FN fibrillogenesis. Interestingly, NMuMG cells expressing PKC{gamma}, high ß1 integrin, and showing increased adhesion to FN and matrix organization were able to grow in an anchorage-independent manner. Many transformation studies, using mainly murine fibroblasts, have shown that fibronectin is down-regulated in transformed cells, inversely correlating with transformation and tumorigenesis (35, 36). However, in support of our findings, it was shown that the survival of murine mammary epithelial cells in soft agar was due to the formation of a rich ECM of FN fibrils within the colonies, in cooperation with growth factor signaling (25). We have not studied FN organization within the colonies of MG-PKC cells; however, their ability to form FN fibrils and to form organized spheroid structures when grown in liquid medium suggests that the mechanism described previously may be operational in MG-PKC cells. Moreover, FN and ß1-integrin can generate mitogenic signals for epithelial tumors in culture and in vivo by activating through integrins the Mek-ERK pathway (20, 22) and FN can also activate through integrins antiapoptotic pathways like PI3K-Akt and Bcl2, promoting survival of different cells types (37, 38). FN fibrils may offer an additional advantage to normal and transformed cells because in fibroblasts and endothelial and carcinoma cells, FN-fibrils can be promitogenic by antagonizing the activation of the growth arresting/proapoptotic pathway p38SAPK (39, 40). The changes observed on PKC{gamma} overexpression suggest that this isoform of PKC may promote malignant conversion of NMuMG cells by inducing an EMT. This is in agreement with previous reports in which TGF-ß may induce EMT in NMuMG cells through a pathway that depends on ß1-integrin activity (41, 42). In agreement with these findings, PKC{gamma} overexpression caused a reduction in E-cadherin protein levels and in E-cadherin-based cell-cell contacts and a strong increase in vimentin expression. These results, taken together, suggest that PKC{gamma} signaling modulates gene expression favoring an EMT conversion.

Another important finding is that MG-PKC cells display enhanced protease (uPA/MMPs) activity. uPA and MMPs overproduction is frequently observed in mammary tumor cells (17) and it is known that these proteases are critical for metastasis formation (43, 44). PKC-induced overproduction of uPA and MMPs has been mainly studied in fibroblasts (17). However, it has been shown that deregulated expression of MMP-3 and MT1-MMP in the mouse mammary epithelium can lead to tumor formation (45, 46). We found that PKC{gamma}-expressing cells can change their ECM organization by inducing FN fibrillogenesis and at the same time increase tissue remodeling by increased proteases production. These results suggest that ECM organization and proteolytic remodeling exist in a dynamic balance. Because cells need to attach to survive and also to invade, it is possible that fine-tuned degradation and reorganization of the ECM provide these conditions in vivo. In support of our findings, highly aggressive human carcinoma cells with high proteolytic activity organize a FN-matrix to stimulate in vivo growth (39). In addition, MG-PKC cells showed not only invasion of the muscle and subepidermal layers, but also were capable to reach and metastasize lungs and lymph nodes, which correlates with increased proteolytic activity and survival capacity of these cells. It is possible that the increased proteolytic activity in the tumor microenvironment may release ECM-bound growth factors (47), favoring tumor proliferation and dissemination in vivo. In summary, we have established a correlation between PKC{gamma} overexpression and the acquisition of properties associated with a malignant phenotype that appears to depend on the induction of an EMT in NMuMG cells.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Reagents and Antibodies
Media for cell culture and agarose were from Life Technologies, Inc. (Rockville, MD). FBS was from GEN (Buenos Aires, Argentina). Plasminogen was purchased from Chromogenix (Molndal, Sweden). Human urokinase was a gift from Serono (Buenos Aires, Argentina). Triton X-100 was obtained from J.T. Baker (Phillipsburg, NJ). Acrylamide is from Sigma Chemical Co. (St. Louis, MO). All other reagents for PAGE were obtained from Bio-Rad (Richmond, CA). Antibodies: Monoclonal antibodies anti-rat PKC{gamma} (clone PK-64), anti ß1-integrin (clone W1B10), anti-vimentin, and mouse anti-cellular fibronectin (IgM isotype, clone FN-3E2) were obtained from Sigma. Polyclonal antibodies goat anti-human fibronectin used in WB were purchased from Pharmacia (Uppsala, Sweden). Monoclonal anti-cytokeratins antibodies (IgG1, clones AE1 and AE3) were from DAKO (Carpinteria, CA). Monoclonal antibodies anti-E-cadherin, anti-phospho-ERK, and anti-cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal anti actin was kindly provided by Dr. E. Farías (New York, USA). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies, Hybond-P membranes for blotting, and chemiluminescence reagents (ECL) were from Amersham (Aylesbury, United Kingdom). Phalloidin-FITC conjugated was from Sigma. H7 and PD96059 were from Sigma. Calphostin C was kindly provided by Dr. Omar Pignataro (Buenos Aires, Argentina). These drugs were diluted in DMSO. No toxicity was observed at the concentrations tested.

Cell Lines, Retroviral Vectors, and Viral Transduction
NMuMG cell line, a normal mammary gland cell line derived from NAMRU mice (29), was maintained in DMEM supplemented with 10% FCS, 260 mU/ml insulin, and 80 µg/ml gentamycin under humidified 5% CO2/95% air at 37°C; these cells, when inoculated into isogenic mice, are able to develop benign cystoadenomas. {psi}2 packaging cells transfected with pZipNeo or pZipNeoPKC{gamma} were kindly provided by O. Finn (Pittsburgh, PA). Packaging cell lines were maintained in DMEM supplemented with 10% FCS, and 80 µg/ml gentamycin under 5% CO2/95% air at 37°C. The development of the PKC{gamma} retroviral vector pZipNeoPKC{gamma} was described previously (48, 49). The plasmids pZipNeo and pZipNeoPKC{gamma} were then transfected into the ecotropic viral packaging cell line {psi}2 and virus-producing cells were established.

For retroviral infection, exponentially growing NMuMG cells were infected with 104–106 G418 CFU. In brief, 4 x 104 NMuMG cells were seeded on 100-mm plastic Petri dishes. After 24 h, cells were infected with 7 ml of retroviral supernatants for 2 h with 8 µg/ml polybrene at 37°C. Subsequently, the monolayer was washed three times with PBS and placed in their usual growth medium. After 48 h, cells were selected by adding 200–400 µg/ml G418 (Life Technologies). After selection, all resistant colonies were pooled to avoid clonal variations. Two cell lines were obtained, MG-Neo, control cell line transfected only with the antibiotic resistance gene, and MG-PKC, a pooled population of PKC{gamma}-expressing cells. Both cell lines grew without evidence of toxicity in levels of G418 between 0.4 and 1 mg/ml. All cell lines were found to be free of Mycoplasma.

Preparation of CM
Secreted uPA and MMP activities were investigated in CM. Briefly, semiconfluent MG-neo or MG-PKC cells were washed three times in PBS to eliminate serum traces. Serum-free medium was added and incubation was continued for 24 h. CM were individually harvested, the remaining monolayers were trypsinized and counted or, after scraping into lysis buffer (1% Triton X-100 in PBS), cell protein content was determined (Bio-Rad Protein Assay). CM samples were centrifuged (600 x g), aliquoted, and stored at -40°C. They were used only once after thawing.

Zymography for MMP Detection and Quantification
Collagenolytic activity was determined on substrate-impregnated gels as previously described (50). Briefly, samples were concentrated four to five times using Centricon-30 (Amicon Inc., Beverly, MA) and separated on gelatin-copolymerized (1 mg/ml, DIFCO, Detroit, MI), SDS-7% polyacrylamide gels under non-reducing conditions, followed by two washes in 2.5% Triton X-100. The gels were then incubated for 30 h at 37°C in 0.25 M Tris, 1 M NaCl, and 25 mM CaCl2 at pH 7.4. After incubation, gels were stained with 0.5% Coomassie G 250 (Bio-Rad) in methanol/acetic acid/H2O (30:10:60). The lytic area of the bands on the stained gels was determined on a digital densitometer GS-700 (Bio-Rad).

Radial Caseinolysis and Zymography for uPA Detection and Quantification
To assay uPA activity, a radial caseinolysis was used as previously described (50). Briefly, 4-mm wells were punched in the plasminogen-rich casein-agarose gels and 10 µl of CM were seeded. Plates were incubated for 24 h at 37°C in a humidified atmosphere. The diameter of lytic zones was measured and the areas of degradation were referenced to a standard curve of purified urokinase. To confirm the identity of the catalytic activity, zymographies for uPA identification were also performed as described previously (51).

Cell Proliferation Assay
Cells (25 x 103) were seeded into 24-well plates in DMEM supplemented with 10% FCS, 260 mU/ml insulin, and 80 µg/ml gentamycin and incubated overnight at 5% CO2/95% air. Cells from duplicate wells were then trypsinized and counted to establish a baseline (day 0). The remaining cells were grown and counted every day using a hemocytometer and trypan blue exclusion.

Colony Formation in Soft Agar
Single-cell suspensions (1 x 105 cells/ml) were plated in 24-well plates in 0.3% agar prepared in DMEM-10% FCS plus 260 mU/ml insulin in the presence or absence of 20 µM H7 or 50 µM PD98059 (added every 3 days), over an underlay of 0.6% agar medium and incubated under standard culture conditions. Cultures were fixed by adding 3.7% formaldehyde in PBS and colonies with more than 20 cells were counted using an inverted microscope at 100x objective lens magnification.

Culture on Bacteriological Plates
Single-cell suspensions (1 x 106 cells/dish) were seeded on 10-mm sterile plastic bacteriological untreated dishes. Cells were grown in 10 ml of growth medium under standard culture conditions as described above. Cell aggregates were counted 48 h later, using an inverted microscope at 100x objective lens magnification. Cultures were followed up to 30 days post-seeding.

Adhesion Assays
Wells of a 96-well microtiter plate were coated with 2 µg fibronectin per well, or 0.5% BSA as control, for 2 h at room temperature and then washed with PBS. Non-specific binding sites were blocked by a 30-min incubation with 0.5% BSA in PBS. Single cell suspensions (2 x 104 cells/well) were seeded in each well. After 45 min, medium was removed and adherent cells were stained with 0.5% crystal violet in 20% methanol. The amount of adherent cells was measured by plate scanning and analysis with Molecular Analysis software (Bio-Rad).

Western Blot
Semiconfluent monolayers were washed three times with ice-cold PBS and then lysed with RIPA buffer [150 mM NaCl, 1% Triton X-100, 0.05% deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.8)]. The samples were denatured by boiling in Laemmli sample buffer with 5% ß-mercaptoethanol and separated by SDS-PAGE, performed as described by Laemmli. Gels were electroblotted to a Hybond-P membrane, blocked with 5% skim milk plus 0.1% Tween 20 in PBS for 1 h and incubated with specific antibodies (anti-pan Erk at 1:5000, anti E-cadherin at 1:2000, anti pErk, anti-rat PKC{gamma} and anti-human FN at 1:1000, anti-cyclin D1 at 1:500, and anti-ß1-integrin at 1:250 dilution in blocking buffer) overnight at 4°C. Secondary antibodies were used diluted 1:1000 for 1 h. After that, detection was performed using ECL reagents. Bands were quantified by scanning on a digital GS-700 densitometer and Molecular Analyst software (Bio-Rad).

Immunocytochemistry and Immunofluorescence
For immunocytochemistry, cells were seeded on glass coverslips. After 48 h, cells were washed twice with ice-cold PBS and fixed with acetone at -20°C for 5 min. Tumor samples were fixed in buffered formaldehyde for 24 h and included in paraffin. Deparaffined cuts mounted on glass slides, or coverslips with cells, were then microwaved to recover antigenicity and non-specific binding was blocked for 20 min with 0.3% BSA in PBS. Samples were then incubated with the commercial primary antibodies (anti-vimentin, anti-cellular FN, or anti-cytokeratin, 1:100–1:200) followed by the incubation with the corresponding biotinylated secondary antibody. Samples were then washed and further incubated with a streptavidin-HRP conjugate (Vector, Burlingame, CA) and revealed with 7% DAB and 3% H2O2 in PBS. Finally, they were counterstained with Harris hematoxylin. Samples were analyzed using a light microscope and photographs were taken using Kodak 100 film.

For immunofluorescence assays, subconfluent monolayers grown in coverslips were fixed with 3.5% formaldehyde, permeabilized with 0.25% Triton X-100 in PBS, and blocked for 1 h with 5% skim milk, 0.1% Tween 20 in PBS. Coverslips were incubated with anti-ß1-integrin or anti-E-cadherin (1:200) overnight at 4°C and anti-mouse IgG-TRITC for 1 h at room temperature. For actin visualization, cells were incubated with 1:500 phalloidin-FITC without blocking. All coverslips were mounted in glycerin-PBS 1:1 and analyzed with a Nikon epifluorescence microscope. Photographs were taken with a Nikon digital camera.

In Vivo Experiments
In two independent experiments, a total of 12 and 18 athymic nude mice was given injections of monocellular suspensions (4 x 106 cells) of MG-Neo or MG-PKC cells, respectively. The animals were observed twice a week. Latency was defined as the period between injection and the detection of a palpable nodule. The average diameter of tumors was established with the aid of a caliper and tumor volume was calculated. All animals were sacrificed between 50 and 80 days post-inoculum and tumors were fixed for histopathological studies. A complete necropsy was performed in 10 MG-PKC-injected and in four MG-Neo-injected mice.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. O. Finn (Pittsburgh, PA) for retroviral constructs, Dr. E. Farías (New York, NY) for anti-actin antibody, and Dr. Omar Pignataro (Buenos Aires, Argentina) for calphostin C.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 University of Buenos Aires, the National Council for Scientific and Technological Research (CONICET), Ramón Carrillo-Arturo Oñativia Fellowship from Ministry of Health and the National Agency for Science and Technology Promotion (ANPCYT), from Argentina (to E.B.K.J.) and by the Charles H. Revson Foundation (J.A.A.G). Note: E.B.K.J. is a member of the CONICET, Argentina. Back

2 Present address: Department of Biology, School of Biological Sciences, New York University, New York, NY 10021. Back

3 Present address: Department of Biomedical Sciences, School of Public Health and Center for Functional Genomics, University at Albany, State University of New York, One University Place Rensselaer, NY 12144-2345. Back

4 Urtreger and Bal de Kier Joffe, unpublished results. Back

Received December 16, 2002; revised June 17, 2003; accepted June 23, 2003.


   References
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 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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