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PNAS 104 (37): 14694-14699

Copyright © 2007 by the National Academy of Sciences.

Suppression of oncogenic properties of c-Myc by LKB1-controlled epithelial organization

Johanna I. Partanen, Anni I. Nieminen, Tomi P. Mäkelä, and Juha Klefstrom*

Cancer Cell Circuitry Laboratory, Institute of Biomedicine/Biochemistry and Genome-Scale Biology Program, Biomedicum Helsinki, University of Helsinki, Room B231a, P.O Box 63, Haartmaninkatu 8, 00014, Helsinki, Finland


Figure 1
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Fig. 1.. Activation of c-Myc at the early stage of acinar morphogenesis results in abnormal acini. (A) (Upper) Phase-contrast micrographs of MCF10A MycERtm acini grown in the presence of 4-OHT or carrier control for the indicated time periods. MCF10A MycERtm or Myc{Delta}ERtm cells were embedded in Matrigel and 4-OHT or carrier control treatment was started on day 1. (Lower) Shown is growth of the acinar size, plotted as mean diameters of the acini at different time points (mean ± SEM). (B) (Upper) Shown are day-20 acini immunostained with Ki-67 antibody to detect cell proliferation. (Lower) Shown are percentages of acini at each time point exhibiting ≥1 Ki67-positive cells. (C) (Upper) Shown are day-20 acini immunostained with an antibody recognizing active caspase-3 to detect apoptosis. (Lower) Percentages of acini containing ≥1 active caspase-3-positive cells are shown. (D) (Top) Day-20 acini immunostained with {alpha}6-integrin antibodies to display acinar morphology and cell polarity. (Middle) The image shows that both the polarized, basal {alpha}6-integrin-expressing ECM facing cells and the inner cells show proliferative activity (Ki-67). (Bottom) The number of ECM facing outer cells, localizing at the rim of acini is shown. Values represent mean ± SEM of three independent experiments, where at least 90 acini per experiment were counted. (Scale bars: 100 µm, A; 20 µm, B–D).

 

Figure 2
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Fig. 2.. c-Myc fails to reinitiate the cell cycle or disrupt architecture in mature MCF10A acinar structures. (A) Phase-contrast micrographs of MCF10A MycERtm acini, which were first allowed to form for 20 days in Matrigel and subsequently treated with 4-OHT for 5 days (20 + 5). (B) Quantitation of Ki-67 positivity. Mature (day 20) MCF10A MycERtm acini were treated with 4-OHT for 3 or 10 days, and Ki-67 positivity was scored in the average-sized acini and the minor fractions of small and large acini. (C) Representative images of Ki-67-immunostained MCF10A MycERtm acini, which were allowed to mature for 20 days and treated with 4-OHT for 5 days. Control shows typical day-10 acinus with Ki-67-positive cells. (D) c-Myc does not disrupt architecture of preformed acinar structures. Representative images of MCF10A MycERtm acini, which were first allowed to form for 20 days and subsequently treated with 4-OHT for 5 days, are shown. (E) MCF10A acini become c-Myc-resistant during morphogenesis. MCF10A MycERtm and Myc{Delta}ERtm cells were embedded in Matrigel on day 0, after which the treatment of developing structures with 4-OHT or carrier control was started on days 1, 5, 10, or 15 (marked as 1+19, 5+15, 10+10, and 15+5, respectively). All samples were fixed and immunostained for Ki-67 on day 20. Values represent mean ± SEM of three independent experiments, where at least 90 acini per experiment were counted. (Scale bars: 100 µm, A; 20 µm, C and D.)

 

Figure 3
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Fig. 3.. c-Myc reinitiates cell cycle in mammary epithelial structures formed in collagen I matrix. (A) Representative images of day-20 MCF10A structures formed either in Matrigel or collagen I matrix. (B) Representative images of MCF10A MycERtm or Myc{Delta}ERtm cells grown in collagen I gel for 20 days and thereafter treated with 4-OHT or carrier control for 10 days. (C) Quantitation of Ki-67 positivity. Day-20 structures were treated with 4-OHT for 3, 5, or 10 days. Structures containing ≥1 Ki-67-positive cells were scored as positive. Values represent mean ± SEM of three independent experiments, where at least 90 structures were counted per experiment. (Scale bars: 20 µm, A and B.)

 

Figure 4
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Fig. 4.. Disruption of architecture by loss of LKB1 allows c-Myc to reinitiate the cell cycle in acinar structures. (A) (Upper) Western blot analysis demonstrating lentiviral shRNA-mediated silencing of LKB1 in MCF10A cells. (Lower) Representative images showing morphology of control and LKB1-deficient acini. (B) The growth of the acinar size is plotted as mean diameters of the acini at different time points (mean ± SEM). Experiments were performed as in Fig. 1. (C) Representative images of day-15 control and LKB1-deficient MCF10A acini grown in the absence of 4-OHT and stained with either beta-catenin, {alpha}6-integrin, or Golgi protein GM130 to visualize architecture and cell polarity. (D) Representative images and Ki-67 quantitation of control and LKB1-deficient MCF10A MycERtm acini grown in Matrigel for 20 days and thereafter treated with 4-OHT for 1, 3, 5, or 10 days. (E) mTOR is required for acinar overgrowth but is dispensable for c-Myc-induced reinitiation of the cell cycle in LKB1-deficient MCF10A acini. The acini were allowed to form in the presence of 20 mM rapamycin, a highly specific mTOR inhibitor. (Upper) Acinar diameters on day 23 are shown. (Lower) c-Myc was activated on day 20, and the acini were cultured for 3 days in the presence of active c-Myc and rapamycin. Shown is the quantitation of Ki-67 positivity in the structures. Values represent mean ± SEM of three independent experiments, where at least 90 structures were counted per experiment. (Magnification: x10, A Lower; scale bars: 20 µm, C and D.)

 

Figure 5
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Fig. 5.. Organized epithelial architecture regulates the apoptotic c-Myc function. (A) (Left) Representative images of MCF10A MycERtm acini immunostained with anti-active caspase-3 antibody. Day-20 MCF10A MycERtm acini were treated with 4-OHT for 24 h, and, subsequently, the acini were treated for 96 h with 0 or 100 ng/ml of TRAIL. TRAIL and 10 µg/ml cycloheximide (CHX) was used as a positive control. (Right) Quantitation of apoptosis. Acini containing >3 active caspase-3-positive cells were scored positive in the assay. (B) Caspase-8, Bid, and Bim mediate the acinar apoptosis induced by c-Myc and TRAIL. Shown is the quantitation of apoptosis in the MCF10A MycERtm acini expressing shRNA constructs silencing either caspase-8, Bid, or Bim. The day-20 acini were treated with 100 ng/ml TRAIL, and the resulting apoptosis was scored as in A. (C) Apoptotic sensitivity of the immature unorganized acini. The day-5 MCF10A MycERtm acini were first treated 24 h with 4-OHT and then either 24 h (Left) or 96 h (Right) with 10 ng/ml TRAIL. The acini containing ≥1 active caspase-3-positive cells were scored as positive. (D) Apoptotic sensitivity of the disorganized Lkb1-deficient acini. (Left) Representative images of the day-20 MCF10A MycERtm shLkb1 acini treated with 4-OHT for 24 h and thereafter for 96 h with 10 ng/ml of TRAIL. (Right) Quantitation of apoptosis. Acini containing >3 active caspase-3-positive cells were scored positive. Values represent mean ± SEM of three independent experiments, where at least 90 acini were counted per experiment. (Scale bars: 20 µm, A and D.)

 


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