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Figure 1. Attenuation of PI3K activity results in phenotypic reversion of HMT-3522 T4-2 human mammary tumor cells cultured in 3D BM. (A) Phase contrast micrographs of 10-d 3D lrBM cultures of phenotypically normal (S-1), malignant (T4-2), and T4-2 cells treated with 8 µM PI3K inhibitor, LY294002 (T4-2+LY). Bar, 20 µm. (B) Cell lysates from 10-d 3D lrBM cultures were analyzed for phosphorylated Akt (serine 473)/total, phosphorylated GSK-3ß (serine 9)/total, and phosphorylated p70 S6 kinase (threonine 389)/total by Western blot. (C) Inhibition of PI3K causes a reduction in cellular proliferation (left, BrdU labeling assay, n = 3), colony size (center, 50 colonies assessed for each experiment, n = 3), and anchorage-independent growth (right, soft agar assay, colonies scored positive when >50 µm, n = 3).
- We found that treatment of T4-2 cells with 8 µM of the PI3K inhibitor LY294002 (which prevented phosphorylation of downstream Akt at serine 473, as well as other downstream signaling mediators glycogen synthase kinase-3ß [GSK-3ß] and p70S6K; Fig. 1 B) did cause phenotypic reversion, as characterized by inhibition of proliferation, decreased colony size, and reduced growth in soft agar cultures (Fig. 1, A and C).
- We find that down-modulation of PI3K activity in the T4-2 cells grown in 3D lrBM causes structural repolarization and reversion to a nonmalignant phenotype (Fig. 1) similar to the effects observed previously from inhibition of EGFR and ß1 integrin (Weaver et al., 1997; Wang et al., 1998), and we additionally show that both the normal and the normalized reverted acinus-like structures had basal polarization of PI3K and its lipid signaling product, PIP3 (Fig. 2 B).
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Figure 2. Inhibition of PI3K results in reestablishment of tissue polarity. (A) Down-modulation of PI3K activity of T4-2 cells was sufficient to repolarize the apicolateral tight junction protein ZO-1 and the basal ECM receptor, {{alpha}}6 integrin, and to result in the reorganization of the actin cytoskeleton. (B) PI3K and its phospholipid product, PIP3, are basolaterally localized in S-1 acini, apolarly distributed in the T4-2 colonies, and normalized in the reverted T4-2 structures. For both A and B, S-1, T4-2, and T4-2+ LY (revertants) were cultured for 10 d in 3D lrBM. {{alpha}}6 integrin, ZO-1, actin, PI3K p85 subunit, and PIP3 were stained by specific antibodies and phalloidin-FITC, and imaged by confocal fluorescence microscopy. Bars, 10 µm.
- Using indirect immunofluorescence, we found that the LY294002-reverted T4-2 cells regained the polarization of the apicolateral tight junction marker ZO-1, the basal marker 6 integrin, and the reorganization of the actin cytoskeleton (Fig. 2 A).
- We found that both PI3K (p85 subunit) and PIP3 are polarized to the basal surface of phenotypically normal S-1 cells grown in 3D lrBM, and that this asymmetric distribution is lost in T4-2 cells (Fig. 2 B).
- However, phenotypic reversion of the T4-2 cells through attenuation of PI3K signaling led to repolarization of these signaling components (Fig. 2 B).
- We find that down-modulation of PI3K activity in the T4-2 cells grown in 3D lrBM causes structural repolarization and reversion to a nonmalignant phenotype (Fig. 1) similar to the effects observed previously from inhibition of EGFR and ß1 integrin (Weaver et al., 1997; Wang et al., 1998), and we additionally show that both the normal and the normalized reverted acinus-like structures had basal polarization of PI3K and its lipid signaling product, PIP3 (Fig. 2 B).
- 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.
- 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).
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Figure 3. Attenuation of PI3K activity results in cross-modulation of other signaling pathways and intermediates. Cell lysates from S-1, T4-2, and T4-2+LY grown in 3D lrBM or on 2D plastic substrata for 10 d were analyzed for expression of (A) EGFR, ß1 integrin, phosphorylated Akt (serine 473)/total, phosphorylated GSK-3ß (serine 9)/total, and (B) PTEN (n = 3); E-cadherin was used as the loading control. It was shown previously that the total level of E-cadherin does not change under these conditions (Weaver et al., 1997).
- 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).
- The evidence that inhibition of PI3K can affect crossmodulation of a number of distinct signaling pathways is a demonstration that pathways downstream of PI3K are integrated into transduction networks when cells are grown in the physiological 3D lrBM; consistent with this model, we found that reversion of the tumor cells to a normal phenotype was associated with increased expression of PTEN, the PI3K antagonist (Fig. 3 B).
- 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).
- 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).
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Figure 4. Expression of Akt increases proliferation but does not affect polarity. (A) Expression of constitutively active Akt in T4-2 cells (Myr-Akt), detected by Western analysis of cells infected with Myr-Akt (+) or vector control (-), and probed with anti-Akt (left) or anti-HA antibodies (right). (B) Activity of Myr-Akt mutant and its downstream target were not affected by PI3K inhibitor LY294002; cell lysates from T4-2 and T4-2+LY expressing Myr-Akt or vector grown in 3D lrBM for 10 d were analyzed for phosphorylated Akt (serine 473)/total and GSK-3ß (serine 9)/total. (C) T4+Myr-Akt colonies were larger than control (T4-2+Vector) colonies, both in the presence and absence of LY294002, as assessed by phase contrast microscopy. Bar, 20 µm. (D) T4-2 + Myr-Akt colonies had more nuclei per spheroid cross section. Total nuclear number at spheroid cross section and spheroid numbers were counted and are presented as cell number per spheroid cross section. Statistical analyses revealed significant differences between Myr-Akt and vector control (mean ± SD, P values calculated using Student's t test; more than 500 colonies from 5 independent experiments were analyzed for each condition). (E) Constitutively active Akt signaling did not affect the basal tissue repolarization when T4-2+Myr-Akt cells were reverted by PI3K inhibitor, as assessed by basal localization of {{alpha}}6 integrin relative to DAPI-stained nuclei. Bar, 10 µm. (F) Quantitative analysis of polarity by percentage of spheroids without polarized distribution of basal {{alpha}}6 integrin. No significant difference was found between Myr-Akt and vector control for each condition (mean ± SD, P > 0.05, Student's t test; more than 600 colonies were analyzed for each condition from 3 independent experiments).
- We examined the possibility that the PI3K inhibitor-mediated reversion was due to reduction of Akt activity by expressing a dominant active Akt construct (Myr-Akt) in the T4-2 cells (Fig. 4 A).
- Expression of this construct blocked the effect of PI3K inhibitor on phosphorylation of Akt and downstream mediators of Akt activity (Fig. 4 B), and substantially increased the proliferation of T4-2 cells (Fig. 4, C and D).
- However, examination of colony polarity revealed that the LY294002-treated T4-Myr-Akt cell spheroids largely retained basal tissue polarity despite their considerably larger size (Fig. 4, E and F).
- Looking for the signaling effectors that controlled the increased proliferation and decreased polarity downstream of PI3K in the T4-2 cells led to identification of Akt as a mediator of increased proliferation (Fig. 4) and Rac1 as an inhibitor of polarization (Fig. 5), and we found that these two genes, when expressed in combination, were sufficient to overcome the inhibition of PI3K (Fig. 6).
- 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.
- 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.
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Figure 5. Expression of Rac1 disrupts tissue polarity but does not prevent proliferation arrest. (A) Rac1 activity correlates with PI3K activity, analyzed by using recombinant PAK-GST-CD fusion protein pulldown from lysates of 10-d 3D lrBM cultures of S-1, T4-2, and T4-2+LY cells. (B) Expression of constitutively active Rac1 L61, detected from lysates of cells infected with Rac1L61 or vector control and probed with anti-HA antibody (left) or immunoprecipitated with anti-HA antibody and blotted by anti-Rac1 antibody (right). (C) Inhibition of PI3K attenuates growth of both T4-2+Rac1 L61 colonies and control (T4-2+Vector) colonies, as assessed by phase contrast microscopy. Bar, 20 µm. (D) Analysis of total nuclear number at spheroid cross section reveals that treatment with LY294002 causes statistically signinficant decrease of colony cell number, but expression of Rac1L61 causes no substantial difference (mean ± SD, P values calculated using Student's t test; more than 300 colonies were analyzed from 3 independent experiments for each condition). (E) Attenuation of PI3K activity does not restore tissue polarity in T4-2 cells that express Rac1 L61, as assessed by immunofluorescence of vector control or transfected cells, stained with antibody against {{alpha}}6 integrin and with DAPI. Bar, 10 µm. (F) Statistical analysis of data in E, in which polarity was assessed by percentage of spheroids without polarized distribution of {{alpha}}6 integrin at basal surface (mean ± SD, P < 0.01, vector control versus Rac1 L61 cells treated with LY294002, Student's t test; more than 700 colonies were analyzed for each condition from 3 independent experiments).
- When we examined the activity of Rac1 in S1, T4-2, and LY294002-reverted T4-2 cells using pull-down assays, we found a high correlation of active Rac1 levels with loss of tissue polarity (Fig. 5 A).
- To test the role of Rac1 in the reversion phenotype, we expressed a dominant active Rac1 construct in T4-2 cells (Rac1L61; Fig. 5 B).
- We found that this construct did not greatly affect the rate of cellular proliferation (Fig. 5, C and D), but did inhibit the restoration of polarity in response to LY294002 (Fig. 5, E and F).
- Looking for the signaling effectors that controlled the increased proliferation and decreased polarity downstream of PI3K in the T4-2 cells led to identification of Akt as a mediator of increased proliferation (Fig. 4) and Rac1 as an inhibitor of polarization (Fig. 5), and we found that these two genes, when expressed in combination, were sufficient to overcome the inhibition of PI3K (Fig. 6).
- 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.
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Figure 6. Simultaneous expression of both Akt and Rac1 is sufficient to prevent reversion induced by attenuation of PI3K activity. (A) Expression of both constitutively active Akt (Myr-Akt) and Rac1 (Rac1 L61) in T4-2 cells was detected by probing Western blots of cell lysates with anti-HA antibody. (B) PI3K inhibition fails to decrease proliferation and to restore tissue organization in Myr-Akt and Rac1 L61-double transfectants, as assessed by phase contrast microscopy and (C) by increased nuclei per spheroid cross section (mean ± SD, P values calculated using Student's t test; more than 300 colonies were analyzed for each condition from three independent experiments). Bar in B, 20 µm. (D) Restoration of basal tissue polarity is disrupted in double transfectants, as assessed by aberrant location of {{alpha}}6 integrin in LY294002-treated T4-2 Myr-Akt+Rac1 L61 cells grown in 3D lrBM for 10 d. Representative images from three independent experiments are shown. Bar, 10 µm. (E) Myr-Akt and Rac1 L61 double transfectants collaborate to increase anchorage-independent cell growth and overcome the inhibitory effects of treatment with PI3K inhibitor. Transfectants were grown in methyl cellulose for 3 wk in the absence or presence of PI3K inhibitor. Representative images are shown from duplicate experiments.
- To determine whether together these two effectors were sufficient to recapitulate the effects of PI3K, T4-2 cells were infected with both constructs (Fig. 6 A).
- Treatment with PI3K inhibitor reduced the increased proliferation only to the level of the malignant vector-transfected cells (Fig. 6, B and C), but it had no repolarizing effect (Fig. 6 D).
- To determine whether these effects were also manifested in a different surrogate tumor malignancy assay, the four cell types (T4-2, T4+Myr-Akt, T4+Rac1L61, and T4+MyrAkt+Rac1L61) were cultured in 3D methylcellulose in the absence or presence of LY294002 (Fig. 6 E, -LY and +LY).
- Looking for the signaling effectors that controlled the increased proliferation and decreased polarity downstream of PI3K in the T4-2 cells led to identification of Akt as a mediator of increased proliferation (Fig. 4) and Rac1 as an inhibitor of polarization (Fig. 5), and we found that these two genes, when expressed in combination, were sufficient to overcome the inhibition of PI3K (Fig. 6).
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Figure 7. Scheme of proposed tumor cell signaling network to control polarity, proliferation, and apoptosis. T4-2 signaling network. PI3K activity is increased in tumor cells as a result of aberrant signaling from cell–ECM and cell–growth factor receptor interactions. The consequent activation of Akt contributes to the increased cellular proliferation through downstream pro-proliferation and anti-apoptotic pathways. Increased PIP3 also leads to up-regulation of Rac1 through activation of Rac1-specific GEF activity, resulting in altered organization of the actin cytoskeleton, formation and maintenance of tight junctions, and directionality of vesicle trafficking, effects that combine to disorganize the tissue structure. Thus, each pathway independently affects cellular behavior, but the synergistic effect leads to the tumor phenotype. ({*}) Cross-modulation in 3D lrBM. Inhibition at any of these pressure points results in normalization of the expression and activity at each of the other points, with concomitant normalization of downstream signaling pathways.
- These results suggest a model in which the key aspects of the early malignant phenotype, growth and disorganization, can be controlled through disruption of signaling pathways that become interconnected and integrated in 3D lrBM (Fig. 7).
- 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).