Raf-induced synthesis of soluble factors leads to activation of EGF-receptor signaling and reduced growth factor dependence in MCF-10A cells. (A) MCF-10A ΔRaf-ER cells were treated with 100 nM 4-OHT or solvent for 48 h and grown in minimal medium for 24 h. Medium was collected and used to stimulate parental MCF-10A cells, which had been cultured in minimal medium for 24 h. To specifically inhibit EGF-receptor tyrosine kinase activity, 300 nM AG1478 was added 30 min before addition of conditioned medium where indicated. Phosphorylation of ErbB1/EGFR, PKB/Akt, and p42^ERK2/p44^ERK1 MAPK was detected by immunoblotting of whole cell lysates using phosphospecific antibodies. (B) MCF-10A ΔRaf-ER cells were treated with 100 nM 4-OHT or solvent for 24 h while being cultured in full medium (FM), minimal medium (MM), or medium supplemented with 10 μg/mL insulin, 5 μg/mL hydrocortisone, and 100 ng/mL cholera toxin (SM) in the presence or absence of 300 nM AG1478. Whole cell lysates were used to detect phosphorylation of PKB/Akt and p42^ERK2/p44^ERK1 MAPK. (C) Growth curves of MCF-10A ΔRaf-ER cells cultured in the presence (solid line, squares) or absence (dashed line, diamonds) of 4-OHT. (Left) Cells grown in full medium; (right) cells grown in medium supplemented with 10 μg/mL insulin, 5 μg/mL hydrocortisone, and 100 ng/mL cholera toxin (suppl. medium). Where indicated, 300 nM AG1478 was added to the culture (solid squares and diamonds).

Activation of Raf induces survival from detachment-induced apoptosis and prevents release of cytochrome c from mitochondria. (A) MCF-10A cells infected with empty vector or stably expressing ΔRaf-ER or V12 Ras were pretreated with 100 nm 4-OHT for 8 or 48 h as indicated, detached by trypsin treatment, and plated on normal (light bars) or poly-HEMA coated dishes (dark bars) in minimal medium in the presence or absence of 4-OHT. After 24 h, DNA fragmentation was measured by Cell Death ELISA. Values represent the mean and standard deviation of three independent experiments. (B) Phosphorylation of p42^ERK2/p44^ERK1 detected with a phosphospecific antibody using cytoplasmic lysates from cells prepared in parallel to the experiment shown in A. (C) Raf activation prevents cytochrome c release in detached cells. MCF-10A ΔRaf-ER cells were pretreated with either 100 nM 4-OHT or solvent for 48 h, detached, and cultivated on normal (A) or poly-HEMA–coated dishes (S) for 24 h. (upper panel) cytochrome c detected in cytoplasmic lysates obtained by disrupting the plasma membrane with digitonin; (lower panel) detachment induced activation of caspase-8 in the presence or absence of 4-OHT. Detection of the cleaved p20 subunit of caspase-8 indicates activation of the protease.

Differential expression of Raf target genes. (A) Northern blot detection of Raf target genes identified by microarray analysis. RNA from empty vector or ΔRaf-ER MCF-10A cells treated with 4-OHT for the indicated times while cultured in minimal medium for 24 h before lysis (in the same way as described for the experiment shown in Table 1) was separated by denaturing electrophoresis, blotted onto nylon membranes, and probed with radioactively labeled cDNA probes for the indicated genes. For comparison, fold change values measured in the microarray experiment (Table 1) are indicated on the left. (B) Whole cells lysates from cells prepared in parallel to samples used for RNA preparation in 3A were used to detect JunB, c-Fos, cyclin D1, and p21^CIP1 by immunoblotting. In addition, whole cell lysates from MCF-10A ΔRaf-ER cells treated with 4-OHT for 7 d were used (lane 7). Activation of ΔRaf-ER by 4-OHT was monitored by detection of p42^ERK2/p44^ERK1 MAPK phosphorylation using a phosphospecific antibody. (C) Same cell lysates as in B were used to detect HB-EGF, TGFα, and amphiregulin by immunoblotting. Amphiregulin was detected as two bands representing the 17-kD precursor and a smaller processed form.

Activation of ΔRaf-ER and changes in cell morphology and growth properties in response to 4-OHT. (A) Detection of MAPK-phosphorylation in MCF-10A human breast epithelial cells stably expressing the ΔRaf-ER fusion protein in response to treatment with 100 nM 4-OHT or 20 ng/mL EGF for the indicated times. Cells were starved in medium containing 5% horse serum for 24 h before treatment. Phosphorylation of p42^ERK2 and p44^ERK1 was detected by immunoblotting with a phosphospecific antibody (upper panel). Expression of p42^ERK2 was monitored by immunoblotting with a pan-ERK antibody (lower panel). (B) Growth curves of MCF-10A ΔRaf-ER cells in medium containing different supplements in the presence (solid line, squares) or absence (dashed line, diamonds) of 4-OHT. (left) cells grown in medium containing 5% horse serum, 20 ng/mL EGF, 10 μg/mL insulin, 5 μg/mL hydrocortisone and 100 ng/mL cholera toxin (full medium); (right) cells grown in medium containing 5% horse serum without supplements (minimal medium). Values shown represent the mean and standard deviation of three replicate experiments. (C) MCF-10A ΔRaf-ER cells after treatment with solvent (0.2 μL/mL ethanol) or 100 nM 4-OHT for 48 h. The morphology of ethanol treated cell is indistinguishable from control or parental cells. (D) MCF-10A ΔRaf-ER grown in collagen gels for 10 d in the presence or absence of 100 nM 4-OHT. Cystic structures in the control image are similar to those described previously for different epithelial cell lines.

Raf-induced survival from detachment induced apoptosis is sensitive to inhibition of EGF receptor tyrosine kinase activity and PI 3-kinase activity. (A) Induction of EGF-like growth factor expression upon Raf activation. MCF-10A ΔRaf-ER cells pretreated with 100 nM 4-OHT for 48 h were detached and plated on normal (A) or poly-HEMA coated dishes (S) in minimal medium in the presence (+) or absence (−) of 100 nM 4-OHT. After 24 h, expression of HB-EGF, amphiregulin, and TGFα and phosphorylation of p42^ERK2/p44^ERK1 MAPK was monitored by immunoblotting of whole cell lysates. Amphiregulin was detected as two bands representing the 17-kD precursor and a smaller processed form. (B) Down-regulation of Bcl-X_L in suspension is prevented by Raf activation. Same lysates as in A were probed for Bcl-X_L and Bcl-2 expression by immunoblotting. (C) EGF-like growth factors provide a survival signal against detachment induced apoptosis. Parental MCF-10A cells were detached and plated on normal (adherent, light bars) or poly-HEMA coated dishes for 24 h (suspension, dark bars) in minimal medium supplemented with EGF, HB-EGF, TGFα, or amphiregulin as indicated. DNA fragmentation was determined by Cell Death ELISA. Values represent the mean and variation of a typical experiment performed in duplicates. (D) MCF-10A ΔRaf-ER cells were detached and plated on normal (A, light bars) or poly-HEMA–coated dishes (S, dark bars) in minimal medium for 24 h in the presence (+) or absence (−) of 100 nM 4-OHT. Selective inhibitors of MEK (PD98059), PI3-kinase (LY294002), EGFR (AG1478, PD168393), or HB-EGF (Glu52-Diphtheria toxin, CRM197) were added as indicated. DNA-fragmentation was determined by Cell Death ELISA. Values represent the mean and variation of a typical experiment performed in triplicates. (E) Whole cell lysates from MCF-10A ΔRaf-ER cells treated in parallel to the experiment shown in D were analyzed for p42^ERK2/p44^ERK1 MAPK phosphorylation by immunoblotting. (F) MCF-10A infected with empty vector or stably expressing V12Ras were cultured in minimal medium with or without 20 ng/mL EGF for 24 h. Whole cell lysates were assayed for PKB/Akt and p42^ERK2/p44^ERK1 MAPK phosphorylation by immunoblotting. (G) MCF-10A infected with empty vector or stably expressing V12Ras were detached and plated on normal (A, light bars) or poly-HEMA–coated dishes (S, dark bars) in minimal medium for 24 h in the presence of 300 nM AG1478, 30 μM PD98059, or 50 μM LY294002 as indicated. DNA-fragmentation was determined by Cell Death ELISA. Values represent the mean and variation of a typical experiment performed in duplicates.