Effects of EGF-R on Signal Transduction and Anti-Apoptotic Cascades in Hematopoietic Cells: EGF-R Activates Multiple Signal Transduction Cascades Which Result in the Prevention of Apoptosis in the Absence of Autocrine Cytokines.
Authors: Shelton John G. , Steelman Linda S. , McMahon Martin , McCubrey James A. 
Published: April 2020

Abstract
v-ErbB is an oncogene related to the Epidermal Growth Factor Receptor (EGF-R) that was initially discovered in the genome of avian erythoblastosis virus. v-ErbB will abrogate the requirement of erythroid progenitor cells for erythropoietin and stem cell factor and block terminal differentiation. EGF-R overexpression has been observed in many pathological situations and there is a truncated form of EGF-R referred to as EGFvIII which resembles v-ErbB in biological properties. EGF-R expression is often constitutive and may occur in the presence of expression of other oncogenes or in the absence of certain tumor suppressor genes. To circumvent these problems, we subcloned v-ErbB into a vector which contained the estrogen receptor hormone binding domain which renders the v-ErbB protein dependent upon the addition of beta-estradiol for activity. v-ErbB:ER conditionally abrogated the cytokine dependence of human (TF-1) and murine (FL5.12 and FDC-P1) hematopoietic cells efficiently. This construct allowed examination of the signal transduction and anti-apoptotic pathways activated by v-ErbB:ER in hematopoietic cells. By determining which signal transduction pathways were activated in response to either v-ErbB:ER or IL-3 in the presence and absence of inhibitors, we could ascertain that v-ErbB:ER expression activated the Jak/STAT, Raf/MEK/ERK, PI3K/Akt/mTOR/p70S6K and JNK pathways. Thus v-ErbB:ER activated a broad spectrum of signal transduction pathways some of which were linked to the prevention of apoptosis. Apoptosis was measured by annexin V/PI binding and activation of caspases 3, 8 & 9. Treatment of v-ErbB:ER cells with the EGF-R inhibitor AG1478 efficiently induced apoptosis in these cells at 100 to 1000 fold lower concentrations than MEK, PI3K/mTOR or JNK inhibitors, and activation of all these signal transduction pathways was inhibited with the EGFR inhibitor. Induction of apoptosis by the EGF-R inhibitor was only observed when the cells were grown in response to v-ErbB:ER activation demonstrating specificity. In contrast, other inhibitors suppressed kinase activation and induced some apoptosis when the cells were grown in response to v-ErbB:ER or IL-3. When the cells were treated with MEK, PI3K/mTOR or JNK inhibitors, only the kinases specific for those pathways were inhibited. When MEK and PI3K/mTOR inhibitors were added a synergistic apoptotic response was observed. In contrast, combinations of Jak or JNK and either MEK or PI3K/mTOR inhibitors did not result in a synergistic apoptotic response. Thus the Raf/MEK/ERK and PI3K/Akt/mTOR/p70S6K pathways are the most important pathways in preventing apoptosis in these cells. FL5.12 cells conditionally transformed to grow in response to activated Raf and Akt, (FL/Akt:ER+Raf-1:AR) were also isolated. Activation of STAT5 by either Raf or Akt did not occur in these cells, but did occur upon IL-3 treatment. Furthermore, Elk activation occurred in response to Raf activation but not IL-3 stimulation in the cells which grew in response to Raf and Akt. v-ErbB:ER is more effective in its ability to abrogate cytokine dependence of hematopoietic cells than Raf or Akt, and it induces multiple signal transduction pathways, only some of which are required for growth and the prevention of apoptosis in tissue culture systems.