Conditional EGFR Promotes Cell Cycle Progression and Prevention of Apoptosis in the Absence of Autocrine Cytokines

Abstract

ABSTRACTv-ErbB is an oncogene related to the Epidermal Growth Factor Receptor (EGFR). EGFR overexpression has been observed in many pathological situations. There is a truncated form of EGFR, referred to as EGFvIII, which resembles v-ErbB in biological properties and is often expressed in certain human tumors. Aberrant EGFR expression in human cancers is often constitutive and may occur in the presence of mutated oncogenes or tumor suppressor genes. To circumvent these problems, we subcloned v-ErbB into a vector which contains the estrogen receptor hormone binding domain (ER) which renders the v-ErbB:ER protein dependent upon β-estradiol for activity. v-ErbB:ER conditionally abrogated the cytokine dependence of hematopoietic cells more efficiently than activated v-Ha-Ras, v-Src, Raf or Akt. Abrogation of cytokine-dependence by v-ErbB:ER was not due to the synthesis of autocrine growth factors. Treatment of v-ErbB:ER cells with the EGFR inhibitor AG1478 efficiently induced apoptosis. Induction of apoptosis and prevention of cell cycle progression by the EGFR inhibitor were only observed when the cells were grown in response to v-ErbB:ER activation demonstrating specificity. In contrast, the other inhibitors suppressed cell cycle progression when the cells were grown in response to v-ErbB:ER or the cytokine interleukin-3. When MEK and either EGFR or PI3K/mTOR inhibitors were added, an enhanced apoptotic response was observed. Thus this conditional ErbB construct is useful to elucidate EGFR signaling and anti-apoptotic pathways in the absence of autocrine cytokine expression.

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

“…[233][234][235][236][237][238] In addition, some melanoma cells carrying B-Raf mutations are sensitive to MEK inhibitors while cells lacking these B-Raf mutations are resistant. 239 We have shown that introduction of activated EGFR mutants into hematopoietic cells renders them sensitive to EGFR inhibitors. 233,240,241 Furthermore, introduction of activated Ras, Raf, MEK genes into hematopoietic cells makes them sensitive to MEK inhibitors.…”

Alteration of Akt activity increases chemotherapeutic drug and hormonal resistance in breast cancer yet confers an achilles heel by sensitization to targeted therapy

“…After allowing the cells to adhere for 24 hrs, the monolayers were washed twice with PBS and then cultured with phenol red free RPMI 1640 containing 0.5% CS-FBS (to avoid endogenous estrogenic like compounds and reduce serum growth factors) for 24 hrs. Cells were then stimulated with 10% FBS for the indicated time intervals and protein lysates prepared as previously described (Shelton et al, 2005b). Western blots were performed with antibodies specific for phospho and total Akt, Ask-1, p70S6K, ERK and Hsp70, as previous described (Shelton et al, 2005b).…”

“…Cells were then stimulated with 10% FBS for the indicated time intervals and protein lysates prepared as previously described (Shelton et al, 2005b). Western blots were performed with antibodies specific for phospho and total Akt, Ask-1, p70S6K, ERK and Hsp70, as previous described (Shelton et al, 2005b). Antibodies used in this study were purchased from Cell Signaling (Beverly, MA).…”

EGF Induces Cell Motility and Multi-Drug Resistance Gene Expression in Breast Cancer Cells

“…13,14 The epidermal growth factor (EGF) receptor (EGFR) pathway is a clear example of a signal transduction pathway that has been associated with development, progression, and resistance to treatment with cytotoxic drugs of a number of human solid tumors including breast. [17][18][19][20][21][22][23][24][25][26] EGFR is part of the tyrosine kinase receptor family that also includes erbB2, erbB3, and erbB4. 20 EGFR are large proteins, which reside in the cell membrane and with the exception of erbB2 each has a specific extracellular ligand-binding domain, a transmembrane and intracellular domain, which has tyrosine kinase activity.…”