Blockade of EGFR and ErbB2 by the novel dual EGFR and ErbB2 tyrosine kinase inhibitor GW572016 sensitizes human colon carcinoma GEO cells to apoptosis.

Coexpression of the epidermal growth factor receptor (EGFR) family receptors is found in a subset of colon cancers, which may cooperatively promote cancer cell growth and survival, as heterodimerization is known to provide for diversification of signal transduction. Recently, efforts have been made to develop novel 4-anilinoquinazoline and pyridopyrimidine derivatives to inhibit EGFR and ErbB2 kinases simultaneously. In this study, we tested the efficacy of a novel reversible dual inhibitor GW572016 compared with the selective EGFR and ErbB2 tyrosine kinase inhibitors (TKI) AG1478 and AG879 and their combination, using the human colon adenocarcinoma GEO mode. GEO cells depend on multiple ErbB receptors for aberrant growth. A synergistic effect on inhibition of cell proliferation associated with induction of apoptosis was observed from the combination of AG1478 and AG879. Compared with AG1478 or AG879, the single TKI compound GW572016 was a more potent inhibitor of GEO cell proliferation and was able to induce apoptosis at lower concentrations. Western blot analysis revealed that AG1478 and AG879 were unable to suppress both EGFR and ErbB2 activation as well as the downstream mitogen-activated protein kinase (MAPK) and AKT pathways as single agents. In contrast, GW572016 suppressed the activation of EGFR, ErbB2, MAPK, and AKT in a concentration-dependent manner. Finally, in vivo studies showed that GW572016 treatment efficiently blocked GEO xenograft growth at a dose range of 30 to 200 mg/kg with a twice-daily schedule. In summary, our study indicates that targeting both EGFR and ErbB2 simultaneously could enhance therapy over that of single agents directed at EGFR or ErbB2 in cancers that can be identified as being primarily heterodimer-dependent.

Reorganization of ErbB family and cell survival signaling after Knock-down of ErbB2 in colon cancer cells.

The role of the ErbB family in supporting the malignant phenotype was characterized by stable transfection of a single chain antibody (ScFv5R) against ErbB2 containing a KDEL endoplasmic reticulum retention sequence into GEO human colon carcinoma cells. The antibody traps ErbB2 in the endoplasmic reticulum, thereby down-regulating cell surface ErbB2. The transfected cells showed inactivation of ErbB2 tyrosine phosphorylation and reduced heterodimerization of ErbB2 and ErbB3. This resulted in greater sensitivity to apoptosis induced by growth deprivation and delayed tumorigenicity in vivo. Furthermore, decreased heterodimerization of ErbB2 and ErbB3 led to a reorganization in ErbB function in transfected cells as heterodimerization between epidermal growth factor receptor (EGFR) and ErbB3 increased, whereas ErbB3 activation remained almost the same. Importantly, elimination of ErbB2 signaling resulted in an increase in EGFR expression and activation in transfected cells. Increased EGFR activation contributed to the sustained cell survival in transfected cells.

The role of transforming growth factor alpha in determining growth factor independence.

Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.