Joan Kyula

Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells.

Activating epidermal growth factor receptor (EGFR) mutations have been linked with sensitivity to gefitinib and erlotinib; however, there are no established predictive markers for response to the combination of EGFR inhibitors with standard chemotherapy in non–small cell lung cancer (NSCLC) patients. In this study, we characterized a panel of human EGFR wild-type and mutant NSCLC cells for their sensitivity to gefitinib alone and in combination with cisplatin or Taxol. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays. Cell cycle distribution was measured by flow cytometry. EGFR expression was measured by flow cytometry, real-time PCR, and Western blotting. EGFR/Her2/Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation were measured by Western blotting. Two of nine EGFR wild type and one of two EGFR mutant NSCLC cells were sensitive to gefitinib, and this was associated with a decrease in phospho (p)–Akt and pErk1/2 following gefitinib exposure. There was no correlation between constitutive EGFR expression or activity and sensitivity to gefitinib nor was there a correlation between Her2/Akt and Erk1/2 activity and gefitinib sensitivity. However, in cells displaying a synergistic interaction between gefitinib and chemotherapy (cisplatin or Taxol), a dose-dependent increase in pEGFR was observed following chemotherapy exposure. In contrast, in cells where no change or a decrease in pEGFR following drug treatment was observed, we found an antagonistic or (at best) an additive interaction between the two compounds. Furthermore, the nature of this interaction was not dependent on the presence of a mutant EGFR. These novel findings suggest that modulation of EGFR activity following drug treatment determines response to gefitinib in combination with chemotherapy in NSCLC cells. [Mol Cancer Ther 2006;5(5):1154–65]

Chemotherapy-induced activation of ADAM-17: a novel mechanism of drug resistance in colorectal cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism. Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting. Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-α, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor. Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC. Clin Cancer Res; 16(13); 3378–89. ©2010 AACR.

Oncogenic Kras Promotes Chemotherapy-Induced Growth Factor Shedding via ADAM17

Oncogenic mutations in Kras occur in 40% to 45% of patients with advanced colorectal cancer (CRC). We have previously shown that chemotherapy acutely activates ADAM17, resulting in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. In this study, we examined the role of mutant Kras in regulating growth factor shedding and ADAM17 activity, using isogenic Kras mutant (MT) and wild-type (WT) HCT116 CRC cells. Significantly higher levels of TGF-α and VEGF were shed from KrasMT HCT116 cells, both basally and following chemotherapy treatment, and this correlated with increased pErk (phosphorylated extracellular signal regulated kinase)1/2 levels and ADAM17 activity. Inhibition of Kras, MEK (MAP/ERK kinase)1/2, or Erk1/2 inhibition abrogated chemotherapy-induced ADAM17 activity and TGF-α shedding. Moreover, we found that these effects were not drug or cell line specific. In addition, MEK1/2 inhibition in KrasMT xenografts resulted in significant decreases in ADAM17 activity and growth factor shedding in vivo, which correlated with dramatically attenuated tumor growth. Furthermore, we found that MEK1/2 inhibition significantly induced apoptosis both alone and when combined with chemotherapy in KrasMT cells. Importantly, we found that sensitivity to MEK1/2 inhibition was ADAM17 dependent in vitro and in vivo. Collectively, our findings indicate that oncogenic Kras regulates ADAM17 activity and thereby growth factor ligand shedding in a MEK1/2/Erk1/2-dependent manner and that KrasMT CRC tumors are vulnerable to MEK1/2 inhibitors, at least in part, due to their dependency on ADAM17 activity.

Src and ADAM-17-mediated shedding of transforming growth factor-alpha is a mechanism of acute resistance to TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL.

Oncogenic Kras-mediated resistance to chemotherapy via increased ADAM17 activity and ligand shedding in colorectal cancer.

e14104 Background: Oncogenic mutations in the small GTPase c-Kirsten (K)-ras occur in 40%-45% of patients with advanced colorectal cancer (CRC) and are challenging in development of anti-cancer the...

Abstract C177: Src and ADAM17‐mediated shedding of heregulin is a mechanism of acute resistance to chemotherapy in gastro‐oesophageal cancer cells

Background: We previously reported that, in response to chemotherapy, gastro‐oesophageal cancer cells (GEC) activate a human epidermal receptor (HER)‐mediated survival response abrogated by HER inhibitors. In order to elucidate potential biomarkers for response to chemotherapy treatment and potential novel targets, we investigated the pathways which mediate chemo‐induced HER activity in GEC cells. Methods: Apoptosis was measured by Flow Cytometry or PARP, Caspase 8/3 cleavage. HER1/HER2/HER3, Akt expression/activity and Heregulin (HRG) expression levels were determined by Western blotting. ADAM17 activity was measured using a fluorometric (MCA) assay kit while sTGF‐α levels were analysed by ELISA. Results: Following treatment with chemotherapy, we found that increased HER1/HER2/HER3 activity was associated with increased Src‐family kinase (SFK) activity. Using the SFK inhibitor AZD0530, we found that chemotherapy‐induced HER1 activation was abolished, indicating that HER1 activation was mediated via SFKs. Furthermore, we found that the dual HER1/HER2 inhibitor lapatinib had no effect on chemo‐induced SFK activity, indicating that SFKs act upstream of HER. Furthermore, we found that chemotherapy treatment resulted in increased ADAM‐17 activity, TGF‐α and heregulin levels in culture medium of GEC cells. ADAM17 inhibition attenuated chemotherapy‐induced TGF‐α and heregulin levels and HER1/3 activity. When cells were co‐treated with heregulin‐neutralizing antibodies, we found strongest inhibition of chemotherapy‐induced HER1/HER3 and Akt activity. In addition, following treatment with AZD0530, we found complete inhibition of chemotherapy‐induced ADAM17 activity and ligand shedding. Finally, when HER1/HER2/HER3, ADAM17 or SFK inhibitors were combined with chemotherapy, we found a potent increase in apoptosis in GEC cells. Conclusions: We provide strong evidence that chemotherapy induces HER1/HER2/HER3 activation via a ligand dependent mechanism in GEC cells and have demonstrated that SFK9s, ADAM17 and heregulin are critical mediators of HER1/HER2/HER3 activation following chemotherapy. We propose a model in which chemotherapy results in SFK activation that in turn activates ADAM17‐mediated shedding of heregulin. Heregulin then activates HER1/HER2/HER3 pro‐survival signalling in an autocrine and paracrine fashion. Thus, inhibiting HER1/HER2/HER3, SFKs or ADAM17 may have therapeutic potential for sensitizing GEC tumors to chemotherapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C177.

Abstract B243: Kras mutation status as biomarker for response to TRAIL treatment in colorectal cancer

Background: Kras is a member of the ras family of GTPase proteins that regulate cellular growth and survival in response to stimulation of cell surface receptors such as the Epidermal Growth Factor Receptor (EGFR). Activating mutations in the Kras gene are found in 30–50% of colorectal tumors (CRC). The presence of mutant Kras in tumors has been shown to correlate with lack of response to EGFR inhibitors. Recently, we found that CRC cells respond to TRAIL with an EGFR/HER2‐mediated survival response and that by blocking this response, apoptosis is enhanced. A phase I/IIa study is currently investigating effect of AMG655 (DR5 agonist) with panitumumab (EGFR monoclonal antibody) in patients with chemorefractory CRC. The aim of this study was to evaluate the role of Kras mutational status in determining response to TRAIL alone and in combination with EGFR‐ or MEK‐targeted therapies in a panel isogenic paired Kras wild type (WT) and mutant (MT) CRC cells. Methods: Erk1/2 phosphorylation and expression were measured by Western blotting. Apoptosis was measured by flow cytometry andWestern blotting for PARP, caspase‐8 and caspase 3 cleavage. Results: Using paired isogenic KrasMT HCT116 and KrasWT HKH‐2 and HKe‐3 cell lines, we found higher basal levels of pErk1/2 in the KrasMT HCT116 cell line compared to the KrasWT HKH‐2 and HKe‐3 cell lines. Furthermore, we found that the HKH‐2 and HKe‐3 KrasWT cells were more sensitive to rhTRAIL compared to the isogenic parental and KrasMT HCT116 cell line. Moreover, we found a statistical significant increase in apoptosis when rhTRAIL was combined with the EGFR monoclonal antibodies panitumumab or cetuximab in KrasWT HKH‐2 and HKe‐3 cell lines but not in the parental KrasMT HCT116 cell line. In contrast, KrasMT HCT116 cell line was more sensitive to combined TRAIL/MEK‐targeted therapy compared to KrasWT HKH‐2 clone. Conclusions: Our findings indicate that Kras mutations can predict response to TRAIL alone and in combination with EGFR‐targeted agents in CRC cells. Inhibition of MEK1/2 in combination with TRAIL‐targeted therapies may provide a strong treatment strategy in Kras mutant CRC tumors. We are currently investigating mechanism of KrasMT induced resistance to TRAIL treatment and these data will be presented during the meeting. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B243.