Floriana Morgillo

Chronic inflammation and oxidative stress in human carcinogenesis

A wide array of chronic inflammatory conditions predispose susceptible cells to neoplastic transformation. In general, the longer the inflammation persists, the higher the risk of cancer. A mutated cell is a sine qua non for carcinogenesis. Inflammatory processes may induce DNA mutations in cells via oxidative/nitrosative stress. This condition occurs when the generation of free radicals and active intermediates in a system exceeds the systems ability to neutralize and eliminate them. Inflammatory cells and cancer cells themselves produce free radicals and soluble mediators such as metabolites of arachidonic acid, cytokines and chemokines, which act by further producing reactive species. These, in turn, strongly recruit inflammatory cells in a vicious circle. Reactive intermediates of oxygen and nitrogen may directly oxidize DNA, or may interfere with mechanisms of DNA repair. These reactive substances may also rapidly react with proteins, carbohydrates and lipids, and the derivative products may induce a high perturbation in the intracellular and intercellular homeostasis, until DNA mutation. The main substances that link inflammation to cancer via oxidative/nitrosative stress are prostaglandins and cytokines. The effectors are represented by an imbalance between pro‐oxidant and antioxidant enzyme activities (lipoxygenase, cyclooxygenase and phospholipid hydroperoxide glutathione‐peroxidase), hydroperoxides and lipoperoxides, aldehydes and peroxinitrite. This review focalizes some of these intricate events by discussing the relationships occurring among oxidative/nitrosative/metabolic stress, inflammation and cancer.

Implications for KRAS status and EGFR-targeted therapies in metastatic CRC

EGFR regulates cancer-cell proliferation, apoptosis and tumor-induced neoangiogenesis, and has been validated as a relevant therapeutic target in several human cancers, including metastatic colorectal cancer (mCRC). The anti-EGFR monoclonal antibodies cetuximab and panitumumab are available for the treatment of patients with mCRC. Although EGFR is expressed in approximately 85% of patients with mCRC, the clinical efficacy of treatment with anti-EGFR antibodies is limited to a subset of patients. A series of potential biomarkers that could be useful in predicting response to EGFR inhibitors has been investigated. In patients with mCRC, activating mutations within KRAS can predict resistance to anti-EGFR monoclonal antibodies. Activating mutations in KRAS, which could result in EGFR-independent intracellular signal transduction activation, are found in approximately 35–40% of patients with mCRC. These mutations are almost exclusively detected in codons 12 and 13 of exon 2. KRAS mutations have been significantly associated with lack of response to cetuximab or panitumumab therapy in patients with mCRC, which suggests that EGFR-independent, constitutive activation of the RAS signaling pathway could impair response to anti-EGFR drugs. We summarize the experimental and clinical evidence supporting the use of KRAS testing for the optimal selection of patients with mCRC to be treated with anti-EGFR monoclonal antibodies.

Implication of the Insulin-like Growth Factor-IR Pathway in the Resistance of Non–small Cell Lung Cancer Cells to Treatment with Gefitinib

Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been found to be effective against lung cancer in vitro, but clinical resistance to these agents has developed as their usage has increased. In this study, we determined whether the insulin-like growth factor I (IGF-I) signaling pathway induces resistance of non–small cell lung cancer (NSCLC) cells to the EGFR tyrosine kinase inhibitor gefitinib. Experimental Design: The effects of gefitinib and cetuximab on NSCLC cells, alone or with an IGF-I receptor (IGF-IR) inhibitor, were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the flow cytometry–based terminal nucleotidyl transferase–mediated nick end labeling assay, coimmunoprecipitation, and Western blot analysis. EGFR and IGFR expression in NSCLC tissues were examined by Western blot analysis. Results: Gefitinib inhibited NSCLC cell proliferation by inducing apoptosis when IGF-IR signaling was suppressed. Treatment with gefitinib, but not cetuximab, induced EGFR:IGF-IR heterodimerization and activation of IGF-IR and its downstream signaling mediators, resulting in increased survivin expression in NSCLC cell lines with high levels of IGF-IR expression. Inhibition of IGF-IR activation and knockdown of survivin expression led to increased apoptosis. In contrast, overexpression of survivin protected cells with low IGF-IR expression from gefitinib-induced apoptosis. Most NSCLC tissues with EGFR overexpression had associated high levels of IGF-IR expression. Conclusions: IGF-IR expression may be useful as a predictive marker for gefitinib treatment of NSCLC. Suppression of IGF-IR signaling pathways may prevent or delay development of gefitinib resistance in patients with NSCLC.

First-Line Erlotinib Followed by Second-Line Cisplatin-Gemcitabine Chemotherapy in Advanced Non–Small-Cell Lung Cancer: The TORCH Randomized Trial

PURPOSE Erlotinib prolonged survival of unselected patients with advanced non-small-cell lung cancer (NSCLC) who were not eligible for further chemotherapy, and two phase II studies suggested it might be an alternative to first-line chemotherapy. A randomized phase III trial was designed to test whether first-line erlotinib followed at progression by cisplatin-gemcitabine was not inferior in terms of survival to the standard inverse sequence. PATIENTS AND METHODS Patients with stage IIIB (with pleural effusion or supraclavicular nodes) to IV NSCLC and performance status of 0 to 1 were eligible. With a 95% CI upper limit of 1.25 for the hazard ratio (HR) for death, 80% power, a one-sided α = .025, and two interim analyses, a sample size of 900 patients was planned. RESULTS At the first planned interim analysis with half the events, the inferiority boundary was crossed, and the Independent Data Monitoring Committee recommended early termination of the study. Seven hundred sixty patients (median age, 62 years; range, 27 to 81 years) had been randomly assigned. Baseline characteristics were balanced between study arms. As of June 1, 2011, median follow-up was 24.3 months, and 536 deaths were recorded (263 in the standard treatment arm and 273 in the experimental arm). Median survival was 11.6 months (95% CI, 10.2 to 13.3 months) in the standard arm and 8.7 months (95% CI, 7.4 to 10.5 months) in the experimental arm. Adjusted HR of death in the experimental arm was 1.24 (95% CI, 1.04 to 1.47). There was no heterogeneity across sex, smoking habit, histotype, and epidermal growth factor receptor (EGFR) mutation. CONCLUSION In unselected patients with advanced NSCLC, first-line erlotinib followed at progression by cisplatin-gemcitabine was significantly inferior in terms of overall survival compared with the standard sequence of first-line chemotherapy followed by erlotinib.

Targeting EGFR in pancreatic cancer treatment.

The prognosis of patients with pancreatic cancer is extremely poor, and current systemic therapies provide marginal survival benefits for treated patients. The era of targeted therapies has offered a new avenue to search for potentially more effective strategies. Epidermal growth factor receptor (EGFR) is a member of the erbB/human epidermal growth factor receptor family of tyrosine kinases, which includes erbB2/HER2, erbB3/HER3 and erbB4/HER4. Epidermal growth factor receptor overexpression may be detected in up to 90% of pancreatic tumors. Two pharmacologic approaches have been successfully used to inhibit epidermal growth factor receptor function in cancer treatment: neutralizing monoclonal antibodies and small molecule tyrosine inhibitors. The randomized trials studying the addition of EGFR targeted agents to gemcitabine compared with gemcitabine alone have been disappointing, although results with the EGFR tyrosine kinase inhibitor erlotinib were statistically significant but clinically of marginal benefit. In this article, we review the epidermal growth factor receptor signaling network in pancreatic cancer, the strategies to increase the effectiveness of epidermal growth factor receptor inhibitors, and the clinical trials of these inhibitors in pancreatic cancer.

Increased TGF-α as a Mechanism of Acquired Resistance to the Anti-EGFR Inhibitor Cetuximab through EGFR–MET Interaction and Activation of MET Signaling in Colon Cancer Cells

Purpose: Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance. Experimental Design: We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab. Results: Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET–dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR–MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells. Conclusions: These results suggest that overexpression of TGF-α through induction of EGFR–MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer. Clin Cancer Res; 19(24); 6751–65. ©2013 AACR.

Elevated Serum Levels of Interleukin-8 in Advanced Non-Small Cell Lung Cancer Patients: Relationship with Prognosis

Interleukin-8 (IL-8) is a pleiotropic cytokine that has also been shown to exert effects relevant to cancer growth and progression. Cancer progression is believed to be contributed to by the ability of this cytokine to promote angiogenesis and mitogenic effects. As IL-8 production at the tumor site may determine elevated serum levels of this cytokine because of hematogenous leakage, it is conceivable that patients with high IL-8 serum levels may have tumors actively producing this cytokine. The aim of this study was, therefore, to assess IL-8 serum levels in 60 non-small cell lung cancer (NSCLC) patients undergoing chemotherapy and to correlate them with prognosis. IL-8 serum levels were found to be significantly elevated in cancer patients with respect to controls. Moreover, IL-8 serum levels were shown to be significantly increased in stage IV patients compared with stage III patients. When basal IL-8 serum levels in cancer patients were analyzed according to response to chemotherapy, responders were shown to have significantly lower IL-8 serum levels than nonresponders. On univariate analysis, the IL-8 serum level was included among the variables capable of affecting both overall survival (OS) and time to treatment failure (TTF). However, multivariate analysis failed to demonstrate an independent prognostic significance for IL-8 serum levels. In conclusion, this study showed that IL-8 serum levels were elevated in advanced NSCLC patients and correlated with both OS and TTF, but they were shown not to be an independent prognostic factor.

Synergistic Antitumor Activity of Sorafenib in Combination with Epidermal Growth Factor Receptor Inhibitors in Colorectal and Lung Cancer Cells

Purpose: Cancer cell survival, invasion, and metastasis depend on cancer cell proliferation and on tumor-induced angiogenesis. We evaluated the efficacy of the combination of sorafenib and erlotinib or cetuximab. Experimental Design: Sorafenib, erlotinib, and cetuximab, alone or in combination, were tested in vitro in a panel of non–small cell lung cancer (NSCLC) and colorectal cancer cell lines and in vivo in H1299 tumor xenografts. Results: Epidermal growth factor receptor (EGFR) ligand mRNAs were expressed in all NSCLC and colorectal cancer cell lines with variable levels ranging from 0.4- to 8.1-fold as compared with GEO colorectal cancer cells. Lung cancer cells had the highest levels of vascular endothelial growth factors (VEGF) A, B, and C, and of VEGF receptors as compared with colorectal cancer cells. Combined treatments of sorafenib with erlotinib or cetuximab produced combination index values between 0.02 and 0.5, suggesting a significant synergistic activity to inhibit soft agar colony formation in all cancer cell lines, which was accompanied by a marked blockade in mitogen-activated protein kinase and AKT signals. The in vitro migration of H1299 cells, which expressed high levels of both VEGF ligands and receptors, was inhibited by treatment with sorafenib, and this effect was significantly increased by the combination with anti-EGFR drugs. In nude mice bearing established human H1299 xenografts, treatment with the combination of sorafenib and erlotinib or cetuximab caused a significant tumor growth delay resulting in 70 to 90 days increase in mice median overall survival as compared with single-agent sorafenib treatment. Conclusions: Combination treatment with sorafenib and erlotinib or cetuximab has synergistic antitumor effects in human colorectal and lung cancer cells. Clin Cancer Res; 16(20); 4990–5001. ©2010 AACR.

Antitumor activity of pimasertib, a selective MEK 1/2 inhibitor, in combination with PI3K/mTOR inhibitors or with multi‐targeted kinase inhibitors in pimasertib‐resistant human lung and colorectal cancer cells

The RAS/RAF/MEK/MAPK and the PTEN/PI3K/AKT/mTOR pathways are key regulators of proliferation and survival in human cancer cells. Selective inhibitors of different transducer molecules in these pathways have been developed as molecular targeted anti‐cancer therapies. The in vitro and in vivo anti‐tumor activity of pimasertib, a selective MEK 1/2 inhibitor, alone or in combination with a PI3K inhibitor (PI3Ki), a mTOR inhibitor (everolimus), or with multi‐targeted kinase inhibitors (sorafenib and regorafenib), that block also BRAF and CRAF, were tested in a panel of eight human lung and colon cancer cell lines. Following pimasertib treatment, cancer cell lines were classified as pimasertib‐sensitive (IC50 for cell growth inhibition of 0.001 µM) or pimasertib‐resistant. Evaluation of basal gene expression profiles by microarrays identified several genes that were up‐regulated in pimasertib‐resistant cancer cells and that were involved in both RAS/RAF/MEK/MAPK and PTEN/PI3K/AKT/mTOR pathways. Therefore, a series of combination experiments with pimasertib and either PI3Ki, everolimus, sorafenib or regorafenib were conducted, demonstrating a synergistic effect in cell growth inhibition and induction of apoptosis with sustained blockade in MAPK‐ and AKT‐dependent signaling pathways in pimasertib‐resistant human colon carcinoma (HCT15) and lung adenocarcinoma (H1975) cells. Finally, in nude mice bearing established HCT15 and H1975 subcutaneous tumor xenografts, the combined treatment with pimasertib and BEZ235 (a dual PI3K/mTOR inhibitor) or with sorafenib caused significant tumor growth delays and increase in mice survival as compared to single agent treatment. These results suggest that dual blockade of MAPK and PI3K pathways could overcome intrinsic resistance to MEK inhibition.

Synergistic effects of metformin treatment in combination with gefitinib, a selective EGFR tyrosine kinase inhibitor, in LKB1 wild-type NSCLC cell lines

Purpose: EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) have been found to be effective against lung cancer, but clinical resistance to these agents has developed as their usage has increased. Metformin is a widely used antidiabetic drug and also displays significant growth-inhibitory and proapoptotic effects in several cancer models, alone or in combination with chemotherapeutic drugs. Experimental Design: The effects of gefitinib, a selective EGFR-TKI, and metformin on a panel of non–small cell lung cancer (NSCLC) cell lines were assessed by using MTT, bromide assay, flow cytometry, anchorage-independent growth, coimmunoprecipitation, and Western blot analysis. Results: The combination of metformin with gefitinib induced a strong antiproliferative and proapoptotic effect in NSCLC cell lines that harbored wild-type LKB1 gene. Treatment with metformin as single agent, however, induced an activation and phosphorylation of mitogen-activated protein kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. The inhibition of EGFR phosphorylation and of downstream signaling by adding gefitinib to metformin treatment abrogated this phenomenon and induced a strong apoptotic effect in vitro and in vivo. Conclusions: Metformin and gefitinib are synergistic in LKB1 wild-type NSCLC cells. However, further studies are required to investigate better the effect of metformin action on the RAS/RAF/MAPK pathway and the best context in which to use metformin in combination with molecular targeted agents. Clin Cancer Res; 19(13); 3508–19. ©2013 AACR.