Vincenzo Damiano

Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Human Cancer Cells with Acquired Resistance to Antiepidermal Growth Factor Receptor Therapy

Purpose: The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small molecule EGFR-selective tyrosine kinase inhibitor, are in advanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase. Experimental Design: The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical significance using the Student’s t test. All Ps were two-sided. Results: Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80–90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pretreated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was observed in mice re-treated with C225 or ZD1839. GEO tumors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO-ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting revealed no major change in the expression of the EGFR ligand transforming growth factor α of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-activated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5–10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474. Conclusions: Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474. These data indicate that inhibition of VEGF signaling has potential as an anticancer strategy, even in tumors that are resistant to EGF inhibitors.

A phase II study of biweekly oxaliplatin plus infusional 5-fluorouracil and folinic acid (FOLFOX-4) as first-line treatment of advanced gastric cancer patients

The aim of the study was to assess the toxicity and the clinical activity of biweekly oxaliplatin in combination with infusional 5-fluorouracil (5-FU) and folinic acid (FA) administered every 2 weeks (FOLFOX-4 regimen) in patients with advanced gastric cancer (AGC). A total of 61 previously untreated AGC patients were treated with oxaliplatin 85 mg m−2 on day 1, FA 200 mg m−2 as a 2 h infusion followed by bolus 5-FU 400 mg m−2 and a 22 h infusion of 5-FU 600 mg m−2, repeated for 2 consecutive days every 2 weeks. All patients were assessable for toxicity and response to treatment. Four (7%) complete responses and 19 partial responses were observed (overall response rate, 38%). Stable disease was observed in 22 (36%) patients, with progressive disease in the other six (10%) patients. Median time to progression (TTP) and median overall survival (OS) were 7.1 and 11.2 months, respectively. National Cancer Institute Common Toxicity Criteria grade 3 and 4 haematologic toxicities were neutropenia, anaemia and thrombocytopenia in 36, 10 and 5% of the patients, respectively. Grade 3 peripheral neuropathy was recorded in three (5%) patients. FOLFOX-4 is an active and well-tolerated chemotherapy. Response rate (RR), TTP and OS were comparable with those of other oxaliplatin-based regimens, suggesting a role for this combination in gastric cancer.

Vascular Endothelial Growth Factor Receptor-1 Contributes to Resistance to Anti–Epidermal Growth Factor Receptor Drugs in Human Cancer Cells

Purpose: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor–resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. Experimental Design: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. Results: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor–resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. Conclusions: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumours sensitive and resistant to anti-EGFR drugs

Inhibition of a single transduction pathway is often inefficient due to activation of alternative signalling. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation, survival and angiogenic pathways and has been implicated in the resistance to EGFR inhibitors. Thus, mTOR blockade is pursued to interfere at multiple levels with tumour growth. We used everolimus (RAD001) to inhibit mTOR, alone or in combination with anti-EGFR drugs gefitinib or cetuximab, on human cancer cell lines sensitive and resistant to EGFR inhibitors, both in vitro and in vivo. We demonstrated that everolimus is active against EGFR-resistant cancer cell lines and partially restores the ability of EGFR inhibitors to inhibit growth and survival. Everolimus reduces the expression of EGFR-related signalling effectors and VEGF production, inhibiting proliferation and capillary tube formation of endothelial cells, both alone and in combination with gefitinib. Finally, combination of everolimus and gefitinib inhibits growth of GEO and GEO-GR (gefitinib resistant) colon cancer xenografts, activation of signalling proteins and VEGF secretion. Targeting mTOR pathway with everolimus overcomes resistance to EGFR inhibitors and produces a cooperative effect with EGFR inhibitors, providing a valid therapeutic strategy to be tested in a clinical setting.

The RIα subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-Receptor

Functional interactions between protein kinase A (PKA) and epidermal growth factor receptor (EGF-R) signalling pathways have been suggested. Unlike the type II isoform of PKA (PKAII), the type I (PKAI) and/or its regulatory subunit RIα are generally overexpressed in cancer cells and are induced following transforming growth factor α (TGFα)/EGF-R-dependent transformation. Downregulation of RIα/PKAI inhibits TGFα expression and EGF-R-dependent signalling. We have previously shown that addition of EGF to quiescent human normal epithelial MCF-10A cells determines PKAI expression and cell membrane translocation before cells enter S phase, while PKAI inhibition prevents S phase entry. Constitutive overexpression of PKAI confers the ability to grow in serum free medium, bypassing EGF requirement. Here we demonstrate a direct interaction of PKAI, but not of PKAII, with the activated EGF-R, that occurs within 5 min following EGF treatment of MCF-10A cells. Moreover, induction of mitogen-activated protein kinase (MAPK) activity following EGF-R activation is mimicked by PKAI overexpression and inhibited by downregulators of PKAI. Finally, the PKAI – EGF-R association occurs through the binding of RIα to the SH3 domain(s) of Grb2 adaptor protein, thus allowing the recruitment of the PKAI holoenzyme to the activated EGF-R. This is the first demonstration of a direct interaction of PKAI with the activated EGF-R macromolecular signalling complex.

Cooperative Antitumor Effect of Multitargeted Kinase Inhibitor ZD6474 and Ionizing Radiation in Glioblastoma

Purpose: Glioblastoma multiforme is an aggressive disease in which vascular endothelial growth factor (VEGF) and the EGF receptor (EGFR) are implicated in tumor growth, relapse, and resistance to radiotherapy and chemotherapy. The VEGF receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR), typically present on endothelial cells, have also been identified in human glioblastoma tissues and cell lines. In addition, EGFR is dysregulated in the majority of human glioblastomas and EGFR overexpression correlates with shorter survival. We have investigated the antitumor and antiangiogenic effect of ZD6474, an inhibitor of both VEGFR and EGFR signaling as a single agent and in combination with ionizing radiation. Experimental Design: We have used ZD6474 and/or ionizing radiation in human glioblastoma cell lines D54 and U251 in vitro and in nude mice bearing established xenografts. The effects of treatment on tumor blood vessels and protein expression were evaluated by Western blot and immunohistochemistry. Results: As single agents, ionizing radiation and ZD6474 caused a dose-dependent inhibition of soft agar growth in D54 and U251 cell lines, whereas a cooperative effect was obtained in combination. Treatment of mice bearing D54 xenografts with either ZD6474 or radiotherapy alone caused tumor growth inhibition that was reversible upon treatment cessation. A cooperative and long-lasting inhibition of tumor growth was obtained with ZD6474 in combination with concomitant radiotherapy. The antiproliferative effect was accompanied by inhibition of VEGF protein expression and inhibition of angiogenesis as measured by vessel counting. Conclusion: This study shows the antitumor activity of ZD6474 in combination with ionizing radiation in glioblastoma both in vitro and in vivo, and provides a scientific rationale to evaluate ZD6474 alone or in combination with radiotherapy in patients affected by this disease.

Rational combination of targeted therapies as a strategy to overcome the mechanisms of resistance to inhibitors of EGFR signaling.

Tumor cell proliferation, de-differentiation, and progression depend on a complex combination of altered intracellular processes including cell cycle regulation, excessive growth factor pathway activation, and decreased apoptosis. Metabolites from these processes result in significant cellular oxidative stress that must be buffered to prevent permanent cell damage and cell death. Tumor cells depend on a complex set of respiratory pathways to generate the necessary energy as well as redox-sensitive pro-survival signaling pathways and factors to cope with and defend against the detrimental effects of oxidative stress. It has been hypothesized that redox-sensitive signaling factors such as thioredoxin reductase-1 (TR) and thioredoxin (TRX) may represent central pro-survival factors that would allow tumor cells to evade the damaging and potentially cytotoxic effects of endogenous and exogenous agents that induce oxidative stress. The overarching theme of this review is an extension of the hypothesis that tumor cells use these redox sensitive pro-survival signaling pathways/factors, which are up-regulated due to increased tumor cell respiration, to evade the cytotoxic effects of anticancer agents. These observations suggest that redox-sensitive signaling factors may be potential novel molecular targets for drug discovery.

Novel Toll-Like Receptor 9 Agonist Induces Epidermal Growth Factor Receptor (EGFR) Inhibition and Synergistic Antitumor Activity with EGFR Inhibitors

Purpose: Immunostimulating Toll-like receptor 9 (TLR9) agonists cause antitumor activity interfering also with cancer proliferation and angiogenesis by mechanisms still incompletely understood. We hypothesized that modified TLR9 agonists could impair epidermal growth factor receptor (EGFR) signaling and, by this means, greatly enhance EGFR inhibitors effect, acting on both the receptor targeting and the immunologic arm. Experimental Design: We used a novel second-generation, modified, immunomodulatory TLR9 agonist (IMO), alone and in combination with the anti-EGFR monoclonal antibody cetuximab or tyrosine kinase inhibitor gefitinib, on the growth of GEO and cetuximab-resistant derivatives GEO-CR colon cancer xenografts. We have also evaluated the expression of several proteins critical for cell proliferation, apoptosis, and angiogenesis, including EGFR, mitogen-activated protein kinase, Akt, bcl-2, cyclooxygenase-2, vascular endothelial growth factor, and nuclear factor-κB. Results: IMO inhibited GEO growth and signaling by EGFR and the other proteins critical for cell proliferation and angiogenesis. IMO plus the anti-EGFR antibody cetuximab synergistically inhibited tumor growth, signaling proteins, and microvessel formation. EGFR signaling inhibition by IMO is relevant because IMO cooperated also with EGFR tyrosine kinase inhibitor gefitinib in GEO tumors, while it was inactive against GEO-CR xenografts. On the other hand, IMO boosted the non-EGFR-dependent cetuximab activity, causing a cooperative antitumor effect in GEO-CR cells. Finally, combination of IMO, cetuximab and chemotherapeutic irinotecan eradicated the tumors in 90% of mice. Conclusion: IMO interferes with EGFR-related signaling and angiogenesis and has a synergistic antitumor effect with EGFR inhibitors, especially with cetuximab, boosting both the EGFR dependent and independent activity of this agent. Moreover, this therapeutic strategy could be translated in patients affected by colorectal cancer.

A novel MDM2 anti-sense oligonucleotide has anti-tumor activity and potentiates cytotoxic drugs acting by different mechanisms in human colon cancer

MDM2 is over‐expressed in several human tumors. Its product is a negative‐feedback regulator of p53, which interferes with the control of cell proliferation and apoptosis, interacting not only with p53 but also with retinoblastoma (Rb) and E2F. Moreover, mutations in the ARF‐Ink4a locus may also allow MDM2 to override p53 functions. In this study, we have used a novel oligonucleotide anti‐sense MDM2, with mixed‐backbone structure and demonstrate that it causes inhibition of MDM2 expression, induction of both p53 and p21/WAF1 expression and a dose‐dependent, growth‐inhibitory effect in human GEO colon‐cancer cells. We also show that anti‐sense MDM2 has a co‐operative growth‐inhibitory effect with different classes of cytotoxic drugs acting by different mechanisms. Moreover, anti‐sense MDM2 induces apoptosis and markedly enhances the apoptotic activity of different cytotoxic drugs. Finally, we show that anti‐sense MDM2 has anti‐tumor activity in vivo in nude mice bearing GEO xenografts and potentiates the anti‐tumor effect of cytotoxic drugs. Indeed, despite the short treatment period, the combination of anti‐sense MDM2 and cytotoxic drugs causes a marked delay in tumor growth and prolongation of mice survival, lasting several months after treatment cessation. The anti‐tumor effect is associated with inhibition of MDM2 expression in tumor specimens of animals treated with anti‐sense MDM2, alone or in combination with a cytotoxic drug. Our results provide the rationale for development of a novel mixed‐backbone anti‐sense MDM2 into a clinical setting in therapeutic combination strategies with conventional cytotoxic drugs. Int. J. Cancer 88:804–809, 2000.

Sphingosine Kinase 1 Overexpression Contributes to Cetuximab Resistance in Human Colorectal Cancer Models

Purpose: Although the anti–EGF receptor (EGFR) monoclonal antibody cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the “sphingolipid rheostat”—the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P)—due to sphingosine kinase 1 (SphK1) overactivation have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK1 and EGFR-dependent signaling pathways have been described. Experimental design: We investigated SphK1 contribution to cetuximab resistance in colorectal cancer, in preclinical in vitro/in vivo models, and in tumor specimens from patients. Results: SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK1 contribution to resistance was supported by the demonstration that SphK1 inhibition by N,N-dimethyl-sphingosine or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents. Moreover, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) antagonist, resulted in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a correlation between SphK1 expression and cetuximab response was found in colorectal cancer patients. Clin Cancer Res; 19(1); 138–47. ©2012 AACR.