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Combined Epidermal Growth Factor Receptor Targeting with the Tyrosine Kinase Inhibitor Gefitinib (ZD1839) and the Monoclonal Antibody Cetuximab (IMC-C225) Superiority Over Single-Agent Receptor Targeting

Purpose: The epidermal growth factor receptor (EGFR) is abnormally activated in cancer and two classes of anti-EGFR agents, monoclonal antibodies and low-molecular-weight tyrosine kinase inhibitors, have shown antitumor activity in patients. Because these two classes of antireceptor agents target the EGFR at different sites, we decided to explore whether the combined administration of gefitinib, a tyrosine kinase inhibitor, and cetuximab, a monoclonal antibody, had superior antitumor activity than either agent given alone. Experimental Design: We studied the effects of the combination of gefitinib and cetuximab in a panel of human cancer cell lines and in an EGFR-dependent human tumor xenograft model (A431). The effects of these two agents on EGFR signaling, proliferation, apoptosis, and vascularization were evaluated. In addition, we analyzed, with cDNA arrays, changes in gene expression profiles induced by both agents. Results: The combined treatment with gefitinib and cetuximab resulted in a synergistic effect on cell proliferation and in superior inhibition of EGFR-dependent signaling and induction of apoptosis. In a series of in vivo experiments, single-agent gefitinib or cetuximab resulted in transient complete tumor remission only at the highest doses. In contrast, suboptimal doses of gefitinib and cetuximab given together resulted in a complete and permanent regression of large tumors. In the combination-treated tumors, there was a superior inhibition of EGFR, mitogen-activated protein kinase, and Akt phosphorylation, as well as greater inhibition of cell proliferation and vascularization and enhanced apoptosis. Using cDNA arrays, we found 59 genes that were coregulated and 45 genes differentially regulated, including genes related to cell proliferation and differentiation, transcription, DNA synthesis and repair, angiogenesis, signaling molecules, cytoskeleton organization, and tumor invasion and metastasis. Conclusions: Our findings suggest both shared and complementary mechanisms of action with gefitinib and cetuximab and support combined EGFR targeting as a clinically exploitable strategy.

Dynamic cross-talk between tumor and immune cells in orchestrating the immunosuppressive network at the tumor microenvironment

Accumulating evidence indicates that a dynamic cross-talk between tumors and the immune system can regulate tumor growth and metastasis. Increased understanding of the biochemical nature of tumor antigens and the molecular mechanisms responsible for innate and adaptive immune cell activation has revolutionized the fields of tumor immunology and immunotherapy. Both the protective effects of the immune system against tumor cells (immunosurveillance) and the evasion of tumor cells from immune attack (tumor-immune escape) have led to the concept of cancer immunoediting, a proposal which infers that a bidirectional interaction between tumor and inflammatory/regulatory cells is ultimately responsible for orchestrating the immunosuppressive network at the tumor site. In this context, a major challenge is the potentiation or redirection of tumor antigen-specific immune responses. The success in reaching this goal is highly dependent on an improved understanding of the interactions and mechanisms operating during the different phases of the cancer immunoediting process. In this review, we discuss the multiple defense and counterattack strategies that tumors have devised in order to evade immune attack and to thwart the effectiveness of several immunotherapeutic approaches.

Th2/Th1 switch induced by a single low dose of cyclophosphamide in a rat metastatic lymphoma model.

Abstract. Cyclophosphamide (Cy) is an alkylating agent widely used in cancer chemotherapy. It has a bimodal effect on the immune system, depending on the dose and schedule of administration. We have previously demonstrated that a single low dose of Cy has an antimetastatic effect, achieved through immunomodulation, in lymphoma bearing rats. Such a treatment reduced the splenic production of IL-10, TGF-β, and NO, restoring the lymphoproliferative capacity. A shift from immunosuppression to immunopotentiation induced by low-dose Cy treatment was mainly mediated by a decrease in IL-10 production. The present study focused on the analysis of the modulation of type-1 cytokine levels by treatment with a single low dose of Cy and the effect these cytokines (IL-2 and IFN-γ) and IL-10 have on primary tumor and metastatic cell growth. Our results suggest that a single low dose of Cy induces a Th2/Th1 shift in the cytokine profile of lymphoma-bearing rats, which may be responsible for its antimetastatic effect. A direct action of IL-10 as a growth factor and IFN-γ as a cytotoxic factor on metastatic cells is also shown.

Inhibitory effect of Lovastatin on spontaneous metastases derived from a rat lymphoma

The HMGCoA reductase inhibitor Lovastatin (LOV) has previously shown to abrogate p21ras farnesylation, which is associated with invasive and metastatic abilities in many tumor models. Considering the scarcity of therapeutic resources against metastasis, our objective was to study LOV as an antimetastatic agent on L-TACB rat lymphoma, which as a syngeneic tumor model resembles more closely the situation in human cancer. We also aimed to analyze the effect of LOV on chemoinvasion, motility, metalloproteases secretion, angiogenic capacity, and adhesion to the reconstituted basement membrane Matrigel®. Our results showed that LOV caused no effect on cell motility, metalloprotease secretion and neovascularization. Conversely, LOV produced a significant inhibition of invasiveness, which could be a consequence of an impaired cell adhesion to the basement membrane observed. These effects could explain, at least in part, the inhibitory action of LOV on L-TACB rat lymphoma metastases.

Down regulation of T-cell-derived IL-10 production by low-dose cyclophosphamide treatment in tumor-bearing rats restores in vitro normal lymphoproliferative response.

In previous reports, we demonstrated an inhibitory effect of a single low-dose of cyclophosphamide (Cy) on spontaneous and experimental metastasis of a rat lymphoma (L-TACB). This antimetastatic effect could be adoptively transferred by immune spleen cells from Cy-treated tumor-bearing rats and it was abrogated by the use of immunosuppressed hosts, suggesting an immunomodulatory effect. Subsequently, we found that increased levels of TGF-beta, IL-10 and NO were involved in tumor-induced immunosuppression by inhibiting lymphocyte proliferation. The treatment of tumor-bearing rats with low-dose Cy reduced the splenic production of these suppressive cytokines, restoring the lymphoproliferative capacity otherwise diminished during tumor growth. Here, we investigated the changes of the cytokines modulated by the Cy therapy that are responsible for the restoration of the lymphoproliferative response and determined the spleen cell type producing TGF-beta, IL-10 and NO in our experimental model. Our current results show that IL-10 and NO are produced exclusively by T lymphocytes and macrophages, respectively, whereas TGF-beta is produced by both cell types. The high level of IL-10 produced by T-cells from tumor-bearing rats is responsible for the inhibition of lymphocyte proliferation. Moreover, our results suggest that the shift from immunosuppression to immunopotentiation induced by treatment of tumor-bearing rats with a single low-dose of Cy is mediated by a reduction in T-cell derived IL-10 production, which would account, to some extent, for the antimetastatic effect of Cy treatment.

MECHANISM OF ANTIMETASTATIC IMMUNOPOTENTIATION BY LOW-DOSE CYCLOPHOSPHAMIDE

We have previously reported the antimetastatic effect of a single low-dose of cyclophosphamide (Cy) on L-TACB rat lymphoma. The phenomenon could be adoptively transferred in immunocompetent rats and is abolished in nude mice, facts for which an immunomodulatory explanation was proposed. The aim of this paper was to identify the mechanism(s) by which spleen cells from Cy-treated tumour-bearing rats could exert this antimetastatic activity. Conditioned media obtained by incubation of spleen cells from Cy-treated and non-treated tumour-bearing rats, under specific or non-specific stimulation, were assayed to evaluate their effect on lymphocyte proliferation. The production of transforming growth factor beta (TGF-beta), interleukin-10 (IL-10) and nitric oxide (NO) by conditioned media was also studied. The restoration of spleen lymphoproliferative responses to normal levels when exposed to media conditioned by splenocytes from Cy-treated tumour-bearing rats, together with a decreased production of suppressive cytokines TGF-beta, IL-10 and NO, suggest an enhancement of host antimetastatic immunity triggered by single low-dose Cy treatment.

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin

Abstract Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in resistance to certain cytotoxic drugs.

Inhibition ofras oncogene: A novel approach to antineoplastic therapy

The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in the ras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21(ras) lead to unregulated, sustained signaling through downstream effectors. The ras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21(ras) farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.

Low molecular weight hyaluronan inhibits colorectal carcinoma growth by decreasing tumor cell proliferation and stimulating immune response.

Hyaluronan modulates cancer progression by multiple mechanisms; nevertheless, its effects remain controversial. In this work, low molecular weight (LMW) hyaluronan but not high molecular weight (HMW) was found to significantly reduce colorectal carcinoma (CRC) growth in vitro and in vivo. Both survival and proliferation of CT26 tumor cells were affected by treatment with low doses of LMW HA, with involvement of Akt signaling mechanisms. We show for the first time that splenocytes isolated from LMW HA-treated animals present significantly higher proliferative capacity upon stimulation with dendritic cells (DCs) pulsed with tumor lysate. Consistently, expression of MHC class II and costimulatory molecules were increased in DCs isolated from the spleen of LMW HA-treated mice. Besides, increased tumor infiltrating lymphocytes was observed in animals treated with LMW HA. Our results suggest that LMW HA in a model of CRC triggers an activation of immune system, which is likely involved in the observed tumor growth inhibition. LMW HA is suggested as a candidate molecule for therapeutic adjuvant treatments in CRC immunotherapy.

A Novel Synergistic Combination of Cyclophosphamide and Gene Transfer of Interleukin-12 Eradicates Colorectal Carcinoma in Mice

Purpose: Interleukin-12 (IL-12) is an immunostimulatory cytokine with potent antitumor effects in several animal models. However, serious toxicity has been associated with its systemic application in humans. Gene transfer has emerged as a tool to specifically express therapeutic genes into the tumor/peritumoral milieu, thus avoiding systemic toxicity. The aim of this study was to analyze whether subtherapeutic doses of an adenovirus encoding IL-12 (AdIL-12) might synergize with low immunopotentiating doses of cyclophosphamide in the treatment of colorectal carcinoma. Experimental Design: The antitumor effect of combining a single low dose of cyclophosphamide with an intratumoral injection of AdIL-12 was evaluated in an in vivo murine colorectal carcinoma model. The immune responses achieved with different treatments were monitored, comparing the effect of combining both therapies with individual treatments. Results: The combined therapy induced a complete tumor regression in >50% of mice in a synergistic fashion, and it significantly prolonged their survival. This strategy was superior to each single treatment in reducing both peripheral and splenic CD4+CD25+Foxp3+ regulatory T cells, increasing the number of activated dendritic cells, and inducing IFN-γ-secreting CD4-positive T lymphocytes. Importantly, the combined treatment generated a powerful tumor-specific CTL response. Consistently, a significant reduction in IL-10 levels was found. Our data suggest that the combination of nontoxic doses of cyclophosphamide with AdIL-12 allows the generation of good antitumoral responses, thus avoiding undesired side effects of both agents. Conclusions: Our data strongly support the use of a combination of cyclophosphamide and AdIL-12 as a novel therapeutic strategy against colorectal carcinoma. (Clin Cancer Res 2009;15(23):7256–65)