Miriam Redrado

Antitumor and antiangiogenic effect of the dual EGFR and HER-2 tyrosine kinase inhibitor lapatinib in a lung cancer model

BackgroundThere is strong evidence demonstrating that activation of epidermal growth factor receptors (EGFRs) leads to tumor growth, progression, invasion and metastasis. Erlotinib and gefitinib, two EGFR-targeted agents, have been shown to be relevant drugs for lung cancer treatment. Recent studies demonstrate that lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER-2 receptors, is clinically effective against HER-2-overexpressing metastatic breast cancer. In this report, we investigated the activity of lapatinib against non-small cell lung cancer (NSCLC).MethodsWe selected the lung cancer cell line A549, which harbors genomic amplification of EGFR and HER-2. Proliferation, cell cycle analysis, clonogenic assays, and signaling cascade analyses (by western blot) were performed in vitro. In vivo experiments with A549 cells xenotransplanted into nude mice treated with lapatinib (with or without radiotherapy) were also carried out.ResultsLapatinib dramatically reduced cell proliferation (P < 0.0001), DNA synthesis (P < 0.006), and colony formation capacity (P < 0.0001) in A549 cells in vitro. Furthermore, lapatinib induced G1 cell cycle arrest (P < 0.0001) and apoptotic cell death (P < 0.0006) and reduced cyclin A and B1 levels, which are regulators of S and G2/M cell cycle stages, respectively. Stimulation of apoptosis in lapatinib-treated A549 cells was correlated with increased cleaved PARP, active caspase-3, and proapoptotic Bak-1 levels, and reduction in the antiapoptic IAP-2 and Bcl-xL protein levels. We also demonstrate that lapatinib altered EGFR/HER-2 signaling pathways reducing p-EGFR, p-HER-2, p-ERK1/2, p-AKT, c-Myc and PCNA levels. In vivo experiments revealed that A549 tumor-bearing mice treated with lapatinib had significantly less active tumors (as assessed by PET analysis) (P < 0.04) and smaller in size than controls. In addition, tumors from lapatinib-treated mice showed a dramatic reduction in angiogenesis (P < 0.0001).ConclusionOverall, these data suggest that lapatinib may be a clinically useful agent for the treatment of lung cancer.

Residual dormant cancer stem-cell foci are responsible for tumor relapse after antiangiogenic metronomic therapy in hepatocellular carcinoma xenografts

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related deaths. Currently available chemotherapeutic options are not curative due in part to tumor resistance to conventional therapies. We generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2rγ null mice by injection of human alpha-feto protein (hAFP)- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. In this model, low-dose metronomic administration of cyclophosphamide (LDM-CTX) caused complete regression of the tumor mass. A significant increase in survival (P<0.0001), reduced aberrant angiogenesis and hyperproliferation, and decrease in the number of circulating tumor cells were found in LDM-CTX-treated animals, in comparison with untreated mice. Co-administration of LDM-CTX with anti-VEGF therapy further improved the therapeutic efficacy. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA− phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, discontinuation of therapy resulted in tumor regrowth. Moreover, in-vitro LDM-CTX treatment reduced hepatosphere formation in both number and size, and the resulting spheres were enriched in CD13+ cells indicating that these cells were particularly resistant to therapy. Co-treatment of the CD13-targeting drug, bestatin, with LDM-CTX leads to slower tumor growth and a decreased tumor volume. Therefore, combining a CD13 inhibitor, which targets the CSC-like population, with LDM-CTX chemotherapy may be used to eradicate minimal residual disease and improve the treatment of liver cancer.

Pilot Clinical Trial of Type 1 Dendritic Cells Loaded with Autologous Tumor Lysates Combined with GM-CSF, Pegylated IFN, and Cyclophosphamide for Metastatic Cancer Patients

Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-α, TNF-α, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day −7), GM-CSF (days 1–4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-γ–ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [111In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.

Differential effects of drugs targeting cancer stem cell (CSC) and non-CSC populations on lung primary tumors and metastasis.

Cancer stem cells (CSCs) are thought to be responsible for tumor initiation and recurrence after chemotherapy. Targeting CSCs and non-CSCs with specific compounds may be an effective approach to reduce lung cancer growth and metastasis. The aim of this study was to investigate the effect of salinomycin, a selective inhibitor of CSCs, with or without combination with paclitaxel, in a metastatic model. To evaluate the effect of these drugs in metastasis and tumor microenvironment we took advantage of the immunocompetent and highly metastatic LLC mouse model. Aldefluor assays were used to analyze the ALDH+/− populations in murine LLC and human H460 and H1299 lung cancer cells. Salinomycin reduced the proportion of ALDH+ CSCs in LLC cells, whereas paclitaxel increased such population. The same effect was observed for the H460 and H1299 cell lines. Salinomycin reduced the tumorsphere formation capacity of LLC by more than 7-fold, but paclitaxel showed no effect. In in vivo experiments, paclitaxel reduced primary tumor volume but increased the number of metastatic nodules (p<0.05), whereas salinomycin had no effect on primary tumors but reduced lung metastasis (p<0.05). Combination of both drugs did not improve the effect of single therapies. ALDH1A1, SOX2, CXCR4 and SDF-1 mRNA levels were higher in metastatic lesions than in primary tumors, and were significantly elevated in both locations by paclitaxel treatment. On the contrary, such levels were reduced (or in some cases did not change) when mice were administered with salinomycin. The number of F4/80+ and CD11b+ cells was also reduced upon administration of both drugs, but particularly in metastasis. These results show that salinomycin targets ALDH+ lung CSCs, which has important therapeutic effects in vivo by reducing metastatic lesions. In contrast, paclitaxel (although reducing primary tumor growth) promotes the selection of ALDH+ cells that likely modify the lung microenvironment to foster metastasis.

Epithelial to mesenchymal transition and cancer stem cell phenotypes leading to liver metastasis are abrogated by the novel TGFβ1-targeting peptides P17 and P144.

Colorectal cancer (CRC) frequently metastasizes to the liver, a phenomenon that involves the participation of transforming-growth-factor-β(1) (TGFβ(1)). Blockade of the protumorigenic effects elicited by TGFβ(1) in advanced CRC could attenuate liver metastasis. We aimed in the present study to assess the antimetastatic effect of TGFβ(1)-blocking peptides P17 and P144, and to study mechanisms responsible for this activity in a mouse model. Colon adenocarcinoma cells expressing luciferase were pretreated with TGFβ(1) (Mc38-luc(TGFβ1) cells), injected into the spleen of mice and monitored for tumor development. TGFβ(1) increased primary tumor growth and liver metastasis, whereas systemic treatment of mice with either P17 or P144 significantly reduced tumor burden (p<0.01). In metastatic nodules, mitotic/apoptotic ratio, mesenchymal traits and angiogenesis (evaluated by CD-31, as well as circulating endothelial and progenitor cells) induced by TGFβ(1) were consistently reduced following injection of peptides. In vitro experiments revealed a direct effect of TGFβ(1) in Mc38 cells, which resulted in activation of Smad2, Smad3 and Smad1/5/8, and increased invasion and transendothelial migration, whereas blockade of TGFβ(1)-signaling reverted these features. Because TGFβ(1)-mediated epithelial-mesenchymal transition (EMT) has been suggested to induce a cancer stem cell (CSC) phenotype, we analyzed the ability of this cytokine to induce tumorsphere formation and the expression of CSC markers. In TGFβ(1)-treated cells, tumorspheres were enriched in CD44 and SOX2, which were diminished in the presence of P17. Our data provide a preclinical rationale to evaluate P17 and P144 as potential therapeutic options for the treatment of metastatic CRC.

Inhibitor of Differentiation-1 as a Novel Prognostic Factor in NSCLC Patients with Adenocarcinoma Histology and Its Potential Contribution to Therapy Resistance

Purpose: High inhibitor of differentiation-1 (Id1) levels have been found in some tumor types. We aimed to study Id1 levels and their prognostic impact in a large series of stages I to IV non-small cell lung cancer (NSCLC) patients. Experiments in cell lines and cells derived from malignant pleural effusions (MPE) were also carried out. Experimental Design: A total of 346 NSCLC samples (three different cohorts), including 65 matched nonmalignant tissues, were evaluated for Id1 expression by using immunohistochemistry. Additional data from a fourth cohort including 111 patients were obtained for Id1 mRNA expression analysis by using publicly available microarrays. In vitro proliferation assays were conducted to characterize the impact of Id1 on growth and treatment sensitivity. Results: Significantly higher Id1 protein levels were found in tumors compared with normal tissues (P < 0.001) and in squamous carcinomas compared with adenocarcinomas (P < 0.001). In radically treated stages I to III patients and stage IV patients treated with chemotherapy, higher Id1 levels were associated with a shorter disease-free survival and overall survival in adenocarcinoma patients in a log-rank test. A Cox model confirmed the independent prognostic value of Id1 levels for both stages I to III and stage IV patients. In silico analysis confirmed a correlation between higher Id1 mRNA levels and poor prognosis for adenocarcinoma subjects. In vitro Id1 silencing in radio/chemotherapy-resistant adenocarcinoma cells from MPEs restored sensitivity to both therapies. Conclusions: In our series, Id1 levels showed an independent prognostic value in patients with adenocarcinoma, regardless of the stage. Id1 silencing may sensitize adenocarcinoma cells to radiotherapy and chemotherapy. Clin Cancer Res; 17(12); 4155–66. ©2011 AACR.

TMPRSS4 regulates levels of integrin α5 in NSCLC through miR-205 activity to promote metastasis.

Background:TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown.Methods:miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC.Results:miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin α5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin α5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin α5 levels.Conclusion:We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin α5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.

Selenoprotein-P is down-regulated in prostate cancer, which results in lack of protection against oxidative damage.

Oxidative stress plays a role in prostate cancer (PrCa) initiation and development. Selenoprotein‐P (SepP; a protein involved in antioxidant defence) mRNA levels are down‐regulated in PrCa. The main goal of our study was to assess whether SepP protects prostate cells from reactive oxygen species (ROS) in prostate carcinogenesis.

Therapeutic Effect of Lenalidomide in a Novel Xenograft Mouse Model of Human Blastic NK Cell Lymphoma/Blastic Plasmacytoid Dendritic Cell Neoplasm

Purpose: Blastic natural killer (NK) cell lymphoma/blastic plasmacytoid dendritic cell neoplasm (BNKL) is a rare and aggressive neoplasia characterized by infiltration of blast CD4+/CD56+ cells in the skin, the bone marrow, and peripheral blood. Currently, more efforts are required to better define molecular and biological mechanisms associated with this pathology. To the best of our knowledge, no mouse model recapitulated human BNKL so far. Experimental Design: Primary bone marrow cells from a BNKL patient were injected in nonobese diabetes/severe combined immunodeficient interleukin (IL) 2rγ−/− mice with the intent to generate the first BNKL orthotopic mouse model. Moreover, because of the lack of efficient treatments for BNKL, we treated mice with lenalidomide, an immunomodulatory and antiangiogenic drug. Results: We generated in mice a fatal disease resembling human BNKL. After lenalidomide treatment, we observed a significant reduction in the number of peripheral blood, bone marrow, and spleen BNKL cells. Tumor reduction parallels with a significant decrease in the number of circulating endothelial and progenitor cells and CD31+ murine endothelial cells. In mice treated with lenalidomide, BNKL levels of active caspase-3 were significantly augmented, thus showing proapoptotic and cytotoxic effects of this drug in vivo. An opposite result was found for proliferating cell nuclear antigen, a proliferation marker. Conclusions: Our BNKL model might better define the cellular and molecular mechanisms involved in this disease, and lenalidomide might be considered for the future therapy of BNKL patients. Clin Cancer Res; 17(19); 6163–73. ©2011 AACR.

Sunitinib reduces tumor hypoxia and angiogenesis, and radiosensitizes prostate cancer stem‐like cells

The need for new treatments for advanced prostate cancer has fostered the experimental use of targeted therapies. Sunitinib is a multi‐tyrosine kinase inhibitor that mainly targets membrane‐bound receptors of cells within the tumor microenvironment, such as endothelial cells and pericytes. However, recent studies suggest a direct effect on tumor cells. In the present study, we have evaluated both direct and indirect effects of Sunitinib in prostate cancer and how this drug regulates hypoxia, using in vitro and in vivo models.