Molecular Mechanisms of Trastuzumab Resistance in HER2 Overexpressing Breast Cancer

The epidermal growth factor receptor 2 (HER2) is a tyrosine kinase overexpressed in nearly 20% to 25% of invasive breast cancers. Trastuzumab is a humanized monoclonal antibody that targets HER2. The majority of patients with metastatic breast cancer initially respond to trastuzumab, however, within 1 year of treatment disease progresses. Several molecular mechanisms have been described as contributing to the development of trastuzumab resistance. They could be grouped as impaired access of trastuzumab to HER2, upregulation of HER2 downstream signaling pathways, signaling of alternative pathways, and impaired immune antitumor mechanisms. However, since many of them have overlapping effects, it would be of great clinical impact to identify the principal signaling pathways involved in drug resistance. Significant efforts are being applied to find other therapeutic modalities besides trastuzumab treatment to be used alone or in combination with current modalities

Rosiglitazone inhibits metastasis development of a murine mammary tumor cell line LMM3

<p>Abstract</p> <p>Background</p> <p>Activation of peroxisome proliferator-activated receptors γ (PPARγ) induces diverse effects on cancer cells. The thiazolidinediones (TZDs), such as troglitazone and ciglitazone, are PPARγ agonists exhibiting antitumor activities; however, the underlying mechanism remains inconclusive. Rosiglitazone (RGZ), a synthetic ligand of PPARγ used in the treatment of Type 2 diabetes, inhibits growth of some tumor cells and is involved in other processes related to cancer progression. Opposing results have also been reported with different ligands on tumor cells. The purpose of this study was to determine if RGZ and 15d-PGJ₂induce antitumor effects <it>in vivo </it>and <it>in vitro </it>on the murine mammary tumor cell line LMM3.</p> <p>Methods</p> <p>The effect on LMM3 cell viability and nitric oxide (NO) production of different doses of RGZ, 15-dPGJ₂, BADGE and GW9662 were determined using the MTS colorimetric assay and the Griess reaction respectively. <it>In vivo </it>effect of orally administration of RGZ on tumor progression was evaluated either on s.c. primary tumors as well as on experimental metastasis. Cell adhesion, migration (wound assay) and invasion in Transwells were performed. Metalloproteinase activity (MMP) was determined by zymography in conditioned media from RGZ treated tumor cells. PPARγ expression was detected by inmunohistochemistry in formalin fixed tumors and by western blot in tumor cell lysates.</p> <p>Results</p> <p>RGZ orally administered to tumor-bearing mice decreased the number of experimental lung metastases without affecting primary s.c. tumor growth. Tumor cell adhesion and migration, as well as metalloproteinase MMP-9 activity, decreased in the presence of 1 μM RGZ (non-cytotoxic dose). RGZ induced PPARγ protein expression in LMM3 tumors. Although metabolic activity -measured by MTS assay- diminished with 1–100 μM RGZ, 1 μM-treated cells recovered their proliferating capacity while 100 μM treated cells died. The PPARγ antagonist Biphenol A diglicydyl ether (BADGE) did not affect RGZ activity. On the contrary, the specific antagonist GW9662 completely abrogated RGZ-induced decrease in cell viability. A decrease in NO levels was detected in the presence of either 1 or 100 μM RGZ. The natural ligand 15d-PGJ₂did not affect metabolic activity although it induced a significant decrease in NO production.</p> <p>Conclusion</p> <p>A significant decrease in the number of experimental LMM3 lung metastasis, but not on primary tumor growth, after oral RGZ administration was observed. <it>In vitro</it>, 100 μMRGZ also reduced cell viability and NO production, while no changes were observed in the presence of 15d-PGJ₂. BADGE did not reverse RGZ effect while the antagonist GW9662 completely abrogated it, suggesting a PPARγ- dependent mechanism. Inhibition of lung metastatic nodules by RGZ administered in vivo, might be associated with the observed decrease in MMP-9 expression, in cell adhesion, migration and invasion. RGZ augmented its expression. PPARγ was detected in cell lysates by western blot and by immunohistochemistry in tumors from RGZ-treated mice. In summary we can suggest that RGZ or any other TZDs might be possible future approaches in the treatment of metastasis of PPARγ-expressing cells.</p

Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice

INTRODUCTION: The role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR). METHODS: Peritoneal macrophages from female BALB/c mice bearing a 7-day LMM3 tumor were inoculated intradermally (3 × 10(5 )cells per site) into syngeneic mice. Before inoculation, TMps were stimulated with the muscarinic agonist carbachol in the absence or presence of different muscarinic antagonists or enzyme inhibitors. Angiogenesis was evaluated by counting vessels per square millimeter of skin. The expression of mAchR, arginase and cyclo-oxygenase (COX) isoforms was analyzed by Western blotting. Arginase and COX activities were evaluated by urea and prostaglandin E(2 )(PGE(2)) production, respectively. RESULTS: TMps, which stimulate neovascularization, express functional mAchR, because carbachol-treated TMps potently increased new blood vessels formation. This response was completely blocked by preincubating TMps with pirenzepine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), M(1 )and M(3 )receptor antagonists, and partly by the M(2 )receptor antagonist methoctramine. M(1 )receptor activation by carbachol in TMps triggers neovascularization through arginase products because N(ω)-hydroxy-L-arginine reversed the agonist action. Preincubation of TMps with methoctramine partly prevented carbachol-stimulated urea formation. In addition, COX-derived liberation of PGE(2 )is responsible for the promotion of TMps angiogenic activity by M(3 )receptor. We also detected a higher expression of vascular endothelial growth factor (VEGF) in TMps than in macrophages from normal mice. Carbachol significantly increased VEGF expression in TMps, and this effect was totally reversed by methoctramine and pirenzepine. Arginase and COX inhibitors partly decreased VEGF derived from TMps. CONCLUSION: TMps themselves induce a potent angiogenic response that is augmented by carbachol action. mAchR activation triggers arginine metabolism, PGE(2 )synthesis and VEGF production, promoting neovascularization

IL-10 expression by CT26 colon carcinoma cells inhibits their malignant phenotype and induces a T cell-mediated tumor rejection in the context of a systemic Th2 response

In spite of the evidence that IL-10 has Th1-immunosuppressive and anti-inflammatory effects, it has been shown that IL-10 may reduce the tumorigenic capacity of certain tumor cell types. In order to characterize the responses elicited by IL-10, we explored the effect of transducing murine CT26 colon carcinoma cells with a recombinant retrovirus expressing mIL-10. IL-10 gene transfer of CT26 cells had no effect on tumor cell growth on plastic surface but inhibited the anchorage-independent growth capacity of tumor cells and their metastatic potential as assessed by their invasive and migration ability. Expression of IL-10 also elicited an antitumor immune response involving both CD4+ and CD8+ T cells. Assessment of the immune status of the animals demonstrated that mice injected with CT26-IL10 cells showed prevalence of a systemic and tumor-specific Th2 response. Spleen cells obtained from these mice showed an increased production of IL-4 and no changes in IFNgamma levels, characteristic of a Th2 response. These results demonstrate that IL-10 affects CT26 tumor cell growth by both inhibiting the malignant phenotype and by recruiting and activating a T cell-mediated antitumor response. This T cell response occurs in the context of a shift towards a Th2 response.Facultad de Ciencias Veterinaria

Angiogenic activity by spleen cell supernatants from tumor‐bearing and tumor‐resected mice

Supernatants of cultured spleen cells (SCS) from small tumor‐bearing mice (STBM) and large tumor‐bearing mice (LTBM) are able to enhance tumor growth as well as accelerate tumor takes in vivo when inoculated 24 h before tumor implant. After tumor resection enhancing activity disappears at a rate depending on the size of the resected tumor. Spleen cells from tumor‐bearing mice also evoke a complex vascular response, lymphocyte‐induced angiogenesis (LIA) elicited via the release of lymphokines from activated T cells. As angiogenic factors promote tumor development we investigated if SCS from tumor‐bearing and tumor‐resected mice contain factors capable of evoking a LIA response. Angiogenic activity was detected by SCS of small and large tumor‐bearing mice as well as in large tumor‐resected mice. On the other hand, SCS from small tumor‐resected mice are not able to evoke an angiogenic response. Possibly, the large antigenic burden in LTRM stimulate the immune system in a different way than in STRM. We can suggest that the different behavior of spleen cells after small and large tumor removal can be explained by quantitative or qualitative changes in the spleen subpopulations.Fil: Davel, Lilia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Miguez, Marta. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Jasnis, María Adela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Eiján, Ana María. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Oisgold Dagá, Sara. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaUnidad documental simpl

Differential nitric oxide release and sensitivity to injury in different murine mammary tumor cell lines

The purpose of this study was to determine whether nitric oxide (NO) production by different mammary tumor cell lines correlated with their sensitivity to NO mediated injury. Three mammary tumor cell lines LM2, LM3 and LMM3 syngeneic to BALB/c mice were cultured in vitro with IFNgamma + LPS. Different levels of NO production among the three lines were detected in culture supernatants. The only tumor cell line which did not produce NO (LM2) showed the highest sensitivity to SNP-derived NO cytotoxicity (87%), while LM3 and LMM3 which both produced higher levels of NO than LM2, showed lower cytotoxicity by SNP (39% and 22% respectively). Spleen cells (SC) from M2 tumor bearing mice (TBM) were able to lyse LM2 cells by NO-dependent mechanisms. SC from M3-TBM exerted cytotoxicity against LM3 cells mainly by NO-independent mechanisms. Thus, we postulate an inverse correlation between NO production and NO mediated cytotoxicity in the three mammary tumor cell lines. It is possible that tumor cells producing NO develop mechanisms to resist NO injury.Fil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Davel, L. E.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Rueda, H. A.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Rozenberg, G.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jasnis, Maria Adela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaUnidad documental simpl

Polyamines prevent DFMO-mediated inhibition of angiogenesis

Tumor growth mainly depend on formation of new blood vessels. DFMO (α-difluoromethylornithine), an inhibitor of polyamine biosynthesis, inhibits tumor growth in many animal tumors. Our investigation was to evaluate the requirement of polyamines for induction of angiogenesis by tumor cells and spleen lymphocytes from tumor-bearing mice. In this regard, we have added DFMO to cell cultures. The neovascular response induced either by tumor cells or spleen lymphocytes was completely abrogated. This inhibition could be reversed by the addition of exogenous putrescine. These findings suggest that the effect of DFMO on angiogenesis is, in part, mediated by the inhibition of polyamine biosynthesis.Fil: Jasnis, Maria Adela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Klein, Slobodanka Mariana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Monte, Martin. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Davel, Lilia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Sacerdote de Lustig, Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Algranati, Israel David. Instituto de Investigaciones Bioquímicas "Fundación Campomar"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaUnidad documental simpl