Identification of a Novel Raf-1 Pathway Activator that Inhibits Gastrointestinal Carcinoid Cell Growth

Endocrine Surgery Research Laboratory, University of Wisconsin, and the University of Wisconsin Carbone Cancer Center, Madison, Wisconsin, USA.
Molecular Cancer Therapeutics (Impact Factor: 5.68). 02/2010; 9(2):429-37. DOI: 10.1158/1535-7163.MCT-09-0718
Source: PubMed


Carcinoids are neuroendocrine tumors (NET) that secrete hormones, including serotonin, resulting in the malignant carcinoid syndrome. In addition to the significant morbidity associated with the syndrome, carcinoids are frequently metastatic at diagnosis, and untreated mortality at 5 years exceeds 70%. Surgery is the only curative option, and the need for other therapies is clear. We have previously shown that activation of Raf-1 inhibits carcinoid cell proliferation. We investigated the ability of leflunomide (LFN), a Food and Drug Administration-approved medication for the treatment of rheumatoid arthritis, and its active metabolite teriflunomide (TFN) as a potential anti-NET treatment. LFN and TFN inhibit the in vitro proliferation of gastrointestinal carcinoid cells and induce G(2)-M phase arrest. Daily oral gavage of nude mice with subcutaneous xenografted carcinoid tumors confirms that LFN can inhibit NET growth in vivo. Treatment with TFN suppresses the cellular levels of serotonin and chromogranin A, a glycopeptide co-secreted with bioactive hormones. Additionally, TFN reduces the level of achaete-scute complex-like 1 (ASCL1), a NET marker correlated with survival. These effects are associated with the activation of the Raf-1/mitiogen-activated protein kinase kinase/extracellular signal-regulated kinase-1/2 pathway, and blockade of mitiogen-activated protein kinase kinase signaling reversed the effects of TFN on markers of the cell cycle and ASCL1 expression. In summary, LFN and TFN inhibit carcinoid cell proliferation in vitro and in vivo and alter the expression of NET markers. This compound thus represents an attractive target for further clinical investigation.

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Available from: Mackenzie Cook, Jan 10, 2014
    • "Table 1 RAF-1 activators in NENs (LFN, leflunomide; TFN, teriflunomide (active metabolite of leflunomide); MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) rapid colorimetric assay (MTT – Sigma Aldrich, St. Louis Mo) to measured cellular growth; ASCL1, achaete-scute homolog 1; CgA, chromogranin A; GSK-3b, Glycogen synthase kinase-3b; MTC, medullary thyroid cancer; TTY, tautomycin; TMC, tautomycetin; ZM336372, a novel Raf-1-activating agent; hASH1, human achaete-scute homolog). Authors Type of study Cell lines Drugs Methods (cell viability) Results in vitro/in vivo Biomarkers results Cook et al., Mol Cancer Ther [42] Preclinical – Human GI carcinoid cancer cells (BON) – Human broncho- pulmonary carcinoid tumor cells LFN -TFN MTT colorimetric assay; NET xenograft; western blot In vitro and in vivo inhibition human GI carcinoid cells tumor cell growth (dose dependent) Suppression of CgA, ASCL1 protein, and serotonin Pinchot et al., Am J Surg [43] Preclinical – Human GI carcinoid cancer cells (BON) – Human broncho- pulmonary carcinoid tumor cells TTY MTT colorimetric assay; western blot In vitro inhibition carcinoid tumor cell growth (dose dependent) Reduction of CgA level Adler et al., Mol Cancer Ther [44] Preclinical Human MTC cells TTY, TMC MTT colorimetric assay; western blot; calcitonin ELISA kit In vitro inhibition MTC cell growth (dose dependent) Reduction of CgA, ASCL1 protein level Van Gompel et al., Mol Cancer Ther [45] Preclinical – Human pulmonary carcinoid cells – Human pancreatic carcinoid tumor cells ZM336372 MTT colorimetric assay; western blot In vitro suppression cell proliferation (dose dependent) Reduction of CgA, hASH1 "
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    ABSTRACT: Neuroendocrine neoplasms are a low-incidence and heterogeneous group of malignancies. In the advanced stage, several therapeutic options can be discussed, including molecular-targeted agents, but biological predicting factors are lacking. A number of molecular targets have been studied over the last decade leading to several phase II studies; however, very few agents progressed to phase III clinical trials. The RAF family of proteins belongs to the mitogen-activated protein kinase (MAPK) pathway, that has a role in several types of cancers, particularly related to BRAF mutations. Indeed BRAF inhibitors have been reported as being effective, mainly in melanoma. However, in neuroendocrine neoplasms BRAF mutations are extremely rare and RAF-1 activation has been reported to inhibit tumor growth in a pre-clinical setting. Therefore, in this field, RAF-1 activators rather than BRAF inhibitors should be clinically investigated. This article reviews the basic science as well as clinical data of RAF signaling in advanced neuroendocrine neoplasms with special emphasis on the potential role of both RAF activators and inhibitors.
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    • "Interestingly, this effect could be inhibited by MEK inhibitors that, however, did not influence ERK1/2 phosphorylation, implicating an ERK1/2-independent pathway (Sippel et al. 2003a,b). While Sippel et al. (2003b) found that RAF1 activation did not to influence human pancreatic (BON) NET cell growth, other studies suggested that the RAF1 activator ZM336372 (as well as the drugs leflunomide and teriflunomide – both also associated with RAF1 pathway activation) suppressed the growth of human pulmonary (H727) and human pancreatic (BON) NET cells in vitro; for leflunomide and teriflunomide, this was also shown in vivo (Van Gompel et al. 2005, Cook et al. 2010). The in vivo reduction in development and growth of human medullary thyroid carcinomas through RAF1 activation has also been reported (Vaccaro et al. 2006). "
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    • "GnrH receptors and the associated Raf-1/MEK/ERK-1/2-pathway are potential targets for analogs in cancer treatment. Activation of the Raf-1/MEK/ERK-1/2-pathway by leflunomide and teriflunomide can inhibit proliferation and growth of BON carcinoid cells in vitro and in vivo and also decrease expression of neuroendocrine markers in both BON and H727 carcinoid cells [45]. Similar effects have been reported for estradiol, ZM336372 and tautomycin [46-50]. "
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