Raf Pathways Research Articles

Abstract A197: Oncogenic KRAS is dependent on the scaffold protein CNKSR1 for cell growth and signaling.

Abstract KRAS is the predominant form of mutated RAS (mut-KRAS) and is found in 25% of patient tumors across many cancer types. Mut-KRAS is well known to play a critical role in driving tumor growth and resistance to therapy, and its effects are so powerful that it overrides the activity of many of the new molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. However, despite extensive effort, there is no effective treatment for mut-KRAS. The effects of mut-KRAS are mediated through multiple downstream signaling pathways which have been independently associated with tumorigenesis, including RAF1, RALGDS, and PI3K. Current efforts to treat mut-KRAS tumors employ concurrent treatment with inhibitors of the RAF and PI3K pathways but this does not address the potential contribution of other pathways for which there are currently no inhibitors. Using a global siRNA screen we searched for genes that when inhibited would block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth using an isogenic MiaPaCa-2 pancreatic cell line with and without oncogenic KRAS, and validated these hits in a similar isogenic HCT-116 colon cell line. From the screen we identified CNKSR1 (connector enhancer of kinase suppressor of Ras 1) as a top hit. CNKSR1 is found associated with KRAS in the RAS membrane associated signaling nanocluster that KRAS has to be associated with to provide growth signals. Knockdown of CNKSR1 with siRNA inhibited the growth of a panel of 10 mut-KRAS non small cell lung cancer (NSCLC) cell lines but not of 4 NSCLC cell lines with wt-KRAS. CNKSR1 is a multidomain protein that has a potentially druggable plekstrin homology (PH) domain responsible for binding to membrane phosphatidylinositols-3-phosphates. In order to demonstrate whether the PH domain of CNKSR1 is necessary for mut-KRAS activity we over expressed the PH domain in H1373 mut-KRAS NSCLC cells and found that it acted as a dominant negative and inhibited cell growth. We suggest that the PH domain fragment competes with the full length CNKSR1 in the cell. We also showed that knockdown of CNKSR1 inhibited KRAS dependent phosphorylation of RAF1 in multiple non small cell lung cancer cell lines. Together our results suggest that the CNKSR1 protein, acting through it's PH domain, is necessary for cell growth and down stream signaling by the KRAS oncogene. The PH domain can be targeted by inhibitors thus potentially providing agents that will selectively block mut-KRAS signaling and cell growth, creating a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A197.

Abstract C152: Mechanisms of KRAS dependency in pancreatic ductal adenocarcinoma.

Abstract Currently, pancreatic ductal adenocarcinoma (PDAC) has a dismal <5% survival rate two years after initial diagnosis. Despite significant advances in development of signal transduction targeted therapies for some cancers, erlotinib, an EGFR inhibitor, in combination with gemcitabine, is the only targeted drug approved for treatment of PDAC. The frequency of KRAS oncogene mutations in PDAC is greater than 90%, thus inhibition of K-Ras4B protein function may have a significant impact on PDAC tumorigenesis. In support of this hypothesis, several recent studies demonstrated that KRAS mutant cancer cell lines display a strong dependence or addiction to mutant K-Ras for survival. Surprisingly, K-Ras addiction was not attributed to either the Raf or phosphoinositide 3-kinase (PI3K) effector pathways, which are two of the best-characterized Ras signaling effectors. These results suggest that other pathways are key for mutant KRAS dependency. Elucidating the effector mechanism(s) necessary for KRAS addiction may implicate signaling pathways, which harbor novel therapeutic targets for future anti-K-Ras drug development. To address this question, we first verified that shRNA silencing of mutant KRAS in a panel of pancreatic cell lines led to a strong reduction in anchorage-independent growth. Notably and in contrast to published studies, this was not due to decreased cell survival or increased anoikis. Using KRAS mutant PDAC cell lines, we purpose to use effector domain mutants of oncogenic K-Ras4B that are differentially impaired in effector interaction and constitutively activated effectors to define the key effector(s) involved in KRAS dependency. These studies will help to define key signaling activities of mutant KRAS that are critical for oncogenesis in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C152.

Ascochlorin suppresses TGF-β1-induced PAI-1 expression through the inhibition of phospho-EGFR in rat kidney fibroblast cells

Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor-β1 (TGF-β1), the growth factor involved in fibrosis, modulates ECM synthesis and accumulation. TGF-β1 enhances the production of stimulators of ECM synthesis such as plasminogen activator inhibitor type 1 (PAI-1). As such, PAI-1 expression directly influences the proteolysis, invasion, and accumulation of ECM. It was shown in this study that ascochlorin, a prenylpenl antiobiotic, prevents the expression of profibrotic factors, such as PAI-1 and collagen type I, and that the TGF-β1-induced PAI-1 promoter activity is inhibited by ascochlorin. Ascochlorin abolishes the phosphorylation of the EGFR-MEK-ERK signaling pathway to regulate the TGF-β1-induced expression of PAI-1 without the inhibition of TβRII phosphorylation. Furthermore, the MEK inhibitor and EGFR siRNA block PAI-1 expression, and the Raf-1, MEK, and ERK signaling pathways for the regulation of PAI-1 expression. Ascochlorin suppresses the matrix metalloproteinases (MMPs) activity to activate the heparin-binding EGF-like growth factor (HB-EGF), to induce the phosphorylation of EGFR, and the MMPs inhibitor suppresses EGFR phosphorylation and the PAI-1 mRNA levels. These results suggest that ascochlorin prevents the expression of PAI-1 via the inhibition of an EGFR-dependent signal transduction pathway activated by MMPs.

Phase II trial of sorafenib in esophageal (E) and gastroesophageal junction (GEJ) cancer: Response and protracted stable disease observed in adenocarcinoma.

4100 Background: Sorafenib is a tyrosine kinase inhibitor targeting VEGFr, PDGFr, Raf and other pathways. Encouraging response and survival were observed in a phase II trial combining sorafenib with chemotherapy in GE cancer (J Clin Oncol 27:2947;2010). We are studying single agent sorafenib in a phase II trial with the primary endpoint to assess progression-free survival (PFS). Secondary endpoints include response and therapy tolerance. Methods: Patients (pts) with measurable metastatic E and GEJ cancer with no more than 3 prior chemotherapy regimens were treated with sorafenib 400 mg BID. CT scans were performed monthly for the first 2 months, then every 2 months. Results: Twenty of 35 pts have been accrued and 16 are currently evaluable, 17 male, 3 female, median KPS 80%, age 59, GEJ 6, E 14, squamous 2, adenocarcinoma (AC) 18. An ongoing complete response (15+ months) was observed in a pt with biopsy proven metastatic neck lymphadenopathy (E primary AC, recurrence after prior chemoradiotherapy and surgery). A second pt (GEJ AC) had protracted stable disease in bulky celiac node disease (19+ months). Grade 3 toxicity was limited to skin rash (1 pt), hand foot reaction (2 pt), and fatigue (1 pt). Only 4 of 16 pts (25%) had early disease progression at 2 months or less. Median PFS 3.7 mos, 4 patients (25%) received drug for more than 7 months. The majority of tumors tested positive for phospho-erk by immunohistochemistry (14/17, 75%). Conclusions: The observation of a durable complete response and protracted stable disease to sorafenib in E cancer is remarkable. Further accrual continues to define PFS. Supported by a grant from Bayer.

Abstract 3563: A highly selective Erk1/2 Inhibitor with in-vivo anti tumor potency

Abstract Mutations in receptor tyrosine kinases, Ras or B-Raf frequently manifest constitutive activation of the Ras/Raf/Mek/Erk pathway in a variety of human cancers. The mitogen activated protein kinase Erk1/2 is an important regulator of cell proliferation and was shown to be over-activated by these dysregulated or mutated upstream signals. AEZS-131 selectively inhibits Erk1/2 with an IC50 of 4 nM, blocks cellular Rsk-1 phosphorylation, modulates downstream cellular substrate activation, arrests tumor cells in G1 and inhibits the growth of multiple human tumor cell lines in the nanomolar range. We have studied the response of human cancer cells (Hct116) in in-vivo mouse xenograft experiments. AEZS-131 significantly inhibited tumor growth at daily doses of 30mg/kg. Currently, AEZS-131 is undergoing in-vivo combination experiments with PI3K inhibitors. Since pharmacological inhibition of Erk1/2 reverses Ras and Raf activation also in cells demonstrating resistancy to common Raf inhibitors like GDC-0879 and PLX4720, it appears likely to put more attention on the downstream kinase Erk1/2 as therapeutic target. Furthermore, in instances of coincident activation of the Raf and PI3K pathways, combinations of AEZS-131 and PI3K inhibitors may prove efficacious. Here we present the in-vitro and in-vivo characterisation of the first in-class Erk1/2 inhibitor showing in-vivo efficacy as single therapy agent. Early development of the Erk1/2 inhibitor AEZS-131 by Æterna Zentaris is integral part of our in-house kinase research program comprising the investigation of different compounds for single Erk1/2 inhibition, single PI3K inhibition and dual kinase inhibitions. All compounds are exclusively synthesized by our Drug Discovery Department. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2011-3563

IL-6 Increases MMP-13 Expression and Motility in Human Chondrosarcoma Cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. IL-6 is a multifunctional cytokine that is associated with the disease status and outcomes of cancers. However, the effect of IL-6 on the migration activity of human chondrosarcoma cells is mostly unknown. Here, we found that IL-6 increased the migration and expression of MMP-13 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of IL-6, which was higher than that in normal cartilage. IL-6-mediated migration and MMP-13 up-regulation were attenuated by anti-IL-6 receptor antibody, Ras, Raf-1, and a MEK inhibitor. Activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathways after IL-6 treatment was demonstrated, and IL-6-induced MMP-13 expression and migration activity were inhibited by the specific inhibitor and mutant Ras, Raf-1, MEK, ERK, and NF-κB cascades. In addition, migration-prone sublines demonstrated that cells with increasing migration ability had greater expression of IL-6 and MMP-13. Taken together, these results indicate that IL-6 and IL-6 receptor interaction enhances migration of chondrosarcoma through an increase in MMP-13 production.

Erratum: Ras and Raf pathways in epidermis development and carcinogenesis

The epidermis is the outermost layer of the body and protects it from environmental insults. This crucial function is sustained by a continuous process of self-renewal involving the carefully balanced proliferation and differentiation of progenitor cells constantly replacing the mature cells at the surface of the epidermis. Genetic changes in the signalling pathways controlling keratinocyte proliferation and differentiation disrupt this balance and lead to pathological changes including carcinogenesis. This review discusses the role of Ras, an oncogene critically involved in the development of skin neoplasia, and its downstream effector Raf in epidermal homeostasis and tumourigenesis. In particular, we will focus on the recently established role of Raf-1 as the decisive element that, by restraining keratinocyte differentiation, allows the development and maintenance of Ras-driven tumours.

Design and Optimization of Potent and Orally Bioavailable Tetrahydronaphthalene Raf Inhibitors

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.

Phase II trial of sorafenib in esophageal (E) and gastroesophageal junction (GEJ) cancer: Response observed in adenocarcinoma.

41 Background: Sorafenib is a tyrosine kinase inhibitor targeting VEGFr, PDGFr, Raf and other pathways. Encouraging response and survival were observed in a phase II trial combining sorafenib with chemotherapy in GE cancer (J Clin Oncol 27:2947;2010). We are studying single agent sorafenib in a phase II trial with the primary endpoint to assess progression free survival (PFS). Secondary endpoints include response and therapy tolerance. Methods: Patients (pts) with measurable metastatic E and GEJ cancer with no more than 3 prior chemotherapy regimens were treated with sorafenib 400 mg BID. CT scans were performed monthly for the first 2 months, then every 2 months. Results: Sixteen of 35 pts have been accrued and 14 are currently evaluable. 13 male, 3 female, median KPS 80%, age 58, GEJ 7, E 9, squamous 2, adenocarcinoma (AC)14. An ongoing complete response (11+ months) was observed in a pt with biopsy proven metastatic neck lymphadenopathy (E primary AC, recurrence after prior chemoradiotherapy and surgery). A second pt (GEJ AC) had protracted stable disease in bulky celiac node disease (15+ months). Grade 3 toxicity was limited to skin rash (1 pt), hand foot reaction (1 pt), and fatigue (1 pt). Only 3 of 14 pts (21%) had early disease progression at 2 months or less. Median PFS 4 mos, 4 patients (29%) continue on therapy for more than 7 months. The majority of tumors tested positive for phospho-erk by immunohistochemistry (11/14, 79%). Conclusions: The observation of a durable complete response and protracted stable disease to sorafenib in E cancer is remarkable. Further accrual continues to define PFS. Supported by a grant from Bayer. [Table: see text]

Ras and Raf pathways in epidermis development and carcinogenesis

The epidermis is the outermost layer of the body and protects it from environmental insults. This crucial function is sustained by a continuous process of self-renewal involving the carefully balanced proliferation and differentiation of progenitor cells constantly replacing the mature cells at the surface of the epidermis. Genetic changes in the signalling pathways controlling keratinocyte proliferation and differentiation disrupt this balance and lead to pathological changes including carcinogenesis. This review discusses the role of Ras, an oncogene critically involved in the development of skin neoplasia, and its downstream effector Raf in epidermal homeostasis and tumourigenesis. In particular, we will focus on the recently established role of Raf-1 as the decisive element that, by restraining keratinocyte differentiation, allows the development and maintenance of Ras-driven tumours.

Raf kinase inhibitor protein suppresses nuclear factor-κB-dependent cancer cell invasion through negative regulation of matrix metalloproteinase expression

Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor-κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP’s function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.

Recurrent BRAF mutations in Langerhans cell histiocytosis

AbstractLangerhans cell histiocytosis (LCH) has a broad spectrum of clinical behaviors; some cases are self-limited, whereas others involve multiple organs and cause significant mortality. Although Langerhans cells in LCH are clonal, their benign morphology and their lack (to date) of reported recurrent genomic abnormalities have suggested that LCH may not be a neoplasm. Here, using 2 orthogonal technologies for detecting cancer-associated mutations in formalin-fixed, paraffin-embedded material, we identified the oncogenic BRAF V600E mutation in 35 of 61 archived specimens (57%). TP53 and MET mutations were also observed in one sample each. BRAF V600E tended to appear in younger patients but was not associated with disease site or stage. Langerhans cells stained for phospho-mitogen–activated protein kinase kinase (phospho-MEK) and phospho-extracellular signal-regulated kinase (phospho-ERK) regardless of mutation status. High prevalence, recurrent BRAF mutations in LCH indicate that it is a neoplastic disease that may respond to RAF pathway inhibitors.

Rearrangements of the RAF kinase pathway in prostate cancer, gastric cancer and melanoma

Although recurrent gene fusions involving erythroblastosis virus E26 transformation-specific (ETS) family transcription factors are common in prostate cancer, their products are considered 'undruggable' by conventional approaches. Recently, rare targetable gene fusions involving the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, have been identified in 1-5% of lung cancers, suggesting that similar rare gene fusions may occur in other common epithelial cancers, including prostate cancer. Here we used paired-end transcriptome sequencing to screen ETS rearrangement-negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF (solute carrier family 45, member 3-v-raf murine sarcoma viral oncogene homolog B1) and ESRP1-RAF1 (epithelial splicing regulatory protein-1-v-raf-1 murine leukemia viral oncogene homolog-1) gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and mitogen-activated protein kinase kinase (MAP2K1) inhibitors. Screening a large cohort of patients, we found that, although rare, recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers and melanoma. Taken together, our results emphasize the key role of RAF family gene rearrangements in cancer, suggest that RAF and MEK inhibitors may be useful in a subset of gene fusion-harboring solid tumors and demonstrate that sequencing of tumor transcriptomes and genomes may lead to the identification of rare targetable fusions across cancer types.

Phase II trial of sorafenib in esophageal (E) and gastroesophageal junction (GEJ) cancer: Response observed in adenocarcinoma.

e14668 Background: Sorafenib is a tyrosine kinase inhibitor targeting VEGFr, PDGFr, Raf and other pathways. Encouraging response and survival were observed in a phase II trial combining sorafenib w...

Abstract 20: Raf kinase inhibitors can induce Raf dimerization, downstream signaling, and cell growth

Abstract Genetic alterations in the Ras/Raf/MEK/ERK pathway are among the most common in human cancers. Up to 70% of melanomas harbor B-Raf mutations, and roughly 90% of pancreatic tumors have K-Ras mutations. To address these Raf pathway-driven cancers, small molecule Raf kinase inhibitors have been developed and are currently under clinical investigation. In B-RafV600E cells, Raf compounds inhibit signaling through MEK and ERK, resulting in the expected anti-proliferative effects. Paradoxically, in wild-type Raf cells and in mutant Ras cells, these compounds induce downstream signaling and can induce cell growth in some settings in vitro. While the induction of downstream signaling has previously been attributed to published Raf pathway feedback loops, this has not been proven directly. In fact, we show here that induction of pMEK and pERK can occur within minutes of Raf compound treatment, even before reported feedback phosphorylation events are seen on B-Raf and C-Raf. Interestingly, the induction of signaling and cell growth both occur in a biphasic pattern, with low compound concentrations (0.01-0.1 uM) causing maximal induction, and higher compound concentrations (1-10 uM) causing less profound induction. Such a biphasic pattern is also observed in biochemical assays with purified wild-type B-Raf or C-Raf. The biphasic pattern is suggestive of a mechanism involving the interaction of two signaling subunits. In addition, recent literature data (Rajakulendran, Nature, 461:542-6) has demonstrated that Raf dimerization can upregulate pMEK, not through trans-phosphorylation of Raf molecules but presumably by conformational activation of the kinase. Consistent with that model, we show that Raf compound treatment induces B/C-Raf dimer formation in cells. In addition, knockdown of A-, B- or C-Raf with siRNA does not abrogate the Raf compound induction of pMEK and pERK, suggesting that induction might be mediated by Raf homo- as well as hetero-dimerization. Notably, knockdown of K-Ras in K-RasMUT cells also does not abolish the induction, implying that this effect is not mediated by Ras. Taken together, these data suggest a model in which compound binding to one Raf molecule induces dimerization and conformational activation of a partner Raf molecule in the dimer. These observations can explain why wild-type Raf and mutant Ras tumors are insensitive to selective Raf kinase inhibitors and might also have important implications for toxicity, since induction of strong mitogenic signaling could lead to hyperproliferation of normal tissues. Understanding the Raf compound induction mechanism may lead not only to the design of improved inhibitors, but also to methods for overcoming the induction seen with current development compounds. Toward that end, we show that combining a MEK inhibitor with a Raf compound causes inhibition of both pERK and cell growth and may therefore have significant advantages in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 20.

Abstract 623: The activation of EGFR-dependent signaling pathways participates in primary and acquired resistance to sorafenib in hepatoma cells

Abstract Context: The multityrosine kinase inhibitor sorafenib is the first and so far only drug to provide overall survival benefit in patients with advanced hepatocellular carcinoma (HCC). Sorafenib blocks the Raf pathway in tumor and endothelial cells as well as VEGFR and PDGFR activation in endothelial cells. Little is known about potential mechanisms that may modify tumor cell sensitivity towards sorafenib. The characterization of such mechanisms could help to alleviate primary or acquired resistance and improve this treatment. Here, we examined whether the activation of EGFR-dependent pathways modulate cell response to sorafenib in responsive or resistant human hepatoma cells. Methods: Five HCC cell lines (HuH7, Hep3B, HepG2, HuH6, PLC/PRF5) were treated with sorafenib alone or in combination with gefitinib, an EGFR TK inhibitor, for up to 72 h. A pool of Hep3B cells resistant to sorafenib (Hep3B-SR) was generated in culture by growing cells in increasing concentrations of sorafenib over a period of several months. Cell viability, DNA synthesis, apoptosis marker expressions (PARP and caspase-3), and pERK/pAKT/cyclin A levels were evaluated. A panel of 42 phosphorylated RTK was analyzed in Hep3B-SR using an antibody array kit (R&D Systems). Results: In the five HCC cell lines, the combination of gefitinib with sorafenib was more efficient in reducing cell viability than each drug alone. Supra-additive anti-proliferative and pro-apoptotic effects were observed in PLC/PRF5 and Hep3B cell lines. At the molecular level, the sorafenib/gefitinib combination markedly reduced pERK and to a lesser extent pAKT. In cell lines, which were the most responsive to combined therapy, sorafenib alone increased pERK, pAKT and cyclin A levels and that these paradoxical effects were abolished in the presence of gefitinib. Consistent with this result, the down-regulation of EGFR expression with a siRNA blunted the stimulatory effect of sorafenib on pERK. The activation of EGFR-dependent pathways under sorafenib treatment did not result from increased EGFR tyrosine phosphorylation. In addition, the analysis of a panel of 42 phosphorylated RTK (including EGFR) did not show any compensatory activation of receptors in the sorafenib-resistant pool Hep3B-SR compared to parental cells. However, Hep3B-SR cells displayed increased sensitivity to gefitinib associated with inhibition of pAKT and pERK levels although both their EGFR protein and mRNA levels were downregulated. Conclusions: these findings indicate that EGFR blockade reinforces the anti-tumoral action of sorafenib in HCC cells. This effect appears to be optimal in cells showing ERK and AKT activation under sorafenib treatment through an EGFR-dependent mechanism. Therefore the combination of EGFR inhibitors to sorafenib might be a new therapeutic strategy able to reduce intrinsic and acquired resistance of HCC to sorafenib. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 623.

CAMP‐dependent pathway(s) directs the B‐Raf MAPK‐Mediated Cytosolic Mislocalization of p27kip‐cyclin D1 in Renal Cancer

The loss of tuberin, the tuberous sclerosis‐2 (Tsc‐2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc‐2EK/+), and has been recently observed in human renal cell carcinoma tissue. Signaling associated with the expression and cytoplasmic mislocalization of p27 is relatively unknown in renal tumor development. TGHQ transformed renal epithelial cells derived from Tsc‐2EK/+ rats (QTRRE cells), that form renal tumors in nude mice, display cytoplasmic mislocalization of p27 and cyclin D1, and also exhibit high ERK, B‐Raf and Raf‐1 kinase activity. Dibutyryl cAMP or phosphodiesterase inhibitors ([PIs]; pentoxifylline or theophylline) cause an increase in cytosolic p27/cyclin D1 protein levels in QTRRE cells. Additionally, treatment of QTRRE cells with both PIs revealed concomitant decreases in p‐p27 T187 and in its E3 ubiquitin ligase skp1; proteosome inhibition resulted in increased p27/cyclin D1 protein levels. Inhibition of Raf kinases with either sorafenib or B‐Raf siRNA resulted in MAPK down‐regulation of p27. Moreover, an associated decrease in cyclin D1 was observed following p27 siRNA knockdown in QTRRE cells. Collectively these data suggest that the cAMP/B‐Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27‐cyclin D1 in tuberous sclerosis‐gene regulated‐renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27‐cyclin D1 cytosolic stabilization during renal tumor formation.

Xanthohumol inhibits the neuroendocrine transcription factor achaete-scute complex-like 1, suppresses proliferation, and induces phosphorylated ERK1/2 in medullary thyroid cancer

Background Achaete-scute complex-like 1 (ASCL1) is a transcription factor important in the malignant development of medullary thyroid cancer (MTC). Activation of Raf-1 signaling is associated with ASCL1 suppression and growth inhibition. Xanthohumol, a natural compound, has recently been shown to have anticancer properties. We thus hypothesized that xanthohumol would suppress growth by activating Raf-1 signaling, thus altering the malignant phenotype of MTC. Methods Human MTC cells were treated with xanthohumol (0–30 μmol/L) for up to 6 days. Proliferation was measured by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) colorimetric assay. Western blot analysis was performed for ASCL1 and markers of Raf-1 pathway activation. Results Treatment of MTC cells with xanthohumol resulted in a dose dependent inhibition of growth. Additionally, induction of phosphorylated ERK1/2 and a reduction of ASCL1 protein was noted. Conclusions Xanthohumol is a potent Raf-1 activator in MTC cells. This compound suppresses MTC growth, alters the malignant phenotype, and warrants further preclinical study.

The Synergistic Effects of the Somatostatin Analog Pasireotide (SOM230) and Raf-1 Pathway Activation in Human Carcinoid Cancer Cells

The Synergistic Effects of the Somatostatin Analog Pasireotide (SOM230) and Raf-1 Pathway Activation in Human Carcinoid Cancer Cells

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

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.