Kanika A. Bowen

mTORC1 and mTORC2 regulate EMT, motility and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways

Activation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with growth and progression of colorectal cancer (CRC). We have previously shown that the mTOR kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis of CRC. However, the contribution of mTOR and its interaction partners toward regulating CRC progression and metastasis remains poorly understood. We found that increased expression of mTOR, Raptor, and Rictor mRNA was noted with advanced stages of CRC, suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor, and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastases compared with normal colon. Inhibition of mTOR signaling, using rapamycin or stable inhibition of mTORC1 (Raptor) and mTORC2 (Rictor), attenuated migration and invasion of CRCs. Furthermore, knockdown of mTORC1 and mTORC2 induced a mesenchymal-epithelial transition (MET) and enhanced chemosensitivity of CRCs to oxaliplatin. We observed increased cell-cell contact and decreased actin cytoskeletal remodeling concomitant with decreased activation of the small GTPases, RhoA and Rac1, upon inhibition of both mTORC1 and mTORC2. Finally, establishment of CRC metastasis in vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating epithelial-mesenchymal transition (EMT), motility, and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC patients.

Targeted inhibition of mammalian target of rapamycin signaling inhibits tumorigenesis of colorectal cancer

Purpose: The mammalian target of rapamycin (mTOR) kinase acts downstream of phosphoinositide 3-kinase/Akt to regulate cellular growth, metabolism, and cytoskeleton. Because ∼60% of sporadic colorectal cancers (CRC) exhibit high levels of activated Akt, we determined whether downstream mTOR signaling pathway components are overexpressed and activated in CRCs. Experimental Design: HCT116, KM20, Caco-2, and SW480 human CRC cells were used to determine the effects of pharmacologic (using rapamycin) or genetic (using RNAi) blockade of mTOR signaling on cell proliferation, apoptosis, cell cycle progression, and subcutaneous growth in vivo. Results: We show that the mTOR complex proteins mTOR, Raptor, and Rictor are overexpressed in CRC. Treatment with rapamycin significantly decreased proliferation of certain CRC cell lines (rapamycin sensitive), whereas other cell lines were resistant to its effects (rapamycin resistant). Transient siRNA-mediated knockdown of the mTORC2 protein, Rictor, significantly decreased proliferation of both rapamycin-sensitive and rapamycin-resistant CRC cells. Stable shRNA-mediated knockdown of both mTORC1 and mTORC2 decreased proliferation, increased apoptosis, and attenuated cell cycle progression in rapamycin-sensitive CRCs. Moreover, stable knockdown of both mTORC1 and mTORC2 decreased proliferation and attenuated cell cycle progression, whereas only mTORC2 knockdown increased apoptosis in rapamycin-resistant CRCs. Finally, knockdown of both mTORC1 and mTORC2 inhibited growth of rapamycin-sensitive and rapamycin-resistant CRCs in vivo when implanted as tumor xenografts. Conclusions: Targeted inhibition of the mTORC2 protein, Rictor, leads to growth inhibition and induces apoptosis in both rapamycin-sensitive and rapamycin-resistant CRCs, suggesting that selective targeting of mTORC2 may represent a novel therapeutic strategy for treatment of CRC.(Clin Cancer Res 2009;15(23):7207–16)

VEGFR-2 expression in carcinoid cancer cells and its role in tumor growth and metastasis

Carcinoid tumors are slow growing and highly vascular neuroendocrine neoplasms that are increasing in incidence. Previously, we showed that carcinoid tumors express vascular endothelial growth factor receptor 2 (VEGFR‐2) in the epithelial compartment of carcinoid tumor sections; yet, its role is not completely understood. The purpose of our study was to: (i) assess the expression of VEGFR‐2 in the novel human carcinoid cell line BON, (ii) to determine the role of PI3K/Akt signaling on VEGFR‐2 expression and (iii) to assess the effect of VEGFR‐2 on BON cell invasion, migration and proliferation. We found that, although VEGFR‐2 is expressed in BON cells, reduction in VEGFR‐2 expression actually enhanced proliferation, invasion, and migration of the BON cell line. Also, expression of VEGFR‐2 was inversely related to PI3K signaling. Carcinoid liver metastases in mice demonstrated decreased VEGFR‐2 expression. Furthermore, the expression of a truncated, soluble form of VEGFR‐2 (sVEGFR‐2), a protein demonstrated to inhibit cell growth, was detected in BON cells. The presence of VEGFR‐2 in the epithelial component of carcinoid tumors and in the BON cell line suggests an alternate role for VEGFR‐2, in addition to its well‐defined role in angiogenesis. The expression of sVEGFR‐2 may explain the inverse relationship between VEGFR‐2 expression and PI3K/Akt signaling and the inhibitory effect VEGFR‐2 has on BON cell proliferation, migration and invasion.

An Analysis of Trends and Growth Factor Receptor Expression of GI Carcinoid Tumors

IntroductionThe purpose of our study was twofold: (1) to determine the incidence, patient and tumor characteristics, and outcome of patients with gastrointestinal carcinoid tumors using the Surveillance, Epidemiology and End Results (SEER) database, and (2) to delineate the expression pattern of growth factor receptors (GFRs) in carcinoid tumors.Materials and methodsThe SEER database search provided information on patients diagnosed with carcinoid tumors from 1990 to 2002. Carcinoid tumor sections (n = 46) were stained for the GFRs: epidermal growth factor receptor, insulin-like growth factor receptor (IGFR), vascular endothelial growth factor receptor (VEGFR), and HER-2/neu.ResultsOver the 12-year analysis period, 18,180 patients were identified with carcinoid tumors of the foregut, midgut, and hindgut; the incidence of carcinoid tumors increased ∼2-fold during this time period. Of the patients with carcinoid tumors, there was a trend of increased expression of VEGFR and IGFR, particularly in the foregut and midgut carcinoids. Analysis of the SEER database confirms that the incidence of carcinoid tumors is increasing with an approximate doubling in the number of carcinoid cases from 1990 to 2002. Furthermore, an increase in VEGFR and IGFR expression suggests that GFR inhibitors may be effective adjuvant therapy for carcinoid cancer.

Recent advances in neuroblastoma.

Purpose of review Although there have been recent advances with multimodal therapy, treatment of neuroblastoma remains a clinical challenge. Despite the identification of several genetic features, there has not been a significant increase in 5-year survival in the last decade. This review will highlight the current operative strategies along with new research developments aimed at improving survival. Recent findings The goal of surgical intervention in the early stages of neuroblastoma is complete curative resection. In advanced-stage disease, tissue biopsy for staging is the initial goal. In recent years, minimally invasive surgery (MIS) is considered in carefully selected patients. Recent advances in neuroblastoma research have focused on tyrosine kinase inhibition, differentiation, pathway inhibition, and immunotherapy. Several of these targets have shown promising results in vivo and are currently under investigation for potential clinical trials. Summary New information on the importance of cell signaling and the targeting of specific genes of interest are providing key insights into neuroblastoma. Only through the discovery of novel treatment strategies made available through the advancement of research will neuroblastoma be survivable for patients with advanced-stage disease.

Tumor necrosis factor–related apoptosis-inducing ligand promotes microvascular endothelial cell hyperpermeability through phosphatidylinositol 3-kinase pathway

BACKGROUND Microvascular hyperpermeability that occurs in hemorrhagic shock and burn trauma is regulated by the apoptotic signaling pathway. We hypothesized that tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL) would promote hyperpermeability directly or by interacting with other signaling pathways. METHODS Rat lung microvascular endothelial cells (RLMECs) grown on Transwell membranes (Corning Life Sciences, Lowell, MA) were treated with recombinant human TRAIL (10, 50, and 100 ng/mL) for 6 hours or TRAIL (100 ng/mL) + LY294002 (a PI3K inhibitor; 20 μmol/L), Z-DEVD-FMK (a caspase-3 inhibitor; 10 μmol/L), or the inhibitors alone. Fluorescein isothiocyanate (FITC)-albumin flux was an indicator of permeability. Caspase-3 activity was measured fluorometrically. Adherens junction integrity was studied using β-catenin immunofluorescence. RESULTS TRAIL + LY294002, but not TRAIL alone, induced monolayer hyperpermeability (P < .05), and caspase-3 activity (P < .05), and disrupted the adherens junctions. Z-DEVD-FMK attenuated hyperpermeability and protected the adherens junctions. CONCLUSIONS TRAIL-induced microvascular hyperpermeability is phosphatidylinositol 3-kinase (PI3K)-dependent and may be mediated by caspase-3 cleavage of the endothelial adherens junctional complex.

Thoracoscopic placement of phrenic nerve pacers for diaphragm pacing in congenital central hypoventilation syndrome

PURPOSE Congenital central hypoventilation syndrome (CCHS), or Ondines curse, is a rare disorder affecting central respiratory drive. Patients with this disorder fail to ventilate adequately and require lifelong ventilatory support. Diaphragm pacing is a form of ventilatory support which can improve mobility and/or remove the tracheostomy from CCHS patients. Little is known about complications and long-term outcomes of this procedure. METHODS A single-center retrospective review was performed of CCHS patients undergoing placement of phrenic nerve electrodes for diaphragm pacing between 2000 and 2012. Data abstracted from the medical record included operation duration, ventilation method, number of trocars required, and postoperative and pacing outcomes. RESULTS Charts of eighteen patients were reviewed. Mean surgical time was 3.3±0.7 hours. In all cases except one, three trocars were utilized for each hemithorax, with no conversions to open procedures. Five patients (27.8%) experienced postoperative complications. The mean ICU stay was 4.3±0.5 days, and the mean hospital stay is 5.7±0.3days. Eleven patients (61.1%) achieved their daily goal pacing times within the follow-up period. CONCLUSIONS Thoracoscopic placement of phrenic nerve electrodes for diaphragmatic pacing is a safe and effective treatment modality for CCHS. Observed complications were temporary, and the majority of patients were able to achieve pacing goals.

Research advances in neuroblastoma immunotherapy

Neuroblastoma is the third most common pediatric cancer in the United States and is responsible for 15% of pediatric cancer-related deaths. Despite major advances in multimodal therapy, the clinical outcome for several patients remains poor. Due to the desperate need for innovativation and improved success in the treatment and management of neuroblastoma, research interests in immunotherapy have been on the rise in recent years. Current immunotherapeutic approaches under investigation include antibodies targeting the neuroblastoma antigen GD2, cytokine stimulation of immune cells, use of immunocytokine conjugates, radioimmunotherapy, and tumor-primed dendritic cells. Immunotherapy could serve as a safe alternative or adjunct to current therapeutic protocols and would presumptively have fewer deleterious effects making it more favorable to patients.

Trophic Effects of Gut Hormones in the Gastrointestinal Tract

Publisher Summary This chapter focuses on the effects of gastrointestinal (GI) hormones on the proliferation and repair of non-neoplastic tissues, and the receptors and signaling pathways, which transmit signals from the cell surface to the nucleus. Gastrin is the GI hormone that stimulates acid secretion from gastric parietal cells, and is the single most important trophic hormone for the gastric mucosa. One of the peptides that stimulates gastric mucosal proliferation is bombesin (BBS)/gastrin-releasing peptide (GRP), which stimulates pancreatic, gastric, and intestinal secretion, gut motility, and smooth muscle contraction, and release of all gut hormones. These peptides can stimulate growth of GI mucosa and pancreas. BBS also stimulates growth of the small bowel mucosa. Administration of BBS effectively prevented mucosal atrophy associated with feeding rats a liquid elemental diet. GI hormone-stimulated signal transduction occurs with the binding of hormones to their cognate cell surface receptors, which are G-protein-coupled receptors (GPCR). These receptors have the typical structural features of G-protein-binding seven-transmembrane receptors, which are G-protein-coupled receptors (GPCR). These receptors have the typical structural features of G-protein-binding seven-transmembrane receptors which can regulate a number of physiological processes, including proliferation, growth, and development. The molecular mechanisms though which GPCRs transduce signals are complex, and likely involve multiple signaling pathways. In addition, the signaling pathways are likely cell-specific, which may explain the diverse physiologic functions controlled by gut hormones, ranging from regulation of secretion, motility, and in some instances growth, depending on the target tissue. Once a trophic GI peptide binds its seven-transmembrane GPCR, signal transduction pathways are activated which ultimately can lead to cell proliferation depending upon cell type.

Abstract A41: Combined inhibition of mTOR and MAPK signaling attenuates the growth and progression of colorectal cancer

The PI3K/Akt/mTOR and Ras/Raf/ERK (MAPK) signaling pathways are constitutively activated and serve critical functions in the pathogenesis of colorectal cancer (CRC). mTOR signaling is involved in cell growth and metabolism; rapamycin inhibits the kinase activity of mTORC1 but leads to feedback activation of upstream PI3K/Akt signaling, resulting in cell survival and chemoresistance. MAPK signaling is involved in cell proliferation and differentiation; sorafenib is a multikinase inhibitor that inhibits Raf kinase. Recent studies suggest that inhibition of mTORC1 with rapamycin leads to feedback induction of MAPK signaling through a S6K-PI3K-Ras feedback loop. Therefore, we investigated whether dual targeting of mTOR and MAPK signaling pathways, using rapamycin and sorafenib, respectively, has therapeutic benefits in CRC. Methods: HCT116 human CRC cells (mutant K-Ras; wild-type B-Raf) were treated with either rapamycin (50 nM) or sorafenib (5 µM) alone or in combination. Effects on cell proliferation (measured by cell counting), apoptosis (measured by detecting the level of histone- associated DNA fragments in mono- and oligonucleosomes), cell cycle progression (measured by fluorescence-activated cell sorting), migration (measured by wound healing assay) and invasion through matrigel (measured by modified Boyden chamber assay) were analyzed. Results: (i) pAktSer473 and pERKThr202/Tyr204 are overexpressed together in stage IV CRC tissues and matched liver metastasis tissues from the same patient (Spearman9s correlation=0.77; p Conclusions: K-Ras mutant CRCs, which display clinical resistance to anti-EGFR therapy, are sensitive to the anti-tumorigenic effect of sorafenib. Furthermore, combinatorial inhibition of mTOR and MAPK signaling pathways, using rapamycin and sorafenib, respectively, significantly potentiates attenuation of growth and progression compared to monotherapy in K-Ras mutant CRCs. This effect may be due to reduced feedback activation of PI3K/Akt and MAPK signaling. CRC is a heterogeneous disease with multiple aberrant signaling pathways driving the oncogenic behavior of tumors; simultaneous inhibition of parallel signaling pathways may be more effective than single pathway inhibition in CRC therapies. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A41.