Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis

Extracellular-signal regulated kinase (ERK) activation by MEK plays a key role in many of the cellular processes that underlie progressive kidney fibrosis including cell proliferation, apoptosis and transforming growth factor β1-mediated epithelial to mesenchymal transition. We therefore assessed the therapeutic impact of ERK1/2 inhibition using a MEK inhibitor in the rat 5/6 subtotal nephrectomy (SNx) model of kidney fibrosis. There was a twentyfold upregulation in phospho-ERK1/2 expression in the kidney after SNx in Male Wistar rats. Rats undergoing SNx became hypertensive, proteinuric and developed progressive kidney failure with reduced creatinine clearance. Treatment with the MEK inhibitor, CI-1040 abolished phospho- ERK1/2 expression in kidney tissue and prevented phospho-ERK1/2 expression in peripheral lymphocytes during the entire course of therapy. CI-1040 had no impact on creatinine clearance, proteinuria, glomerular and tubular fibrosis, and α-smooth muscle actin expression. However, inhibition of ERK1/2 activation led to significant compensatory upregulation of the MAP kinases, p38 and JNK in kidney tissue. CI-1040 also increased the expression of plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasmin-dependent matrix metalloproteinases. Thus inhibition of ERK1/2 activation has no therapeutic effect on kidney fibrosis in SNx possibly due to increased compensatory activation of the p38 and JNK signalling pathways with subsequent upregulation of PAI-1

Depression in Patients with Mastocytosis: Prevalence, Features and Effects of Masitinib Therapy

Depression in patients with mastocytosis is often reported but its prevalence and characteristics are not precisely described. In addition, the impact of therapies targeting mast cells proliferation, differentiation and degranulation on psychic symptoms of depression have never been investigated. Our objective was to determine the prevalence and to describe features of depression in a large cohort of mastocytosis patients (n = 288) and to investigate the therapeutic impact of the protein kinase inhibitor masitinib in depression symptoms. The description of depression was based on the analysis of a database with Hamilton scores using Principal Component Analysis (PCA). Efficacy of masitinib therapy was evaluated using non parametric Wilcoxon test for paired data within a three months period (n = 35). Our results show that patients with indolent mastocytosis present an elevated prevalence of depression (64%). Depression was moderate in 56% but severe in 8% of cases. Core symptoms (such as psychic anxiety, depressed mood, work and interests) characterized depression in mastocytosis patients. Masitinib therapy was associated with significant improvement (67% of the cases) of overall depression, with 75% of recovery cases. Global Quality of Life slightly improved after masitinib therapy and did not predicted depression improvement. In conclusion, depression is very frequent in mastocytosis patients and masitinib therapy is associated with the reduction its psychic experiences. We conclude that depression in mastocytosis may originate from processes related to mast cells activation. Masitinib could therefore be a useful treatment for mastocytosis patients with depression and anxiety symptoms

The clinical implications of sunitinib-induced hypothyroidism: a prospective evaluation

Sunitinib is approved for the treatment of metastatic renal cell carcinoma (RCC) and imatinib-resistant or -intolerant gastrointestinal stromal tumours (GIST). Several studies have identified unexpected rates of thyroid dysfunction with sunitinib treatment. We performed a prospective observational study with the aim of more accurately defining the incidence and severity of hypothyroidism in RCC or GIST patients receiving sunitinib. Thyroid function was assessed at baseline and on days 1 and 28 of each treatment cycle. Thyroid antibodies were assessed at baseline and during follow-up if abnormal thyroid function tests were recorded. Sixteen patients (27%) developed sub- or clinical hypothyroidism and required hormone replacement and 20 patients (34%) showed at least one elevated thyroid-stimulating hormone not requiring therapeutic intervention. Twenty patients (34%) did not develop any biochemical thyroid abnormality. Thus, sunitinib can induce (sub-) clinical hypothyroidism, warranting close monitoring of thyroid function. We propose a new algorithm for managing this side effect in clinical practise

Upregulation of MAPK pathway is associated with survival in castrate-resistant prostate cancer

BACKGROUND: Recent evidence has implicated the MAP kinase (MAPK) pathway with the development of castrate-resistant prostate cancer (CRPC). We have previously reported gene amplification of critical members of this pathway with the development of castrate-resistant disease. In addition, we have shown that rising Raf-1 expression, with the development of CRPC, influences time to biochemical relapse. We therefore sought to further analyse the role of both Raf-1 and its downstream target MAPK in the molecular pathogenesis of CRPC. METHODS: Protein expression of Raf-1 and MAPK, including their activation status, was analysed using immunohistochemistry in a database of 65 paired tumour specimens obtained before and after the development of CRPC and correlated with other members of the pathway. RESULTS: Patients whose nuclear expression of MAPK rose with the development of CRPC had a significantly shorter median time to death following biochemical relapse (1.40 vs 3.00 years, P=0.0255) as well as reduced disease-specific survival when compared with those whose expression fell or remained unchanged (1.16 vs 2.62 years, P=0.0005). Significant correlations were observed between protein expression of Raf-1 and MAPK with the type 1 receptor tyrosine kinases, Her2 and epidermal growth factor receptor, as well as the transcription factor AP-1 in CRPC tumours. CONCLUSION: We conclude that the Her2/Raf-1/MAPK/AP-1 axis may promote the development of CRPC, leading to early relapse, and reduced disease-specific survival. In addition, members of the pathway may act as novel therapeutic and/or diagnostic targets for prostate cancer. British Journal of Cancer (2011) 104, 1920-1928. doi:10.1038/bjc.2011.163 www.bjcancer.com Published online 10 May 2011 (C) 2011 Cancer Research U

Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

<p>Abstract</p> <p>Background</p> <p>Nerve growth factor (NGF) is a potent growth factor that plays a key role in neuronal cell differentiation and may also play a role in hematopoietic differentiation. It has been shown that NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with macrophage-colony stimulating factor (M-CSF or CSF-1), or stem cell factor (SCF). However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells.</p> <p>Results</p> <p>To study the signal transduction pathways induced by NGF treatment in hematopoietic cells, we utilized the mastocytoma cell line HMC-1(V560G c-Kit) which expresses the NGF receptor, tropomyosin-receptor-kinase (Trk)A, as well as the constitutively activated SCF receptor, V560G c-Kit, which can be inhibited completely by treatment with the potent tyrosine kinase inhibitor imatinib mesylate (imatinib). NGF rescues HMC-1(V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation. To examine the NGF mediated proliferation and survival in these cells, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit activity for 4 h) by transcriptome analysis. The following conclusions can be drawn from the microarray data: Firstly, gene expression profiling reveals 50% overlap of genes induced by NGF-TrkA with genes expressed downstream of V560G c-Kit. Secondly, NGF treatment does not enhance expression of genes involved in immune related functions that were down regulated by imatinib treatment. Thirdly, more than 55% of common upregulated genes are involved in cell proliferation and survival. Fourthly, we found Kruppel-like factor (KLF) 2 and Smad family member 7 (SMAD7) as the NGF mediated novel downstream genes in hematopoietic cells. Finally, the downregulation of KLF2 gene enhanced imatinib induced apoptosis.</p> <p>Conclusion</p> <p>NGF does not induce genes which are involved in immune related functions, but induces proliferation and survival signals in HMC-1(V560G c-Kit) cells. Furthermore, the current data provide novel candidate genes, KLF2 and SMAD7 which are induced by NGF/TrkA activation in hematopoietic cells. Since the depletion of KLF2 causes enhanced apoptosis of HMC-1(V560G c-Kit), KLF2 may play a role in the NGF mediated survival signal.</p

Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using Fluorescence in situ Hybridization (FISH) and immunohistochemistry

<p>Abstract</p> <p>Background</p> <p>Esophageal squamous cell carcinoma (ESCC) is the sixth most frequent neoplasia in Brazil. It is usually associated with a poor prognosis because it is often at an advanced stage when diagnosed and there is a high frequency of lymph node metastases. It is important to know what prognostic factors can facilitate diagnosis, optimize therapeutic decisions, and improve the survival of these patients. A member of the epidermal growth factor receptor (EGFR) family, c-erbB-2, has received much attention because of its therapeutic implications; however, few studies involving fluorescence <it>in situ </it>hybridization (FISH) analysis of HER-2/neu gene amplification and protein expression in ESCC have been conducted. The aim of this study was to verify the presence of HER-2/neu gene amplification using FISH, and to correlate the results with immunohistochemical expression and clinical-pathological findings.</p> <p>Methods</p> <p>One hundred and ninety-nine ESCC cases were evaluated using the Tissue Microarray (TMA) technique. A polyclonal antibody against c-erbB-2 was used for immunohistochemistry. Analyses were based on the membrane staining pattern. The results were classified according to the Herceptest criteria (DAKO): negative (0/1+), potential positive (2+) and positive (3+). The FISH reactions were performed according to the FISH HER2 PharmDx (DAKO) protocol. In each case, 100 tumor nuclei were evaluated. Cases showing a gene/CEN17 fluorescence ratio ≥ 2 were considered positive for gene amplification.</p> <p>Results</p> <p>The c-erbB-2 expression was negative in 117/185 cases (63.2%) and positive in 68 (36.8%), of which 56 (30.3%) were 2+ and 12 (6.5%) were 3+. No significant associations were found among protein expression, clinicopathological data and overall survival. Among the 47 cases analyzed, 38 (80.9%) showed no gene amplification while 9 (19.1%) showed amplification, as demonstrated by FISH. Cases that were negative (0/1+) and potential positive (2+) for c-erbB-2 expression by immunohistochemistry showed no gene amplification. However, all cases with gene amplification were positive (3+) by immunohistochemistry. According to univariate analysis, there was a significant difference (p = 0.003) in survival rates when cases with and without HER-2/neu amplification were compared.</p> <p>Conclusion</p> <p>Our data demonstrate the correspondence between gene amplification and protein expression of HER-2/neu. Gene amplification is an indicator of poor prognosis in ESCC.</p

The Adnectin CT-322 is a novel VEGF receptor 2 inhibitor that decreases tumor burden in an orthotopic mouse model of pancreatic cancer

<p>Abstract</p> <p>Background</p> <p>Pancreatic cancer continues to have a 5-year survival of less than 5%. Therefore, more effective therapies are necessary to improve prognosis in this disease. Angiogenesis is required for tumor growth, and subsequently, mediators of angiogenesis are attractive targets for therapy. Vascular endothelial growth factor (VEGF) is a well-characterized mediator of tumor angiogenesis that functions primarily by binding and activating VEGF receptor 2 (VEGFR2). In this study, we evaluate the use of CT-322, a novel biologic (Adnectin). This small protein is based on a human fibronectin domain and has beneficial properties in that it is fully human, stable, and is produced in bacteria. CT-322 binds to and inhibits activation of VEGFR2.</p> <p>Methods</p> <p>The efficacy of CT-322 was evaluated <it>in vivo </it>using two orthotopic pancreatic tumor models. The first model was a human tumor xenograft where MiaPaCa-2 cells were injected into the tail of the pancreas of nude mice. The second model was a syngeneic tumor using Pan02 cells injected into pancreas of C57BL/6J mice. In both models, therapy was initiated once primary tumors were established. Mice bearing MiaPaCa-2 tumors were treated with vehicle or CT-322 alone. Gemcitabine alone or in combination with CT-322 was added to the treatment regimen of mice bearing Pan02 tumors. Therapy was given twice a week for six weeks, after which the animals were sacrificed and evaluated (grossly and histologically) for primary and metastatic tumor burden. Primary tumors were also evaluated by immunohistochemistry for the level of apoptosis (TUNEL), microvessel density (MECA-32), and VEGF-activated blood vessels (Gv39M).</p> <p>Results</p> <p>Treatment with CT-322 was effective at preventing pancreatic tumor growth and metastasis in orthotopic xenograft and syngeneic models of pancreatic cancer. Additionally, CT-322 treatment increased apoptosis, reduced microvessel density and reduced the number of VEGF-activated blood vessels in tumors. Finally, CT-322, in combination with gemcitabine was safe and effective at controlling the growth of syngeneic pancreatic tumors in immunocompetent mice.</p> <p>Conclusion</p> <p>We conclude that CT-322 is an effective anti-VEGFR2 agent and that further investigation of CT-322 for the treatment of pancreatic cancer is warranted.</p

Severe paraneoplastic hypoglycemia in a patient with a gastrointestinal stromal tumor with an exon 9 mutation: a case report

BACKGROUND: Non-islet cell tumor induced hypoglycemia (NICTH) is a very rare phenomenon, but even more so in gastrointestinal stromal tumors. It tends to present in large or metastatic tumors, and can appear at any time in the progression of the disease. We present herein a case of NICTH in a GIST tumor and report an exon 9 mutation associated to it. CASE PRESENTATION: A thirty nine year-old man with a recurrent, metastatic gastrointestinal stromal tumor presented to the hospital with nausea, dizziness, loss of consciousness, and profound hypoglycemia (20 mg/dL). There was no evidence of factitious hypoglycemia. He was stabilized with a continuous glucose infusion and following selective vascular embolization, the patient underwent debulking of a multicentric 40 cm × 25 cm × 10 cm gastrointestinal stromal tumor. After resection, the patient became euglycemic and returned to his normal activities. Tumor analysis confirmed excessive production of insulin-like growth factor II m-RNA and the precursor protein, "big" insulin-like growth factor II. Mutational analysis also identified a rare, 6 bp tandem repeat insert (gcctat) at position 1530 in exon 9 of KIT. CONCLUSION: Optimal management of gastrointestinal stromal tumor-induced hypoglycemia requires a multidisciplinary approach, and surgical debulking is the treatment of choice to obtain immediate symptom relief. Imatinib or combinations of glucocorticoids and growth hormone are alternative palliative strategies for symptomatic hypoglycemia. In addition, mutations in exon 9 of the tyrosine kinase receptor KIT occur in 11–20% of GIST and are often associated with poor patient outcomes. The association of this KIT mutation with non-islet cell tumor induced hypoglycemia has yet to be established

Lymphatic vessel density and VEGF-C expression are significantly different among benign and malignant thyroid lesions

Thyroid cancer is the most frequent endocrine neoplasia worldwide. The route for metastasis and loco-regional invasion preferentially occurs by lymphatic vessels. For this reason, the assessment of lymphatic vessel density (LVD) is supposed to represent both a prognostic parameter and also a potential therapeutic target. In order to evaluate the value of LVD in benign and malignant thyroid lesions, we analyzed 110 thyroidectomy specimens using D2-40, a specific marker for lymphatic vessels and vascular endothelial growth factor C (VEGF-C), the most potent molecule of lymphatic proliferation. LVD was significantly different between papillary and follicular carcinomas in total (p = 0.045) and peritumoral area (p = 0.042). Follicular adenoma and follicular carcinoma showed an important difference of intra- (p = 0.019) and peritumoral (p = 0.033) LVD. VEGF-C was more markedly expressed in malignancies than in benign lesions (p = 0.0001). Almost all cancers with high positive VEGF-C expression also exhibited increased peritumoral LVD (p = 0.049) when compared with the benign lesions. Indeed, the high peritumoral LVD of malignant thyroid lesions is an important finding for surgery planning and supports the practice of total thyroidectomy in malignant thyroid neoplasm's since the lymphatic peritumoral vessels definitely are an escape path for tumor cells