Downregulation Of p-ERK Research Articles

A phase I study of systemic sorafenib in combination with isolated limb infusion with melphalan (ILI-M) in patients (pts) with locally advanced in-transit melanoma

9065 Background: Sorafenib is a multi-kinase inhibitor that may also enhance the cytotoxicity of concurrently administered chemotherapy. In a preclinical model of regionally advanced melanoma, the combination of systemic Sorafenib and regional melphalan led to augmented tumor responses. Methods: A Phase I multicenter study was performed to evaluate the safety and pharmacokinetics (PK) of systemic Sorafenib in combination with ILI-M in patients with measurable in-transit melanoma of the extremity. Sorafenib dose escalation cohorts consisted of 200mg, 300mg, and 400mg administered systemically twice daily for one week prior and one week after a standard dose ILI-M corrected for ideal body weight. Tumor biopsies pre-therapy and pre-ILI were obtained to assess molecular changes associated with Sorafenib pretreatment. Response was defined at 3 months using RECIST. Results: Nine pts with high disease burden, including 7 previous ILI-M alone failures, have been treated; 3 in the first cohort and 6 in the second cohort. There were no grade 5 toxicities. Four patients had CTCAE Grade 4 toxicities including neutropenia (2), CPK elevation (1), and skin ulceration (1). In the remaining 5 patients, there were no >grade 3 toxicities. The maximally tolerated dose (MTD) has not yet been defined. Initial in field response determination in 6 of the 9 patients out at least 3 months includes 2 partial responses and 4 disease progressions. Conclusions: Systemic Sorafenib administered pre and post ILI-M is a well tolerated, novel targeted therapy approach to regionally advanced melanoma. An additional 10 patients will be enrolled to define the MTD. Correlation of response with drug PK, Sorafenib downregulation of pErk and Mcl1, and a melphalan resistance signature is in progress. No significant financial relationships to disclose.

Recombinant Human Erythropoietin Protects against Experimental Spinal Cord Trauma Injury by Regulating Expression of the Proteins MKP-1 and p-ERK

The present study explored the tissue-protective effect of erythropoietin in rats after experimental spinal cord injury (SCI) produced by dropping a weight onto surgically exposed spinal cord. Sixty rats were randomized to sham operation (spinal cord exposure; control), SCI plus intraperitoneal saline injection, or SCI plus intraperitoneal erythropoietin injection. Locomotor function was evaluated with Basso, Beattie and Bresnahan scores 1 day (24 h) and 7 days later, and rats were then killed for analysis of lesion site tissue. Compared with saline-treated SCI rats, erythropoietin-treated SCI rats showed significantly less locomotor dysfunction and faster locomotor recovery. Immunohistochemistry showed that erythropoietin-treated SCI rats had a significantly lower phospho-extracellular signal-regulated kinase (p-ERK) protein expression and a significantly higher mitogen-activated protein kinase phosphatase-1 (MKP-1) protein expression than saline-treated SCI rats. Haematoxylin-eosin staining showed progressive disruption of dorsal white matter and neuron loss after SCI; lesions were less severe and there was more neuron regeneration in the erythropoietin group than in the saline group. It is concluded that erythropoietin reduces pathological changes and SCI severity via down-regulation of p-ERK and up-regulation of MKP-1.

Combination of Sorafenib, Idarubicin, and Cytarabine Has a High Response Rate in Patients with Newly Diagnosed Acute Myeloid Leukemia (AML) Younger Than 65 Years

Background: Sorafenib is an orally active multi-kinase inhibitor with potent activity against the Raf/ERK/MEK pathway, VEGFR, PDGFR-β, and c-KIT. In vitro, it has growth-inhibitory effects in several AML cell lines with or without constitutive activation of ERK signaling. Sorafenib selectively induces cell growth arrest and apoptosis in FLT3-mutant human AML cell lines at nM concentrations. In a phase I study of single agent sorafenib in patients (pts) with AML escalating doses were well tolerated with no myelosuppression and with significant clinical activity predominantly (but not exclusively) in FLT3 mutated pts.Methods: This study was conducted to determine the tolerability and efficacy of combination of sorafenib with cytarabine 1.5 g/m2 iv over 24 hours daily × 4 (× 3 for pts over 60) and idarubicin 12 mg/m2 iv daily × 3. In the phase I portion of study, pts with relapsed AML were treated with escalating doses of sorafenib po (400 mg qod, 400 mg daily and 400 mg bid) for 7 days during induction, and 400 mg bid was established as a safe dose for phase II evaluation. Pts achieving CR receive up to 5 courses of consolidation with idarubicin 8 mg/m2 iv daily × 2 and cytarabine 0.75 g/m2 iv daily × 3 in addition to continuous sorafenib 400 mg po bid for up to 28 days per cycle. Maintenance with sorafenib 400 mg bid would continue for up to a year after consolidation.Results: Ten pts (median age 34 years, range 21–58) with relapsed AML (median prior therapy 2, range 1–6) were treated on the phase I portion. Seven had FLT3-ITD mutation (5 with high mutation burden, 2 with low), and 3 were negative. Four achieved CR, and 6 failed. In the phase II portion, 30 pts (including 8 with FLT3-ITD and 2 with FLT3-TKD) have been treated. Median age is 53 years (range 18 – 65) Cytogenetics were diploid in 13, +8 in 3, −5/−7 in 3, t(9;11) in 1, miscellaneous in 6, and unavailable in 4. The median presentation WBC was 4.6 × 109/L (range 1.5 –122.7 × 109/L). FLT3 mutation burden was low in blasts from 4 pts, and high in 6). Five pts were FLT3-ITD+/NPM1-. Among 25 evaluable pts, 22 (88%) have achieved CR (n=19), or CRi (n=3); 1 achieved PR, 1 died at induction from pneumonia, 1 was resistant; 5 pts are too early. The regimen is well tolerated and grade 3 adverse events thought to be possibly related to the study combination have included elevation of transaminases (3), hyperbilirubinemia (4), small bowel obstruction (1), diarrhea (2), rash (2), pericarditis (1), elevated creatinine (1), and atrial fibrillation (1). Median follow-up is 8 weeks (range, 1 – 28) with the probability of survival at 6 months of 87%; 2 pts have relapsed with CR durations of 2 and 3 months. Samples from 8 pts were studied prior to and 24–48 hours post sorafenib administration, and prior to chemotherapy. In six pts (75%), sorafenib alone induced apoptosis in peripheral blood blasts and in CD33/CD34 positive leukemia progenitor cells as determined by flow cytometry. Expression of phospho-ERK (pERK) was detectable by flow cytometry in 5 out of 7 samples tested at baseline; 24-hour exposure to sorafenib resulted in >50% downregulation of pERK in 3 of the 5 samples. Plasma inhibitory assay was performed using day 7 samples from 10 pts; mutant FLT3 was suppressed by all samples with 5-fold more potent suppression against mutant versus wildtype FLT3.Conclusions: Combination of sorafenib with idarubicin and cytarabine is safe and has a high CR rate in frontline therapy of younger pts with AML. Correlative studies confirm potent activity of sorafenib against ERK and FLT3 signaling.

Raf-1 Is Over Expressed in Chronic Lymphocytic Leukemia and Promotes Cell Survival by Phosphorylation of ERK and BAD

Introduction: The serine threonine kinase Raf-1 plays a protective role in many cell types, but its expression and function in CLL cells has not been studied in detail. In the present study, we analyzed Raf-1 expression and tested the hypothesis that Raf-1 is critical for CLL cell survival.Materials and Methods: By using qRT-PCR and western blot, we compared the expression of Raf-1 of mRNA and protein levels in purified B cells from 45 CLL cases and CD38 negative B cells from 5 reactive tonsils. By western blot, we analyzed the activity of phospho-Raf-1 (ser338) and its downstream targets (phospho-ERK1/2, phospho-BAD) in 23 CLL cases and 4 CLL cell lines (JVM-3, MEC-1, MEC-2 and MO1043) before and after IgM stimulation. By immunoprecipitation, we analyzed if Raf-1 co-localizes with Bcl-2. We correlated the change in phosphorylation status (Raf-1, ERK and BAD) in response to IgM stimulation with the ZAP-70/SYK mRNA ratio, which was detected by qRT-PCR in purified B cells from CLL cases. After using specific inhibitors, including the Raf-1 inhibitor GW5074, the Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137, we investigated apoptosis by Annexin V flowcytometry in CLL cases and CLL cell lines as well as cell cycle changes in the 4 cell lines by flowcytometry.Results: In comparison to normal resting (CD38 negative) B cells, there was a strongoverexpression of Raf-1 in CLL cells, both at at the mRNA and protein level. Using qRT-PCR there was an almost linear correlation between Raf-1 and Bcl-2 expression. Moreover, the phosphorylation status of Raf-1 and ERK in response to IgM stimulation strongly correlated with the ZAP-70/SYK mRNA ratio. Using immunoprecipitation and confocal miscroscopy we found colocalization of Raf-1 with Bcl-2, which might account for the observed constitutive activation of BAD in CLL cells. The Raf-1 inhibitor GW5074, Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137 all led to p-Raf-1 inhibition as well as downregulation of p-ERK and p-BAD. Additionally, all three inhibitors downregulated cyclin D3 and cyclin E, which are important for G0/G1 transition. We also found that GW5074 induced apoptosis in CLL cell lines and primary cells of CLL cases.Conclusion/Discussion: In conclusion, our study identifies Raf-1 as a critical anti-apoptotic and cell cycle regulating kinase in CLL cells.

Inhibitory role of focal adhesion kinase on anoikis in the lung cancer cell A549

Resistance to anoikis is a characteristic of malignant cells with increased tumorigenesis and metastasis. Altered FAK activity has been strongly implicated in the development, growth, progression, and metastasis of human cancers, but the mechanism of FAK in regulating anoikis is unknown. In this study, the resistance anoikis role of FAK and its downstream mediators was evaluated in the human lung cancer cell line A549. It has been shown that down regulation of FAK stimulates the apoptosis of cells and the down-regulation of p-ERK, p-PI3K, p-Src, and p-p38. Furthermore, in detached A549 cells, increased FAK phosphorylations (Tyr397, Tyr861, Tyr925) were detected in a time-dependent manner, and the specific inhibitors of MEK1, PI3K, and Src (PD98059, LY294002, and PP2) partly abolished the resistance to the anoikis characteristic of cancer cells. Altogether, our data suggested that Src is involved in the progress of detachment-induced FAK activation in lung tumor cells. PI3K/AKT, MAPK-ERK, and perhaps MAPK-p38 but not MAPK-JNK, appear to be the key downstream effectors of FAK in mediating cell survival. The increased FAK activity upon cell detachment may contribute to the metastasis potential of malignant tumors.

Effects of Oxymatrine on the apoptosis of human esophageal carcinoma Eca109 cell line and its mechanism

The effects of Oxymatrine (Oxy) on the proliferation and apoptosis of human esophageal carcinoma Eca109 cell line and the mechanism were investigated. The human esophageal carcinoma Eca109 cells were cultured in vitro. The Oxy-induced apoptosis of Eca 109 cells was assayed by using flow cytometry. The expressions of p-ERK(1/2), Cyclin D1, p21(waf/cip1), Bax and Bcl-2 were detected by Western blot. Flow cytometry revealed that Oxy could induce the apoptosis of Eca109 cells. Western blot showed that Oxy of different concentrations suppressed the expressions of p-ERK(1/2), Cyclin D1 and Bcl-2, but up-regulated the expression of p21(waf/cip1) and Bax, and the ratio of Bax/Bcl-2 was increased. It was suggested the Oxy could induce the apoptosis of Eca109 cells, which might be related to the upregulation of p21(waf/cip1) and the downregulation of p-ERK(1/2), Cyclin D1 and p21(waf/cip1). The possible pathway may be related to Bcl-2/Bax.

Targeting CCR1 for the Treatment of Osteolytic Bone Disease in Multiple Myeloma.

Osteolytic bone disease (OBD) is a frequent complication of multiple myeloma (MM), affecting 70 to 80% of the patients. OBD is characterized by imbalanced bone remodeling, due to decreased osteoblast (OB) number and increased osteoclast (OC) formation and activity. MM cells secrete osteoclastogenic factors, such as receptor activator of nuclear factor kappa B ligand (RANKL) and CCL3. In turn, OC support MM cell proliferation and survival, thus promoting a positive feedback that exacerbates bone resorption. Chemokines modulate osteoclastogenesis and promote MM cell proliferation, in particular CCL3 and its receptor CCR1 play an important role in mediating OBD in MM. MLN3897 (Millennium Pharmaceuticals, Cambridge) is a novel small molecule specific antagonist of human CCR1 (IC50 0.8 nM). It has a favorable toxicity profile in healthy volunteers and is currently undergoing phase II clinical trials in rheumatoid arthritis and multiple sclerosis. Here we evaluate the effects of MLN3897 on OC function and activity, as well as OC-MM cell interactions. Our in vitro data demonstrates a dual mechanism of action for MLN3897: it inhibits osteoclastogenesis and also overcomes the protective effects conferred by OC on MM cells. Our data further shows inhibition of OC formation and function by 40 and 70%, respectively, following MLN3897 treatment. This is mediated via inhibition of the fusion process and is accompanied by downregulation of pERK and c-fos signaling. To analyze its effect on MM cells, we verified CCR1 and CCR5 expression levels on MM1.S (15% and 3.6%) and OPM1 (3.8 and 0.7%). Our data show that OC secrete high levels of CCL3 which triggers MM cell migration; and that MLN3897 abrogates these effects by inhibiting the PI3K/Akt pathway. Moreover, MLN3897 overcomes the proliferative advantage conferred by OC on MM cells, as demonstrated in INA6, MM1.S and MM patient derived primary cells. OC induced MM cell proliferation is mediated by adhesion and cytokine secretion, and MLN3897 abrogates both MM cell-to-OC adhesion and interleukin-6 (IL6) secretion by OC in a co-culture system, thereby resulting in decreased MM cell survival and proliferation. To confirm these in vitro results, in vivo studies in a SCID-hu mouse model are underway. Implanted SCID-Hu INA-6 bearing mice are treated with twice daily oral MLN3897 for 3 weeks. The evaluation of osteolytic lesions and OC, OB and endothelial cell number; and tumor burden will be presented. Our in vitro results therefore show novel biologic sequelae of CCL3 and its inhibition on both osteoclastogenesis and MM cell growth. Our in vivo experiments will further validate the role of CCR1 in a human BM microenvironment-MM model, providing the framework for clinical trials of MLN3897 for the treatment of OBD in MM.

Somatostatin analogues stimulate p27 expression and inhibit the MAP kinase pathway in pituitary tumours

Somatostatin (SST) analogues play an important role in the medical management of somatotroph pituitary adenomas and new agonists have the potential to be effective in a wider group of pituitary and other tumours. The anti-proliferative effect of SST occurs through multiple mechanisms, one of which is cell-cycle arrest, where p27, a cyclin-dependent kinase inhibitor, is an important regulator. We hypothesised that SST may upregulate p27 protein levels and downregulate the MAP kinase pathway in these tumours. Human pituitary adenoma cells and rat pituitary cell line (GH3) were cultured and treated in vitro with octreotide and the broad-spectrum SST agonist SOM230 (pasireotide). Immunoblotting for p27 and phospho-ERK (pERK) was performed and proliferation assessed by [3H]-thymidine incorporation. Histological samples from acromegalic patients treated with octreotide before surgery were immunostained for p27 and compared to samples from untreated patients matched for sex, age, tumour size, extension and invasiveness. We detected upregulation of p27 protein levels with SST analogue treatment in vitro in human pituitary adenoma samples. pERK1/2 was inhibited by SST analogues in both the human samples and GH3 cells. SST and its analogues inhibited the proliferation of GH3 cells. p27 immunostaining was stronger in samples from patients with longer preoperative octreotide treatment (more than 6 months) than in samples from patients with shorter treatment periods. This study demonstrates that SST-mediated growth inhibition is associated with the downregulation of pERK and upregulation of p27. More potent and broader-spectrum SST analogues are likely to play an increasing role in the treatment of tumours, where the MAP kinase pathway is overactivated.

Novel proteomics approach for functional identification of regulators of cancer drug response and apoptosis in ex vivo tissue cultures of human colorectal cancer

9658 Background: It is notoriously difficult to study the molecular effects of anticancer drug treatment in vivo. So far, this has been only possible by performing serial biopsies in tumor patients undergoing treatment. An approach to address this problem is to perform ex vivo tissue cultures, where patient tumor tissue is obtained at the occasion of a routine operation and then processed in vitro. The molecular program of apoptosis and the activation status of various signal transduction pathways has been identified as a major determinant of chemotherapy responsiveness in cancer. Methods: To analyze genetic pathways potentially involved in anticancer drug response, we have treated tumor samples from colorectal cancer patients ex vivo with antitumor agents with various mechanisms of action. Cytotoxicity was quantified using a colorimetric dye assay. Proteome profiles of the untreated/treated and normal/cancerous patient samples have been generated by reverse-phase protein microarrays, which are a novel approach to analyze signaling pathways using small numbers of cells microdissected from biopsy specimens. For the feasibility phase of the project, we looked at well established growth factor activation pathways such as p-ERK, p-SAPK/JNK, p-AKT, p-p38, p-JNK, p-IkBa, p-STAT3, proliferation regulatory pathways such as p-histone H3, p-Rb, p-p53, cyclin D1, and pathways involved in apoptosis regulation such as cleaved caspase 3, cleaved PARP, and Bcl-2. Results: We found higher cytotoxic activity of most anticancer agents in cancer tissue as compared to normal tissue of the same organ (colon or liver). There was a prominent down-regulation of p-SAPK/JNK and a less pronounced down-regulation of p-ERK and p-p38 with chemotherapy in tumor samples but not in normal tissue samples. Conclusions: This culture technique allows to treat human tissue with various agents ex vivo to simulate the in vivo treatment situation and to assess cytotoxicity by a simple colorimetric dye assay. With the reverse-phase protein microarray technology activated signalling transduction pathways can be identified on low quantity tissue samples. No significant financial relationships to disclose.