The development of cigarette smoke induced chronic pancreatitis in mice is associated with increased expression of K-Ras and NF-κB.

INTRODUCTION AND OBJECTIVE: Epidemiological studies have demonstrated a strong association between cigarette smoking (CS) and chronic pancreatitis (CP); however, the exact mechanisms of this phenomenon remains unknown. The authors have previously shown that increased Ras expression activates the NF-κB mediated pathway and promotes development of CP. However, it is unclear whether a similar phenomenon occurs in CS-induced CP. Therefore, the aim of the study was to determine whether CS increases the expression of K-Ras, and promotes the development of CP in mice exposed to repeated episodes of acute pancreatitis (AP). MATERIAL AND METHODS: C57BL6/cmdb mice were exposed to CS or a sham treatment for 12 weeks. After one week of exposure, half of the animals from both groups were additionally subjected to repeated cerulein treatment (once a week, for 10 consecutive weeks) to mimic recurrent episodes of AP. Extension of pancreatic damage was determined histologically by H&E and Trichrome staining. The expression of K-Ras protein and downstream components (NF-κB, Cox-2, TGF-ß) was evaluated by immunohistochemistry. RESULTS: C57BL6/cmdb mice exposed to CS or cerulein alone did not develop any chronic pancreatic damage. However, concomitant treatment with both of these agents caused focal acinar atrophy, with slight intralobular and perivascular areas of fibrosis, and inflammatory cells infiltration resembling mild CP. Moreover, immunohistochemistry examinations revealed increased pancreatic expression of K-Ras and NF-κB only in mice treated both with CS and cerulein. CONCLUSIONS: CS promotes development of CP in mice exposed to repeated episodes of AP. This process may be, at least partially, related to increased expression of K-Ras and NF-κB protein.

Prolonged clinical response with regorafenib administered as second-line therapy in an elderly patient suffering from peritoneal carcinomatosis of colon cancer.

Peritoneal carcinomatosis from colorectal cancer (CRC) has a poor prognosis with median survival and clinical responses that are worse than for other metastatic sites, and even more so in pretreated patients proposed for regorafenib therapy. Thus, patients with these characteristics are a therapeutic challange. The present study reports the case of an 83-year-old woman with diffuse peritoneal carcinomatosis from CRC, RAS-mutated, and treated with second-line therapy with the off-label administration of regorafenib at full dose (160 mg once daily, for the first 21 days of each 4-week cycle), refusing conventional chemotherapy. The patient reported an unexpected clinical response, reduced toxicity, excellent adherence to therapy and remained progression-free for 30 months from the start of treatment. In clinical practice, an earlier use of regorafenib and a different selection of patients could be the subject of future studies.

The effectiveness of cetuximab and panitumumab when combined with FOLFIRI in second-line treatment of KRAS wild type metastatic colorectal cancers. Single centre experience.

Panitumumab and cetuximab are monoclonal antibodies known to be effective in metastatic colorectal cancer (mCRC). Although the survival benefits when combined with chemotherapy have been determined, there are no studies comparing the two agents with chemotherapy in the second-line treatment. In this study, we aimed to compare the efficacy of cetuximab vs panitumumab in patients who previously received chemotherapy. Who progressed after first-line treatment for K-ras wild type mCRC were analyzed. The efficacy of cetuximab vs panitumumab on overall survival (OS) and progression-free survival (PFS) when combined with FOLFIRI regimen was compared retrospectively. Median PFS was 6.9 months in the cetuximab group and 4.7 months in the panitumumab group. Median OS cetuximab and panitumumab groups were 18.4 and 12.2 months, respectively. In the second-line treatment of K-ras wild type mCRC, both PFS and OS were found to be longer in patients receiving cetuximab than in patients receiving panitumumab, but no statistically significant difference was found.

Anti-tumor Properties of Picrasma quassioides Extracts in H-RasG12V Liver Cancer Are Mediated Through ROS-dependent Mitochondrial Dysfunction.

BACKGROUND: Picrasma quassioides (PQ) is a traditional Asian herbal medicine with anti-tumor properties that can inhibit the viability of HepG2 liver cancer cells. H-Ras is often mutated in liver cancer, however, the effect of PQ treatment on H-Ras mutated liver cancer is unclear. This study aimed to investigate the role of PQ on ROS accumulation and mitochondrial dysfunction in H-ras mutated HepG2 (HepG2G12V) cells. MATERIALS AND METHODS: PQ ethanol extract-induced HepG2G12V apoptosis was analyzed by the MTT assay, fluorescence microscopy, flow cytometry and western blotting. RESULTS: PQ treatment affected cell migration and colony formation in HepG2G12V cells. Cleaved-caspase-3, cleaved-caspase-9 and BCL2 associated agonist of cell death (BAD) expression levels were increased, while the levels of B-cell lymphoma-extra large (Bcl-xL) were decreased with PQ treatment. PQ treatment led to a reduction of H-Ras expression levels in liver cancer cells, thus reducing their abnormal proliferation. Furthermore, it led to increased expression levels of Peroxiredoxin VI, which regulates the redox signal in cells. CONCLUSION: Taken together these results provide a new functional significance for the role of PQ in treating HepG2G12V liver cancer.

ROC-1, P21 and CAIX as markers of tumor aggressiveness in bladder carcinoma in Egyptian patients.

BACKGROUND: Bladder cancer (BC) is one of the most common malignancies in Egypt, representing about 8.7% of cancers in both sexes with more predominance in males, making identification of valuable predictive and prognostic markers, mandatory. Cullin-RING ligases (CRL) play an important role in the ubiquitination of cell cycle-related proteins or other proteins (e.g., DNA replication protein, signal transduction protein). Regulator of Cullins-1 (ROC-1) is a key subunit of CRL. P21 belongs to the family of cyclin dependent kinase inhibitors (CKIs) which regulates cell cycle by inactivating Cyclin- Dependent Kinases key regulators of the cell cycle. CAIX a highly active member of the family of carbonic anhydrases has gained much interest as a hypoxic marker. Hypoxia is a consequence of the rapid growth of many tumors, including bladder cancer, and is an important regulator of gene expression and resistance to chemotherapy and radiotherapy. Therefore the purpose of this study is to evaluate the role of ROC-1, CAIX and P21 and its relationship with the clinico-pathological features of bladder cancer in Egyptian patients. METHODS: Using the standard immunohistochemical technique, ROC-1, CAIX and P21 expression in 80 primary bladder carcinomas and 15 normal bladder specimens as control group were assessed. The bladder carcinoma cases included 50 cases with muscle invasive bladder cancer and 30 cases with non-muscle invasive bladder cancer. RESULTS: Over expression of ROC-1, CAIX and P21 in BC were significantly associated with muscularis propria invasion and high grade BC. ROC-1, CAIX and P21, showed significant inverse relationship in primary BC cases. CAIX expression was significantly higher in BC compared with controls. Regarding the survival analysis, expression of ROC-1, CAIX and P21 didn't affect the survival of BC patients. CONCLUSIONS: High expression of ROC-1, CAIX and P21 could be promising potential biomarkers for identifying patients with poor prognostic factors in bladder cancer serving as potential targets for cancer therapy.

The proprotein convertase furin is a pro-oncogenic driver in KRAS and BRAF driven colorectal cancer.

Mutations in KRAS and/or BRAF that activate the ERK kinase are frequently found in colorectal cancer (CRC) and drive resistance to targeted therapies. Therefore, the identification of therapeutic targets that affect multiple signaling pathways simultaneously is crucial for improving the treatment of patients with KRAS or BRAF mutations. The proprotein convertase furin activates several oncogenic protein precursors involved in the ERK-MAPK pathway by endoproteolytic cleavage. Here we show that genetic inactivation of furin suppresses tumorigenic growth, proliferation, and migration in KRAS or BRAF mutant CRC cell lines but not in wild-type KRAS and BRAF cells. In a mouse xenograft model, these KRAS or BRAF mutant cells lacking furin displayed reduced growth and angiogenesis, and increased apoptosis. Mechanistically, furin inactivation prevents the processing of various protein pecursors including proIGF1R, proIR, proc-MET, proTGF-ß1 and NOTCH1 leading to potent and durable ERK-MAPK pathway suppression in KRAS or BRAF mutant cells. Furthermore, we identified genes involved in activating the ERK-MAPK pathway, such as PTGS2, which are downregulated in the KRAS or BRAF mutant cells after furin inactivation but upregulated in wild-type KRAS and BRAF cells. Analysis of human colorectal tumor samples reveals a positive correlation between enhanced furin expression and KRAS or BRAF expression. These results indicate that furin plays an important role in KRAS or BRAF-associated ERK-MAPK pathway activation and tumorigenesis, providing a potential target for personalized treatment.

An Application of pET SUMO Protein Expression System in Escherichia coli: Cloning, Expression, Purification, and Characterisation of Native Kras4BG12V Oncoprotein.

Being an important regulator of cell growth and survival, a point mutation at glycine-12 residue of Kras4B to valine (V), renders Kras4BG12V oncogenic. Kras4B recombinant protein is used as a bait to fish its potential ligands in the attempt of drugging this oncoprotein and to validate its pharmacologically relevant ligand in protein-ligand interaction studies. Nevertheless, synthesis of Kras4B recombinant protein is challenging as it was reported being susceptible to aggregation into inclusion bodies in the bacterial host, resulting in a poor yield of recombinant protein. Here, we describe a novel method to produce native Kras4BG12V protein by using pET SUMO protein expression system as a solution to the formation of inclusion bodies. Kras4BG12V oncogene was cloned into pET SUMO vector, followed by a 12 h chemically induced protein expression in Escherichia coli at 20 °C. Native Kras4BG12V protein was produced in a series of protein purification steps involving immobilised nickel ion-affinity column chromatography, SUMO fusion protein and polyhistidine tag removal, and size exclusion column chromatography. The identity of the purified Kras4BG12V protein was validated by immunoblot analysis. The purified protein exhibited self-dimerising, indicating that the purified protein structurally resembles Kras4B. Its physical interaction with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a known binder of Kras4B, confirms the identity of the purified protein as Kras4BG12V. The native Kras4BG12V protein was successfully purified in a substantial amount by using the pET SUMO protein expression system.

Moderate alcohol intake promotes pancreatic ductal adenocarcinoma development in mice expressing oncogenic Kras.

Kras mutations are associated with pancreatic ductal adenocarcinoma (PDAC). Although tobacco smoking, pancreatitis, and obesity are known environmental risk factors for PDAC, the contribution of moderate alcohol intake to PDAC remains elusive. In the present study, we tested whether a combination of risk factors or moderate alcohol intake induces PDAC development in mice. Control Pdx1Cre and Pdx1Cre;LSL-KrasG12D mutant mice were fed a Western alcohol diet containing high levels of cholesterol and saturated fat, 3.5% alcohol, and lipopolysaccharide for 5 mo. In addition, mice were treated with cerulein, for induction of pancreatitis, and nicotine every month. Treatment with all of these risk factors promoted development of advanced pancreatic neoplasia and PDAC in the Pdx1Cre;LSL-KrasG12D mice but not in the control Pdx1Cre mice. Moderate alcohol intake or Western diet feeding also significantly promoted advanced neoplasia and PDAC development in Pdx1Cre;LSL-KrasG12D mice compared with mice fed a regular chow. Alcohol, but not Western diet, increased tumor development in the liver in the Pdx1Cre;LSL-KrasG12D mice, but its origin remained elusive due to leakiness of Pdx1Cre in hepatocytes. RNA-seq analysis revealed that alcohol feeding increases expression of markers for tumors (Epcam, Krt19, Prom1, Wt1, and Wwtr1), stroma (Dcn, Fn1, and Tnc), and cytokines (Tgfb1 and Tnf) and decreases expression of Fgf21 and Il6 in the pancreatic tumor tissues. Immunostaining showed heterogeneous expression of nephronectin, S100 calcium-binding protein A6, and vascular cell adhesion molecule 1 in pancreatic tumors surrounded by podoplanin-positive stromal cells. Our data indicate that moderate alcohol drinking is a risk factor for development of PDAC.NEW & NOTEWORTHY Heavy alcohol intake has been suspected to be a risk factor of pancreatic ductal adenocarcinoma (PDAC) in humans. However, the contribution of moderate alcohol intake to PDAC development remains elusive. In the present study, we experimentally show that moderate alcohol feeding significantly induces advanced stages of pancreatic intraepithelial neoplasia development and invasive PDAC in Pdx1Cre;LSL-KrasG12D mutant mice. Our data indicate that moderate alcohol drinking is a risk factor for PDAC.

KRasG12D expression in the bone marrow vascular niche affects hematopoiesis with inflammatory signals.

The bone marrow (BM) niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained. Previous studies have indicated that genetic mutations in various components of the niche can affect hematopoiesis and promote hematologic abnormalities, but the impact of abnormal BM endothelial cells (BMECs), a crucial niche component, on hematopoiesis remains incompletely understood. To dissect how genetic alterations in BMECs could affect hematopoiesis, we have employed a novel inducible Tie2-CreERT2 mouse model, with a tdTomato fluorescent reporter, to introduce an oncogenic KRasG12D mutation specifically in the adult endothelial cells. Tie2-CreERT2;KRasG12D mice had significantly more leukocytes and myeloid cells in the blood with mostly normal BM HSPC populations and developed splenomegaly. Genotyping polymerase chain reaction revealed KRasG12D activation in BMECs but not hematopoietic cells, confirming that the phenotype is due to the aberrant BMECs. Competitive transplant assays revealed that BM cells from the KRasG12D mice contained significantly fewer functional hematopoietic stem cells, and immunofluorescence imaging showed that the hematopoietic stem cells in the mutant mice were localized farther away from BM vasculature and closer to the endosteal area. RNA sequencing analyses found an inflammatory gene network, especially tumor necrosis factor α, as a possible contributor. Together, our results implicate an abnormal endothelial niche in compromising normal hematopoiesis.

Synthesis of Nano-Paramagnetic Oleuropein to Induce KRAS Over-Expression: A New Mechanism to Inhibit AGS Cancer Cells.

Background and objectives: Human gastric adenocarcinoma (AGS) is one of the most common malignant cancers worldwide. The present study aimed to transfer oleuropein into cancer cells using synthetic paramagnetic nanoparticles and study their effect on the AGS (ATCC® CRL1739™) cell line. Materials and Methods: Paramagnetic nano-oleuropein was synthesized using four-stage co-precipitation by developing NH-connected bridges and was evaluated by EDS, SEM and FTIR methods. Different concentrations of magnetic oleuropein (0, 0.15, 0.45, 1.37, 4.12, 12.35, 37.04, 111.11, 333.33, 1000 µg/mL) were used to treat the AGS cell line in a completely randomized design using a statistical framework with three replicates. The relative expression rate of miR-200 and KRAS oncogenes was evaluated using real-time PCR. The inhibition rate of the AGS cells was assessed using the MTT test at 24, 48 and 72 h intervals. Results: The results showed that there was a significant difference between the inhibition rates of magnetic nano-oleuropein at IC50-24h (23.6 µg/mL), IC50-48h (15.2 µg/mL) and IC50-72h (9.2 µg/mL). Real-time PCR indicated that the relative expression of KRAS and miR-200 genes was highest at IC50 at these intervals. Conclusions: Magnetic nano-oleuropein can be subjected to objective testing and clinical evaluations as a natural antioxidant to prevent and treat gastric adenocarcinoma.

Production of Farnesylated and Methylated Proteins in an Engineered Insect Cell System.

Protein prenylation is a common posttranslational modification that enhances the ability of proteins to interact with membrane components within the cell. In many cases, these prenylated proteins are involved in important human diseases, including aging-related disorders and cancer. To effectively study these proteins or develop therapeutics, large quantities of properly modified proteins are required. Historically, production of fully modified farnesylated and methylated proteins at high yield has been challenging. Recently, an engineered insect cell system which is capable of producing authentically modified KRAS protein was used to generate material for structural studies and assay development. Here we describe protocols for extending this work to other farnesylated and methylated substrates.

Distinct oncogenes drive different genome and epigenome alterations in human mammary epithelial cells.

Molecular subtypes of breast cancer are defined on the basis of gene expression and genomic/epigenetic pattern differences. Different subtypes are thought to originate from distinct cell lineages, but the early activation of an oncogene could also play a role. It is difficult to discriminate the respective inputs of oncogene activation or cell type of origin. In this work, we wished to determine whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. To this aim, we transduced shp53 immortalized HMECs in parallel with the CCNE1, WNT1 and RASv12 oncogenes which activate distinct oncogenic pathways and characterized them at sequential stages of transformation for changes in their genetic and epigenetic profiles. We show that initial activation of CCNE1, WNT1 and RASv12, in shp53 HMECs results in different and reproducible changes in mRNA and micro-RNA expression, copy number alterations (CNA) and DNA methylation profiles. Noticeably, HMECs transformed by RAS bore very specific profiles of CNAs and DNA methylation, clearly distinct from those shown by CCNE1 and WNT1 transformed HMECs. Genes impacted by CNAs and CpG methylation in the RAS and the CCNE1/WNT1 clusters showed clear differences, illustrating the activation of distinct pathways. Our data show that early activation of distinct oncogenic pathways leads to active adaptive events resulting in specific sets of CNAs and DNA methylation changes. We, thus, propose that activation of different oncogenes could have a role in reshaping the genetic landscape of breast cancer subtypes.

COX-2/C-MET/KRAS status-based prognostic nomogram for colorectal cancer: A multicenter cohort study.

BACKGROUND/AIM: To construct quantitative prognostic models for colorectal cancer (CRC) based on COX-2/C-MET/KRAS expression status in clinical practice. PATIENTS AND METHODS: Clinical factors and COX-2/C-MET/KRAS expression status of 578 eligible patients from two Chinese hospitals were included. The patients were randomly allocated into training and validation datasets. We created several models using Cox proportional hazard models: SignatureC contained clinical factors, SignatureG contained COX-2/C-MET/KRAS expression status, and SignatureCG contained both. After comparing their accuracy, nomograms for progression-free survival (PFS) and overall survival (OS) were built for the best signatures, with their concordance index and calibration tested. Further, patients were subgrouped by the median of the best signatures, and survival differences between the subgroups were compared. RESULTS: For PFS, among the three signatures, SignaturePFS-CG had the best area under the curve (AUC), with the 1-, 2- and 3-year AUCs being 0.70, 0.73 and 0.89 in the training dataset, respectively and 0.67, 0.73 and 0.87 in the validation dataset, respectively. For OS, the AUCs of SignatureOS-CG for 1-, 2- and 3-years were 0.63, 0.71 and 0.81 in the training dataset, respectively and 0.68, 0.71 and 0.76 in validation dataset, respectively. The nomograms based on SignaturePFS-CG and SignatureOS-CG had good calibrations. Subsequent stratification analysis demonstrated that the subgroups were significantly different for both PFS (training:P < 0.001; validation:P< 0.001) and OS (training:P < 0.001; validation:P < 0.001). CONCLUSIONS: Combining clinical factors and COX-2/C-MET/KRAS expression status, our models provided accurate prognostic information in CRC. They can be used to aid treatment decisions in clinical practice.

RANKL, OPG, TRAIL, KRas, and c-Fos expression in relation to central lymph node metastases in papillary thyroid carcinoma.

PURPOSE: RANKL, OPG and TRAIL have long been pursued in cancer. Mutated KRas proteins and c-Fos overexpression - well-recognized oncogenic events - have been conceived as coordinators of RANKL, OPG and TRAIL pathways. Considering the paucity in the relevant literature, the purpose of the present study was to investigate whether the expression of these molecules configures a distinct papillary thyroid carcinoma (PTC) subgroup with adverse clinicopathological characteristics. METHODS: RANKL, OPG, TRAIL, KRas, and c-Fos immunohistochemical expression in relation to clinicopathological characteristics of PTC was assessed retrospectively in paraffin-embedded PTC specimens from 114 patients who underwent total thyroidectomy with simultaneous central lymph node dissection (CLND). RESULTS: Expression of RANKL, OPG, TRAIL, Kras and c- Fos was revealed in 78.6, 63.2, 61.4, 47.4, and 73.7% of PTC, respectively. As predominant KRas-expressing PTC histotype emerged the classical PTC (cPTC), comprising 66.7% of PTC. A significant correlation was demonstrated of RANKL, OPG, and TRAIL expression with central lymph node metastasis CLNM (p=0.007, p<0.001, and p=0.002, respectively), concerning especially cPTC as regards to RANKL (p=0.027) and OPG (p=0.006), and both cPTC (p=0.043) and follicular variant of PTC (FVPTC) (p=0.049) with regard to TRAIL. OPG expression associated significantly with multifocality (p=0.045). Multivariable-adjusted logistic regression models characterized TRAIL as independent predictor of CLNM (OR=10.335, 95% CI: 1.23-86.87). CLNM correlated significantly with six pairs of coexpressions: TRAIL-KRas (p=0.011), TRAIL-c-Fos (p=0.006), OPG-c-Fos (p=0.024), RANKL-TRAIL (p<0.001), RANKL-OPG (p<0.001), TRAIL- OPG (p<0.001). CONCLUSION: The present study suggested for the first time that OPG, RANKL, TRAIL expressions, either alone or in concert involving c-Fos and KRas expression, are related to CLNM. Further research is warranted to elucidate whether the examined molecules can be endorsed as indicators of aggressive PTC behavior and guide a personalized therapeutic intervention.

RAS-expanded Mutations and HER2 Expression in Metastatic Colorectal Cancer: A New Step of Precision Medicine.

Cetuximab and panitumumab monoclonal antibodies are a milestone in the history of treatment of metastatic colorectal cancer (mCRC) and point toward future directions for personalized treatment. Recent studies have shown that broader RAS testing is needed to select patients for targeted therapy. The objectives of our study were to identify the prevalence of RAS mutations and evaluate human epidermal growth factor receptor 2 (HER2) expression in KRAS exon 2 wild-type (WT) mCRC patients, correlating the findings with objective response rate, progression-free survival, and overall survival. In total, 29 mCRC patients undergoing treatment with cetuximab therapy were enrolled in this study. By pyrosequencing, mutations were found in 17% of nonresponder patients, in KRAS codon 146 and NRAS codon 12. HER2 positivity was limited to only 1 responder carcinoma specimen. There was no correlation between RAS mutation, HER2/neu expression, and clinicopathologic findings. We highlighted significantly the differences between objective response rate and RAS gene status. The overall survival and progression-free survival of RAS WT patients were higher compared with those with RAS-mutated disease. Clinical response to cetuximab therapy is impaired in the presence of RAS-expanded mutations. In fact, our finding of 5 mutations in RAS-expanded genes allowed us to understand the resistance to cetuximab in 33% of KRAS WT exon 2 nonresponder patients. HER2 does not seem to be a potential biomarker for cetuximab-targeted therapy. These analyses suggest that the assessment of other biomarkers is needed to determine the best treatment for patients with mCRC, to maximize benefit and minimize harm.

Characterizing the interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) and KRAS expression.

Insulin-like growth factor 2 mRNA-binding protein-1 (IMP1) has high affinity for KRAS mRNA, and it can regulate KRAS expression in cells. We first characterized the molecular interaction between IMP1 and KRAS mRNA. Using IMP1 variants with a point mutation in the GXXG motif at each KH domain, we showed that all KH domains play a critical role in the binding of KRAS RNA. We mapped the IMP1-binding sites on KRAS mRNA and show that IMP1 has the highest affinity for nts 1-185. Although it has lower affinity, IMP1 does bind to other coding regions and the 3'-UTR of KRAS mRNA. Eight antisense oligonucleotides (AONs) were designed against KRAS RNA in the nts 1-185 region, but only two, SM6 and SM7, show potent inhibition of the IMP1-KRAS RNA interaction in vitro To test the activity of these two AONs in SW480 human colon cancer cells, we used 2'-O-methyl-modified versions of SM6 and SM7 in an attempt to down-regulate KRAS expression. To our surprise, both SM6 and SM7 had no effect on KRAS mRNA and protein expression, but significantly inhibited IMP1 protein expression without altering IMP1 mRNA level. On the other hand, knockdown of IMP1 using siRNA lowered the expression of KRAS. Using Renilla luciferase as a reporter, we found that IMP1 translation is significantly reduced in SM7-treated cells with no change in let-7a levels. The present study shows that the regulation of KRAS expression by IMP1 is complex and may involve both the IMP1 protein and its mRNA transcript.

Production of authentic geranylgeranylated KRAS4b using an engineered baculovirus system.

Protein prenylation is a vital eukaryotic post-translational modification which permits interaction of proteins with cellular membranes. Prenylated proteins are involved in a number of human diseases, and play a major role in cancers driven by the oncogene KRAS, which is normally farnesylated. In cases where the farnesylation machinery is inhibited, however, KRAS eludes inactivation by using an alternative prenylation pathway in which the protein is geranylgeranylated. In order to study this alternative prenylation, large quantities of accurately processed protein are required. We have developed a system to permit high-yield production of geranylgeranylated KRAS which utilizes an engineered baculovirus system. The development of this system helped to elucidate a potential metabolic bottleneck in insect cell production that should enable better production of any geranylgeranylated proteins using this system.

Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis.

Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis.IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as a biomarker of disease progression.

Molecular Testing Guideline for the Selection of Patients With Lung Cancer for Treatment With Targeted Tyrosine Kinase Inhibitors: American Society of Clinical Oncology Endorsement of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology Clinical Practice Guideline Update.

Purpose In response to advances in the field, the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) recently updated their recommendations for molecular testing for the selection of patients with lung cancer for treatment with targeted tyrosine kinase inhibitors. ASCO has a policy and set of procedures for endorsing clinical practice guidelines that have been developed by other professional organizations. Methods The molecular testing guideline was reviewed for developmental rigor by methodologists. Then an ASCO Expert Panel reviewed the content and the recommendations. Results The ASCO Expert Panel determined that the recommendations from the CAP/IASLC/AMP molecular testing guideline are clear, thorough, and based upon the most relevant scientific evidence. ASCO endorsed the guideline with minor modifications. Recommendations This update clarifies that any sample with adequate cellularity and preservation may be tested and that analytical methods must be able to detect mutation in a sample with as little as 20% cancer cells. It strongly recommends against evaluating epidermal growth factor receptor (EGFR) expression by immunohistochemistry for selection of patients for EGFR-targeted therapy. New for 2018 are recommendations for stand-alone ROS1 testing with additional confirmation testing in all patients with advanced lung adenocarcinoma, and RET, ERBB2 (HER2), KRAS, and MET testing as part of larger panels. ASCO also recommends stand-alone BRAF testing in patients with advanced lung adenocarcinoma. Recommendations are also provided for testing methods for lung cancers that have a nonadenocarcinoma non-small-cell component, for patients with targetable mutations who have relapsed on targeted therapy, and for testing the presence of circulating cell-free DNA. Additional information is available at www.asco.org/thoracic-cancer-guidelines and www.asco.org/guidelineswiki .

A cross-sectional study examining the expression of splice variants K-RAS4A and K-RAS4B in advanced non-small-cell lung cancer patients.

OBJECTIVES: Mammalian cells differently express 4 RAS isoforms: H-RAS, N-RAS, K-RAS4A and K-RAS4B, which are important in promoting oncogenic processes when mutated. In lung cancer, the K-RAS isoform is the most frequently altered RAS protein, being also a difficult therapeutic target. Interestingly, there are two K-RAS splice variants (K-RAS4A and K-RAS4B) and little is known about the role of K-RAS4A. Most studies targeting K-RAS, or analysing it as a prognostic factor, have not taken into account the two isoforms. Consequently, the in-depth investigation of them is needed. METHODS: The present study analysed 98 specimens from advanced non-small cell lung cancer (NSCLC) adenocarcinoma patients originated from Brazil. The alterations present in K-RAS at the DNA level (Sanger sequencing) as well as the expression of the splicing isoforms at the RNA (qRT-PCR) and protein levels (immunohistochemistry analysis), were evaluated. Possible associations between clinicopathological features and the molecular findings were also investigated. RESULTS: Our results showed that in the non-smoking population, the cancer incidence was higher among women. In contrast, in smokers and former smokers, the incidence was higher among men. Regarding sequencing results, 10.5% of valid samples presented mutations in exon 2, being all wild-type for exon 3, and the most frequently occurring base change was the transversion G → T. Our qRT-PCR and immunohistochemical analysis showed that both, K-RAS4A and K-RAS4B, were differently expressed in NSCLC tumour samples. For example, tumour specimens showed higher K-RAS4A mRNA expression in relation to commercial normal lung control than did K-RAS4B. In addition, K-RAS4B protein expression was frequently stronger than K-RAS4A in the patients analysed. CONCLUSION: Our results highlight the differential expression of K-RAS4A and K-RAS4B in advanced adenocarcinoma NSCLC patients and underline the need to further clarify the enigma behind their biological significance in various cancer types, including NSCLC.