miR-328 mediates a metabolic shift in colon cancer cells by targeting SLC2A1/GLUT1.

PURPOSE: Increasing evidence shows that altered metabolism is a critical hallmark in colon cancer. There is a strong need to explore the molecular mechanisms underlying cancer metabolism. Whether the aberrant expression of microRNAs contributes to cancer metabolism is not fully understood. miR-328 is a putative potential target of SLC2A1, but the regulating mechanism between them remains unknown. We have examined whether miR-328 directly regulates SLC2A1/GLUT1 expression in colon cancer cells. METHODS: We performed in silico bioinformatic analyses to identify miR-328-mediated molecular pathways and targets. We also performed luciferase assays and western blot analyses in LOVO and SW480 colon cancer cell lines. In addition, we assessed miR-328 expression in 47 paired tumor and normal tissue specimens from resected colon cancer patients. RESULTS: Luciferase reporter assays showed that miR-328 directly targeted SLC2A1 3'-untranslated region (UTR), with a significant decrease in luciferase activity in both LOVO and SW480 cell lines. These results were validated by western blot. miR-328 expression was significantly downregulated in tumor tissue compared with paired normal tissue. CONCLUSIONS: Our results show that miR-328 targets SLC2A1/GLUT1. We suggest that miR-328 may be involved in the orchestration of the Warburg effect in colon cancer cells. Furthermore, miR-328 expression is reduced in colon cancer patients and thus inversely correlates with the classically reported upregulated SLC2A1/GLUT1 expression in tumors.

miR-203 and miR-221 regulate SOCS1 and SOCS3 in essential thrombocythemia.

The biological basis of essential thrombocythemia (ET) patients lacking known mutations is still unknown. MicroRNAs (miRNA) regulate hematopoietic differentiation and are deregulated in several hematopoietic malignancies. However, miRNA expression in ET patients has been poorly explored. We performed miRNA profiling in platelets from 19 ET patients and 10 healthy controls. Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic 70-miRNA signature (P<0.0001), 68 of which were downregulated. According to the mutational status, three differentially expressed miRNAs, miR-15a (P=0.045), miR-150 (P=0.001) and miR-519a (P=0.036), were identified. A 40-miRNA signature was identified characterizing JAK2V617F-positive ET patients. Eight genes, whose interaction with the miRNAs could activate the JAK/STAT pathway were identified. An inverse correlation was observed between miRNAs expression and their target genes for SOCS1 and miR-221, SOCS3 and miR-221, SOCS3 and miR-203, and PTPN11 and miR-23a. All three miRNAs were upregulated in JAK2V617F-negative ET patients. SOCS1 and SOCS3 were validated as targets of miR-221 and miR-203, respectively. In summary, our study shows that platelets from JAK2V617F-negative ET patients harbor a specific miRNA signature that can participate in the modulation of the JAK/STAT pathway through regulation of key genes as SOCS1 and SOCS3.

Quality of life in caregivers of children with asthma: Validity and reliability of the IFABI-R questionnaire.

BACKGROUND: Parents'/caregivers' quality of life is an important aspect to consider when handling paediatric asthma, but there is a paucity of valid and reliable instruments to measure it. The Family Impact of Childhood Bronchial Asthma (IFABI-R) is a recently developed questionnaire to facilitate the assessment of asthma-related parents'/caregivers' quality of life. This study researches the psychometric properties of IFABI-R. METHODS: Parents/main caregivers of 462 children between 4 and 14 years of age with active asthma were included in the sample. IFABI-R was administered on two different occasions and a number of other variables related to the parents'/caregivers' quality of life were measured: child's asthma control, family functioning, and parents'/caregivers' perception of asthma symptoms in the child. IFABI-R evaluative and discriminative properties were analysed, and the minimal important change in the IFABI-R score was identified. RESULTS: IFABI-R showed high internal consistency (Cronbach's alpha=0.941), cross-sectional construct validity (correlation with the degree of child's asthma control, family functioning and parent/caregiver perception of the child's asthma symptoms), longitudinal construct validity (correlation of changes in the IFABI-R with changes in asthma control and changes in the perception of symptoms), sensitivity to change and test-retest reliability. An absolute change of 0.3 units in IFABI-R related to a minimal significant change in the parents'/caregivers' quality of life. CONCLUSIONS: IFABI-R is a reliable and valid instrument to study the quality of life of parents/caregivers of children with asthma.

The expression level of BAALC-associated microRNA miR-3151 is an independent prognostic factor in younger patients with cytogenetic intermediate-risk acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (⩾60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P=0.0025), shorter leukemia-free survival (P=0.026) and higher cumulative incidence of relapse (P=0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P=0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML.

MicroRNA expression at diagnosis adds relevant prognostic information to molecular categorization in patients with intermediate-risk cytogenetic acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease, and optimal treatment varies according to cytogenetic risk factors and molecular markers. Several studies have demonstrated the prognostic importance of microRNAs (miRNAs) in AML. Here we report a potential association between miRNA expression and clinical outcome in 238 intermediate-risk cytogenetic AML (IR-AML) patients from 16 institutions in the CETLAM cooperative group. We first profiled 670 miRNAs in a subset of 85 IR-AML patients from a single institution and identified 10 outcome-related miRNAs. We then validated these 10 miRNAs by individual assays in the total cohort and confirmed the prognostic impact of 4 miRNAs. High levels of miR-196b and miR-644 were independently associated with shorter overall survival, and low levels of miR-135a and miR-409-3p with a higher risk of relapse. Interestingly, miR-135a and miR-409-3p maintained their independent prognostic value within the unfavorable molecular subcategory (wild-type NPM1 and CEBPA and/or FLT3-ITD), and miR-644 retained its value within the favorable molecular subcategory. miR-409-3p, miR-135a, miR-196b and mir-644 arose as prognostic markers for IR-AML, both overall and within specific molecular subgroups.

Acute myeloid leukemia with translocation (8;16)(p11;p13) and MYST3-CREBBP rearrangement harbors a distinctive microRNA signature targeting RET proto-oncogene.

Acute myeloid leukemia (AML) with t(8;16)(p11;p13) (t(8;16) AML) has unique clinico-biological characteristics, but its microRNA pattern is unknown. We analyzed 670 microRNAs in seven patients with t(8;16) AML and 113 with other AML subtypes. Hierarchical cluster analysis showed that all t(8;16) AML patients grouped in an independent cluster. Supervised analysis revealed a distinctive signature of 94-microRNAs, most of which were downregulated, including miR-21 and cluster miR-17-92. The mRNA expression analysis of two known transcription factors of these microRNAs (STAT3 and c-Myc, respectively) showed significant downregulation of STAT3 (P=0.04). A bioinformatic analysis showed that 29 of the downregulated microRNAs might be regulated by methylation; we treated a t(8;16) AML sample with 5-aza-2'-deoxycytidine (5-AZA-dC) and trichostatin A and found that 27 microRNAs were re-expressed after treatment. However, there was no difference in methylation status between t(8;16) and other AML subtypes, either overall or in the microRNA promoter. Cross-correlation of mRNA and microRNA expression identified RET as a potential target of several microRNAs. A Renilla-luciferase assay and flow cytometry after transfection with pre-microRNAs confirmed that RET is regulated by miR-218, miR-128, miR-27b, miR-15a and miR-195. In conclusion, t(8;16) AML harbors a specific microRNA signature that is partially epigenetically regulated and targets RET proto-oncogene.

Expression of myosin heavy chain isoforms in the human supraspinatus muscle: variations related to age and sex.

The contractile function of skeletal muscles is primarily regulated by the expression of myosin heavy chain (MHC) isoforms. Adult human skeletal muscles express three MHC isoforms (MHC-I, MHC-IIa and MHC-IIx). The muscles mainly expressing MHC-I are slow but resistant to fatigue, while those with major expression of MHC-IIa and MHC-IIx are fast and powerful but less resistant to fatigue. In this study, mRNA levels of the MHC isoforms were assessed in 24 human supraspinatus muscles by reverse-transcription polymerase chain reaction. The average expression of the MHC-I isoform was 36.72%, that of the MHC-IIa isoform was 33.52%, and the average expression of the MHC-IIx isoform was 29.76%. The higher average expression of the two MHC-II isoforms taken together (63.28%) indicates that the human supraspinatus muscle is a powerful, fast muscle with relatively low resistance to fatigue, in accordance with its role in the elevation of the upper extremity. In women, and more markedly in older women, the trend towards upregulation of the fast MHC-II isoforms and downregulation of the slow MHC-I isoform, which is absent in males, may improve our understanding of possible causes of the subacromial impingement syndrome.

Surgical technique in cadaveric donors for partial hand allotransplant in patients with rheumatoid arthritis.

For patients with severe hand deformities due to rheumatoid arthritis, we propose an allotransplantation of an osteomyotendinose structure (OMTS), preserving the recipient's skin and sensory nerves. Our objective was to develop the surgical technique in a 10 cadavers, five as donors and five as recipients. The donor's hand was 10% to 15% smaller than the recipient's. Dissections were performed by two surgical teams under magnification. In the donor, the OMTS was procured at the distal third of the forearm, maintaining the integrity of the arterial system, with its concomitant veins and motor branches of the median and ulnar nerves, leaving the skin envelope. In the recipient, the OMTS was removed, taking care to preserve the cutaneous cover with the digital arteries in continuity with the superficial palmar arch and radial and ulnar arteries. Also, the digital nerves were maintained in the skin flap, in continuity with the median and ulnar nerves. Their motor branches were divided after emergence from the main nerves. The superficial dorsal veins and radial nerve were kept adhered to the cutaneous cover. Then, the donor OMTS was placed within the recipient cutaneous flap; all the anatomic structures were repaired. The average surgical time was 780 minutes. Methylene blue was present in the digital arteries. There were no difficulties in the anatomic repair. The surgical technique is quite laborious, especially the dissection of the recipient interdigital spaces. Due to the requirement for arterial system integrity, the cutaneous flap must be viable. Also, the allotransplanted OMTS has all necessary conditions to obtain good tissue perfusion for subsequent function. Procurement without skin permits a greater opportunity to find donors, and greater social and personal acceptance by the recipient.

Tumour CD133 mRNA expression and clinical outcome in surgically resected colorectal cancer patients.

BACKGROUND: Human prominin-1 (CD133) is a novel pentaspan membrane protein which was originally classified as a marker of primitive haematopoietic and neural stem cells. Cancer stem cells have been isolated and expanded from leukaemia and several solid tumours, and have been associated with metastasis, chemoresistance and relapse. CD133 is recognised as a stem cell marker and is capable of identifying a tumour-initiating subpopulation in brain, colon, melanoma and other solid tumours. METHODS: We assessed CD133 mRNA expression levels by RT-QPCR in tumour and matched normal tissue from 64 stages I-III colorectal cancer (CRC) patients and correlated tumour CD133 levels with clinicopathological characteristics and clinical outcome. RESULTS: In four patients, CD133 mRNA was not expressed in tumour or in normal tissue. In the remaining 60 patients, expression levels were higher in tumour than in normal tissue (p=0.001). Higher levels of CD133 expression were associated with shorter relapse-free interval (RFI) (p=0.004) and overall survival (OS) (p<0.0001). In the multivariate analyses, CD133 levels emerged as a prognostic marker for RFI and OS. CONCLUSIONS: We have observed longer RFI and OS in patients with lower levels of CD133, regardless of adjuvant treatment and other clinical characteristics. If these findings are confirmed in larger prospective studies, CD133 assessment may prove useful for new diagnostic and therapeutic procedures for CRC patients.

Epigenetic events in normal colonic mucosa surrounding colorectal cancer lesions.

Gene inactivation by promoter hypermethylation has been demonstrated in the colonic mucosa of colorectal cancer (CRC) patients. However, current data do not prove direct involvement of this epigenetic modification in the early stages of CRC. Promoter methylation profiles of E-cadherin, hMLH1, MGMT, p16(INK4a), p15(INK4b) and p14(ARF); mutations of K-ras, B-raf and TP53 and microsatellite instability (MSI) were examined in normal and cancerous colonic mucosal tissue in 82 CRC patients using methylation-specific PCR assays. Methylation of hMLH1 and MGMT in normal mucosa correlated significantly with MSI and K-ras activation in neighbouring cancerous mucosal tissues. Similarly, poorly differentiated tumours were associated with methylated p16(INK4a) and E-cadherin in neighbouring normal colonic tissues (NCTs). Our results indicate that epigenetic changes in mucosa surrounding colorectal neoplastic lesions may describe a 'field cancerisation' phenomenon that may occur previous to genetic alterations in early stages of carcinogenesis.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

The role of chemotherapy in early non-small-cell lung cancer management.

Great advances have been made in chemotherapy in advanced and metastatic non-small-cell lung cancer (NSCLC), and a major milestone was reached with the administration of neoadjuvant chemotherapy in stage IIIA N2 disease. The systemic nature of lung cancer has been confirmed by many genetic analyses documenting micrometastases in negative lymph nodes and bone marrow, and mRNA gene overexpression as a surrogate of cancer cells has been identified in peripheral blood. Furthermore, serum or plasma cell-free tumor DNA has been observed even in tumors with a diameter of less than 2 cm. Pharmacogenetic screening can lead to tailored chemotherapy even in patients with early disease through the use of a genetic tool kit that will allow us to optimize the use of chemotherapy by using serial measurements of serum DNA that can help to detect residual disease and re-assess the chemosensitivity of sub-clinical micrometastatic disease. The ongoing (neo)adjuvant taxol/carboplatin hope (NATCH) trial is testing the value of three cycles of chemotherapy given pre- or post-operatively compared with surgery alone and will analyze genetic abnormalities in serum DNA at three different points during patient follow-up. Our major concern in this review is to analyze the pros and cons of chemotherapy in NSCLC. Although this review is not a formal meta-analysis, we have discussed the most relevant published studies in this field. We conclude that not only is there no evidence of detrimental effects of chemotherapy, in fact, there are many indications that chemotherapy induces response in up to 80% of patients and downgrades N2 disease in up to 50% of patients. This translates into significantly better survival when accompanied by complete resection. Since at least 50% of patients with stage IB disease develop distant metastases, it seems logical to explore the role of chemotherapy in early disease.

Types and localisation of p53 gene mutations: a report on 332 non-small cell lung cancer patients.

Mutations of p53 suppressor gene are among the most common molecular abnormalities in human malignancies. We demonstrated earlier significant differences in mutational profiles between NSCLC patients from Poland and Spain. These differences were most probably related to ethnic and/or geographical factors. In the present study we analyzed the types and location of p53 gene mutations in a large group of 332 operated NSCLC patients from two institutions in Northern Poland. Within the last decades this region has been characterized by the highest incidence of lung cancer in Poland. We used both frozen and paraffin-embedded tumor samples and the screened region included exons from 5 to 8. A total of 96 samples (29%) were positive for p53 gene mutation. The proportion of mutations in particular exons was as follows: exon 5-33%, exon 6-22%, exon 7-16%, and exon 8-29%. Three 'hot spots' were located in codons 176,245 and 248. Evolutionary conserved domains were much more frequently affected than the regions outside domains. The majority of mutations (73%) were missense type, followed by null and silent mutations (21 and 6%, respectively). In all six silent mutations substituted was the third base in codon. There were no major differences in the types and locations of mutations between patients from the two institutions. This homogeneity, together with our earlier findings, may confirm the impact of ethnic and geographical factors on the mutational profile of p53 gene in NSCLC.

Drug resistance in non-small cell lung cancer.

There is an underlying feeling in the oncology community that chemotherapy has reached a therapeutic glass ceiling in non-small cell lung cancer (NSCLC) and that we will never attain the acceptable survival rates that are just beyond our reach. Multiple clinical trials have tested doublets, triplets and sequential chemotherapy in what is often regarded as a futile attempt to break through this ceiling. Recent large randomized trials have not demonstrated that any of these combinations is superior to any of the others. Nevertheless, the analysis of DNA or RNA from patients can permit us to assess genes that may be specific targets of certain cytotoxic agents and others that may be markers of multidrug resistance. With this in mind, the Spanish Lung Cancer Group has designed a trial to test the involvement of various genes in resistance to gemcitabine and cisplatin.

Prognostic value of K-ras mutations and allelic imbalance on chromosome 18q in patients with resected colorectal cancer.

PURPOSE: We designed this study to assess the frequency of K-ras mutations in patients with resected colorectal tumors and their association with survival. A second objective was to analyze the prognostic value of different K-ras genotypes. In a subgroup of patients we also investigated the presence of allelic imbalance on chromosome 18q and its relationship to clinical outcome. METHODS: One hundred fourteen colorectal tumors resected between 1983 and 1986 were analyzed to detect K-ras point mutations at codons 12, 13, and 61 by polymerase chain reaction followed by allele specific oligonucleotide hybridization. A subgroup of 77 tumors was further screened to detect loss of heterozygosity on chromosome 18q using three polymorphic microsatellite markers (D18S67, D18S474 and D18S58). RESULTS: K-ras mutations were detected in 29 percent (33/114) of patients. K-ras mutations correlated with age and preoperative carcinoembryonic antigen levels, and there was some indication that they may be linked to poor survival, especially in Stage II tumors, where a subgroup of patients with aspartic and serine mutations showed significantly reduced survival (P = 0.03) compared with K-ras-negative patients. 18q loss of heterozygosity was present in 39 percent (25/63) of tumors. A multivariate analysis of Stage II tumors showed that 18q loss of heterozygosity was significantly associated with a worse prognosis (P = 0.006). A significant decrease in survival was identified in ten patients harboring both genetic alterations (K-i mutations and 18q loss of heterozygosity; P = 0.02). CONCLUSIONS: In colorectal tumors, K-ras mutations and 18q loss of heterozygosity are two genetic markers which may identify patients with more aggressive behavior, mainly in Stage II tumors. These findings warrant further research, because they can be useful in customizing adjuvant chemotherapy.

Novel approaches in the treatment of non-small-cell lung cancer.

A wealth of data indicates that certain genetic abnormalities can target specific cytotoxic drugs and intervene at an early step as a mechanism of resistance in the treatment of non-small-cell lung cancer. Therefore prescribing certain combinations of cytotoxic anticancer agents to a vast majority of these patients is futile. Genetic abnormalities have been found to be useful surrogate markers for response, particularly in colorectal cancer: thymidylate synthase mRNA and ERCC1 mRNA levels. In addition, beta-tubulin mutations may also confer paclitaxel resistance in patients. An important target to be explored for gemcitabine resistance is the assessment of a particular region in chromosome 11p15.5 wherein lies the ribonucleotide reductase gene that could affect gemcitabine metabolism. Shedding light on this genetic framework, several proposed customized chemotherapy studies could help validate the relevance of these markers.

Chromosomal alterations in lung adenocarcinoma from smokers and nonsmokers.

The etiology of lung tumors arising in nonsmokers remains unclear. Although mutations in the K-ras and p53 genes have been reported to be significantly higher in smoking-related lung carcinomas, in the present study we performed a more comprehensive analysis in search of additional genetic changes between lung adenocarcinoma from tobacco- and non-tobacco-exposed patients. We selected a matched cohort of 18 lifetime nonsmoking and 27 smoking patients diagnosed with primary adenocarcinoma of the lung and searched for chromosomal alterations in each tumor by testing normal and tumor tissue with 54 highly polymorphic microsatellite markers located on 28 different chromosomal arms. Allelic losses or gains at chromosomal arms 3p (37 versus 6%), 6q (46 versus 12%), 9p (65 versus 22%), 16p (28 versus 0%), 17p (45 versus 11%), and 19p (58 versus 16%) were present significantly more often in adenocarcinomas from smokers than from nonsmokers. Chromosomal arms showing allelic imbalance in lung tumors from nonsmokers were rare but occurred more often at 19q (22%), 12p (22%), and 9p (22%). The FAL (fractional allelic loss or gain) is defined as the percentage of chromosomal arm losses/gains among the total informative chromosomal arms. Tumors from smokers harbored higher levels of FAL (13 (48%) of 27 showed FAL > or = 0.3) compared with the lung tumors from the nonsmoker patients (2 (11%) of 18 showed FAL > or = 0.3; P = 0.02; odds ratio, 0.13; 95% confidence interval, 0.01-0.79). Our data demonstrate that widespread chromosomal abnormalities are frequent in lung adenocarcinoma from smokers, whereas these abnormalities are infrequent in such tumors arising in nonsmokers. These observations support the notion that lung cancers in nonsmokers arise through genetic alterations distinct from the common events observed in tumors from smokers.

A novel anti-apoptosis gene: Re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers.

PURPOSE: The survivin gene is a novel apoptosis inhibitor, related to the baculovirus gene, which is believed to play a pivotal role in fetal development and in cancer. We hypothesised that survivin would be expressed in tumors of patients with non-small-cell lung cancer (NSCLC), and we attempted to determine the influence of survivin re-expression on clinical outcome in patients with up to stage IIIA NSCLC who had undergone radical surgery. METHODS: We designed a reverse transcriptase polymerase chain reaction (RT-PCR) assay to study the expression of the survivin gene in 83 NSCLC tumor samples and compared the results with relevant clinical and pathologic data. RESULTS: The RT-PCR identified survivin gene transcript in 71 (85. 5%) of the tumor samples and in only 10 (12%) of the paired, histopathologically normal lung samples. There was no relationship between histologic subtype (squamous v nonsquamous) and survivin gene expression. The 12 patients without survivin expression had significantly better overall survival than the 71 patients with survivin expression (P =.01 by univariate analysis; relative risk, 2. 1). There was no significant correlation between survivin expression and age, sex, cigarette smoking, histologic subtype, tumor differentiation, tumor size, or the presence of mediastinal lymph node metastases in surgical specimens. CONCLUSION: The survivin gene was expressed in a vast majority of NSCLC tumors. We conclude that survivin transcript is a defining diagnostic marker for NSCLC that may also yield prognostic information and, as an apoptosis inhibitor, be an important target in cancer therapy.