The emerging role of microRNAs in resistance to lung cancer treatments

One of the major challenges in the treatment of lung cancer is the development of drug resistance. This represents a major obstacle in the treatment of patients, limiting the efficacy of both conventional chemotherapy and biological therapies. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and in developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in resistance to various cancer treatments. MicroRNAs are a family of small non-coding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified to date. While as little as one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance to a number of cancer treatments, thereby modulating the sensitivity of cancer cells to such therapies. Therefore, targeting miRNAs may be an attractive strategy for developing novel and more effective individualized therapies, improving drug efficiency, and for predicting patient response to different treatments. In this review, we provide an overview on the role of miRNAs in resistance to current lung cancer therapies and novel biological agents.

Lung cancer stem cells: The root of resistance

In the absence of specific treatable mutations, platinum-based chemotherapy remains the gold standard of treatment for lung cancer patients. However, 5-year survival rates remain poor due to the development of resistance and eventual relapse. Resistance to conventional cytotoxic therapies presents a significant clinical challenge in the treatment of this disease. The cancer stem cell (CSC) hypothesis suggests that tumors are arranged in a hierarchical structure, with the presence of a small subset of stem-like cells that are responsible for tumor initiation and growth. This CSC population has a number of key properties such as the ability to asymmetrically divide, differentiate and self-renew, in addition to having increased intrinsic resistance to therapy. While cytotoxic chemotherapy kills the bulk of tumor cells, CSCs are spared and have the ability to recapitulate the heterogenic tumor mass. The identification of lung CSCs and their role in tumor biology and treatment resistance may lead to innovative targeted therapies that may ultimately improve clinical outcomes in lung cancer patients. This review will focus on lung CSC markers, their role in resistance and their relevance as targets for future therapies.

Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials

Background We aimed to assess the effect of afatinib on overall survival of patients with EGFR mutation-positive lung adenocarcinoma through an analysis of data from two open-label, randomised, phase 3 trials. Methods Previously untreated patients with EGFR mutation-positive stage IIIB or IV lung adenocarcinoma were enrolled in LUX-Lung 3 (n=345) and LUX-Lung 6 (n=364). These patients were randomly assigned in a 2:1 ratio to receive afatinib or chemotherapy (pemetrexed-cisplatin [LUX-Lung 3] or gemcitabine-cisplatin [LUX-Lung 6]), stratified by EGFR mutation (exon 19 deletion [del19], Leu858Arg, or other) and ethnic origin (LUX-Lung 3 only). We planned analyses of mature overall survival data in the intention-to-treat population after 209 (LUX-Lung 3) and 237 (LUX-Lung 6) deaths. These ongoing studies are registered with ClinicalTrials.gov, numbers NCT00949650 and NCT01121393. Findings Median follow-up in LUX-Lung 3 was 41 months (IQR 35–44); 213 (62%) of 345 patients had died. Median follow-up in LUX-Lung 6 was 33 months (IQR 31–37); 246 (68%) of 364 patients had died. In LUX-Lung 3, median overall survival was 28·2 months (95% CI 24·6–33·6) in the afatinib group and 28·2 months (20·7–33·2) in the pemetrexed-cisplatin group (HR 0·88, 95% CI 0·66–1·17, p=0·39). In LUX-Lung 6, median overall survival was 23·1 months (95% CI 20·4–27·3) in the afatinib group and 23·5 months (18·0–25·6) in the gemcitabine-cisplatin group (HR 0·93, 95% CI 0·72–1·22, p=0·61). However, in preplanned analyses, overall survival was significantly longer for patients with del19-positive tumours in the afatinib group than in the chemotherapy group in both trials: in LUX-Lung 3, median overall survival was 33·3 months (95% CI 26·8–41·5) in the afatinib group versus 21·1 months (16·3–30·7) in the chemotherapy group (HR 0·54, 95% CI 0·36–0·79, p=0·0015); in LUX-Lung 6, it was 31·4 months (95% CI 24·2–35·3) versus 18·4 months (14·6–25·6), respectively (HR 0·64, 95% CI 0·44–0·94, p=0·023). By contrast, there were no significant differences by treatment group for patients with EGFR Leu858Arg-positive tumours in either trial: in LUX-Lung 3, median overall survival was 27·6 months (19·8–41·7) in the afatinib group versus 40·3 months (24·3–not estimable) in the chemotherapy group (HR 1·30, 95% CI 0·80–2·11, p=0·29); in LUX-Lung 6, it was 19·6 months (95% CI 17·0–22·1) versus 24·3 months (19·0–27·0), respectively (HR 1·22, 95% CI 0·81–1·83, p=0·34). In both trials, the most common afatinib-related grade 3–4 adverse events were rash or acne (37 [16%] of 229 patients in LUX-Lung 3 and 35 [15%] of 239 patients in LUX-Lung 6), diarrhoea (33 [14%] and 13 [5%]), paronychia (26 [11%] in LUX-Lung 3 only), and stomatitis or mucositis (13 [5%] in LUX-Lung 6 only). In LUX-Lung 3, neutropenia (20 [18%] of 111 patients), fatigue (14 [13%]) and leucopenia (nine [8%]) were the most common chemotherapy-related grade 3–4 adverse events, while in LUX-Lung 6, the most common chemotherapy-related grade 3–4 adverse events were neutropenia (30 [27%] of 113 patients), vomiting (22 [19%]), and leucopenia (17 [15%]). Interpretation Although afatinib did not improve overall survival in the whole population of either trial, overall survival was improved with the drug for patients with del19 EGFR mutations. The absence of an effect in patients with Leu858Arg EGFR mutations suggests that EGFR del19-positive disease might be distinct from Leu858Arg-positive disease and that these subgroups should be analysed separately in future trials.

Mutational analysis of the insulin‑like growth factor 1 receptor tyrosine kinase domain in non-small cell lung cancer patients

The insulin‑like growth factor 1 receptor (IGF1R) pathway plays an important role in the pathogenesis of non‑small cell lung cancer (NSCLC) and also provides a mechanism of resistance to targeted therapies. IGF1R is therefore an ideal therapeutic target and several inhibitors have entered clinical trials. However, thus far the response to these inhibitors has been poor, highlighting the importance of predictive biomarkers to identify patient cohorts who will benefit from these targeted agents. It is well‑documented that mutations and/or deletions in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain predict sensitivity of NSCLC patients to EGFR TK inhibitors. Single‑nucleotide polymorphisms (SNPs) in the IGF pathway have been associated with disease, including breast and prostate cancer. The aim of the present study was to elucidate whether the IGF1R TK domain harbours SNPs, somatic mutations or deletions in NSCLC patients and correlates the mutation status to patient clinicopathological data and prognosis. Initially 100 NSCLC patients were screened for mutations/deletions in the IGF1R TK domain (exons 16‑21) by sequencing analysis. Following the identification of SNP rs2229765, a further 98 NSCLC patients and 866 healthy disease‑free control patients were genotyped using an SNP assay. The synonymous SNP (rs2229765) was the only aberrant base change identified in the IGF1R TK domain of 100 NSCLC patients initially analysed. SNP rs2229765 was detected in exon 16 and was found to have no significant association between IGF1R expression and survival. The GA genotype was identified in 53.5 and 49.4% of NSCLC patients and control individuals, respectively. No significant difference was found in the genotype (P=0.5487) or allele (P=0.9082) frequencies between the case and control group. The present findings indicate that in contrast to the EGFR TK domain, the IGF1R TK domain is not frequently mutated in NSCLC patients. The synonymous SNP (rs2229765) had no significant association between IGF1R expression and survival in the cohort of NSCLC patients.

Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis

The incorporation of dUMP during replication or the deamination of cytosine in DNA results in the occurrence of uracils in genomes. To maintain genomic integrity, uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base-excision repair pathway. Here, we cloned, purified and biochemically characterized a family 5 UDG, UdgB, from Mycobacterium smegmatis to allow us to use it as a model organism to investigate the physiological significance of the novel enzyme. Studies with knockout strains showed that compared with the wild-type parent, the mutation rate of the udgB(-) strain was approximately twofold higher, whereas the mutation rate of a strain deficient in the family 1 UDG (ung(-)) was found to be similar to 8.4-fold higher. Interestingly, the mutation rate of the double-knockout (ung(-)ludgB(-)) strain was remarkably high, at similar to 19.6-fold. While CG to TA mutations predominated in the ung(-) and ung(-)/udgb(-) strains, AT to GC mutations were enhanced in the udgB(-) strain. The ung(-)/udgB(-) strain was notably more sensitive to acidified nitrite and hydrogen peroxide stresses compared with the single knockouts (ung(-) or udgB(-)). These observations reveal a synergistic effect of UdgB and Ung in DNA repair, and could have implications for the generation of attenuated strains of Mycobacterium tuberculosis.

Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli

Uracil DNA glycosylase (Ung)initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by similar to 7- and similar to 170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed similar to 5-, similar to 3.0- and similar to 2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair.

Resistance responses blocking systemic movement of Potato virus A in potato

Peruna kestää A-virusta estämällä sen leviämistä Peruna on maissin ohella maailman kolmanneksi tärkein ravintokasvi vehnän ja riisin jälkeen. Perunaa lisätään kasvullisesti mukuloita istuttamalla, jolloin virukset siirtyvät sairaiden siemenmukuloiden välityksellä kasvukaudesta toiseen. Virustauteja voi torjua ainoastaan terveen siemenperunan ja kestävien lajikkeiden avulla. Kestävyys perustuu usein siihen, että kasvi estää viruksen leviämisen tartuntakohdasta välttyäkseen virustaudilta. Tässä työssä tutkittiin kolmea perunan A-viruksen (PVA) liikkumista estävää kestävyysmekanismia perunassa. Lisäksi työn kokeelliseen osaan oleellisesti kuuluvaa virustartutusta varten kehitettiin uusi paranneltu versio geenipyssystä. Tämä itse rakennettu laite optimoitiin PVA:n tartuttamiseen mahdollisimman helposti ja pienin käyttökustannuksin. Tutkimuksen kohteena olleessa perunan risteytysjälkeläistössä oli PVA:ta kestäviä kasveja (ryhmä nnr), jotka estivät viruksen liikkumisen aiheuttamatta oireita tartutuskohdassa, sekä kasveja, joissa PVA aiheutti kuolioläikkinä näkyvän yliherkkyysvasteen (ryhmä HR). Molemmissa kestävyystyypeissä virus pystyi monistumaan ja leviämään solusta soluun paikallisesti, mutta liikkuminen muihin kasvinosiin nilan kautta estyi. Ryhmän nnr kasveissa PVA-tartunta ei aiheuttanut tilastollisesti merkitsevää muutosta useimpien geenien ilmenemiseen tartuntakohdassa. Ainoastaan geeniperhe, joka ilmentää tiettyä proteinaasi-inhibiittoria (PI), reagoi PVA:han 24 tuntia tartutuksesta. Kun tämän PVA:han reagoivan geeniperheen jäsenet hiljennettiin nnr- perunalinjoissa, ne muuttuivat alttiiksi PVA:lle ja virus levisi tartuntakohdasta muihin kasvinosiin. Tulos osoittaa, että PI on viruskestävyystekijä. Lisäksi muut tutkimuksessa saadut tulokset tukevat mahdollisuutta, että PI estää PVA:n P1-proteinaasin toimintaa. HR-linjoissa todettiin erilaisiin puolustusvasteisiin liittyvien PR-geenien aktivoitumista PVA-tartunnan seurauksena, mutta myös ilman sitä kasvien kasvettua mullassa noin neljä viikkoa. Sen sijaan solukkoviljelyssä tai vasta kaksi viikkoa mullassa kasvaneissa kasveissa vastaavaa ei vielä todettu. Tulos viittaa siihen, että HR-perunat reagoivat herkemmin ympäristöön ja/tai kasvin kehitysasteeseen laukaisten puolustusvasteita, jotka saattavat parantaa kestävyyttä taudinaiheuttajia vastaan. Kolmas tutkittu kestävyystyyppi havaittiin Pito-perunalajikkeessa. Se muistutti nnr-kestävyyttä siten, että myös siinä viruksen liikkuminen nilassa muihin kasvinosiin estyi. PVA:n todettiin pysähtyvän vasta lehtiruodin tyvelle muodostuvaan irtoamisvyöhykkeeseen, mitä havainnollistettiin käyttämällä muunnettua PVA-rotua, joka tuotti UV-valossa fluoresoivaa vihreää valoa. Tulos viittaa siihen, että virus ei pääse kulkemaan vyöhykkeeseen kuuluvan suojaavan kerroksen läpi, jollei sillä ole pääsyä nilaan. Tällainen kestävyys on tarpeen, jotta virus ei voi korvata nilakuljetusta solusta soluun leviämisellä. Tulokset tuovat uusia näkökulmia kasvien viruskestävyyteen ja auttavat selittämään viruksen nilakuljetuksen estymistä sekä solusta soluun leviämisen pysähtymistä kestävissä kasveissa.

Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetyl-glucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. beta-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.

The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins

Formylation of the initiator tRNA is essential for normal growth of Escherichia coil, The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon, However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation, In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient, The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms.

Voltage delay during constant-current or constant-resistance discharge of Mg-MnO2 dry cells: A comparative study

A theoretical approach has been developed to relate the voltage delay transients of the Mg-MnO2 dry cell observed during discharge by two commonly employed modes, viz., (1) at constant current, and (2) across a constant resistance. The approach has been verified by comparison of experimentally obtained transients with those generated from theory. The method may be used to predict the delay parameters of the Mg-MnO2 dry cell under the two modes of discharge and can, in principle, be extended to lithium batteries.

Tracking Random Walk of Individual Domain Walls in Cylindrical Nanomagnets with Resistance Noise

The stochasticity of domain-wall (DW) motion in magnetic nanowires has been probed by measuring slow fluctuations, or noise, in electrical resistance at small magnetic fields. By controlled injection of DWs into isolated cylindrical nanowires of nickel, we have been able to track the motion of the DWs between the electrical leads by discrete steps in the resistance. Closer inspection of the time dependence of noise reveals a diffusive random walk of the DWs with a universal kinetic exponent. Our experiments outline a method with which electrical resistance is able to detect the kinetic state of the DWs inside the nanowires, which can be useful in DW-based memory designs.

Isobaric electrical resistance along the critical line in nickel: An experimental test of universality

High-precision measurement of the electrical resistance of nickel along its critical line, a first attempt of this kind, as a function of pressure to 47.5 kbar is reported. Our analysis yields the values of the critical exponents α=α’=-0.115±0.005 and the amplitude ratios ‖A/A’‖=1.17±0.07 and ‖D/D’‖=1.2±0.1. These values are in close agreement with those predicted by renormalization-group (RG) theory. Moreover, this investigation provides an unambiguous experimental verification to one of the key consequences of RG theory that the critical exponents and amplitudes ratios are insensitive to pressure variation in nickel, a Heisenberg ferromagnet.