Genetic modifiers of EGFR dependence in non-small cell lung cancer

Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.The term “oncogene addiction” has been used to describe the phenomenon whereby tumor cells exhibit singular reliance on an oncogene or oncogenic pathway for their survival, despite the accumulation of multiple genetic lesions (1). In non-small cell lung cancer (NSCLC), this principle is perhaps best exemplified with the finding that epidermal growth factor receptor (EGFR) mutations predict response to EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, and thus represent a dependency in the subset of tumors harboring these alterations (2–6). However, though EGFR-mutant NSCLCs typically respond dramatically to EGFR TKIs, clinical responses are not universal, even within this genetically defined cohort, with the rate of objective response estimated to be ∼71% (5, 6). Furthermore, the overwhelming majority of patients who initially respond to EGFR inhibitors ultimately develop resistance to therapy (7). A deeper understanding of the genetic underpinnings of EGFR addiction, and how EGFR-mutant cells can overcome reliance on EGFR, may improve clinical outcomes.Here, we have applied an unbiased screening approach to identify genetic modifiers of EGFR dependence in NSCLC. Mounting evidence supports the existence of several genetic modifiers of EGFR dependence in EGFR-mutant NSCLC, which can reduce the degree to which these tumors rely on EGFR and thereby contribute to EGFR TKI resistance (8). Examples include amplification of the MET receptor tyrosine kinase (RTK) (9), activation of the NF-κB pathway (8), amplification of the HER2 (ERBB2) RTK (10), amplification of the CRKL gene (11), and activation of the AXL kinase (12). Notably, MET bypass can be reciprocally achieved via EGFR activation in MET-dependent cells (13), and analogous examples of reciprocal kinase switching have been reported in other kinase-driven cancer models (14, 15). These and other findings suggest that compensatory kinase switching may be a more general way in which oncogene-dependent cancers overcome reliance on their primary driver kinase (14, 16), but the full-range of kinases capable of mediating EGFR bypass has not been systematically studied.Recent advances in large-scale functional genetic libraries have made it possible to query a wide range of genetic perturbations for their ability to modulate specific cellular phenotypes in mammalian systems (17, 18). Using the model of EGFR-mutant, erlotinib-sensitive NSCLC cells, we have performed a systematic ORF-based screen to identify kinase and kinase-related genes whose overexpression can complement loss of EGFR activity in an EGFR-dependent context. Our findings indicate broad potential for EGFR substitution in the setting of EGFR dependence, with compensatory mechanisms commonly conferring EGFR-independent activation of the PI3K-AKT and MEK-ERK signaling pathways. Importantly, this approach has recovered known mechanisms of erlotinib resistance as well as identified novel mediators of EGFR bypass in EGFR-mutant NSCLC. These data support the idea that the EGFR-dependent state can be redundantly driven by diverse genetic inputs that commonly converge on shared downstream signaling nodes.     

Organization of sensory input to the nociceptive‐specific cutaneous trunk muscle reflex in rat,an effective experimental system for examining nociception and plasticity

Detailed characterization of neural circuitries furthers our understanding of how nervous systems perform specific functions and allows the use of those systems to test hypotheses. We have characterized the sensory input to the cutaneous trunk muscle (CTM; also cutaneus trunci [rat] or cutaneus maximus [mouse]) reflex (CTMR), which manifests as a puckering of the dorsal thoracolumbar skin and is selectively driven by noxious stimuli. CTM electromyography and neurogram recordings in naïve rats revealed that CTMR responses were elicited by natural stimuli and electrical stimulation of all segments from C4 to L6, a much greater extent of segmental drive to the CTMR than previously described. Stimulation of some subcutaneous paraspinal tissue can also elicit this reflex. Using a selective neurotoxin, we also demonstrate differential drive of the CTMR by trkA‐expressing and nonexpressing small‐diameter afferents. These observations highlight aspects of the organization of the CTMR system that make it attractive for studies of nociception and anesthesiology and plasticity of primary afferents, motoneurons, and the propriospinal system. We use the CTMR system to demonstrate qualitatively and quantitatively that experimental pharmacological treatments can be compared with controls applied either to the contralateral side or to another segment, with the remaining segments providing controls for systemic or other treatment effects. These data indicate the potential for using the CTMR system as both an invasive and a noninvasive quantitative assessment tool providing improved statistical power and reduced animal use. J. Comp. Neurol. 522:1048–1071, 2014. © 2013 Wiley Periodicals, Inc.     

Design and evaluation of small interfering RNAs that target expression of the N-methyl-D-aspartate receptor NR1 subunit gene in the spinal cord dorsal horn

NR1 is an essential subunit of the N-methyl-D-aspartate (NMDA) receptor, which at the spinal level is involved in injury-induced pain hypersensitivity and morphine tolerance. An in vitro luciferase assay was used to identify candidate and control (inactive) short interfering RNA (siRNA) sequences that are expressed by a recombinant adeno-associated virus (rAAV) plasmid. rAAV vectors targeting the NR1 subunit were prepared that express active or control (mismatch) siRNA sequences and injected into the mouse spinal cord dorsal horn (SCDH). Three weeks after vector administration, green fluorescent protein labeling of the ipsilateral SCDH confirmed the spatial localization of the viral transduction. Active siRNAs resulted in a 60 to 75% knockdown of NR1 mRNA and protein in the area of the virus injection. The spatial knockdown persisted for at least 6 months after a single administration of the vector. Neither the active nor the mismatch siRNAs resulted in cellular toxicity as measured by nuclear staining and cell integrity. The vector-derived knockdown of NR1 expression in SCDH did not alter acute thermal or mechanical stimulus paw-withdrawal thresholds. However, the vector-derived siRNA prevented the mechanical allodynia measured at 24 and 48 h after injection into the paw of the inflammatory agent, Complete Freund's adjuvant. These results demonstrate that vector-derived siRNAs can be used to produce an in vivo spatial knockdown of the expression and function of the NMDA receptor that is confined to the ipsilateral SCDH. Vector-derived siRNAs may have therapeutic potential for the management of injury-induced pain resulting from the activation of NMDA receptors in the SCDH.     

三种大鼠骨质疏松模型的比较研究

用维Ａ酸、摘除卵巢及糖皮质激素诱导大鼠骨质疏松症，以血清碱性磷酸酶（ｓ－ＡＬＰ），血清钙（ｓ－Ｃａ）、磷（ｓ－Ｐ），尿钙／肌酐（ｕ－Ｃａ／Ｃｒ）及羟脯氨酸／肌酐（ｕ－Ｈｏｐ／Ｃｒ）等为指标，引入骨表观线密度（Ｗ／Ｌ）、面密度（Ｗ／Ｌφ）两个新的骨指数，对该三种较常用的大鼠骨质疏松模型进行了比较研究。结果显示大剂量维Ａ酸组（９０、１００、１２０ｍｇ／ｋｇ）及摘除卵巢组骨重（Ｗ）减轻，Ｗ／Ｌ、Ｗ／Ｌφ明显降低（ｐ＜０．０１，ｐ＜０．０５），形成典型的大鼠骨质疏松症；糖皮质激素组ｓ－ＡＬＰ、ｕ－Ｃａ／Ｃｒ及ｕ－Ｈｏｐ／Ｃｒ显著升高（ｐ＜０．０１，ｐ＜０．０１），但Ｗ／Ｌ、Ｗ／Ｌφ没有明显差异     

Influence of rugby injuries on players' subsequent health and lifestyle: beginning a long term follow up

OBJECTIVES: To describe the current rugby playing status of a cohort of 1,169 men who had previously participated in an epidemiological survey of rugby injuries during the 1993-1994 season, and assess the consequences of rugby injuries sustained. METHODS: In May 1998, 911 (78%) men completed a questionnaire reporting their current involvement in rugby and the influence that the 324 (71%) injuries they had sustained four years earlier had since had on their health and wellbeing. RESULTS: The most common reasons given by the 390 (43%) ex-players for ceasing to play rugby were family (10%), employment (25%), and an injury sustained while playing rugby (26%), 80% of which were dislocations, strains, and sprains, mainly to the knee (35%), back (14%), and shoulder (9%). A significantly (chi2 test 21.7, df = 1, p<0.001) higher proportion of current players (90%) undertook (non-rugby) sporting activities compared with ex-players (78%). Few ex-players undertook coaching (12%) and refereeing (2%). Only 22 (9%) men reported significant negative effects to employment, family life, and health up to mid-1998 from injuries that occurred during the 1993-1994 season, although the impact on their lifestyle had been substantial in some cases. CONCLUSIONS: With the recent increase in the incidence of dislocation, strain, and sprain injuries in rugby football, the findings of this follow up could have a great impact on the game in the future. Although this survey has shown that, so far, only a small proportion of players suffer significant effects of rugby injuries, four years is not long enough to assess the long term effects. This cohort of rugby players need to be followed up for at least a further 20 years to determine whether there is a higher incidence of subsequent degenerative joint disease or other long term sequelae to injuries sustained while playing rugby.     

BDNF-induced facilitation of afferent-evoked responses in lamina II neurons is reduced after neonatal spinal cord contusion injury

We previously reported that brain-derived neurotrophic factor (BDNF), a pronociceptive neurotransmitter, induces synaptic facilitation of excitatory postsynaptic current (EPSC) in lamina II neurons of neonatal rats up to P14 in a N-methyl-d-aspartate (NMDA) receptor-dependent manner. Here we used the patch-clamp technique to study synaptic and NMDA-evoked responses in transverse spinal slices in the lumbar enlargement as well as the ability of BDNF to modify these responses from 1 day to 6 wk after neonatal contusion. In older uninjured animals (>P14), BDNF continued to evoke synaptic facilitation although superfusion of NMDA (in TTX) induced inward current of significantly smaller amplitude than that observed in younger rats. After contusion injury, BDNF was unable to facilitate dorsal root-evoked EPSCs in lamina II neurons despite the finding that NMDA-evoked currents were only slightly smaller than those observed in age-matched uninjured animals. These findings suggest that although BDNF-induced facilitation of the AMPA/kainate receptor-mediated response to dorsal root stimulation is maintained in the mature dorsal horn from intact rats, BDNF may no longer elicit these pronociceptive actions after neonatal contusion injury. The lack of change in NMDA-evoked currents in contused cords suggests that diminished NMDA receptor function is not the major cause of the decline in BDNF action after contusion. It seems more likely that diminished trkB expression and enhanced expression of truncated trkB receptors in the contused cord play a significant role in determining the reduced effect of BDNF under these conditions.     

The changing pattern of hypertension and the declining incidence of stroke

We studied whether changes in recognition and control of hypertension could be detected in the population of Rochester, Minn, from 1950 to 1979, a period in which a major decrease in the incidence rate of stroke was observed. Prevalence of diastolic blood pressure greater than or equal to 105 mm Hg fell 26% and 70% in men and women, respectively, between 1950 to 1959 and 1970 to 1979. Prevalence of pressures greater than or equal to 95 mm Hg decreased 5% in men and 58% in women. Increasing control of hypertension had an almost inverse linear relationship with the decreasing incidence of stroke in women, but the incidence of stroke in men did not decrease until ten years after improvement in the control of blood pressure began. We conclude that improvements in the detection and control of hypertension contributed to the declining incidence of stroke and that differences in management of hypertension could account for the difference between men and women in the trend of stroke decline.