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Scorpions and assassin bugs are arthropod predators that may impact in trophic chain, coexisting in similar microhabitats and exhibiting antagonistic interactions. This study reports the first case of predation of the scorpion Physoctonus debilis by the assassin bug Microtomus tibialis in a Brazilian Seasonal Dry Tropical Forest biome. During a nocturnal collection, an adult individual of M. tibialis was observed, with the head and body facing downwards, while holding a female of P. debilis, with the aid of the mouthparts under the vegetation. Reports like these are important to understand the evolutionary dynamics and natural history of predators that cohabit in the same microhabitat.

In Dichotomius genus, transposable elements (TE) have been related to chromosome remodeling, genomic evolution, and, possibly, to the speciation process. The objective of this study was to verify the interpopulational and interspecific conservation/variation of Tc1-Mariner elements (possibly autonomous) in Dichotomius species, aiming to identify possible contributions in the speciation process of this group. The analysis was performed on four species of Dichotomius, belonging to the Selenocopris subgenus. We verified the presence of the DsPogo_8 and DsTc1_5 elements by PCR and sequencing. We also isolated and sequenced the 28S and 16S rRNA genes aiming at the phylogenetic reconstruction of the analyzed species. Chromosomal mapping of TEs DsTc1_5 and DsPogo_8 was performed by fluorescent in situ hybridization. The results revealed the presence of the elements in the different species analyzed, except for DsTc1_5 in D. (S.) geminatus. These results suggest a vertical inheritance, with the presence of these elements in the common ancestor of these species. In the analyzed species, the nucleotide similarity of DsTc1_5 was higher than that of the 28S and 16S rRNA genes, suggesting the occurrence of horizontal transfer. The phylogenetic tree indicated that the absence of DsTc1_5 in D. (S.) geminatus is related to stochastic loss of this TE. Chromosomal mapping revealed dispersed signals, with predominance in euchromatic regions and wide variation in the chromosomal localization pattern of DsTc1_5 and DsPogo_8, both interpopulational and interspecific. This variation indicates that DsTc1_5 and DsPogo_8 may have contributed to prezygotic and postzygotic isolation, thus contributing to the speciation of these species.

We developed a combined experimental and theoretical study that gives mechanistic insights into the silver-catalyzed direct selanylation of indoles.

After identifying a captive herd of white-tailed deer in central Texas with >94% seroprevalence with SARS-CoV-2 neutralizing antibodies in September 2021, we worked retrospectively through archived serum samples of 21 deer and detected seroconversion of all animals between December 2020 and January 2021. We then collected prospective samples to conclude that the duration of persistence of neutralizing antibodies is at least 13 months for 19 (90.5%) of the animals, with two animals converting to seronegative after six and eight months. Antibody titres generally waned over this time frame, but three deer had a temporary 4- to 8-fold increases in plaque reduction neutralization test titres over a month after seroconversion; anamnestic response cannot be ruled out.

A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA–vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA–vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA–vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA–vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.

Knowledge on the diet of the Black-backed Woodpecker (Picoides arcticus Swainson, 1832) is fragmentary and relies on a limited number of studies. Gaps remain in our understanding of the plasticity of its diet, particularly in the eastern part of its range. The main objective of this study was to assess the diet of Black-backed Woodpeckers in burned and unburned habitats and among sexes and ages in Québec. We collected feces and fecal bags from unburned and burned habitats in the Central Laurentians ecoregion of the eastern boreal shield ecozone, and assessed diets based on identified prey items. Buprestidae and Cerambycidae of the subfamily Lamiinae were predominant prey for adult Black-backed Woodpeckers in burned habitats, and the Pythidae Pytho niger (Kirby, 1837) and Lamiinae were the most prevalent prey in unburned habitats. Lamiinae were the most predominant prey items provisioned to nestling in burned habitat while P. niger was their predominant food in unburned habitat, followed by Cerambycidae (without Lamiinae) and Lamiinae. Our results present new insights into Black-backed Woodpecker diet where parents feed their offspring with the largest prey available, potentially providing higher fitness for their offspring. Furthermore, our study confirms that Black-backed Woodpeckers, at least in the eastern part of its range, are not restricted to feed on Lamiinae, but are rather opportunistic in taking advantage from resource-pulse interactions provided by recently disturbed habitats, especially from recently burned habitats

Multiresistant pathogens pose a serious threat to human health. The genus candida is one class of human pathogenic yeasts responsible for infections affecting healthy and immunocompromised patients. In this context, plant essential oils emerged as a future natural alternative to control the diseases caused by these pathogens. Here, it highlighted antimicrobial activity and the mechanisms of action of the essential oil extracted from Croton pluriglandulosus Carn.-Torres & Riina (CpEO) leaves on human pathogenic microorganisms in planktonic and biofilm lifestyles. In addition, for the first time, the oil composition was revealed by GC-MS analysis and the toxicity to human red blood cells (HRBC). Twenty-six chemical compounds were identified in EOCp, Elemicin, Bicyclogermacrene, caryophyllene, brevifolin and 2,4,6-trimethoxy-styrene. Through hemolytic assay, it was shown that CpEO has no toxicity to human RBCs. At the concentration of 50 μg mL-1, CpEO did not show great antibacterial potential. However, promising data were found for C. krusei and C. parapsilosis inhibiting by 89.3% and 80.7% of planktonic cell growth and 83.5% and 77.9% the biofilm formation, respectively. Furthermore, the mechanisms of action CpEO were elucidated by fluorescence. Scanning electron microscopy revealed damage to the cell membrane and pore formation, ROS overproduction, and Induction of apoptosis in candida cells. Our results reinforce the potential of CpEO as an effective alternative molecule of pharmaceutical interest.

West Nile virus (WNV) is the primary mosquito-borne disease in the United States and has had case reports every year since its introduction in 1999. As such, it is critical that we characterize the distribution of WNV vectors. Estimates of Culex tarsalis Coquillett species distribution, a major WNV vector, are scarce. We used ensemble distribution modeling to estimate habitat suitability for this species across the contiguous United States by consolidating presence data from four publicly available mosquito trapping data servers. The central plains region and much of the western US were estimated to have high habitat suitability. We identified multiple metrics of temperature and precipitation to be important in predicting the occurrence of Cx. tarsalis in a given geographic area. Furthermore, we observed habitat suitability for Cx. tarsalis to be significantly higher in areas with a high incidence of West Nile neuroinvasive disease compared to areas with low WN disease incidence, suggesting that Cx. tarsalis is present in regions with a high incidence of disease.

The spatial organization of eukaryotic genomes is linked to their biological functions, although it is not clear how this impacts the overall evolution of a genome. Here, we uncover the three-dimensional (3D) genome organization of the phytopathogenVerticillium dahliae, known to possess distinct genomic regions, designated adaptive genomic regions (AGRs), enriched in transposable elements and genes that mediate host infection. Short-range DNA interactions form clear topologically associating domains (TADs) with gene-rich boundaries that show reduced levels of gene expression and reduced genomic variation. Intriguingly, TADs are less clearly structured in in AGRs than in the core genome. At a global scale, the genome contains bipartite long-range interactions, particularly enriched for AGRs and more generally containing segmental duplications. Notably, the patterns observed forV. dahliaeare also present in otherVerticilliumspecies. Thus, our analysis links 3D genome organization to evolutionary features conserved throughout theVerticilliumgenus.

The emergence of resistance to EGF receptor (EGFR) inhibitor therapy is a major clinical problem for patients with non–small cell lung cancer (NSCLC). The mechanisms underlying tumor resistance to inhibitors of the kinase activity of EGFR are not fully understood. Here, we found that inhibition of EGFR by erlotinib induces STAT3 phosphorylation at Tyr705 in association with increased Bcl2/Bcl-XL at both mRNA and protein levels in various human lung cancer cells. PTPMeg2 is a physiologic STAT3 phosphatase that can directly dephosphorylate STAT3 at the Tyr705 site. Intriguingly, treatment of cells with erlotinib results in downregulation of PTPMeg2 without activation of STAT3 kinases [i.e., Janus-activated kinase (JAK2) or c-Src], suggesting that erlotinib-enhanced phosphorylation of STAT3 may occur, at least in part, from suppression of PTPMeg2 expression. Because elevated levels of phosphorylated STAT3 (pSTAT3), Bcl2, and Bcl-XL were observed in erlotinib-resistant lung cancer (HCC827/ER) cells as compared with erlotinib-sensitive parental HCC827 cells, we postulate that the erlotinib-activated STAT3/Bcl2/Bcl-XL survival pathway may contribute to acquired resistance to erlotinib. Both blockage of Tyr705 phosphorylation of STAT3 by niclosamide and depletion of STAT3 by RNA interference in HCC827/ER cells reverse erlotinib resistance. Niclosamide in combination with erlotinib potently represses erlotinib-resistant lung cancer xenografts in association with increased apoptosis in tumor tissues, suggesting that niclosamide can restore sensitivity to erlotinib. These findings uncover a novel mechanism of erlotinib resistance and provide a novel approach to overcome resistance by blocking the STAT3/Bcl2/Bcl-XL survival signaling pathway in human lung cancer. Mol Cancer Ther; 12(10); 2200–12. ©2013 AACR.

PDF file - 218K, Ionizing radiation (IR) induces activation of the JAK2/STAT3/Bcl2/Bcl-XL survival signaling pathway in various human lung cancer cells.

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A major challenge affecting the outcomes of patients with lung cancer is the development of acquired radioresistance. However, the mechanisms underlying the development of resistance to therapy are not fully understood. Here, we discovered that ionizing radiation induces phosphorylation of Janus-associated kinase (JAK)-2 and STAT3 in association with increased levels of Bcl2/Bcl-XL in various human lung cancer cells. To uncover new mechanism(s) of radioresistance of lung cancer, we established lung cancer cell model systems with acquired radioresistance. As compared with radiosensitive parental lung cancer cells (i.e., A549, H358, and H157), the JAK2/STAT3/Bcl2/Bcl-XL survival pathway is significantly more activated in acquired radioresistant lung cancer cells (i.e., A549-IRR, H358-IRR, and H157-IRR). Higher levels of STAT3 were found to be accumulated in the nucleus of radioresistant lung cancer cells. Niclosamide, a potent STAT3 inhibitor, can reduce STAT3 nuclear localization in radioresistant lung cancer cells. Intriguingly, either inhibition of STAT3 activity by niclosamide or depletion of STAT3 by RNA interference reverses radioresistance in vitro. Niclosamide alone or in combination with radiation overcame radioresistance in lung cancer xenografts. These findings uncover a novel mechanism of radioresistance and provide a more effective approach to overcome radioresistance by blocking the STAT3/Bcl2/Bcl-XL survival signaling pathway, which may potentially improve lung cancer outcome, especially for those patients who have resistance to radiotherapy. Mol Cancer Ther; 13(3); 606–16. ©2013 AACR.

PDF file - 47K, Differentially methylated genes with beta values (3KT vs carcinogen exposed).

PDF file, 171K, Inhibition of EGFR by erlotinib (Erlo) results in STAT3 phosphorylation at Tyr 705 and increased Bcl2/Bcl-XL in human lung cancer H1650 and H1975 cells.

PDF file - 74K, Niclosamide (Niclo) reduces STAT3 nuclear localization in radioresistant human lung cancer A549-IRR cells.

PDF file, 46K, Structures of erlotinib and niclosamide.

PDF file - 65K, Pathway analysis of differentially methylated genes.

DNA methylation is an early event in bronchial carcinogenesis and increased DNA methyltransferase (DNMT)1 protein expression is a crucial step in the oncogenic transformation of epithelia. Here, we investigate the role of class I histone deacetylases (HDAC) 1 to 3 in the stabilization of DNMT1 protein and as a potential therapeutic target for lung cancer chemoprevention. Long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens led to oncogenic transformation, increased HDAC expression, cell-cycle independent increased DNMT1 stability, and DNA hypermethylation. Overexpression of HDACs was associated with increased DNMT1 stability and knockdown of HDACs reduced DNMT1 protein levels and induced DNMT1 acetylation. This suggests a causal relationship among increased class I HDACs levels, upregulation of DNMT1 protein, and subsequent promoter hypermethylation. Targeting of class I HDACs with valproic acid (VPA) was associated with reduced HDAC expression and a profound reduction of DNMT1 protein level. Treatment of transformed bronchial epithelial cells with VPA resulted in reduced colony formation, demethylation of the aberrantly methylated SFRP2 promoter, and derepression of SFRP2 transcription. These data suggest that inhibition of HDAC activity may reverse or prevent carcinogen-induced transformation. Finally, immunohistochemistry on human lung cancer specimens revealed a significant increase in DNMT1, HDAC1, HDAC2, and HDAC3 expression, supporting our hypotheses that class I HDACs are mediators of DNMT1 stability. In summary, our study provides evidence for an important role of class I HDACs in controlling the stability of DNMT1 and suggests that HDAC inhibition could be an attractive approach for lung cancer chemoprevention. Cancer Prev Res; 7(3); 351–61. ©2014 AACR.

PDF file - 190K, Niclosamide increases the proportion of the cell population in the G0/G1 phase in A549 parental and radioresistant A549 (A549-IRR) cells.

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PDF file, 78K, Treatment of mice with human lung cancer HCC827/ER xenografts using a combination of erlotinib and niclosamide results in a long term tumor-free survival.

PDF file, 152K, Toxicity analysis for treatments with erlotinib and niclosamide in mice bearing HCC827/ER xenografts.

PDF file - 1302K, S1: Primary transcript qPCR indicates HDAC1 and 2 are regulated transcriptionally, HDAC3 is not. S2: Analysis of golden gate methylation array identifies 42/807 genes hypermethylated after long term carcinogen exposure. S3: Cycloheximide 2ug/ml final was added to cultures of 3kt or T31 cells for the indicated times (hours). Cycloheximide blocks the VPA induced degradation of DNMT1. S4: HEK293 cells transiently transfected with pDest51-V5tagged HDAC1, 2 demonstrates protective role of HDACs on DNMT1. % indicates percent fold change of DNMT1 expression over parental cell. S5: 3kt cells stably transfected with HDAC3 demonstrates protective role on DNMT1. % indicates percent fold change of DNMT1 expression over parental cell. S6: Overexpression of HDAC1 or HDAC3 do not increase cellular proliferation compared to empty vector control. S7: 6 tumor samples and matched resected histologically normal lungs were stained for PCNA. PCNA staining intensity is quantified by weighted index. S8: AKT phosphorylation (S473) was assessed with and without VPA exposure in several different passages of carcinogen exposure. VPA had no effect on AKT phosphorylation. S9: T31 wells were grown in triplicates in logarithmic phase and counted manually. VPA's effects on proliferation were assessed at 0.1 mM and 0.5 mM. No discernable effect of particularly the low dose of VPA on growth was observed. S10: carcinogen-transformed T31 cells were passaged for 28 days in the presence of VPA (0.5mM). Bisulfite sequencing of the RASSF1 locus was performed on cells derived from day 0 and day 28.

PDF file - 109K, Niclosamide-induced growth inhibition is diminished after specific knockdown of STAT3.

PDF file, 161K, Toxicity analysis for treatments with erlotinib and niclosamide in mice bearing HCC827 xenografts.

PDF file - 142K, Niclosamide or in combination with IR inhibits cell proliferation in tumor tissues.

A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non–small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo. Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001–12. ©2017 AACR.

Supplementary Figures. Supplemental Figure S1: Representative sections showing changes in expression between baseline biopsy and post-treatment surgical specimens. Supplemental Figure S2: Correlogram showing the correlation between changes in protein expression immunoscore, metabolic activity on PET and tumor size by CT scan.

Mcl-1 is a unique antiapoptotic Bcl2 family protein that functions as a gatekeeper in manipulating apoptosis and survival in cancer cells. Akt is an oncogenic kinase that regulates multiple cellular functions and its activity is significantly elevated in human cancers. Here we discovered a cross-talk between Mcl-1 and Akt in promoting lung cancer cell growth. Depletion of endogenous Mcl-1 from human lung cancer cells using CRISPR/Cas9 or Mcl-1 shRNA significantly decreased Akt activity, leading to suppression of lung cancer cell growth in vitro and in xenografts. Mechanistically, Mcl-1 directly interacted via its PEST domain with Akt at the pleckstrin homology (PH) domain. It is known that the interactions between the PH domain and kinase domain (KD) are important for maintaining Akt in an inactive state. The binding of Mcl-1/PH domain disrupted intramolecular PH/KD interactions to activate Akt. Intriguingly, Mcl-1 expression correlated with Akt activity in tumor tissues from patients with non–small cell lung cancer. Using the Mcl-1–binding PH domain of Akt as a docking site, we identified a novel small molecule, PH-687, that directly targets the PH domain and disrupts Mcl-1/Akt binding, leading to suppression of Akt activity and growth inhibition of lung cancer in vitro and in vivo. By targeting the Mcl-1/Akt interaction, this mechanism-driven agent provides a highly attractive strategy for the treatment of lung cancer.Significance:These findings indicate that targeting Mcl-1/Akt interaction by employing small molecules such as PH-687 represents a potentially new and effective strategy for cancer treatment.

Supplementary Figure 1. Knockout of Mcl-1 from H1299 by CRISPR/Cas9 system results in decreased cell proliferation and increased caspase 3/7 activity. Supplementary Figure 2. Expression of constitutive active form of Akt in H1299 Mcl-1 knockout cells restores cell growth. Supplementary Figure 3. Depletion of Mcl-1 by shRNA retards the growth of H460 lung xenografts. Supplementary Figure 4. Expression of WT Mcl-1 but not the PEST deletion mutant Mcl-1 in H1299 Mcl-1 knockout cells restores Akt/PDK1 and Akt/mTORC2 interactions. Supplementary Figure 5. Treatment of H1299 cells with PH-687 results in increased intramolecular PH/KD interactions in Akt and decreased interaction of Akt with PDK1 or mTORC2. Supplementary Figure 6. Mcl-1/Akt signaling pathway is essential for PH-687 inhibition of cancer cell growth. Supplementary Figure 7. Knockout of Mcl-1 or treatment of cells with PH-687 inhibits growth factor (s)-stimulated Akt activation. Supplementary Figure 8. In vivo toxicity analysis. Supplementary Methods

Supplementary Data from Tumor Microenvironment of Metastasis in Human Breast Carcinoma: A Potential Prognostic Marker Linked to Hematogenous Dissemination

Purpose: The altered PI3K/mTOR pathway is implicated in lung cancer, but mTOR inhibitors have failed to demonstrate efficacy in advanced lung cancer. We studied the pharmacodynamic effects of everolimus in resectable non–small cell lung cancer (NSCLC) to inform further development of these agents in lung cancer.Experimental Design: We enrolled 33 patients and obtained baseline tumor biopsy and 2[18F]fluoro-2-deoxy-D-glucose-positron emission tomography/computed tomography (FDG-PET/CT) imaging followed by everolimus treatment (5 or 10 mg daily, up to 28 days), or without intervening treatment for controls. Target modulation by everolimus was quantified in vivo and ex vivo by comparing metabolic activity on paired PET scans and expression of active phosphorylated forms of mTOR, Akt, S6, eIF4e, p70S6K, 4EBP1, and total Bim protein between pretreatment and posttreatment tissue samples.Results: There were 23 patients on the treatment arm and 10 controls; median age 64 years; 22 tumors (67%) were adenocarcinomas. There was a dose-dependent reduction in metabolic activity (SUVmax: 29.0%, −21%, −24%; P = 0.014), tumor size (10.1%, 5.8%, −11.6%; P = 0.047), and modulation of S6 (−36.1, −13.7, −77.0; P = 0.071) and pS6 (−41.25, −61.57, −47.21; P = 0.063) in patients treated in the control, 5-mg, and 10-mg cohorts, respectively. Targeted DNA sequencing in all patients along with exome and whole transcriptome RNA-seq in an index patient with hypersensitive tumor was employed to further elucidate the mechanism of everolimus activity.Conclusions: This “window-of-opportunity” study demonstrated measurable, dose-dependent, biologic, metabolic, and antitumor activity of everolimus in early-stage NSCLC. Clin Cancer Res; 21(8); 1859–68. ©2015 AACR.

Purpose: Multiphoton-based intravital imaging has shown that invasive carcinoma cells in mouse and rat mammary tumors intravasate when associated with perivascular macrophages, identifying a potential tumor microenvironment of metastasis (TMEM). We define TMEM as the tripartite arrangement of an invasive carcinoma cell, a macrophage, and an endothelial cell. The aim of this study was to determine if TMEM density in human breast carcinoma samples predicts the development of systemic, hematogenous metastases.Experimental Design: A case-control study of 30 patients who developed metastatic breast cancer and 30 patients without metastatic disease was done. Cases were matched to controls based on currently used prognostic criteria. Paraffin-embedded primary breast cancer samples were stained using a triple immunohistochemical method allowing simultaneous identification of carcinoma cells, macrophages, and endothelial cells. Two pathologists, blinded to outcome, evaluated the number of TMEM per 20 high-power fields.Results: No association was seen between TMEM density and tumor size or grade, lymph node metastasis, lymphovascular invasion, or hormone receptor status. TMEM density was greater in the group of patients who developed systemic metastases compared with the patients with only localized breast cancer (median, 105 versus 50, respectively; P = 0.00006). For every 10-unit increase in TMEM density, the odds ratio for systemic metastasis was 1.9 (95% confidence interval, 1.1-3.4).Conclusions: TMEM density predicted the development of systemic, hematogenous metastases. The ability of TMEM to predict distant metastasis was independent of lymph node status and other currently used prognosticators. Quantitation of TMEM may be a useful new prognostic marker for breast cancer patients.

Figure S1. Bak BH3 peptide position in Bak protein. Figure S2. Dephosphorylation of Bax by lambdaPPase in vitro. Figure S3. Bax activators CYD-2-11 and SMBA1 failed to induce Bak oligomerization. Figure S4. Analysis of CYD-2-11 toxicity in vivo. Figure S5. CYD-2-11 has no significant effect on total Bax expression in SCLC PDX models. Figure S6. Toxicity analysis of CYD-2-11 in 2 PDX models. Figure S7. Bax phosphorylation is up-regulated in KRAS driven lung cancer. Figure S8. Quantification of tumor burden. Figure S9. H&E staining of normal tissues from various organs after CYD-2-11 treatment of KL mice. Figure S10. The dual PI3K/mTOR inhibitor BEZ235 inhibits ATK activity leading to down-regulation of Bax phosphorylation. Figure S11. CYD-2-11 synergizes with RAD001 to reverse rapalog resistance of lung cancer cells. Figure S12. RAD001 and CYD-2-11 did not affect total Bax in A549 xenografts. Figure S13. Toxicity analysis following treatment with RAD001, CYD-2-11 or in combination in A549 xenografts. Figure S14. CYD-2-11 synergizes with RAD001 against lung cancer in PDX model.

Supplementary Table S1. Differentially expressed genes

PDF file - 53K, ROC curve to determine the best cutoff value for CHFR intensity

Purpose: Currently, there is no clinically validated test for the prediction of response to tubulin-targeting agents in non–small cell lung cancer (NSCLC). Here, we investigated the significance of nuclear expression of the mitotic checkpoint gene checkpoint with forkhead and ringfinger domains (CHFR) as predictor of response and overall survival with taxane-based first-line chemotherapy in advanced stage NSCLC.Methods: We studied a cohort of 41 patients (median age 63 years) with advanced NSCLC treated at the Atlanta VAMC between 1999 and 2010. CHFR expression by immunohistochemistry (score 0–4) was correlated with clinical outcome using chi-square test and Cox proportional models. A cutoff score of “3” was determined by receiver operator characteristics analysis for “low” CHFR expression. Results were validated in an additional 20 patients who received taxane-based chemotherapy at Emory University Hospital and the Atlanta VAMC.Results: High expression (score = 4) of CHFR is strongly associated with adverse outcomes: the risk for progressive disease after first-line chemotherapy with carboplatin–paclitaxel was 52% in patients with CHFR-high versus only 19% in those with CHFR-low tumors (P = 0.033). Median overall survival was strongly correlated with CHFR expression status (CHFR low: 9.9 months; CHFR high: 6.2 months; P = 0.002). After multivariate adjustment, reduced CHFR expression remained a powerful predictor of improved overall survival (HR = 0.24; 95% CI, 0.1–0.58%; P = 0.002). In the validation set, low CHFR expression was associated with higher likelihood of clinical benefit (P = 0.03) and improved overall survival (P = 0.038).Conclusions: CHFR expression is a novel predictive marker of response and overall survival in NSCLC patients treated with taxane-containing chemotherapy. Clin Cancer Res; 19(6); 1603–11. ©2013 AACR.

Bcl-XL is a major antiapoptotic protein in the Bcl-2 family whose overexpression is more widely observed in human lung cancer cells than that of Bcl-2, suggesting that Bcl-XL is more biologically relevant and therefore a better therapeutic target for lung cancer. Here, we screened small molecules that selectively target the BH3 domain (aa 90–98) binding pocket of Bcl-XL using the UCSF DOCK 6.1 program suite and the NCI chemical library database. We identified two new Bcl-XL inhibitors (BXI-61 and BXI-72) that exhibit selective toxicity against lung cancer cells compared with normal human bronchial epithelial cells. Fluorescence polarization assay reveals that BXI-61 and BXI-72 preferentially bind to Bcl-XL protein but not Bcl2, Bcl-w, Bfl-1/A1, or Mcl-1 in vitro with high binding affinities. Treatment of cells with BXI-72 results in disruption of Bcl-XL/Bak or Bcl-XL/Bax interaction, oligomerization of Bak, and cytochrome c release from mitochondria. Importantly, BXI-61 and BXI-72 exhibit more potent efficacy against human lung cancer than ABT-737 but less degree in platelet reduction in vivo. BXI-72 overcomes acquired radioresistance of lung cancer. On the basis of our findings, the development of BXI(s) as a new class of anticancer agents is warranted and represents a novel strategy for improving lung cancer outcome. Cancer Res; 73(17); 5485–96. ©2013 AACR.

PDF file, 198K, Analysis of toxicity for combination of BXI-72 and IR in vivo.

PDF file, 73K, Irradiation resistance (IRR) is associated with elevated levels of Bcl-XL and Bcl-2 in human lung cancer cells.

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