Tumor Microenvironment Enriches the Stemness Features: The Architectural Event of Therapy Resistance and Metastasis

Cancer divergence has many facets other than being considered a genetic term. It is a tremendous challenge to understand the metastasis and therapy response in cancer biology; however, it postulates the opportunity to explore the possible mechanism in the surrounding tumor environment. Most deadly solid malignancies are distinctly characterized by their tumor microenvironment (TME). TME consists of stromal components such as immune, inflammatory, endothelial, adipocytes, and fibroblast cells. Cancer stem cells (CSCs) or cancer stem-like cells are a small sub-set of the population within cancer cells believed to be a responsible player in the self-renewal, metastasis, and therapy response of cancer cells. The correlation between TME and CSCs remains an enigma in understanding the events of metastasis and therapy resistance in cancer biology. Recent evidence suggests that TME dictates the CSCs maintenance to arbitrate cancer progression and metastasis. The immune, inflammatory, endothelial, adipocyte, and fibroblast cells in the TME release growth factors, cytokines, chemokines, microRNAs, and exosomes that provide cues for the gain and maintenance of CSC features. These intricate cross-talks are fueled to evolve into aggressive, invasive, migratory phenotypes for cancer development. In this review, we have abridged the recent developments in the role of the TME factors in CSC maintenance and how these events influence the transition of tumor progression to further translate into metastasis and therapy resistance in cancer

Afatinib and Temozolomide Combination Inhibits Tumorigenesis by Targeting EGFRvIII-cMet Signaling in Glioblastoma Cells

BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor with universal recurrence and poor prognosis. The recurrence is largely driven by chemoradiation resistant cancer stem cells (CSCs). Epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are amplified in ~ 60% and ~ 30% of GBM patients, respectively; however, therapies targeting EGFR have failed to improve disease outcome. EGFRvIII-mediated cross-activation of tyrosine kinase receptor, cMET, regulates GBM CSC maintenance and promote tumor recurrence. Here, we evaluated the efficacy of pan-EGFR inhibitor afatinib and Temozolomide (TMZ) combination on GBM in vitro and in vivo. METHODS: We analyzed the effect of afatinib and temozolomide (TMZ) combination on GBM cells U87MG and U251 engineered to express wild type (WT) EGFR, EGFRvIII or EGFRvIII dead kinase, CSCs isolated from U87 and U87EGFRvIII in vitro. The therapeutic utility of the drug combination was investigated on tumor growth and progression using intracranially injected U87EGFRvIII GBM xenografts. RESULTS: Afatinib and TMZ combination synergistically inhibited the proliferation, clonogenic survival, motility, invasion and induced senescence of GBM cells compared to monotherapy. Mechanistically, afatinib decreased U87EGFRvIII GBM cell proliferation and motility/invasion by inhibiting EGFRvIII/AKT, EGFRvIII/JAK2/STAT3, and focal adhesion kinase (FAK) signaling pathways respectively. Interestingly, afatinib specifically inhibited EGFRvIII-cMET crosstalk in CSCs, resulting in decreased expression of Nanog and Oct3/4, and in combination with TMZ significantly decreased their self-renewal property in vitro. More interestingly, afatinib and TMZ combination significantly decreased the xenograft growth and progression compared to single drug alone. CONCLUSION: Our study demonstrated significant inhibition of GBM tumorigenicity, CSC maintenance in vitro, and delayed tumor growth and progression in vivo by combination of afatinib and TMZ. Our results warrant evaluation of this drug combination in EGFR and EGFRvIII amplified GBM patients

Genome-Wide Differentiation of Various Melon Horticultural Groups for Use in GWAS for Fruit Firmness and Construction of a High Resolution Genetic Map

Ajuts: Funding support is provided by Gus R. Douglass Institute (Evans Allen Project to Nimmakayala) and USDA-NIFA (2010-02247 and 2012-02511).Melon (Cucumis melo L.) is a phenotypically diverse eudicot diploid (2n = 2x = 24) has climacteric and non-climacteric morphotypes and show wide variation for fruit firmness, an important trait for transportation and shelf life. We generated 13,789 SNP markers using genotyping-by-sequencing (GBS) and anchored them to chromosomes to understand genome-wide fixation indices (Fst) between various melon morphotypes and genomewide linkage disequilibrium (LD) decay. The FST between accessions of cantalupensis and inodorus was 0.23. The FST between cantalupensis and various agrestis accessions was in a range of 0.19-0.53 and between inodorus and agrestis accessions was in a range of 0.21-0.59 indicating sporadic to wide ranging introgression. The EM (Expectation Maximization) algorithm was used for estimation of 1436 haplotypes. Average genome-wide LD decay for the melon genome was noted to be 9.27 Kb. In the current research, we focused on the genome-wide divergence underlying diverse melon horticultural groups. A high-resolution genetic map with 7153 loci was constructed. Genome-wide segregation distortion and recombination rate across various chromosomes were characterized. Melon has climacteric and non-climacteric morphotypes and wide variation for fruit firmness, a very important trait for transportation and shelf life. Various levels of QTLs were identified with high to moderate stringency and linked to fruit firmness using both genome-wide association study (GWAS) and biparental mapping. Gene annotation revealed some of the SNPs are located in β-D-xylosidase, glyoxysomal malate synthase, chloroplastic anthranilate phosphoribosyltransferase, and histidine kinase, the genes that were previously characterized for fruit ripening and softening in other crops

Elevated PAF1-RAD52 Axis Confers Chemoresistance to Human Cancers

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers

Disruption of FDPS/Rac1 Axis Radiosensitizes Pancreatic Ductal Adenocarcinoma by Attenuating DNA Damage Response and Immunosuppressive Signalling

BACKGROUND: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells. METHODS: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient\u27s clinical study. FINDINGS: FDPS overexpression in PDAC tissues and cells (P \u3c 0.01 and P \u3c 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P \u3c 0.05, P \u3c 0.01, and P \u3c 0.001) and in vivo (P \u3c 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment. INTERPRETATION: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers. FUNDING: National Institutes of Health (P50, P01, and R01)

Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits

[EN] Cucurbita pepo contains two cultivated subspecies, each of which encompasses four fruit-shape morphotypes (cultivar groups). The Pumpkin, Vegetable Marrow, Cocozelle, and Zucchini Groups are of subsp. pepo and the Acorn, Crookneck, Scallop, and Straightneck Groups are of subsp. ovifera. Recently, a de novo assembly of the C. pepo subsp. pepo Zucchini genome was published, providing insights into its evolution. To expand our knowledge of evolutionary processes within C. pepo and to identify variants associated with particular morphotypes, we performed whole-genome resequencing of seven of these eight C. pepo morphotypes. We report for the first time whole-genome resequencing of the four subsp. pepo (Pumpkin, Vegetable Marrow, Cocozelle, green Zucchini, and yellow Zucchini) morphotypes and three of the subsp. ovifera (Acorn, Crookneck, and Scallop) morphotypes. A high-depth resequencing approach was followed, using the BGISEQ-500 platform that enables the identification of rare variants, with an average of 33.5X. Approximately 94.5% of the clean reads were mapped against the reference Zucchini genome. In total, 3,823,977 high confidence single-nucleotide polymorphisms (SNPs) were identified. Within each accession, SNPs varied from 636,918 in green Zucchini to 2,656,513 in Crookneck, and were distributed homogeneously along the chromosomes. Clear differences between subspecies pepo and ovifera in genetic variation and linkage disequilibrium are highlighted. In fact, comparison between subspecies pepo and ovifera indicated 5710 genes (22.5%) with Fst > 0.80 and 1059 genes (4.1%) with Fst = 1.00 as potential candidate genes that were fixed during the independent evolution and domestication of the two subspecies. Linkage disequilibrium was greater in subsp. ovifera than in subsp. pepo, perhaps reflective of the earlier differentiation of morphotypes within subsp. ovifera. Some morphotype-specific genes have been localized. Our results offer new clues that may provide an improved understanding of the underlying genomic regions involved in the independent evolution and domestication of the two subspecies. Comparisons among SNPs unique to particular subspecies or morphotypes may provide candidate genes responsible for traits of high economic importance.This work has been supported by Hellenic Agricultural Organization (ELGO) Demeter. Furthermore, we thank the Conselleria de Educacio, Investigacio, Cultura i Esport (Generalitat Valenciana) for funding Project Prometeo 2017/078 "Seleccion de Variedades Tradicionales y Desarrollo de Nuevas Variedades de Cucurbitaceas Adaptadas a la Produccion Ecologica". Also, this work was supported by Chiang Mai University.Xanthopoulou, A.; Montero-Pau, J.; Mellidou, I.; Kissoudis, C.; Blanca Postigo, JM.; Picó Sirvent, MB.; Tsaballa, A.... (2019). Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits. Horticulture Research. 6:1-17. https://doi.org/10.1038/s41438-019-0176-9S1176Maynard, D. & Paris, H. in The Encyclopedia of Fruits & Nuts (eds Paull, R. E. & Janick, J.) 276–313 (CABI, New Jersey, U.S.A., 2018).Paris, H. S. in Genetics and Genomics of Cucurbitaceae, Grumet, Rebecca, Katzir, Nurit, Garcia-Mas, Jordi (Eds.) 111–154 (Springer, New York, U.S.A., 2016).Whitaker, T. W. & Davis, G. N. Cucurbits (Leonard Hill (Books) Ltd., London, and Interscience Publishers Inc., New York, 1962).Paris, H. S. History of the cultivar-groups of Cucurbita pepo. Hortic. Rev. 25, 71–170 (2001).Paris, H. S. A proposed subspecific classifiaction for Cucurbita pepo. Phytologia (USA) 61, 133–138 (1986).Lira, R., Andres, T. C. & Nee, M. in Systematic and Ecogeographic Studies on Crop Genepools, Vol. 9, 1–115 (International Plant Genetic Resources Institute, Roma, Italia, 1995).Castellanos-Morales, G. Historical biogeography and phylogeny of Cucurbita: insights from ancestral area reconstruction and niche evolution. Mol. Phylogenet. Evol. 128, 38–54 (2018).Paris, H. S., Lebeda, A., Křistkova, E., Andres, T. C. & Nee, M. H. Parallel evolution under domestication and phenotypic differentiation of the cultivated subspecies of Cucurbita pepo (Cucurbitaceae). Econ. Bot. 66, 71–90 (2012).Dong, W., Wu, D., Li, G., Wu, D. & Wang, Z. Next-generation sequencing from bulked segregant analysis identifies a dwarfism gene in watermelon. Sci. Rep. 8, 2908 (2018).Galpaz, N. et al. Deciphering genetic factors that determine melon fruit‐quality traits using RNA‐Seq‐based high‐resolution QTL and eQTL mapping. Plant J. 94, 169–191 (2018).Gur, A. et al. Genome-wide linkage-disequilibrium mapping to the candidate gene level in melon (Cucumis melo). Sci. Rep. 7, 9770 (2017).Blanca, J. et al. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genom. 12, 104 (2011).Esteras, C. et al. High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genom. 13, 80 (2012).Montero-Pau, J. et al. An SNP-based saturated genetic map and QTL analysis of fruit-related traits in Zucchini using genotyping-by-sequencing. BMC Genom. 18, 94 (2017).Vicente-Dólera, N. et al. First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement. PLoS ONE 9, e112743 (2014).Wyatt, L. E., Strickler, S. R., Mueller, L. A. & Mazourek, M. An acorn squash (Cucurbita pepo ssp. ovifera) fruit and seed transcriptome as a resource for the study of fruit traits in Cucurbita. Hortic. Res. 2, 14070 (2015).Xanthopoulou, A. et al. De novo comparative transcriptome analysis of genes involved in fruit morphology of pumpkin cultivars with extreme size difference and development of EST-SSR markers. Gene 622, 50–66 (2017).Montero‐Pau, J. et al. De novo assembly of the zucchini genome reveals a whole‐genome duplication associated with the origin of the Cucurbita genus. Plant Biotechnol. J. 16, 1161–1171 (2018).Garcia-Mas, J. et al. Cloning and mapping of resistance gene homologues in melon. Plant Sci. 161, 165–172 (2001).Xanthopoulou, A. et al. Comparative analysis of genetic diversity in Greek Genebank collection of summer squash (‘Cucurbita pepo’) landraces using start codon targeted (SCoT) polymorphism and ISSR markers. Aust. J. Crop Sci. 9, 14 (2015).Huang, J. et al. A reference human genome dataset of the BGISEQ-500 sequencer. Gigascience 6, gix024 (2017).Natarajan, K. N. et al. Comparative analysis of sequencing technologies for single-cell transcriptomics. Genome Biol. 20, 70 (2019).Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).Tian, L. et al. Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots. BMC Plant Biol. 9, 1 (2009).Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).Bradbury, P. J. et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635 (2007).Chang, C. C. et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 4, 7 (2015).Team, R. C. (2015). http://www.r-project.org/ .Krzywinski, M. I. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).Kosman, E. & Leonard, K. J. Similarity coefficients for molecular markers in studies of genetic relationships between individuals for haploid, diploid, and polyploid species. Mol. Ecol. 14, 415–424 (2005).Huson, D. H. & Bryant, D. Estimating Phylogenetic Trees and Networks Using SplitsTree 4. www.splitstree.org (2005).Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strainw1118; iso-2; iso-3. Fly 6, 80–92 (2012).Wu, S. et al. A common genetic mechanism underlies morphological diversity in fruits and other plant organs. Nat. Commun. 9, 4734 (2018).Drevensek, S. et al. The Arabidopsis TRM1–TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes. Plant Cell 24, 178–191 (2012).Sievers, F. et al. Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2014).Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587 (2017).Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2017).Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).Leida, C. et al. Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon (Cucumis melo L.). BMC Genet. 16, 28 (2015).Esteras, C. et al. SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium. Theor. Appl. Genet. 126, 1285–1303 (2013).Maria José Gonzalo et al. Re-evaluation of the role of Indian germplasm as center of melon diversification based on genotyping-by-sequencing analysis. BMC Genom. 20, p. 448 (2019).Nimmakayala, P. et al. Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon. BMC Genom. 15, 767 (2014).Gonzalo, M. J. & Monforte, A. J. in Genetics and Genomics of Cucurbitaceae, Grumet, Rebecca, Katzir, Nurit, Garcia-Mas, Jordi (Eds.) 269–290 (Springer, New York, U.S.A., 2016).Pomares-Viciana, T. et al. First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.). BMC Plant Biol. 19, 61 (2019).Lu, S. et al. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulation. Plant Cell 18, 3594–3605 (2006).Jin, B., Kim, J., Jung, J., Kim, D. & Park, Y. Characterization of IQ domain gene homologs as common candidate genes for elongated fruit shape in cucurbits. Hortic. Sci. Technol. 36, 85–97 (2018).van der Knaap, E. et al. What lies beyond the eye: the molecular mechanisms regulating tomato fruit weight and shape. Front. Plant Sci. 5, 227 (2014).Xiao, H., Jiang, N., Schaffner, E., Stockinger, E. J. & Van Der Knaap, E. A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit. Science 319, 1527–1530 (2008).Dou, J. et al. Genetic mapping reveals a candidate gene (ClFS1) for fruit shape in watermelon (Citrullus lanatus L.). Theor. Appl. Genet. 131, 947–958 (2018).Pan, Y. et al. Round fruit shape in WI7239 cucumber is controlled by two interacting quantitative trait loci with one putatively encoding a tomato SUN homolog. Theor. Appl. Genet. 130, 573–586 (2017).Liu, J. et al. Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening. PLoS ONE 10, e0123870 (2015).Liu, J. et al. Mu MADS 1 and Ma OFP 1 regulate fruit quality in a tomato ovate mutant. Plant Biotechnol. J. 16, 989–1001 (2018).Cong, B., Barrero, L. S. & Tanksley, S. D. Regulatory change in YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication. Nat. Genet. 40, 800 (2008).Huang, Z., Van Houten, J., Gonzalez, G., Xiao, H. & van der Knaap, E. Genome-wide identification, phylogeny and expression analysis of SUN, OFP and YABBY gene family in tomato. Mol. Genet. Genom. 288, 111–129 (2013).Bowman, J. L. The YABBY gene family and abaxial cell fate. Curr. Opin. Plant Biol. 3, 17–22 (2000).Liu, J., Van Eck, J., Cong, B. & Tanksley, S. D. A new class of regulatory genes underlying the cause of pear-shaped tomato fruit. Proc. Natl Acad. Sci. USA 99, 13302–13306 (2002).Tsaballa, A., Pasentsis, K., Darzentas, N. & Tsaftaris, A. S. Multiple evidence for the role of an Ovate-like gene in determining fruit shape in pepper. BMC Plant Biol. 11, 46 (2011).Wang, S., Chang, Y., Guo, J. & Chen, J. G. Arabidopsis Ovate family protein 1 is a transcriptional repressor that suppresses cell elongation. Plant J. 50, 858–872 (2007).Lazzaro, M. D., Wu, S., Snouffer, A., Wang, Y. & Van Der Knaap, E. Plant organ shapes are regulated by protein interactions and associations with microtubules. Front. Plant Sci. 9, 1766 (2018)