DataSheet1_Integrated transcriptome and cell phenotype analysis suggest involvement of PARP1 cleavage, Hippo/Wnt, TGF-β and MAPK signaling pathways in ovarian cancer cells response to cannabis and PARP1 inhibitor treatment.docx

Introduction:Cannabis sativa is utilized mainly for palliative care worldwide. Ovarian cancer (OC) is a lethal gynecologic cancer. A particular cannabis extract fraction ('F7′) and the Poly(ADP-Ribose) Polymerase 1 (PARP1) inhibitor niraparib act synergistically to promote OC cell apoptosis. Here we identified genetic pathways that are altered by the synergistic treatment in OC cell lines Caov3 and OVCAR3.Materials and methods: Gene expression profiles were determined by RNA sequencing and quantitative PCR. Microscopy was used to determine actin arrangement, a scratch assay to determine cell migration and flow cytometry to determine apoptosis, cell cycle and aldehyde dehydrogenase (ALDH) activity. Western blotting was used to determine protein levels.Results: Gene expression results suggested variations in gene expression between the two cell lines examined. Multiple genetic pathways, including Hippo/Wnt, TGF-β/Activin and MAPK were enriched with genes differentially expressed by niraparib and/or F7 treatments in both cell lines. Niraparib + F7 treatment led to cell cycle arrest and endoplasmic reticulum (ER) stress, inhibited cell migration, reduced the % of ALDH positive cells in the population and enhanced PARP1 cleavage.Conclusion: The synergistic effect of the niraparib + F7 may result from the treatment affecting multiple genetic pathways involving cell death and reducing mesenchymal characteristics.</p

Table1_Integrated transcriptome and cell phenotype analysis suggest involvement of PARP1 cleavage, Hippo/Wnt, TGF-β and MAPK signaling pathways in ovarian cancer cells response to cannabis and PARP1 inhibitor treatment.XLSX

Introduction:Cannabis sativa is utilized mainly for palliative care worldwide. Ovarian cancer (OC) is a lethal gynecologic cancer. A particular cannabis extract fraction ('F7′) and the Poly(ADP-Ribose) Polymerase 1 (PARP1) inhibitor niraparib act synergistically to promote OC cell apoptosis. Here we identified genetic pathways that are altered by the synergistic treatment in OC cell lines Caov3 and OVCAR3.Materials and methods: Gene expression profiles were determined by RNA sequencing and quantitative PCR. Microscopy was used to determine actin arrangement, a scratch assay to determine cell migration and flow cytometry to determine apoptosis, cell cycle and aldehyde dehydrogenase (ALDH) activity. Western blotting was used to determine protein levels.Results: Gene expression results suggested variations in gene expression between the two cell lines examined. Multiple genetic pathways, including Hippo/Wnt, TGF-β/Activin and MAPK were enriched with genes differentially expressed by niraparib and/or F7 treatments in both cell lines. Niraparib + F7 treatment led to cell cycle arrest and endoplasmic reticulum (ER) stress, inhibited cell migration, reduced the % of ALDH positive cells in the population and enhanced PARP1 cleavage.Conclusion: The synergistic effect of the niraparib + F7 may result from the treatment affecting multiple genetic pathways involving cell death and reducing mesenchymal characteristics.</p

Table of demographics.

<p>Table of demographics.</p

A 15-loci MIRU-VNTR derived neighbour joining tree (right) of 142 strains of the LAM clade (lineage 4) for which both whole genome sequence data were available labelled according to Institut Pasteur website www.miru-vntrplus.org and compared to a whole genome sequence neighbour joining tree (left) demonstrating the misclassification of strains into groups at high definition based on MIRU alone.

<p>A 15-loci MIRU-VNTR derived neighbour joining tree (right) of 142 strains of the LAM clade (lineage 4) for which both whole genome sequence data were available labelled according to Institut Pasteur website <a href="http://www.miru-vntrplus.org/" target="_blank">www.miru-vntrplus.org</a> and compared to a whole genome sequence neighbour joining tree (left) demonstrating the misclassification of strains into groups at high definition based on MIRU alone.</p

Manhattan plot of the polymorphisms most influential of phylogenetic structure with the most significant genes labelled.

<p>Manhattan plot of the polymorphisms most influential of phylogenetic structure with the most significant genes labelled.</p

Phylogeny of study strains with clade names and position of homoplastic sites identified in the study.

<p>Phylogeny of study strains with clade names and position of homoplastic sites identified in the study.</p