br Available online February br Fig Low SUV H
Available online 14 February 2019
Fig. 1. Low SUV39H1 expression characterises cervical cancer migratory states. a. Schematic representation of an in vitro transwell set up, used to obtain populations of migrated and non migrated SiHa cells. b. Western blot showing abundance of SUV39H1 in migrated populations of NPS-2143 (n = 3). c. Quantitation of SUV39H1 knockdown efficiency, 48 h post transfection, using western blotting (n = 3). d. Quantitation of migration and invasion in SUV39H1 kd cells using a transwell assay (n = 3, *, p < 0.05). e. Cleaved PARP estimation by western blotting under anoikis conditions (n = 4) f. Estimation of clonogenic potential using clonogenic assay (n = 3, *, p < 0.05). Western blots depict mean fold intensity ± S.E.M.
Mesenchymal Transition (EMT) . While these approaches have been informative, alternative modalities of cell migration have also been reported in solid cancers , and in other contexts, EMT is not associated with migration, and is instead linked to functions such as chemoresistance [12,13]. To study chromatin changes directly asso-ciated with migratory traits, we analysed migrated and non migrated populations of cervical cancer cells, functionally segregated with the help of an in vitro transwell set up (Fig. 1a). As invasive and pre-me-tastatic traits prominently emerge during the carcinoma in situ stage, we used the SiHa cell line, which is derived from a primary cervical car-cinoma. Combining this approach with an analysis of TCGA SUV39H1-low tumours, immunohistochemistry, genomics, imaging and RNAi, we demonstrate that a SUV39H1-low chromatin state associates with, and promotes, migratory cell populations in cervical cancers.
2.1. Low SUV39H1 expression characterises cervical cancer migratory states in vitro
Initial observations showed a depletion of SUV39H1 in CD66 en-riched spheroid cultures , as well as in CD66 +ve cells sorted from these cultures (Fig. S1a). Given these findings, we sought to determine whether SUV39H1 depletion is directly linked to a migratory pheno-type, and whether this depletion enhances cell migration. To examine cell populations showing enhanced migration, we sorted migrated and non migrated populations of SiHa cells using a transwell set up (Fig. 1a). 1.9-fold lower SUV39H1 protein was noted in sorted migrated populations, relative to non-migrated populations (Fig. 1b). Next, we
used endoribonuclease-prepared silencer RNAs (esiRNAs) to knock down SUV39H1 in SiHa cells, to avoid off-target effects using individual siRNAs. SUV39H1 knockdown led to a 3 and 2-fold enhanced transwell migration and Matrigel invasion, respectively, and 2-fold lower cleaved PARP under anoikis conditions (Fig. 1c–e). Under proliferation cues, these cells also show 2.7-fold enhanced clonogenic potential (Fig. 1f, Fig. S1b). These phenotypes were accompanied by a 1.4-fold upregu-lation of CD66 at a protein level (Fig. S1c). We also used a retroviral shRNA approach to knockdown SUV39H1. qPCR indicated that the SUV39H1 shRNA knockdown was successful (Fig. S1d). Additionally, this approach further confirmed that a knockdown of SUV39H1 en-hances cell migration (Fig. S1e). On the other hand, overexpression of SUV39H1 resulted in a small decrease in migratory ability (1.2-fold) (Fig. S1f, g). Thus, SUV39H1 protein is depleted in migrated popula-tions in vitro, and its knockdown results in enhanced migration and invasion.
2.2. Low SUV39H1 expression correlates with poor clinical outcomes in cervical cancers
Given that low SUV39H1 was noted in migrated populations, and SUV39H1 knockdown resulted in enhanced tumorigenic properties in vitro, we next asked if SUV39H1 abundance in tumours correlated with disease outcomes in human cervical cancers. We examined publicly available RNA-seq data from primary human cervical tumours (TCGA cervical squamous cell carcinoma and endocervical adenocarcinoma, CESC) [14,15]. Squamous Cell Carcinoma (SCC) cases were sorted into SUV39H1-low and SUV39H1-high groups, based on tumoural SUV39H1 mRNA expression. The SUV39H1-low group showed significantly lower