Revealing the heterogeneity in neuroblastoma cells via nanopillar-guided subnuclear deformation.

Neuroblastoma is a hard-to-treat childhood cancer that is well known for the heterogeneity of its clinical phenotypes. Although the risk levels of neuroblastoma have been defined from a complex matrix of clinical and tumor biological factors to guide treatment, the accuracy in predicting cancer relapse and related fatality is still poor in many cases, where heterogeneity with subpopulations in highly malignant or drug-resistant tumors is believed to be underestimated by the current analysis methods. Therefore, new technologies to probe neuroblastoma heterogeneity are needed for the improvement of risk stratification. In this study, we introduce the nanopillar-guided subnuclear morphology as an effective indicator for heterogeneity evaluation among individual neuroblastoma cells. Nuclear polymorphisms, especially the generation of subnuclear irregularities, are well-known markers of high cancer metastasis risk and poor prognosis. By quantitatively evaluating the orientation of nanopillar-guided nuclear envelope features in neuroblastoma cells, we identified two subpopulations with differential motilities and EMT marker levels. Moreover, with endogenous expression, cells with high levels of the nuclear structure protein lamin A exhibit anisotropic deformation on nanopillars and migrate faster than low-lamin A cells, indicating a greater potential for metastasis. Overexpression of lamin A, however, reduces both the coherency and migration speed, suggesting that subpopulations with similar lamin A levels may have different metastatic potentials. We further verified that nanopillar-generated nuclear deformation patterns can quantitatively reveal individual cells' responses to anti-cancer drug treatment. Overall, we envision that the nanopillar-based assessment of subnuclear irregularities brings new additions to our toolkits for both precise risk stratification in neuroblastoma and the evaluation of related anti-cancer therapeutics.

The chromatin structuring protein HMGA2 influences human subtelomere stability and cancer chemosensitivity.

The transient build-up of DNA supercoiling during the translocation of replication forks threatens genome stability and is controlled by DNA topoisomerases (TOPs). This crucial process has been exploited with TOP poisons for cancer chemotherapy. However, pinpointing cellular determinants of the best clinical response to TOP poisons still remains enigmatic. Here, we present an integrated approach and demonstrate that endogenous and exogenous expression of the oncofetal high-mobility group AT-hook 2 (HMGA2) protein exhibited broad protection against the formation of hydroxyurea-induced DNA breaks in various cancer cells, thus corroborating our previously proposed model in which HMGA2 functions as a replication fork chaperone that forms a protective DNA scaffold at or close to stalled replication forks. We now further demonstrate that high levels of HMGA2 also protected cancer cells against DNA breaks triggered by the clinically important TOP1 poison irinotecan. This protection is most likely due to the recently identified DNA supercoil constraining function of HMGA2 in combination with exclusion of TOP1 from binding to supercoiled substrate DNA. In contrast, low to moderate HMGA2 protein levels surprisingly potentiated the formation of irinotecan-induced genotoxic covalent TOP1-DNA cleavage complexes. Our data from cell-based and several in vitro assays indicate that, mechanistically, this potentiating role involves enhanced drug-target interactions mediated by HMGA2 in ternary complexes with supercoiled DNA. Subtelomeric regions were found to be extraordinarily vulnerable to these genotoxic challenges induced by TOP1 poisoning, pointing at strong DNA topological barriers located at human telomeres. These findings were corroborated by an increased irinotecan sensitivity of patient-derived xenografts of colorectal cancers exhibiting low to moderate HMGA2 levels. Collectively, we uncovered a therapeutically important control mechanism of transient changes in chromosomal DNA topology that ultimately leads to enhanced human subtelomere stability.

The RNA interactome of human telomerase RNA reveals a coding-independent role for a histone mRNA in telomere homeostasis.

Telomerase RNA (TR) provides the template for DNA repeat synthesis at telomeres and is essential for genome stability in continuously dividing cells. We mapped the RNA interactome of human TR (hTR) and identified a set of non-coding and coding hTR-interacting RNAs, including the histone 1C mRNA (HIST1H1C). Disruption of the hTR-HIST1H1C RNA association resulted in markedly increased telomere elongation without affecting telomerase enzymatic activity. Conversely, over-expression of HIST1H1C led to telomere attrition. By using a combination of mutations to disentangle the effects of histone 1 RNA synthesis, protein expression, and hTR interaction, we show that HIST1H1C RNA negatively regulates telomere length independently of its protein coding potential. Taken together, our data provide important insights into a surprisingly complex hTR-RNA interaction network and define an unexpected non-coding RNA role for HIST1H1C in regulating telomere length homeostasis, thus offering a glimpse into the mostly uncharted, vast space of non-canonical messenger RNA functions.