Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification.

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.

Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Epigenomic landscape of human colorectal cancer unveils an aberrant core of pan-cancer enhancers orchestrated by YAP/TAZ.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.

Effect of donepezil on the expression and responsiveness to LPS of CHRNA7 and CHRFAM7A in macrophages: A possible link to the cholinergic anti-inflammatory pathway.

The α7 nicotinic acetylcholine receptor (CHRNA7) modulates the inflammatory response by activating the cholinergic anti-inflammatory pathway. CHRFAM7A, the human-restricted duplicated form of CHRNA7, has a negative effect on the functioning of α7 receptors, suggesting that CHRFAM7A expression regulation may be a key step in the modulation of inflammation in the human setting. The analysis of the CHRFAM7A gene's regulatory region reveals some of the mechanisms driving its expression and responsiveness to LPS in human immune cell models. Moreover, given the immunomodulatory potential of donepezil we show that it differently modulates CHRFAM7A and CHRNA7 responsiveness to LPS, thus contributing to its therapeutic potential.

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster.

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance.

Lymphoblastoid cell lines as a model to understand amyotrophic lateral sclerosis disease mechanisms.

In the past, amyotrophic lateral sclerosis (ALS) has been considered a 'neurocentric' disease; however, new evidence suggests that it should instead be looked at from a 'multisystemic' or 'non-neurocentric' point of view. From 2006, we focused on the study of non-neural cells: ALS patients' peripheral blood mononuclear cells (PMBCs) and lymphoblastoid cell lines (LCLs). Here, we characterize LCLs of sporadic ALS (sALS) and patients carrying SOD1, TARDBP and FUS mutations to identify an ALS biologically relevant molecular signature, and determine whether and how mutations differentially affect ALS-linked pathways. Although LCLs are different from motor neurons (MNs), in LCLs we found some features typical of degenerating MNs in ALS, i.e. protein aggregation and mitochondrial dysfunction. Moreover, different gene mutations have different effects on ALS cellular mechanisms. TARDBP and FUS mutations imbalance mitochondrial dynamism toward increased fusion, whereas sALS and SOD1 mutations mainly affect fission. With regards to protein aggregation and/or mislocalization, TARDBP and SOD1 mutations show the presence of aggregates, whereas FUS mutation does not induce protein aggregation and/or mislocalization. Finally, all LCLs, independently from mutation, are not able to work in a condition of excessive energy request, suggesting that mitochondria from ALS patients are characterized by a significant metabolic defect. Taken together, these data indicate that LCLs could be a valid cellular model in ALS research in the identification and study of specific pathological pathways.

Glucocorticoids downregulate TLR4 signaling activity via its direct targeting by miR-511-5p.

Endotoxin tolerance assures proper regulation of the TLR4 signaling pathway and avoids uncontrolled inflammation, limiting tissue damage and endotoxin shock development. Though underlying molecular mechanisms are still undefined, evidence indicates the involvement of microRNAs, which represent a new layer of regulation of inflammatory pathways. Here, we report that LPS and other inflammatory stimuli repress miR-511-5p expression in human monocytes, while anti-inflammatory stimuli, such as TGF-ß and glucocorticoids, have the opposite effect. MiR-511-5p levels selectively influenced cell activation when endotoxin was used, while biological activity of other TLR agonists was unaffected. Consistent with this, TLR4 was validated as the miR-511-5p direct target responsible for glucocorticoids- and TGF-ß-mediated inhibition of pro-inflammatory cytokines production observed in endotoxin tolerant monocytes. MiR-511-5p thus acts as an intracellular mediator of glucocorticoids and TGF-ß for the induction of endotoxin tolerance in human monocytes.