Biology subjects

La Rocca, G.

Publications and source records attributed to La Rocca, G..

7 recordsLinked to original sources

bioRxiv2025

miRISC inhibition causes mitotic defects and synergizes with genotoxic agents in cancers

Although individual microRNAs (miRNAs) can have tumorigenic or tumor-suppressive properties, their overall role in cancer remains controversial. Here, we show that cancer tissues and cell lines are characterized by preferential accumulation of the high-molecular-weight miRNA-induced silencing complex (HMWR), the functionally active form of the effector complex responsible for miRNA-mediated gene repression. Experimentally induced disassembly of the HMWR impairs the growth of human tumor xenografts and of autochthonous tumors in mouse models of human cancer in vivo. Furthermore, disassembly of the HMWR increases chromosome mis-segregation, which synergized with genotoxic agents to potentiate cancer cell vulnerability and improve therapeutic response. These findings suggest pharmacologic inhibition of HMWR as a novel anti-cancer strategy.

cancer biology↗

bioRxiv2023

Immortalization and transformation of primary cells mediated by engineered ecDNAs

Focal gene amplifications are among the most common cancer-associated mutations, but their evolution and contribution to tumorigenesis have proven challenging to recapitulate in primary cells and model organisms. Here we describe a general approach to engineer large (>1 Mbp) focal amplifications mediated by extrachromosomal circular DNAs (ecDNAs, also known as "double minutes") in a spatiotemporally controlled manner in cancer cell lines and in primary cells derived from genetically engineered mice. With this strategy, ecDNA formation can be coupled with expression of fluorescent reporters or other selectable markers to enable the identification and tracking of ecDNA-containing cells. We demonstrate the feasibility of this approach by engineering MDM2-containing ecDNAs in near-diploid human cells, showing that GFP expression can be used to track ecDNA dynamics under physiological conditions or in the presence of specific selective pressures. We also apply this approach to generate mice harboring inducible Myc- and Mdm2-containing ecDNAs analogous to those spontaneously occurring in human cancers. We show that the engineered ecDNAs rapidly accumulate in primary cells derived from these animals, promoting proliferation, immortalization, and transformation.

cancer biology↗

bioRxiv2022

AGO2 silences mobile transposons in the nucleus of quiescent cells

Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway with essential roles in development and disease. Yet, little is known about its regulation in vivo. Here, we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells where mTOR signaling is low, AGO2 accumulates in the nucleus where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.

molecular biology↗

bioRxiv2021

Folate pathway metabolites are altered in the plasma of subjects with Down syndrome: relation to chromosomal dosage

Down syndrome (DS) is the most common chromosomal disorder and it is caused by trisomy of chromosome 21 (Hsa21). Subjects with DS show a large heterogeneity of phenotypes and the most constant clinical features present are typical facies and intellectual disability (ID). Several studies demonstrated that trisomy 21 causes an alteration in the metabolic profile, involving among all one-carbon cycle. We performed enzyme-linked immunosorbent assays (ELISAs) to identify the concentration of 5 different intermediates of the one-carbon cycle in plasma samples obtained from a total of 164 subjects with DS compared to 54 euploid subjects. We investigated: tetrahydrofolate (THF; DS n=108, control n=41), 5-methyltetrahydrofolate (5-methyl-THF; DS n=140, control n=34), 5-formyltetrahydrofolate (5-formyl-THF; DS n=80, control n=21), S-adenosyl-homocysteine (SAH; DS n=94, control n=20) and S-adenosyl-methionine (SAM; DS n=24, control n=15). Results highlight specific alterations of THF with a median concentration ratio DS/control of 2:3, a decrease of a necessary molecule perfectly consistent with a chromosomal dosage effect. Moreover, SAM and SAH show a ratio DS/control of 1.82:1 and 3.6:1, respectively. The relevance of these results for the biology of intelligence and its impairment in trisomy 21 is discussed, leading to the final proposal of 5-methyl-THF as the best candidate for a clinical trial aimed at restoring the dysregulation of one-carbon cycle in trisomy 21, possibly improving cognitive skills of subjects with DS.

genetics↗

bioRxiv2021

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss of function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

molecular biology↗

bioRxiv2020

mir-21 is associated with inactive low molecular weight Argonaute complexes in thyroid cancer cell lines

Thyroid cancer is the most prevalent endocrine malignancy. We and others have shown that several microRNAs, which are post-transcriptional gene regulators, are aberrantly expressed in anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) tissues, as well as cell lines derived from these cancers. In the cell, miRNAs are bound to Argonaute (AGO) proteins as what could be termed low molecular weight RNA-Induced Silencing Complexes (LMW-RISCs) that can assemble with additional proteins, mRNA, and translation machinery into high molecular weight RISCs (HMW-RISCs) that exert regulatory function. In this study, we sought to analyze the association of miRNAs with RISC complexes in ATC and PTC. For ATC and PTC lines, miRNA species were enriched in both HMW-RISC and LMW-RISC cellular fractions, compared with intermediate molecular weight fractions and very low molecular weight (AGO-poor) fractions. Furthermore, 60% of all miRNAs were slightly more abundant in LMW-RISC versus HMW-RISC fractions by ~2-4 fold. Surprisingly, miR-21-5p, one of the most abundant miRNAs in both ATC and PTC lines and one of the most widely studied oncogenic miRNAs in many solid tumors, was consistently one the least abundant miRNAs in HMW-RISC and the most enriched miRNA in LMW-RISC fractions. These findings may suggest that miR-21 has a role or roles distinct from canonical post-transcriptional regulation in cancer. Furthermore, the methodology described here is a useful way to assess the distribution of miR-21 between HMW and LMW-RISCs and may help to reveal the true roles of this miRNA in thyroid cancer development, progression, and treatment.

molecular biology↗

bioRxiv2019

High-resolution in vivo identification of miRNA targets by Halo-Enhanced Ago2 Pulldown

The identification of miRNA targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily translated to an in vivo setting. To overcome these limitations and to facilitate the investigation of miRNA functions in mice, we have developed a method (HEAP: for Halo-Enhanced Ago2 Pulldown) to map miRNA-mRNA binding sites. This method is based on a novel genetically engineered mouse harboring a conditional, Cre-regulated, Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be efficiently purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, in developing embryos, in adult tissues and in autochthonous mouse models of human brain and lung cancers.\n\nThe tools and the datasets we have generated will serve as a valuable resource to the scientific community and will facilitate the characterization of miRNA functions under physiological and pathological conditions.

molecular biology↗