The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors.

Background: Papillary thyroid cancers (PTCs) are common, usually indolent malignancies. Still, a small but significant percentage of patients have aggressive tumors and develop distant metastases leading to death. Currently, it is not possible to discriminate aggressive lesions due to lack of prognostic markers. Long noncoding RNAs (lncRNAs), which are selectively expressed in a context-dependent manner, are expected to represent a new landscape to search for molecular discriminants. Transforming growth factor β (TGFβ) is a multifunctional cytokine that fosters epithelial-to-mesenchymal transition and metastatic spreading. In PTCs, it triggers the expression of the metastatic marker Cadherin 6 (CDH6). Here, we investigated the TGFβ-dependent lncRNAs that may cooperate to potentiate PTC aggressiveness. Methods: We used a genome-wide approach to map enhancer (ENH)-associated lncRNAs under TGFβ control. Linc00941 was selected and validated using functional in vitro assays. A combined approach using bioinformatic analyses of the thyroid cancer (THCA)—the cancer genome atlas (TCGA) dataset and RNA-seq analysis was used to identify the processes in which linc00941 was involved in and the genes under its regulation. Correlation with clinical data was performed to evaluate the potential of this lncRNA and its targets as prognostic markers in THCA. Results: Linc00941 was identified as transcribed starting from one of the TGFβ-induced ENHs. Linc00941 expression was significantly higher in aggressive cancer both in the TCGA dataset and in a separate validation cohort from our institution. Loss of function assays for linc00941 showed that it promotes response to stimuli and invasiveness while restraining proliferation in PTC cells, a typical phenotype of metastatic cells. From the integration of TCGA data and linc00941 knockdown RNA-seq profiling, we identified 77 genes under the regulation of this lncRNA. Among these, we found the prometastatic gene CDH6 . Linc00941 knockdown partially recapitulates the effects observed upon CDH6 silencing, promoting cell cytoskeleton and membrane adhesions rearrangements and autophagy. The combined expression of CDH6 and linc00941 is a distinctive feature of highly aggressive PTC lesions. Conclusions: Our data provide new insights into the biology driving metastasis in PTCs and highlight how lncRNAs cooperate with coding transcripts to sustain these processes.

Antisense RNAs (asRNAs) represent an underappreciated yet crucial layer of gene expression regulation. Generally thought to modulate their sense genes in cis through sequence complementarity or their act of transcription, asRNAs can also regulate different molecular targets in trans, in the nucleus or in the cytoplasm. Here, we performed an in-depth molecular characterization of NFYCAntisense1 (NFYC-AS1), the asRNA transcribed head-to-head to NFYC subunit of the proliferation-associated NF-Y transcription factor. Our results show that NFYC-AS1 is a prevalently nuclear asRNA peaking early in the cell cycle. Comparative genomics suggests a narrow phylogenetic distribution, with a probable origin in the common ancestor of mammalian lineages. NFYC-AS1 is overexpressed pancancer, preferentially in association with RB1 mutations. Knockdown of NFYC-AS1 by antisense oligonucleotides impairs cell growth in lung squamous cell carcinoma and small cell lung cancer cells, a phenotype recapitulated by CRISPR/Cas9-deletion of its transcription start site. Surprisingly, expression of the sense gene is affected only when endogenous transcription of NFYC-AS1 is manipulated. This suggests that regulation of cell proliferation is at least in part independent of the in cis transcription-mediated effect on NFYC and is possibly exerted by RNA-dependent in trans effects converging on the regulation of G2/M cell cycle phase genes. Accordingly, NFYC-AS1-depleted cells are stuck in mitosis, indicating defects in mitotic progression. Overall, NFYC-AS1 emerged as a cell cycle-regulating asRNA with dual action, holding therapeutic potential in different cancer types, including the very aggressive RB1-mutated tumors.

The control of gene expression at a transcriptional level requires a widespread landscape of regulatory elements. Central to these regulatory circuits are enhancers (ENHs), which are defined as cis-acting DNA elements able to increase the transcription of a target gene in a distance- and orientation-independent manner. ENHs are not independent functional elements but work in a complex and dynamic cooperative network, constituting the building blocks of multimodular domains of gene expression regulation. The information from each of these elements converges on the target promoter, contributing to improving the precision and sharpness of gene modulation. ENHs’ interplay varies in its nature and extent, ranging from an additive to redundant effect depending on contexts. Moving from super-enhancers that drive the high expression levels of identity genes, to shadow-enhancers, whose redundant functions contribute to buffering the variation in gene expression, this review aims to describe the different modalities of ENHs’ interaction and their role in the regulation of complex biological processes like cancer development.

Recent insights into the mechanisms controlling gene expression identified enhancer-associated long non-coding RNAs (elncRNAs) as master players of transcription in cancers. RUNX2, a mammalian RUNT-related transcription factor, is increasingly recognized in cancer biology for its role in supporting survival and progression also in thyroid cancer (TC). We recently identified, within the RUNX2 locus, a novel elncRNA that we named RAIN (RUNX2 associated intergenic lncRNA). We showed that RAIN and RUNX2 expression correlate in TC, both in vitro and in vivo, and that RAIN promotes RUNX2 expression by interacting with and affecting the activity of the RUNX2 P2 promoter through two distinct mechanisms. Here, we took forward these observations to explore the genome-wide transcriptional function of RAIN and its contribution to the RUNX2-dependent gene expression program in TC. By combining multiple omics data, we demonstrated that RAIN functionally cooperates with RUNX2 to the regulation of a subset of functionally related genes involved in promoting matrix remodeling, migration, and loss of differentiation. We showed that RAIN interacts with RUNX2 and its expression is required for the efficient recruitment of this TF to its target regulatory regions. In addition, our data revealed that besides RUNX2, RAIN governs a hierarchically organized complex transcriptional program by controlling a core of cancer-associated TFs that, in turn, orchestrate the expression of downstream genes. This evidence indicates that the functional cooperation observed between RAIN and RUNX2 can be a diffuse work mechanism for this elncRNA.