MicroRNAs (miRNAs) control cell cycle progression by targeting the transcripts encoding for cyclins, CDKs and CDK inhibitors, such as p27^KIP1 (p27). p27 expression is controlled by multiple transcriptional and posttranscriptional mechanisms, including translational inhibition by miR-221/222 and posttranslational regulation by the SCF^SKP2 complex. The oncosuppressor activity of miR-340 has been recently characterized in breast, colorectal and osteosarcoma tumor cells. However, the mechanisms underlying miR-340-induced cell growth arrest have not been elucidated. Here, we describe miR-340 as a novel tumor suppressor in non-small cell lung cancer (NSCLC). Starting from the observation that the growth-inhibitory and proapoptotic effects of miR-340 correlate with the accumulation of p27 in lung adenocarcinoma and glioblastoma cells, we have analyzed the functional relationship between miR-340 and p27 expression. miR-340 targets three key negative regulators of p27. The miR-340-mediated inhibition of both Pumilio family RNA-binding proteins (PUM1 and PUM2), required for the miR-221/222 interaction with the p27 3′-UTR, antagonizes the miRNA-dependent downregulation of p27. At the same time, miR-340 induces the stabilization of p27 by targeting SKP2, the key posttranslational regulator of p27. Therefore, miR-340 controls p27 at both translational and posttranslational levels. Accordingly, the inhibition of either PUM1 or SKP2 partially recapitulates the miR-340 effect on cell proliferation and apoptosis. In addition to the effect on tumor cell proliferation, miR-340 also inhibits intercellular adhesion and motility in lung cancer cells. These changes correlate with the miR-340-mediated inhibition of previously validated (MET and ROCK1) and potentially novel (RHOA and CDH1) miR-340 target transcripts. Finally, we show that in a small cohort of NSCLC patients (n=23), representative of all four stages of lung cancer, miR-340 expression inversely correlates with clinical staging, thus suggesting that miR-340 downregulation contributes to the disease progression.

Pre–B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial–mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-β. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-β. PREP1 modulates the cellular sensitivity to TGF-β by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-β pathway, EMT, and metastasis in NSCLC. Significance Epithelial–mesenchymal transition (EMT) is a transdifferentiation program implicated in tumor cell dissemination, controlled by networks of transcription complexes responsive to paracrine factors, such as TGF-β. Pre–B-cell leukemia homeobox (Pbx)-regulating protein-1 (PREP1) is a ubiquitous homeodomain transcription factor involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. PREP1 is a haploinsufficient oncosuppressor in mouse tumorigenesis. By characterizing PREP1 as a novel regulator of EMT in human lung adenocarcinoma, we show that PREP1 also harbors prometastatic properties. While autosustaining its activity by stabilizing its transcriptional partner PBX1, PREP1 modulates the responsiveness of lung cancer cells to TGF-β by controlling the expression of two proinvasive transcription factors (SMAD3 and Fos-related antigen 1) implicated in metastasis mechanisms. Thus, PREP1 represents a novel, promising therapeutic target in non-small cell lung cancer.

Among the major mechanisms involved in tumorigenesis, DNA methylation is an important epigenetic modification impacting both genomic stability and gene expression. Methylation of promoter-proximal CpG islands (CGIs) and transcriptional silencing of tumor suppressors represent the best characterized epigenetic changes in neoplastic cells. The global cancer-associated effects of DNA hypomethylation influence chromatin architecture and reactivation of repetitive elements. Moreover, recent analyses of cancer cell methylomes highlight the role of the DNA hypomethylation of super-enhancer regions critically controlling the expression of key oncogenic players. We will first summarize some basic aspects of DNA methylation in tumorigenesis, along with the role of dysregulated DNA methyltransferases and TET (Ten-Eleven Translocation)-family methylcytosine dioxygenases. We will then examine the potential contribution of epimutations to causality and heritability of cancer. By reviewing some representative genes subjected to hypermethylation-mediated silencing, we will survey their oncosuppressor functions and roles as biomarkers in various types of cancer. Epithelial-to-mesenchymal transition (EMT) and the gain of stem-like properties are critically involved in cancer cell dissemination, metastasis, and therapeutic resistance. However, the driver vs passenger roles of epigenetic changes, such as DNA methylation in EMT, are still poorly understood. Therefore, we will focus our attention on several aspects of DNA methylation in control of EMT and metastasis suppressors, including both protein-coding and noncoding genes.

The genetic and epigenetic changes affecting transcription factors, coactivators, and chromatin modifiers are key determinants of the hallmarks of cancer. The acquired dependence on oncogenic transcriptional regulators, representing a major determinant of cancer cell vulnerability, points to transcription factors as ideal therapeutic targets. However, given the unavailability of catalytic activities or binding pockets for small-molecule inhibitors, transcription factors are generally regarded as undruggable proteins. Among components of the AP-1 complex, the FOS-family transcription factor Fra-1, encoded by FOSL1, has emerged as a prominent therapeutic target. Fra-1 is overexpressed in most solid tumors, in response to the BRAF-MAPK, Wnt-beta-catenin, Hippo-YAP, IL-6-Stat3, and other major oncogenic pathways. In vitro functional analyses, validated in onco-mouse models and corroborated by prognostic correlations, show that Fra-1-containing dimers control tumor growth and disease progression. Fra-1 participates in key mechanisms of cancer cell invasion, Epithelial-to-Mesenchymal Transition, and metastatic spreading, by driving the expression of EMT-inducing transcription factors, cytokines, and microRNAs. Here we survey various strategies aimed at inhibiting tumor growth, metastatic dissemination, and drug resistance by interfering with Fra-1 expression, stability, and transcriptional activity. We summarize several tools aimed at the design and tumor-specific delivery of Fra-1/AP-1-specific drugs. Along with RNA-based therapeutics targeting the FOSL1 gene, its mRNA, or cognate regulatory circRNAs, we will examine the exploitation of blocking peptides, small molecule inhibitors, and innovative Fra-1 protein degraders. We also consider the possible caveats concerning Fra-1 inhibition in specific therapeutic contexts. Finally, we discuss a recent suicide gene therapy-based approach, aimed at selectively killing the Fra-1-overexpressing neoplastic cells.

Among FOS-related components of the dimeric AP-1 transcription factor, the oncoprotein FRA-1 (encoded by FOSL1) is a key regulator of invasion and metastasis. The well-established FRA-1 pro-invasive activity in breast cancer, in which FOSL1 is overexpressed in the TNBC (Triple Negative Breast Cancer)/basal subtypes, correlates with the FRA-1-dependent transcriptional regulation of EMT (Epithelial-to-Mesenchymal Transition). After summarizing the major findings on FRA-1 in breast cancer invasiveness, we discuss the FRA-1 mechanistic links with EMT and cancer cell stemness, mediated by transcriptional and posttranscriptional interactions between FOSL1/FRA-1 and EMT-regulating transcription factors, miRNAs, RNA binding proteins and cytokines, along with other target genes involved in EMT. In addition to the FRA-1/AP-1 effects on the architecture of target promoters, we discuss the diagnostic and prognostic significance of the EMT-related FRA-1 transcriptome, along with therapeutic implications. Finally, we consider several novel perspectives regarding the less explored roles of FRA-1 in the tumor microenvironment and in control of the recently characterized hybrid EMT correlated with cancer cell plasticity, stemness, and metastatic potential. We will also examine the application of emerging technologies, such as single-cell analyses, along with animal models of TNBC and tumor-derived CTCs and PDXs (Circulating Tumor Cells and Patient-Derived Xenografts) for studying the FRA-1-mediated mechanisms in in vivo systems of EMT and metastasis.