FRA-1 as a Regulator of EMT and Metastasis in Breast Cancer

Abstract

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.

Keywords: AP-1 transcription factors; EMT; FOSL1; FRA-1; TNBC.

Figure 2

FRA-1 interactions with core EMT-TFs, mRNAs, and EMT-related target genes. Circled numbers indicate the following regulatory interactions. (1) FOSL1 is transcriptionally induced by SNAIL (and TWIST), binding to the FOSL1 intronic enhancer [19]. (2) The FRA-1/AP-1 heterodimers cooperate with SNAIL for transactivating ZEB1/2 [19]. (3) In addition to core EMT-TFs, the FRA-1/AP-1 dimers positively control many target genes encoding for multiple components implicated in EMT in breast cancer: cytokines (IL6, TGFB1/2); GPI-anchored (PLAUR); G-protein-coupled (ADORA2B) and tyrosine kinase receptors (AXL); extracellular proteases (MMPs, PLAU, SERPINE1); mitosis-regulating protein-kinases (AURKA); cyclins (CCND1, CCNA2); non-histone chromatin components (HMGA1); and miRNAs [7,8,9,66]. (4) The FRA-1/AP-1-mediated repression of CLCA2 downregulates the expression of the calcium channel component CLCA2 [8], which also participates in cell-cell junctions, thus representing a negative EMT regulator, as discussed in Section 3.4. (5) The double-negative feedback loops formed by the FOSL1 and SNAI1 transcripts, targeted by the same oncosuppressor miRNA family (miR-34), which is a strong EMT inhibitor in BCCs. (6) In addition to the above illustrated transcriptional and posttranscriptional interactions, both FRA-1 and SNAIL are posttranslationally controlled by phosphorylation: while the ERK2-mediated phosphorylation stabilizes FRA-1 in response to the RAS-RAF-MEK pathway, GSK3-beta positively controls SNAIL ubiquitylation and degradation [67].

Figure 1

Molecular Classification of Breast Cancer. The five intrinsic breast cancer subtypes (luminal-A, normal-like, luminal-B, HER2-enriched, and basal-like) based on the PAM50 expression signature are shown in the left column [1,2]. Below is the IHC-based classification of the TNBC surrogate subtype: the basal-like subtype is commonly referred to as TNBC, although not all basal-like are triple-negative and vice versa. TNBCs are further characterized by expression profiling into six molecular subtypes (BL1, BL2, ML, MSL, IM, LAR). The vertical bracket on the side of claudin-low subtype indicates that the claudin-low tumors, rather than representing a subfraction of TNBC, can pervade the five intrinsic subtypes. FRA-1 overexpression is associated with the TNBC/Basal-like subtype. FOSL1 (along with ZEB1 and YAP) overexpression marks the claudin-low subtype, which exhibits EMT-like and stem-like expression signatures, along with high levels of MAPK pathway activation and stromal infiltration. The FRA-1 transcriptomes and cistromes have been characterized in the mesenchymal-like (ML) BT-549 and the mesenchymal-stem-like (MSL) MDA-MB-231 cell lines, both representing the claudin-low subtype [6,7,8,9].

Figure 3

FRA-1/AP-1 controls ZEB2 accumulation and activity by direct and indirect mechanisms. FRA-1/AP-1 drives the transcription from both proximal (1b) and distal (1a) ZEB2 promoters by mediating DNA looping and long-range interactions [68]. The FRA-1/AP-1 dimers also induce the expression of the co-transcribed miRNAs miR-221 and miR-222 through binding to the transcriptional promoter of the MIR222HG (miR-221/222 cluster Host Gene). The FRA-1-induced miR-221 and miR-222, in turn, downregulate the transcript encoding for the EMT inhibitor TRPS1, acting as a transcriptional repressor of GATA-regulated genes, including ZEB2. Therefore, the FRA-1-induced miR-221 and miR-222, by inhibiting the TRPS1 expression, relieve the repression and induce ZEB2 by an indirect mechanism that synergizes with the direct FRA-1-mediated ZEB2 transcriptional induction [69].

Figure 4

The FRA-1/AP-1 complex acts as a relay for the selection of the ZEB1-induced vs. the ZEB1-repressed target genes. The model (modified from Feldker et al., EMBO J., 2020 [6]) represents the cooperation between FRA-1/AP-1, TEAD4/YAP, and ZEB1 in EMT transcriptional regulation. Upper drawing: the extensive overlap of low-affinity monomeric ZEB1 binding sites with AP-1 and YAP motifs, along with physical interactions between FRA-1/c-JUN and ZEB1, have been elucidated by genome-wide analyses in the claudin-low MDA-MB-231 cell line. FRA-1/AP-1 and TEAD4/YAP predominantly recruit ZEB1 to distal regulatory regions through TEAD4-AP-1 DNA binding sites to positively regulate transcription of tumor-promoting genes. Lower drawing: the ZEB1-mediated repression of epithelial genes depends on direct ZEB1 binding to bipartite high-affinity Z-boxes, not overlapping with AP-1 and YAP binding sites and preferentially localized in promoter regions of target genes. The complexes interacting with the ZEB-only binding sites inhibit the expression of epithelial target genes (e.g., CDH1) by recruiting transcriptional repressors, such as CTBP.

Figure 5

Multiple autocrine and paracrine mechanisms of FRA-1-mediated induction of TGF-beta and IL-6 in breast tumors [72]. Upper drawing: Dual action of FRA-1 on the TGF-beta-SMAD pathway. The FRA-1/AP-1-mediated transactivation of TGFB1 results in the accumulation and secretion of TGF-beta, which contributes to the autocrine EMT induction in BCCs. Moreover, FRA-1 specifically interacts and cooperates with SMAD2/3 on target promoters (e.g., SERPINE1), thus contributing to the convergence between the RAS-MAPK-AP-1 and TGF-beta-SMAD pathways in transcriptional control of EMT. Lower drawing: FRA-1 contributes to the EMT paracrine mechanisms mediated by TAMs in breast cancer. In response to signals released in the neoplastic microenvironment, FRA-1 is accumulated in TAMs, in which the FRA-1/AP-1 dimers transactivate the IL6 promoter and trigger interleukin-6 secretion. The consequent induction of the IL6-STAT3 pathway in BCCs, in cooperation with the FRA-1/AP-1 complexes, stimulates the transcription and release of MMP9, TGF-beta, and VEGF, and subsequent cancer cell invasion and mesenchymal transformation, along with increased tumor angiogenesis.