Over the past decade, long noncoding RNAs (lncRNAs) have been identified as significant players in gene regulation. They are often differentially expressed and widely-associated with a majority of cancer types. The aberrant expression of these transcripts has been linked to tumorigenesis, metastasis, cancer stage progression and patient survival. Despite their apparent link to cancer, it has been challenging to gain a mechanistic understanding of how they contribute to cancer, partially due the difficulty in discriminating functional RNAs from other noncoding transcription events. However, there are several well-studied lncRNAs where specific mechanisms have been more clearly defined, leading to new discoveries into how these RNAs function. One major observation that has come to light is the context-dependence of lncRNA mechanisms, where they often have unique function in specific cell types and environment. Here, we review the molecular mechanisms of lncRNAs with a focus on cancer pathways, illustrating a few informative examples. Together, this type of detailed insight will lead to a greater understanding of the potential for the application of lncRNAs as targets of cancer therapies and diagnostics.

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

The human long noncoding RNA (lncRNA) HOTAIR acts in trans to recruit the Polycomb repressive complex 2 (PRC2) to the HOXD gene cluster and to promote gene silencing during development. In breast cancers, overexpression of HOTAIR increases metastatic potential via the repression of many additional genes. It has remained unclear what factors determine HOTAIR-dependent PRC2 activity at specific genomic loci, particularly when high levels of HOTAIR result in aberrant gene silencing. To identify additional proteins that contribute to the specific action of HOTAIR, we performed a quantitative proteomic analysis of the HOTAIR interactome. We found that the most specific interaction was between HOTAIR and the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, a member of a family of proteins involved in nascent mRNA processing and RNA matchmaking. Our data suggest that A2/B1 are key contributors to HOTAIR-mediated chromatin regulation in breast cancer cells: A2/B1 knockdown reduces HOTAIR-dependent breast cancer cell invasion and decreases PRC2 activity at the majority of HOTAIR-dependent loci. We found that the B1 isoform, which differs from A2 by 12 additional amino acids, binds with highest specificity to HOTAIR. B1 also binds chromatin and associates preferentially with RNA transcripts of HOTAIR gene targets. We furthermore demonstrate a direct RNA–RNA interaction between HOTAIR and a target transcript that is enhanced by B1 binding. Together, these results suggest a model in which B1 matches HOTAIR with transcripts of target genes on chromatin, leading to repression by PRC2.

A trigger for gene silencing is caused by RNAs pairing together via structural changes.

Long noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

Collagens comprise a large, diverse family of proteins that are abundantly expressed throughout most tissues. As a main component of the extracellular matrix (ECM), it is becoming increasingly appreciated how vital collagens are to tumor development, progression, and metastasis. COL6A3, which encodes the alpha 3 chain of type VI collagen, is a unique member of the collagen family that encodes a C-terminal peptide with powerful signaling capabilities, named endotrophin (ETP). Expression of COL6A3 is required for the survival, migration, and invasion of many cancer cell lines, including breast, bladder, liver, and colorectal cancers. ETP, which was originally discovered to be enriched in the adipocytes of mammary tumors, is a powerful oncopeptide that can alter signaling of tumor and stromal cells. ETP has greater signaling potential than the parental COL6A3 as it can induce EMT and promote chemoresistance, metastasis, and stemness in an TGF-β-like manner. ETP can also function independently of TGF-β to recruit endothelial cells and macrophages. In this review, we examine the molecular implications of COL6A3 and ETP expression and their effects on tumor growth, metastasis, and therapeutic response. Finally, we speculate on the potential of serum ETP as a prognostic biomarker in both carcinomas and sarcomas. In summary, COL6A3 and ETP are powerful drivers of tumor growth that have potential as noninvasive diagnostic and prognostic tools for the clinical management of cancer.