Transposon DNA sequences facilitate the tissue-specific gene transfer of circulating tumor DNA between human cells.

The exchange of genes between cells is known to play an important physiological and pathological role in many organisms. We show that circulating tumor DNA (ctDNA) facilitates cell-specific gene transfer between human cancer cells and explain part of the mechanisms behind this phenomenon. As ctDNA migrates into the nucleus, genetic information is transferred. Cell targeting and ctDNA integration require ERVL, SINE or LINE DNA sequences. Chemically manufactured AluSp and MER11C sequences replicated multiple myeloma (MM) ctDNA cell targeting and integration. Additionally, we found that ctDNA may alter the treatment response of MM and pancreatic cancer models. This study shows that retrotransposon DNA sequences promote cancer gene transfer. However, because cell-free DNA has been detected in physiological and other pathological conditions, our findings have a broader impact than just cancer. Furthermore, the discovery that transposon DNA sequences mediate tissue-specific targeting will open up a new avenue for the delivery of genes and therapies.

A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis.

Introduction: We have demonstrated that transposons derived from ctDNA can be transferred between cancer cells. The present research aimed to investigate the cellular uptake and intracellular trafficking of Multiple Myeloma-zip code (MM-ZC), a cell-specific zip code, in myeloma cell lines. We demonstrated that MM-ZC uptake by myeloma cells was concentration-, time- and cell-type-dependent. Methods: Flow cytometry and confocal microscopy methods were used to identify the level of internalization of the zip codes in MM cells. To screen for the mechanism of internalization, we used multiple inhibitors of endocytosis. These experiments were followed by biotin pulldown and confocal microscopy for validation. Single interference RNA (siRNA) targeting some of the proteins involved in endocytosis was used to validate the role of this pathway in ZC cell internalization. Results: Endocytosis inhibitors identified that Monensin and Chlorpromazine hydrochloride significantly reduced MM-ZC internalization. These findings suggested that Clathrin-mediated endocytosis and endosomal maturation play a crucial role in the cellular uptake of MM-ZC. Biotin pulldown and confocal microscopic studies revealed the involvement of proteins such as Clathrin, Rab5a, Syntaxin-6, and RCAS1 in facilitating the internalization of MM-ZC. Knockdown of Rab5a and Clathrin proteins reduced cellular uptake of MM-ZC and conclusively demonstrated the involvement of Clathrin-Rab5a pathways in MM-ZC endocytosis. Furthermore, both Rab5a and Clathrin reciprocally affected their association with MM-ZC when we depleted their proteins by siRNAs. Additionally, the loss of Rab5a decreased the Syntaxin-6 association with MMZC but not vice versa. Conversely, MM-ZC treatment enhanced the association between Clathrin and Rab5a. Conclusion: Overall, the current study provides valuable insights into the cellular uptake and intracellular trafficking of MM-ZC in myeloma cells. Identifying these mechanisms and molecular players involved in MM-ZC uptake contributes to a better understanding of the delivery and potential applications of cell-specific Zip-Codes in gene delivery and drug targeting in cancer research.

Discovery and characterization of anti-cancer peptides from a random peptide library.

We performed a forward genetic screen to discover peptides that specifically target breast cancer cells using a Penetratin tagged, random 15mer peptide library. We identified a group of novel peptides that specifically inhibited the proliferation and survival of breast cancer cells without affecting normal primary mammary epithelial cells or fibroblasts. The intrinsic apoptotic pathway is activated by these peptides in the face of abnormal expression of numerous cell cycle regulatory genes. Associated alterations in histone marks, nuclear structure, and levels of critical RNA binding proteins vary in a peptide specific manner. This study demonstrates a novel method for the discovery of new potential therapeutic peptides.

hnRNPK-derived cell-penetratingpeptide inhibits cancer cell survival.

hnRNPK is a multifunctional protein that plays an important role in cancer cell proliferation and metastasis via its RNA- and DNA-binding properties. Previously we showed that cell-penetrating peptides derived from the RGG RNA-binding domain of SAFA (hnRNPU) disrupt cancer cell proliferation and survival. Here we explore the efficacy of a peptide derived from the RGG domain of hnRNPK. This peptide acts in a dominant-negative manner on several hnRNPK functions to induce death of multiple types of cancer cells. The peptide phenocopies the effect of hnRNPK knockdown on its mRNA-stability targets such as KLF4 and EGR1 and alters the levels and locations of long non-coding RNAs (lncRNAs) and proteins required for nuclear and paraspeckle formation and function. The RGG-derived peptide also decreases euchromatin as evidenced by loss of active marks and polymerase II occupancy. Our findings reveal the potential therapeutic utility of the hnRNPK RGG-derived peptide in a range of cancers.

Inhibiting an RBM39/MLL1 epigenomic regulatory complex with dominant-negative peptides disrupts cancer cell transcription and proliferation.

RBM39 is a known splicing factor and coactivator. Here, we report that RBM39 functions as a master transcriptional regulator that interacts with the MLL1 complex to facilitate chromatin binding and H3K4 trimethylation in breast cancer cells. We identify RBM39 functional domains required for DNA and complex binding and show that the loss of RBM39 has widespread effects on H3K4me3 and gene expression, including key oncogenic pathways. RBM39's RNA recognition motif 3 (RRM3) functions as a dominant-negative domain; namely, it disrupts the complex and H3K4me trimethylation and expression of RBM/MLL1 target genes. RRM3-derived cell-penetrating peptides phenocopy the effects of the loss of RBM39 to decrease growth and survival of all major subtypes of breast cancer and yet are nontoxic to normal cells. These findings establish RBM39/MLL1 as a major contributor to the abnormal epigenetic landscape in breast cancer and lay the foundation for peptide-mediated cancer-specific therapy based on disruption of RBM39 epigenomic functions.

Novel Cell-Penetrating Peptides Derived From Scaffold-Attachment- Factor A Inhibits Cancer Cell Proliferation and Survival.

Scaffold-attachment-factor A (SAFA) has important roles in many normal and pathologic cellular processes but the scope of its function in cancer cells is unknown. Here, we report dominant-negative activity of novel peptides derived from the SAP and RGG-domains of SAFA and their effects on proliferation, survival and the epigenetic landscape in a range of cancer cell types. The RGG-derived peptide dysregulates SAFA binding and regulation of alternatively spliced targets and decreases levels of key spliceosome proteins in a cell-type specific manner. In contrast, the SAP-derived peptide reduces active histone marks, promotes chromatin compaction, and activates the DNA damage response and cell death in a subset of cancer cell types. Our findings reveal an unprecedented function of SAFA-derived peptides in regulating diverse SAFA molecular functions as a tumor suppressive mechanism and demonstrate the potential therapeutic utility of SAFA-peptides in a wide range of cancer cells.

Tbx3-Mediated Regulation of Cardiac Conduction System Development and Function: Potential Contributions of Alternative RNA Processing.

In this article, we provide a brief summary of work by us and others to discover the molecular underpinnings of early conduction system development and function. We focus on how the multifunctional protein Tbx3 contributes to acquisition and homeostasis of the tissue-specific properties of the sinoatrial and atrioventricular nodes. We also provide unpublished, preliminary findings supporting the role of Tbx3-regulated alternative RNA processing in the developing conduction system.

LncRNAs Regulatory Networks in Cellular Senescence.

Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides with no open reading frame. They play a key role in the regulation of cellular processes such as genome integrity, chromatin organization, gene expression, translation regulation, and signal transduction. Recent studies indicated that lncRNAs are not only dysregulated in different types of diseases but also function as direct effectors or mediators for many pathological symptoms. This review focuses on the current findings of the lncRNAs and their dysregulated signaling pathways in senescence. Different functional mechanisms of lncRNAs and their downstream signaling pathways are integrated to provide a bird's-eye view of lncRNA networks in senescence. This review not only highlights the role of lncRNAs in cell fate decision but also discusses how several feedback loops are interconnected to execute persistent senescence response. Finally, the significance of lncRNAs in senescence-associated diseases and their therapeutic and diagnostic potentials are highlighted.

Discovery and Characterization of piRNAs in the Human Fetal Ovary.

Piwi-interacting RNAs (piRNAs), a class of 26- to 32-nt non-coding RNAs (ncRNAs), function in germline development, transposon silencing, and epigenetic regulation. We performed deep sequencing and annotation of untreated and periodate-treated small RNA cDNA libraries from human fetal and adult germline and reference somatic tissues. This revealed abundant piRNAs originating from 150 piRNA-encoding genes, including some exhibiting gender-specific expression, in fetal ovary and adult testis-developmental periods coinciding with mitotic cell divisions expanding fetal germ cells prior to meiotic divisions. The absence of reads mapping uniquely to annotated piRNA genes demonstrated their paucity in fetal testis and adult ovary and absence in somatic tissues. We curated human piRNA-expressing regions and defined their precise borders and observed piRNA-guided cleavage of transcripts antisense to some piRNA-producing genes. This study provides insights into sex-specific mammalian piRNA expression and function and serves as a reference for human piRNA analysis and annotation.

Long noncoding RNA PANDA and scaffold-attachment-factor SAFA control senescence entry and exit.

Cellular senescence is a stable cell cycle arrest that limits the proliferation of pre-cancerous cells. Here we demonstrate that scaffold-attachment-factor A (SAFA) and the long noncoding RNA PANDA differentially interact with polycomb repressive complexes (PRC1 and PRC2) and the transcription factor NF-YA to either promote or suppress senescence. In proliferating cells, SAFA and PANDA recruit PRC complexes to repress the transcription of senescence-promoting genes. Conversely, the loss of SAFA-PANDA-PRC interactions allows expression of the senescence programme. Accordingly, we find that depleting either SAFA or PANDA in proliferating cells induces senescence. However, in senescent cells where PANDA sequesters transcription factor NF-YA and limits the expression of NF-YA-E2F-coregulated proliferation-promoting genes, PANDA depletion leads to an exit from senescence. Together, our results demonstrate that PANDA confines cells to their existing proliferative state and that modulating its level of expression can cause entry or exit from senescence.