I-CBP112 declines overexpression of ATP-binding cassette transporters and sensitized drug-resistant MDA-MB-231 and A549 cell lines to chemotherapy drugs.

Despite extensive efforts and ongoing progress in personalized anticancer approaches, chemotherapy remains the first line or the only treatment for some tumors that may develop resistance to chemotherapeutics in time due to inter alia overexpression of ATP-binding cassette transporters. Using clinically-relevant resistant models of triple negative breast cancer (MDA-MB-231; TNBC) as well as non-small cell lung cancer (A549; NSCLC), we tested the efficacy of I-CBP112 - CBP/EP300 bromodomain inhibitor to overcome drug resistance by declining ABC gene transcription. I-CBP112 significantly reduced ABCB1, ABCC1, ABCC2, ABCC3, ABCC5 and ABCG2 in all resistant lines, as well as ABCC10 in TNBC and ABCC4 in paclitaxel-resistant NSCLC, thereby increasing intracellular drug accumulation and cytotoxicity in 2D and 3D cultures. This was phenocopied only by the joint effect of ABC inhibitors such as tariquidar (ABCB1 - P-glycoprotein and ABCG2) and MK-571 (ABCC), whereas single inhibition of ABCB1/ABCG2 or ABCC proteins did not affect drug accumulation, thereby implying the need of simultaneous deficiency in activity of majority of drug pumps for enhanced drug retention. I-CBP112 failed to directly inhibit activity of ABCB1, ABCG2 and ABCC subfamily members at the same time. Importantly, I-CBP112 treated cancer cells polarized human macrophages into proinflammatory phenotypes. Moreover, I-CBP112 remained non-toxic to primary cell lines, nor did it enhance anticancer drug toxicity to blood-immune cells. In silico assay of ADMET properties confirmed the desired pharmacokinetic features of I-CBP112. The results suggest that the CBP/p300 inhibitor is a promising co-adjuvant to chemotherapy in drug-resistant cancer phenotypes, capable of decreasing ABC transporter expression.

Activation of ABCC Genes by Cisplatin Depends on the CoREST Occurrence at Their Promoters in A549 and MDA-MB-231 Cell Lines.

Although cisplatin-based therapies are common among anticancer approaches, they are often associated with the development of cancer drug resistance. This phenomenon is, among others, caused by the overexpression of ATP-binding cassette, membrane-anchored transporters (ABC proteins), which utilize ATP to remove, e.g., chemotherapeutics from intracellular compartments. To test the possible molecular basis of increased expression of ABCC subfamily members in a cisplatin therapy mimicking model, we generated two cisplatin-resistant cell lines derived from non-small cell lung cancer cells (A549) and triple-negative breast cancer cells (MDA-MB-231). Analysis of data for A549 cells deposited in UCSC Genome Browser provided evidence on the negative interdependence between the occurrence of the CoREST complex at the gene promoters and the overexpression of ABCC genes in cisplatin-resistant lung cancer cells. Pharmacological inhibition of CoREST enzymatic subunits-LSD1 and HDACs-restored gene responsiveness to cisplatin. Overexpression of CoREST-free ABCC10 in cisplatin-resistant phenotypes was caused by the activity of EP300 that was enriched at the ABCC10 promoter in drug-treated cells. Cisplatin-induced and EP300-dependent transcriptional activation of ABCC10 was only possible in the presence of p53. In summary, the CoREST complex prevents the overexpression of some multidrug resistance proteins from the ABCC subfamily in cancer cells exposed to cisplatin. p53-mediated activation of some ABCC genes by EP300 occurs once their promoters are devoid of the CoREST complex.

LSD1 Facilitates Pro-Inflammatory Polarization of Macrophages by Repressing Catalase.

The increased level of hydrogen peroxide accompanies some modes of macrophage specification and is linked to ROS-based antimicrobial activity of these phagocytes. In this study, we show that activation of toll-like receptors with bacterial components such as LPS is accompanied by the decline in transcription of hydrogen peroxide decomposing enzyme-catalase, suppression of which facilitates the polarization of human macrophages towards the pro-inflammatory phenotype. The chromatin remodeling at the CAT promoter involves LSD1 and HDAC1, but activity of the first enzyme defines abundance of the two proteins on chromatin, histone acetylation status and the CAT transcription. LSD1 inhibition prior to macrophage activation with LPS prevents CAT repression by enhancing the LSD1 and interfering with the HDAC1 recruitment to the gene promoter. The maintenance of catalase level with LSD1 inhibitors during M1 polarization considerably limits LPS-triggered expression of some pro-inflammatory cytokines and markers such as IL1ß, TNFα, COX2, CD14, TLR2, and IFNAR, but the effect of LSD1 inhibitors is lost upon catalase deficiency. Summarizing, activity of LSD1 allows for the CAT repression in LPS stimulated macrophages, which negatively controls expression of some key pro-inflammatory markers. LSD1 inhibitors can be considered as possible immunosuppressive drugs capable of limiting macrophage M1 specialization.

Contribution of BRG1-dependent SWI/SNF complexes to determining the phenotype of cancer cell / Udzial kompleksów SWI/SNF opartych na aktywnosci BRG1 w determinowaniu fenotypu komórek nowotworowych.

ATP-dependent chromatin remodeling complexes are documented as indispensible element of epigenetic mechanisms, which control transcription. These multiprotein functional units are capable of insertion, deletion and sliding of nucleosomes at the gene regulatory elements thereby defining DNA accessibility to transcription machinery. SWI/SNF is one out of four identified and described complexes. The enzymatic role in SWI/SNF molecular "motors" is assigned to two ATP-ases: BRM and BRG1. Accumulating evidence suggests the link between BRG1 and pathophysiology of some types of cancer. BRG1 has been documented as an activator of genes encoding factors responsible for i.a. proliferation, DNA repair, transmembrane transport and metabolism. Therefore, inhibitors of BRG1 and co-operating enzymes, which modulate activity of this ATP-ase or mark histones for shuttling to/from the chromatin, may turn out as an alternative to the compounds that are currently used to suppress the growth of tumors or as supplements that increase cell vulnerability to anticancer drugs.

The Role of PARP1 in Monocyte and Macrophage Commitment and Specification: Future Perspectives and Limitations for the Treatment of Monocyte and Macrophage Relevant Diseases with PARP Inhibitors.

Modulation of PARP1 expression, changes in its enzymatic activity, post-translational modifications, and inflammasome-dependent cleavage play an important role in the development of monocytes and numerous subtypes of highly specialized macrophages. Transcription of PARP1 is governed by the proliferation status of cells at each step of their development. Higher abundance of PARP1 in embryonic stem cells and in hematopoietic precursors supports their self-renewal and pluri-/multipotency, whereas a low level of the enzyme in monocytes determines the pattern of surface receptors and signal transducers that are functionally linked to the NFκB pathway. In macrophages, the involvement of PARP1 in regulation of transcription, signaling, inflammasome activity, metabolism, and redox balance supports macrophage polarization towards the pro-inflammatory phenotype (M1), which drives host defense against pathogens. On the other hand, it seems to limit the development of a variety of subsets of anti-inflammatory myeloid effectors (M2), which help to remove tissue debris and achieve healing. PARP inhibitors, which prevent protein ADP-ribosylation, and PARP1‒DNA traps, which capture the enzyme on chromatin, may allow us to modulate immune responses and the development of particular cell types. They can be also effective in the treatment of monocytic leukemia and other cancers by reverting the anti- to the proinflammatory phenotype in tumor-associated macrophages.

BRG1 Activates Proliferation and Transcription of Cell Cycle-Dependent Genes in Breast Cancer Cells.

Cancer malignancy is usually characterized by unlimited self-renewal. In some types of advanced tumors that are rapidly dividing, gene expression profiles depict elevations in pro-proliferative genes accompanied by coordinately elevated transcription of factors responsible for removal of DNA lesions. In our studies, fast proliferating breast cancer cell lines (MDA-MB-231 and MCF7), BRG1, a component of the SWI/SNF complex, emerges as an activator of functionally-linked genes responsible for activities such as mitotic cell divisions and DNA repair. Products of at least some of them are considerably overrepresented in breast cancer cells and BRG1 facilitates growth of MCF7 and MDA-MB-231 cell lines. BRG1 occurs at the promoters of genes such as CDK4, LIG1, and NEIL3, which are transcriptionally controlled by cell cycle progression and highly acetylated by EP300 in proliferating cells. As previously documented, in dividing cells BRG1 directly activates gene transcription by evicting EP300 modified nucleosomes from the promoters and, thereby, relaxing chromatin. However, the deficiency of BRG1 or EP300 activity for 48 h leads to cell growth arrest and to chromatin compaction, but also to the assembly of RB1/HDAC1/EZH2 complexes at the studied cell cycle-dependent gene promoters. Epigenetic changes include histone deacetylation and accumulation of H3K27me trimethylation, both known to repress transcription. Cell cycle arrest in G1 by inhibition of CDK4/6 phenocopies the effect of the long-term BRG1 inhibition on the chromatin structure. These results suggest that BRG1 may control gene transcription also by promoting expression of genes responsible for cell cycle progression in the studied breast cancer cells. In the current study, we show that BRG1 binding occurs at the promoters of functionally linked genes in proliferating breast cancer cells, revealing a new mechanism by which BRG1 defines gene transcription.

PARP1 Co-Regulates EP300-BRG1-Dependent Transcription of Genes Involved in Breast Cancer Cell Proliferation and DNA Repair.

BRG1, an active subunit of the SWI/SNF chromatin-remodeling complex, enables the EP300-dependent transcription of proliferation and DNA repair genes from their E2F/CpG-driven promoters in breast cancer cells. In the current study, we show that BRG1-EP300 complexes are accompanied by poly-ADP-ribose polymerase 1 (PARP1), which emerges as the functional component of the promoter-bound multiprotein units that are capable of controlling gene expression. This enzyme is co-distributed with BRG1 at highly acetylated promoters of genes such as CDK4, LIG1, or NEIL3, which are responsible for cancer cell growth and the removal of DNA damage. ADP-ribosylation is necessary to maintain active transcription, since it ensures an open chromatin structure that allows high acetylation and low histone density. PARP1-mediated modification of BRG1 and EP300 does not affect the association of enzymes with gene promoters; however, it does activate EP300, which acetylates nucleosomes, leading to their eviction by BRG1, thus allowing mRNA synthesis. Although PARP1 was found at BRG1 positive/H3K27ac negative promoters of highly expressed genes in a transformed breast cancer cell line, its transcriptional activity was limited to genes simultaneously controlled by BRG1 and EP300, indicating that the ADP-ribosylation of EP300 plays a dominant role in the regulation of BRG1-EP300-driven transcription. In conclusion, PARP1 directs the transcription of some proliferation and DNA repair genes in breast cancer cells by the ADP-ribosylation of EP300, thereby causing its activation and marking nucleosomes for displacement by BRG1. PARP1 in rapidly dividing cells facilitates the expression of genes that confer a cancer cell phenotype. Our study shows a new mechanism that links PARP1 with the removal of DNA damage in breast cancer cells via the regulation of BRG1-EP300-dependent transcription of genes involved in DNA repair pathways.