Doxorubicin is believed to cause dose-dependent cardiotoxicity through redox cycling and the generation of reactive oxygen species (ROS). Here we show that cardiomyocyte-specific deletion of Top2b ...(encoding topoisomerase-IIβ) protects cardiomyocytes from doxorubicin-induced DNA double-strand breaks and transcriptome changes that are responsible for defective mitochondrial biogenesis and ROS formation. Furthermore, cardiomyocyte-specific deletion of Top2b protects mice from the development of doxorubicin-induced progressive heart failure, suggesting that doxorubicin-induced cardiotoxicity is mediated by topoisomerase-IIβ in cardiomyocytes.
Breast cancer subtypes expressing hormone receptors (HR+ BCa) have a good prognosis and respond to first-line endocrine therapy (ET). However, the majority of HR+ BCa patients exhibit intrinsic or ...acquired ET resistance (ET-R) and rapid onset of incurable metastatic BCa. With the failure of conventional ET, limited targeted therapy exists for ET-R HR+ BCa patients. The androgen receptor (AR) in HR-negative BCa subtypes is emerging as an attractive alternative target for therapy. The AR drives Luminal AR (LAR) triple-negative breast cancer progression, and LAR patients consistently exhibit positive clinical benefits with AR antagonists in clinical trials. In contrast, the function of the AR in HR+ BCa is more conflicting. AR in HR+ BCa correlates with a favorable prognosis, and yet, the AR supports the development of ET-R BCa. While AR antagonists were ineffective, ongoing clinical trials with a selective AR modulator have shown promise for HR+ BCa patients. To understand the incongruent actions of ARs in HR+ BCa, the current review discusses how the structure and post-translational modification impact AR function. Additionally, completed and ongoing clinical trials with FDA-approved AR-targeting agents for BCa are presented. Finally, we identify promising investigational small molecules and chimera drugs for future HR+ BCa therapy.
Hormone receptor positive (HR+) breast cancer (BCa) is the most frequently diagnosed subtype. Acquired and intrinsic resistance to conventional endocrine therapy (ET) commonly occurs and prompts ...incurable metastatic disease. Hence, ET-resistant (ET-R) HR+ BCa presents a therapeutic challenge. Previous studies show elevated androgen receptor (AR) that supports resistance to ET tamoxifen and correlates with HR+ BCa metastasis. Yet surprisingly, studies with AR-blocker enzalutamide (Enz) in ET-R HR+ BCa present conflicting results. We now report that a constitutively active, unique from canonical Enz-targeted, AR accumulates in endocrine resistant HR+ BCa cells.
AR protein profiles in acquired and intrinsic ET-R HR + -BCa were defined with cell-free modification tests, in-house in-vivo SUMOylation assays, and PLA imaging. Genomic activity of native AR and modified-AR mimetic was tested with reporter assays and limited transcriptome analysis. Spheroid growth and migration studies were used to evaluate inhibitory actions of Enz and combinatorial therapy.
Sustained higher molecular weight SUMO-modified AR (SUMO-AR) persists in acquired and intrinsic ET-R BCa cell lines. Concurrently, SUMO isoforms and global SUMO-modified proteome also accumulates in the same cell lines. We identified AR as a novel substrate for the SUMO-E3 ligase HSPB1/Hsp27. Independent of ligand, SUMO-AR is resilient to ubiquitin-mediated proteasomal degradation, enriched in the nucleus, readily chromatin-bound, and transcriptionally active. Constitutive SUMO-AR initiates a gene-expression profile that favors epithelial-mesenchymal transition. Enz combined with a SUMO inhibitor attenuates migration and metastatic phenotype of ET-R HR+ BCa.
Targeting both unmodified and SUMO-modified AR prevents the metastatic progression of HR+ BCa with ET-R. Video abstract.
Two Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) variants are naturally expressed in breast epithelia. Breast cancer (BCa) onset down-regulates the short SENP7 splice ...variant (SENP7S) and enhances the long transcript (SENP7L). Here, we show that SENP7L induction promotes gene expression profiles that favor aberrant proliferation and initiate epithelial-mesenchymal transition (EMT). SENP7L exhibits an interaction domain for the epigenetic remodeler heterochromatin protein 1 α (HP1α) and isopeptidase activity against SUMO-modified HP1α. Loss of this interaction domain, as observed with SENP7S, favors HP1α SUMOylation. SUMOylated HP1α is enriched at E2F-responsive and mesenchymal gene promoters, silences transcription of these genes, and promotes cellular senescence. Elevated SENP7L renders HP1α hypo-SUMOylated, which relieves transcriptional repression of the same genes and concurrently decreases transcription of epithelial-promoting genes via an HP1α-independent mechanism. Consequently, SENP7L levels correlate with EMT, motility, and invasiveness of BCa cells. Stable knockdown of elevated SENP7L levels lessens the dissemination of highly metastatic BCa cells to the lungs from primary implantation sites in in vivo studies. Thus, differential splicing of the SENP7 regulates either tumor suppression or progression.
SUMOylation has been shown to modulate DNA replication/repair, cell cycle progression, signal transduction, and the hypoxic response. SUMO (small ubiquitin-like modifier)-specific proteases regulate ...SUMOylation, but how changes in the expression of these proteases contribute to physiological and/or pathophysiological events remains undefined. Here, we show that SENP1 (sentrin/SUMO-specific protease 1) is highly expressed in human prostate cancer specimens and correlates with hypoxia-inducing factor 1α (HIF1α) expression. Mechanistic studies in a mouse model indicate that androgen-driven expression of murine SENP1 leads to HIF1α stabilization, enhanced vascular endothelial growth factor production, and angiogenesis. Further pathological assessment of the mouse indicates that SENP1 overexpression induces transformation of the normal prostate gland and gradually facilitates the onset of high-grade prostatic intraepithelial neoplasia. Consistent with cell culture studies, SENP1 enhances prostate epithelial cell proliferation via modulating the androgen receptor and cyclin D1. These results demonstrate that deSUMOylation plays a critical role in prostate pathogenesis through induction of HIF1α-dependent angiogenesis and enhanced cell proliferation.
Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors which regulate the expression of lipid and cholesterol metabolism genes. Moreover, LXRs ...and their ligands have been shown to inhibit tumor growth in a variety of cancers. We have previously identified the small molecule compound GAC0001E5 (1E5) as an LXR inverse agonist and a potent inhibitor of pancreatic cancer cells. Transcriptomic and metabolomic studies showed that 1E5 disrupts glutamine metabolism, an essential metabolic pathway commonly reprogrammed during malignant transformation, including in breast cancers. To determine the role of LXRs and potential application of 1E5 in breast cancer, we examined LXR expression in publicly available clinical samples, and found that LXR expression is elevated in breast tumors as compared to normal tissues. In luminal A, endocrine therapy-resistant, and triple-negative breast cancer cells, 1E5 exhibited LXR inverse agonist and "degrader" activity and strongly inhibited cell proliferation and colony formation. Treatments with 1E5 downregulated the transcription of key glutaminolysis genes, and, correspondingly, biochemical assays indicated that 1E5 lowered intracellular glutamate and glutathione levels and increased reactive oxygen species. These results indicate that novel LXR ligand 1E5 is an inhibitor of glutamine metabolism and redox homeostasis in breast cancers and suggest that modulating LXR activity and expression in tumor cells is a promising strategy for targeting metabolic reprogramming in breast cancer therapeutics.
Origin recognition complex 2 (ORC2), a subunit of the ORC, is essential for DNA replication initiation in eukaryotic cells. In addition to a role in DNA replication initiation at the G1/S phase, ORC2 ...has been shown to localize to the centromere during the G2/M phase. Here, we show that ORC2 is modified by small ubiquitin-like modifier 2 (SUMO2), but not SUMO1, at the G2/M phase of the cell cycle. SUMO2-modification of ORC2 is important for the recruitment of KDM5A in order to convert H3K4me3 to H3K4me2, a “permissive” histone marker for α-satellite transcription at the centromere. Persistent expression of SUMO-less ORC2 led to reduced α-satellite transcription and impaired pericentric heterochromatin silencing, which resulted in re-replication of heterochromatin DNA. DNA re-replication eventually activated the DNA damage response, causing the bypass of mitosis and the formation of polyploid cells. Thus, ORC2 sustains genomic stability by recruiting KDM5A to maintain centromere histone methylation in order to prevent DNA re-replication.
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•ORC2 is SUMOylated from the late-S to G2/M phase of the cell cycle•ORC2 SUMOylation promotes recruitment of KDM5A to centromeres•Centromeric non-coding RNA transcription is deregulated in ORC2 SUMOylation mutant cells•ORC2 SUMOylation defect results in bypass of mitosis and polyploidy
Huang et al. demonstrate that ORC2 SUMOylation is required for the recruitment of KDM5A to centromeres to maintain normal histone H3K4 methylation. Loss of ORC2 SUMOylation results in abnormal centromeric H3K4 methylation, leading to re-replication of heterochromatin DNA, abnormal mitosis, and formation of polyploidy.
Long, noncoding RNAs (lncRNAs) are indispensable for normal cell physiology and, consequently, are tightly regulated in human cells. Yet, unlike mRNA, substantially less is known about the mechanisms ...for lncRNA degradation. It is important to delineate the regulatory control of lncRNA degradation, particularly for lncRNA telomeric repeat-containing RNA (TERRA), as the TERRA-telomere R-loops dictate cell cycle progression and genomic stability. We now report that the exosome complex component Exosc9 degrades lncRNA TERRA in human mammary epithelial cells. Heterochromatin protein 1 alpha (HP1α) recruits Exosc9 to the telomeres; specifically, the SUMO-modified form of HP1α supports interaction with Exosc9 and, as previously reported, lncRNA TERRA. The telomeric enrichment of Exosc9 is cell cycle-dependent and consistent with the loss of telomeric TERRA in the S/G2 phase. Elevated Exosc9 is frequently observed and drives the growth of endocrine therapy-resistant (ET-R) HR+ breast cancer (BCa) cells. Specifically, the knockdown of Exosc9 inversely impacts telomeric R-loops and the integrity of the chromosome ends of ET-R cells. Consistently, Exosc9 levels dictate DNA damage and the sensitivity of ET-R BCa cells to PARP inhibitors. In this regard, Exosc9 may serve as a promising biomarker for predicting the response to PARP inhibitors as a targeted monotherapy for ET-R HR+ BCa.
SUMO posttranslational modification directs gene transcription and epigenetic programming to support normal cell function. The dynamic nature of SUMO-modification makes it difficult to identify ...endogenous protein substrates. Isolation of chromatin-bound SUMO targets is exceptionally challenging, as conventional immunoprecipitation assays are inefficient at concentrating this protein population. This chapter describes a protocol that effectively precipitates chromatin-associated fractions of SUMOylated heterochromatin protein 1α in cultured cells. Techniques to enrich endogenous SUMO substrates at the chromatin are also demonstrated and discussed. This approach could be adapted to evaluate chromatin-bound SUMO targets in additional in vivo systems.
Prostate cancer, the most frequently diagnosed carcinoma in males, is readily modulated via the transcriptional activity of androgen receptors. Our recent publication reported that androgen ...receptor-dependent transcription is significantly elevated with expression of the human sentrin/SUMO-specific protease (SENP1) in the androgen-sensitive human prostate cancer cell line (LNCaP). In situ hybridization studies indicated an elevation of SENP1 message in prostatic intraepithelial neoplasia and prostate cancer lesions as compared with normal prostate epithelia. This study aimed to delineate the mechanism for the regulation of SENP1 message and to determine the pathophysiological consequence of SENP1 induction with respect to prostate cancer. Real-time PCR confirmed the elevation of SENP1 mRNA in prostate cancer cells as compared with normal prostate epithelial cells. Chronic androgen exposure of LNCaP cells prompted an enhancement in the SENP1 transcript selectively. This androgen-mediated augmentation of SENP1 was absent with co-administration of the androgen receptor antagonist bicalutamide and in androgen receptor-negative prostate cancer PC-3 cells, indicating an androgen receptor-dependent event. Activation of the androgen receptor was required for binding an identified androgen response element and positively regulating SENP1 promoter activity. Abrogation of elevated SENP1 mRNA in prostate cancer cells significantly decreased androgen-mediated cell growth. Because increased SENP1 expression directly modulated androgen receptor-dependent cell proliferation and transcription, SENP1 could play an important role in prostate carcinogenesis.
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