R-Loops and R-Loop-Binding Proteins in Cancer Progression and Drug Resistance.

R-loops are three-stranded DNA/RNA hybrids that form by the annealing of the mRNA transcript to its coding template while displacing the non-coding strand. While R-loop formation regulates physiological genomic and mitochondrial transcription and DNA damage response, imbalanced R-loop formation can be a threat to the genomic integrity of the cell. As such, R-loop formation is a double-edged sword in cancer progression, and perturbed R-loop homeostasis is observed across various malignancies. Here, we discuss the interplay between R-loops and tumor suppressors and oncogenes, with a focus on BRCA1/2 and ATR. R-loop imbalances contribute to cancer propagation and the development of chemotherapy drug resistance. We explore how R-loop formation can cause cancer cell death in response to chemotherapeutics and be used to circumvent drug resistance. As R-loop formation is tightly linked to mRNA transcription, their formation is unavoidable in cancer cells and can thus be explored in novel cancer therapeutics.

Multi-Omics Database Analysis of Aminoacyl-tRNA Synthetases in Cancer.

Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in the mRNA translation machinery, yet they possess numerous non-canonical functions developed during the evolution of complex organisms. The aaRSs and aaRS-interacting multi-functional proteins (AIMPs) are continually being implicated in tumorigenesis, but these connections are often limited in scope, focusing on specific aaRSs in distinct cancer subtypes. Here, we analyze publicly available genomic and transcriptomic data on human cytoplasmic and mitochondrial aaRSs across many cancer types. As high-throughput technologies have improved exponentially, large-scale projects have systematically quantified genetic alteration and expression from thousands of cancer patient samples. One such project is the Cancer Genome Atlas (TCGA), which processed over 20,000 primary cancer and matched normal samples from 33 cancer types. The wealth of knowledge provided from this undertaking has streamlined the identification of cancer drivers and suppressors. We examined aaRS expression data produced by the TCGA project and combined this with patient survival data to recognize trends in aaRSs' impact on cancer both molecularly and prognostically. We further compared these trends to an established tumor suppressor and a proto-oncogene. We observed apparent upregulation of many tRNA synthetase genes with aggressive cancer types, yet, at the individual gene level, some aaRSs resemble a tumor suppressor while others show similarities to an oncogene. This study provides an unbiased, overarching perspective on the relationship of aaRSs with cancers and identifies certain aaRS family members as promising therapeutic targets or potential leads for developing biological therapy for cancer.

Structural and functional analysis of cystatin E reveals enzymologically relevant dimer and amyloid fibril states.

Protein activity is often regulated by altering the oligomerization state. One mechanism of multimerization involves domain swapping, wherein proteins exchange parts of their structures and thereby form long-lived dimers or multimers. Domain swapping has been specifically observed in amyloidogenic proteins, for example the cystatin superfamily of cysteine protease inhibitors. Cystatins are twin-headed inhibitors, simultaneously targeting the lysosomal cathepsins and legumain, with important roles in cancer progression and Alzheimer's disease. Although cystatin E is the most potent legumain inhibitor identified so far, nothing is known about its propensity to oligomerize. In this study, we show that conformational destabilization of cystatin E leads to the formation of a domain-swapped dimer with increased conformational stability. This dimer was active as a legumain inhibitor by forming a trimeric complex. By contrast, the binding sites toward papain-like proteases were buried within the cystatin E dimer. We also showed that the dimers could further convert to amyloid fibrils. Unexpectedly, cystatin E amyloid fibrils contained functional protein, which inhibited both legumain and papain-like enzymes. Fibril formation was further regulated by glycosylation. We speculate that cystatin amyloid fibrils might serve as a binding platform to stabilize the pH-sensitive legumain and cathepsins in the extracellular environment, contributing to their physiological and pathological functions.

Structural Elucidation of a Nonpeptidic Inhibitor Specific for the Human Immunoproteasome.

Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a nonpeptidic scaffold with the absence of an electrophile was reported in a patent. Here, we investigated the mode of action of the lead compound by using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the ß5i substrate-binding channel. Distinct interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.

Selective Inhibition of the Immunoproteasome by Structure-Based Targeting of a Non-catalytic Cysteine.

Clinically applied proteasome inhibitors induce cell death by concomitant blockage of constitutive and immunoproteasomes. In contrast, selective immunoproteasome inhibition is less cytotoxic and has the potential to modulate chronic inflammation and autoimmune diseases. In this study, we rationally designed decarboxylated peptides that covalently target a non-catalytic cysteine of the immunoproteasome subunit ß5i with α-chloroacetamide-containing sidechains. The enhanced isoform specificity decreased cytotoxic effects and the compound suppressed the production of inflammatory cytokines. Structure-based optimization led to over 150-fold selectivity for subunit ß5i over ß5c. This new compound class provides a promising starting point for the development of selective immunoproteasome inhibitors as potential anti-inflammatory agents.

Targeted Delivery of Proteasome Inhibitors to Somatostatin-Receptor-Expressing Cancer Cells by Octreotide Conjugation.

Clinical application of proteasome inhibitors (PIs) is so far limited to peripheral blood cancers due to the pronounced cytotoxicity towards all cell types. Targeted delivery of PIs could permit the treatment of other cancers along with decreasing side effects. Herein we describe the first small-molecule proteasome inhibitor conjugate for targeted delivery, created by fusing PIs to a synthetic ligand of somatostatin receptors, which are highly expressed in a variety of tumors. X-ray crystallographic studies and in vitro IC50 measurements demonstrated that addition of the cyclopeptide octreotide as a targeting vehicle does not affect the PI's binding mode. The cytotoxicity of the conjugate against somatostatin-receptor-expressing cells was up to 11-fold higher than that of a non-targeting surrogate. We have therefore established PIs as a new payload for drug conjugates and have shown that targeted delivery thereof could be a promising approach for the broader application of this FDA-approved class of compounds.

TIMP-1 signaling via CD63 triggers granulopoiesis and neutrophilia in mice.

The homeostasis of neutrophil granulocytes can affect the outcome of several inflammation-associated diseases including cancer. The regulation of this homeostasis is still not completely understood. We previously found that elevated systemic levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) induce an increase of neutrophils in the liver, which in turn strongly promotes liver metastasis. Here, we report that increasing systemic TIMP-1 levels were sufficient to induce neutrophilia in mice. This was not attributed to prolonged survival or direct mobilization of neutrophils. However, TIMP-1 induced enrichment of myeloid progenitors and concomitant upregulation of granulopoiesis-associated genes in the bone marrow compartment. BrdU pulse-labeling confirmed that proliferating progenitors accounted for TIMP-1-induced neutrophilia. TIMP-1 variants that dissect its protease-inhibitory from its CD63 binding function relevant for cell signaling revealed that the TIMP-1 signaling domain was necessary and sufficient to augment granulopoiesis. Consequently, ablation of the TIMP-1 receptor CD63 abolished both neutrophilia and TIMP-1-enhanced granulopoiesis in the bone marrow. Our findings reveal that elevated levels of TIMP-1 impact on neutrophil homeostasis via signaling through CD63. This may provide a link to clinical observations, where TIMP-1 correlates with high severity and bad prognosis in inflammation-associated diseases.

Tissue inhibitor of metalloproteinases (TIMP)-1 creates a premetastatic niche in the liver through SDF-1/CXCR4-dependent neutrophil recruitment in mice.

UNLABELLED: Due to its ability to inhibit prometastatic matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP)-1 has been thought to suppress tumor metastasis. However, elevated systemic levels of TIMP-1 correlate with poor prognosis in cancer patients, suggesting a metastasis-stimulating role of TIMP-1. In colorectal cancer patients, tumor as well as plasma TIMP-1 levels were correlated with synchronous liver metastasis or distant metastasis-associated disease relapse. In mice, high systemic TIMP-1 levels increased the liver susceptibility towards metastasis by triggering the formation of a premetastatic niche. This promoted hepatic metastasis independent of origin or intrinsic metastatic potential of tumor cells. High systemic TIMP-1 led to increased hepatic SDF-1 levels, which in turn promoted recruitment of neutrophils to the liver. Both inhibition of SDF-1-mediated neutrophil recruitment and systemic depletion of neutrophils reduced TIMP-1-induced increased liver susceptibility towards metastasis. This indicates a crucial functional role of neutrophils in the TIMP-1-induced premetastatic niche. CONCLUSION: Our results identify TIMP-1 as an essential promoter of hepatic premetastatic niche formation.

Tetraspanin CD63 acts as a pro-metastatic factor via ß-catenin stabilization.

The tetraspanin CD63 is implicated in pro-metastatic signaling pathways but, so far, it is unclear, how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial-like phenotype concomitant with increased E-cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N-cadherin. In addition, ß-catenin protein was markedly reduced, negatively affecting expression of the target genes MMP-2 and PAI-1. ß-catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of ß-catenin upstream regulators PI3K/AKT or GSK3ß could rescue the mesenchymal phenotype underlining the importance of the ß-catenin pathway in CD63-regulated cell plasticity. CD63 knock down-induced phenotypical changes correlated with a decrease of experimental metastasis whereas CD63 overexpression enhanced the tumor cell-intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity.

Selective inhibition of the immunoproteasome by ligand-induced crosslinking of the active site.

The concept of proteasome inhibition ranks among the latest achievements in the treatment of blood cancer and represents a promising strategy for modulating autoimmune diseases. In this study, we describe peptidic sulfonyl fluoride inhibitors that selectively block the catalytic ß5 subunit of the immunoproteasome by inducing only marginal cytotoxic effects. Structural and mass spectrometric analyses revealed a novel reaction mechanism involving polarity inversion and irreversible crosslinking of the proteasomal active site. We thus identified the sulfonyl fluoride headgroup for the development and optimization of immunoproteasome selective compounds and their possible application in autoimmune disorders.

Systematic comparison of peptidic proteasome inhibitors highlights the α-ketoamide electrophile as an auspicious reversible lead motif.

The ubiquitin-proteasome system (UPS) has been successfully targeted by both academia and the pharmaceutical industry for oncological and immunological applications. Typical proteasome inhibitors are based on a peptidic backbone endowed with an electrophilic C-terminus by which they react with the active proteolytic sites. Although the peptide moiety has attracted much attention in terms of subunit selectivity, the target specificity and biological stability of the compounds are largely determined by the reactive warheads. In this study, we have carried out a systematic investigation of described electrophiles by a combination of in vitro, in vivo, and structural methods in order to disclose the implications of altered functionality and chemical reactivity. Thereby, we were able to introduce and characterize the class of α-ketoamides as the most potent reversible inhibitors with possible applications for the therapy of solid tumors as well as autoimmune disorders.

On the Pro-Metastatic Stress Response to Cancer Therapies: Evidence for a Positive Co-Operation between TIMP-1, HIF-1α, and miR-210.

In contrast to expectations in the past that tumor starvation or unselective inhibition of proteolytic activity would cure cancer, there is accumulating evidence that microenvironmental stress, such as hypoxia or broad-spectrum inhibition of metalloproteinases can promote metastasis. In fact, malignant tumor cells, due to their genetic and epigenetic instability, are predisposed to react to stress by adaptation and, if the stress persists, by escape and formation of metastasis. Recent recognition of the concepts of dynamic evolution as well as population and systems biology is extremely helpful to understand the disappointments of clinical trials with new drugs and may lead to paradigm-shifts in therapy strategies. This must be complemented by an increased understanding of molecular mechanism involved in stress response. Here, we review new roles of Hypoxia-inducible factor-1 (HIF-1), one transcription factor regulating stress response-related gene expression: HIF-1 is crucial for invasion and metastasis, independent from its pro-survival function. In addition, HIF-1 mediates pro-metastatic microenvironmental changes of the proteolytic balance as triggered by high systemic levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), typical for many aggressive cancers, and regulates the metabolic switch to glycolysis, notably via activation of the microRNA miR-210. There is preliminary evidence that TIMP-1 also induces miR-210. Such positive-regulatory co-operation of HIF-1α, miR-210, and TIMP-1, all described to correlate with bad prognosis of cancer patients, opens new perspectives of gaining insight into molecular mechanisms of metastasis-inducing evasion of tumor cells from stress.