Longitudinal analysis of circulating tumor cell numbers improves tracking metastatic breast cancer progression.

Hormone-responsive breast cancer represents the most common type and has the best prognosis, but still approximately 40% of patients with this type can develop distant metastases, dramatically worsening the patient's survival. Monitoring metastatic breast cancer (mBC) for signs of progression is an important part of disease management. Circulating tumor cell (CTC) detection and molecular characteristics gain importance as a diagnostic tool, but do not represent a clinical standard and its value as a predictor of progression is not yet established. The main objective of this study was to estimate the prognostic value of not only the CTC numbers, but also the dynamics of the CTC numbers in the same patient during the continuous evaluation of CTCs in patients with advanced breast cancer. The other objective was to assess the molecular changes in CTCs compared to primary tumor samples by genetic analysis of the seven genes associated with estrogen signaling pathway, mutations in which are often responsible for the resistance to endocrine therapy, and subsequent progression. This approach was taken to evaluate if genetic analysis of CTCs can be used in tracking the resistance, signaling that hormonal therapy should be replaced. Consequently, this report presents the results of a longitudinal CTC study based on three subsequent blood collections from 135 patients with metastatic breast cancer, followed by molecular analysis of the isolated single CTCs. CTCs were detected and isolated using an image-based, EpCAM-independent system CytoTrack; this approach allowed evaluation of EpCAM expression in detected CTCs. Isolated CTCs were subjected to NGS analysis to assess mutational changes. The results confirm the importance of the status of the CTC for progression-free survival and overall survival and provide new data on the dynamics of the CTC during a long monitoring period and in relation to clinical progression, highlighting the advantage of constant monitoring over the single count of CTC. Furthermore, high genetic and phenotypic inter- and intrapatient heterogeneity observed in CTCs suggest that metastatic lesions are divergent. High genetic heterogeneity in the matching CTC/primary tumor samples may indicate early dissemination. The tendency towards the accumulation of activating/oncogenic mutation in CTCs, leading to anti-estrogen resistant disease, was not confirmed in this study.

HAX1: A versatile, intrinsically disordered regulatory protein.

HAX1 is a relatively small, ubiquitously expressed, predominantly mitochondrial, intrinsically disordered protein. It has been implicated in the regulation of apoptosis, cell migration, calcium cycling, proteostasis, angiogenesis, autophagy and translation. A wide spectrum of functions, numerous interactions and still elusive molecular mechanisms of action make HAX1 an intriguing subject of research. Moreover, HAX1 is involved in the pathogenesis of diseases; its deficiency leads to neutropenia and its overexpression is associated with cancer. In this review we aim to describe the characteristics of HAX1 gene and protein, and comprehensively discuss its multiple functions, highlighting the emerging role of HAX1 in protection from stress and apoptosis through maintaining cellular proteostasis and homeostasis.

The RNA-Binding Landscape of HAX1 Protein Indicates Its Involvement in Translation and Ribosome Assembly.

HAX1 is a human protein with no known homologues or structural domains. Mutations in the HAX1 gene cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported the RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here, we report the transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in translation, ribosome biogenesis, and rRNA processing. Using CRISPR knockouts, we find that HAX1 RNA targets partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. The functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

Protein Binding to Cis-Motifs in mRNAs Coding Sequence Is Common and Regulates Transcript Stability and the Rate of Translation.

Protein binding to the non-coding regions of mRNAs is relatively well characterized and its functionality has been described in many examples. New results obtained by high-throughput methods indicate that binding to the coding sequence (CDS) by RNA-binding proteins is also quite common, but the functions thereof are more obscure. As described in this review, CDS binding has a role in the regulation of mRNA stability, but it has also a more intriguing role in the regulation of translational efficiency. Global approaches, which suggest the significance of CDS binding along with specific examples of CDS-binding RBPs and their modes of action, are outlined here, pointing to the existence of a relatively less-known regulatory network controlling mRNA stability and translation on yet another level.

Adhesion of Triple-Negative Breast Cancer Cells under Fluorescent and Soft X-ray Contact Microscopy.

Understanding cancer cell adhesion could help to diminish tumor progression and metastasis. Adhesion mechanisms are currently the main therapeutic target of TNBC-resistant cells. This work shows the distribution and size of adhesive complexes determined with a common fluorescence microscopy technique and soft X-ray contact microscopy (SXCM). The results presented here demonstrate the potential of applying SXCM for imaging cell protrusions with high resolution when the cells are still alive in a physiological buffer. The possibility to observe the internal components of cells at a pristine and hydrated state with nanometer resolution distinguishes SXCM from the other more commonly used techniques for cell imaging. Thus, SXCM can be a promising technique for investigating the adhesion and organization of the actin cytoskeleton in cancer cells.

The interactome of multifunctional HAX1 protein suggests its role in the regulation of energy metabolism, de-aggregation, cytoskeleton organization and RNA-processing.

HCLS1-associated protein X-1 (HAX1) is a multifunctional protein involved in many cellular processes, including apoptosis, cell migration and calcium homeostasis, but its mode of action still remains obscure. Multiple HAX1 protein partners have been identified, but they are involved in many distinct pathways, form different complexes and do not constitute a coherent group. By characterizing HAX1 protein interactome using targeted approach, we attempt to explain HAX1 multiple functions and its role in the cell. Presented analyses indicate that HAX1 interacts weakly with a wide spectrum of proteins and its interactome tends to be cell-specific, which conforms to a profile of intrinsically disordered protein (IDP). Moreover, we have identified a mitochondrial subset of HAX1 protein partners and preliminarily characterized its involvement in the cellular response to oxidative stress and aggregation.

Water-soluble galactosamine derivative of ß-cyclodextrin as protective ligand and targeted carrier for delivery of toxic anthracycline drug.

ß-cyclodextrin modified with an electron-rich aromatic triazole linker and targeting moiety (galactosamine) was synthesized and studied as a carrier for the anticancer drug, doxorubicin (DOX), with the aim of targeting the pathological cells, reducing the cardiotoxic side effects and increasing the binding of the drug to DNA. The ß-cyclodextrins modified with galactosamine (ßCDGAL) are non-toxic and highly soluble in aqueous medium compared to the native ßCD and ßCD modified only with aromatic moiety, such as triazole linker. Molecular modelling and NMR study gave a deeper insight into the ligand structure, providing an explanation for its increased solubility, and the drug-ligand interactions. The triazole linker strengthened the drug binding and introduced pH dependence of the complex stability constants for ßCDGAL derivative, as confirmed by the voltammetry measurements. Spectroscopic studies have shown that entrapment of the DOX in ßCDGAL cavity reduces the stability constant of the DOX:Fe(III) complex responsible for the production of cardiotoxic reactive oxygen species and additionally supports the binding of the drug to the double strand DNA. The MTT assay and confocal microscopy results showed that despite encapsulation of the drug in the cyclodextrin molecule, its cytotoxic effect on the liver cancer cell line (HepG2) is comparable to that of the free, non-protected drug.

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers.

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

HAX1 impact on collective cell migration, cell adhesion, and cell shape is linked to the regulation of actomyosin contractility.

HAX1 protein is involved in the regulation of apoptosis, cell motility and calcium homeostasis. Its overexpression was reported in several tumors, including breast cancer. This study demonstrates that HAX1 has an impact on collective, but not single-cell migration, thus indicating the importance of cell-cell contacts for the HAX1-mediated effect. Accordingly, it was shown that HAX1 knockdown affects cell-cell junctions, substrate adhesion, and epithelial cell layer integrity. As demonstrated here, these effects can be attributed to the modulation of actomyosin contractility through changes in RhoA and septin signaling. Additionally, it was shown that HAX1 does not influence invasive potential in the breast cancer cell line, suggesting that its role in breast cancer progression may be linked instead to collective invasion of the epithelial cells but not single-cell dissemination.

RNA-protein interactions: disorder, moonlighting and junk contribute to eukaryotic complexity.

RNA-protein interactions are crucial for most biological processes in all organisms. However, it appears that the complexity of RNA-based regulation increases with the complexity of the organism, creating additional regulatory circuits, the scope of which is only now being revealed. It is becoming apparent that previously unappreciated features, such as disordered structural regions in proteins or non-coding regions in DNA leading to higher plasticity and pliability in RNA-protein complexes, are in fact essential for complex, precise and fine-tuned regulation. This review addresses the issue of the role of RNA-protein interactions in generating eukaryotic complexity, focusing on the newly characterized disordered RNA-binding motifs, moonlighting of metabolic enzymes, RNA-binding proteins interactions with different RNA species and their participation in regulatory networks of higher order.

Cytoplasmic HAX1 Is an Independent Risk Factor for Breast Cancer Metastasis.

HAX1 is an antiapoptotic factor involved in the regulation of cell migration and calcium homeostasis, overexpressed in several cancers, including breast cancer. It has been suggested that HAX1 is also implicated in metastasis. Herein we report the results of meta-analysis of HAX1 expression, based on publicly available data, which confirms its significant overexpression in breast cancer and demonstrates copy number gain and prognostic value of HAX1 overexpression for metastatic relapse in ER+ tumors. IHC analysis reported here also reveals its significant overexpression in breast cancer samples from primary tumors, indicating significantly higher HAX1 protein levels in a group of patients who developed distant metastases in a disease course. Moreover, we demonstrate that HAX1 localization is important for the prediction of metastatic relapse and that cytoplasmic but not nuclear HAX1 is an independent risk factor for breast cancer metastasis.