Improving the Prognostic and Predictive Value of Circulating Tumor Cell Enumeration: Is Longitudinal Monitoring the Answer?

Circulating tumor cell (CTC) numbers in the blood of cancer patients can indicate the progression and invasiveness of tumors, and their prognostic and predictive value has been repeatedly demonstrated. However, the standard baseline CTC count at the beginning of treatment, while informative, is not completely reliable and may not adequately reflect the state of the disease. A growing number of studies indicate that the long-term monitoring of CTC numbers in the same patient provides more comprehensive prognostic data and should be incorporated into clinical practice, as a factor that contributes to therapeutic decisions. This review describes the current status of CTC enumeration as a prognostic and predictive factor, highlights the shortcomings of current solutions, and advocates for longitudinal CTC analysis as a more effective method of the evaluation of developing disease, treatment efficacy, and the long term-monitoring of the minimal residual disease. As evidenced by the described reports, the longitudinal monitoring of CTCs should provide a better and more sensitive prediction of the course of the disease, and its incorporation in clinical practice should be beneficial.

Circulating Tumor Cells: Pathological, Molecular and Functional Characteristics.

This Special Issue, 'Circulating Tumor Cells: Pathological, Molecular and Functional Characteristics 1 [...].

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.

New 2-[(4-Amino-6-N-substituted-1,3,5-triazin-2-yl)methylthio]-N-(imidazolidin-2-ylidene)-4-chloro-5-methylbenzenesulfonamide Derivatives, Design, Synthesis and Anticancer Evaluation.

In the search for new compounds with antitumor activity, new potential anticancer agents were designed as molecular hybrids containing the structures of a triazine ring and a sulfonamide fragment. Applying the synthesis in solution, a base of new sulfonamide derivatives 20-162 was obtained by the reaction of the corresponding esters 11-19 with appropriate biguanide hydrochlorides. The structures of the compounds were confirmed by spectroscopy (IR, NMR), mass spectrometry (HRMS or MALDI-TOF/TOF), elemental analysis (C,H,N) and X-ray crystallography. The cytotoxic activity of the obtained compounds toward three tumor cell lines, HCT-116, MCF-7 and HeLa, was examined. The results showed that some of the most active compounds belonged to the R1 = 4-trifluoromethylbenzyl and R1 = 3,5-bis(trifluoromethyl)benzyl series and exhibited IC50 values ranging from 3.6 µM to 11.0 µM. The SAR relationships were described, indicating the key role of the R2 = 4-phenylpiperazin-1-yl substituent for the cytotoxic activity against the HCT-116 and MCF-7 lines. The studies regarding the mechanism of action of the active compounds included the assessment of the inhibition of MDM2-p53 interactions, cell cycle analysis and apoptosis induction examination. The results indicated that the studied compounds did not inhibit MDM2-p53 interactions but induced G0/G1 and G2/M cell cycle arrest in a p53-independent manner. Furthermore, the active compounds induced apoptosis in cells harboring wild-type and mutant p53. The compound design was conducted step by step and assisted by QSAR models that correlated the activity of the compounds against the HCT-116 cell line with molecular descriptors.

Cell Death by Entosis: Triggers, Molecular Mechanisms and Clinical Significance.

Entosis-a homotypic insertion of one cell into another, resulting in a death of the invading cell-has been described in many reports, but crucial aspects of its molecular mechanisms and clinical significance still remain controversial. While actomyosin contractility of the invading cell is very well established as a driving force in the initial phase, and autophagy induced in the outer cell is determined as the main mechanism of degradation of the inner cell, many details remain unresolved. The multitude of triggering factors and crisscrossing molecular pathways described in entosis regulation make interpretations difficult. The question of the physiological role of entosis also remains unanswered. In this review, we summarize the knowledge of molecular mechanisms and clinical data concerning entosis accumulated so far, highlighting both coherent explanations and controversies.

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.

The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1.

Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells. TAKE AWAY: EspO homologues are found in EHEC, Shigella, S. typhimurium and some EPEC. EspO homologues interact with HAX-1. EspO protects infected cells from apoptosis. EspO joins a growing list of T3SS effectors that manipulate cell death pathways.

How to Predict Metastasis in Luminal Breast Cancer? Current Solutions and Future Prospects.

Breast cancer metastasis is the main cause of breast cancer mortality. Luminal breast cancer represents the majority of breast cancer cases and, despite relatively good prognosis, its heterogeneity creates problems with a proper stratification of patients and correct identification of the group with a high risk of metastatic relapse. Current prognostic tools are based on the analysis of the primary tumor and, despite their undisputed power of prediction, they might be insufficient to foresee the relapse in an accurate and precise manner, especially if the relapse occurs after a long period of dormancy, which is very common in luminal breast cancer. New approaches tend to rely on body fluid analyses, which have the advantage of being non-invasive and versatile and may be repeated and used for monitoring the disease in the long run. In this review we describe the current, newly-developed, and only-just-discovered methods which are or may become useful in the assessment of the probability of the relapse.

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.

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.

Impact of Medium pH on DOX Toxicity toward HeLa and A498 Cell Lines.

The influence of the pH of the multicomponent cell medium on the performance of doxorubicin (DOX), an anticancer drug, was studied on the examples of cervical (HeLa) and kidney (A498) cancer cell lines. The change of pH of the cell medium to more acidic led to a decrease of DOX toxicity on both cell lines due to the change of drug permeability across the cell membrane as a result of drug protonation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) studies and lactate dehydrogenase (LDH) release tests have shown low toxicity of the drug, especially in the case of A498 cells, which are characterized by an extremely high glycolytic metabolism. The behavior was ascribed primarily to the increased proton concentration in the peripheral blood follicle in the presence of products of the acidic glycolytic metabolism. It is not observed in the measurements performed in commercially available media since they usually have a neutral pH. In earlier reports on kidney cancer, several mechanisms were discussed, including the metabolism of DOX to its less toxic derivative, doxorubicinol, overexpression of ATP binding cassette subfamily B member 1 (ABCB1) transporters, that remove DOX from the inside of cells; however, there was no focus on the simple but very important contribution of drug protonation described in the present study. Drug pH-dependent equilibria in the cell medium should be considered since changes in the drug form may be an additional reason for multidrug resistance.

Circulating Tumor Cells in Early and Advanced Breast Cancer; Biology and Prognostic Value.

Breast cancer metastasis is the leading cause of cancer deaths in women and is difficult to combat due to the long periods in which disseminated cells retain a potential to be re-activated and start the relapse. Assessing the number and molecular profile of circulating tumor cells (CTCs) in breast cancer patients, especially in early breast cancer, should help in identifying the possibility of relapse in time for therapeutic intervention to prevent or delay recurrence. While metastatic breast cancer is considered incurable, molecular analysis of CTCs still have a potential to define particular susceptibilities of the cells representing the current tumor burden, which may differ considerably from the cells of the primary tumor, and offer more tailored therapy to the patients. In this review we inspect the routes to metastasis and how they can be linked to specific features of CTCs, how CTC analysis may be used in therapy, and what is the current status of the research and efforts to include CTC analysis in clinical practice.

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.

Calcium-Binding Proteins with Disordered Structure and Their Role in Secretion, Storage, and Cellular Signaling.

Calcium is one of the most important second messengers and its intracellular signaling regulates many aspects of cell physiology. Calcium ions, like phosphate ions, are highly charged and thus are able to alter protein conformation upon binding; thereby they constitute key factors in signal transduction. One of the most common calcium-binding structural motifs is the EF-hand, a well-defined helix-loop-helix structural domain, present in many calcium-binding proteins (CBPs). Nonetheless, some CBPs contain non-canonical, disordered motifs, which usually bind calcium with high capacity and low affinity, and which represent a subset of proteins with specific functions, but these functions rarely involve signaling. When compared with phosphorylation-mediated signal transduction, the role of intrinsic disorder in calcium signaling is significantly less prominent and not direct. The list of known examples of intrinsically disordered CBPs is relatively short and the disorder in these examples seems to be linked to secretion and storage. Calcium-sensitive phosphatase calcineurin is an exception, but it represents an example of transient disorder, which is, nevertheless, vital to the functioning of this protein. The underlying reason for the different role of disordered proteins in the two main cellular signaling systems appears to be linked to the gradient of calcium concentration, present in all living cells.

The calcium binding properties and structure prediction of the Hax-1 protein.

Hax-1 is a protein involved in regulation of different cellular processes, but its properties and exact mechanisms of action remain unknown. In this work, using purified, recombinant Hax-1 and by applying an in vitro autoradiography assay we have shown that this protein binds Ca2+. Additionally, we performed structure prediction analysis which shows that Hax-1 displays definitive structural features, such as two α-helices, short ß-strands and four disordered segments.

HAX-1: a novel p-body protein.

HAX-1, a multifunctional protein involved in the regulation of apoptosis, cell migration, and calcium homeostasis, binds the 3' untranslated region motifs of specific transcripts. This suggests that HAX-1 plays a role in post-transcriptional regulation, at the level of mRNA stability/transport or translation. In this study, we analyze in detail HAX-1 colocalization with processing bodies (P-bodies) and its dependence on mRNA availability. Endogenous P-body markers DCP1 and Rck/p54 were shown to colocalize with endogenous HAX-1, but in case of the overexpressed proteins, only DCP1 displayed unperturbed colocalization with HAX-1. HAX-1 colocalization with DCP1 was observed in most of the cell lines studied, but its presence was not required for P-body formation, and its silencing caused an increase in P-body number. Preliminary mapping suggested that HAX-1 has more than one short P-body-targeting sequence. The pools of P-body-localized HAX-1 and cytosolic HAX-1 were demonstrated to dynamically exchange, suggesting steady flow of the protein. Active transcription was shown to be a factor in the localization of HAX-1 to P-bodies. Also, it was observed that HAX-1 localizes to some unidentified foci, which do not contain DCP1. In addition, it was demonstrated that HAX-1 status influences vimentin expression levels. Overall, HAX-1 was shown to colocalize with P-body markers and influence P-body number per cell in a manner dependent on mRNA availability. Presented data support the hypothesis that HAX-1 is involved in mRNA processing as an element of P-body interaction network.

Exploring the anti-apoptotic role of HAX-1 versus BCL-XL in cytokine-dependent bone marrow-derived cells from mice.

HS-1-associated protein X-1 (HAX-1) is a multi-functional protein that has been implicated in the regulation of apoptosis, cell motility and calcium homeostasis. In the present study, we set out to assess the postulated functional resemblance of HAX-1 to the BCL-2 family of anti-apoptotic proteins using non-transformed, cytokine-dependent murine bone marrow cells as a model system. BCL-X(L), but not HAX-1 protected against cytokine withdrawal-induced apoptosis while HAX-1 and BCL-X(L) significantly reduced thapsigargin-triggered (calcium-dependent) apoptosis. The data argue in favor of cell type- and stimulus-specific roles of HAX-1 in regulation of cell survival.