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.

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.

Overall survival analysis of > 65-year-old patients with breast cancer based on their molecular, clinicopathological and laboratory factors.

Introduction: The objective of the present study was to characterize > 65-year-old patients with breast cancer according to clinicopathological, molecular and laboratory factors. Methods: A total of 723 breast cancer patients, who had been diagnosed and treated during 2005-2019, were retrospectively reviewed. Patients > 65 years of age (92 patients) were compared with < 50-year-old women (306 patients). We analyzed 398 women from 723 patients. Results: Overall survival analysis was conducted for both groups, separately and combined. Patients with BC aged > 65 years were characterized by G1-2, higher lymphocyte values, lower platelet (PLT) counts and lower NLR or PLR values than patients < 50 years of age. Conclusions: Age > 65 years is a negative prognostic factor independent of other factors.

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.

Clinicopathological characteristics of breast cancer patients with NOD2 mutation according to age.

INTRODUCTION: The purpose of the present study was to characterise patients with breast cancer (BC) and NOD2-mutation (age ≥ 50 years) according to their clinicopathological factors or family history. Patients aged ≥ 50 years were compared with the control group and with NOD2-mutation carriers aged < 50 years. MATERIAL AND METHODS: Prognostic factors were analysed in patients with BC with confirmed NOD2 c.3016_3017insC (n = 150) mutations. The control group was selected from patients with BC without mutations (n = 376). RESULTS: There were significant differences between NOD2-mutation carriers and the control group aged ≥ 50 years, according to HER2 overexpression (p = 0.0001), ER (-) (p = 0.007), PR (-) (p = 0.003), T1-T2 (p = 0.011), and G3 (p = 0.036). Similarly, significant differences were observed between NOD2-mutation carriers and the control group aged < 50 years, according to HER2 overexpression (p = 0.0001), ER (-) (p = 0.049), and N (+) (p = 0.038). In patients aged ≥ 50 years, family history of cancer, including BC, was observed more often in NOD2-mutation carriers compared with the control group of patients (OR = 1.66; p = 0.072, for BC in family history: OR = 2.65; p = 0.002). NOD2-mutation carriers aged ≥ 50 years had significantly less frequent G3 (p = 0.004) and HER2 overexpression (p = 0.043) compared with patients with NOD2 mutation aged < 50 years. CONCLUSIONS: The presence of the NOD2 mutation is not only characteristic of younger patients but also in patients > 50 years of age. In NOD2-mutation carriers aged ≥ 50 years, the presence of larger tumour size, G3, or HER2 overexpression were lower compared with younger patients with NOD2 mutation.

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.

Genetic 3'UTR variations and clinical factors significantly contribute to survival prediction and clinical response in breast cancer patients.

The study describes a relationship between the 3'UTR variants, clinicopathological parameters and response to chemotherapy. We analyzed 33 germline polymorphisms in 3'UTRs of ADME genes in 305 breast cancer women treated with FAC regime. Clinical endpoints of this study were: overall survival (OS), progression-free survival (PFS), recurrence-free survival (RFS) and overall response defined as treatment failure-free survival (TFFS). The shortened OS was connected with the presence of NR1/2 rs3732359 AA, SLC22A16 rs7756222 CC, as well as SLC22A16 rs9487402 allele G and clinical factors belonging to TNM classification: tumor size >1 cm, nodal involvement and presence of metastases. PFS was related to two polymorphisms PGR rs1824125 GG, PGR rs12224560 CC and SLC22A16 rs7756222 CC as well as preexisting metastases. The RFS was shortened due to the DPYD rs291593 CC, AKR1C3 rs3209896 AG and negative expression of PGR. The presence of ALDH5A1 rs1054899 allele A, lack of pre-chemotherapy surgery and negative status of PGR correlated with worse treatment response. The germline variants commonly present in the population are important factors determining the response to treatment. We observed the effect of the accumulation of genetic and clinical factors on poor survival prognosis and overall treatment response.

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.

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.

BRCA1 mutation in breast cancer patients: Analysis of prognostic factors and survival.

The presence of BRCA1 mutations is associated with an increased risk of breast and ovarian cancer. The present study compared clinicopathological characteristics and overall survival (OS) of hereditary and sporadic breast cancer. Using data collected from a previous study conducted between 2007-2016 at the Maria Sklodowska Curie Cancer Center and Institute of Oncology (Gliwice, Poland), the prognostic factors and survival in 60 breast cancer mutation carriers were analyzed. A control group was selected from the breast cancer patients without BRCA mutations (n=386). BRCA mutation carriers had significantly worse survival when compared with non-carriers (P=0.017). The 10-year OS rate was 78.0% for all analyzed groups: 65.9% for BRCA mutation carriers and 81.1% for non-carriers. In the univariate analyses, BRCA mutation carriers had a significantly higher risk of mortality in comparison to non-carriers [hazard ratio (HR)=1.87; 95% confidence interval (CI) 1.08-3.25]. Increased tumor size (HR=3.64), lymph node metastases (HR=2.45) and higher histological grade (HR=2.84) were significant factors for worse OS. Positive estrogen receptor status was associated with a better OS (HR=0.49, P=0.022). Age ≤40 years (HR=0.48, P=0.081) was an insignificantly favorable factor. The 10-year survival rate was significantly decreased in patients with BRCA1 mutation. Therefore, negative factors for OS in mutation carriers included lymph nodes metastases, negative steroid receptor status and increased tumor size.