MicroRNAs (miRNAs) and their isoforms, known as isomiRs, are important regulators of tumorigenesis that act as post-transcriptional modulators of gene expression. Among these, 5’-isomiRs—generated through imprecise cleavage during miRNA biogenesis—exhibit altered seed regions compared to their canonical counterparts, potentially leading to distinct targetomes. Consequently, 5’-isomiRs may exert biological functions that differ substantially from those of the corresponding canonical miRNAs. Despite growing recognition of their potential significance, the functional roles of 5’-isomiRs remain largely uncharacterized for most miRNAs. In this study, we investigated the targetome divergence between canonical miRNAs and their 5’-isomiRs, focusing on hsa-miR-93-5p, a miRNA with a well-established oncogenic role in prostate adenocarcinoma. Target transcripts of the 5’-isomiRs were identified using a shRNA-based overexpression system. Bioinformatic analysis revealed a substantial overlap between the targets of the 5’-isomiRs and the canonical miRNA. This overlap was attributed to the co-occurrence of both canonical and shifted seed motifs within the same mRNA targets. Notably, hsa-miR-93-5p ranked among the top miRNAs with a relatively high number of targets transcripts containing both seed motifs, suggesting a unique dual-targeting capacity.

Metastasis is a hallmark of malignancy and poses a formidable challenge for oncologists. This complex process begins when the primary malignant cells start to proliferate unlimitedly. Once the tumor reaches a certain size, cancer cells detach from the primary tumor mass and the basal lamina to which they are anchored, eventually invading nearby blood vessels. Within the bloodstream, these tumor cells have to survive and attach to the endothelium at distant sites. Cell‐to‐cell and cell‐to‐matrix interactions play an important role during these stages, in which integrins—a family of cell adhesion molecules (CAMs)—stand out as functionally centrally involved. Their expression on the tumor cell surface needs to be dynamically regulated throughout the process of metastasis. During the attachment phase to the endothelium, another group of CAMs, such as E‐/P‐selectins—primarily expressed on endothelial cells—may also play a critical role in certain malignancies. Additionally, the interplay between integrins and selectins may influence the tumor microenvironment. This review focuses on the role of integrins and their interplay with selectins in metastasis, emphasizing findings from in vivo studies.

IntroductionMetastasis formation poses a significant challenge to oncologists, as it severely limits the survival of colorectal cancer (CRC) patients. Recently, we demonstrated that CD44 promotes spontaneous distant metastasis in a CRC xenograft model. The depletion of CD44 was associated with reduction in hypoxia, EMT, as well as improved mitochondrial metabolism in primary tumor. Collectively, these effects decreased the metastatic potential of the CRC xenograft tumors under investigation. In this study we explore the molecular mechanisms by which CD44 knockdown (kd) leads to such substantial changes of tumor properties.MethodsUsing miRNA-Seq data combined with bioinformatic analysis, we investigated the role of miRNA expression changes in the metastasis prevention observed with CD44 kd.ResultsAmong the differentially expressed miRNAs, three members of Let-7 family (let-7a-5p, let-7b-5p, and let-7c-5p), two isoforms of miR-203a (canonical miR-203a-3p and its +1 5’-isoform), miR-101-3p, miR-200b-3p|+1 5’-isoform, miR-125a-5p, and miR-185-5p were identified as potentially involved in regulating CD44-mediated metastasis. Gene set analysis of differentially expressed mRNA targets of these miRNAs, along with an examination of key regulators driving the observed changes in both mRNA and miRNA expression profiles, suggests that the CD44-STAT3-Let-7 miRNA axis as one of the most relevant in regulation of colon cancer metastasis via the CD44 receptor.DiscussionOur findings suggest a regulatory relationship between CD44, Let-7 miRNAs, and STAT3 in HT-29 tumors. Additionally, we propose the potential involvement of both isoforms of miR-203a (canonical and its +1 5’-isoform) in this regulatory network and suggest a role for miR-101-3p and miR-125a-5p in metastasis regulation through CD44 kd.

Cancer chemoresistance presents a challenge in oncology, often leading to treatment failure and disease progression. CD44, a multifunctional cell surface glycoprotein, has garnered attention for its involvement in various aspects of cancer biology. Through alternative splicing, CD44 can form isoforms with the inclusion of only standard exons, typical for normal tissue, or with the addition of variant exons, frequently expressed in cancer tissue and associated with chemoresistance. The functions of CD44 involved in regulation of cancer signaling pathways are being actively studied, and the significance of specific variant exons in modulating cell death pathways, central to the response of cancer cells to chemotherapy, begins to become apparent. This review provides a comprehensive analysis of the association of CD44 variant exons/total CD44 with clinical outcomes of patients undergoing chemotherapy. The role of CD44 variant exons v6, v9 and others with a significant effect on patient chemotherapy outcomes by means of key cellular death pathways such as apoptosis, ferroptosis and autophagy modulation is further identified, and their impact on drug resistance is highlighted. An overview of clinical trials aimed at targeting variant exon-containing isoforms is provided, and possible directions for further development of CD44-targeted therapeutic strategies are discussed.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related morbidity and mortality worldwide. Tumor sidedness, distinguishing left- (LCRC) and right-sided (RCRC) cancers, has emerged as a critical clinical determinant, influencing patient prognosis and therapeutic response. However, the cellular and molecular mechanisms underlying these differences remain poorly understood. Recent advances in single-cell RNA sequencing (sc-seq) provide high-resolution insights into CRC heterogeneity, revealing distinct tumor, immune, and stromal cell populations and their context-specific interactions. In this review, we synthesize sc-seq studies that dissect the molecular programs driving progression, therapy resistance, and metastasis in CRC. We highlight malignant subclusters characterized by metabolic reprogramming and spatially organized oncogenic signaling; specialized immune cell states, including macrophage subsets, exhausted T cells, and mast cells, that shape tumor immunity; and stromal elements such as cancer-associated fibroblasts and endothelial tip cells that remodel the extracellular matrix, promote angiogenesis, and foster immune evasion. Importantly, sc-seq demonstrates that LCRC and RCRC represent distinct multicellular ecosystems with differential immune recruitment and stromal signaling, underscoring the need for sidedness-informed therapeutic strategies. We propose that future interventions should target cell-cell communication networks and spatially defined tumor–microenvironment interactions to overcome heterogeneity and improve clinical outcomes.

shRNA-mediated strategy of miRNA overexpression based on RNA Polymerase III (Pol III) expression cassettes is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that expression of miRNAs longer than 19 nt (e.g. 23 nt in length hsa-miR-93-5p) using such approach could be accompanied by undesired predominant generation of 5' end miRNA isoforms (5'-isomiRs). Extra U residues (up to five) added by Pol III at the 3' end of the transcribed shRNA during transcription termination could cause a shift in the Dicer cleavage position of the shRNA. This results in the formation of 5'-isomiRs, which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. We demonstrated that the commonly used qPCR method is insensitive to the formation of 5'-isomiRs and cannot be used to confirm miRNA overexpression. However, the predominant expression of 5'-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5'-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. Overall, the presented results show that structures of shRNAs for stable overexpression of miRNAs requires careful design to avoid generation of undesired 5'-isomiRs.

Regulation of alternative splicing is carried out by RNA-binding proteins. Each alternative splicing event is controlled by several RNA-binding proteins, which in combination create the distribution of alternative splicing products in a given cell type. Transmembrane protein CD44 plays an important role at various stages of the metastatic cascade and is considered as a promising molecule for the therapy of tumor diseases and the construction of prognostic classifiers. However, the functions of specific isoforms of this protein may differ significantly. In this work, we performed a bioinformatic search of RNA-binding proteins that can determine the expression of clinically significant isoforms 3 and 4 of CD44 protein. The analysis revealed five RNA-binding proteins, three of which (OAS1, ZFP36L2, and DHX58) are shown for the first time as potential regulators of the studied process.

Hematogenous metastasis limits the survival of colorectal cancer (CRC) patients. Here, we illuminated the roles of CD44 isoforms in this process. Isoforms 3 and 4 were predominantly expressed in CRC patients. CD44 isoform 4 indicated poor outcome and correlated with epithelial–mesenchymal transition (EMT) and decreased oxidative phosphorylation (OxPhos) in patients; opposite associations were found for isoform 3. Pan‐CD44 knockdown (kd) independently impaired primary tumor formation and abrogated distant metastasis in CRC xenografts. The xenograft tumors mainly expressed the clinically relevant CD44 isoforms 3 and 4. Both isoforms were enhanced in the paranecrotic, hypoxic tumor regions but were generally absent in lung metastases. Upon CD44 kd, tumor angiogenesis was increased in the paranecrotic areas, accompanied by reduced hypoxia‐inducible factor‐1α and CEACAM5 but increased E‐cadherin expression. Mitochondrial genes and proteins were induced upon pan‐CD44 kd, as were OxPhos genes. Hypoxia increased VEGF release from tumor spheres, particularly upon CD44 kd. Genes affected upon CD44 kd in xenografts specifically overlapped concordantly with genes correlating with CD44 isoform 4 (but not isoform 3) in patients, validating the clinical relevance of the used model and highlighting the metastasis‐promoting role of CD44 isoform 4.

Since its original discovery over a decade ago, extracellular RNA (exRNA) has been found in all biological fluids. Furthermore, extracellular microRNA has been shown to be involved in communication between various cell types. Importantly, the exRNA is protected from RNases degradation by certain carriers including membrane vesicles and non-vesicular protein nanoparticles. Each type of carrier has its unique exRNA profile, which may vary depending on cell type and physiological conditions. To clarify putative mechanisms of intercellular communication mediated by exRNA, the RNA profile of each carrier has to be characterized. While current methods of biofluids fractionation are continuously improving, they fail to completely separate exRNA carriers. Likewise, most popular library preparation approaches for RNA sequencing do not allow obtaining exhaustive and unbiased data on exRNA transcriptome. In this mini review we discuss ongoing progress in the field of exRNA, with the focus on exRNA carriers, analyze the key methodological challenges and provide recommendations on how the latter could be overcome.

Alternative splicing is often deregulated in cancer, and cancer-specific isoform switches are part of the oncogenic transformation of cells. Accumulating evidence indicates that isoforms of the multifunctional cell-surface glycoprotein CD44 play different roles in cancer cells as compared to normal cells. In particular, the shift of CD44 isoforms is required for epithelial to mesenchymal transition (EMT) and is crucial for the maintenance of pluripotency in normal human cells and the acquisition of cancer stem cells phenotype for malignant cells. The growing and seemingly promising use of splicing inhibitors for treating cancer and other pathologies gives hope for the prospect of using such an approach to regulate CD44 alternative splicing. This review integrates current knowledge about regulating CD44 alternative splicing by RNA-binding proteins.

A widely used procedure for selecting significant miRNA-mRNA or isomiR-mRNA pairs out of predicted interactions involves calculating the correlation between expression levels of miRNAs/isomiRs and mRNAs in a series of samples. In this manuscript, we aimed to assess the validity of this procedure by comparing isomiR-mRNA correlation profiles in sets of sequence-based predicted target mRNAs and non-target mRNAs (negative controls). Target prediction was carried out using RNA22 and TargetScan algorithms. Spearman’s correlation analysis was conducted using miRNA and mRNA sequencing data of The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) project. Luminal A, luminal B, basal-like breast cancer subtypes, and adjacent normal tissue samples were analyzed separately. Using the sets of putative targets and non-targets, we introduced adjusted isomiR targeting activity (ITA)—the number of negatively correlated potential isomiR targets adjusted by the background (estimated using non-target mRNAs). We found that for most isomiRs a significant negative correlation between isomiR-mRNA expression levels appeared more often in a set of predicted targets compared to the non-targets. This trend was detected for both classical seed region binding types (8mer, 7mer-m8, 7mer-A1, 6mer) predicted by TargetScan and the non-classical ones (G:U wobbles and up to one mismatch or unpaired nucleotide within seed sequence) predicted by RNA22. Adjusted ITA distributions were similar for target sites located in 3′-UTRs and coding mRNA sequences, while 5′-UTRs had much lower scores. Finally, we observed strong cancer subtype-specific patterns of isomiR activity, highlighting the differences between breast cancer molecular subtypes and normal tissues. Surprisingly, our target prediction- and correlation-based estimates of isomiR activities were practically non-correlated with the average isomiR expression levels neither in cancerous nor in normal samples.

Viscumin, a lectin used in anti-cancer therapy, was originally considered as βGal recognizing protein; later, an ability to bind 6'-sialyl N-acetyllactosamine (6'SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galβ1-4GlcNAc, was observed. Unexpectedly, only weak interaction was detected with parent neutral β-galactosides including LN-LN-LN and branched (LN)2LN oligolactosamines; in the light of these data, one should not confidently classify viscumin as a β-galactoside-binding lectin. Carrying out PGA in the presence of neutral or sulfated/sialylated glycan, together with sequential elution from lactose-sepharose and consideration of the protein structure, lead to the conclusion that two glycan-binding sites of viscumin have different specificities, one of which prefers charged sulfated and sialylated moieties.

Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We assume that the higher conformational plasticity of RTA is favorable for the ER-mediated translocation pathway, which leads to a higher rate of toxin penetration into the cytoplasm.

The expression profile of CD44 isoforms in 55 colorectal cancer (CRC) cell lines has been evaluated based on mRNA sequencing data from the Cancer Cell Line Encyclopedia database. The distribution of CD44 mRNA isoforms differs significantly between CRC lines. In 13 studied lines, including Caco-2 and RKO, CD44 expression was not detected. In most other lines CD44 mRNA isoform 3 was the most abundant; however, the level of its expression varied, and it was absent in the MDST8 and SNU503 lines. The highest level of isoform 3 was observed in CW2 and T84 lines. The next most frequent was isoform 4 with its expression level lower than that of isoform 3, except for the HCT116, SNU81, NCIH508 and SNUC4 lines. The highest expression of isoform 4 was detected in the MDST8 line, the only line in which isoform 6 was also expressed. Isoform 2 was also present in CRC cell lines; its highest expression level was found in the SNU503 line. Isoforms 1, 5, and 7 did not show expression in any of the studied lines. It is necessary to take into account the mRNA expression profile of specific CD44 isoforms when choosing a cell model to study its role in CRC. colorectal cancer, CD44 isoforms, mRNA sequencing, CCLE The study was funded by the Russian Science Foundation (project no. 17-14-01338).

Detection of colorectal cancer biomarkers (CRC) remains an urgent task for the diagnosis and prediction of the disease course. A promising approach is the study of cancer stem cell markers. The cell surface glycoprotein CD44 is very important for CRC and its stem cells. Alternative splicing of 9 variable exons of CD44 mRNA leads to the formation of various isoforms of the protein with different roles in the progression of cancer. Studies of the functions of CD44 isoforms require adequate models considering the distribution of CD44 isoforms in real tumor samples. In the present study, the expression profile of CD44 isoforms in CRC was assessed based on the publicly available mRNA sequencing data of patient tumors from the TCGA-COAD database. It was shown that normal tissues predominantly expressed isoforms 3 and 4 at nearly equal levels, whereas tumors mainly expressed isoforms 2, 3, and 4; isoform 3 was expressed at the highest level. Further, the most relevant cell lines for studying the role of CD44 in CRC were identified based on the analysis of mRNA sequencing data of 55 CRC cell lines form CCLE database.

Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.

Intestinal epithelial cells exist in physiological hypoxia, leading to hypoxia-inducible factor (HIF) activation and supporting barrier function and cell metabolism of the intestinal epithelium. In contrast, pathological hypoxia is a common feature of some chronic disorders, including inflammatory bowel disease (IBD). This work was aimed at studying HIF-associated changes in the intestinal epithelium in IBD. In the first step, a list of genes responding to chemical activation of hypoxia was obtained in an in vitro intestinal cell model with RNA sequencing. Cobalt (II) chloride and oxyquinoline treatment of both undifferentiated and differentiated Caco-2 cells activate the HIF-signaling pathway according to gene set enrichment analysis. The core gene set responding to chemical hypoxia stimulation in the intestinal model included 115 upregulated and 69 downregulated genes. Of this set, protein product was detected for 32 genes, and fold changes in proteome and RNA sequencing significantly correlate. Analysis of publicly available RNA sequencing set of the intestinal epithelial cells of patients with IBD confirmed HIF-1 signaling pathway activation in sigmoid colon of patients with ulcerative colitis and terminal ileum of patients with Crohn’s disease. Of the core gene set from the gut hypoxia model, expression activation of ITGA5 and PLAUR genes encoding integrin α5 and urokinase-type plasminogen activator receptor (uPAR) was detected in IBD specimens. The interaction of these molecules can activate cell migration and regenerative processes in the epithelium. Transcription factor analysis with the previously developed miRGTF tool revealed the possible role of HIF1A and NFATC1 in the regulation of ITGA5 and PLAUR gene expression. Detected genes can serve as markers of IBD progression and intestinal hypoxia.

PURPOSE:The transcription factor Fra-2 affects the invasive potential of breast cancer cells by dysregulating adhesion molecules in vitro. Previous results suggested that it upregulates the expression of E- and P-selectin ligands. Such selectin ligands are important members of the leukocyte adhesion cascade, which govern the adhesion and transmigration of cancer cells into the stroma of the host organ of metastasis. As so far, no in vivo data are available, this study was designed to elucidate the role of Fra-2 expression in a spontaneous breast cancer metastasis xenograft model.METHODS:The effect of Fra-2 overexpression in two stable Fra-2 overexpressing clones of the human breast cancer cell line MDA MB231 on survival and metastatic load was studied after subcutaneous injection into scid and E- and P-selectin-deficient scid mice.RESULTS:Fra-2 overexpression leads to a significantly shorter overall survival and a higher amount of spontaneous lung metastases not only in scid mice, but also in E- and P-deficient mice, indicating that it regulates not only selectin ligands, but also selectin-independent adhesion processes.CONCLUSION:Thus, Fra-2 expression influences the metastatic potential of breast cancer cells by changing the expression of adhesion molecules, resulting in increased adherence to endothelial cells in a breast cancer xenograft model.

In this study we analyzed expression of CD24 in a cohort of colorectal cancer patients using immunohistochemistry staining of CD24. We found a significant association between absence or low expression of CD24 (10% of membranous and 55% of cytoplasmic staining) and shortened patient survival. Protein localization played a crucial role in the prognosis: membranous form was the major and prognostic one in primary tumors, while cytoplasmic expression was elevated in liver metastases compared to the primary tumors and contained prognostic information. Then, using The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) RNA-seq data, we showed that CD24 mRNA level was two-fold decreased in primary colorectal cancers compared to adjacent normal mucosa. Like the protein staining data, ten percent of patients with the lowest mRNA expression levels of CD24 in primary tumors had reduced survival compared to the ones with higher expression. To explain these findings mechanistically, shRNA-mediated CD24 knockdown was performed in HT-29 colorectal cancer cells. It resulted in the increase of cell migration in vitro, no changes in proliferation and apoptosis, and a slight decrease in cell invasion. As increased cell migration is a hallmark of metastasis formation, this finding corroborates the association of a decreased CD24 expression with poor prognosis. Differential gene expression analysis revealed upregulation of genes involved in cell migration in the group of patients with low CD24 expression, including integrin subunit α3 and α3, β3 subunits of laminin 332. Further co-expression analysis identified SPI1, STAT1 and IRF1 transcription factors as putative master-regulators in this group.