AbstractThe molecular mechanisms underlying cell migration remain incompletely understood. Here, we show that knock-out cells for NHSL3, the most recently identified member of the Nance-Horan Syndrome family, are more persistent than parental cells in single cell migration, but that, in wound healing, follower cells are impaired in their ability to follow leader cells. The NHSL3 locus encodes several isoforms. We identify the partner repertoire of each isoform using proteomics and predict direct partners and their binding sites using an AlphaFold2-based pipeline. Rescue with specific isoforms, and lack of rescue when relevant binding sites are mutated, establish that the interaction of a long isoform with MENA/VASP proteins is critical at cell-cell junctions for collective migration, while the interaction of a short one with 14-3-3θ in lamellipodia is critical for single cell migration. Taken together, these results demonstrate that NHSL3 regulates single and collective cell migration through distinct mechanisms.

Breast cancer develops upon sequential acquisition of driver mutations in mammary epithelial cells; however, how these mutations collaborate to transform normal cells remains unclear in most cases. We aimed to reconstitute this process in a particular case. To this end, we combined the activated form of the PI 3-kinase harboring the H1047R mutation with the inactivation of the histone lysine methyl-transferase KMT2D in the non-tumorigenic human mammary epithelial cell line MCF10A. We found that PI 3-kinase activation promoted cell-cycle progression, especially when growth signals were limiting, as well as cell migration, both in a collective monolayer and as single cells. Furthermore, we showed that KMT2D inactivation had relatively little influence on these processes, except for single-cell migration, which KMT2D inactivation promoted in synergy with PI 3-kinase activation. The combination of these two genetic alterations induced expression of the ARPC5L gene that encodes a subunit of the Arp2/3 complex. ARPC5L depletion fully abolished the enhanced migration persistence exhibited by double-mutant cells. Our reconstitution approach in MCF10A has thus revealed both the cell function and the single-cell migration, and the underlying Arp2/3-dependent mechanism, which are synergistically regulated when KMT2D inactivation is combined with the activation of the PI 3-kinase.

Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in PTEN and ZFHX3 tumor suppressor genes (TSGs). We generated single and combined PTEN and ZFHX3 knock-outs (KOs) in the immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of PTEN, but not ZFHX3, induced the formation of large colonies in soft agar. ZFHX3 inactivation in PTEN KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon PTEN and ZFHX3 KO. Inactivation of PTEN enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast, ZFHX3 knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes.

uPAR is a membrane receptor that binds extracellular protease urokinase, contributes to matrix remodeling and plays a crucial role in cellular adhesion, proliferation, survival, and migration. uPAR overexpression in tumor cells promotes mitogenesis, opening a prospective avenue for targeted therapy. However, uPAR targeting in cancer has potential risks. We have recently shown that uPAR downregulation in neuroblastoma promotes epithelial-mesenchymal transition (EMT), potentially associated with metastasis and chemoresistance. We used data mining to evaluate the role of uPAR expression in primary and relapsed human neuroblastomas. To model the decreased uPAR expression, we targeted uPAR using CRISPR/Cas9 and shRNA in neuroblastoma Neuro2a cells and evaluated their chemosensitivity in vitro as well as tumor growth and metastasis in vivo. We demonstrate that the initially high PLAUR expression predicts poor survival in human neuroblastoma. However, relapsed neuroblastomas have a significantly decreased PLAUR expression. uPAR targeting in neuroblastoma Neuro2a cells leads to p38 activation and an increased p21 expression (suggesting a dormant phenotype). The dormancy in neuroblastoma cells can be triggered by the disruption of uPAR-integrin interaction. uPAR-deficient cells are less sensitive to cisplatin and doxorubicin treatment and exhibit lower p53 activation. Finally, low uPAR-expressing Neuro2a cells formed smaller primary tumors, but more frequent metastasis in mice. To the best of our knowledge, this is the first study revealing the pathological role of dormant uPAR-deficient cancer cells having a chemoresistant and motile phenotype.

Urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored receptor of urokinase (uPA), which is involved in brain development, nerve regeneration, wound healing and tissue remodeling. We have recently shown that Plaur, which encodes uPAR, is an early response gene in murine brain. Assumingly, diverse functions of Plaur might be attributed to hypothetical, unidentified microRNAs encoded within introns of the Plaur gene. Using a bioinformatic approach we identified novel small RNAs within the Plaur gene and named them Plaur-miR1-3p and Plaur-miR1-5p. We confirmed Plaur-dependent expression of Plaur-miR1-3p and Plaur-miR1-5p in the mouse brain and mouse neuroblastoma Neuro2a cells. Utilizing an in silico MR-microT algorithm in DianaTools we selected two target genes - Mef2d and Emx2 with the highest binding scores to small RNAs selected from identified Plaur-Pre-miR1. Furthermore, sequencing of mouse brain samples for Plaur-miR1-5p target genes revealed two more genes-Nrip3 and Snrnp200. The expression of Emx2, Mef2d, and Snrnp200 in the mouse brain and Mef2d and Snrnp200 in Neuro2a cells correlated with expression of Plaur and small RNAs-Plaur-miR1-3p and Plaur-miR1-5p. Finally, we demonstrated elevated MEF2D protein expression in the mouse brain after Plaur induction and displayed activating effects of Plaur-miR1-5p on Mef2d expression in Neuro2a cells using Luciferase reporter assay. In conclusion, we have identified Plaur-miR1-3p and Plaur-miR1-5p as novel small RNAs encoded in the Plaur gene. This finding expands the current understanding of Plaur function in brain development and functioning.

By 2003, the Human Genome project had been completed; however, it turned out that 97% of genome sequences did not encode proteins. The explanation came later when it was found the untranslated DNA contain sequences for short microRNAs (miRNAs) and long noncoding RNAs that did not produce any mRNAs or tRNAs, but instead were involved in the regulation of gene expression. Initially identified in the cytoplasm, miRNAs have been found in all cell compartments, where their functions are not limited to the degradation of target mRNAs. miRNAs that are secreted into the extracellular space as components of exosomes or as complexes with proteins, participate in morphogenesis, regeneration, oncogenesis, metastasis, and chemoresistance of tumor cells. miRNAs play a dual role in oncogenesis: on one hand, they act as oncogene suppressors; on the other hand, they function as oncogenes themselves and inactivate oncosuppressors, stimulate tumor neoangiogenesis, and mediate immunosuppressive processes in the tumors, The review presents current concepts of the miRNA biogenesis and their functions in the cytoplasm and nucleus with special focus on the noncanonical mechanisms of gene regulation by miRNAs and involvement of miRNAs in oncogenesis, as well as the authors’ opinion on the role of miRNAs in metastasis and formation of the premetastatic niche.

By 2003, the Human Genome project had been completed; however, it turned out that 97% of genome sequences did not encode proteins. The explanation came later when it was found the untranslated DNA contain sequences for short microRNAs (miRNAs) and long noncoding RNAs that did not produce any mRNAs or tRNAs, but instead were involved in the regulation of gene expression. Initially identified in the cytoplasm, miRNAs have been found in all cell compartments, where their functions are not limited to the degradation of target mRNAs. miRNAs that are secreted into the extracellular space as components of exosomes or as complexes with proteins, participate in morphogenesis, regeneration, oncogenesis, metastasis, and chemoresistance of tumor cells. miRNAs play a dual role in oncogenesis: on one hand, they act as oncogene suppressors; on the other hand, they function as oncogenes themselves and inactivate oncosuppressors, stimulate tumor neoangiogenesis, and mediate immunosuppressive processes in the tumors, The review presents current concepts of the miRNA biogenesis and their functions in the cytoplasm and nucleus with special focus on the noncanonical mechanisms of gene regulation by miRNAs and involvement of miRNAs in oncogenesis, as well as the authors’ opinion on the role of miRNAs in metastasis and formation of the premetastatic niche.

The urokinase system is involved in a variety of physiological processes, such as fibrinolysis, matrix remodeling, wound healing, and regeneration. Upon binding to its cognate receptor urokinase‐type plasminogen activator receptor (uPAR), urokinase‐type plasminogen activator (uPA) catalyzes the conversion of plasminogen to plasmin and the activation of matrix metalloproteases. Apart from this, uPA–uPAR interaction can lead to the activation of transcription factors, mitogen‐activated protein kinase signaling pathways and RTK cascades. Elevated expression of uPA and uPAR is markedly associated with cancer progression and metastasis and correlates with a poor prognosis in clinics. Targeting the urokinase system has proved to be effective in experimental models in vitro and in vivo, however, in clinics the inhibition of the uPA/uPAR system has fallen short of expectations, suggesting that the question of the functional relevance of uPA/uPAR system is far from being moot. Recently, using CRISPR/Cas9 technology, we have shown that uPAR knockout decreases the proliferation of neuroblastoma Neuro2a cells in vitro. In the present study we demonstrate that uPAR expression is essential for maintaining the epithelial phenotype in Neuro2a cells and that uPAR silencing promotes epithelial‐mesenchymal transition (EMT) and increased cell migration. Accordingly, uPAR knockout results in the downregulation of epithelial markers (E‐cadherin, occludin, and claudin‐5) and in the increase of mesenchymal markers (N‐cadherin, α‐smooth muscle actin, and interleukin‐6). In search of the molecular mechanism underlying these changes, we identified uPA as a key component. Two key insights emerged as a result of this work: in the absence of uPAR, uPA is translocated into the nucleus where it is presumably involved in the activation of transcription factors (nuclear factor κB and Snail) resulting in EMT. In uPAR‐expressing cells, uPAR functions as a uPA “trap” that binds uPA on the cell surface and promotes controlled uPA internalization and degradation in lysosomes.

PURPOSE:Urokinase receptor (uPAR) promotes extracellular matrix proteolysis, regulates adhesion and cell migration, transduces intracellular signals through interactions with the lateral partners. The expression of uPAR and urokinase (uPA) is significantly upregulated in peripheral nerves after injury, however, little is known about uPAR function in nerve regeneration or the molecular mechanisms involved. The purpose of this study is to investigate the role of uPAR in nerve regeneration after traumatic injury of n. Peroneus communis in uPA-/-, uPAR-/- or control mice (WT) and in neuritogenesis in an in vitro Neuro 2A cell model.RESULTS:Electrophysiological analysis indicates that nerve recovery is significantly impaired in uPAR-/- mice, but not in uPA-/- mice. These data correlate with the reduced amount of NF200-positive axons in regenerating nerves from uPAR-/- mice compared to uPA-/- or control mice. There is an increase in uPAR expression and remarkable colocalization of uPAR with α5 and β1 integrin in uPA-/- mice in recovering nerves, pointing to a potential link between uPAR and its lateral partner α5β1-integrin. Using an in vitro model of neuritogenesis and α325 blocking peptide, which abrogates uPAR-α5β1 interaction in Neuro 2A cells but has no effect on their function, we have further confirmed the significance of uPAR-α5β1 interaction.CONCLUSION:Taken together, we report evidence pointing to an important role of uPAR, rather than uPA, in peripheral nerve recovery and neuritogenesis.

Urokinase-type plasminogen activator uPA and its receptor (uPAR) are the central players in extracellular matrix proteolysis, which facilitates cancer invasion and metastasis. EGFR is one of the important components of uPAR interactome. uPAR/EGFR interaction controls signaling pathways that regulate cell survival, proliferation and migration. We have previously established that uPA binding to uPAR stimulates neurite elongation in neuroblastoma cells, while blocking uPA/uPAR interaction induces neurite branching and new neurite formation. Here we demonstrate that blocking the uPA binding to uPAR with anti-uPAR antibody decreases the level of pEGFR and its downstream pERK1/2, but does increase phosphorylation of Akt, p38 and c-Src Since long-term uPAR blocking results in a severe DNA damage, accompanied by PARP-1 proteolysis and Neuro2a cell death, we surmise that Akt, p38 and c-Src activation transmits a pro-apoptotic signal, rather than a survival. Serum deprivation resulting in enhanced neuritogenesis is accompanied by an upregulated uPAR mRNA expression, while EGFR mRNA remains unchanged. EGFR activation by EGF stimulates neurite growth only in uPAR-overexpressing cells but not in control or uPAR-deficient cells. In addition, AG1478-mediated inhibition of EGFR activity impedes neurite growth in control and uPAR-deficient cells, but not in uPAR-overexpressing cells. Altogether these data implicate uPAR as an important regulator of EGFR and ERK1/2 signaling, representing a novel mechanism which implicates urokinase system in neuroblastoma cell survival and differentiation.

Epileptogenesis progressively leads to the rearrangement of normal neuronal networks into more excitable ones and can be viewed as a form of neuroplasticity, the molecular mechanisms of which still remain obscure. Here, we studied pentylenetetrazole seizure‐induced regulation of genes for plasminogen activator system in the mouse brain. We found that expression of tissue plasminogen activator (tPA) and urokinase receptor (uPAR) mRNA was strongly increased in the mouse cerebral cortex, hippocampus, striatum and amygdala as early as 3 hr after pentylenetetrazole seizures. Such early activity‐induced expression of uPAR in the central nervous system has not been demonstrated before. uPAR mRNA accumulation was followed by elevation of uPAR protein, indicating a complete transcription–translation process. Both tPA gene induction and uPAR gene induction were independent of the protein synthesis, suggesting that they are regulated by neural activity as immediate‐early genes. In contrast to tPA and uPAR genes, the expression of which returned to the basal level 6 hr following seizures, urokinase and plasminogen activator inhibitor‐1 gene expression showed a delayed activation only at 3 days after seizures. In conclusion, our results suggest an important sensitivity of the brain plasminogen activator system to seizure activity which raises the question of its role in activity‐dependent neural tissue remodeling in pathological and normal conditions.

Purpose: Urokinase receptor (uPAR) promotes extracellular matrix proteolysis, regulates adhesion and cell migration, transduces intracellular signals through interactions with the lateral partners. The expression of uPAR and urokinase (uPA) is significantly upregulated in peripheral nerves after injury, however, little is known about uPAR function in nerve regeneration or the molecular mechanisms involved. The purpose of this study is to investigate the role of uPAR in nerve regeneration after traumatic injury of n. Peroneus communis in uPA-/-, uPAR-/- or control mice (WT) and in neuritogenesis in an in vitro Neuro 2A cell model. Results: Electrophysiological analysis indicates that nerve recovery is significantly impaired in uPAR-/- mice, but not in uPA-/- mice. These data correlate with the reduced amount of NF200-positive axons in regenerating nerves from uPAR-/- mice compared to uPA-/- or control mice. There is an increase in uPAR expression and remarkable colocalization of uPAR with alpha 5 and beta 1 integrin in uPA-/- mice in recovering nerves, pointing to a potential link between uPAR and its lateral partner alpha 5 beta 1-integrin. Using an in vitro model of neuritogenesis and alpha 325 blocking peptide, which abrogates uPAR-alpha 5 beta 1 interaction in Neuro 2A cells but has no effect on their function, we have further confirmed the significance of uPAR-alpha 5 beta 1 interaction. Conclusion: Taken together, we report evidence pointing to an important role of uPAR, rather than uPA, in peripheral nerve recovery and neuritogenesis.

IMPACT STATEMENT:Cell lines represent convenient models to elucidate specific causes of multigenetic and pluricausal diseases, to test breakthrough regenerative technologies. Most commonly used cell lines surpass diploid cells in their accessibility for delivery of large DNA molecules and genome editing, but the main obstacles for obtaining cell models with knockout-targeted protein from aneuploid cells are multiple allele copies and karyotype/phenotype heterogeneity. In the study, we report an original approach to CRISPR-/Cas9-mediated genome modification of aneuploid cell cultures to create functional cell models, achieving highly efficient targeted protein knockout and avoiding "clonal effect" (for the first time to our knowledge).

Neuroblastoma is a tumor arising from pluripotent sympathoadrenal precursor cells of neural cell origin. Neuroblastoma is one of the most aggressive childhood tumors with highly invasive and metastatic potential. The increased expression of urokinase and its receptor is often associated with a negative prognosis in neuroblastoma patients. We have shown that targeting of the Plaur gene in mouse neuroblastoma Neuro 2A cells by CRISPR/Cas9n results in ~60% decrease in cell proliferation (p<0.05), reduction in the number of Ki-67 positive cells, caspase 3 activation and PARP-1 cleavage. Knockout of uPAR leads to downregulation of mRNA encoding full-length TrkC receptor, which is involved in p38MAPK and Akt signalling pathways. This finding provides a rationale to study a role of uPAR in neuroblastoma progression, since uPAR could be considered a potential therapeutic target in neuroblastoma treatment.