CtBP modulates Snail-mediated tumor invasion in Drosophila

Cancer is one of the most fatal diseases that threaten human health, whereas more than 90% mortality of cancer patients is caused by tumor metastasis, rather than the growth of primary tumors. Thus, how to effectively control or even reverse the migration of tumor cells is of great significance for cancer therapy. CtBP, a transcriptional cofactor displaying high expression in a variety of human cancers, has become one of the main targets for cancer prediction, diagnosis, and treatment. The roles of CtBP in promoting tumorigenesis have been well studied in vitro, mostly based on gain-of-function, while its physiological functions in tumor invasion and the underlying mechanism remain largely elusive. Snail (Sna) is a well-known transcription factor involved in epithelial-to-mesenchymal transition (EMT) and tumor invasion, yet the mechanism that regulates Sna activity has not been fully understood. Using Drosophila as a model organism, we found that depletion of CtBP or snail (sna) suppressed RasV12/lgl-/--triggered tumor growth and invasion, and disrupted cell polarity-induced invasive cell migration. In addition, loss of CtBP inhibits RasV12/Sna-induced tumor invasion and Sna-mediated invasive cell migration. Furthermore, both CtBP and Sna are physiologically required for developmental cell migration during thorax closure. Finally, Sna activates the JNK signaling and promotes JNK-dependent cell invasion. Given that CtBP physically interacts with Sna, our data suggest that CtBP and Sna may form a transcriptional complex that regulates JNK-dependent tumor invasion and cell migration in vivo.

Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

3D matrix adhesion composition facilitates nuclear force coupling to drive invasive cell migration

Cell invasion and metastasis is a multi-step process, initialised through the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembranespanning integrin adhesion complexes, which are well characterised in cells plated on 2D surfaces, yet much less is known about them in cells embedded in 3D matrices. We establish a technique to determine the composition of cell matrix adhesion complexes of invasive breast cancer cells in 3D matrices and on 2D surfaces and we identify an interaction complex enriched in 3D adhesive sites required for 3D invasive migration. Depletion of β-PIX-Myosin18A (Myo18A) abolishes cancer cell invasion, without negatively affecting matrix degradation, Rho GTPase signalling, or protrusion formation in collagen matrices. Instead, in a mechanism only seen in cells moving through 3D matrix, β-PIX and Myo18A drive the polarised recruitment of non-muscle Myosin 2A (NM2A) to the tips of protrusions. This recruitment of NM2A is required for the creation of an NM2A-NM2B isoform gradient, which ranges from the protrusion to the nucleus. We observe a requirement for active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. We postulate that the establishment of the NM2A-NM2B actomyosin gradient facilitates the coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)+ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner

Glioblastoma multiforme (GBM) recurrences after temozolomide (TMZ) treatment result from the expansion of drug-resistant and potentially invasive GBM cells. This process is facilitated by O6-Methylguanine-DNA Methyltransferase (MGMT), which counteracts alkylating TMZ activity. We traced the expansion of invasive cell lineages under persistent chemotherapeutic stress in MGMTlow (U87) and MGMThigh (T98G) GBM populations to look into the mechanisms of TMZ-induced microevolution of GBM invasiveness. TMZ treatment induced short-term, pro-invasive phenotypic shifts of U87 cells, in the absence of Snail-1 activation. They were illustrated by a transient induction of their motility and followed by the hypertrophy and the signs of senescence in scarce U87 sub-populations that survived long-term TMZ stress. In turn, MGMThigh T98G cells reacted to the long-term TMZ treatment with the permanent induction of invasiveness. Ectopic Snail-1 down-regulation attenuated this effect, whereas its up-regulation augmented T98G invasiveness. MGMTlow and MGMThigh cells both reacted to the long-term TMZ stress with the induction of Cx43 expression. However, only in MGMThigh T98G populations, Cx43 was directly involved in the induction of invasiveness, as manifested by the induction of T98G invasiveness after ectopic Cx43 up-regulation and by the opposite effect after Cx43 down-regulation. Collectively, Snail-1/Cx43-dependent signaling participates in the long-term TMZ-induced microevolution of the invasive GBM front. High MGMT activity remains a prerequisite for this process, even though MGMT-related GBM chemoresistance is not necessary for its initiation.

A molecular clock controls periodically driven cell migration in confined spaces

Navigation through dense, physically confining extracellular matrix is common in invasive cell spread and tissue re-organization, but is still poorly understood. Here, we show that this migration is mediated by cyclic changes in the activity of a small GTP-ase RhoA, dependent on the oscillatory changes in the activity and abundance of the RhoA Guanine Exchange Factor, GEF-H1, triggered by a persistent increase in the intracellular Ca2+ levels. We show that the molecular clock driving these cyclic changes is mediated by two coupled negative feedback loops, dependent on the microtubule dynamics, with the frequency that can be experimentally modulated based on a predictive mathematical model. We further demonstrate that an increasing frequency of the clock translates into a faster cell migration within physically confining spaces. This work lays the foundation for a better understanding of the molecular mechanisms dynamically driving cell migration in complex environments.