Chorein sensitive microtubule organization in tumor cells

Introduction

Chorein proteins are large proteins that share structural features (Segeletz et al., 2019). Various cells and tissues express differentially chorein, which is the eventual protein product of Vacuolar Protein Sorting Protein 13A (VPS13A) (Velayos-Baeza et al., 2004; Segeletz et al., 2019). Lack of this protein causes/leads to the development of an autosomal and progressive neurodegenerative disease called chorea-acanthocytosis (ChAc) (Dobson-Stone et al., 2004; Tomiyasu et al., 2011; Dobson-Stone et al., 2002; Ueno et al., 2001). This disease is escorted by severe movement’s disorders and variable erythrocyte acanthocytosis (Velayos Baeza et al., 1993; Saiki et al., 2007; Adjobo-Hermans, Cluitmans & Bosman, 2015). Previous studies revealed that this protein is also linked with regulation of various functional cell responses (Velayos-Baeza et al., 2004; Ueno et al., 2001). These include exocytosis (Lang et al., 2017; Pelzl et al., 2017), secretion and aggregation (Schmidt et al., 2013), endothelial cell stiffness (Alesutan et al., 2013), cytoskeletal organization (Honisch et al., 2015a; Shiokawa et al., 2013) and cell survival (Honisch et al., 2015b). From these studies, it became evident that chorein may be implicated in various additional pathophysiological disorders besides neurodegenerative diseases.

Recent studies partially elucidated the molecular footing of the cytoskeletal alterations governed by chorein. Reportedly, chorein binds to phosphatidylinositol lipids (Foller et al., 2012). This interaction activates a downstream cascade involving phosphoinositide-3-kinase (PI3K)-p85-subunit, Ras-related C3 botulinum toxin substrate 1 (RAC1) activation followed by phosphorylation of p21-activated kinase 1 (PAK1) (Foller et al., 2012). These findings documented that chorein may be critical in regulating cytoskeletal architecture.

In contrast, alterations of cytoskeleton organization and signaling, as depicted formerly in various tumor cells (Stournaras et al., 1996; Wang et al., 2022), appears to be a crucial dedifferentiation step (Araki et al., 2015; Stournaras et al., 2014; Kotula, 2012; Papakonstanti & Stournaras, 2008). This interface may in turn govern key cellular functions of tumor cells. These include cell growth and apoptosis (Grzanka, Grzanka & Orlikowska, 2003; Papadopoulou et al., 2009), motility and invasiveness (Nürnberg, Kollmannsperger & Grosse, 2014; Jiang, Enomoto & Takahashi, 2009; Yamazaki, Kurisu & Takenawa, 2005), Epithelial to Mesenchymal Transition (EMT) (Yilmaz & Christofori, 2009) and ion channel activity (Lang & Stournaras, 2014; Alevizopoulos et al., 2014).

We have previously studied in detail chorein-actin cytoskeleton interactions, both in malignant and non-malignant cells (Schmidt et al., 2013; Foller et al., 2012). In addition, the function of chorein protein in modulating organization of microtubular and intermediate filaments has been investigated in non-malignant cells (Honisch et al., 2015a). Functional analysis revealed that chorein participates in the regulation of diverse cellular functions (Saiki et al., 2007) including exocytosis (Pelzl et al., 2017), cytoskeletal organization (Honisch et al., 2015a). In this study, we reported disordered microtubular cytoskeleton, and cytokeratins and desmin in fibroblasts extracted from patients with chorea-acanthocytosis (ChAc), when evaluated against fibroblasts from that of non-diseased donors (Honisch et al., 2015a). These data further supported a meaningful function of chorein protein as cytoskeletal regulators. However, up to now it is still elusive whether chorein may also be involved in the modulation of the microtubular architecture in tumor cells. In this research study, we thus addressed a potential part that chorein may play in regulating tubulin architecture in tumor cells. We report here that chorein protein silencing induced large deregulation of microtubules organization in diverse human rhabdomyosarcoma cancer cells, establishing a substantial role of chorein in tumors.

Materials & Methods

Results

We have analyzed microtubules architecture in different rhabdomyosarcoma cell types. In ZF cells, analysis of morphological changes using confocal laser microscopy failed to establish a clear reorganization of microtubular structures (Fig. 1), although diminished intensities in fluorescence staining of microtubules were apparent in chorein-silenced cells. Similar observations became evident in RD cells (Fig. 2).

In contrast, confocal laser microscopy revealed a clear microtubules reorganization in chorein-silenced RH30 rhabdomyosarcoma cells in contrast to untreated RH30 cells (Fig. 3). In order to further analyze this interesting finding, we performed quantification analysis of engagement of microtubules by using FACS based tubulin-tracker fluorescence measurements. As depicted in Fig. 4, a sizable decrease of tubulin based microtubular fluorescence staining became evident upon chorein silencing (Fig. 4), fully supporting the morphological observations (Fig. 3). This finding implies clearly depolymerization of microtubules upon silencing of chorein in RH30 rhabdomyosarcoma cells.

Interestingly, the morphological alterations in microtubular structures in chorein-silenced RH30 rhabdomyosarcoma cells were not paralleled by transcriptional control of tubulin gene. Indeed, as displayed in (Fig. 5), both alpha- and beta- tubulin RNA transcripts were not statistically different in wild type and chorein-silenced RH30 cells, conforming with previously reported findings. In this report, tubulin gene transcription in chorein-deficient human fibroblasts retrieved from ChAc patients was similar to fibroblast from non-diseased donors (Honisch et al., 2015a).

Discussion

Genetic alterations like mutation in chorine gene mostly lead to a rare neurodegenerative disease (Segeletz et al., 2019). In the current research, we are the first one to report that decrease in chorein protein, by silencing, downregulates the microtubular organization in tumor cells. This became evident in human rhabdomyosarcoma tumor cells with poorly differentiation. Those cells have previously been shown to possess high chorein expression (Honisch et al., 2015b). The most abundant effect was witnessed in the RH30 rhabdomyosarcoma cell line. Here, confocal laser scanning microscopy documented clearly the breakdown of microtubule organization upon chorein silencing (Fig. 3). Quantitation analysis by specific FACS based tubulin-tracker fluorescence measurement (Fig. 4) fully supported this effect in RH30 cells. The observed depolymerization of microtubules was however, much less prominent in ZF- and RD- rhabdomyosarcoma cells, although moderate diminished fluorescence staining was still observed by confocal microscopic analysis in both cell lines (Figs. 1 and 2). However, these moderate effects could not be supported by FACS based tubulin-tracker fluorescence analysis (data not shown). Since previous studies established differential expression levels of chorein in the three rhabdomyosarcoma cell lines (Honisch et al., 2015b), our present observations may pointing out a possible correlation between chorein expression levels and interaction to the microtubular network. Conversely, previous studies established that reorganization of cytoskeletal structures including actin microfilaments and modulation of cytoskeletal gene signaling in tumor cells may regulate pivotal cellular functions including cell survival and motility (Yamazaki, Kurisu & Takenawa, 2005; Yilmaz & Christofori, 2009; Jaya, 2020; Ong et al., 2020; Li et al., 2022). Accordingly, our study indicates that the microtubule reorganization might contribute to the reported regulation of apoptosis in rhabdomyosarcoma cells upon chorein silencing, as described before (Honisch et al., 2015b). Further studies are now necessary to confirm this interesting finding in additional tumor cell lines, expressing high levels of chorein.

Another interesting finding of the present work was the lack of transcriptional regulation of the tubulin gene upon chorein silencing. This result indicates that chorein downregulation modulates the polymerization equilibrium of microtubules rather than the protein gene expression.

This observation agrees with earlier research reports from human fibroblasts. Indeed, in these experiments tubulin gene transcription was not altered in fibroblast extracted from chorein protein deficient ChAc patients in comparison with the tubulin gene expression patterns in fibroblasts from non-diseased controls (Honisch et al., 2015a).

Our present findings showing a powerful regulation of tubulin-based cytoskeleton in rhabdomyosarcoma cells are associated with the recently reported interaction of chorein with actin cytoskeleton dynamics in the same cells (Yu et al., 2016). These findings imply a critical function of chorein protein in governing actin- and tubulin-base cytoskeletal architecture in tumor cells.

Besides tubulin and actin, chorein interacts as well with additional cytoskeletal proteins in erythrocytes or human fibroblasts. Indeed, a recent study on ChAc patients reported intracellular localization of both β-adducin and β-actin with chorein in human embryonic kidney 293 (HEK293) cells and erythrocytes expressing high levels of chorein protein. Since adducin along with actin regulate the function of synapsis, it was concluded that this co-localization might contribute to ChAc pathophysiology (Shiokawa et al., 2013). Another study reported that chorein may also interact with the intermediate filament structures of desmin and cytokeratins (Honisch et al., 2015a). Indeed, it was previously reported that actin microfilaments are depolymerized in erythrocytes, fibroblasts and blood platelets isolated from chorea-acanthocytosis (ChAc) patients (Foller et al., 2012; Pelzl et al., 2017). Furthermore, experiments performed in preparations of human fibroblasts extracted from patients with ChAc showed weakened expression of cytokeratins and desmin gene, along with reorganization of staining of cytokeratin- and desmin-intermediate filaments (Honisch et al., 2015a). Taken together, these data strongly imply that chorein protein is a potent regulator of numerous cytoskeletal elements. However, it remains elusive whether chorein interactions with additional cytoskeletal elements are likewise relevant in rhabdomyosarcoma tumor cells.

Conclusions

This work elucidates for the first time the interaction between chorein and tubulin cytoskeleton in human RH30 rhabdomyosarcoma cell line. The effect was mainly manifested by the observed structural reorganization of microtubules following depolymerization. There was no experimental evidence directing the control of gene transcription of tubulin by chorein. Our findings establish an essential function of chorein protein in governing cytoskeletal structure in poorly differentiated tumor cells. Further studies are now needed to establish the clinical potential of those observations.

Supplemental Information