Chorein addiction in VPS13A overexpressing rhabdomyosarcoma cells.

Chorein encoded by VPS13A (vacuolar protein sorting-associated protein 13A) is defective in chorea-acanthocytosis. Chorein fosters neuronal cell survival, cortical actin polymerization and cell stiffness. In view of its anti-apoptotic effect in neurons, we explored whether chorein is expressed in cancer cells and influences cancer cell survival. RT-PCR was employed to determine transcript levels, specific siRNA to silence chorein, FACS analysis to follow apoptosis and Western blotting to quantify protein abundance. Chorein transcripts were detected in various cancer cell types. The mRNA coding for chorein and chorein protein were most abundant in drug resistant, poorly differentiated human rhabdomyosarcoma cells. Chorein silencing significantly reduced the ratio of phosphorylated (and thus activated) to total phosphoinositide 3 kinase (PI-3K), pointing to inactivation of this crucial pro-survival signaling molecule. Moreover, chorein silencing diminished transcript levels and protein expression of anti-apoptotic BCL-2 and enhanced transcript levels of pro-apoptotic Bax. Silencing of chorein in rhabdomyosarcoma cells was followed by mitochondrial depolarization, caspase 3 activation and stimulation of early and late apoptosis. In conclusion, chorein is expressed in various cancer cells. In cells with high chorein expression levels chorein silencing promotes apoptotic cell death, an effect paralleled by down-regulation of PI-3K activity and BCL-2/Bax expression ratio.

Chorein expression is not restricted to brain and erythrocytes, but is observed in diverse further tissues [13][14][15]. Chorein in blood platelets participates in the regulation of secretion and aggregation [15]. Moreover, chorein expressed in endothelial cells contributes to the regulation of endothelial cell stiffness [14]. Clearly, much www.impactjournals.com/oncotarget is to be learned on the functional significance of this protein.
In view of the anti-apoptotic effect of chorein, we explored whether chorein is expressed in tumor cells and, if so, whether it modifies the survival of those cells. To this end, the chorein transcript and protein levels were determined in several tumor cells. As a result, chorein is expressed in some tumor cells with highest transcript levels found in ZF rhabdomyosarcoma cells. The impact of chorein on the survival of those cells was elucidated by chorein silencing and subsequent analysis of apoptosis and apoptotic signaling.

results
The present study explored whether chorein is expressed in tumor cells and impacts on the survival of those cells. As illustrated in Fig.1A, chorein is expressed in several tumor cell lines. mRNA coding for chorein was predominantly transcribed in drug resistant, poorly differentiated ZF rhabdomyosarcoma cells (Fig.  1A). Other cancer cell lines such as the human colon carcinoma CaCo2 cells expressed significantly lower levels of chorein (Fig. 1A). The differences in chorein transcript levels between ZF rhabdomyosarcoma cells and CaCo2 cells were paralleled by similar differences in protein expression. Accordimg to both, Western-blot and confocal scanning analysis chorein protein expression was markedly higher in ZF rhabdomyosarcoma cells than in CaCo2 cells (Fig, 2). According to confocal microscopy chorein protein is mainly localized in the cytoplasm (Fig.  2B).
Additional experiments addressed the impact of chorein on the expression of anti-apoptotic protein B-cell lymphoma 2 (BCL-2) and of pro-apoptotic protein Bax. As illustrated in Fig. 3A, chorein silencing significantly decreased the BCL-2 (Fig. 3A) and increased the Bax (Fig. 3B) transcript levels in ZF rhabdomyosarcoma cells. Furthermore, chorein silencing decreased the BCL-2 protein abundance in ZF rhabdomyosarcoma cells (Fig.  3C).
Caspase 3 activity was determined to elucidate whether chorein influenced apoptotic signaling. As illustrated in Fig. 4A, chorein silencing significantly enhanced caspase-3 activity in ZF rhabdomyosarcoma cells, an observation pointing to triggering of apoptosis. In line with this observation, chorein silencing of ZF rhabdomyosarcoma cells was followed by significant mitochondrial depolarization (Fig. 4B). Mitochondrial depolarization reached statistical significance already 24 h after chorein silencing and persisted throughout 72 h (Suppl. Fig. 2C).
Apoptosis was evidenced from Annexin V and propidium iodide binding. As illustrated in Fig. 5A, B, chorein significantly enhanced the percentage of ZF rhabdomyosarcoma cells in early or late apoptosis. In contrast, chorein silencing of Caco2 cells (Fig. Suppl.  Fig. 1A), which express only low levels of chorein (demonstrated in Fig. 1A) had almost no effect on apoptosis of these cells (Fig. 6A,B).
Additional experiments addressed the effect of separate and combined chorein silencing and treatment with the cytotoxic drug Doxorubicin (500 nM). As shown in Suppl. Fig. 3, both, chorein silencing and doxorubicin Cells were stained with conjugated inhibitor of active Caspase-3 (FITC-DEVD-FMK) and measured by FACS. Shown are arithmetic means (left) ± SE (n=4) and representative original histograms (right) demonstrating caspase activity in ZF cells transfected with control siRNA (siNeg) or siRNA for chorein (siVPS13A). * significant difference (p<0.05; unpaired t-test). B. Arithmetic means (left) ± SEM (n=4) and representative original histograms (right) of mitochondrial depolarization measured by FACS in ZF cells transfected with negative control siRNA (siNeg) or siRNA for chorein (siVPS13A).*** indicates significant difference (p<0.001, unpaired t-test).

Figure 3: chorein sensitive bcl-2 and bax gene transcription and protein expression in ZF rhabdomyosarcoma cells.
Arithmetic means ± SEM (n=6) of the BCL-2 (A) and Bax (B) mRNA levels relative to GAPDH mRNA levels in ZF cells transfected with control siRNA (siNeg) or siRNA for chorein (siVPS13A). ** significant difference (p<0.01; unpaired t-test) C. Left: Arithmetic means ± SEM (n=6) of the BCL-2 protein levels compared to GAPDH levels in ZF cells transfected with control siRNA (siNeg) and siRNA for chorein (siVPS13A). Right: original Western blots of BCL-2 and GAPDH as loading control in ZF cells transfected with control siRNA (siNeg) or siRNA for chorein (siVPS13A). * significant difference (p<0.05; unpaired t-test) www.impactjournals.com/oncotarget  treatment are followed by triggering of apoptosis. However, the combined treatment with chorein silencing and doxorubicin did not lead to further significant increase of apoptosis (Suppl. Fig. 3A middle). Chorein silencing did not significantly modify FITC dextran uptake (Suppl. Fig. 4).

dIscussIon
The present study discloses a completely novel regulator of tumor cell survival, i.e. chorein, the protein which is defective in patients with chorea-acanthocytosis. We show that chorein is expressed in several tumor cell lines with particularly high expression in ZF rhabdomyosarcoma cells. We further show that survival of those cells, but not of colon carcinoma cells with low levels of chorein expression is highly sensitive to the presence of chorein.
In view of the extra-cerebral expression and functions of chorein, chorea-acanthocytosis is a systemic disease presumably affecting a wide variety of functions. Chorein expression is particularly high in testis, kidney, spleen and brain [13]. Apparently, function and survival of neurons and skeletal muscle cells are particularly dependent on chorein [5, 10]. According to the present observations, some tumor cells similarly depend on chorein expression for survival. A more detailed analysis is expected to disclose chorein sensitive functions and survival of further cell types.
In view of the present observations, the lack of chorein in chorea-acanthocytosis may confer some protection against the development of specific malignancies. It is tempting to speculate that pharmacological interference with the interaction between chorein and PI3K may enhance the susceptibility of chorein rich tumor cells to treatment. PI3K inhibitors are effective in the treatment of malignancy but create considerable side effects due to disruption of the multiple PI3K dependent cellular functions [27,64]. Chorein inhibition would not be expected to completely disrupt PIK3 signaling and compromise function and survival only of those cells expressing high levels of chorein and depending on chorein sensitive PI3K signaling. Along those lines complete lack of chorein in patients suffering from chorea-acanthocytosis requires decades to generate clinically relevant disorders [4][5][6][7][8][9].
In conclusion, the present observations reveal a novel function of chorein, i.e. the stimulation of tumor cell survival. Future studies will be required to define the impact of chorein expression on malignancy and therapy resistance of defined malignancies.

MAterIAls And Methods cells
Chorein transcript levels were determined in ZF rhabdomyosarcoma cells (established at the Children's Hospital Tuebingen from a multifocal, alveolar rhabdomyosarcoma of an eight year old girl) UW228-3 medulloblastoma cells (kindly provided by S Pfister, DKFZ Heidelberg) CaCo2 (ATCC, USA.) colon carcinoma cells, 451Lu melanoma (kindly provided by T. Sinnberg, Dermatology Tübingen), HF normal dermal skin fibroblasts (Promocell) and MCF-7 breast carcinoma cells (from ATCC, USA). All cells were seeded at 3x10 5 cells/ ml and grown for 48 h before RNA isolation in DMEM high glucose medium (Gibco) containing 10% FBS and 1% penicillin/streptomycin.

rt-Pcr
To determine transcript levels, total RNA was isolated 24 h, 48 h and 72 h after transfection using the Trifast Reagent (Peqlab, Erlangen, Germany). Two µg RNA was reverse-transcribed using oligo(dT) 12-18 primers and GoScript Reverse Transcriptase Kit (Promega) according to the manufacturer's protocol. Quantitative real-time PCR was performed with the BioRad iCycler iQ TM Real-Time PCR Detection System (Bio-Rad Laboratories) using GoTaq Sybr Green Master Mix (Promega). The reaction was applied in a final volume of 20 µl containing 2 µl of cDNA under following conditions: an initial incubation at 95°C for 5 min, 40 cycles at 95°C for 15 s, 59°C for 20 s and 72°C for 30 s. Specificity of the PCR products was verified by melting curve analysis. The subsequent primers were used (5'→3' orientation): VPS13A fw: AGTGGGACGACGTCTGTACAC VPS13A rev:AGTTCTCATCTTCTGGCTTCAG BCL-2 fw: TGGATGACTGAGTACCTGAACCG BCL-2 rev: TGAGCAGAGTCTTCAGAGACAGC BAX fw: ACTGGACAGTAACATGGAGCTG BAX rev: AGCCCATGATGGTTCTGATCAG GAPDH fw: TGAGTACGTCGTGGAGTCCACTG GAPDH rev: GGTGCTAAGCAGTTGGTGGTG The mRNA expression levels of the respective genes were normalized to the expression levels of GAPDH in the same cDNA sample. Relative quantification of gene expression was calculated according to the ΔΔCt method.

FAcs analysis
To determine the apoptotic response we used the Annexin V Apoptosis Detection Kit (MabTag, Germany). After silencing cells were harvested from the 6 well plates by treatment with trypsin-EDTA (Sigma-Aldrich, Germany) for 10 min and washed once with cell culture medium. After a further wash with PBS and centrifugation at 1600 RPM for 3 min at RT cells were suspended in 100 µl binding buffer containing Annexin V -FITC and propidium iodide and incubated for 20 min in the dark at RT. In the following the cells were washed once, resuspended in 200 µl binding buffer and measured immediately using the BD FACS Calibur (BD Biosciences, USA). For Doxorubicin treated cells TO-PRO®-3 Iodide (1:1000, Life Technologies, USA) was used instead of propidium iodide. Active caspase-3 was estimated utilizing CaspGlow Fluorescein Active Caspase-3 Staining Kit (BioVision, USA) according to the manufacturer's instructions. After detaching and one wash with cell culture medium cells were suspended in 300 µl of complete DMEM (Gibco, USA) including 1 µl of FITC conjugated inhibitor of active Caspase-3 (FITC-DEVD-FMK). After 1h incubation and two washes with supplied wash buffer, cells were resuspended in 300 µl of wash buffer and analyzed by flow cytometry (BD FACS Calibur, BD Biosciences, USA).
Depolarization of the outer mitochondrial membrane was measured by incubating 10 5 cells in 10 ng/ml JC9 (Life Technologies, USA) in the dark for 10 min at 37 o C. The cells were washed once in PBS at 1600 g for 3 min. A potential-dependent shift of fluorescence emission from 525 nm (FL1) to 590 nm (FL2) in the mitochondria was measured immediately by flow cytometry (BD FACS Calibur, BD Biosciences, USA).
FITC-dextran uptake was measured after 2 hours incubation with FITC-conjugated dextran (500 µg/ml, Sigma-Aldrich, Germany) in serum free DMEM medium at 37°C and on ice (background). The intake was stopped by adding ice-cold PBS to the cells. After three washes with ice-cold PBS, cells were re-suspended in 200 µl of PBS and analyzed by flow cytometry (BD FACS Calibur, BD Biosciences, USA). For the calculation, mean fluorescence values of the samples incubated on ice were subtracted from corresponding values incubated at 37°C.

confocal laser scanning microscopy
For chorein staining, ZF and CaCo2 cells were cultured on glass cover slips for 24 h. After washing twice with PBS, cells were fixed with 4% PFA for 15 min on RT and then permeabilized with 0,03% Triton-X100 for 10 min. After blocking with 3% BSA in PBST cells were incubated at 4°C overnight with anti-VPS13Aantibody (1:300, Sigma-Aldrich, Germany). The cells were rinsed three times with PBST and incubated with secondary goat anti-rabbit CF TM 488 antibody (1:300, Sigma-Aldrich, Germany) for 2 h at room temperature. Additional cells were incubated 30 min in the dark with rhodamine-phalloidin (1:200, Life Technologies, USA) for F-actin staining and with DRAQ-5 dye (1:3000, Biostatus, Leicestershire, UK) for nuclei staining. After three washing steps all slides and coverslips were mounted with ProLong Gold antifade reagent (Life Technologies, USA). Images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser scanning microscope (Carl Zeiss, Germany) with a water immersion Plan-Neofluar 63/1.3 NA DIC.

statistics
Data are expressed as arithmetic means ± SEM. Statistical analysis was made by unpaired t-test. A p<0.05 value was considered statistically significant.