The tumor suppressor RhoBTB1 controls Golgi integrity and breast cancer cell invasion through METTL7B

Full details of antibodies, oligonucleotides and plasmids used in this study are given in Additional file 1.

HeLa, HEK293T, MDA-MB-231, MCF7 and T47D cells were cultured in DMEM containing 10% heat-inactivated fetal bovine serum. HMT-S1 and MCF10A cells were cultured as previously described [17, 18]. HeLa cells were transfected with plasmids and siRNA oligonucleotides using calcium phosphate [16].

RNA was isolated from cells using the TRIzol extraction method (Invitrogen) and 40 μg of purified RNA used for reverse transcription using Omniscript RTase (Qiagen) for 1 h at 37 °C. cDNAs were then subjected to real-time PCR using DyNAmo Flash SYBR Green (Finnzymes). Amplification was performed using an Opticon 2 thermocycler (MJ Research) and data was analyzed using the comparative Ct method.

Cells were fixed for 15 min in 4% paraformaldehyde in PBS and then permeabilized in 0.2% Triton X-100 in PBS for 5 min. Cells were then incubated with 0.1% sodium borohydride for 10 min. Primary antibodies were incubated with cells in 1% BSA for 1 h followed by secondary antibodies for 45 min. The cells were stained with 2 μg/ml DAPI for 10 min and mounted over MOWIOL 4–88 (Calbiochem) containing 0.6% 1,4-diazabicyclo-(2.2.2) octane as an anti-photobleaching agent. Confocal microscopy was performed using a Leica TCS-NT confocal laser-scanning microscope with an attached Leica DMRBE upright epifluorescence microscope under a PlanApo x63/1.32 oil-immersion objective. A series of images were taken at 0.5 μm intervals through the Z-plane of the cells and processed to form a projected image.

Fragmentation of the Golgi ribbon was scored blind in cells stained for the Golgi marker giantin. Fifty cells were scored from each condition to give the percentage of cells with a fragmented Golgi. Data from three independent experiments were processed to give the mean.

Expression of RhoBTB1 was restored in T47D cells by stable integration of a lentiviral RhoBTB1 construct. mCherry-tagged RhoBTB1 was subcloned into pHR’SIN-cPPT-SEW [19]. Virus was generated by transfection of HEK293T cells as described [19]. Briefly, 40 μg of RhoBTB1 vector was mixed with 10 μg of envelope plasmid pMDG, 30 μg of packaging plasmid psPAX2 and 1 μl of 1 mM polyethylenimine (Sigma) in OptiMEM (Invitrogen). This transfection mixture was replaced 4 h later with 15 ml of normal culture medium. The cells were then incubated for 48 h to allow virus production. After the incubation, the medium was removed and centrifuged for 10 min at 2,600 x g. The supernatant was then passed through a 0.45 μm filter and this filtrate was used as the virus stock. T47D cells were transduced with virus stock by overnight incubation.

T47D cells were grown to confluence in chamber slides (Ibidi). The confluent monolayer was scratched with a sterile pipette tip and migration was followed by brightfield imaging at 37 °C for 14 h using a Leica AF6000 live cell imaging workstation and x40 objective. Multipoint revisiting allowed for parallel imaging of samples. Average cell velocity was calculated using ImageJ (NIH). For quantification of Golgi polarization, cells were fixed 1 h after making the scratch and stained for giantin. Cell polarization was assessed by dividing cells into quadrants centered on the nucleus. Cells were judged to have polarized if the majority of their Golgi apparatus resided within the quadrant facing the scratch edge.

Invasion of T47D cells was quantified using BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer’s protocol. Briefly, T47D cells in serum-free medium were plated at 1 × 10⁵ cells per chamber. Medium containing 10% fetal bovine serum was used as the chemoattractant in the lower chamber. After 24 h, non-invaded cells were removed from the upper chamber using a cotton swab. Cells that invaded through the Matrigel to the bottom of the insert were fixed and stained by incubation with Diff-Quick (BD Biosciences) for 10 min. These stained cells were washed with PBS, air-dried and counted.

Our previous microarray screen identified METTL7A as a potential target of RhoBTB2-mediated transcriptional regulation [16]. To validate this, we used two independent siRNAs to silence RhoBTB2 in HeLa cells and measured METTL7A expression by RT-PCR. We also examined the expression of METTL7B, a closely-related protein that shares 59% sequence identity with METTL7A. Silencing of RhoBTB2 reduced the expression of METTL7A by approximately 30% (Fig. 1a). Previously, we showed that both RhoBTB1 and RhoBTB2 are required for expression of CXCL14 [16]; however, silencing of RhoBTB1 had no effect on METTLA expression (Fig. 1b). Interestingly, RhoBTB1 instead regulated the expression of METTL7B (Fig. 1b). We conclude that the two human METTL7 isoforms are differentially regulated by these two RhoBTBs.

METTL7B was originally identified as a Golgi-associated methyltransferase of unknown function [20]. Previous work has shown that inhibition of cellular methylation leads to fragmentation of Golgi, although the identity of the methyltransferase involved is unknown [21]. We hypothesized that the RhoBTBs might act through METTL7 proteins to control some aspect of Golgi function. To examine this, we silenced RhoBTB1 and RhoBTB2 expression and examined the basic morphology of the Golgi by staining cells for the well-characterized Golgi marker, giantin. In control cells, the Golgi was present as a characteristic ribbon situated next to the nucleus. Silencing of RhoBTB2 had a negligible effect on Golgi morphology; however, silencing of RhoBTB1 caused profound fragmentation of the Golgi (Fig. 2a, b).

We designed specific siRNAs to each METTL7 isoform to allow us to investigate their role in the regulation of Golgi integrity (Fig. 3a, b). Interestingly, silencing of either isoform caused significant fragmentation of the Golgi (Fig. 3c, d). This raised the possibility that downregulation of METTL7B might underlie the effects of RhoBTB1 depletion on Golgi integrity. To test this, we silenced RhoBTB1 and then restored METTL7B expression by transfection. This re-expression of METTL7B was able to significantly rescue Golgi integrity (Fig. 3e, f). Intriguingly, expression of METTL7A also promoted recue of normal Golgi morphology (Fig. 3e, f). We conclude that the effects of RhoBTB1 depletion on Golgi integrity are mediated through the consequent reduced expression of METTL7B, but that METTL7A overexpression can substitute for this activity.

METTL7B has been reported as being localized to the Golgi [20], whereas METTL7A (a.k.a. AAM-B) has previously been shown to localize to the endoplasmic reticulum (ER) and to lipid droplets [22]. It was unclear why both isoforms should affect Golgi morphology, given their different locations. We examined the cellular localization of both proteins. In contrast to the previous report [20], we found that both METTL7s localized to the ER in HeLa cells, with no apparent Golgi localization (Fig. 4a, b). We conclude that the relevant target(s) for METTL7 action is likely to be an ER resident protein, or a protein that actively shuttles between the two compartments.

Many studies have reported downregulation of RhoBTB2 expression in breast cancer, most commonly due to methylation of the RhoBTB2 promoter [7, 10, 11, 23, 24]. We examined the relative expression of RhoBTB1 and RhoBTB2 in a selection of human breast cancer cell lines (MDA-MB-231, T47D, MCF7) in comparison to two well-characterized normal human mammary epithelial cell lines – MCF10A [25] and HMT-3522 S1 [26]. All three breast cancer cell lines had reduced RhoBTB1 expression (Fig. 5a). T47D and MDA-MB-231 cells also showed reduced expression of RhoBTB2, whereas expression was normal in MCF7 cells (Fig. 5b). We examined the expression of METTL7A and METTL7B in the same cell lines. Interestingly, both isoforms showed reduced expression in the breast cancer cell lines, with T47D cells having the lowest overall expression of METTL7 isoforms (Fig. 5c, d). T47D cells have previously been shown to undergone loss of RhoBTB2 expression [7]. They also had the lowest expression of RhoBTB1 (Fig. 5a). Intriguingly, whereas the normal mammary cell lines had a compact Golgi ribbon, T47D cells showed extreme fragmentation of the Golgi (Figs. 5e, 6b), raising the possibility of a functional link between loss of RhoBTB1 expression and Golgi morphology in this breast cancer line.

To explore the effect of loss of RhoBTB1 expression on Golgi integrity in T47D cells, we restored RhoBTB1 expression using lentiviral transduction. The resultant stable cell line had near normal levels of RhoBTB1, but still had dramatically lower levels of RhoBTB2 (Fig. 6a). Restoration of RhoBTB1 expression led to a significant rescue of Golgi integrity (Fig. 6b).

Fragmentation of the Golgi has little effect on the rate of protein secretion in cells [27]. In keeping with this, we saw no apparent change in the rate of trafficking of VSV-G through the secretory pathway in cells depleted of RhoBTB1 (data not shown). One proposed function of the Golgi ribbon structure is to facilitate the cellular localization of this organelle. Polarized epithelial cells orientate their Golgi towards the apical surface of the cell, and this is important for polarized secretion and for maintenance of the apical-basolateral axis [28]. In migrating cells, the Golgi is polarized towards the leading edge of the cell in the majority of cell types and this reinforces directional migration [29]. Dysregulation of cell polarity pathways is an important component in the conversion of cancer cells into a migratory, invasive phenotype. We examined whether restoration of RhoBTB1 expression and normalization of Golgi morphology would affect these processes in T47D breast cancer cells. We used a scratch migration assay to measure Golgi polarization and the directional migration of cells. T47D cells failed to polarize their Golgi effectively in the scratch assay; whereas restoration of RhoBTB1 expression resulted in strong polarization of the Golgi towards the migration front (Fig. 6d). Restoration of RhoBTB1 expression had no effect on T47D cell migration, however, suggesting that the Golgi fragmentation observed in T47D cells does not affect their migration in 2D (Fig. 6e). We next examined the invasive capacity of these cells. A critical early stage in breast cancer progression is breakdown of the highly polarized organization of cells in a mammary duct and their invasion through the basement lamina and into the surrounding stroma. We measured the ability of T47D breast cancer cells to invade through a 3D layer of extracellular matrix that mimics the composition of the basement lamina [30]. Restoration of RhoBTB1 expression dramatically inhibited cell invasion (Fig. 6f). We conclude that loss of RhoBTB1 expression in T47D breast cancer cells contributes to a pro-invasion phenotype. Our hypothesis is that this is a result of reduced METTL7B expression, leading to Golgi fragmentation and a breakdown of normal cell polarity.