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

Abstract

Background: RhoBTB1 and 2 are atypical members of the Rho GTPase family of signaling proteins. Unlike other Rho GTPases, RhoBTB1 and 2 undergo silencing or mutation in a wide range of epithelial cancers; however, little is known about the consequences of this loss of function.

Methods: We analyzed transcriptome data to identify transcriptional targets of RhoBTB2. We verified these using Q-PCR and then used gene silencing and cell imaging to determine the cellular function of these targets downstream of RhoBTB signaling.

Results: RhoBTB1 and 2 regulate the expression of the methyltransferases METTL7B and METTL7A, respectively. RhoBTB1 regulates the integrity of the Golgi complex through METTL7B. RhoBTB1 is required for expression of METTL7B and silencing of either protein leads to fragmentation of the Golgi. Loss of RhoBTB1 expression is linked to Golgi fragmentation in breast cancer cells. Restoration of normal RhoBTB1 expression rescues Golgi morphology and dramatically inhibits breast cancer cell invasion.

Conclusion: Loss of RhoBTB1 expression in breast cancer cells leads to Golgi fragmentation and hence loss of normal polarity.

Keywords: BTB domain; Cell invasion; Cell migration; Golgi fragmentation; Methyltransferase; Rho GTPases; RhoBTB1.

Fig. 1

RhoBTBs differentially control METTL7 expression. a HeLa cells were transfected with siRNAs targeting RhoBTB1 or RhoBTB2, or the lamin siRNA control. Two independent siRNAs were used for each target. The efficiency of these siRNAs in HeLa cells was quantified in our previous study [16]. After 48 h, RNA was prepared from the cells and the expression of METTL7A was quantified by RT-PCR. Silencing of RhoBTB2 significantly reduced the expression of METTL7A. b Expression of METTL7B was quantified in the same samples. Silencing of RhoBTB1 significantly reduced the expression of METTL7B. Data are means ± SEM (n = 3). Data were normalized to mock-transfected HeLa cells. Comparisons are to the lamin siRNA control; *P <0.05, **P <0.01, ****P <0.0001

Fig. 2

Silencing of RhoBTB1 causes Golgi fragmentation. HeLa cells were transfected with siRNAs targeting RhoBTB1 or RhoBTB2. After 48 h, the cells were fixed and stained for the Golgi marker giantin (green). a Shows representative images of normal and RhoBTB1 depleted cells. Bar = 10 μm. b Silencing of RhoBTB1 caused a marked and significant fragmentation of the Golgi. Data are means ± SEM (n = 3). Data were normalized to mock-transfected HeLa cells. Comparisons are to the lamin siRNA control; *P <0.05, **P <0.01

Fig. 3

RhoBTB1 regulates Golgi integrity through METTL7 proteins. a-b HeLa cells were transfected with siRNAs targeting METTL7A, METTLB or both. After 48 h, RNA was prepared from the cells and the expression of each METTL7 was quantified by RT-PCR. Data are means ± SEM (n = 3). c HeLa cells were transfected with the same siRNAs and fixed and stained for the Golgi marker giantin. Silencing of either methyltransferase caused a marked and significant fragmentation of the Golgi. Data are means ± SEM (n = 3). Comparisons are to the control lamin siRNA. d Shows representative images of the cells. Golgi are stained in green; nuclei in blue. e HeLa cells were transfected with siRNAs targeting RhoBTB1 and with plasmids expressing myc-tagged METTL7A or METTL7B. After 48 h, the cells were fixed and stained for the Golgi marker giantin (green) and for the transfected methyltransferases (red). Bars = 10 μm. f Quantification of transfected cells showed that expression of either METTL7 rescued Golgi morphology in cells depleted of RhoBTB1. Data are means ± SEM (n = 3); comparisons are as indicated. *P <0.05; **P <0.01, ***P <0.001, ****P <0.0001

Fig. 4

METTL7 proteins are localized to the endoplasmic reticulum. a HeLa cells were transfected with myc-tagged METTL7A (green) and fixed and stained for the ER marker calnexin (red) or the Golgi marker giantin (red). METTL7A showed no significant localization to the Golgi, but instead was present in the ER and also to small punctate structures. b The experiment was repeated with myc-tagged METTL7B. METTL7B also localized to the ER and small puncta. Bar = 10 μm

Fig. 5

RhoBTB1 is expression is downregulated in breast cancer cell lines. a-d The expression of RhoBTB1, RhoBTB2, METTL7A and METTL7B was measured by RT-PCR in three well-characterized breast cancer cell lines: MDA-MB-231, T47D and MCF7. Expression was compared with two well-characterized normal mammary epithelial cell lines: MCF-10A and HMT-S1. Data are means ± SEM (n = 3). Data were normalized to HeLa cell samples. Comparisons are to the MCF10A control; *P <0.05; **P <0.01, ***P <0.001, ****P <0.0001. All three breast cancer lines showed a markedly reduced RhoBTB1 expression, as well as reduction in expression of METTL7A and METTL7B. T47D cells had the lowest levels of both RhoBTB1 and RhoBTB2. e the breast cancer cell lines were fixed and stained for giantin (green) and Golgi morphology compared to the normal mammary epithelial HMT-S1 line. T47D cells showed marked fragmentation of the Golgi. Bar = 10 μm

Fig. 6

Restoration of RhoBTB1 expression restores normal Golgi morphology to T47D cells and inhibits invasion. a T47D cells were transduced with a lentiviral RhoBTB1 construct to restore RhoBTB1 expression. Expression of RhoBTB1 and RhoBTB2 was measured by RT-PCR. Transduction with RhoBTB1 restored expression to near normal levels, but did not affect expression of RhoBTB2. Data are means ± SEM (n = 2). b Cells were fixed and stained for the Golgi marker giantin. Restoration of RhoBTB1 expression caused a significant reduction in Golgi fragmentation. Data are means ± SEM (n = 3). c shows representative images of control T47D cells, and the T47D line expressing RhoBTB1. Golgi are stained in green; nuclei in blue. Bar = 10 μm. d T47D cells were grown to confluence and cell migration was initiated by scratching the coverslip to remove a strip of cells. Cells were fixed at 1 h and stained for Golgi (giantin, green). The dashed line indicates the cell front. Quantification of Golgi polarization showed that T47D cells had little ability to polarize (25% polarization corresponds to random orientation in this assay); whereas restoration of RhoBTB1 supported robust polarization. Data are means ± SEM (n = 3). e Migration was measured in the same assay over 14 h. Data are means ± SEM (n = 3). Restoration of RhoBTB1 expression had no effect on T47D cell migration in 2D. f T47D invasion through 3D extracellular matrix was measured in Matrigel invasion chambers over 24 h. Restoration of RhoBTB1 expression markedly reduced invasive capacity. Data are means ± SEM (n = 3). *P <0.05; **P <0.01, ***P <0.001, ****P <0.0001