RAL GTPases are linchpin modulators of human tumour-cell proliferation and survival

Abstract

The monomeric RAL (RAS-like) GTPases have been indirectly implicated in mitogenic regulation and cell transformation. Here, we show that RALA and RALB collaborate to maintain tumorigenicity through regulation of both proliferation and survival. Remarkably, this task is divided between these highly homologous isoforms. RALB is specifically required for survival of tumour cells but not normal cells. RALA is dispensable for survival, but is required for anchorage-independent proliferation. Reducing the 'oncogenic burden' in human tumour cells relieves the sensitivity to loss of RALB. These observations establish RAL GTPases as crucial components of the cellular machinery that are exploited by factors that drive oncogenic transformation.

Figure 1

Small-interfering-RNA-mediated inhibition of RAL isoform expression. The indicated cell lines were transfected with small interfering RNAs (siRNAs) that were designed to selectively target RALA or RALB. Whole-cell lysates were prepared 72 h post-transfection and equivalent amounts of total protein were analysed by SDS–polyacrylamide gel electrophoresis for the indicated proteins. Extracellular-signal-regulated protein kinase 1/2 (ERK1/2) was used as a loading control. Similar results were obtained using two independent siRNA sequences for both RALA and RALB. HMECs, human-mammary epithelial cells; HMEC-hTERT, human diploid mammary epithelial cell line immortalized by hTERT expression; PrECs, primary human-prostate epithelial cells; RAL, RAS-like.

Figure 2

RALB is required for cell survival. (A) HeLa cells were transfected with the indicated small interfering RNAs (siRNAs) and incubated in the presence or absence of 50 μM zVAD-FMK. Ninety-six hours post-transfection, cells were fixed and stained with DAPI (4′,6-diamidino-2-phenylindole) to visualize nuclei. Representative fields of view are shown for each treatment. (B) HeLa and SW480 cells were transfected as described above. Seventy-two hours post-transfection, cells were labelled by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP–biotin nick-end labelling) to detect fragmented DNA, or with annexin V to detect surface phosphatidyl serine. The percentages of annexin-V-positive and TUNEL-positive cells were quantified by microscopic observation. Bars indicate s.e.m.s for three independent experiments. Panels showing TUNEL labelling from a representative experiment are shown below the graphs. RAL, RAS-like.

Figure 3

Tumour-derived cell lines are sensitized to RALB-dependent survival pathways. DNA content in propidium-iodide-treated cells was analysed by fluorescence-activated cell-sorting (FACS) 96 h after transfection with the indicated siRNAs. Asynchronous, proliferating, adherent cell cultures were used, except where indicated. MCF7 and SW480 cell lines are aneuploid and give multiple peaks. DNA fragmentation results in a shift of the population of events towards reduced signal intensities (hypodiploid). Inhibition of RALA and/or RALB expression was verified by western blotting. The data shown are representative of three independent experiments. HMECs, human-mammary epithelial cells; PrECs, primary human-prostate epithelial cells; RAL, RAS-like.

Figure 4

RALA is required for anchorage-independent proliferation of transformed cells. (A) The indicated cell lines were transiently transfected with MYC–RBD (a fusion of a MYC epitope to the RAL-binding domain (RBD) of RAL-binding protein 1) or empty vector. Twenty-four hours after transfection, cells were incubated with BrdU (5-bromo-2-deoxyuridine) for another 24 h in adherent (attached (Att.)) or suspension (Susp.) cultures. The percentages of RBD-expressing cells that incorporated BrdU are shown. More than 100 transfected cells were analysed for each experimental group. Transfection efficiencies ranged from 30% to 50%. Bars represent s.e.m.s for three independent experiments. An overlay image showing the detection of RBD expression and BrdU incorporation from a representative experiment is shown. (B) MCF7 and SW480 cells were transfected with control small interfering RNAs (siRNAs) that targeted mouse caveolin 1, or with RALA siRNAs. Seventy-two hours after transfection, cells were incubated with BrdU as described above. Quantitation of BrdU incorporation was carried out as described in (A). RAL, RAS-like.

Figure 5

Oncogenic RAS sensitizes mammary epithelial cells to loss of RALB. (A) HMEC–hTERT:H-RAS-G12V cells were transiently transfected with MYC–RBD (a fusion of a MYC epitope to the RAL-binding domain (RBD) of RAL-binding protein 1) or empty vector. Proliferation assays were carried out as described in Fig. 4B. HMEC-hTERT and HMEC-hTERT:H-RAS-G12V cells were transfected with the indiciated small interfering RNAs (siRNAs). Seventy-two hours post-transfection, cells were stained with DAPI (4′,6-diamidino-2-phenylindole) to visualize chromatin structure. Cells with condensed, picnotic nuclei were scored as apoptotic. HMEC–hTERT, human diploid mammary epithelial cell line immortalized by hTERT expression; RAL, RAS-like.