The growth and tumor suppressors NORE1A and RASSF1A are targets for calpain-mediated proteolysis

Abstract

Background: NORE1A and RASSF1A are growth and tumour suppressors inactivated in a variety of cancers. Methylation of NORE1A and RASSF1A promoters is the predominant mechanism for downregulation of these proteins; however, other mechanisms are likely to exist.

Methodology/principal findings: Here we describe a proteolysis of NORE1A and RASSF1A by calpains as alternative mechanism of their downregulation. Extracts of H358 cell line, a human bronchoalveolar carcinoma, and H460, a large cell carcinoma, were capable of proteolysis of NORE1A protein in the calpain-dependent manner. Likewise, RASSF1A tumor suppressor was proteolyzed by the H358 cell extract. Addition of calpain inhibitor to H358 and H460 cells growing in tissue culture resulted in re-expression of endogenous NORE1A. A survey of 10 human lung tumours revealed that three of them contain an activity capable of inducing NORE1A degradation.

Conclusions/significance: Thus, degradation by calpains is a novel mechanism for downregulation of NORE1A and RASSF1A proteins and might be the mechanism allowing cancer cells to escape growth suppression.

Figure 1. Cleavage of NORE1A and RASSF1A by an activity expressed in some tumor cell lines.

Full-length NORE1A and RASSF1A, tagged at the N-terminus with the FLAG tag, were expressed in HEK293 cells, immunopurified on FLAG beads and beads were eluted with FLAG-peptide. NORE1A (lanes 1–4) or RASSF1A (lanes 5–8) were incubated with the Buffer A (lanes 1, 5) or H358 cell lysate (lanes 2, 6), or H460 cell lysates (lanes 3, 7), or A549 cell lysates (lanes 4, 8) for 30 minutes at 37°C. After incubation, samples were subjected to gel electrophoresis followed by Western blotting with anti-FLAG antibodies.

Figure 2. NORE1A cleavage occurs at the N-terminus of the protein.

A: Full-length NORE1A (lanes 1, 2) or its fragments aa 1–364 (lanes 3, 4) or aa 1–190 (lanes 5, 6), tagged N-terminally with the FLAG tag, immunopurified as described in Figure 1, were incubated with the lysis buffer (lanes 1, 3, 5) or with H358 cell lysate (lanes 2, 4, 6) for 30 minutes at 37°C. After incubation, samples were subjected to gel electrophoresis followed by Western blotting with anti-FLAG antibodies. Asterisks denote non-specific bands. B: NORE1A fragments, amino acids 18–190, 43–190 and 78–190 were synthesized in vitro using Promega TnT T7 Quick for PCR DNA kit in the presence of ³⁵S-Methionine. Each fragment contained an additional methionine as start codon and two extra methionines at the C-terminus to facilitate detection. An aliquot of translation mixture was incubated with 4× excess (v/v) of H358 cell extract or with Buffer A for 1 hour at 37°C, resolved on SDS gel and transferred to Immobilon. Shown is the autoradiogram of the membrane obtained by Phosphoimager.

Figure 3

A: Cleavage of NORE1A and RASSF1A requires calcium and is sensitive to calpain inhibitors. NORE1A, tagged N-terminally with the FLAG tag, was expressed in HEK293 cells and adsorbed on FLAG beads (lanes 1–4). RASSF1A, tagged N-terminally with the FLAG tag, was expressed as described in Figure 1 (lanes 5–8). Equal amount of beads containing NORE1A, or RASSF1A were incubated for 30 min at 37°C with H358 cells extract with no additions (lanes 2, 6) or with 1 µM of calpain inhibitor ALLN (lanes 3, 7) or with 50 mM EDTA (lanes 4, 8). NORE1A and RASSF1A, eluted from beads with the FLAG peptide without cleavage, was used as controls (lanes 1 and 5, respectively). After incubation, NORE1A-containing beads were extensively washed. Samples were subjected to gel electrophoresis followed by Western blotting with anti-FLAG antibodies. Asterisk denoted IgG heavy chain eluted from FLAG beads. B, Cathepsin B and Cathepsin K are not responsible for NORE1A cleavage by H358 and H460 cell lysates: NORE1A, tagged N-terminally with the FLAG tag, was expressed as described in Methods and adsorbed on FLAG beads. Equal amounts of NORE1A were incubated for 30 min at 37°C with H358 cells extract (lanes 2–5) or H460 cell extract (lanes 6–9) with no additions (lanes 2, 6) or with ALLN (1 µM, lanes 3, 7) or with inhibitor of Cathepsin B (10 µM, lanes 4, 8) or with inhibitor of Cathepsin K (2 µM, lanes 5, 9). NORE1A eluted from beads with the FLAG peptide was used as control (lane 1). After incubation, beads were extensively washed and proteins retained on them were subjected to gel electrophoresis followed by Western blotting with anti-FLAG antibodies.

Figure 4

A, The addition of a calpain inhibitor ALLN to cultured cells prevents NORE1A degradation: NORE1A was re-expressed in H157 cells (lanes 1, 6), H358 cells (lanes 3, 8) and H460 cells (lanes 5, 10) using a vector with a drug-resistance marker with subsequent selection for the drug. Lanes 2, 7 represent H358 parental cells and lanes 4, 9 - H460 parental cells. Cells were cultured for 3 days without inhibitors (top panel) or with 1 µM ALLN (bottom panel). Cell extracts, equalized by α-tubulin, were probed with 10F10 antibody by Western Blotting. At 1 µM concentration ALLN did not induce apoptotic response in H358 and H460 cells (data not shown). B, NORE1A expression at the mRNA level in H358 and H460 cells: NORE1A cDNA was re-expressed in H358 and H460 cells using a vector with a drug-resistance marker. Total mRNA was extracted from pools of drug-resistant cells emerged after selection as indicated and analyzed by RT-PCR for NORE1A expression. Note that these cells did not express NORE1A protein (Fig 4A, lanes 3 and 5). NORE1A, cDNA of vector used for transduction was included as a positive control. M, DNA molecular weight markers. C: Extracts of human tumors are capable of degrading NORE1A. Human lung tumors from patients 3, 9, 10 and 8 and matching normal lung tissues were extracted into buffer for cell extraction and normalized by total amount of cellular protein. Equal amount of extracts from tumors (T) and normal tissues (N) were mixed with FLAG-tagged NORE1A, immunopurified as described in Methods. After thirty minute incubation at 37°C, electrophoretic sample buffer was added to 1× concentration, samples were boiled and subjected to Western blotting with anti-FLAG antibodies. As positive control for NORE1A degradation, NORE1A incubated with H460 cell extract (lane 2) was used.