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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

BMC Cancer 1/2018

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter

Zeitschrift:
BMC Cancer > Ausgabe 1/2018
Autoren:
Walter Blum, László Pecze, Janine Wörthmüller Rodriguez, Martine Steinauer, Beat Schwaller
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12885-018-4385-7) contains supplementary material, which is available to authorized users.

Abstract

Background

The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood.

Methods

We searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments.

Results

Septin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7.

Conclusions

Our results demonstrate septin 7 not only serving as a “cytoskeletal” protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.
Zusatzmaterial
Additional file 1: Figure S1. MTT assay of different MM cell lines exposed to butyrate (Bt). Relative MTT signals for A) MSTO-211H, B) ZL55 and C) ZL5 MM cells exposed to various Bt concentrations ranging from 0.33 to 5 mM. Results are from 3 independent experiments (each sample in triplicate). The value of untreated cells in each experiment was defined as 100%. Results represent mean±SEM. Figure S2. Point mutations in the PubMed database sequence (CALB2; gene ID: 794) of the CALB2 promoter region containing BRE7-13 in comparison to human Met-5A and ZL55 cells are boxed in green (Met-5A) or yellow (ZL55). Insertions or deletions found in the sequence of all analyzed cell lines are boxed in cyan. None of the mutations concern the 7 BRE listed in Fig. 2. Figure S3. Top panel: Ponceau-Red stained membrane used for the Western blot shown in Fig. 4a. Sizes of marker proteins range from 17 kDa (faintly stained lowest band marking gel front) to 100 kDa (most upper band). Middle panel: Ponceau-Red stained membrane used for the Western blot shown in Fig. 4d. The size of marker proteins ranges from 20 kDa to 135 kDa. Lower panel: Ponceau-Red stained membrane used for the Western blot shown in Fig. 5c. The size of marker proteins ranges from 20 kDa to 135 kDa. (DOCX 1083 kb)
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