BackgroundThe genetic program, as manifested as the cellular phenotype, is in large part dictated by the cell’s protein composition. Since characterisation of the proteome remains technically laborious it is attractive to define the genetic expression profile using the transcriptome. However, the transcriptional landscape is complex and it is unclear as to what extent it reflects the ribosome associated mRNA population (the translatome). This is particularly pertinent for genes using multiple transcriptional start sites (TSS) generating mRNAs with heterogeneous 5′ transcript leaders (5′TL). Furthermore, the relative abundance of the TSS gene variants is frequently cell-type specific. Indeed, promoter switches have been reported in pathologies such as cancer. The consequences of this 5′TL heterogeneity within the transcriptome for the translatome remain unresolved. This is not a moot point because the 5′TL plays a key role in regulating mRNA recruitment onto polysomes.ResultsIn this article, we have characterised both the transcriptome and translatome of the MCF7 (tumoural) and MCF10A (non-tumoural) cell lines. We identified ~550 genes exhibiting differential translation efficiency (TE). In itself, this is maybe not surprising. However, by focusing on genes exhibiting TSS heterogeneity we observed distinct differential promoter usage patterns in both the transcriptome and translatome. Only a minor fraction of these genes belonged to those exhibiting differential TE. Nonetheless, reporter assays demonstrated that the TSS variants impacted on the translational readout both quantitatively (the overall amount of protein expressed) and qualitatively (the nature of the proteins expressed).ConclusionsThe results point to considerable and distinct cell-specific 5′TL heterogeneity within both the transcriptome and translatome of the two cell lines analysed. This observation is in-line with the ribosome filter hypothesis which posits that the ribosomal machine can selectively filter information from within the transcriptome. As such it cautions against the simple extrapolation transcriptome → proteome. Furthermore, polysomal occupancy of specific gene 5′TL variants may also serve as novel disease biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2179-8) contains supplementary material, which is available to authorized users.

Osteosarcoma is characterized by frequent relapse and metastatic disease associated with resistance to chemotherapy. We previously showed that syndecan-2 is a mediator of the antioncogenic effect of chemotherapeutic drugs. The purpose of this work was to elucidate molecular mechanisms responsible for the low expression of syndecan-2 in osteosarcoma. We compared the regulatory activity of cis-acting DNA sequences of the syndecan-2 gene in osteosarcoma and osteoblastic cell lines. We identified a DNA region that negatively regulates syndecan-2 transcription in the osteosarcoma cells. T-cell factors (TCF) bind to this sequence in vivo. Wnt3a stimulation, β-catenin activation, and TCF overexpression resulted in syndecan-2 repression, whereas Wnt inhibition using sFRP-1 increased syndecan-2 expression in U2OS cells. RhoA activation blunted the stimulatory effect of sFRP-1 on syndecan-2 transcription, whereas RhoA inhibition enhanced syndecan-2 expression. These results indicate that Wnt/β-catenin and Wnt/RhoA signaling contribute to syndecan-2 repression. The alteration of syndecan-2 expression in osteosarcoma cell lines also seemed to be related to a higher shedding, controlled by Wnt/RhoA. Conversely, syndecan-2 was found to activate its own expression in U2OS cells through RhoA inhibition. These data identify a molecular network that may contribute to the low expression of the proapoptotic proteoglycan syndecan-2 in osteosarcoma cells. The high activity of the canonical Wnt pathway in the different osteosarcoma cells induces a constitutive repression of syndecan-2 transcription, whereas Wnt/RhoA signaling blocks the amplification loop of syndecan-2 expression. Our results identify syndecan-2 as a Wnt target and bring new insights into a possible pathologic role of Wnt signaling in osteosarcoma. Cancer Res; 70(13); 5399-408. ©2010 AACR.

BackgroundThe molecular mechanisms that are involved in the growth and invasiveness of osteosarcoma, an aggressive and invasive primary bone tumor, are not fully understood. The transcriptional co-factor FHL2 (four and a half LIM domains protein 2) acts as an oncoprotein or as a tumor suppressor depending on the tissue context. In this study, we investigated the role of FHL2 in tumorigenesis in osteosarcoma model.Methodology/Principal FindingsWestern blot analyses showed that FHL2 is expressed above normal in most human and murine osteosarcoma cells. Tissue microarray analysis revealed that FHL2 protein expression is high in human osteosarcoma and correlates with osteosarcoma aggressiveness. In murine osteosarcoma cells, FHL2 silencing using shRNA decreased canonical Wnt/β-catenin signaling and reduced the expression of Wnt responsive genes as well as of the key Wnt molecules Wnt5a and Wnt10b. This effect resulted in inhibition of osteosarcoma cell proliferation, invasion and migration in vitro. Using xenograft experiments, we showed that FHL2 silencing markedly reduced tumor growth and lung metastasis occurence in mice. The anti-oncogenic effect of FHL2 silencing in vivo was associated with reduced cell proliferation and decreased Wnt signaling in the tumors.Conclusion/SignificanceOur findings demonstrate that FHL2 acts as an oncogene in osteosarcoma cells and contributes to tumorigenesis through Wnt signaling. More importantly, FHL2 depletion greatly reduces tumor cell growth and metastasis, which raises the potential therapeutic interest of targeting FHL2 to efficiently impact primary bone tumors.