Reptin regulates insulin-stimulated Akt phosphorylation in hepatocellular carcinoma via the regulation of SHP-1/PTPN6.

Hepatocellular carcinoma (HCC) is the main primary cancer of the liver. Many studies have shown that insulin resistance is a risk factor for HCC. We previously discovered the overexpression and oncogenic role of the Reptin/RUVBL2 ATPase in HCC. Here, we found that Reptin silencing enhanced insulin sensitivity in 2 HCC cell lines, as shown by a large potentiation of insulin-induced AKT phosphorylation on Ser473 and Thr308, and of downstream signalling. Reptin silencing did not affect the tyrosine phosphorylation of the insulin receptor nor of IRS1, but it enhanced the tyrosine phosphorylation of the p85 subunit of PI3K. The expression of the SHP-1/PTPN6 phosphatase, which dephosphorylates p85, was reduced after Reptin depletion. Forced expression of SHP-1 restored a normal AKT phosphorylation after insulin treatment in cells where Reptin was silenced, demonstrating that the downregulation of SHP1 is mechanistically linked to increased Akt phosphorylation. In conclusion, we have uncovered a new function for Reptin in regulating insulin signalling in HCC cells via the regulation of SHP-1 expression. We suggest that the regulation of insulin sensitivity by Reptin contributes to its oncogenic action in the liver.

Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin.

Hepatocellular carcinoma is associated with a high rate of intra-hepatic invasion that carries a poor prognosis. Meprin alpha (Mep1A) is a secreted metalloproteinase with many substrates relevant to cancer invasion. We found that Mep1A was a target of Reptin, a protein that is oncogenic in HCC. We studied Mep1A regulation by Reptin, its role in HCC, and whether it mediates Reptin oncogenic effects.MepA and Reptin expression was measured in human HCC by qRT-PCR and in cultured cells by PCR, western blot and enzymatic activity measurements. Cell growth was assessed by counting and MTS assay. Cell migration was measured in Boyden chambers and wound healing assays, and cell invasion in Boyden chambers.Silencing Reptin decreased Mep1A expression and activity, without affecting meprin ß. Mep1A, but not meprin ß, was overexpressed in a series of 242 human HCC (2.04 fold, p < 0.0001), and a high expression correlated with a poor prognosis. Mep1A and Reptin expressions were positively correlated (r = 0.39, p < 0.0001). Silencing Mep1A had little effect on cell proliferation, but decreased cell migration and invasion of HuH7 and Hep3B cells. Conversely, overexpression of Mep1A or addition of recombinant Mep1A increased migration and invasion. Finally, overexpression of Mep1A restored a normal cell migration in cells where Reptin was depleted.Mep1A is overexpressed in most HCC and induces HCC cell migration and invasion. Mep1A expression is regulated by Reptin, and Mep1A mediates Reptin-induced migration. Overall, we suggest that Mep1A may be a useful target in HCC.

HnRNP A1 tethers KSRP to an exon splicing silencer that inhibits an erythroid-specific splicing event in PU.1-induced erythroleukemia.

Exon 16 inclusion is a critical splicing event that triggers the production of a functional protein 4.1R in mature normal erythroblasts, and is obviated in PU.1-induced erythroleukemia cells. Exon 16 contains an exonic splicing silencer (ESS16) that interacts with hnRNP A/B in heterologous cell context. We here show that ESS16 promotes the recruitment of a protein complex containing hnRNP A1 and a 79-kDa protein in nuclear extracts from either proliferative erythroleukemia cells or cells induced to terminal differentiation. By using 2D gel fractionation and mass spectrometry, we unambiguously identified KSRP as the 79-kDa component interacting with ESS16. Furthermore, we show that KSRP slightly decreases in erythroleukemia cells induced to terminal erythroid differentiation. Yet, KSRP inducible knockdown, through stable transfection of small hairpin KSRP RNA, did not alter exon 16 splicing, suggesting that KSRP alone does not modulate the splicing event. Interestingly, absence of hnRNP A1 prevented KSRP from binding to ESS16. Reciprocally, KSRP interaction with ESS16 was recovered when hnRNP A1 expression is restored in hnRNP A1-null cells. Collectively, this study establishes that hnRNPA1 is part of a KSRP-containing RNP complex, and emphasizes that, aside from its function in AU-rich element-mediated mRNA decay and its role in microRNA biogenesis, KSRP associates with hnRNP A1 to bind an ESS. These findings further support the role of members of the KH-domain protein family in organizing large RNA-protein complex formation, rather than primarily in modulating specific splicing events.

Combined inhibition of PI3K and activation of MAPK p38 signaling pathways trigger erythroid alternative splicing switch of 4.1R pre-mRNA in DMSO-induced erythroleukemia cells.

There is increasing evidence showing that many extracellular cues modulate pre-mRNA alternative splicing, through different signaling pathways. We here show that 4.1R exon 16 splicing is altered in response to specific signals. The switch from erythroblastic isoform lacking exon 16 to mature erythrocytic isoform containing this exon is tightly regulated during late erythroid differentiation, and blocage of this splicing switch in erythroleukemia cells is seen as a consequence of the deregulation of important regulatory pathways. We support that combined inhibition of PI3K and activation of p38 signaling pathways impinge on erythroid 4.1R pre-mRNA alternative splicing switch, and on cell differentiation as witnessed by hemoglobin production. By contrast, MEK/ERK signaling appeared not to affect neither cell hemoglobin production nor erythroid 4.1R pre-mRNA splicing. We also found that the signal-induced alternative splicing is not typically distinctive of EPO-non-responsive cells, but operates in EPO-responsive cells as well. Pre-mRNA splicing is a major regulatory mechanism at the crossroad between transcription and translation. We here provide evidence that inhibition of PI3K activates the splicing switch in a promoter-dependent manner, whereas p38 activation induces this event in a promoter-independent fashion. Our data further support that constitutive activation of EPO-R by the viral protein gp55 and the short form of the tyrosine kinase receptor Stk, transduces PI3K proliferation signal, but not MAPK p38 differentiation signal. Concurrently, this work lend credence to the concept that DMSO triggers transient activation of p38 signaling and irreversible inhibition of PI3K/AKT signaling pathway, hence uncovering an old conundrum regarding the mechanism by which DMSO induces erythroleukemia cell differentiation.

Proteasome-mediated proteolysis of SRSF5 splicing factor intriguingly co-occurs with SRSF5 mRNA upregulation during late erythroid differentiation.

SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development.

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis.

Defining the function of the genes that, like RUNX1, are deregulated in blood cell malignancies represents an important challenge. Myeloid leukemia factors (MLFs) constitute a poorly characterized family of conserved proteins whose founding member, MLF1, has been associated with acute myeloid leukemia in humans. To gain insight into the functions of this family, we investigated the role of the Drosophila MLF homolog during blood cell development. Here we report that mlf controls the homeostasis of the Drosophila hematopoietic system. Notably, mlf participates in a positive feedback loop to fine tune the activity of the RUNX transcription factor Lozenge (LZ) during development of the crystal cells, one of the two main blood cell lineages in Drosophila. At the molecular level, our data in cell cultures and in vivo strongly suggest that MLF controls the number of crystal cells by protecting LZ from degradation. Remarkably, it appears that the human MLF1 protein can substitute for MLF in the crystal cell lineage. In addition, MLF stabilizes the human oncogenic fusion protein RUNX1-ETO and is required for RUNX1-ETO-induced blood cell disorders in a Drosophila model of leukemia. Finally, using the human leukemic blood cell line Kasumi-1, we show that MLF1 depletion impairs RUNX1-ETO accumulation and reduces RUNX1-ETO-dependent proliferation. Thus, we propose that the regulation of RUNX protein levels is a conserved feature of MLF family members that could be critical for normal and pathological blood cell development.