Theo van Laar

VEGF and inhibitors of TGFβ type-I receptor kinase synergistically promote blood-vessel formation by inducing α5-integrin expression

Vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGFβ) are potent regulators of angiogenesis. How VEGF and TGFβ signaling pathways crosstalk is not well understood. Therefore, we analyzed the effects of the TGFβ type-I-receptor inhibitors (SB-431542 and LY-2157299) and VEGF on endothelial cell (EC) function and angiogenesis. We show that SB-431542 dramatically enhances VEGF-induced formation of EC sheets from fetal mouse metatarsals. Sub-optimal doses of VEGF and SB-431542 synergistically induced EC migration and sprouting of EC spheroids, whereas overexpression of a constitutively active form of TGFβ type-I receptor had opposite effects. Using quantitative PCR, we demonstrated that VEGF and SB-431542 synergistically upregulated the mRNA expression of genes involved in angiogenesis, including the integrins α5 and β3. Specific downregulation of α5-integrin expression or functional blocking of α5 integrin with a specific neutralizing antibody inhibited the cooperative effect of VEGF and SB-431542 on EC sprouting. In vivo, LY-2157299 induced angiogenesis and enhanced VEGF- and basic-fibroblast-growth-factor-induced angiogenesis in a Matrigel-plug assay, whereas adding an α5-integrin-neutralizing antibody to the Matrigel selectively inhibited this enhanced response. Thus, induction of α5-integrin expression is a key determinant by which inhibitors of TGFβ type-I receptor kinase and VEGF synergistically promote angiogenesis.

Snail and Slug, key regulators of TGF-β-induced EMT, are sufficient for the induction of single-cell invasion.

TGF-β plays a dual role in cancer; in early stages it inhibits tumor growth, whereas later it promotes invasion and metastasis. TGF-β is thought to be pro-invasive by inducing epithelial-to-mesenchymal transition (EMT) via induction of transcriptional repressors, including Slug and Snail. In this study, we investigated the role of Snail and Slug in TGF-β-induced invasion in an in vitro invasion assay and in an embryonic zebrafish xenograft model. Ectopic expression of Slug or Snail promoted invasion of single, rounded amoeboid cells in vitro. In an embryonic zebrafish xenograft model, forced expression of Slug and Snail promoted single cell invasion and metastasis. Slug and Snail are sufficient for the induction of single-cell invasion in an in vitro invasion assay and in an embryonic zebrafish xenograft model.

Mutations of the PU.1 Ets domain are specifically associated with murine radiation-induced, but not human therapy-related, acute myeloid leukaemia

Murine radiation-induced acute myeloid leukaemia (AML) is characterized by loss of one copy of chromosome 2. Previously, we positioned the critical haematopoietic-specific transcription factor PU.1 within a minimally deleted region. We now report a high frequency (>65%) of missense mutation at codon 235 in the DNA-binding Ets domain of PU.1 in murine AML. Earlier studies, outside the context of malignancy, determined that conversion of arginine 235 (R235) to any other amino-acid residue leads to ablation of DNA-binding function and loss of expression of downstream targets. We show that mutation of R235 does not lead to protein loss, and occurs specifically in those AMLs showing loss of one copy of PU.1 (P=0.001, Fishers exact test). PU.1 mutations were not found in the coding region, UTRs or promoter of human therapy-related AMLs. Potentially regulatory elements upstream of PU.1 were located but no mutations found. In conclusion, we have identified the cause of murine radiation-induced AML and have shown that loss of one copy of PU.1, as a consequence of flanking radiation-sensitive fragile domains on chromosome 2, and subsequent R235 conversion are highly specific to this mouse model. Such a mechanism does not operate, or is extremely rare, in human AML.

The novel MMS-inducible gene Mif1/KIAA0025 is a target of the unfolded protein response pathway

In a search for genes induced by DNA‐damaging agents, we identified two genes that are activated by methyl methanesulfonate (MMS). Expression of both genes is regulated after endoplasmic reticulum (ER) stress via the unfolded protein response (UPR) pathway. The first gene of those identified is the molecular chaperone BiP/GRP78. The second gene, Mif1, is identical to the anonymous cDNA KIAA0025. Treatment with the glycosylation inhibitor tunicamycin both enhances the synthesis of Mif1 mRNA and protein. The Mif1 5′ flanking region contains a functional ER stress‐responsive element which is sufficient for induction by tunicamycin. MMS, on the other hand, activates Mif1 via an UPR‐independent pathway. The gene encodes a 52 kDa protein with homology to the human DNA repair protein HHR23A and contains an ubiquitin‐like domain. Overexpressed Mif1 protein is localized in the ER.

A role for Rad23 proteins in 26S proteasome-dependent protein degradation?

Treatment of cells with genotoxic agents affects protein degradation in both positive and negative ways. Exposure of S. cerevisiae to the alkylating agent MMS resulted in activation of genes that are involved in ubiquitin- and 26S proteasome-dependent protein degradation. This process partially overlaps with the activation of the ER-associated protein degradation pathway. The DNA repair protein Rad23p and its mammalian homologues have been shown to inhibit degradation of specific substrates in response to DNA damage. Particularly the recently identified inhibition of degradation by mouse Rad23 protein (mHR23) of the associated nucleotide excision repair protein XPC was shown to stimulate DNA repair.Recently, it was shown that Rad23p and the mouse homologue mHR23B also associate with Png1p, a deglycosylation enzyme. Png1p-mediated deglycosylation plays a role in ER-associated protein degradation after accumulation of malfolded proteins in the endoplasmic reticulum. Thus, if stabilization of proteins that are associated with the C-terminus of Rad23p is a general phenomenon, then Rad23 might be implicated in the stimulation of ER-associated protein degradation as well. Interestingly, the recently identified HHR23-like protein Mif1 is also thought to play a role in ER-associated protein degradation. The MIF1 gene is strongly activated in response to ER-stress. Mif1 contains a ubiquitin-like domain which is most probably involved in binding to S5a, a subunit of the 19S regulatory complex of the 26S proteasome. On the basis of its localization in the ER-membrane, it is hypothesized that Mif1 could play a role in the translocation of the 26S proteasome towards the ER-membrane, thereby enhancing ER-associated protein degradation.

Mif1: A Missing Link between the Unfolded Protein Response Pathway and ER-Associated Protein Degradation?

Eukaryotic cells have three different mechanisms to deal with the accumulation of unfolded proteins in the endoplasmic reticulum: (1) In cells in which unfolded polypeptides accumulate, translation initiation is inhibited to prevent further accumulation of unfolded proteins. (2) Expression of proteins involved in polypeptide folding is strongly enhanced by a process called the Unfolded Protein Response (UPR). (3) Proteins missing the proper tertiary structure are degraded by the ER-Associated protein Degradation (ERAD) mechanism. Recent studies in S. cerevisiae have shown that the processes of UPR and ERAD are functionally linked to each other. Cells lacking a functional ERAD show a constitutive activation of UPR. In addition, many of the components of ERAD are under the direct transcriptional control of UPR. Finally, while neither UPR nor ERAD are essential for cell viability, deletion of both pathways results in severe growth impairment. UPR and ERAD are conserved between yeast and mammalian cells. One of the components of mammalian UPR is the protease presenilin-1. Mutations in the gene for presenilin-1 cause early-onset familial Alzheimer disease. Interestingly, inhibition of proteolysis by the ubiquitin-26S proteasome system has also been described for Alzheimer s disease. This suggests a link between UPR and ERAD in mammalian cells. The recently identified gene Mif1 is a possible candidate to form a direct link between UPR and ERAD in mammalian cells. The Mif1 gene is under the direct control of UPR. Mif1 is a trans-ER-membrane protein, with both the N- and the C-termini facing the cytoplasmic side of the ER membrane. It contains an N-terminal ubiquitin-like domain. It is anticipated that Mif1 may associate through its ubiquitin-like domain with the 26S proteasome, in this way connecting the protein degradation machinery to the ER membrane and resulting in an efficient ERAD.

Induction of the SAPK activator MIG‐6 by the alkylating agent methyl methanesulfonate

The alkylating agent methylmethanesulfonate (MMS) activates the c‐jun N‐terminal kinase (JNK)/stress‐activated protein kinase (SAPK) and the p38 mitogen‐activated protein kinase (p38MAPK) pathways via different mechanisms of action. Activation of p38MAPK by MMS involves the pp125 focal adhesion kinase–related tyrosine kinase RAFTK and the MAPK kinase 3. The way in which MMS can activate JNK/SAPK has not been elucidated. Here we describe the identification by differential display of human mitogen‐activated gene‐6 (MIG‐6) as a novel MMS‐inducible gene. Induction of MIG‐6 by MMS was found in human diploid skin fibroblasts and in simian virus 40–transformed skin fibroblasts, indicating that the enhanced expression of MIG‐6 after MMS‐treatment did not require p53. The signal leading to activation of MIG‐6 appeared to be independent of DNA damage. High MIG‐6 expression was found in the liver, lung, and placenta. MIG‐6 is an adapter protein that binds to the activated form of cdc42Hs and to 14‐3‐3 proteins, thereby activating JNK/SAPKs. Our results suggest that activation of JNK/SAPKs by MMS may involve the induction of MIG‐6.

Spheroid Assay to Measure TGF-β-induced Invasion

TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage(1,2). Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis (3,4). The mechanisms by which TGF-β induces invasion are not well understood. TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes(5). In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways(6). To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells(7), the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice(8), and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung(9). This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background. For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid(10). Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures(11). This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy.

Temperature-sensitive mutant p53 (ala143) interferes transiently with DNA-synthesis and cell-cycle progression in Saos-2 cells.

It has been demonstrated that temperature-sensitive mutant p53 (val-->ala143) inhibits cell-proliferation at the permissive temperature, albeit to a lesser extent than wild-type p53 (Zhang et al.: EMBO J 13:2535-2544, 1994). We have studied its effect on the cell-cycle by dual-parameter flow cytometry, extended pulse-labeling, and pulse-chase experiments. p53ala143 interferes in Saos-2 cells at three levels with cell-cycle progression at permissive temperatures: it caused a G1-arrest, a reduced rate of DNA synthesis during S, and a prolonged G2/M. Strikingly, all these effects are transient. Continued culturing at 32 degrees C resulted in normal cell-cycle progression. Abrogation of the G1-block occurred even in the presence of high p21Waf1 protein levels, a negative cell-cycle regulator of which the expression is induced by wild-type p53.