Carl-Henrik Heldin

Differential ubiquitination defines the functional status of the tumor suppressor Smad4

Smad4 is an essential signal transducer of all transforming growth factor-β (TGF-β) superfamily pathways that regulate cell growth and differentiation, and it becomes inactivated in human cancers. Receptor-activated (R-) Smads can be poly-ubiquitinated in the cytoplasm or the nucleus, and this regulates their steady state levels or shutdown of the signaling pathway. Oncogenic mutations in Smad4 and other Smads have been linked to protein destabilization and proteasomal degradation. We analyzed a panel of missense mutants derived from human cancers that map in the N-terminal Mad homology (MH) 1 domain of Smad4 and result in protein instability. We demonstrate that all mutants exhibit enhanced poly-ubiquitination and proteasomal degradation. In contrast, wild type Smad4 is a relatively stable protein that undergoes mono- or oligo-ubiquitination, a modification not linked to protein degradation. Analysis of Smad4 deletion mutants indicated efficient mono- or oligo-ubiquitination of the C-terminal MH2 domain. Mass spectrometric analysis of mono-ubiquitinated Smad4 MH2 domain identified lysine 507 as a major target for ubiquitination. Lysine 507 resides in the conserved L3 loop of Smad4 and participates in R-Smad C-terminal phosphoserine recognition. Mono- or oligo-ubiquitinated Smad4 exhibited enhanced ability to oligomerize with R-Smads, whereas mutagenesis of lysine 507 led to inefficient Smad4/R-Smad hetero-oligomerization and defective transcriptional activity. Finally, overexpression of a mutant ubiquitin that only leads to mono-ubiquitination of Smad4 enhanced Smad transcriptional activity. These data suggest that oligo-ubiquitination positively regulates Smad4 function, whereas poly-ubiquitination primarily occurs in unstable cancer mutants and leads to protein degradation.

Role of Platelet-Derived Growth Factor in Vivo

Platelet-derived growth factor (PDGF) was originally identified as a mitogen for fibroblasts, smooth muscle cells, and glial cells (Kohler and Lipton, 1974; Ross et al., 1974; Westermark and Wasteson, 1976). PDGF was subsequently purified from human platelets (Antoniades et al., 1979; Deuel et al., 1981; Heldin et al., 1979; Raines and Ross, 1982). More recent studies have shown that PDGF is synthesized by a number of cell types and also acts on many different cell types (for reviews on PDGF, see Heldin and Westermark, 1990; Raines et al., 1990).

Transforming Growth Factor (TGF-β)-specific Signaling by Chimeric TGF-β Type II Receptor with Intracellular Domain of Activin Type IIB Receptor

Members of the transforming growth factor-β (TGF-β) superfamily signal via different heteromeric complexes of two sequentially acting serine/threonine kinase receptors, i.e.type I and type II receptors. We generated two different chimeric TGF-β superfamily receptors, i.e. TβR-I/BMPR-IB, containing the extracellular domain of TGF-β type I receptor (TβR-I) and the intracellular domain of bone morphogenetic protein type IB receptor (BMPR-IB), and TβR-II/ActR-IIB, containing the extracellular domain of TGF-β type II receptor (TβR-II) and the intracellular domain of activin type IIB receptor (ActR-IIB). In the presence of TGF-β1, TβR-I/BMPR-IB and TβR-II/ActR-IIB formed heteromeric complexes with wild-type TβR-II and TβR-I, respectively, upon stable transfection in mink lung epithelial cell lines. We show that TβR-II/ActR-IIB restored the responsiveness upon transfection in mutant cell lines lacking functional TβR-II with respect to TGF-β-mediated activation of a transcriptional signal, extracellular matrix formation, growth inhibition, and Smad phosphorylation. Moreover, TβR-I/BMPR-IB and TβR-II/ActR-IIB formed a functional complex in response to TGF-β and induced phosphorylation of Smad1. However, complex formation is not enough for signal propagation, which is shown by the inability of TβR-I/BMPR-IB to restore responsiveness to TGF-β in cell lines deficient in functional TβR-I. The fact that the TGF-β1-induced complex between TβR-II/ActR-IIB and TβR-I stimulated endogenous Smad2 phosphorylation, a TGF-β-like response, is in agreement with the current model for receptor activation in which the type I receptor determines signal specificity.

Ras and TGF-β signaling enhance cancer progression by promoting the ΔNp63 transcriptional program

In tumors with mutant p53, both Ras and TGF-β promote metastatic disease by stimulating ΔNp63 activity. Metastatic convergence at ΔNp63 The p53 family of transcription factors, which includes p53, p63, and p73, is implicated in both tumor-suppressive and tumorigenic functions. Activation of the Ras and transforming growth factor–β (TGF-β) signaling pathways are similarly enigmatic with both tumor-suppressing and tumor-promoting activity. Deciphering their roles in various stages of tumor development is critical to developing targeted therapeutics. Vasilaki et al. found that these factors are all connected in the development of some cancers. Activation of Ras or TGF-β signaling stimulated the transcriptional activity of the isoform ΔNp63 in breast or squamous cancer cells by suppressing the abundance of the mutant form of p53, an inhibitor of ΔNp63 and also a feature implicated in early tumorigenesis. Increased abundance of ΔNp63 in breast cancer cells stimulated metastatic behaviors in culture and in mice and correlated with poor prognosis in patients with mutant p53–positive tumors. These findings provide some insight into dichotic, stage-specific signals in tumorigenesis. Identification of this downstream common effector could also offer new therapeutic opportunities for some advanced Ras- or TGF-β–driven tumors. The p53 family of transcription factors includes p63, which is a master regulator of gene expression in epithelial cells. Determining whether p63 is tumor-suppressive or tumorigenic is complicated by isoform-specific and cellular context–dependent protein associations, as well as antagonism from mutant p53. ΔNp63 is an amino-terminal–truncated isoform, that is, the predominant isoform expressed in cancer cells of epithelial origin. In HaCaT keratinocytes, which have mutant p53 and ΔNp63, we found that mutant p53 antagonized ΔNp63 transcriptional activity but that activation of Ras or transforming growth factor–β (TGF-β) signaling pathways reduced the abundance of mutant p53 and strengthened target gene binding and activity of ΔNp63. Among the products of ΔNp63-induced genes was dual-specificity phosphatase 6 (DUSP6), which promoted the degradation of mutant p53, likely by dephosphorylating p53. Knocking down all forms of p63 or DUSP6 and DUSP7 (DUSP6/7) inhibited the basal or TGF-β–induced or epidermal growth factor (which activates Ras)–induced migration and invasion in cultures of p53-mutant breast cancer and squamous skin cancer cells. Alternatively, overexpressing ΔNp63 in the breast cancer cells increased their capacity to colonize various tissues upon intracardiac injection in mice, and this was inhibited by knocking down DUSP6/7 in these ΔNp63-overexpressing cells. High abundance of ΔNp63 in various tumors correlated with poor prognosis in patients, and this correlation was stronger in patients whose tumors also had a mutation in the gene encoding p53. Thus, oncogenic Ras and TGF-β signaling stimulate cancer progression through activation of the ΔNp63 transcriptional program.

JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion

Abstract It is well established that transforming growth factor-β (TGFβ) switches its function from being a tumor suppressor to a tumor promoter during the course of tumorigenesis, which involves both cell-intrinsic and environment-mediated mechanisms. We are interested in breast cancer cells, in which SMAD mutations are rare and interactions between SMAD and other transcription factors define pro-oncogenic events. Here, we have performed chromatin immunoprecipitation (ChIP)-sequencing analyses which indicate that the genome-wide landscape of SMAD2/3 binding is altered after prolonged TGFβ stimulation. De novo motif analyses of the SMAD2/3 binding regions predict enrichment of binding motifs for activator protein (AP)1 in addition to SMAD motifs. TGFβ-induced expression of the AP1 component JUNB was required for expression of many late invasion-mediating genes, creating a feed-forward regulatory network. Moreover, we found that several components in the WNT pathway were enriched among the late TGFβ-target genes, including the invasion-inducing WNT7 proteins. Consistently, overexpression of WNT7A or WNT7B enhanced and potentiated TGFβ-induced breast cancer cell invasion, while inhibition of the WNT pathway reduced this process. Our study thereby helps to explain how accumulation of pro-oncogenic stimuli switches and stabilizes TGFβ-induced cellular phenotypes of epithelial cells.

Regulation of bone morphogenetic protein signaling by ADP-ribosylation

We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation.

The protein kinase SIK downregulates the polarity protein Par3

The multifunctional cytokine transforming growth factor β (TGFβ) controls homeostasis and disease during embryonic and adult life. TGFβ alters epithelial cell differentiation by inducing epithelial-mesenchymal transition (EMT), which involves downregulation of several cell-cell junctional constituents. Little is understood about the mechanism of tight junction disassembly by TGFβ. We found that one of the newly identified gene targets of TGFβ, encoding the serine/threonine kinase salt-inducible kinase 1 (SIK), controls tight junction dynamics. We provide bioinformatic and biochemical evidence that SIK can potentially phosphorylate the polarity complex protein Par3, an established regulator of tight junction assembly. SIK associates with Par3, and induces degradation of Par3 that can be prevented by proteasomal and lysosomal inhibition or by mutation of Ser885, a putative phosphorylation site on Par3. Functionally, this mechanism impacts on tight junction downregulation. Furthermore, SIK contributes to the loss of epithelial polarity and examination of advanced and invasive human cancers of diverse origin displayed high levels of SIK expression and a corresponding low expression of Par3 protein. High SIK mRNA expression also correlates with lower chance for survival in various carcinomas. In specific human breast cancer samples, aneuploidy of tumor cells best correlated with cytoplasmic SIK distribution, and SIK expression correlated with TGFβ/Smad signaling activity and low or undetectable expression of Par3. Our model suggests that SIK can act directly on the polarity protein Par3 to regulate tight junction assembly.

Snail regulates BMP and TGF beta pathways to control the differentiation status of glioma-initiating cells

Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor β (TGFβ) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFβ1 signaling activity. Exogenous TGFβ1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFβ pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.