Identification of a Novel Link between the Protein Kinase NDR1 and TGFβ Signaling in Epithelial Cells

Transforming growth factor-beta (TGFβ) is a secreted polypeptide that plays essential roles in cellular development and homeostasis. Although mechanisms of TGFβ-induced responses have been characterized, our understanding of TGFβ signaling remains incomplete. Here, we uncover a novel function for the protein kinase NDR1 (nuclear Dbf2-related 1) in TGFβ responses. Using an immunopurification approach, we find that NDR1 associates with SnoN, a key component of TGFβ signaling. Knockdown of NDR1 by RNA interference promotes the ability of TGFβ to induce transcription and cell cycle arrest in NMuMG mammary epithelial cells. Conversely, expression of NDR1 represses TGFβ-induced transcription and inhibits the ability of TGFβ to induce cell cycle arrest in NMuMG cells. Mechanistically, we find that NDR1 acts in a kinase-dependent manner to suppress the ability of TGFβ to induce the phosphorylation and consequent nuclear accumulation of Smad2, which is critical for TGFβ-induced transcription and responses. Strikingly, we also find that TGFβ reciprocally regulates NDR1, whereby TGFβ triggers the degradation of NDR1 protein. Collectively, our findings define a novel and intimate link between the protein kinase NDR1 and TGFβ signaling. NDR1 suppresses TGFβ-induced transcription and cell cycle arrest, and counteracting NDR1's negative regulation, TGFβ signaling induces the downregulation of NDR1 protein. These findings advance our understanding of TGFβ signaling, with important implications in development and tumorigenesis

Identifying Novel SUMO Regulators of TGF Beta-induced EMT and Breast Cancer Invasion

Epithelial-mesenchymal transition (EMT) is a fundamental developmental process, which is reactivated in cancer contributing to tumor invasiveness and metastasis. The secreted factor transforming growth factor β (TGFβ) is a potent inducer of EMT in development and cancer. The transcriptional regulator SnoN and E3 ubiquitin ligase Smurf2 control TGFβ signalling in a complex manner with implications for cancer development and progression. The focus of my doctoral studies has been to examine the nature and regulation of SnoN and Smurf2 functions in TGFβ-induced EMT. In this regard, I have characterized the role of SUMO pathway in modifying both SnoN and Smurf2, thereby regulating their roles in controlling EMT. I employed three-dimensional (3D) model systems to follow EMT in mammary epithelial cells and invasive growth behavior of breast cancer cells to increase the chance of getting results with in-vivo relevance. My studies have led to the identification of the protein TIF1γ as a novel SUMO E3 ligase that promotes SnoN sumoylation. Importantly, my data show that TIF1γ acts via SnoN sumoylation to suppress TGFβ-induced EMT in 3D-mammary epithelial cell-derived acini. Next, I found that Smurf2 suppresses EMT in 3D cultures of epithelial cells. Also, I discovered that the SUMO pathway modifies Smurf2 at specific lysine residues through the SUMO E3 ligase PIAS3. Importantly, my data suggest that sumoylation is critical for Smurf2 to suppress TGFβ-induced EMT. Mechanistically, I found that sumoylation promotes Smurf2-induced degradation of TGFβ receptors, leading to suppression of TGFβ signalling and EMT. Lastly, my latest findings suggest that the PIAS3-Smurf2 sumoylation pathway suppresses TGFβ-induced invasive growth of 3D-breast cancer cell-derived spheroids. Future studies, using a xenograft cancer model will aim to investigate role of the PIAS3-Smurf2 sumoylation pathway in tumorigenesis. Overall, our research questions have led to the identification of novel regulators of TGFβ-induced EMT and potentially breast cancer invasion and metastasis. Importantly, SUMO-based pathways promote the ability of both SnoN and Smurf2 to suppress TGFβ-induced EMT in mammary epithelial cells, with relevance to cancer cell invasion and metastasis. Our findings could help in providing potential new diagnostic or druggable targets for treatment of breast tumors

TGFβ signaling promotes NDR1 turnover.

<p><b>A.</b> Lysates of untreated or 48h-TGFβ-treated NMuMG cells in the absence or presence of the TGFβ type I receptor kinase inhibitor SB431542 (KI) were subjected to immunoblotting with the NDR1 or actin antibody. <b>B.</b> Protein abundance of NDR1 and actin in immunoblots, including those shown in A, were quantified and percent reduction of NDR1 (normalized to actin) in response to TGFβ was analyzed. Data are presented as the mean+SEM (n = 4) of percent decrease in protein abundance of NDR1 in NMuMG cells in response to TGFβ. TGFβ treatment decreased the protein abundance of NDR1 in NMuMG cells. <b>C.</b> TGFβ does not repress NDR1 mRNA expression. RNA extracts from untreated or 48h-TGFβ-treated NMuMG cells were analyzed by quantitative RT-PCR for NDR1 and GAPDH mRNA abundance. Data are presented as the mean+SEM (n = 3) of relative mRNA abundance of NDR1 in NMuMG cells. TGFβ did not reduce relative abundance of NDR1 mRNA. Significant differences are indicated in B and C as determined by unpaired, two-tailed t-test. <b>D.</b> Lysates of NMuMG cells left untreated or incubated with 10 µg/ml cycloheximide for different times, alone or together with 100 pM TGFβ, were subjected to immunoblotting using the NDR1 or actin antibody. <b>E.</b> Protein abundance of NDR1 in immunoblots, including the one shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067178#pone-0067178-g006" target="_blank">Figure 6D</a>, were quantified and normalized to actin. Data are presented as the mean±SEM (n = 3) of normalized protein abundance of NDR1 expressed relative to that at time 0 for the respective minus or plus TGFβ group. Data interpolation indicated that NDR1's half-life was greater than 16 h. TGFβ reduced NDR1's half-life to approximately 9 h. <b>F.</b> Lysates of untreated or 24 h-TGFβ-preincubated NMuMG cells followed by exposure to cycloheximide for different time points, were subjected to immunoblotting using NDR1 or actin antibody. <b>G.</b> Protein abundance of NDR1 in immunoblots as described and including the one shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067178#pone-0067178-g006" target="_blank">Figure 6F</a> was quantified as described in E. Data are presented as the mean±SEM (n = 4) of relative NDR1 levels. TGFβ reduced the half-life of NDR1 from greater than 16 h to approximately 2.5 h. <b>H.</b> TGFβ signaling enhances the ubiquitination of NDR1. Lysates of 293T cells expressing FLAG-NDR1, HA-ubiquitin, and constitutively active TGFβ type I receptor, were subjected to immunoprecipitation using the FLAG antibody, followed by immunoblotting with the HA or NDR1 antibody. Cell lysates were also immunoblotted with the FLAG or actin antibody. HC refers to the heavy chain of the FLAG antibody. I. Lysates of 293T cells transfected with FLAG-NDR1 alone or together with constitutively active TGFβ type I receptor and treated without or with 0.5 µM MG132 (Sigma) for 7 hours were subjected to immunoblotting with the FLAG or actin antibody. *, **, or *** in E and G indicates significant difference from respective control at P<0.05, p<0.01, or p<0.001, respectively (ANOVA). # indicates significant difference from control (p<0.05, unpaired, one tailed t-test).</p

NDR1 represses TGFβ-induction of endogenous PAI-1 gene expression.

<p><b>A.</b> RNA extracts of untreated or TGFβ-treated NMuMG cells transfected with a control RNAi plasmid or the combination of NDR1i-1 and NDR1i-2 RNAi plasmids were subjected to quantitative RT-PCR to determine the abundance of PAI-1 mRNA, where GAPDH mRNA was used as an internal control. Data are presented as the mean+SEM (n = 5) of GAPDH-normalized PAI-1 mRNA abundance relative to untreated control. <b>B.</b> Lysates of NMuMG cells expressing FLAG-tagged wild type NDR1 (WT) or kinase-inactive NDR1 in which Lysine 118 is mutated to arginine (KR), or vector control were subjected to immunoblotting using the NDR1 or actin antibody, with the latter serving as a loading control. <b>C.</b> RNA extracts from untreated or TGFβ-treated NMuMG cells expressing wild type or kinase-inactive NDR1 or the vector control were subjected to quantitative RT-PCR analysis of PAI-1 and GAPDH mRNA as described in A. Data are presented as the mean+SEM (n = 3) of relative GAPDH-normalized PAI-1 mRNA abundance as in A. *, **, or *** indicates significant difference from the TGFβ-treated control at p<0.05, p<0.01, or p<0.001, respectively (ANOVA).</p

Mass spectrometry data of SnoN-interacting proteins in HaCaT cells.

a<p>Identified proteins represent peptides only present in the SnoN-expressing samples, and not in the control cells.</p>b<p>Trypsin, immunoglobulin and keratin are considered contaminants and are omitted from this list.</p>c<p>only identified proteins with a score of ≥50 are shown here.</p>d<p>These identified proteins were also present in the control cells, but the number of peptides matched and overall score was much lower in the control cells than in the SnoN-expressing samples.</p

NDR1 is a novel SnoN-interacting protein.

<p><b>A.</b> Lysates of untreated or TGFβ-treated HaCaT cells expressing FLAG, HA-SnoN or control vector were immunoblotted with the SnoN or actin antibody. TGFβ similarly reduced the abundance of endogenous and exogenous SnoN in HaCaT cells. <b>B.</b> Lysates of 293T expressing MYC-SnoN alone or together with HA-NDR1 were subjected to immunoprecipitation with the HA antibody followed by immunoblotting with the SnoN or HA antibody. Total lysates were also subjected to immunoblotting with the SnoN or actin antibody, the latter to serve as a loading control. <b>C.</b> Lysates of 293T cells expressing HA-NDR1 alone or together with MYC-SnoN were subjected to immunoprecipitation with the SnoN antibody followed by immunoblotting with the HA or SnoN antibody. Lysates were also immunoblotted with the HA or actin antibody. NDR1 formed a complex with SnoN. <b>D.</b> A schematic diagram showing the wild type (amino acid (aa) 1–684) and four deletion mutants of SnoN. The dotted area represents the ski/sno/dac (DACH) domain, the shaded area the SAND domain, and the striped areas the helical dimerization domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067178#pone.0067178-Pot1" target="_blank">[33]</a>. <b>E.</b> Lysates of 293T cells expressing Rluc in fusion with wild type or a series of SnoN mutants, as shown in D, alone or together with HA-NDR1 were subjected to immunoprecipitation with the HA antibody followed by luciferase assays to determine the levels of Rluc-SnoN fusion proteins in the NDR1 immunoprecipitates. Aliquots of cell lysates were also assayed for luciferase activity as a measure of Rluc-SnoN expression. The expression of HA-NDR1 in aliquots of immunoprecipitates (10%) and cell lysates was confirmed by immunoblotting using the HA antibody (data not shown). NDR1-associated Rluc-SnoN luciferase activity was normalized to Rluc-SnoN and NDR1 expression. Data are presented as the mean+SEM (n = 4) of NDR1-associated Rluc activity relative to Rluc activity associated with NDR1 in the case of the wild type Rluc-SnoN fusion protein. <b>F.</b> A schematic diagram showing the wild type (aa1–465) and three deletion mutants of NDR1. The dotted area represents the N-terminal regulatory domain, the shaded area the kinase domain, and the striped area the C-terminal regulatory domain. <b>G.</b> Lysates of 293T cells expressing Rluc in fusion with wild type or a series of NDR1 mutants, as in F, alone or together with MYC-SnoN, were subjected to immunoprecipitation using the MYC or SnoN antibody. Immunoprecipitates and cell lysates were subjected to luciferase assays, SnoN immunoblotting (Data not shown), and data analyses as described in E. Data are presented as the mean+SEM (n = 7) of SnoN-associated Rluc activity expressed relative to SnoN-associated Rluc activity in the case of wild type NDR1-Rluc. *, or *** indicates significant difference as compared to wild type SnoN-Rluc (E) or NDR1-Rluc (G) at p<0.05 or p<0.001, respectively (ANOVA).</p