The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition.

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor ß (TGFß) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFß, including TGFß auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFß signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFß type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFß family members.

Promotion of squamous cell carcinoma tumorigenesis by oncogene-mediated THG-1/TSC22D4 phosphorylation.

Carcinoma cells possess high proliferative and invasive potentials and exhibit a resilience against stresses, metabolic disorder, and therapeutic efforts. These properties are mainly acquired by genetic alterations including driver gene mutations. However, the detailed molecular mechanisms have not been fully elucidated. Here, we provide a novel mechanism connecting oncogenic signaling and the tumorigenic properties by a transforming growth factor-ß1-stimulated clone 22 (TSC-22) family protein, THG-1 (also called as TSC22D4). THG-1 is localized at the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas (SCCs). THG-1 knockdown suppressed SCC cell proliferation, invasiveness, and xenograft tumor formation. In contrast, THG-1 overexpression promoted the EGF-induced proliferation and stratified epithelium formation. Furthermore, THG-1 is phosphorylated by the receptor tyrosine kinase (RTK)-RAS-ERK pathway, which promoted the oncogene-mediated tumorigenesis. Moreover, THG-1 involves in the alternative splicing of CD44 variants, a regulator of invasiveness, stemness, and oxidative stress resistance under the RTK pathway. These findings highlight the pivotal roles of THG-1 as a novel effector of SCC tumorigenesis, and the detection of THG-1 phosphorylation by our established specific antibody could contribute to cancer diagnosis and therapy.

PMEPA1/TMEPAI isoforms function via its PY and Smad-interaction motifs for tumorigenic activities of breast cancer cells.

PMEPA1 (prostate transmembrane protein, androgen-induced 1)/TMEPAI (transmembrane prostate androgen-induced protein) is highly expressed in diverse cancers, including breast, lung and prostate cancers. It consists of four isoforms with distinct extracellular regions (isoforms a-d). The expression and function of these isoforms are still poorly understood. Hence, we aimed to identify the preferentially expressed isoforms in breast cancer cells and analyze possible differences in tumorigenic functions. In this study, we used 5' Rapid Amplification of cDNA Ends (RACE) and Western blot analyses to identify the mRNA variants and protein isoforms of TMEPAI and found that TMEPAI isoform d as the major isoform expressed by TGF-ß stimulation in breast cancer cells. We then generated CRISPR/Cas9-mediated TMEPAI knockout (KO) breast cancer cell lines and used a lentiviral expression system to complement each isoform individually. Although there were no clear functional differences between isoforms, double PPxY (PY) motifs and a Smad-interaction motif (SIM) of TMEPAI were both essential for colony and sphere formation. Collectively, our results provide a novel insight into TMEPAI isoforms in breast cancer cells and showed that coordination between double PY motifs and a SIM of TMEPAI are essential for colony and sphere formation but not for monolayer cell proliferation.

TMEPAI, a transmembrane TGF-beta-inducible protein, sequesters Smad proteins from active participation in TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.

Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

We previously established a mechanism of negative regulation of transforming growth factor ß 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.

C18 ORF1, a novel negative regulator of transforming growth factor-ß signaling.

Transforming growth factor (TGF)-ß signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-ß signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-ß signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-ß signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-ß type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-ß-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-ß. Consistently, TGF-ß-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-ß signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-ß signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-ß signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-ß signaling in a coordinated manner when cells are stimulated with high levels of TGF-ß.

Cooperative induction of transmembrane prostate androgen induced protein TMEPAI/PMEPA1 by transforming growth factor-ß and epidermal growth factor signaling.

TMEPAI/PMEPA1 (transmembrane prostate androgen induced-RNA/prostate transmembrane protein, androgen induced 1) is a pro-tumorigenic factor induced by TGF-ß signaling and constitutive TMEPAI expression in lung cancer cells depends on activated autocrine TGF-ß signaling. Here we demonstrate a novel mechanism of TMEPAI transcriptional co-regulation by EGF signaling. Interestingly, we found that ELK-1, downstream of EGFR/Ras/MAPK pathway, modulates TMEPAI expression. ELK-1 binds to the first intron (+1037 to +1294) of the TMEPAI gene together with TGF-ß activated Smad3 and enhances the transcription of TMEPAI. Furthermore, TMEPAI gene activation by EGF and TGF-ß signaling was reduced by the MEK inhibitor U0126. Together, EGF signaling collaboratively regulates TGF-ß-induced TMEPAI expression.

TMEPAI/PMEPA1 enhances tumorigenic activities in lung cancer cells.

TMEPAI/PMEPA1 is a transmembrane protein that was originally identified as a prostatic RNA, the synthesis of which is induced by testosterone or its derivatives. We have recently identified TMEPAI as a direct target gene of transforming growth factor-ß (TGF-ß)/Smad signaling that participates in negative feedback control of the duration and intensity of TGF-ß/Smad signaling. TMEPAI is constitutively and highly expressed in many types of cancer and is associated with poor prognosis. Here, we report that TMEPAI is highly expressed in the lung adenocarcinoma cell lines Calu3, NCI-H23, and RERF-LC-KJ. Expression of TMEPAI in these cancer cells was significantly suppressed by a TGF-ß receptor kinase antagonist, SB208, and by TGF-ß neutralizing antibodies. These results suggest that constitutive expression of TMEPAI in these cancer cells depends on autocrine TGF-ß stimulation. Knockdown of TMEPAI in Calu3 and NCI-H23 cells enhanced levels of Smad2 phosphorylation and significantly suppressed cell proliferation in the presence of TGF-ß, indicating that highly expressed TMEPAI suppresses levels of Smad phosphorylation in these cancer cells and reduces the growth inhibitory effects of TGF-ß/Smad signaling. Furthermore, knockdown of TMEPAI in Calu3 and NCI-H23 cells suppressed sphere formation in vitro and tumor formation in s.c. tissues and in lungs after tail vein injection in NOD-SCID mice in vivo. Together, these experiments indicate that TMEPAI promotes tumorigenic activities in lung cancer cells.

Transcriptional induction of salt-inducible kinase 1 by transforming growth factor ß leads to negative regulation of type I receptor signaling in cooperation with the Smurf2 ubiquitin ligase.

Transforming growth factor ß (TGFß) regulates many physiological processes and requires control mechanisms to safeguard proper and timely action. We have previously described how negative regulation of TGFß signaling is controlled by the serine/threonine kinase salt-inducible kinase 1 (SIK1). SIK1 forms complexes with the TGFß type I receptor and with the inhibitory Smad7 and down-regulates the type I receptor. We now demonstrate that TGFß induces SIK1 levels via a direct transcriptional mechanism that implicates the Smad proteins, and we have mapped a putative enhancer element on the SIK1 gene. We provide evidence that the ubiquitin ligase Smurf2 forms complexes and functionally cooperates with SIK1. Both the kinase activity of SIK1 and the ubiquitin ligase activity of Smurf2 are important for proper type I receptor turnover. We also show that knockdown of endogenous SIK1 and Smurf2 enhances physiological signaling by TGFß that leads to epithelial growth arrest. In conclusion, TGFß induces expression of Smad7, Smurf2, and SIK1, the products of which physically and functionally interlink to control the activity of this pathway.

Requirement of TCF7L2 for TGF-beta-dependent transcriptional activation of the TMEPAI gene.

The TGF-ß and Wnt pathways are involved in cell fate and tumorigenicity. A recent report indicated that a TGF-ß target gene, TMEPAI (transmembrane prostate androgen-induced RNA), is possibly also a downstream target of Wnt signaling. Although TMEPAI was believed to be involved in tumorigenicity because of its blockage of TGF-ß signaling, how TGF-ß and Wnt signals affect the activation of the TMEPAI gene is not well understood. Herein, we show that the TMEPAI promoter is regulated synergistically by TGF-ß/Smad and Wnt/ß-catenin/T cell factor (TCF) 7L2. The critical cis-element for dual signals, termed TGF-ß-responsive TCF7L2-binding element (TTE), is located in intron 1 of the TMEPAI gene. TCF7L2, but not Smad proteins, bound to TTE, whereas the disruption of TTE by mutagenesis remarkably counteracted both TGF-ß and TCF7L2 responses. The introduction of mutations in critical Smad-binding elements blocked the activation of the TMEPAI promoter by TCF7L2. Furthermore, our DNA-protein interaction experiments revealed the indirect binding of TCF7L2 to Smad-binding elements via Smad3 upon TGF-ß stimulation as well as its TGF-ß-dependent association with TTE. We demonstrate that the Wnt/ß-catenin/TCF7L2 pathway is preferentially able to alter the transcriptional regulation of the TGF-ß-target gene, TMEPAI.

TGF-ß-Induced TMEPAI Promotes Epithelial-Mesenchymal Transition in Doxorubicin-Treated Triple-Negative Breast Cancer Cells via SMAD3 and PI3K/AKT Pathway Alteration.

INTRODUCTION: Epithelial-mesenchymal transition (EMT) and overexpression of drug efflux transporters have been reported to cause doxorubicin resistance. Our previous study indicated that TMEPAI (transmembrane prostate androgen-induced protein) attenuated doxorubicin sensitivity in triple-negative breast cancer cells. However, how TMEPAI contributes to doxorubicin resistance in TNBC remains unclear. Thus, the present study aimed to elucidate the mechanism of TMEPAI in doxorubicin resistance in triple-negative breast cancer cells. METHODS: We used BT549, triple-negative cells wild type (WT), and BT549 TMEPAI knock-out. Both cells were treated with TGF-ß 2 ng/mL for 24 hours, followed by TGF-ß 2 ng/mL and doxorubicin 12.9 nM for another 24 hours. Afterward, the cells were harvested and counted. Cells were further lysed and used for RT-PCR and Western blot analysis. We determined the expression levels of proliferation, apoptosis, EMT markers, and drug efflux transporters. Additionally, we investigated the expressions of PI3K as well as SMAD3 and AKT phosphorylation. RESULTS: TNBC cells were shown to be less sensitive to doxorubicin in the presence of TMEPAI. TMEPAI was shown to alleviate the mRNA expressions of apoptosis markers: Bax, Bcl2, Caspase-3, and Caspase-9. Our results indicated that the presence of TMEPAI greatly amplifies EMT and increases drug efflux transporter expressions after doxorubicin treatment. Furthermore, our findings demonstrated that TMEPAI reduced the action of doxorubicin in inhibiting SMAD3 phosphorylation. TMEPAI was also shown to modify the effect of doxorubicin by reducing PI3K expressions and Akt phosphorylation in triple-negative breast cancer cells. CONCLUSION: Our findings indicate that TMEPAI promotes EMT and drug efflux transporters at least in part by shifting doxorubicin action from SMAD3 phosphorylation reduction to PI3K/AKT inhibition in triple-negative breast cancer cells.

PMEPA1/TMEPAI Is a Unique Tumorigenic Activator of AKT Promoting Proteasomal Degradation of PHLPP1 in Triple-Negative Breast Cancer Cells.

Transmembrane prostate androgen-induced protein (TMEPAI), also known as PMEPA1, is highly expressed in many types of cancer and promotes oncogenic abilities. However, the mechanisms whereby TMEPAI facilitates tumorigenesis are not fully understood. We previously established TMEPAI-knockout (KO) cells from human triple-negative breast cancer (TNBC) cell lines and found that TMEPAI-KO cells showed reduced tumorigenic abilities. Here, we report that TMEPAI-KO cells upregulated the expression of pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and suppressed AKT Ser473 phosphorylation, which was consistent with TCGA dataset analysis. Additionally, the knockdown (KD) of PHLPP1 in TMEPAI-KO cells partially but significantly rescued AKT Ser473 phosphorylation, as well as in vitro and in vivo tumorigenic activities, thus showing that TMEPAI functions as an oncogenic protein through the regulation of PHLPP1 subsequent to AKT activation. Furthermore, we demonstrated that TMEPAI PPxY (PY) motifs are essential for binding to NEDD4-2, an E3 ubiquitin ligase, and PHLPP1-downregulatory ability. Moreover, TMEPAI enhanced the complex formation of PHLPP1 with NEDD4-2 and PHLPP1 polyubiquitination, which leads to its proteasomal degradation. These findings indicate that the PY motifs of TMEPAI suppress the amount of PHLPP1 and maintain AKT Ser473 phosphorylation at high levels to enhance the tumorigenic potentiality of TNBC.

TGF-ß-Induced TMEPAI Attenuates the Response of Triple-Negative Breast Cancer Cells to Doxorubicin and Paclitaxel.

PURPOSE: Triple-negative breast cancer (TNBC) is a refractory type of breast cancer with poor prognosis and limited choice for treatment. Previous studies had shown that TNBC has high expressions of transmembrane prostate androgen-induced protein (TMEPAI). TMEPAI was known to be induced by TGF-ß/Smad signaling and have tumorigenic functions that converting TGF-ß from tumor suppressor to tumor promoter and inducing epithelial-mesenchymal transition (EMT). Therefore, we aimed to define the role of TMEPAI in triple-negative breast cancer cells treatment using several anti-cancers in the presence of TGF-ß. METHODS: TMEPAI-knock out (KO) was carried out in a triple-negative breast cancer cell, BT549. TMEPAI editing was developed using the CRISPR-Cas9 system using two combinations of sgRNA to remove exon 4 of the TMEPAI gene entirely. Genotyping and proteomic analysis were performed to check the establishment of the TMEPAI-KO cells. Wild type (WT) and KO cells were used to determine inhibitory concentration 50% (IC50) of several anti-cancers: doxorubicin, cisplatin, paclitaxel, and bicalutamide in the presence of TGF-ß treatment. RESULTS: KO cells were successfully established by completely removing the TMEPAI gene, which was proven in genomic and proteomic analysis. Further, in TMEPAI-KO cells, we found a significant reduction of IC50 for doxorubicin and paclitaxel, and minimal effects were seen for cisplatin and bicalutamide. Our findings suggest that TGF-ß-induced TMEPAI attenuates the response of TNBC to doxorubicin and paclitaxel, but not to cisplatin and bicalutamide. CONCLUSION: TGF-ß induced TMEPAI contributes to the reduced response of TNBC treatment to doxorubicin and paclitaxel, but minimal on cisplatin and bicalutamide. Further study is needed to confirm our findings in other growth factor-induced cells, as well as in in vivo model.

PMEPA1/TMEPAI knockout impairs tumour growth and lung metastasis in MDA-MB-231 cells without changing monolayer culture cell growth.

Prostate transmembrane protein androgen-induced 1 (PMEPA1)/transmembrane prostate androgen-induced protein (TMEPAI), a direct target and a negative regulator of transforming growth factor beta signalling, has an oncogenic role in many cancers. We observed that knockout (KO) of PMEPA1 in human breast cancer cell line MDA-MB-231 using a CRISPR-Cas9 system resulted in reduction of in vivo tumour growth and lung metastasis but not of in vitro monolayer growth capacity of these KO cell lines. This phenomenon was associated with PMEPA1 KO-mediated downregulation of the key proangiogenic factors vascular endothelial growth factor alpha (VEGFA) and interleukin-8 (IL8) that are essential for in vivo but not in vitro growing cells and are also substantial for initiation of lung metastasis.

TMEPAI/PMEPA1 inhibits Wnt signaling by regulating ß-catenin stability and nuclear accumulation in triple negative breast cancer cells.

Transmembrane prostate androgen-induced protein (TMEPAI) is a type I transmembrane protein induced by several intracellular signaling pathways such as androgen, TGF-ß, EGF, and Wnt signaling. It has been reported that TMEPAI functions to suppress TGF-ß and androgen signaling but here, we report a novel function of TMEPAI in Wnt signaling suppression. First, we show that TMEPAI significantly inhibits TCF/LEF transcriptional activity stimulated by Wnt3A, LiCl, and ß-catenin. Mechanistically, TMEPAI overexpression prevented ß-catenin accumulation in the nucleus and TMEPAI knockout in triple negative breast cancer cell lines promoted ß-catenin stability and nuclear accumulation together with increased mRNA levels of Wnt target genes AXIN2 and c-MYC. The presence of TGF-ß type I receptor kinase inhibitor did not affect the enhanced mRNA expression of AXIN2 in TMEPAI knockout cells. These data suggest that TMEPAI suppresses Wnt signaling by interfering with ß-catenin stability and nuclear translocation in a TGF-ß signaling-independent manner.

Glycoprotein nmb Is Exposed on the Surface of Dormant Breast Cancer Cells and Induces Stem Cell-like Properties.

Glycoprotein nmb (GPNMB) is a type I transmembrane protein that contributes to the initiation and malignant progression of breast cancer through induction of epithelial-mesenchymal transition (EMT). Although it is known that EMT is associated with not only cancer invasion but also acquisition of cancer stem cell (CSC) properties, the function of GPNMB in this acquisition of CSC properties has yet to be elucidated. To address this issue, we utilized a three-dimensional (3D) sphere culture method to examine the correlation between GPNMB and CSC properties in breast cancer cells. Three-dimensional sphere cultures induced higher expression of CSC genes and EMT-inducing transcription factor (EMT-TF) genes than the 2D monolayer cultures. Three-dimensional culture also induced cell surface expression of GPNMB on limited numbers of cells in the spheres, whereas the 2D cultures did not. Therefore, we isolated cell surface-GPNMBhigh and -GPNMBlow cells from the spheres. Cell surface-GPNMBhigh cells expressed high levels of CSC genes and EMT-TF genes, had significantly higher sphere-forming frequencies than the cell surface-GPNMBlow cells, and showed no detectable levels of proliferation marker genes. Similar results were obtained from transplanted breast tumors. Furthermore, wild-type GPNMB, but not mutant GPNMB (YF), which lacks tumorigenic activity, induced CSC-like properties in breast epithelial cells. These findings suggest that GPNMB is exposed on the surface of dormant breast cancer cells and its activity contributes to the acquisition of stem cell-like properties.Significance: These findings suggest that cell surface expression of GPNMB could serve as a marker and promising therapeutic target of breast cancer cells with stem cell-like properties. Cancer Res; 78(22); 6424-35. ©2018 AACR.

Snail regulates BMP and TGFß 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 ß (TGFß) 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 TGFß1 signaling activity. Exogenous TGFß1 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 TGFß pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.

The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling.

The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease.

Fine-tuning of Smad protein function by poly(ADP-ribose) polymerases and poly(ADP-ribose) glycohydrolase during transforming growth factor ß signaling.

BACKGROUND: Initiation, amplitude, duration and termination of transforming growth factor ß (TGFß) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFß signaling. METHODS: A robust cell model of TGFß signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFß stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference. RESULTS: TGFß signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFß target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFß-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1. CONCLUSION: Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFß signaling.