Post-Transcriptional Regulation of PARP7 Protein Stability Is Controlled by Androgen Signaling.

Poly-ADP-ribose polymerases (PARPs) are enzymes that catalyze ADP-ribosylation and play critical roles in normal and disease settings. The PARP family member, PARP7, is a mono-ADP-ribosyltransferase that has been suggested to play a tumor suppressive role in breast, ovarian, and colorectal cancer. Here, we have investigated how androgen signaling regulates PARP7 homeostasis in prostate cancer cells, where PARP7 is a direct target gene of AR. We found that the PARP7 protein is extremely short-lived, with a half-life of 4.5 min. We show that in addition to its transcriptional regulation by AR, PARP7 is subject to androgen-dependent post-transcriptional regulation that increases its half-life to 25.6 min. This contrasts with PARP1, PARP2, PARP9, and PARP14, which do not display rapid turnover and are not regulated by androgen signaling. Androgen- and AR-dependent stabilization of PARP7 leads to accumulation in the nucleus, which we suggest is a major site of action. Mutations in the catalytic domain, the Cys3His1 zinc finger, and WWE (tryptophan-tryptophan-glutamate) domains in PARP7 each reduce the degradation rate of PARP7, suggesting the overall structure of the protein is tuned for its rapid turnover. Our finding that PARP7 is regulated by AR signaling both transcriptionally and post-transcriptionally in prostate cancer cells suggests the dosage of PARP7 protein is subject to tight regulation.

TGIF transcription factors repress acetyl CoA metabolic gene expression and promote intestinal tumor growth.

Tgif1 (thymine-guanine-interacting factor 1) and Tgif2 repress gene expression by binding directly to DNA or interacting with transforming growth factor (TGF) ß-responsive SMADs. Tgifs are essential for embryogenesis and may function in tumor progression. By analyzing both gain and loss of Tgif function in a well-established mouse model of intestinal cancer, we show that Tgifs promote adenoma growth in the context of mutant Apc (adenomatous polyposis coli). Despite the tumor-suppressive role of TGFß signaling, transcriptome profiling of colon tumors suggests minimal effect of Tgifs on the TGFß pathway. Instead, it appears that Tgifs, which are up-regulated in Apc mutant colon tumors, contribute to reprogramming metabolic gene expression. Integrating gene expression data from colon tumors with other gene expression and chromatin-binding data identifies a set of direct Tgif target genes encoding proteins involved in acetyl CoA and pyruvate metabolism. Analysis of both tumor and nontumor tissues indicates that these genes are targets of Tgif repression in multiple settings, suggesting that this is a core Tgif function. We propose that Tgifs play an important role in regulating basic energy metabolism in normal cells, and that this function of Tgifs is amplified in some cancers.

Myt1 and Myt1l transcription factors limit proliferation in GBM cells by repressing YAP1 expression.

Myelin transcription factor 1 (Myt1) and Myt1l (Myt1-like) are zinc finger transcription factors that regulate neuronal differentiation. Reduced Myt1l expression has been implicated in glioblastoma (GBM), and the related St18 was originally identified as a potential tumor suppressor for breast cancer. We previously analyzed changes in gene expression in a human GBM cell line with re-expression of either Myt1 or Myt1l. This revealed largely overlapping gene expression changes, suggesting similar function in these cells. Here we show that re-expression of Myt1 or Myt1l reduces proliferation in two different GBM cell lines, activates gene expression programs associated with neuronal differentiation, and limits expression of proliferative and epithelial to mesenchymal transition gene-sets. Consistent with this, expression of both MYT1 and MYT1L is lower in more aggressive glioma sub-types. Examination of the gene expression changes in cells expressing Myt1 or Myt1l suggests that both repress expression of the YAP1 transcriptional coactivator, which functions primarily in the Hippo signaling pathway. Expression of YAP1 and its target genes is reduced in Myt-expressing cells, and there is an inverse correlation between YAP1 and MYT1/MYT1L expression in human brain cancer datasets. Proliferation of GBM cell lines is reduced by lowering YAP1 expression and increased with YAP1 over-expression, which overcomes the anti-proliferative effect of Myt1/Myt1l expression. Finally we show that reducing YAP1 expression in a GBM cell line slows the growth of orthotopic tumor xenografts. Together, our data suggest that Myt1 and Myt1l directly repress expression of YAP1, a protein which promotes proliferation and GBM growth.

TGFß signaling limits lineage plasticity in prostate cancer.

Although treatment options for localized prostate cancer (CaP) are initially effective, the five-year survival for metastatic CaP is below 30%. Mutation or deletion of the PTEN tumor suppressor is a frequent event in metastatic CaP, and inactivation of the transforming growth factor (TGF) ß signaling pathway is associated with more advanced disease. We previously demonstrated that mouse models of CaP based on inactivation of Pten and the TGFß type II receptor (Tgfbr2) rapidly become invasive and metastatic. Here we show that mouse prostate tumors lacking Pten and Tgfbr2 have higher expression of stem cell markers and genes indicative of basal epithelial cells, and that basal cell proliferation is increased compared to Pten mutants. To better model the primarily luminal phenotype of human CaP we mutated Pten and Tgfbr2 specifically in luminal cells, and found that these tumors also progress to invasive and metastatic cancer. Accompanying the transition to invasive cancer we observed de-differentiation of luminal tumor cells to an intermediate cell type with both basal and luminal markers, as well as differentiation to basal cells. Proliferation rates in these de-differentiated cells were lower than in either basal or luminal cells. However, de-differentiated cells account for the majority of cells in micro-metastases consistent with a preferential contribution to metastasis. We suggest that active TGFß signaling limits lineage plasticity in prostate luminal cells, and that de-differentiation of luminal tumor cells can drive progression to metastatic disease.

Analysis of transcriptional activity by the Myt1 and Myt1l transcription factors.

Myt1 and Myt1l (Myelin transcription factor 1, and Myt1-like) are members of a small family of closely related zinc finger transcription factors, characterized by two clusters of C2HC zinc fingers. Both are widely expressed during early embryogenesis, but are largely restricted to expression within the brain in the adult. Myt1l, as part of a three transcription factor mix, can reprogram fibroblasts to neurons and plays a role in maintaining neuronal identity. Previous analyses have indicated roles in both transcriptional activation and repression and suggested that Myt1 and Myt1l may have opposing functions in gene expression. We show that when targeted to DNA via multiple copies of the consensus Myt1/Myt1l binding site Myt1 represses transcription, whereas Myt1l activates. By targeting via a heterologous DNA binding domain we mapped an activation function in Myt1l to an amino-terminal region that is poorly conserved in Myt1. However, genome wide analyses of the effects of Myt1 and Myt1l expression in a glioblastoma cell line suggest that the two proteins have largely similar effects on endogenous gene expression. Transcriptional repression is likely mediated by binding to DNA via the known consensus site, whereas this site is not associated with the transcriptional start sites of genes with higher expression in the presence of Myt1 or Myt1l. This work suggests that these two proteins function similarly, despite differences observed in analyses based on synthetic reporter constructs.

The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

BACKGROUND: Phosphoinositide-3 (PI-3) kinase signaling has a pervasive role in cancer. One of the key effectors of PI-3 kinase signaling is AKT, a kinase that promotes growth and survival in a variety of cancers. Genetically engineered mouse models of prostate cancer have shown that AKT signaling is sufficient to induce prostatic epithelial neoplasia (PIN), but insufficient for progression to adenocarcinoma. This contrasts with the phenotype of mice with prostate-specific deletion of Pten, where excessive PI-3 kinase signaling induces both PIN and locally invasive carcinoma. We reasoned that additional PI-3 kinase effector kinases promote prostate cancer progression via activities that provide biological complementarity to AKT. We focused on the PKN kinase family members, which undergo activation in response to PI-3 kinase signaling, show expression changes in prostate cancer, and contribute to cell motility pathways in cancer cells. METHODS: PKN kinase activity was measured by incorporation of 32 P into protein substrates. Phosphorylation of the turn-motif (TM) in PKN proteins by mTOR was analyzed using the TORC2-specific inhibitor torin and a PKN1 phospho-TM-specific antibody. Amino acid substitutions in the TM of PKN were engineered and assayed for effects on kinase activity. Cell motility-related functions and PKN localization was analyzed by depletion approaches and immunofluorescence microscopy, respectively. The contribution of PKN proteins to prostate tumorigenesis was characterized in several mouse models that express PKN transgenes. The requirement for PKN activity in prostate cancer initiated by loss of phosphatase and tensin homolog deleted on chromosome 10 (Pten), and the potential redundancy between PKN isoforms, was analyzed by prostate-specific deletion of Pkn1, Pkn2, and Pten. RESULTS AND CONCLUSIONS: PKN1 and PKN2 contribute to motility pathways in human prostate cancer cells. PKN1 and PKN2 kinase activity is regulated by TORC2-dependent phosphorylation of the TM, which together with published data indicates that PKN proteins receive multiple PI-3 kinase-dependent inputs. Transgenic expression of active AKT and PKN1 is not sufficient for progression beyond PIN. Moreover, Pkn1 is not required for tumorigenesis initiated by loss of Pten. Triple knockout of Pten, Pkn1, and Pkn2 in mouse prostate results in squamous cell carcinoma, an uncommon but therapy-resistant form of prostate cancer.

Genetic and Molecular Analyses indicate independent effects of TGIFs on Nodal and Gli3 in neural tube patterning.

Holoprosencephaly (HPE) is a prevalent craniofacial developmental disorder that has both genetic and environmental causes. The gene encoding TG-interacting factor 1 (TGIF1) is among those that are routinely screened in HPE patients. However, the mechanisms by which TGIF1 variants cause HPE are not fully understood. TGIF1 is a transcriptional repressor that limits the output of the Transforming Growth Factor ß (TGFß)/Nodal signaling pathway, and HPE in patients with TGIF1 variants has been suggested to be due to increased Nodal signaling. Mice lacking both Tgif1 and its paralog, Tgif2, have HPE, and embryos lacking Tgif function do not survive past mid-gestation. Here, we show that in the presence of a Nodal heterozygous mutation, proliferation defects are rescued and a proportion of embryos lacking all Tgif function survive to late gestation. However, these embryos have a classic HPE phenotype, suggesting that this is a Nodal-independent effect of Tgif loss of function. Further, we show that the Gli3 gene is a direct target for repression by Tgifs, independent of TGFß/Nodal signaling, consistent with Tgif mutations causing HPE via Nodal-independent effects on the Sonic Hedgehog (Shh) pathway. Based on this work, we propose a model for distinct functions of Tgifs in the Nodal and Shh/Gli3 pathways during forebrain development.

Prostate cancer induced by loss of Apc is restrained by TGFß signaling.

Recent work with mouse models of prostate cancer (CaP) has shown that inactivation of TGFß signaling in prostate epithelium can cooperate with deletion of the Pten tumor suppressor to drive locally aggressive cancer and metastatic disease. Here, we show that inactivating the TGFß pathway by deleting the gene encoding the TGFß type II receptor (Tgfbr2) in combination with a deletion of the Apc tumor suppressor gene specifically in mouse prostate epithelium, results in the rapid onset of invasive CaP. Micro-metastases were observed in the lymph nodes and lungs of a proportion of the double mutant mice, whereas no metastases were observed in Apc single mutant mice. Prostate-specific Apc;Tgfbr2 mutants had a lower frequency of metastasis and survived significantly longer than Pten;Tgfbr2 double mutants. However, all Apc;Tgfbr2 mutants developed invasive cancer by 30 weeks of age, whereas invasive cancer was rarely observed in Apc single mutant animals, even by one year of age. Further comparison of the Pten and Apc models of CaP revealed additional differences, including adenosquamous carcinoma in the Apc;Tgfbr2 mutants that was not seen in the Pten model, and a lack of robust induction of the TGFß pathway in Apc null prostate. In addition to causing high-grade prostate intra-epithelial neoplasia (HGPIN), deletion of either Pten or Apc induced senescence in affected prostate ducts, and this restraint was overcome by loss of Tgfbr2. In summary, this work demonstrates that TGFß signaling restrains the progression of CaP induced by different tumor suppressor mutations, suggesting that TGFß signaling exerts a general tumor suppressive effect in prostate.

TG-interacting factor 1 acts as a transcriptional repressor of sterol O-acyltransferase 2.

Acat2 [gene name: sterol O-acyltransferase 2 (SOAT2)] esterifies cholesterol in enterocytes and hepatocytes. This study aims to identify repressor elements in the human SOAT2 promoter and evaluate their in vivo relevance. We identified TG-interacting factor 1 (Tgif1) to function as an important repressor of SOAT2. Tgif1 could also block the induction of the SOAT2 promoter activity by hepatocyte nuclear factor 1α and 4α. Women have ∼ 30% higher hepatic TGIF1 mRNA compared with men. Depletion of Tgif1 in mice increased the hepatic Soat2 expression and resulted in higher hepatic lipid accumulation and plasma cholesterol levels. Tgif1 is a new player in human cholesterol metabolism.

Inhibition of CtBP1 activity by Akt-mediated phosphorylation.

Pc2 (Cbx4) is a member of the chromobox family of polycomb proteins, and is a SUMO E3 ligase for the transcriptional corepressor CtBP1. Here, we show that both CtBP1 and Pc2 are phosphorylated by the kinase Akt1, which is activated by growth factor signaling via the PI3-kinase pathway. In the presence of Pc2, phosphorylation of CtBP1 is increased, and this requires interaction of both CtBP1 and Akt1 with Pc2. Pc2 promotes CtBP1 phosphorylation by recruiting Akt1 and, in part, by preventing de-phosphorylation of activated Akt1. Alteration of the Akt-phosphorylated residue in CtBP1 to a phosphomimetic results in decreased CtBP1 dimerization, but does not prevent interaction with other transcriptional regulators. The phosphomimetic mutant of CtBP1 is expressed at a lower level than the wild type protein, resulting in decreased transcriptional repression. We show that this CtBP1 mutant is targeted for poly-ubiquitylation and is less stable than the wild type protein. Co-expression of Pc2 and Akt1 results in both phosphorylation and ubiquitylation of CtBP1, thereby targeting CtBP1 for degradation. This work suggests that Pc2 might coordinate multiple enzymatic activities to regulate CtBP1 function.

Tgif1 and Tgif2 regulate Nodal signaling and are required for gastrulation.

Tgif1 and Tgif2 are transcriptional co-repressors that limit the response to TGFbeta signaling and play a role in regulating retinoic-acid-mediated gene expression. Mutations in human TGIF1 are associated with holoprosencephaly, but it is unclear whether this is a result of deregulation of TGFbeta/Nodal signaling, or of effects on other pathways. Surprisingly, mutation of Tgif1 in mice results in only relatively mild developmental phenotypes in most strain backgrounds. Here, we show that loss-of-function mutations in both Tgif1 and Tgif2 result in a failure of gastrulation. By conditionally deleting Tgif1 in the epiblast, we demonstrate that a single wild-type allele of Tgif1 in the extra-embryonic tissue allows the double null embryos to gastrulate and begin organogenesis, suggesting that extra-embryonic Tgif function is required for patterning the epiblast. Genetically reducing the dose of Nodal in embryos lacking all Tgif function results in partial rescue of the gastrulation defects. Conditional double null embryos have defects in left-right asymmetry, which are also alleviated by reducing the dose of Nodal. Together, these data show that Tgif function is required for gastrulation, and provide the first clear evidence that Tgifs limit the transcriptional response to Nodal signaling during early embryogenesis.

The Tgif2 gene contains a retained intron within the coding sequence.

BACKGROUND: TGIF and TGIF2 are homeodomain proteins, which act as TGFbeta specific Smad transcriptional corepressors. TGIF recruits general repressors including mSin3 and CtBP. The related TGIF2 protein functions in a similar manner, but does not bind CtBP. In addition to repressing TGFbeta activated gene expression, TGIF and TGIF2 repress gene expression by binding directly to DNA. TGIF and TGIF2 share two major blocks of similarity, encompassing the homeodomain, and a conserved carboxyl terminal repression domain. Here we characterize two splice variants of the Tgif2 gene from mouse and demonstrate that the Tgif2 gene contains a retained intron. RESULTS: By PCR from mouse cDNA, we identified two alternate splice forms of the Tgif2 gene. One splice variant encodes the full length 237 amino acid Tgif2, whereas the shorter form results in the removal of 39 codons from the centre of the coding region. The generation of this alternate splice form occurs with the mouse RNA, but not the human, and both splice forms are present in all mouse tissues analyzed. Human and mouse Tgif2 coding sequences contain a retained intron, which in mouse Tgif2 is removed by splicing from around 25-50% of RNAs, as assessed by RT-PCR. This splicing event is dependent on sequences within the mouse Tgif2 coding sequence. Both splice forms of mouse Tgif2 encode proteins which are active transcriptional repressors, and can repress both TGFbeta dependent and independent transcription. In addition, we show that human and mouse Tgif2 interact with the transcriptional corepressor mSin3. CONCLUSION: These data demonstrate that the Tgif2 gene contains a retained intron, within the second coding exon. This retained intron is not removed from the human mRNA at a detectable level, but is spliced out in a significant proportion of mouse RNAs. This alternate splicing is dependent entirely on sequences within the mouse Tgif2 coding sequence, suggesting the presence of an exonic splicing enhancer. Both splice forms of mouse Tgif2 produce proteins which are functional transcriptional repressors.

TGIF inhibits retinoid signaling.

TGIF (TG-interacting factor) represses transforming growth factor beta (TGF-beta)-activated gene expression and can repress transcription via a specific retinoid response element. Mutations in human TGIF are associated with holoprosencephaly, a severe defect of craniofacial development with both genetic and environmental causes. Both TGF-beta and retinoic acid signaling are implicated in craniofacial development. Here, we analyze the role of TGIF in regulating retinoid responsive gene expression. We demonstrate that TGIF interacts with the ligand binding domain of the RXRalpha retinoid receptor and represses transcription from retinoid response elements. TGIF recruits the general corepressor, CtBP, to RXRalpha, and this recruitment is required for full repression by TGIF. Interaction between TGIF and RXRalpha is reduced by the addition of retinoic acid, consistent with a role for TGIF as an RXRalpha transcriptional corepressor. We created a Tgif null mutation in mice and tested the sensitivity of mutant mice to increased levels of retinoic acid. Tgif mutant embryos are more sensitive to retinoic acid-induced teratogenesis, and retinoid target genes are expressed at a higher level in tissues from Tgif null mice. These results demonstrate an important role for TGIF as a transcriptional corepressor, which regulates developmental signaling by retinoic acid, and raises the possibility that TGIF may repress other RXR-dependent transcriptional responses.