RNA-binding motif protein 10 inactivates c-Myc by partnering with ribosomal proteins uL18 and uL5.

RNA-binding motif protein 10 (RBM10) is a frequently mutated tumor suppressor in lung adenocarcinoma (LUAD). Yet, it remains unknown whether cancer-derived mutant RBM10 compromises its tumor suppression function and, if so, the molecular insight of the underlying mechanisms. Here, we show that wild-type RBM10 suppresses lung cancer cell growth and proliferation by inactivating c-Myc that is essential for cancer cell survival. RBM10 directly binds to c-Myc and promotes c-Myc's ubiquitin-dependent degradation, while RBM10 knockdown leads to the induction of c-Myc level and activity. This negative action on c-Myc is further boosted by ribosomal proteins (RPs) uL18 (RPL5) and uL5 (RPL11) via their direct binding to RBM10. Cancer-derived mutant RBM10-I316F fails to bind to uL18 and uL5 and to inactivate c-Myc, thus incapable of suppressing tumorigenesis. Our findings uncover RBM10 as a pivotal c-Myc repressor by cooperating with uL18 and uL5 in lung cancer cells, as its failure to do so upon mutation favors tumorigenesis.

Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells.

Cancer cells survive chemotherapy and cause lethal relapse by entering a senescent state that facilitates expression of many phagocytosis/macrophage-related genes that engender a novel cannibalism phenotype. We used biosensors and live-cell imaging to reveal the basic steps and mechanisms of engulfment by senescent human and mouse tumor cells. We show filamentous actin in predator cells was localized to the prey cell throughout the process of engulfment. Biosensors to various phosphoinositide (PI) species revealed increased concentration and distinct localization of predator PI(4) P and PI(4,5)P2 at the prey cell during early stages of engulfment, followed by a transient burst of PI(3) P before and following internalization. PIK3C2B, the kinase responsible for generating PI(3)P, was required for complete engulfment. Inhibition or knockdown of Clathrin, known to associate with PIK3C2B and PI(4,5)P2, severely impaired engulfment. In sum, our data reveal the most fundamental cellular processes of senescent cell engulfment, including the precise localizations and dynamics of actin and PI species throughout the entire process.

Mouse model and human patient data reveal critical roles for Pten and p53 in suppressing POLE mutant tumor development.

Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole +/P286R;Trp53 +/- mice showed accelerated cancer mortality compared to Pole +/P286R;Trp53 +/+ mice. Cells from Pole +/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. However, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V, was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole +/P286R mice that spontaneously acquired PtenF341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Re-analysis of human TCGA (The Cancer Genome Atlas) data showed that all PTEN-F341V mutations occurred in tumors with mutations in POLE. Taken together with recent published work, our results support the idea that development of POLE mutant tumors may involve disabling surveillance of nuclear DNA damage in addition to POLE-mediated hypermutagenesis.

Pregnancy reprograms the epigenome of mammary epithelial cells and blocks the development of premalignant lesions.

Pregnancy causes a series of cellular and molecular changes in mammary epithelial cells (MECs) of female adults. In addition, pregnancy can also modify the predisposition of rodent and human MECs to initiate oncogenesis. Here, we investigate how pregnancy reprograms enhancer chromatin in the mammary epithelium of mice and influences the transcriptional output of the oncogenic transcription factor cMYC. We find that pregnancy induces an expansion of the active cis-regulatory landscape of MECs, which influences the activation of pregnancy-related programs during re-exposure to pregnancy hormones in vivo and in vitro. Using inducible cMYC overexpression, we demonstrate that post-pregnancy MECs are resistant to the downstream molecular programs induced by cMYC, a response that blunts carcinoma initiation, but does not perturb the normal pregnancy-induced epigenomic landscape. cMYC overexpression drives post-pregnancy MECs into a senescence-like state, and perturbations of this state increase malignant phenotypic changes. Taken together, our findings provide further insight into the cell-autonomous signals in post-pregnancy MECs that underpin the regulation of gene expression, cellular activation, and resistance to malignant development.

BH3 mimetics selectively eliminate chemotherapy-induced senescent cells and improve response in TP53 wild-type breast cancer.

TP53 wild-type breast tumors rarely undergo a complete pathological response after chemotherapy treatment. These patients have an extremely poor survival rate and studies show these tumors preferentially undergo senescence instead of apoptosis. These senescent cells persist after chemotherapy and secrete cytokines and chemokines comprising the senescence associated secretory phenotype, which promotes survival, proliferation, and metastasis. We hypothesized that eliminating senescent tumor cells would improve chemotherapy response and extend survival. Previous studies have shown "senolytic" agents selectively kill senescent normal cells, but their efficacy in killing chemotherapy-induced senescent cancer cells is unknown. We show that ABT-263, a BH3 mimetic that targets antiapoptotic proteins BCL2/BCL-XL/BCL-W, had no effect on proliferating cells, but rapidly and selectively induced apoptosis in a subset of chemotherapy-treated cancer cells, though sensitivity required days to develop. Low NOXA expression conferred resistance to ABT-263 in some cells, necessitating additional MCL1 inhibition. Gene editing confirmed breast cancer cells relied on BCL-XL or BCL-XL/MCL1 for survival in senescence. In a mouse model of breast cancer, ABT-263 treatment following chemotherapy led to apoptosis, greater tumor regression, and longer survival. Our results reveal cancer cells that have survived chemotherapy by entering senescence can be eliminated using BH3 mimetic drugs that target BCL-XL or BCL-XL/MCL1. These drugs could help minimize residual disease and extend survival in breast cancer patients that otherwise have a poor prognosis and are most in need of improved therapies.

Allele-specific enhancer interaction at the Peg3 imprinted domain.

The parental allele specificity of mammalian imprinted genes has been evolutionarily well conserved, although its functional constraints and associated mechanisms are not fully understood. In the current study, we generated a mouse mutant with switched active alleles driving the switch from paternal-to-maternal expression for Peg3 and the maternal-to-paternal expression for Zim1. The expression levels of Peg3 and Zim1, but not the spatial expression patterns, within the brain showed clear differences between wild type and mutant animals. We identified putative enhancers localized upstream of Peg3 that displayed allele-biased DNA methylation, and that also participate in allele-biased chromosomal conformations with regional promoters. Most importantly, these data suggest for the first time that long-distance enhancers may contribute to allelic expression within imprinted domains through allele-biased interactions with regional promoters.

Chemotherapy-induced senescent cancer cells engulf other cells to enhance their survival.

In chemotherapy-treated breast cancer, wild-type p53 preferentially induces senescence over apoptosis, resulting in a persisting cell population constituting residual disease that drives relapse and poor patient survival via the senescence-associated secretory phenotype. Understanding the properties of tumor cells that allow survival after chemotherapy treatment is paramount. Using time-lapse and confocal microscopy to observe interactions of cells in treated tumors, we show here that chemotherapy-induced senescent cells frequently engulf both neighboring senescent or nonsenescent tumor cells at a remarkable frequency. Engulfed cells are processed through the lysosome and broken down, and cells that have engulfed others obtain a survival advantage. Gene expression analysis showed a marked up-regulation of conserved macrophage-like program of engulfment in chemotherapy-induced senescent cell lines and tumors. Our data suggest compelling explanations for how senescent cells persist in dormancy, how they manage the metabolically expensive process of cytokine production that drives relapse in those tumors that respond the worst, and a function for their expanded lysosomal compartment.

Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment.

BACKGROUND: Previous studies on the role of TP53 mutation in breast cancer treatment response and survival are contradictory and inconclusive, limited by the use of different endpoints to determine clinical significance and by small sample sizes that prohibit stratification by treatment. METHODS: We utilized large datasets to examine overall survival according to TP53 mutation status in patients across multiple clinical features and treatments. RESULTS: Confirming other studies, we found that in all patients and in hormone therapy-treated patients, TP53 wild-type status conferred superior 5-year overall survival, but survival curves crossed at 10 or more years. In contrast, further stratification within the large dataset revealed that in patients receiving chemotherapy and no hormone therapy, wild-type TP53 status conferred remarkably poor overall survival. This previously unrecognized inferior survival is consistent with p53 inducing arrest/senescence instead of apoptosis. Addition of hormone therapy to chemotherapy improved survival notably in patients with TP53 wild-type tumors, but not mutant, suggesting hormone therapy could eradicate arrested/senescent cells. Testing this, we found that estrogen receptor-positive, TP53 wild-type breast cancer cells that were made senescent by doxorubicin treatment were sensitive to tamoxifen. CONCLUSIONS: The poor survival of chemotherapy-treated patients with TP53 wild-type tumors may be improved by strategies to eliminate senescent cells, including the addition of hormone therapy when appropriate.

Oxytocin receptor is regulated by Peg3.

Mouse Peg3 encodes a DNA-binding protein involved in the milk letdown process. In the current study, we tested whether PEG3 controls the expression of the oxytocin receptor gene. According to the results, PEG3 directly binds to a genomic region within the 3rd exon of Oxtr, which contains a DNA-binding motif for PEG3. In nursing female mice, removal of PEG3 resulted in the increased expression of Oxtr in mammary epithelial cells and also in the hypothalamus. This suggests a repressor role of PEG3 in the expression of Oxtr in these tissues. Overall, this study suggests that Peg3 may function as a direct transcriptional regulator for Oxtr expression that acts to moderate the milk letdown process.

p53 Mediates Vast Gene Expression Changes That Contribute to Poor Chemotherapeutic Response in a Mouse Model of Breast Cancer.

p53 is a transcription factor that regulates expression of genes involved in cell cycle arrest, senescence, and apoptosis. TP53 harbors mutations that inactivate its transcriptional activity in roughly 30% of breast cancers, and these tumors are much more likely to undergo a pathological complete response to chemotherapy. Thus, the gene expression program activated by wild-type p53 contributes to a poor response. We used an in vivo genetic model system to comprehensively define the p53- and p21-dependent genes and pathways modulated in tumors following doxorubicin treatment. We identified genes differentially expressed in spontaneous mammary tumors harvested from treated MMTV-Wnt1 mice that respond poorly (Trp53+/+) or favorably (Trp53-null) and those that lack the critical senescence/arrest p53 target gene Cdkn1a. Trp53 wild-type tumors differentially expressed nearly 10-fold more genes than Trp53-null tumors after treatment. Pathway analyses showed that genes involved in cell cycle, senescence, and inflammation were enriched in treated Trp53 wild-type tumors; however, no genes/pathways were identified that adequately explain the superior cell death/tumor regression observed in Trp53-null tumors. Cdkn1a-null tumors that retained arrest capacity (responded poorly) and those that proliferated (responded well) after treatment had remarkably different gene regulation. For instance, Cdkn1a-null tumors that arrested upregulated Cdkn2a (p16), suggesting an alternative, p21-independent route to arrest. Live animal imaging of longitudinal gene expression of a senescence/inflammation gene reporter in Trp53+/+ tumors showed induction during and after chemotherapy treatment, while tumors were arrested, but expression rapidly diminished immediately upon relapse.

Allele and dosage specificity of the Peg3 imprinted domain.

The biological impetus for gene dosage and allele specificity of mammalian imprinted genes is not fully understood. To address this, we generated and analyzed four sets of mice from a single breeding scheme with varying allelic expression and gene dosage of the Peg3 domain. The mutants with abrogation of the two paternally expressed genes, Peg3 and Usp29, showed a significant decrease in growth rates for both males and females, while the mutants with biallelic expression of Peg3 and Usp29 resulted in an increased growth rate of female mice only. The mutant cohort with biallelic expression of Peg3 and Usp29 tended to have greater numbers of pups compared to the other genotypes. The mutants with switched active alleles displayed overall similar phenotypes to the wild type, but did show some differences in gene expression, suggesting potential non-redundant roles contributed by the maternal and paternal alleles. Overall, this study demonstrates a novel in vivo approach to investigate the allele and dosage specificity of mammalian imprinted domains.

BPTF Maintains Chromatin Accessibility and the Self-Renewal Capacity of Mammary Gland Stem Cells.

Chromatin remodeling is a key requirement for transcriptional control of cellular differentiation. However, the factors that alter chromatin architecture in mammary stem cells (MaSCs) are poorly understood. Here, we show that BPTF, the largest subunit of the NURF chromatin remodeling complex, is essential for MaSC self-renewal and differentiation of mammary epithelial cells (MECs). BPTF depletion arrests cells at a previously undefined stage of epithelial differentiation that is associated with an incapacity to achieve the luminal cell fate. Moreover, genome-wide analysis of DNA accessibility following genetic or chemical inhibition, suggests a role for BPTF in maintaining the open chromatin landscape at enhancers regions in MECs. Collectively, our study implicates BPTF in maintaining the unique epigenetic state of MaSCs.

Tissue-Specific Contributions of Paternally Expressed Gene 3 in Lactation and Maternal Care of Mus musculus.

Paternally Expressed Gene 3 (Peg3) is an imprinted gene that controls milk letdown and maternal-caring behaviors. In this study, a conditional knockout allele has been developed in Mus musculus to further characterize these known functions of Peg3 in a tissue-specific manner. The mutant line was first crossed with a germline Cre. The progeny of this cross displayed growth retardation phenotypes. This is consistent with those seen in the previous mutant lines of Peg3, confirming the usefulness of the new mutant allele. The mutant line was subsequently crossed individually with MMTV- and Nkx2.1-Cre lines to test Peg3's roles in the mammary gland and hypothalamus, respectively. According to the results, the milk letdown process was impaired in the nursing females with the Peg3 mutation in the mammary gland, but not in the hypothalamus. This suggests that Peg3's roles in the milk letdown process are more critical in the mammary gland than in the hypothalamus. In contrast, one of the maternal-caring behaviors, nest-building, was interrupted in the females with the mutation in both MMTV- and Nkx2.1-driven lines. Overall, this is the first study to introduce a conditional knockout allele of Peg3 and to further dissect its contribution to mammalian reproduction in a tissue-specific manner.

APeg3: regulation of Peg3 through an evolutionarily conserved ncRNA.

Mammalian APeg3 is an antisense gene that is localized within the 3'-untranslated region of the imprinted gene, Peg3. APeg3 is expressed only in the vasopressinergic neurons of the hypothalamus, thus is predicted to play significant roles in this specific area of the brain. In the current study, we investigate the functions of APeg3 with comparative genomics and cell line-based functional approaches. The transcribed region of APeg3 displays high levels of sequence conservation among placental mammals, but without any obvious open reading frame, suggesting that APeg3 may have been selected as a ncRNA gene during eutherian evolution. This has been further supported by the detection of a conserved local RNA secondary structure within APeg3. RNA secondary structure analyses indicate a single conserved hairpin-loop structure towards the 5' end of the transcript. The results from cell line-based transfection experiments demonstrate that APeg3 has the potential to down-regulate the transcription and protein levels of Peg3. The observed down-regulation by APeg3 is also somewhat orientation-independent. Overall, these results suggest that APeg3 has evolved as a ncRNA gene and controls the function of its sense gene Peg3 within specific neuronal cells.

Identification of an evolutionarily conserved cis-regulatory element controlling the Peg3 imprinted domain.

The mammalian Peg3 domain harbors more than 20 evolutionarily conserved regions (ECRs) that are spread over the 250-kb genomic interval. The majority of these ECRs are marked with two histone modifications, H3K4me1 and H3K27ac, suggesting potential roles as distant regulatory elements for the transcription of the nearby imprinted genes. In the current study, the chromatin conformation capture (3C) method was utilized to detect potential interactions of these ECRs with the imprinted genes. According to the results, one region, ECR18, located 200-kb upstream of Peg3 interacts with the two promoter regions of Peg3 and Zim2. The observed interaction is most prominent in brain, but was also detected in testis. Histone modification and DNA methylation on ECR18 show no allele bias, implying that this region is likely functional on both alleles. In vitro assays also reveal ECR18 as a potential enhancer or repressor for the promoter of Peg3. Overall, these results indicate that the promoters of several imprinted genes in the Peg3 domain interact with one evolutionarily conserved region, ECR18, and further suggest that ECR18 may play key roles in the transcription and imprinting control of the Peg3 domain as a distant regulatory element.

Peg3 mutational effects on reproduction and placenta-specific gene families.

Peg3 (paternally expressed gene 3) is an imprinted gene encoding a DNA-binding protein. This gene plays important roles in controlling fetal growth rates and nurturing behaviors. In the current study, a new mutant mouse model has been generated to further characterize the functions of this DNA-binding protein. Besides known phenotypes, this new mutant model also revealed potential roles of Peg3 in mammalian reproduction. Female heterozygotes produce a much smaller number of mature oocytes than the wild-type littermates, resulting in reduced litter sizes. According to genome-wide expression analyses, several placenta-specific gene families are de-repressed in the brain of Peg3 heterozygous embryos, including prolactin, cathepsin and carcinoembryonic antigen cell adhesion molecule (Ceacam) families. The observed de-repression is more pronounced in females than in males. The de-repression of several members of these gene families is observed even in the adult brain, suggesting potential defects in epigenetic setting of the placenta-specific gene families in the Peg3 mutants. Overall, these results indicate that Peg3 likely controls the transcription of several placenta-specific gene families, and further suggest that this predicted transcriptional control by Peg3 might be mediated through unknown epigenetic mechanisms.

Imprinting control region (ICR) of the Peg3 domain.

The imprinting and transcription of the 500 kb genomic region surrounding the mouse Peg3 is predicted to be regulated by the Peg3-differentially methylated region (DMR). In the current study, this prediction was tested using a mutant mouse line lacking this potential imprinting control region (ICR). At the organismal level, paternal and maternal transmission of this knockout (KO) allele caused either reduced or increased growth rates in the mouse, respectively. In terms of the imprinting control, the paternal transmission of the KO allele resulted in bi-allelic expression of the normally maternally expressed Zim2, whereas the maternal transmission switched the transcriptionally dominant allele for Zfp264 (paternal to maternal). However, the allele-specific DNA methylation patterns of the DMRs of Peg3, Zim2 and Zim3 were not affected in the mice that inherited the KO allele either paternally or maternally. In terms of the transcriptional control, the paternal transmission caused a dramatic down-regulation in Peg3 expression, but overall up-regulation in the other nearby imprinted genes. Taken together, deletion of the Peg3-DMR caused global changes in the imprinting and transcription of the Peg3 domain, confirming that the Peg3-DMR is an ICR for this imprinted domain.

The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles.

Aquatic photosynthetic organisms, such as the green alga Chlamydomonas reinhardtii, respond to low CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Carbonic anhydrases (CAs) are important components of the CCM. CAs are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO(2) and HCO(3)(-). In C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three alpha, six beta, and three gamma or gamma-like CAs. The expression of the three alpha and six beta genes has been measured from cells grown on elevated CO(2) (having no active CCM) versus cells growing on low levels of CO(2) (with an active CCM) using northern blots, differential hybridization to DNA chips and quantitative RT-PCR. Recent RNA-seq profiles add to our knowledge of the expression of all of the CA genes. In addition, protein content for some of the CA isoforms was estimated using antibodies corresponding to the specific CA isoforms: CAH1/2, CAH3, CAH4/5, CAH6, and CAH7. The intracellular location of each of the CA isoforms was elucidated using immunolocalization and cell fractionation techniques. Combining these results with previous studies using CA mutant strains, we will discuss possible physiological roles of the CA isoforms concentrating on how these CAs might contribute to the acquisition and retention of CO(2) in C. reinhardtii.