Dynamically generating T32 training documents using structured data.

BACKGROUND: The US National Institutes of Health (NIH) funds academic institutions for training doctoral (PhD) students and postdoctoral fellows. These training grants, known as T32 grants, require schools to create, in a particular format, seven or eight Word documents describing the program and its participants. Weill Cornell Medicine aimed to use structured name and citation data to dynamically generate tables, thus saving administrators time. CASE PRESENTATION: The author's team collected identity and publication metadata from existing systems of record, including our student information system and previous T32 submissions. These data were fed into our ReCiter author disambiguation engine. Well-structured bibliographic metadata, including the rank of the target author, were output and stored in a MySQL database. We then ran a database query that output a Word extensible markup (XML) document according to NIH's specifications. We generated the T32 training document using a query that ties faculty listed on a grant submission with publications that they and their mentees authored, bolding author names as required. Because our source data are well-structured and well-defined, the only parameter needed in the query is a single identifier for the grant itself. The open source code for producing this document is at http://dx.doi.org/10.5281/zenodo.2593545. CONCLUSIONS: Manually writing a table for T32 grant submissions is a substantial administrative burden; some documents generated in this manner exceed 150 pages. Provided they have a source for structured identity and publication data, administrators can use the T32 Table Generator to readily output a table.

The uterine epithelial loss of Pten is inefficient to induce endometrial cancer with intact stromal Pten.

Mutation of the tumor suppressor Pten often leads to tumorigenesis in various organs including the uterus. We previously showed that Pten deletion in the mouse uterus using a Pgr-Cre driver (Ptenf/fPgrCre/+) results in rapid development of endometrial carcinoma (EMC) with full penetration. We also reported that Pten deletion in the stroma and myometrium using Amhr2-Cre failed to initiate EMC. Since the Ptenf/fPgrCre/+ uterine epithelium was primarily affected by tumorigenesis despite its loss in both the epithelium and stroma, we wanted to know if Pten deletion in epithelia alone will induce tumorigenesis. We found that mice with uterine epithelial loss of Pten under a Ltf-iCre driver (Ptenf/f/LtfCre/+) develop uterine complex atypical hyperplasia (CAH), but rarely EMC even at 6 months of age. We observed that Ptenf/fPgrCre/+ uteri exhibit a unique population of cytokeratin 5 (CK5) and transformation related protein 63 (p63)-positive epithelial cells; these cells mark stratified epithelia and squamous differentiation. In contrast, Ptenf/fLtfCre/+ hyperplastic epithelia do not undergo stratification, but extensive epithelial cell apoptosis. This increased apoptosis is associated with elevation of TGFß levels and activation of downstream effectors, SMAD2/3 in the uterine stroma. Our results suggest that stromal PTEN via TGFß signaling restrains epithelial cell transformation from hyperplasia to carcinoma. In conclusion, this study, using tissue-specific deletion of Pten, highlights the epithelial-mesenchymal cross-talk in the genesis of endometrial carcinoma.

PI3K/PTEN/AKT Genetic Mouse Models of Endometrial Carcinoma.

The PI3K/PTEN/AKT pathway is the most frequently mutated pathway in endometrial carcinoma. Mouse models are invaluable tools to understand, at the molecular level, the contributions of components of this pathway towards initiation and progression of endometrial carcinoma. This chapter summarizes results of germline and tissue specific knockout mouse models generated to understand how mutations in components of this pathway lead to development of carcinoma and its interactions with other frequently altered pathways like mismatch repair and estrogen signaling. The mouse models show that loss of both alleles of Pten is necessary and sufficient for complex atypical hyperplasia (CAH) to develop but insufficient for progression to carcinoma. Additional events like mutations in Pik3ca or mismatch repair deficiency are required for progression to carcinoma. The models show that the interaction between Pten and estrogen signaling is complex. In the absence of estrogen, Pten loss is sufficient for development of CAH. Additionally, lack of ERα on a background of Pten loss leads to the development of carcinoma.

Activated mutant p110α causes endometrial carcinoma in the setting of biallelic Pten deletion.

PTEN and PIK3CA mutations occur with high frequency in uterine endometrioid carcinoma (UEC). Although PTEN mutations are present in complex atypical hyperplasia and carcinoma, PIK3CA mutations are restricted to carcinoma. We generated mouse models harboring Pten loss and/or activated Pik3ca in the endometrial epithelium to investigate their respective roles in the pathogenesis of UEC. Presence of an activated mutant Pik3ca on the background of Pten loss led to aggressive disease, with 100% of mice exhibiting carcinoma. Expression of Pik3ca with E545K mutation alone was unable to cause hyperplasia or cancer in the uterus and did not activate Akt as effectively as Pten deletion in short-term cultures of mouse endometrial epithelium, likely explaining the lack of phenotype in vivo. We also report that nuclear localization of FOXO1 correlated with PTEN mutational status irrespective of the PIK3CA status in endometrial cancer cell lines. Furthermore, gene expression profiles resulting from Pten loss or activation of Pik3ca in primary mouse endometrial epithelial cells exhibit minimal overlap. Thus, Pten and Pik3ca have distinct consequences on the activation of the phosphatidylinositol 3-kinase pathway in endometrial epithelium and are likely to affect other nonoverlapping cellular mechanisms involved in the development and progression of the most common type of uterine cancer.

Molecular alterations of PIK3CA in uterine carcinosarcoma, clear cell, and serous tumors.

OBJECTIVES: Type II endometrial carcinomas-uterine carcinosarcomas or uterine malignant mesodermal mixed tumors (UMMMTs), clear cell carcinomas (UCCs), and uterine serous carcinomas (USCs)-are aggressive malignancies that present with advanced disease and have high mortality rates. PIK3CA mutations are commonly found in endometrial cancers. The objective of the study was to characterize molecular alterations in the PIK3CA gene in these tumors. METHODS: A total of 84 cases (20 UMMMTs, 18 UCCs, and 46 USCs) were selected from the surgical pathology files of Weill Cornell Medical College and Johns Hopkins Hospital. The diagnoses were confirmed by gynecologic pathologists (L.H.E. and A.Y.). DNA was extracted from paraffin-embedded tissue. Polymerase chain reaction was performed for mutational analysis. All the studies were performed in accordance with approved Institutional Review Board protocols. RESULTS: Mutations in the PIK3CA gene were identified in 3 (15%) of 20 UMMMT, 3 (16.7%) of 18 UCC, and 10 (21.7%) of 46 USC cases. We report novel mutations in PIK3CA in uterine carcinosarcoma. CONCLUSIONS: A significant percentage of UMMMTs, UCCs, and USCs have mutations in PIK3CA. Further investigation is needed to develop targeted therapies for these aggressive uterine cancers.

Endometrial tumorigenesis in Pten(+/-) mice is independent of coexistence of estrogen and estrogen receptor α.

Numerous studies support the role for mutations in the phosphatase and tensin homologue (PTEN) tumor suppressor gene and unopposed estrogen stimulation in the pathogenesis of uterine endometrioid carcinoma. However, the relation between PTEN signaling and estrogen/estrogen receptor in endometrial tumorigenesis remains unresolved. We used genetically engineered mice as a model to address this relation. Mice with a single deleted Pten allele (Pten(+/-)) spontaneously develop complex atypical hyperplasia and ~20% develop endometrial cancer. To determine the effect of removing endogenous estrogen, we performed oophorectomies on Pten(+/-) mice. Although there was a reduction in the number and severity of hyperplastic lesions, the endometrial phenotype persisted, suggesting that Pten mutation, independent of estrogen, can initiate the development of complex atypical hyperplasia. To recapitulate the situation in women with unopposed estrogen, we implanted 17ß-estradiol pellets in adult female Pten heterozygous mice, resulting in increased carcinoma incidence. Because studies have shown that estrogen largely acts on the endometrium via estrogen receptor ERα, we generated Pten(+/-)ERα(-/-) mice. Strikingly, 88.9% of Pten(+/-)ERα(-/-) mice developed endometrial hyperplasia/carcinoma. Furthermore, Pten(+/-)ERα(-/-) mice showed a higher incidence of in situ and invasive carcinoma, suggesting that endometrial tumorigenesis can progress in the absence of ERα. Thus, the relation between Pten alterations and estrogen signaling in the development of endometrial carcinoma is complex; the results presented herein have important implications for the treatment of endometrial hyperplasia and carcinoma in women.

Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma.

Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten(+/-) mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten(+/-) mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ERα as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

Oviduct-specific glycoprotein is a molecular marker for invasion in endometrial tumorigenesis identified using a relevant mouse model.

The light microscopic distinction between complex atypical hyperplasia (CAH) and invasive endometrioid carcinoma (UEC) on endometrial sampling is problematic and often has significant clinical implications. Using mouse models of endometrial tumorigenesis based on two of the most common molecular alterations found in primary human UEC we sought to characterize the transition from CAH to carcinoma to identify clinically useful biomarkers. We used the previously described Pten(+/-); Mlh1(-/-) mouse model. DNA was isolated from microdissected lesions (CAH and carcinoma) and analyzed for LOH and mutations of Pten and additional candidate genes. To identify novel candidate genes associated with invasion, global gene expression profiles were compared from uteri with extensive CAH and carcinoma. The majority of CAHs and carcinomas, arising in this model showed biallelic inactivation of Pten mediated through LOH or intragenic mutation of the wild-type allele suggesting that complete loss of Pten is insufficient for the development of carcinoma. The global gene expression studies detected increased expression of oviduct-specific glycoprotein (OGP) in carcinoma as compared with CAHs. This finding was validated using immunohistochemical staining in a collection of primary human UECs and CAHs. Our studies identify a molecular marker for invasive endometrial cancer that may have clinical significance, and highlight the usefulness of this mouse model in not only understanding the genetic underpinnings of endometrial carcinoma, but as a tool to develop clinically relevant biomarkers.

Distinct properties of cyclin-dependent kinase complexes containing cyclin A1 and cyclin A2.

The distinct expression patterns of the two A-type cyclins during spermatogenesis and the absolute requirement for cyclin A1 in this biological process in vivo suggest that they may confer distinct biochemical properties to their CDK partners. We therefore compared human cyclin A1- and cyclin A2-containing CDK complexes in vitro by determining kinetic constants and by examining the complexes for their ability to phosphorylate pRb and p53. Differences in biochemical activity were observed in CDK2 but not CDK1 when complexed with cyclin A1 versus cyclin A2. Further, CDK1/cyclin A1 is a better kinase complex for phosphorylating potentially physiologically relevant substrates pRb and p53 than CDK2/cyclin A2. The activity of CDKs can therefore be regulated depending upon which A-type cyclin they bind and CDK1/cyclin A1 might be preferred in vivo.

Conditional loss of uterine Pten unfailingly and rapidly induces endometrial cancer in mice.

Etiology of endometrial cancer (EMC) is not fully understood. Animal models with rapidly and spontaneously developing EMC will help explore mechanisms of cancer initiation and progression. Pten(+/-) mice are currently being used as a model to study EMC. These females develop atypical endometrial hyperplasia of which approximately 20% progresses to EMC. In addition, tumors develop in other organs, complicating the use of this model to specifically study EMC. Here, we show that conditional deletion of endometrial Pten results in EMC in all female mice as early as age 1 month with myometrial invasion occurring by 3 months. In contrast, conditional deletion of endometrial p53 had no phenotype within this time frame. Whereas mice with endometrial Pten deletion had a life span of approximately 5 months, mice with combined deletion of endometrial Pten and p53 had a shorter life span with an exacerbated disease state. Such rapid development of EMC from homozygous loss of endometrial Pten suggests that this organ is very sensitive to this tumor suppressor gene for tumor development. All lesions at early stages exhibited elevated Cox-2 and phospho-Akt levels, hallmarks of solid tumors. More interestingly, levels of two microRNAs miR-199a(*) and miR-101a that posttranscriptionally inhibit Cox-2 expression were down-regulated in tumors in parallel with Cox-2 up-regulation. This mouse model in which the loxP-Cre system has been used to delete endometrial Pten and/or p53 allows us to study in detail the initiation and progression of EMC. These mouse models have the added advantage because they mimic several features of human EMC.

Cyclin A1-deficient mice lack histone H3 serine 10 phosphorylation and exhibit altered aurora B dynamics in late prophase of male meiosis.

Male mice lacking cyclin A1 protein are sterile. Their sterility results from an arrest in the meiotic cell cycle of spermatocytes, which we now identify as occurring at late diplotene, immediately before diakinesis. The stage of arrest in cyclin A1-deficient mice is distinct from the arrest seen in spermatocytes that are deficient in its putative catalytic partner Cdk2, which occurs much earlier in pachytene. The arrest in cyclin A1-deficient spermatocytes is also accompanied by an unusual clustering of centromeric heterochromatin. Consistent with a possible defect in the centromeric region, immunofluorescent staining of cyclin A1 protein shows localization in the region of the centromere. Phosphorylation of histone H3 at serine 10 in pericentromeric heterochromatin, which normally occurs in late diplotene, is reduced in spermatocytes from heterozygous Ccna1(+/-) testes and completely absent in spermatocytes with no cyclin A1 protein. Concomitantly, the levels of pericentromeric aurora B kinase, known to phosphorylate histone H3 during meiosis, are partially reduced in spermatocytes from testes of heterozygous mice and further reduced in homozygous null spermatocytes. These data suggest a critical and concentration-dependent function for cyclin A1 in the pericentromeric region in late diplotene of meiosis, perhaps in assembly or function of the passenger protein complex.

Insights into male germ cell apoptosis due to depletion of gonadotropins caused by GnRH antagonists.

The role of pituitary gonadotropins in the regulation of spermatogenesis has been unequivocally demonstrated, although, the precise mechanism of this regulation is not clearly understood. Previous studies have shown that specific immunoneutralization of LH/testosterone caused apoptotic cell death of meiotic and post-meiotic germ cells while that of FSH resulted in similar death of meiotic cells. In the present study, the death process of germ cells has been characterized by depleting both FSH and testosterone by administering two different potent GnRH antagonists, Cetrorelix and Acyline to both rats and mice. Pro-survival factors like Bcl-2 and Bcl-x/l were unaltered in germ cells due to GnRH antagonist treatment, although a significant increase in several pro-apoptotic markers including Fas and Bax were evident at both protein and RNA levels. This culminated in cytochrome C release from mitochondria and eventually increase in the activity of caspase-8 and caspase-3. These data suggest that both extrinsic and intrinsic apoptotic death pathways are operative in the germ cells death following decrease in FSH and testosterone levels. Multiple injections of GnRH antagonist resulted in complete disappearance of germ cells except the spermatogonial cells and discontinuation of the treatment resulted in full recovery of spermatogenesis. In conclusion our present data suggest that the principal role of FSH and testosterone is to maintain spermatogenic homeostasis by inhibiting death signals for the germ cells.

Expression of apoptotic genes in testicular germ cells and their modulation by luteinizing hormone.

Gonadotropins regulate spermatogenesis by promoting survival and differentiation of germ cells. The molecular markers that are modulated by these hormones to ensure survival however have not been described in great detail. Immunoneutralization of LH in particular leads to apoptotic cell death of the spermatocytes and the round spermatids. In the present study, the expression pattern and regulation of apoptotic markers after specific immunoneutralization of LH in germ cells purified from rats has been investigated at the RNA and protein level. Of the several markers tested, Bax, caspases 1 and 2 and Fadd exhibit differential expression, with the round spermatids expressing higher levels of caspases 1 and 2, and the spermatocytes expressing higher levels of Bax and Fadd. The two cell types therefore exhibit differential expression of apoptotic markers. The cell types also differ with respect to their response to LH antiserum treatment. Fas and Bax both are up-regulated in the round spermatids after 24h of antiserum treatment. In the spermatocytes, Fas was up-regulated as early as 12h after antiserum treatment while Bax was up-regulated after 2 days. These results demonstrate that LH regulates survival of germ cells by modulating the levels of pro and anti-apoptotic proteins.

Induction of apoptosis involving multiple pathways is a primary response to cyclin A1-deficiency in male meiosis.

The meiotic arrest in male mice null for the cyclin A1 gene (Ccna1) was associated with apoptosis of spermatocytes. To determine whether the apoptosis in spermatocytes was triggered in response to the arrest at G2/M phase, as opposed to being a secondary response to overall disruption of spermatogenesis, we examined testes during the first wave of spermatogenesis by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining. We observed enhanced apoptosis coinciding with the arrest point in postnatal day 22 tubules, with no overt degeneration. Along with activation of caspase-3, an increase in the levels and change of subcellular localization of Bax protein was observed in cyclin A1-deficient spermatocytes, which coincided with the detection of apoptosis. As p53 is implicated in the activation of Bax-mediated cell death, we generated mice lacking both cyclin A1 and p53. Although the absence of p53 did not rescue the meiotic arrest, there was a decrease in the number of apoptotic cells in the double-mutant testes. This finding suggested that p53 may be involved in the process by which the arrested germ cells are removed from the seminiferous tubules but that other pathways function as well to ensure removal of the arrested spermatocytes.