Faculty Bibliography

PTPRD is a receptor protein tyrosine phosphatase that is genetically associated with neurodevelopmental disorders. Here, we asked whether Ptprd mutations cause aberrant neural development by perturbing neurogenesis in the murine cortex. We show that loss of Ptprd causes increases in neurogenic transit-amplifying intermediate progenitor cells and cortical neurons and perturbations in neuronal localization. These effects are intrinsic to neural precursor cells since acute Ptprd knockdown causes similar perturbations. PTPRD mediates these effects by dephosphorylating receptor tyrosine kinases, including TrkB and PDGFRβ, and loss of Ptprd causes the hyperactivation of TrkB and PDGFRβ and their downstream MEK-ERK signaling pathway in neural precursor cells. Moreover, inhibition of aberrant TrkB or MEK activation rescues the increased neurogenesis caused by knockdown or homozygous loss of Ptprd. These results suggest that PTPRD regulates receptor tyrosine kinases to ensure appropriate numbers of intermediate progenitor cells and neurons, suggesting a mechanism for its genetic association with neurodevelopmental disorders.

SHP2, encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germline mutations in PTPN11 gene responsible for catalytic gain- or loss- of function of SHP2 cause two disorders with multiple organ defects, respectively Noonan syndrome (NS) and NS with Multiple Lentigines (NSML). Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in two mouse models carrying PTPN11 mutations responsible for these two syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists, and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and a_IIbb₃ integrin signaling, platelet secretion and TXA₂ generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice associated with a significant increase in the tail bleeding time. In contrast, NSML mice platelets exhibited increased platelet activation following GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress-dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects and provides important information for the medical care of patients with NS in situations at risk of bleeding.

The cell-of-origin of high grade serous ovarian carcinoma (HGSOC) remains controversial, with fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) both considered candidates. Here, by using genetically engineered mouse models and organoids, we assessed the tumor-forming properties of FTE and OSE harboring the same oncogenic abnormalities. Combined RB family inactivation and Tp53 mutation in Pax8 + FTE caused Serous Tubal Intraepithelial Carcinoma (STIC), which metastasized rapidly to the ovarian surface. These events were recapitulated by orthotopic injection of mutant FTE organoids. Engineering the same genetic lesions into Lgr5 + OSE or OSE-derived organoids also caused metastatic HGSOC, although with longer latency and lower penetrance. FTE- and OSE-derived tumors had distinct transcriptomes, and comparative transcriptomics and genomics suggest that human HGSOC arises from both cell types. Finally, FTE- and OSE-derived organoids exhibited differential chemosensitivity. Our results comport with a dualistic origin for HGSOC and suggest that the cell-of-origin might influence therapeutic response.

The cell-of-origin of high grade serous ovarian carcinoma (HGSC) has been a focus of debate. Here, by using genetic mouse models as well as cognate organoid systems, we assessed the tumor forming capacity and properties of the fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) bearing the same oncogenic abnormalities. Combined RB family inactivation (via T121 expression) and Tp53 mutation in Pax8+ fallopian tube secretory cells causes transformation and characteristics of Serous Tubal Intraepithelial Carcinoma (STIC). This genetically engineered mouse HGSC model is faithfully recapitulated in fallopian organoids, from which serous ovarian cancer with broad abdominal metastasis is generated upon orthotopic injection. The same genetic events in Lgr5+ OSE cells organdies derived from these cells also result in an apparent neoplastic process, expressing markers of early serous carcinoma (but not Pax8), which subsequently develop into serous-like tumors. Hence, both Pax8+ fallopian tube epithelial cells and Lgr5+ ovarian surface epithelial cells can undergo similar neoplastic transformation, suggesting that HGSC might derive from distinct cell and tissue sources. Similar organoid systems can be used to rapidly model other gene combinations associated with HGSC

Introduction/Background Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumour heterogeneity of OC are limited and hard to establish. Methodology We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Results Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra-and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumour subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Conclusion Taken together, this demonstrates their potential application for research and personalized medicine. (Figure Presented)

High grade serous ovarian cancer (HGSC) is typically diagnosed at an advanced stage and the vast majority of patients relapse and die within 5 years of diagnosis. Significant clinical problems in HGSC include wide-spread abdominal dissemination of disease and chemotherapy resistance. Cancer-associated fibroblasts (CAFs) have been shown to play a role in promotion of cancer cell proliferation and invasion, and mediation of chemotherapy resistance. To interrogate the molecular properties of CAFs in HGSC we used fluorescence activated cell sorting to isolate CAFs directly from primary tumor samples and performed gene expression profiling. We found that patients stratify into two classes based on their CAF gene signatures: One with high expression of Fibroblast Activation Protein (FAP-High; FH) and one with low expression of FAP (FAP-Low; FL). FH CAFs express classical CAF genes whereas FL CAFs possesses a preadipocyte-like molecular signature. The FL phenotype has remained largely unnoticed as it is generally out-competed in vitro by FH cells when grown under classical CAF culture conditions. Patients from The Cancer Genome Atlas (TCGA), as well as from our own institute, can be stratified into FH and FL subtypes; in both cohorts patients with FH CAFs have a significantly shorter disease-free and overall survival. In vitro and in vivo functional assays performed with isolated CAFs of both types indicate that FH CAFs aggressively promote proliferation, invasion and therapy resistance of cancer cells, whereas FL CAFs do not. Finally, we identified TCF21, a transcriptional repressor, as a FL-specific transcription factor. Analysis of published TCF21 ChIP-Seq data indicates that TCF21 targets a large number of genes specific to FH CAFs. Overexpression of TCF21 in FH CAFs partially reversed their ability to promote cancer cell invasion and tumor growth. Our discovery of CAF heterogeneity in HGSC highlights the need to personalize patient treatment with respect to both cancer and stromal phenotypes. FH patients may benefit from inhibition of cancer-stroma interactions or from epigenetic modulators that reprogram cancer-promoting FH CAFs into the non-supportive FL state

RAS-ERK signaling pathway regulates large numbers of biological processes. Thus, germline mutations in RAS-ERK signaling pathway are associated with developmental disorders, collectively called RASopathy. BRAF is a direct downstream effector of RAS and germline mutations of BRAF accounts for over 70% of cardio-facio-cutaneous (CFC) syndrome which severely impairs cognitive functions. However, the cellular and molecular basis for the brain abnormalities in CFC syndrome remains largely unknown. Here, we investigated how the dysregulated RAS-ERK signaling affects malformation of central nervous system (CNS) and cognitive deficits by using conditional knockin mice harboring RASopathy-associated mutations in Braf. Neural stem cell-specific expression of Braf mutations significantly impaired hippocampus-dependent learning and memory in Morris water maze. Histological examination and transcriptome analyses revealed changes in specific cell types although mutants were expressed in neural stem cells, suggesting that the impacts of expressing Braf mutations are sensitive to specific cell types as previously shown in other RASopathy mouse models. Our study will contribute to untangle the complexity of RAS-ERK signaling network in brain development and cognitive functions.
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Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction-oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose-based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich H₂O₂-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain-containing phosphatase-2 and PKM2 oxidation in cells stimulated with platelet-derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.-Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells' transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumor's aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirmed these findings by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival.