Genomic Landscape of Lynch Syndrome Colorectal Neoplasia Identifies Shared Mutated Neoantigens for Immunoprevention.

BACKGROUND & AIMS: Lynch syndrome (LS) carriers develop mismatch repair-deficient neoplasia with high neoantigen (neoAg) rates. No detailed information on targetable neoAgs from LS precancers exists, which is crucial for vaccine development and immune-interception strategies. We report a focused somatic mutation and frameshift-neoAg landscape of microsatellite loci from colorectal polyps without malignant potential (PWOMP), precancers, and early-stage cancers in LS carriers. METHODS: We generated paired whole-exome and transcriptomic sequencing data from 8 colorectal PWOMP, 41 precancers, 8 advanced precancers, and 12 early-stage cancers of 43 LS carriers. A computational pipeline was developed to predict, rank, and prioritize the top 100 detected mutated neoAgs that were validated in vitro using ELISpot and tetramer assays. RESULTS: Mutation calling revealed >10 mut/Mb in 83% of cancers, 63% of advanced precancers, and 20% of precancers. Cancers displayed an average of 616 MHC-I neoAgs/sample, 294 in advanced precancers, and 107 in precancers. No neoAgs were detected in PWOMP. A total of 65% of our top 100 predicted neoAgs were immunogenic in vitro, and were present in 92% of cancers, 50% of advanced precancers, and 29% of precancers. We observed increased levels of naïve CD8+ and memory CD4+ T cells in mismatch repair-deficient cancers and precancers via transcriptomics analysis. CONCLUSIONS: Shared frameshift-neoAgs are generated within unstable microsatellite loci at initial stages of LS carcinogenesis and can induce T-cell responses, generating opportunities for vaccine development, targeting LS precancers and early-stage cancers.

Comparative molecular genomic analyses of a spontaneous rhesus macaque model of mismatch repair-deficient colorectal cancer.

Colorectal cancer (CRC) remains the third most common cancer in the US with 15% of cases displaying Microsatellite Instability (MSI) secondary to Lynch Syndrome (LS) or somatic hypermethylation of the MLH1 promoter. A cohort of rhesus macaques from our institution developed spontaneous mismatch repair deficient (MMRd) CRC with a notable fraction harboring a pathogenic germline mutation in MLH1 (c.1029C

Current Trends in Vaccine Development for Hereditary Colorectal Cancer Syndromes.

The coming of age for cancer treatment has experienced exponential growth in the last decade with the addition of immunotherapy as the fourth pillar to the fundamentals of cancer treatment-chemotherapy, surgery, and radiation-taking oncology to an astounding new frontier. In this time, rapid developments in computational biology coupled with immunology have led to the exploration of priming the host immune system through vaccination to prevent and treat certain subsets of cancer such as melanoma and hereditary colorectal cancer. By targeting the immune system through tumor-specific antigens-namely, neoantigens (neoAgs)-the future of cancer prevention may lie within arm's reach by employing neoAg vaccines as an immune-preventive modality for hereditary cancer syndromes like Lynch syndrome. In this review, we discuss the history, current trends, utilization, and future direction of neoAg-based vaccines in the setting of hereditary colorectal cancer.

Enhancement of myogenic potential of muscle progenitor cells and muscle healing during pregnancy.

The decline of muscle regenerative potential with age has been attributed to a diminished responsiveness of muscle progenitor cells (MPCs). Heterochronic parabiosis has been used as a model to study the effects of aging on stem cells and their niches. These studies have demonstrated that, by exposing old mice to a young systemic environment, aged progenitor cells can be rejuvenated. One interesting idea is that pregnancy represents a unique biological model of a naturally shared circulatory system between developing and mature organisms. To test this hypothesis, we evaluated the muscle regeneration potential of pregnant mice using a cardiotoxin (CTX) injury mouse model. Our results indicate that the pregnant mice demonstrate accelerated muscle healing compared to nonpregnant control mice following muscle injury based on improved muscle histology, superior muscle regeneration, and a reduction in inflammation and necrosis. Additionally, we found that MPCs isolated from pregnant mice display a significant improvement of myogenic differentiation capacity in vitro and muscle regeneration in vivo when compared to the MPCs from nonpregnant mice. Furthermore, MPCs from nonpregnant mice display enhanced myogenic capacity when cultured in the presence of serum obtained from pregnant mice. Our proteomics data from these studies provides potential therapeutic targets to enhance the myogenic potential of progenitor cells and muscle repair.

Hypoxia-inducible factor 1α (HIF-1α) is a major determinant in the enhanced function of muscle-derived progenitors from MRL/MpJ mice.

Although the mouse strain Murphy Roths Large (MRL/MpJ) possesses high regenerative potential, the mechanism of tissue regeneration, including skeletal muscle, in MRL/MpJ mice after injury is still unclear. Our previous studies have shown that muscle-derived stem/progenitor cell (MDSPC) function is significantly enhanced in MRL/MpJ mice when compared with MDSPCs isolated from age-matched wild-type (WT) mice. Using mass spectrometry-based proteomic analysis, we identified increased expression of hypoxia-inducible factor (HIF) 1α target genes (expression of glycolytic factors and antioxidants) in sera from MRL/MpJ mice compared with WT mice. Therefore, we hypothesized that HIF-1α promotes the high muscle healing capacity of MRL/MpJ mice by increasing the potency of MDSPCs. We demonstrated that treating MRL/MpJ MDSPCs with dimethyloxalylglycine and CoCl2 increased the expression of HIF-1α and target genes, including angiogenic and cell survival genes. We also observed that HIF-1α activated the expression of paired box (Pax)7 through direct interaction with the Pax7 promoter. Furthermore, we also observed a higher myogenic potential of MDSPCs derived from prolyl hydroxylase (Phd) 3-knockout (Phd3-/-) mice, which displayed higher stability of HIF-1α. Taken together, our findings suggest that HIF-1α is a major determinant in the increased MDSPC function of MRL/MpJ mice through enhancement of cell survival, proliferation, and myogenic differentiation.-Sinha, K. M., Tseng, C., Guo, P., Lu, A., Pan, H., Gao, X., Andrews, R., Eltzschig, H., Huard, J. Hypoxia-inducible factor 1α (HIF-1α) is a major determinant in the enhanced function of muscle-derived progenitors from MRL/MpJ mice.

A novel dual kinase function of the RET proto-oncogene negatively regulates activating transcription factor 4-mediated apoptosis.

The RET proto-oncogene, a tyrosine kinase receptor, is widely known for its essential role in cell survival. Germ line missense mutations, which give rise to constitutively active oncogenic RET, were found to cause multiple endocrine neoplasia type 2, a dominant inherited cancer syndrome that affects neuroendocrine organs. However, the mechanisms by which RET promotes cell survival and prevents cell death remain elusive. We demonstrate that in addition to cytoplasmic localization, RET is localized in the nucleus and functions as a tyrosine-threonine dual specificity kinase. Knockdown of RET by shRNA in medullary thyroid cancer-derived cells stimulated expression of activating transcription factor 4 (ATF4), a master transcription factor for stress-induced apoptosis, through activation of its target proapoptotic genes NOXA and PUMA. RET knockdown also increased sensitivity to cisplatin-induced apoptosis. We observed that RET physically interacted with and phosphorylated ATF4 at tyrosine and threonine residues. Indeed, RET kinase activity was required to inhibit the ATF4-dependent activation of the NOXA gene because the site-specific substitution mutations that block threonine phosphorylation increased ATF4 stability and activated its targets NOXA and PUMA. Moreover, chromatin immunoprecipitation assays revealed that ATF4 occupancy increased at the NOXA promoter in TT cells treated with tyrosine kinase inhibitors or the ATF4 inducer eeyarestatin as well as in RET-depleted TT cells. Together these findings reveal RET as a novel dual kinase with nuclear localization and provide mechanisms by which RET represses the proapoptotic genes through direct interaction with and phosphorylation-dependent inactivation of ATF4 during the pathogenesis of medullary thyroid cancer.

Structural insights into histone demethylase NO66 in interaction with osteoblast-specific transcription factor osterix and gene repression.

Osterix (Osx) is an osteoblast-specific transcriptional factor and is required for osteoblast differentiation and bone formation. A JmjC domain-containing protein NO66 was previously found to participate in regulation of Osx transcriptional activity and plays an important role in osteoblast differentiation through interaction with Osx. Here, we report the crystal structure of NO66 forming in a functional tetramer. A hinge domain links the N-terminal JmjC domain and C-terminal winged helix-turn-helix domain of NO66, and both domains are essential for tetrameric assembly. The oligomerization interface of NO66 interacts with a conserved fragment of Osx. We show that the hinge domain-dependent oligomerization of NO66 is essential for inhibition of Osx-dependent gene activation. Our findings suggest that homo-oligomerization of JmjC domain containing proteins might play a physiological role through interactions with other regulatory factors during gene expression.

Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase.

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Osx null mice develop a normal cartilage skeleton but fail to form bone and to express osteoblast-specific marker genes. To better understand the control of transcriptional regulation by Osx, we identified Osx-interacting proteins using proteomics approaches. Here, we report that a Jumonji C (JmjC)-domain containing protein, called NO66, directly interacts with Osx and inhibits Osx-mediated promoter activation. The knockdown of NO66 in preosteoblast cells triggered accelerated osteoblast differentiation and mineralization, and markedly stimulated the expression of Osx target genes. A JmjC-dependent histone demethylase activity was exhibited by NO66, which was specific for both H3K4me and H3K36me in vitro and in vivo, and this activity was needed for the regulation of osteoblast-specific promoters. During BMP-2-induced differentiation of preosteoblasts, decreased NO66 occupancy correlates with increased Osx occupancy at Osx-target promoters. Our results indicate that interactions between NO66 and Osx regulate Osx-target genes in osteoblasts by modulating histone methylation states.

Immunoprevention Strategies for Colorectal Cancer in Lynch Syndrome Carriers.

ABSTRACT: The immune revolution that swept the field of oncology in the mid-2010s with the advent of checkpoint inhibitors has led to a paradigm shift in approaches toward adapting new cancer prevention modalities. Cancer vaccines have emerged from this era with astounding potential as a durable intervention to prevent cancers especially for patients with hereditary susceptibilities such as Lynch syndrome carriers. This review covers new insights in the immunoprevention landscape for patients living with Lynch syndrome including highlights ranging from clinical trials exploring the use of chemoprevention agents to boost immune cellularity to investigative studies using novel vaccine approaches to induce long-term antitumor immunity.

MONET: a database for prediction of neoantigens derived from microsatellite loci.

Background: Microsatellite instability (MSI) secondary to mismatch repair (MMR) deficiency is characterized by insertions and deletions (indels) in short DNA sequences across the genome. These indels can generate neoantigens, which are ideal targets for precision immune interception. However, current neoantigen databases lack information on neoantigens arising from coding microsatellites. To address this gap, we introduce The MicrOsatellite Neoantigen Discovery Tool (MONET). Method: MONET identifies potential mutated tumor-specific neoantigens (neoAgs) by predicting frameshift mutations in coding microsatellite sequences of the human genome. Then MONET annotates these neoAgs with key features such as binding affinity, stability, expression, frequency, and potential pathogenicity using established algorithms, tools, and public databases. A user-friendly web interface (https://monet.mdanderson.org/) facilitates access to these predictions. Results: MONET predicts over 4 million and 15 million Class I and Class II potential frameshift neoAgs, respectively. Compared to existing databases, MONET demonstrates superior coverage (>85% vs. <25%) using a set of experimentally validated neoAgs. Conclusion: MONET is a freely available, user-friendly web tool that leverages publicly available resources to identify neoAgs derived from microsatellite loci. This systems biology approach empowers researchers in the field of precision immune interception.

Genetic and molecular control of osterix in skeletal formation.

Osteoblast differentiation is a multi-step process where mesenchymal cells differentiate into osteoblast lineage cells including osteocytes. Osterix (Osx) is an osteoblast-specific transcription factor which activates a repertoire of genes during differentiation of preosteoblasts into mature osteoblasts and osteocytes. The essential role of Osx in the genetic program of bone formation and in bone homeostasis is well established. Osx mutant embryos do not form bone and fail to express osteoblast-specific marker genes. Inactivation of Osx in mice after birth causes multiple skeletal phenotypes including lack of new bone formation, absence of resorption of mineralized cartilage, and defects in osteocyte maturation and function. Since Osx is a major effector in skeletal formation, studies on Osx gained momentum over the last 5-7 years and implicated its important function in tooth formation as well as in healing of bone fractures. This review outlines mouse genetic studies that establish the essential role of Osx in bone and tooth formation as well as in healing of bone fractures. We also discuss the recent advances in regulation of Osx expression, which is under control of a transcriptional network, signaling pathways, and epigenetic regulation. Finally, we summarize important findings on the positive and negative regulation of Osx's transcriptional activity through protein-protein interactions in expression of its target genes during osteoblast differentiation. In particular, the identification of the histone demethylase NO66 as an Osx-interacting protein, which negatively regulates Osx activity opens further avenues in studying epigenetic control of Osx target genes during differentiation and maturation of osteoblasts.

Osteoblast-Specific Overexpression of Nucleolar Protein NO66/RIOX1 in Mouse Embryos Leads to Osteoporosis in Adult Mice.

In previous study, we showed that nucleolar protein 66 (NO66) is a chromatin modifier and negatively regulates Osterix activity as well as mesenchymal progenitor differentiation. Genetic ablation of the NO66 (RIOX1) gene in cells of the Prx1-expressing mesenchymal lineage leads to acceleration of osteochondrogenic differentiation and a larger skeleton in adult mice, whereas mesenchyme-specific overexpression of NO66 inhibits osteochondrogenesis resulting in dwarfism and osteopenia. However, the impact of NO66 overexpression in cells of the osteoblast lineage in vivo remains largely undefined. Here, we generated osteoblast-specific transgenic mice overexpressing a FLAG-tagged NO66 transgene driven by the 2.3 kB alpha-1type I collagen (Col1a1) promoter. We found that overexpression of NO66 in cells of the osteoblast lineage did not cause overt defects in developmental bones but led to osteoporosis in the long bones of adult mice. This includes decreased bone volume (BV), bone volume density (bone volume/total volume, BV/TV), and bone mineral density (BMD) in cancellous compartment of long bones, along with the accumulation of fatty droplets in bone marrow. Ex vivo culture of the bone marrow mesenchymal stem/stromal cells (BMSCs) from adult Col1a1-NO66 transgenic mice showed an increase in adipogenesis and a decrease in osteogenesis. Taken together, these data demonstrate a crucial role for NO66 in adult bone formation and homeostasis. Our Col1a1-NO66 transgenic mice provide a novel animal model for the mechanistic and therapeutic study of NO66 in osteoporosis.

Advances in vaccine development for cancer prevention and treatment in Lynch Syndrome.

Lynch Syndrome (LS) is one of the most common hereditary cancer syndromes, and is caused by mutations in one of the four DNA mismatch repair (MMR) genes, namely MLH1, MSH2, MSH6 and PMS2. Tumors developed by LS carriers display high levels of microsatellite instability, which leads to the accumulation of large numbers of mutations, among which frameshift insertion/deletions (indels) within microsatellite (MS) loci are the most common. As a result, MMR-deficient (MMRd) cells generate increased rates of tumor-specific neoantigens (neoAgs) that can be recognized by the immune system to activate cancer cell killing. In this context, LS is an ideal disease to leverage immune-interception strategies. Therefore, the identification of these neoAgs is an ongoing effort for the development of LS cancer preventive vaccines. In this review, we summarize the computational methods used for in silico neoAg prediction, including their challenges, and the experimental techniques used for in vitro validation of their immunogenicity. In addition, we outline results from past and on-going vaccine clinical trials and highlight avenues for improvement and future directions.

Naproxen chemoprevention induces proliferation of cytotoxic lymphocytes in Lynch Syndrome colorectal mucosa.

Background: Recent clinical trial data from Lynch Syndrome (LS) carriers demonstrated that naproxen administered for 6-months is a safe primary chemoprevention that promotes activation of different resident immune cell types without increasing lymphoid cellularity. While intriguing, the precise immune cell types enriched by naproxen remained unanswered. Here, we have utilized cutting-edge technology to elucidate the immune cell types activated by naproxen in mucosal tissue of LS patients. Methods: Normal colorectal mucosa samples (pre- and post-treatment) from a subset of patients enrolled in the randomized and placebo-controlled 'Naproxen Study' were obtained and subjected to a tissue microarray for image mass cytometry (IMC) analysis. IMC data was processed using tissue segmentation and functional markers to ascertain cell type abundance. Computational outputs were then used to quantitatively compare immune cell abundance in pre- and post-naproxen specimens. Results: Using data-driven exploration, unsupervised clustering identified four populations of immune cell types with statistically significant changes between treatment and control groups. These four populations collectively describe a unique cell population of proliferating lymphocytes within mucosal samples from LS patients exposed to naproxen. Conclusions: Our findings show that daily exposure of naproxen promotes T-cell proliferation in the colonic mucosa, which paves way for developing combination of immunoprevention strategies including naproxen for LS patients.

Exercise Training Reduces the Inflammatory Response and Promotes Intestinal Mucosa-Associated Immunity in Lynch Syndrome.

PURPOSE: Lynch syndrome (LS) is a hereditary condition with a high lifetime risk of colorectal and endometrial cancers. Exercise is a non-pharmacologic intervention to reduce cancer risk, though its impact on patients with LS has not been prospectively studied. Here, we evaluated the impact of a 12-month aerobic exercise cycling intervention in the biology of the immune system in LS carriers. PATIENTS AND METHODS: To address this, we enrolled 21 patients with LS onto a non-randomized, sequential intervention assignation, clinical trial to assess the effect of a 12-month exercise program that included cycling classes 3 times weekly for 45 minutes versus usual care with a one-time exercise counseling session as control. We analyzed the effects of exercise on cardiorespiratory fitness, circulating, and colorectal-tissue biomarkers using metabolomics, gene expression by bulk mRNA sequencing, and spatial transcriptomics by NanoString GeoMx. RESULTS: We observed a significant increase in oxygen consumption (VO2peak) as a primary outcome of the exercise and a decrease in inflammatory markers (prostaglandin E) in colon and blood as the secondary outcomes in the exercise versus usual care group. Gene expression profiling and spatial transcriptomics on available colon biopsies revealed an increase in the colonic mucosa levels of natural killer and CD8+ T cells in the exercise group that were further confirmed by IHC studies. CONCLUSIONS: Together these data have important implications for cancer interception in LS, and document for the first-time biological effects of exercise in the immune system of a target organ in patients at-risk for cancer.

Combination of Sulindac and Bexarotene for Prevention of Intestinal Carcinogenesis in Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer syndrome, which results in the development of hundreds of adenomatous polyps carpeting the gastrointestinal tract. NSAIDs have reduced polyp burden in patients with FAP and synthetic rexinoids have demonstrated the ability to modulate cytokine-mediated inflammation and WNT signaling. This study examined the use of the combination of an NSAID (sulindac) and a rexinoid (bexarotene) as a durable approach for reducing FAP colonic polyposis to prevent colorectal cancer development. Whole transcriptomic analysis of colorectal polyps and matched normal mucosa in a cohort of patients with FAP to identify potential targets for prevention in FAP was performed. Drug-dose synergism of sulindac and bexarotene in cell lines and patient-derived organoids was assessed, and the drug combination was tested in two different mouse models. This work explored mRNA as a potential predictive serum biomarker for this combination in FAP. Overall, transcriptomic analysis revealed significant activation of inflammatory and cell proliferation pathways. A synergistic effect of sulindac (300 µmol/L) and bexarotene (40 µmol/L) was observed in FAP colonic organoids with primary targeting of polyp tissue compared with normal mucosa. This combination translated into a significant reduction in polyp development in ApcMin/+ and ApcLoxP/+-Cdx2 mice. Finally, the reported data suggest miRNA-21 could serve as a predictive serum biomarker for polyposis burden in patients with FAP. These findings support the clinical development of the combination of sulindac and bexarotene as a treatment modality for patients with FAP. PREVENTION RELEVANCE: This study identified a novel chemopreventive regimen combining sulindac and bexarotene to reduce polyposis in patients with FAP using in silico tools, ex vivo, and in vivo models. This investigation provides the essential groundwork for moving this drug combination forward into a clinical trial.

The Transcriptomic Landscape of Mismatch Repair-Deficient Intestinal Stem Cells.

Lynch syndrome is the most common cause of hereditary colorectal cancer and is secondary to germline alterations in one of four DNA mismatch repair (MMR) genes. Here we aimed to provide novel insights into the initiation of MMR-deficient (MMRd) colorectal carcinogenesis by characterizing the expression profile of MMRd intestinal stem cells (ISC). A tissue-specific MMRd mouse model (Villin-Cre;Msh2 LoxP/LoxP ) was crossed with a reporter mouse (Lgr5-EGFP-IRES-creERT2) to trace and isolate ISCs (Lgr5+) using flow cytometry. Three different ISC genotypes (Msh2-KO, Msh2-HET, and Msh2-WT) were isolated and processed for mRNA-seq and mass spectrometry, followed by bioinformatic analyses to identify expression signatures of complete MMRd and haplo-insufficiency. These findings were validated using qRT-PCR, IHC, and whole transcriptomic sequencing in mouse tissues, organoids, and a cohort of human samples, including normal colorectal mucosa, premalignant lesions, and early-stage colorectal cancers from patients with Lynch syndrome and patients with familial adenomatous polyposis (FAP) as controls. Msh2-KO ISCs clustered together with differentiated intestinal epithelial cells from all genotypes. Gene-set enrichment analysis indicated inhibition of replication, cell-cycle progression, and the Wnt pathway and activation of epithelial signaling and immune reaction. An expression signature derived from MMRd ISCs successfully distinguished MMRd neoplastic lesions of patients with Lynch syndrome from FAP controls. SPP1 was specifically upregulated in MMRd ISCs and colocalized with LGR5 in Lynch syndrome colorectal premalignant lesions and tumors. These results show that expression signatures of MMRd ISC recapitulate the initial steps of Lynch syndrome carcinogenesis and have the potential to unveil novel biomarkers of early cancer initiation. SIGNIFICANCE: The transcriptomic and proteomic profile of MMR-deficient intestinal stem cells displays a unique set of genes with potential roles as biomarkers of cancer initiation and early progression.

Combinations of Tyrosine Kinase Inhibitor and ERAD Inhibitor Promote Oxidative Stress-Induced Apoptosis through ATF4 and KLF9 in Medullary Thyroid Cancer.

Medullary thyroid carcinoma (MTC) originates from the C cells of the thyroid gland, which secrete calcitonin. Lymph node and distant metastases are frequently present at diagnosis. Activating mutations of RET, a driver oncogene in MTC that encodes a tyrosine kinase receptor, prevents apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum (ER) stress. We hypothesized that the combination of a tyrosine kinase inhibitor (TKI) and an ATF4 inducer promotes cell death by triggering catastrophic oxidative stress and apoptotic cell death. Here, we report that the ER-associated protein degradation (ERAD) inhibitor eeyarestatin sensitized MTC cells to the TKIs, sunitinib and vandetanib, thereby leading to synergistic upregulation of ATF4 expression, accumulation of reactive oxygen species, and subsequent cell death. Genome-wide analysis of ATF4 interaction sites by chromatin immunoprecipitation (ChIP) sequencing revealed that among ATF4 target genes was KLF9 (Kruppel-like factor 9), which induces MTC apoptosis. ChIP assays revealed that ATF4 occupancy at the KLF9 promoter was increased in MTC cells treated with eeyarestatin or vandetanib alone and was further enhanced in cells treated with both drugs, leading to increased KLF9 transcription. Depletion of ATF4 by shRNA led to downregulation of KLF9 expression and prevented oxidative stress-induced cell death. Furthermore, we identified ATF4 target genes (LZTFL1, MKNK2, and SIAH1 with known tumor suppressor function) that were synergistically upregulated with the combination of TKI and ERAD inhibitor. IMPLICATIONS: These findings reveal a combination therapy that induces reactive oxygen species-dependent catastrophic cell death through induction of ATF4 and KLF9 transcriptional activity.

Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer.

Epigenetic changes that cause dysregulated gene expression during progression of androgen-independent prostate cancer (PCa) and metastatic skeletal lesions remain elusive. Here, we explored the role of histone demethylase NO66 in the pathogenesis of PCa and bone metastasis-related skeletal lesions. Tissue and cDNA microarrays of PCa were analyzed for NO66 mRNA and protein levels. We examined the effects of gain and loss of NO66 function on cell viability, colony formation, migration, invasion, and tumor-induced skeletal lesions in femoral bone. RNAseq and ChIPseq were performed to elucidate NO66-target genes in PCa. We report that NO66 levels were upregulated in advanced primary prostate tumors compared to normal tissue or tumors with low Gleason scores. Forced expression of NO66 promoted cell survival and invasion of PCa cells; whereas, knockdown of NO66 resulted in decreased cell survival and increased sensitivity to docetaxel. NO66-overexpressing PC3 cells implanted into the femoral bone of male SCID mice caused massive bone loss and stimulation of mouse osteoclast-promoting genes, including Dickkopf1, Cathepsin K, Nf-kß,; and Calcr, suggesting a role for NO66 in tumor growth in bone and osteoclast activity. Combined RNAseq and ChIP-seq revealed that NO66 activates the survival gene MCL1, the invasion-associated genes IGFBP5 and MMP3, the pro-oncogenic genes CTNNB1 and CCND1, and the epigenetic modifier gene KMT2A in androgen-independent PCa. Our findings uncover the role of NO66 as a key oncogenic driver in PCa, causing osteolytic lesions through upstream epigenetic regulation of key genes for survival, invasion and metastasis, and pro-osteoclastic factors.

Specificity Protein 7 Is Required for Proliferation and Differentiation of Ameloblasts and Odontoblasts.

The Sp7/Osterix transcription factor is essential for bone development. Mutations of the Sp7 gene in humans are associated with craniofacial anomalies and osteogenesis imperfecta. However, the role of Sp7 in embryonic tooth development remains unknown. Here we identified the functional requirement of Sp7 for dentin synthesis and tooth development. Sp7-null mice exhibit craniofacial dysmorphogenesis and are completely void of alveolar bone. Surprisingly, initial tooth morphogenesis progressed normally in Sp7-null mice. Thus the formation of alveolar bone is not a prerequisite for tooth morphogenesis. Sp7 is required for mineralization of palatal tissue but is not essential for palatal fusion. The reduced proliferative capacity of Sp7-deficient ectomesenchyme results in small and misshapen teeth with randomly arranged cuboidal preodontoblasts and preameloblasts. Sp7 promotes functional maturation and polarization of odontoblasts. Markers of mature odontoblast (Col1a, Oc, Dspp, Dmp1) and ameloblast (Enam, Amelx, Mmp20, Amtn, Klk4) are barely expressed in incisors and molar tissues of Sp7-null mice. Consequently, dentin and enamel matrix are absent in the Sp7-null littermates. Interestingly, the Sp7 expression is restricted to cells of the dental mesenchyme indicating the effect on oral epithelium-derived ameloblasts is cell-nonautonomous. Abundant expression of Fgf3 and Fgf8 ligand was noted in the developing tooth of wild-type mice. Both ligands were remarkably absent in the Sp7-null incisor and molar, suggesting cross-signaling between mesenchyme and epithelium is disrupted. Finally, promoter-reporter assays revealed that Sp7 directly controls the expression of Fgf-ligands. Together, our data demonstrate that Sp7 is obligatory for the differentiation of both ameloblasts and odontoblasts but not for the initial tooth morphogenesis. © 2018 American Society for Bone and Mineral Research.