Ann M. Decker

DNMT1 Regulates Epithelial-Mesenchymal Transition and Cancer Stem Cells, Which Promotes Prostate Cancer Metastasis

Cancer metastasis is a multistep process associated with the induction of an epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs). Although significant progress has been made in understanding the molecular mechanisms regulating EMT and the CSC phenotype, little is known of how these processes are regulated by epigenetics. Here we demonstrate that reduced expression of DNA methyltransferase 1 (DNMT1) plays an important role in the induction of EMT and the CSC phenotype by prostate cancer (PCa) cells, with enhanced tumorigenesis and metastasis. First, we observed that reduction of DNMT1 by 5-azacitidine (5-Aza) promotes EMT induction as well as CSCs and sphere formation in vitro. Reduced expression of DNMT1 significantly increased PCa migratory potential. We showed that the increase of EMT and CSC activities by reduction of DNMT1 is associated with the increase of protein kinase C. Furthermore, we confirmed that silencing DNMT1 is correlated with enhancement of the induction of EMT and the CSC phenotype in PCa cells. Additionally, chromatin immunoprecipitation assay reveals that reduction of DNMT1 promotes the suppression of H3K9me3 and H3K27me3 on the Zeb2 and KLF4 promoter region in PCa cells. Critically, we found in an animal model that significant tumor growth and more disseminated tumor cells in most osseous tissues were observed following injection of 5-Aza pretreated–PCa cells compared with vehicle-pretreated PCa cells. Our results suggest that epigenetic alteration of histone demethylation regulated by reduction of DNMT1 may control induction of EMT and the CSC phenotype, which facilitates tumorigenesis in PCa cells and has important therapeutic implications in targeting epigenetic regulation.

Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow.

Disseminated prostate cancer (PCa) cells in the marrow survive for years without evidence of proliferation, while maintaining the capacity to develop into metastatic lesions. These dormant disseminated tumor cells (DTCs) may reside in close proximity to osteoblasts, while expressing high levels of Axl, one of the tyrosine kinase receptors for growth arrest specific 6 (Gas6). Yet how Axl regulates DTC proliferation in marrow remains undefined. Here, we explored the impact of the loss of Axl in PCa cells (PC3 and DU145) on the induction of their dormancy when they are co-cultured with a pre-osteoblastic cell line, MC3T3-E1. MC3T3-E1 cells dramatically decrease the proliferation of PCa cells, however this suppressive effect of osteoblasts is significantly reduced by the reduction of Axl expression in PCa cells. Interestingly, expression of both TGF-β and its receptors were regulated by Axl expression in PCa cells, while specific blockade of TGF-β signaling limited the ability of the osteoblasts to induce dormancy of PCa cells. Finally, we found that both Gas6 and Axl are required for TGF-β2-mediated cell growth suppression. Taken together, these data suggest that a loop between the Gas6/Axl axis and TGF-β2 signaling plays a significant role in the induction of PCa cell dormancy.

The role of hematopoietic stem cell niche in prostate cancer bone metastasis

Approximately 80% of prostate cancers exhibit some degree of bone metastasis. The role of the bone marrow and the hematopoietic stem cell (HSC) niche in attracting metastatic cells and maintaining dormancy of disseminated tumor cells (DTCs) is an increasingly important topic towards the development of novel prostate cancer therapies. This paper reviews aspects of the HSC niche that lead to prostate cancer cell homing and dormancy in the bone marrow. This review also discusses the role of DTCs in the niche environment and discusses the role of erythropoietin in targeting DTCs within the HSC niche.

Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

Clinical observations have identified an association between psychologic stress and cancer relapse, suggesting that the sympathetic nervous system/norepinephrine (NE) plays a role in reactivation of dormant disseminated tumor cells (DTC) in the bone marrow niche. Here, the mechanism by which NE regulates prostate cancer DTCs in the marrow is explored. NE directly stimulated prostate cancer cell proliferation through β2-adrenergic receptors (ADRB2). NE also altered prostate cancer proliferation in the marrow niche by indirectly downregulating the secretion of the dormancy inducing molecule growth arrest specific-6 (GAS6) expressed by osteoblasts. These observations were confirmed in cocultures of prostate cancer cells expressing the fluorescent ubiquitination-based cell-cycle reporters (FUCCI) and osteoblasts isolated from GAS6-deficient (GAS6−/−) animals. A novel ex vivo model system, using femurs harvested from GAS6+/+ or GAS6−/− mice, was used to confirm these results. As in coculture, when prostate cancer cells were injected into the marrow cavities of GAS6+/+ femurs, NE altered the prostate cancer cell cycle. However, NE had less of an impact on prostate cancer cells in femur explants isolated from GAS6−/− mice. Together, this study demonstrates that NE reactivates prostate cancer cell cycling through both a direct action on prostate cancer cells and indirectly on adjacent niche cells. Implications: Identification of mechanisms that target DTCs may provide novel therapeutic approaches to prevent or treat cancer metastases more effectively. Mol Cancer Res; 15(12); 1644–55. ©2017 AACR.

Mer Tyrosine Kinase Regulates Disseminated Prostate Cancer Cellular Dormancy

Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of P‐Erk1/2 to P‐p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intra‐cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891–902, 2017.

Human Stem Cells Overexpressing miR-21 Promote Angiogenesis in Critical Limb Ischemia by Targeting CHIP to Enhance HIF-1α Activity

Critical limb ischemia (CLI) is a severe blockage in the arteries of the lower extremities. However, the effective and optimal treatment for CLI remains to be elucidated. Previous therapeutic research is mainly focused on proangiogenic growth factors administrations. Recently, miR‐21 has been revealed to play a crucial role in angiogenesis. Thus, we hypothesize that miR‐21 over‐expression in human umbilical cord blood‐derived mesenchymal stem cells (UCBMSCs) can effectively treat CLI. Herein, UCBMSCs were transduced with lentivirus‐miR‐21‐Luciferase (Lenti‐miR‐21) or lentivirus‐ LacZ‐Luciferase (Lenti‐LacZ). The results indicated that miR‐21 induced UCBMSCs proliferation, migration, and angiogenesis in vitro. Subsequently, general observation and laser Doppler perfusion imaging were introduced to detect perfusion in muscles of CLI‐nude mice on 1, 4, 7, 14, and 28 day postoperation. There was a significant improvement in blood vessels of the ischemic limb in Lenti‐miR‐21 group at 7 day compared with the saline or Lenti‐LacZ groups. At 28 day, histological analysis confirmed that UCBMSCs over‐expressing miR‐21 increased neovascularization in CLI. Furthermore, carboxyl terminus of Hsc70‐interacting protein (CHIP) was found to be the target gene for miR‐21‐mediated activation of hypoxia‐inducible factor 1α (HIF‐1α) in UCBMSCs. In summary, our study demonstrated that over‐expressing miR‐21 in UCBMSCs could improve neovascularization in CLI through enhancing HIF‐1α activity by targeting CHIP, which may hold great therapeutic promise in treating CLI. Stem Cells 2016;34:924–934

Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow

GAS6 and its receptors (Tryo 3, Axl, Mer or “TAM”) are known to play a role in regulating tumor progression in a number of settings. Previously we have demonstrated that GAS6 signaling regulates invasion, proliferation, chemotherapy-induced apoptosis of prostate cancer (PCa) cells. We have also demonstrated that GAS6 secreted from osteoblasts in the bone marrow environment plays a critical role in establishing prostate tumor cell dormancy. Here we investigated the role that endogenous GAS6 and Mer receptor signaling plays in establishing prostate cancer stem cells in the bone marrow microenvironment. We first observed that high levels of endogenous GAS6 are expressed by disseminated tumor cells (DTCs) in the bone marrow, whereas relatively low levels of endogenous GAS6 are expressed in PCa tumors grown in a s.c. setting. Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133–/CD44–) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G1) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo. Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.

The Role of Occlusion in Implant Therapy: A Comprehensive Updated Review

Purpose:Occlusal overload may cause implant biomechanical failures, marginal bone loss, or even complete loss of osseointegration. Thus, it is important for clinicians to understand the role of occlusion in implant long-term stability. This systematic review updates the understanding of occlusion on dental implants, the impact on the surrounding peri-implant tissues, and the effects of occlusal overload on implants. Additionally, recommendations of occlusal scheme for implant prostheses and designs were formulated. Materials and Methods:Two reviewers completed a literature search using the PubMed database and a manual search of relevant journals. Relevant articles from January 1950 to September 20, 2015 published in the English language were considered. Results:Recommendations for implant occlusion are lacking in the literature. Despite this, implant occlusion should be carefully addressed. Conclusion:Recommendations for occlusal schemes for single implants or fixed partial denture supported by implants include a mutually protected occlusion with anterior guidance and evenly distributed contacts with wide freedom in centric relation. Suggestions to reduce occlusal overload include reducing cantilevers, increasing the number of implants, increasing contact points, monitoring for parafunctional habits, narrowing the occlusal table, decreasing cuspal inclines, and using progressive loading in patients with poor bone quality. Protecting the implant and surrounding peri-implant bone requires an understanding of how occlusion plays a role in influencing long-term implant stability.

Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G1 Arrest/S Phase Delay and Inhibition of Apoptosis During Chemotherapy in Bone Marrow

Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest‐specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the fraction of cells in G1 and the duration of phase in PCa cells. Importantly, the effect of GAS6 on G1 is potentiated during docetaxel chemotherapy. GAS6 altered the levels of several key cell cycle regulators, including the downregulation of Cyclin B1 (G2/M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry), while the upregulation of cell cycle inhibitors p27 and p21, Cyclin D1, and CDK4. Importantly, these changes became further accentuated during docetaxel treatment in the presence of GAS6. Moreover, GAS6 alters the apoptotic response of PCa cells during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis is efficiently suppressed in PCa cell culture in the presence of GAS6 or GAS6 secreted from co‐cultured osteoblasts. Similarly, the GAS6‐expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. Docetaxel induced significant levels of Caspase‐3 and PARP cleavage in PCa cells, while GAS6 protected PCa cells from docetaxel‐induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which will have important implications for targeting metastatic disease. J. Cell. Biochem. 117: 2815–2824, 2016.

Personalized Medicine Approaches to the Prevention, Diagnosis, and Treatment of Chronic Periodontitis

Over the years, the multifactorial and interactive nature of periodontal disease challenged clinicians in an attempt to delineate a pattern for its destructive progression. Periodontal disease is caused by persistent inflammation of the periodontium in response to the colonization of microbes upon the tooth surface near the gingival margin and a subsequent biofilm formation resulting in irreversible attachment loss of marginal alveolar bone and associated periodontal ligament and migration of the junctional epithelium. In spite of the infectious nature of periodontal disease, identification of the oral microbiome and a complete understanding of its pathogenic pathway seem the most logical step toward the development of newer and effective approaches for periodontal therapy.