Wnt/ß-catenin-C-kit axis may play a role in adenoid cystic carcinoma prognostication.

Adenoid cystic carcinoma (ACC) is one of the most common malignant salivary gland tumors. ACC is composed of myoepithelial and epithelial neoplastic cells which grow slowly and have a tendency for neural invasion. The long term prognosis is still relatively poor. Although several gene abnormalities, such as fusions involving MYB or MYBL1 oncogenes and the transcription factor gene NFIB, and overexpression of KIT have been reported in ACC, their precise functions in the pathogenesis of ACC remain unclear. We recently demonstrated that the elevated expression of Semaphorin 3A (SEMA3A), specifically expressed in myoepithelial neoplastic cells, might function as a novel oncogene-related molecule to enhance cell proliferation through activated AKT signaling in 9/10 (90%) ACC cases. In the current study, the patient with ACC whose tumor was negative for SEMA3A in the previous study, revisited our hospital with late metastasis of ACC to the cervical lymph node eight years after surgical resection of the primary tumor. We characterized this recurrent ACC, and compared it with the primary ACC using immunohistochemical methods. In the recurrent ACC, the duct lining epithelial cells, not myoepithelial neoplastic cells, showed an elevated Ki-67 index and increased cell membrane expression of C-kit, along with the expression of phosphorylated ERK. Late metastasis ACC specimens were not positive for ß-catenin and lymphocyte enhancer binding factor 1 (LEF1), which were detected in the nuclei of perineural infiltrating cells in primary ACC cells. In addition, experiments with the GSK-3 inhibitor revealed that ß-catenin pathway suppressed not only KIT expression but also proliferation of ACC cells. Moreover, stem cell factor (SCF; also known as KIT ligand, KITL) induced ERK activation in ACC cells. These results suggest that inactivation of Wnt/ß-catenin signaling may promote C-kit-ERK signaling and cell proliferation of in metastatic ACC.

Stem cells in clinical dentistry.

BACKGROUND: Stem cells are present in most of the tissues in the craniofacial complex and play a major role in tissue homeostasis and repair. These cells are characterized by their capacity to differentiate into multiple cell types and to self-renew to maintain a stem cell pool throughout the life of the tissue. TYPES OF STUDIES REVIEWED: The authors discuss original data from experiments and comparative analyses and review articles describing the identification and characterization of stem cells of the oral cavity. RESULTS: Every oral tissue except enamel, dentin, and cementum contains stem cells for the entire life span. These stem cells self-renew to maintain a pool of cells that can be activated to replace terminally differentiated cells (for example, odontoblasts) or to enable wound healing (for example, dentin bridge in pulp exposures and healing of periodontal tissues after surgery). In addition, dental stem cells can differentiate into functional blood vessels and nerves. Initial clinical trials have shown that transplanting dental pulp stem cells into disinfected necrotic teeth has allowed for the recovery of tooth vitality and vertical and horizontal root growth in immature teeth with incomplete root formation. PRACTICAL IMPLICATIONS: As a consequence of these groundbreaking discoveries, stem cell banks are now offering services for the cryopreservation of dental stem cells. The future use of stem cell-based therapies in the clinic will depend on the collaboration of clinicians and researchers in projects designed to understand whether these treatments are safe, efficacious, and clinically feasible.

Therapeutic inhibition of Bmi-1 ablates chemoresistant cancer stem cells in adenoid cystic carcinoma.

OBJECTIVES: Adenoid Cystic Carcinomas (ACC) typically show modest responseto cytotoxic therapy. Cancer stem cells (CSC) have been implicated in chemoresistance and tumor relapse. However, their role in ACC remains unknown. The purpose of this work was to evaluate the impact of targeting ACC CSCs with Bmi-1 inhibitors on resistance to cytotoxic therapy and tumor relapse. MATERIALS AND METHODS: Therapeutic efficacy of a small molecule inhibitor of Bmi-1 (PTC596; Unesbulin) and/or Cisplatin on ACC stemness was evaluated in immunodeficient mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in human ACC cell-lines (UM-HACC-2A,-14) or low passage primary human ACC cells (UM-HACC-6). The effect of therapy on stemness was examined by salisphere assays, flow cytometry for ALDH activity and CD44 expression, and Western blots for Bmi-1 (self-renewal marker) and Oct4 (embryonic stem cell marker) expression. RESULTS: Platinum-based agents (Cisplatin, Carboplatin) induced Bmi-1 and Oct4 expression, increased salisphere formation and the CSC fraction in vitro and in vivo. In contrast, PTC596 inhibited expression of Bmi-1, Oct4 and pro-survival proteins Mcl-1 and Claspin; decreased the number of salispheres, and the fraction of ACC CSCs in vitro. Silencing Claspin decreased salisphere formation and CSC fraction. Both, single agent PTC596 and PTC596/Cisplatin combination decreased the CSC fraction in PDX ACC tumors. Notably, short-term combination therapy (2 weeks) with PTC596/Cisplatin prevented tumor relapse for 150 days in a preclinical trial in mice. CONCLUSION: Therapeutic inhibition of Bmi-1 ablates chemoresistant CSCs and prevents ACC tumor relapse. Collectively, these results suggest that ACC patients might benefit from Bmi-1-targeted therapies.

Naringenin stimulates osteogenic/odontogenic differentiation and migration of human dental pulp stem cells.

Background/purpose: Naringenin, a naturally occurring flavanone in citrus fruits, regulates bone formation by bone marrow-derived mesenchymal stem cells. The purpose of this study was to characterize the effects of naringenin on some biological behaviors of human dental pulp stem cells (HDPSCs). Materials and methods: HDPSCs were cultured in osteogenic differentiation medium and osteo/odontogenic differentiation and mineralization were analyzed by alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining. The migration of HDPSCs was evaluated by transwell chemotactic migration assays and scratch wound healing migration assay. Using tooth slice/scaffold model, we assessed the in vivo odontogenic differentiation potential of HDPSCs. Results: We have demonstrated that naringenin increases the osteogenic/odontogenic differentiation of HDPSCs through regulation of osteogenic-related proteins and the migratory ability of HDPSCs through stromal cell derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis. Moreover, naringenin promotes the expression of dentin matrix acidic phosphoprotein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) in HDPSCs seeded on tooth slice/scaffolds that are subcutaneously implanted into immunodeficient mice. Conclusion: Our present study suggests that naringenin promotes migration and osteogenic/odontogenic differentiation of HDPSCs and may serve as a promising candidate in dental tissue engineering and bone regeneration.

Gelatin methacryloyl hydrogel as an injectable scaffold with multi-therapeutic effects to promote antimicrobial disinfection and angiogenesis for regenerative endodontics.

Regenerative endodontics represents a paradigm shift in dental pulp therapy for necrotic young permanent teeth. However, there are still challenges associated with attaining maximum root canal disinfection while supporting angiogenesis and preserving resident stem cells viability and differentiation capacity. Here, we developed a hydrogel system by incorporating antibiotic-eluting fiber-based microparticles in gelatin methacryloyl (GelMA) hydrogel to gather antimicrobial and angiogenic properties while prompting minimum cell toxicity. Minocycline (MINO) or clindamycin (CLIN) was introduced into a polymer solution and electrospun into fibers, which were further cryomilled to attain MINO- or CLIN-eluting fibrous microparticles. To obtain hydrogels with multi-therapeutic effects, MINO- or CLIN-eluting microparticles were suspended in GelMA at distinct concentrations. The engineered hydrogels demonstrated antibiotic-dependent swelling and degradability while inhibiting bacterial growth with minimum toxicity in dental-derived stem cells. Notably, compared to MINO, CLIN hydrogels enhanced the formation of capillary-like networks of endothelial cells in vitro and the presence of widespread vascularization with functioning blood vessels in vivo. Our data shed new light onto the clinical potential of antibiotic-eluting gelatin methacryloyl hydrogel as an injectable scaffold with multi-therapeutic effects to promote antimicrobial disinfection and angiogenesis for regenerative endodontics.

Bmi-1: A master regulator of head and neck cancer stemness.

Head and neck cancers are composed of a diverse group of malignancies, many of which exhibit an unacceptably low patient survival, high morbidity and poor treatment outcomes. The cancer stem cell (CSC) hypothesis provides an explanation for the substantial patient morbidity associated with treatment resistance and the high frequency of tumor recurrence/metastasis. Stem cells are a unique population of cells capable of recapitulating a heterogenous organ from a single cell, due to their capacity to self-renew and differentiate into progenitor cells. CSCs share these attributes, in addition to playing a pivotal role in cancer initiation and progression by means of their high tumorigenic potential. CSCs constitute only a small fraction of tumor cells but play a major role in tumor initiation and therapeutic evasion. The shift towards stem-like phenotype fuels many malignant features of a cancer cell and mediates resistance to conventional chemotherapy. Bmi-1 is a master regulator of stem cell self-renewal as part of the polycomb repressive complex 1 (PRC1) and has emerged as a prominent player in cancer stem cell biology. Bmi-1 expression is upregulated in CSCs, which is augmented by tumor-promoting factors and various conventional chemotherapies. Bmi-1+ CSCs mediate chemoresistance and metastasis. On the other hand, inhibiting Bmi-1 rescinds CSC function and re-sensitizes cancer cells to chemotherapy. Therefore, elucidating the functional role of Bmi-1 in CSC-mediated cancer progression may unveil an attractive target for mechanism-based, developmental therapeutics. In this review, we discuss the parallels in the role of Bmi-1 in stem cell biology of health and disease and explore how this can be leveraged to advance clinical treatment strategies for head and neck cancer.

PDGF-BB signaling via PDGFR-ß regulates the maturation of blood vessels generated upon vasculogenic differentiation of dental pulp stem cells.

A functional vascular network requires that blood vessels are invested by mural cells. We have shown that dental pulp stem cells (DPSC) can undergo vasculogenic differentiation, and that the resulting vessels anastomize with the host vasculature and become functional (blood carrying) vessels. However, the mechanisms underlying the maturation of DPSC-derived blood vessels remains unclear. Here, we performed a series of studies to understand the process of mural cell investment of blood vessels generated upon vasculogenic differentiation of dental pulp stem cells. Primary human DPSC were co-cultured with primary human umbilical artery smooth muscle cells (HUASMC) in 3D gels in presence of vasculogenic differentiation medium. We observed DPSC capillary sprout formation and SMC recruitment, alignment and remodeling that resulted in complex vascular networks. While HUASMC enhanced the number of capillary sprouts and stabilized the capillary network when co-cultured with DPSC, HUASMC by themselves were unable to form capillary sprouts. In vivo, GFP transduced human DPSC seeded in biodegradable scaffolds and transplanted into immunodeficient mice generated functional human blood vessels invested with murine smooth muscle actin (SMA)-positive, GFP-negative cells. Inhibition of PDGFR-ß signaling prevented the SMC investment of DPSC-derived capillary sprouts in vitro and of DPSC-derived blood vessels in vivo. In contrast, inhibition of Tie-2 signaling did not have a significant effect on the SMC recruitment in DPSC-derived vascular structures. Collectively, these results demonstrate that PDGF-BB signaling via PDGFR-ß regulates the process of maturation (mural investment) of blood vessels generated upon vasculogenic differentiation of human dental pulp stem cells.

p53 Inhibits Bmi-1-driven Self-Renewal and Defines Salivary Gland Cancer Stemness.

PURPOSE: Mucoepidermoid carcinoma (MEC) is a poorly understood salivary gland malignancy with limited therapeutic options. Cancer stem cells (CSC) are considered drivers of cancer progression by mediating tumor recurrence and metastasis. We have shown that clinically relevant small molecule inhibitors of MDM2-p53 interaction activate p53 signaling and reduce the fraction of CSC in MEC. Here we examined the functional role of p53 in the plasticity and self-renewal of MEC CSC. EXPERIMENTAL DESIGN: Using gene silencing and therapeutic activation of p53, we analyzed the cell-cycle profiles and apoptosis levels of CSCs in MEC cell lines (UM-HMC-1, -3A, -3B) via flow cytometry and looked at the effects on survival/self-renewal of the CSCs through sphere assays. We evaluated the effect of p53 on tumor development (N = 51) and disease recurrence (N = 17) using in vivo subcutaneous and orthotopic murine models of MEC. Recurrence was followed for 250 days after tumor resection. RESULTS: Although p53 activation does not induce MEC CSC apoptosis, it reduces stemness properties such as self-renewal by regulating Bmi-1 expression and driving CSC towards differentiation. In contrast, downregulation of p53 causes expansion of the CSC population while promoting tumor growth. Remarkably, therapeutic activation of p53 prevented CSC-mediated tumor recurrence in preclinical trials. CONCLUSIONS: Collectively, these results demonstrate that p53 defines the stemness of MEC and suggest that therapeutic activation of p53 might have clinical utility in patients with salivary gland MEC.

Adenoid Cystic Carcinoma from the salivary and lacrimal glands and the breast: Different clinical outcomes to the same tumor.

Adenoid cystic carcinoma (ACC) is a biphasic malignant lesion that can develop at various anatomical sites. Salivary and lacrimal ACC lesions have a high risk of local invasion, metastasis, and poor prognosis. In more distant organs, such as the breast, ACC is a rarer and less aggressive lesion. One of the major predictors of mortality of ACC is perineural invasion, which can be seen in 30 % of breast lesions, 85% of salivary lesions, and almost 100 % of lacrimal gland tumors. The biological differences between these three ACC tumors are still poorly understood. We focused on the current understanding of the genetic variations observed on ACC tumors and prognostic differences associated with distinct anatomical sites. A special effort was made to present the currently available therapies alongside the emerging strategies under development.

Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells.

AIM: To evaluate the potential use of Cephaeline as a therapeutic strategy to manage mucoepidermoid carcinomas (MEC) of the salivary glands. MATERIAL AND METHODS: UM-HMC-1, UM-HMC-2, and UM-HMC-3A MEC cell lines were used to establish the effects of Cephaeline over tumor viability determined by MTT assay. In vitro wound healing scratch assays were performed to address cellular migration while immunofluorescence staining for histone H3 lysine 9 (H3k9ac) was used to identify the acetylation status of tumor cells upon Cephaeline administration. The presence of cancer stem cells was evaluated by the identification of ALDH enzymatic activity by flow cytometry and through functional assays using in vitro tumorsphere formation. RESULTS: A single administration of Cephaeline resulted in reduced viability of MEC cells along with the halt on tumor growth and cellular migration potential. Administration of Cephaeline resulted in chromatin histone acetylation as judged by the increased levels of H3K9ac and disruption of tumorspheres formation. Interestingly, ALDH levels were increased in UM-HMC-1 and UM-HMC-3A cell lines, while UM-HMC-2 showed a reduced enzymatic activity. CONCLUSION: Cephaeline has shown anti-cancer properties in all MEC cell lines tested by regulating tumor cells' viability, migration, proliferation, and disrupting the ability of cancer cells to generate tumorspheres.

Crosstalk between cancer stem cells and the tumor microenvironment drives progression of premalignant oral epithelium.

Cancer stem cells (CSC) are a subpopulation of cancer cells that exhibit properties of self-renewal and differentiation and have been implicated in metastasis and treatment failures. There is mounting evidence that carcinogen-initiated mucosal epithelial stem cells acquire the CSC phenotype following exposure to environmental or infectious mutagens and are responsible for promoting the malignant transformation of premalignant (dysplastic) epithelium. CSC further contribute to the progression of dysplasia by activating signaling pathways through crosstalk with various cell populations in the tumor microenvironment. Two cell types, tumor-associated macrophages (TAM) and vascular endothelial cells (EC) nurture CSC development, support CSC stemness, and contribute to tumor progression. Despite mounting evidence implicating CSC in the initiation and progression of dysplastic oral epithelium to squamous cell carcinoma (SCC), the molecular mechanisms underlying these synergistic biological processes remain unclear. This review will examine the mechanisms that underlie the transformation of normal epithelial stem cells into CSC and the mechanistic link between CSC, TAM, and EC in the growth and the malignant conversation of dysplastic oral epithelium.

Cancer-specific type-I interferon receptor signaling promotes cancer stemness and effector CD8+ T-cell exhaustion.

Type-I interferon (IFN-I) signaling is critical to maintaining antigen-presenting cell function for anti-tumor immunity. However, recent studies have suggested that IFN-I signaling may also contribute to more aggressive phenotypes, raising the possibility that IFN-I downstream signaling in cancer and myeloid cells may exert dichotomous functions.We analyzed the clinicopathologic correlation of cancer-specific IFN-I activation in 195 head and neck squamous cell carcinoma patients. We also characterized the immune impact of IFN-I receptor (IFNAR1)-deficiency in syngeneic tumor models using biochemistry, flow cytometry, and single-cell RNA-Seq. We stained HNSCC tissue microarrays with a sensitive IFN-I downstream signaling activation marker, MX1, and quantitated cancer cell-specific MX1 staining. Kaplan-Meier analysis revealed that MX1-high tumors exhibited worse survival, a phenotype that depends on the number of CD8+ intratumoral T-cells. We found that cancer-specific IFNAR1 engagement promotes cancer stemness and higher expression levels of suppressive immune checkpoint receptor ligands in cancer-derived exosomes. Notably, mice bearing Ifnar1-deficient tumors exhibited lower tumor burden, increased T-cell infiltration, reduced exhausted CD4+PD1high T-cells, and increased effector population CD8+IFN-γ+ T-cells. Then, we performed single-cell RNA-sequencing and discovered that cancer-specific IFN-I signaling not only restricts effector cells expansion but also dampens their functional fitness.The beneficial role of IFN-I activation is largely dependent on the myeloid compartment. Cancer-specific IFN-I receptor engagement promotes cancer stemness and the release of cancer-derived exosomes with high expression levels of immune checkpoint receptor ligands. Cancer-specific IFN-I activation is associated with poor immunogenicity and worse clinical outcomes in HNSCC.

Pulpbow: A Method to Study the Vasculogenic Potential of Mesenchymal Stem Cells from the Dental Pulp.

Understanding how Mesenchymal Stem Cells (MSCs) form blood vessels is critical for creating mechanism-based approaches for the therapeutic use of these cells. In addition, understanding the determinants and factors involved in lineage hierarchy is fundamental to creating accurate and reliable techniques for the study of stem cells in tissue engineering and repair. Dental Pulp Stem Cells (DPSC) from permanent teeth and Stem cells from Human Exfoliated Deciduous teeth (SHED) are particularly interesting sources for tissue engineering as they are easily accessible and expandable. Previously, we have shown that DPSCs and SHEDs can differentiate into endothelial cells and form functional blood vessels through vasculogenesis. Here, we described how we created the "pulpbow" (pulp + rainbow), a multicolor tag experimental model that is stable, permanent, unique to each cell and passed through generations. We used the pulpbow to understand how dental pulp stem cells contributed to blood vessel formation in 3D models in in vitro and ex vivo live cell tracking, and in vivo transplantation assays. Simultaneous tracking of cells during sprout formation revealed that no single multicolor-tagged cell was more prone to vasculogenesis. During this process, there was intense cell motility with minimal proliferation in early time points. In later stages, when the availability of undifferentiated cells around the forming sprout decreased, there was local clonal proliferation mediated by proximity. These results unveiled that the vasculogenesis process mediated by dental pulp stem cells is dynamic and proximity to the sprouting area is critical for cell fate decisions.

Systemic therapies for salivary gland adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a slow growing, but relentless cancer. Due to its rarity and lack of understanding of its molecular etiology, no standard chemotherapy for ACC currently exists and many patients suffer from recurrent and/or metastatic disease. As such, development of safe and effective therapies is imperative. To describe and summarize existing clinical trial studies and preclinical discoveries, we surveyed the PubMed on developmental therapeutics for ACC. Objective response rates to monotherapy with cytotoxic agents were approximately 10% with cisplatin, 5-FU, gemcitabine, mitoxantrone, epirubicin, vinorelbine and paclitaxel. The most studied combination therapies were cyclophosphamide-doxorubicin-cisplatin (CAP) and cisplatin-vinorelbine, with an objective response rate of 18-31%. Among molecularly targeted drugs, the most studied drugs are inhibitors targeting the vascular endothelial growth factor receptor (VEGFR) to inhibit tumor angiogenesis. Among those, lenvatinib and axitinib showed a relatively high objective response rate of 11-16% and 9-17%, respectively. Given high recurrence rates and chemoresistance of ACC, treatments targeting cancer stem cells (CSC), which function as tumor-initiating cells and drive chemoresistance, may be particularly valuable. CSC have been shown to be targetable via MYB, Notch1, p53 and epigenetic mechanisms. Myb overexpression is characteristic in ACC but was previously thought to present a difficult target due to its nature as a transcription factor. However, due to the development Myb-targeted inhibitors and an ongoing clinical trial of MYB-targeted cancer vaccine therapy, MYB is becoming an increasingly attractive therapeutic target. Drugs targeting NOTCH signaling demonstrated 5-17% response rate in phase I clinical trials. Within the field of epigenetics, treatment with PRMT5 inhibitors has shown 21% partial response rate in phase I clinical trial. Immunotherapies, such as PD-1 inhibitors, are also associated with CSC, but have not been effective against ACC. However, clinical trials of cancer vaccine therapies are actively being conducted. In addition to conventional chemotherapies and inhibitors of angiogenesis, the emergence of new therapies such as immunotherapy and those targeting cancer stemness is expected to bring clinical benefits to patients in the future.

The IL-6R and Bmi-1 axis controls self-renewal and chemoresistance of head and neck cancer stem cells.

Despite major progress in elucidating the pathobiology of head and neck squamous cell carcinoma (HNSCC), the high frequency of disease relapse correlates with unacceptably deficient patient survival. We previously showed that cancer stem-like cells (CSCs) drive tumorigenesis and progression of HNSCC. Although CSCs constitute only 2-5% of total tumor cells, CSCs contribute to tumor progression by virtue of their high tumorigenic potential and their resistance to chemo-, radio-, and immunotherapy. Not only are CSCs resistant to therapy, but cytotoxic agents actually enhance cancer stemness by activating transcription of pluripotency factors and by inducing expression of Bmi-1, a master regulator of stem cell self-renewal. We hypothesized therapeutic inhibition of interleukin-6 receptor (IL-6R) suppresses Bmi-1 to overcome intrinsic chemoresistance of CSCs. We observed that high Bmi-1 expression correlates with decreased (p = 0.04) recurrence-free survival time in HNSCC patients (n = 216). Blockade of IL-6R by lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, secondary sphere formation, and to decrease the CSC fraction even in Cisplatin-resistant HNSCC cells. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant HNSCC cells. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function and inhibits cancer stemness. These results suggest therapeutic inhibition of IL-6R might be a viable strategy to overcome the CSC-mediated chemoresistance typically observed in HNSCC patients.

Inverse and reciprocal regulation of p53/p21 and Bmi-1 modulates vasculogenic differentiation of dental pulp stem cells.

Dental pulp stem cells (DPSC) are capable of differentiating into vascular endothelial cells. Although the capacity of vascular endothelial growth factor (VEGF) to induce endothelial differentiation of stem cells is well established, mechanisms that maintain stemness and prevent vasculogenic differentiation remain unclear. Here, we tested the hypothesis that p53 signaling through p21 and Bmi-1 maintains stemness and inhibits vasculogenic differentiation. To address this hypothesis, we used primary human DPSC from permanent teeth and Stem cells from Human Exfoliated Deciduous (SHED) teeth as models of postnatal mesenchymal stem cells. DPSC seeded in biodegradable scaffolds and transplanted into immunodeficient mice generated mature human blood vessels invested with smooth muscle actin-positive mural cells. Knockdown of p53 was sufficient to induce vasculogenic differentiation of DPSC (without vasculogenic differentiation medium containing VEGF), as shown by increased expression of endothelial markers (VEGFR2, Tie-2, CD31, VE-cadherin), increased capillary sprouting in vitro; and increased DPSC-derived blood vessel density in vivo. Conversely, induction of p53 expression with small molecule inhibitors of the p53-MDM2 binding (MI-773, APG-115) was sufficient to inhibit VEGF-induced vasculogenic differentiation. Considering that p21 is a major downstream effector of p53, we knocked down p21 in DPSC and observed an increase in capillary sprouting that mimicked results observed when p53 was knocked down. Stabilization of ubiquitin activity was sufficient to induce p53 and p21 expression and reduce capillary sprouting. Interestingly, we observed an inverse and reciprocal correlation between p53/p21 and the expression of Bmi-1, a major regulator of stem cell self-renewal. Further, direct inhibition of Bmi-1 with PTC-209 resulted in blockade of capillary-like sprout formation. Collectively, these data demonstrate that p53/p21 functions through Bmi-1 to prevent the vasculogenic differentiation of DPSC.

Propolis reduces the stemness of head and neck squamous cell carcinoma.

OBJECTIVE: To evaluate the effect of Brazilian propolis on head and neck cancer stem cells in vitro. METHODS: Head and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-17B and UM-SCC-74A), human keratinocytes (HK), and primary human dermal microvascular endothelial cells (HDMEC) were treated with 0.5, 5.0, or 50 µg/mL green, brown or red Brazilian propolis or vehicle control for 24, 36, and 72 h. Cell viability was evaluated by Sulforhodamine B assay. Western blots evaluated expression of cancer stem cell (CSC) markers (i.e. ALDH, CD44, Oct-4, Bmi-1) and flow cytometry was performed to determine the impact of propolis in the fraction of CSC, defined as ALDHhighCD44high cells. RESULTS: propolis significantly reduced cell viability of HNSCC and HDMEC cells, but not HK. Notably, red propolis caused a significant reduction in the percentage of CSC, reduced the number of orospheres, and downregulated the expression of stem cell markers. CONCLUSIONS: Collectively, our data demonstrate an anti-CSC effect of propolis, and suggest that propolis (i.e. red propolis) might be beneficial for patients with head and neck cancer.

Survival of salivary gland cancer stem cells requires mTOR signaling.

Advanced salivary gland mucoepidermoid carcinoma (MEC) is a relentless cancer that exhibits resistance to conventional chemotherapy. As such, treatment for patients with advanced MEC is tipically radical surgery and radiotherapy. Facial disfigurement and poor quality of life are frequent treatment challenges, and many patients succumb to loco-regional recurrence and/or metastasis. We know that cancer stem-like cells (CSC) drive MEC tumorigenesis. The current study tests the hypothesis that MEC CSC are sensitive to therapeutic inhibition of mTOR. Here, we report a correlation between the long-term clinical outcomes of 17 MEC patients and the intratumoral expression of p-mTOR (p = 0.00294) and p-S6K1 (p = 0.00357). In vitro, we observed that MEC CSC exhibit constitutive activation of the mTOR signaling pathway (i.e., mTOR, AKT, and S6K1), unveiling a potential strategy for targeted ablation of these cells. Using a panel of inhibitors of the mTOR pathway, i.e., rapamycin and temsirolimus (mTOR inhibitors), buparlisib and LY294002 (AKT inhibitors), and PF4708671 (S6K1 inhibitor), we observed consistently dose-dependent decrease in the fraction of CSC, as well as inhibition of secondary sphere formation and self-renewal in three human MEC cell lines (UM-HMC-1,-3A,-3B). Notably, therapeutic inhibition of mTOR with rapamycin or temsirolimus induced preferential apoptosis of CSC, when compared to bulk tumor cells. In contrast, conventional chemotherapeutic drugs (cisplatin, paclitaxel) induced preferential apoptosis of bulk tumor cells and accumulation of CSC. In vivo, therapeutic inhibition of mTOR with temsirolimus caused ablation of CSC and downregulation of Bmi-1 expression (major inducer of stem cell self-renewal) in MEC xenografts. Transplantation of MEC cells genetically silenced for mTOR into immunodeficient mice corroborated the results obtained with temsirolimus. Collectively, these data demonstrated that mTOR signaling is required for CSC survival, and unveiled the therapeutic potential of targeting the mTOR pathway for elimination of highly tumorigenic cancer stem-like cells in salivary gland mucoepidermoid carcinoma.