Establishment and characterization of cMYB-expressing human salivary adenoid cystic carcinoma cell lines (UM-HACC-14, UM-HACC-6) and matching patient-derived xenograft model (UM-PDX-HACC-14).

OBJECTIVE: Limited availability of authentic human adenoid cystic carcinoma (ACC) cell lines has hindered progress in understanding mechanisms underpinning the biology of this disease and the development of safe and effective therapies. STUDY DESIGN: Surgical human ACC specimens (UM-HACC-6, UM-HACC-14) were dissociated into single cell suspensions and cultured in fibronectin-coated flasks. Alternatively, tumor fragments were transplanted subcutaneously into female immunodeficient (SCID) mice to establish patient-derived xenograft tumors (PDX; UM-PDX-HACC-14). RESULTS: Both ACC cell lines showed continuous growth in monolayers for over 100 passages. Total RNA-Seq, RT-PCR, and FISH analysis revealed that both are MYB-NFIB fusion negative. Western blots revealed passage-dependent expression of E-Cadherin, PCNA, p63, phospho-c-MYB, and NFIB. Both, UM-HACC-14 and UM-HACC-6 cells exhibited tumorigenic potential when injected orthotopically into mouse submandibular glands. CONCLUSION: UM-HACC-14, patient-matching UM-PDX-HACC-14, and the UM-HACC-6 cell line are new, authenticated preclinical models of ACC that are well suited for mechanistic and developmental therapeutics studies.

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.

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.

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.

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.