Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling.

Endothelial cell-derived interleukin-6 regulates tumor growth.

BACKGROUND: Endothelial cells play a complex role in the pathobiology of cancer. This role is not limited to the making of blood vessels to allow for influx of oxygen and nutrients required for the high metabolic demands of tumor cells. Indeed, it has been recently shown that tumor-associated endothelial cells secrete molecules that enhance tumor cell survival and cancer stem cell self-renewal. The hypothesis underlying this work is that specific disruption of endothelial cell-initiated signaling inhibits tumor growth. METHODS: Conditioned medium from primary human dermal microvascular endothelial cells (HDMEC) stably transduced with silencing RNA for IL-6 (or controls) was used to evaluate the role of endothelial-derived IL-6 on the activation of key signaling pathways in tumor cells. In addition, these endothelial cells were co-transplanted with tumor cells into immunodefficient mice to determine the impact of endothelial cell-derived IL-6 on tumor growth and angiogenesis. RESULTS: We observed that tumor cells adjacent to blood vessels show strong phosphorylation of STAT3, a key mediator of tumor progression. In search for a possible mechanism for the activation of the STAT3 signaling pathway, we observed that silencing interleukin (IL)-6 in tumor-associated endothelial cells inhibited STAT3 phosphorylation in tumor cells. Notably, tumors vascularized with IL-6-silenced endothelial cells showed lower intratumoral microvessel density, lower tumor cell proliferation, and slower growth than tumors vascularized with control endothelial cells. CONCLUSIONS: Collectively, these results demonstrate that IL-6 secreted by endothelial cells enhance tumor growth, and suggest that cancer patients might benefit from targeted approaches that block signaling events initiated by endothelial cells.

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.

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.

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.

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.

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.

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.

UM-HACC-2A: MYB-NFIB fusion-positive human adenoid cystic carcinoma cell line.

OBJECTIVES: Limited availability of validated human adenoid cystic carcinoma (ACC) cell lines has hindered the mechanistic understanding of the pathobiology of this malignancy and the development of effective therapies. The purpose of this work was to generate and characterize a human ACC cell line. MATERIAL AND METHODS: Immediately after surgery, a tumor fragment from a minor salivary gland from the tongue of a female Caucasian was minced, dissociated, and a single cell suspension was plated in fibronectin-coated flasks. A culture medium containing bovine brain extract and rhEGF was optimized for these cells. Whole exome sequencing was used to evaluate the presence of MYB-NFIB translocation. RESULTS: The University of Michigan-Human Adenoid Cystic Carcinoma (UM-HACC)-2A cells showed continuous growth in monolayers for at least 180 in vitro passages while maintaining epithelial morphology. Short-tandem repeat (STR) profiling confirmed a 100% match to patient DNA. Whole exome sequencing revealed the presence of the MYB-NFIB fusion in UM-HACC-2A cells, which was confirmed by PCR analysis. Western blots revealed high expression of epithelial markers (e.g. E-cadherin, EGFR, pan-cytokeratin) and proteins associated with ACC (e.g. c-Myb, p63). Developmental therapeutic studies showed that UM-HACC-2A cells were resistant to cisplatin (IC50 = 44.7 µM) while more responsive to paclitaxel (IC50 = 0.0006 µM). In a pilot study, we observed that UM-HACC-2A cells survived orthotopic transplantation into the submandibular gland. Notably, one of the mice injected with UM-HACC-2A cells exhibited lung metastasis after 6 months. CONCLUSION: UM-HACC-2A is a MYB-NFIB fusion-positive ACC cell line that is suitable for mechanistic and developmental therapeutics studies.

Targeting histone deacetylase and NFκB signaling as a novel therapy for Mucoepidermoid Carcinomas.

Malignancies from the salivary glands are rare and represent 11% of all cancers from the oropharyngeal anatomical area. Mucoepidermoid Carcinomas (MEC) is the most common malignancy from the salivary glands. Low survival rates of high-grade Mucoepidermoid Carcinomas (MEC) are particularly associated with the presence of positive lymph nodes, extracapsular lymph node spread, and perineural invasion. Most recently, the presence of cancer stem cells (CSC), and the activation of the NFκB signaling pathway have been suggested as cues for an acquired resistance phenotype. We have previously shown that NFκB signaling is very active in MEC tumors. Herein, we explore the efficacy of NFκB inhibition in combination with class I and II HDAC inhibitor to deplete the population of CSC and to destroy MEC tumor cells. Our finding suggests that disruption of NFκB signaling along with the administration of HDAC inhibitors constitute an effective strategy to manage MEC tumors.

Therapeutic Inhibition of the MDM2-p53 Interaction Prevents Recurrence of Adenoid Cystic Carcinomas.

Purpose: Conventional chemotherapy has modest efficacy in advanced adenoid cystic carcinomas (ACC). Tumor recurrence is a major challenge in the management of ACC patients. Here, we evaluated the antitumor effect of a novel small-molecule inhibitor of the MDM2-p53 interaction (MI-773) combined with cisplatin in patient-derived xenograft (PDX) ACC tumors.Experimental Design: Therapeutic strategies with MI-773 and/or cisplatin were evaluated in SCID mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in low passage primary human ACC cells (UM-HACC-2A, -2B, -5, -6) in vitro The effect of therapy on the fraction of cancer stem cells (CSC) was determined by flow cytometry for ALDH activity and CD44 expression.Results: Combined therapy with MI-773 with cisplatin caused p53 activation, induction of apoptosis, and regression of ACC PDX tumors. Western blots revealed induction of MDM2, p53 and downstream p21 expression, and regulation of apoptosis-related proteins PUMA, BAX, Bcl-2, Bcl-xL, and active caspase-9 upon MI-773 treatment. Both single-agent MI-773 and MI-773 combined with cisplatin decreased the fraction of CSCs in PDX ACC tumors. Notably, neoadjuvant MI-773 and surgery eliminated tumor recurrences during a postsurgical follow-up of more than 300 days. In contrast, 62.5% of mice that received vehicle control presented with palpable tumor recurrences within this time period (P = 0.0097).Conclusions: Collectively, these data demonstrate that therapeutic inhibition of MDM2-p53 interaction by MI-773 decreased the CSC fraction, sensitized ACC xenograft tumors to cisplatin, and eliminated tumor recurrence. These results suggest that patients with ACC might benefit from the therapeutic inhibition of the MDM2-p53 interaction. Clin Cancer Res; 23(4); 1036-48. ©2016 AACR.

Endothelial-derived interleukin-6 induces cancer stem cell motility by generating a chemotactic gradient towards blood vessels.

Recent evidence suggests that the metastatic spread of head and neck squamous cell carcinomas (HNSCC) requires the function of cancer stem cells endowed with multipotency, self-renewal, and high tumorigenic potential. We demonstrated that cancer stem cells reside in perivascular niches and are characterized by high aldehyde dehydrogenase (ALDH) activity and high CD44 expression (ALDHhighCD44high) in HNSCC. Here, we hypothesize that endothelial cell-secreted interleukin-6 (IL-6) contributes to tumor progression by enhancing the migratory phenotype and survival of cancer stem cells. Analysis of tissue microarrays generated from the invasive fronts of 77 HNSCC patients followed-up for up to 11 years revealed that high expression of IL-6 receptor (IL-6R) (p=0.0217) or co-receptor gp130 (p=0.0422) correlates with low HNSCC patient survival. We observed that endothelial cell-secreted factors induce epithelial to mesenchymal transition (EMT) and enhance invasive capacity of HNSCC cancer stem cells. Conditioned medium from CRISPR/Cas9-mediated IL-6 knockout primary human endothelial cells is less chemotactic for cancer stem cells in a microfluidics-based system than medium from control endothelial cells (p<0.05). Blockade of the IL-6 pathway with a humanized anti-IL-6R antibody (tocilizumab) inhibited endothelial cell-induced motility in vitro and decreased the fraction of cancer stem cells in vivo. Notably, xenograft HNSCC tumors vascularized with IL-6-knockout endothelial cells exhibited slower tumor growth and smaller cancer stem cell fraction. These findings demonstrate that endothelial cell-secreted IL-6 enhances the motility and survival of highly tumorigenic cancer stem cells, suggesting that endothelial cells can create a chemotactic gradient that enables the movement of carcinoma cells towards blood vessels.

5T4-Targeted Therapy Ablates Cancer Stem Cells and Prevents Recurrence of Head and Neck Squamous Cell Carcinoma.

Purpose: Locoregional recurrence is a frequent treatment outcome for patients with advanced head and neck squamous cell carcinoma (HNSCC). Emerging evidence suggests that tumor recurrence is mediated by a small subpopulation of uniquely tumorigenic cells, that is, cancer stem cells (CSC), that are resistant to conventional chemotherapy, endowed with self-renewal and multipotency.Experimental Design: Here, we evaluated the efficacy of MEDI0641, a novel antibody-drug conjugate targeted to 5T4 and carrying a DNA-damaging "payload" (pyrrolobenzodiazepine) in preclinical models of HNSCC.Results: Analysis of a tissue microarray containing 77 HNSCC with follow-up of up to 12 years revealed that patients with 5T4high tumors displayed lower overall survival than those with 5T4low tumors (P = 0.038). 5T4 is more highly expressed in head and neck CSC (ALDHhighCD44high) than in control cells (non-CSC). Treatment with MEDI0641 caused a significant reduction in the CSC fraction in HNSCC cells (UM-SCC-11B, UM-SCC-22B) in vitro Notably, a single intravenous dose of 1 mg/kg MEDI0641 caused long-lasting tumor regression in three patient-derived xenograft (PDX) models of HNSCC. MEDI0641 ablated CSC in the PDX-SCC-M0 model, reduced it by five-fold in the PDX-SCC-M1, and two-fold in the PDX-SCC-M11 model. Importantly, mice (n = 12) treated with neoadjuvant, single administration of MEDI0641 prior to surgical tumor removal showed no recurrence for more than 200 days, whereas the control group had 7 recurrences (in 12 mice; P = 0.0047).Conclusions: Collectively, these findings demonstrate that an anti-5T4 antibody-drug conjugate reduces the fraction of CSCs and prevents local recurrence and suggest a novel therapeutic approach for patients with HNSCC. Clin Cancer Res; 23(10); 2516-27. ©2016 AACR.

Patient-derived xenograft (PDX) tumors increase growth rate with time.

Patient-derived xenograft (PDX) models are frequently used for translational cancer research, and are assumed to behave consistently as the tumor ages. However, growth rate constancy as a function of time is unclear. Notably, variable PDX growth rates over time might have implications for the interpretation of translational studies. We characterized four PDX models through several in vivo passages from primary human head and neck squamous cell carcinoma and salivary gland adenoid cystic carcinoma. We developed a mathematical approach to merge growth data from different passages into a single measure of relative tumor volume normalized to study initiation size. We analyzed log-relative tumor volume increase with linear mixed effect models. Two oral pathologists analyzed the PDX tissues to determine if histopathological feature changes occurred over in vivo passages. Tumor growth rate increased over time. This was determined by repeated measures linear regression statistical analysis in four different PDX models. A quadratic statistical model for the temporal effect predicted the log-relative tumor volume significantly better than a linear time effect model. We found a significant correlation between passage number and histopathological features of higher tumor grade. Our mathematical treatment of PDX data allows statistical analysis of tumor growth data over long periods of time, including over multiple passages. Non-linear tumor growth in our regression models revealed the exponential growth rate increased over time. The dynamic tumor growth rates correlated with quantifiable histopathological changes that related to passage number in multiple types of cancer.

Overcoming adaptive resistance in mucoepidermoid carcinoma through inhibition of the IKK-ß/IκBα/NFκB axis.

Patients with mucoepidermoid carcinoma (MEC) experience low survival rates and high morbidity following treatment, yet the intrinsic resistance of MEC cells to ionizing radiation (IR) and the mechanisms underlying acquired resistance remain unexplored. Herein, we demonstrated that low doses of IR intrinsically activated NFκB in resistant MEC cell lines. Moreover, resistance was significantly enhanced in IR-sensitive cell lines when NFκB pathway was stimulated. Pharmacological inhibition of the IKK-ß/IκBα/NFκB axis, using a single dose of FDA-approved Emetine, led to a striking sensitization of MEC cells to IR and a reduction in cancer stem cells. We achieved a major step towards better understanding the basic mechanisms involved in IR-adaptive resistance in MEC cell lines and how to efficiently overcome this critical problem.

Sensitizing mucoepidermoid carcinomas to chemotherapy by targeted disruption of cancer stem cells.

Mucoepidermoid carcinoma (MEC) is the most common malignancy of salivary glands. The response of MEC to chemotherapy is unpredictable, and recent advances in cancer biology suggest the involvement of cancer stem cells (CSCs) in tumor progression and chemoresistance and radioresistance phenotype. We found that histone acetyltransferase inhibitors (HDACi) were capable of disrupting CSCs in MEC. Furthermore, administration of HDACi prior to Cisplatin (two-hit approach) disrupts CSCs and sensitizes tumor cells to Cisplatin. Our findings corroborate to emerging evidence that CSCs play a key role in tumor resistance to chemotherapy, and highlights a pharmacological two-hit approach that disrupts tumor resistance to conventional therapy.

IL-6 Inhibition With MEDI5117 Decreases The Fraction of Head and Neck Cancer Stem Cells and Prevents Tumor Recurrence.

Head and neck squamous cell carcinomas (HNSCC) exhibit a small population of uniquely tumorigenic cancer stem cells (CSC) endowed with self-renewal and multipotency. We have recently shown that IL-6 enhances the survival and tumorigenic potential of head and neck cancer stem cells (i.e. ALDH(high)CD44(high) cells). Here, we characterized the effect of therapeutic inhibition of IL-6 with a novel humanized anti-IL-6 antibody (MEDI5117) using three low-passage patient-derived xenograft (PDX) models of HNSCC. We observed that single agent MEDI5117 inhibited the growth of PDX-SCC-M1 tumors (P < .05). This PDX model was generated from a previously untreated HNSCC. In contrast, MEDI5117 was not effective at reducing overall tumor volume for PDX models representing resistant disease (PDX-SCC-M0, PDX-SCC-M11). Low dose MEDI5117 (3 mg/kg) consistently decreased the fraction of cancer stem cells in PDX models of HNSCC when compared to IgG-treated controls, as follows: PDX-SCC-M0 (P < .001), PDX-SCC-M1 (P < .001), PDX-SCC-M11 (P = .04). Interestingly, high dose MEDI5117 (30 mg/kg) decreased the CSC fraction in the PDX-SCC-M11 model (P = .002), but not in PDX-SCC-M0 and PDX-SCC-M1. MEDI5117 mediated a dose-dependent decrease in the number of orospheres generated by ALDH(high)CD44(high) cells cultured in ultra-low attachment plates (P < .05), supporting an inhibitory effect on head and neck cancer stem cells. Notably, single agent MEDI5117 reduced the overall recurrence rate of PDX-SCC-M0, a PDX generated from the local recurrence of human HNSCC. Collectively, these data demonstrate that therapeutic inhibition of IL-6 with low-dose MEDI5117 decreases the fraction of cancer stem cells, and that adjuvant MEDI5117 inhibits recurrence in preclinical models of HNSCC.

Targeting MDM2 for Treatment of Adenoid Cystic Carcinoma.

PURPOSE: There are no effective treatment options for patients with advanced adenoid cystic carcinoma (ACC). Here, we evaluated the effect of a new small molecule inhibitor of the MDM2-p53 interaction (MI-773) in preclinical models of ACC. EXPERIMENTAL DESIGN: To evaluate the anti-tumor effect of MI-773, we administered it to mice harboring three different patient-derived xenograft (PDX) models of ACC expressing functional p53. The effect of MI-773 on MDM2, p53, phospho-p53, and p21 was examined by Western blots in 5 low passage primary human ACC cell lines and in MI-773-treated PDX tumors. RESULTS: Single-agent MI-773 caused tumor regression in the 3 PDX models of ACC studied here. For example, we observed a tumor growth inhibition index of 127% in UM-PDX-HACC-5 tumors that was associated with an increase in the fraction of apoptotic cells (P = 0.015). The number of p53-positive cells was increased in MI-773-treated PDX tumors (P < 0.001), with a correspondent shift in p53 localization from the nucleus to the cytoplasm. Western blots demonstrated that MI-773 potently induced expression of p53 and its downstream targets p21, MDM2, and induced phosphorylation of p53 (serine 392) in low passage primary human ACC cells. Notably, MI-773 induced a dose-dependent increase in the fraction of apoptotic ACC cells and in the fraction of cells in the G1 phase of cell cycle (P < 0.05). CONCLUSIONS: Collectively, these data demonstrate that therapeutic inhibition of the MDM2-p53 interaction with MI-773 activates downstream effectors of apoptosis and causes robust tumor regression in preclinical models of ACC. Clin Cancer Res; 22(14); 3550-9. ©2016 AACR.

ABCC5, ERCC2, XPA and XRCC1 transcript abundance levels correlate with cisplatin chemoresistance in non-small cell lung cancer cell lines.

BACKGROUND: Although 40-50% of non-small cell lung cancer (NSCLC) tumors respond to cisplatin chemotherapy, there currently is no way to prospectively identify potential responders. The purpose of this study was to determine whether transcript abundance (TA) levels of twelve selected DNA repair or multi-drug resistance genes (LIG1, ERCC2, ERCC3, DDIT3, ABCC1, ABCC4, ABCC5, ABCC10, GTF2H2, XPA, XPC and XRCC1) were associated with cisplatin chemoresistance and could therefore contribute to the development of a predictive marker. Standardized RT (StaRT)-PCR, was employed to assess these genes in a set of NSCLC cell lines with a previously published range of sensitivity to cisplatin. Data were obtained in the form of target gene molecules relative to 106 beta-actin (ACTB) molecules. To cancel the effect of ACTB variation among the different cell lines individual gene expression values were incorporated into ratios of one gene to another. Each two-gene ratio was compared as a single variable to chemoresistance for each of eight NSCLC cell lines using multiple regression. In an effort to validate these results, six additional lines then were evaluated. RESULTS: Following validation, single variable models best correlated with chemoresistance (p < 0.001), were ERCC2/XPC, ABCC5/GTF2H2, ERCC2/GTF2H2, XPA/XPC and XRCC1/XPC. All single variable models were examined hierarchically to achieve two variable models. The two variable model with the highest correlation was (ABCC5/GTF2H2, ERCC2/GTF2H2) with an R2 value of 0.96 (p < 0.001). CONCLUSION: These results provide markers suitable for assessment of small fine needle aspirate biopsies in an effort to prospectively identify cisplatin resistant tumors.