Brain derived neutrophic factor (BDNF) coordinates lympho-vascular metastasis through a fibroblast-governed paracrine axis in the tumor microenvironment.

It has long been known that the tumor microenvironment contributes to the proliferation and survival of neoplasms through the constant interaction with the stromal and immune compartments. In this investigation, we explored the role of cancer-associated fibroblasts (CAFs) in the regulation of the tumor microenvironment in head and neck squamous cell carcinoma (HNSCC) though a complex intercellular BDNF-TrkB signaling system. Our studies show that conditioned media derived from patient-derived CAFs promoted HNSCC cell proliferation, in vitro cell migration, cell invasion and chemotherapy resistance, compared to normal fibroblasts. Furthermore, examination of the in vivo impact of CAF pathophysiology in the tumor microenvironment in animal xenograft models revealed that HNSCC cell lines in combination with CAFs promoted tumor growth and increased incidence of lymphovascular metastasis as compared to injection of tumor cells or CAF cells alone. Using pharmacological and genetic alterations, we mechanistically demonstrate the critical importance of BDNF-TrkB signaling in the tumor microenvironment. These investigations further support the rationale for BDNF/TRKB targeted therapy against in the treatment of HNSCC.

Human epidermal growth factor receptor 2/neu as a novel therapeutic target in sinonasal undifferentiated carcinoma.

BACKGROUND: Sinonasal undifferentiated carcinoma (SNUC) is a rare and aggressive cancer. Despite multimodal therapy, the prognosis in SNUC remains poor, and new therapies are needed. Thus, the purpose of this study was to explore potential therapeutic targets in SNUC. METHODS: Using the human-derived SNUC MDA8788-6 cell line, we performed whole genome single nucleotide polymorphism (SNP) analysis to identify copy number changes in this line. Protein expression levels were evaluated by Western blotting. Cell growth inhibition was assessed by methylthiazol tetrazolium (MTT) and clonogenic assays. The mouse flank model was used to examine the effect of growth inhibition in vivo. RESULTS: The ERBB2 gene was highly amplified and cell extracts showed human epidermal growth factor receptor 2 (HER2) was overexpressed and phosphorylated in MDA8788-6. Lapatinib effectively inhibited the HER2 signaling pathway in our SNUC cell line. HER2 inhibition successfully suppressed the cell growth of MDA8788-6 cells both in vitro and in vivo. CONCLUSION: Targeting HER2 may be a promising avenue for the development of novel therapies for SNUC. © 2016 Wiley Periodicals, Inc. Head Neck 38: E1926-E1934, 2016.

Coordinated targeting of the EGFR signaling axis by microRNA-27a*.

Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.

Establishment and characterization of novel cell lines from sinonasal undifferentiated carcinoma.

PURPOSE: Sinonasal undifferentiated carcinoma (SNUC) is a rare and aggressive cancer. Despite the use of multimodality treatment, the overall prognosis remains poor. To better understand the biologic features of SNUC and help develop new therapies for the disease, we established SNUC cell lines and characterized their biologic behaviors. EXPERIMENTAL DESIGN: Cell lines were established from a patient with a T4N0M0 SNUC of the right maxillary sinus who was treated with surgical resection at our center. Tumor colonies were harvested and were sequentially replated onto larger plates. Two populations were developed and labeled MDA8788-6 and MDA8788-7. These cell lines were characterized with molecular, biomarker, functional, and histologic analyses. RESULTS: Short tandem repeat genotyping revealed that the cell line is isogenic to the parental tumor, and cytogenetic analysis identified 12 chromosomal translocations. The SNUC cell lines do not form colonies in soft agar but are tumorigenic and nonmetastatic in an orthotopic mouse model of sinonasal cancer. Western blot analysis revealed that both MDA8788 cell lines express epithelial markers but do not express mesenchymal markers or the endocrine marker synaptophysin. CONCLUSIONS: This is the first report of the establishment of stable human-derived SNUC cell lines. The lines were highly tumorigenic and maintain the histologic and molecular features of the original tumor. These cell lines should serve as useful tools for the future study of SNUC biology and the development and testing of novel therapies for this deadly disease.

A novel role for BDNF-TrkB in the regulation of chemotherapy resistance in head and neck squamous cell carcinoma.

Mechanisms of resistance for HNSCC to cisplatin (CDDP), the foundational chemotherapeutic agent in the treatment of this disease, remain poorly understood. We previously demonstrated that cisplatin resistance (CR) can be overcome by targeting Trk receptor. In the current study, we explored the potential mechanistic role of the BDNF-TrkB signaling system in the development of CDDP resistance in HNSCC. Utilizing an in vitro system of acquired CR, we confirmed a substantial up-regulation of both BDNF and TrkB at the protein and mRNA levels in CR cells, suggesting an autocrine pathway dysregulation in this system. Exogenous BDNF stimulation led to an enhanced expression of the drug-resistance and anti-apoptotic proteins MDR1 and XiAP, respectively, in a dose-dependently manner, demonstrating a key role for BDNF-TrkB signaling in modulating the response to cytotoxic agents. In addition, modulation of TrkB expression induced an enhanced sensitivity of cells to CDDP in HNSCC. Moreover, genetic suppression of TrkB resulted in changes in expression of Bim, XiAP, and MDR1 contributing to HNSCC survival. To elucidate intracellular signaling pathways responsible for mechanisms underlying BDNF/TrkB induced CDDP-resistance, we analyzed expression levels of these molecules following inhibition of Akt. Inhibition of Akt eliminated BDNF effect on MDR1 and Bim expression in OSC-19P cells as well as modulated expressions of MDR1, Bim, and XiAP in OSC-19CR cells. These results suggest BDNF/TrkB system plays critical roles in CDDP-resistance development by utilizing Akt-dependent signaling pathways.

Targeting PKC-mediated signal transduction pathways using enzastaurin to promote apoptosis in acute myeloid leukemia-derived cell lines and blast cells.

Recent studies in acute myeloid leukemia (AML) suggest activation of pro-proliferative signaling cascades including those mediated by protein kinase C (PKC) represent a poor prognostic factor for patients. The classical PKC isoforms α and ß generally support survival signaling and have emerged as important targets for anti-cancer therapy. Enzastaurin is a PKC ß inhibitor and is in clinical trials for lymphomas, gliomas, and lung cancer. Presently, it is not known if enzastaurin could be effective against AML. In the current study, we found that high dose enzastaurin was found to promote apoptosis in the AML-derived cell lines and in blast cells from AML patients. The mechanism of cell death, however, likely does not involve PKC ß as another PKC ß inhibitor was not toxic to AML cell lines and did not promote enzastaurin-induced cell killing. While enzastaurin is fairly specific for PKC ß, the agent can inhibit other PKC isoforms at higher concentrations. Enzastaurin was effective at inhibiting PKC α phosphorylation and membrane localization in the AML cell lines and suppressed phosphorylation of BCL2. Furthermore, enzastaurin suppressed activation of ERK (which can be activated by PKC α). Analysis of the serine/threonine phosphorylation profile in HL60 cells after enzastaurin treatment revealed that the drug inhibits the phosphorylation of a distinct set of proteins while promoting phosphorylation of another set of proteins. This suggests the drug may regulate multiple signaling pathways. Taken together, these findings suggest that enzastaurin could be effective in the therapy of AML.

Theraputic targeting of Trk supresses tumor proliferation and enhances cisplatin activity in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is a biologically aggressive disease that has been modestly impacted by improvements in therapeutic strategies. Several lines of evidence support the role of TrkB for invasion and metastasis in various solid tumor models, and we have shown an important function of this receptor in HNSCC tumor biology. Therapeutic modulation of TrkB function has been supported in the literature by the development of small molecule inhibitors (SMI) with minimal success. To assess the validity of targeting TrkB in HNSCC, we tested a novel agent, AZ64 and show significant dose and time-dependent inhibition of cellular proliferation in cell lines. Genetic studies revealed the specificity of this compound for the TrkB receptor, as exposure of cells that had genetic suppression of TrkB did not demonstrate abrogated oncogenic signaling. We next assessed the impact of AZ64 as a chemotherapy-sensitizer and identified an enhancement of cisplatin-mediated anti-proliferation across all cell lines. We then demonstrated that AZ64 can overcome chemotherapy resistance in a novel model of cisplatin resistance in HNSCC. Modulation of the pro-oncogenic STAT3 and Src pathways was identified, suggesting molecular mechanisms of action for AZ64. In this study, we demonstrate the feasibility of targeting TrkB and suggest a novel approach for the treatment of some chemotherapy-resistant HNSCC.

Ceramide promotes apoptosis in chronic myelogenous leukemia-derived K562 cells by a mechanism involving caspase-8 and JNK.

Ceramide is a sphingolipid that activates stress kinases such as p38 and c-JUN N-Terminal Kinase (JNK). Though Chronic Myelogenous Leukemia (CML) derived K562 cells resist killing by short chain C2-ceramide, we report here that longer chain C6-ceramide promotes apoptosis in these cells. C6-ceramide induces cleavage of Caspase-8 and Caspase-9, but only Caspase-8 is required for apoptosis. The sphingolipid killed CML derived KBM5 cells and, to a lesser extent, imatinib-resistant KBM5-STI cells suggesting that BCR-ABL can not completely block C6-ceramide-induced apoptosis but the kinase may regulate the process. BCR-ABL is known to suppress Protein Phosphatase 2A (PP2A) in CML cells. While C6-ceramide can activate PP2A in acute leukemia cells, the sphingolipid did not activate the phosphatase in K562 cells. C6-ceramide did not activate p38 kinase but did promote JNK activation and phosphorylation of JUN. Inhibition of JNK by pharmacological agent protected K562 cells from C6-ceramide suggesting that JNK plays an essential role in C6-ceramide mediated apoptosis. Furthermore, the sphingolipid promoted MCL-1 phosphorylation by a mechanism that, at least in part, involves JNK. The findings presented here suggest that Caspase-8, JNK, and perhaps MCL-1 may play important roles in regulating cell death and may represent new targets for therapeutic strategies for CML.

Mitochondrial protein phosphatase 2A regulates cell death induced by simulated ischemia in kidney NRK-52E cells.

Acute renal failure can occur after an ischemic injury and results in significant mortality. The stress-signaling pathways that are activated during renal ischemia are unknown. PP2A has emerged as an important regulator of cell death. To study the role of PP2A in ischemia-induced cell death, we used an in vitro model of simulated ischemia. In the present study, simulated ischemia in rat renal tubule epithelial NRK-52E cells (A) results in cell death that involves both necrosis and apoptosis, (B) activates PP2A, and (C) up-regulates the PP2A B56 alpha regulatory subunit. Previous data have shown that PKC alpha negatively regulates B56 alpha protein expression. Consistent with this finding, simulated ischemia suppressed PKC alpha and up-regulated B56 alpha. Treatment of NRK-52E cells with ceramide suppressed PKC alpha and activated PP2A in a manner that mimicked simulated ischemia. A role for PP2A in simulated ischemia-induced cell death is likely since inhibition of PP2A protected NRK-52E cells. In addition, overexpression of exogenous B56 alpha but not B55 in NRK-52E cells enhanced simulated ischemia-induced cell death. These findings suggest that activation of a PP2A isoform that contains the B56 alpha regulatory subunit is required for ischemia-induced cell death in kidney epithelial proximal tubule cells.

Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia.

BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

Ceramide promotes apoptosis in lung cancer-derived A549 cells by a mechanism involving c-Jun NH2-terminal kinase.

Ceramide regulates diverse signaling pathways involving cell senescence, the cell cycle, and apoptosis. Ceramide is known to potently activate a number of stress-regulated enzymes, including the c-Jun NH(2)-terminal kinase (JNK). Although ceramide promotes apoptosis in human lung cancer-derived A549 cells, a role for JNK in this process is unknown. Here, we report that ceramide promotes apoptosis in A549 cells by a mechanism involving JNK. The JNK inhibitor SP600125 proved effective at protecting cells from the lethal effects of ceramide. To understand which JNK-mediated pathway may be involved, a number of JNK target proteins were examined, including the transcription factor, c-Jun, and the apoptotic regulatory proteins Bcl-X(L) and Bim. A549 cells exhibited basal levels of phosphorylated c-Jun in nuclear fractions, revealing that active c-Jun is present in these cells. Ceramide was found to inhibit c-Jun phosphorylation, suggesting that JNK-mediated phosphorylation of c-Jun is not likely involved in ceramide-induced apoptosis. Ceramide did not promote Bcl-X(L) phosphorylation. On the other hand, ceramide promoted phosphorylation of Bim and induced translocation of active JNK from the nucleus to the cytoplasm and mitochondrial fraction. Ceramide-mediated changes in localization of JNK were consistent with the observed changes in phosphorylation status of c-Jun and Bim. Furthermore, ceramide promoted Bim translocation to the mitochondria. Mitochondrial localization of Bim has been shown recently to promote apoptosis. These results suggest that JNK may participate in ceramide-induced apoptosis in A549 cells by a mechanism involving Bim.