Search

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

CAFs exhibit increased LC3 puncta per cell compared to NF.

CAF secreted HGF mediates HNSCC glycolysis.

Includes legends for all supplementary figures. S1: CAFs exhibit increased LC3 puncta per cell compared to NF. S2: CAF autophagy inhibition significantly decreases HNSCC in vitro progression. S3: IL-6 and IL-8 induce fibroblast autophagy. S4: bFGF treatment increases autophagy regulated secreted factors. S5: Autophagy inhibition reduces HNSCC in combination therapy, and does not affect CAF proliferation.

Despite therapeutic advancements, there has been little change in the survival of patients with head and neck squamous cell carcinoma (HNSCC). Recent results suggest that cancer-associated fibroblasts (CAF) drive progression of this disease. Here, we report that autophagy is upregulated in HNSCC-associated CAFs, where it is responsible for key pathogenic contributions in this disease. Autophagy is fundamentally involved in cell degradation, but there is emerging evidence that suggests it is also important for cellular secretion. Thus, we hypothesized that autophagy-dependent secretion of tumor-promoting factors by HNSCC-associated CAFs may explain their role in malignant development. In support of this hypothesis, we observed a reduction in CAF-facilitated HNSCC progression after blocking CAF autophagy. Studies of cell growth media conditioned after autophagy blockade revealed levels of secreted IL6, IL8, and other cytokines were modulated by autophagy. Notably, when HNSCC cells were cocultured with normal fibroblasts, they upregulated autophagy through IL6, IL8, and basic fibroblast growth factor. In a mouse xenograft model of HNSCC, pharmacologic inhibition of Vps34, a key mediator of autophagy, enhanced the antitumor efficacy of cisplatin. Our results establish an oncogenic function for secretory autophagy in HNSCC stromal cells that promotes malignant progression. Cancer Res; 77(23); 6679–91. ©2017 AACR.

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR.Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769–82. ©2018 AACR.

Legends for all supplemental figures

Objectives: This study seeks to (1) demonstrate how machine learning (ML) can be used for prediction modeling by predicting the treatment patients with T1–2, N0–N1 oropharyngeal squamous cell carcinoma (OPSCC) receive and (2) assess the impact patient, socioeconomic, regional, and institutional factors have in the treatment of this population. Methods: A retrospective cohort of adults diagnosed with T1–2, N0–N1 OPSCC from 2004 to 2013 was obtained using the National Cancer Database. The data was split into 80/20 distribution for training and testing, respectively. Various ML algorithms were explored for development. Area under the curve (AUC), accuracy, precision, and recall were calculated for the final model. Results: Among the 19,111 patients in the study, the mean (standard deviation) age was 61.3 (10.8) years, 14,034 (73%) were male, and 17,292 (91%) were white. Surgery was the primary treatment in 9,533 (50%) cases and radiation in 9,578 (50%) cases. The model heavily utilized T-stage, primary site, N-stage, grade, and type of treatment facility to predict the primary treatment modality. The final model yielded an AUC of 78% (95% CI, 77–79%), accuracy of 71%, precision of 72%, and recall of 71%. Conclusion: This study created a ML model utilizing clinical variables to predict primary treatment modality for T1–2, N0–N1 OPSCC. This study demonstrates how ML can be used for prediction modeling while also highlighting that tumor and facility realted variables impact the decision making process on a national level.

Aim Healthcare workers (HCWs) were among the first group of people vaccinated with the Pfizer-BioNTech Covid-19 vaccine (BNT162b2). Characterization of the kinetics of antibody response to vaccination is important to devise future vaccination strategies. To better characterize the antibody response to BNT162b2, we analyzed the kinetics of IgG and IgM antibody response to 5 different SARS-CoV-2 epitopes over a period of 6 months. Methods and results An observational single-centered study was conducted to evaluate the temporal dynamics of anti-SARS-CoV-2 antibodies following immunization with two doses of BNT162b2. Anti-SARS-CoV-2 antibodies were assessed using the Maverick SARS-CoV-2 multi-antigen panel (Genalyte Inc.). Healthcare workers aged ≥18 receiving BNT162b2 vaccination who self-reported no prior symptoms of COVID-19 nor prior COVID-19 PCR test positivity, were included in this study. HCWs developed an IgG antibody response to SARS-CoV-2 Spike S1, Spike S1 receptor binding domain (RBD), Spike S1S2 and Spike S2 after vaccination. IgG response was observed at two weeks following immunization in most participant samples and continued to increase at week 4, but subsequently decreased significantly starting at 3 months and up to 6 months. In contrast, IgM response to respective epitopes was minimal. Conclusion Multiplex results demonstrate that, contrary to natural infection, immunization with BNT162b2 produces minimal anti-Spike IgM response. Polyclonal IgG response to Spike declined at 3 months and continued to do so up to 6 months.

Prescription drug stewardship is critical. Autopopulation(AP) of medication quantities may influence prescriber behaviour. We investigate the impact of AP removal(APR) on opioid prescribing.Inpatient and emergency department(ED) discharges with opioid pain medications 2 years before and after APR were identified. Milligrams of morphine equivalents(MMEs) prescribed were recorded. Group comparisons were performed using Mann-Whitney U tests. Spearman's rho was used to analyse correlations between pain level and quantity of prescribed opioids. Mann-Kendall tests assessed trends in prescription patterns. Generalized estimating equations assessed trends in total quantity of prescribed MME.A total of 53 608 patient encounters were included for analysis. In surgical patients, there were no trends in the frequency of prescriptions below, at or above the AP quantity pre-APR. Post-APR, there was a decrease in the percentage of prescriptions written for the AP quantity(τ = -.493, P = .001) and an increase in prescriptions for30 tablets(τ = .468,P = .001). In non-operative patients, the pre-APR period was associated with a lower percentage of prescriptions30 tablets and a greater percentage of prescriptions for30 tablets. Interestingly, APR reversed this trend in prescriptions for30 tablets and resulted in an increase in larger prescriptions. Multivariate analysis of the total prescribed quantity of MME found no significant trend across months for inpatients prior to and after APR (0.997, P = .065 and 1.003, P = .142; respectively). The ED model found a monthly downward trend in amount of prescribed MME prior to and after APR (0.986, P .001 and 0.990, P .001; respectively). In the inpatient setting, pain was positively correlated to discharge MME (ρ = .028, P .001); with those reporting the highest pain receiving the greatest amount of opioids both pre- and post-APR. Interestingly, in the ED, this finding was negatively correlated (ρ = -.086, P .001); with those reporting the lowest pain receiving the greatest amount of opioids both pre- and post-APR.AP removal may have unintended consequences, such as increased prescriptions for greater quantities. To drive down prescription amounts, lower anchor values may be of more utility than APR. The poor correlation of pain values with prescribed medications warrants further investigation.

Although best understood as a degradative pathway, recent evidence demonstrates pronounced involvement of the macroautophagic/autophagic molecular machinery in cellular secretion. With either overexpression or inhibition of autophagy mediators, dramatic alterations in the cellular secretory profile occur. This affects secretion of a plethora of factors ranging from cytokines, to granule contents, and even viral particles. Encompassing a wide range of secreted factors, autophagy-dependent secretion is implicated in diseases ranging from cancer to neurodegeneration. With a growing body of evidence shedding light onto the molecular mediators, this review delineates the molecular machinery involved in selective targeting of the autophagosome for either degradation or secretion. In addition, we summarize the current understanding of factors and cargo secreted through this unconventional route, and describe the implications of this pathway in both health and disease. Abbreviations: BECN1, beclin 1; CAF, cancer associated fibroblast; CUPS, compartment for unconventional protein secretion; CXCL, C-X-C motif chemokine ligand; ER, endoplasmic reticulum; FGF2, fibroblast growth factor 2; HMGB1, high mobility group box 1; IDE, insulin degrading enzyme; IL, Interleukin; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAPS, misfolding associated protein secretion; MEF, mouse embryonic fibroblast; MTORC1, MTOR complex I; PtdIns, phosphatidyl inositol; SEC22B, SEC22 homolog B, vesicle trafficking protein (gene/pseudogene); SFV, Semliki forest virus; SNCA, synuclein alpha; SQSTM1, sequestosome 1; STX, Syntaxin; TASCC, TOR-associated spatial coupling compartment; TGFB, transforming growth factor beta; TRIM16, tripartite motif containing 16; UPS, unconventional protein secretion; VWF, von Willebrand factor.

Hearing loss (HL) is the most common neurodegenerative disease worldwide. Despite its prevalence, clinical testing does not yield a cell or molecular based identification of the underlying etiology of hearing loss making development of pharmacological or molecular treatments challenging. A key to improving the diagnosis of inner ear disorders is the development of reliable biomarkers for different inner ear diseases. Analysis of microRNAs (miRNA) in tissue and body fluid samples has gained significant momentum as a diagnostic tool for a wide variety of diseases. In previous work, we have shown that miRNA profiling in inner ear perilymph is feasible and may demonstrate distinctive miRNA expression profiles unique to different diseases. A first step in developing miRNAs as biomarkers for inner ear disease is linking patterns of miRNA expression in perilymph to clinically available metrics. Using machine learning (ML), we demonstrate we can build disease specific algorithms that predict the presence of sensorineural hearing loss using only miRNA expression profiles. This methodology not only affords the opportunity to understand what is occurring on a molecular level, but may offer an approach to diagnosing patients with active inner ear disease.

To provide a state of the art review of artificial intelligence (AI), including its subfields of machine learning and natural language processing, as it applies to otolaryngology and to discuss current applications, future impact, and limitations of these technologies.PubMed and Medline search engines.A structured search of the current literature was performed (up to and including September 2018). Search terms related to topics of AI in otolaryngology were identified and queried to identify relevant articles.AI is at the forefront of conversation in academic research and popular culture. In recent years, it has been touted for its potential to revolutionize health care delivery. Yet, to date, it has made few contributions to actual medical practice or patient care. Future adoption of AI technologies in otolaryngology practice may be hindered by misconceptions of what AI is and a fear that machine errors may compromise patient care. However, with potential clinical and economic benefits, it is vital for otolaryngologists to understand the principles and scope of AI.In the coming years, AI is likely to have a major impact on biomedical research and the practice of medicine. Otolaryngologists are key stakeholders in the development and clinical integration of meaningful AI technologies that will improve patient care. High-quality data collection is essential for the development of AI technologies, and otolaryngologists should seek opportunities to collaborate with data scientists to guide them toward the most impactful clinical questions.

Head and neck squamous cell carcinoma (HNSCC) is associated with low survival, and the current aggressive therapies result in high morbidity. Nutraceuticals are dietary compounds with few side effects. However, limited antitumor efficacy has restricted their application for cancer therapy. Here, we examine combining nutraceuticals, establishing a combination therapy that is more potent than any singular component, and delineate the mechanism of action. Three formulations were tested: GZ17-S (combined plant extracts from Arum palaestinum, Peganum harmala and Curcuma longa); GZ17-05.00 (16 synthetic components of GZ17-S); and GZ17-6.02 (3 synthetic components of GZ17S; curcumin, harmine and isovanillin). We tested the formulations on HNSCC proliferation, migration, invasion, angiogenesis, macrophage viability and infiltration into the tumor and tumor apoptosis. GZ17-6.02, the most effective formulation, significantly reduced in vitro assessments of HNSCC progression. When combined with cisplatin, GZ17-6.02 enhanced anti-proliferative effects. Molecular signaling cascades inhibited by GZ17-6.02 include EGFR, ERK1/2, and AKT, and molecular docking analyses demonstrate GZ17-6.02 components bind at distinct binding sites. GZ17-6.02 significantly inhibited growth of HNSCC cell line, patient-derived xenografts, and murine syngeneic tumors in vivo (P

Juvenile nasopharyngeal angiofibroma (JNA) is a highly vascularized and locally aggressive tumor that typically presents in adolescent males. The molecular biology of this tumor remains understudied. We sought to identify differentially expressed genes in the JNA transcriptome through messenger RNA sequencing of primary fibroblasts from 2 tumor explants and tonsil tissue from tumor-free subjects. In total, 1088 significant, differentially expressed genes were identified with 749 upregulated and 339 downregulated. Pathway analysis identified a number of activated signaling pathways, most notably, the vascular endothelial growth factor (VEGF) pathway (adjusted overlap P = .03). VEGF-A showed a 4.4-fold upregulation in JNA samples. In addition, the angiogenic receptor, fibroblast growth factor receptor 2 (FGFR2), was not present in tumor-free samples but increased in JNA. We validate these findings with immunohistochemistry, demonstrating upregulation of VEGF and FGFR2 in patient sections. Inhibition of the VEGF or FGFR signaling axes may have therapeutic potential in the treatment of JNA.

Background Juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor that presents in adolescent males. Although surgical excision is the mainstay of treatment, recurrences complicate treatment. There is a need to develop less invasive approaches for management. JNA tumors are composed of fibroblasts and vascular endothelial cells. We identified fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor (VEGF) expression in JNA-derived fibroblasts. FGFR influences fibroblast proliferation and VEGF is necessary for angiogenesis. We hypothesized that targeting FGFR would mitigate JNA fibroblast proliferation, invasion, and migration, and that targeting the VEGF receptor would attenuate endothelial tubule formation. Methods After informed consent, fibroblasts from JNA explants of 3 patients were isolated. Fibroblasts were treated with FGFR inhibitor AZD4547, 0 to 25 μg/mL for 72 hours and proliferation was quantified using CyQuant assay. Migration and invasion of JNA were assessed using 24-hour transwell assays with subsequent fixation and quantification. Mitigation of FGFR and downstream signaling was evaluated by immunoblotting. Tubule formation was assessed in human umbilical vein endothelial cells (HUVECs) treated with vehicle control (dimethylsulfoxide [DMSO]) or semaxanib (SU5416) as well as in serum-free media (SFM) or JNA conditioned media (CM). Tubule length was compared between treatment groups. Results Compared to control, AZD4547 inhibited JNA fibroblast proliferation, migration, and invasion through inhibition of FGFR and downstream signaling, specifically phosphorylation of - p44/42 mitogen activated protein kinase (p44/42 MAPK). JNA fibroblast CM significantly increased HUVEC tubule formation (p = 0.0039). Conclusion AZD4547 effectively mitigates FGFR signaling and decreases JNA fibroblast proliferation, migration, and invasion. SU5416 attenuated JNA fibroblast-induced tubule formation. AZD4547 may have therapeutic potential in the treatment of JNA.

Importance Predicting survival of oral squamous cell carcinoma through the use of prediction modeling has been underused, and the development of prediction models would augment clinicians’ ability to provide absolute risk estimates for individual patients. Objectives To develop a prediction model using machine learning for 5-year overall survival among patients with oral squamous cell carcinoma and compare this model with a prediction model created from the TNM (Tumor, Node, Metastasis) clinical and pathologic stage. Design, Setting, and Participants A retrospective cohort study was conducted of 33 065 patients with oral squamous cell carcinoma from the National Cancer Data Base between January 1, 2004, and December 31, 2011. Patients were excluded if the treatment was considered palliative, staging demonstrated T0 or Tis, or survival or staging data were missing. Patient, tumor, treatment, and outcome information were obtained from the National Cancer Data Base. The data were split into a distribution of 80% for training and 20% for testing. The model was created using 2-class decision forest architecture. Permutation feature importance scores were used to determine the variables that were used in the model’s prediction and their order of significance. Statistical analysis was conducted from August 1, 2018, to January 10, 2019. Main Outcomes and Measures Ability to predict 5-year overall survival assessed through area under the curve, accuracy, precision, and recall. Results Among the 33 065 patients in the study, the mean (SD) age was 64.6 (14.0) years, 19 791 were men (59.9%), 13 274 were women (40.1%), and 29 783 (90.1%) were white. At 60 months, there were 16 745 deaths (50.6%). The median time of follow-up was 56.8 months (range, 0-155.6 months). Age, pathologic T stage, positive margins at the time of surgery, lymph node size, and institutional identification were identified among the most significant variables. The calculated area under the curve for this machine learning model was 0.80 (95% CI, 0.79-0.81), accuracy was 71%, precision was 71%, and recall was 68%. In comparison, the calculated area under the curve of the TNM staging system was 0.68 (95% CI, 0.67-0.70), accuracy was 65%, precision was 69%, and recall was 52%. Conclusions and Relevance Using machine learning algorithms, a prediction model was created based on patient social, demographic, clinical, and pathologic features. The developed prediction model proved to be better than a prediction model that exclusively used TNM pathologic and clinical stage according to all performance metrics. This study highlights the role that machine learning may play in individual patient risk estimation in the era of big data.

To apply a novel methodology with machine learning (ML) to a large national cancer registry to help identify patients who are high risk for delayed adjuvant radiation.Observational cohort study.National Cancer Database (NCDB).A total of 76,573 patients were identified from the NCDB who had invasive head and neck cancer and underwent surgery, followed by radiation. The model was constructed from 80% of the patient data and subsequently evaluated and scored with the remaining 20%. Permutation feature importance analysis was used to understand the weighted model construction.A total of 76,573 patients met inclusion and exclusion criteria. Our ML model was able to predict whether patients would start adjuvant therapy beyond 50 days after surgery with an overall accuracy of 64.41% and a precision of 58.5%. The 2 most important variables used to build the model were treating facility and urban versus rural demographics.Statistics can provide inferences within an overall system, while ML is a novel methodology that can make predictions. We can identify patients who are "high risk" for delayed radiation using information from75,000 patient experiences, which has the potential for a direct impact on clinical care. Our inability to achieve greater accuracy is due to limitations of the data captured by the NCDB, and we need to continue to identify new variables that are correlated with delayed radiation therapy. ML will prove to be a valuable clinical tool in years to come, but its utility is limited by available data.

Background Pain gained recognition as a vital sign in the early 2000s, underscoring the importance of accurate documentation, characterization, and treatment of pain. No prior studies have demonstrated the utility of the 0–10 pain scale with respect to discharge opioid prescriptions, nor characterized the most influential factors in discharge prescriptions. Methods Inpatient and emergency department(ED) encounters from July 1, 2012 to April 1, 2018 resulting in a discharge prescription for tablet opioid medications were identified. The primary outcome was to determine if pain levels in 24 h prior to discharge correlated with opioids (in milligrams of morphine equivalents (MME)) prescribed. Secondary outcomes included the impact of patient and prescriber demographics, demographics. A generalized linear model was created to investigate factors affecting the quantity of prescribed opioids. Results n = 78,691 patient encounters. Overall mean adjusted MME for non-ED visits was 378 versus 197 for ED visits. Whites received the highest quantities; those identifying as non-white and non-black received the lowest. Women received significantly fewer discharge MMEs in both the ED and inpatient cohorts. Provider prescribing patterns exhibited the most profound effect on discharge MMEs. The most prolific (≥300 prescriptions over the study period) writing the largest amount. In the ED, there was a significant negative correlation between documented pain levels and discharge MMEs(ρ = 0.074,p Conclusions Pain scale was significantly negatively correlated with discharge MMEs in the ED and positively correlated in the inpatient population. Individual prescriber characteristics were the more influential variable, with prolific high prescribers writing for the largest MME amounts. The inverse association of pain and MMEs at discharge in the ED, and the large effect pre-existing prescriber patterns exhibited, both improved methodology for assessing and appropriately treating pain, and effective prescriber-targeted interventions, must be a priority.

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR. Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769–82. ©2018 AACR.

Despite therapeutic advancements, there has been little change in the survival of patients with head and neck squamous cell carcinoma (HNSCC). Recent results suggest that cancer-associated fibroblasts (CAF) drive progression of this disease. Here, we report that autophagy is upregulated in HNSCC-associated CAFs, where it is responsible for key pathogenic contributions in this disease. Autophagy is fundamentally involved in cell degradation, but there is emerging evidence that suggests it is also important for cellular secretion. Thus, we hypothesized that autophagy-dependent secretion of tumor-promoting factors by HNSCC-associated CAFs may explain their role in malignant development. In support of this hypothesis, we observed a reduction in CAF-facilitated HNSCC progression after blocking CAF autophagy. Studies of cell growth media conditioned after autophagy blockade revealed levels of secreted IL6, IL8, and other cytokines were modulated by autophagy. Notably, when HNSCC cells were cocultured with normal fibroblasts, they upregulated autophagy through IL6, IL8, and basic fibroblast growth factor. In a mouse xenograft model of HNSCC, pharmacologic inhibition of Vps34, a key mediator of autophagy, enhanced the antitumor efficacy of cisplatin. Our results establish an oncogenic function for secretory autophagy in HNSCC stromal cells that promotes malignant progression. Cancer Res; 77(23); 6679–91. ©2017 AACR.

Juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor that presents in adolescent males. Although surgical excision is the mainstay of treatment, recurrences complicate treatment. There is a need to develop less invasive approaches for management. JNA tumors are composed of fibroblasts and vascular endothelial cells. We identified fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor (VEGF) expression in JNA-derived fibroblasts. FGFR influences fibroblast proliferation and VEGF is necessary for angiogenesis. We hypothesized that targeting FGFR would mitigate JNA fibroblast proliferation, invasion, and migration, and that targeting the VEGF receptor would attenuate endothelial tubule formation.After informed consent, fibroblasts from JNA explants of 3 patients were isolated. Fibroblasts were treated with FGFR inhibitor AZD4547, 0 to 25 μg/mL for 72 hours and proliferation was quantified using CyQuant assay. Migration and invasion of JNA were assessed using 24-hour transwell assays with subsequent fixation and quantification. Mitigation of FGFR and downstream signaling was evaluated by immunoblotting. Tubule formation was assessed in human umbilical vein endothelial cells (HUVECs) treated with vehicle control (dimethylsulfoxide [DMSO]) or semaxanib (SU5416) as well as in serum-free media (SFM) or JNA conditioned media (CM). Tubule length was compared between treatment groups.Compared to control, AZD4547 inhibited JNA fibroblast proliferation, migration, and invasion through inhibition of FGFR and downstream signaling, specifically phosphorylation of - p44/42 mitogen activated protein kinase (p44/42 MAPK). JNA fibroblast CM significantly increased HUVEC tubule formation (p = 0.0039).AZD4547 effectively mitigates FGFR signaling and decreases JNA fibroblast proliferation, migration, and invasion. SU5416 attenuated JNA fibroblast-induced tubule formation. AZD4547 may have therapeutic potential in the treatment of JNA.

Background mTOR inhibition of aromatase inhibitor (AI)-resistant breast cancer is currently under evaluation in the clinic. Everolimus/RAD001 (Afinitor®) has had limited efficacy as a solo agent but is projected to become part of combination therapy for AI-resistant breast cancer. This study was conducted to investigate the anti-proliferative and resistance mechanisms of everolimus in AI-resistant breast cancer cells. Methods In this study we utilized two AI-resistant breast cancer cell lines, MCF-7:5C and MCF-7:2A, which were clonally derived from estrogen receptor positive (ER+) MCF-7 breast cancer cells following long-term estrogen deprivation. Cell viability assay, colony formation assay, cell cycle analysis and soft agar anchorage-independent growth assay were used to determine the efficacy of everolimus in inhibiting the proliferation and tumor forming potential of MCF-7, MCF-7:5C, MCF-7:2A and MCF10A cells. Confocal microscopy and transmission electron microscopy were used to evaluate LC3-II production and autophagosome formation, while ERE-luciferase reporter, Western blot, and RT-PCR analyses were used to assess ER expression and transcriptional activity. Results Everolimus inhibited the proliferation of MCF-7:5C and MCF-7:2A cells with relatively equal efficiency to parental MCF-7 breast cancer cells. The inhibitory effect of everolimus was due to G1 arrest as a result of downregulation of cyclin D1 and p21. Everolimus also dramatically reduced estrogen receptor (ER) expression (mRNA and protein) and transcriptional activity in addition to the ER chaperone, heat shock protein 90 protein (HSP90). Everolimus restored 4-hydroxy-tamoxifen (4OHT) sensitivity in MCF-7:5C cells and enhanced 4OHT sensitivity in MCF-7 and MCF-7:2A cells. Notably, we found that autophagy is one method of everolimus insensitivity in MCF-7 breast cancer cell lines. Conclusion This study provides additional insight into the mechanism(s) of action of everolimus that can be used to enhance the utility of mTOR inhibitors as part of combination therapy for AI-resistant breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2490-z) contains supplementary material, which is available to authorized users.

Tumor-associated fibroblasts (TAFs) are the most abundant cell type in the microenvironment of head and neck squamous cell carcinoma (HNSCC). We and others previously reported that secreted factors from TAFs increase the proliferation, migration, and invasion of HNSCC. Further studies revealed that HNSCC-secreted factors induce secretory autophagy in TAFs. However, the contribution of TAF autophagy to immune-evasion in the HNSCC microenvironment is unknown. Natural killer (NK) cells are an essential arm of anti-tumor immunity; thus, our study set out to determine the role of TAF autophagy in suppressing NK cell effector functions against HNSCC. To address this, we treated the NK cell line NK-92MI with conditioned media from primary HNSCC TAFs or normal fibroblasts derived from cancer-free subjects and assessed changes in IFN-γ production and cytotoxicity, measures of NK cell activation. We then assessed the role of TAF autophagy in mediating this effect by repeating the studies using conditioned media from TAFs whose autophagy had either been inhibited by chloroquine treatment or knocked down by siRNA. Finally, we assessed the mechanisms by which autophagy in the TAF affects NK cell function. Our data demonstrate HNSCC tumor cells orchestrate a complex mechanism of NK cell inhibition through the modulation of TAFs in the microenvironment. Treatment with TAF-conditioned media induced IFN-γ mRNA expression in NK-92MI cells. Interestingly, TAF-conditioned media also reduced NK cell-mediated cytotoxicity as compared to conditioned media from normal fibroblasts. Knockdown of key regulators in the secretory autophagy pathway by siRNA abrogated release of TAF-secreted factors, including hepatocyte growth factor (HGF). HGF induced NK-92MI expression of IFN-γ mRNA, which could be abrogated by an inhibitor of the HGF receptor, c-MET. In conclusion, these data describe a previously undefined effect of TAFs on NK cell function in the HNSCC microenvironment. Citation Format: Jonathan David Enders, Jacob New, Rana Aliani, Sufi Mary Thomas. Secretory autophagy in tumor-associated fibroblasts alter effector function of natural killer cells against HNSCC [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B72.

Radiation-induced fibrosis (RIF) is a major side effect of radiotherapy in cancer patients with no effective therapeutic options. RIF involves excess deposition and aberrant remodeling of the extracellular matrix (ECM) leading to stiffness in tissues and organ failure. Development of preclinical models of RIF is crucial to elucidate the molecular mechanisms regulating fibrosis and to develop therapeutic approaches. In addition to radiation, the main molecular perpetrators of fibrotic reactions are cytokines, including transforming growth factor-β (TGF-β). We hypothesized that human oral fibroblasts would develop an in vitro fibrotic reaction in response to radiation and TGF-β. We demonstrate here that fibroblasts exposed to radiation followed by TGF-β exhibit a fibrotic phenotype with increased collagen deposition, cell proliferation, migration and invasion. In this in vitro model of RIF (RIFiv), the early biological processes involved in fibrosis are demonstrated, along with increased levels of several molecules including collagen 1α1, collagen XIα1, integrin-α2 and cyclin D1 mRNA in irradiated cells. A clinically relevant antifibrotic agent, pentoxifylline, and a curcumin analogue both mitigated collagen deposition in irradiated fibroblast cultures. In summary, we have established an in vitro model for RIF that facilitates the elucidation of molecular mechanisms in radiation-induced fibrosis and the development of effective therapeutic approaches.

Epithelial ovarian cancer (EOC) has poor prognosis and rapid recurrence because of widespread dissemination of peritoneal metastases at diagnosis. Multiple pathways contribute to the aggressiveness of ovarian cancer, including hypoxic signaling mechanisms. In this study, we have determined that the hypoxia-inducible histone demethylase KDM4B is expressed in ∼60% of EOC tumors assayed, including primary and matched metastatic tumors. Expression of KDM4B in tumors is positively correlated with expression of the tumor hypoxia marker CA-IX, and is robustly induced in EOC cell lines exposed to hypoxia. KDM4B regulates expression of metastatic genes and pathways, and loss of KDM4B increases H3K9 trimethylation at the promoters of target genes like LOXL2, LCN2 and PDGFB. Suppressing KDM4B inhibits ovarian cancer cell invasion, migration and spheroid formation in vitro. KDM4B also regulates seeding and growth of peritoneal tumors in vivo, where its expression corresponds to hypoxic regions. This is the first demonstration that a Jumonji-domain histone demethylase regulates cellular processes required for peritoneal dissemination of cancer cells, one of the predominant factors affecting prognosis of EOC. The pathways regulated by KDM4B may present novel opportunities to develop combinatorial therapies to improve existing therapies for EOC patients.

Radiation-induced fibrosis (RIF) is a long-term side effect of external beam radiation therapy for the treatment of cancer. It results in a multitude of symptoms that significantly impact quality of life. Understanding the mechanisms of RIF-induced changes is essential to developing effective strategies to prevent long-term disability and discomfort following radiation therapy. In this review, we describe the current understanding of the etiology, clinical presentation, pathogenesis, treatment, and directions of future therapy for this condition.A literature review of publications describing mechanisms or treatments of RIF was performed. Specific databases utilized included PubMed and clinicaltrials.gov, using keywords "Radiation-Induced Fibrosis," "Radiotherapy Complications," "Fibrosis Therapy," and other closely related terms.RIF is the result of a misguided wound healing response. In addition to causing direct DNA damage, ionizing radiation generates reactive oxygen and nitrogen species that lead to localized inflammation. This inflammatory process ultimately evolves into a fibrotic one characterized by increased collagen deposition, poor vascularity, and scarring. Tumor growth factor beta serves as the primary mediator in this response along with a host of other cytokines and growth factors. Current therapies have largely been directed toward these molecular targets and their associated signaling pathways.Although RIF is widely prevalent among patients undergoing radiation therapy and significantly impacts quality of life, there is still much to learn about its pathogenesis and mechanisms. Current treatments have stemmed from this understanding, and it is anticipated that further elucidation will be essential for the development of more effective therapies.

Head and neck squamous cell carcinoma (HNSCC) affects 40,000 patients annually and is associated with Citation Format: Dhruv Kumar, Jacob New, Vikalp Vishwakarma, Hemant Chavan, Partha Kasturi, Sufi M. Thomas. Mitigating tumor-stroma metabolic symbiosis for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2017-2970

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC), which affects 50,000 new patients annually in the United States, is associated with less than 50% 5-year survival. HNSCC tumors display increased glycolysis, even in the presence of oxygen. Consequently, there is an increase in lactic acid (LA) production. However, the effect of lactic acid in the tumor microenvironment and the mechanisms whereby HNSCC tumors survive in highly acidic conditions remain unknown. HNSCC consist of up to 80% tumor-associated fibroblasts (TAFs). We previously reported that activation of receptor tyrosine kinase, c-Met, by TAF-secreted hepatocyte growth factor (HGF) contributes to HNSCC progression (1,2). The mechanism associated with tumor-stroma metabolic symbiosis is not well understood. On this basis, we hypothesized that TAF-secreted HGF regulates HNSCC glycolysis. We demonstrate that HNSCC-secreted basic fibroblast growth factor (bFGF) induces oxidative phosphorylation (OXPHOS) in TAFs. In addition, bFGF regulates secretion of HGF from TAFs. TAF-secreted HGF increases HNSCC glycolysis and induces bFGF secretion from HNSCC. Thus, HNSCC and TAFs engage in reciprocal signaling through paracrine effects of HGF and bFGF. Inhibition of c-Met with small molecule inhibitor PF-02341066 or specific knockdown with c-Met siRNA decreased TAF-facilitated HNSCC glycolysis, lactic acid production and bFGF expression. In addition, inhibition of FGFR with small molecule inhibitor AZD-4547 decreased OXPHOS and HGF expression in TAFs. Inhibition of FGFR also reduced HNSCC-stimulated phosphorylation of p42/44 mitogen activated protein kinase, proliferation and migration in TAFs. Further, we tested the efficacy of AZD-4547 in combination with c-Met inhibitor PF-02341066 in mitigating TAF-induced HNSCC growth in vitro and in vivo. Combined treatment with AZD-4547 and PF-02341066 significantly inhibited TAF-induced proliferation of HNSCC in vitro compared to the vehicle control treated cells (p 1. Wheeler SE, Shi H, Lin F, Dasari S, Bednash J, Thorne S, et al. Enhancement of head and neck squamous cell carcinoma proliferation, invasion, and metastasis by tumor-associated fibroblasts in preclinical models. Head & neck 2014;36(3):385-92. 2. Knowles LM, Stabile LP, Egloff AM, Rothstein ME, Thomas SM, Gubish CT, et al. HGF and c-Met participate in paracrine tumorigenic pathways in head and neck squamous cell cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15(11):3740-50. Citation Format: Dhruv Kumar, Vikalp Vishwakarma, Jacob New, Wade Gutierrez, Hemant Chavan, Partha Kasturi, Ossama Tawfik, Douglas Girod, Bennett Van Houten, George Leef, Radhika Joshi, Shary Shelton, Jeffrey Straub, Yelizaveta Shnayder, Kiran Kakarala, Terance Tsue, Fangchen Lin, Sumana Dasari, Sufi Thomas. Targeting tumor-stroma metabolic symbiosis for head and neck cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B37.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy affecting 40,000 cases in the USA annually. Despite aggressive therapies HNSCC is associated with less than a 50% 5-year survival rate. Limited treatment options and high morbidity necessitate the development of new treatments. HNSCC tumors frequently consist of up to 80% tumor-associated fibroblasts (TAFs). We reported that tumor associated fibroblasts (TAFs) facilitate HNSCC progression. However, the molecular mechanisms that facilitate TAF-mediated tumor growth and metastasis remain unknown. Unlike normal cells, malignant cells often display increased glycolysis, even in the presence of oxygen; a phenomenon known as the Warburg effect. As a consequence, there is an increase in lactic acid (LA) production. It has been reported that HNSCC tumors have high LA levels that correlate with reduced survival. The mechanisms whereby HNSCC tumors survive in highly acidic conditions remain unknown. We previously reported that TAF-secreted hepatocyte growth factor (HGF) facilitates HNSCC progression. Here we demonstrate that TAF-secreted HGF regulates HNSCC glycolysis and increases basic fibroblast growth factor (bFGF) secretion from HNSCC. Further, HNSCC-secreted bFGF regulates mitochondrial oxidative phosphorylation (OXPHOS) in TAFs. Inhibition of c-Met by PF-02341066 or knockdown by siRNA decreases glycolysis and bFGF expression in HNSCC. In addition, inhibition of FGFR by AZD-4547 decreased OXPHOS and HGF expression in TAFs. Thus, HNSCC and TAFs engage in reciprocal signaling through paracrine effects of HGF and bFGF. Combined treatment with AZD-4547 and PF-02341066 significantly inhibited TAF-induced proliferation of HNSCC in vitro compared to the vehicle control treated cells. For the first time, we tested FGFR inhibitor AZD-4547 in combination with c-Met inhibitor PF-02341066 both in vitro and in vivo xenograft models. The combination treatment significantly inhibited proliferation of HNSCC in vitro and effectively reduced HNSCC tumor growth compared to either agent alone in vivo (p Citation Format: Dhruv Kumar, Vikalp Vishwakarma, Jacob New, Radhika Joshi, Fangchen Lin, Sumana Dasari, Wade Gutierrez, Hemant Chavan, Ossama Tawfik, Shary Shelton, Yelizaveta Shnayder, Kiran Kakarala, Terance Tsue, Douglas Girod, Bennett Van Houten, Partha Kasturi, Sufi M. Thomas. Targeting tumor-stroma metabolic symbiosis for head and neck cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1030.

Although Arum palaestinum has been used as a natural prevention and treatment of cancer in Palestine for many years, few studies have examined its efficacy and mechanism of action. Chemically synthesized components of Arum palaestinum extract mixed in a ratio that was present in the natural extract were tested on head and neck squamous cell carcinoma (HNSCC), which affects annually 50,000 new people and causes 10,000 deaths a year in the US alone. We examined the effects of the nutraceutical on cell lines and patient-derived HNSCC tumors, and we delineated the mechanism of action. Among three formulations tested of Arum palaestinum (GZ17-S, a fortified version of the plant extract; GZ17-05.00, 16 components of the original plant extract; and GZ17-06.02, 3 components of the original plant extract), we found formulation GZ17-06.02 to be the most effective at reducing cancer cell proliferation, migration, and invasion. Further, GZ17-06.02 decreased angiogenesis in a tubule formation assay. When combined with a standard chemotherapeutic, cisplatin, GZ17-06.02 decreased cancer cell proliferation to a similar level of cisplatin, and enhanced the effect of cisplatin when used in combination. In comparison to radiotherapy, GZ17-06.02 performed better at reducing HNSCC cell proliferation than 3, 6, or 9 gy of radiation alone, and sequential treatment of HNSCC with radiation followed by GZ17-06.02 did not enhance the cytotoxic effects compared to GZ17-06.02 alone. We identified molecular signaling cascades inhibited by GZ17-06.02, including Src, ERK1/2, EGFR, AKT1/2/3, STAT-2, and Chk2, and we performed molecular docking analyses between GZ17-06.02 components and EGFR and AKT. We found that GZ17-06.02 components bind simultaneously at distinct binding sites of the kinase domains of EGFR and AKT. We found GZ17-06.02 induced significant apoptosis in HNSCC cells, increasing cleaved PARP, and activating caspase-3. Intratumoral injections of GZ17-06.02 were well tolerated in mice, and inhibited the growth and induced necrosis of HNSCC xenografts when injected intratumorally. In addition, orally administered GZ17-06.02 not only mitigated the growth of patient-derived HNSCC xenografts, but significantly decreased the tumor volume compared to the initially implanted tumor. Response to treatment correlated with a reduction in ERK1/2, which establishes it as an excellent biomarker for future clinical trials. Taken together, we demonstrate GZ17-06.02 as a highly effective nutraceutical, and pave the way for future clinical application in HNSCC. Citation Format: Jacob New, Vikalp Vishwakarma, Dhruv Kumar, Kyle Crooker, Vusala Snyder, Yelizaveta Shnayder, Kiran Kakarala, Terance T. Tsue, Douglas Girod, Lisa Stehno-Bittel, Sufi M. Thomas. Mechanistic insights into the antitumor efficacy of nutraceutical GZ17-06.02, a highly effective formulation of Arum palaestinum extract, on head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B116.