DCLK1-Mediated Regulation of Invadopodia Dynamics and Matrix Metalloproteinase Trafficking Drives Invasive Progression in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major health concern due to its high mortality from poor treatment responses and locoregional tumor invasion into life sustaining structures in the head and neck. A deeper comprehension of HNSCC invasion mechanisms holds the potential to inform targeted therapies that may enhance patient survival. We previously reported that doublecortin like kinase 1 (DCLK1) regulates invasion of HNSCC cells. Here, we tested the hypothesis that DCLK1 regulates proteins within invadopodia to facilitate HNSCC invasion. Invadopodia are specialized subcellular protrusions secreting matrix metalloproteinases that degrade the extracellular matrix (ECM). Through a comprehensive proteome analysis comparing DCLK1 control and shDCLK1 conditions, our findings reveal that DCLK1 plays a pivotal role in regulating proteins that orchestrate cytoskeletal and ECM remodeling, contributing to cell invasion. Further, we demonstrate in TCGA datasets that DCLK1 levels correlate with increasing histological grade and lymph node metastasis. We identified higher expression of DCLK1 in the leading edge of HNSCC tissue. Knockdown of DCLK1 in HNSCC reduced the number of invadopodia, cell adhesion and colony formation. Using super resolution microscopy, we demonstrate localization of DCLK1 in invadopodia and colocalization with mature invadopodia markers TKS4, TKS5, cortactin and MT1-MMP. We carried out phosphoproteomics and validated using immunofluorescence and proximity ligation assays, the interaction between DCLK1 and motor protein KIF16B. Pharmacological inhibition or knockdown of DCLK1 reduced interaction with KIF16B, secretion of MMPs, and cell invasion. This research unveils a novel function of DCLK1 within invadopodia to regulate the trafficking of matrix degrading cargo. The work highlights the impact of targeting DCLK1 to inhibit locoregional invasion, a life-threatening attribute of HNSCC.

Acryl-3,5-bis(2,4-difluorobenzylidene)-4-piperidone targeting cellular JUN proto-oncogene, AP-1 transcription factor subunit inhibits head and neck squamous cell carcinoma progression.

Aim: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with a survival rate below fifty percent. Addressing meager therapeutic options, a series of small molecule inhibitors were screened for antitumor efficacy. The most potent analog, acryl-3,5-bis(2,4-difluorobenzylidene)-4-piperidone (DiFiD; A-DiFiD), demonstrated strong cellular JUN proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (JUN, c-Jun) antagonism. c-Jun, an oncogenic transcription factor, promotes cancer progression, invasion, and adhesion; high (JUN) mRNA expression correlates with poorer HNSCC survival. Methods: Four new small molecules were generated for cytotoxicity screening in HNSCC cell lines. A-DiFiD-treated HNSCC cells were assessed for cytotoxicity, colony formation, invasion, migration, and adhesion. Dot blot array was used to identify targets. Phospho-c-Jun (p-c-Jun) expression was analyzed using immunoblotting. The Cancer Genome Atlas (TCGA) head and neck cancer datasets were utilized to determine overall patient survival. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) datasets interfaced with University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) were analyzed to determine protein levels of c-Jun in HNSCC patients and correlate levels with patient. Results: Of the small molecules tested, A-DiFiD was the most potent in HNSCC lines, while demonstrating low half-maximal drug inhibitory concentration (IC50) in non-malignant Het-1A cells. Additionally, A-DiFiD abrogated cell invasion, migration, and colony formation. Phospho-kinase in vitro array demonstrated A-DiFiD reduced p-c-Jun. Likewise, a time dependent reduction in p-c-Jun was observed starting at 3 min post A-DiFiD treatment. TCGA Firehose Legacy vs. recurrent and metastatic head and neck cancer reveal a nearly 3% DNA amplification in recurrent/metastatic tumor compared to below 1% in primary tumors that had no lymph node metastasis. CPTAC analysis show higher tumor c-Jun levels compared to normal. Patients with high JUN expression had significantly reduced 3-year survival. Conclusions: A-DiFiD targets c-Jun, a clinical HNSCC driver, with potent anti-tumor effects.

The Role of STATs in Ovarian Cancer: Exploring Their Potential for Therapy.

Ovarian cancer (OvCa) is a deadly gynecologic malignancy that presents many clinical challenges due to late-stage diagnoses and the development of acquired resistance to standard-of-care treatment protocols. There is an increasing body of evidence suggesting that STATs may play a critical role in OvCa progression, resistance, and disease recurrence, and thus we sought to compile a comprehensive review to summarize the current state of knowledge on the topic. We have examined peer reviewed literature to delineate the role of STATs in both cancer cells and cells within the tumor microenvironment. In addition to summarizing the current knowledge of STAT biology in OvCa, we have also examined the capacity of small molecule inhibitor development to target specific STATs and progress toward clinical applications. From our research, the best studied and targeted factors are STAT3 and STAT5, which has resulted in the development of several inhibitors that are under current evaluation in clinical trials. There remain gaps in understanding the role of STAT1, STAT2, STAT4, and STAT6, due to limited reports in the current literature; as such, further studies to establish their implications in OvCa are necessitated. Moreover, due to the deficiency in our understanding of these STATs, selective inhibitors also remain elusive, and therefore present opportunities for discovery.

Cancer-Associated Fibroblasts: Master Tumor Microenvironment Modifiers.

Cancer cells rely on the tumor microenvironment (TME), a composite of non-malignant cells, and extracellular matrix (ECM), for survival, growth, and metastasis. The ECM contributes to the biomechanical properties of the surrounding tissue, in addition to providing signals for tissue development. Cancer-associated fibroblasts (CAFs) are stromal cells in the TME that are integral to cancer progression. Subtypes of CAFs across a variety of cancers have been revealed, and each play a different role in cancer progression or suppression. CAFs secrete signaling molecules and remodel the surrounding ECM by depositing its constituents as well as degrading enzymes. In cancer, a remodeled ECM can lead to tumor-promoting effects. Not only does the remodeled ECM promote growth and allow for easier metastasis, but it can also modulate the immune system. A better understanding of how CAFs remodel the ECM will likely yield novel therapeutic targets. In this review, we summarize the key factors secreted by CAFs that facilitate tumor progression, ECM remodeling, and immune suppression.

The Contribution of the Human Oral Microbiome to Oral Disease: A Review.

The oral microbiome is an emerging field that has been a topic of discussion since the development of next generation sequencing and the implementation of the human microbiome project. This article reviews the current literature surrounding the oral microbiome, briefly highlighting most recent methods of microbiome characterization including cutting edge omics, databases for the microbiome, and areas with current gaps in knowledge. This article also describes reports on microorganisms contained in the oral microbiome which include viruses, archaea, fungi, and bacteria, and provides an in-depth analysis of their significant roles in tissue homeostasis. Finally, we detail key bacteria involved in oral disease, including oral cancer, and the current research surrounding their role in stimulation of inflammatory cytokines, the role of gingival crevicular fluid in periodontal disease, the creation of a network of interactions between microorganisms, the influence of the planktonic microbiome and cospecies biofilms, and the implications of antibiotic resistance. This paper provides a comprehensive literature analysis while also identifying gaps in knowledge to enable future studies to be conducted.

Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

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.

Vasopressin Receptor Type-2 Mediated Signaling in Renal Cell Carcinoma Stimulates Stromal Fibroblast Activation.

Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R signaling within human ccRCC tumor cells (Caki1 cells) stimulates stromal cancer-associated fibroblasts (CAFs). We found that cell culture conditioned media from Caki1 cells increased activation, migration, and proliferation of fibroblasts in vitro, which was inhibited by V2R gene silencing in Caki1 cells. Analysis of the conditioned media and mRNA of the V2R gene silenced and control Caki1 cells showed that V2R regulates the production of CAF-activating factors. Some of these factors were also found to be regulated by YAP in these Caki1 cells. YAP expression colocalized and correlated with V2R expression in ccRCC tumor tissue. V2R gene silencing or V2R antagonist significantly reduced YAP in Caki1 cells. Moreover, the V2R antagonist reduced YAP expression and myofibroblasts in mouse xenograft tumors. These results suggest that V2R plays an important role in secreting pro-fibrotic factors that stimulate fibroblast activation by a YAP-dependent mechanism in ccRCC tumors. Our results demonstrate a novel role for the V2R-YAP axis in the regulation of myofibroblasts in ccRCC and a potential therapeutic target.

Immunoglobin A Deficiency and Squamous Cell Carcinoma With a Rare Presentation as Anal Cancer.

Selective immunoglobin A deficiency (IgAD) is the most common immunodeficiency disorder in the western world. Cancer is the most common cause of death in these individuals. Various cases have been reported of squamous cell carcinoma (SCC) in IgAD at sites like skin, oral cavity, and lung. Here we present a rare case of SCC occurring as anal cancer. No other reports to our knowledge describe this rare presentation. A 54-year-old Caucasian woman with asymptomatic partial IgAD presented with a palpable anal mass. Further evaluation showed stage IIIa SCC anal cancer (T1N1M0). Additional workup showed positive human papilloma virus (HPV) serology and positive HPV immunohistochemistry studies. The patient achieved complete response with chemoradiation with her most recent imaging and anorectal exam showing no evidence of cancer recurrence at 3 years follow-up. This case highlights the association between IgAD and malignancy. Although IgAD is the most common primary antibody deficiency, this patient's case presents a rare instance of anal SCC in an IgA-deficient individual. Studies show an association between HPV infection and SCC, but few include IgA-deficient individuals. Patients with IgAD and other immunodeficiencies are at higher risk for HPV infection and therefore may be at a higher risk of SCC. With widespread use of the HPV vaccine, the medical community should be aware of its importance in cancer prevention for these patients. Further studies are needed to evaluate relationships between IgAD, HPV infections, SCC cancer, and the role that the HPV vaccine has in cancer prophylaxis.

Mitogen-activated protein kinase-activated protein kinase-2 (MK2) and its role in cell survival, inflammatory signaling, and migration in promoting cancer.

Cancer and the immune system share an intimate relationship. Chronic inflammation increases the risk of cancer occurrence and can also drive inflammatory mediators into the tumor microenvironment enhancing tumor growth and survival. The p38 MAPK pathway is activated both acutely and chronically by stress, inflammatory chemokines, chronic inflammatory conditions, and cancer. These properties have led to extensive efforts to find effective drugs targeting p38, which have been unsuccessful. The immediate downstream serine/threonine kinase and substrate of p38 MAPK, mitogen-activated-protein-kinase-activated-protein-kinase-2 (MK2) protects cells against stressors by regulating the DNA damage response, transcription, protein and messenger RNA stability, and motility. The phosphorylation of downstream substrates by MK2 increases inflammatory cytokine production, drives an immune response, and contributes to wound healing. By binding directly to p38 MAPK, MK2 is responsible for the export of p38 MAPK from the nucleus which gives MK2 properties that make it unique among the large number of p38 MAPK substrates. Many of the substrates of both p38 MAPK and MK2 are separated between the cytosol and nucleus and interfering with MK2 and altering this intracellular translocation has implications for the actions of both p38 MAPK and MK2. The inhibition of MK2 has shown promise in combination with both chemotherapy and radiotherapy as a method for controlling cancer growth and metastasis in a variety of cancers. Whereas the current data are encouraging the field requires the development of selective and well tolerated drugs to target MK2 and a better understanding of its effects for effective clinical use.

DNAJA1 promotes cancer metastasis through interaction with mutant p53.

Accumulation of mutant p53 (mutp53) is crucial for its oncogenic gain of function activity. DNAJA1, a member of J-domain containing proteins or heat shock protein 40, is shown to prevent unfolded mutp53 from proteasomal degradation. However, the biological function of DNAJA1 remains largely unknown. Here we show that DNAJA1 promotes tumor metastasis by accumulating unfolded mutp53. Levels of DNAJA1 in head and neck squamous cell carcinoma (HNSCC) tissues were higher than those in normal tissues. Knockdown of DNAJA1 in HNSCC cell lines carrying unfolded mutp53 significantly decreased the levels of mutp53, filopodia/lamellipodia formation, migratory potential, and active forms of CDC42/RAC1, which were not observed in HNSCC cells with DNA contact mutp53, wild-type p53, or p53 null. Such mutp53-dependent functions of DNAJA1 were supported by the observation that DNAJA1 selectively bound to unfolded mutp53. Moreover, DNAJA1 knockdown in HNSCC cells carrying unfolded mutp53 inhibited primary tumor growth and metastases to the lymph nodes and lungs. Our study suggests that DNAJA1 promotes HNSCC metastasis mainly in a manner dependent on mutp53 status, suggesting DNAJA1 as a potential therapeutic target for HNSCC harboring unfolded mutp53.

Mechanical Properties in the Glioma Microenvironment: Emerging Insights and Theranostic Opportunities.

Gliomas represent the most common malignant primary brain tumors, and a high-grade subset of these tumors including glioblastoma are particularly refractory to current standard-of-care therapies including maximal surgical resection and chemoradiation. The prognosis of patients with these tumors continues to be poor with existing treatments and understanding treatment failure is required. The dynamic interplay between the tumor and its microenvironment has been increasingly recognized as a key mechanism by which cellular adaptation, tumor heterogeneity, and treatment resistance develops. Beyond ongoing lines of investigation into the peritumoral cellular milieu and microenvironmental architecture, recent studies have identified the growing role of mechanical properties of the microenvironment. Elucidating the impact of these biophysical factors on disease heterogeneity is crucial for designing durable therapies and may offer novel approaches for intervention and disease monitoring. Specifically, pharmacologic targeting of mechanical signal transduction substrates such as specific ion channels that have been implicated in glioma progression or the development of agents that alter the mechanical properties of the microenvironment to halt disease progression have the potential to be promising treatment strategies based on early studies. Similarly, the development of technology to measure mechanical properties of the microenvironment in vitro and in vivo and simulate these properties in bioengineered models may facilitate the use of mechanical properties as diagnostic or prognostic biomarkers that can guide treatment. Here, we review current perspectives on the influence of mechanical properties in glioma with a focus on biophysical features of tumor-adjacent tissue, the role of fluid mechanics, and mechanisms of mechanical signal transduction. We highlight the implications of recent discoveries for novel diagnostics, therapeutic targets, and accurate preclinical modeling of glioma.

Multicellular contractility contributes to the emergence of mesothelioma nodules.

Malignant pleural mesothelioma (MPM) has an overall poor prognosis and unsatisfactory treatment options. MPM nodules, protruding into the pleural cavity may have growth and spreading dynamics distinct that of other solid tumors. We demonstrate that multicellular aggregates can develop spontaneously in the majority of tested MPM cell lines when cultured at high cell density. Surprisingly, the nodule-like aggregates do not arise by excessive local cell proliferation, but by myosin II-driven cell contractility. Prominent actin cables, spanning several cells, are abundant both in cultured aggregates and in MPM surgical specimens. We propose a computational model for in vitro MPM nodule development. Such a self-tensioned Maxwell fluid exhibits a pattern-forming instability that was studied by analytical tools and computer simulations. Altogether, our findings may underline a rational for targeting the actomyosin system in MPM.

DNA repair gene expression is increased in HPV positive head and neck squamous cell carcinomas.

The incidence of head and neck squamous cell carcinomas (HNSCCs) is rising in developed countries. This is driven by an increase in HNSCCs caused by high-risk human papillomavirus (HPV) infections or HPV + HNSCCs. Compared to HNSCCs not caused by HPV (HPV- HNSCCs), HPV + HNSCCs are more responsive to therapy and associated with better oncologic outcomes. As a result, the HPV status of an HNSCC is an important determinant in medical management. One method to determine the HPV status of an HNSCC is increased expression of p16 caused by the HPV E7 oncogene. We identified novel expression changes in HPV + HNSCCs. A comparison of gene expression among HPV+ and HPV- HNSCCs in The Cancer Genome Atlas demonstrated increased DNA repair gene expression in HPV + HNSCCs. Further, DNA repair gene expression correlated with HNSCC survival. Immunohistochemical analysis of a novel HNSCC microarray confirmed that DNA repair protein abundance is elevated in HPV + HNSCCs.

Cucurbitacin B and I inhibits colon cancer growth by targeting the Notch signaling pathway.

Cancer stem cells (CSCs) have the ability to self-renew and induce drug resistance and recurrence in colorectal cancer (CRC). As current chemotherapy doesn't eliminate CSCs completely, there is a need to identify novel agents to target them. We investigated the effects of cucurbitacin B (C-B) or I (C-I), a natural compound that exists in edible plants (bitter melons, cucumbers, pumpkins and zucchini), against CRC. C-B or C-I inhibited proliferation, clonogenicity, induced G2/M cell-cycle arrest and caspase-mediated-apoptosis of CRC cells. C-B or C-I suppressed colonosphere formation and inhibited expression of CD44, DCLK1 and LGR5. These compounds inhibited notch signaling by reducing the expression of Notch 1-4 receptors, their ligands (Jagged 1-2, DLL1,3,4), γ-secretase complex proteins (Presenilin 1, Nicastrin), and downstream target Hes-1. Molecular docking showed that C-B or C-I binds to the ankyrin domain of Notch receptor, which was confirmed using the cellular thermal shift assay. Finally, C-B or C-I inhibited tumor xenograft growth in nude mice and decreased the expression of CSC-markers and notch signaling proteins in tumor tissues. Together, our study suggests that C-B and C-I inhibit colon cancer growth by inhibiting Notch signaling pathway.

Targeting the vasopressin type-2 receptor for renal cell carcinoma therapy.

Arginine vasopressin (AVP) and its type-2 receptor (V2R) play an essential role in the regulation of salt and water homeostasis by the kidneys. V2R activation also stimulates proliferation of renal cell carcinoma (RCC) cell lines in vitro. The current studies investigated V2R expression and activity in human RCC tumors, and its role in RCC tumor growth. Examination of the cancer genome atlas (TCGA) database, and analysis of human RCC tumor tissue microarrays, cDNA arrays and tumor biopsy samples demonstrated V2R expression and activity in clear cell RCC (ccRCC). In vitro, V2R antagonists OPC31260 and Tolvaptan, or V2R gene silencing reduced wound closure and cell viability of 786-O and Caki-1 human ccRCC cell lines. Similarly in mouse xenograft models, Tolvaptan and OPC31260 decreased RCC tumor growth by reducing cell proliferation and angiogenesis, while increasing apoptosis. In contrast, the V2R agonist dDAVP significantly increased tumor growth. High intracellular cAMP levels and ERK1/2 activation were observed in human ccRCC tumors. In mouse tumors and Caki-1 cells, V2R agonists reduced cAMP and ERK1/2 activation, while dDAVP treatment had the reverse effect. V2R gene silencing in Caki-1 cells also reduced cAMP and ERK1/2 activation. These results provide novel evidence for a pathogenic role of V2R signaling in ccRCC, and suggest that inhibitors of the AVP-V2R pathway, including the FDA-approved drug Tolvaptan, could be utilized as novel ccRCC therapeutics.

Diphenylbutylpiperidine Antipsychotic Drugs Inhibit Prolactin Receptor Signaling to Reduce Growth of Pancreatic Ductal Adenocarcinoma in Mice.

BACKGROUND & AIMS: Prolactin (PRL) signaling is up-regulated in hormone-responsive cancers. The PRL receptor (PRLR) is a class I cytokine receptor that signals via the Janus kinase (JAK)-signal transducer and activator of transcription and mitogen-activated protein kinase pathways to regulate cell proliferation, migration, stem cell features, and apoptosis. Patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of PRL. We investigated whether PRLR signaling contributes to the growth of pancreatic tumors in mice. METHODS: We used immunohistochemical analyses to compare levels of PRL and PRLR in multitumor tissue microarrays. We used structure-based virtual screening and fragment-based drug discovery to identify compounds likely to bind PRLR and interfere with its signaling. Human pancreatic cell lines (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2), with or without knockdown of PRLR (clustered regularly interspaced short palindromic repeats or small hairpin RNA), were incubated with PRL or penfluridol and analyzed in proliferation and spheroid formation. C57BL/6 mice were given injections of UNKC-6141 cells, with or without knockdown of PRLR, into pancreas, and tumor development was monitored for 4 weeks, with some mice receiving penfluridol treatment for 21 days. Human pancreatic tumor tissues were implanted into interscapular fat pads of NSG mice, and mice were given injections of penfluridol daily for 28 days. Nude mice were given injections of Panc-1 cells, xenograft tumors were grown for 2 weeks, and mice were then given intraperitoneal penfluridol for 35 days. Tumors were collected from mice and analyzed by histology, immunohistochemistry, and immunoblots. RESULTS: Levels of PRLR were increased in PDAC compared with nontumor pancreatic tissues. Incubation of pancreatic cell lines with PRL activated signaling via JAK2-signal transducer and activator of transcription 3 and extracellular signal-regulated kinase, as well as formation of pancospheres and cell migration; these activities were not observed in cells with PRLR knockdown. Pancreatic cancer cells with PRLR knockdown formed significantly smaller tumors in mice. We identified several diphenylbutylpiperidine-class antipsychotic drugs as agents that decreased PRL-induced JAK2 signaling; incubation of pancreatic cancer cells with these compounds reduced their proliferation and formation of panco spheres. Injections of 1 of these compounds, penfluridol, slowed the growth of xenograft tumors in the different mouse models, reducing proliferation and inducing autophagy of the tumor cells. CONCLUSIONS: Levels of PRLR are increased in PDAC, and exposure to PRL increases proliferation and migration of pancreatic cancer cells. Antipsychotic drugs, such as penfluridol, block PRL signaling in pancreatic cancer cells to reduce their proliferation, induce autophagy, and slow the growth of xenograft tumors in mice. These drugs might be tested in patients with PDAC.

Occupational exposure of oropharyngeal human papillomavirus amongst otolaryngologists.

OBJECTIVES/HYPOTHESIS: Human papillomavirus (HPV) is a DNA virus that causes cancer in multiple sites. Although sexual activity is the primary means of oropharyngeal HPV acquisition, studies suggest HPV transmission through occupational exposure from medical instruments and surgical fumes. We assess if aerosolization of HPV16 DNA via electrocautery places otolaryngologists at risk for exposure. STUDY DESIGN: Animal and human laboratory model. METHODS: Plasmid (pLXSN16E6E7) expressing HPV p16 E6/E7 genes was transformed into DH5α Escherichia coli cells using the heat shock method. Miniprep and maxiprep purification of transformed DNA with subsequent restriction enzyme double digestion confirmed presence of E6E7 fragment. We injected 2 µg plasmid DNA in 20 µL TE (Tris and ethylenediaminetetraacetic acid) buffer intradermally into freshly severed mouse tail then cauterized for 5 to 10 seconds. Generated fumes were collected through a suction tube fitted with Whatman filter paper. Filter paper was placed in 100 µL TE buffer. Additionally, six patients undergoing transoral robotic surgery for resection of oropharyngeal cancer were identified, three with p16-negative tumors and three with p16-positive tumors. Intraoperatively, Whatman filter paper was exposed to electrocautery fumes, then placed in 100 uL TE buffer. Additional samples were collected from the suction tubing and filter, the surgical mask of the surgeon at head of the bed, and the robot arm. RESULTS: Samples were analyzed via polymerase chain reaction with an assay sensitivity of 1.5 ng E6E7 DNA. None of the patient or mouse tail samples yielded detectable HPV16 DNA in the electrocautery fumes. We did not detect HPV16 DNA on the surgical masks, suction apparatus, or robot arm intraoperatively. CONCLUSIONS: There is likely minimal risk of occupational exposure to HPV16 via electrocautery fumes. LEVEL OF EVIDENCE: NA Laryngoscope, 130:2366-2371, 2020.

The role of the innate and adaptive immune response in HPV-associated oropharyngeal squamous cell carcinoma.

Human papilloma virus (HPV) has been implicated in the development of oropharyngeal squamous cell carcinoma (OPSCC) and is directly attributed to its increasing incidence. The immune microenvironment surrounding HPV-associated OPSCC tumors is complex and plays a critical role in the carcinogenic process. The neoplastic mechanism includes cells of the innate immunity such as macrophages, and dendritic cells as well as cells of the adaptive immune process such as CD8+ T-cells. The intricate interactions between these two arms of the immune system allow for a pro-inflammatory and pro-tumorigenic environment. Intensive efforts are underway to gain a greater understanding of the mechanisms involved in the immune system's role in tumor development. This study seeks to summarize the current knowledge pertaining to role of the innate and adaptive immune response in HPV-associated OPSCC. LEVEL OF EVIDENCE: 3a.

Targeting transcription factor TCF4 by γ-Mangostin, a natural xanthone.

Given that colon cancer is the third most common cancer in incidence and cause of death in the United States, and current treatment modalities are insufficient, there is a need to develop novel agents. Towards this, here we focus on γ-Mangostin, a bioactive compound present in the Mangosteen (Garcinia mangostana) fruit. γ-Mangostin suppressed proliferation and colony formation, and induced cell cycle arrest and apoptosis of colon cancer cell lines. Further, γ-Mangostin inhibited colonosphere formation. Molecular docking and CETSA (Cellular thermal shift assay) binding assays demonstrated that γ-Mangostin interacts with transcription factor TCF4 (T-Cell Factor 4) at the ß-catenin binding domain with the binding energy of -5.5 Kcal/mol. Moreover, γ-Mangostin treatment decreased TCF4 expression and reduced TCF reporter activity. The compound also suppressed the expression of Wnt signaling target proteins cyclin D1 and c-Myc, and stem cell markers such as LGR5, DCLK1 and CD44. To determine the effect of γ-Mangostin on tumor growth in vivo, we administered nude mice harboring HCT116 tumor xenografts with 5 mg/Kg of γ-Mangostin intraperitoneally for 21 days. γ-Mangostin treatment significantly suppressed tumor growth, with notably lowered tumor volume and weight. In addition, western blot analysis revealed a significant decrease in the expression of TCF4 and its downstream targets such as cyclin D1 and c-Myc. Together, these data suggest that γ-Mangostin inhibits colon cancer growth through targeting TCF4. γ-Mangostin may be a potential therapeutic agent for colon cancer.

The Histone Demethylase KDM3A, Increased in Human Pancreatic Tumors, Regulates Expression of DCLK1 and Promotes Tumorigenesis in Mice.

BACKGROUND & AIMS: The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells. METHODS: We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time. RESULTS: Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients. CONCLUSIONS: We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.