Head and Neck Cancer Invasion: Contributions of Actin Regulatory Proteins and the Microenvironment

Metastasis of primary tumor lesions is the leading cause of cancer-related death. In head and neck cancer, a local-regional disease, metastasis is achieved mainly through invasion into surrounding tissue and spreads to cervical lymph nodes. Movement from the initial tumor site requires dynamic reorganization of the actin cytoskeleton, which utilizes the coordinated action of many actin regulatory proteins. However, there is increasing evidence that the tumor microenvironment is also a driver of invasion. This work aims to determine the contributions of proteins which regulate the actin cytoskeleton during head and neck cancer invasion both in vitro and in vivo, and provide details on how the HNSCC tumor microenvironment influences progression. This was accomplished, by the following Studies. In Study one, the actin binding protein coronin 1B is found to be amplified and overexpressed in invasive HNSCC patient samples, and a novel function in the regulation of protrusive membrane structures called invadopodia is described. Study two defines an in vivo role for the actin regulatory protein cortactin, which has been previously associated with more aggressive cancers in vitro and in patients. This work finds that cortactin expression is dispensable for tongue tumor invasion in a transgenic model of oral cancer, implicating the tumor microenvironment as being the major contributor to driving oral cancer invasion. Study three describes a technique for monitoring and biopsying cervical lymph nodes of mice using high frequency ultrasound. By using this technique, alterations in cervical lymph node size and blood flow were discovered in mice given the carcinogen 4-NQO to induce oral carcinogenesis. Collectively, these studies shed light on the importance of choosing comprehensive model systems for studying roles of actin binding proteins in cancer invasion

Recently Identified Biomarkers That Promote Lymph Node Metastasis in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous cancer that arises in the upper aerodigestive tract. Despite advances in knowledge and treatment of this disease, the five-year survival rate after diagnosis of advanced (stage 3 and 4) HNSCC remains approximately 50%. One reason for the large degree of mortality associated with late stage HNSCC is the intrinsic ability of tumor cells to undergo locoregional invasion. Lymph nodes in the cervical region are the primary sites of metastasis for HNSCC, occurring before the formation of distant metastases. The presence of lymph node metastases is strongly associated with poor patient outcome, resulting in increased consideration being given to the development and implementation of anti-invasive strategies. In this review, we focus on select proteins that have been recently identified as promoters of lymph node metastasis in HNSCC. The discussed proteins are involved in a wide range of critical cellular functions, and offer a more comprehensive understanding of the factors involved in HNSCC metastasis while additionally providing increased options for consideration in the design of future therapeutic intervention strategies

Use of High Frequency Ultrasound to Monitor Cervical Lymph Node Alterations in Mice

<div><p>Cervical lymph node evaluation by clinical ultrasound is a non-invasive procedure used in diagnosing nodal status, and when combined with fine-needle aspiration cytology (FNAC), provides an effective method to assess nodal pathologies. Development of high-frequency ultrasound (HF US) allows real-time monitoring of lymph node alterations in animal models. While HF US is frequently used in animal models of tumor biology, use of HF US for studying cervical lymph nodes alterations associated with murine models of head and neck cancer, or any other model of lymphadenopathy, is lacking. Here we utilize HF US to monitor cervical lymph nodes changes in mice following exposure to the oral cancer-inducing carcinogen 4-nitroquinoline-1-oxide (4-NQO) and in mice with systemic autoimmunity. 4-NQO induces tumors within the mouse oral cavity as early as 19 wks that recapitulate HNSCC. Monitoring of cervical (mandibular) lymph nodes by gray scale and power Doppler sonography revealed changes in lymph node size eight weeks after 4-NQO treatment, prior to tumor formation. 4-NQO causes changes in cervical node blood flow resulting from oral tumor progression. Histological evaluation indicated that the early 4-NQO induced changes in lymph node volume were due to specific hyperproliferation of T-cell enriched zones in the paracortex. We also show that HF US can be used to perform image-guided fine needle aspirate (FNA) biopsies on mice with enlarged mandibular lymph nodes due to genetic mutation of Fas ligand (Fasl). Collectively these studies indicate that HF US is an effective technique for the non-invasive study of cervical lymph node alterations in live mouse models of oral cancer and other mouse models containing cervical lymphadenopathy.</p></div

Image-guided fine needle biopsy of Fasl mandibular lymph nodes.

<p><b>A.</b> Transverse section of a Fasl mouse neck imaged with HF US. The enlarged cervical mandibular node is evident as an oval hypoechoic region near the skin surface (circumscribed in yellow). Scale bar  = 1 mm. <b>B.</b> Frames from fine needle biopsy of a Fasl mandibular node guided by HF US. Images show the position of the sampling hyperechoic needle tip prior to cervical skin penetration (<i>left</i>), position of the needle during tissue removal (<i>middle</i>), and following needle withdrawal (<i>right</i>). Note the break in the skin following needle withdrawal (arrow). The angle and trajectory of the dorsal needle surface is denoted by the yellow dotted line. Scale bar  = 1 mm. The entire procedure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s003" target="_blank">Video S1</a>. <b>C.</b> Examples of lymph tissue obtained by HF US guided FNA of a Fasl cervical mandibular node following staining and processing by cytospin. Scale bar  = 100 µm. LT; lymph tissue, RF; reticular fibers, L; individual lymphocytes.</p

Mapping of mouse cervical lymph nodes by high frequency ultrasound.

<p><b>A.</b> Overview image of the HF US platform for cervical lymph node evaluation. An anesthetized B6 mouse is shown positioned on the Vevo 2100 heated imaging platform with the ventral side exposed. Each paw is tapped to a monitoring electrode and the rectal probe (blue) secured to the stage. The transducer (white) is positioned over the ventral neck area. <b>B.</b> Diagram showing relative locations of murine cervical lymph nodes. Individual neck sections visualized by HF US imaging and histology are indicated by dashed lines. Arrows denote specific positions of each mapped section relative to corresponding ultrasound and histology images. Each imaged anatomical location is numbered. M, mandibular node. AM, accessory mandibular node. SP, superficial parotid node. <b>C.</b> Serial transverse sections of the mouse neck imaged by HF US corresponding to the indicated anatomic regions in (B). <b>D.</b> Transverse cervical H&E stained histological sections corresponding to the HF US sections in (C). Arrows labeled “2” denote mandibular node as diagrammed in B. Scale bar  = 1 mm. CP, cheek pouch. VT, ventral tongue. DT, dorsal tongue. E, esophagus.</p

4-NQO treatment induces paracortical/T-cell zone hyperplasia in mandibular lymph nodes.

<p>Representative examples of H&E stained, dissected mandibular lymph nodes from age-matched (<b>A</b>) control and (<b>B</b>) 4-NQO-treated (28 wk) mice. T-cell zone expansions in each node are circumscribed in yellow. Scale bar  = 250 µm. <b>C.</b> Distribution of nodal paracortical/T-cell zone hyperplasia. Mandibular lymph nodes were pathologically scored and grouped according to relative scale of T-cell zone involvement, using the following scale: None, absent to focal limited expansion; Modest, multifocal or focal up to moderate expansion; Robust, multifocal moderate expansion and/or confluence of paracortical subregions.</p

4-NQO exposure induces precancerous alterations in mouse mandibular lymph nodes.

<p><b>A–C.</b> Images of dissected H&E and whole animal HF US (ultrasound) mandibular lymph nodes from representative age-matched (AM) control (<b>A</b>), 4-NQO-treated (28 wk) (<b>B</b>) and Fasl (<b>C</b>) mice. Lymph node borders in the HF US images are indicated in yellow. Vascular flow identified by power Doppler imaging is shown in red. Power Doppler flow dynamics for each condition are visualized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s004" target="_blank">Video S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s006" target="_blank">S4</a>. H&E scale bar  = 250 µm, ultrasound scale bar  = 1 mm. CP, Cheek Pouch. <b>D&E.</b> Analysis of lymph nodes by HF US. 4-NQO treated mice at 0 and 28 wk were imaged after 8 week 4-NQO treatment and study end point. B6 age-matched (AM) Ctl 0 and 28 wk mice were imaged at the same age as 4-NQO treated mice. The Fasl lymph node data is included for comparison. <b>D.</b> 4-NQO exposure induces increased mandibular lymph node volume. <b>E.</b> 4-NQO exposure increases vascular flow in mandibular nodes. N = 6 lymph nodes from 3 mice per group, except for the controls, where N = 8 lymph nodes were analyzed from 4 mice. Box and whisker plots show minimum, 25^th, median, 75^th and maximum values, respectively. *, p≤0.05.</p

FGF21 is required for the metabolic benefits of IKKε/TBK1 inhibition

The protein kinases IKKε and TBK1 are activated in liver and fat in mouse models of obesity. We have previously demonstrated that treatment with the IKKε/TBK1 inhibitor amlexanox produces weight loss and relieves insulin resistance in obese animals and patients. While amlexanox treatment caused a transient reduction in food intake, long-term weight loss was attributable to increased energy expenditure via FGF21-dependent beiging of white adipose tissue (WAT). Amlexanox increased FGF21 synthesis and secretion in several tissues. Interestingly, although hepatic secretion determined circulating levels, it was dispensable for regulating energy expenditure. In contrast, adipocyte-secreted FGF21 may have acted as an autocrine factor that led to adipose tissue browning and weight loss in obese mice. Moreover, increased energy expenditure was an important determinant of improved insulin sensitivity by amlexanox. Conversely, the immediate reductions in fasting blood glucose observed with acute amlexanox treatment were mediated by the suppression of hepatic glucose production via activation of STAT3 by adipocyte-secreted IL-6. These findings demonstrate that amlexanox improved metabolic health via FGF21 action in adipocytes to increase energy expenditure via WAT beiging and that adipocyte-derived IL-6 has an endocrine role in decreasing gluconeogenesis via hepatic STAT3 activation, thereby producing a coordinated improvement in metabolic parameters