Amy S. Whigham

Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Invadopodia are branched actin-rich structures associated with extracellular matrix (ECM) degradation that collectively form the invasive machinery of aggressive cancer cells. Cortactin is a prominent component and a specific marker of invadopodia. Amplification of cortactin is associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), possibly because of its activity in invadopodia. Although the role of cortactin in invadopodia has been attributed to signaling and actin assembly, it is incompletely understood. We made HNSCC cells deficient in cortactin by RNA interference knockdown methods. In these cortactin knockdown cells, invadopodia were reduced in number and lost their ability to degrade ECM. In the reverse experiment, overexpression of cortactin dramatically increased ECM degradation, far above and beyond the effect on formation of actin/Arp3-positive invadopodia puncta. Secretion of matrix metalloproteinases (MMP) MMP-2 and MMP-9, as well as plasma membrane delivery of MT1-MMP correlated closely with cortactin expression levels. MMP inhibitor treatment of control cells mimicked the cortactin knockdown phenotype, with abolished ECM degradation and fewer invadopodia, suggesting a positive feedback loop in which degradation products from MMP activity promote new invadopodia formation. Collectively, these data suggest that a major role of cortactin in invadopodia is to regulate the secretion of MMPs and point to a novel mechanism coupling dynamic actin assembly to the secretory machinery, producing enhanced ECM degradation and invasiveness. Furthermore, these data provide a possible explanation for the observed association between cortactin overexpression and enhanced invasiveness and poor prognosis in HNSCC patients.

Aggressiveness of HNSCC tumors depends on expression levels of cortactin, a gene in the 11q13 amplicon

11q13 amplification is a late-stage event in several cancers that is often associated with poor prognosis. Among 11q13-amplified genes, the actin assembly protein cortactin/CTTN is considered a likely candidate for direct involvement in tumor progression because of its cell motility-enhancing functions. We modulated cortactin expression in head and neck squamous cell carcinoma (HNSCC) cell lines. Cortactin expression levels directly correlated with tumor size, vascularization and cell proliferation in an orthotopic HNSCC in vivo model. In contrast, under normal in vitro culture conditions, cortactin expression levels had no effect on cell proliferation. However, cell lines in which cortactin expression was reduced by knockdown (KD) grew poorly in vitro under harsh conditions of growth factor deprivation, anchorage independence and space constraint. In contrast, overexpression of cortactin enhanced in vitro growth under the same harsh conditions. Surprisingly, defects in growth factor-independent proliferation of cortactin-KD cells were rescued by coculture with cortactin-expressing cells. As the cocultured cells are separated by permeable filters, cortactin-expressing cells must secrete growth-supporting autocrine factors to rescue the cortactin-KD cells. Overall, cortactin expression modulates multiple cellular traits that may allow survival in a tumor environment, suggesting that the frequent overexpression of cortactin in tumors is not an epiphenomenon but rather promotes tumor aggressiveness.

Human-in-mouse modeling of primary head and neck squamous cell carcinoma.

To develop a reliable modeling system for head and neck squamous cell carcinoma (HNSCC).

Observation of Viable Nontypeable Haemophilus Influenzae Bacteria within Neutrophil Extracellular Traps in Clinical Samples from Chronic Otitis Media

Bacterial otitis media is an inherently inflammatory condition and often features an influx of neutrophils and other phagocytes into the middle-ear chamber. Neutrophils undergo a specific death pathway that results in formation of Neutrophil Extracellular Traps or “NETs”, which have been postulated as an antimicrobial defense mechanism that both mediates direct bacterial killing and facilitates phagocytic uptake and killing. However, we have shown that some mucosal pathogens within the airway including nontypeable Haemophilus influenzae survive within NETs in animal and in vitro models. In this report, we utilize exudate samples obtained from patients with chronic otitis media to show that nontypeable H. influenzae bacteria survive within NET structures within human patients.

Respiratory events after adenotonsillectomy requiring escalated admission status in children with obstructive sleep apnea

OBJECTIVES To characterize postoperative respiratory complications following adenotonsillectomy (AT) in children with obstructive sleep apnea (OSA) and to identify variables associated with pediatric intensive care unit (PICU) admission. METHODS Retrospective analysis of 133 pediatric OSA patients with prior AT. Assessment of the postoperative hospital course informed patient stratification based on respiratory event severity, PICU admission status, and unscheduled escalation of care. RESULTS Thirty-six (26.8%) patients were admitted to the PICU. Compared to non-PICU admissions, these patients were significantly younger and with greater preoperative apnea-hypopnea indices, comorbidities, and percentage of post-anesthesia care unit (PACU) time requiring supplemental oxygen. Seventy-one respiratory events occurred in 59 patients, with 60.6% affecting PICU patients. Fifteen severe events occurred, affecting 31% of PICU patients. Of 14 unscheduled escalations of care, 7 were PICU admissions who, compared to planned PICU admissions, spent significantly more time in the PACU and exhibited a trend towards greater PACU time on supplemental oxygen. CONCLUSIONS Pediatric patients requiring post-AT PICU care have more risk factors for respiratory compromise. Total PACU time and total PACU time requiring supplemental oxygen may indicate patient risk for postoperative respiratory complications and need for intensive care. Future work includes prospective determination of appropriate post-AT PICU admission.

Planned Neck Dissection after CCR for HNSCC Is It Necessary

tions are subject to a same-day discharge algorithm that incorporates post-anesthesia-care-unit (PACU) rapid PTH as the major discharge criterion. Patients with PTH 30 (pg/ml) are eligible for same-day discharge from PACU without supplementation, PTH 20 are eligible for discharge but receive calcium supplementation, and PTH 20 are observed overnight (23 hours) with calcium and vitamin D supplementation. RESULTS: 180 patients (mean age 48.9, 83.3% female) underwent total (77.2%) or completion (22.7%) thyroidectomy with or without node dissection (42.2% minimally invasive video assisted). Seventy-six percent (137/180) of patients had a PTH 20, meeting PTH discharge criterion. Sixty-nine percent of those eligible (95/137) were discharged same-day (53.1% of total). Of the 95 patients undergoing outpatient surgery, none were admitted, seen, or called with symptoms of hypocalcemia. Mean PACU PTH was not significantly different from pre-operative PTH (43.4 vs. 38.7, p 0.10). Mean adjusted total calcium in PACU was 8.7 /0.5 (mg/dl). Of the 85 patients observed overnight, 11.1% were hypocalcemic on post-op day (POD) 1. Of the 42 patients observed despite PTH 20, 7.1% had asymptomatic POD1 hypocalcemia. All 180 patients were eucalcemic (Ca 8) at POD7 and POD30 office visits. No patients were hypoparathyroid at POD30. CONCLUSION: A PTH-based discharge algorithm can safely facilitate outpatient completion or total thyroidectomy, with minimal risk of clinically significant outpatient hypocalcemia.

Prediction of Nodal Metastases from Genomic Analyses of the Primary Tumor

Due to the associated poor prognostic significance, the finding of nodal metastases during the evaluation and staging of patients with head and neck cancer drives therapeutic decisions. Advances in imaging have enabled the detection of smaller cervical metastases with more certainty; however, an unacceptable portion (a minority) of patients with negative clinical and radiographic evaluation for cervical disease actually has metastases upon pathologic evaluation of resected nodes. Clinicians are left in the uncomfortable position of treating cervical nodal basins in patients with greater than 20% risk of metastases based on historical data, despite the fact that, in the majority of patients, this intervention may not be beneficial. This treatment frequently results in patient morbidity and is a health care expenditure that could be redirected if physicians could better predict metastases. On the cellular level, metastasis is a rare event due to its complex nature requiring that the metastatic tumor cells acquire a number of disparate behaviors through gene alterations. Recent data suggest that gene expression profiling of the primary tumor can predict metastatic potential. Because primary tumor biopsies provide adequate material for gene expression profiling, one can imagine that data derived from a diagnostic biopsy could be used to direct therapeutic decisions.

Short-Term Culture and In-Vivo Modeling of Primary HNSCC

Problem Head and neck squamous cell carcinoma (HNSCC) accounts for 4% of annual U.S. cancer deaths. In-vivo models exist using established HNSCC lines, but currently there is no such model that allows consistent growth of HNSCC from primary tumors. Methods Primary HNSCC tissue was obtained from 103 patients at biopsy/resection, disaggregated and seeded onto collagen-coated plates in keratinocyte growth media with 10% FBS, additives and antibiotics. After short-term growth in culture, cells were transferred to denuded rat tracheas and implanted subcutaneously in nude mice. Indirect immunofluorescent staining using antibodies specific to cytokeratin, vimentin and nuclear Ku was performed to determine cell lineage and origin. Results Cultured cells exhibited morphology consistent with epithelial or stromal derivation. 80% of cultures had viable cells present at 10 days and 24% were maintained 30 days or longer. 5 cultures (5%) proliferated after multiple passages and thrived on uncoated plates in the absence of mesenchymal cells. The xenograft model was able to successfully establish tumors in vivo from 59% of primary tumors. Immunostaining for nuclear Ku and cytokeratin confirmed human origin and epithelial cell lineage, respectively. Conclusion The high success rate indicates that selective growth and survival pressures for short-term culture of primary HNSCC may be considerably less than for establishment of cell lines. Additionally, the techniques permit tumor-derived epithelial and mesenchymal cells to be cultured simultaneously. Preliminary data for the in-vivo trachea xenograft model is promising. A luciferase lentiviral system has been created to allow monitoring of tumor growth in vivo with serial live animal imaging. Significance These short-term culture techniques may more accurately characterize both the biological diversity of HNSCC and tumor-stromal cell interactions. Once optimized, the trachea xenograft model can be used to determine the in-vivo response of a heterogeneous group of HNSCC to standard and novel therapies. Support Funds provided by an endowment for the Barry Baker Laboratory for Head and Neck Oncology, the Vanderbilt Ingram Cancer Center Endowed Professorship Fund, and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.