Jennifer R. Grandis

The Mutational Landscape of Head and Neck Squamous Cell Carcinoma

The mutational profile of head and neck cancer is complex and may pose challenges to the development of targeted therapies. Head and neck squamous cell carcinoma (HNSCC) is a common, morbid, and frequently lethal malignancy. To uncover its mutational spectrum, we analyzed whole-exome sequencing data from 74 tumor-normal pairs. The majority exhibited a mutational profile consistent with tobacco exposure; human papillomavirus was detectable by sequencing DNA from infected tumors. In addition to identifying previously known HNSCC genes (TP53, CDKN2A, PTEN, PIK3CA, and HRAS), our analysis revealed many genes not previously implicated in this malignancy. At least 30% of cases harbored mutations in genes that regulate squamous differentiation (for example, NOTCH1, IRF6, and TP63), implicating its dysregulation as a major driver of HNSCC carcinogenesis. More generally, the results indicate the ability of large-scale sequencing to reveal fundamental tumorigenic mechanisms.

Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor- mediated cell growth In vitro.

Stimulation of epidermal growth factor receptor (EGFR) by ligand(s) leads to activation of signaling molecules including Stat1 and Stat3, two members of the signal transducers and activators of transcription (STAT) protein family. Activation of Stat1 and Stat3 was constitutive in transformed squamous epithelial cells, which produce elevated levels of TGF-alpha, and was enhanced by the addition of exogenous TGF-alpha. Targeting of Stat3 using antisense oligonucleotides directed against the translation initiation site, resulted in significant growth inhibition. In addition, cells stably transfected with dominant negative mutant Stat3 constructs failed to proliferate in vitro. In contrast, targeting of Stat1 using either antisense or dominant-negative strategies had no effect on cell growth. Thus, TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat3 but not Stat1.

Epidermal Growth Factor Receptor Biology in Head and Neck Cancer

Epidermal growth factor receptor (EGFR) is overexpressed in several epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC), which exhibits EGFR overexpression in up to 90% of tumors. EGFR ligands such as transforming growth factor alpha are also overexpressed in HNSCC. EGFR plays a critical role in HNSCC growth, invasion, metastasis and angiogenesis. However, EGFR inhibitors as monotherapy have yielded only modest clinical outcomes. Potential mechanisms for lack of response to EGFR inhibition in HNSCC include constitutive activation of signaling pathways independent of EGFR, as well as genetic aberrations causing dysregulation of the cell cycle. EGFR-directed therapy may be optimized by identifying and selecting those HNSCC patients most likely to benefit from EGFR inhibition. Resistance to EGFR inhibition may be circumvented by combination therapy employing EGFR inhibitors together with other treatment modalities.

Mutant Epidermal Growth Factor Receptor (EGFRvIII) Contributes to Head and Neck Cancer Growth and Resistance to EGFR Targeting

Purpose: Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where expression levels correlate with decreased survival. Therapies that block EGFR have shown limited efficacy in clinical trials and primarily when combined with standard therapy. The most common form of mutant EGFR (EGFRvIII) has been described in several cancers, chiefly glioblastoma. The present study was undertaken to determine the incidence of EGFRvIII expression in HNSCC and the biological consequences of EGFRvIII on tumor growth in response to EGFR targeting. Experimental Design: Thirty-three HNSCC tumors were evaluated by immunostaining and reverse transcription-PCR for EGFRvIII expression. A representative HNSCC cell line was stably transfected with an EGFRvIII expression construct. EGFRvIII-expressing cells and vector-transfected controls were compared for growth rates in vitro and in vivo as well as chemotherapy-induced apoptosis and the consequences of EGFR inhibition using the chimeric monoclonal antibody C225/cetuximab/Erbitux. Results: EGFRvIII expression was detected in 42% of HNSCC tumors where EGFRvIII was always found in conjunction with wild-type EGFR. HNSCC cells expressing EGFRvIII showed increased proliferation in vitro and increased tumor volumes in vivo compared with vector-transfected controls. Furthermore, EGFRvIII-transfected HNSCC cells showed decreased apoptosis in response to cisplatin and decreased growth inhibition following treatment with C225 compared with vector-transfected control cells. Conclusions: EGFRvIII is expressed in HNSCC where it contributes to enhanced growth and resistance to targeting wild-type EGFR. The antitumor efficacy of EGFR targeting strategies may be enhanced by the addition of EGFRvIII-specific blockade.

STAT signaling in head and neck cancer

The upper aerodigestive tract is predisposed to the formation of multiple primary tumors due to field cancerization. TGF-α/EGFR autocrine signaling appears to play an important role in squamous cell carcinoma of the head and neck (SCCHN) and upregulation of TGF-α and EGFR is an early event in SCCHN carcinogenesis. STAT proteins, including Stat3, are activated by TGF-α and EGFR and strategies that downmodulate TGF-α or EGFR inhibit SCCHN cell proliferation and abrogate Stat3 activation. Targeting Stat3 leads to SCCHN growth inhibition, increases apoptosis and a downmodulation of Bcl-xL expression in head and neck tumors. These studies support the role of Stat3 as an oncogene, which is activated early in SCCHN carcinogenesis, and efforts to understand EGFR-mediated Stat3 signaling could facilitate novel strategies that will interfere with this growth promoting pathway.

Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth

The transcription factor signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers including squamous cell carcinoma of the head and neck (SCCHN). Previous investigations have demonstrated that activated Stat3 contributes to a loss of growth control and transformation. To investigate the therapeutic potential of blocking Stat3 in cancer cells, we developed a transcription factor decoy to selectively abrogate activated Stat3. The Stat3 decoy was composed of a 15-mer double-stranded oligonucleotide, which corresponded closely to the Stat3 response element within the c-fos promoter. The Stat3 decoy bound specifically to activated Stat3 and blocked binding of Stat3 to a radiolabeled Stat3 binding element. By contrast, a mutated version of the decoy that differed by only a single base pair did not bind the activated Stat3 protein. Treatment of head and neck cancer cells with the Stat3 decoy inhibited proliferation and Stat3-mediated gene expression, but did not decrease the proliferation of normal oral keratinocytes. Thus, disruption of activated Stat3 by using a transcription factor decoy approach may serve as a novel therapeutic strategy for cancers characterized by constitutive Stat3 activation.

Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers

Genomic findings underscore the heterogeneity of head and neck squamous cell carcinoma (HNSCC). Identification of mutations that predict therapeutic response would be a major advance. We determined the mutationally altered, targetable mitogenic pathways in a large HNSCC cohort. Analysis of whole-exome sequencing data from 151 tumors revealed the phosphoinositide 3-kinase (PI3K) pathway to be the most frequently mutated oncogenic pathway (30.5%). PI3K pathway-mutated HNSCC tumors harbored a significantly higher rate of mutations in known cancer genes. In a subset of human papillomavirus-positive tumors, PIK3CA or PIK3R1 was the only mutated cancer gene. Strikingly, all tumors with concurrent mutation of multiple PI3K pathway genes were advanced (stage IV), implicating concerted PI3K pathway aberrations in HNSCC progression. Patient-derived tumorgrafts with canonical and noncanonical PIK3CA mutations were sensitive to an mTOR/PI3K inhibitor (BEZ-235), in contrast to PIK3CA-wild-type tumorgrafts. These results suggest that PI3K pathway mutations may serve as predictive biomarkers for treatment selection.

STAT3 signaling: anticancer strategies and challenges.

Multiple lines of evidence place STAT3 at a central node in the development, progression, and maintenance of many human tumors, and STAT3 has been validated as an anti-cancer target in several contexts. STAT3 modulates the transcription of a variety of genes involved in the regulation of critical functions, including cell differentiation, proliferation, apoptosis, angiogenesis, metastasis, and immune responses. For many cancers, elevated levels of activated STAT3 have been associated with a poor prognosis. We review approaches that have been pursued to target STAT3, and we highlight some of the promises and challenges associated with developing an anticancer drug that might therapeutically inhibit the STAT3 signaling pathway.

STAT‐mediated EGFR signaling in cancer

The epidermal growth factor receptor (EGFR) and signal transducers and activators of transcription (STATs) are commonly expressed and activated in many malignancies. EGFR is an upstream activator of several pathways involved in tumor progression, and STATs activate selected genes involved in oncogenesis. There are several different mechanisms by which STAT proteins can mediate intracellular EGFR signaling, including direct activation of STATs by EGFR binding and indirect activation of STATs through Src‐mediated EGFR signaling. EGFR likely activates STAT in a manner distinctive from other mechanisms of STAT activation; STAT5 can be phosphorylated in an EGF‐dependent manner at unique sites, conferring novel functions. Cumulative evidence suggests that targeting EGFR signaling pathways at several levels may demonstrate synergistic therapeutic effects compared with targeting the upstream receptor alone. Thus, methods to inhibit EGFR in conjunction with oncogenic STATs may represent a novel therapeutic strategy for cancers characterized by upregulation of EGFR signaling. J. Cell. Biochem. 102: 311–319, 2007.

The changing face of malignant (necrotising) external otitis: clinical, radiological, and anatomic correlations

Malignant (necrotising) external otitis is an invasive infection of the external auditory canal. Although elderly patients with diabetes remain the population most commonly affected, immunosuppressed individuals (eg, from HIV infection, chemotherapy, etc) are also susceptible to malignant external otitis. Pseudomonas aeruginosa is isolated from the aural drainage in more than 90% of cases. The pathophysiology is incompletely understood although aural water exposure (eg, irrigation for cerumen impaction) has been reported as a potential iatrogenic factor. The typical patient presents with exquisitely painful otorrhoea. If untreated, cranial neuropathies (most commonly of the facial nerve) can develop due to subtemporal extension of the infection. The diagnosis of malignant external otitis is based on a combination of clinical findings, an increased erythrocyte sedimentation rate, and radiographic evidence of soft tissue with or without bone erosion in the external canal and infratemporal fossa. Treatment consists of prolonged administration (6-8 weeks) of an antipseudomonal agent (typically an orally administered quinolone). With the introduction and widespread use of both oral and topical quinolones, there are reports of less severe presentation of malignant external otitis and even the emergence of ciprofloxacin resistance. Reservation of systemic quinolones for the treatment of invasive ear infections is recommended.