Lin Wang

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death in the US. The protein kinase D (PKD) family has emerged as a promising target for cancer therapy with PKD1 being most intensively studied; however, its role in HNSCC has not been investigated. The expression of PKD was evaluated in human HNSCC by quantitative RT-PCR, Western blot and immunohistochemistry. Cell proliferation, wound healing, and matrigel invasion assays were performed upon siRNA-mediated knockdown of PKD1 in HNSCC cells, and subcutaneous xenograft mouse model was established by implantation of the stable doxycycline (Dox)-inducible PKD1 expression cell lines for analysis of tumorigenic activity in vivo. PKD1 was frequently downregulated in HNSCC cell lines at both transcript and protein levels. In human HNSCC tissues, PKD1 was significantly down-regulated in localized tumors and metastases, and in patient-paired tumor tissues as compared to their normal counterparts, which was in part due to epigenetic modification of the PRKD1 gene. The function of PKD1 in HNSCC was analyzed using stable doxycycline-inducible cell lines that express native or constitutive-active PKD1. Upon induction, the rate of proliferation, survival, migration and invasion of HNSCC cells did not differ significantly between the control and PKD1 overexpressing cells in the basal state, and depletion of endogenous PKD1 did not impact the proliferation of HNSCC cells. However, the median growth rate of the subcutaneous HNSCC tumor xenografts over time was elevated with PKD1 induction, and the final tumor weight was significantly increased in Dox-induced vs. the non-induced tumors. Moreover, induced expression of PKD1 promoted bombesin-induced cell proliferation of HNSCC and resulted in sustained ERK1/2 activation in response to gastrin-releasing peptide or bombesin stimulation, suggesting that PKD1 potentiates GRP/bombesin-induced mitogenic response through the activation of ERK1/2 in HSNCC cells. Our study has identified PKD1 as a frequently downregulated gene in HNSCC, and functionally, under certain cellular context, may play a role in GRP/bombesin-induced oncogenesis in HNSCC.

BACKGROUND. EGFR and Src family kinases are upregulated in head and neck squamous cell carcinoma (HNSCC). EGFR interacts with Src to activate STAT3 signaling, and dual EGFR-Src targeting is synergistic in HNSCC preclinical models. pSrc overexpression predicted resistance to the EGFR inhibitor, erlotinib, in a prior window trial. We conducted a 4-arm window trial to identify biomarkers associated with response to EGFR and/or Src inhibition. METHODS. Patients with operable stage II–IVa HNSCC were randomized to 7–21 days of neoadjuvant erlotinib, the Src inhibitor dasatinib, the combination of both, or placebo. Paired tumor specimens were collected before and after treatment. Pharmacodynamic expression of EGFR and Src pathway components was evaluated by IHC of tissue microarrays and reverse-phase protein array of tissue lysates. Candidate biomarkers were assessed for correlation with change in tumor size. RESULTS. From April 2009 to December 2012, 58 patients were randomized and 55 were treated. There was a significant decrease in tumor size in both erlotinib arms ( P = 0.0014); however, no effect was seen with dasatinib alone ( P = 0.24). High baseline pMAPK expression was associated with response to erlotinib ( P = 0.03). High baseline pSTAT3 was associated with resistance to dasatinib ( P = 0.099). CONCLUSIONS. Brief exposure to erlotinib significantly decreased tumor size in operable HNSCC, with no additive effect from dasatinib. Baseline pMAPK expression warrants further study as a response biomarker for anti-EGFR therapy. Basal expression of pSTAT3 may be independent of Src, explain therapeutic resistance, and preclude development of dasatinib in biomarker-unselected cohorts. TRIAL REGISTRATION. NCT00779389. FUNDING. National Cancer Institute, American Cancer Society, Pennsylvania Department of Health, V Foundation for Cancer Research, Bristol-Myers Squibb, and Astellas Pharma.

There is current interest in anti-angiogenesis therapies for head and neck squamous cell carcinomas (HNSCC), although the utility of these therapies in human papillomavirus (HPV) positive and HPV-negative HNSCC is unclear. Therefore, we explored heterogeneity in expression of a distal factor in angiogenesis (EGFR, the epidermal growth factor receptor), a proximal factor in angiogenesis (VEGF, the vascular endothelial growth factor) and a putative factor in angiogenesis (NOTCH1) in a HNSCC case series using immunohistochemistry in N = 67 cases (27 HPV-positive, 40 HPV-negative, by in situ hybridization). Box plots and the Wilcoxon rank sum or Kruskal–Wallis tests were used to compare staining scores (intensity × percent of cells staining) by HPV status and lifestyle factors. Associations between EGFR, VEGF, and NOTCH1 were assessed using box plots and Spearman correlation (ρ) in all cases, and stratified by HPV status. HPV-negative HNSCC over-expressed EGFR [median (range): 30 (0–300)] relative to HPV-positive HNSCC [7.5 (0–200)] (P = 0.006). VEGF and NOTCH1 were unrelated to HPV status (P > 0.05). EGFR was associated with VEGF in HPV-negative (ρ = 0.40, P = 0.01) but not HPV-positive HNSCC (ρ = 0.25, P = 0.20). NOTCH1 and VEGF were associated in HPV-negative (ρ = 0.40, P = 0.01) but not HPV-positive tumors (ρ = −0.12, P = 0.57). NOTCH1 was not associated with EGFR (P > 0.05). Our results are suggestive of heterogeneity in HNSCC angiogenesis. Future studies should explore angiogenesis mechanisms in HPV-positive and HPV-negative HNSCC.