Enhanced antiproliferative effect of resveratrol in head and neck squamous cell carcinoma using GE11 peptide conjugated liposome.

Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma

Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. To identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant intercell line differences for 42 kinases (p < 0.05), and loss of function experiments using siRNA in high and low expressing cell lines identified kinases including EGFR, NEK9, LYN, JAK1, WEE1, and EPHA2 involved in cell survival and proliferation. EGFR inhibition by the small molecule inhibitors lapatinib, gefitinib, and erlotinib as well as siRNA led to strong reduction of viability in high but not low expressing lines, confirming EGFR as a drug target in 10-20% of HNSCC cell lines. Similarly, high, but not low EPHA2-expressing cells showed strongly reduced viability concomitant with down-regulation of AKT and ERK signaling following EPHA2 siRNA treatment or EPHA1-Fc ligand exposure, suggesting that EPHA2 is a novel drug target in HNSCC. This notion is underscored by immunohistochemical analyses showing that high EPHA2 expression is detected in a subset of HNSCC tissues and is associated with poor prognosis. Given that the approved pan-SRC family kinase inhibitor dasatinib is also a very potent inhibitor of EPHA2, our findings may lead to new therapeutic options for HNSCC patients. Importantly, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy.

BackgroundErlotinib is a commonly used epidermal growth factor receptor (EGFR)-targeted therapeutic choice for head and neck squamous cell carcinoma; however, its efficacy is largely compromised by cancer cell resistance. Understanding and targeting the erlotinib adaptive mechanisms of squamous cell carcinoma of the head and neck (HNSCC) cancer cells are still pressing challenges. This study aimed to elucidate the cooperative erlotinib-sensitizing mechanisms of histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K) co-inhibition, which will be helpful in gaining a better understanding of the mechanism of EGFR-tyrosine kinase inhibitor (TKI) resistance in head and neck cancer cells.MethodsHigh-content screening (HCS) was performed to analyze the cell counts of different treatment groups and their drug-sensitizing effect phenotype. Western blotting and immunofluorescence staining assays were used to measure and locate the expression of proteins in the FaDu and TU212 cells. Annexin V/PI and DAPI staining were also used to determine the ratio of apoptotic cells and different cell cycle phases.ResultsThe expression of phosphor-EGFRTyr992 was significantly increased in erlotinib-treated FaDu cells compared with dimethyl sulfoxide (DMSO)-treated FaDu cells. Meanwhile, erlotinib + vorinostat + copanlisib jointly attenuated the expression of phosphor-EGFRTyr1068 and phosphor-EGFRTyr992, but stimulated the expression of E-cadherin. Moreover, we found that the tri-drug group also impaired the expression of phosphor-STAT3Ser727 and its relevant activators, including phosphor-SrcTyr416.ConclusionsThese findings indicate that HDACs and PI3K co-inhibition sensitizes erlotinib via inactivation of the phosphor-EGFRTyr1068-induced RTK-STAT3 axis. However, PI3K inhibition was sufficient to sensitize TU212 cells to erlotinib, providing new perspectives for the further clinical study of erlotinib + vorinostat + copanlisib as a potential combination therapeutic solution for EGFR responsive reactivation-induced resistance to erlotinib.

Understanding of p53 family protein (p53, p63 and p73) networks in head and neck squamous cell carcinoma (HNSCC), can positively influence in cancer screening, diagnosis, treatment and prevention. P53 family proteins are regulating diverse cell signalling pathways at diverse conditions to determine cell fate. At each condition of cells state, how these proteins are controlling cell fate is more complexed due to the presense of several isoforms for each p53 family proteins and their multifaceted interactions. Like other solid tumours, the p53 pathway is disabled by several mechanisms in HNSCC. Despite of the convincing evidence of a high frequency of p53 mutations in HNSCC, a subset of cancers arise in the absence of mutations. The molecular mechaninsms through which p53 lacking mutations subvert its tumor supressor functions in HNSCC still remain uncertain. In fact, some cancers and established cancer cell lines are over-expressing wild type p53 protein makes questionable of p53 role as only a tumor suppressor or it has some other additional functions, even in cancer cells. For an answer of this hypothesis, we have performed detailed molecular characterization, in an invitro model of head and neck cancer, in a broad panel of 12 newly established HNSCC cell lines. In our studies, we found that some head and neck cancer cell lines are accumulating wild p53 protein hyperphosphorylated at serine15 and 392 and it leads to the over expression of DNp73, the mechanism already reported from our laboratory in HPV38 immortalized keratinocytes. To better understand the functions of accumulated wild p53 role in HPV positive and negative cancer cell lines, we have performed p53 knock down by siRNA and the results have showed that p53 knock down inhibited cell proliferation in both HPV positive and negative cancer cells. Moreover, p53 knock down induced significant morphological changes and senescence associated beta galactosidase activity in these cells. Our results pinpoint, wild-type p53 protein accumulated in transformed cell lines has an additional role in cell proliferation other than its well known tumor supressor activity. Moreover, the key role of p53 family network in modulating epidermal growth factor receptor (EGFR) expression and in controlling cell proliferation and apoptosis arises the question of whether p53 family proteins status influences the efficacy of EGFR inhibitors, investigating its ability to reduce cell growth, to induce apoptosis and to modulate cell cycle and various EGFR pathwayrelated targets in head and neck cancer. Because, recently improved understanding of the pathogenesis of human head and neck squamous cell carcinoma has led to the development of new, molecular based therapeutic strategies, one of the most promising is the utilization of tyrosine kinase (TK) inhibitors, targeting EGFR. The comparison between the targets analysed and gefitinib effectiveness evidenced the absence of a clear relationship, exluding them as predictive factors for gefitinib efficacy. Our results confirmed the in vitro efficacy of an anti-EGFR approach, but other targets than those analysed here should be characterized in order to identify valid predictive factors for gefitinib utilization in head and neck cancer. We have also performed chemoresistance analysis in our invitro model of HNSCC cell lilnes and found that p53 family network has less predictive role in HNSCC chemoresistance but ABCG2, a multidrug resistance protein, has over-expressed in HNSCC.

Phosphoproteomic Analysis of Signaling Pathways in Head and Neck Squamous Cell Carcinoma Patient Samples

&lt;div&gt;Abstract&lt;p&gt;Cisplatin and its analogues are the most commonly used agents in the treatment of head and neck squamous cell carcinoma. In this study, we investigated a possible role of epidermal growth factor (EGF) receptor (EGFR) phosphorylation and degradation in cisplatin-induced cytotoxicity. Cisplatin treatment led to an increase in initial EGFR phosphorylation at Y1045, the binding site of ubiquitin ligase, Casitas B-lineage lymphoma (c-Cbl), followed by ubiquitination in the relatively cisplatin-sensitive cell lines. However, cisplatin-resistant cell lines underwent minimal EGFR phosphorylation at the Y1045 site and minimal ubiquitination. We found that EGFR degradation in response to cisplatin was highly correlated with cytotoxicity in seven head and neck cancer cell lines. Pretreatment with EGF enhanced cisplatin-induced EGFR degradation and cytotoxicity, whereas erlotinib pretreatment blocked EGFR phosphorylation, degradation, and cisplatin-induced cytotoxicity. Expression of a mutant Y1045F EGFR, which is relatively resistant to c-Cbl–mediated degradation, in Chinese hamster ovary cells and the UMSCC11B human head and neck cancer cell line protected EGFR from cisplatin-induced degradation and enhanced cell survival compared with wild-type (WT) EGFR. Transfection of WT c-Cbl enhanced EGFR degradation and cisplatin-induced cytotoxicity compared with control vector. These results show that cisplatin-induced EGFR phosphorylation and subsequent ubiquitination and degradation is an important determinant of cisplatin sensitivity. Our findings suggest that treatment with an EGFR inhibitor before cisplatin would be antagonistic, as EGFR inhibition would protect EGFR from cisplatin-mediated phosphorylation and subsequent ubiquitination and degradation, which may explain the negative results of several recent clinical trials. Furthermore, they suggest that EGFR degradation is worth exploring as an early biomarker of response and as a target to improve outcome. Cancer Res; 70(7); 2862–9&lt;/p&gt;&lt;/div&gt;

Cisplatin and its analogues are the most commonly used agents in the treatment of head and neck squamous cell carcinoma. In this study, we investigated a possible role of epidermal growth factor (EGF) receptor (EGFR) phosphorylation and degradation in cisplatin-induced cytotoxicity. Cisplatin treatment led to an increase in initial EGFR phosphorylation at Y1045, the binding site of ubiquitin ligase, Casitas B-lineage lymphoma (c-Cbl), followed by ubiquitination in the relatively cisplatin-sensitive cell lines. However, cisplatin-resistant cell lines underwent minimal EGFR phosphorylation at the Y1045 site and minimal ubiquitination. We found that EGFR degradation in response to cisplatin was highly correlated with cytotoxicity in seven head and neck cancer cell lines. Pretreatment with EGF enhanced cisplatin-induced EGFR degradation and cytotoxicity, whereas erlotinib pretreatment blocked EGFR phosphorylation, degradation, and cisplatin-induced cytotoxicity. Expression of a mutant Y1045F EGFR, which is relatively resistant to c-Cbl-mediated degradation, in Chinese hamster ovary cells and the UMSCC11B human head and neck cancer cell line protected EGFR from cisplatin-induced degradation and enhanced cell survival compared with wild-type (WT) EGFR. Transfection of WT c-Cbl enhanced EGFR degradation and cisplatin-induced cytotoxicity compared with control vector. These results show that cisplatin-induced EGFR phosphorylation and subsequent ubiquitination and degradation is an important determinant of cisplatin sensitivity. Our findings suggest that treatment with an EGFR inhibitor before cisplatin would be antagonistic, as EGFR inhibition would protect EGFR from cisplatin-mediated phosphorylation and subsequent ubiquitination and degradation, which may explain the negative results of several recent clinical trials. Furthermore, they suggest that EGFR degradation is worth exploring as an early biomarker of response and as a target to improve outcome.

&lt;div&gt;Abstract&lt;p&gt;Cisplatin and its analogues are the most commonly used agents in the treatment of head and neck squamous cell carcinoma. In this study, we investigated a possible role of epidermal growth factor (EGF) receptor (EGFR) phosphorylation and degradation in cisplatin-induced cytotoxicity. Cisplatin treatment led to an increase in initial EGFR phosphorylation at Y1045, the binding site of ubiquitin ligase, Casitas B-lineage lymphoma (c-Cbl), followed by ubiquitination in the relatively cisplatin-sensitive cell lines. However, cisplatin-resistant cell lines underwent minimal EGFR phosphorylation at the Y1045 site and minimal ubiquitination. We found that EGFR degradation in response to cisplatin was highly correlated with cytotoxicity in seven head and neck cancer cell lines. Pretreatment with EGF enhanced cisplatin-induced EGFR degradation and cytotoxicity, whereas erlotinib pretreatment blocked EGFR phosphorylation, degradation, and cisplatin-induced cytotoxicity. Expression of a mutant Y1045F EGFR, which is relatively resistant to c-Cbl–mediated degradation, in Chinese hamster ovary cells and the UMSCC11B human head and neck cancer cell line protected EGFR from cisplatin-induced degradation and enhanced cell survival compared with wild-type (WT) EGFR. Transfection of WT c-Cbl enhanced EGFR degradation and cisplatin-induced cytotoxicity compared with control vector. These results show that cisplatin-induced EGFR phosphorylation and subsequent ubiquitination and degradation is an important determinant of cisplatin sensitivity. Our findings suggest that treatment with an EGFR inhibitor before cisplatin would be antagonistic, as EGFR inhibition would protect EGFR from cisplatin-mediated phosphorylation and subsequent ubiquitination and degradation, which may explain the negative results of several recent clinical trials. Furthermore, they suggest that EGFR degradation is worth exploring as an early biomarker of response and as a target to improve outcome. Cancer Res; 70(7); 2862–9&lt;/p&gt;&lt;/div&gt;

Head and Neck Cancer

Purpose: Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), is approved for use in the treatment of head and neck cancer (HNC). One recent study (SPECTRUM) suggested that patient with recurrent or metastatic HPV+ HNC do not benefit from treatment with EGFR targeted therapy. However, most studies of EGFR inhibition have been performed with HPV-negative cells and clinical trials have been performed without regard to HPV status. We performed this study to determine whether HPV+ HNC cells and patient-derived tumorgrafts respond to cetuximab and to examine the mechanisms through which cetuximab affects HPV+ HNC. Methods: Four HPV+ cell lines (UD-SCC2, UM-SCC47, UPCI-SCC90, 93-VU-147T) were assessed for EGFR expression by western blot. Sensitivity to cetuximab was tested by assessing cell density 2, 4, 6, and 8 days after cetuximab treatment and by assessing colony formation 2 weeks after plating. Apoptosis was measured by caspase activation, flow cytometry for Annexin V and propidium iodide staining, and immunoblot. Cell cycle was assessed by immunoblot for cyclin D1, cyclin B1, and p27Kip-1 and confirmed by flow cytometry for propidium iodide stained cells. Subcutaneous flank xenografts and tumorgrafts were performed in Hsd:athymic Nude-Foxn1nu female mice treated with intraperitoneal cetuximab (0.2mg/mouse) delivered twice weekly for 2 weeks. Time to tumor quadrupling from baseline was assessed by Kaplan-Meier method. Results: Significant variation in EGFR expression was seen in HPV+ cells. Cetuximab treatment resulted in significant delay in cell proliferation (p&amp;lt;0.005 for all lines) and decrease in colony formation (p&amp;lt;0.04 for all lines). In these cell lines, cetuximab caused an increase in apoptosis as measured by caspase activity, Bax activation, and Annexin V labeling. In addition, as previously seen in HPV- cell lines, cetuximab increased p27Kip-1 and decreased cyclin B1 and cyclin D1 coinciding with in a G1 cell cycle arrest. Using both a cell line xenograft model and a direct-from-patient tumorgraft model of HPV+ HNCs, cetuximab resulted in significant tumor growth delay (median time to tumor quadrupling: 15 vs. 24 days, p=0.02; and 42 vs. 89 days, p=0.0001, respectively). Conclusions: Epidermal growth factor receptor inhibition by cetuximab appears to be effective in slowing proliferation and inducing apoptosis in HPV+ HNC. The proposed mechanism of action appears to be similar to that shown over a decade ago in HPV- HNC. These results suggest that cetuximab may play a role in the management of patients with HPV+ HNC. Citation Format: Grace C. Blitzer, Molly A. Smith, Alexandra D. Torres, Eric A. Armstrong, Paul M. Harari, Paul F. Lambert, Randall J. Kimple. Epidermal growth factor inhibition of HPV positive head and neck cancer cells and primary tumorgrafts results in significant growth inhibition mediated by apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5474. doi:10.1158/1538-7445.AM2013-5474

Background: An overexpression of the epidermal growth factor (EGF) receptor has been found in a wide variety of malignancies including squamous cell carcinomas of the head and neck. The overexpression of EGF receptor is of increasing interest because of a possible contribution to metastasis. Primary tumors and metastasis may differ in the expression of EGF receptor and provide a basis for metastasis. Material and Methods: This study examined the expression of the cell-surface receptor for EGF on 30 cervical lymph node metastases and on 30 primary squamous cell carcinomas of the oropharynx and the oral cavity. Immunoreactive receptor was localized using a mouse monoclonal antibody which reacts with sequences in the external domain of the receptor. Results: We saw a significant higher expression of EGF receptor in lymph node metastases than in primary tumors (p = 0.005). Examining the primary tumors, there was no correlation between the EGF receptor level and tumor localization or TNM-stage. On the other hand, we did find an interesting correlation between EGF receptor level and grading of the tumors, EGF receptor expression being significantly higher in G3 than in G1-G2 tumors (p = 0.001). Conclusions: EGF receptor system may play an important role in regulating the growth of head and neck cancer, and the process of metastasis and elevated EGF receptor level might characterize more metastatic tumors. The significant correlation between EGF receptor level and the histological grading suggests that EGF receptor expression may identify biologically more aggressive tumors.

To determine the frequency of expression of epidermal growth factor receptor (EGFR) by immunohistochemistry in head and neck squamous cell carcinoma (HNSCC). Cross-sectional study. Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2015 to March 2016. Fifty-two cases of head and neck squamous cell carcinoma diagnosed on H&E stain were included in the study. Patients' gender and age were noted. Immunohistochemistry for EGFR was applied and the results were recorded. The data were analyzed by using computer software program SPSS version 19. Descriptive statistics, frequencies and percentages were calculated. Out of the 52 patients of HNSCC, 37 patients were males and 15 females. The age of the patients was between 21 and 80 years with an average age of 58.58 ±12.63. Out of 52 cases, 45 cases (86.53%) were positive for EGFR while 7 cases (13.46%) were negative for EGFR. Significant statistical association was not seen between the tumour grade and EGFR expression (p=0.162). The high expression of EGFR in head and neck cancers among Pakistani patients suggests its value as a therapeutic target. EGFR inhibitors have become well-known part of HNSCC treatment; therefore, patients with EGFR positive HNSCC can be benefitted from the therapy.

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; Chemoradiation forms the major line of treatment in advanced head and neck squamous cell carcinoma, but the benefit of chemotherapeutic agents is at the expense of various toxicities. Curcumin has demonstrated promising results in in-vivo and in-vitro studies as a radiosensitiser. The objective of the study was to determine the role of curcumin as an adjuvant in patients undergoing chemo radiation for advanced head and neck cancers.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; Study involved 21 patients who underwent chemo radiotherapy for advanced head and neck cancers. They were randomized into two groups. Group A received 500 mg of curcumin while, Group B received placebo along with chemoradiation. The response was assessed using RECIST criteria at three months post treatment using contrast enhanced computerized tomography scan. &lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; Overall 58.3% patients had partial response and 41.7% patients had stable disease in group A. In group B, 33.3% patients had a partial response and 66.6% patient had a stable disease.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; Patients receiving curcumin along with chemoradiation had a marginal decrease in tumour volume and 58.3% patients had partial response and 41.7% had stable disease. A statistical significance could not be achieved due to lack of stage-match controls. Further studies are required to validate the role of curcumin as an adjuvant in the treatment of head and neck squamous cell carcinomas.&lt;/p&gt;

Somatic Mutation of Epidermal Growth Factor Receptor in a Small Subset of Cutaneous Squamous Cell Carcinoma

Overexpression of epidermal growth factor receptor (EGFR) is frequently observed in many solid tumor types, including head and neck squamous cell carcinomas (HNSCC). Recent laboratory experiments have demonstrated that high EGFR levels correlate with increased tumor resistance to radiation. This study investigated the relationship between EGFR expression levels and radiosensitivity in 5 HNSCC cell lines (HSC2, HSC3, HSC4, SCC25, and Ca9-22) and whether treatment with ZD1839 ('Iressa'), a selective EGFR-tyrosine kinase inhibitor (TKI), would improve tumor cell response to radiotherapy. ZD1839 suppressed the growth of HNSCC cell lines in a dose- and time-dependent manner. Radiosensitivity of these HNSCC cell lines, assessed by a clonogenic survival assay, differed greatly and the expression of EGFR varied. EGFR expression levels (EGFR numbers/cell) correlated with increased tumor resistance to radiation (f[x]= 4.54 X, R2 = 0.715; f[x]: EGFR numbers/cell, X: radiosensitivity; D10). Following exposure of the HNSCC cells to 1.0 microM ZD1839 and radiation (0-10 Gy), greater than additive growth inhibitory effects were observed. These results suggest that ZD1839 could enhance tumor radiosensitivity and inhibit tumor growth after radiation, indicating that this combination could have clinical potential in the treatment of patients with head and neck cancer.