Refractory lympho-epithelial carcinoma of the nasopharynx: a case report illustrating a protracted clinical course

Nasopharyngeal carcinoma is an uncommon cancer in North America. Its clinical course is typified by locally advanced disease at diagnosis and has a high propensity for both regional and distant spread. It is, therefore, typically treated with a combination of radiation and chemotherapy. This report describes our 10-year clinical and radiological findings in a 48-year-old Vietnamese male patient with locally-advanced T4N1M0 lympho-epithelial carcinoma of the nasopharynx. Despite a long remission period after his initial course of aggressive chemoradiation, his tumor recurred locally after 4 years. Thereafter, throughout a period of over 10 years, he has been treated with multiple courses of re-irradiation and three different trials of chemotherapy. He was ultimately provided with over 30 months of progression-free tumor control with the epidermal growth factor receptor (EGFR)-inhibitor cetuximab. This case illustrates the commonly protracted course of this disease and its responsiveness to multiple treatment modalities

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression

MARCKS phosphorylation is modulated by a peptide mimetic of MARCKS effector domain leading to increased radiation sensitivity in lung cancer cell lines

Lung cancer is the leading cause of cancer-associated mortality in the United States. Kinase hyperactivation is a known mechanism of tumorigenesis. The phosphorylation status of the plasma membrane-associated protein myristoylated alanine rich C-kinase substrate (MARCKS) effector domain (ED) was previously established as being important in the sensitivity of lung cancer to radiation. Specifically, when MARCKS ED was in a non-phosphorylated state, lung cancer cells were more susceptible to ionizing radiation and experienced prolonged double-strand DNA breaks. Additional studies demonstrated that the phosphorylation status of MARCKS ED is important for gene expression and in vivo tumor growth. The present study used a peptide mimetic of MARCKS ED as a therapeutic intervention to modulate MARCKS phosphorylation. Culturing A549, H1792 and H1975 lung cancer cell lines with the MARCKS ED peptide led to reduced levels of phosphorylated MARCKS and phosphorylated Akt serine/threonine kinase 1. Further investigation demonstrated that the peptide therapy was able to reduce lung cancer cell proliferation and increase radiation sensitivity. In addition, the MARCKS peptide therapy was able to prolong double-strand DNA breaks following ionizing radiation exposure. The results of the present study demonstrate that a peptide mimetic of MARCKS ED is able to modulate MARCKS phosphorylation, leading to an increase in sensitivity to radiation. Keywords: lung cancer, myristoylated alanine rich C-kinase substrate, radiation sensitivity, effector domain, peptide mimeti

Kinomic exploration of temozolomide and radiation resistance in Glioblastoma multiforme xenolines

Glioblastoma multiforme (GBM) represents the most common and deadly primary brain malignancy, particularly due to temozolomide (TMZ) and radiation (RT) resistance. To better understand resistance mechanisms, we examined global kinase activity (kinomic profiling) in both treatment sensitive and resistant human GBM patient-derived xenografts (PDX or “xenolines”)

Kinomic Profiling of Electromagnetic Navigational Bronchoscopy Specimens: A New Approach for Personalized Medicine

Purpose Researchers are currently seeking relevant lung cancer biomarkers in order to make informed decisions regarding therapeutic selection for patients in so-called “precision medicine.” However, there are challenges to obtaining adequate lung cancer tissue for molecular analyses. Furthermore, current molecular testing of tumors at the genomic or transcriptomic level are very indirect measures of biological response to a drug, particularly for small molecule inhibitors that target kinases. Kinase activity profiling is therefore theorized to be more reflective of in vivo biology than many current molecular analysis techniques. As a result, this study seeks to prove the feasibility of combining a novel minimally invasive biopsy technique that expands the number of lesions amenable for biopsy with subsequent ex vivo kinase activity analysis. Methods Eight patients with lung lesions of varying location and size were biopsied using the novel electromagnetic navigational bronchoscopy (ENB) technique. Basal kinase activity (kinomic) profiles and ex vivo interrogation of samples in combination with tyrosine kinase inhibitors erlotinib, crizotinib, and lapatinib were performed by PamStation 12 microarray analysis. Results Kinomic profiling qualitatively identified patient specific kinase activity profiles as well as patient and drug specific changes in kinase activity profiles following exposure to inhibitor. Thus, the study has verified the feasibility of ENB as a method for obtaining tissue in adequate quantities for kinomic analysis and has demonstrated the possible use of this tissue acquisition and analysis technique as a method for future study of lung cancer biomarkers. Conclusions We demonstrate the feasibility of using ENB-derived biopsies to perform kinase activity assessment in lung cancer patients

Targeting the effector domain of the myristoylated alanine rich C-kinase substrate enhances lung cancer radiation sensitivity

Lung cancer is the leading cause of cancer related deaths. Common molecular drivers of lung cancer are mutations in receptor tyrosine kinases (RTKs) leading to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pro-growth, pro-survival signaling pathways. Myristoylated alanine rich C-kinase substrate (MARCKS) is a protein that has the ability to mitigate this signaling cascade by sequestering the target of PI3K, phosphatidylinositol (4,5)-bisphosphate (PIP2). As such, MARCKS has been implicated as a tumor suppressor, though there is some evidence that MARCKS may be tumor promoting in certain cancer types. Since the MARCKS function depends on its phosphorylation status, which impacts its subcellular location, MARCKS role in cancer may depend highly on the signaling context. Currently, the importance of MARCKS in lung cancer biology is limited. Thus, we investigated MARCKS in both clinical specimens and cell culture models. Immunohistochemistry scoring of MARCKS protein expression in a diverse lung tumor tissue array revealed that the majority of squamous cell carcinomas stained positive for MARCKS while other histologies, such as adenocarcinomas, had lower levels. To study the importance of MARCKS in lung cancer biology, we used inducible overexpression of wild-type (WT) and non-phosphorylatable (NP)-MARCKS in A549 lung cancer cells that had a low level of endogenous MARCKS. We found that NP-MARCKS expression, but not WT-MARCKS, enhanced the radiosensitivity of A549 cells in part by inhibiting DNA repair as evidenced by prolonged radiation-induced DNA double strand breaks. We confirmed the importance of MARCKS phosphorylation status by treating several lung cancer cell lines with a peptide mimetic of the phosphorylation domain, the effector domain (ED), which effectively attenuated cell growth as measured by cell index. Thus, the MARCKS ED appears to be an important target for lung cancer therapeutic development

MARCKS regulates growth and radiation sensitivity and is a novel prognostic factor for glioma

Purpose This study assessed whether Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) can regulate glioblastoma (GBM) growth, radiation sensitivity and clinical outcome. Experimental Design MARCKS protein levels were analyzed in five GBM explant cell lines and eight patient-derived xenograft tumors by immunoblot, and these levels were correlated to proliferation rates and intracranial growth rates, respectively. Manipulation of MARCKS protein levels was assessed by lentiviral-mediated shRNA knockdown in the U251 cell line and MARCKS over-expression in the U87 cell line. The effect of manipulation of MARCKS on proliferation, radiation sensitivity and senescence was assessed. MARCKS gene expression was correlated with survival outcomes in the Repository of Molecular Brain Neoplasia Data (REMBRANDT) Database and The Cancer Genome Atlas (TCGA). Results MARCKS protein expression was inversely correlated with GBM proliferation and intracranial xenograft growth rates. Genetic silencing of MARCKS promoted GBM proliferation and radiation resistance, while MARCKS overexpression greatly reduced GBM growth potential and induced senescence. We found MARCKS gene expression to be directly correlated with survival in both the REMBRANDT and TCGA databases. Specifically, patients with high MARCKS expressing tumors of the Proneural molecular subtype had significantly increased survival rates. This effect was most pronounced in tumors with unmethylated O6-methylguanine DNA methyltransferase (MGMT) promoters, a traditionally poor prognostic factor. Conclusions MARCKS levels impact GBM growth and radiation sensitivity. High MARCKS expressing GBM tumors are associated with improved survival, particularly with unmethylated MGMT promoters. These findings suggest the use of MARCKS as a novel target and biomarker for prognosis in the Proneural subtype of GBM

Spleen tyrosine kinase-mediated autophagy is required for epithelial-mesenchymal plasticity and metastasis in breast cancer

The ability of breast cancer cells to transiently transition between epithelial and mesenchymal states contributes to their metastatic potential. Therefore, driving tumor cells into a stable mesenchymal state, as opposed to complete tumor cell eradication, presents an opportunity to pharmacologically limit disease progression by promoting an asymptomatic state of dormancy. Here we compare a reversible model of epithelial-mesenchymal transition (EMT) induced by TGF-β to a stable mesenchymal phenotype induced by chronic exposure to the ErbB kinase inhibitor lapatinib. Only cells capable of returning to an epithelial phenotype resulted in skeletal metastasis. Gene expression analyses of the two mesenchymal states indicated similar transition expression profiles. A potently downregulated gene in both datasets was spleen tyrosine kinase (SYK). In contrast to this similar diminution in mRNA, kinome analyses using a peptide array and DNA-conjugated peptide substrates showed a robust increase in SYK activity upon TGF-β-induced EMT only. SYK was present in cytoplasmic RNA processing depots known as P-bodies formed during the onset of EMT, and SYK activity was required for autophagy-mediated clearance of P-bodies during mesenchymal-epithelial transition (MET). Genetic knockout of autophagy related 7 (ATG7) or pharmacological inhibition of SYK activity with fostamatib, a clinically approved inhibitor of SYK, prevented P-body clearance and MET, inhibiting metastatic tumor outgrowth. Overall, the current study suggests assessment of SYK activity as a biomarker for metastatic disease and the use of fostamatinib as a means to stabilize the latency of disseminated tumor cells

Expression Signature of IFN/STAT1 Signaling Genes Predicts Poor Survival Outcome in Glioblastoma Multiforme in a Subtype-Specific Manner

Previous reports have implicated an induction of genes in IFN/STAT1 (Interferon/STAT1) signaling in radiation resistant and prosurvival tumor phenotypes in a number of cancer cell lines, and we have hypothesized that upregulation of these genes may be predictive of poor survival outcome and/or treatment response in Glioblastoma Multiforme (GBM) patients. We have developed a list of 8 genes related to IFN/STAT1 that we hypothesize to be predictive of poor survival in GBM patients. Our working hypothesis that over-expression of this gene signature predicts poor survival outcome in GBM patients was confirmed, and in addition, it was demonstrated that the survival model was highly subtype-dependent, with strong dependence in the Proneural subtype and no detected dependence in the Classical and Mesenchymal subtypes. We developed a specific multi-gene survival model for the Proneural subtype in the TCGA (the Cancer Genome Atlas) discovery set which we have validated in the TCGA validation set. In addition, we have performed network analysis in the form of Bayesian Network discovery and Ingenuity Pathway Analysis to further dissect the underlying biology of this gene signature in the etiology of GBM. We theorize that the strong predictive value of the IFN/STAT1 gene signature in the Proneural subtype may be due to chemotherapy and/or radiation resistance induced through prolonged constitutive signaling of these genes during the course of the illness. The results of this study have implications both for better prediction models for survival outcome in GBM and for improved understanding of the underlying subtype-specific molecular mechanisms for GBM tumor progression and treatment response