Weg M. Ongkeko

Octamer 4 (Oct4) mediates chemotherapeutic drug resistance in liver cancer cells through a potential Oct4–AKT–ATP‐binding cassette G2 pathway

Chemoresistance presents a major obstacle to the efficacy of chemotherapeutic treatment of cancers. Using chemotherapeutic drugs to select drug‐resistant cancer cells in hepatocellular carcinoma (HCC) and several other cancer cell lines, we demonstrate that chemoresistant cells displayed cancer stem cell features, such as increased self‐renewal ability, cell motility, multiple drug resistance, and tumorigenicity. Octamer 4 (Oct4) messenger RNA (mRNA) levels were dramatically increased in chemoresistant cancer cells due to DNA demethylation regulation of Oct4. By functional study, Oct4 overexpression enhanced whereas Oct4 knockdown reduced liver cancer cell resistance to chemotherapeutic drugs in vitro and in xenograft tumors. It is known that the Oct4‐TCL1‐AKT pathway acts on embryonic stem cells and cancer stem cells in cell proliferation through inhibition of apoptosis. We further demonstrate that Oct4 overexpression induced activation of TCL1, AKT, and ABCG2 to mediate chemoresistance, which can be overcome by addition of the PI3K/AKT inhibitor; therefore, a direct pathway of Oct4‐TCL1‐AKT‐ABCG2 or a combination of Oct4‐TCL1‐AKT with the AKT‐ABCG2 pathway could be a potential new mechanism involved in liver cancer cell chemoresistance. Moreover, the clinical significance of the Oct4‐AKT‐ABCG2 pathway can be demonstrated in HCC patients, with a strong correlation of expression patterns in human HCC tumors. The role of the Oct4‐AKT‐ABCG2 axis in cancer cell chemoresistant machinery suggests that AKT pathway inhibition (PI3K inhibitors) not only inhibits cancer cell proliferation, but may also enhance chemosensitivity by target potential chemoresistant cells. Conclusion: Oct4, a transcriptional factor of pluripotent cells, can mediate chemoresistance through a potential Oct4‐AKT‐ABCG2 pathway. (HEPATOLOGY 2010;)

ABCG2: the key to chemoresistance in cancer stem cells?

Multi-drug chemoresistance remains one of the most common reasons for chemotherapy failure. The membrane transporter protein ABCG2/BCRP1 has been shown in vitro to effectively reduce the intracellular concentrations of several prominent anticancer chemotherapeutic agents such as mitoxantrone and doxorubicin. Intriguingly, cancer stem cells are known to be characterized by multi-drug chemoresistance. Taking into account that the ABCG2+ subset of tumor cells are often enriched with cells with cancer stem-like phenotypes, it has been proposed that ABCG2 activity underlies the ability of cancer cells to regenerate post-chemotherapy. Furthermore, we also review evidence suggesting that tyrosine kinase inhibitors, including imatinib and gefitinib, are both direct and downstream inactivators of ABCG2 and, therefore, serve as candidates to reverse cancer stem cell chemoresistance and potentially target cancer stem cells.

Expression of Protein Tyrosine Kinases in Head and Neck Squamous Cell Carcinomas

Protein tyrosine kinases (TKs) are overexpressed in many carcinomas and sarcomas. We studied the expression of the following TKs in head and neck squamous cell carcinoma (HNSCC): platelet-derived growth factor receptor (PDGFR), c-kit, epidermal growth factor receptor (EGFR), and a serine-threonine kinase, Akt. Formalin-fixed, paraffin-embedded tumor blocks from 44 consecutive patients with primary HNSCC and 5 specimens of benign pharyngeal and laryngeal mucosa were retrieved for immunohistochemical analysis. Of the specimens, 38 had enough material to stain for all 4 antibodies. The study included 21 pharyngeal (base of tongue, 14; tonsil, 6; soft palate, 1), 16 laryngeal, and 1 floor of the mouth carcinoma. All 4 kinases in the tumor samples were expressed highly (PDGFR, 95%-100%; EGFR, 38%-43%; c-kit, 50%-86%; p-Akt, 57%-81%), with EGFR, c-kit, and p-Akt significantly higher than in benign samples. None of the kinase expressions correlated with disease-free survival. The expression of the kinases raises the possibility of treatment of HNSCC by tyrosine and serine-threonine kinase inhibitors.

EGFR regulates the side population in head and neck squamous cell carcinoma.

Objective: To identify the presence of side population (SP) cells in established head and neck squamous carcinoma cell (HNSCC) lines and to determine the role of EGFR in the regulation of the side population of these cells.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

OBJECTIVES Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. MATERIALS AND METHODS HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. RESULTS E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. CONCLUSION E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed.

EGFR Kinase Promotes Acquisition of Stem Cell-Like Properties: A Potential Therapeutic Target in Head and Neck Squamous Cell Carcinoma Stem Cells

Members of the EGFR/ErbB family of tyrosine kinases are found to be highly expressed and deregulated in many cancers, including head and neck squamous cell carcinoma (HNSCC). The ErbB family, including EGFR, has been demonstrated to play key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSCs) which are believed to be responsible for tumor initiation and maintenance. In this study, we investigated the possible role of EGFR as a regulator of “stemness” in HNSCC cells. Activation of EGFR by the addition of EGF ligand or ectopic expression of EGFR in two established HNSCC cell lines (UMSCC-22B and HN-1) resulted in the induction of CD44, BMI-1, Oct-4, NANOG, CXCR4, and SDF-1. Activation of EGFR also resulted in increased tumorsphere formation, a characteristic ability of cancer stem cells. Conversely, treatment with the EGFR kinase inhibitor, Gefinitib (Iressa), resulted in decreased expression of the aforementioned genes, and loss of tumorsphere-forming ability. Similar trends were observed in a 99.9% CD44 positive stem cell culture derived from a fresh HNSCC tumor, confirming our findings for the cell lines. Additionally, we found that these putative cancer stem cells, when treated with Gefitinib, possessed a lower capacity to invade and became more sensitive to cisplatin-induced death in vitro. These results suggest that EGFR plays critical roles in the survival, maintenance, and function of cancer stem cells. Drugs that target EGFR, perhaps administered in combination with conventional chemotherapy, might be an effective treatment for HNSCC.

ErbB and Nrg: potential molecular targets for vestibular schwannoma pharmacotherapy.

Objective: Identify molecular targets for development of tumor-specific pharmacotherapeutics aimed at treating vestibular schwannomas (VSs). Activated epidermal growth factor receptor B (ErbB) 2 and ErbB3 are abundantly expressed in VS. ErbB2 signaling is essential for Schwann cell differentiation, survival, and proliferation. VS arise after loss of functional merlin, a putative tumor suppressor. Merlin internalizes ErbB2 receptors in rodent Schwann cells.Unregulated ErbB signaling may contribute to VS tumorigenesis. Study Design: Molecular analyses, retrospective clinical correlation. Setting: Tertiary referral center. Patients: Thirty-eight specimens from patients operated for sporadic (n = 21) and neurofibromatosis (NF) 2-related (n = 17) VS. Intervention(s): VS analyses via real-time polymerase chain reaction, immunohistochemistry, and correlation with patient clinical data. Main Outcome Measure(s): ErbB signaling molecule expression, tumor size, age, and NF2 status. Results: VS upregulated epidermal growth factor (EGF) receptor in 68% (62% sporadic and 75% NF2-associated VS) and ErbB2 in 84% (76% sporadic and 94% NF2-related VS). ErbB3 was upregulated in 34%, and ErbB4 is downregulated in NF2-related VS. Of EGF receptor (EGFR) ligands, EGF was upregulated in all NF2-related VS, but none of the sporadic VS (p < 0.01), and transforming growth factor &agr; and &bgr;-cellulin showed upregulation in 67% of NF2-related VS but not sporadic VS (p = 0.02 and p = 0.01, respectively). Neuregulin (Nrg) was upregulated in 86% of sporadic VS versus 19% of NF2-related VS (p < 0.01). EGFR expression levels correlated directly with VS tumor size and inversely with patient age, whereas Nrg expression correlated directly with age (p = 0.0005). EGF expression predicts NF2 status, whereas Nrg predicts non-NF2 status (p < 0.01). Conclusion: These findings implicate the ErbB pathway in VS growth and as potential molecular targets for VS pharmacotherapy.

Salinomycin induces cell death and differentiation in head and neck squamous cell carcinoma stem cells despite activation of epithelial-mesenchymal transition and Akt

BackgroundCancer stem cells (CSC) are believed to play a crucial role in cancer recurrence due to their resistance to conventional chemotherapy and capacity for self-renewal. Recent studies have reported that salinomycin, a livestock antibiotic, selectively targets breast cancer stem cells 100-fold more effectively than paclitaxel. In our study we sought to determine the effects of salinomycin on head and neck squamous cell carcinoma (HNSCC) stem cells.MethodsMTS and TUNEL assays were used to study cell proliferation and apoptosis as a function of salinomycin exposure in JLO-1, a putative HNSCC stem cell culture. MTS and trypan blue dye exclusion assays were performed to investigate potential drug interactions between salinomycin and cisplatin or paclitaxel. Stem cell-like phenotype was measured by mRNA expression of stem cell markers, sphere-forming capacity, and matrigel invasion assays. Immunoblotting was also used to determine expression of epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation. Arrays by Illumina, Inc. were used to profile microRNA expression as a function of salinomycin dose.ResultsIn putative HNSCC stem cells, salinomycin was found to significantly inhibit cell viability, induce a 71.5% increase in levels of apoptosis, elevate the Bax/Bcl-2 ratio, and work synergistically with cisplatin and paclitaxel in inducing cell death. It was observed that salinomycin significantly inhibited sphere forming-capability and repressed the expression of CD44 and BMI-1 by 3.2-fold and 6.2-fold, respectively. Furthermore, salinomycin reduced invasion of HNSCC stem cells by 2.1 fold. Contrary to expectations, salinomycin induced the expression of EMT markers Snail, vimentin, and Zeb-1, decreased expression of E-cadherin, and also induced phosphorylation of Akt and its downstream targets GSK3-β and mTOR.ConclusionsThese results demonstrate that in HNSCC cancer stem cells, salinomycin can cause cell death and decrease stem cell properties despite activation of both EMT and Akt.

Cigarette Smoke Promotes Drug Resistance and Expansion of Cancer Stem Cell-Like Side Population

It is well known that many patients continue to smoke cigarettes after being diagnosed with cancer. Although smoking cessation has typically been presumed to possess little therapeutic value for cancer, a growing body of evidence suggests that continued smoking is associated with reduced efficacy of treatment and a higher incidence of recurrence. We therefore investigated the effect of cigarette smoke condensate (CSC) on drug resistance in the lung cancer and head and neck cancer cell lines A549 and UMSCC-10B, respectively. Our results showed that CSC significantly increased the cellular efflux of doxorubicin and mitoxantrone. This was accompanied by membrane localization and increased expression of the multi-drug transporter ABCG2. The induced efflux of doxorubicin was reversed upon addition of the specific ABCG2 inhibitor Fumitremorgin C, confirming the role of ABCG2. Treatment with CSC increased the concentration of phosphorylated Akt, while addition of the PI3K inhibitor LY294002 blocked doxorubicin extrusion, suggesting that Akt activation is required for CSC-induced drug efflux. In addition, CSC was found to promote resistance to doxorubicin as determined by MTS assays. This CSC-induced doxurbicin-resistance was mitigated by mecamylamine, a nicotinic acetylcholine receptor inhibitor, suggesting that nicotine is at least partially responsible for the effect of CSC. Lastly, CSC increased the size of the side population (SP), which has been linked to a cancer stem cell-like phenotype. In summary, CSC promotes chemoresistance via Akt-mediated regulation of ABCG2 activity, and may also increase the proportion of cancer stem-like cells, contributing to tumor resilience. These findings underscore the importance of smoking cessation following a diagnosis of cancer, and elucidate the mechanisms of continued smoking that may be detrimental to treatment.

STI‐571 (Gleevec) Potentiates the Effect of Cisplatin in Inhibiting the Proliferation of Head and Neck Squamous Cell Carcinoma In Vitro

Objective: The objective of this study was to determine whether STI‐571 (Gleevec; imatinib mesylate) could sensitize established head and neck squamous cell carcinoma (HNSCC) cell lines to the effects of cisplatin.