Jay Patrick Lopez

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;)

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.

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.

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.

Imatinib-Mediated Inactivation of Akt Regulates ABCG2 Function in Head and Neck Squamous Cell Carcinoma

OBJECTIVE To investigate whether the mechanism for the reversal of ABCG2 (also known as ABCP, MXR, and BCRP)-mediated drug resistance by imatinib mesylate (Gleevec, STI571; Novartis Pharmaceuticals Corp, East Hanover, New Jersey) is caused by the downregulation of Akt kinase. The adenosine triphosphatase-binding cassette protein ABCG2 has been suggested to be involved in the resistance against various anticancer drugs. Recent studies show that imatinib reverses ABCG2-mediated drug resistance to topotecan hydrochloride and SN-38. In addition, we have previously reported that imatinib downregulates Akt kinase activity, which is elevated in head and neck squamous cell carcinoma. DESIGN Flow cytometric analysis was used to determine the levels of drug or dye extrusion from the cells. RESULTS We used Akt kinase inhibitors, transfection with short interfering RNA (siRNA) Akt, and the tyrosine kinase inhibitor imatinib to show that these treatments decreased the side population by 50% to 70% in Hoechst 33342 extrusion studies. Doxorubicin hydrochloride extrusion experiments also demonstrated 20% to 26% decrease in doxorubicin efflux on cells treated with imatinib, 1L6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, and transfection with siRNA Akt. With Western blot and immunofluorescence experiments, our data suggest that ABCG2 translocation is the mechanism by which imatinib and Akt regulate drug resistance. Clonogenic survival assays performed with imatinib-treated cells resulted in a dose-dependent decrease in cell survival compared with the control population. CONCLUSION Our findings demonstrate that imatinib confers greater doxorubicin retention, presumably via inhibition of Akt, which regulates ABCG2 function.

Gefitinib (Iressa) Potentiates the Effect of Ionizing Radiation in Thyroid Cancer Cell Lines

Objectives/Hypothesis: To determine whether inactivation of epidermal growth factor receptor (EGFR) kinase activity will sensitize thyroid cancer cell lines to ionizing radiation‐induced death.

Potential role of imatinib mesylate (Gleevec, STI-571) in the treatment of vestibular schwannoma.

Hypothesis: To determine the expression of the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and c-Kit in vestibular schwannoma (VS) and to determine the potential role of imatinib mesylate (Gleevec) in regulating the growth and cell death of this tumor. Background: Protein tyrosine kinases are transmembrane tyrosine kinase receptors that transduce signals from inside and outside the cell and function as relay points for signaling pathways. They have a key role in numerous processes that affect cell proliferation, tumorigenesis, cancer invasion, metastasis, and modulation of apoptosis. A few of these kinases have been demonstrated to be overexpressed and dysregulated in many carcinomas, sarcomas, and benign tumors. Methods: Immunohistochemical staining was used to investigate the expression of PDGFR and c-Kit in archived acoustic neuroma tissue. Clinical data including size of tumors, age, sex, and symptoms were correlated with kinase expression, whereas Western blot analysis and immunofluorescence were performed to demonstrate the expression and localization of PDGFR and c-Kit in HEI193, an immortalized VS cell line. Clonogenic survival assays were performed to assess proliferation inhibition by Gleevec. Gleevecs effect on the cell cycle profile also was investigated via flow cytometry analysis. Results: Expression of PDGFR in the formalin-fixed VS tumor tissue was observed in 23 (67.5%) of the 34 samples. C-kit was expressed in 18 (52.9%) of the 34 samples. Western blot analysis demonstrates positive expression of c-Kit and PDGFR-Q in HEI193 and a primary VS culture. Western blot analysis showed downregulation of phospho-c-kit and phospho-PDGFR-Q with 5 and 10 uM Gleevec. Immunofluorescent staining of this cell line also reveals that PDGFR-&bgr; is localized primarily in the cytoplasm, whereas c-Kit is both nuclear and cytoplasmic. Cell cycle analysis of HEI193 96 hours after incubation with Gleevec indicates a dose-dependent increase in G1 from 61.6% to 70.7% and 74% at 5 and 10 uM of Gleevec, respectively. Colony formation assays demonstrate dose-dependent growth inhibition by Gleevec, in the HEI193 cell line as well as in a VS cell culture derived from a fresh tumor. Conclusion: The expression of PDGFR-Q and c-Kit in VS tissue may indicate novel molecular targets involved in the development of this tumor. Direct inhibition of these molecules by Gleevec may have relevant therapeutic applications.

p73 expression and function in vestibular schwannoma.

OBJECTIVES To determine the expression of the p53 family member p73 in vestibular schwannoma (VS) and to determine the potential role of this tumor suppressor in regulating the proliferation of HEI193, a human papillomavirus E6-E7 immortalized VS cell line. METHODS Immunohistochemical staining was used to investigate the expression of p73 in 34 cases of archived VS tissue, while Western blot analysis and immunofluorescence were performed to demonstrate the expression and localization of p73 in HEI193. After transfection of a full-length p73 plasmid (TAp73alpha), flow cytometry analysis was performed to determine the effect of p73 expression on cell cycle distribution, while annexin V-FITC (fluorescein isothiocyanate) analysis and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay were used to measure apoptosis. The effect of p73 expression on ionizing radiation-induced cell death was also investigated with annexin V staining, TUNEL assay, and flow cytometry analysis. RESULTS Of the 34 vestibular schwannoma tissues examined, p73 was expressed in 14 (41%) but was not expressed in HEI193. Transfection of p73 alone resulted in increased apoptosis and necrosis, and G(1) accumulation with concomitant induction of p21. The presence of p73 also significantly increased early apoptosis (P = .046), late apoptosis (P < .001), and necrosis (P = .009) on exposure of the HEI193 cells to ionizing radiation. CONCLUSION Forced expression of p73, perhaps by gene therapy, to induce apoptosis directly or to sensitize VS tumors to ionizing radiation may have relevant therapeutic applications.