Jay Patrick Lopez

University of California, San Diego, San Diego, CA, United States

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Publications (9)20.06 Total impact

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    ABSTRACT: 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.
    PLoS ONE 01/2012; 7(2):e32459. · 3.53 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 01/2012; 7(11):e47919. · 3.53 Impact Factor
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    ABSTRACT: 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. 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. 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. Gleevec's effect on the cell cycle profile also was investigated via flow cytometry analysis. 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-β 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. 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.
    Otology & neurotology: official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology 01/2011; 32(1):163-70. · 1.44 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Archives of otolaryngology--head & neck surgery 08/2009; 135(7):662-9. · 1.92 Impact Factor
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    ABSTRACT: 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. Flow cytometric analysis was used to determine the levels of drug or dye extrusion from the cells. 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. Our findings demonstrate that imatinib confers greater doxorubicin retention, presumably via inhibition of Akt, which regulates ABCG2 function.
    Archives of otolaryngology--head & neck surgery 10/2008; 134(9):979-84. · 1.92 Impact Factor
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    ABSTRACT: To determine whether inactivation of epidermal growth factor receptor (EGFR) kinase activity will sensitize thyroid cancer cell lines to ionizing radiation-induced death. Established human thyroid cancer cells lines were studied. Colony formation assay was used to determine the effect of Gefitinib, a small molecule inhibitor of EGFR, on anaplastic (ARO) and follicular (WRO) thyroid cancer cell lines. In addition, colony formation assay was used to determine the effect of ionizing radiation in the presence or absence of Gefitinib. EGFR protein expression on the cell lines and inactivation of EGFR kinase by Gefitinib was analyzed by Western blot. Immunohistochemistry was performed on archived thyroid cancer tissue to demonstrate expression of EGFR. Incubation with Gefitinib caused decreased phosphorylation of EGFR protein in established thyroid cancer cell lines as measured by Western blot. Inhibition of EGFR kinase activity by Gefitinib resulted in a dose-dependent decrease in colony formation in both ARO and WRO thyroid cancer cell lines. Addition of Gefitinib in combination with ionizing radiation reduced cell proliferation in ARO (P = .0084) and WRO (P = .0252) as measured by colony formation assay. Inactivation of the EGFR kinase by Gefitinib potentiates the ionizing radiation-induced inhibition of cell proliferation in thyroid cancer cell lines. Use of this combination treatment of Gefitinib and ionizing radiation may be a promising therapy for anaplastic thyroid and metastatic follicular thyroid cancer and should be extended into animal models.
    The Laryngoscope 06/2008; 118(8):1372-6. · 1.98 Impact Factor
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    ABSTRACT: To investigate the regulation of the breast cancer resistance protein ABCG2/BRCP1 drug transporter by epidermal growth factor receptor (EGFR) kinase activity, and to determine whether gefitinib, an EGFR small molecule inhibitor, will modulate the effects of doxorubicin hydrochloride by inhibiting its extrusion from thyroid cancer cells. Extrusion assays using flow cytometry analysis were used to determine the ability of thyroid cancer cells to extrude the chemotherapy drug, doxorubicin, via the ABCG2 drug transporter in the presence or absence of gefitinib. Immunofluorescence was employed to determine the cellular expression of ABCG2. The ABCG2 expression in ARO and WRO cell lines was analyzed by Western blot analysis. Inactivation of EGFR kinase by gefitinib was analyzed by Western blot analysis and immunofluorescence. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay was performed to demonstrate ABCG2-mediated apoptosis in the presence of doxorubicin. Colony formation assays were performed to determine the effect of gefitinib on thyroid cancer cell survival in response to gefitinib, doxorubicin, or the combination of both drugs. Inhibition of EGFR kinase activity by gefitinib causes the translocation of the ABCG2 drug transporter away from the plasma membrane, resulting in a concomitant decrease in doxorubicin extrusion in thyroid cancer cell lines. Both ARO and WRO demonstrated differential ABCG2 expression, whereas both were sensitized to doxorubicin-induced apoptosis on ABCG2 knockdown with short interfering RNA. The addition of gefitinib increases doxorubicin-induced cell death in thyroid cancer cells as measured by colony formation assay. Epidermal growth factor receptor regulates the function of the drug transporter ABCG2/BCRP1 and correlates with ABCG2 protein expression levels. Inactivation of the EGFR kinase by gefitinib potentiates the cytotoxic effect of doxorubicin in thyroid cancer, most likely by decreasing the ability of the cell to extrude doxorubicin. The expression of ABCG2 may explain in part the ineffectiveness of doxorubicin as a single modality treatment for anaplastic thyroid cancer or for treatment of metastatic follicular thyroid cancer. Use of this combination treatment of gefitinib and doxorubicin may be a promising therapy for anaplastic thyroid and metastatic follicular thyroid cancer and needs to be investigated further.
    Archives of Otolaryngology - Head and Neck Surgery 11/2007; 133(10):1022-7. · 1.78 Impact Factor
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    ABSTRACT: 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. Western blot analysis and immunofluorescence were used to examine the expression of the tyrosine kinases that are known targets of Gleevec, including c-kit, c-Abl, and platelet-derived growth factor receptor, on the cell lines, and immunohistochemistry was performed to determine the expression of these kinases in human HNSCC tissue. Once these targets were confirmed, clonogenic cell survival assays were performed to determine the effect STI-571 had on growth and proliferation when used in combination with cisplatin compared with STI-571 alone or cisplatin alone. Cells were incubated with a range of doses of STI-571 24 hours before the addition of cisplatin. Flow cytometry analysis was performed to determine cell-cycle distribution and to measure apoptosis caused by the various treatments. An annexin V assay was also used to further measure apoptosis. Our results indicate that STI-571 potentiates the effect of cisplatin and leads to a significant decrease in cell proliferation and colony formation compared with cisplatin alone in a dose-dependent fashion. Surprisingly, there was a slight decrease in the level of apoptosis when Gleevec was used in combination with cisplatin compared with cisplatin alone. Gleevec alone resulted in a slight increase in G1 phase of the cell cycle, whereas cisplatin alone resulted in a G2 arrest. The addition of Gleevec to cisplatin resulted in an enhanced S/G2 phase accumulation. Although we did not demonstrate an increase in cisplatin-induced cell death, we postulate that the increased S/G2 arrest resulting from the DNA damage in the presence of Gleevec results in decreased proliferation of HNSCC, resulting in a net decrease in colony formation. The small molecule inhibitor Gleevec, which targets specific tyrosine kinases that are expressed in HNSCC, can significantly potentiate the antiproliferative effects of cisplatin. Because Gleevec alone has minimal side effects, treatment with the combination treatment of cisplatin and Gleevec may result in increased efficacy of cisplatin in treating this cancer. Additional studies are warranted, keeping in mind that drug combinations may result in unexpected toxicities that are not frequently seen with either drug alone.
    The Laryngoscope 09/2006; 116(8):1409-16. · 1.98 Impact Factor
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    ABSTRACT: 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. SP cells were identified using flow cytometry analysis by the ability of these cells to extrude the Hoechst 33342 dye via the drug transporter BCRP1/ABCG2. Effect of EGFR on the side population was determined also by difference in Hoechst extrusion and by immunofluorescence. Immunohistochemical staining was performed to show the presence of the BCRP1/ABCG2 transporter and the phosphorylated form of EGFR in HNSCC tissue. SP cells are present in HNSCC cell lines. With the Hoechst 33342 extrusion assay, SP cells were found to comprise an average of 0.69% of the UMSCC10B cells and 0.91% of HN12 cells. Addition of the EGF ligand increased the SP population while inactivation of the EGFR kinase by Iressa significantly decreased SP. In established head and neck squamous cell carcinoma cell lines, SP cells were found using methods that determine expression and function of the drug transporter BCRP1/ABCG2. Activation of EGFR, a gene implicated in tumorigenesis in HNSCC leads to increased SP, and conversely, inhibition of EGFR leads to decrease in SP. This finding could help explain the role of EGFR in regulating cancer stem cells and thus tumorigenesis in HNSCC.
    The Laryngoscope 04/2006; 116(3):401-6. · 1.98 Impact Factor

Publication Stats

122 Citations
20.06 Total Impact Points

Institutions

  • 2006–2012
    • University of California, San Diego
      • • Division of Otolaryngology-Head and Neck Surgery
      • • Department of Surgery
      San Diego, CA, United States
    • VA San Diego Healthcare System
      San Diego, California, United States
  • 2007
    • CSU Mentor
      Long Beach, California, United States