Eric L. Abhold

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.

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.

Parathyroid hormone related-protein promotes epithelial-to-mesenchymal transition in prostate cancer.

Parathyroid hormone-related protein (PTHrP) possesses a variety of physiological and developmental functions and is also known to facilitate the progression of many common cancers, notably their skeletal invasion, primarily by increasing bone resorption. The purpose of this study was to determine whether PTHrP could promote epithelial-to-mesenchymal transition (EMT), a process implicated in cancer stem cells that is critically involved in cancer invasion and metastasis. EMT was observed in DU 145 prostate cancer cells stably overexpressing either the 1-141 or 1-173 isoform of PTHrP, where there was upregulation of Snail and vimentin and downregulation of E-cadherin relative to parental DU 145. By contrast, the opposite effect was observed in PC-3 prostate cancer cells where high levels of PTHrP were knocked-down via lentiviral siRNA transduction. Increased tumor progression was observed in PTHrP-overexpressing DU 145 cells while decreased progression was observed in PTHrP-knockdown PC-3 cells. PTHrP-overexpressing DU 145 formed larger tumors when implanted orthoptopically into nude mice and in one case resulted in spinal metastasis, an effect not observed among mice injected with parental DU 145 cells. PTHrP-overexpressing DU 145 cells also caused significant bone destruction when injected into the tibiae of nude mice, while parental DU 145 cells caused little to no destruction of bone. Together, these results suggest that PTHrP may work through EMT to promote an aggressive and metastatic phenotype in prostate cancer, a pathway of importance in cancer stem cells. Thus, continued efforts to elucidate the pathways involved in PTHrP-induced EMT as well as to develop ways to specifically target PTHrP signaling may lead to more effective therapies for prostate cancer.

Recombinant human erythropoietin promotes the acquisition of a malignant phenotype in head and neck squamous cell carcinoma cell lines in vitro

BackgroundRecent studies indicate an increase in tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC) of cancer patients taking recombinant human erythropoietin (rhEpo) for anemia. This study was undertaken to investigate the potential role of rhEpo in invasion, proliferation, and cisplatin-induced cell death in HNSCC cell lines.MethodsThe following experiments were performed with two HNSCC cell lines, UMSCC-10B and UMSCC-22B. Presence of EpoR in both cell lines was determined by western blot and quantitative PCR. Colorimetric MTS assays and clonogenic assays were used to study the effect of rhEpo at pharmacologically relevant doses on cell proliferation. Matrigel invasion assays were performed in order to determine effects of exogenous rhEpo on invasive abilities. Clonogenic assays were also used to study potential cytoprotective effects of rhEpo against cisplatin. Immunoblotting was done to analyze the effect of rhEpo on Akt phosphorylation. Finally, MTS and TUNEL assays were performed to test our hypothesis that Akt activation by PI3K was involved in rhEpo-mediated cisplatin resistance.ResultsHNSCC cell lines were shown to express Epo receptor (EpoR). RhEpo increased invasion 1.8-fold in UMSCC-10B and 2.6-fold in UMSCC-22B compared to control. RhEpo at 10 U/ml increased cell proliferation by 41% and 53% in UMSCC-10B and UMSCC-22B, respectively, and colony formation by 1.5-fold and 1.8-fold. UMSCC-10B treated with cisplatin and exposed to rhEpo at 1 and 10 U/ml resulted in a 1.7-fold and 3.0-fold increase in colony number compared to control, respectively. UMSCC-22B treated with cisplatin and rhEpo at 1 or 10 U/ml resulted in ~2.5-fold increase in colony number. A TUNEL assay demonstrated a 30.5% and 76.5% increase in survival in UMSCC-10B and UMSCC-22B cells, respectively, in cisplatin and rhEpo-treated cells compared to cisplatin alone. MTS assay showed similar cytoprotective effects. Western blot revealed increased phosphorylation of Akt upon exposure of HNSCC cell lines to rhEpo. MTS assay and TUNEL analyses implicate Akt as a likely contributor to regulation of rhEpo-mediated cytoprotection.ConclusionsThe results demonstrate that, in HNSCC cells expressing functional EpoR, rhEpo promotes invasion, cell proliferation, and induces resistance to cisplatin, which may contribute to tumor progression.

Activation of PDGFR and EGFR promotes the acquisition of a stem cell-like phenotype in schwannomas.

Objectives Vestibular schwannomas (VS) are benign tumors that arise from unregulated growth of Schwann cells. Both benign and malignant tumors are believed to contain tumor stem cells that are hypothesized to originate from dysregulation of tumor suppressors and oncogenes. We aimed to determine if schwannoma cells express stem cell genes and markers and if activation of the proto-oncogenes epidermal growth factor receptor and platelet-derived growth factor receptor would regulate the stem cell properties of these cells. Methods Immunohistochemical staining was used to determine the expression of stem cell genes in archived VS tissue, immunofluorescence was used to investigate the expression in cell lines, and Western blot analysis was used to measure PDGFR expression in vestibular schwannoma tissue. Upon activation of PDGFR or EGFR in schwannoma cell lines using specific ligands, flow cytometry was used to quantify the side population (SP), stem cell genes were measured using quantitative PCR, and tumorsphere-forming ability was determined. Results Stem cell genes are expressed in vestibular schwannoma tissue and schwannoma cell lines. Activation of both EGFR and PDGFR resulted in increase in the induction of the expression of the stem cell genes Oct-4 and Nanog and marked increase in tumorsphere-forming ability, but only PDGFR activation resulted in an increase in the side population in JS1 cells. Conclusion Dysregulation of EGFR and PDGFR promotes the acquisition of a stem cell-like phenotype in schwannnoma cells that may be critical in vestibular schwannoma tumorigenesis.

Abstract 3378: Metformin protects head and neck cancer stem cells against DNA damage induced apoptosis

The cancer stem cell hypothesis posits that within a tumor exists a distinct subpopulation of cells responsible for tumor initiation, progression, and maintenance. These cancer stem cells (CSCs) tend to be resistant to conventional chemotherapy and, like normal stem cells, possess the ability to self-renew and differentiate, thereby effecting tumor recurrence. Metformin, the most widely prescribed drug for the treatment of diabetes, has received attention in recent years as a potential anticancer agent capable of targeting cancer stem cells through such means as inhibiting cell proliferation and abrogating chemo-resistance. In the current study, we sought to determine the effects of Metformin on a putative head and neck squamous cell carcinoma (HNSCC) cancer stem cell culture. In contrast to the findings of previous studies, our data suggests that Metformin promotes properties of a cancer stem cell phenotype in HNSCC in vitro. Treatment with Metformin resulted in a dose-dependent induction of the stem cell genes CD44, BMI-1, Oct-4, and Nanog, as measured by qPCR. These results were supported by immunofluorescence data. Metformin treatment promoted self-renewal capacity of HNSCC stem cells, as demonstrated by the increase in size and number of tumorspheres formed in non-adherent and non-differentiating conditions. At various doses, treatment with Metformin alone had no effect on cell proliferation, as measured by MTS assay. Furthermore, when administered in combination with cisplatin, Metformin significantly protected against cisplatin-induced cell death, as demonstrated by MTS and TUNEL assays. Immunoblot experiments demonstrating a decrease in Akt phosphorylation upon treatment with Metformin suggests Metformin-mediated cytoprotection is independent of the Akt pathway. Other possible mechanisms are currently under investigation, including Metformin9s ability to regulate autophagy. Although further in vivo studies are necessary, taken together, our findings suggest that Metformin may not be an effective therapeutic option for patients with HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3378. doi:1538-7445.AM2012-3378

Abstract 422: Nanog promotes PI3K/Akt and mTOR-mediated invasion in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) affects nearly 40,000 new patients annually in the United States with approximately 50% of these patients having a poor prognosis. Frequent metastasis of the primary tumor to nearby cervical lymph nodes and local critical structures poses a major problem in the treatment of HNSCC. To date, several molecular mediators of invasion and metastasis in HNSCC have been studied; however, with the emergence of the cancer stem cell hypothesis, other genes normally associated with self-renewal and pluripotency in stem cells would also need to be evaluated for their role in tumor invasion and metastasis. In the present study, we used various functional assays to determine the role of Nanog in HNSCC invasion and metastasis as well as identify the underlying pathway through which Nanog acts to regulate invasion and metastasis. We observed that Nanog overexpression increased invasion and cell migration in two cell lines (UMSCC-10B and UMSCC-22B) while Nanog knockdown using siRNA decreased invasion and cell migration in both cell lines and in a putative HNSCC cancer stem cell culture which expressed high levels of Nanog. Treatment with LY294002 or Rapamycin using doses that did not significantly affect cell viability and proliferation dramatically reversed both invasion and migration conferred by Nanog overexpression, indicating the effect is most likely dependent upon the PI3K/Akt/mTOR pathway. By western blot, we found that Nanog-overexpressing cells expressed higher levels of p-Akt and p-mTOR compared to control cells or Nanog-knockdown cells, confirming regulation of Akt by Nanog. In addition, Nanog overexpression resulted in the induction of MMP-2/MMP-9 as well as EMT genes Snail, Vimentin, and Twist. This enhanced expression of EMT was consistent with the observed increased migration as demonstrated by a wound healing assay. Nanog regulation in a mouse model is currently under way to confirm our results in vivo. The results of our study support the possibility of screening for Nanog expression as a prognostic tool. Meanwhile, further efforts aimed at elucidating the signaling pathway between Nanog and Akt may reveal novel treatment targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 422. doi:1538-7445.AM2012-422

Abstract 4368: Targeting of head and neck cancer stem cells using salinomycin

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cancer stem cells are believed to play a crucial role in cancer recurrence due to their resistance to conventional chemotherapy and capacity for self-renewal. Gupta and colleagues recently reported that salinomycin, a livestock antibiotic, selectively targets breast cancer stem cells. In our study we sought to determine the effect of salinomycin on head and neck squamous cell carcinoma (HNSCC) stem cells. We observed that salinomycin inhibits self-renewal capacity in HNSCC stem cells, as shown by a dose-dependent decrease in sphere formation. In addition, treatment with salinomycin resulted in a decrease in mRNA expression of the stem cell markers CD44 and BMI-1, both of which are critical for maintaining tumorigenicity in HNSCC. In combination with the chemotherapeutic agents cisplatin and doxorubicin, salinomycin synergistically killed HNSCC cancer stem cells more effectively compared to either drug alone, as demonstrated by cell proliferation and TUNEL assays. High-throughput analysis revealed a set of differentially expressed microRNAs in salinomycin-treated HNSCC stem cells, warranting further investigation into the possibility of microRNA-mediated pathways that are regulated by salinomycin. Taken together, our findings indicate promise for using salinomycin or related derivatives as a novel treatment for HNSCC, although further studies are needed to confirm efficacy in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4368. doi:10.1158/1538-7445.AM2011-4368

Abstract 4262: Characterization and regulation of a CXCR4-expressing subpopulation in head and neck cancer stem cells

The cancer stem cell (CSC) hypothesis proposes that only the CSCs within a tumor possess the ability to self-renew and to generate new tumors. Under this model, CSCs are presumed responsible for cellular invasion and metastasis, as well as the recurrence of cancers following chemotherapy. In head and neck squamous cell carcinoma (HNSCC), cells which are CD44 positive and lineage negative (CD44+/Lin-) have been shown to possess properties of CSCs. These cells form tumors in NOD/SCID mice at a much greater efficiency compared to CD44-/Lin- cells, and express the proto-oncogene BMI-1. More recently, it has been shown in pancreatic cancer that a distinct subpopulation of CSCs expressing the chemokine receptor CXCR4 is essential for tumor metastasis. Here we demonstrate the existence of a highly invasive CXCR4+ subpopulation in HNSCC cancer stem cells and elucidate the pathways through which this subpopulation could be regulated. For this study putative CSC cultures derived from primary HNSCC tissue were used from which CXCR4-expressing subpopulations were isolated. A matrigel invasion assay revealed that the CXCR4+ fraction of CSCs was significantly more invasive compared to the CXCR4- fraction. We also surveyed the mRNA expression of these two fractions by qRT-PCR, and found that expression of the proteinases MMP2 and MMP9 was markedly higher in CXCR4+ cells. Interestingly, treatment with nicotine increased the expression of CXCR4 and its ligand SDF-1 in the parental CSC culture, suggesting a possible mechanism by which CXCR4 could be regulated within the CSC population. Nicotine, a major component of tobacco, is known to have proliferative, anti-apoptotic, and angiogenic effects, and has been shown to promote epithelial-to-mesenchymal transition in a variety of human cancers. Mortality from HNSCC remains high due primarily to cancer invasion of vital structures and development of distant metastases. Our findings shed light on the mechanisms by which CXCR4 confers an invasive and metastatic CSC phenotype. In addition, the observed effects of nicotine could provide novel insights towards the regulation of the CXCR4/SDF-1 axis in HNSCC, for which tobacco use has been identified as the leading risk factor. Continued efforts in mapping the molecular mechanisms surrounding CXCR4-mediated invasion and metastasis of CSCs could lead to new and effective treatment strategies for HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4262.