Selena Z. Kuo

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.

Transcriptome sequencing uncovers novel long noncoding and small nucleolar RNAs dysregulated in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma persists as one of the most common and deadly malignancies, with early detection and effective treatment still posing formidable challenges. To expand our currently sparse knowledge of the noncoding alterations involved in the disease and identify potential biomarkers and therapeutic targets, we globally profiled the dysregulation of small nucleolar and long noncoding RNAs in head and neck tumors. Using next-generation RNA-sequencing data from 40 pairs of tumor and matched normal tissues, we found 2808 long noncoding RNA (lncRNA) transcripts significantly differentially expressed by a fold change magnitude ≥2. Meanwhile, RNA-sequencing analysis of 31 tumor-normal pairs yielded 33 significantly dysregulated small nucleolar RNAs (snoRNA). In particular, we identified two dramatically down-regulated lncRNAs and one down-regulated snoRNA whose expression levels correlated significantly with overall patient survival, suggesting their functional significance and clinical relevance in head and neck cancer pathogenesis. We confirmed the dysregulation of these noncoding RNAs in head and neck cancer cell lines derived from different anatomic sites, and determined that ectopic expression of the two lncRNAs inhibited key EMT and stem cell genes and reduced cellular proliferation and migration. As a whole, noncoding RNAs are pervasively dysregulated in head and squamous cell carcinoma. The precise molecular roles of the three transcripts identified warrants further characterization, but our data suggest that they are likely to play substantial roles in head and neck cancer pathogenesis and are significantly associated with patient survival.

The non-coding landscape of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. To enhance molecular knowledge of HNSCC and define a non-coding RNA (ncRNA) landscape of the disease, we profiled the transcriptome-wide dysregulation of long non-coding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) using RNA-sequencing data from 422 HNSCC patients in The Cancer Genome Atlas (TCGA). 307 non-coding transcripts differentially expressed in HNSCC were significantly correlated with patient survival, and associated with mutations in TP53, CDKN2A, CASP8, PRDM9, and FBXW7 and copy number variations in chromosomes 3, 5, 7, and 18. We also observed widespread ncRNA correlation to concurrent TP53 and chromosome 3p loss, a compelling predictor of poor prognosis in HNSCCs. Three selected ncRNAs were additionally associated with tumor stage, HPV status, and other clinical characteristics, and modulation of their expression in vitro reveals differential regulation of genes involved in epithelial-mesenchymal transition and apoptotic response. This comprehensive characterization of the HNSCC non-coding transcriptome introduces new layers of understanding for the disease, and nominates a novel panel of transcripts with potential utility as prognostic markers or therapeutic targets.

Alcohol-dysregulated miR-30a and miR-934 in head and neck squamous cell carcinoma.

BackgroundAlcohol consumption is a well-established risk factor for head and neck squamous cell carcinoma (HNSCC); however, the molecular mechanisms by which alcohol promotes HNSCC pathogenesis and progression remain poorly understood. Our study sought to identify microRNAs that are dysregulated in alcohol-associated HNSCC and investigate their contribution to the malignant phenotype.MethodUsing RNA-sequencing data from 136 HNSCC patients, we compared the expression levels of 1,046 microRNAs between drinking and non-drinking cohorts. Dysregulated microRNAs were verified by qRT-PCR in normal oral keratinocytes treated with biologically relevant doses of ethanol and acetaldehyde. The most promising microRNA candidates were investigated for their effects on cellular proliferation and invasion, sensitivity to cisplatin, and expression of cancer stem cell genes. Finally, putative target genes were identified and evaluated in vitro to further establish roles for these miRNAs in alcohol-associated HNSCC.ResultsFrom RNA-sequencing analysis we identified 8 miRNAs to be significantly upregulated in alcohol-associated HNSCCs. qRT-PCR experiments determined that among these candidates, miR-30a and miR-934 were the most highly upregulated in vitro by alcohol and acetaldehyde. Overexpression of miR-30a and miR-934 in normal and HNSCC cell lines produced up to a 2-fold increase in cellular proliferation, as well as induction of the anti-apoptotic gene BCL-2. Upon inhibition of these miRNAs, HNSCC cell lines exhibited increased sensitivity to cisplatin and reduced matrigel invasion. miRNA knockdown also indicated direct targeting of several tumor suppressor genes by miR-30a and miR-934.ConclusionsAlcohol induces the dysregulation of miR-30a and miR-934, which may play crucial roles in HNSCC pathogenesis and progression. Future investigation of the alcohol-mediated pathways effecting these transformations will prove valuable for furthering the understanding and treatment of alcohol-associated HNSCC.

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.

Evaluation of Non-Coding RNAs as Potential Targets in Head and Neck Squamous Cell Carcinoma Cancer Stem Cells

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and has not seen improved survival rates over the past few decades. Current treatment plans include surgery, radiation therapy, and chemotherapy, but these are relatively ineffective options for recurrent or metastatic tumors. Therefore, there is a high priority for new therapies that specifically target the resistant HNSCC cancer stem cells (CSCs), a subpopulation responsible for tumor initiation and metastasis. Given their vast effects on gene expression and biological processes, including stem cell capabilities, non-coding RNAs (ncRNAs) have become a promising new repertoire of genes to investigate as potential diagnostic or therapeutic targets. This review presents a comprehensive overview of current investigative studies that can contribute to our understanding of the still tentative link between ncRNA and the biology of HNSCC cancer stem cells. In doing so, we aim to analyze the potential role of stem cell-related ncRNAs in the development of molecularly targeted cancer therapy for HNSCC. Although the majority of updated knowledge on HNSCC and ncRNA focuses heavily on microRNA, we chose to give considerable attention to the promise of other classes of ncRNAs (lncRNA, piRNA, and snoRNA), many of which are not yet well characterized or are yet to be discovered, and thus represent a potentially exciting and untapped pool of molecular targets or biomarkers in HNSCC therapy.

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