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Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry,and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling,migration,viability,and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression,whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling,cell viability,motility/migration in vitro,and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S),juxtamembrane (T1010I/R988C),and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with textgreater10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression,increased gene copy number,and mutations. MET inhibition abrogated MET functions,including proliferation,migration/motility,and angiogenesis. MET is a promising,novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored. View Publication

Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds,we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia,myeloma,and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore,oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition,oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models,thereby establishing its ability to target leukemia stem cells. Mechanistically,CPX bound intracellular iron,and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance,CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus,in summary,CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore,CPX could be rapidly repurposed for the treatment of malignancies,including leukemia and myeloma. View Publication

Aldehyde dehydrogenase 1 A1 (ALDH1A1) has recently been suggested as a marker for cancer stem or stem-like cancer cells of some human malignancies. The purpose of this study was to investigate the stem cell-related function and clinical significance of the ALDH1A1 in bladder urothelial cell carcinoma. Aldefluor assay was used to isolate ALDH1A1+ cells from bladder cancer cells. Stem cell characteristics of the ALDH1A1+ cells were then investigated by in vitro and in vivo approaches. Immunohistochemistry was done for evaluating ALDH1A1 expression on 22 normal bladder tissues and 216 bladder tumor specimens of different stage and grade. The ALDH1A1+ cancer cells displayed higher in vitro tumorigenicity compared with isogenic ALDH1A1- cells. The ALDH1A1+ cancer cells could generate xenograft tumors that resembled the histopathologic characteristics and heterogeneity of the parental cells. High ALDH1A1 expression was found in 26% (56 of 216) of human bladder tumor specimens and significantly related to advanced pathologic stage,high histologic grade,recurrence and progression,and metastasis of bladder urothelial cell carcinomas (all P textless 0.05). Furthermore,ALDH1A1 expression was inversely associated with cancer-specific and overall survivals of the patients (P = 0.027 and 0.030,respectively). Therefore,ALDH1A1+ cell population could be enriched in tumor-initiating cells. ALDH1A1 may serve as a useful marker for monitoring the progression of bladder tumor and identifying bladder cancer patients with poor prognosis who might benefit from adjuvant and effective treatments. View Publication

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However,our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore,we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types,n = 792) by immunohistochemical staining. Using the ALDEFUOR assay,ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition,an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct,and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers,we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439,p = 0.0036). Finally,ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker,ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast,lung,ovarian or colon cancer. View Publication

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency,and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3,H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers,such as OCT4 (POU5F1),TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors. View Publication

Use of animal feeder layers and serum containing media in the derivation and propagation of induced pluripotent stem cells (iPSCs) can hinder clinical translation,because of the presence of xeno-material/pathogens. A defined and standardized system would be ideal for generating a homogenous population of iPSCs,which closely resembles human embryonic stem cells (hESCs). This article presents a novel and extensive comparison between in-house produced iPSCs and hESCs under feeder" and "feeder-free" conditions� View Publication

We isolated neural stem cells/neural progenitors (NSC) from 1-day-old rat pups born to mothers fed diets that were deficient or supplemented with n-3 polyunsaturated fatty acids (PUFAs) and compared their proliferation and differentiation in vitro. The cells isolated from the n-3PUFA-deficient pups consistently proliferated more slowly than cells that were isolated from n-3PUFA-supplemented pups,despite the fact that both were cultured under the same conditions. The differences in the proliferation rates were evaluated up until 40 days of culture and were highly significant. When the cells were allowed to differentiate,the deficient cells exhibited a higher degree of neuronal maturation in response to the addition of PUFAs in the medium,as demonstrated by an increase in neurite length,whereas the neurons derived from the supplemented pups showed no change. This result was consistent,regardless of the age of the culture. The properties of the NSC were durably modified throughout the length of the culture,although the membrane phospholipid compositions were similar. We examined the differential expression of selected mRNAs and micro RNAs. We found significant differences in the gene expression of proliferating and differentiating cells,and a group of genes involved in neurogenesis was specifically modified by n-3 PUFA treatment. We conclude that n-3 PUFA levels in the maternal diet can induce persistent modifications of the proliferation and differentiation of NSCs and of their transcriptome. Therefore,the n-3 supply received in utero may condition on a long-term basis cell renewal in the brain. View Publication

BACKGROUND Glycophorin C (GPC) is necessary in the maintenance of red blood cell structure. Severe autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn (HDFN) have been associated with Gerbich (Ge) blood group system antigens expressed on GPC. Previous in vitro studies with cord blood progenitor cells have shown that anti-Ge suppresses erythropoiesis. STUDY DESIGN AND METHODS Here,we evaluated the K562 erythroleukemic cell line to study the cellular effects of a murine anti-GPC. Cell proliferation was evaluated after treatment with anti-GPC. Flow cytometry was used to evaluate exofacial phosphatidylserine (PS) expression and cell viability (propidium iodide binding). Cell morphology was evaluated under light microscopy with cytospin preparations stained with May-Grünwald Giemsa. RESULTS Anti-GPC dramatically inhibited K562 proliferation and increased PS expression,consistent with cytoplasmic blebbing,suggesting evidence of apoptosis. Z-VAD-FMK,an inhibitor of classical apoptosis,was unable to reverse the suppressive effect of anti-GPC. However,hemin was able to attenuate growth suppression. CONCLUSION Together,the data suggest that anti-GPC suppresses erythroid proliferation through the induction of nonclassical apoptosis. View Publication

The incidence of human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) has rapidly increased over the past 30 years,prompting the suggestion that an epidemic maybe on the horizon. Therefore,there is a clinical need to develop alternate therapeutic strategies to manage the growing number of HPV-positive HNSCC patients. High-risk HPV E6 inactivates p53 through two distinct mechanisms; association with E6AP to degrade p53 and association with p300 to block p300-mediated p53 acetylation and activation. In this study,we determined if targeting the E6-p300 interaction is an effective approach to reactivate p53 in HPV-positive HNSCC. Ectopic expression of the CH1 domain of p300 in HPV-positive HNSCC blocks the association between E6 and p300,increases total and acetylated p53 levels and enhances p53 transcriptional activity. Moreover,expression of p21,miR-34a and miR-200c are increased,demonstrating functional p53 reactivation. CH1 overexpression in HPV-positive HNSCC has a global anticancer effect resulting in a decrease in cell proliferation and clonogenic survival and an increase in apoptosis. The in vivo tumor-initiating ability of HPV-positive HNSCC is severely compromised with CH1 overexpression,in part through a reduction in the cancer-initiating cell population. A novel small-molecule CH1 inhibitor,CH1iB,reactivates p53 and potentiates the anticancer activity of cis-platinum in HPV-positive HNSCC cells. Our work shows that CH1-domain inhibitors represent a novel class of p53-reactivation therapeutics for managing HPV-positive HNSCC patients. View Publication

Electrospun poly-l-lactic acid (PLLA) nanofiber mats carrying surface amine groups,previously introduced by nitrogen atmospheric pressure nonequilibrium plasma,are embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1,a biocompatible polyamidoamine with arg-gly-asp (RGD)-reminiscent repeating units. The resultant mixture is finally cured giving PLLA-AGMA1 hydrogel composites that absorb large amounts of water and,in the swollen state,are translucent,soft,and pliable,yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant,since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds,the composites prove capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions. View Publication