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Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab,the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance. View Publication

PURPOSE: We have previously demonstrated that in pancreatic ductal adenocarcinoma (PDAC)-derived cell lines,the common Src family kinase inhibitors PP2 and PP1 effectively inhibited morphologic alterations associated with TGFβ1-mediated epithelial-to-mesenchymal transition (EMT) by blocking the kinase activity of the TGF-β type I receptor ALK5 rather than Src (Ungefroren et al. in Curr Cancer Drug Targets 11:524,2011). In this report,the ability of PP2 and PP1,the more specific Src inhibitor SU6656,and the ALK5 inhibitor SB431542 to functionally block TGF-β1-dependent EMT and cell motility in established PDAC (Panc-1,Colo 357) and primary NSCLC (Tu459) cell lines were investigated. METHODS: The effects of PP2,PP1,SU6656,and SB431542 on TGF-β1-dependent cell scattering/EMT,cell migration/invasion,and expression of invasion-associated genes were measured by using the real-time cell analysis assay on the xCELLigence system and quantitative real-time RT-PCR,respectively. RESULTS: In all three cell lines tested,PP1,PP2,and SB431542 effectively blocked TGF-β1-induced cell scattering/EMT,migration,and invasion and in Colo 357 cells inhibited the induction of the invasion-associated MMP2 and MMP9 genes. In contrast,SU6656 only blocked TGF-β1-induced invasion in Panc-1 and Tu459 but not Colo 357 cells. PP1,and to a greater extent PP2,also inhibited the high spontaneous migratory activity of Panc-1 cells expressing a kinase-active ALK5 mutant. CONCLUSIONS: These data provide evidence that PP2 and PP1 are powerful inhibitors of TGF-β-induced cell migration and invasion in vitro and directly target ALK5. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics to prevent metastatic spread in late-stage PDAC and NSCLC. View Publication

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted,yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence,in this study,we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2),mild dysplasia (DOK),severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44,p75(NTR),CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44,p75(NTR),CD24 antigens and ALDH activity (ALDEFLUOR(®) assay),with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer,increased FOXA1 and decreased FOXA2 expression correlated with disease severity,but OCT3/4,Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. View Publication

INTRODUCTION: Lung cancer contains a small population of cancer stem cells that contribute to its initiation and progression. We investigated the biological function and clinical significance of aldehyde dehydrogenase 1A1 (ALDH1A1) in non-small-cell lung carcinoma (NSCLC). METHODS: ALDH1A1 assay or small interfering RNA transfection was employed to isolate ALDH1A1+ cells or knock down ALDH1A1 expression in H2087 cells,respectively. Biological functions of ALDH1A1+ and ALDH1A1 silenced cells were investigated using in vitro and in vivo methods. ALDH1A1 expression was analyzed using immunohistochemistry on tissue microarrays with 179 lung cancer tissues and 26 normal lung tissues. RESULTS: The abilities of clone formation,proliferation,cell growth,and migration were increased in ALDH1A1+ and ALDH1A1 silenced cells. ALDH1A1+ lung cancer cells initiated tumors that resembled the histopathologic characteristics and heterogeneity of the parental lung cancer cells in mice. The silencing of ALDH1A1 expression in H2087 lung cancer cells inhibited cell proliferation and migration significantly. ALDH1A1 was expressed in 42% of normal lung tissues (11 of 26),with strong expression in the basal cells and globular cells of the normal bronchus and weak expression in the alveolar epithelial cells. Compared with normal lung tissues,45% of NSCLC samples (81 of 179) were read as positive for ALDH1A1. Positive ALDH1A1 expression was correlated with patients' smoking status (p = 0.022),lymph-node metastasis (p = 0.006),clinical stage (p = 0.004),and a decreased overall survival time (p textless 0.001). Positive ALDH1A1 expression in lung cancer tissues was an independent prognostic factor for NSCLC (odds ratio = 5.232,p textless 0.001). CONCLUSION: Elucidating the biological functions of ALDH1A1 could be helpful in studying lung tumorigenesis and for developing new therapeutic approaches. View Publication

PURPOSE: Prostate cancer cells include a small population of cancer stem-like/cancer initiating cells,which have roles in cancer initiation and progression. Recently aldehyde dehydrogenase activity was used to isolate stem cells of various cancer and normal cells. We evaluated the aldehyde dehydrogenase activity of the human prostate cancer cell line 22Rv1 (ATCC®) with the ALDEFLUOR® assay and determined its potency as prostate cancer stem-like/cancer initiating cells. MATERIALS AND METHODS: The human prostate cancer cell line 22Rv1 was labeled with ALDEFLUOR reagent and analyzed by flow cytometry. ALDH1(high) and ALDH1(low) cells were isolated and tumorigenicity was evaluated by xenograft transplantation into NOD/SCID mice. Tumor sphere forming ability was evaluated by culturing in a floating condition. Invasion capability was evaluated by the Matrigel™ invasion assay. Gene expression profiling was assessed by microarrays and reverse transcriptase-polymerase chain reaction. RESULTS: ALDH1(high) cells were detected in 6.8% of 22Rv1 cells,which showed significantly higher tumorigenicity than ALDH1(low) cells in NOD/SCID mice (p textless 0.05). Gene expression profiling revealed higher expression of the stem cell related genes PROM1 and NKX3-1 in ALDH1(high) cells than in ALDH1(low) cells. ALDH1(high) cells also showed higher invasive capability and sphere forming capability than ALDH1(low) cells. CONCLUSIONS: Results indicate that cancer stem-like/cancer initiating cells are enriched in the ALDH1(high) population of the prostate cancer cell line 22Rv1. This approach may provide a breakthrough to further clarify prostate cancer stem-like/cancer initiating cells. To our knowledge this is the first report of cancer stem-like/cancer initiating cells of 22Rv1 using the aldehyde dehydrogenase activity assay. View Publication

PURPOSE: There are no effective therapies for patients with poorly differentiated papillary thyroid cancer (PTC) or anaplastic thyroid cancer (ATC),and metastasis to the bone represents a significantly worse prognosis. Src family kinases (SFKs) are overexpressed and activated in numerous tumor types and have emerged as a promising therapeutic target,especially in relation to metastasis. We recently showed that Src is overexpressed and activated in thyroid cancer. We therefore tested whether inhibition of Src with dasatinib (BMS-354825) blocks thyroid cancer growth and metastasis. EXPERIMENTAL DESIGN: The effects of dasatinib on thyroid cancer growth,signaling,cell cycle,and apoptosis were evaluated in vitro. The therapeutic efficacy of dasatinib was further tested in vivo using an orthotopic and a novel experimental metastasis model. Expression and activation of SFKs in thyroid cancer cells was characterized,and selectivity of dasatinib was determined using an Src gatekeeper mutant. RESULTS: Dasatinib treatment inhibited Src signaling,decreased growth,and induced cell-cycle arrest and apoptosis in a subset of thyroid cancer cells. Immunoblotting showed that c-Src and Lyn are expressed in thyroid cancer cells and that c-Src is the predominant SFK activated. Treatment with dasatinib blocked PTC tumor growth in an orthotopic model by more than 90% (P = 0.0014). Adjuvant and posttreatment approaches with dasatinib significantly inhibited metastasis (P = 0.016 and P = 0.004,respectively). CONCLUSION: These data provide the first evidence that Src is a central mediator of thyroid cancer growth and metastasis,indicating that Src inhibitors may have a higher therapeutic efficacy in thyroid cancer,as both antitumor and antimetastatic agents. View Publication

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM,a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells,GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However,the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature,which defines aggressiveness and invasion,and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1,which encode a key regulator of selective autophagy,p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs,DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation,thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast,autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally,DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism,in particular reduced ATP and lactate levels. Taken together,these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells. View Publication

This study evaluated the effects of a mammalian target of mTOR inhibitor everolimus alone or in combination with trastuzumab on stem cells from HER2-overexpressing primary breast cancer cells and the BT474 breast cancer cell line in vitro and in vivo. For the in vitro studies,we sorted ESA(+)CD44(+)CD24(-/low) cells as stem cells from primary breast cancer cells and BT474 cells using flow cytometry. The MTT assay was used to quantify the inhibitory effect of the drugs on total cells and stem cells specifically. Stem cell apoptosis,cell cycle distributions,and their tumorigenicity after treatment were investigated by flow cytometry or soft agar colony formation assays. For the in vivo studies,BALB/c mice were injected with BT474 stem cells,and the different treatments were administered. After necropsy,the expression of Ki67,CD31,AKT1,and phospho-AKT (Thr308) was analyzed by immunohistochemistry. For the in vitro studies,Treatment with everolimus resulted in stem cell growth inhibition in a dose-dependent manner. The combination of everolimus with trastuzumab was more effective at inhibiting cell growth (P textless 0.001) and tumorigenicity (P textless 0.001) compared with single-agent therapy. In addition,an increase in G1 cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either of the single-agent groups (P textless 0.01). For the in vivo studies,everolimus plus trastuzumab therapy was much more effective at reducing tumor volume in mice compared with either single agent alone (P textless 0.05). Compared with everolimus alone,the combination of everolimus and trastuzumab reduced the expression of Ki67,AKT1,and phospho-AKT (Thr308) (P textless 0.05). We conclude that everolimus has effective inhibitory effects on HER2-overexpressing stem cells in vitro and vivo. Everolimus plus trastuzumab is a rational combination treatment that may be promising in human clinical trials. View Publication

Glioblastoma (GBM) and other malignant gliomas are aggressive primary neoplasms of the brain that exhibit notable refractivity to standard treatment regimens. Recent large-scale molecular profiling has revealed distinct disease subclasses within malignant gliomas whose defining genomic features highlight dysregulated molecular networks as potential targets for therapeutic development. The proneural" designation represents the largest and most heterogeneous of these subclasses View Publication

Most studies of cancer stem cells (CSC) involve the inoculation of cells from human tumors into immunosuppressed mice,preventing an assessment on the immunologic interactions and effects of CSCs. In this study,we examined the vaccination effects produced by CSC-enriched populations from histologically distinct murine tumors after their inoculation into different syngeneic immunocompetent hosts. Enriched CSCs were immunogenic and more effective as an antigen source than unselected tumor cells in inducing protective antitumor immunity. Immune sera from CSC-vaccinated hosts contained high levels of IgG which bound to CSCs,resulting in CSC lysis in the presence of complement. CTLs generated from peripheral blood mononuclear cells or splenocytes harvested from CSC-vaccinated hosts were capable of killing CSCs in vitro. Mechanistic investigations established that CSC-primed antibodies and T cells were capable of selective targeting CSCs and conferring antitumor immunity. Together,these proof-of-concept results provide a rationale for a new type of cancer immunotherapy based on the development of CSC vaccines that can specifically target CSCs. View Publication

Reversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower,less toxic,doses. Experimentally,high doses induce rapid DNA damage and cytotoxicity,which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses,without causing immediate cytotoxicity,produce an antitumor memory" response View Publication

BACKGROUND: Previously,we enriched a subpopulation of head and neck cancer-derived tumor initiating cells (HNC-TICs) presented high tumorigenic,chemo-radioresistant,and coupled with epithelial-mesenchymal transition (EMT) properties. The purpose of this study was to investigate the therapeutic effect and molecular mechanisms of quercetin on HNC-TICs. METHOD: ALDH1 activity of head and neck cancer cells with quercetin treatment was assessed by the Aldefluor assay flow cytometry analysis. Self-renewal,invasiveness,and EMT capability of HNC-TICs with different doses of quercetin was presented. RESULTS: We first observed that the treatment of quercetin significantly downregulated the ALDH1 activity of head and neck cancer cells in a dose-dependent manner (p textless .05). Moreover,quercetin reduced self-renewal property and stemness signatures expression in head and neck cancer-derived sphere cells. The migration ability of head and neck cancer-derived sphere cells was lessened under quercetin treatment partially due to the decreased productions of Twist,N-cadherin,and vimentin. CONCLUSION: Quercetin suppressing HNC-TICs characteristics may therefore be valuable therapeutics clinically in combination with standard treatment modalities. View Publication