Scientific Resources

BACKGROUND: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer,as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence,novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. METHODS: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance,irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover,we identified and isolated CD44(+)CD24(+)ESA(+) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. RESULTS: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore,GLV-1h68 also showed preferential replication in CD44(+)CD24(+)ESA(+) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44(+)CD24(-)ESA(+) cells. CONCLUSIONS: Taken together,our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus,GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors,especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors. View Publication

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

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

INTRODUCTION: The expression of proinflammatory protein tissue transglutaminase 2 (TG2) is frequently upregulated in multiple cancer cell types. However,the exact role of TG2 in cancer cells is not well-understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted cancer stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells. METHODS: Using various mutant constructs,we analyzed the activity of TG2 that is essential for promoting the EMT-CSC phenotype. RESULTS: Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT-CSC in mammary epithelial cells. In contrast,overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover,TG2-dependent activation of the proinflammatory transcription factor NF-κB is deemed essential for promoting the EMT-CSC phenotype in mammary epithelial cells. CONCLUSIONS: Our results suggest that the transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide a strong rationale for developing small-molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways,reversing drug resistance and inhibiting the metastasis of cancer cells. View Publication

The aim is to investigate the clinical implications of the Oct-4 and Nestin protein in human breast cancers. A total of 346 cases including 26 fresh and 320 paraffin-embedded tumor tissues were selected for characterizing the frequency of CD44(+)CD24(-) tumor cells by flow cytometry and the differential expression of the stem cell-related genes between CD44(+)CD24(-) and non-CD44(+)CD24(-) tumor cells was analyzed by PCR Array and immunofluorescence. In comparison with the non-CD44(+)CD24(-) tumor cells,the CD44(+)CD24(-),particularly for those with high percentage of Oct-4(+) and Nestin(+),tumor cells had higher tumorigenicity by forming mammospheres in vitro. More importantly,42 (13.125%) out of 320 tumor tissues were positive for Oct-4 and Nestin staining. Universal analysis and multivariate analysis revealed that the expression of Oct-4 and Nestin was associated significantly with younger age,pathogenic degrees,lymph node metastasis and triple-negative breast cancer independently (P textless 0.05) as well as shorter survival (P = 0.001). Oct-4 and Nestin were important regulators of the development of breast cancer,and Oct-4 and Nestin may be used as predictors for the prognosis of breast cancers. View Publication

PURPOSE: To investigate the distribution of CD44(+)/CD24(-) cells in breast cancers in relation to tumor size before and after the administration of neoadjuvant chemotherapy. METHODS: CD44(+)/CD24(-) tumor cells obtained from breast cancer specimens were characterized in vivo and in vitro using tumor formation assays and mammosphere generation assays,respectively. The distribution of CD44+/CD24- tumor cells in 78 breast cancer specimens following administration of neoadjuvant chemotherapy was also evaluated using immunofluorescence assays,and this distribution was compared with the extent of tumor invasion predicted by Response Evaluation Criteria in Solid Tumours (RECIST). RESULTS: In 27/78 cases,complete remission (CR) was identified using RECIST. However,18 of these CR cases were associated with a scattered distribution of tumor stem cells in the outline of the original tumor prior to neoadjuvant chemotherapy. After neoadjuvant chemotherapy,24 cases involved cancer cells that were confined to the tumor outline,and 21 cases had tumor cells or tumor stem cells overlapping the tumor outline. In addition,there were 6 patients who were insensitive to chemotherapy,and in these cases,both cancer cells and stem cells were detected outside the contours of the tumor volume imaged prior to chemotherapy. CONCLUSION: CD44+/CD24- tumor cells may be an additional parameter to evaluate when determining the extent of breast cancer invasion. View Publication

Mammary cancer stem cells (MaCSCs) have been identified as a rare population of cells capable of self-renewal to drive mammary tumorigenesis and metastasis. Nevertheless,relatively little is known about the intracellular signaling pathways regulating self-renewal and metastatic activities of MaCSCs in vivo. Using a recently developed breast cancer mouse model with focal adhesion kinase (FAK) deletion in mammary tumor cells (MFCKO-MT mice),here we present evidence suggesting a compensatory function of Pyk2,a FAK-related kinase,in the regulation of MaCSCs and metastasis in these mice. Increased expression of Pyk2 was found selectively in pulmonary metastatic nodules of MFCKO-MT mice,and its inhibition significantly reduced mammary tumor development and metastasis in these mice. Consistent with the idea of metastasis driven by MaCSCs,we detected selective up-regulation of Pyk2 in MaCSCs,but not bulk mammary tumor cells,of primary tumors developed in MFCKO-MT mice. We further showed that inhibition of Pyk2 in FAK-null MaCSCs significantly decreased their tumorsphere formation and migration in vitro as well as self-renewal,tumorigenicity,and metastatic activity in vivo. Last,we identified PI3K/Akt signaling as a major mediator of FAK regulation of MaCSCs as well as a target for the compensatory function of Pyk2 in FAK-null MaCSCs. Together,these results further advance our understanding of FAK and its related tyrosine kinase Pyk2 in regulation of MaCSCs in breast cancer and suggest that pharmaceutically targeting these kinases may hold promise as a novel treatment for the disease by targeting and eradicating MaCSCs. View Publication

Little is known about the origin of basal-like breast cancers,an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues,and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless,we found that BRCA1 interacts with the specific p63 isoform ΔNp63γ along with transcription factor isoforms AP-2α and AP-2γ. BRCA1 required ΔNp63γ and AP-2γ to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the ΔNp63 isoforms. In mammary stem/progenitor cells,siRNA-mediated knockdown of ΔNp63 expression resulted in genomic instability,increased cell proliferation,loss of DNA damage checkpoint control,and impaired growth control. Together,our findings establish that transcriptional upregulation of ΔNp63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-ΔNp63 signaling are key events in the pathogenesis of basal-like breast cancer. View Publication

Purpose: The bone marrow microenvironment is considered a critical component in the dissemination and fate of cancer cells in the metastatic process. We explored the possible correlation between bone marrow mesenchymal stem cells (BM-MSC) and disseminated breast cancer-initiating cells (BCIC) in primary breast cancer patients. Experimental design: Bone marrow mononuclear cells (BM-MNC) were collected at the time of primary surgery in 12 breast cancer patients. BM-MNC was immunophenotyped and BCIC was defined as epithelial cells (CD326+CD45-) with a stem-like" phenotype (CD44+CD24low/-� View Publication

BACKGROUND: Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44(+)CD24(lo) that exhibit aldehyde dehydrogenase activity (Aldefluor(+)). We hypothesised that if CSCs are responsible for tumour dissemination,disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore,we assessed the presence of Aldefluor(+) epithelial (CD326(+)CD45(dim)) cells for the presence of the CD44(+)CD24(lo) phenotype in BM of patients with primary breast cancer (PBC). METHODS: BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor(+) epithelial cells with or without CD44(+)CD24(lo) expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls. RESULTS: Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population above the highest level of HD. Furthermore,patients who received NACT were more likely to have percentages of Aldefluor(+) epithelial cells than the highest level of HD (P=0.004). CONCLUSION: The percentage of CD44(+)CD24(lo) CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor(+) epithelial cells by NACT may represent an opportunity to target these cells with novel therapies. View Publication

BACKGROUND: Cancer stem cells (CSCs) have been shown to be a small stem cell-like cell population which appears to drive tumorigenesis,tumor recurrence and metastasis. Thus,identification and characterization of CSCs may be critical to defining effective anticancer therapies. In prostate cancer (PCa),the CD44(+) cell population appears to have stem cell-like properties including being tumorigenic. The enzyme aldehyde dehydrogenase (ALDH) has been found to identify hematopoietic stem cells and our aim was to determine the utility of ALDH activity and CD44 in identifying PCa stem cell-like cells in PCa cell lines. MATERIALS AND METHODS: LNCaP cells and PC-3 cells were sorted based on their expression of CD44 and ALDH activity. The cell populations were investigated using colony-forming assays,invasion assays,sphere formation experiments in a non-adherent environment and 3-D Matrigel matrix culture to observe the in vitro stem-cell like properties. Different sorted cell populations were injected subcutaneously into NOD/SCID mice to determine the corresponding tumorigenic capacities. RESULTS: ALDH(hi) CD44(+) cells exhibit a higher proliferative,clonogenic and metastatic capacity in vitro and demonstrate higher tumorigenicity capacity in vivo than did ALDH(lo) CD44(-) cells. The tumors recapitulated the population of the original cell line. However,ALDHlo CD44(-) cells were able to develop tumors,albeit with longer latency periods. CONCLUSION: ALDH activity and CD44 do not appear to identify PCa stem cells; however,they do indicate increased tumorigenic and metastatic potential,indicating their potential importance for further exploration. View Publication